rms , directly from experiment. charge migration along dna helices may be biologically important because extended electronic states could play a role in the processes of sensing of dna damage and/or dna repair via long-range charge transfer. we measured conductivity and other physical characteristics of several models of natural and diversely damaged molecules of dna. dna polymers were mimicked by various sequences of nucleotide-long double helices with fully watson-crick (wc) paired bases, with several central bases mismatched, and also with chemical modifications that included removal of bases from a few central nucleotides (abasic dna), and neutralization of phosphate charges by their derivatization. the model dnas were investigated by scanning tunneling microscopy, time-resolved thz spectroscopy, raman spectroscopy, circular dichroism, and modeled by molecular dynamics simulations. dna has the highest conductivity in its biologically most relevant double helical form with wc base pairs and negatively charged phosphates equilibrated with counterions. mismatches and all chemical modifications always lower the conductivity. the mechanism of charge transfer is consistent with electron or hole hopping between parallel stacked bases. these observations and data showing that the natural dna has also the most regular double helical form suggest that the continuous base stacking is critical for charge transfer. to control the passage across the bacterial cell wall nature created a large number of "nano"-channels which may act as selective gates for water soluble molecules. here we focus on porins from e. coli which control permeation through interactions with the channel surface. comparing single channel temperature dependent conductance measurements with all atom modeling allow conclusions on the mode of permeation. for example, surprisingly modeling ompf-conductance revealed not only a good agreement with the experiment over a broad range of temperature but also the selectivity for ions. the primary task of porins is to provide facilitated diffusion. we investigated facilitated diffusion of maltooligosaccharide or antibiotics. time resolved conductance measurements allows conclusion on the flux and molecular modeling identifies the limiting interactions with the surface, reveal potential barriers and pathways. exploiting the selectivity of natural or bioengineered channels has promising applications for detecting molecules, characterizing molecular interaction, sequencing dna, protein folding etc. traditionally, studies of diffusion-controlled reaction of biological macromolecules have been made in diluted solutions. however, the high concentration of macromolecules in intracellular environments results into non-specific interactions (macromolecular crowding), which have a great importance on the kinetics and thermodynamics of possible reactions that occur in these systems. in the literature there are studies concerning monte carlo (mc) simulations, giving results that are satisfactory agreement with experimental data, showing, for example, that the protein diffusion in cell cytoplasm is reduced considerably. in addition, there are mc studies about enzymatic reaction, which predict a temporal dependency of the velocity constant in macromolecular crowding. in this work, we try to compare the predicted behavior by mc simulation with the results obtained from the study of the diffusion and reaction of a model protein (alpha-chymotrypsin) using spectroscopic techniques in highly confined media in order to study experimentally the temporal dependence of its diffusion and reaction coefficients. a. paciaroni , a. orecchini , c. petrillo , a. de francesco , f. sacchetti university of perugia, italy, cnr-infm, genova, italy the single-particle and collective dynamical properties of protein hydration water have been studied by neutron scattering experiments in a wide temperature and hydration range. an unprecedented accuracy has been achieved thanks to the availability of a large amount of fully deuterated protein powder and the use of the high-flux spectrometers in and brisp. the protein under investigation was the maltose binding protein (mbp), which is a well-known and widely studied model of biosensor systems. we found that the low-temperature single particle dynamics of mbp hydration water shows clear features that can be traced back to amorphous systems. more in detail, its vibrational density of states is simply described as the superposition of the contributions of low-density and high-density amorphous ice. the quasielastic signal, which appears at the higher temperatures, can be excellently described with a fractional power law which may put in relationship with the peculiarities of fractal systems. quite strikingly, there is a strong similarity, on both the qualitative and quantitative point of view, with the behaviour of hydrated proteins. the collective dynamics of protein hydration water is characterised by the presence of two modes, whose dispersion curves are reminiscent of those of bulk water. however, the relevant damping factors suggest a strong similarity with glassy systems. m. malferrari , f. francia , s. sacquin-mora , g. venturoli università di bologna, bologna, italy, cnrs upr , paris, france the coupling between electron transfer and protein dynamics has been compared in reaction centers (rc) from the wild type (wt) and the carotenoid-less mutant r , by combining brownian dynamics simulations and the kinetic analysis of charge recombination. upon incorporation of the rc into a progressively dehydrated trehalose matrix the electron transfer between the primary photoreduced quinone and the photoxidized donor accelerates progressively and becomes broadly distributed. this behaviour reflects the hindrance of protein relaxation following charge separation and the inhibition of interconversion between conformational substates. in extensively dehydrated matrices the recombination kinetics is two-times faster and three-times more distributed in the wt rc, indicating a larger inhibition of the internal protein dynamics. in line with this findings brownian dynamics simulations reveal a larger rigidity of the carotenoid-containing structure, in which a cluster of residues close to the quinone acceptors is stiffened as compared to the r rc. the in silico and experimental results indicate that the introduction of an internal void in the rc structure has long-range effects on the protein dynamics and that the coupling between the glassy matrix and the rc interior depends markedly on the local mechanical properties of the protein. n. maghelli , v. krstic , n. pavin , f. julicher , i. tolic-norrelykke mpi-cbg, dresden, germany, mpi-pks, dresden, germany in the fission yeast schizosaccharomyces pombe, the nucleus is positioned at the cell center. since the nucleus determines the cell division site, keeping the nucleus at the center is crucial for ensuring symmetrical cell division ( ) . microtubules push against the cell ends and exert force on the nucleus ( ), but how the cell regulates these forces in order to center the nucleus remains unknown. here we tackle this problem by using a combination of live cell imaging, cell manipulations by optical tweezers, and a theoretical model. we show that microtubule pushing forces can center the nucleus because of a larger number of contacts between the microtubules and the proximal cell end than the distal one. moreover, kinesin- motors (klp / ) increase the rate of microtubule catastrophe (transition from growth to shrinkage) in a microtubule length-and contact-dependent manner. thus, the motor behavior results in a longer contact between a microtubule and the proximal than the distal cell end. taken together, our experimental and theoretical results provide a novel centering mechanism, where kinesin- motors increase the efficiency of nuclear centering. electropermeabilization is a commonly used physical method which can induce a transient permeabilization of the cell membrane allowing the entry of therapeutic molecules into the cell and is thus of great interest in the fields of cancer treatment and gene therapy [ ] . however, very little is known about the mechanisms occurring at molecular level. there is clearly some microscopic reorganization of the membrane which is responsible for this change in its transverse transport properties. rather than studying the change of these transport properties, we adopt a simple strategy based on the use of giant unilamellar vesicles and videomicroscopy, as described below. we apply a series of permeabilizing electric pulses to the liposomes, and we observe a size decrease down to a critical radius beyond which their size no longer changes. this decrease in size points to the fact that during the physical processes leading to electropermeabilization, lipids are lost from the vesicles. our results published in [ ] suggest different possible modes for lipid loss, which can be small vesicles, pores, or tubules formation. imaging of brain activation using core techniques as fmri, pet and synchrotron radiation in parallel c. poitry-yamate, g. margaritondo, r. gruetter ecole polytechnique fédérale de lausanne, switzerland functional magnetic resonance imaging (fmri), magnetic resonance spectroscopy (mrs), positron emission tomography (pet) and synchrotron x-ray emission imaging form a highly complementary set of imaging core technologies for studying brain function and energy metabolism. owing to differences in the information each conveys and the temporal and spatial scales on which they measure labeled substances, a single working hypothesis can be tested from different angles to provide a cross-validated, consistent and coherent explanation. the cibm is a unique research facility in europe for advancing our understanding of biomedical processes in health and disease. the housing of a -tesla human magnet, . and . -tesla animal magnets, an animal pet imaging facility and fully-equipped neurochemistry and rf laboratories has enabled us to develop and perform well-targeted experiments around one research theme. in parallel, a longterm collaborative project using synchrotron x-ray transmission and emission imaging at elettra enables us to combine these core technologies towards understanding brain function in vitro and in vivo, from tissue to cells. a brief presentation of these methods will be followed by their application in studying the visual system from man to mouse. p. picone , r. carrotta , d. giacomazza , m. di carlo dip. chimica e tecnologie farmaceutiche, università di palermo, italia, ibf -cnr, palermo, italia, ibim -cnr, palermo, italia diabetes and alzheimer's disease are connected in a way that still is not completely known. diabetes has been implicated as a risk factor for developing alzheimer's disease. some diabetes drugs appear to decrease the cognitive decline associated with alzheimer's disease. it has been recently demonstrated that extracellular injection of insulin is able to protect neurons against a-beta amyloid cell death. one of the proposed theories to explain such an effect is that the hematic glucose levels affect the metabolism of the hippocampus, a part of the brain (associated with memory, emotion and motor skills), which is strongly damaged in alzheimer's patients. the aim of this study is to investigate the effect of insulin on the a-beta induced degeneration and oxidative stress on the neuroblastoma lan cell line. in particular, the present study looks into the role of insulin in the inhibition of abeta specific degenerative apoptotic pathways. preliminary results indicate that insulin dissolved in culture medium in its hexameric form (as tested by absolute scale light scattering) is able to reduce neurodegeneration induced by a-beta amyloid in a dose dependent manner. the link between diabetes and alzheimer's disease may provide new targets for future alzheimer's treatments. moreover, due to the increased incidence of diabetes in western countries, a deeper understanding of such a link is relevant in order to control the escalation in the number of people dealing with dementia. a. pelizzola dipartimento di fisica, politecnico di torino, torino, italy many features of protein folding have been shown to be described by an ising-like model (one-dimensional, with longrange, multispin interactions) whose equilibrium thermodynamics is exactly solvable [ ] [ ] [ ] . we have generalized such a model to the problem of mechanical unfolding. the equilibrium thermodynamics is still exactly solvable, and the characteristic kinetic responses found in force ramp and force clamp experiments are well reproduced [ , ] . unfolding and refolding pathways and intermediates can also be studied, again with good agreement with experiments [ ]. applications to various proteins and rna fragments will be discussed. [ the key role of water in living systems has been widely studied in literature, along with its anomalies, consequence of the extensive three-dimensional hydrogen bonding of water molecules. moreover protein-water interactions take place at protein surface where cell water has been recognized to behave differently from bulky water. the two-states theory of water assumes that water is a mixture of microdomains of different structure and density, the low-density water (ldw) and the high-density water (hdw) domains, and ions partition selectively into ldw or hdw domains. the idea developed in this work was to explore the ordered water structure by measuring delayed luminescence (dl) from salt aqueous solutions in which water structuring is anticipated. it appeared that dl signal from salt solutions is significantly relevant when prevalence of ldw domains is foreseen, with a decay time probability distribution function characterized by a broad maximum in the microsecond range. the obtained results support the ability of dl to reveal the different properties of ldw and hdw domains induced by salt molecules. moreover, the results reveal the existence of clusters, whose characteristics strongly depend on the specific ion effects, of surprisingly long lifetimes not observed till now. this could give new insight into biological water properties. self assembly of patchy particles and dnafunctionalized dendrimers f. sciortino dipartimento di fisica e infm-cnr-soft università 'la sapienza', roma, italy i will report numerical results on the phase behavior of very simple models of patchy particles with the aim of understanding the interplay between phase separation and selfassembly and how the fraction of surface allowing for attractive interactions controls the collective behavior of the system. the case of janus particles, particles characterized by a surface divided evenly into two areas of different chemical composition, will be discussed. i will also discuss the self-assembly of a simple model for four single strands of dna tethered to a central core, and show that the model exhibits a rich phase diagram that includes at least four thermodynamically distinct amorphous phases (polyamorphism) in a one-component system. the dense phases consist of a hierarchy of interpenetrating networks, reminiscent of a woven cloth. peptide dimer motifs in the phospholipid environment -structure, interaction and molecular design p. e. schneggenburger , a. beerlink , t. salditt , u. diederichsen iobc, universität göttingen, germany., irp, universität göttingen, germany. based on recently reported homodimeric peptide pores with a d,l-alternating configuration a novel double helical hairpin-motif of a membrane active gramicidin a analog was designed. [ , ] the cd spectroscopic analyses of the peptide-lipid complexes revealed the structural preservation and elucidated the importance of a zwitterionic interaction of the peptide termini. [ ] the peptide design was enhanced regarding the versatile functionalization with analytical probes as well as molecular recognition moieties like peptide nucleic acids (pnas) to observe the effects of aggregation and specific organization within model lipid membranes even at high peptide-to-lipid ratios. [ ] x-ray reflectivity on lipid bilayer stacks in combination with heavy atom labeling and spectroscopic studies of vesicle systems provides information about the peptide structure and interaction in the native fluid state of the membrane system. [ , ] for this, the fmoc-diiodo-allylglycine building block was created to serve as a novel iodine label pinpointing at a certain position with respect to the membrane normal. we study thermal undulations of giant unilamellar vesicles (guvs) of lipids by flickering spectroscopy. getting values for the mechanical parameters of lipid bilayers requires the experimental fluctuation spectra to be scrutinized in view of the classical helfrich's theory. pure bending modes are revealed unable in predicting the large fluctuations systematically found at high wavevectors. hybrid curvature-dilational modes have been invoked as a more efficient mode of motion in producing high curvatures. a bimodal spectrum of the thermal undulations has been theoretically developed for the shell-like topology. from this new description, two important consequences emerge a priori, the dependence of the fluctuation dynamics on either vesicle size and on bending/compression parameters. for popc and dopc vesicles containing cholesterol the experimental fluctuation spectra are well described by the new spectrum. reconciliation between experiments and theory is achieved when this bimodal spectrum is considered. the new theory opens enormous possibilities for better exploring membrane mechanics in guv models. under normal conditions, platelets circulate in the vascular system having very low interaction with each other and with other cells. the platelets become activated when the biological system is disturbed, for instance by vascular damage in which blood gets in contact with collagen. upon activation, different types of receptors/molecules are exposed on the cell membrane to support adhesion, spreading and aggregation of the platelets onto the damaged vessel. the measurement of altered platelet function is particularly important in cardiovascular diseases such as thrombosis. we are investigating biosensor technologies for the detection of functional properties of platelets. an important study is the specific and non-specific stimulation of platelets in a biosensor cartridge. we will present experimental results on biosensor platelet activation using the platelet-specific membrane markers p-selectin and gp b. multi-joint analysis of locomotion in the first neonatal rats flown in space d. sulica, j. vinersan "carol davila" university of medicine and pharmacy, bucharest, romania the first mammalian neonatal animals in space were the rats flown on the space shuttle endeavor during a -day mission, sts- . the development of locomotion in weightlessness was evaluated using two litters of neonatal rats, launched at postnatal days and . age-and cage-matched animals were used as ground controls. free walking was videotaped from the landing day. although preliminary analysis of walking showed differences in both hindlimb and forelimb joint angles and a hyperextension of the hindlimbs was apparent, the numerical values reached the significance level only for the ankle angle measured at specific moments of the step cycle: foot contact, maximum loading with weight, foot lift and maximum flexion during swing. we report here on the behavior of all the joints during the whole step cycle, by computing the integral of these angles over the step cycle. the results were affected by the differences in the walking speed (the young animals walked faster than the controls), so we scaled the integrals by the step cycle duration. we found that, besides the ankle, the knee was also more extended throughout the whole step cycle in both groups of animals. moreover, all the joints (including the toe and the hip) were affected in the same way (hyperextended), since the differences were still significant when we added together these angles. the animals recovered slowly, with significant differences remaining after days of readaptation. the effect of dextran concentration on red blood cell deposit formation j. strzelecka, b. grzegorzewski department of biophysics, collegium medicum in bydgoszcz, nicolaus copernicus university - bydgoszcz, poland red blood cell (rbc) deposit formation was examined by means of an optical method. blood was obtained from healthy donors and measurements were performed at initial hematocrit %. the intensity of scattered light was measured during sedimentation of rbcs suspended in saline -dextran solutions at different polymer concentrations ( - g/dl). the changes in the intensity of the scattered light manifest rbc aggregate formation, their sedimentation and the process of deposit formation. the deposit formation curve was determined. it is shown that the concentration of dextran affects the deposit formation. an empirical model has been used to describe the experimental data. the parameters of the deposit formation curve as a function of dextran concentration are analyzed. water is essential to life and a major scientific interest lies in a detailed understanding of how it interacts with biological macromolecules in cells. we studied water dynamics in whole cells with neutron scattering [ , , ] . the cellular environment is extremely crowded with macromolecules and water molecules are permanently in close contact to biological interfaces. we measured water dynamics in e. coli and human red blood cells with neutron scattering [ , ] . the data revealed two populations of water in the cells: a major fraction which has dynamical properties similar to those of bulk water (relaxation times ∼ps) and a minor fraction in the order of ∼ % which is interpreted as bound hydration water with significantly slower dynamics (relaxation times > ps). in this contribution we report on investigation of model membrane dynamics by means of quasi elastic and inelastic incoherent neutron scattering and on the effect of membrane inserted pore forming peptide gramicidin. model membrane are realized by highly oriented, hydrated phospholipid bilayer stacks of dmpc ( , -dimyristoyl-snglycero- -phoshatidylcholine) hydrated with d o in excess of solvent condition. the bilayer were supported on mica substrates and prepared at different concentrations of gramicidin, a -residue oligopeptide showing antimicrobial activity by forming pores on the membrane surfaces which allow water and small ions to permeate across the membrane. incoherent qens and ins spectra, measured on in and in spectrometer at ill, allows to obtaining information on the mean dynamics of the hydrogen atoms in the system. moreover, by proper orientations of the membrane plane respect to the scattering wave vector q, we were able to derive information on in plane and out of plane motions of the phospholipids. the using of media products for the creating attracted bioenergetic brain rhythmus advertisement v. i. vlastopulo, v. g. nikolajev str. gen. petrova , app. , odessa, ukraine the technical efficiency of bioenergetical influence of advertisement is present with assistance of consciousness of memory at revision and hearing of advertisement on tv, radio stations, mobile phones, at supermarkets and other places. in a basis of useful model it is put a task to improve the method of creating the attracted advertisement, in which the creation of bioenergetical influence by the oscillation of not less electromagnetic fields or video-images is introduced with their creating as the base on the spatial or flat structure formative macro matrix or matrixes with repeatable structure with the brain α-rhythm frequency of extreme attention and δ-rhythm frequency of meditation. the point of the patent on the device is in the bioenergetical influence increases in addition to the information influence by advertisement of pictures and audio oscillations the bioenergetical influence increases at the consciousness contribution of human memory at the moment of watching or hearing of television, radio stations, working in the internet, music in the supermarkets, banks, clubs, metro and other places. cell adhesion and motility are processes involved in fundamental biological phenomena. they imply multimolecular scaffolds as anchorage points and actin cytoskeleton filaments to build internal stress and eventually crawl onto the substrate. these processes, very dynamic by nature are out of equilibrium. we study cell adhesion on micro-patterned substrates where the introduction of a finite distance between the possible anchorage points of the cell modifies drastically the organization of the cytoskeleton and the anchorage point's distribution. because statistical quantification shows that some shapes are more likely than other, we believe they represent particular organizations of the system which should minimize the energy dissipation. we checked this hypothesis by using the cellular potts model. shapes obtained by simulation are in excellent qualitative agreement with experimental shapes. they depend on phenomenological parameters such as interaction between cells and the extra cellular matrix, a line tension and an elastic modulus. the aim of this work is to link model parameters to physico-chemical properties of cells and to establish phenomenological relations between relevant biochemical regulators controlling the cytoskeleton organization. c. canale, d. ferrera, f. benfenati, l. gasparini the italian institute of technology, genova, italy alzheimer disease (ad) is characterized by cerebral extracellular deposits of β-amyloid (aβ) fibrils. aβ aggregation is a multi-step process involving the formation of various conformational species including soluble intermediate species (i.e. aβ oligomers), protofibrils and fibrils. such aggregates may have various effects on neuronal and glial function and differentially contribute to ad neurodegeneration. aim of this study was to investigate the structural properties of distinct aβ aggregated species and dissect out their effects on neuronal viability. recombinant aβ and aβ peptides were aggregated in vitro in conditions differing by ionic strength, temperature and ph and were analyzed by gel electrophoresis, thioflavin t binding assay and atomic force microscopy (afm). afm analysis was performed using both hydrophilic and hydrophobic substrates, to analyze the full spectrum of structural species. stable low molecular weight oligomers were obtained when aβ was incubated at • c for days in low salt concentration buffer. doughnut-shaped conformational species were detected by afm alongside globular aggregates ( . - . nm height range). acidic ph promoted aggregation of aβ into thioflavin-positive fibrils and protofibrils. protofibrils appeared as beaded chains having a mean height of . ± . nm. effects of aβ on viability of mouse hypppocampal neurons were assessed and correlated with their conformational features. a. bellova , e. bystrenova , m. koneracka , p. kopcansky , f. valle , j. bagelova , f. biscarini , z. gazova institute of experimental physics, slovak academy of sciences, kosice, slovakia, ismn cnr, bologna, italy peptide amyloid aggregation is a hallmark of amyloid diseases including azheimer's disease or type ii diabetes. recent works have addressed the potential of nanoparticles to affect amyloid aggregation. the experimental data are very controversial suggesting that particle characteristics markedly influence the final effect of nanoparticles on the amyloid aggregation (initiation, acceleration or inhibition of amyloid aggregation). we investigate the ability of electrostatically stabilized magnetic nanoparticles of fe o to affect the amyloid aggregation of lysozyme, as a prototype amyloidogenic protein. we have used a combination of spectroscopic (tht fluorescence) and local microscopic techniques (afm). we found, that the ability of magnetic nanoparticles to inhibit formation of amyloid aggregates or destroy pre-formed amyloids exhibit concentrationdependence. the values of inhibition ic and depolymerization dc were determined suggesting that nanoparticles interfere with lysozyme aggregation at stoichiometric concentrations. the observed features make magnetic nanoparticles of potential interest as a therapeutical agent against amyloid diseases. (this work was supported by project of esf and by slovak academy of sciences in frame of cex nanofluid, vega grants , and and eu-strp biodot.). a. bellova , l. balogova , b. chelli , e. bystrenova , f. valle , j. imrich , p. kristian , l. drajna , j. bagelova , f. biscarini , z. gazova institute of experimental physics, sas, kosice, slovakia, faculty of sciences, p. j. safarik university, kosice, slovakia, ismn cnr, bologna, italy numerous diseases have been linked to a common pathogenic process called amyloidosis, whereby proteins or peptide clump together to form amyloid aggregates in the body. an attractive strategy to develop therapies for these diseases seems to be reduction of polypeptide aggregation. we have tested several acridine derivatives characterized by various glycosyl groups for their potential to affect the lysozyme amyloid aggregation in vitro. the ability of glycosyl acridines to interfere with lysozyme aggregation was investigated by tht assay. we found that structure of acridine side chain is factor affecting their anti-aggregation activity significantly. for the most effective compounds the values of ic and dc were obtained. the reduction of protein aggregation was confirmed by afm. to investigate influence of the glycosyl acridines on the cell processes we examined effect of compounds on cell viability. we performed glycosyl acridines characterization by high anti-aggregation activity and low toxicity suggesting their possible application for therapeutical purpose. (this work was supported by project of esf and by slovak academy of sciences in frame of cex nanofluid and vega grants , and and eu-strp biodot.). a. j. beevers, a. m. dixon department of chemistry, university of warwick, uk due to the immense medical importance of proteins which span the membrane of cells, detailed molecular structural information of these systems is essential. practical difficulties in employing high-resolution structural elucidation techniques have resulted in a relative paucity of fully resolved membrane protein structures. therefore a variety of lower-resolution techniques are used to determine structural information of the transmembrane (tm) domains of proteins. one example of such a membrane protein is erbb- , a receptor tyrosine kinase responsible for triggering cell division and which is prone to a mutation in its transmembrane domain resulting in permanent activation and oncogenic effects. we have predicted an interface for the mutated tm domain dimer using site-specific infrared spectroscopy containing a repeating sequence of ile, val and leu . applying the in vivo toxcat assay to the tm domain sequence and to specific mutants of it, confirms this proposed interface whilst another proposed interface is discounted. current studies are focussing on the effect of the tm mutation to the activation of the erbb- receptor and to any possible change in this interface. bacteriophages are complex molecular assemblies which multiplication relies on bacteria infection. the process starts with the binding of the phage on its specific host receptor and the injection of its genome into the host cytoplasm. our work aims to determine the physical mechanisms and forces driving the dna transfer from the phage capsid. the in vitro dna ejection has been analyzed by using light scattering and gel electrophoresis measurements for three phages (t , spp and lambda) belonging to the same family (syphoviridae). our results reveal two forces contributing to drive the dna transfer: the first one is originated from the pressurization due to the strong confinement of dna into the capsid; the second one comes from a pulling mechanism originated by the presence of condensed dna outside the capsid. these two contributions were characterized in in vitro conditions but they likely play a role in the in vivo transfer. the ejection kinetics was also analysed and the characteristic time of the mechanism was studied as a function of the temperature. it appears to follow an arrhenius law, allowing the determination of the activation energy that governs the transfer. the energy values are close for the different phages, suggesting that the mechanism regulating the ejection is common for a given phage family. below these general features, our studies also reveal differences between the three phages. the effect of &beta-amyloid peptide on polymer cushioned membranes s. dante , r. steitz , t. hauss , c. canale , n. a. dencher istituto italiano di tecnologia, genova, italy, bensc, helmholtz center berlin, germany, tu-darmstadt germany beta-amyloid (aβ) is a peptide implicated in the neurodegenerative process characteristic of the alzheimer's disease (ad). to clarify its mechanism of action it is crucial to elucidate the interaction of aβ with the neural membrane. in previous work we demonstrated the capability of aβ to penetrate and perturb stacked lipid bilayers. in this study we considered polymer cushioned lipid bilayers as a model for neural membranes. the polymer cushion is aimed to preserve the membrane natural fluidity; it is obtained depositing charged polyelectrolites layer-by-layer; the lipid membrane is built on the top of the polymer film by fusion of unilamellar vesicles. the floating membranes were kept always in contact to the subphase. the kinetics of adsorption of the lipid double layer at the polymer/water interface was monitored by neutron reflectivity; different experimental conditions to obtain the best surface coverage were exploited. after administration of aβ to the subphase the lipid membrane still adhered to the polymer cushion, but its structure was modified by the interaction with aβ. neutron reflectivity showed a change of the scattering density profile in the direction perpendicular to the membrane plane, suggesting a penetration of aβ inside the double layer. a change in the surface morphology was detected by afm imaging; afm film-rupture experiments showed that aβ weakens the lipid packing. x. cheng , r. pacheco-gomez , a. rodger , h. matthew , d. i. roper university of warwick, u.k., university of birmingham, u.k. ftsz, the ancestral homolog of eukaryotic tubulins, is a gt-pase that assembles into a cytokinetic ring structure (z ring) essential for cell division in prokaryotic cells. the z ring also recruits other proteins (e.g. zapa, ygfe, zipa) to the division site, where they participate in formation of the septum that separates the two daughter cells. we have studied ftsz polymerization and its dynamic behaviour in real time by right angle light scattering. similar to tubulin, ftsz polymerizes into dynamic protofilaments in the presence of gtp; polymer assembly is accompanied by gtp hydrolysis. the kinetics of inorganic phosphate (p i ) released from the gtp hydrolysis have been studied as well, employing a fast and sensitive colourimetric assay. at ph . , approximate % of the p i was released into the media within minutes of gtp addition. the effects of gtp, ph, k + , and mg + were studied in both cases, and the results were used to build up a model for the mechanism of fibre assembly and disassembly. ygfe, a ftsz accessory protein, is identified as a functional zapa orthologue. finally, we have studied the ygfe bundling to ftsz polymers. it strongly promotes ftsz bundling and is an inhibitor of the gtpase activity. many genomes of viruses encode small membrane spanning proteins which are proposed to modify membrane permeability for ions and small molecules. these channel or pore forming proteins are getting into the focus for antiviral therapy since they are essential for some of the viruses. one of the general themes of the mechanism of function of the proteins is to self-assemble to form the functional form. we present a study on the open reading frame (orf) a membrane protein encoded in structural region of human severe acute respiratory syndrome coronavirus (sars-covs). the full length orf a protein is residues long and contains a single transmembrane (tm) domain. full length protein is synthesized using solid phase peptide synthesis and reconstituted into artificial lipid bilayers forms cation-selective ion channels. the bilayer recordings show cation selection channel activity with a major conductance level of around . ps also at elevated temperatures ( . • c). in silico studies with a amino acid tm domain are done to assess conformational space of the monomeric orf a helix. with this monomeric helix homooligomeric helical bundle models are built and embedded in a fully hydrated -palmitoyl- -oleoyl-sn-glycerol- phosphatidylcholine (popc) bilayer. results of both experimental channel recordings and computational modeling show sars orf a to act as a channel forming protein. -biomolecular self-assembly - microtubules are involved in many vital processes. their rigid structure can resist high forces while their intrinsic ability to switch stochastically between growth and shrinkage phases allows them dynamically to reorganise. in cells, a sizeable network of microtubule binding proteins control and regulate microtubule dynamics. alp and dis are members of the dis / xmap family that are major players in s.pombe. the deletion phenotypes of alp and dis are similar, but nonetheless distinct. both are involved in the formation of spindles but alp is also involved in the maintenance of cytosolic microtubules in interphase. the restrictive temperatures of alp -deletion and dis -deletion mutants are different. alp interacts with alp , a potential member of the tacc protein family. i am working to reconstitute alp /dis -dependent microtubule dynamics in vitro, using purified s. pombe tubulin. both alp and dis express well in insect cells and can be readily purified. preliminary data show that both proteins bind tubulin at low salt concentrations and that both influence the dynamics of pig brain microtubules. my goal is a complete functional analysis of alp and dis , individually and in combination, to test candidate molecular mechanisms for alp /dis -catalysis of s. pombe microtubule dynamics. the syphoviridae coliphage t is a well-suited model to study the assembly of large viral capsids. biochemical and biophysical approaches were used to reconstitute in vitro the assembly pathway of its capsid. the t structure was recently solved from cryo-em and image reconstruction. its icosahedral capsid (t = ) is built from the major head protein (pb , copies) forming both the pentons and hexons and from the portal protein (pb , copies) located at one vertex. its assembly proceeds by steps. pb and pb first assemble into a precursor structure called prohead i, which is converted to prohead ii by proteolysis of pb and pb by a head maturation protease. packaging of the kbp dsdna is then driven through the portal pore by a molecular motor, the terminase. this promotes expansion of prohead ii leading to the mature capsid. the different assembly steps and the conformational changes accompanying capsid maturation were characterized using proheads i either self-assembled from the overproduced and purified capsid proteins or isolated from a phage mutant. these precursor capsid structures were analysed by small angle x-ray scattering. the d structure of prohead ii and of the expanded capsid were solved from cryo-em. our data show that the assembly process of a large icosahedral capsid can be efficiently reconstituted in vitro. amyloid beta peptide fibril formation modulated by phospholipid membranes e. hellstrand , e. sparr , s. linse lund univ., department of biophysical chemistry, sweden, lund univ., department of physical chemistry, sweden disease-causing amyloid fibril formation can be modulated by many factors including interactions with biological lipid membranes. an increasing amount of evidence suggests that the process of fibril formation in vivo and the mechanism of toxicity both involve membrane interactions. alzheimer's is probably the most well-known amyloid disease and the associated amyloid beta peptide originates from the membrane incorporated amyloid precursor protein (app). we use recombinant abeta m - and abeta m - produced in escherichia coli, which allows us to perform large scale kinetics assays with good statistics where the amyloid formation process is followed in means of thioflavin t fluorescence. the lipid membranes are introduced in the system as large unilamellar vesicles composed of dopc, dppc and sphingomyelin, with and without incorporation of cholesterol. we find that the phase behanviour of the membrane in the vesicles has a large effect on the lag time of the amyloid formation process for both abeta m - and m - . all membranes increase the lagtime to some degree but dppc has the largest effect. by comparing different phases we can conclude that the translational diffusion in the membrane seems to be more important than the acyl chain ordering. furthermore, electrostatics, concentration dependence and membrane addition at different time points in the amyloid formation process have been investigated. equilibrium/non-equilibrium transitions in macromolecule interactions p. dumas , g. gibrat , s. bernacchi , e. ennifar ibmc-cnrs, strasbourg, france, llb (cea/cnrs), saclay, france usually, so called 'relaxation phenomena' occur on a fast time-scale and 'p-jump' or 't-jump' techniques are required to follow such events lasting (much) less than ms. we report that, during stability studies of proteins or nucleic acids, such relaxation events can be observed on astonishing long time-scales. we first performed 'melting studies' with nucleic-acid duplexes by using linear variations of temperature (t) with time (t). we observed that even very low rates dt/dt could lead to a frozen state for temperature values below a sharp temperature range, and relaxation to equilibrium beyond that range. this allows defining a 'relaxation temperature' t r separating the two regimes. numerical simulations very accurately described the related hysteresis phenomenon observed upon a heating-cooling cycle, which is the hallmark of departure from equilibrium. analogous observations were made with protein oligomers submitted to either a variable pressure, or variable concentration in denaturant. importantly, a single theoretical frame predicts that the critical relaxation value x r (x standing indifferently for temperature, pressure or denaturant concentration) depends on ln(dx/dt). one may ask whether some thermosensor rnas known for switching on or off genetic expression by 'feeling' a temperature variation, might also 'feel' dt/dt. if true, the exact switching temperature would depend on dt/dt and faster temperature changes would increase t r . -biomolecular self-assembly - the major component of amyloid plaques in the gerstmann-sträussler-scheinker disease is a prion peptide fragment from - to - residues. here, we present a structural study of prp - in form of oligomers and fibrils by fourier transform infrared spectroscopy (ftir) and atomic force microscopy (afm). after incubation at • c, the unfolded peptide was found to aggregate into oligomers characterized by intermolecular β-sheet infrared bands and by a wide distribution of oligomer volumes. after a lag phase, a conformational rearrangement of oligomers into fibrils, with a parallel orientation of the cross β-sheet structures, was observed. by afm, different morphologies were also detected for fibrils that displayed high heterogeneity in their twisting periodicity and a complex hierarchical assembly. in addition, we also studied thermal and random aggregation. the prp - peptide was found to undergo several aggregation pathways, whose end products display different structural properties and intermolecular interactions. these findings underline the high plasticity of the prion peptide, a peculiar feature of prion proteins to overcome species barriers (natalello et al. j.mol.biol. ; : - ). the role of proline isomerisation in the aggregation process and fibril formation of alpha-synuclein j. meuvis , m. gerard , v. baekelandt , y. engelborghs lab.of biomolecular dynamics, ku leuven, belgium, lab.of biochemistry, campus kortrijk, belgium, lab.for neurobiology & gene therapy, ku leuven, belgium alpha-synuclein (α-syn) plays a central role in parkinson's disease. the aggregation of this protein, which contains five proline residues (p ,p ,p ,p ,p ), is accelerated in vitro by fk binding proteins (fkbps), a family of enzymes with a peptidyl-prolyl cis-trans isomerase activity (ppiase). fkbps catalyze the cis-trans conformational change of proline, often a rate limiting step in protein folding. to elucidate the role of the proline residues in aggregation, we constructed a mutant p( , , , , )a α-syn . the kinetics of the aggregation of the mutant were studied with turbidity and thioflavin t fluorescence (tht). turbidity measurements show the formation of early, tht negative aggregates which is as fast for both wt and mutant. fibril formation however is faster for the proline-deficient mutant. we also studied the effect of fkbp on the aggregation of the mutant. although wt α-syn early aggregate formation is accelerated by the addition of µm fkbp , this effect disappears in the mutant. addition of ( pm- µm) fkbp accelerates the fiber formation of wt α-syn, which is abolished in the mutant. we can conclude that α-syn fiber formation is accelerated for the proline-deficient mutant, which suggests a role for the proline residues in fiber formation. furthermore all accelerating effects of fkbp are abolished in the mutant which suggests that the ppiase activity of fkbp is responsible for the accelerating effect on the aggregation of wt α-syn. materials used as gene delivery vehicles must be able to condense dna into small sizes to facilitate transport and crossing various barriers. one of the polycations investigated for dna compaction is chitosan, which has the advantage of being safe and biodegradable. as a step towards reducing the aggregation behaviour of dna-chitosan complexes, chitosans were modified by grafting peg-chains on the backbone. it is known that the transfection efficacy depends on the chitosan chain length. additionally, the degree of pegylation might influence the condensation process. here a systematic biophysical study of pegylated chitosans and how the interplay between chitosan chain length and degree of pegylation affect the compaction of dna in terms of particle size and structure, stability in pbs and when exposed to serum, and transfection efficacy is presented. three different chain lengths of chitosans are employed, and for each sample three pegylation degrees are investigated and the properties of the dna-pegchitosan complexes compared to complexes formed when employing the original, chitosan for dna compaction. it is found pegylation of chitosans can be used to increase both the stability of the dna-chitosan complexes when exposed to serum as well as increase their transfection efficacy in hek cells. max-planck institute for polymer research, mainz, germany model membranes mimic the essential function of a natural membrane. however, the complexity is reduced in order to allow the study of fundamental processes. tethered membranes consist in principle of a lipid bilayer that is covalently linked to a solid support through a spacer group. this architecture allows the characterization of the membrane itself as well as of incorporated membrane proteins using surface analytical techniques. we have established a versatile system of various anchor lipids, which allow membrane formation on different surfaces. the architectures have been characterized by surface plasmon techniques, neutron reflectivity and electrochemical methods. the membranes are electrically insulating and allow for the functional incorporation of ion channel proteins. polymerizable lipids allow to pattern the membrane and to study lateral diffusion processes. furthermore, the membranes can be used as a sensing platform, where embedded membrane proteins act as actual sensing units. a. perico, s. pietronave, l. arcesi, c. d'arrigo consiglio nazionale delle ricerche (cnr), institute for macromolecular studies (ismac) the electrostatic free energy (fe) of two parallel rigid likecharged polyelectrolytes (pes) is given as a function of the separation distance. for high linear charge density, z , the fe shows a minimum due to the increasing of the counterion condensation and condensation volume as the two pes approach. the interaction fe is governed by a critical linear charge density, z c , inversely proportional to the counterion valence. for highly charged pes (z > z c , like dna), the pes attract the stronger the smaller is the counterion valence, because the fe is dominated by the entropic term due to condensation of counterions in a volume displaying a maximum at short distances. for weakly charged pes (z < z c / ) the pes remain undercritical in the whole separation range and therefore repel. for moderately charged pes (z c / < z < z c ), the infinitely separated pes are undercritical but become supercritical as they approach a critical distance and charge condensation and condensation volume expansion start: in these circumstances the pes may attract if the counterion valence is sufficiently large. in the case of many dna rods, hexagonal clusters may be formed. upon interaction with hydrophobic surfaces, proteins show a tendency to expose regions that are normally buried in the hydrophobic core. unfolding is generally perceived as an undesired process in studies aimed to anchor functional proteins at surfaces. upon an upset of perspective the fine control of the unfolding/re-assembly process could be regarded as a strategy to build up molecular nanostructures for the development of organic-inorganic assemblies. we show that molecular layers patterned at the nanoscale, with longrange order properties extending over the microscopic scale, can be obtained upon adsorption of proteins onto the hydrophobic and ordered surface of pyrolytic graphite. upon adsorption, proteins lose their native folding and polypeptide chains re-assemble on the surface in a layered fashion, forming a molecular bilayer. the first layer, in contact with the substrate, and the second molecular layer show corduroy-like nanopatterns of different periodicity, with a relative orientation between the first and second layer patterns of • . surface-induced protein unfolding and polypeptide chain reassembly according to a layered ordered structure is a rather general phenomenon since it is observed for different proteins irrespectively of their specific structural properties. the possibility of using these ordered molecular structures as templates for the subsequent patterned deposition of supramolecular aggregates will be discussed. understanding protein-protein interactions and assemblies to control the hierarchical building of well-ordered supramolecular structures is highly relevant to new tailormade biomaterials. we previously evidenced that contrary to native calcium-loaded α-lactalbumin (holo α-la), calcium-depleted form (apo α-la) has the ability to selfassemble with lysozyme (lys) to form different supramolecular structures in temperature-dependent manner. in the present work, the events occurring at molecular scale were explored using fluorescence techniques. fluorescence anisotropy and fluorescence lifetime measurements provide a powerful and sensitive mean to measure intermolecular interactions. we showed that lys interacts with both apo α-la and holo α-la to form oligomers, assumed to be heterodimers, at • c and • c. the dissociation constants for dimerization, found to be in the µm range, were sensitive to the ionic strength. correlation time calculations suggest that formed heterodimers holo α-la/lys and apo α−la/lys differed in their shape and/or conformation. such conformation differences could explain why holo α-la/lys complexes are trapped as heterodimers while the apo α-la/lys complexes have the ability to further self-assemble into previously reported various supramolecular structures. polyglutamine aggregation and neurodegeneration g. nicastro, l. masino, a. pastore national institute for medical research, the ridgeway, nw aa london, u.k. polyglutamine (polyq) diseases are rare but dominant misfolding diseases linked to neurodegeneration. they are caused by the expansion of cag codon repeats, which encode polyq tracts in the corresponding gene products. aggregation of polyq proteins is thought to be triggered by polyq expansion but be strongly modulated by the protein context. in the attempt of understanding the molecular bases of polyq diseases, we are studying the structures, interactomes and aggregation properties of selected polyq proteins. here, we present recent work on ataxin- , taken as a representative example of the whole family. ataxin- is a ubiquitin specific cysteine protease, involved in the ubiquitinproteasome pathway and known to bind poly-ubiquitin chains of four or more subunits. the enzymatic site resides in the n-terminal josephin domain of ataxin- . we have characterized, using different biophysical techniques, the structure in solution and the aggregation properties of josephin both in isolation and in a ubiquitin complex. we demonstrate that interaction with ubiquitin strongly modulates the aggregation properties of ataxin- and suggest the importance of protein-protein interactions in preventing aggregation. our study also provides new insights into the molecular mechanisms which determine ataxin- specificity for poly-ubiquitin chains of the correct length and cross-linking. förster resonant energy transfer (fret) from an optically excited to a non-excited molecule has been widely used to probe molecular interactions in living cells. changes in the molecular makeup of a cellular region occurring during the acquisition of fluorescence images place tight constraints on the fret technology and data analysis, which could not be addressed satisfactorily until recently. we will describe a method for imaging protein complex distributions in living cells with sub-cellular spatial resolution, which relies on a spectrally resolved two-photon microscope (raicu et al, , nature photon. : - ) and a simple theory of fret in oligomeric complexes (v. raicu, , j. biol. phys. : - ) . then, we will overview recent results on the determination of the supra-molecular structure and distributions in living cells of oligomeric complexes of some g protein-coupled receptors. observing protein aggregates on surfaces m. rabe, d. verdes, s. seeger institute of physical chemistry, university of zurich, switzerland protein aggregation is an important topic of current protein research as it is associated with several human diseases including alzheimer's disease, parkinson's disease, and type ii diabetes. although protein aggregation mechanisms and conditions have been comprehensively investigated, studies on the formation and the fate of protein aggregates in contact with solid interfaces are scarce. we have comprehensively investigated the structure of protein assemblies that form spontaneously upon protein adsorption on solid interfaces using a surface sensitive fluorescence imaging technique based on super critical angle fluorescence (saf) detection. combining this technique with fret we not only succeeded to detect protein aggregates deposited on surfaces but also to characterize their behavior in real time, i.e., their emergence, growth, or spreading. the model protein bsa, for instance, was found to exhibit a certain tendency for aggregation in the buffer solution. these protein clusters can deposit onto solid surfaces and spread resulting in a large, flat structure after some time. a different possibility how protein aggregates emerge on surfaces consists of a direct deposition of protein monomers to pre existing aggregates. such a growth of protein aggregates on the surface has been observed in a model system using the protein α-synuclein, which is tightly associated with the parkinson's syndrome. we present the results of a spectroscopic ellipsometry (se) study of the adsorption process of yeast cytochrome c (ycc) on gold and graphite substrates, according to methods already applied to study the growth dynamics of organosulphur sams on gold [ ] . se investigation was carried out both in situ, at room temperature during protein deposition, and ex situ. on gold, se data demonstrate the formation of an about - nm thick layer, consistent with the formation of a dense monolayer of ycc molecules, confirmed by afm inspection. both in situ and ex situ measurements were characterized by well defined spectral features related to the soret band. analysis of the fine position of this feature allowed to obtain information on the oxidation state of the iron ion of the heme group. se data suggest that proteins have preserved their native structure. a completely different adsorption mechanism was observed on highly oriented pyrolytic graphite (hopg) [ ] . ex-situ se data on ycc/hopg, supported by afm observations, indicate the formation of an ultrathin molecular layer (∼ . nm) related to complete protein unfolding at the hydrophobic surface. the role of phospholipid anisotropy in the stability of inverted hexagonal phase was considered. the equilibrium configuration of the system was determined by the minimum of the free energy involving the contribution of the isotropic and deviatoric bending and the interstitial energy of phospholipid monolayers. the shape and local interactions of a single lipid molecule were taken into account. the minimization with respect to the configuration of the lipid layers was performed by the monte carlo simulated annealing method. at high enough temperature the lipid molecules attain a shape exhibiting higher intrinsic mean and deviatoric curvatures which fits better into the inverted hexagonal phase than into the lamellar phase. for the mathematical model the advanced geometry with non-spherical cross-section of inverted hexagonal phase was calculated, resulting in lower energy in non-spherical cross-section. theoretical results are in a good agreement with the small angle x-ray scattering experimental data. for a long while the conventional view has been that alzheimer disease is brought about by the beta-amyloid fibrils found in the senile plaques, but more recently it has been suggested that the main neurotoxic species would be the soluble oligomeric species, apparently prone to interact with cell structures and macromolecules potentially inducing neuronal dysfunction. peptide-peptide interactions resulting in self-assembly phenomena of beta-amyloid yielding fibrils can be modulated and influenced by small organic molecules that might also be effective therapeutic tools to ideally target both oligomeric and fibrillar species. in this perspective, polycyclic aromatic molecules are of special interest because they might disrupt the molecular architectures precursors of beta-amyloid fibrils by means of weak, non-covalent aromatic interactions, like stacking interactions. we have performed an in vitro spectroscopic study (light scattering, circular dichroism, ftir and fluorescence) of the effects on beta-amyloid fibrillogenesis of the natural pigment hypericin extracted from hypericum perforatum. our results show that, thanks to its structural characteristics and peculiar spectroscopic features, hypericin can be easily used to in vitro monitor the appearance of initial aggregation states of beta-amyloid peptides and, more importantly, that hypericin can interfere with the early stages of polymerization process, playing the role of an aggregation inhibitor. peptaibols are peculiar peptides produced by fungi associated to plants. they are composed by to amminoacidic residues and exhibit antibiotic and antifungal properties. due to their amphypatic nature, they can form ion channels in biological membranes. by making use of experimental models of biological membranes (biomimetic membranes) currently employed in the laboratory of bioelectrochemistry, and models of plant membranes (corn seed root), that are used in the international laboratory of plant neurobiology (linv), we characterized synthetic peptides such as trichogin gaiv and its shorter homologues ( and residues). we studied these peptaibols in a dioleoylphosphatidylcholine monolayer supported by hg using different electrochemical techniques (ac,vc,eis). the experimental technique employed in the linv (clark microelectrode coupled to mife system) allows to measure oxygen flux in the solution contacting plant cell membranes, after treatment with different peptide concentrations. preliminary results might indicate that short peptides can influence the whole metabolism of the plant and can therefore be used as "elicitors" in order to induce an acquired systemic resistance. supported biomimetic membranes (sbm) were developed for protein-membrane interactions studies. phospholipid vesicles were chemically linked onto amine grafted gold or glass surfaces; after an osmotic choc and liposomes fusion a continuous membrane bilayer was formed. the anchoring phospholipid molecule (dspe-peg-nhs) incorporated into the vesicles allowed the formation of a water-filled compartment between the surface and the bilayer. this first sbm model was used to monitor the membranes binding properties (dependent of calcium) of the adenylate cyclase toxin (cyaa) from bordetella pertussis. the sbm model was improved in order to study the translocation of the catalytic domain of cyaa across the bilayer. naturally, the cyaa catalytic domain, when it reaches the target cell cytosol, associates with intracellular calmodulin (cam) an activator of the adenylate cyclase activity of cyaa. to mimic this biological phenomenon, cam was first immobilized on the surface (gold or glass) and in a second step membrane construction was performed over the cam layer. the formation of the biomimetic membrane onto the cam layer was monitored by spr while membrane fluidity and continuity were analysed by fluorescence. our results demonstrated the potentialities of sbm for the study of protein insertion into and translocation across biological membranes. a multi-resolution approach to the structure and function of integrin αiibβ m. rocco , c. rosano , j. w. weisel , d. horita , r. r. hantgan istituto nazionale per la ricerca sul cancro (ist), genova, italy, university of pennsylvania, philadelphia, pa, usa, wake forest university, winston-salem, nc, usa. integrins are heterodimeric transmembrane receptors involved in mechanical anchoring and two-way signaling. each α and β subunit has a modular structure with a large extracellular portion, a single transmembrane region, and a cytoplasmic domain. integrins activation mechanism is regulated by controversial conformational changes: while crystallography revealed similar bent shapes for resting and primed extracellular region constructs, ligand binding-induced large structural rearrangements in smaller constructs suggested extension, "opening" and tails separation. in a multiresolution approach, we used experimental and computed hydrodynamics to discriminate among αiibβ integrin models built on x-ray, nmr, and em data. in contrast with xray data and d em maps, an extension is needed to match the hydrodynamics of full-length, solubilized αiibβ ; an electron tomography-based model fares better. consistent with that, and with our averaged d em images, a conformational change in the head region (β hybrid domain swingout) coupled to a simple transmembrane helices shift matches priming agents-induced frictional changes in full-length αiibβ . our multi-resolution study thus suggests that in integrins extension and immediate tail separation are uncoupled from head domain rearrangements following activation. -biomolecular self-assembly - stabilizing effects of α s -casein, a natively unfolded protein, on the aggregation of biomolecules a. trapani, r. carrotta, p. l. san biagio, d. bulone ibf-cnr palermo, italy α s -casein is one of the four types of caseins, a group of calcium phosphate-binding proteins that, in the form of micellar aggregates, makes up the largest protein component of bovine milk. the structure of α s -casein is that of a triblock polymer with a hydrophilic tract interposed between two hydrophobic blocks. due to the lack of a compact folded conformation, this protein can be classified as one of the intrinsically disordered (or natively unfolded) proteins. this class of proteins is known to exert a stabilizing activity on biomolecules through specific interaction with hydrophobic surfaces that partially unfolded molecules may expose to the solvent. here we present results on α s -casein effects on the thermally induced aggregation of gluthathione stransferase, a ligand-binding anzyme, and - β-amyloid peptide involved in alzheimer's disease. by means of light scattering and circular dichroism experiments, we attempt to reveal the molecular details of α s -biomolecules interaction. bacterial protein self-assembly on surfaces of well-defined chemistry s-layers are one of the most common cell envelope components of prokaryotic organisms and represent the simplest biological membrane developed during evolution. these (glyco)proteins, which can self-assemble into -d crystalline nanostructures on lipid films, liposomes, and polymers, play already an important role in nanobiotechnology. in this work, we present new findings concerning the recrystallization of bacterial proteins on substrates with defined chemistry. manipulation of the protein-sample interaction was carried out by changing the relative height of oh and ch terminated moieties in self-assembled monolayers (sams). we have found that differences in chain length lead to: i) protein bilayer-protein monolayer transition, ii) preferential protein side adsorption, and iii) increase of the crystal lattice parameters. further manipulation of the protein-sample interaction was achieved by using silane chemistry. we will show that sample hidrophobicity speeds up recrystallization kinetics and reduces the crystalline domain size (and layer compliance). the protein-adsorbed mass per unit area on these substrates is reported for the first time. self-assembly of phenylalanine oligopeptides: insights from experimental and computational studies p. tamamis , l. adler-abramovich , m. reches , k. marshall , p. sikorski , l. serpell , e. gazit , g. archontis dpt. of physics, univ. of cyprus, cyprus, dpt. of molecular microbiology and biotechnology, tel aviv univ., israel, dpt. of biochemistry, univ. of sussex, u.k. the diphenylalanine peptide (ff) forms well ordered nanotubes and its derivatives form nano-assemblies of various morphologies with promising material applications. we demonstrate for the first time by electron and laser microscopy and ftir spectroscopy that the related, triphenylalanine peptide (fff) assembles into rather planar nanostructures, rich in β-sheet. in addition, we conduct . -µs replica exchange m.d. simulations of aqueous ff and fff solutions in implicit solvent. the peptides coalesce into aggregates and participate frequently in open or ring-like linear networks, as well as elementary and network-containing structures with β-sheet characteristics. polar and nonpolar interactions, as well as the surrounding aggregate medium contribute to the network stabilities. within a network, consecutive peptides are linked by head-to-tail interactions; the aromatic sidechains of neighbor peptides assume approximately "t-shaped" orientations. these features are observed in ff crystals and could characterize early formations, or stabilize the mature nanostructures. the fff aggregates acquire higher stability and peptide-network propensity compared to the ff aggregates due to energetic contributions , . chlorophyll biosynthesis is light-dependent in angiosperms because the reduction of protochlorophyllide (pchlide) into chlorophyllide is driven by a photoenzyme, nadph:pchlide oxidoreductase (por). the unique properties of por are due to its ability to assemble into dimers and oligomers within the prolamellar body (plb) membranes of etioplasts studied mainly in leaves of dark-grown seedlings under laboratory conditions. we extended these studies to plant organs developed in the nature: cabbage heads, leaf primordia inside buds, pericarp-covered regions of sunflower cotyledons, potato tubers and seedlings germinating under the soil. in electron microscopic and fluorescence spectroscopic studies we found in many of these organs poorly developed plbs in which por was mainly in monomer state. as a consequence, the chlorophyll accumulation was slow and photo-oxidation processes occurred at illumination. in vitro we artificially induced the aggregation of por monomers into oligomers in glycerol and sucrose containing buffers. this resulted in the increase of the photoreduction rate at the expense of photo-oxidation. these results underline the importance of the self-assembly of por and the plbs in chloroplast development and chlorophyll synthesis in nature. v. vetri , g. ossato , v. militello , m. a. digman , m. leone , e. gratton dsfa, university of palermo, palermo, italy, lfd, university of california, irvine, ca, usa we report an experimental study on concanavalina (cona) aggregation in live cells. in vitro, close to physiological temperature, cona readily forms fibrils involving secondary structure changes leading to β-aggregate structures. the effect of cona on cell cultures and formation of protein aggregates were measured by confocal fluorescence microscopy. in particular, we monitored protein aggregation in live cells by means n&b analysis, cross-n&b and rics. n&b showed the aggregation kinetic and the progressive formation of cona oligomers at cell surface. this suggests that, at cell membrane where local concentration is higher, nucleation sites for aggregation are provided. in parallel, the morphology of the cells changes indicating the progressive cell compaction and death. aggregation and binding of small aggregates to the cell surface were assessed by rics: it is possible to distinguish regions where small aggregates are diffusing and regions where they are bound to the cell. oligomers formation may stimulate non-specific cellular responses due to the exposure of reactive regions of protein structure and of progressive formation of cross−β structures. moreover, aggregates stoichiometry was measured during the kinetic by cross-variance n&b. the two conductive pathways of p x purinergic receptor: different modulation and selectivity r. barbieri , s. alloisio , a. di garbo , m. nobile institute of biophysics, cnr, via de marini , genoa, italy, institute of biophysics, cnr, via g. moruzzi , pisa, italy the p x purinoceptor (p x r) is an atp-gated cation channel that is able to activate a cell permeabilizing pore. p x r cytosolic c-terminal tail is thought to modulate this function. this study was aimed to characterise the biophysical properties of p x r compared to those of the variant lacking the entire c-terminus tail (trp x r) by measuring whole-cell currents and intracellular ca + variations. a mathematical model is used to describe the experimental results. in p x r expressing hek- cells, the potent agonist '-o-( -benzoyl)benzoyl adenosine '-triphosphate (bzatp) -evoked ionic currents depending on concentration and frequency of agonist applications. the currents were strongly inhibited by extracellular mg + in a noncompetitive way. by contrast, in trp x r cells, only high bzatp concentrations elicited small currents not affected by mg + . interestingly, bzatp-induced ca + influx was present both in p x r and in trp x r cells, albeit in the latter the intracellular ca + elevation was smaller. importantly, in trp x r the intracellular ca + rise maintained a competitive mechanism of mg + inhibition similar to that observed in p x r. the experimental data and the modelling findings support the tenet of a functional structure of p x r possessing two distinct conductive pathways. the review of our data on the effects of physical and chemical weak signals on physicochemical properties of water, cell volume, activity and the number of membrane proteins (receptors, ionic channels and enzymes, na + /k + pump and na + /ca + exchanger), intracellular signal systems in norm and pathology (cancer and nerve disorders) would be presented. light microscopic, cell voltage-and patch-clamp, isotope, standard biochemical and genetic engineering methods were used. weak signal-induced effects on cell functional activity (intracellular enzymes activity, the number of functionally active membrane proteins) are realized by changing the physicochemical properties of extra-and intracellular aqua medium. the latter induces the modulation of na + /k + pump-induced cell hydration, which serves as a primary mechanism through which the autoregulation of pump and regulation of membrane excitability and chemosensitivity are realized. by genetic engineering method in oocytes it was shown that the correlation between na + /k + pump and na + /ca + exchanger, which is realized through intracellular messenger systems, plays a crucial role in weak signals transduction in cells and determination of aging-induced increase of cell pathology. listeriolysin pore forming ability in planar lipid membranes at different ph listeriolysin o (llo) is a cholesterol-dependent cytolysin secreted by the intracellular pathogen listeria monocytogenes. its main task is to enable escape of bacteria from the phagosomal vacuole into the cytoplasm. llo exhibits optimal cytolytic activity at low ph but it is still able to bind membranes at physiological or even slightly basic ph values in a cholesterol-dependent fashion. high cholesterol concentrations in the membrane restore the low activity of llo at high ph values. based on this broad ph activity we investigated the electrophysiological properties of pores formed by llo at room temperature and at different phs using planar lipid bilayer technique. llo is able to form pores both at ph . and . with a similar permeabilizing ability and similar heterogeneous conductances in the range of picosiemes to nanosiemens. cholesterol content directly correlates with llo activity but it does not change the pore characteristics. collectively, our results demonstrate that llo activity at physiological ph cannot be neglected and that its action at sites distal to cell entry may have important physiological consequences for listeria pathogenesis. s. aimon, g. toombes, p. bassereau institut curie, paris, france the physics of membrane/channel and channel/channel interactions is difficult to investigate in cells where it is nearly impossible to modify relevant parameters to deduce physics laws. to overcome these difficulties we built a model system in which voltage gated ion channels were reconstituted in giant unilamellar vesicles (guvs) for the first time. as a first step, we successfully expressed kvap (a voltage gated potassium channel) in e-coli. the channel was purified, fluorescently labelled and reconstituted in small liposomes. its functionality was checked with electrophysiology via fusion of these liposomes into black lipid membranes (blm). as a second step, guvs were formed from these small proteoliposomes using electroformation in a buffer containing mm kcl salt. the proper incorporation of proteins into guvs was controlled using confocal microscopy. functional proteins were detected using the patch clamp technique. with our protocol, we are thus able to prepare guvs containing functional voltage-gated ion channels. one goal is now to study the effect of channel activity on its spatial distribution in these guvs. ryr activation in cultured shr cardiomyocytes at the end of the prehypertensive period the rate of [ca + ] i elevation after the ryanodine receptor (ryr) activation by -chloro-m-cresol ( -cmc) and l-type ca + channels (dhpr) activation by bay k was studied in cultured ( days) cardiomyocytes of spontaneously hypertensive (shr) and normotensive rats (wky, wistar) during weeks of postnatal development. the differences in dh-prs and ryrs activities began to be evident after weeks age when cicr formation has finished and became more expressed at the end of prehypertensive period ( weeks). in response to -cmc ( mm), a drastic increase in the rate of [ca + ] i accumulation ( . ± . times) in shr myocytes was registered after days age versus a decrease in the rates of ca + efflux from the sarcoplasmic reticulum of wistar and wky rat cardiomyocytes. bayk ( µm) also induced more sharp [ca + ] i elevation in shr myocytes ( . ± . times) as compared with wistar ( . ± . times) and wky ( . ± . times) ones of the same age. our results argue that in shr and wky cardiomyocytes, as opposed to normotensive wistar rats, gradual growth of dhpr activity is observed, which follows in parallel with cicr formation in the excitation-contraction coupling during early postnatal ontogenesis, and drastic activation of ryr in shr myocytes after the process termination. cytochrome ba from thermus thermophilus belongs to the large family of structurally related heme-copper oxidases. it accepts electrons from cytochrome c at the p-side of the membrane and uses them to reduce oxygen to water. the energy released in this reaction is used for proton pumping across the membrane to form of an electrochemical proton gradient, used by the cell for formation of atp. in this work we followed the kinetics of single-electron injection into the oxidized nonrelaxed state (o h →e h ) of cytochrome ba by time-resolved optical spectroscopy. two main phases of electron transfer were resolved. the first (τ∼ µs) includes oxidation of cu a and simultaneous reduction of both low and high spin hemes. the second (τ∼ µs) reflects reoxidation of both hemes by cu b . this is in significant contrast to the o h →e h transition of aa -type oxidases, where the fastest phase is due to transient reduction of the low-spin heme a only. on the other hand, the single-electron reduction of the relaxed o state in ba oxidase consisted of only rapid electron transfer from cu a to heme b, which is similar to that in aa oxidase. this indicates a functional difference between the relaxed o and the pulsed o h states of cytochrome ba . as opposed to the phospholamban pentamer, sarcolipin forms anion-selective channels in biomembranes l. becucci , c. b. karim , d. d. thomas , g. veglia , r. guidelli chemistry department, florence university, florence,italy, chemistry department, university of minnesota, minneapolis, mn , department of biochemistry, molecular biology, and biophysics, university of minnesota, minneapolis, mn sarcolipin (sln) and the phospholamban pentamer (pln) are two membrane proteins that inhibit ca-atpase of the sarcoplasmic reticulum at low concentrations. in contrast to pln, sln stimulates maximal ca + uptake rates. sln and pln were incorporated in a bilayer lipid membrane (tblm) tethered to a mercury electrode through a hydrophilic spacer. electrochemical impedance spectroscopy measurements show that sln forms channels permeable to chloride ion, weakly permeable to phosphate ion and impermeable to inorganic cations such as na + and k + . a relationship between this property of sln and its regulatory function on ca-atpase of sarcoplasmic reticulum is proposed. atp increases the permeability of a tblm incorporating sln to phosphate ion by associating to sln with an association constant of . µm. an explanation for this behavior is provided. sln can be identified with the " p i transporter" described by a.g. lee et al. conversely, both electrochemical impedance spectroscopy measurements and molecular dynamics simulations provide strong evidence that the pore of the pentameric form of pln does not act as a chloride channel. "social" domain organization and dynamics of nicotinic acetylcholine receptor at the cell membrane f. j. barrantes unesco chair biophys. & mol. neurobiology. univ. nac. sur, b. blanca, argentina a combination of experimental techniques (patch-clamp, confocal frap and fcs, single-particle tracking, highresolution fluorescence microscopy) has been used to analyze the supramolecular organization of the acetylcholine receptor (achr), the dynamics of the receptor at the cell surface, and the kinetics of receptor internalization. changes in cholesterol (chol) content affected muscle and neuronal-type achr organization and dynamics at the cell surface. chol depletion produced gain-of-function of single-channel dwell time. submicron-sized (∼ nm) domains, stable over a period of hours at the cell membrane, could be resolved into achr "nano-clusters" with a peak size distribution of ∼ nm by sted microscopy. chol depletion reduced the number of nanoclusters, increasing their size, and changed their supramolecular "social" organization on larger scales ( . - . microns). frap, fcs and spt experiments provided information on the dynamics of achr nanoclusters, disclosing the dependence of their mobility on chol content and cortical cytoskeleton. chol content at the plasmalemma may thus modulate cell-surface organization and dynamics of receptor domains, and fine-tune receptor channel function to temporarily compensate for acute achr losses from the cell surface. m. czaplinska, k. gwozdzinski, a. koceva-chyla division of research of structure of biopolymers university of lodz, lodz, poland doxorubicin (dox) and paclitaxel (ptx) are anticancer drugs commonly used in chemotherapy of breast cancer therapy, however, the use of these drugs is limited by the risk of developing heart failure. generation of reactive oxygen species contributes to the cardiotoxicity of doxorubicin. nitroxides are low molecular weight, stable free radicals reacting with ros and they present antioxidant properties. the aim of this study was to analyze the effects of pirolid (pd) on the oxidative stress induced by dox and taxane in mcf- breast cancer cell line. results from mtt test revealed that ptx is more cytotoxic towards mcf- cells than dox. the ic was . µm and µm, respectively. pd alone does not influence cell viability. pd in combination with both drugs did not change viability of cells. both drugs increased the level of carbonyl groups in cells. the highest level of peroxide was observed in cells incubated with dox (approx. -fold). nitroxide alone did not influence the level of peroxide in the whole range of concentrations. combination of pd with dox and ptx reduced the level of carbonyls depending on its concentration. pd did not affected on the level of peroxide in cells suspension. dox and ptx increased ( -fold) the level of carbonyls. pd decreased the level of peroxides in cells treated with dox and ptx. the lowest concentration of peroxide was observed at µm of pd. these results show that pd protect mcf- cells against oxidative stress induced by drugs. open channel structure of mscl from fret microscopy and simulation mechanosensitive channels open in response to membrane bilayer deformations occurring in physiological processes such as touch, hearing and osmoregulation. here, we have determined the likely structure of the open state of the mechanosensitive channel of large conductance from e. coli (mscl) in a natural environment using a combination of patch-clamp studies, fret spectroscopy, epr data, molecular and brownian dynamics simulation. structural rearrangements of the protein are measured while controlling the state of the pore by modifying lipid bilayer morphology. fret efficiency changes can be related to distance changes using a monte carlo analysis program in conjunction with detailed orientational analysis. these measurements are used as restraints in all atom molecular simulations in order to determine the likely structure of the open state, whose probable conductance is derived from brownian dynamics simulations. transition to the open state occurs via large rearrangements throughout the protein that create a wide pore nearly a in diameter. both transmembrane helices are found to line part of the pore. the n terminal helix is found to lie along the face of the membrane where it can act to sense membrane tension and directly transfer this to the pore lining helices. the method of coupling spectroscopic data with simulations is likely to be of great value for studying conformational changes in a range of membrane proteins. putative potassium channels in synechocystis sp. pcc v. checchetto, m. zanetti, g. m. giacometti, i. szabò, e. bergantino department of biology, university of padova, italy we are interested in the identification and characterization of potassium channels in the cyanobacterium synechocystis sp. pcc , an organism which is considered the ancestor of plant chloroplasts. a bioinformatic screening of synechocystis proteome identified, among others, two proteins on which we focused our attention. the first one (syncak) displays sequence homology to mthk, a ca + -dependent potassium channel from m . thermoautotrophicum. the second one (synk) is predicted to contain six transmembrane regions and the typical selectivity filter of all potassium channels. our goal is to understand their roles in the physiology of cyanobacteria. we cloned their coding sequences in fusion with gfp and the hybrid proteins were expressed in chinese hamster ovary cells. we evaluated the presence of both proteins in plasma membrane by fluorescence microscopy and then we proceeded to their functional characterization using patch clamp technique.this analysis will allow us to gain information about channel activity, regulation and pharmacology. we also plan to evaluate the importance of syncak and synk channels in photosynthesis. to test the hypothesis that they could be involved in regulating this process, we will produce deletion and site-specific mutants in synechocystis. finally we would also identify the homologous of these channels in the higher plant a. thaliana and obtain some information about their localization and function. j. braunagel max-planck institute for polymer research, ackermannweg , mainz, germany the cyclododecadepsipeptide valinomycin is composed of two amino acids (l-valine and dvaline) and two hydroxyl acids (d-α-hydroxy-isovaleric acid and l-lactic acid). they form a membered ring of alternating amino and hydroxyl acids. in the cyclic structure, the polar groups are oriented towards the central cavity, whereas the rest of the molecule is relatively nonpolar. this enables the complexation of ions and valinomycin acts as a selective ion transporter (k + ) in lipid membranes. when one of the amino acids is exchanged, e.g one l-valine by an l-lysine, selected functionality can be engineered into the depsipeptide while maintaining its ion conducting properties. we induced several modifications into valinomycin, i.e. a biotin binding unit or a ferrocene group to induce an electrochemical active center. the ion conducting properties of the modified ion carriers have been probed in planar lipid bilayers as well as in solid supported membranes. the role of the membrane dipole potential (ϕ d ) is of a particular interest due to a powerful impact of this potential on the membrane permeability and lipid-protein interactions. channel forming activity of gramicidin a, alamethicin, syringomycin e, hpa peptide, and ompf porin are influenced by ϕ d . we have studied the effect of the membrane dipole modifier, phloretin, on the properties of single channels formed by a wild-type alpha-hemolysin in planar lipid bilayers. the single channel of a ∼ ps conductance exhibits transitions into a number of low-conductance states as the transmembrane voltage exceeds ∼ mv (regardless of the voltage polarity). the phloretin addition to the bathing solutions ( µm) (after the hemolysin channel was formed in the membrane) shifts dramatically the channel voltagedependence. transitions to the low-conductance states are observed at ∼ mv. the effect of the phloretin addition was not observed in the case when it was introduced into the bathing solution before alpha-toxin. since phloretin reduces ϕ d , the data may report on the influence of the electric potential profile on the energy of the low-conductance state of the alpha-hemolysin channel. the alternative explanation of this effect consists in a specific interaction between the phloretin and toxin channel. the work is supported in part by rfbr (# - - ), ss (# . . ) , and the program of the ras «molecular and cell biology». atypical mechanism of conduction in potassium channels c. domene , s. furini physical and theoretical chemistry laboratory, department of chemistry, university of oxford, oxford, u.k., department of electronics, computer science and systems, university of bologna, bologna, italy potassium channels can conduct k + ions with rates of up to ∼ ions per second at physiological conditions, and they are selective to these species by a factor of over na + ions. ion conduction has been proposed to involve transitions between two main states, with two or three k + ions occupying the selectivity filter separated by an intervening water molecule. the largest free energy barrier of such a process was reported to be of the order of - kcal mol − . here, we present an alternative mechanism for conduction of k + in k + channels where site vacancies are involved, and we propose that coexistence of several ion permeation mechanisms is energetically possible. conduction can be described as a more anarchic phenomenon than previously characterized by the concerted translocations of k + -water-k + . we exploited the au-deposited self-assembled monolayers of the type: [−s−(ch ) n −ch ] (where n = , and ) with hydrophobically adsorbed redox protein -azurin to verify intrinsic electron transfer mechanisms according to the charge-transfer theory. the enthalpies and volumes of activation were determined through the variation of temperature ( - o c) and pressure ( - mpa) and experimental values were compared with those expected on the theoretical grounds. for the case of n = the activation enthalpy definitely contains a large contribution originated from frictional-like dynamics of protein, and the activation volume has a small positive value. for n = the value of activation enthalpy directly matches / of that for the reorganization energy, and the activation volume attains substantially negative value. for n = we observed the intermediary performance. the whole kinetic pattern is consistent with the smooth changeover between adiabatic and nonadiabatic mechanisms of electron transfer. two gating modalities in the pore of the miniature k + channel kcv kcv is a viral protein that forms functional k + channel in heterologous systems. because of its miniature size ( amino acids) we use kcv as a model system to study and manipulate basic properties of the k + channel pore. by analysing single-channel recordings we highlighted two voltage-dependent modalities of gating in kcv: a slow and a fast gating. the presence of a slow gating is revealed by the very low (in the order of - %) mean open probability. slow gating is not related to the presence of a bundle crossing, as shown by accessibility of the cavity to mts reagents. channel opening might involve the transient formation of salt bridges between residues at the n and c termini of the channel, as suggested by mutational experiments inspired by molecular dynamics simulations of kcv. fast gating, analyzed by beta distributions, is responsible for the negative slope conductance in the single-channel i/v curve at extreme potentials and can be explained by depletion-aggravated instability of the filter region. aca is a type b caatpase with a regulatory n-terminus whose autoinhibitory action can be suppressed by binding of calmodulin (cam). aca n-terminus is able to bind a region of the small cytoplasmic loop connecting transmembrane domains and . to define the role of this interaction in autoinhibition we have analysed a number of single point mutants produced by mutagenesis of aca e -n sequence. mutation to ala of any of acidic residues (e , d , d , d , e , d ) originates an enzyme with normal activity in the presence of cam, but less camstimulated. these results highlight the relevance of a negative charge of the surface area of the small cytoplasmic loop in aca autoinhibition. the most deregulated mutant is d a aca , which is less activated also by controlled proteolysis or by acidic phospholipids; moreover, the phenotype of the d a mutant is stronger than that of d n aca suggesting a more direct involvement of this residue in autoinhibition. of the other mutants (i a, n a, p a, p a, v a, n a), only p a aca has a basal activity higher than that of the wt. these results provide the first evidence that the small cytoplasmic loop of a type b caatpase plays a role in the attainment of the autoinhibited state. complex i, the first member of the respiratory chain, serves as a proton pump catalyzing transfer of two electrons from nadh to ubiquinone coupled with the translocation of four protons across the membrane. so far the mechanism of energy transduction by complex i is unknown. the nadh-binding cavity of complex i has a very prominent feature -the presence of two invariant amino acid residues, glutamate and tyrosine, that are exposed to the solvent and located in the vicinity of the fmn, the primary electron acceptor in the enzyme. it was suggested that they might be involved in the binding of nadh through interaction with its nicotinamide moiety. in this work we assessed the function of corresponding glu from the nuof subunit of e. coli complex i by mutation for glutamine. we showed that the negative charge of glutamate in the catalytic site is needed for the electrostatic repulsion of negatively charged phosphates of nucleotides. this process facilitates release of the product nad + and, as a result, accelerates turnover of complex i. we also found that glutamate, as one of the four negatively charged amino acid residues surrounding the isoalloxazine ring of the fmn at a distance of - Å, has a share of mv of the overall mv depression of the midpoint potential of this redox cofactor. l. erokhova , p. kügler , p. pohl institute of biophysics, johannes kepler university, linz, austria, ricam, austrian academy of sciences, linz, austria the mechanism of water transport through epithelia is still under debate. in the present work we tested the hypothesis of isosmolal water transport using mdck cells stably expressing the human sodium-glucose cotransporter (hsglt ). tagging hsglt with egfp enabled determination of its abundance in the plasma membrane by fluorescence correlation spectroscopy (fcs). by monitoring tiny shifts in the concentration of water-soluble dyes in the vicinity of epithelia, fcs also allowed assessment of the water fluxes through confluent cell monolayers grown on permeable supports. fitting the set of differential equations for the osmotic drift and for the back diffusion to the experimentally determined dye distribution permitted calculation of water flow both in the presence and in the absence of an osmotic gradient. from the calorimetric measurements of glucose transported across the cell monolayer, the water:glucose stoichiometry was derived. dividing the increment in osmotic water flux due to hsglt expression by the number of hsglt copies in the plasma membrane resulted in a single transporter water permeability p f of . x − cm /sec. thus, p f is close to the single channel water permeability of aquaporin- . consequently, even small osmolyte concentration differences between the cytoplasm and the basolateral buffer solution are sufficient to drive a substantial water flux. m. emre, s. kavak cukurova university school of medicine, department of biophysics, balcali-adana, turkey experimental studies have shown that the at -receptor antagonists telmisartan (tel) has a ppar-activating property, but there does not appear to be a class effect. to test telmisartan's importance, we investigated its effect on electrical activities (ea) in diabetic (d) rats. the purpose of this study was to investigate the effects of the tel on ea of diabetic papillary muscle (dpm) with stz-induced. in this study, we used four groups: ( ) nondiabetic control (ndc) group (c), ( ) tel-treated ndc group (c+tel), ( ) diabetic group (d), and ( ) tel-treated d group (d+tel). diabetes was induced by a single i.v injection of stz. in the study, membrane potential (mp) and action potential (ap) recorded after the establishment of diabetes. ) mp was decreased significantly in both tel-treated c and d rats (from - , ± , to - , ± , mv and from - , ± , to - , ± , mv). ) ap unchanged in d group, whereas c+tel and d+tel groups showed increase in ap compared with c and d groups. ) repolarization time was prolonged in diabetic rats. ) in c+tel and d+tel groups depolarization rate values increased significantly. on the other hand, in d group repolarization rate values descreased increased significantly compared to baseline values in tel solution. as a result, our data suggest that the beneficial effects of tel-treatment on the ea of the dpm appear to be due to the diminished k + currents. cytochrome c oxidase is a terminal complex (cco, complex iv) of a respiratory chain that is located in an internal membrane of mitochondria or plasma membrane of bacteria. cco is an electron transfer enzyme that reduces o and uses the redox energy of the o reduction for the proton translocation across the membrane. the electron-and proton-transfer generate a transmembrane electrochemical gradient (∆µh + ) that is used for atp synthesis and all other kinds of work for the cell needs. the proton translocation mechanism of cco requires 'channels' for the h + uptake and expulsion within the enzyme. the proton transfer occurs on a time-scale of micro-to-milliseconds. in order to study the proton transfer in cco, a flow-flash approach based on a time-resolved ftir spectroscopy was developed and applied. our ftir flow-flash approach (the measurement of the reaction of cco with o ) allows to reach a time resolution up to tens milliseconds. with this approach and site-specific mutants of cco where the catalysis is slowed down, separate steps of the proton transfer were studied. the results showed that a unique cross-linked tyr- (in a combination with a time-resolved visible spectroscopy and electrometry) serves as a proton donor for the dioxygen bond cleavage during the o reduction by cco. furthermore, the protolytic transitions of glu- -a key amino acid in the proton transfer mechanism in cco -were shown for the first time. role of calcium ions in nickel potentiation of nmda currents p. gavazzo, i. zanardi, p. guida, c. marchetti biophysics institute ,cnr, genova, italy nmda receptors are glutamate-gated channels distributed throughout the brain in the excitatory synapses and are critical for the nervous system function. they are assembled from two types of subunits, the essential nr and at least one nr (a,b,c,d). nickel (ni) modulates the current flowing through nmda receptors in a different way depending on the nr subunit present. we have recently identified several domains of the channel involved in ni interaction, but many aspects of this modulation remain elusive. in this work we intended to determine the role of calcium (ca) ions in the potentiation induced by ni on the current through nr /nr b recombinant nmda receptors. when ni was applied in the presence of the physiological concentration of ca ( . mm) , a voltage-independent potentiation of the current was observed with a kp of . µm. this effect was progressively reduced by decreasing ca concentrations and it was no more detectable with . mm ca or in the presence of barium (ba, . mm). in this last case the effect of ni on nr /nr b receptors was mainly inhibitory (ki(- mv)= µm). therefore a physiological concentration of ca is necessary to induce ni amplification of the current. many data in the literature indicate a correlation between ca ion entrance through the channel, nmda current facilitation and cytoskeleton; however, in our experiments, the application of the actin perturbing agent cytochalasin-d did not produce major modifications in ni effect. heteromerization properties of voltage dependent potassium channels f. gambale , l. pedemonte , a. naso , i. testa , c. usai , a. diaspro , c. picco istituto di biofisica, cnr, genova, italy, lambs-microscobio, department of physics, genova, italy voltage-gated potassium channels are either homomeric or heteromeric tetramers composed of four α-subunits. in order to bring a contribution to the comprehension of channel heteromerization we have been investigating the properties of two plant voltage-gated k + -channels by using electrophysiological and fluorescence techniques. experiments were focussed on kdc and kdc , coexpressed in xenopus laevis oocytes. kdc , the first potassium channel cloned from daucus carota, belongs to the subfamily of α-modulatory silent channels as it doesn't form functional homomeric channels by itself. on the contrary kdc forms functional heteromeric channels when coexpressed with homologous subunits. kdc , the last k + channel cloned from d. carota, belongs to the kat family and shares an overall identity of % with kat . to correlate kdc functional properties with its localization in oocytes, kdc and/or kdc subunits were labelled with gfp and their properties investigated by confocal microscopy and voltage-clamp. we found that the kdc -egfp fusion protein is not targeted to the plasma membrane unless it is coexpressed with kdc . moreover electrophysiological experiments demonstrated that the heteromeric kdc -kdc channel has altered selectivity and activation properties with respect to homomeric kdc channel. circulating leukocyte sequestration in pulmonary capillaries is arguably the initiating event of lung injury in acute respiratory distress syndrome (ards) [ ] . we present a microfluidic investigation of the roles of actin organization and myosin ii activity during the different stages of leukocyte trafficking through narrow capillaries using specific drugs. the deformation rate during entry reveals that cell stiffness depends strongly on f-actin organization and hardly on myosin ii activity, supporting microfilament role in leukocyte sequestration. in the transit stage, cell friction is influenced by stiffness, demonstrating that the actin network is not completely broken after a forced entry into a capillary. conversely, membrane unfolding was independent of leukocyte stiffness. the surface area of sequestered leukocytes increased by up to % in absence of myosin ii activity, showing the major role of molecular motors on microvilli wrinkling and zipping. finally, cell shape relaxation was largely independent of both actin organization and myosin ii activity, whereas a deformed state was required for normal trafficking through capillary segments [ ] . [ ] g.s. worthen et al., science, , - ( ) excitation-contraction coupling in skeletal and cardiac muscle is tightly regulated by the calcium release channel of the sarcoplasmic reticulum, the ryanodine receptor (ryr). we could previously show that suramin is a potent activator of the ryr via the calmodulin binding site. calmodulin shows dualistic action, i.e. activation or inhibition of the ryanodine receptor, depending on the absence or presence of ca + . screening of suramin analogues identified nf as a use-dependent inhibitor of the skeletal muscle ryr (ryr ). here we show that nf inhibits high affinity [ h]ryanodine binding and single channel recordings of the purified ryr . nf induced a reduction of open probabilities in a concentration dependent manner, with no effect on current amplitude and unitary conductance. importantly, nf triggers flickering episodes of channel openings and closings before the ryr is frozen in a complete non-conducting state, which is fully reactivated by the ryr agonist atp. moreover, zwitterionic behaviour of nf facilitates plasma membrane permeation, which prevented caffeine induced ca + transients in skeletal muscle cells and cardiomyocytes. conversely, ip mediated ca + signals were not altered by nf . this work was supported by herzfelder'sche familienstiftung and fwf . beyond steady-state protein dynamics t. hauß , j. pieper , a. buchsteiner , r. e. lechner , n. a. dencher helmholtz-zentrum berlin für materialien und energie, berlin, germany, technische universität darmstadt, germany, technische universität berlin, germany to study protein dynamics beyond steady-state experiments we have developed a novel laser-pump:neutron-probe experiment which allows us to monitor temporal changes in protein dynamics during a working cycle of a protein. protein dynamics has been extensively studied, but so far, the correlation of internal protein dynamics with the function of proteins was investigated only indirectly in steady-state experiments by variation of external parameters by variation of external parameters like temperature or hydration. the method comprises of an in-situ optical activation of a protein and a time-dependent sampling of the dymamic response using quasi-elastic neutron scattering. with the membrane protein bacteriorhodopsin, a light driven proton pump, we can demonstrate for the first time temporary alterations in the protein dynamics after triggering the working cycle. this observation is a direct proof for the functional significance of protein structural flexibility, in connection with the largescale conformational changes in the protein structure occurring during the operation of a "molecular machine". the slow vacuolar (sv) channels are ubiquitous in all tissues of higher plants. the sv channel is a non-selective cation channel permeable to both monovalent and divalent cations. sv currents recorded in a typical patch-clamp experiment require unphysiologically high cytosolic and low vacuolar calcium concentrations for full activation. we aim at looking for endogenous plant substances which might be able to modify or shift the voltage activation threshold of this channel towards more physiological conditions. flavonoid naringenin [nar] is present in all plant species where it plays a central role in the flavonoid biosynthetic pathway. nar is stored in the vacuoles in glycosylated form called naringin. when nar was added to cytosolic bath solution, we recorded a dose-dependent reversible decrease in sv channel activity. when we investigated the effect of nar on the voltage dependence of the channel, we observed that the activation threshold of the sv channel is shifted towards more positive voltages. our group has evidences that approximately % of the total sv current at high (e.g.> mv) positive voltages is mediated by calcium. therefore, in order to verify whether nar affects both potassium and calcium conductance, we performed experiments by combining the patch clamp technique with fluorescence measurements using the fluorophore fura- : both sv currents and calcium signals were abolished by mm [nar]. determination of calcium currents in cation channels using a novel fluorescence/patch-clamp approach p. v. k. gutla, a. gradogna, a. carpaneto istituto di biofisica, consiglio nazionale delle ricerche, via de marini , genova, italy the patch-clamp technique combined with fura- fluorescence detection is suitable to investigate calcium fluxes. we used the excised patch configuration and focused the photomultiplier to the tip of the recording pipette where the fluorescent dye was present (fluoresence combined with excised patch = flep). this configuration has several advantages, i.e. absence of delay in loading the fluorophore, of interference by endogenous calcium buffers and of photobleaching. here we present an application for the determination of fractional calcium currents (pf) in a plant non-selective cation channel, showing that pf can be modulated by cytosolic calcium and potassium. flep is very efficient for measuring small calcium currents (< pa) of sufficiently long duration; fluorescence signals are amplified by integration in time, as the calcium/fura- complex accumulates at the tip of the recording pipette and diffuses slowly. we propose this technique not only for the study of calcium transport pathways, but also for other transporters of divalent cations as nickel and manganese known to quench fluorescence thus reducing both and nm components. moreover, using the appropriate fluorophore the technique may be extended to further ion species, e.g. bcecf for investigating proton transport pathways. ref: we introduced an original method for the monitoring of the changes in the electrostatic surface potential, using the quenching of the intrinsic tryptophan fluorescence by acrylamide or iodide. this approach opens new way to understanding the dynamic processes within the proteins. our experiments revealed that the conformation of the na + /k + -atpase large cytoplasmic loop (c ) in the presence of the atp (without magnesium) substantially differed from the conformation in the presence of mg + or mgatp or in the absence of any ligand not only in the sense of geometry but also in the sense of the electrostatic surface potential. moreover, our data indicate that the effect of the ligand binding is not restricted only to the close environment of the binding site and that the information is in fact transmitted also to the distal parts of the molecule. this property could be important for the communication between the cytoplasmic headpiece and the cation binding sites located within the transmembrane domain. influx of antibiotics into the periplasm of gram-negative bacteria is facilitated by porins that form channels in the outer membrane. we propose that certain natural antibiotics have been optimized by co-evolution to take advantage of the charge distribution in non-specific porins to achieve binding and thereby facilitating their uptake in bacteria. we investigate the permeation pathways of antibiotics into bacteria by reconstitution of a single porin into an artificial lipid bilayer and measuring the binding of antibiotic molecules through the time-resolved modulation of a small ion current. we have been able to characterize facilitated translocation of several antibiotics through escherichia coli and enterobacter aerogenes porins. noise analysis of ion currents through a porin in the presence of effective antibiotics revealed binding kinetics at a single molecule level. we report for the first time temperature dependent antibiotic translocation that revealed complete energy profile. combining these results with microbiological assays and molecular dynamics simulations, we conclude the molecular mechanism of antibiotic permeation. our approach may contribute to the rational design of new antibiotics against clinical bacterial strains for the most efficient delivery to target sites. mitochondria regulate ca + influx and determine patterns of er ca + refilling in acinar cel o. kopach, i. kruglikov, t. pivneva, n. voitenko, n. fedirko bogomoletz institute of physiology, kiev, ukraine store-operated ca + entry (soce) is mediated by activation of soc-channels of plasma membrane following the emptying of endoplasmic reticulum (er) ca + stores. the soce is required for calcium signaling, secretion of neurotransmitters and proteins, but the mechanisms of natural soce regulation are not well understood. we utilized several imaging methods to measure ca + signals in cytoplasm ( ] mit transients, observed both in egta-and bapta-buffering inside solutions. these data suggest that ca + sequestration by mit is associated with the formation of microdomains and prevents ca + -dependent socc inactivation. we also found that inhibition of mit under prolonged cell stimulation resulted in complete inhibition of soce as well as decrease and deceleration of er refilling. thus, mit regulate calcium recycling and maintain the soce controlling dynamic interplay between soce and sustained er refilling under prolonged stimulation. bioelectrochemical devices composed of au electrodes coated by self-assembled monolayers (sams) of different composition and thickness are ideal systems to probe et patterns and mechanisms for redox proteins. representative proteins, cytochrome c and azurin were studied by using the combinations of four different strategies including the variation of sam thickness ([−s−(ch ) n −ω], with n running over the range to , throughout), solution viscosity (varied by adding of the viscose additive -glucose), temperature ( to o c) and hydrostatic pressure (up to mpa), aiming the identification of different intrinsic et patterns and interplay between them in the framework of generalized charge-transfer theory. we demonstrated the full adiabatic (frictional) control for the case of thinner sams, the intermediate (mixed) regime, and the complete changeover to the nonadiabatic mechanism (long-range tunneling) for the case of thick sams owing to the variation of electronic coupling, in a nice agreement with theoretical predictions. h. nury , f. manon , b. arnou , m. le maire , e. pebay-peyroula , c. ebel institut de biologie structurale (ibs) cea cnrs ujf grenoble france, cea ibitec cnrs ura univ. paris-sud gif-sur-yvette france adp/atp carriers (aacs) are major and essential constituents of the inner mitochondrial membrane. they drive the import of adp and the export of newly synthesized atp. they were described as functional dimers from the s until the structures of the aac shed doubt on this consensus. we aimed to ascertain the published biophysical data claiming that aacs are dimers and to characterize the oligomeric state of the protein before crystallization. analytical ultracentrifugation sedimentation velocity experiments clearly show that the bovine aac is a monomer in -laurylamido-n,n dimethylpropylaminoxide (lapao), whereas in triton x- and reduced triton x- , higher molecular mass species can also be identified. neutron scattering data for monomeric bovine aac in lapao does not give definite conclusions on the association state, because the large amount of detergent and lipids is imperfectly matched by contrast methods. we discuss a possible way to integrate previously published biochemical evidence in favor of assemblies, the lack of well-defined multimers that we observe, and the information from the high-resolution structures, considering supramolecular organizations of aacs within the mitochondrial membrane. local anaesthetic binding to shaker channels: role of aromatic residues j. nilsson, h. ullman, k. sahlholm, p. arhem the nobel institute for neurophysiology, department of neuroscience, karolinska institutet, se- stockholm, sweden local anaesthetic, antiepileptic and antiarrhythmic drugs acting on nav and herg channels have been assumed to bind to aromatic residues in the internal vestibule; to f and y in nav (ragsdale et al., and to y and f in herg (mitcheson et al., ) . despite a lack of such residues in kv channels, local anaesthetics, antiepileptic and antiarrhythmics bind to kv channels with a considerable affinity. to explore the role of aromatic residues for the binding we investigated the effect of bupivacaine, benzocaine, phenytoin and quinidine on shaker channels mutated to residues corresponding to the most c-terminal of the two aromatic residues in the s segment of the nav and the herg channels, (v y and p f respectively). the channels were expressed in xenopus oocytes and the currents measured with the two-electrode voltage-clamp technique. the results suggest that aromatic residues do not increase the binding affinity of the studied compounds to kv channels. rather, the affinity decreases (as reflected in typical k d values for bupivacaine on v f, p f and wildtype channels, being , and µm, respectively). thus, aromatic residues seem not to be necessary for high-affinity binding of the studied compounds to kv channels. how this relates to their suggested roles in the nav and herg channels, remains to be evaluated. (molina et al, ) . the occurrence of a similar behavior in other channels points out to clustering and coupled gating as a potentially important drug target to modulate channel activity. we have identified molecular determinants involved in single and coupled channel gating in kcsa. first, we detected that clustering and coupled gating of kcsa is modulated by anionic lipid. also, a model for the interaction between two kcsa open channels was built. the docking predicts intermolecular sites which includes the non-annular lipid binding site. this explains how an excess of anionic lipid disrupts interactions between channels, destabilizing clustering and coupled gating in kcsa. in addition, the docking model reveals molecular determinants involved in single and coupled channel gating. this interaction involves w , which affects the neighbouring channel through specific interactions in the extracellular mouth stabilizing the selectivity filter in an open conformation. the coupled gating is also explained since this mechanism affects the opposite channel in a mutual manner. finally, mutants kcsa e a and kcsa w a disrupt the coupled gating of kcsa, thus, supporting the model. we think that this coupled gating phenomenon could correspond to the second gate previously detected by fluorescence methods (blunck, et al, ) . supported by grants from the spanish bfu - /bmc and csd - . a. kumar, e. hajjar, p. ruggerone, m. ceccarelli university of cagliari, monserrato, italy the striking presence of outer membrane (om) in gramnegative bacteria of e.coli represents a strong barrier for any molecule to penetrate inside bacteria. in particular for β-lactam antibiotics, which have their target located inside the bacteria; the first step towards reaching inner part of bacterium is the cellular uptake. ubiquitous presence of porins (such as for instance ompf, ompc) in the om, function as a channel facilitating the transport of molecules (such as, for instance antibiotics) across the om. bacteria can exhibit resistant towards antibiotics by: (i) decreasing their uptake by under-expressing the porins or/and (ii) production of inactivating enzymes such as ß-lactamases. to combat the latter mechanism ß-lactamase inhibitors (such as, sulbactam for instance) are prescribed together with the antibiotics. like the antibiotics, the inhibitors must penetrate the om, the main path being through porins. it is thus evident the biological relevance of investigating the mechanisms by which porins can regulate entry/exit across the om. to achieve this goal, molecular dynamics simulations were performed to explore the structure and dynamics of pores formed by ompf and ompc porin. from the analysis of data obtained from our simulations, we identified the key residues buried behind the l loop, which may play be crucial for porins to exert their biological role. as a case study, we report results about the diffusion of sulbactam through the two porins. the pentadecapeptide gramicidin forms a cation-specific ion channel in membrane environment. the two main conformations are the head-to-head helical dimer (hd) known as the channel conformation and the intertwined double helical form (dh) often referred to as non-channel conformation. in this comparative study [ ] , the energetics of single potassium ion permeation by means of the potential of mean force (pmf) for both gramicidin conformations embedded in a dmpc bilayer has been addressed by molecular dynamics simulations. a significantly decreased free energy barrier by ∼ kj/mol for potassium ion passage through dh as compared to hd is reported. favorable electrostatic side chain-cation interactions in hd are overcompensated by phospholipid-cation interactions in dh. the latter are coupled to an increased accessibility of the channel entrance in dh due to distributed tryptophans along the channel axis. this result underscores the importance of the lipid environment of this channel not only for the equilibrium between the different conformations but also for their function as cation channels. y. sawada , m. murase , m. sokabe dept. physiol. nagoya univ. grad. sch. med., nagoya, japan, icorp/sorst cell mechanosensing, jst, nagoya, japan the bacterial mechanosensitive channel of large conductance mscl is constituted of homopentamer of a subunit with two transmembrane inner and outer α-helices, and its d structure of the closed state has been resolved. the major issue of mscl is to understand the gating mechanism driven by tension in the membrane. although several models for the opening process have been proposed with molecular dynamics (md) simulations, as they do not include mscllipid interactions, it remains unclear which amino acids sense membrane tension and how the sensed force induces channel opening. we performed md simulations for the mechano-gating of mscl embedded in the lipid bilayer. upon tension in the bilayer, phe in the outer helix was dragged by lipids, leading to a tilting of the helices. among amino acids in the outer helix facing the bilayer, phe at the water-lipid interface showed the strongest interaction with lipids, thus may work as a major tension sensor. neighboring inner helices cross each other in the inner leaflet, forming the most constricted part of the pore. as tension increases, the crossings move toward the cytoplasm associated with an expansion of the constricted part. during the movement, a hydrophobic water block environment around the constricted part was broken followed by water penetration and permeation. the k + channel kcsa is an integral membrane protein from s.lividans, used as a model system for studies on ion channels and oligomeric membrane proteins. its atomic structure has been solved by x-ray diffraction, which shows an assembly of four identical subunits around a central aqueous pore, including the so-called selectivity filter. this channel is able to permeate k + at high flux rate and it is blocked by na + (physiological blocker). fluorescence, circular dichroism and fourier transform infrared experiments carried out in our laboratory demonstrated that k + and na + are able to bind to kcsa in a competitive manner. this binding is indeed associated with channel conformational changes, which seem to be related to the permeation and blockade processes. to further investigate this phenomenon we carried out a detailed study of chemical and thermal denaturation of wild-type and mutant kcsa channels. these two types of experiments were in agreement and indicate that both cations are able to stabilize the channel through conformational changes, being k + the more efficient one, even more when lipids are present. particularly, mutant channels with a structurally altered selectivity filter show that ion and lipid-induced global conformational changes are intimately associated to the conformation of this selectivity filter (conductive and non-conductive forms). modulation of the voltage-gated sodium channel nav . by rcssii, a toxin from the scorpion centruroides suffusus c. picco , g. corzo , l. d. possani , g. prestipino institute of biophysics, cnr, genova, italy, instituto de biotecnologia, unam, cuernavaca, mexico the main cardiac voltage-gating sodium channel, na v . , generates the fast depolarization of the cardiac action potential and plays a key role in cardiac conduction. its importance for normal cardiac function has been exemplified by the description of numerous naturally occurring genetic variants of the gene scn a, which encodes na v . , that are linked to various cardiac deseases. subsequently, studies of this channel localization have led to its identification in immature and denervated skeletal muscle and in the brain neurons. in our effort to identify high affinity ligands for this channel, we have investigated the effects of the recombinant cssii (rc-ssii), a four disulfide-bridged scorpion toxin isolated from the venom of the scorpion centruroides suffusus. human cardiac sodium channel α subunit scn a was expressed in cho cells and macroscopic na + currents were recorded with patchclamp technique in whole cell configuration. the electrophysiological experiments have highlighted a strong affinity for the channel at low nanomolar concentration. compared with control conditions, rcssii toxin affects in reversible way the kinetics of activation and inactivation and marked decrease the peak na + influx. the extrusion mechanism of substrates in rnd family efflux pumps: a molecular dynamics study a. v the rnd transporters of the acrab-tolc (e.coli) and mexab-oprm (p.aeruginosa) systems are able to export structurally and chemically different substrates outside bacteria through the membrane, being responsible of multidrug resistance. on the basis of crystallographic information, an extrusion process conceived as a three-cyclic peristaltic pumping has been proposed, but further microscopically well-funded investigations are needed to understand the mechanism. using different computational methods like adaptive bias force (abf) and targeted molecular dynamics (tmd), we have investigated the mechanism of substrate uptake and pumping at a molecular level. with the first method we have investigated the passage of antibiotics from the periplasm into the internal pore of the pump, while tmd has been used to assess the effect of conformational changes on the extrusion of drugs (which have been located into one of the proposed binding pockets). comparison between the active pumps acrb and mexb (which show different resistance patterns despite their homology) provide insights into the microscopic details of their functioning. in arabidopsis thaliana there are twenty genes, grouped into three subfamilies, encoding for homologues of animal ionotropic glutamate receptors (iglrs). each protein displays a pore-forming loop, flanked by two conserved helices (plus a third c-terminal helix), a glutamate-binding domain and an n-terminal region. through pharmacological and/or genetic approaches, many physiological functions have been attributed to plant glurs, such as the regulation of cytosolic calcium, photomorphogenesis, water balance and carbon/nitrogen sensing and assimilation. according to the endosymbiotic theory, the cyanobacteria are considered to represent the precursors of the present chloroplasts. the first prokaryotic glutamate receptor (glur ) was identified in the cyanobacterium synechocystis. the putative products of the atglr . and atglr . genes display a possible targeting sequence for chloroplast location and show a high degree of homology with glur . using specific antibodies and confocal microscopy, we have localized the two members of the atglr subgroup , glr . and glr . (splicing variant) , to the chloroplast in arabidopsis and to the inner envelope membrane in spinach. electrophysiological experiments indicate the presence of an activity which is compatible with that of glutamate receptors. furthermore, oxygen evolution measurements suggest that chloroplast-located glutamate receptors may play a role in the regulation of photosynthesis. under extreme conditions many cells control their volume responding to osmotic challenges by unloading or loading solutes to recover their original volume. a faster volume regulatory role triggered by membrane tension has been disclosed for aquaporins in kidney proximal tubule cells, where aqp is the main water channel, using isolated brush border membranes. in conventional osmotic studies in animal cells it is common to disregard internal hydrostatic pressures because they are insignificant compared to osmotic forces. however, by using low osmolarity buffers in small radii vesicles, we detected a rise on the internal pressure that creates surface tension and causes membrane stress, with a negative outcome on aquaporin water permeability. these findings suggested a mechanism for volume regulation in kidney proximal tubule epithelia where massive solute and fluid transport occurs. to further explore aquaporin regulation by membrane tension, yeast cells were used as a model that could bare surface tension some orders of magnitude higher than animal cells due to the existence of a cell wall. the effect of increasing levels of membrane tension on yeast water channel activity was evaluated. an impairment of aquaporin activity correlated with the increase of membrane tension corroborates the volume regulatory role of aquaporin in different cells. deuterium isotope effects on fast gating of the chloride channel clc- g. zifarelli, a. r. murgia, p. soliani, p. michael istituto di biofisica, cnr, via de marini, , i- genova, italy gating of the torpedo cl − channel clc- is modulated by intracellular and extracellular ph, but the mechanism responsible for this regulation has remained so far elusive. using inside-out patch clamp measurements we studied the dependence of the fast gate on ph int and [cl − ] int . only the closing rate, but not the opening rate showed a strong dependence on these intracellular factors. using mutagenesis we excluded several candidate residues as mediators of the ph int dependence. we propose a model in which a proton generated by the dissociation of an intrapore water molecule protonates e leading to channel opening. deuterium isotope effects confirm that proton transfer is rate limiting for gate opening and that channel closure depends mostly on [oh − ]. the model is in natural agreement with the finding that only the closing rate constant, but not the opening rate constant, depends on ph int and [cl − ] int . deletion of the c-terminus destabilizes phosphorylated na/k pump state containing na ions n. vedovato, d. c. gadsby the rockefeller university, new york, u.s.a. the na/k pump's extended c-terminus (compared to the serca ca pump's) links transmembrane helices, and its truncation lowers cytoplasmic na affinity for forming the occluded e p(na ) state. here we test the effects of c-terminal truncations on interactions with external na. we deleted the last (yy) or (kesyy) residues in xenopus α β pumps made ouabain resistant by mutations q r-n d (rd) or c y (c-y), and then used two-microelectrode voltageclamp recording in xenopus oocytes to measure pump currents as mm ouabain-sensitive currents while endogenous na/k pumps were silenced with µm ouabain. inhibition by external na of steady outward pump current ([k] o = mm) at large negative voltages was somewhat weaker in both rd and c-y pumps than in wt pumps, but was severely impaired in all c-terminal truncated pumps. consistent with this, the voltage dependence of transient charge movements under na/na exchange conditions ([k] o = mm) was strongly shifted to more negative potentials in the truncated pumps relative to the parent rd or c-y pumps, shifts comparable to those seen in wt pumps on decreasing [na] o several-fold. together, the results suggest that these c-terminal deletions lower the apparent affinity for external na ions to bind and become occluded in the na/k pump. the c-terminus therefore provides contacts important for stabilizing the occluded e p(na ) conformation, regardless of the route of na ion entry into the binding pocket. muscle contraction is driven by molecular motors that adapt their energy utilization according to the demands made on them. we test the hypothesis that rate constants controlling the biochemical steps involved in atp hydrolysis by myosin atpase are affected by the force of the muscle. here we use fluorescence lifetime imaging microscopy (flim) of a fluorescently labelled atp analogue to investigate changes in the environment of the myosin atpase, caused by different loads applied to skeletal muscle. single muscle fibres were subjected to cycles of stretches and releases in the presence of rigor solution and µm of coumarin-labelled atp. flim acquisition was synchronised with stretch/release cycles and force measurements, which allow us to investigate the effect of strain on the lifetime of the labelled atp bound to the actomyosin complex. characterization of the fluorescence decay by a bi-exponential function resolved the time constant of two populations, namely, free fluorophore (τ = . ± . ns; mean ± s.d.) and fluorescent nucleotide bound to the actomyosin complex (τ = . ± . ns at low strain). these experiments showed that while the time constant of the free fluorophore did not change with force, the time constant of the fluorescent nucleotide bound to actomyosin showed a linear dependence with the force applied to the muscle of . ± . ps/kpa. neck linker docking coordinates the kinetics of kinesin´s heads i. derenyi, a. czovek, g. j. szollosi department of biological physics, eotvos university, pazmany p. stny. a, h- budapest, hungary conventional kinesin is a two-headed motor protein, which is able to walk along microtubules processively by hydrolyzing atp. its neck linkers, which connect the two motor domains and can undergo a docking/undocking transition, are widely believed to play the key role in the coordination of the chemical cycles of the two motor domains and, consequently, in force production and directional stepping. although many experiments, often complemented with partial kinetic modeling of specific pathways, support this idea, the ultimate test of the viability of this hypothesis requires the construction of a complete kinetic model. considering the two neck linkers as entropic springs that are allowed to dock to their head domains and incorporating only the few most relevant kinetic and structural properties of the individual heads, we have developed the first detailed, thermodynamically consistent model of kinesin that can (i) explain the cooperation of the heads during walking and (ii) reproduce much of the available experimental data (speed, dwell time distribution, randomness, processivity, hydrolysis rate, etc.) under a wide range of conditions (nucleotide concentrations, loading force, neck linker length and composition, etc.). besides revealing the mechanism by which kinesin operates, our model also makes it possible to look into the experimentally inaccessible details of the mechanochemical cycle and predict how certain changes in the protein affect its motion. the positive role of noise on the transport efficiency of na, k atpase c.-h. chang , t. y. tsong institute of physics, national chiao tung university & physics division, national center for theoretical sciences, hsinchu, taiwan, institute of physics, academy of sciences, taipei , taiwan na, k atpase is a molecular motor which is able to transport ions through cell membranes, even against the transmembrane ion concentration gradient. while in vivo this nanoscale soft machine consumes atp, it may be driven by external fluctuating electric fields, no matter they are periodic or random. theoretically, the motor conformations can be described by a conformation vector v(t) governed by a multi-dimensional kinetic equation. given an oscillating electric field with a slight fluctuation, the boltzmann distributions of these conformations will change with time. the instantaneous transported ion flux is a functional of the quasi-cyclic trajectory v(t) of this non-autonomous dynamical system. various interesting dynamical properties of this ion pump, including stochastic resonance, can be studied theoretically, some of which have good agreement with recent experimental findings. in situ measurements of the molecular motor of muscle with nanometer-microsecond resolution in a contracting muscle, arrays of the dimeric motor protein myosin ii pull the actin filament towards the centre of the sarcomere during cyclical atp driven interactions. when the external load is smaller than the array force, the sarcomere works as a motor, converting metabolic energy into mechanical work; when the external load is larger than the array force, the sarcomere acts as a brake resisting the load with reduced metabolic cost. to investigate the molecular basis of the work production and the braking action of muscle, we use sarcomere-level mechanics and x-ray interferometry in intact single cells from frog skeletal muscle. during isometric contraction, each motor bears a force of about pn. during shortening against high and moderate loads, the number of myosin motors attached to actin reduces in proportion to the external load while the force per attached motor is maintained similar to the isometric value (piazzesi et al., cell , - , ) . rapid stretches of - nm between each overlapping set of myosin and actin filaments in a muscle sarcomere cause the stiffness of the array of myosin motors to increase up to twice the isometric value within ms (brunello et al., pnas usa , - , ) , indicating that the high resistance of active muscle to stretch is due to recruitment of the second motor domain of the myosin molecules with the first domain already attached to actin. supported by miur and ente crf (italy), nih (usa), mrc (uk), embl, esrf. kinesin- is a molecular motor that moves cellular cargo along microtubules. its functional mechanism is well understood for individual motors. however, the way that many kinesin- motor proteins bound to the same cargo move together is not. we addressed the structural basis for this phenomenon using video microscopy of single microtubulebound full-length motors and various spectroscopy methods were employed to study synthetic peptides derived from hinge- region. these peptides show an unexpected profile of secondary structure forming propensities. video microscopy of single microtubule-bound full-length motors reveal the sporadic occurrence of high compliance states alternating with longer-lived, low compliance states. the deletion of hinge- abolishes transitions to the high compliance state. from the results we hypothesize that strain accumulated during multiple kinesin motility populates the high compliance state by unfolding helical secondary structure in the central hinge domain flanked by unordered regions, thereby preventing the motors from interfering with each other in multiple motor situations. titin is a giant protein of vertebrate skeletal and cardiac muscles. cardiac titin is expressed in two main isoforms: short n b (∼ kda) and long n ba (∼ kda). we have studied changes of titin isoform composition in myocardium of hibernating ground squirrels and spontaneously hypertensive rats (shr). using electrophoresis we have revealed considerable decrease (by - times) in the content of titin relative to myosin heavy chains in shr heart as compared with that for normotensive rats. surprisingly that the data of qrt-pcr showed the increase in mrna content of n ba and n b-isoforms in hypertrophic heart more than times in comparison with norm. we suppose that such a result is an effect of depressed translation of mrnatitin in pathology. we have observed the decrease (by , times) of total titin amount in heart of hibernating animals in comparison with that for summer active animals. however n ba/n b ratio in the heart upon hibernation was increased by times. similar trend was not revealed for the mrna level of corresponding isoforms, although we have showed the decrease of mrna of both titin isoforms in heart of hibernating ground squirrels as compared to their content of summer animals. the decrease in total mrna level may be explained by repressed transcription or mrna degradation in the cell during hibernation. these discrepancies in protein and mrna levels may be considered as the posttranscriptional regulation of titin isoforms expression. actomyosin cross-bridges formed when the globular heads of myosins bind to actin filaments are the molecular engines that drive muscle contraction, fuelled by atp hydrolysis. critical to this process is the change in shape of the cross-bridge and the change in the interactions with actin, in response to force applied to the muscle, and to the status of the nucleotide in the binding pocket. although molecular detail is known from x-ray crystallography and biochemistry, understanding of the interplay between cross-bridge shape and chemical state requires studies in muscle fibres generating force. we use fluorescence life time imaging microscopy (flim) as a probe of the cross-bridge environment.with a fluorescent analogue of atp , fluorescence life-time (flt) changes when the crossbridge binds to actin. now, we show preliminary experiments on the effect of force on flt. the essential light chain of myosin (elc) is a ∼ kda peptide that wraps around a nmlong α-helix of the myosin cross-bridge known as the lever arm which tilts during force generation. using a recombinant elc, labelled with a fluorophore at a strategic cys, we replace the native elc and introduce the fluorescent elc in muscle fibres. preliminary experiments demonstrate that the elc fluorophore also is sensitive to force applied to the muscle fibre. in addition, förster resonance energy transfer occurs between the nucleotide and elc fluorophores, opening the way for studying structural changes in cross-bridges during force generation by fret. muscle contraction: pitfalls in the determination of the contractile response e. grazi dipartimento di biochimica e biologia molecolare, università di ferrara, ferrara, italy the contractile response of an active muscle depends on the load. the load is a force /cross-section. there are three fundamental dimensions: the mass, m; the space, l; and the time, t. from these three dimensions are built up all the physical dimensions. as an example the acceleration, a, is given by, a=l.t − . once the direction and versus are settled the modulus fully defines the physical effect of the acceleration. what about the force? the force, f, is given by, f=m.a. at variance with the acceleration, once the direction and versus are settled, the modulus does not define the physical and the biological effects of the force: the same force is generated by an infinite number of mass-acceleration couples that display different physical and biological effects. the same occurs with the load. thus defining the load that opposes the contractile force does not define the contractile system. in the studies on muscle contraction the acceleration of the load is not considered nor it is provided a way to extract this information. thus these systems are poorly defined from the physical as well as from the biological point of view. models of muscle contraction that consider explicitly both the mass and the acceleration of the load show that, at the same load, the decrease of the acceleration of the load significantly delays the pre-steady state of the contraction and decreases the stiffness of the active fibre. r. shahapure , f. difato , a. laio , d. cojoc , e. ferrari , j. laishram , g. bisson , v. torre int. school for advanced studies, trieste, italy, iit-sissa unit, trieste, italy, lab. nazionale tasc, trieste, italy polymerization of actin filaments is the main source of motility in lamellipodia and is controlled by many regulatory proteins. the underlying molecular mechanisms are only partially understood and now a determination of the dynamical properties of force generation is needed. using optical tweezers we measured with millisecond temporal resolution and pn sensitivity the force-velocity (fv) relationship and the power dissipated by lamellipodia of dorsal root ganglia neurons. when force and velocity are averaged over - s, fv relationships can be flat. on a finer time scale, random occurrence of fast growths and sub-second retractions become predominant. maximal power dissipated by lamellipodia over a silica bead with a diameter of µm is − w. due to the presence of adhesion forces, beads in close contact with a lamellipodium can seal on its membrane reducing the amplitude of brownian fluctuations often by more than times. under these conditions, when lamellipodia grow and push the beads, discrete jumps varying from about to nm are detected. when lamellipodia retract, pulling the beads, no discrete events are observed. our results on the dynamical properties of force generation are: a) force generation is a probabilistic process; b) underlying biological events have a bandwidth up to at least hz; c) fast growths of lamellipodia leading edge alternate with local retractions; d) force generation is produced in discrete steps with varying amplitude up to . pn. pushing on microtubules: dominant spindle centering mechanism in c. elegans embryo? j. pecreaux, s. redemann, a. a. hyman, j. howard mpi-cbg, pfotenhauerstr , dresden, germany asymmetric cell division, where the content of the two daughter cells -as well as their sizes -differ, is found in many organisms. strikingly, the spindle, first centered, starts to be displaced out of the center only in late metaphase. in c elegans embryo, the spindle rocks and is posteriorly displaced during anaphase by force generators asymmetrically localized on cell cortex. prior to anaphase onset, the spindle is usually assumed to be centered by the same pulling force. it thus requires the force generators to be carefully repressed to distribute forces symmetrically. on live embryos, we measured positional fluctuations of centrosomes during metaphase with nm accuracy. fourier analysis shows an extremely accurate centering respect to the number of force generators and microtubules. furthermore, spectrum is close to a lorentzian, modeled by a spring and a dashpot, suggesting a spindle centering more likely by pushing on microtubules than pulling. deviation at high frequencies indicates a subdominant pulling force. rnai of gpr- / , known to control force generation, increases slightly centering accuracy; this result supports the hypothesis of an independent centering mechanism. conversely, zyg- (rnai), a microtubule growing factor, decreases centering accuracy, modeled spring stiffness and damping modulus. conclusion: first, the spindle centering mechanism is independent of cortical pulling force generator. second, microtubules pushing is likely to center the spindle. mechanical forces are important in the regulation of cellular adhesion and migration. the focal adhesion kinase (fak) has been suggested to transduce cellular forces and govern cell migration. to obtain more insight in the functioning of fak, a fret-based optical biosensor for fak was designed to relate integrin-mediated conformational changes in its ferm domain to focal adhesion behavior during cell spreading and migration in living cells. imaging of the kinetics of ferm-based fak conformational changes in spreading cells revealed two consecutive stages of focal adhesion activation. heterogeneous ferm conformational responses were observed in individual focal adhesions of adherent motile cells, with the active ferm conformation being enriched in growing and sliding fas, but not in stable and shrinking focal adhesions. inhibition of the cellular actomyosin system revealed the involvement of rho-rock rather than mlckinduced tension signaling in the modulation of the ferm response. our results place the ferm conformational change of fak at the interface between integrin and force sensing. the time course of inorganic phosphate release in permeabilized cardiac trabeculae of the rat c. mansfield, t. west, m. a. ferenczi imperial college london, u.k. the rate of p i release was determined in permeabilized rat trabeculae. contraction was elicited at • c by laser-flash photolysis of npe-caged atp, and time-resolved p i release was monitored using mdcc-pbp, a coumarin-labelled phosphate binding protein, which increases its fluorescence intensity five-fold upon p i binding. the atpase rate during the first turnover of the total crossbridges (assuming µm myosin heads) was s − . the rate decreased to a steady state of s − after the eighth turnover ( . - . s after activation). this steady state rate is comparable to published values of - s − , made ∼ s after activation using an nadh-linked enzyme assay of adp release. the advantage of using mdcc-pbp is that the control of mechanochemical coupling can be examined from the onset of force production and as it progresses toward the steady state. force production and p i release were simulated using a seven step scheme. force was attributed to the states in the sequence a.m.adp.p i ↔ a.m.adp ↔ a.m.adp, with strain sensitivity incorporated into the isomerisation of a.m.adp. the a.m.adp.p i and a.m.adp states populated rapidly as force was increasing. in contrast, the a.m.adp state accumulated slowly after the force plateau was reached and became the dominant force bearing state at the time of the eighth crossbridge turnover. experiments are on-going to examine how the distribution of a.m states changes in response to rapid length-changes. pulling as a factor in forming the heterophasic structure of immunoglobulin proteins structure of proteins of immunoglobulin superfamily: human igg kuc and muscle protein titin, has been investigated by methods of electron microscopy and diffraction with the use synchrotron radiation. super elasticity of titin, the protein of immunoglobulin superfamily, is a key parameter that determines the mechanical properties of muscle. however, the structural-physical mechanism of titin elasticity under tension remains poorly understood. here both tension transduction and high elasticity of titin are explained in terms of crystalline polymer physics. x-ray data suggest a model of titin as a nanoscale, morphological, aperiodical array of rigid ig-and fn -type domains covalently-connected by conformationally variable short loops. the line group symmetry of the model can be defined as s m with axial translation τ ∞ . homologous domains would have similar stability, but the structure of different domains on stretching is subject to different forces because they have different orientations relative to the axis of the molecule. under the force influence the structure of any domain can become either rigid or flexible depending on its orientation in the titin strand. pulling geometry forms an active axial structure from latent isotropic random coil structure of titin strand. we are suddenly faced with nanophase-separated morphology of igg kuc. study was supported by rfbr grant - - . strain response of myosin essential light chain in permeabilized skeletal muscle fibres d. s. ushakov, d. ibanez-garcia, t. g. west, p. m. w. french, m. a. ferenczi imperial college london, uk we applied fluorescence lifetime imaging microscopy (flim) to investigate the relation between conformation of myosin head and mechanical force in skeletal muscle fibres. recombinant myosin essential light chain (elc) was expressed in e.coli and labelled at cys- with coumarin. the labelled elc was exchanged with native elc in single permeabilized rabbit m.psoas fibres. fluorescence lifetime was measured using leica sp upright confocal microscope equipped with becker & hickl time-correlated single photon counting module and x . na leica planapo dipping objective, with the two-photon fluorescence excitation at nm by sapphire pulsed laser. after acquiring flim images of muscle fibres in relaxed state, the solution was changed to ca-free rigor. further images were acquired in rigor with or without . - % stretch applied by a motor. both single and double exponential fluorescence decay analysis showed that the lifetime in rigor was lower compared to relaxed (about ps difference for single exponential fit) and to rigor fibres under strain (about ps). these data suggest a change in the microenvironment of coumarin induced by nucleotide binding and strain. this change is likely to be due to interaction between c-terminal domain of elc and the n-terminal domain of myosin heavy chain related to the lever arm re-orientation process. supported by bbsrc. alpha-synuclein and its a p mutant affects the actin cytoskeleton structure and dynamics v. sousa , s. bellani , g. ronzitti , f. valtorta , j. meldolesi , e. chieregatti department of neuroscience, hsr, milano, italy, department of neuroscience, iit, genova, italy alpha-synuclein (syn) is a soluble protein abundant in the brain, primarily enriched at pre-synapses. syn overexpression and the expression of its a p mutant participate in the pathogenesis of parkinson's disease. many roles have been proposed for syn, including the regulation of synaptic vesicle pools and of neurotransmitter release. the actin cytoskeleton regulates many aspects of synaptic function and its dysregulation may be a cause of neurodegeneration. working both in cell-free and in vivo conditions we demonstrate that syn and the a p mutant have different effects on the actin cytoskeleton dynamics. our results show that syn binds actin, and decreases actin polymerization rate probably by monomer sequestration. on the contrary, a p accelerates actin polymerization in vitro and disrupts the cytoskeleton of intact cells. in particular, during dynamic cytoskeleton remodeling, a p induces the assembly of discrete actin-rich foci. actin trapping and the impairment of filaments reassembly lead to inhibition of cell movement and of the re-establishment of cell-cell contacts. in a p expressing cells cytoskeleton-based processes, such as cell migration and the exo/endocytic traffic are inhibited. elucidating the dynamics of syn interaction with actin may contribute to the understanding of its role in neuronal physiology as well as in neurodegeneration. on the physics of muscle contraction m. l. shur ural state university, yekaterinburg, russian federation whichever energy source is chosen as an engine, its force will decrease with increasing velocity. this is connected with a limited power of any engine. thus, we state that hill's formula is a mere sequence of the law of energy conservation. to derive a mathematical dependence "force-velocity", all the means of consumption of fuel energy should be determined -in our case, the energy of the atp hydrolyze. moreover, the conformation energy of the crossbridges attached serves as the force source as well. we state that part of the energy release transforms into the energy of oscillations of myosin proteins; the other part goes into thermal energy of the sarcoplasmic solution. interaction of the oscillating myosin system with the sarcoplasmic solution controls the process of force generation by a muscle. it is just this interaction that leads to the temperature dependence of force. the presentation is devoted to constructing a theory based on these simple considerations. a discussion and constructive critic is especially wanted. -biological motility and molecular motors - meiotic nuclear oscillations in the fission yeast schizosaccharomyces pombe are crucial for proper chromosome pairing and recombination. we report a mechanism of these oscillations based on collective behavior of dynein motors linking the cell cortex and dynamic microtubules that extend from the spindle pole body in opposite directions. by combining quantitative live cell imaging and laser ablation with a theoretical description, we show that dynein dynamically redistributes in the cell in response to load forces, resulting in more dynein attached to the leading than to the trailing microtubules. the redistribution of motors introduces an asymmetry of motor forces pulling in opposite directions, leading to the generation of oscillations. our work provides the first direct in vivo observation of self-organized dynamic dynein distributions, which, due to the intrinsic motor properties, generate regular large-scale movements in the cell ( ) m. versaevel, s. gabriele, p. damman university mons-hainaut, mons, belgium the remodeling of blood vessels in response to changes in blood flow is mainly realized by endothelial cells (ecs) that convert mechanical stimuli from flowing blood into changes in cell signaling through a process called mechanotransduction. many of the biological responses to external forces originate at two types of microscale structures: focal adhesions linking cells to their extracellular matrix and adherens junctions that link adjacent cells. this study aims to elucidate the role of the cytoskeleton, cell-matrix and cell-cell junctions in transducing fluid shear stress into intracellular signals in ecs. by using microcontact printing of proteins, we design substrates with defined adhesive islands in order to control shapes of living cells. this confinement of ecs allows to study the organization and the contractile activity of the cytoskeleton in order to redistribute their intracellular forces in response to externally applied forces. we design microfluidic channels with sizes and geometries close to small blood vessels to apply a physiological range of shear stresses on ecs. our results indicate that cells deposited on a precisely defined adhesive area inside microchannels and subjected to shear stress reorganize their cytoskeleton, their focal adhesions and adherens junctions in response to blood flow. drugs interfering with the cytoskeleton are used to underline the role of its different components in the cellular adaptation to the mechanical environment. s. mahdavi , b. ranjbar , s. gharibzadeh , m. toosi , m. javan tarbiat modares university, tehran, iran, amirkabir university of technology, tehran, iran multiple sclerosis (ms) is the main known pathology of myelinating cells. an autoimmune reaction occurs against myelin sheets of neurons, so action potential (ap) propagation along the affected nerve fibers has been destroyed and it causes various disorders. here, we propose a novel strategy for ms symptoms treatment. we modeled neuron by orcad software and simulated the action potential propagation along the axon in normal condition. our model simulated normal neuronal behavior. then we destroyed the myelin sheet as it occurs in ms and observed destroyed ap propagation as it was reported in ms disease. we investigated the effect of changes in the voltage-gated sodium channel (vgsc) threshold on the efficiency of ap propagation. the results demonstrated that reduction of vgsc threshold improves the propagation of ap by increasing the amount of sodium flux during ap propagation. although, some researches have proposed vgsc blocker as ms symptom treatment, our result suggests that the increase of sodium current produced by reduction of vgsc threshold, improves ap propagation and probably cure some ms symptoms. so, we suggest that vgsc gating modifiers can be considered as novel strategy for ms treatment. surely, this results needs to be confirmed by experimental studies. a. gradogna, e. babini, a. picollo, m. pusch istituto di biofisica, consiglio nazionale delle ricerche, via de marini , genova, italy clc-ka and clc-kb are highly homologous cl − channels expressed in the kidney and the inner ear where they mediate transepithelial chloride transport. both channels heteromerize with the beta subunit barttin. mutations in clc-kb and barttin genes lead to bartter's syndrome. we analyzed the modulatory effect of extracellular ca + and h + on clc-k channels using the xenopus oocyte expression system. clc-ka currents increased with increasing [ca + ] ext without full saturation for [ca + ] ext up to mm. however, in the virtual absence of ca + , clc-ka currents are about % of currents measured in mm [ca + ] ext , demonstrating that ca + is not strictly essential for opening. vice versa, clc-ka was blocked by increasing the [h] + ext with an almost complete block at ph . among various reaction models tested, the model that best fitted all state-steady data predicts an allosteric regulation of channel opening by separate binding sites for ca + and h + . moreover, the best fit suggests that one ca + and two h + bind to the channel. kinetic analysis of current responses upon [ca + ] ext and ph jumps confirmed the allosteric character of modulation. in support of the presence of two separate binding sites we identified several mutations that selectively altered ca + or h + sensitivity. our data represent a first step towards a molecular picture of ca + and proton regulation of clc-k channels and suggest that it is of physiological relevance. the extracellular matrix molecule hyaluronic acid modulates l-type voltage-dependent ca + channels e. dvoretskova , g. kochlamazashvili , o. bukalo , c. henneberger , d. rusakov , m. schachner , a. dityatev italian institute of technology, genova, italy, centre for molecular neurobiology, hamburg, germany, institute of neurology, university college london, london, uk we studied the effects of hyaluronic acid (ha), a major extracellular matrix molecule, on activity of l-vdccs in a heterologous expression system and in hippocampal slices. we recorded currents mediated by a major neuronal subtype of l-vdccs (ca v . c, β b, and α δ ) expressed in cho cells. a five-minute application of . mg/ml ha potentiated l-vdcc currents at - , - , and + mv by approximately %. analysis of boltzmann curves showed that ha increased maximal conductance rather than other parameters (v . or k ). treatment with hyaluronidase removed endogenous ha in murine hippocampal slices and specifically impaired long-term potentiation (ltp) induced at ca -ca synapses by repetitive theta-burst stimulation. blockade of l-vdccs reduced ltp in control slices to the levels seen after hyaluronidase treatment. a potentiation of l-vdccs with bay k fully restored ltp after hyaluronidase treatment. removal of ha reduced ca + transients elicited by backpropagating action potentials in individual dendritic shafts and spines of ca pyramidal cells, whereas pretreatment with nifedipine fully occluded this effect. thus, ha potentiates postsynaptic l-vdccs and by this way influences use-dependent synaptic plasticity. whole-cell patch clamp recordings from a variety of human cancer cells showed that functional voltage-gated sodium channel (vgsc) expression occurred specifically with strongly metastatic cells. in addition, where studied, this was accompanied by down-regulation of outward (mainly potassium) currents. this has led to the celex ("cellular excitability") hypothesis of cancer according to which metastatic cell membranes are excitable and this promotes their hyperactivity. importantly, the vgsc genes expressed are embryonic splice variants, which are normally developmentally regulated, hence the phenomenon is 'oncofetal'. in breast cancer, where the predominant vgsc is nav . the neonatal and adult forms are significantly different and this is reflected in channel activity whereby the neonatal vgsc has much slower inactivation kinetics. the double-charge change at position is critical for this difference. the slow kinetics results in much greater influx of na + into cells and one consequence of this is activation of protein kinase a. this is a tonic effect and, under steady-state resting conditions, it results in a positive feedback effect promoting post-translational trafficking of vgsc protein to plasma membrane. the unique amino acid sequence of the spliced region has enabled the production of a polyclonal blocking antibody specific to neonatal nav . . it is concluded that vgscs represent novel biophysical targets for clinical management of metastatic disease. m. pusch cnr, istituto di biofisica, genoa, italy clc proteins form an evolutionary conserved gene-family that comprises members in mammals. four of the human clcs are passive plasma membrane cl − ion channels. the other clcs are expressed in intracellular organelles. clc- and clc- , mutations of which lead to dent's disease, are secondary active cl − /h + antiporters, similar to the bacterial clc-ec , and with identical cl − : h + stoichiometry. cl − and h + transport activity of the exchanger clcs depends on two glut residues. mutating a 'gating glutamate' (e in clc- ) converts the exchanger into anion conductances. neutralizing the 'proton glutamate' (e ), but not its replacement by some other titratable groups, abolishes cl − and h + transport. noise analysis indicated that clc- switches between silent and transporting states with an apparent unitary conductance of . ps, indicating a very large transport turnover. no − uncouples h + transport but mutating the highly conserved s to p, as found in the plant no − / h + antiporter atclca, led to coupled no − : h + exchange. clc proteins are a fascinating example of how a very similar protein architecture can be used to provide either a passive electrodiffusive permeation pathway or a strictly coupled secondary active ion transporter. (supported by telethon italy -grant ggp ). the role of ion dynamics in zebrafish fin regeneration the specific and directional ion transport across cell membranes or tissue layers results in differential accumulation of ions and endogenous electric currents. these phenomena have been shown to be important for vertebrate organs regeneration. however, the specific ion nature of such electric currents remains unknown, as well as the role of cellular ion dynamics during regeneration and the molecular signalling pathway that transduces electric cues into cellular responses. we use zebrafish caudal fin as an adult regeneration model to unveil the specific ion composition of the currents associated with wound healing and regeneration, using a non-invasive ion-specific scanning microprobe setup. our data suggests a role for potassium (k + ), calcium (ca + ) and protons (h + ) at different stages of the regeneration process. k + and ca + extracellular effluxes have both been detected during the wound healing stage. h + efflux is triggered during wound healing and is maintained throughout regeneration. we are validating these data with genetic and pharmacological approaches, as well as advanced ion imaging. overall, our results suggest ion-driven mechanisms underlie adult tissue regeneration and its comprehension may open way for new therapeutic strategies, both in regenerative and developmental medicine and in cancer therapy. cardiac effects of anabolic steroids: an electrophysiological approach anabolic androgenic steroids (aas) have been used by athletes and non athletes for almost five decades in order to improve performance. however, the illicit abuse of high-doses of aas has been attributed as a main cause of several cardiovascular disorders such as arterial hypertension, lipid profile abnormalities, heart failure, hypertrophic cardiomyopathy, arrhythmia and sudden death. the aim of this study was to investigate qt interval and transient outward potassium current (i to ) changes in rats treated with nandrolone decanoate (deca). male wistar rats received weekly mg/kg of deca (n= ) or vehicle (control, n= ). electrocardiogram was recorded weekly, and qt interval was measured. after weeks hearts were excised and single myocytes were isolated from the ventricles of animals. i to was recorded by means of the whole cell patch clamp technique. qt interval was larger in deca group from th to th week (p < . ). analysis of i to showed a decreased current density (p < . ) in ventricular cardiomyocytes of deca group, compared to control group. in conclusion, our results show that alterations on ventricular repolarizaton may constitute an early consequence of the chronic administration of high doses of anabolic steroids in rats, and demonstrated qt prolongation and i to density reduction, which may constitute an important marker of arrhythmia vulnerability and sudden death. -ion channels in channelopathies and cancer - denitrifying bacteria control no and no cytosolic levels by regulating the expression of denitrification gene clusters via redox signalling of specific transcriptional factors that may act as no sensors in vivo. a protein belonging to the subclass dnr (dissimilative nitrate respiration regulator) from pseudomonas aeruginosa has been recently suggested to be a heme containing protein. very recently the three dimensional structure of the apo-form of dnr (in the absence of heme) has been determined by x-ray crystallography, whereas the holo-form (in the presence of heme) has not yet been crystallized. we have investigated the heme local structure in solution of ferric, ferrous, co bound and no bound holo-dnr by x-ray aborption spectroscopy (xas) and we added a kinetic study of the co bound form by means of a flash photolysis setup using uv-visible absorption as a spectroscopic probe. the combination of fe k-edge xanes fingerprints and kinetic study reveal a heme pocket able to bind exogenous ligands like no and co with increased plasticity, thus supporting its role as the cofactor involved in no sensing activity. molecular examination of motifs that lead to the formation of s-nitrosylated proteins i. alicea, e. r. schreiter university of puerto rico rio piedras, san juan, puerto rico physiologically, a wide range of proteins experience structural and functional modifications after the addition of a nitric oxide (no) moiety to cysteine thiol. this posttranslational modification, known as s-nitrosylation, regulates a large number of cellular processes like vasodilatation, cell signaling and others, and the products of s-nitrosylation can be involved during the development of different human diseases. however, little is known about the mechanism by which different proteins specifically bind the no moiety to their cysteine. here we show a bio-statistical analysis of some properties of cysteine that are s-nitrosylated in different proteins, including the pk, electrostatic environment, solvent accessibility of the target cysteine and identity of surrounding amino acids. we also chose model proteins (human thioredoxin and s protein) to make specific targeted amino acid substitutions around selected cysteines to alter the properties described above. the reactivity and stability of these mutant proteins towards s-nitrosylation will be examined. sampling the flexibility of ppar-γ s. aci-sèche , n. garnier , d. genest , s. bourg , c. marot , l. morin-allory , m. genest upr cnrs , orléans, france, fr pcv cnrs , orléans, france, umr cnrs , université d´orléans, france a promising approach to consider the flexibility of proteins in docking studies consists in performing multiple rigid docking on a representative set of the receptor conformations. molecular dynamic (md) simulation is one of the best adapted methods for structural sampling, but exploring the conformational diversity of a protein is computationally expensive. we present a protocol for generating a wide range of conformational states of a receptor using restrained md and a partitioning protocol to select a few representative conformations of the binding site from this md. a way to speed up efficiently md calculation is using an implicit model to represent the solute-solvent interactions. we explore a protocol using a distance-dependant permittivity function to represent solvent effect and an ensemble of controlled restraints applied on a subset of specific atoms in order to prevent artefactual structural distortions, but preserving receptor's flexibility. ten ns simulations have been performed using different sets of parameters and compared to a reference ns simulation with explicit solvent. to select a representative set of conformations, partitioning (k -means algorithm) was applied on the ensemble of simulated conformations. this methodology was applied to the ligand binding domain of peroxysome proliferator-activated receptor-γ. department of biomedical sciences, university of antwerp, antwerp, belgium the complex system of cavities identified in human neuroglobin (ngb) has been postulated to be of functional significance to the putative no dioxygenase activity of the protein. the interconnected hydrophobic cavities may support this catalytic activity by acting as reservoir for reactants and providing preferential pathways assisting product removal from the active site. we thus decided to investigate co rebinding kinetics to ngb embedded in silica gels to expose ligand migration processes in the geminate phase. encapsulation of the co complexes of reduced neuroglobin, leads to a slight increase in geminate recombination after nanosecond laser photolysis. increasing the viscosity of the medium, by soaking the gels in glycerol, completely inhibits escape of the photodissociated ligand to the solvent, and highlights a complex, multiphasic kinetic pattern. this finding can be rationalized by assuming the existence of a discrete set of temporary docking sites, capable of trapping the photodissociated ligand for very long times, up to a few ms after photolysis. g. bartolommei, f. tadini-buoninsegni, m. r. moncelli bioelectrolab -department of chemistry, university of florence, italy ion pumps are integral membrane proteins devoted to ion transport through a lipid membrane phase. the ion pumps ca-atpase and na,k-atpase are prominent members of the p-type atpases family. due to fundamental physiological roles of these proteins, they are very promising drug targets. bioelectrolab has a wide expertise in the study of ion transport by these proteins [ ] . our attention has been recently focused on the interaction of these enzymes with molecules of potential pharmacological interest. frequently, drugs exert an inhibitory action on the transport activity of an ion pump, usually confining it in an inactive conformation. molecules like thapsigargin and cyclopiazonic acid belong to high (nanom) affinity inhibitors of the ca-atpase, whereas clotrimazole and curcumin are medium (microm) affinity inhibitors of both ca-atpase and na,k-atpase. for each of these compounds a mechanism of action is proposed. moreover, recent results concerning ca-atpase inhibition by clotrimazole analogues will be shown: the relevance of this type of molecules is due to their potential employment as an alternative to traditional drugs against malaria parasite. financial support of ente cassa di risparmio di firenze and of miur (prin project) is gratefully acknowledged. [ ] tadini-buoninsegni f., bartolommei g., moncelli m.r., fendler k. . arch. biochem. biophys. : - (review). s. asthana , s. shukla , g. giliberti , f. luliano , m. ceccarelli , r. loddu , p. ruggerone , p. la colla department of biomedical science and technology, università di cagliari, cagliari, italy, department of physics, università di cagliari, cagliari, italy studies of protein-inhibitors interactions are helpful to elucidate the mode of action of ligands and thereby providing clues for rational drug design. bvdv, is an important target of drug discovery activities largely because it is essential for viral replication. in bvdv rdrp no specific nni binding site has been reported till now. experimental results have shown that different class of inhibitors (benzimidazole, imidazoquinolines and pyridoxyquinolines), have resistant mutations located in the finger domain of the rdrp. all the reported mutations are spatially very close to each other. thereby, indicating that binding sites of nni's may lie in the finger domain for these different class of inhibitors.herein, we have utilized docking procedure to investigate binding sites, binding modes as well as binding affinity of different class of inhibitors.we then used all atom molecular dynamics (md) simulations to investigate the stabilizing interaction between inhibitor-receptor pairs. our md results are in good agreement with experimental data and provide deep insights into the dynamical features of the high affinity inhibitorreceptor binding. thus identifying the binding modes of our inhibitors and mechanism leading to inactivity of the enzyme can help us to build a microscopically well-funded picture of the functioning of these enzymes. we present an investigation of the molecular basis of ligand binding and reactivity of heme proteins using computer simulation. a combination of classical molecular dynamics and hybrid quantum-classical (qm-mm) calculations are applied to explore distal and proximal effects on diatomic ligand binding to the heme. trends in binding energies and in the kinetic constants are illustrated through a number of selected examples. an investigation of the interplay between ligand migration and protein dynamics obtained through classical molecular dynamics techniques in combination with advanced sampling tools is also presented to yield information about free energy profiles and possible secondary docking sites. results for truncated n hemoglobin of mycobacterium tuberculosis, presented as an illustrative example, suggest that the truncated hemoglobin n has evolved a dual-path mechanism for selective/distinct migration of o and no to the heme, to achieve efficient no detoxification. finally, we present also an analysis of the molecular basis of hexacoordination in human neuroglobin, which suggest that the flexibility of the cd plays a key role in determining the ligand binding properties. thermodynamic bases of nucleoplasmin-histone complexes recognition by the nuclear transport machinery i. arregi, j. falces, s. bañuelos, m. a. urbaneja, s. g. taneva unidad de biofísica (csic/upv-ehu), departamento de bioquímica y biología molecular, universidad del país vasco, spain the nuclear transport of the chromatin remodeling (nucleoplasmin) and chromatin building (histones) proteins is mediated by importins. nucleoplasmin (np) contains a classical bipartite nuclear localization signal (nls) that is recognized by importin α, while histones present multiple sequence elements (nls-like motifs) for nuclear targeting. besides, ternary importin/np/histone complexes might represent a putative coimport pathway for nuclear import of linker (h ), nucleosomal core (h ah b) histones and their chaperone protein np, enhancing the histone import efficiency. to better understand np and histone recognition by the transport machinery we studied the thermodynamics of complex formation of importin α (a truncated form) and importin β with histones and np, and with np/histone binary complexes by means of isothermal titration calorimetry. data show that importins interact with the two histone types and np, and that importin and histones can simultaneously bind to np. analysis of the binding energetics reveals an enthalpy driven formation of high affinity binary and ternary complexes. we demonstrate that different amount of importin molecules can be loaded on np/binary complexes dependent on the histone type, linker or core, and the amount of the bound histones. g. breuzard, i. di maïo, p. barbier, d. allegro, c. brault, v. peyrot cro inserm u , ufr de pharmacie, marseille, france since a decade, our laboratory has already studied the interaction of tau variant with tubulin (tub) or microtubules (mts) and phosphorylation process on this interaction. indeed, fret assay was a powerful tool to achieve binding parameters between tau and tub in vitro: / with mts stabilized by fluorescein-coupled taxol (flutax- ) as donor and rhodamine-labelled tau (rho-tau) as acceptor, and / in living cells by confocal laser scanning microscopy (clsm) with tau/α-tub fused to egfp/mcherry, respectively. results revealed ± % energy transfer efficiency from flutax- to rho-tau and a donor-to-acceptor distance of ± Å. by titration, the dissociation constant of tau was determined to . ± . µm. a cleavage procedure of αβ-tub was performed to determine the influence of the c-term tails of αβ-tub on the tau-mt interaction. no difference in distances and binding parameters was observed. clsm images displayed a heightened concentration of fluorescent tau in patches along mts. fret experiments revealed in particular higher efficiencies between gfp-tau to mcherry-α tub proteins in these locations. overall, our results suggested no involvement of the hypervariable and highly acidic c-term tails of tub in mt/tau binding. a molecular model is proposed in which flutax- is directly accessible to tau molecules. besides, the modified distribution of fluorescent tau could be implicated in local change of mechanical properties of mts. a. boreham , k. winkler , c. gebhard , k. rueck-braun , p. henklein , e. michalsky , r. preissner , r. misselwitz , a. ziegler , u. alexiev freie universität berlin, berlin, germany, technische universität berlin, berlin, germany, charité-universitätsmedizin berlin, berlin, germany peptide presentation by major histocompatibility complex (mhc) molecules is crucial for immune responses. photocontrol of peptide dynamics by means of photo-switchable peptide analogs will provide insights in peptide dynamics and its dependence on mhc polymorphism ( ) ( ) ( ) . we have designed a hemithioindigo (mhti) photo-switch bearing peptide using the viral epitope rrrwrrltv (plmp ) as a template. incorporation of mhti in the peptide backbone should result only in a minor change of the overall peptide structure given the relaxed conformation of the zisomer. indeed, the human mhc molecule hla-b* can tolerate nonpeptidic elements in plmp , as evidenced by normal peptide binding. using the autofluorescent properties of mhti we determined the stability of the hla-b* /mhti-plmp complex. photoswitching from z →e results in a decrease of hla-complex stability. based on computer modeling this decrease is due to a reduced interaction of the peptide c-terminus with hla-b* . truncated hemoglobins (trhbs) are heme proteins present in bacteria, unicellular eukaryotes, and higher plants. three phylogenetic groups (n, o, and p) have been identified in trhbs. the crystal structure of truncated hemoglobin o of b.subtilis, does not show an evident tunnel/cavity system connecting the protein active site with the solvent, a fact that cannot be easily rationalized considering the very high oxygen association rate. moreover, resonant raman results of the co bound protein, showed that a complex hydrogen bond network exists in the distal cavity, making it difficult to assign unambiguously the residues involved in the stabilization of the bound ligand. for these reasons we performed classical molecular dynamics simulations of the oxy, carboxy and deoxy protein, and computed the free energy profiles associated with ligand migration to the active site. our results suggest that there is a key residue, glne , that may present an alternate conformation in which a wide ligand migration tunnel is formed, consistently with the kinetic data. the results for the co and o bound protein show also that glne is directly involved in the stabilization of the coordinated ligand, playing a similar role as tyrb , and trpg in other trhbs. our results not only reconcile the structural data with the kinetic information, but also provide additional insight about the general behaviour of trhbs. patterned functionalization of surfaces for guided transport on molecular motors tracks m. bhagawati , s. ghosh , t. surrey , j. piehler department of biophysics, university of osnabrueck, osnabrueck, germany, european molecular biology laboratory, cell biology and biophysics unit, heidelberg, germany chelator head groups with multiple nitrilotriacetic acid (nta) moieties have been very well characterized and successfully utilized as high affinity adapters for functional immobilization of oligohistidine tagged proteins to surfaces. we have recently established a generic method for patterning nta functionalized surfaces by selective photodestruction via a light induced fenton reaction. efficiency of different transition metal ions for catalyzing this reaction was tested. functionality of the patterned protein was confirmed using the interaction between interferonα and its receptor. implementation of this technique in a confocal laser scanning microscope allowed us to control surface density of binding sites, providing the possibility to vary the surface concentration of immobilized proteins in a spatially resolved manner. we also applied this approach for exploring guided transport of microtubules by kinesin selectively immobilized onto trisnta patterns. rheumatoid arthritis (ra) is an autoimmune disorder, leading to pathological damage at the level of joints, associated with the hla class ii allele hla-dr . although etiology of ra is unknown, type ii collagen (cii) is a potential antigen candidate and it is believed that t cell responses in collagendependent ra are directed towards the immunodominant pathogenic epitope cii( - ). despite recent advances in characterization of class ii major histocompatibility complex (mhc) and t-cell receptor (tcr) contacts in this epitope, the atomic details of tcr-cii( - )-mhc complex are not known. here, homology modeling and molecular docking studies have been used to derive a three-dimensional model of tcr β chains, obtained from a dr + subject, in complex with cii( - )/hla-dr . the best complex from docking was further refined using molecular dynamics simulations for ns. the proposed model represents a reasonable structural basis for understanding cii( - )-mhcii complex recognition by tcr and for rational design of inhibitors targeting tcr-pmhc interface. probing bio-molecular bonds with magnetic force for biosensor applications a. m. de jong , a. jacob , x. j. janssen , j. m. van noorloos , l. j. van ijzendoorn , m. w. prins eindhoven university of technology, eindhoven, the netherlands, philips research laboratories, eindhoven, the netherlands we investigate new technologies to be applied in next generation biosensors, which not only measure biomarker concentrations but also probe bio-molecular interactions. the concept is based on the response of ligand-receptor pairs to an applied force or torque [ , ] . we use functionalized magnetic beads and magnetic fields to apply translational and rotational forces on the molecular bonds. in a model experiment, polystyrene surfaces were coated with anti-biotin and beads were coated with biotin. after incubation, a constant magnetic force was applied to the beads and the number of bound beads was measured as a function of time. this was repeated for a range of forces. the dissociation rate (k o f f ) is determined for each force and k o f f at zero force is extracted from these data. rotational forces were exerted on protein-g coated beads bound to igg antibodies immobilized on a surface. rotating fields revealed an oscillating behavior, which can be understood from the balance between the applied magnetic torque and the torque due to the deformation of the biological bond. studying interactions between cop regulatory protein and hy transcription factor d. s. dalafave the college of new jersey, ewing, nj , usa this work addresses important questions of protein-ligand interactions and selective protein recognition. proteinligand bindings are crucial for many cellular processes. dependable methods for predicting binding sites would lead to a better understanding of proteins' selective recognition and would, in turn, help research on fighting diseases. presented here is a study of interactions between the wd domain found in a regulatory protein cop and the motif v-p-e/d-Φ-g (Φ=hydrophobic residue) found in a transcription factor hy . cop and hy proteins have opposing roles in developmental regulation. cop can repress hy by directly binding with it. the repression involves specific interactions between the wd domain and the motif v-p-e/d-Φ-g. previous experimental research showed that mutations in the motif's v-p pair resulted in a large decrease in hy repression. to study effects of similar mutations, residues in the motif v-p-e/d-Φ-g were systematically substituted with other residues. interactions between the mutated motif and the wd domain were studied. distributions of binding sites, bond lengths, local shape complementarity, and interaction potentials were modeled for each residue substitution. the study identified binding sites critical for the cop -hy binding. some hy residues in the vicinity of the motif were also found to be important in the binding. the significance of the results for understanding selective protein recognition is discussed. determination of protein-ligand binding thermodynamics by thermal shift assay p. cimmperman, a. zubriene, l. baranauskiene, e. kazlauskas, j. matuliene, d. matulis laboratory of biothermodynamics and drug design, institute of biotechnology, vilnius, lithuania thermal shift assay determines the effect of ligand binding on the protein thermal denaturation equilibrium. several models have been derived to describe the general cases of protein-ligand binding. first, the model describing protein stabilization and destabilization by ligand binding to the native and unfolded states. second, the split of protein melting transitions by tightly binding ligands is presented when the transition of free protein and ligand-bound protein occur separately. mathematical equations describing the protein unfolding and ligand binding thermodynamic parameters at various temperatures are presented. the protein melting temperature shift can be determined by various techniques such as differential scanning calorimetry, circular dichroism, and intrinsic or extrinsic fluorescence. the advantages of fluorescent techniques are presented. the melting temperature shift caused by ligand binding is dependent on the thermodynamic parameters of protein unfolding and ligand binding, including enthalpy, entropy, and heat capacity, thus allowing determination of binding thermodynamics. application of the models in the design of hsp chaperone and carbonic anhydrase inhibitors is discussed. comparison with isothermal titration calorimetry data is presented. recent reports have shown that the bacterial redox protein azurin can enter into cancer cells and induce apoptosis by stabilizing p . the formation of a complex between the two proteins has been demonstrated, but little is known about binding features. for the first time, we show here that azurin binds to the n-terminal region of p with a dissociation constant in the - µm range. trp phosphorescence lifetime measurements revealed conformational changes of azurin induced by the interaction with p ( - ). acrylamide quenching of trp phosphorescence also indicated a significant increase of the overall flexibility of azurin upon binding to p . no change of the fluorescence emission of p ( - ) was detected in the presence of azurin. the latter finding suggests that w of p is not directly involved in domain binding to azurin, indicating that the binding site is distinct from that of mdm . the present results may assist the design of novel cancer treatments based on p stabilization by azurin. a. eleta , r. georgieva , h. bäumler , j. l. toca-herrera cic biomagune, donostia-san sebastián, spain, charité-universitätsmedizin berlin, berlin, germany human serum albumin (hsa) is the most abundant nonglycosilated plasma protein in the human body. this multifunctional protein has ligand-binding and transport properties, antioxidant functions and enzymatic activity [ ] . bilirubin interacts with albumin in order to be transported from the blood to the liver where it is secreted. the interaction occurs specifically in hsa i-domain of its three domains [ ] . in our work, we investigate the interaction between hsa and bilirubin by quartz crystal microbalance with dissipation (qcm-d) and atomic force microscopy (afm) [ , ] . albumin was adsorbed on negatively charged silicon oxide. however, hsa was removed after rinsing with pbs. hsa adsorbed on positively charged polyelectrolyte multilayers leads to a stable layer of surface mass density of ng/cm . cross linked albumin with glutaraldehyde after its adsorption on nh -terminated thiols is also stable reaching surface mass density of ng/cm . a preliminary bilirubin adsorption results on cross linked hsa substrate show that ng/cm is immobilized. taking into account that hsa-bilirubin stoichiometry is : , the outcome demonstrates that the % of hsa i-domains remain active. antimicrobial peptides (amps) are short positively charged polypeptides. they are important due to their potential to provide an alternative to conventional therapy against bacterial infections. rbpi is a kda peptide based on the nterminal region of the neutrophil bactericidal/permeabilityincreasing protein (bpi). it was shown that this amp possesses bactericidal effects on gram-negative bacteria and higher affinity for lipopolysaccharide (lps), neutralizing its effect. the peptide use against meningitis, is in phase iii clinical trials. here, we demonstrate that rbpi promotes aggregation of negatively charged large unilamellar vesicles (luv) and lps aggregates, by dynamic light scattering, while for zwitterionic phosphatidylcholine (popc) luv the size remains unchanged. the aggregation increases with peptide concentration until peptide promotes massive aggregation followed by sample flocculation/precipitation. with the rbpi -lipid interaction there is a progressive change in the zeta-potential of the luv systems and lps aggregates. luv systems composed of phosphatidylglycerol (popg) and popc:popg mixtures have higher zeta-potential variations than popc luv. for lps aggregates, rbpi neutralizes the surface charge and at higher peptide concentrations overcompensates it. results demonstrate that the interaction of the peptide rbpi with lps aggregates and luv systems has electrostatic and hydrophobic contributions. serratia marcescens heme acquisition system: heme transport and protein:protein interactions m. delepierre unité de rmn des biomolécules cnrs ura , institut pasteur, paris, france heme transport systems in bacteria are required and might be potential target for antibacterial drugs. the heme acquisition system, has, exists in pathogenic as well as in opportunistic bacteria but only for the latter one extensive studies have been conducted, constituting as such a model system. the outer membrane receptor hasr, the central component of this system, functions in synergy with a secreted high affinity heme binding protein, the hemophore hasa. hasa extracts heme from host hemoproteins and returns it to hasr. then, the energy given by a protein complex of the inner membrane is used to allow heme entrance across the bacterial membrane and to eject the empty hemophore from the receptor. reconstitution of this heme acquisition system in e coli, overexpression and purification of its various components have allowed us to obtain sufficient amount of protein to perform nmr and biophysical studies to analyse at the molecular level the different steps of heme acquisition by hasr. protein-protein interactions (ppi) are the central pillar supporting most of biological functional activity on the molecular level. a binding event between two proteins typically consists of two stages: ) diffusional search of the binding partners for each other, and ) specific recognition of the compatible binding surfaces followed by the formation of the complex. we focus here on the non-specific component of ppi, which refers to all physico-chemical properties of the binding partners (such as size, charge, isoelectric point, hydrophilicity etc.) that are independent of the exact details of their binding sites, but which could in turn affect their localization or diffusional search for one another. it is known that proteins co-localize due to segregation into different cellular compartments, sequestration via anchor and scaffold proteins or even chemical modifications. we suggest that the non-specific component of ppi determines in part the co-localization and clustering of the binding partners, which then directly in a non-specific fashion influences their interactions. we examine the possibility that such signature might be encoded within the experimental d structures of a large set of known mutually interacting proteins. we provide preliminary evidence that this indeed may be the case, and corroborate our findings by using different statistical tests to compare those features of the known interacting partners, and ascertain correlations and commonalities between them. a link between hinge-bending domain motions and the temperature dependence of catalysis in ipmdh i. hajdú, a. szilágyi, j. kardos, p. závodszky institute of enzymology, hung acad sci, budapest, hungary enzyme function depends on specific conformational motions. since conformational flexibility strongly depends on temperature, temperature dependent enzyme kinetic studies with measurements related to dynamics can give us some insight at atomic level into these functionally relevant motions. the catalytic efficiency (k cat /k m ) of -isopropylmalate dehydrogenase for its substrate (ipm) has unusual temperature dependence, showing a local minimum at • c. in search of an explanation, we measured the individual constants k cat and k m,ipm as a function of temperature, and found that the van't hoff plot of k m,ipm shows a sigmoid-like transition in the - • c temperature range. by means of various measurements including h-d exchange and fret, we showed that the conformational fluctuations, including hinge-bending domain motions increase more steeply with temperature above • c. the thermodynamic parameters of ligand binding determined by itc as a function of temperature were found to be strongly correlated to the conformational fluctuations of the enzyme. because the binding of ipm is associated with a hinge-bending domain closure, the more intense hinge-bending fluctuations at higher temperatures increasingly interfere with ipm binding, thereby abruptly increasing its dissociation constant and leading to the observed unusual temperature dependence of the catalytic efficiency. a simulation approach to multiple sclerosis: study of a peptide with a pharmaceutical potential c. guardiani , s. marsili , p. procacci , r. livi centro dinamiche complesse, università di firenze, italy, dipartimento di chimica, università di firenze, italy, dipartimento di fisica, università di firenze, italy multiple sclerosis (ms) is an autoimmune disease of the central nervous system, leading to premature death. one of the potential targets of the autoimmune reaction is the myelin protein mog that has been crystallized in complex with the - c ms-autoantibody. the analysis of contacts and buried surface area combined with an alanine scanning computation reveals the key role of mog fragment - for the interaction with - c . a docking simulation shows that the - fragment, excised from mog, and kept in crystal-like conformation, is still capable of fitting into the binding pocket of the antibody. we then studied, through replica exchange molecular dynamics simulations, the structural equilibrium distribution of the free peptide and of a number of analogs stabilized by a disulfide bond. we found that the free peptide yields a significant fraction of crystal-like conformations and the proportion of native-like structures is further increased by the disulfide bridge. when we tried to dock the centroids of the most populated clusters to - c , we discovered the existence of a docking funnel whose bottom is populated by stable complexes where the peptide occupies the same spatial region as in the crystal. we therefore conclude that the mog - fragment may be used to develop a diagnostic assay or a drug for ms. the escherichia coli membrane insertase yidc reversibly binds its substrate pf coat protein u. gerken, s. winterfeld, a. kuhn institute of microbiology and molecular biology, university of hohenheim, germany the membrane insertase yidc of e. coli belongs to the oxa family of mitochondria and plays an essential role in facilitating the insertion and assembly of membrane proteins. we have previously shown with detergent-solubilized (c pc) yidc, labelled with ans, and pf coat that the initial step of the membrane insertion process, the binding of the substrate pf coat to yidc, is reversible [biochemistry , - ( ) ]. the dissociation constant k d for that particular system is about µm. in order to obtain data for the native system we used in this study membrane-reconstituted (dopc and dope/dopg) yidc. the effect of the initial binding was examined in vitro by fluorescence quenching of the tryptophan (trp) residues of yidc which are highly sensitive fluorescent probes for changes of the tertiary structure. quenching of the trp fluorescence after titration with a trp-free pf mutant indicates a change in the yidcs tertiary structure upon binding to its substrate. the binding data show a k d value in the range of . - . µm. the influence of different environments (lipid membranes, ddm micelles) on the secondary structure of yidc as well as on the yidc large periplasmic domain p was investigated by circular dichroism (cd). the cd data show that the secondary structure of yidc changes upon reconstitution into a membranes when compared to the detergent solubilized state. particularly, the p domain is considerably affected by the detergent c pc. b. karasulu, b. erman, o. keskin koc university, istanbul, turkey histone proteins are fundamental to the cells since they are involved in cell regulatory processes, such as chromatin regulation, gene silencing and transcription, cell cycle control, and epigenetics, which are controlled via post-transcriptional modifications of the histone protein tails. these modifications are categorized under four main groups: methylation, acetylation, ubiquitination, and phosphorylation. among them methylation has been very recently proven to be reversible with the discovery of histone demethylase proteins and this modification type has been extensively studied, because the abnormal methylation rates cause the excess proliferation of the cell, which, in turn, triggers the cancer. therefore, understanding of the details of reaction mechanisms of histone methylation/demethylation dynamics provide the required knowledge basis for preventing the abnormal methylation rates by designing proper inhibitor (drug) molecules. in this study, we display possible reaction mechanisms (such as amine oxidation via lsd ) for the demethylation of specific histone tail proteins. we try to explain the role/importance of residues that take place in or near to the reaction pocket for the demethylation reaction. we also carry out md simulations and reaction path (free energy profile) analysis using free energy perturbation (fep) method for qm/mm hybrid systems in order to compare different possible reaction pathways. the sonic hedgehog (shh) signalling pathway plays an important role both in embryonic development and in adult stem cell function. inappropriate regulation of this pathway is often due to dysfunction between two membrane receptors patched (ptc) and smoothened (smo) , which lead to birth defects, cancer or neurodegenerative diseases. however, little is known about ptc, the receptor of the shh protein, and the way ptc regulates smo, the receptor responsible for the transduction of the signal. to develop structure-function studies of these receptors, we expressed human ptc (hptc) in the yeast saccharomyces cerevisiae. we demonstrated that hptc expressed in a yeast membrane fraction is able to interact with its purified ligand shh, indicating that hptc is produced in yeast in its native conformational state. using surface plasmon resonance technology, we showed that fluorinated surfactants preserve the ability of hptc to interact with its ligand after purification. this is the first report on the heterologous expression and the purification of a native and stable conformation of the human receptor ptc. this work will allow the scale-up of hptc production enabling its biochemical characterization, allowing the development of new therapeutic approaches against diseases induced by shh signalling dysfunction. dissecting the colicin translocon c. l. johnson , a. solovyova , p. callow , s. a. holt , l. a. clifton , k. weiss , j. h. lakey inst. for cell and molecular biosciences, newcastle univ., uk, inst. laue langevin, grenoble, france, isis, rutherford appleton lab., didcot, uk, oak ridge national lab., centre for structural molecular biology, oak ridge, usa pore-forming colicin n hijacks e. coli outer membrane protein ompf and exploits it as both a receptor and translocator to cross the outer membrane [ ] . it is currently a matter of debate if the translocation route is through the ompf lumen or the interface between ompf and the lipid bilayer. recent electron microscopy data from our laboratory suggests the latter route for translocation [ ] . the colicin n/ompf complex in detergent has been studied by sans to examine the translocation pathway undertaken by colicin n. by using a combination of deuterated ompf and hydrogenated colicin we have been able to derive a low resolution structure of individual proteins in the binary complex. low resolution structural studies supplemented by targeted mutagenesis and screening techniques including itc, potassium efflux assays and auc have allowed us further our understanding of colicin n translocation. dual-color fluorescence cross correlation spectroscopy (fccs) has been used to explore the molecular dynamics at immune cell surfaces, with a particular focus towards the regulation mechanisms of natural killer (nk) lymphocytes. nk cells are critical mediators of anti-viral immunity and protectors against cancer spread. their activity is governed by a fine-tuned balance between inhibitory and activating receptors, where ly a and kir receptors represents the inhibitory ones. their ligands are mhc class i receptors. fcs is a technique based on the analysis of intensity fluctuations of fluorescent molecules excited by a focused laser beam. the technique offers information about molecular dynamics at the single molecular level, in the nanosecond to millisecond range. dual color fccs expands fcs by correlating the intensity from two different colors. by labeling two potential interaction partners with dyes emitting at different wavelengths, the amount of interaction can be determined. here, we will report on recent fccs data exploring the interaction between the inhibitory receptors and their ligands, as well as different labeling strategies used to enable these measurements. effect of osmolytes on the dhfr activity, structure and dynamics b. legrand , s. renaud , m. collen , c. tascon , s. bonnassie , e. gautier , j. mellet , c. blanco , e. le rumeur , j.-f. hubert , a. bondon rmn-ilp, duals, cbp, umr cnrs , univ. de rennes , france osmolytes are small molecules accumulated by a wide variety of organisms in response to hyperosmotic stress. they contribute to save the cellular integrity and to stabilize the macromolecules from environmental stress. dihydrofolate reductase activity is inhibited by several osmolytes. we studied the impact of osmolytes on the dhfr structure and dynamics by various techniques. we observe that substrate (dhf) and cofactor (nadph) diffusions are quite different in glycerol and betaine despite similar viscosities. we demonstrate that the overall structure is maintained at high osmolyte concentrations while no direct interactions can be detected with the enzyme. the k o f f of substrate analogues decreases with increasing the osmolyte concentrations. the enzyme dynamics, in various media, has been compared with the dhfr behaviour in water described in the literature. the osmolyte impact appears only partly conditioned by its viscogenic properties which reduce the molecules diffusion and the k o f f of the product controlled by the m loop of the dhfr. we suggest that the osmolytes decrease m loop mobility. comparing the results obtained with different osmolytes, we offer a better understanding of the osmolyte nature dependence of the dhfr inhibition. estrogen receptor (er) is a well characterized member of the nuclear receptor superfamily that modulates the expression of estrogen-responsive target genes in response to estradiol and other natural and synthetic chemicals mimicking the estradiol structure. in human, two ers, erα and erβ, lied on two distinct chromosomes, are known. er exhibits several functional domains: two conserved domains, a short domain c involved in dna-binding and a large domain e/f responsible for ligand-binding and hormone-dependent transcription activation, are linked by a hinge domain d; a poorly conserved a/b domain, at the n terminus, mediating interactions with the general transcription machinery, is involved in hormone-independent transcription activation. upon estrogen binding, ers can specifically bind to a dna fragment, called estrogen response element ere, and activate the transcription. the optimal ere sequence consists of two six base-pair half-sites, aggtca, organized as inverted repeats with a three base-pair spacing. in this study, we have investigated, by fluorescence methods, the effect of kcl concentrations, on the protein conformational flexibility and the thermodynamic stability of hers -eres complexes. we show, here, that electrostatic interactions, inside hers, contribute to its conformational flexibility and its thermal stability. moreover, the specific interaction between hers and eres is poorly sensitive to changes in ionic strength, in opposite to unspecific complexes. kinetic and structural explanation for the low enantioselectivity of human -phosphoglycerate kinase p. lallemand , j. rouhana , l. chaloin , b. roy , s. arold , t. barman , c. lionne cpbs umr , bd henri iv cs , montpellier cedex , france, ibmm umr , cbs umr l-nucleosides comprise a new class of antiviral and anticancer agents that are converted to pharmacologically active nucleoside triphosphates in vivo. the last step of the cascade may be catalyzed by -phosphoglycerate kinase (pgk), an enzyme that has low specificity for nucleoside diphosphate: ndp + , -bisphosphoglycerate ↔ ntp + -phosphoglycerate. here we compare the kinetics of formation of the complexes of human pgk with different d-and their mirror images, l-nucleoside diphosphates, and the effect of -phosphoglycerate thereon. two types of experiment were carried out: equilibrium experiments allow the estimation of dissociation constants, and stopped-flow experiments the transient kinetics of the interactions. in addition, by the rapid-quench-flow technique, we compare the kinetics of the phospho-transfer and product release steps with each d-or l-nucleotide. crystallographic and molecular modelling studies allow defining the structural reasons for the low enantioselectivity of pgk. the aim of this basic work on the mechanism of action of human pgk with non-natural nucleotides is to obtain information for the optimization of therapeutic nucleoside analogues that are poorly phosphorylated by pgk. anrs is gratefully acknowledged for financial support. a new itc assay for measuring high-and lowaffinity protein-ligand interactions g. krainer , j. fanghänel , s. keller leibniz institute of molecular pharmacology (fmp), berlin, germany, bayer schering pharma ag, berlin, germany isothermal titration calorimetry (itc) is the gold standard for the quantitative characterisation of protein-ligand and protein-protein interactions [ ] . however, reliable determination of the dissociation constant (kd) is typically limited to the range µm > kd > nm. nevertheless, interactions characterised by a higher or lower kd can be assessed indirectly, provided that a suitable competitive ligand is available whose kd falls within the directly accessible window [ ] . unfortunately, the established competitive itc assay requires that the high-affinity ligand be soluble at high concentrations in aqueous buffer containing only minimal amounts of organic solvent. this poses serious problems when studying protein binding of small-molecule ligands taken from compound libraries dissolved in organic solvents, as is usually the case during screening or drug development. here we introduce a new itc competition assay that overcomes this limitation, thus allowing for a precise thermodynamic description of high-and low-affinity protein-ligand interactions involving poorly water-soluble compounds. we discuss the theoretical background of the approach and demonstrate some practical applications using examples of both high-affinity (kd < nm) and low-affinity (kd > µm) protein-ligand interactions. a molecular dynamics study of the cftr nucleotide binding domains interaction v. martorana , r. noto , o. moran cnr-istituto di biofisica, palermo, italy, cnr-istituto di biofisica, genova, italy the cystic fibrosis transmembrane conductance regulator (cftr), the dysfunctional protein in cystic fibrosis, contains two transmembrane domains, two nucleotide-binding domains (nbds), and a regulatory domain. opening of the pore have been linked to the atp-driven tight dimerisation of nbd. we have studied the nbd -nbd interactions on wild type and cystic fibrosis-related mutations by steered molecular dynamics simulations (smd). a fully solvated dimer, including the two bound atps, was separated by pulling one monomer with an external, increasing force. interestingly, the force needed to break the mutated (g d) dimer is significantly smaller than in the wild type case. the effect of a cftr potentiator, the genistein, has also been tested by repeating the smd simulations with the small molecule docked at the interface between the two nbd domains. to test the validity of our results we have repeated the separation process for different simulation lengths and force strengths. the amount of distortion on the pulled nbd domain has also been studied. this work is partially supported by the italian cystic fibrosis foundation (prog ffc # / ), with the contribution of "mille bambini a via margutta"onlus"blunotte", "lega italiana fc toscana" stability and aggregation studies of human septin (sept ) j. n. a. macêdo, r. c. garratt, a. p. u. araújo instituto de física de são carlos, usp, são carlos, brazil the septins are a conserved family of nucleotide binding proteins firstly identified in saccharomyces cerevisae as proteins required for the completion of the cell cycle. septins are implicated in several cellular functions such as cytokinesis, vesicle trafficking, exocytosis, cytoeskeletal dynamics, cell polarity and sperm motility. furthermore, they are associated with alzheimer's and parkinson's disease. sept was identified in rat brain being highly enriched in presynaptic nerve terminals. it colocalizes with synaptophysin and dynamin i and is associated with synaptic vesicle. in this work, human sept without its n-terminal domain (sept gc) was expressed in e. coli and purified by affinity and size exclusion cromatographies. structural stability studies were performed with recombinant sept gc using circular dichroism spectroscopy (cd), right-angle light scattering, and fluorescence spectroscopy. the sept gc cd spectrum showed a conformational transition from a predominantly α-helical starting structure to one dominated by β-sheet, just above physiological temperatures. the formation of irreversible aggregates, detected by light scattering, and their ability to bind thioflavin-t suggested that sept forms amyloidlike structures, as has been previously observed for human septins and . our data suggest that amyloid formation by isolated septins in vitro may be a general phenomenon whose physiological relevance needs to be further investigated. pln is an antimicrobial peptide produced from lactobacillus plantarum nric . analog pln (pln a) and a ser-derived (pln s, tyr to ser replacement) were synthesized on solid phase and their interactions with biomembrane model systems and inhibitory property on s.aureus and p.aeruginosa were investigated by cd, leakage assays, fluorescence spectroscopy, and differential scanning calorimetry. both peptides share the same unordered structure-like cd spectrum in aqueous solution, but a helicoidal induction in the presence of negative vesicles were observed, however pln s showed lower helical content than pln a. the ser-derived peptide presented % decrease of its leakage activity in different liposome compositions, a threefold increase in the dissociation constant from the liposomes than pln a, and close to % reduction for the inhibitory activity against p. aeruginosa growth. we can conclude that besides the electrostatic contacts between the amphipathic &alpha-helix, formed by the cationic residues from pln and negatively charged phospholipids, the pln -membrane binding is a process driven for the hydrophobic interactions from non-polar residues. in this case, there was a significant contribution of the tyr residue, which must be allocated in a lipid interface, described as the preferential position to this residue in membrane proteins. supported: fapesp label free detection using deep-uv laser-based fluorescence lifetime imaging microscopy q. li, s. seeger physikalisch-chemisches institut, universität zürich, winterthurerstrasse , ch- zürich, switzerland label free detection based on native fluorescence excited at uv region shows great potential for the life sciences. it offers simple, low-cost and fast method for sensitive detection of important biological analytes without modification. in this contribution we present a deep uv fluorescence lifetime imaging microscopy system (duv-flim) based on a picosecond deep uv laser using time-correlated single-photon counting method. the described setup is well-suited for biological applications for ultrasensitive detection. we have showed single uv dye (bmq) and single protein (ecβ gal ) molecules detection using duv-flim. further, the label free detection of protein interaction between ecβ gal and monoclonal anti-ecβ gal has been demonstrated by means of steady-state and time-resolved fluorescence spectroscopy. we achieved detection sensitivity for the ecβ gal/ anti-ecβ gal pair down to the nanomolar concentration range. we also extended this method to study the interaction of therapeutic drug porphyrin with bsa protein. fluorescence resonance energy transfer between protein and alexa fluor has been investigated using duv-flim. the intrinsic fluorescence and fluorescence lifetime changes of donor biotin β-galactosidase have been measured. energy transfer efficiency and donor acceptor distance have been obtained. fluorescence images of acceptor af due to fret have been observed when excited at nm. the monomeric heme-containing indoleamine , dioxygenase (ido) catalyses the oxidative cleavage of the indole moiety of l-tryptophan (l-trp). enhanced l-trp degradation contributes to various physiological disorders including depression or failure of the immuno-regulating system. the regulation of ido by inhibitors is extensively studied. however, the catalytic mechanism of ido on the molecular level is still unknown. in addition to l-trp, a wide range of substrates are degraded including d-tryptophan, melatonine or tryptamine. in contrast, indole or histidine do not function as substrates for ido. to understand the determinants for substrate specificity, we investigate the interaction of the heme iron, the heme-bound ligand and the substrate. we use (time-resolved) uv/visible and fourier transform infrared (ftir) spectroscopy over a wide temperature range ( - k) to monitor the binding of diatomic ligands to the heme iron and the binding of different substrates to coligated ido. changes in the spectra upon addition of l-trp are analyzed and compared to those induced by other substrates or inhibitors. archaeal protoglobin d-structure: novel ligand diffusion paths and heme-reactivity modulation protoglobin (pgb) from methanosarcina acetivorans c a, a strictly anaerobic methanogenic archaea, is the latest entry in the hemoglobin superfamily. our previous crystallographic studies on pgb have shown that protoglobinspecific loops and a n-terminal extension completely bury the heme within the protein matrix ( ) . access of o , co, and no to the heme is granted by protoglobin-specific apolar tunnels reaching the heme distal site from locations at the b/g and b/e helix interfaces. here we report structural and kinetic data on pgb mutants engineered to probe the protein structural and kinetic properties. six crystal structures (pgb mutants: ∆ (missing nter residues), y(b ) →a, y(b ) →w, f(b ) →w, f(g ) →w, i(g ) →f) show that the mutations engineered essentially restrict access to ligand tunnel . an accurate molecular level description of the signaling mechanism in ca + transducers necessitates the knowledge of the kinetics and energetics of conformational changes associated with ca + binding to various calcium binding proteins. with this in mind, we have developed an approach that combines the laser-induced photolysis of photolabile "caged" ca + compound, dm-nitrophen, with the photothermal beam deflection (pbd) technique to determine thermodynamic profiles associated with the ligand binding to calcium chelator, edta, and ca + sensor, calmodulin. this approach allows us to monitor time profiles of volume and enthalpy changes on the microsecond to millisecond timescale. the initial pbd study of ca + photo-relase from ca + loaded dm-nitrophen reveals the presence of two phases. the first step takes place within first µs upon and is associated with a volume decrease of - ml mol − and enthalpy change of kcal mol − . on the longer timescale (τ = µs), the second event with a positive volume change of ml mol − and enthalpy change of kcal mol − was detected. on the other hand, ca + photorelease in the presence of calmodulin is accompanied with an additional phase with a distinct lifetime and volume and enthalpy changes that reflects the metal binding to calmodulin and concomitant structural changes. antimicrobial peptides: linking partition, activity and high membrane-bound concentrations antimicrobial peptides (amps) have been intensively studied at micro and macroscopic levels for over twenty years. knowledge from these two domains has, however, contributed little to a comprehensive understanding of amp action; rather, in vivo amp performance has been only remotely correlated to biophysical properties. we focus on the assessment of peptide accumulation on bacterial membranes as an example of this separation: amp-bilayer interactions have been subject to extensive biophysical characterization, but conversion of that information into educated estimates of in vivo membrane-bound amp concentrations is lacking. this has led to the overlooking of important factors for activity. using simple partition models we were able to analyze available information on amp activity and interaction with membranes to show that unexpectedly high membranebound peptide concentrations are likely in vivo and may, in some cases, be required for triggering bacterial death. e. e. schäfer , s. schuy , a. janshoff georg august-university göttingen, germany, johannes-gutenberg-university mainz, germany retrovirus entry into cells occurs through fusion of the lipid bilayers that surround the virus and the lipid bilayer of the host cell. fusion proteins, present on the surface of the virus membrane play an essential role in the early stage of virus entry. understanding of the molecular mechanism is important for the design and function of modern fusion inhibitors. in this project we analyze the fusion of active conformation of the envelope gp from the human and simian immunodeficiency viruses (hiv and siv). during the infection process gp undergoes a sequence of conformational changes where the n -terminal nhr develop a trimer pre-hairpin intermediate. afterwards the three chr fold back towards the central nhr and a six helix bundle is formed. this rearrangement forces the viral and the host membrane into close contact and fusion pores may induce membrane fusion. this decisive molecular step in retroviral fusion has been modeled by rational design of lipopeptide assemblies that mimic a coiled-coil structure serving as a receptor for potential antagonists. for this purpose, sslbs were functionalized in an in situ coupling reaction with peptides originating from the nhr (n-peptides) of siv and hiv to monitor the interactions with the specific chr peptides (c and t ). binding of antagonists to surface confined coiled-coil structures has been quantified by ellipsometry, quartz crystal microbalance and was visualized by atomic force microscopy. a. rupprecht , e. sokolenko , e. e. pohl institute of cell biology and neurobiology, charité -universitätsmedizin, berlin, germany, frumkin institute of physical chemistry and electrochemistry russian academy of sciences, moscow, russia the production of reactive oxygen species (ros) in mitochondria is very sensitive to the proton motive force and can be decreased by mild uncoupling, mediated e.g. by uncoupling proteins (ucp) . in contrast, the activation of uncoupling proteins by ros as a negative feedback loop is a highly controversial hypothesis. ucp activation in mitochondria by -hydroxy- -nonenal (hne, aldehydic product of lipid peroxidation) was first demonstrated by echtay et al. . here we investigate the ability of hne to activate and/or to regulate the expression of ucp in two different systems: (i) in lipid membranes reconstituted with recombinant ucp and (ii) in primary neuronal cells. total bilayer conductance was enhanced in the presence of hne, but this effect was independent on ucp and ucp . the results concerning the hne-mediated ucp expression after induction of ros-production and/or after exogenous addition of hne are discussed for three brain-associated proteins (ucp , ucp , ucp ) in view of their possible functions. . beck, v et al. . faseb j. : - . . echtay, k. s. et al. . t. rudack, j. schlitter, k. gerwert ruhr university, department of biophysics, nd north, bochum, germany the gtpase ras p which is linked to the membrane via a lipid anchor, is a crucial switch in the cellular signal transduction processes that control cell growth and proliferation. docking simulations can be seriously hampered by the great difficulty to accurately estimate the ligand-protein binding affinity constant k, which is usually derived via the computation of the binding free energy ∆g. unfortunately, due to the involved logarithmic relationship, errors of less than . kcal/mol in the computation of ∆g result in about one order of magnitude inaccuracy on k. this can hinder computational methods from discriminating micromolar from nanomolar compounds. to improve the reliability of docking prediction, we make use of enhanced sampling methods, ranging from steered molecular dynamics to metadynamics . we also test several descriptors, such as the recently developed path collective variables , to identify the most suitable reaction coordinates accounting for binding and unbinding processes. using these approaches we investigate ligand docking and undocking and we attempt to describe at an atomistic level the kinetics of binding, which we intend to exploit for drug design purposes. here, an example of application in drug design is reported. . isralewitz, b. et al.; j. mol. graph. model. , , - . . laio, a. and parrinello, m.; pnas , , - . . branduardi, d.et al.; j. chem. phys. , , . prediction of protein-protein complex structures using wang-landau simulations a. solernou, barcelona supercomputing center, jordi girona , barcelona, spain protein-protein interactions are essential in the majority of life processes, so they have keen interest in several knowledge areas. however, experimental data on protein complexes is being produced at a quite low pace in comparison to that of the individual components. thus, in recent years attention has focused into computational approaches to the proteinprotein docking problem. a variety of docking protocols have been recently reported, sharing usually the following strategy: fast rigid-body search of the interacting subunits, followed by scoring and refinement of the interfaces. although this kind of strategy has proven some good results in the capri blind test it has two main limitations. on one hand one should include full flexibility on the protein structures. on the other, the evaluation should be made with free energy calculations instead of using a scoring function. in this work we propose to search the native complex structure in the minimum of the free energy landscape. we use the coarse grained potential unres to get the potential energy of the possible conformations. they are generated changing the dihedral angles, the side chain rotamers, and lastly the mutual orientation using a new sampling protocol we have developed (rotation-based uniform sampling; rotbus). finally, the free energy calculations are performed using an omp parallelization of the wang-landau algorithm. s. shukla , s. asthana , g. giliberti , f. luliano , m. ceccarelli , r. loddu , p. ruggerone , p. la colla department of biomedical science and technology, università di cagliari, cagliari, italy, department of physics, università di cagliari, cagliari, italy the virus encoded rdrp has emerged as a prime target in the search for specific hcv and other flaviviridae antivirals. recently, the determination of the hcv rdrp structure in complex with certain benzimidazoles has been reported, these nni's bind to the surface of the thumb domain, thereby disrupting its interaction with the finger domain, which is necessary for catalytic activity. on the other hand, we have found that, in bvdv, the mutations conferring resistance to same class of inhibitors lie in the finger domain of rdrp, indicating that the mode of inhibition of benzimidazole class of compounds is different in both hcv and bvdv rdrp. herein, we have applied docking approaches (binding orientation), molecular dynamics (md) simulations combined with metadynamics to elucidate the microscopic mechanism of the interactions between the ligand and the receptor in order to identify features barely seen in experiment. the recently designed algorithm overcomes the time scale problem by accelerating properly defined reaction coordinates. it mimics the real dynamics of a ligand staying or leaving the receptor and in doing so reconstructs the free energy surface, which in turn gives an idea of the residence time of the inhibitor in the cavity.finally we identified the binding modes and different mechanism of inhibition of benzimidazole class of compounds in rdrp of two closely related rna viruses. . c. seifert , w. stacklies , f. graeter protein mechanics and evolution, bioquant, inf bq , heidelberg, germany, ag graeter, picb, yueyang lu, shanghai , china we use a new method that detects force distribution in proteins. based on molecular dynamic simulations, changes in inter-atomic forces are calculated, here caused by different ligands. these changes will then be analyzed to detect a signal transfer through a protein initiated by the binding of a ligand to the protein. chaperones are ubiquitous proteins, which help other proteins to fold into a native conformation. they are able to refold misfolded proteins with a great variety of mechanisms. in this project, our group focuses on molecular dynamic simulations of htpg, an e. coli homolog of the human hsp (heat shock protein kda). the full structure of htpg was published by shiau et al. in , but the mechanism of how htpg performs its function is still not understood. the folding mechanism is an atp driven reaction cycle, in which the functional entity is a homodimer of htpg. we separate the cycle in three main states: apo, adp-and atpbound state. conventional molecular dynamics simulations are used to build a stable simulation system and provide structure trajectories and primary information about the behavior of htpg in its different states of the folding process. experiments indicate that the molecular movement is atp driven. we use force distribution analysis to elucidate how atp effects htpg conformation and dynamics. the small size of myoglobin makes it the preferred candidate to investigate the structure-function paradigm. in its interior five docking sites have been identified and for long time these xenon cavities have been classified as packing defects. recently, it was shown that they might be involved in ligands migration path. however, some questions regarding its role as oxygen carrier no scavenger remain yet open as well as the microscopic mechanism regulating these biological functions. in this work we made use of standard md simulations of solvated myoglobin to characterize internal cavities. our principal results is that we have found several secondary cavities showing volume and occurrence at least comparable to that of xenon cavities. in order to analyze and rationalize internal cavities we applied special cluster-analysis: we classified all cavities with respect to the position, size and occurrence ascribing them to different clusters. this analysis highlights possible intrinsic migration paths for small ligands within the protein matrix controlled by spontaneous fluctuations of the protein itself. moreover, we identified some key residues playing a fundamental role in controlling internal pathways. our suggestion that the secondary cavities constitute the preferred path for ligand escape is also supported by explicit metadynamics simulations of ligand escape. a. varga , p. lallemand , j. szabó , p. závodszky , m. vas , t. barman , l. chaloin , c. lionne institute of enzymology, brc, hungarian academy of sciences, budapest, hungary, cnrs-université montpellier -université montpellier , institut de biologie, umr , montpellier, france -phosphoglycerate kinase (pgk) is a promising candidate for the activation of nucleotide analogues used in antiviral and anticancer therapies. pgk is a key enzyme in glycolysis; it catalyses the reversible reaction , -bisphosphoglycerate + adp ↔ -phosphoglycerate + atp. here we explored the catalytic role in human pgk of the highly conserved lys that has been proposed to be essential for pgk function, by a transient and equilibrium kinetic study with the active site mutant k a. by the stopped-flow method we show that the kinetics of substrate binding and the associated protein isomerization steps are fast and identical for the wild-type pgk and mutant k a. by the use of a chemical sampling method (rapid-quench-flow) under multi and single turnover conditions and in both directions of the reaction, we show that the rate-limiting step with wild type pgk follows product formation, whereas with the mutant it is the phospho-transfer step itself that is rate limiting. these data are supported by saxs measurements which showed no direct role of lys in the domain closure of the enzyme i.e. the isomerisation step of the reaction. the results are explained by the structural data of the enzyme. characterization of different recombinant nrp proteins and interactions with heparin k. a. uniewicz, y. ahmed, d. g. fernig school of biological sciences and centre for glycobiology, biosciences building university of liverpool, l zb liverpool, u.k. neuropilin- (nrp ) is a mammalian membrane glycoprotein involved in tip sprouting processes like angiogenesis and neurogenesis. it has been shown that the interaction of nrp with heparin/heparan sulfate is implicated in its enhancement of growth factor signalling, however the mechanism is not yet known. commercially available extracellular domain of nrp is available either as a truncated his-tagged human protein (hnrp ) or as the rat protein fused to histagged human fc and expressed as a dimer (fcrnrp ). biochemical properties such as kinetics of heparin binding and structural requirements for sugar binding together with biochemical tests of protein properties were performed in order to characterise these commercial proteins. fc rnrp shown high affinity to heparin (kd= . nm) and required a minimum of dp to effectively compete with fc rnrp binding to immobilised heparin. competition experiments with various modified sugars show that interaction of fc rnrp with heparin is highly ionic and dependent on the position of sulfate groups along the heparin chain. the hnrp did not bind to heparin immobilised via nhs-biotin, though it did bind to some heparin affinity chromatography matrices. organic compounds of tin are among wide spread environmental pollutants. due to physical and/or chemical actions, poly-substituted alkyltins speciate into less substituted, more toxic species. tetra-or tri-alkyltins show a marked delay in their toxic action with respect to the corresponding di-and mono-alkylated analogs. it has been hypothesized that the delayed toxicity may results from the progressive dealkylation of alkyltins in more toxic di-and mono-derivatives, which bind and inhibit essential enzymes. it has been proposed that alkyltins preferentially target enzyme sulphydryl groups. previously, we showed that a nine amino acid linear peptide (i lgcwcylr ) containing a cxc motif is able to bind and dealkylate tri-substituted alkyltin compounds into the corresponding dialkyl derivatives. here, we investigated the time dependence of the degradation of the most common alkyltin derivatives by this peptide. we monitored the reaction kinetics using the intrinsic fluorescence of the tryptophan residue in position of the peptide. we found that all of the alkyltins analyzed are progressively degraded to dialkyl derivatives, following a pseudo-enzymatic reaction mechanism. the end-point of the reactions was the formation of a covalent complex between the disubstituted alkyltin and the peptide. these data agree with the speciation profiles proposed for polysubstituted alkyltins in the environment and reveal a possible biotic degradation pathway for these toxic compounds. parkinson's disease (pd) is a multifactorial neurodegenerative condition characterized by the progressive loss of dopaminergic neurons in the substantia nigra and by the presence of intracellular inclusions, composed predominantly of fibrillar alpha-synuclein (as). post-mortem studies indicate the presence of oxidative damage in the nigral neurons. dopamine oxidation, which leads to the formation of highly reactive quinones (daq), may account for the specificity neurodegeneration observed in pd. daq have many potential protein targets for chemical modifications. among them, we focused our attention on dj- , of which mutated forms have been found in familial cases of pd. a possible function of dj- is its redox-dependent chaperone activity that could prevent as aggregation and fibril formation. in the present work, we analyzed the structural and functional modification induced on dj- by daq. n-hsqc spectra of dj- were recorded in the presence of different amounts of daq and chemical shift perturbations were used to identify the dj- residues target of daq and their relative reactivity toward daq. aggregation assays were also performed to evaluate the functional effects of the daq modifications on the chaperone activity of dj- . rabies virus (rabv) infects neurons exclusively and causes lethal encephalitis. pathogenic rabv strains favor neuronal survival, whereas non-pathogen strains lead to neuronal apoptosis. the use of recombinant rabv showed: / the g protein determined the induction of the survival or death phenotypes; / the last cooh amino acids of the g protein cytoplasmic domain (cytog) are critical. these residues form a binding site for pdz domain (pdz-bs). one of the amino acid differences between survival and death gproteins are located in this pdz-bs. results of two-hybrid experiments showed that cytog survival interacted pdz domain of ser/thr kinases (mast), while cytog death interacted also with additional pdz containing host proteins. to understand the fine structural basis for the specificity of the pdz-cytog complexes, we determined the d structure and the dynamics of the mast-cytog complexes by nmr. the structures, as well as the affinities and constant kinetics, of the mast pdz complexes with both cytog are similar. we conclude that difference by one aa in the pdz-bs of the two strains cannot drastically modify the interaction with mast -pdz, in agreement with the two-hybrid data. preliminary results suggest that the interaction of cytog death with one additional cellular partner blurs the pro-survival signals engaging the infected cells through apoptotic trails. functional protein immobilization on glass-type surfaces s. waichman, m. bhagawati, y. podoplelova, j. piehler institute of biology, department of biophysics,university of osnabrück , barbara st. osnabrück, germany the immobilization of membrane proteins onto solid supports enables protein interactions and conformational changes to be probed by spectroscopic techniques under highly defined conditions. here, we present a novel method for covalent protein immobilization on glass-type surfaces using a bottom-up approach. in this approach the 'phosphopantetheinyl group of coenzyme a (coa) was transferred to the acyl carrier protein-derived ybbr tag of the target protein by means of the phosphopantetheinyl transferase sfp. the glass-support was rendered biocompatible by coating it with an ultrathin layer of peg (polyethylene glycol), followed by functionalization with coa through maleimide chemistry. immobilization of ybbr-tagged proteins in presence of sfp was followed in real time by label-free detection using reflectance interference spectroscopy. the immobilization procedure was thus systematically optimized by means of binding specificity, enzyme activity and functionality of the immobilized protein. this approach was employed for immobilizing the type i interferon ifnα in order to probe ligand recognition by ifnar and ifnar and ligand-induced ternary complex formation. the versatility of this technique was further enhanced by its combination with photopatterning methods. this immobilization technique can provide a beneficial tool for bioanalytical and biophysical applications at the single molecule level. r. vijayan, p. c. biggin department of biochemistry, university of oxford, south parks road, oxford, ox qu, uk ionotropic glutamate receptors mediate excitatory synaptic transmission in the brain and are heavily implicated in memory and learning as well as in numerous neuropathological conditions. one family of iglurs, the kainate receptors, show unusual sensitivity to changes in external ion species resulting in an apparent requirement for both sodium and chloride ions for activation. our recent work revealed the location and selectivity of the cation binding sites. despite this progress, it is still unclear how the cation binding sites confer sodium selectivity and how apparent affinity for chloride is influenced by the presence of cations. we have attempted to address these questions by performing extensive free energy calculations using all-atom molecular dynamics simulations. the rank order of cation binding obtained from relative binding free energy calculations is in agreement with experimental measurements of apparent affinity. these calculations also reveal that the pair of cation binding sites in the dimer interface act independently. binding free energy calculations performed using a reduced model of the binding site show that cation selectivity can been attributed to both the rigidity and high charge density of the binding sites. finally, a potential of mean force derived from umbrella sampling simulations indicate that the presence of cations stabilize the anion binding site considerably. the effect of toxins on the inorganic phosphate release during the actin filament formation a. vig, t. kupi, g. hild, m. nyitrai university of pécs, faculty of medicine, department of biophysics, szigeti str. , pécs, hungary actin can be found in monomeric (g-actin) and filamentous (f-actin) form in eukaryote cells under physiological circumstances. the first step of actin polymerisation is the formation of actin nuclei by atp-binding actin monomers. the next step in is the elongation when monomers are associated to the previously formed nuclei or to the ends of the growing filaments. after the association of monomers the bound atp is hydrolysed to adp.p i , and with first order kinetics the release of inorganic phosphate occures. the rate constant of the release is , s − , which is a slower process than the hydrolysis itself ( , s − ).phalloidin, a cyclic peptide from amanita phalloides can bind to the filaments and stabilizes their structure. jasplakinolide is another cyclic peptide from marine sponge (jaspis johnstoni) which binds actin filaments. the aim of this study was to investigate whether the binding of toxins to the newly created filaments has an effect on the kinetics of the inorganic phosphate release or not. we used absorption photometry measurements to measure the rate of phosphate release. phalloidin decreased the rate of the release substantially. although the effect of jasplakinolide was weaker, the results showed that the binding of these toxins to the actin can modify the rate of the release of inorganic phosphate from the filaments. these observations are in agreement with the molecular mechanisms by which these toxins stabilise the actin filamens. fluorescent proteins (fps) are invaluable fluorescent markers in cell biology. however, their use is often limited by photobleaching of the chromophore, notably in single-molecule, time-resolved or super-resolution imaging studies. we will present the crystallographic studies at near atomic resolution of a photo-activatable fluorescent protein irisfp that has been observed in a transient radical state en route to photobleaching. we took advantage of x-rays to populate the radical, which, under illumination with visible light, presumably forms with low probability from the triplet state. the combined x-ray diffraction and in crystallo spectroscopic data (from uv-vis and raman spectroscopies) reveal that radical formation in irisfp involves strong but reversible distortion of the chromophore, suggesting a transient loss of pi-conjugation. these results will help unravel the mechanisms of blinking and photobleaching in fps, which is of importance to rationally design variants of higher photostability. optimizing photoactivatable fluorescent proteins for live-cell imaging recently, novel fluorescent proteins (fps) have been reported which perform spectral changes in response to irradiation with light of a particular wavelength. reversibly photoactivatable proteins switch between a bright and a dim fluorescent state. this process is accompanied by photoisomerization of the protein chromophore. irreversible photoactivation results from photochemical processes within the protein, e.g., photolysis of an amino acid side chain, or an extension of the alternating π-electron system of the chromophore by a β-elimination reaction. fps have became valuable tools in live-cell imaging because they allow intracellular protein labeling by using them as fusion tag, and photoactivatable fps are powerful tools for application in novel subdiffraction imaging techniques. however, the available fps still offer potential for improvement in various ways. they frequently show a tendency to aggregate or oligomerize, incomplete chromophore maturation, fluctuating emission and low photostability. here, we will present our recent progress towards engineering the 'perfect' fp. simultaneous intracellular chloride and ph measurements using gfp-based sensor in ldcv d. arosio , f. ricci , l. marchetti , l. albertazzi , f. beltram italian institute of technology, udr pisa, italy, nest, scuola normale superiore, pisa, italy, nest, infm cnr, pisa, italy chloride ion participates in many physiological functions including control of neuronal resting potential, charge balance during endosome acidification, and regulation of cell volume. as a consequence dysfunctions in regulating membrane chloride permeability lead to severe diseases including motor disorders, cystic fibrosis and epilepsy. at present processes regulating intracellular chloride ion concentrations are still widely unexplored mainly as a consequence of limiting methods to quantify chloride fluxes in living cells. in the present work a highly specific, genetically encoded sensor is developed for detecting simultaneously intracellular ph and chloride concentration. the sensor is obtained by fusion of a red fluorescent protein (dsred-monomer), insensitive to chloride and ph, to a gfp variant containing a specific chloride-binding site (gfp-chl). dsred-gfp-chl binds the chloride ion following a fluorescence static quenching mechanism, which allows measurements of intracellular ph in a chlorideindependent manner. the sensor has been successfully tested in different living cells, in a ph range - and chloride concentration up to mm. for the first time, to the best of our knowledge, it allowed to measure the chloride concentration of dense core vesicles in the secretory pathway. applicability to high-throughput screening, range of validity and accuracy of time-lapse maps will be discussed. we aim to understand the basis of the photophysical changes in fluorescent proteins (fp) induced by reactive oxygen species (ros). indeed, ros might be involved in photochemistry of fp, leading to their photobleaching or photoconversion. in addition, fp may be used to investigate cellular events like phagocytosis or mitochondrial activity, where ros are naturally produced. in the latter cases, an accurate analysis of fp's fluorescence signals requires the full knowledge of reactions between ros and fp. in the future, this work may help in developing either photoresistant or photoswitchable fp and improving their use for imaging under oxidative stress conditions. using γ-radiolysis as a quantitative source of ros, we investigated the reactions of oh and o − radicals on the cyan fluorescent protein (cfp) and the modifications of the cfp's photophysical properties by oh radicals were explored in detail (submitted). in order to address the corresponding chemical changes in the protein, we devised a mild proteolysis protocol that for the first time offers a peptide mass fingerprint almost covering the cfp sequence (alvarez et al. biochemistry ). then, we achieved the meticulous characterization of the cfp oxidation products by mass spectrometry and proposed a mechanism to account for their formation by pulse radiolysis. since the cloning of the green fluorescent protein from aequoria victoria, numerous screens have been performed to improve the brightness of this protein, its spectral variants and fluorescent proteins from other species. the improvement is evaluated by comparing fluorescence intensity of individual bacteria or colonies. in this way also expression level, folding and maturation efficiency, and thickness of the bacterial colony contribute. here we report a screening method that, in addition to fluorescence intensity, quantifies the excited state lifetime of a fluorescent protein, which is independent of intensity or expression level, and provides a direct measure for the quantum efficiency of the fluorescent protein. the novel approach was used to screen a library of cyan fluorescent protein (cfp) variants randomly mutated at position , and , yielding an improved bright cyan fluorescent protein named, mposeidon, with a markedly increased fluorescence lifetime and quantum yield, increased photostability and improved fluorescence intensity in vivo. it is shown that mposeidon is the brightest cyan fluorescent protein in mammalian cells. in addition, several lifetime variants were identified that can be used for lifetime unmixing. it is demonstrated that three cfp variants can be separated and their distribution quantified in a single detection channel. a. r. faro , p. carpentier , d. bourgeois , e. rosny irtsv, cea, ufj, grenoble, france, ibs, cea, cnrs, ufj, grenoble, france, ibs, cea, cnrs, ufj, grenoble, france, ibs, cnrs, ufj, grenoble, france enhanced yellow fluorescent protein (eyfp) is extensively used as a fluorescent marker. like other photo activatable fluorescence proteins (pafp), it exhibits photo-switching properties. however, the mechanism by which fluorescence can be swiched on or off upon light irradiation is not fully understood at the molecular level. the bright to dark conversion involves a protonation step and structural rearrangements of the chromophore, but it is not clear which of these two steps is the triggering event. to answer this question, we carried out photo-switching experiments at cryotemperatures. our data suggest that a photo-induced protonation step (probably in the triplet state) is the primary event in the bright to dark conversion. our results may bear relevance to other pafps, such as iris, eos, dronpa, padron or kaede. how misfolding and aggregation of proteins constitutes a toxic insult to neurons remains largely unknown. in order to obtain insight into the molecular biology of neurodegenerative disease, we have developed a number of gfp-based biosensors for the detection and quantification of cellular clearing mechanisms for aggregated proteins. the high load on these protein quality control mechanisms, and their failure to meet normal physiological demand ultimately results in a "de-compensation" condition from which the nerve cell cannot recover. our fret/flim-based bioassays visualize protein ubiquitination and degradation; proteasomal activity; foldase activity using a folding-impaired gfp mutant which gains fluorescence conditional on the upregulation of chaperone activity; chaperone binding to unfolded proteins; and autophagosome formation/lysosomal integrity via the targeted and sensitive fret-based measurement of ph changes. these sensors are employed in cellular model systems for parkinson's and alzheimer's disease, and amyotrophic lateral sclerosis (als) to delineate the molecular pathway of cellular demise, and to gain a mechanistic understanding of the toxicity of protein aggregates and the basis for the vulnerability of neurons. millisecond photo-switching dynamics of e q gfp mutants for sensor and imaging applications m. collini , v. quercioli , l. d'alfonso , g. baldini , b. campanini , s. bettati , g. chirico dipartimento di fisica, università di milano bicocca, italy, dipartimento di biochimica e biologia molecolare, università di parma, parma, italy e q mutants of the green fluorescent protein are known to possess photo-chromic properties: the anionic emission, primed by a pump nm laser beam, can be switched between two levels of different brightness by irradiation with a blue, ∼ = nm, probe laser light. we have studied here the amplitude and the dynamics of the brightness enhancement of the e q mutant of gfpmut . the fluorescence emission increases almost threefold, under saturating probe laser excitation, for pump excitation intensity in the linear regime. two characteristic activation times, estimated by means of modulated two colour fluorescence correlation spectroscopy, are detected in the - ms range, independent of solution temperature and viscosity. the brightness enhancement factor and the characteristic activation times depend markedly on the solution ph. these results indicate that this mutant can be used as a high sensitive intracellular marker for local proton concentration and for modulated excitation imaging. the advent of fluorescent proteins (fp) gave researchers the opportunity to study proteins in situ. fluorescence resonance energy transfer (fret) benefited from this. cell fixation is a commonly used approach when working with microscopy. however, we have found that fret efficiency (e) in cells transfected with cerulean and venus chimeras could not be reproducibly measured after fixation. to evaluate this problem in detail, we measured e of cerulean-venus standard constructs by acceptor photobleaching fret, intensity-based ratiometric fret and flim-fret. the constructs were produced as standards (biophys j, , , ) with , and % e values, comprising donor-acceptor separations of , and amino acids, respectively. transient transfection of the fusion plasmids was performed into hela cells and e was measured in live and pfa or methanol fixed cells. literature e values were reproduced when measuring live cells. conversely, cell fixation caused a deviation of e values. methanol fixed cells showed e between - % for all the constructs. the effect of pfa fixation on both fluorescence intensity and fret varied vastly among independent experiments regardless of the measurement modality. thus, fixation should be avoided due to the effects it has on fp's fluorescence and consequently on fret efficiency. to explain the improvement in the fluorescence properties of cerulean when compared to ecfp, we have determined the x-ray crystallographic structures of these two proteins at physiological ph, and performed molecular dynamics simulations. both proteins exhibit a structural heterogeneity in the nterminal half of their seventh strand, which forms a specific set of van der waals interactions with the chromophore. the critical h d mutation present in cerulean induces a modification of these interactions, and allows the chromophore to be more planar and better packed, albeit only intermittently. as a consequence, the probability of non-radiative decay is significantly decreased. our results highlight the considerable dynamical flexibility that exists in the vicinity of the tryptophan-based chromophore of these engineered fluorescent proteins, and provide insights which should allow the design of mutants with enhanced optical properties. the fluorescence lifetime of green fluorescent protein g. jung biophysical chemistry, saarland university, saarbruecken, germany biotechnological design of the green fluorescent protein (gfp) and the discovery of other proteins boosted the development of the life sciences in the past decade. tracking protein movements and high resolution microscopy are only a few recent applications which were realized by fluorescent protein technology. among these examples, the switching between two chromophore state is exploited. our aim is to establish fluorescent proteins for bioanalytical fluorescence lifetime measurements. despite the progress in other fields, quantification with gfp still imposes practical problems [ ] . in the past, we observed that the uv-light driven decarboxylation of the nearby aminoacids glu distorts the fluorescence lifetime of gfp [ ] . we found out that this chemical reaction also occurs under excitation of the anionic chromophore state with a high quantum yield [ ] . recently, we could show by time-resolved spectroscopy that, indeed, the more susceptible state for this kind of photoconversion is the anionic chromophore state [ ] . suppression of this reaction therefore enables the design of autofluorescent proteins which can be used e.g. for the quantification of ions and which are beneficial as donors in fret applications. the fluorescent properties of tryptophan residues (w) in proteins are highly dependent on their immediate protein environment. however, the multi exponential decay of single w proteins is not completely understood. the most cited hypothesis contributes a multi exponential decay to the existence of several micro conformations (rotamers) of the w residue within the protein matrix. to determine rotamers we apply a method based on dead-end elimination (dee) and molecular dynamics simulations (md). low energy rotamers are calculated by dee while dynamics and further refinement is accomplished using md. the method was applied on several test cases including the protein mutant bc-csp l e from bacillus caldolyticus, which contains a solvent exposed w residue. as resolved by x-ray crystallography, this w residue occupies two conformations. using dee and md we were able to retrieve the w conformations found in the x-ray structure. the results demonstrate the ability of the method to obtain valuable w rotamers, both for solvent shielded as exposed residues. the determined conformations were compatible with the findings based on a method using replica exchange simulations. k. nienhaus , h. nar , r. heilker , j. wiedenmann , g. u. nienhaus inst. of biophysics, universität ulm, ulm, germany, dept. of lead discovery, boehringer ingelheim, biberach/riss, germany, national oceanography center, university of southampton, southampton, uk, inst. of applied physics, universität karlsruhe (th), karlsruhe, germany eqfp is a red fluorescent protein (rfp) with the chromophore in a co-planar trans orientation, whereas the cis isomer is preferred by most other rfps. by using x-ray crystallography, we determined the structures of the dimeric variants d eqfp and d eqfp at high resolution (up to . Å). for d eqfp , we had previously seen a redshifted species upon irradiation with -nm light. concomitant changes in the raman spectrum were interpreted as evidence of a trans-cis isomerization of the chromophore. here we have combined x-ray crystallography and site-directed mutagenesis to assess whether we can create a stable fluorescent, red-shifted eqfp variant with a cis chromophore. in a first step, we introduced the n s substitution. this variant, d rfp , is highly fluorescent, with the absorption (emission) maximum red-shifted by ( ) nm. with an additional s c mutation, the chromophore is found completely in the cis form. the variant, rfp , is highly fluorescent, with excitation and emission maxima at and nm. still further red shifts appear to be in reach. k. nienhaus , v. adam , d. bourgeois , g. u. nienhaus of biophysics, universität ulm, ulm, germany, esrf, grenoble, france, institute of applied physics, universität karlsruhe (th), karlsruhe, germany dendra is an engineered, monomeric gfp-like protein that belongs to a sub-class of fluorescent proteins undergoing irreversible photoconversion from a green-to a red-emitting state upon exposure to purple-blue light. this process occurs in the neutral state of the chromophore and is known to result from backbone cleavage accompanied by an extension of the delocalized π-electron system. we have measured the x-ray structure of green dendra and performed a comprehensive characterization of the optical absorption and fluorescence properties of the protein in both its green and red forms. the structure, which is very similar to those reported for the closely related proteins eosfp and kaede, revealed a local structural change next to the chromophore, involving mainly arg and a water molecule. we propose that this structural change explains the blue shift of the absorption and emission bands, as well as the markedly higher pks of the hydroxyphenyl moiety of the chromophore. the -fold enhancement of the neutral species in dendra at physiological ph accounts for the observed higher photoconversion yield of this protein in comparison to eosfp. photochromic green fluorescent protein mutants: chromophore states unveiled by raman spectroscopy s. luin, v. voliani, g. lanza, r. bizzarri, r. nifosì, p. amat, v. tozzini, m. serresi, f. beltram nest, scuola normale superiore, cnr-infm and italian institute of technology, pisa, italy the most widespread genetically-encodable fluorescent markers used for studies in living cells and tissues belong to the green fluorescent protein (gfp) family. reversibly switchable fluorescent proteins (rsfps) were developed that can undergo repeated transitions between different states, e.g. bright and dark forms. this property makes rsfps particularly attractive as active labels in biological systems for selective photolabeling applications or subdiffraction imaging. we shall present pre-resonant raman results unveiling the photophysical mechanism underlying the observed photochromic behavior. the variation of spectral properties before and after photoconversion of chemically-synthesized isolated chromophores under different protonation and/or isomerization have been analyzed, and compared to results obtained for the case of complete folded proteins comprising the same chromophores. experimental results have been analyzed within a time-dependent density functional theory, allowing us to assign all relevant vibrational modes. these results make it possible to discriminate between the effect of cis-trans isomerization and of diverse protonation states of the chromophore in the photoproducts of these proteins. s. luin et al, j. am. chem. soc. , - ( ). r. bizzarri et al., anal. bioanal. chem. , - . a combined study of the interaction of outer membrane proteins with cephalosporin antibiotics m. lovelle, i. barroso, m. j. feio, p. gameiro requimte , fac. ciências, universidade do porto, portugal gram-negative bacteria characteristically are protected by an outer membrane that serves as a selective permeation barrier. most of the β-lactam antibiotics appear to penetrate the outer membrane through these non-specific channels, and it becomes important to understand the possible interactions between β-lactams and the porin. fluorescence techniques have been largely used to characterize both the conformation and the dynamic behavior of large biological structures such as membranes and proteins. the fluorescence of the tryptophan residues is quenched in the presence of the different cephalosporin antibiotics. this reaction between the excited state of the fluorophores and the drug can be described as a formation of a non-fluorescent complex. the dependence of the fluorescence intensity upon quencher concentration for static quenching is proportional to the binding constant for complex formation. since β-lactam susceptibility is closely related to the presence of these non-specific porins, minimum inhibitory concentration (mic) by micro-broth dilution in microplate were used to assess the bactericidal activity of cephalosporin antibiotics upon on escherichia coli strain bl (de ) and a series of bl (de ) mutated in different outer membrane proteins. this combined study of the interactions at single molecular level and at in vivo level provides new insights for a better understanding of the antibiotic translocation. when used in combination with e.g. a cyan fluorescent protein, keima offers the unique opportunity to perform dual color fluorescence cross-correlation spectroscopy using a single laser line to excite both fluorophores. the molecular determinants of the large stokes shift of mkeima have been characterized structurally by combining x-ray crystallography with in crystallo uv-visible absorption, fluorescence and raman spectroscopy. our results reveal a ph-dependant "reverse chromophore protonation" of mkeima, driven by the key residue asp . moreover, the chromophore protonation state is shown to be coupled with different chromophore conformations, cis conformation at ph . , and mostly trans conformation at ph . . these results will help unravel the mechanisms of fluorescence in fps, which is of importance to rationally design and develop new fluorescent markers. recently reversibly switchable fluorescent proteins (rsfps) have become a new branch of the green fluorescent protein (gfp) like family. the rsfps may be reversibly switched between a fluorescent and a non-fluorescent state by irradiation with light of distinct wavelengths. the key process of this switching behaviour is a light induced change of the chromophore between a cis-and a trans-isomeric state. the proteins are becoming increasingly important for diverse applications like protein tracking, sub-diffraction resolution microscopy and data storage. based on the switching mechanism, we created novel rsfps with unique and improved characteristics. padron and bs-dronpa are two of these new switchable proteins. padron features a reversed switching mechanism as compared to all other green rsfps known to date while bsdronpa exhibits a very broad absorption spectrum extending into the uv. utilizing the characteristics of both proteins, we performed multi label single detection colour microscopy as well as dual colour sub-diffraction microscopy, the latter with a resolution of nm. further, we recently introduced the first monomeric rsfp exhibiting red fluorescence: using a semi rational mutagenesis approach on the non-switchable mcherry, we generated the switchable monomeric protein rscherryrev. the use of this protein in single molecule switching microscopy allowed us to record time-lapse live-cell images of the endoplasmic reticulum with sub-diffraction resolution. a spectroscopic approach to the study of chromophoric dissolved organic matter (cdom) in the sea c. santinelli, r. lavezza, l. nannicini, a. seritti cnr-ibf, via moruzzi , pisa, italy dissolved organic matter (dom) in the ocean represents the largest reservoir of reactive carbon on the earth. it is produce at each level of the marine food web and it represents the food for heterotrophic bacteria, which can use the different pools of dom (labile, semi-labile, refractory) with a large range of turn-over times (from minutes to millennia). dom plays a central role in the global carbon cycle and it has a high ecological significance. chromophoric dom (cdom) is the fraction of dom capable of adsorbing light at uv and visible spectral regions. it determines the underwater light availability in open and coastal regions with important implication on primary production and biological activity. it is photodegraded to co , co, with a significant impact on the role of the ocean as source and/or sink of atmospheric co and to highly reactive compounds, dangerous for marine organisms. in its pool "humic-like" and "protein-like" fluorophores have been individuated. cdom optical properties (absorption and fluorescence) together with doc data collected in some key regions of the mediterranean sea will be presented and discussed, in order to (i) investigate the powerful of cdom optical properties to get information on cdom "quality" (i) asses the role of dom in carbon export at depth, (iii) underline the main unresolved question on dom and cdom dynamics in the ocean, with particular emphasis to their biological lability. mechanism and applications of photo-and redox-switchable fluorescent proteins s. j. remington, j. n. henderson, x. shu, j. lohman institute of molecular biology, university of oregon, u.s.a. photoswitchable fluorescent proteins have significant advantages over conventional fluorescent labels, and in a revolutionary application, now allow cell biologists to exceed the diffraction limit in light microscopy by a factor of ten. atomic resolution crystal structures and time resolved spectroscopy of both reversible (mtfp . ) and irreversible (pa-gfp) photoswitchable fluorescent proteins in the light and dark states explain the long term stability of either state, as well as how illumination at the appropriate wavelength causes the molecules to switch between states. the photoswitching mechanisms will be discussed in terms of photochemistry, light induced chromophore isomerization and excited state proton transfer (espt). mutagenesis of espt pathways provide insight into the nature of the ratedetermining steps in proton transfer and lead to practical applications, such as redox-sensitive gfp biosensors. k. i. willig, b. hein, s. w. hell max-planck-institute for biophysical chemistry, goettingen, germany stimulated emission depletion (sted) nanoscopy is a light microscopic technique offering a resolution far beyond the diffraction limit: the excitation beam is overlapped with a doughnut-shaped, red-shifted sted beam, which switches off the ability of the molecules to fluoresce in the outer region of the excitation spot. scanning the nanosized focal spot through the sample renders sub-diffraction images with a sub-second frame rate. we used the yellow fluorescent protein citrine to image individual structural elements of living mammalian cells: vimentin and the tubular network, structures of the cytoskeleton, were recorded with a lateral (x,y) resolution < nm inside the living cell, corresponding to a -fold improvement over that of a confocal microscope. also, time lapse sted imaging of dendritic spines of yfppositive hippocampal neurons in organotypic slices outperforms confocal microscopy in revealing important structural details. as an alternative to fluorescent proteins we used a genetically encoded protein tag which can be labelled with modified organic dyes within living samples. thus nanoscale imaging of structures in the interior of living cells greatly expands the scope of light microscopy in cell biology. pulling membrane tubes from solid-supported lipid bilayers with atomic force microscopy j. w. armond , j. v. macpherson , m. s. turner department of physics, university of warwick, u.k., department of chemistry, university of warwick, u.k. information on the elastic and dynamic properties of membranes is essential for a thorough understanding of biological processes such as exocytosis, endocytosis, cell division, and pore formation. we use an atomic force microscope to pull on solid-supported lipid bilayers and observe that an energy barrier must be overcome prior to the formation of membrane tubes. we have modified elastic models of lipid bilayer vesicles to describe the free energy of a planar lipid bilayer in adhesive contact with a surface. from this model we are able to extract force-distance curves for the formation of a membrane tether, including the associated energy barrier. the experimental data can thus be understood in a quantitative fashion. this work enables the atomic force microscope as a quantitative instrument for measuring membrane pulling mechanics, and in future work will allow two-dimensional mapping of elastic properties under tension. from pores to micelles -a peptide-membrane interaction study t. l. andresen, j. r. henriksen technical university of denmark, dtu nanotech, frederiksborgvej , b , roskilde, denmark. email: thomas.andresen@nanotech.dtu.dk. research in the partitioning of peptides into lipid membranes has been intense for several years. along with scattering techniques and nmr, isothermal titration calorimetry (itc) has proven to be a powerful tool for thermodynamic characterization of peptide-membrane interactions. we have studied two antimicrobial peptides, mastoparan-x and melittin, and found that these peptides induce a range of structural transitions of popc and popc/popg membrane systems at different peptide-lipid ratios. we have found that itc can be used to elucidate the threshold where transitions occur, including the threshold for pore formation and micellation. this has been achieved using a thermodynamic model based on gouy-chapman theory, which provides the partitioning constant of the peptide-membrane interaction and thereby the concentration of peptide on the membrane surface. the structural changes have furthermore been visualized by cryo-tem. we have further investigated the pore formation in detail and found that the thermodynamic parameters of pore formation can be fully characterized using a system specific temperature where the enthalpy of peptide partitioning becomes zero. this allows for an exclusive study of the pore formation process. lactoferrin is a glycoprotein with two globular lobes, with of two domains each. since its discovery, the research on antimicrobial properties has been extended to peptides derived from this protein. the largest family studied so far is known as lactoferricin b, obtained from the protein by digestion with pepsin. more recently, a new family of antimicrobial peptides derived from lactoferrin was discovered by bolcher et al and named lactoferrampin (lfampin). the original sequence of lfampin contained residues - from the n domain of lactoferrin. we studied peptides derived from lfampin obtained by extension and/or truncation at the c-or n-terminal sides, keeping the essential characteristics, in order to unravel the main structural features responsible for antimicrobial action. the peptides were tested against model membranes. the ability to adopt helical conformation was followed by cd, the perturbation of the membrane phase transition by dsc, the energetics of interaction by isothermal titration calorimetry (itc), the partition of the peptide to the membrane by trfs and the importance of charge effects assessed by zeta potential measurements. the results are discussed and compared to the antimicrobial and hemolytic activities obtained by microbiology techniques. defensins are small cysteine-rich cationic proteins or peptides found in both vertebrates and invertebrates. they can be active against bacteria, fungi and many enveloped and non-enveloped viruses; thus, being also classified as antimicrobial peptides (amps). in the present work a comparative study between psd (a plant defensin with antifungic properties obtained from the garden pea pisum sativum) and hnp (a human neutrophils defensin) was conducted, in order to shed some light on the mechanism of action at the molecular level of these defensins. large unillamelar vesicles with different lipid compositions were used for this purpose as biomembranes model systems; namely, popc/cholesterol (characteristic of the outer leaflet of vertebrates cell membranes) and popc/ergosterol (fungal) mixtures. changes on the intrinsic fluorescence of the tryptophan residues present in these peptides upon membrane binding/insertion were followed by fluorescence spectroscopy. experimental results show the affinity of both defensins for mammalian and fungal sterols. the partitioning behavior of psd showed a high selectivity for ergosterol rich membranes, while hnp has preference for cholesterol rich membranes. preliminary characterization of atomistic computer models of galactolipid bilayers k. baczynski, m. pasenkiewicz-gierula faculty of biochemistry, biophysics and biotechnology, jagiellonian university, krakow, poland the main lipid component of thylakoid membranes are galactolipids, which constitute more than % of its lipid composition. the most common types of galactolipids found in thylakoid are monogalactosyldiacylglycerol(mgdg), whose headgroup consists of a single molecule of beta-d-galactose and digalactosyldiacylglycerol(dgdg), whose headgroup consists of two galactose molecules: alpha-d-galactose and beta-d-galactose linked by o-glycosidic bond majorty of thylakoid galactolipid have gamma-linolenic acid both in the sn- and sn- position. atomistic computer models of mgdg and dgdg molecules have been constructed and parametrized in the opls-aa force field. the molecules were used to construct three bilayer systems for molecular dynamics (md) simulations:( ) composed entirely of dgdg molecules,( ) composed entirely of mgdg molecules,( ) composed of a mixture of dgdg and mgdg molecules the ratio : . the systems have been md simulated using the gromacs . . package. the generated trajectories were analysed to determine a number of structural parameters among them: membrane thickness, average area per lipid, electron density profile accross the bilayer, order parameters, organisation of the bilayer/water interface as well as several dynamic parametrers like diffusion coefficients, lifetimes of conformational states, lifetimes od lipid lipid interactions. single mixed-lipid guv method reveals interaction of viper venom with lipid membranes n. m. ayvazyan, n. a. zaqaryan, n. a. ghazaryan dpt. biophysics, yerevan state university, armenia studies on the interaction of snake venom and organized lipid interfaces have been conducted using a variety of systems, including bilayer lipid membranes (blms), small and large unilamellar vesicles. giant unilamellar vesicles (guvs) with a mean diameter of µm have a minimum curvature and mimic cell membranes in this respect. guvs are ideal for studying lipid/lipid and lipid/protein interactions using microscopy techniques with membrane fluorescence probes. guvs were formed from the total lipid fraction from bovine brain by the electroformation method, developed by angelova and dimitrov ( ) . vipera lebetina obtusa venom was added to the sample chamber before the vesicles were formed. the membrane fluorescence probes, ans and pyrene, were used to assess the state of the membrane and specifically mark the phospholipid domains. ans and pyrene allows us to quantify the fluidity changes in the membrane by measuring of the fluorescence intensity. the presence of viper venom in guvs media reveals a noticable decreasing of membrane fluidity compare the control, while the binding of fluorophores with guvs modified by venom lead to appearance of channel activity. it was recognized early that the vipers' venom components preferred an organized lipid substrate near the lipid's phase transition and were particularly active against micellar lipids. these studies also emphasize the importance of a membrane surface curvature for its interaction with enzymatic components of venom. the attenuated total reflection fourier transform infrared (atr-ftir) spectroscopy is ideal for highly absorbing samples such as water suspensions or even bulk water due to the small light penetration depth. it is also suited for experiments on lipid membranes in excess water. for example, we have studied the hydrogen bonding (h-bonding) between cholesterol (ch) and phosphatidylcholine (pc) or sphingomyelin (sm), which could be important for the stabilization of the cholesterol-rich lipid domains. the atr-ftir method enabled comparison of the carbonyl band shape for pc/ch samples in excess h o or d o, and has confirmed similar behavior for both [ ] . secondly, we were able to analyze the amide ii band for sm/ch samples in excess h o. the observations confirm the presence of h-bonds between ch and n-h group in sm [ ] . another example is the study of the interlamellar water structure, which could influence the water-mediated phenomena in membranes. h-bonds in interlamellar water in partially hydrated lipid multibilayers are stronger with respect to bulk water. in contrast, we show by atr-ftir spectroscopy that the h-bonds are weaker in multibilayers in excess water [ ] . the bactericidal activity of antimicrobial peptides is linked to their ability to perturb the permeability of bacterial cells. they often show α-helical conformation, and many peptides have a kink in the middle of this structure, caused by pro or gly. in order to understand the role of the kink we designed various analogues of p , in which the central pro residue was moved from its central position, or removed altogether (in analogue p f). displacement of the pro residue caused a decrease of the antimicrobial activity, and an increase in the toxicity against erythrocytes, with the less active and more toxic peptide being p f. fluorescence studies suggest that both p and p f bind on the membrane surface. fluorescence experiments show that the activities of the two analogues correlate with their affinity for different kinds of lipid bilayers: the kinked p peptide has a dramatically higher affinity for negatively charged vesicles (mimicking the composition of bacterial membranes) than for neutral liposomes (similar to mammalian cells), while analogue p f exhibits comparable affinities for both membranes. therefore, our data suggest that the central pro-induced kink is involved in selectivity, inhibiting peptide binding to the membranes of eukaryotic cells. d. behn , h. hoffmeister , r. witzgall , c. steinem institute for organic and biomolecular chemistry, university of göttingen, germany, institute for molecular and cellular anatomy, university of regensburg, germany polycystin- , encoded by pkd , is an integral membrane protein with a size of kda and amino acids. the protein, which is known to be a ca + permeable, non selective cation channel, interacts with several proteins such as polycystin- , pigea or pacs- /- etc. the interaction takes place through a proposed coiled-coil domain of polycystin- located at the c-terminus of the protein.if mutation of either pkd or pkd (gene product of polycystin- ) occurs, the interaction between the proteins is disturbed leading to increased formation of renal cysts. this autosomal polycystic kidney disease (adpkd) is one of the most common genetic diseases causing renal failure due to the enormous cyst formation. in this study, the interaction of the c-terminus of polycystin- with its postulated specific interaction partners has been investigated in terms of its biological relevance. binding affinities as well as kinetic parameters of the interaction were determined. in particular, the interaction of c-polycystin- and pigea immobilized on solid supported membranes with c-polycystin- has been quantified by means of the quartz crystal microbalance (qcm) method. a dissociation constant of about nm was obtained. the results are compared with those obtained by surface plasmon resonance (spr) technique using a different immobilization strategy. correlation of the lateral structure of lipid bilayers and monolayers using two photon excitation fluorescence microscopy l. a. bagatolli membrane biophysics and biophotonics group/memphys -center for biomembrane physics, bmb, university of southern denmark most of the reported fluorescence microscopy applications on langmuir lipid films are focused in obtaining fluorescence intensity images using particular fluorescence probes. in this type of experiments the probes are generally utilized to obtain "contrast" between membrane regions (lipid domains) displaying dissimilar physical properties. the obtained information largely depends on the partition of the fluorescent probes for the lipid domains and the obtained fluorescence intensity pictures are not providing any information about the local physical properties of the lipid film. local physical properties of these distinct regions can be determined by exploring fluorescent probe related parameters such lifetime, emission shift, polarization. however these fluorescent probe's properties are almost unexplored in this type of experiments. this presentation will focus in describing a new experimental setup that includes a specially designed film balance on top of a custom built multiphoton excitation fluorescence microscope. this setup allows obtaining laur-dan gp images ( ) many studies on phase separations of lipids in bilayers and their leaflet asymmetry make use of fluorescence techniques. we used a dithionite assay, steady-state fluorescence anisotropy and fluorescence resonance energy transfer (fret) for characterization. dithionite assay was performed to measure the fluorophore distribution in the inner and outer leaflets of symmetric membranes. for low concentrations, the fluorophore is distributed almost homogeneously, whereas for concentrations > . mol % it accumulates in the outer leaflet. we discuss these effects taking into account the presence of multilamellar liposomes and dynamic effects produced by higher local bending elasticities. the results point out possible artifacts in the use of fluorophores to characterize bilayers under the assumption of their homogeneous distribution. dithionite relative bilayer permeability is discussed. the results won by fret and steady-state fluorescence anisotropy regarding lipid phase transitions are in good coincidence to each other and to results reported in the literature. the photosensitizing properties of three chlorins are compared in solution and when incorporated in dioleoylphosphatidylcholine vesicules. in solution, they possess a similar efficacy to generate singlet oxygen and a similar ability to induce the peroxidation. however, the role of the photosensitizer localization within the lipidic bilayer is strongly highlighted, when chlorins are incorporated in liposomes, both by the changes in order of peroxidation efficacy but by the measurements of the photoinduced permeation of the liposomes. the results are discussed in relation with the technology of photochemical internalization, pci. then, using giant unilamellar vesicles, we asymmetrically oxidize the membranes. we observed different shape transitions, such as budding, typical of membrane curvature modifications. the asymmetry of the shape transitions are in accordance to a lowered effective spontaneous curvature of the leaflet targeted. this effect is interpreted as a decreased preferred area of the targeted leaflet compared to the other, due to the secondary products of oxidation. permeabilization of guv by photooxidation is interpret as the opening of a pore above a critical membrane tension due to the budding of vesicles. the effective spontaneous curvature of photosensitized vesicle at lysis was estimated. additionally photooxidation was shown to be fusogenic. influence of polyphenol extracts from fruits on biological and model membranes d. bonarska-kujawa, h. pruchnik, j. sarapuk, j. oszmiański, h. kleszczyńska univ. of environmental and life sciences, wroc law, poland biological activity of polyphenol extracts from apple, strawberry and chokeberry was studied. polyphenols were shown to be good antioxidants and to act as antyhemolytic agents. both the activities are the result of polyphenols incorporation into biological membranes. to check potential changes they induce in membranes some experiments were performed with the use of erythrocytes, and lipid membranes. it was found that the extracts studied induced shape transitions of erythrocytes. they were classified according to the bessis-brecher scale. strawberry extract induced mainly discocytes and discoechinocytes. populations of discocytes, echinocytes and some discoechinocytes were found on applying apple extract, while chokeberry induced mainly the formation of echinocytes and spheroechinocytes. the results evidence that the polyphenols incorporate into the external monolayer of lipid bilayer of the erythrocyte membrane. the results of the fluorimetric experiments showed that all the extracts changed fluidity in the hydrophilic part of rbc membrane. the changes observed were biggest for chokeberry extract and smallest for strawberry one. incorporation was also followed by changes in electrical resistance of black lipid membranes and in shifting the temperatures of main transition (t m ) and pretransition (t p ) in liposomes. this work was sponsored by ministry of science and education, scientific project no. n n . a. boll , n. czudnochowski , m. geyer , c. steinem institue of organic and biomolecular chemistry, university of göttingen, germany, mpi for molecular physiology, dortmund, germany hiv- tat belongs to the accessory proteins of hiv and has regulatory functions. tat is concentrated in the nucleus and nucleolus of infected cells. the protein is composed of amino acids with a molecular weight of kda. tat is a transcriptional activator protein, which stimulates rna polymerase ii-mediated transcription elongation. therefore, tat interacts with cyclin t and binds to the tar rna element. tat has different domains. with respect to the interaction with lipid membranes, the most important structural motif is its basic region, including arginine and lysine residues. the peptide derived from this basic region belongs to the cell-penetrating peptides and is able to translocate through lipid membranes. the major aim of this study is to investigate the influence of full length hiv- tat on artificial lipid membranes. as a starting point solid-supported membranes composed of -palmitoyl- -oleoyl-sn-glycero- phosphocholine (popc) immobilized on silicon dioxide were used. the influence of the lipid head groups on the interaction with tat was analysed by using membranes composed of the negatively charged lipid -palmitoyl- -oleoyl-sn-glycero- -[phospho-l-serine] (pops) in a mixture with popc. fluorescently labelled tat was used to localise the protein in the membrane. the impact of tat on lipid membranes was investigated by fluorescence and atomic force microscopy, showing that it is capable of perturbing lipid membranes. g. bocchinfuso , a. palleschi , b. orioni , g. grande , f. formaggio , c. toniolo , y. park , k. s. hahm , l. stella univ. of rome tor vergata, dept. of chemistry, italy, dept. of chemistry, univ. of padova and cnr, italy, chosun univ., rcpm, south korea. most antimicrobial peptides exert their activity by perturbing the permeability of bacterial membranes, but the molecular details of this process are still debated. here, we compare fluorescence experiments and molecular dynamics simulations regarding the interaction of two antimicrobial peptides (pmap- and trichogin ga iv) with lipid membranes, showing that their mechanisms of bilayer perturbation are significantly different. pmap- , a cationic peptide member of the cathelicidin family, associates to the membrane close to its surface and parallel to it, and in this arrangement it causes a severe perturbation to the bilayer, both regarding its surface tension and lipid order. on the other hand, trichogin ga iv, a neutral peptide member of the peptaibol family, undergoes a transition from a surface-bound state to an inserted orientation. in the first arrangement it does not cause any strong membrane perturbation, while in the second orientation it may span the bilayer from one side to the other, despite its relatively short length, by causing a significant thinning of the membrane. lipopolysaccharide (lps) is the main component of the outer membrane of gram negative bacteria. lps is also known as endotoxin because of its potency to induce sepsis, a serious source of mortality in many clinical cases. among lps-neutralizing agents, polymyxin b (pxb) is considered as the "gold standard" due to its high efficiency of binding and detoxification of endotoxin. in this work, we have further explored the interaction of pxb to lps from the rough mutant of salmonella minnesota (re-lps) both in the gel and in the liquid crystalline phase, using isothermal titration calorimetry (itc) and fluorescence based techniques. the effect of pxb binding on lps-membrane integrity was determined with a fluorescence quenching assay treating vesicles of lps labeled with nbd-pe with dithionite. thermodynamic parameters associated with the binding process as well as binding stoichiometry were obtained from itc experiments. finally, itc was conducted with enterococcus faecalis and salmonella typhimurium, as representative examples of a gram negative and a gram positive bacterium respectively. pressure jumps -an accessible trigger for biomolecular transformations high pressure can be used to induce many structural changes in biological systems: from triggering phase changes in model membranes to causing protein unfolding, in fact any change involving a volume reduction is promoted by pressure. as well as being broadly applicable, pressure changes can be applied very quickly both up and down in contrast to other structure change triggers such as temperature jumps. fast pressure jumps allow the trigger to be decoupled from structural changes, so with fast structure probe techniques such as time resolved x-ray diffraction, the out-of-equilibrium evolution of these systems can be monitored. despite great advantages, high pressure remains under-utilised primarily due to its technical difficulty. in response to this technology vacuum a high pressure user facility based around a pressure jump cell for small and wide angle x-ray diffraction has been commissioned at beamline i , diamond light source, uk and will be freely available to the user community. the cell is highly robust requiring virtually no user intervention during an experiment and the pressure system is computer controlled with a graphical user interface and is integrated with the beamline. pressures between and . gpa are accessible and jumps can be carried out in approximately ms at temperatures from - to • c. sample changing has been made simple and fast with a dedicated sample loading port and modular sample holders allow optimisation for a broad range of samples. the transformation of vesicle and lateral distribution of mobile membrane inclusions b. bozic institute of biophysics, faculty of medicine, university of ljubljana, lipičeva , si- ljubljana, slovenia membrane inclusions such as membrane embedded peptides or proteins exhibit curvature dependent interaction with the surrounding lipid matrix due to the mismatch between their intrinsic curvature and the local membrane curvature. this interaction causes an inhomogeneous lateral distribution of the inclusions and a corresponding adjustment of the vesicle shape. by taking into consideration that the membrane free energy includes elastic energy of the lipid bilayer and a contribution due to an inclusion-membrane interaction, the configurations of lipid vesicles with mobile inclusions have been studied theoretically. the variational problem to calculate equilibrium vesicle shapes is solved by applying a ritz method based on an expansion in spherical harmonics. in general, vesicle shapes adjust to the presence of inclusions by increasing regions with favorable curvature and decreasing regions of unfavorable curvature in such a way that the lateral distribution of inclusions becomes inhomogeneous. if the number of inclusions or the inclusion-membrane interaction exceeds a certain value, the prolate shapes become globally stable. investigating the structure of pores formed by antimicrobial peptides using epr spectroscopy m. bortolus , k.-s. hahm , a. l. maniero universita' di padova, padova, italy, chosun university, kwangju, south korea spin label electron paramagnetic resonance (epr) is a spectroscopical technique effective to study the molecular mobility of membrane components and the membranepeptide interactions, as the timescale of epr is optimally matched to the rotational motions of lipids in membranes. we applied epr to study the pore-forming mechanism of two antimicrobial peptides (amp) that create pores of different dimensions when interacting with liposomes. hp ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) is derived from the n-terminus of helicobacter pylori ribosomal protein l , and hpa is an hp ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) analogue where gln and asp at positions and were substituted by trp. we studied the interaction of the two amp with zwitterionic and negatively charged vesicles, doped with phospholipids spin labelled in the lipid head or at the c or c positions; the different phospholipids allow us to study the peptide-membrane interaction at different depths relative to the membrane surface. we studied the interaction of the amp with vesicles following the influence of amp on label mobility as a function of temperature and membrane depth. we also prepared spin-labelled dmpc/dhpc bicelles, doped with lanthanide ions (dy + /tm + ) that allow us to macroscopically orient the system using the magnetic field of the epr spectrometer. we studied the interaction of amp with the oriented bicellar system monitoring the effect of amp on the order parameter of the phase. a. chattopadhyay centre for cellular and molecular biology, hyderabad, india we addressed the organization and dynamics of the human serotonin a receptor fused to enhanced yellow fluorescent protein (serotonin a r-eyfp) expressed in cho cells. serotonin a receptors are prototypical members of the gprotein coupled receptor superfamily and represent a prime target for therapeutic actions of several anxiolytic and antidepressant drugs. interestingly, we observed significant retention in fluorescence of serotonin a receptors upon triton x- treatment of intact cells at low temperature demonstrating their detergent insolubility. we analyzed the role of cholesterol in the plasma membrane organization of the serotonin a receptor by fluorescence recovery after photobleaching (frap) measurements with varying bleach spot sizes. our results show that lateral diffusion parameters of serotonin a receptors are altered in cholesterol-depleted cells in a manner that is consistent with dynamic confinement of serotonin a receptors in the plasma membrane. interestingly, results from frap measurements performed under conditions of mild cytoskeletal destabilization suggest that receptor signaling is correlated with receptor mobility, in agreement with the 'mobile receptor hypothesis'. our current work is focused on exploring the oligomerization of the receptor using photobleaching anisotropy measurements and indicates the presence of constitutive oligomers of the serotonin a receptor in live cells. expression and reconstitution of connexin in pore-suspending membranes c. carnarius , s. kaufmann , m. tanaka , c. steinem institute of organic and biomolecular chemistry, university of göttingen, tammannstrasse , göttingen, germany, institute of physical chemistry, university of heidelberg, im neuenheimer feld , heidelberg, germany the intercellular communication and electronic coupling between adjacent cells of vertebrates are mediated by gap junctions. these proteins are composed of two connexon hemichannels, whereas each connexon consists of six connexin subunits. each subunit is characterized by two conserved motives: two extracellular loops and four transmembrane α-helices. in this study, we focused on cx . to obtain large amounts of this protein, we expressed cx and cx +gfp in a rather new expression system: dictyostelium discoideum. in contrast to human tissue cultures, the system allows for high cell densities up to million cells per ml and the cells can be cultivated by fermentation. cx +gfp was successfully visualized in d. discoideum by confocal laser scanning microscopy, where it was preferentially found in the plasma membrane.after the cells were harvested, plasma membranes were prepared and both proteins (cx and cx +gfp) were verified by western blot analysis. the proteins were solubilized by addition of % n-octyl-β-d-glucopyranoside and purified by ion metal chelate affinity chromatography. the activities of both proteins were confirmed by a cytochrome c assay. after the purification of both proteins, they were reconstituted in µm-sized pore-suspending membranes. in the near future, we plan to determine the mobility of cx and cx +gfp in these membranes by fluorescence recovery after photobleaching. o. cañadas, c. casals dpt. biochemistry and molecular biology i, and ciber enfermedades respiratorias, complutense university of madrid, madrid, spain inhaled bacterial lipopolysaccharide (lps) may incorporate into the lung surfactant monolayer. in this study, the effect of smooth lps (s-lps) on the surface activity of lung surfactant was evaluated. to that end we investigated the behavior of dppc films containing s-lps, with and without surfactant protein a (sp-a) in the subphase, using epifluorescence microscopy combined with a surface balance. our data show that s-lps injected into the subphase incorporated into dppc films forming mixed dppc/s-lps monolayers. cospread s-lps fluidized the dppc monolayer as demonstrated by epifluorescence images and changes in the compressibility modulus of the monolayer as a function of s-lps molar fraction (x s−lps ). the presence of low amounts of s-lps in the monolayer promoted early collapse, preventing high surface pressures to be reached. moreover, s-lps hampered the re-spreading of dppc molecules during dynamic compression at s-lps concentrations as small as x s−lps = . . such inhibitory effects could not be relieved by repeated compression-expansion cycles or by adding surfactant protein a. however, sp-a facilitated the squeeze-out of s-lps from dppc/s-lps mixed monolayers, suggesting that sp-a is an s-lps scavenger. cholesterol displaces ceramide from its tight packing with sphingomyelin in the absence of ld phase j. v. busto, j. sot, j. requejo-isidro, f. m. goñi, a. alonso unidad de biofísica (csic-upv/ehu) and departamento de bioquímica, u. país vasco (upv/ehu), spain a set of biophysical approaches have been applied to study the phase behaviour of palmitoylsphingomyelin (psm)/cholesterol (chol) model membranes upon palmitoylceramide (pcer) addition. fluorescence spectroscopy of di- -aneppdhq-stained psm/chol vesicles reveals no segregation of large liquid-ordered (l o ) microdomains. in contrast, the formation of disperse, compositionally homogeneous l o psm/chol ( : ) nanodomains over a psm gel (l β ) phase is proposed. dsc measurements show that pcer addition to vesicles with coexisting l o and l β phases (low [chol] ) induces the formation of large gel-like psm/pcer microdomains, coexisting with a l o psm/chol phase. the ∆h for the psm/pcer phase at high psm/(chol+pcer) ratio is close to that of the binary mixture in the absence of chol, supporting immiscibility, but no displacement, between chol and pcer-rich phases. on the contrary, both confocal microscopy of guvs and the dsc data coincide in showing that a rise in pcer increases the gel-like phase to a lower extent than in the pure psm/pcer mixture, revealing some cholinduced restriction. in the presence of a pure l o psm/chol phase (high [chol] ), pcer addition is unable to induce the formation of large psm/pcer microdomains. the present data support the role of chol as the key determinant in controlling its own displacement from l o domains by ceramide. hydroquinones modifying lipid membrane morphology c. di vitta , l. marzorati , v. rebbin , s. s. funari iqusp, university of são paulo, são paulo, brasil, hasylab, hamburg, germany quinones are important molecules in cells. flavina, ubiquinone and coenzyme q, coq, are associated with electron transfer. coq, having a hydrophobic tail, is soluble in the internal lipid membrane of mitochondria. their reduction leads to the equivalent hydroquinones. we synthesized novel alkylthioquinones aths, to investigate their interaction with phospholipids. one or more long hydrophobic chains attached to the quinone ring alter their hydrophobicity and electron availability. we aimed at their influence on the structure of membranes. different phs highlights the charge/polarity effect on the interaction and on the morphology of the bilayer. for that, pope and popc, lipids with different charges, but identical chains were selected. x-rays diffraction on pope/ , bath, / at different temperatures and ph, showed cubic phases coexisting with the usual phases formed by simply hydrated pope, at different phs. for investigation of the charge/polarity, we turned to the popc/ , bath system (smaller charge/polarity). despite decrease in temperature of phase transition and dimensions of the lattice, the structures were the same as in hydrated lipid, illustrating a less significant effect than on the similar lipid pope. another additive, , ath, mixed with pope showed the effect of multiple thioalky chains. no morphology change was seen, compared to pure lipid. interestingly, despite both additives differ by one thioalkyl chain only, their influence on the pope matrix is so different. cross-linking of phospholipid membranes by calcium-sensitive synaptotagmins synaptotagmins are vesicular proteins implicated in many membrane trafficking events. they are highly conserved in evolution and the mammalian family contains isoforms. we now show that the tandem c domains of several calcium-sensitive synaptotagmin isoforms tested, including drosophila synaptotagmin, rapidly cross-link phospholipid membranes. in contrast to the tandem structure, individual c domains failed to trigger membrane cross-linking in several novel assays. large-scale liposomal aggregation driven by tandem c domains in response to calcium was confirmed by the following techniques: turbidity assay, dynamic lightscattering and both confocal and negative stain electron microscopy. high-resolution cryo-electron microscopy revealed that membrane cross-linking by tandem c domains results in a constant distance of approximately nm between the apposed membranes. our findings show the conserved nature of this important property of synaptotagmin, demonstrate the significance of the tandem c domain structure and provide a plausible explanation for the accelerating effect of synaptotagmins on membrane fusion. membrane proteins can be challenging samples to work with and as such often require the use of multiple techniques. here we present an insight into the structure of the antimicrobial peptide melittin in lipid membranes using various techniques. we explore the conformational changes of membrane-bound melittin and its interaction with model membrane lipid systems with a series of spectroscopic methods that can be used in parallel. the techniques used include linear dichroism and ft-ir for orientation information and circular dichroism for conformation information. we use dynamic light scattering for molecular sizing, fluorescence emission for information on peptide environment, thin-layer chromatography for lipid identification, analytical ultracentrifugation to identify oligomerisation state and calorimetry to investigate the thermodynamics. we observe how the physical properties of both the peptide and the membranes affect the insertion kinetics of the peptide in the membrane. phospholipid membranes dynamics: molecular dynamics vs neutron scattering v. conti nibali , m. tarek , u. wanderlingh , g. d'angelo department of physics, faculty of science, university of messina, italy, unité mixte de recherche cnrs/uhp , université henri poincaré, nancy, france collective dynamics and single-particle dynamics of hydrated multilamellar phospholipid bilayers ( , -dimyristoylsn-glycero- -phosphatidylcholine, dmpc) have been studied by means of all-atom molecular dynamics simulations. here we report results of a gel phase bilayer at k and of a liquid crystal phase bilayer at k. coherent and incoherent dynamic structure factors and meansquare displacements have been calculated from the trajectories for both the inplane and out-of-plane lipid dynamics. moreover, the results have been compared to recent quasi-elastic and inelastic neutron scattering data. optical tweezers allow trapping of particles of different types in a wide range of sizes [ ] . among these, unilamellar vesicles are of interest as they are known to be effective vectors in drug delivery and they are also studied as models for cell membranes. this work focuses on the interactions between optically confined unilamellar vesicles and their cross-linking by proteins [ ] . synaptotagmins are vesicular proteins implicated in many membrane trafficking events having an accelerating effect on the membrane fusion. calcium-sensitive synaptotagmins are thought to confer calcium sensitivity to the fusion of secretory vesicles with target membranes [ ] . the novel optical assay reported here allowed us to visualize the cross-linking of nm liposomes mediated by synaptotagmin and calcium. the use of the optical tweezers approach to investigate the function of other fusogenic proteins related to exocytosis (i.e., snare proteins) is discussed as well. antimicrobial peptides (amps) have received increasing interest as the search for new potential antibiotics has become imperative due to increasing bacterial multiresistance. acylating the natural occurring polymyxin b (pmb) significantly enhances antibacterial activity towards two representative gram-negative bacteria e. coli and k. pneumonia compared to the nonacylated pmb. the aim of the presented work was to study various biophysical parameters, such as partitioning coefficients, zeta potentials and effective charges of a selected amp, mastoparan-x (mpx) and a propanoylated (pampx) and octanoylated (oampx) analogue, respectively. fluorescence spectroscopy, isothermal titration calorimetry and ζ-potential measurements were the main techniques used for the measurements. we employed different luv model systems; a partially charged (popg/popc : ) and a neutral system (popc). for the neutral luvs there was an increase in partitioning with increasing length of the acyl chain, whereas partioning into the partially charged luvs was governed by a balance of hydrophobic and electrostatic contributions. the selectivity for the partially charged luvs over the neutral luvs was in the order pampx, mpx and oampx. the modeled effective charge for the peptide followed the same trend as the partitioning coefficient for the three peptides for the respective luv systems. does the lysosomal membrane need triglycerides? a spectroscopic study of a simple model membrane l. duelund , k. pakkanen , m. vuento , j. h. ipsen memphys -center for biomembrane physics, university of southern denmark, odense, denmark, nanoscience center, university of jyväskylä, jyväskylä, finland lysosomes are intracellular organelles in which proteins and other macromolecules are degraded. morphological and functional changes in different compartments of the endocytic pathway are connected to several diseases. a crucial step in understanding biogenesis of lysosomes and their role in disease conditions, is to characterise the properties of the lysosomal membrane. by the use of tlc we have found that lysosomes contain non-neglible amounts of triglycerides (tg). to investigate how the presence of tgs could influence the lysosomal membrane, we have investigated the properties of a mixture of popc and triolein, as a simple model for the lysosomal membrane. we found the system to form two types of popc-rich membranes. these were determined as co-existing phases based on their spontaneous and stable separation and named heavy and light phase according to their sedimentation behaviour. by using epr and fluorescence spectroscopy the physical properties, including order, fluidity and water penetration, of these phases were found to differ markedly despite of their almost identical composition. the results suggest that presence of tgs on lysosomal membranes could have a crucial effect in the barrier functions and thus, the integrity of the organelle. model membranes with the capacity to align in magnetic fields such as bicelles and magnetically sensitive vesicles are of high interest, since their macroscopically alignment allows for the determination of structure, dynamics and topology of molecules within the membrane [ ] . we performed p solid state nmr on a magnetically sensitive lipid possessing a large positive magnetic anisotropy introduced in form of a biphenyl unit during lipid synthesis in one of its acyl chains ( -tetradecanoyl- -( -( biphenyl) butanoyl)-snglycero- -phosphocholine (tbbpc)). the phosphorus lineshapes of tbbpc mlvs studied at various magnetic fields ( . t, . t, . t, . t), revealed a drastic change in shape upon exposure to fields > . tesla: resonances resulting from phospholipid molecules oriented perpendicular to the magnetic field decrease whereas resonances resulting from parallel oriented molecules increase. this is a sign for magnetically induced vesicle deformation from vesicle to oblate ellipsoid shape. factors influencing this drastic deformation, such as magnetic anisotropy, membrane elasticity, lipid chain length and field dependency are discussed based on existing theories [ ] . hepatitis g virus (gbv-c/hgv) is an enveloped viruses belonging to the flaviviridae family. clinical findings have suggested that in people co-infected with gbv-c/hgv and human immunodeficiency virus (hiv), delayed progression of aids has been observed ( ). the mechanism by which this virus may inhibit the progression of aids remain to be elucidated. enveloped viruses acquire their lipid membranes by budding through host cellular membranes ( ) , in this process; fusion peptides play an important role. study of the interaction of hgv-c peptides with lipid membranes could lead us useful information about the mechanism that takes place. in this work we present a study on the effects of e peptides on the fluidity of and polarizability of model membranes (dmpc and dppc liposomes) by dsc and fluorescence polarization. both techniques showed that the presence of the studied sequences in phospholipid mixtures already affected the thermotropic properties of the gel to liquid-crystalline phase transition. systems were thermodynamically charac- small angle neutron scattering (sans) studies have been performed to study the structural changes induced in membranes of vesicles prepared from phospholipid and mixed phospholipid-sterol mixtures, in the presence of different concentrations of the anti-fungal drug, amphotericin b (amb).the vesicles, sonicated to a mean size∼ nm, were prepared using dimyristoylphosphatidylcholine(dmpc) or dmpc-cholesterol or dmpc-ergosterol mixtures -with both of the mixed systems involving mol% sterol. analysis of the sans data show that when the concentration of amb added is just above the drug's cmc (∼ µm) there is an increase in the membrane thickness of both the dmpc-chol and dmpc-erg vesicles (both cases + Å), but the thickness of the pure dmpc vesicle membranes remains the same as in the absence of amb. when amb is added at a concentration in excess of its cmc (∼ µm), the mixed-sterol vesicles show the same changes in membrane thickness as observed with the lower amb concentration, and the pure dmpc vesicles again remain unaffected. on the basis of these studies, therefore, there appears to be no difference in the structural changes induced by the insertion of amb into the model fungal cell membranes (mimicked by dmpc-erg vesicles and those resulting from its insertion into the model mammalian cell membranes (mimicked by dmpc-chol vesicles). the third transmembrane helix of bacteriorhodopsin, also known as phlip, is a unique model system for studying the interactions of a natural transmembrane domain with lipid membranes: depending on ph, the water-soluble peptide either adsorbs superficially or inserts as a transmembrane helix on addition of lipid vesicles [ ] . published values for the free energies of these processes were based on a stoichiometric model invoking two distinct sets of binding sites [ ] . however, discrepancies between data obtained from different experimental techniques and inconsistencies between experimental and expected temperature dependencies suggest that these values should be taken with caution. we therefore reassessed membrane interactions of phlip using titration calorimetry and fluorescence spectroscopy. if electrostatic effects at the membrane surface are taken into account, the data can be described quantitatively by a partition equilibrium, but not by a stoichiometric binding model. the thermodynamics of membrane partitioning differ substantially from those determined previously [ ] and draw a different picture of peptide-lipid interactions. beyond deepening our insights into the first step of the two-stage model of membrane protein folding, this also sheds light on the ability of phlip to drag cargo molecules across lipid membranes. adsorption of monolysine and polylysines at the surface of lipid membranes of varied composition was studied by two methods. both methods -the electrokinetic one, measured the surface potential of liposomes, and the intramembranouse field compensation sensitive to boundary potential of planar bilayer lipid membranes -detected the positive changes of the potentials for membranes with negatively charged component (cardiolipin, cl). neither monolysine, nor polylysines adsorbed at neutral membranes (phosphatidylcholine, pc). pentalysine show the difference between these potentials for the membranes composed from cl/pc mixtures. this difference is attributed to dipole part of boundary potential and indicates the changes in lipid packing. polylysines show high affinity to the membrane and saturation with plateau. these saturation levels correspond to surface charge densities . and . c/m for oligomers with and units, and . c/m for polymers with and units. these values do not depend on the ionic strength of background electrolyte but proportional to the content of negatively charged components in the lipid bilayer. the polylysine layer at the mica surface was studied by atomic-force microscope (afm) technique. it was shown that pentalysine molecules cover the surface by the layer of . nm thickness and polylysines of high molecular weight by the layer up to nm. effects of lysolipids on the mechanical stability of lipid membranes j. r. henriksen , l. feldborg , j. h. ipsen , t. l. andresen technical university of denmark, dtu nanotech, frederiksborgvej , b , roskilde, denmark., university of southern denmark, department of physics and chemistry, memphys center, campusvej , odense m, denmark lysolipids (lpcs) and fatty acids (fas) are the natural products formed by the hydrolysis of phospholipids. lpcs and fas form micelles in solution and thus act as detergents in the presence of lipid membranes. in the present study we investigate the detergent strength of a homologous series of lysolipids (lpcs) on popc lipid membranes by use of isothermal titration calorimetry (itc) and vesicle fluctuation analysis (vfa). the membrane partition coefficient (k) and critical micelle concentration (cmc) are determined and found to obey an inverse proportionality relation (cmc x k ∼ constant). the partition coefficient and critical micelle concentration are used for the analysis of lpc's effect on the membrane bending rigidity. the dependence of the bending rigidity on the lpc membrane coverage has been analyzed in terms a phenomenological model based on continuum elastic theory which yield information about the curvature inducing properties of the lpc molecule. the results reveal: (i) an increase in the partition coefficient with lpc acyl-chain length and (ii) the degree in acyl chain mismatch between lpc and popc determines the magnitude of the membrane mechanical perturbation per lpc molecule on the membrane. patch clamp electrophysiology remains the gold-standard for ion channel research because of the information richness of the data produced. automated planar patch clamp devices, with their higher throughput and high data information content, have made the technique accessible to a wider audience. nanion technologies offers two planar patch clamp workstations combining higher throughput with high data quality. the port-a-patch records from a single cell at a time and the patchliner from up to cells simultaneously with high success rates (typically - %). both, the port-a-patch and the patchliner are bench top patch clamp rigs which uses a planar borosilicate glass chip for obtaining a giga-seal for electrophysiological recordings. suction applied from the underside of the chip is used to attract a single cell to the recording site without the need for optical visualization. both workstations have been successfully used for whole cell, perforated patch, and cell attached recordings as well as for guv-bilayer recordings. due to the versatility of nanion`s products it is possible to study a wide variety of ion channels including nav, kv, cav, ligandgated, herg or reconstituted proteins such as ksca, cx , ompc. special features unique to nanion products, including but not limited to internal solution exchange and temperature control, expand the experimental possibilities. waves on lipid monolayers j. griesbauer, s. boessinger, a. wixforth, s. f. matthias univiversity of augsburg, experimental physics i, biological physics group, augsburg, germany " for the sake of illustration we shall try to provide a physical basis for the equations, but must emphasize that the interpretation given is unlikely to provide a correct picture of the membrane." [ hodgkin&huxley, ] to explain the occurrence of reversible heat production during nerve pulse propagation it has been suggested by multiple authors [wilke,kaufmann,heimburg] that travelling sound waves and not ion channels may offer a better explanation than the hodgkin&huxley theory. therefore, if sound waves are an essential feature of the nerve membrane they should also appear on lipid monolayers where ion conductivity is evidently absent. here we demonstrate that sound waves can be excited on a lipid monolayer by using a set of planar electrodes incorporated into the monolayer and driven by an alternating voltage. not only do our results indicate propagating waves on lipid monolayers in accordance with their thermodynamic predictions, but importantly, no significant attenuation is detected proposing an adiabatic phenomenon. in order to provide evidence that our physical explanation provides a correct picture of the membrane, direct detection of the waves was done, whereas a clear transduction of signals was shown. finally, the impact of toxins, neuropharmaca and anaesthetics needs to be integrated in our physical picture of the nerve membranes, what can easily be done now and could deliver a new approach for understanding their physical mechanism. our earlier studies showed that organometallic compounds (otc) in the presence of uvc radiation enhanced the degree of phosphatidylcholine (pc) liposome oxidation, whereas quercetin effectively protected the membrane. the present investigation is concerned with the effect of uvb and otc (chlorides of diphenyl-and dibutyltin, triphenyl-and tributyltin), both in combined action and separately, on oxidation of erythrocyte proteins, pc liposome and albumin. the degree of oxidation of proteins and liposomes induced by otc and radiation, also in the presence of selected antioxidants (trolox, quercetin) was determined on the basis of changes in the number of sulfhydryl groups, carbonyl groups and malone dialdehyde, respectively. the studies indicate that uvb induces pc liposome and erythrocyte oxidation, whereas in the case of albumin it causes both an increase in the number of c=o groups and free sh groups (most probably, braking sulphonic bridges). otc compounds interact with membrane biomolecules both as weak pro-and antioxidants, also in combined action (uvb plus otc). the prooxidative effects are markedly diminished by application of antioxidants. the action of quercetin results from its ability to incorporate into membranes and formation of complexes with otc (liposome>erythrocyte>albumine). this work was supported by grant n n . a phospholipase c/ sphingomyelinase from pseudomonas aeruginosa has been assayed on giant unilamellar vesicles (guv) consisting of phosphatidylcholine, sphingomyelin, phosphatidylethanolamine, and cholesterol, at equimolar ratios. the enzyme activity modifies the chemical composition, thus the physical properties of the bilayer, and conversely the latter influence the enzyme activity. biochemical assays of enzyme activity, together with confocal fluorescence microscopy examination of guv provide novel information about the system. the original lipid composition in the absence of enzyme gives rise to lateral phase separation of liquid-ordered and liquid-disordered domains in the guv. the two enzyme end-products, diacylglycerol and ceramide, have opposite effects on the bilayer physical properties, the former abolishes lateral phase separation, while the latter generates a new gel phase. morphological examination of individual guv shows that the enzyme binds preferentially the more fluid (or more disordered) domains, and that, in most cases, it causes the fluidification (disordering) of the other domains.. cholesterol incorporation into lipid bilayers, in the form of multilamellar vesicles or extruded large unilamellar vesicles, has been quantitated. to this aim, the cholesterol contents of bilayers prepared from phospholipid:cholesterol mixtures ( - mol% cholesterol) have been measured and compared with the original mixture before lipid hydration. there is a great diversity of cases, but under most conditions the actual cholesterol proportion present in the bilayers is much lower than expected. the maximum solubility of chol in bilayers containing saturated pc or pc with less than four unsaturations is - mol%, while in polyunsaturated pc, e. g. dic : , and in sphingomyelin the maximum chol contents is mol%. a quantitative analysis of the vesicles is thus required before any experimental study is undertaken. membrane fusion assay based on poresuspending lipid bilayers i. höfer, c. steinem institute of organic and biomolecular chemistry, university of göttingen, germany membrane fusion has attracted significant interest due to its biological relevance. thus in recent years a great variety of fusion assays has been developed. since the process of membrane fusion is not yet fully understood, our aim is to develop a new fusion assay based on pore-spanning membranes, which have been proven mechanically stable, highly insulating and rather tension free. the application of these so-called micro-blms should allow simultaneous monitoring of lipid mixing, content release as well as electrical readouts. furthermore both membrane sides can be addressed individually to apply transmembrane potential or fusion modulating compounds. first results show that the micro-blms provide the opportunity to investigate lipid bilayer fusion by means of fluorescence microscopy. the formation of porespanning membranes is achieved by the painting technique of dphpc doped with oregon green dhpe dissolved in ndecane. the addition of large unilamellar vesicles doped with texas red dhpe allows direct observation of single fusion events. lipid mixing during fusion leads to a decrease of the donor fluorescence (oregon green) and an increase in acceptor fluorescence intensity (texas red) in the plane of the planar bilayer due to fret. in future work simultaneous monitoring of lipid-mixing and content release combined with electrical measurements are planned to gain further insight into different intermediate steps of membrane fusion. the kinetics of membrane-peptide folding and orientation m. r. hicks department of chemistry, university of warwick, uk. understanding interactions of peptides with lipid membranes is essential if we are to be able to design new and better antibiotic peptides. there are different steps in these interactions and following the kinetics of these processes requires that we combine the information from different biophysical techniques. here we present data on the kinetics of peptidemembrane interactions using circular dichroism to report on conformation and linear dichroism to report on orientation of the peptides in/on membranes. these are combined with other techniques such as dynamic light scattering and fluorescence spectroscopy to elucidate the mechanisms of action of the peptides. one of the most important aspects of membrane-active peptide design is that of specificity. we investigate specificity for different types of membranes by using libraries of lipids with different properties of charge, chain saturation and curvature stress. in this way one can test for effects of e.g. negatively charged head groups found in bacterial membranes or cholesterol found in animal and human membranes. using these approaches it is proposed that we will be able to modify peptide sequences, test what part of the kinetic processes are affected and subsequently use this information to design new and better antibiotics. k. kubica, h. misiak wroclaw university of technology, wroclaw, poland reversible membrane electroporation, that can be observed as a effect of electric field influence on biological membranes, finds application in cell delivery of biologically active compounds. the membrane susceptibility for creation of stable pores, depends on electric field parameters applied to the membrane as well as on the membrane environment (ionic strength, ph, temperature) and membrane molecular composition. we have already shown that the changes of van der waals lipid chain interaction effect the process of pore formation. there are known factors which increase or decrease the membrane respond to electric field. because pores appear in lipid membrane matrix it make sense to study this phenomenon using model planar lipid membranes. as lipids are very receptive on oxidizing factors we decided to study the relation between lipid oxidation and membrane reaction on electric field. with the use of four electrode galvanostat we are examining the influence of factors initiating lipid oxidation (uv, fe + ) and the efficiency of some antioxidant compounds of lipid protection on electric properties of lipid membrane. the product of lipid oxidation is determined by characteristic reaction with tiobarbituric acid. we want to determine the role of polyunsaturated fatty acids on membrane properties also. results of our work will be useful in optimization of medicine distribution, aided by electric field, into cells. budding of giant phospholipid vesicles induced by β -glycoprotein i j. kovacic, b. bozic, s. svetina institute of biophysics, faculty of medicine, university of ljubljana, ljubljana, slovenia β -glycoprotein i (β gpi) is classified among amphitropic proteins: in its inactive form it is dissolved in plasma and in its active form it is bound to membrane. in comparison to other amphitropic proteins it exhibits a distinct membrane interaction behavior. a patch of positively charged amino acid residues contacts anionic phospholipids via electrostatic interactions, and a hydrophobic loop anchores the protein into the outer leaflet of the membrane via hydrophobic interactions. the binding constant depends on physical properties of the membrane such as membrane lipid composition, surface potential, lipid packing density and curvature. the binding of an amphitropic protein alters the membrane's spontaneous curvature as well as the difference between the equilibrium areas of membrane leaflets. theoretical model has been developed to predict vesicles shape transformations realized by the formation of buds. it was shown that the effects are stronger for flaccid vesicles which were therefore chosen for our experimental investigations. vesicles were composed of % pops and % popc, and flaccidity was achieved by an osmotic adjustment. the solution containing β gpi was subsequently injected to a chamber with flaccid vesicles by a syringe pump. observation took place under a phase-contrast microscope. experiments showed a concentration dependent occurrence of buds. their number and size were related to the degree of vesicle flaccidness. the interplay between detergent cohesion and peptide adsorption on the structure and dynamics of a glycophorin a tm-detergent complex j. khao, j.-p. duneau, j. n. sturgis lism, cnrs -aix marseille university, marseille, france in spite of the major interest of membrane proteins at functional, genomic and therapeutic scales, their biochemical and structural study remain challenging as they require, solubilization in detergent micelles. the complexity of this task arises from the structural dependence of membrane proteins on their anisotropic environment and in particular by a delicate balance between different physico-chemical properties. in order to study these properties in a small protein detergent complex, we have used molecular dynamics simulations on the transmembrane part of glycophorin a (gpatm) solubilized in micelles of the detergent di-hexanoyl-phosphatidylcholine(d pc). we show that the molecular aggregates organizes to give distinct populations of detergent molecules. those molecules which loosely interact with the peptide are preferentially involved in highly cohesive inter-detergent interactions that impose a global bilayer structure to the micelle. interaction profiles of other detergents with gpatm depend upon the nature of residues along the surface of the peptide. this topology dependence leads to different modes and strength of interactions that ultimately constrain the orientation of the micelles around the peptide. this simple model illustrates how differential detergent selectivity for faces and strong constraints coming from purely environmental features could influence transmembrane helix packing, membrane protein structure and assembly. in this paper we present a study of a new family of bolaamphiphiles. these amphiphiles are unsymmetrical bolalipids having one sugar polar head and the second glycine betaine polar head. they are potentially useful for pharmaceutical or cosmetics applications as vectors for drugs. therefore it is important to investigate their self-assembled properties. the chemical variations that we introduced in this new family concern the length of the main chain that connects two polar heads as well as the length of the side chain placed on the position of the sugar moiety. another variation concerns the introduction of a diacetylenic unit into the main chain in order to rigidify it. we have performed the saxs (small angle x-ray scattering) measurements on the dehydrated compounds as a function of temperature and observed the lamellar liquid crystalline structures. we also measured the saxs spectra of aqueous solutions of these compounds that have shown lamellar l α structure in all cases. these measurements are compared with polarised optical microscopy measurements that confirmed our interpretation. formation of membrane tubular structures induced by phase separation in giant vesicles y. li, r. lipowsky, r. dimova max planck institute of colloids and interfaces, science park golm, potsdam, germany tubular membrane structures (also known as tethers) exist widely in eukaryotes. abundant work has been done on tube extrusion from cells and model membranes under the application of external forces. we present a novel system allowing tube formation in the absence of external forces. aqueous solutions of two chemically dissimilar polymers, polyethylene glycol (peg) and dextran are encapsulated in giant vesicles, a cell-size model system. the exposure of vesicles to hypertonic solutions induces phase separation of the internal aqueous polymer solution. the excess membrane area created by this vesicle deflation, engages in the formation of tubular structures. membrane tube formation and phase separation are coupled processes. hydrodynamic flows and changes in the membrane spontaneous curvature during phase separation might be the driving forces for tube formation. the tubes are rather stable: without external perturbation, they can exist for several days. they prefer to be located in the peg-rich phase at low polymer concentration. at high concentration, they are absorbed at the interface of the liquid phases to lower the surface energy of the system by decreasing the contact area between the liquid phases. the membrane tubes can be retracted back to the vesicle surface by increasing the membrane tension via vesicle aspiration. membrane tubes, which can form and be retracted easily, might be relevant to lipid storage in cells. insight into the antimicrobial mechanism of a de novo auto-assembling peptide b. legrand , m. laurencin , e. duval , c. zatylny , j. henry , m. baudy-floc´h , a. bondon rmn-ilp, umr cnrs -univ. de rennes , france, icmv, umr cnrs -univ. de rennes , france, pemm, umr ifremer -univ. de caen, france a short ( residues) de novo antimicrobial peptide (k ) composed of a cationic polar head and a hydrophobic tail was studied. it exhibits a broad spectrum of antimicrobial activity on bacteria. no haemolytic activity or cytotoxicity on eukaryotic cells are observed at mic. when bacteria are lysed by the k , spherical objects are observed on the sem micrograph. k was structurally studied using various membrane mimetic media such as different micelles and small unilamellar vesicles (suv) of different composition. cd revealed that k adopt various structures (random, β-turn, α-helix) in the presence or the absence of detergents or phospholipids. nmr structures confirmed the α-helical structure of k hydrophobic tail in presence of sds. k self-assembles at high concentration as observed by sem and dls. we suggest that k may act as a surfactant building mixed microsomes composed of peptide and lipids. this destabilization mechanism of the bacterial membrane support the "detergent like model" previously described in the literature. oxidative stress and the membrane dipole potential; modulation with tocopherol s. le-nen-davey , b. m. davis , j. l. richens , k. a. vere , m. w. tilley , p. g. petrov , c. p. winlove , p. o'shea biomedical physics group, university of exeter, uk, cell biophysics group, university of nottingham, uk tocopherol, a component of vitamin e well know for its antioxidant properties, has recently been thought also to influence the structure of cellular membranes. tocopherol treatment reduces hyperglycaemia induced oxidative stress and the associated endothelial dysfunction which is a precursor to the vascular complications of diabetes. tocopherol and insulin interactions are also modified by hyperglycaemia. it is unclear whether these clinically important effects arise from the anti-oxidant and/or structural properties of tocopherol. we have therefore measured the dipole and surface potentials of phosphatidylcholine vesicles containing different amounts of cholesterol, ketocholesterol and tocopherol. we have also investigated the effects of hyperglycaemia, ketocholesterol and tocopherol, alone and in combination, on microdomain formation and interactions of insulin within the membranes of cultured endothelial cells. both sets of experiments indicate that tocopherol causes significant modifications of the membrane dipole and surface potentials. the physiological significance of these changes will be discussed. t. d. lazzara , a. janshoff , c. steinem georg-august university göttingen, institute for organic and biomolecular chemistry, tammannstr. , , germany, georg-august university göttingen, institute for physical chemistry, tammannstr. , , germany cell penetrating peptides (cpp) have been shown to penetrate cellular membranes. they have been of interest for their ability to translocate not only themselves through cellular membranes, but also carry along with them, cargo as large as iron nanoparticles. the exact entry mechanism remains unclear, but has been shown to vary with peptide sequence. their translocation properties have been demonstrated through different experiments involving vesicles, cells and living animals. we plan on using nano-black lipid membranes (nano-blm), which span the pores of nanoporous materials as a model system to study the interaction between cpp and lipid membranes. the nanopores can be used as cellular containers whose interior surface can be functionalized with receptors for biotin-streptavidin recognition. we hope that this system will provide greater control over experimental variables, such as the type of cpp and lipid used, as well as provide kinetic data that can be used to evaluate cpp activity and the kinetics of cargo transport. ethanol induces phospholipid acyl chain interdigitation. while much is known, important issues remain unclear, such as the role of lipid domains. the main purpose of this study was to follow, in real time, the changes induced by ethanol on supported lipid bilayers with nano to microdomains by in situ atomic force microscopy. to this goal, a pure lipid in the fluid phase (dopc), a pure lipid in the gel phase (dppc), binary phospholipid mixtures with gel/fluid phase coexistence, and cholesterol-containing, raft -forming mixtures (dopc/dppc/cholesterol and dopc/sphingomyelin/cholesterol) were investigated. from the height differences observed upon ethanol addition to pure lipids (dppc and dopc), and to dopc/dppc mixtures, it is shown that in the binary system the interdigitation of the fluid phase occurs prior to the gel phase. however, for the lipid rafts mixtures the simultaneous interdigitation of both raft and non raft portions of the membrane is observed both in mica and silicon substrates. for all compositions studied, domain formation or rearrangement accompanied by lipid bilayer expansion occurs as a consequence of interdigitation. these results show the ability of ethanol to influence the bilayer properties in different ways according to membrane composition. ethanol may exert its biological effects by reducing bilayer thickness, and also by changing membrane proteins conformation and lateral distribution, as a consequence of the altered properties of the lipid bilayer. interactions between non-steroidal anti-inflammatory drugs and a pc/cholesterol bilayer m. markiewicz , t. librowski , p. serda , m. pasenkiewicz-gierula faculty of biochemistry, biophysics and biotechnology, jagiellonian university " department of pharmacodynamics, medical college, jagiellonian university " regional laboratory, jagiellonian university, krakow, poland. the non-steroidal anti-inflammatory drugs (nsaids) are among the most frequently prescribed and used drugs [ ] . there are several side effects connected with frequent use of nsaid, mainly gastrointestinal ulcers and bleedings. a plausible molecular mechanism of these effects are direct interactions of nsaids with gastric phospholipids [ , ] . the influence of three well-known non-steroidal antiinflammatory drugs with a diverse gastric toxicity (aspirin, ketoprofen and piroxicam) and three newly-synthesized xanthone derivatives (belonging to the nsaids) on the structure and dynamics of lipid bilayers was studied using small angle x-ray scattering and molecular dynamics simulations. the results showed some correlation between nsaid toxicity and its binding to the lipid polar groups. this binding increases membrane fluidity by reducing its density due to an increased membrane surface area. a reduced lipid packing in the membrane most likely increases gastric mucosa permeability, which can result in a decreased resistance of the gastric mucosa to luminal acid. we studied the partition of the anionic amphiphile pyrenesulfonate (psa) into mlv and luv (produced by extrusion), composed by zwitterionic popc and zwitterionic/anionic mixtures with pops (until mol %). psa is an anionic amphiphile that mimics several xenobiotics (e.g. pharmaceuticals, pesticides) and endogenous substrates that interact with biological membranes. we found increasing b. lorenz, s. schuy, a. janshoff georg august university, göttingen, germany cell-cell and cell-virus interactions are ubiquitous in living organisms. the analysis of the forces acting between two cells or a cell and a virus gives insight into details of these interaction processes such as their stochastics, cooperativity, reversibility, and energy landscape. using colloidal probe microscopy in conjunction with solid-supported lipid bilayer techniques, we mimic the contact between two membranous compounds displaying sponge glycolipids or viral peptides on their surfaces. after spreading functionalized lipid bilayers on both -a colloidal probe and a silicon wafer surface -the molecular interactions are quantified by means of force-distance curves. by probing the dynamic interaction strength between the viral peptides n and c we aim for a deeper understanding of the role of these peptides in the complex process of the formation of the prehairpin intermediate as the key step in retroviral fusion. as far as the cellular interaction of marine sponge cells is concerned, we intend to investigate the strength, specificity, and the ca + dependency of the self-recognition between sponge glycans. the systems advantages are the flexibility of the membrane composition and the control over the distribution of receptor molecules in the membranes. since adhesion in biological systems relies heavily on cluster formation within the biomembrane, we plan to mimic the clustering by "printing" interaction domains and comparing the results to homogeneous samples. development of video-rate imaging microscope using laurdan and its applications to lipid raft t. ohba, k.-i. muto, t. kiuchi, k. ohki department of physics, graduate school of science, aobaku, sendai, japan - , ohki@bio.phys.tohoku.ac.jp various biological functions are located on cell membranes, and the biomembranes maintain heterogeneity as microdomain even in its dynamic structure. existence of such domain was reported as phase-separated 'rafts' in a cell. and the bio-functions of membrane protein are affected by physical property of its surrounding lipid bilayers. laurdan is a useful fluorescence dye to monitor membrane fluidity, and we have developed an instrument to image spatial and temporal change in membrane fluidity by use of laurdan. in order to examine role of 'micro-domain' in biomembrane, we applied this imaging instrument to a giant liposome, cho cells and pc d cells. fluorescence microscope was equipped with a home-build dual-view optical unit. microscopic image of membranes stained with laurdan is separated into an image at nm and an image at nm by the combination of monochromatic filters and dichroic mirrors. the each image is focused on an image plane of a ccd camera side by side. and generalized polarization (g.p.) image is calculated from those images by personal computer according to the definition of g.p. the g.p. imaging at video rate was applied to a giant liposome of composed of dmpc and dmpe in order to observe phase separation. it was also examined that specific interaction of sphingomyelin and cholesterol in living cho cells and neuritis protrusion from raft region of pc d cells stimulated by neuron growth factor. current fluctuations in biological lipid membranes from human cell lines s. nuschele, a. wixforth, m. f. schneider university of augsburg, experimental physics , univer-sitätsstrasse , d - augsburg, germany lipid membranes can undergo phase transitions at physiological temperatures. not only do single lipid component membranes show melting behaviour but also biological lipid membranes from eukaryotes as well as from prokaryotes. performing calorimetric and monolayer studies we are able to detect phase transitions in extracted membranes from different human cell lines (e.g. keratinocytes and melanoma cells). close to and in the melting transition regime we measured distinct channel like current fluctuations. the opening times of these current fluctuations can be predicted based on the lateral compressibility measured from the monolayer isotherm and agree well with the experimental data [*]. the applied method of extraction excludes the presence of functional proteins in the membrane rendering the lipid bilayer as the source of the observed current fluctuations. the molecules are composed of single hydrophobic tail and two hydrophilic aldonamide-type groupings (gluconyl c -dga or lactobionyl c -dla) linked by the propylene chain at the nitrogen atom. the micellization processes of c -dga and c -dla were studied by means of itc. the critical micelle concentrations, the enthalpies (∆h m ) and the entropies (∆s m ) of micellization as well as the contributions of the headgroups to the gibbs free energies (∆g m (hy)) were calculated. qspr analysis was also used to predict cmc of studied compounds. the interactions of c -dga and c -dla with model membranes (dppc and dppc/chol. bilayers) were studied by means of dsc. using quantum computations some basic molecular properties were calculated. the conformational space was explored using molecular mechanics. obtained results were compared with those for analogical compounds with single head groups, e.g. with also synthesised by us n-alkanoyl-nmethyllactitolamines (c n mela) and common sugar-based surfactants c gluc and mega- . enfuvirtide (t- ) was the first hiv- fusion inhibitor peptide approved for clinical use. t- is an inhibitor still under evaluation. previous studies, based on tryptophan intrinsic fluorescence, showed that the peptides interact differentially with membrane model systems (luv) with different lipid compositions. studies with human blood cells were necessary to further establish the role of membranes on these peptides mode of action. an experimental strategy was applied based on the membrane dipole potential, as measured by the fluorescent probe di- -anepps. human erythrocytes and peripheral blood mononuclear cells (pbmc) were successfully labeled. for both systems, a fusion inhibitor concentration-dependent decrease on di- -anepps fluorescence excitation ratio (a measure of the spectral shift and dipole potential) was observed. the quantitative analysis of these variations indicated that t- has an approximately ten-fold higher affinity towards erythrocyte and pbmc, when compared with enfuvirtide. this is in agreement with the previously known adsorption of t- on cholesterol-rich membrane domains and with its higher partition constants. hiv associates with erythrocytes in vivo, which can constitute a route to deliver peptide to the viral membranes (also rich in cholesterol). lymphocyte membranes can concentrate and accelerate the drug interactions with its molecular target, gp in its exposed conformation. oxidation of low density lipoprotein (ldl) is known to be a key step in atherogenesis, leading to inflammation, proliferation and apoptosis of cells of the arterial wall. these effects are largely exerted by oxidatively fragmented phospholipids, which are highly exchangeable between cells, tissues and lipoproteins. in particular, pgpc has been identified in minimally modified ldl and has been reported to elicit a wide range of pathophysiological responses in vascular cells, e.g. the activation of apoptotic signaling pathways. we investigated here the behavior of the fluorescent oxidized pgpe-alexa compared to dhpe-bodipy in artificial supported lipid bilayers with different cholesterol contents. the two labeled lipids differ in the type of membrane insertion: while dhpe-bodipy is anchored to the membrane via two fatty acids, pgpe is incorporated with only one fatty acid. the second chain is an acyl fragment in sn- position, which represents the oxidation product of an unsaturated acyl chain. with increasing cholesterol content we observed a decrease in the diffusion coefficient for both lipids. interestingly, the diffusion of the oxidized lipid was reduced in a higher degree compared to that of the non-oxidized lipid. the calculated ratios of the diffusion constants of pgpe-alexa and dhpe-bodipy suggest a different type of interaction with cholesterol. membrane proteins account for a third of all proteins encoded for by the human genome, and play a vital role in a number of cellular processes. few membrane protein structures have been determined to date in comparison to soluble proteins. this discrepancy is due to experimental difficulties in preparing membrane protein samples for structural analysis. traditional techniques for studying membrane proteins by x-ray crystallography or solution nmr use detergent solubilised proteins which can differ from their native confirmations. solid-state nmr allows the study of transmembrane proteins in lipid bilayers, representing a more native like environment in which to obtain biologically relevant structural information. we have been working on the development of reliable methods for reconstitution of transmembrane peptide into liposomes using glycophorin a as a model tm protein. using reconstitution methods based on the removal of detergent by bio-beads, we have used electron microscopy to screen the ideal conditions for insertion of gpa into liposomes in preparation for mas nmr experiments. em has allowed us to identify conditions favourable for insertion of peptide into lipid vesicles and those that result in aggregation. in order to confirm the secondary structure and insertion of gpa into lipid vesicles, techniques such as cd, ocd, ftir and dls have been used to provide quantitative information in addition to the visual results from em. nonesterified fatty acids regulate a broad spectrum of metabolic activities and are involved in many physiological, pathological and/or pharmacological processes in living cells. they spontaneously transfer between donors and acceptors such as fatty acid binding proteins and lipid membranes. we focus on protein-lipid dispersions of human serum albumin (hsa) and sterically stabilized liposomes (ssl) composed of dppc and appropriate amount of peg: -dppe in which stearic acids (sa) are inserted either in the protein or in the ssl. exploiting the fact that hsa has a single tryptophan residue and that the intrinsic trp-fluorescence emission signal is quenced by the presence of sa, the kinetics of sa transport between hsa and peg: -grafted dppc membranes is studied by means of fluorescence. it is found that the transfer of sa between hsa and ssl is a first-order process and the kinetics of transfer depends on the type of donor and acceptor matrix, on the temperature (i.e., on the physical state of the lipid bilayers), and on the grafting density of the peg-lipids at the protein/lipid interface. indeed, in the absence of polymer-lipids, the rate of transfer increases with temperature in both directions of transfer and it is faster for the passage from dppc bilayers to hsa. the presence of polymer-lipids reduces the rate of transfer both in the mushroom and in the brush regime of the polymer-chains, especially for lipid membranes in the fluid phase. a. orth , w. römer , l. johannes , c. steinem institute for organic and biomolecular chemistry, university of goettingen, germany, laboratoire trafic et signalisation, institut curie, paris, france shiga toxin (stx) from shigella dysenteriae is an ab -class bacterial toxin. infections with stx lead to the haemolyticuraemic syndrome, which is known to be a major cause for renal failure at an early age. the interaction of the homopentameric b-subunit (stxb), which is responsible for binding and intracellular transport of the holotoxin, with its cellular receptor, the glycosphingolipid gb , is the first step for endocytosis of the toxin. one b-subunit can bind up to gb -molecules to form stxb-gb -clusters causing negative curvature of a membrane. the binding of stxb to giant unilamellar vesicles, composed of dopc, cholesterol and gb induces tubular membrane invaginations, which were also found in experiments with energy-depleted hela-cells. in recent studies, protein and lipid reorganization processes after attaching stxb to solid supported membranes, composed of dopc/sphingomyelin/cholesterol/gb have been investigated. the compaction of gb -molecules led to an additional stxb-gb -enriched phase, which was also observed in lipid monolayers at the air/water interface. in this study, the influence of stxb on model membranes will be investigated combining the advantages of free-standing lipid membranes with those of ssms. the impact of stxb on pore-suspending membranes will be followed by confocal laser scanning mi croscopy and atomic force microscopy. a. robaszkiewicz , c. spickett , p. sicinska , g. bartosz , m. soszynski department of molecular biophysics, university of lodz, lodz, poland, strathclyde institute of pharmacy and biomedical sciences, glasgow, u.k. hypochlorite generated in vivo under pathological conditions is a known oxidant, able to initiate lipid peroxidation process, which affects the stability of biological membranes. the aim of this study was the analysis of the products formed during the reaction of hypochlorite with phosphatidylcholines containing unsuturated fatty acid residues ( -stearoyl- -oleoyl-, -stearoyl- -linoleoyl-, stearoyl- -arachidonylphosphatidylcholine) and their effects on the human erythrocytes. using electrospray mass spectrometry we observed complete conversion of the lipids into chlorohydrins, which resulted in the decrease of the rotational correlation time and rotational motion freedom of liposomes estimated by epr using spin probes ( -and doxylstearic acid). unilamellar chlorohydrin liposomes had lower diffusion coefficient for calcein than liposomes made of parent lipids. flow cytometry demonstrated fast incorporation of uni-and multilamellar chlorohydrin liposomes labeled with nbd-pe into erythrocytes. this effect was accompanied by the formation of the erythrocyte subpopulations of higher volume, decrease of the rate of fluorescein diacetate hydrolysis, estimated by flow cytometry, and increase of affinity and maximal velocity of the membrane enzyme acetylcholinesterase. extensive bilayer perforation coupled with the phase transition region of an anionic phospholipid k. a. riske , l. q. amaral , m. t. lamy depto. biofisica, universidade federal de sao paulo, sao paulo, brazil, instituto de fisica, universidade de sao paulo, sao paulo, brazil at low ionic strength dimyristoylphosphatidylglycerol (dmpg) exhibits a broad phase transition region characterized by several superimposed calorimetric peaks. peculiar properties, such as sample transparency, are observed only in the transition region. we use differential scanning calorimetry, turbidity and optical microscopy to study the narrowing of the transition region with the increase of ionic strength. upon addition of salt, the temperature extension of the transition region is reduced and the number of calorimetric peaks decreases until a single cooperative event is observed in the presence of mm nacl. the transition region is always coupled with a decrease in turbidity, but a transparent region is detected within the melting process only in the presence of up to mm nacl. optical microscopy of giant vesicles shows that bilayers first rupture when the transition region is reached and subsequently lose optical contrast. fluorescence microscopy reveals a blurry image in the transparent region, suggesting a different lipid self-assembly. overall sample turbidity can be related to the bilayer optical contrast. our observations are discussed in terms of the bilayer being perforated along the transition region. in the transparent region the perforation is extensive and the bilayer completely loses the optical contrast. financial support: fapesp. label-free imaging of biological membranes using surface imaging techniques j. l. richens, k.-a. vere, p. o'shea cell biophysics group, university of nottingham, nottingham, uk surface plasmon resonance (spr) is a detection technique traditionally used for specific protein detection, which is now exploited routinely as a generic label-free sensor. spr responds to changes on a metal surface conditioned to sense the binding of analytes. thus, the composition of the external medium and metal surface will be fundamental to the signal output. here we use a model phospholipid membrane system to investigate the effect of altered buffer and surface compositions on spr signals. comparisons are undertaken between continuous gold surfaces and gold nanoparticles. we demonstrate that surface electrostatics and the salt composition and molarity of a buffer all have significant impacts on spr output. the association of respiratory syncytial virus matrix protein with membrane microdomains the association of the matrix protein from respiratory syncytical virus with membranes has been characterised by tensiometry, brewster angle microscopy, and atomic force microscopy following deposition of langmuir monolayers onto modified silica substrates. association of the protein with monolayers containing phosphocholines and cholesterol leads to the formation of materials with new properties that differ from those of either of the pure components. the behaviour of the protein in monolayers rich in cholesterol and sphingomyelin exhibits a significant concentration dependence. at low concentrations, the protein exhibits a simple partioning behaviour. at higher concentrations, lipids are extruded from the monolayer below a critical surface area. these findings are discussed in relation to the recently published structure of the protein (pnas, , , - ) , the documented formation of viral filaments during key stages of the infection cycle and the isolation of the protein from detergent-resistant membrane fractions. structure and dynamics of closed melting membranes v. m. rondelli , s. santorsola , p. brocca , e. del favero , l. cantù , m. zimbone dep. of chemistry, biochemistry and biotechnologies for medicine, university of milan, segrate, italy., dep. of physics, university of catania, catania, italy. we studied the dynamical and structural properties of large unilamellar vesicles (≈ nm luvs) of phospholipids (dmpc, dc pc and dmpc : dc pc = : molar mixture) in the temperature range around the chain-melting transition, ≈ • c wide, with . • c resolution and . • c accuracy. small-angle (saxs) and wide-angle x-ray scattering (waxs) measurements show that across the transition the vesicle behaves as an 'evolving membrane', passing through several different states, each of them being characterized by different proportions of coexisting fluid-and gelchains molecules. noteworthy, no kinetics has been detected. on the same samples, a unique and very sensitive laser light scattering technique allows to determine the characteristic times of thermally induced shape fluctuations, connected to the elastic properties of the membranes. results indicate a clear softening of the membranes in correspondence to the chain-melting transition, as indicated by a manifold increase of the corresponding fluctuation characteristic time. meanwhile the overall size of the vesicle is not sensibly changed. this softening is likely to be due to the presence of structural defects, eventually driving to local morphological modifications. thermodynamics characterization of isolated lung surfactant assembled as lamellar bodies e. x. rodriguez , r. alvarez , r. barrio , c. irles , a. ortega biochemistry depto., school of medicine, inst. of physics unam, nat. inst. of perinatology, mexico city, mexico lungs are a large extension of ∼ m of one layer cells, which is structured as compacted sacs called alveoli, where lung function takes place: the gas exchange. alveolar endotelium is formed by two kinds of cells: neumocyte type i (nti), which covers ∼ % of the alveolar surface where gas exchange occurs and, ntii where lung surfactant (ls) is produced. ls are a mixture that lay over a water film in the luminal surface of the alveoli and it is thought to decrease the surface tension to avoid alveolar collapse during expiration. ls are made of phospholipids and proteins, which are determinant for the structure of ls under particular conditions of pressure and temperature. ls inside the cell is packed in organelles called lamellar bodies (lb), and is released to the water/air interphase in other conformation. in the present study we isolate lb from pig's lung and study lb thermodynamic characteristics by microcalorimetry in the presence and in the absence of structural proteins. phase transition profile of lb with and without proteins is basically the same; while ls in the alveolar lumen have a higher transition temperature (tm). mayor changes in tm are observed between lb and ls from alveolar lumen. although ls lipid composition in and outside the cell is assumed to be the same, the ground for the differences in tm under these two conditions is unknown. m. rodrigues , g. rádis-baptista , b. g. de la torre , m. castanho , d. andreu , n. c. santos instituto de medicina molecular, portugal, pompeu fabra university, spain, federal university of cearà, brasil nucleolar-targeting peptides (nrtps) have been recently designed by structural dissection of crotamine, a toxin from the venom of a south america rattlesnake (radis-baptista et al. . j med chem : ) . at µm concentration, nrtps penetrate different cell types and exhibit exquisite nucleolar localization. the aim of this work is to decipher the molecular mechanism for the translocation of nrtps into cells. quantification of partition into membranes was carried out, based on intrinsic tyrosine fluorescence. the role of the bilayer phase, anionic lipids, reducing agents and peptide concentration on the extent and kinetics of partition were studied. both nrtp and nrtp exhibited high partition to popc (neutral) lipid vesicles (k p ≈ × ), which was enhanced by the anionic lipid popg for nrtp , but not nrtp . the peptides showed a decrease in partition for popc:cholesterol (liquid ordered state) or dppc (gel) membranes. depending on the lipid composition, the peptides either increased or decreased their quantum yield upon membrane insertion. quenching experiments with acrylamide showed no peptide aggregation in solution. once the translocation mechanism is fully understood we will test nrtps as carriers of relevant cellular cargos, evaluating their potential clinical application in drug delivery or gene therapy among other applications. the ingestion of trans fatty acids (tfa) formed during the partial hydrogenation of vegetable oils has been linked to a detrimental impact on health by an, as yet, unknown mechanism. we synthesized deuterated analogs of -elaidoyl- -stearoylphosphatidylcholine (t : - : pc) that contains a single "unnatural" trans double bond and -oleoyl- -stearoylphosphatidylcholine (c : - : pc) that contains a single "natural" cis double bond. solid state h nmr, complemented by molecular dynamics (md) simulations, was then employed to compare molecular organization in model membranes prepared from these isomeric molecules. analysis of spectra recorded as a function of temperature reveals a higher chain melting temperature for the trans isomer, indicating tighter molecular packing in the gel state. in the liquid crystalline, however, the difference between the trans and cis isomers is subtle. order as probed by the perdeuterated [ h ] : sn- chain, and corroborated by computer simulation, coincides within < %. only in the conformation of the double bond is an appreciable difference implied. thus, our results contradict the conventional view that tfa resemble saturated fatty acids, which is > % more ordered. (supported by acs, prf -ac .) photooxidation and lateral membrane diffusion of dipole molecules v. s. sokolov , e. a. sokolenko , a. a. lents , p. pohl a.n frumkin institute of physical chemistry and electrochemistry, russian academy of science, moscow, russia, institut fuer biophysik, johannes kepler universitaet linz, austria the photodynamic oxidation of phloridzin, of the styryl dye di- -anepps and of unsaturated lipids was monitored "online" by measuring the collapse of the dipole potential which has been introduced to the membrane by these molecules. their photodamage occurred at different rates when the target molecules and the singlet oxygen generating photosensitizer (phthalocyanin) were adsorbed to the same or to opposite sides of the planar lipid bilayer. the difference in the oxidation rates were attribute to singlet oxygen transport through lipid bilayer and therefore we were able to estimate the permeability of lipid bilayers to singlet oxygen. however, the apparent permeabilities derived from experiments with different targets were differ from each other. therefore, we tested the hypothesis that the lateral membrane diffusion of target molecules and oxidation product may have biased our analysis. in line with this anticipation we found that the apparent permeability is dependent on the size of the planar bilayer. the development of a new mathematical model, which takes the mobility of all reacting species in the aqueous and lipid environments into account, allowed estimation of the real membrane permeability to singlet oxygen. it appeared to be very close to that of oxygen in the ground state. a number of complex three-dimensional lyotropic liquid crystal phases are already known, such as the bicontinuous cubic phases, but so far only a single example has been found -a cubic phase of spacegroup fd m -of a structure based upon a complex close packing of inverse micelles ( ) . we now report the discovery ( ) of a novel lyotropic liquid crystal phase, of space group, p /mmc, whose structure is based upon a hexagonal close packing of identical quasi-spherical inverse micelles. the model membrane system consists of a hydrated mixture of dioleoylphosphatidylcholine, dioleoylglycerol, and cholesterol. this novel phase has a number of unique features which may render it useful for a range of applications. firstly, it is the only known self-assembled lyotropic phase whose structure consists of a periodic close packing of identical inverse micelles. secondly, it is stable in excess aqueous solution, which is very important for potential biological or biomedical applications. references ( ) v. luzzati, v., r. vargas, a. gulik, p. mariani, j.m. seddon, and e. rivas, biochemistry , - ( ) . dystrophin is a rod-shaped muscular subsarcolemal protein. its deficiency is one of the root causes of duchenne muscular dystrophy. dystrophin rod domain contains homologous repeats, where the sub-domain constituted by the repeats to (r - ) was reported to bind actin and membrane lipids. we analyzed the interaction of r - with lipid monolayers. to better understand the assembly mechanism of this protein with lipids, we studied its adsorption behavior at the air-liquid and lipid/liquid interface using ellipsometry, surface pressure, and atomic force microscopy (afm). using two different mixtures of phospholipids, ellipsometry and pressure surface data show that r - interacts with the lipid monolayers, but is inserted into the monolayer formed by dopc-dops while it lies below the monolayer of dopc-dope. this indicates that r - interacts more strongly with dopc-dops than with dopc-dope. afm images show that the pressure of lipid monolayer influences the organization of r - . when the lipid surface pressure is mn/m, r - forms a striking network, indicating a protein-protein interaction in addition to the protein-lipid interaction. this unique behavior of one part of the central domain of dystrophin may explain its key role in muscle cell. studies on polyphenol extracts from helichrysum l., fagopyrum mill., crataegus l. and hypericum l. were performed in order to find if they may be applied as a natural free radical scavengers protecting biological membranes against peroxidation. ghosts erythrocytes were used in the experiments. they were suspended in tris-edta solution, ph , , then.irradiated with a bactericidal lamp without (control) or with a proper amount of the extracts studied. the product of lipid peroxidation was malonic dialdehyde (ma). the colour reaction of ma with thiobarbituric acid (tba) enables to determine the concentration of ma spectrophotometrically. it was found that peroxidation increased with the irradiation time. however, it significantly decreased when concentrations of poliphenols increased. the best antioxidtive property was found for hypericum l. the antioxidative efficiency sequence of the plant extracts studied was the following: hypericum l. > crataegus l. > fagopyrum mill. > helichrysum l. the results obtained indicate that polyphenol extracts exhibit excelent antioxidative properties that make them good free radical scavengers for efficient protection of biological membranes. this work was sponsored by ministry of science and education, scientific project no. n n . interaction of cationic porphyrins with neutral and negatively charged liposomes i. voszka , g. corradi , p. maillard , h.-j. steinhoff , g. csík institute of biophysics and radiation biology, semmelweis university budapest, hungary, research institute for solid state physics and optics, hungarian academy of sciences, budapest, hungary, institute curie, section de biologie, orsay, france, fachbereich physik, universitat osnabrück, germany porphyrin derivatives are used in photodynamic therapy of tumors. the knowledge of the photosensitizer location within the cell is important. the effect of porphyrin derivatives containing cationic side groups was examined on neutral and negatively charged liposomes by esr. the esr signal was first examined as a function of temperature and porphyrin concentration in the dark. a significant change related to the appearance of quasi free spin labels was obtained for spin probes at the th carbon atom and was more expressed for the asymmetrical derivative. illuminating the samples the esr amplitude decreased for all positions of the spin probe but to different extent. the effect was more expressed in case of the symmetrical derivative, especially for label positions and . for the asymmetrical derivative the effect changed from moderate to weak from the th to the th position. this indicates that the asymmetrical derivative is incorporated nearer to the lipid head groups while the symmetrical one may be located deeper in the membrane. under oxygen-free conditions both derivatives showed weaker but still pronounced effects.. single channel recording of α-hemolysin in nanopore-spanning tethered bilayer lipid membranes n. t. thet , i. pfeiffer , w. knoll , i. köper max planck institute for polymer research (mpi-p), ackermannweg , mainz, austrian research centers gmbh -arc, tech gate vienna, vienna, austria artificial lipid bilayer membranes mimic biological cell membranes in many aspects and can be used to study functional processes such as ion channeling, signal transducing, transport of nutrients etc. as most of the functions of a cell are accomplished by membrane proteins, research has been ongoing in studying and characterization of membrane proteins embedded in model lipid bilayer membranes. black lipid membranes (blms) were studied for decades but their potential for practical applications is hindered, mainly due to their lack of stability and limited lifespan. recently, tethered bilayer lipid membranes (tblms) proved to have long life span of up to several months without significant changes in membrane resistance and capacitance. in order to combine advantages of blms and tblms, we have designed a membrane system which is freely spanned across a single nanopore in a silicon nitride membrane. this system not only mimics cell membranes, but also it allows control over the chemical composition of buffer with unlimited ionic reservoirs on both sides of the membrane. this freestanding tblm maintains structural stability and lifetime of up to hours without significant decrease in its structural integrity and electrical sealing. for the validation of the tblm formation, we have inserted well known α-hl pores. we are able to control the amount of α-hl pores insertion and measure single channel ion transport across the tblm by α-hl pores using a capacitor feedback amplifier. the language of shape: biological reactions are dramatically affected by the shape of lipid membrane d. stamou university of copenhagen, copenhagen, denmark a plethora of biological process are taking place on the surface of lipid membranes. as a rule membranes in vivo are curved in a variety of complex geometries. here i will present a quantitave study on the influence of membrane curvature on protein-membrane and membrane-membrane interactions. to gain systematic access to a continuum of membrane curvatures we immobilized liposomes on a surface at dilute densities. using confocal fluorescence microscopy we imaged single liposomes of different size, and therefore different curvature, and monitored their interaction with a binding partner (proteins or other liposomes). i will discuss unpublished data on two important classes of biomolecular interactions that exhibited dramatic curvature dependence: a) snare-mediated docking and fusion b) anchoring of peripheral proteins. the following references provide partial information on the single-liposome assay: a. zettergren, c. gudmundsson, t. nylander, e. sparr physical chemistry , lund university, lund, sweden there is accumulating evidence of substantial amounts of phospholipids in the cell nuclei , although the function of these lipids is still not fully understood. it has been shown that the chromatin complex composed of dna, rna and proteins also includes phospholipids, and that rna colocalize with these . although the rna-phospholipid interactions may have important implications to biological function, in gene therapy and in medicine, very little work has been dedicated to the characterization of rna interaction with phospholipids. the objective of this work is to investigate the adsorption behavior of short single stranded bases long rna (ssrna ) molecules (similar to mirna) to lipid monolayers at the air-water interface as well as to study how the presence of rna affect the domain formation in the monolayers using fluorescence microscopy. monolayer studies have shown adsorption of ssrna to monolayers consisting of zwitterionic dppc as well as to monolayers consisting of cationic dodab. the adsorption behavior of these very short nucleic acids differ significantly from the adsorption process for longer nucleic acids as for example a base pairs long ds dna (dsdna ) which has been used as a reference system . the presence of ssrna significantly changes the compression isotherm of both dppc and dodab monolayers. plant defensins are cysteine-rich antimicrobial peptides found in various plant species. they share a common threedimensional structure, stabilized by eight disulphide-linked cysteines consisting of three antiparallel β-strands and one α-helix. most plant defensins show antifungal activity with no effect on mammalian and plant cells. expression of these peptides in plant tissue is induced by pathogen infection. the mechanism of defensin action is based on membrane permeabilization. this occurs through an interaction with high affinity binding sites on fungal membranes, resulting in alteration of membrane potential. the genes encoding for a peach ppdfn and a grape vvamp defensins were expressed in e. coli and purified to homogeneity. they were tested for antimicrobial activity against some fungi and showed to have an inhibitory effect on the spore germination. biophysical analysis showed that defensins were able to interact with artificial membranes. binding of defensins to membranes was dependent on lipid composition, increasing with the sphingolipids content. interaction between peptides and sphingolipids could lead to insertion of the defensins into the membrane resulting in its destabilization. extracts of the plant perilla frutescens have many uses in the asian kitchen, e.g. as a popular garnish used in sushi. the plant is also employed in eastern traditional medicine to treat a variety of ailments including colds, food allergy and depression. two of the main constituents of the plant are limonene and perilla aldehyde. by bio-oxidation, these molecules can be converted to perillic alcohol and perillic acid. these cyclic terpenes possess antibacterial and anticarcinogenic properties. the modes of action for these compounds are at present not understood, but their remarkably diverse pharmacological properties suggest that they might target the phospholipid matrix of the cellular lipid membrane. indeed, the cyclic terpenes can bind to, and alter the physiochemical properties of the lipid bilayer of the membrane, the effects of which can cascade down to several essential cellular processes. here, we use molecular dynamics (md) simulations to investigate the effect of limonene and its derivatives on the properties of lipid bilayers including the changes in acyl chain order parameters, bilayer thickness and the area per lipid. md can afford molecular-scale dynamic information, often not easily accessible from experimental measurements. this information can be used to interpret existing experimental data obtained by e.g. isothermal titration calorimetry, electron paramagnetic spectroscopy and differential scanning calorimetry. catalysis in the membrane: interfacial mechanism of phospholipase a h. p. wacklin , r. k. thomas institut laue langevin, grenoble, france, physical and theoretical chemistry laboratory, oxford university, u.k. phospholipase a cleaves the sn- acyl chains of membrane phospholipids and performs a range of physiological functions. one of the least well understood aspects of the its mechanism is how its activity is regulated by the interaction with the substrate membrane. we have used neutron reflection to monitor changes in membrane structure during lipid hydrolysis [ , ] . the penetration depth of pla depends on lipid packing and increases during the lag phase of porcine pancreatic pla . by using a selectively deuterated lipid substrate, d -popc, we determined the relative membrane-water partitioning of the lipid products in-situ. the lyso-lipid product partitions into the solution phase, while fatty acid accumulates in the membrane and increases the affinity of pla [ ] . pla is inhibited at ph , which is consistent with protonation of the catalytic histidine. however, irrespective of ph, pla is fully activated by me-betacyclodextrin, which facilitates the release of the lyso-lipid from the enzyme-substrate complex. me-beta-cyclodextrin does not interact directly with the membrane surface or the substrate lipids, indicating that product release occurs outside the immediate membrane-water interface. diffraction limits resolution in optical microscopy, but many interesting biological problems occur on shorter (molecular) length scales. recently, methods to circumvent the diffraction limit have been presented. fluorescence photoactivation localization microscopy (fpalm) uses activation of many small subsets of photoactivatable or photoswitchable fluorescent probes (pafps) to generate images with effective resolution in the tens of nanometers. pafp molecules are photoactivated, imaged, localized, and photobleached in small numbers. the process is repeated for many subsets to build up data on thousands to many hundreds of thousands of molecules. the positions of all localized molecules are used to construct an image of the sample with resolution limited not by diffraction, but by the localization precision and molecular density. results will be shown from a variety of biological systems, including live and fixed cells expressing a variety of pafp-tagged proteins. bi-plane fpalm can image in three dimensions with demonstrated resolution of nm x nm x nm. polarization fpalm can image both molecular positions and anisotropies simultaneously with lateral resolution of ∼ - nm. using these powerful capabilities, many potentially interesting biological problems can be addressed. binding of the hiv- ncp on oligonucleotides at the single-molecule level j. godet, p. didier, a. jouonang, y. arntz, y. mély laboratory of biophotonics and pharmacology, umr cnrs , university of strasbourg, france the nucleocapsid protein (ncp ) of the hiv- is a small basic protein which plays key functions in the viral life cycle. the activity of ncp mainly rely on its potent rna-and dna-chaperone activities that direct the rearrangement of numerous nucleic acid molecules into their most stable conformation. two main features of nc's chaperone activity are its abilities to aggregate and destabilize nucleic acids. in addition, the rapid kinetics of ncp interaction with nucleic acids was recently proposed as another major component of nc's chaperone function based on the apparent correlation between an indirect measurement of the nucleic acid dissociation kinetics of ncp and its overall chaperone activity. but so far, no direct measurement of the on/off rates of ncp binding to oligonucleotides was performed. in the present work, we realized single molecule fluorescence resonance energy transfer (smfret) measurements to probe the transient interactions of a tmr-labelled ncp with a short cy -labelled dna oligonucleotide confined into nanovesicles. after confirming the efficiency of the ncp /odn complex encapsulation into the nanovesicles by fcs, the vesicles were tethered to the surface for immobilization. integrity of the entrapping vesicles attached on the surface was confirmed by afm. finally, the association and dissociation constants retrieved from these smfret measurements were discussed in the context of the dna-chaperoning activity of the protein. high-resolution spatiotemporal organization of the integrin lfa- m. f. garcia-parajo , t. s. van zanten , g.-j. bakker , r. diez-ahedo , a. cambi , c. g. figdor bionanophotonics group; ibec, barcelona, spain, tumour immunology dept; ncmls, nijmegen, the netherlands lfa- is a leukocyte-specific integrin involved in different steps of the immune response. on monocytes, lfa- plays a key role in the regulation of monocyte-endothelial interaction during rolling, arrest and extravasation into the underlying tissue. i n-vivo experiments showed that blood borne lymphocytes can 'switch' within seconds from rolling to arrest. furthermore, tem observations of pro-active, ligandindependent nanoclusters confirmed that affinity and clustering are complementary processes required in adhesion. yet, the mechanisms leading to fast-switching remain obscure. in our group, we used a combination of single molecule fluorescence techniques to study the spatiotemporal organization of lfa- on monocytes. we performed optical nanoimaging of lfa- nanoclusters in relation to membrane rafts with a resolution of nm and accuracy of ∼ nm. in quiescent cells, lfa- nanoclusters do not associate with membrane rafts and diffuse freely on the membrane. binding of the integrin to its ligand icam- induces the formation of microclusters that further associate with rafts and exhibit reduced mobility, consistent with cytoskeleton interactions. our work highlights the markedly different spatiotemporal organization of lfa- that might explain its concerted action to form larger and stable platforms on the cell surface required for rapid and effective cell adhesion. c. ciobanasu, u. kubitscheck rheinische friedrich-wilhelms-universität bonn, germany cell penetrating peptides like the hiv tat peptide have the property to rapidly translocate the cell membranes and the capability to deliver a wide range of cargoes. the mechanism of the membrane translocation is still under investigation and object of considerable controversy. we applied and single-molecule and confocal laser scanning microscopy (lsm) to study peptide-membrane interactions. electron-multiplying ccd cameras yield images of single fluorescent molecules with a time resolution in the range of a few milliseconds only, which allows the tracking of fluorescently labelled peptides and lipids at bio-interfaces in realtime with a localization precision of a few nanometers. we formed giant unilamellar vesicles (guvs) from different lipid mixtures and examined their interaction with fluorescently labeled tat peptides. we found that the passive peptide internalization process depends on lipid composition, charge of the lipid bilayer, and the ionic properties of the medium. a translocation of cationic tat peptides was observed in membranes containing at least mol% of lipids with a phosphatidyl ethanolamine or a high mol fraction of the phosphatidyl serine head group. in salt-free solution tat efficiently bound to guvs, however, in a physiological nacl solution tat binding was completely abrogated, but the peptides efficiently equilibrated across the guv membrane. new approaches to measure interactions in the live cell plasma membrane g. j. schütz biophysics institute, johannes kepler university linz, austria in my lecture, i will show examples how to obtain insights into the organization of the cellular nanocosm by single molecule experiments. our primary goal is an understanding of the role of such structures for immune recognition. brightness and single molecule colocalization analysis allows us to study stable or transient molecular associations in vivo. in particular, i will present results on the interaction between antigen-loaded mhc and the t cell receptor directly in the interface region of a t cell with a surrogate antigenpresenting cell. in addition, we developed a method for in vivo micropatterning of plasma membrane proteins to measure molecular interactions. the method allows identifying and characterizing interactions between an arbitrary fluorescence labeled protein ("prey") and a membrane protein ("bait") directly in living cells. cells transfected with a fluorescent fusion protein of the prey are plated on micropatterned surfaces functionalized with specific antibodies to the extracellular domain of the bait; the fluorescence copatterning is used as readout for the interaction. we applied this tool for the study of the interaction between cd -the major coreceptor for t cell activation -and lck, an important tyrosine kinase in early t cell signaling. in addition to the well-known zinc-clasp structure, we found strong contributions of lck membrane anchorage for the binding of the two proteins. developing a fluorescent redox sensor for monitoring metal-ion mediated catalysis in biosystems a. rybina , a. kiel , b. thaler , a. sprödefeld , r. krämer , d. p. herten bioquant and, department of inorganic chemistry, heidelberg university, germany a fluorescent redox sensor is an electron photo-switching device that can be used for the characterization of the redox state of a given environment. it combines a fluorescent fragment with a redox-active unit that senses the media by a redox reaction and controls the light emitting properties of the fluorophore. such reversible sensor can help to examine the electrochemical state and changes in biological systems during biochemical processes in real time. recently a new fluorescent molecular sensor with a redox-active hydroquinoneunit covalently linked to fluorophore rhodamine b was developed. (kierat r.m. et al., bioorg. med. chem. lett., -accepted) . the reduced hydroquinone-form of the sensor is fluorescent while its oxidation to benzoquinone-derivative leads to a significant decrease of the fluorescence. although the above method shows great promise for applications in biological systems, the exact mechanism of this process is not fully understood yet. we use fluorescence spectroscopy to investigate oxidation reactions on the ensemble and single-molecule level and study kinetic rates. the proposed strategy is to use cu (ii) complex as oxidation mediator immobilized on surface via dna linker to examine the oxidation mechanism. fluorescently labeled atp as a probe of the outer mitochondrial membrane barrier: role of vdac fluorescence correlation spectroscopy (fcs) was applied for studying the distribution of fluorescently labeled atp (bodipy-atp) in isolated mitochondria. the setup and peak intensity analysis (pia) was described in our recent paper (perevoshchikova et al. biochim.biophys.acta : . the binding of bodipy-atp to mitochondria was maximal in the non-energized state, whereas the addition of succinate (respiratoty substrate) or atractyloside (adenine nucleotide translocase inhibitor) led to a decrease in the binding. nadh reduced the fcs signal from bodipy-atp added to non-energized mitochondria more than nad+ did under the same conditions suggesting the control of nucleotide transport through voltage-dependent anion channel (vdac) residing in the outer membrane. konig's polyanion also decreased the bodipy-atp binding to mitochondria with the effect being reduced by alamethicin or digitonin. control experiments showed that bodipy-atp did not bind to liposomes showing minor role of unspecific binding. it was suggested that bodipy-atp in combination with fcs can be used to monitor the functional state of mitochondrial vdac which is considered to be a principal regulator of mitochondrial function. fluorescence correlation spectroscopy studies of lysozyme partition to phospholipid vesicles a. m. melo , a. coutinho , m. prieto cqfm and in, ist, - lisboa, portugal, dqb, fcul, - , lisboa, portugal binding to membrane lipids has been increasingly recognized as an important step in the aggregation and cytotoxicity of several amyloidogenic proteins [ ] . in addition, it has been recently reported that membranes containing negatively-charged phospholipids can also trigger rapid amyloid-like fiber formation by a variety of several nonamyloidogenic proteins, such as cytochrome c and lysozyme [ ] . our study aims to elucidate the factors that govern the formation of these lipid-protein complexes. given the importance of electrostatic interactions between the proteins and the acidic phospholipids in the putative membrane-induced protein misfolding step, it is essential to first characterize quantitatively the protein partition behavior towards liposomes prepared with variable anionic lipid content. in this study, lysozyme was chosen as a model protein and fluorescence correlation spectroscopy (fcs) was used to monitor its binding to liposomes after its conjugation to alexa fluor . most organic dyes labelling techniques produce a mixture of populations of molecules labelled with a different number of fluorophores. the influence of this poli-dispersity of labelled molecules on the protein partition behaviour will be explored, namely the ability of the fcs technique to detect the production of non-competent membrane-binding species. t. wohland , p. liu , x. shi , y. h. foo , t. sudhaharan , s.-w. chong , v. korzh , s. ahmed chemistry dept., singapore nat. univ., medical biology inst., singapore, molecular & cell biology inst., singapore biomolecular interactions have been measured mostly under in vitro conditions because of higher accuracy and ease of measurement. however, it has become clear that the cellular environment has an important influence on these interactions. it was therefore necessary to develop new tools to allow the measurement of interactions in cells and organisms. recently, we have developed a modality of fluorescence cross-correlation spectroscopy (fccs) called single wavelength fccs (sw-fccs), which uses one-photon excitation to excite two fluorophores with overlapping absorption but separable emission spectra. sw-fccs has been used to determine e.g. dimer fractions of proteins in live cells. here we aim to extend the use of sw-fccs to cells and organisms. in the first part we determine the dissociation constants of a small rho-gtpase (cdc ) with an effector domain (crib) and two effectors (n-wasp or irsp ). in the second part we measure the binding between cdc and a scaffolding protein (iqgap ) in dependence of their expression levels in cho cells and in live zebrafish embryos. by using gfp/mrfp fusion proteins we can excite both fluorophores at nm and detect them separately in two different wavelength channels. we quantitatively determine the dissociation constants and compare their differences in vitro, in cells, and in embryos. these experiments demonstrate that sw-fccs is a powerful biophysical tool for the quantitation of biomolecular interactions in cells and organisms. addressing plasma membrane structure at the nanoscopic length-scale s. wieser, m. axmann, g. j. schütz biophysics institute, johannes kepler university linz, altenbergerstr. , a- linz, austria there is increasing interest in a detailed understanding of the structure and dynamics of the cellular plasma membrane, primarily based on recognizing its essential role for controlling cellular signaling processes. we employed single molecule fluorescence microscopy to study diffusion of cd , a gpi-anchored protein, in the plasma membrane of living t cells at sub-wavelength resolution, both on the cell body and on tunneling nanotubules connecting cells. the lateral motion of this single fluorescence labeled molecule was imaged on a millisecond time scale. within the experimental errors, no indications for confined diffusion for cd on the cell body in t cells have been found. furthermore by separating longitudinal and transversal mobility, we found isotropic diffusion behavior on the surface of tunneling nanotubules. in both studies we analyzed the mean square displacement as a function of the time-lag and the distribution of displacement steps. however, a closed analytical theory for these analysis is only available for the simplest models. to address a suspected diffusion process we reasoned that a full analytical description may not be required; it may well be sufficient to compare the experimental data with monte carlo simulations of the process. we demonstrated the working principle for this simulation based analysis for free diffusion, hop diffusion and transient binding of the tracer molecule to slowly moving receptors. n. chakroun , f. fraternali , m. malfois , h. rezaei , c. a. dreiss king's college london, u.k., diamond light source, didcot, oxfordshire, u.k., inra, jouy-en-josas, france prion(prp) diseases are fatal neurodegenerative diseases affecting mammals including human and sheep.they are characterized by the accumulation of extracellular βrich fibrillar deposits of a structurally modified form (prp sc ) of the cellular prp c .despite the increasing interest for prp diseases,the mechanism of prp c /prp sc conversion is still unknown.studies on prp diseases suggest that neurotoxicity arises from small pre-fibrillar oligomers.we have used a range of biophysical techniques combined with molecular dynamics simulations (md) to resolve the oligomerization pathways of sheep prp (sprp).we have shown that under well established conditions, sprp oligomerizes into three oligomers, which form in parallel.in addition, we have now identified the minimal region of sprp leading to the same oligomerization profile of the entire sprp,namely h h .low resolution shapes of sprp, h h and resulting oligomers have been determined by small-angle x-ray scattering.time-resolved studies have been used to follow the oligomerization of sprp and h h monomers into the oligomers.the conversion of sprp sc at the molecular scale was studied by md.simulations of the h h region recreating experimental conditions revealed a complete unfolding of h helix followed by h helix.these crucial steps are followed by the formation of β-sheet structures leading to a stable βrich double hairpin structure. single-molecule force spectroscopy investigation of the conformational equilibria of alphasynuclein m. brucale , a. rampioni , m. sandal , i. tessari , l. tosatto , l. bubacco , b. samorì istituto di biochimica g.moruzzi, università di bologna (italy), dipartimento di biologia, università di padova (italy) alpha-synuclein (asyn) is an abundant intrinsically disordered protein (idp) primarily located at presynaptic terminals. mutations in the gene encoding asyn have been linked to early-onset parkinson's disease (pd). by means of single molecule force spectroscopy (smfs) experiment, we show how the conformational equilibrium of monomeric wild type (wt) asyn shifts toward more compact structures in several unrelated conditions linked to pd pathogenicity [ ] . the conformational heterogeneity of pathological alpha-syn mutants a p, a t and e k has also been characterized, revealing marked differences in the conformational behaviors of the mutants with respect to wt asyn [ ] . all the mutants show a distinctively higher propensity, with respect to wt, to acquire a monomeric compact conformation that is compatible with the acquiring of beta structure. the same smfs experimental methodology is then used to characterize the conformational behavior of wt and mutant asyn in a variety of conditions, in an attempt to gain insight about the multiple and contrasting parameters controlling the equilibrium. in vitro protein folding studies using chemical denaturants have contributed tremendously to our understanding of the folding thermodynamics and kinetics of water-soluble proteins. this is not the case for integral membrane proteins, which constitute about one third of all eukaryotic proteins and more than half of all validated and potential drug targets. fully reversible denaturant-induced unfolding remains limited to a few β-barrel porins, whereas the much larger and more relevant class of α-helical membrane proteins has thus far evaded this approach. we report here the first example of an α-helical membrane protein that can be unfolded completely and reversibly by a chemical denaturant: mistic, a -residue protein from bacillus subtilis [ ] , dissociates from detergent micelles or lipid vesicles and assumes an unfolded monomeric state on titration with urea. using spectroscopic and microcalorimetric techniques, we exploited this unique property to provide (i) a quantitative comparison of membrane protein stability in different membrane-mimetic systems; (ii) an experimental test of controversial predictions [ ] regarding the folding core of mistic; and (iii) a convenient setup to study the spontaneous, translocon-independent membrane insertion of this unusual membrane protein. the mechanical functioning of biological tissues is important from many viewpoints such as diseases, clothing and even food. the protein collagen makes up the greater part of these tissues, and is remarkable for its many uses in the body, however there are at least two other major components. one of the most interesting properties of these tissues is their non-linear behavior under stress. this behavior is essential to prevent a catastrophic failure such as an aneurysm. at least three major theories have been proposed within the past few years to explain this behavior, but have been impossible to verify. in order to determine a correct description of the mechanical structure of the tissue we have been using cutting edge technological solutions to address the single molecules within the extra cellular matrix. this technique combines optical methods with single molecule force spectroscopy, allowing stiffness measurements over the nanoscale as well as determining the individual protein tensions within the extra cellular matrix. the results show that this method can be used to determine the network properties even in the complicated aortic wall enabling better understanding of disease states, which in this case include marfan's syndrome and ascending aortic aneurysms. beta amyloid peptide abetapy - shows a faster aggregation kinetics than abeta - c. d'arrigo , m. tabaton , a. perico institute for macromolecular studies, national research council, genova, italy, department of neurosciences, university of genova, genova, italy we test directly the differences in the aggregation kinetics of three important beta amyloid peptides, the full-length abeta - and the two n-terminal truncated and pyroglutamil modified abetapy - and abetapy - , found in different relative concentrations in the brains in normal aging and in alzheimer disease. we find by following the cd signal and the tht fluorescence of the solution in phosphate buffer, a substantial faster aggregation kinetics for abetapy - . this behavior is due to the particular sequence of this peptide which is also responsible of the specific oligomeric aggregation states, found by tem, during the fibrillization process which are very different from those of abeta - , more prone to fibril formation. in addition abetapy - is found here to have an inhibitory effect on abeta - fibrillogenesis, coherently with its known greater infective power. this is an indication of the important role of this peptide in the aggregation process of beta-peptides in alzheimer disease. the puzzle of the anomalously long denaturation kinetics of green fluorescent protein (gfp) mutants still is largely unveiled. in this study we have followed the effect of mutation h g on the stability of gfpmut (mut g) in the presence of guanidinium hydrochloride (gdnhcl). different techniques of fluorescence spectroscopy have been employed in order to obtain information concerning the unfolding event: time resolved fluorescence, fluorescence correlation spectroscopy (fcs), and fluorescence anisotropy. the substitution of the histidine with glycine affects protein stability versus ph: in particular mut g kinetics is not ph dependent and at basic ph values the protein is less stable. the fluorescence properties (quantum yield, lifetime) and the rotational correlation time are unchanged during the unfolding dynamics, while the number of fluorescent proteins decreases exponentially. an extrinsic probe, bound to cysteine , has been employed in order to gain more insights on the unfolding process, monitoring the stability of a different region of the protein. in particular, it has been found that a softer region is present around cysteine in both gfp variants, showing that the unfolding process does not follow a simple two step mechanism. recently, negatively-charged membranes were reported to catalyze amyloid fiber formation by amyloidogenic peptides/proteins and also to induce formation of "amyloidlike" fibrils by nonamyloidogenic proteins. here, we used an approach which combines steady-state and time-resolved fluorescence measurements to obtain structural information about these supramolecular assemblies and to gain insights about the factors that control their formation. by exploring a wide range of lipid concentrations, the interaction of alexa -lysozyme with phosphatidylserine-containing membranes was found to be a complex multi-step process, critically dependent upon the protein-to-lipid molar ratio (p/l) used. upon increasing the total lipid concentration in solution, there was a balance between an increased overall protein binding to the lipid vesicles and a progressive protein dilution on the membrane surface. as the surface potential of the vesicles decreased upon increasing the protein interfacial coverage of the liposomes, the protein binding mode was found to switch from a peripheral binding of lysozyme to the anionic headgroups (at low to intermediate p/l) to a partial insertion of the basic protein into the hydrophobic core of the membrane (at a high p/l). it is hypothesized that disruption of the protein tertiary structure might be a stepwise process beginning with loosening of the structure caused by its deeper insertion in the membrane bilayer. unexpected scaling laws in the mechanical unfolding of single protein molecules m. clusel, e. i. corwin, h. lannon, j. brujic center for soft matter research, physics department, new york university, new york, ny, usa it is a question of fundamental importance to understand the response of proteins to a stretching force, particularly in the case of mechanically active proteins, such as those in muscle fibers. we aim to understand how the structure and topology of a protein affect its resilience to external forces and presumably its function. owing to recent advances in single molecule force-clamp spectroscopy using the atomic force microscope (afm), we are now able to probe the structure and dynamics of single proteins under a constant stretching force by measuring their end-to-end length over time. the probability distribution of unfolding times at a given force allows us to estimate the strength of the protein in terms of a characteristic energy barrier, while the shape of the distribution provides a window into the microscopic mechanism by which the protein breaks apart. here we show a novel scaling of the unfolding kinetics with the stretching force, which deviates significantly from the currently accepted bell model. instead, we propose a physical picture for forced unfolding that is analogous to the mechanics of fracture. v. garcía-gonzález, j. mas-oliva instituto de fisiología celular. universidad nacional autónoma de méxico. méxico, d.f. méxico. studies focused on the thermodynamic and kinetic analysis have demonstrated that transfer of neutral lipids such as cholesterol esters through an aqueous phase is a highly costly biophysical event. therefore, nature has developed a series of lipid transfer proteins such as the cholesterylester transfer protein (cetp) designed to efficiently lower the energy barrier for transfer of cholesterol-esters between lipoproteins through an aqueous environment. employing circular dichroism we evaluated the secondary structure stability of a small peptide derived from the carboxy-end of cetp (helix y ) in a wide range of ph's. the percentage of α-helix is diminished only at extreme temperatures and acidic ph's in a reversible way. we report that while a mixture of phosphatidylcholine/cholesteryl-ester forms large aggregated particles independently of ph, inclusion of helix y to the mixtures close to neutral ph's allows the formation of small micellar-like structures confirmed by dynamic light scattering and electron microscopy. these results suggest that helix y when close to physiological ph values presents the ability to organize a micellar structure around itself. this type of organization allows the process to dramatically lower the energetic barrier for lipid transfer through aqueous media, phenomenon directly related with the facilitation of lipid transfer between lipoproteins. mimicking metastasis by a novel microfluidic approach there is increasing evidence that cancer metastasis shares commonalities with thrombosis. the von-willebrand-factor (vwf), a mechanical active blood clotting protein appears to be a particular potent candidate to bridge the gap between clotting and cancer extravasation. modeling the crucial physiological conditions of the blood circulatory system, for an in situ study of blood clotting-metastasis connections is not only, absolutely necessary, but also a challenging task. here, we present acoustically driven flow as a novel microfluidics method for mimicking the blood flow. this method enjoys very beneficial advantages of possibility of handling very little volumes of fluids, together with freedom to model most of the geometries present in our microcirculatory system. one technologically challenging, yet physiologically important factor, is the hydrodynamic condition in a bifurcated vessel, where complex shear profiles arise. we present a model to mimic these conditions and discuss the impact of hydrodynamics on vwf mediated cancer cell adhesion in bifurcated vessels of our microcirculatory system. protein structural changes occurring in flows stresses inherent to viscous fluid flow have previously been associated with protein unfolding, although structural changes have not been well documented as a function of relevant hydrodynamic parameters. we have used raman spectroscopy to monitor the structure of various protein solutions in situ for multiple flow scenarios within a concentric cylinder fluidic device ( ) . the flows, which ranged from circular couette to wavy taylor-couette flow, were characterised experimentally using particle image velocimetry. several proteins were observed to change conformation when exposed to these flows, although the nature of these changes was protein specific. shearing hen egg white lysozyme in water altered the protein backbone structure, while similar shear rates in a % glycerol, % water solution affected the solvent exposure of the side chain residues near the exterior of the α-domain. the solventdependent response may be due to the flow topology, viscous stress, or the surface hydration properties. comparison of spectra acquired at different time points, including before and after flow, confirmed that the observed changes are reversible and independent of fluid stress exposure time. the scripps research institute, la jolla, ca, usa. intrinsically disordered proteins are increasingly found to play major roles in cell biology and disease. we are utilizing single-molecule fluorescence methods to probe these complex and highly dynamic molecules, allowing more direct measurements of structural distributions and dynamics, while avoiding loss of information due to ensemble averaging. in one example, we investigated the structural dynamics of sup -nm, whose regulatable amyloid formation is believed to have functional significance in yeast. using a combination of single-molecule fret as a molecular ruler, coincidence to interrogate intermolecular interactions, and correlation analysis to probe conformational dynamics, we showed that the monomeric protein populates an ensemble of compact and rapidly interconverting conformations. a particularly interesting feature of intrinsically disordered proteins is that they are relatively unstructured in isolation, but can gain stable structure by interaction with binding partners. in this context, we used single-molecule fluorescence to characterize the complex folding pathway for the parkinson's-related idp alpha-synuclein induced by binding to a lipid-mimic. this combined single-molecule fluorescence methodology provides a powerful approach for detailed studies of the coupling of folding and dynamics with interactions and biology of this important class of proteins. m. ito , j. johansson , r. stromberg , l. nilsson department of biosciences and nutrirtion, karolinska institutet, huddinge, sweden, department of anatomy, physiology and biochemistry, swedish university of agricultural sciences, the biomedical centre, uppsala, sweden amyloid β-peptide (aβ) is a - amino acid polypeptide and known to aggregate and form insoluble amyloid fibril, which is regarded as a primary cause of alzheimer's disease (ad). the discovery of practical and effective treatments and drugs for ad has been waited eagerly. in a recent experimental study, it was suggested that stabilization of the helical conformation of the aβ middle region, which strongly favors collapsed coil formation in the extracellular environment, would reduce the aβ fibril formation. based on the experimental evidence, inhibition of the unfolding of the aβ α-helix can be a forceful strategy to repress the aβ fibril formation resulting in prevention of ad. however, the detailed mechanism of the unfolding of the aβ α-helix has remained unclear, because the x-ray or the nmr structure of the aβ α-helix in aqueous solution has not been reported due to its instability. the aim of this study is to find effective ways to inhibit the unfolding of the aβ middle region, which is a prerequisite for the aβ fibril formation. in this study, we attempted to elucidate the molecular mechanism of the aβ unfolding by molecular modeling and molecular dynamics (md) simulations. the md simulations were performed for α-helical structures of a wild-type aβ( - ) model and a mutant aβ( - ) model. linker average hydrophilicity as a tool to discriminate between extended and non-extended calcium binding proteins a. isvoran , e. quiniou , c. craescu , l. mouawad west university of timisoara, department of chemistry, pestalozzi , timisoara, romania, inserm u , centre universitaire paris-sud, bâtiment , orsay, france the ef-hand calcium binding proteins (cabps) may exist either in an extended or a compact conformation, sometimes correlated with their functions. for the cabps with know structure and function, calcium sensors are usually extended and calcium buffers compact, hence the interest to predict the form of the protein starting from its sequence. in this study we used two different procedures, the sosuidumbbell algorithm and a novel procedure that is based on the linker average hydrophilicity (lah). the two procedures were tested on known-structure cabps and then applied to unknown-structure centrins. the so-suidumbbell algorithm yielded the right conformations for of the known-structure proteins and predicted that all centrins should are compact. the lah procedure discriminated well between the extended and non-extended forms of all the known-structure cabps and it reflected well the phylogenetic classification of centrins being a simple and powerful means to discriminate between extended and nonextended forms of cabps. only few residues that constitute the linker are responsible for the form of the cabp, showing that this form is mainly governed by short-range interactions. (http://u .curie.u-psud.fr/modelisation/lah) lipid and protein organization of hepatitis b antigen characterized by fluorescence spectroscopy v. greiner , c. egelé , s. oncul , f. ronzon , c. manin , a. klymchenko , y. mély laboratoire de biophotonique et pharmacologie, umr cnrs, faculté de pharmacie, université de strasbourg, france, sanofi pasteur, av. marcel mérieux, marcy l'étoile, france. hepatitis b surface antigen (hbsag) particles are nm lipoprotein particles, mainly composed of the major s surface viral protein containing trp residues and yeast-derived lipids. since the structure of these particles is still missing, we further characterized them by fluorescence techniques. fcs indicated that the particles diffuse mainly as monomers and contain about proteins per particle. fluorescence quenching and time-resolved fluorescence experiments showed that the fluorescence signal is largely dominated by the trp residues at the protein surface. moreover, time-resolved anisotropy measurements indicate that the protein motion is restricted and that the surface trp residues exhibit both local and segmental motions. the lipid part of the particles has been studied by environment sensitive -hydroxyflavone probes and viscosity-sensitive dphbased probes, and compared to lipid bilayers and low density lipoproteins (ldls), taken as models. the results suggest that the lipid part of hbsag is closer to ldls than to model lipid bilayers. we present an extensive calorimetric study of bovine alphalactalbumin for various ca++ content. equilibrium dsc raw data are analyzed and the melting temperature tm, the specific heat jump deltacp, the heat of unfolding deltahm are directly extracted. the binding of calcium on the native (n) state greatly stabilizes the protein, essentially by the enthalpic difference between the unfolded (u) and n states. we show that subsequent addition of calcium in the mm range stabilizes further the n state. the equilibrium calorimetric measurements are completed with out of equilibrium stopped flow refolding kinetics by cd spectroscopy performed at different temperature and ca++ concentrations. we discuss the possible stabilization mechanisms compatible with our measurements. protein unfolding/refolding in cellular compartments: application of luciferase constructs our studies show that a reporter enzyme, firefly luciferase, can be used for evaluation of the stress-induced proteotoxicity within different cellular compartments such as the nucleus, cytoplasm or mitochondria. in transfected mammalian cells, firefly luciferase is localized in microsomes. we engineered plasmid constructs encoding luciferase with inserted specific sequences that ensure its cytoplasmic or intranuclear, or intramitochondrial localization. in addition, we fused luciferase to the green fluorescent protein (gfp) that enables to visualize patterns of the compartment-targeted product. using such gfp-labeled constructs we had a possibility to monitor protein unfolding, aggregation and refolding in the cytoplasm, nucleus and mitochondria of transfectants exposed to either stressful conditions. gfp-luciferase expressed in mammalian cells behaves as a relatively labile protein which can undergo reversible unfolding and aggregation in response to heat shock, atp depletion or action of toxic agents. in the case of cell recovery, refolding of denatured luciferase is carried out at the chaperone machine. we explored unfolding/refolding of gfp-luciferase in the cytoplasm, nucleus and mitochondria of ischemia-stressed rat cardiac cells and in several cancer cell lines treated with hyperthermia or some chemotherapeutic drugs. the results obtained have revealed intriguing correlations between the proteotoxic impact within either compartment and the viability of treated cells. amyloidogenic and conformational properties of proiapp and iapp in the presence of lipid bilayers s. jha , d. sellin , r. seidel , r. winter biophysical chemistry, department of chemistry, tu dortmund university, otto-hahn str. , d- , dortmund, max-planck-institute for molecular physiology, otto-hahn str. , d- , dortmund, germany the islet amyloid polypeptide (iapp), which is considered as the primary culprit for β-cell loss in type diabetes mellitus patients, is synthesized in the β-cells of the pancreas from its precursor, the pro-islet amyloid polypeptide (proiapp). proiapp is co-processed in the secretory granules and co-secreted to the extracellular matrix together with insulin as iapp. here, we compare the amyloidogenic and conformational properties of proiapp and iapp in the presence of lipid membranes, which have been discussed as loci of initiation of the fibrillation reaction. the two peptides show an enhanced amyloidogenic propensity in the presence of negatively charged membranes and similar secondary structural properties. however, proiapp shows a much less amyloidogenic propensity, probably due to the increased net charge on proiapp, compared to iapp. unlike iapp, proiapp forms small oligomeric structures at the lipid interface, having heights of ∼ . nm. this morphological distinction can be attributed to the presence of the pro-region, flanking the amyloidogenic iapp. the addition of proiapp to iapp marginally delays iapp fibrillation, probably by interfering with the interaction between amyloidogenic iapp cores of distinct iapp molecules. thus, it appears reasonable to speculate that the pro-region of the proiapp could serve to delay the fibrillogenesis of iapp at negatively charged lipid bilayers. the role of transmembrane domain interactions in the kinetics and folding of cpt z. a. jenei , k. borthwick , v. a. zammit , a. m. dixon chemistry dept., univ. of warwick, coventry, uk, clinicalȃsciencesȃres.ȃinst., warwick univ., coventry, uk carnitine palmitoyltransferase i (cpt ) enzymes are polytopic integral membrane proteins in the outer membrane of mitochondria (omm). cpt controls the rate of entry of long-chain fatty acids into the mitochondrial matrix for βoxidation. the two catalytically active isoforms, cpt a and cpt b, are different in their inhibitor binding kinetics and structure (interaction between n-and c-segments, interactions of transmembrane domains (tmd)). it has been suggested that inter-and intramolecular tmds interactions are important for cpt a, but not for cpt b function. cpt a has also been implicated in formation of oligomeric complexes through its tm segments. the study of tm helix-helix interactions in cpt isoforms could lead to a better understanding of their function and inhibitor binding kinetics, and will contribute towards the design of pharmacological strategies aimed at modulating the activities of cpt enzymes in conditions such as diabetes. to investigate the ability of the tmd in cpt to self-associate and the order of any oligomers formed, several biochemical and biophysical techniques have been used. we found the self-association of rcpt a tmds (tm , tm ) to be different as measured using the in vivo tox-cat assay. chemical cross-linking and analytical ultracentrifugation studies demonstrated formation of both trimers and hexamers by the rcpt a tm peptide. these results provide further evidence that tm plays role in formation of a channel in the omm. self-assembly of transmembrane domains in cpt : role of gxxxg(a) motif in possible channel formation z. a. jenei , k. borthwick , v. a. zammit , a. m. dixon department of chemistry and, warwick medical school, university of warwick, coventry, uk carnitine palmitoyltransferase a (cpt a), a membrane protein that controls the rate of oxidation of long-chain fatty acids, is of key importance in diabetes and has recently been reported to exist as an oligomer in vivo. we have investigated full-length cpt a and find that the protein exists as a hexamer in liver mitochondria. using mutants of cpt a expressed in yeast mitochondria, we have localised key protein interactions in the hexamer to the transmembrane (tm) domains of the protein. detailed study of the tm domains in isolation, in both e.coli membranes and detergent micelles, demonstrated that while tm shows little self-assembly, tm displayed significant self-association. biophysical analyses of a tm -derived synthetic peptide revealed oligomerization behaviour identical to native cpt a in mitochondria, providing a strong link between tm helixhelix interactions and cpt a hexamer formation. this is significant in light of a recent suggestion that cpt a oligomerization may lead to formation of a channel in the mitochondrial outer membrane through which acylcarnitine gains access to the inter-membrane space. our data supports this new theory, and we go on to demonstrate experimentally the structural determinants of hexamer (channel) formation, specifically gxxxg(a) motifs in the tm domain which pack favourably in the hexamer and stabilize the oligomer. investigation of flexible loop role in structure and thermodynamic stability of firefly luciferase p. maghami, b. ranjbar, s. hosseinkhani department of biochemistry and biophysics, faculty of basic sciences, tarbiat modares university, tehran, iran protein folding, like any chemical process, consists of two fundamental components, thermodynamics and kinetics, which determine the stability of the folded state and the pathway of folding, respectively. these processes are currently too difficult to be solved de novo by purely computational methods. experimental evidence is required to simplify the problem via protein engineering .in this research, the wild type firefly luciferase (photinus pyralis) and some of its mutants were over expressed and purified. then their unfolding thermodynamics were examined, using circular dichroism and conventional fluorescence measurements. the unfolding equilibrium constant were measured over a complete rang of denaturant conditions. the measurements were shown structural and physico-chemical changes between wild-type and mutant proteins. exploring intrinsic disorder of unstructured membrane proteins by surface polymer physics intrinsically unstructured proteins (iups) are considered as a separate class within the protein world because they lack a well-defined folded structure. because iup's function is indeed directly linked to structural disorder, they are assumed to be natively unfolded. several physicochemical techniques are available to discriminate the degree of disorder. however a clear structural classification is still lacking. in this context, polymer physics emerges as a powerful tool for getting inside on the conformational abilities directly related to structural disordered of iups. in the present contribution, surface pressure and ellipsometry experiments in conjunction with polymer physics have been used to infer structural data in terms of molecular conformation and flexibility of a membrane protein essential for bacterial division, zipa. this protein has been pointed to posses a high molecular flexibility and to adopt a random coil conformation. folding dynamics of peptides studied by timeresolved infrared spectroscopy c. krejtschi , o. ridderbusch , r. huang , l. wu , t. a. keiderling , k. hauser institute of biophysics, university of frankfurt, germany, department of chemistry, university of illinois at chicago, usa peptides are ideal model systems to study protein folding mechanisms. ir techniques provide both the necessary time resolution as well as the structural sensitivity. we initiate rapid heating by laser-excited ns temperature jumps (∼ • c) and study fast ns-to-µs relaxation dynamics [ ] . the dynamics of the α-helix to random coil transition of polyglutamic acid was analyzed under reversible folding/refolding ph-conditions. the observed relaxation kinetics allowed separation of the folding and unfolding process with additional use of ftir measurements in thermal equilibrium. sitespecific dynamics were monitored by use of isotopic labeling for a β-hairpin peptide whose conformation is stabilized by a hydrophobic core. various single and cross-strand isotopically labeled variants were analyzed. the isotope-edited kinetics show variations in local structural stability of the hairpin backbone. our data support a multistate dynamic behavior, and the site-specific kinetics are consistent with a hydrophobic collapse hypothesis for hairpin folding [ ] . small heat shock protein hsp was predicted to belong to the family of intrinsically disordered proteins. one of the features of these proteins is that they do not demonstrate cooperative thermal transitions on heating. we applied different methods (dsc, ftir and intrinsic tryptophan fluorescence) to investigate the thermal unfolding of hsp . dsc results have shown that thermal denaturation of hsp begins from o c and occurs, with very low cooperativity, within a broad temperature region (up to o c and above). the thermal unfolding of hsp is fully reversible. the ftir data show that heating of hsp from to o c results in complete disappearance of α-helices (from - % to ) and the decrease in β-sheets content from to %. studies on the temperature dependences of tryptophan fluorescence have shown a significant red shift of the spectrum. these changes occurred within temperature region from to o c with midpoint at ∼ o c. probably, this transition can be explained by destruction of β-sheets around trp , the only trp residue of the α-crystallin domain of hsp (other trp residues of hsp are localized in the n-terminal domain). the data obtained confirm the suggestion that hsp is a protein, whose significant part is intrinsically disordered. we propose that, on heating, the α-crystallin domain containing β-sheets melts at higher temperature than the n-terminal domain containing the most of α-helices. t. rosenkranz , a. katranidis , d. atta , j. enderlein , i. gregor , m. grzelakowski , p. rigler , w. meier , j. fitter isb- : molecular biophysics, research centre jülich, germany, institute of physics, biophysics/complex systems, georg august university, göttingen, germany, institut für physikalische chemie; universität basel, basel, swizerland the protein folding mechanism is the missing link in the biological flow of information from the dna to its specific function. since most of proteins within a cell consist of more than one domain studies on this protein class are of major importance. it is a common feature of multidomain proteins to aggregate under refolding conditions, which hinders a refolding. molecular encapsulation of single molecules prevents aggregation. by immobilizing the nanocapsule the observation period in a wide field microscope will be extended, so that slow or rare folding events can be detected. a major goal of this study is to investigate polymeric vesicles with respect to their suitability for protein folding studies [ ] . polymer vesicles maintain their structural integrity even under harsh unfolding conditions. furthermore the nanocontainer proved to be permeable to guanidinium hydrochloride. using encapsulated phoshoglycerate kinase, labeled with atto- , a dye which experiences fluorescence quenching by photo-induced electron transfer (pet) with tryptophans, we demonstrate the remarkable properties of polymeric nanocontainers. applying pet as a folding probe we detected multiple unfolding/refolding transitions of single proteins. proteins frequently become irreversibly modified by carbonylation, a process of introducing the carbonyl group (carbon monoxide) in a reaction with reactive oxygen species (ros) such as superoxide, peroxide or ozone. the main targets for carbonylation in proteins are amino-acid side chains of lysine, arginine and proline. products of carbonylation are aminoadipic semialdehyde from lysine (asa) and glutamic semialdehyde (gsa) from arginine and proline. importantly, carbonylated proteins are marked for proteolysis by the proteasome, but can escape degradation and form aggregates that can be cytotoxic. carbonylation increases with the age of cells and it is associated with ageing and age related disorders such as alzheimer's disease, parkinson's disease and cancer. we have used the molecular dynamics method to study the stability of carbonylated proteins villin headpiece and ubiquitin. simulations were run after mutations of arginine, proline and lysine into gsa and asa had been performed. in addition, we have used thermodynamic integration on lysine, arginine, proline, asa and gsa residues in order to estimate their solvation free energy (related to relative hydrophobicity and hydrophilicity). our results suggest that carbonylation markedly decreases the overall stability of proteins, and that one potential reason for that may be a disruption of the balance between hydrophilic and hydrophobic regions in the protein. a. martino, d. crane, i. m. feavers, b. bolgiano division of bacteriology, national institute for biological standards and control, potters bar, uk the sensitivity to protein's secondary structure and progress in computational calculations have made circular dichroism (cd) an attractive technique to explore the optical properties of three promising vaccine candidates to neisseria meningitidis. clinical batches of a c-term deleted form of nada (genome-derived neisseria antigen -gna ) and the fusion proteins gna - (fp- ) and gna - (fp- ) were therefore studied. increases in temperature and denaturant concentration on secondary structures and folding/unfolding profile were monitored by cd in the far and near uv regions and complemented by trp fluorescence spectroscopy data. furthermore, epitope conformational changes on binding activities to immune-sera were investigated. the calculated secondary structure content was in broad agreement with the available predicted or solved protein structures. upon temperature incubation, a structural transition from a highly α-helical nada to a more unordered conformation, with a mid point at ∼ • c, was observed. fp- and fp- maintained their conformation up to • c or m guhcl in the case of fp- . unfolding was not always reversible. reductions in binding to monoclonal ab titrated along with increasing unfolding. folding/unfolding studies have proven useful in better understanding the solution behaviour and extent of folding of proteins. cold denaturation of yfh offers the clue to understand the effect of alcohols on protein stability s. r. martin , v. esposito , p. de los rios , a. pastore , p. a. temussi national institute for medical research, the ridgeway, nw aa london, u.k., laboratoire de biophysique statistique, sb/itp, ecole polytechnique fédérale de lausanne (epfl), ch- , lausanne, switzerland, dipartimento di chimica, università di napoli federico ii, via cinthia, i- napoli, italy although alcohols are well known to be protein denaturants when present at high concentrations, their effect on proteins at low concentrations is much less well characterized. here we present a study of the effects of alcohols on protein stability using yfh . exploiting the unusual property of this protein of undergoing cold denaturation around • c without any ad hoc destabilization, we determined the stability curve on the basis of both high and low temperature unfolding in the presence of three commonly used alcohols: trifluoroethanol,ethanol methanol. in all cases, we observed an extended temperature range of protein stability as determined by a modest increase of the high temperature of unfolding but an appreciable decrease in the low temperature of unfolding. we suggest that alcohols, at low concentration and physiological ph, stabilize proteins by greatly widening the range of temperatures over which the protein is stable. our results also clarify the molecular mechanism of the interaction and validate the current theoretical interpretation of the mechanism of cold denaturation. biomolecular sciences and biotechnology tor vergata moro , rome, italy cholesterol plays an important role in regulating the structural properties of phospholipid and non-phospholipid membranes. in this study we have applied in situ energy dispersive x-ray diffraction (edxd) to investigate the effect of cholesterol on the structure of different phospholipid and non-phospholipid oriented membranes. in detail, phosphatidylcholine (pc) bilayers and niosomal membranes, made of a non-ionic surfactant centre for bioactive chemistry, department of chemistry department of chemistry this process is initiated at nuclear envelope remnants (ners) in the presence of atp and gtp. the mvs can be divided in two main populations: mv and mv . mv has a classical lipid composition while mv is enriched in phosphoinositides (pips: pi, pip, pip and pip ). ners have an unusual lipid composition, enriched both in cholesterol and pips. physicochemical properties of the pips were investigated as a function of ph and temperature (t) using nmr, saxs and dls to map out their phase state. pips-water dispersions are observed in lamellar, hexagonal or isotropic phases depending on t and ph. in parallel, model membranes mimicking mv and ners lipid composition were studied by h and p nmr. mv modelling shows a complex behaviour of pips on pc membranes: they order or disorder membranes, whereas the order of pc/pi/pip/pip membrane is lower than that of pc or pc/pi membranes c. manzo, t. s. van zanten, m. f. garcia-parajo bionanophotonics group, ibec-institut de bioenginyeria de catalunya, barcelona, spain membrane proteins play a fundamental role in intra-and inter-cellular functions. in particular, the proteins lateral mobility in the fluid membrane environment is crucial for the regulation of several mechanisms, as receptor-mediated signal transduction and establishment of immunological synapses. these mechanisms are controlled through protein crowding and reduced lateral diffusion, which induce macromolecular associations and limit the application of conventional single molecule fluorescence techniques. to measure proteins mobility on living cells membrane, we developed a fluorescent correlation spectroscopy (fcs) setup in which the sample illumination is obtained through near-field scanning optical microscopy (nsom) probes. the use of nsom probes is particularly suited for the observation of dynamics on the cell membrane and overcomes the drawbacks of other techniques. in fact, through a shear-force-based position control, the probe is kept at a fixed distance from the membrane and its sub-wavelength aperture (∼ nm) reduces the illumination area, allowing the observation of highly crowded regions of the membrane. on the basis of preliminary results, the nsom-fcs is expected to provide an additional insight on the proteins trafficking at the membrane level. the technique also presents several potential developments, as the further reduction of the illumination area and two-colors correlation. single molecule fluorescence microscopy of the store-operated calcium channel subunit orai j. madl, j. weghuber, d. bergmair, m. fahrner, m. muik, c. romanin, g. j. schütz johannes kepler university, institute for biophysics, linz, austria store-operated calcium entry (soce) is essential for many cellular signalling processes. the essential pore forming subunit of soce channels in the plasma membrane is orai . here we present single molecule fluorescence microscopy of orai which was performed in order to directly visualize the stoichiometry of mobile orai pores. the protein was fluorescently labeled with monomeric gfp. a novel single molecule fluorescence approach, toccsl (thinning out clusters while conserving the stoichiometry of labeling), was used for the determination of the stoichiometry. this technique allows reducing the density of fluorescently labeled molecules without affecting the stoichiometry of labeling. density reduction is achieved by completely photobleaching a defined area within the plasma membrane; nonbleached gfp-orai aggregates enter the bleached region subsequently by diffusion. our data indicate that there are different populations of orai present in the cell: most of orai is located in the plasma membrane. a second population of orai -mgfp was found to be localized in intracellular vesicles. a significant fraction of the plasma membrane orai exhibits a diffusive movement. we found by analyzing the bleaching characteristics of single orai -mgfp aggregates that in resting cells mobile orai is predominantly dimeric. a. kobitski , a. nierth , m. helm , a. jäschke , g. u. nienhaus university of ulm, germany, university of heidelberg, germany, university of karlsruhe, germany rna molecules have attracted enormous attention in recent years, and various novel roles were revealed for rna in biological processes. ribozymes are a class of rna molecules capable of catalyzing chemical reactions. we have studied a diels-alderase (dase) ribozyme, a small artificial -mer ribozyme, which is capable of catalyzing carbon-carbon bond formation between an anthracene diene and a maleimide dienophile in multiple turnovers. single-molecule fluorescence resonance energy transfer was employed to investigate the intramolecular dynamics of this rna molecule as a function of mg + ion concentration. folding into a functional state occurs via an intermediate state, and continuous fluctuations between these two states were observed on the ms time-scale at the midpoint concentration of mg + ions. an effect of substrates binding on the folding and catalytic reaction of the dase ribozyme is in the focus of our recent research with the ultimate goal to obtain a detailed structural view of the single-molecule conformational changes that accompany the catalytic reaction. a. katranidis , r. schlesinger , k. nierhaus , i. gregor , m. gerrits , g. bueldt , j. numerous studies showed that protein folding and maturation can differ substantially between de novo synthesized proteins and in vitro refolded proteins. here we present an approach employing a two color single molecule sensitive fluorescence wide-field microscope in order to visualize surface tethered fluorescently labeled ribosomes and de novo synthesized gfp molecules in real time [ ] . fluorescence of co-translational folded proteins was observed from mature fluorescent gfp molecules which carry additional amino acids at the c terminus remaining linked to the ribosome. thus it was possible to co-localize fluorescence from labeled ribosomes and from gfp molecules. we demonstrate that the green fluorescence protein mutant gfp emerald is produced with a characteristic time of five minutes. the fastest gfp molecules appeared already within one minute. processes precedent to chromophore formation, such as polypeptide synthesis and protein folding, are fast and last not longer than one minute. in fluorescence spectroscopy, photobleaching is a process which leads to irreversible loss of fluorescent properties of a dye molecule, usually due to photochemical reactions. it is especially important for fcs experiments on slow-diffusion systems since for high excitation intensities it can have a strong impact on fluorescence intensity correlation function. usually it is observed as apparent shortening of the mean diffusion time of the dye molecules. the behavior of tmr-labeled fd-virus rods in water solution under various excitation conditions was investigated. the experiments were conducted for low ( : ) and high ( : ) tmr:virus ratios and for increasing laser intensities. the correlation function was measured in the experiments. the results were fitted using origin software to estimate the influence of photobleaching, and compared with computer simulations. a strong effect of photobleaching was visible for rods labeled with a single dye molecule, while rods labeled with tmr molecules showed little to no bleaching. a prolongation of characteristic diffusion times for highly labeled virus rods in comparison to low-labeled ones was also observed. k. toth , a. gansen , a. valeri , v. böhm , c. a. seidel , j. langowski abt. biophysik der makromoleküle, deutsches krebsforschungszentrum, heidelberg, germany, lehrstuhl für molekulare physikalische chemie, heinrich heine universität, düsseldorf, germanythe nucleosome has a central role in the compaction of genomic dna and the control of dna accessibility for transcription and replication. we studied the effect of dna sequence and selective histone acetylation on the structure, stability and disassembly of the mononucleosomes. quantitative single molecule fret measurements between dyes attached to different parts of the nucleosome permitted us to detect the equilibrium between several subpopulations of reconstituted nucleosomes in solution. we obtained that the heterogeneity and stability of the samples are correlated with each other and influenced both by the dna sequence and the histone acetylation. the path of the linker dna is more sensitive to all studied effects than the dna on the core. intermediates of the disassembly pathway were identified and characterized. j. strömqvist , s. johansson , y. ohsugi , k. andersson , l. xu , m. kinjo , p. höglund , j. widengren experimental biomolecular physics, kth, stockholm, sweden, department of microbiology and cell biology, karolinska institutet, stockholm, sweden, laboratory of molecular cell dynamics, hokkaido university, sapporo, japan dual-color fluorescence cross correlation spectroscopy (fccs) has been used to explore the molecular dynamics at immune cell surfaces, with a particular focus towards the regulation mechanisms of natural killer (nk) lymphocytes. nk cells are critical mediators of anti-viral immunity and protectors against cancer spread. their activity is governed by a fine-tuned balance between inhibitory and activating receptors, where ly a and kir receptors represents the inhibitory ones. their ligands are mhc class i receptors. fcs is a technique based on the analysis of intensity fluctuations of fluorescent molecules excited by a focused laser beam. the technique offers information about molecular dynamics at the single molecular level, in the nanosecond to millisecond range. dual color fccs expands fcs by correlating the intensity from two different colors. by labeling two potential interaction partners with dyes emitting at different wavelengths, the amount of interaction can be determined.here, we will report on recent fccs data exploring the interaction between the inhibitory receptors and their ligands, as well as different labeling strategies used to enable these measurements. dynamic multiple-target tracing probes spatiotemporal cartography of cell membranes in order to decipher the non random and non homogeneity of the plasma membrane organization, we had performed fluorescence correlation spectroscopy measurements on live cells. this allowed us to establish the presence of nanoscale confining structures and to demonstrate their implication in signaling process . complementing these studies, we present here a new analytical method, namely multiple-target tracing (mtt) which takes advantage of the high resolution provided by singlemolecule sensitivity to generate dynamic maps at high densities of tracked particles. introducing deflation by subtracting detected peaks allows detecting peaks of lower intensity. we achieved an exhaustive detection of particles with performances reaching theoretical limits, and a reconnection of trajectories integrating the statistical information from past trajectories. we demonstrate the potential of this new method of analysis by applying it to the epidermal growth factor receptor labeled with quantum dots, in the plasma membrane of live cells. this has allowed us to build up a global representation of molecular dynamics in cell membranes. dual polarisation interferometry (dpi) is a surface analytical technique capable of dynamically measuring biophysical parameters of conformational change in biomolecular interactions. the technique measures three key parameters, namely layer thickness, layer density (ri) and mass, thereby enabling the resolution of conformational changes involved during binding. a number of different applications are presented. protein-protein interactions: understanding the biophysical nature of protein interactions can deliver insights into the mechanisms by which proteins interact, thereby elucidating protein function. dpi enables correlation between binding affinity and conformational change, greatly enhancing the study of structure-function relationships. lipid layers: the birefringence mode of dpi can be used to study the formation of lipid bilayers and biomolecular self-assembly. it is possible to use a combination of bilayer refringence and mass to study phase transitions associated with protein or peptide binding to the lipid bilayer. the individual stages of adsorption, absorption and micellisation can be distinguished. carbohydrate interactions: dpi uses a planar glass surface and a wide range of coupling chemistries. a carbohydratespecific surface can be used to study a wide range of biomolecular interactions, such as lectins, acidic proteins, extracellular matrix signaling and interactions with complex membranes. the experimental characterization of the elementary conformational steps constituting the protein folding pathway remains a big challenge. quenching of the triplet state of tryptophan by close contact with cysteine has been shown to provide a new tool for measuring the rate of intramolecular contact formation -one of the most elementary events in the folding process -in peptides and proteins using only natural probes. here we show an extensive study on a stabilized mutant of the second beta-hairpin of gb domain. steady state fluorescence and kinetics of contact formation between a natural tryptophan and a cysteine added to the c-terminal are measured for different temperatures and solvent conditions. we separately address the contributions of different structural elements to the overall stability of the hairpin. we extract kinetics parameter for contact formations in the unfolded state, formation of the hydrophobic core and tails pairing in the folded state. by means of a fragment peptide terminated with a tryptophan and a cysteine, we also estimate the structural propensity of the turn region. the data coherently combine with a simple model previously developed to describe the dynamics of unstructured chain [biophys. j. , ( )], here modified with the addition of attractive interactions between specific residues. catalytic power of partially denatured enzymes: implementation of molten-globule-like states m. shushanyan , d. e. khoshtariya , m. makharadze , t. tretyakova , r. van eldik institute of molecular biology and biophysics, gotua , tbilisi, georgia, department of physics, i. javakhishvili tbilisi state university, tbilisi, georgia, department of chemistry and pharmacy, university of erlangen-nürnberg, germany impact of nonspecific moderate denaturants, urea and dmso, on the kinetic (functional) and thermodynamic (stability) patterns of a hydrolytic enzyme, α-chymotrypsin (α-ct) has been investigated. furthermore, an impact of urea and guhcl in combination with of temperature on the kinetic pattern of carboxypeptidase a has been examined. for the case of α-ct, in particular, we have observed about tenfold increase of the apparent mickaelis constant when going from to m urea ( o c), whereas the value of catalytic constant remained almost unchanged, indicating that the protein is not unfolded even under those severe conditions. the matching microcalorimetric experiments revealed that both the temperature-induced melting temperature and transition enthalpy decrease gradually with the increase of the additive concentration. with m urea the peak-shaped calorimetric feature disappears totally. however, catalytic power of α-ct was preserved owing to its catalytic constant. for other studied enzyme/substrate/denaturant arrays diverse kinetic peculiarities due to mgls have been observed. rubredoxins are a class of iron-containing proteins whose biological role on electron transfer processes and metal binding is still unclear. the unfolding dynamics of the rubredoxin mutant rda c from the mesophile desulfovibrio vulgaris (dv) was studied on the temperature range from • c to • c and along time at • c. resonance energy transfer (ret) was used to determine the donor (d; trp ) to acceptor (a; , -iaedans) distance. from • c to • c the d-a distance increased Å. however the random coil expected d-a distance was only achieved after heating the protein solution during . hours at • c. from uv-vis absorption data it's clear that this protein is capable of maintaining its iron-sulfur center at • c. by melting the protein at the same temperature all iron-centers disintegrate and the protein unfold after . hour. the trp fluorescence also shifts nm to the red reflecting the partial exposition of the indole ring to the solvent. from fluorescence and anisotropy decay curves a breathing type movement of the protein structure was observed between • c to • c without lost of significant secondary structure. this structure flexibility should play an important role on the thermal stability of the dvrd the antimicrobial peptide novicidin (nc) -modified from the sheep self defense peptide smap- , for reduced mammalian cytotoxicity -is a cationic peptide (net charge +∼ . ) that adopts random coil structure in solution, but an α-helix in the presence of lipid vesicles. the conformation of nc in presence of the lipids dlpc, dlpg, dmpc, dmpg, dopc, dopg, dope, and dops in different combinations reveal the lipid selectivity, affinity, and phase dependent changes with varying l/p ratios and temperatures, observed from cd spectroscopy. it is understood that the conformational change is dependent on chain length and head group of the lipid. apart from the in vivo results on the nc activity, studies using qcm-d, dual polarisation interferometry, and calcein release assay reveal the kinetics and concentration dependent activity of nc in lipid bilayers and vesicles. preliminary studies on orientation of nc in various lipid environments using ssnmr, lsnmr, and molecular dynamics simulations apparently suggest that nc may form toroidal pores/detergent effects depending on the chain length of the lipids. further experiments on nc in presence of lipids using dsc, itc, ssnmr, oriented cd, ld, and confocal microscopy to determine the structure, thermal stability, orientation in lipid bilayers, and thereof the action of nc will help in proposing a comprehensive model for its mechanism of action in model membranes. dielectric method for measuring glass transition and denaturation temperatures of hydrated proteins g. e. thomas , s. bone , g. drago institute for bioelectronic and molecular microsystems, bangor university, gwynedd, uk., applied enzyme technology, pontypool, uk.the flexibility of protein structures is important in allowing the variety of motions, covering a wide range of magnitudes and frequencies, essential to biological activity. high frequency dielectric measurements can be used to study the flexibility of proteins by probing the relaxation of dipolar constituents of their structures. hydrated proteins exhibit a broad dielectric loss extending over the frequency range from mhz to ghz which can be decomposed into a number of constituent dispersions. one of these dispersions, with a relaxation time of ∼ ns, has been attributed to the relaxation of protein backbone peptide groups in the protein interior. in the work reported here, this dielectric dispersion was investigated as a function of temperature for the enzyme glucose oxidase. two critical temperatures were identified as the glass transition and denaturation temperatures, both of which were found to decrease with increasing protein water content. the results were consistent with a scheme in which the hydrated glassy protein undergoes a change in structural mobility at the glass transition temperature and experiences an irreversible change in conformation at a higher denaturation temperature. both glass transition and denaturation temperatures are key indicators of protein stability and are important in the production and storage of protein based pharmaceuticals. a. szymańska, k. kacprzyk, g.Ślósarek department of molecular biophysics, a. mickiewicz university, umultowska , - poznań, poland aggregation dynamics of proteins plays an important role in molecular biology and medicine as it permits explanation of several disease states. an interesting problem is to find out in which conditions the interactions of the protein molecules lead to formation of ordered structures and in which to disordered ones. in this study, dynamic light scattering, circular dichroism and also congo red dye experiments were performed to analyze various structural states of lysozyme induced under different concentration of ethanol solvent. at low ethanol concentration the attractive interaction between the protein macromolecules dominate. after addition of more ethanol solvent, the translational diffusion coefficient was much smaller than that for lysozyme solution at zero ethanol concentration. it can be explained by the structural transformation of the polypeptide chain leading to a partially folded conformation needed for oligomerization and fibrillation process. on the basis of the cd experiments we concluded that the ethanol solvent induces changes in secondary structure of lysozyme solution. on addition of % v/v ethanol solution the intramolecular hydrogen bonds were destabilized. above this ethanol concentration, β -sheet were the dominant secondary structure of lysozyme in solution. the phase diagram illustrating the formation of: monomers, oligomers at various structural states, protofilament formation state, protofilament and amyloid fibrils was constructed. key: cord- - r ndzv authors: nan title: posters date: - - journal: glia doi: . /glia. sha: doc_id: cord_uid: r ndzv nan university of colorado school of medicine, aurora, united states oligodendrocyte progenitor cells (opcs) migrate long distances before differentiating and myelinating axons. it is well established that both short and long-range soluble guidance factors, as well as contact with the surrounding extracellular matrix (ecm) and neighboring cells, can modulate opc migration and differentiation. however, the influence of cell-matrix interactions on opc responses to guidance molecules is less clear. previous in vitro studies of opc migration in response to semaphorins and netrin- yielded inconsistent results, but these studies were performed on a variety of ecm substrates, i.e., explants, collagen, poly-d-lysine. in the current study, we systematically studied opc migration in response to guidance factors on several different ecm substrates, to better understand the influence of ecm interactions on opc responses to chemotropic molecules. understanding the regulation of opc migration and differentiation has important implications for the treatment of demyelinating diseases such as multiple sclerosis (ms). for example, semaphorins a and f are expressed in active, but not chronic, ms lesions, and in demyelination/remyelination studies in animal models, semaphorins influence opc migration into lesions, affecting the rate at which remyelination occurs. in addition, aberrant expression of ecm ligands occurs in and around ms lesions. we performed live imaging experiments of opc migration on different ecm substrates in response to gradients of chemotropic molecules. these studies are combined with ihc, co-ip, western blot and rt-pcr analyses to determine the effects of ecm substrates on opc migration and signaling pathway responses to chemotropic molecules. our preliminarily results suggest that ecm substrate can regulate both the direction and rate of opc migration. semaphorin a is repulsive to opcs on laminin , while on fibronectin opc migration remains uniform in the presence of semaphorin a. since laminin is the ligand for a b integrin heterodimers, while opc migration on fibronectin is mediated by avb integrins, the repulsive effect of semaphorin a appears likely to function through b integrin. thus, signaling through the neuropilin-plexin receptor complex that underlies the repulsive effects of semaphorin a on opc migration may be modulated by interactions with b integrins. x. bai university of saarland, homburg, germany secondary injury processes after acute brain trauma involve activation of different cell types like astrocytes, oligodendrocytes and microglia cells. a complex and yet not completely understood sequence of cellular responses initiate functional recovery after the neurodegeneration process. by using double-transgenic mice gcpb (gfap-egfp plp-dsred ) mice, we were able to identify a particular type of activated glia expressing astro-and oligodendroglial properties simultaneously (ao cells) that transiently appeared after three types of acute cortical injuries (stab wound injury, pial vessel disruption and middle cerebral artery occlusion). ao cells could be labeled by oligodendrocyte precursor cell (opc) markers olig and sox , but not for markers of mature astrocytes or neurons. two-photon live imaging of gcpb mice revealed that ao cells originated from plp-dsred-positive oligodendrocyte lineage cells. electrophysiological inspection of ao cells in acutely isolated brain slices revealed the expression of delayed rectifying, voltage gated k currents, a typical property of ng glia. therefore, we classified ao cells as opcs. to follow the fate of ao cells which disappeared about days after the injury, we generated gfap-n-cre plp-c-cre rosa eyfp triple transgenic (crec) mice. under non-injury conditions, few recombined cells were observed. however, numerous recombined cells appeared after week of stab wound injury adjacent to the lesion site, and lasted over weeks. we found that - % of recombined cells were gfap-positive, - % of them were pdgfra-positive and - % were positive for the oligodendrocyte marker gstp. in line with that, about % of newly generated gfap-positive astrocytes were observed from ng -creert x rosa tdtomato mice after week of stab wound injury. these results provide a strong evidence for opcs giving rise to astrocytes after acute cortical injuries. to further understand the molecular mechanism, we performed intra-cerebral injection of bone morphogenetic protein (bmp , known to promote astrocyte, but blocking oligodendrocyte differentiation) into gcpb and crec mice. bmp , but not leukemia inhibitory factor (lif), significantly increased the number of ao cells as well as astrocytes in the respective mice. in conclusion, ng /opcs display strong potential to differentiate into astrocytes after acute cortical injury. for instance, sox and sox have a crucial role in opc specification and differentiation, respectively. using microarray analysis of oligodendrocyte lineage cells, we previously identified sox as a major regulator of oligodendrocyte development (sohn et al., ) . in opc cultures, sox expression was maximal at developmental corresponding to cell cycle exit and onset of differentiation. these findings, suggest that sox has critical functions in the controlling of opc cell cycle exit and differentiation. in order to decipher the functional role of sox in oligodendrocyte development, we generated a tet-sox :sox rtta double transgenic mouse line. this mouse strain allows a sox overexpression specifically in sox cells in a doxycycline dependant manner (tet-on system). in the cns of dox-treated transgenic animals, we showed that sox overexpression was specifically targeted in sox oligodendroglial cells. in the developing cns, sox overexpression was targeted in pdgfra /nkx . opcs and in olig /cc differentiated oligodendrocytes. to assess the effect of sox gain-of-function on oligodendrocyte development and myelination, we analysed opc proliferation, apoptosis and differentiation, after doxycycline induction from e . to p . in the embryonic spinal cord, we showed that overexpression of sox did not modify cell proliferation or the number of pdgfra opcs. these data strongly suggest that sox is not involved in early steps of oligodendrocyte development. interestingly, we found that sox gain-of-function drastically reduces the density of cc mature oligodendrocytes at postnatal stages, correlating with a delay in myelination in transgenic mouse spinal cords. altogether, our data reveal critical functions of sox in oligodendrocyte lineage progression and myelination. supports: arsep and nmss (usa). m.f is a phd fellow of the french mesr (ed c). m. rocha, p. fernandes, a.c. manhães, p.c. barradas, f. tenorio universidade do estado rio de janeiro, instituto de biologia, rio de janeiro, brazil nutrient restriction during the perinatal period exerts a profound influence on brain development. previous studies using an undernutrition protocol ( % protein diet) during the first half of the lactation period in rats showed that the offspring presented an altered feeding pattern, which reflected a metabolic imprinting effect on feeding behavior. there is a delay in the pattern of npy expression in the arcuateparaventricular (arc-pvn) pathway, reflecting an effect on the development of the hypothalamic circuitry, leading to metabolic imprinting. here we studied the effects of undernutrition on glial differentiation using the same model. we used rats from to postnatal (p) days of age whose dams were either fed a % protein diet (dg) or a normoprotein diet (cg) from p to p . we assessed ki , vimentin, gfap, ed- and cnpase distribution in hypothalamic nuclei using immunohistochemistry. our results showed impairment in cell proliferation, more evident in the pvn and in the lateral hypothalamus (lh), where dg animals showed a lower number of ki- positive (ki ) cells at p when compared to cg. however, at p and p , the number of ki cells was significantly increased in dg. from p onwards, staining intensity remained relatively stable and similar in all groups. dg animals showed a delay in astroglial differentiation, presenting a decrease in gfap expression and a peak of vimentin expression at p and p accompanied by an increase in vimentin/ki cells. an increase in the number of ed positive cells next to the third ventricle was also shown at the same ages in dg. cnpase staining was very faint in most nuclei and did not show any obvious difference in dg animals. our results suggest that glial differentiation is delayed in dg, which may contribute to the changes in hypothalamic circuitry that causes metabolic imprinting. recent studies have demonstrated that neural precursor cells (npcs) residing in the adult subventricular zone also exhibit the capacity to generate new oligodendrocytes following experimental demyelination. the relative capacities of these two cell types to contribute to oligodendrogenesis in this context remain largely unexplored. we have adopted transgenic approaches that enable either the specific labeling of npcs or oligodendrocytes and the interrogation of their subsequent fate within the corpus callosi of mice subjected to central demyelination induced by cuprizone. adult nestin-creer t : r r-eyfp mice were administered tamoxifen ( . g/kg/day) for days by oral gavage, inducing the permanent expression of yellow fluorescent protein (yfp) in npcs and their progeny. mice were subsequently fed . % cuprizone for weeks followed by weeks recovery on normal food to enable the analysis of remyelination. expression of yfp and other cellular markers was examined immunohistochemically to determine the fate and migratory potential of npcs in the remyelinating corpus callosum (cc). rostrocaudal analyses of cc in the cuprizonechallenged mice demonstrated that approximately % of yfp npcs commit to an oligodendroglial fate. there was robust recruitment of npc-derived oligodendroglial cells, with a -fold increase in yfp sox cells compared to unchallenged mice. most of these cells were mature oligodendrocytes (yfp cc ) and their density in the remyelinating cc was highest adjacent to the dorsolateral corner of svz and decreased proportionally with distance in the mediolateral axis. on the other hand, fate mapping of opcs using pdgfracreer t : r r-eyfp mice revealed that oligodendrocytes derived from parenchymal opcs were distributed in a converse manner to svz-derived oligodendrocytes. in addition, we found that, independent of the cellular source, many oligodendrocytes repopulated the corpus callosum as linear arrays of clonally derived cells, in a manner that recapitulated the postnatal development of these structures. these results demonstrate that lineage determination and maturation of oligodendroglia from npcs occurs efficiently in situ. we also reveal that remyelination occurs in a coordinated way, most likely secondary to interactions between a restricted pool of axons and a sentinel opc induced to proliferate in situ. play an important role in developmental oligodendrogenesis and myelination and mounting evidence from rodent models of demyelination suggest that they also promote remyelination. the therapeutic application of thyroid hormone to demyelinating disorders is limited by the potential for cardiac side effects mediated by thyroid hormone receptor (thr) a signaling. here we report that gc- , a thyromimetic with selective thrb action promotes in vitro oligodendrogenesis from both rodent and human oligodendrocyte progenitor cells. in addition, we used pdgfar-creer;rosa -eyfp double-transgenic mice to examine the effect of gc- on the fate of oligodendrocyte progenitor cells and find that treatment with gc- during developmental myelination promotes oligodendrogenesis in vivo. these results indicate that a b receptor selective thyromimetic can enhance ol differentiation in vitro and during developmental myelination and warrants further study in demyelinating models. the olfactory epithelial layer contains multipotent horizontal basal cells (hbcs) that differentiate into olfactory sensory neurons. we generated olfactory sphere (os) cells in cultures that were derived from adult rat olfactory mucosa. fluorescence-activated cell sorting and immunofluorescence analyses showed that os cells were derived from hbcs. os cells underwent neuronal and glial differentiation in vitro. to examine multipotent differentiation in vivo, os cells were transplanted into injured rat spinal cords. the transplanted cells integrated into host tissue and underwent glial differentiation. our data showed the stem cell properties of hbcs. oligodendrocyte progenitor cells (opcs) comprise the main cycling cell population of the cns parenchyma during the early postnatal period and at adult stages. however, the molecular mechanisms implicated in opc divisions are still by large obscure. with the aims to unveil i) whether opc divisions exploit the same molecular machinery of neuronal precursors, and ii) whether distinct opc subsets exist with different cell division machineries, we focused on a mutant mouse where citron-kinase, a crucial regulator of cytokinesis in neuronal precursors, is germinally ablated (cit-k ko). these mice have severe cns developmental abnormalities, reduction of selected neuronal populations due to apoptosis triggered by defective divisions, display ataxia, epileptic seizures and die by the third postnatal week. interestingly, they were reported to have myelin defects, suggesting that the cit-ko affects oligodendroglial cells. notably, already early after birth we found a -fold decrease in opc density in both the cortical grey (gm) and white matter (wm), compared to wild-type mice (wt). here, most opcs were hypertrophic and multinucleated, indicating a cytokinesis failure after nuclear division. at later ages, opcs progressively disappeared from the cortical gm, while in both wm and ventral areas of the telencephalon (i.e. striatum, hypothalamus), uni-and multi-nucleated opcs could be detected, although at lower densities compared to wt. these data suggests opc heterogeneity in both cit-k requirement for cytokinesis and susceptibility to death. however, even where normal uninucleated opcs persisted, we could not find pre-myelinating or myelinating cells, in line with additional differentiation defects. to discriminate the contribution of cell-autonomous vs. environmental factors in differentiation impairment, we performed homochronic grafts of wt fluorescently tagged (gfp ) cells into perinatal cit-ko mice. strikingly, gfp opcs invaded the whole cit-k ko brain and actively divided. however, they hardly ever differentiated into more mature cells at difference with cells grafted in the wt, indicating that altered environmental signals contribute to myelination defects in cit-k ko. in conclusion, our results show that cit-k is involved also in glial cell divisions and suggest distinct cit-k requirement and proneness to death of dorsal and ventral opcs. additionally, cit-k ablation results in pervasive nervous tissue alterations affecting opc maturation. further studies will clarify the molecular mechanisms underlying these alterations. knowledge of how these cells act in repair is lacking, partly due to a limited understanding of their behaviour in normal developmental processes. radial glia cells are a unique population of neural progenitors that have recently been suggested to function in neurogenesis in the cerebral cortex. however, many questions still remain regarding their specific role in the developing spinal cord. using an organotypic spinal cord slice culture model and two-photon microscopy we directly visualized radial glial cell behaviour in the developing spinal cord. mm slices of rat embryonic spinal cord (e -e ) were cultured in order to preserve the d cytoarchitecture of the cellular environment. using electroporation, slices were transfected with a brain-lipid binding protein (blbp) driven gfp expression plasmid, which specifically labels endogenous radial glial cells. slice cultures were then imaged using two-photon time-lapse microscopy, allowing for high spatially and temporally resolved d imaging. we were able to follow individual gfp expressing radial glial cells over time ( hours to days) and characterized precise morphological changes. at early developmental ages, gfp expressing cells initially exhibited a radial morphology with the soma located in the ventricular zone and processes extending out to the pial surface. over time, many cells developed a multipolar morphology and migrated away from the lumen, primarily using somal translocation. using immunohistochemistry we identified populations of transfected cells which also expressed gfap, representing the differentiation of blbp-expressing radial glia cells into astrocytes. we also found that radial glial cells differentiate into oligodendrocytes (olig and cnpase positive) but found little evidence for gfp co-localization with neuronal markers (neun, dcx, biiitubulin). using this model we can directly observe complex developmental behaviours of specific cell populations and elucidate the relevant genetic regulation and molecular mechanisms that govern spinal cord development. by understanding these intricate events, we will gain insight into how a simple tube of undifferentiated neuroepithelial cells generates different cell populations that will eventually develop into the adult spinal cord. methods: we used the new cre-expressing mouse strain cx cr creer to express a diphtheria toxin receptor (dtr) specifically on microglia/macrophages. in these mice, administration of tamoxifen leads to cre-mediated excision of a loxp flanked transcriptional stop element in both macrophages and microglia, thus allowing for the expression of the dtr as well as an yfp reporter. due to their fast turnover macrophages are replaced by unaffected precursors whereas microglia persist in the modified stage. these mice were used for in vivo and ex vivo analysis of microglia by facs and histology. results: our optimized protocol with two tamoxifen injections at the age of weeks resulted in - % of reporter-positive microglia in adulthood, whereas all peripheral macrophages were reporter-negative. with additional three successive dt injections we could achieve a microglia ablation efficiency of %. interestingly, histological as well as facs analysis revealed % of microglia repopulation already days after depletion. two weeks after depletion microglia numbers were even higher compared to unaffected controls. on day , we found a cd . high ly c population, indicating replenishment by peripheral monocytes. in bm chimera experiments, where peripheral cells were labeled with a congenic marker, we obtained striking evidence showing that almost all of the repopulating cells are of peripheral origin. conclusions: after depletion microglia were rapidly repopulated, which emphasizes their critical role in brain homeostasis and function. even though ms has an onset in young patients, it persists throughout life and progresses along adulthood and aging. therefore, mscs from aged individuals should be tested for their pro-oligodendrogenic activity before moving into the clinical trials. this is insofar of clinical relevance, since the brain's capacity for self-repair and remyelination strongly declines with aging. thus, the aim of this study was to determine whether mscs from aged individuals maintain their known prooligodendrogenic activity. mscs were obtained from bone marrow of young ( months) as well as old ( - months) rats and analyzed their stem cell properties as well as their pro-oligodendrogenic activity. as expected, mscs derived from aged bone marrow presented diminished stem cell properties and differentiation capacity. to test the msc prooligodendrogenic activity, npcs were incubated with msc conditioned medium. surprisingly, soluble factors derived from old rats mscs maintain their ability to induce an increase of mbp-expressing oligodendrocytes on npcs. moreover, aged mscs conserve their oligodendrogenic activity also on npcs derived from old donors ( months). these results indicate that mscs keep their oligodendrogenic activity regardless of age and, therefore, this study provides a rationale for clinical trials of autologous msc transplantation in ms therapy. munich cluster for systems neurology (synergy), munich, germany astrocytes fulfill essential functions under physiological conditions and are involved in various beneficial and adverse functions after brain injury (reviewed by sofroniew ). in order to improve functional recovery after injury, it is essential to delineate the beneficial from adverse effects. however, little is yet to know which extent distinct sets of astrocytes perform distinct functions or whether all astrocytes behave in a uniform manner. live imaging of astrocytes after stab wound injury in the adult cerebral cortex in vivo revealed a striking heterogeneity of astrocyte behaviour with distinct subsets extending long processes towards the site of injury, others proliferating and yet other subtypes undergoing only hypertrophy. most strikingly, the vast majority of astrocytes proliferating were located with their somata directly at blood vessels (capillaries or post-capillary vessels). this close proximity was confirmed at ultrastructural level and identified as juxtavascular positions, sometimes adjacent to pericytes located in the perivascular space. this novel population of reactive astrocytes at juxtavascular positions is of particular interest, as the capacity of astrocytes to reactivate proliferation ( cell stem cell, in press). therefore we examined whether this population of juxtavascular astrocytes would also preferentially proliferate in even more severe injury conditions, such as hour of middle cerebral artery occlusion (mcao). the proliferation of juxtavasular astrocyte was analyzed immunohistochemically in fixed brain sections co-stained for s b/gfap/ki /cd at different time points after injury. at days post injury, % of all astrocytes were actively dividing (ki ). strikingly the majority of these was again located at juxtavascular positions ( %), suggesting a general bias of astrocytes at this position to proliferate after injury, while astrocytes further distant from blood vessels virtually fail to divide. we shall further present data from conditional knock-out mice with reduced proliferative activity of juxtavascular astrocytes in order to elucidate the function of this highly specific astrocyte population and their proliferative reaction after injury. a.d. rivera, a. butt university of portsmouth, portsmouth, united kingdom the generation of oligodendrocytes (ols) from their precursors (opcs) occurs throughout adult life and is particularly important following demyelinating insults, such as occurs in multiple sclerosis (ms). glycogen synthase kinase -b (gsk b) acts as a constitutively active negative regulator of multiple pathways that regulate proliferation, survival and differentiation of opcs. lithium is a potent inhibitor of gsk- and is used for the treatment of bipolar disorder, but in addition is neuroprotective in a wide range of diseases, including stroke, amyotrophic lateral sclerosis, and alzheimer's disease. lithium inhibits gsk- by binding directly to the enzyme's magnesium-sensitive site and indirectly by enhancing its phosphorylation. notably, by inhibiting gsk b, lithium acts as a wnt mimetic to promote b-catenin-dependent transcriptional events, including enhancement of genes involved in apoptotic inhibition. here, we have examined the effects of lithium on oligodendrocytes in the adult mouse optic nerve, a typical cns white matter tract; all procedures were in accordance with the animals scientific procedures act ( ) . transgenic mice aged postnatal day (p) in the developing and adult cns multipotent neural stem cells reside in distinct niches. specific carbohydrates and glycoproteins are expressed in these niche microenvironments that are important regulators of cell fate determination and stem cell maintenance. in previous studies we described lewisx (lex) glycan as a specific marker of neural stem precursor cells (nspcs) in developing brain and showed it to be involved in nspcs migration and proliferation. using lex glycosylation as a biomarker we aimed to identify the lex carrier glycoproteins which could have a functional relevance for cns development. in addition to already known lex carriers we have found lrp as new major lex carrier and for the first time showed it's expression in nspcs and developing brain. lrp is essential for the normal neuronal function in adult cns, whereas the role of lrp in development is still unclear, mainly due to the lethality of the lrp knock-out in mice. in the current study we investigated the basic properties of lrp knock-out nspcs created by means of cre-loxp mediated recombination. the elimination of lrp in vitro was induced by the addition of cell permeable cre-recombinase to nspcs derived from embryonic brain of lrp flox/flox mice. the functional status of lrp -deficient cells was subsequently studied using proliferation, migration and differentiation assays. lrp knock-out cells maintained as neurospheres, retained the ability to migrate and differentiate. interestingly, lrp knock-out nspcs generated -times less opcs in comparison to lrp wt/wt cells. this suggests that lrp is involved in the control of the oligodendroglial differentiation. astrocytes are multifaceted cells in the adult central nervous system and react to pathology of the adult brain with a finely graded continuum of morphological and behavioral changes. given that astrocytes in healthy adult brain rarely divide outside the stem cell niches, but activate both proliferation as well as a stem cell potential after injury (for review see robel et al., ) , it is important to understand their exact mode of cell division. indeed, a defining hallmark of a stem cell is the capacity to self-renew -often by asymmetric cell division. therefore we set out to image reactive astrocyte divisions in vitro by developing a primary culture system for reactive astrocytes obtained from the somatosensory cortex of adult mouse at days after stab wound injury. continuous live imaging and single cell tracking (costa et al., (costa et al., , allowed us observing the mode of cell division mode as well the cell fate of their immediate progeny. results of our study reveal (i) that reactive astrocytes can undergo multiple rounds of cell division, (ii) the shh signaling has a profound influence on this behavior, (iii) reactive astrocytes divide with distinct modes of cell division, which can be influenced by the local microenvironment. taken together, this new culture system allows close examination of signals on the mode and multitude of reactive astrocyte cell divisions and provides a great tool to identify signals derived from the extracellular matrix, other cell types or as diffusible signals. interestingly, even without injury temporary depletion of proliferating opcs leads to behavioral deficits which will be described. to identify the molecular mechanisms by which ng cells may react and influence other cells after brain injury, we isolated sox -gfp cells by facs either from the intact or the lesioned cerebral cortex at three days after stab wound injury. comparative genome-wide expression profiling revealed exciting candidates for opc function in the physiological and pathological brain. oligodendrocytes are the myelin forming cells of the central nervous system (cns) that enable fast salutatory signalling transduction and mediate trophic support and protection of axons. oligodendrocytes originate from a population of precursors cells (oligodendrocyte precursor cells, opcs) that are formed at distinct developmental stages. the biological process leading to opc differentiation involves a cascade of molecular events that eventually constitute the complex and multifaceted cellular program that determines the generation of mature oligodendrocytes. to identify the earliest detectable transcriptional correlates of differentiation we used a microarray approach investigating changes in gene expression occurring in primary rat opcs within the first hours after purification. amongst the most regulated factors that were detected was hairless (hr), a nuclear receptor co-repressor that has been associated with thyroid hormone signalling in the neonatal cns. validation of the microarray results by rt-qpcr, western blot and immunochemistry confirmed a rapid increase of hr expression during opc differentiation in media containing thyroid hormone. in the absence of thyroid hormone, hr expression was suppressed and associated with impaired opc differentiation. similarly, rnai mediated gene silencing of hr induced an impairment of opc differentiation demonstrating an important functional role of hr in the differentiation program. thyroid hormones regulate hr expression via the thyroid hormone receptors thra and thrb as rnai-mediated gene silencing of each of the receptors resulted in a decrease in hr expression. hr is known to negatively regulate rar-related orphan receptor alpha (rora) and vitamin d receptor (vdr). however, rnai-mediated gene silencing of rora and vdr did not change the course of opc differentiation. interestingly, an interaction between hr and histone de-acetylases (hdacs), of which hdac and are known regulators of opc differentiation, has been noted in the past. using an in situ protein-protein interaction assay we were able to demonstrate a direct interaction of hr with hdac and hdac in differentiating opcs. moreover, chip qpcr analysis revealed hr-hdac recruitment to the opc differentiation inhibitor hes . taken together these data show that thyroid hormone promotes opc differentiation via hr by modulating hdac and function. as iodine deficiency results in hypothyriodism our results give an example of a developmental disease that is triggered by environmental factors (the absence of iodine) causing epigenetic dysregulation. c. stagni, a.d. rivera, a. butt university of portsmouth, portsmouth, united kingdom astrocytes respond to all forms of cns insults by reactive astrogliosis, which involves changes in their molecular expression and morphology, and in most severe cases glial scar formation. reactive astrogliosis is potentially neuroprotective, but can also act as chemical and physical barrier to axon growth and repair. however, mechanisms underlying this process are still poorly understood. the enzyme glycogen synthase kinase -b (gsk- b) is a key regulator of many signalling pathways involved in cell proliferation, survival and differentiation, including wnt signalling. lithium is a potent inhibitor of gsk- and acts as a wnt mimetic to promote b-catenin-dependent transcriptional events. wnt signalling is altered following cns injury and in a range of neurodegenerative diseases, such as stroke, amyotrophic lateral sclerosis, and alzheimer's disease. notably, lithium is neuroprotective in these same diseases. here, we have examined the effects of wnt a and lithium on astrocytes of the adult mouse optic nerve, a typical cns white matter tract; all procedures were in accordance with the animals scientific procedures act ( ). we used transgenic mice aged postnatal day (p) inwhich gfap drives the expression of egfp. optic nerves (ons) were isolated with the retina intact and maintained in organotypic culture for days in vitro (div), in control medium, or medium containing lithium chloride, or wnt a. both agents resulted in a significant increase in the number of astrocytes, compared to controls (p < . , unrelated t-tests), but lithium and wnts a had markedly divergent effects on astrocyte morphology. in the presence of lithium, astrocytes became highly polarised, extending long thin processes that traversed the nerve perpendicularly to the long axis. in contrast, wnt a treatment resulted in the generation of morphologically simple astrocytes, with short, fine branching processes that extended radially from a centrally located cell body. genome wide microarray analysis indicated wnt a and lithium differentially altered chondroitin sulphate proteoglycans (cspgs), a family of extracellular matrix molecules highly expressed at cns injury sites. further experiments are required to define the molecular phenotypes of the novel astrocytes generated by these treatments, but the results provide evidence that wnt signalling regulates astrogenesis in situ and is therefore a potential molecular target to modulate astrogliosis. the contrasting effects of lithium suggest it is acting via other pathways, which require further study. studies of mouse schwann cell culture. the proliferation of scs is regulated by axonal signals and several growth factors. growth factors that are essential for rodent sc survival in culture include heregulin-b and forskolin. in the case of rat scs, these factors are also sufficient to allow a robust proliferation. in contrast, the proliferation of mousederived scs in culture requires the presence of other growth factors. we studied effect of several factors known to be expressed in scs after nerve injury on mouse sc proliferation. these included fibroblast growth factor type (fgf), pituitary extract (pe), epidermal growth factor, platelet-derived growth factor bb, and transforming growth factor b . we found that heregulin-b and forskolin are essential for mouse sc to survive in culture, fgf and pe were the best combination of growth factors promoting the mouse sc proliferation, and that neuronal axons provide effective mitogenic environment for mouse scs. the expression of the large myelin-associated glycoprotein in pns was studied in scs cultured alone and in cocultures of scs and drgns. when schwann cells are alone without axons, expression of the cytoplasmic domain of l-mag is seen in the nucleus of schwann cells. when schwann cells make contact with neuronal axons, the location of the expression shifts out of the nucleus to the perinuclear and cytoplasmic regions; the extracellular domain is not visible. until the schwann cells initiate the myelination programme. these results support the hypothesis that l-mag might have role in the regulation of the myelination programme in the pns. it has been postulated that trophic support provided by either stem cells or precursors is actuallyone of the most beneficial effects in the cell-based therapies. to address this issue, a comparison between the neuroprotective properties of opcs and the stem cells derived from warton jelly was carried out in the co-culture system with ischemically injured organotypic hippocampal slices. those two cell types were selected in respect of the advancement in their differentiation process. while the mesenchymal stem cells residing in warthon jelly are still pluripotent, the opcs are already the glia-committed precursors. to evaluate their neuroprotective properties, we set -up an ex vivo co-culture system of either stem cells or opcs isolated from neonatal rat brain with organotypic hippocampal slices subjected to a short oxygenglucose deprivation (ogd). the cell death, as well as the number of the newborn cells has been quantified in slices co-cultured for one week. a microarray analysis of a broad spectrum of trophic factors and cytokines expressed by opcs was performed for the purpose of finding the factor(s) contributing to the observed effect. three of them were selected for the subsequent blocking experiments with specificl antibodies to verify their influence on the injured tissue. the results obtained in the study prove that both cell types secrete soluble factors and are potent in exerting the neuroprotective effect in a paracrine-like manner, promoting cell survival and proliferation in damaged hippocampal slices. in conclusion, the observed advantageous qualities of stem cells and oligodendroglia-biased progenitors significantly contribute to anticipating a clinical improvement in cell replacement therapies. ng is a type i transmembrane glycoprotein and also known as chondroitin sulphate proteoglycan (cspg ). in the central nervous system ng -expressing cells have been identified as a novel type of glia with a strong potential to generate oligodendrocytes in the developing white matter. for temporally-controlled gene targeting of ng glia in vivo, we generated a mouse line in which the open reading frame of the tamoxifen-inducible form of cre recombinase (creert ) was inserted into the ng locus by homologous recombination. here, we investigated the differentiation potential of ng glia at different developmental stages of the forebrain. cre recombinase activity was induced at embryonic day . , postnatal day (p ), p , p , p and p by intraperitoneal tamoxifen injections into novel tgh(ng -creert ) mice crossbred to rosa -tdtomato and rosa -eyfp reporter lines that helped to identify ng glia and its progeny. recombination and cell identification were determined to days after the first tamoxifen injection. induction of recombination at embryonic stages revealed that embryonic ng cells mainly generated more ng glia and oligodendrocytes, however, a significant number of astrocytes could be detected as well. recombined cells were predominantly restricted to the ventral brain. in contrast, after tamoxifen injections at p and p recombined cells were widely found in all brain regions, thereby suggesting the presence of a distinct subpopulation of ng cells after birth. interestingly, only very few recombined astrocytes could be found, which are probably derived from few remaining embryonic ng glia. starting from p , ng glia stopped generating astrocytes, with the progeny largely restricted to the oligodendrocyte lineage. however, consistently, we found recombined neun cells with the morphology of neurons in the ventral cortex after administration of tamoxifen at p . even more of such cells were present in the whole cortex when cre activity was induced in adult mice. the morphology, the presence of neun immunoreactivity and our electrophysiological characterization that demonstrated bona fide action potentials clearly identify these cells as functional neurons. our results suggest that ng cells display a broad differentiation potential that appears to be highly age-dependent during development. brdu-assay) and the number of surviving cells (by counting dapi-stained nuclei). after we had observed, that the choice of the basal medium (neurobasal, rpmi or dmem) exerts a pronounced effect on opc proliferation we here investigated the effect of cell density on opc proliferation. starting from a small seeding density ( . cells/cm ) a tenfold increase in seeding density only resulted in a fourfold augmentation of the density of surviving cells after days in culture. using plating densities of . ; . and . cells/cm we observed, that an increase in seeding density led to a decrease in the proliferation rate, as tested with a brduassay and monitored after days in culture. similarly, the percentage of a b -positive, immature, cells was lower, the higher the cell density. we then tested whether this finding is due to proliferation inhibiting factors secreted in cultures with high seeding densities and whether the remaining microglia population could be involved. to this aim we seeded opcs at low density ( . cells/cm ) and cultivated them with the medium supernatant of cultures with high density ( . cells/ cm ). furthermore we seeded opcs in low density ( . cells/cm ) and co-cultivated them with an added density of microglia as counted in cultures of high density ( . cells/cm ). the resulting cell counts after days showed, that the percentage of brdu positive cells after cultivation with the supernatant was significantly reduced compared with control cultures, although the number of a b -positive cells was not influenced by this treatment. the addition of microglia to the cultures lead to a strong reduction of surviving cells, even though the percentage of brdu and a b positive cells were not altered. so apoptosis, necrosis or phagocytosis might be responsible for the reduction of surviving cells. our findings suggest that the reduction of surviving opcs in high density cultures is due to secreted factors inhibiting opc proliferation and that microglia could be involved in this effect. in neuropathological studies, it is important to detect microglial cells; however, a specific microglia marker has yet to be determined. [methods] in this study, we examined and compared the usefulness of various microglia markers, including human glucose transporter- (glut- ), mhc class ii, cd , and iba- . [results] all of the microglia markers consistently stained for microglia in paraffin sections fixed with formalin. cd staining of microglia in the gray matter was not as well defined as the other microglia markers. moreover, cd did not clearly stain the microglial processes. with regard to mhc class ii, it was difficult to observe the microglial processes in the white matter. in contrast, both glut- and iba- clearly stained for microglia in the gray and white matter, and provided detailed staining of microglial processes. although it has previously been shown that perivascular cells are negative for glut- , we found perivascular macrophages to be glut- positive. [conclusions] nevertheless, glut- and iba- may be considered as markers suitable for routine histopathological staining procedures. neurons and glia of the enteric nervous system (ens) originate from a pool of sox progenitors. in addition to being a scaffold for enteric neurons, enteric glial cells (egcs) have been suggested to function in mucosal integrity, neuroprotection, adult neurogenesis, neuro-immune interactions and synaptic transmission. currently, diverse glial populations are known to reside within the ens. however, whether, specific functions are carried out by specialized glial subtypes, with characteristic morphologies and molecular markers and within specific locations in the gut remains elusive. to address this question we used a repertoire of genetic tools, immunostaining and imaging techniques. for high single-cell resolution study of egcs, we mosaic analysis with double markers (madm) in conjunction with sox -cre. we analyzed adult gut tissue to characterize the different glial populations based on morphology, position in the ens and their relationship with the intestinal epithelial and vascular system. moreover, we used reporter mice (sox -icreer t ; r r-yfp and gfap-icreer t ; r r-yfp) to quantify the expression of three glial markers -gfap, s b and sox in myenteric plexus (mp) preparations of adult mice. our investigation on madm-mp preparations, revealed three subtypes of enteric glia in the mp of adult mice. type i and type ii glia, present in the primary plexus of mouse ens have been previously defined. in addition, we characterized a third subtype in the extraganglionic space of the mp of adult mice, at present termed as type iii-extraganglionic glia. furthermore, our analyses using d-imaging, on madm-gut sections allowed us to characterize the relationship of enteric glia located within the mucosa, with the intestinal epithelium and vascular system of the villi. our marker expression analysis showed that while the majority of glial cells co-expressed gfap and s b, a large fraction of glia within the myenteric ganglia was positive for either s b ($ %) or gfap ($ %). we also found that s b /gfapglia were abundant outside the ganglia. most glial cells co-expressed sox with gfap and s b yet a significant proportion ($ %) of mainly extra-ganglionic glia (type iii) expressed only sox . surprisingly, $ % of the cells that were s band sox displayed high gfap expression. use of gfap-icreer t ; r r-yfp mice showed that about $ % of yfp-labelled cells did not express gfap, implying dynamic regulation of gfap expression. overall, our high resolution studies reveal extensive heterogeneity of enteric glial cells in terms of morphology, position and expression of molecular markers suggesting differential functional roles. our future experiments will address whether different physiological roles of egcs can be assigned to specific subtypes. university of washington, seattle, united states m€ uller glia are the major type of glia in the retina, spanning its entire thickness. it is known that bmp signaling promotes astroglial differentiation in cortex, but its role in m€ uller glial development in the retina has not been studied. the purpose of this study was to investigate the role of bmp-smad / / signaling in m€ uller glial development. c bl/ mouse retinas were collected at various ages between postnatal day (p ) and p in order to analyze the expression and activation of smad / / . smad / / was transiently but robustly activated in the inner nuclear layer, including developing m€ uller glia between p -p . the timing of this transient activation of smad / / corresponds with the timing of m€ uller glial differentiation and maturation. to test the hypothesis that activation of bmp-smad / / signaling is essential for m€ uller glial development, retinas were explanted at p or p and treated with bmp or a bmp inhibitor dorsomorphin (dm) for days in order to activate or inhibit smad / / , respectively. when p and p retinal explants were treated with dm for days in vitro, there was a significant reduction in m€ uller glial markers (cralbp, gs, sox , and sox ). smad / / activation and cralbp expression were also significantly attenuated in vivo day after intraocular injection of a bmp inhibitor dm at p . our data suggest that activation of bmp-smad / / signaling is necessary for the development of m€ uller glia. this work was supported by r ey to tar. huazhong university of science and technology, wuhan, china rho-associated kinase (rock) has been identified as an important regulator of proliferation and cell cycle progression in a number of cell types. although its effects on astrocyte proliferation have not been well characterized, rock has been reported to play important roles in gap junction formation, morphology, and migration of astrocytes. in the present study, our aim was to investigate the effect of rock inhibition by [( )-(r)trans- -( -aminoethyl)-n-( -pyridyl) cyclohexanecarboxamide dihydrochloride] (y ) on proliferation and dna synthesis in cultured astrocytes from rat spinal cord and the possible mechanism involved. western blots showed that treatment of astrocytes with y increased their expression of cyclin d , cdk , and cyclin e, thereby causing cell cycle progression. furthermore, y -induced astrocyte proliferation was mediated through the extracellular-signalregulated kinase signaling cascade. these results indicate the importance of rock in astrocyte proliferation. here we used cell transplantation experiments to determine to which extent this differential behavior is regulated by environmental signals differing between the wm and gm, or intrinsic fate determinants restricted to one or the other cell population. these experiments indicate that the wm promotes differentiation of opcs to mature oligodendrocytes, as the rate of differentiation of gm cells increased when exposed to this environment. interestingly, cells derived from the wm retained their higher differentiation rate also in a less supportive environment like the gm. these results reveal not only important environmental differences for opc maturation, but also support the concept of intrinsic heterogeneity among adult opcs, persisting even when exposed to more inhibitory environments. thus, this work provides the basis to identify molecular pathways regulated by distinct niche/environmental signals and involved in the heterogeneity of adult opcs. multiple sclerosis (ms) is a chronic inflammatory and neurodegenerative demyelinating disease of the central nervous system (cns) characterized by inflammation, which leads to formation of demyelinating areas due to loss of oligodendrocytes, astrogliosis and, finally, axonal degeneration. different studies have suggested that - -cyclic adenosine monophosphate (camp) levels might play an important role in neuroprotection and neuroinflammatory response so the modulation of this nucleotide intracellular levels might control the neuroinflammatory pathological process and, consequently, to delay the progression of ms. intracellular camp levels depend on its synthesis by adenylyl cyclases, and on its degradation by cyclic nucleotide , -phosphodiesterases (pdes). specific inhibitors for the different isoforms of pdes family, particularly camp-specific pde, emerge as putative treatments of this kind of disease. pde has emerged as a new therapeutic target, not only for a variety of immunological and immunodeficiency conditions to alleviate chronic inflammation, but also for several neurodegenerative disorders, including ms, in which both immune system and cns are implicated. in the present work, we have detected the expression of pde in oligodendrocyte precursor cells (opcs) isolated from cerebral cortex of p and p mice and from adult human samples derived from neurosurgery of epilepsy. opcs are abundant in the mice and human cns during development but they also represent - % of the total number of cells in the adult cns, where they serve as a source of new oligodendrocytes to replace those which die. however, in ms these endogenous opcs are not able to effectively replace dead oligodendrocytes. in vitro, we have demonstrated that two newly-developed pde selective inhibitors (tc . and vp . ) favour opc survival and differentiation towards mature oligodendrocytes, being both more effective than the commercial pde inhibitor brl- . erk intracellular pathway has been identified as a key in these cellular processes related with the camp/pka/creb pathway. moreover, we have observed that both specific inhibitors (vp . and tc . ) increase the differentiation of human opcs. these findings, combined with the already known anti-inflammatory effect carried out by these pde inhibitors, point them as potential therapeutic agents to treat ms. the knowledge of factors that affect the biology of murine opcs and even more, human opcs, are critical for possible remyelination in this kind of pathologies. their role in cns remyelination has not been fully investigated. previous studies using genetic fate mapping techniques revealed their recruitment into injured spinal cord and differentiation into astrocytes and oligodendrocytes. in this study, we used aninducible cre-floxed-stop-rosa -yfp mediated lineage tracing strategy to characterize the fate of foxj expressing ependymal cells during remyelination of toxin-induced demyelination. we found that in unlesioned spinal cord, foxj labelled ependymal cells. however, it also labeled a population of cells in the spinal roots with immnochemical features of fibroblasts. following lysolecithin-induced demyelination in the dorsal funiculus, there were few yfp expressing cells in the lesion area at and days post lesion (dpl) indicating that the ependymal cells were not recruited in demyelinated area. however, in ventral white matter lesions, which were adjacent to damaged nerve roots, many cells were detected expressing yfp. the distribution of yfp positive cells overlapped that of remyelinating schwann cells and a number of yfp expressing cells colocalised with mature schwann cell marker periaxin. these data revealed a foxj expressing population in peripheral nerve which migrates into demyelinated lesions andcontributes to cns remyelinaiton. the generation of myelinating cells from multipotential neural stem cells in the cns requires the initiation of specific gene expression programs in oligodendrocytes (ols). we reasoned that micrornas (mirnas) could play an important role in this process by regulating genes crucial for ol development. here we identified mir- a as one of the highly enriched mirnas in oligodendrocyte precursor cells (opcs), overexpression of which in either neural progenitor cells (npcs) or embryonic mouse cortex promoted the generation of ol lineage cells. blocking the function of mir- a in differentiating npcs led to a reduction in ol number and an expansion of neuronal populations simultaneously. we also found that overexpression of this mirna in purified opc cultures promoted cell proliferation and inhibited further maturation. in addition, mir- a might exert the effects just mentioned partially by directly repressing proneuronal differentiation factors including pax and neurod , or prool genes involved in oligodendrocyte maturation. these results suggest that mirna pathway is essential in determining cell fate commitment for ols and thus providing a new strategy for modulating this process in ol loss diseases. a. guzman de la fuente , j. huang new sheaths can be regenerated following the recruitment of adult neural progenitor cells (opc) into areas of injury and their differentiation into myelinating oligodendrocytes. although this regenerative process (called remyelination) occurs efficiently in the early stages of multiple sclerosis, its efficiency gradually declines leaving axons demyelinated and vulnerable to degeneration. several signalling pathways are involved in regulating opc differentiation and subsequent myelination. understanding their mechanisms is necessary to design regenerative therapies to overcome remyelination failure. we have identified rxrc as a key positive regulator of opc differentiation and remyelination (huang et al., ). rxrc is a nuclear receptor forms a heterodimer with other nuclear receptors to activate its downstream signalling cascades. here we describe rxrc binding partners expressed in both opc and oligodendrocytes, and the binding of vdr, rarb and pparc to rxrc within oligodendrocyte lineage cells by coimmunoprecipitation. vdr-rxrc heterodimer is necessary for the oligodendrocyte differentiation. treatment of oligodendrocytes with vdr antagonist inhibits opc differentiation and abrogates the effect of -cis retinoic acid, an rxrc agonist, in opc differentiation. our data suggests a model in which rxrc is an essential anchor for different nuclear receptors, including vdr, with a shifting and complex rxrc signaling in oligodendrocyte lineage cells. cleavage of type i membrane proteins by a-and c-secretases to yield biologically active fragments is best exemplified for the evolutionarily conserved protein notch. here we show that the protein ng , a type i membrane protein which has homologues in mouse (ng ) and drosophila (kontiki) and is expressed by multiple immature cell types including glia, is processed in a similar fashion to notch. in both mouse and drosophila this generates a major kd shedded ectodomain, a c-terminal fragment (ctf) and a small intracellular domain (icd). the ng icd is present after overexpression in cells of biochemical isolates of nuclear fractions, and can be visualized in the nucleus by immunofluorescence of cultured murine glial cells. in glial nuclei of developing drosophila larva, a striking intranuclear staining of the kontiki icd is observed. levels of expression of ng mrna and protein in mouse glial cells are regulated by the neurotransmitter glutamate and overexpression of cleaved fragments modulates protein expression in murine glial cells. these results demonstrate that cleavage of ng takes place in mouse and drosophila glia and that this generates fragments with signaling properties. modulation of ng expression by glutamate provides a way for neuronal activity to regulate ng signalling. our goal is to understand the glial signaling that controls inflammatory responses in the central nervous system (cns). this glial response can play both a detrimental and beneficial role. toll like receptor (tlr) signaling is implicated in responses to pathogens or endogenous signals. tlr signaling mediates immune response by inducing cytokines, including type i interferons (ifn). ifn-beta, a member of the type i ifn family, is a first-line therapeutic for multiple sclerosis. type i ifns signal through a common receptor, ifnar. the aim of the present study was to investigate the in vivo response of microglia and astrocytes to cns administration of tlr ligand/agonist, and to examine whether this response involves type i ifn signaling. mice were administered tlr ligands/agonists by injection to the cisterna magna. we analyzed leukocyte entry to the cns by flow cytometry, and their localization by immunostaining. the induction of ifn-beta was examined by in vivo imaging of an ifn-beta reporter mouse. astrocytes and microglia were sorted by facs and gene expression was measured using quantitative real time-pcr. injection of ligands/agonists for tlr , and resulted in infiltration of cd leukocytes after hrs, most strongly by tlr . immunostaining showed parenchymal localization of infiltrating cells in cerebellum. facs sorted astrocytes expressed equivalent levels of tlr mrna to microglia but lower levels of tlr or . astrocytes were induced by tlr signaling to express interferon regulatory factor , which regulates the induction of type i ifn. the induction of ifnbeta in cns in response to tlr signaling was verified in ifn-beta reporter mice. tlr , and signaling led to increased levels of mrna for glial cxcl . together these results suggest the involvement of type i ifn signaling. however, unlike cxcl gene expression, that was dependent on ifnar signaling, tlr -induced leukocyte infiltration was not affected in ifnar deficient mice. these studies point to a role for tlr signaling in the innate glial response that regulates cns inflammation. microglial phagocytosis plays a key role in neuroprotective and neurodegenerative responses of the innate immune system in the brain. here we investigated the regulatory function of the signaling protein phosphoinositide -kinasec (pi kc) in phagocytosis of bacteria and zymosan particles by mouse brain microglia in vitro and in vivo. using genetic and pharmacological approaches our data revealed pi kc as an essential mediator of microglial phagocytosis. unexpectedly, microglia expressing lipid kinase deficient mutant pi kc exhibited similar phagocytosis as wild-type cells. detailed analysis disclosed pi kc dependent stimulation of camp phosphodiesterase as a crucial mediator of phagocytosis. both stimulation of protein kinase a and epac were shown to convey inhibitory effects of camp on phagocytosis. together our findings indicate pi kc-dependent suppression of camp signaling as an indispensible regulatory element of microglial phagocytosis. v. kovaleva, e. berezhnaya, m. rudkovskii, a. uzdensky southern federal university, rostov-on-don, russian federation photodynamic therapy (pdt) is currently used in oncology, particularly, in treatment of brain tumors. the possible role of no-mediated signaling in photodynamic injury and protection of neurons and surrounding glial cells (gc) was studied. crayfish stretch receptor consisting of a single neuron enveloped by gc was photosensitized with alumophthalocyanine photosens ( nm, min incubation) and irradiated with laser diode ( nm, . w/cm ). application of no generators notably mm sodium nitroprusside and mm nonoate reduced pdtinduced necrosis of gc and showed the same tendency in neuronal necrosis. nonoate significantly increased pdt-induced apoptosis of gc. inhibitor of neuronal no-synthase l-name ( mm) significantly increased the percentage of necrotic glial cells after pdt but did not influence neuronal necrosis. this confirmed the anti-necrotic effect of no in glial cells and involvement of neuronal no synthase in protection of glial cells. mm l-name and mm l-nna (another inhibitor of neuronal no-synthase) as well as lm s-methhylisothioharnstoff sulfat (inhibitor of inducible no synthase) protected gc from pdtinduced apoptosis. therefore no may be involved in pdt-induced apoptosis of glial cells. inhibition of no-activated protein kinase g with lm kt decreased the percentage of necrotic glial cells but not neurons. therefore protein kinase g appeared to be involved in pdtinduced necrosis of gc, possibly independently on no. kt also increased the level of apoptosis of gc indicating the anti-apoptotic role of protein kinase g. thus no is involved in regulation of pdt-induced necrosis of neurons and glial cells as well as in apoptosis of glial cells. g. tyzack, t. cymes, n. lau, a. lakatos university of cambridge, cambridge, united kingdom background and question. emerging evidence suggests that signalling by damaged neurones leads to reactive astrocyte response that may have a fundamental impact on synaptic reorganisation. soluble factors, such as cytokines, have been described to induce such astrocyte activation. however, rapid contact dependent signalling between damaged neurones and astrocytes is also a possibility. in light of recent findings of ephb upregulation on injured neurons closely surrounded by reactive astrocytes, we hypothesized that ephb can signal to ephrin-b expressing astrocytes. methods. to test this in simplified in vitro assays, purified astrocyte cultures were treated with clustered-ephb -fc, and the characteristic features of glial activation, such as cytoskeletal protein expression, stat phosphorylation, and p-stat nuclear transfer were monitored. results. we have demonstrated that ) astrocytes express ephrin-b , ) ephb triggers a two-fold increase in gfap and ezrin expression, ) ephb induces both stat phosphorylation and nuclear transfer by a three-fold value, and ) all effects were significantly diminished by knocking down ephrin-b in astrocytes. conclusions. our data show that ephb induces astrocyte activation by triggering ephrinb -mediated reverse signalling via stat activation. this work therefore suggests a potential novel route in neuron-astrocyte communication after injury. a further characterisation of this signalling pathway may provide a better understanding of mechanisms underlying glial activation and its role in plasticity. n. kramann , r. pf€ ortner , u.-k. hanisch , k. hagemeier , t. kuhlmann , w. br€ uck , c. wegner university medical center g€ ottingen, department of neuropathology, g€ ottingen, germany university hospital m€ unster, institute of neuropathology, m€ unster, germany introduction: laquinimod (laq) is an oral well-tolerated molecule that has been shown to reduce brain atrophy, disability progression and relapse rate in patients with relapsing-remitting multiple sclerosis. recent experimental data indicate that laq minimizes demyelination, inflammation and axonal damage in mice with cuprizone challenge. aim: since astrocytic nfjb activation plays a crucial role in cuprizone-induced demyelination, we investigated the effects of laq on cns cells in vitro and in vivo. methods: in vitro experiments used primary cells to test effects of laq on oligodendroglial survival as well as on cytokine secretion and nfjb activation in astrocytes and microglia. primary mouse astrocytes and microglia were pre-treated with , . and . mm laq and stimulated by pro-inflammatory cytokines. a dual-luciferase reporter assay was used to measure nfjb activity. for in vivo experiments, -week-old male c bl/ j mice were challenged with . % cuprizone and treated daily with laq ( mg/ kg) or water. to assess astrocytic and microglial nfkb activation in vivo, nuclear translocation of nfjb p was assessed in astrocytes and microglia by double immunofluorescence with antibodies against p and iba or gfap. results: in vitro, astrocytic but not microglial nfjb activation was markedly reduced by laq as evidenced by nfjb reporter assay. in astrocytes, pre-treatment with . and . mm laq significantly reduced the induced nfjb activity after tnfa stimulation compared to stimulated controls. pre-treatment with . mm laq also significantly reduced nfjb activation after stimulation with the combination of il- b and ifnc compared to untreated stimulated controls. oligodendroglial viability and survival were not affected by laq treatment. to confirm the in vivo relevance of these findings, we also examined astrocytic and microglial p translocation in mice with and without laq treatment after cuprizone challenge. the proportion of astrocytes with nuclear p immunoreactivity was significantly reduced in laqtreated mice ( . % . %) compared to untreated controls ( . % . %). microglia did not display marked translocation of nfjb p after cuprizone challenge. conclusion: our data indicate that laq prevents cuprizoneinduced demyelination by attenuating astrocytic nfjb activation. in vitro, laq reduced the astrocytic nfjb activation by up to % as evidenced by nfjb reporter assay. similar quantitative findings were obtained in vivo when laq treatment also led to a % reduction of astrocytes with nfjb activation, as evidenced by nuclear translocation of p . these findings suggest that targeting the astrocytic nfjb pathway might have therapeutic effects in demyelinating cns disorders. . we asked whether brain-derived neurotrophic factor (bdnf), known to be highly expressed in a circadian manner in scn, might be involved in the mechanisms governing these astroglial rearrangements. using an analog-sensitive kinase allele murine model (trkb f a ) and semi-quantitative electron microscopy, we found that the pharmacological blockade of the tropomyosin-related kinase receptor type b (trkb), the high affinity receptor of bdnf, abolished the day/night changes in glial coverage of vip dendrites. the bdnf/trkb signaling pathway therefore exerts a permissive role on the ultrastructural rearrangements that occur in scn across the day/night cycle. in contrast, the extent of glial coverage of non-vip dendrites was not different at daytime and nighttime in trkb f a mice submitted to trkb inactivation or not receiving any pharmacological treatment. considering the well-established role of bdnf in the clock's photic synchronization process, these dataprovide strong support to the view that the daily astroglial rearrangements in scn could be involved in the photic entrainment of circadian rhythms. above all, they highly suggest that they are necessary for the modulatory action of bdnf on the scn responses to light. features of this form of neurodegenerative ataxia, including a striking reduction in lifespan when polyq atrophins are expressed in the glia. a sensible hypothesis is that affected glia will act non-autonomously on neurons as in other neurodegenerative pathologies. this is an emerging field that yields new and important therapeutic possibilities. we now wish to elucidate the mechanisms through which degenerating glia impairs organism functions, and identify genes involved cell-autonomously and non-autonomously in generating this aspect of the pathology. first, we investigated the impact of glial polyq atrophin on lifespan. using different glia marker, we have demonstrated that the expression of polyq atrophin in glia, or specifically in astrocyte-like cortex glia, has a dramatic effect on drosophila lifespan while brood brain barrier glia do not have any phenotype. we will then investigate the effect of other subtypes of glia looking at lifespan and motility to identify the relevance of each subtype in drpla. second, to identify new regulators of gliopathology we will conduct unbiased genetic screens for mutations in neurons that modify the lifespan of drpla flies, expressing polyq atrophin within the glia. our studies will be paramount in deciphering the complex glia-neurons interactions in ataxias and may be beneficial for several ataxias. they will open up new avenues for therapies aimed at targeting glianeuron interactions during disease progression. in alzheimer's disease, microglial clearance of beta-amyloid (ab) is considered neuroprotective. whether ab uptake regulates microglial cell responsiveness, and whether macrophages participate, remains unclear. here we investigated whether in vivo phagocytosis of endogenously-produced ab disturbs the turnover of microglia and macrophages by changing rates of cellular proliferation and apoptosis. using app swe /ps de tg mice, we show that plaque-associated microglia and cd b cd high macrophages bind and ingest endogenously-produced ab in vivo. plaqueassociated microglia and cd leukocyte-like cells were also observed in brains from patients with alzheimer's disease. phagocytic microglia and macrophages had a reduced rate of proliferation compared to abcells. instead, high proportions of apoptotic annexin v microglia and macrophages were found in aged app/ps tg mice. phagocytic microglia and macrophages had greater caspase activity and increased p expression after in vivo uptake of ab, than cells that did not phagocytose ab. in vitro, we found that ab uptake induced caspase activation in microglia, whereas blocking ab uptake triggered apoptosis-induced cell death in macrophages. our data demonstrate that ab uptake impairs microglial/ macrophage viability and responsiveness. amyloid clearance may fail as phagocytic microglia and macrophages undergo ab-induced apoptosis. f. michetti, s. ceccariglia, a. d'altocolle, f. pizzolante, m. barba, a. delf a, c. gangitano universit a cattolica del s. cuore, rome, italy trimethyltin (tmt) is a neurotoxicant producing neuronal degeneration in the mammalian central nervous system , especially in the hippocampus (for reviews, , ). magnetic resonance imaging (mri) investigation in tmt-treated rats has evidenced dilation of lateral ventricles, possibly correlated to alterations in blood brain barrier permeability. we have investigated in the hippocampus and cortex of rats the expression of aquaporin (aqp ), a glial water channel protein which is regarded to play a role in brain oedematous conditions, to explore the molecular mechanisms involved in the phenomenon. tmt-treated aqp expression was tested both by real-time pcr and western blotting analysis in hippocampus and cortex homogenates. to confirm molecular results and visualize the aqp cell distribution, double-label immunofluorescence for aqp and gfap was performed. real-time pcr and western blotting data show a significant upregulation of aqp starting from days of tmt treatment in the hippocampus and, at a lower degree, in the cortex. accordingly, the immunofluorescence shows an intense astrogliosis and aqp immunoreactivity diffusely pronounced in the hippocampal and cortex areas starting from days after intoxication. aqp immunolabelling was localized in astrocytic end-feet encircling the blood vessels, as expected. the study of the rhodamine b fluorescent tracer, intraperitoneally administered, also revealed an intense vascular reaction, characterized by hypertrophic vessels with abnormal course and dimensions in the brain of tmt-treated rats, indicating a vascular involvement in the tmt-induced neurodegenerative processes. aqp over-expression and the concurrent astrogliosis occurring in the brain of tmt-treated rats might be candidated to play a role in alterations of vascular permeability and brain oedema formation evidenced by mri studies. amyotrophic lateral sclerosis (als) is a neurodegenerative disease affecting predominantly motoneurons but with a particular pathological role of non-neuronal cells. previous research suggested that increased concentration of extracellular potassium could lead to motoneuron dysfunction. the na /k -atpase should regulate the homeostasis of potassium ions and thus help maintain regular neuronal activity. this homeostasis may be regulated by na /k -atpase in both neurons and astrocytes. in order to reveal the possible role of the na /k -atpase in als pathology, we explored the expression of the alpha catalytic subunit of na /k -atpase in neurons and astrocytes from different brain regions of the sod g a rat model of als. labeling immunofluorescently the alpha isoform of the na /k -atpase revealed a decreased signal in the trigeminal nucleus and cerebellum of the als rat. double immunofluorecence with neun for neurons showed that the reduced alpha was on the neuronal surface in the trigeminal nucleus. in addition, the alpha ring-like staining of cerebellar granule neurons was lower in als as compared to the wild type rat. in comparison to neurons, astrocytes from the wild type rat were characterized with a lower expression of alpha . in the examined brain regions we did not observe a prominent difference in alpha expression between astrocytes of wild type and als rats. based on the findings of this study the prominent reduction of na /k -atpase level observed in neurons and not in astrocytes could contribute to further understaning of impaired ion homeostasis affecting proper neuronal physiology in als. the role of glial na /k -atpase needs to be further studied with markers of other alpha subunit isoforms. although commonly used as nutritional supplements, excessive intake of bcaas might favour the establishment of neurotoxic conditions as indicated by the severe neurological symptoms characterising inherited disorders of bcaa catabolism such as maple syrup urine disease (msud). recent evidence indicates that bcaas induce excitotoxicity through mechanisms that require the presence of astrocytes. in the present study, we evaluated the effects of bcaas on microglia, the main immune cells of the brain. as an experimental model we used primary microglial cells harvested from mixed glial cultures that had been kept in normal or high bcaa medium (h-bcaa). we show that h-bcaa microglial cells exhibit a peculiar phenotype characterized by a partial skewing toward the m state, with enhanced il- expression and phagocytic activity but also increased free radical generation and decreased neuroprotective functions. we suggest that such an intermediate m /m phenotype might result in a less efficient microglial response, which would promote the establishment of a low grade chronic inflammation and increase the likelihood of neurodegeneration. although based on in vitro evidence, our study adds on to an increasing literature indicating that the increasing use of dietary integrators might deserve consideration for the possible drawbacks. in addition to excitotoxicity, the altered immune profile of microglia might represent a further mechanism by which bcaas might turn into toxicants and facilitate neurodegeneration. the proteolipid protein gene (plp ), located on the x-chromosome, undergoes alternative splicing to produce the most abundant proteins of central nervous system myelin: plp and dm . related to the extent of myelin defect, mutations in the plp gene in humans are associated with a spectrum of x-linked disorders, from the severe pelizaeus-merzbacher disease (pmd), to the mild form spastic paraplegia type (spg ). phenotype-genotype correlation exists, large duplications of plp gene are predominantly found in pmd and null mutations in spg . plp is almost % conserved across mammalian species, and the large spectrum of phenotype severity is also found plp transgenic mice. experiments on these models have contributed to our understanding in the pathophysiology of plp related disorders. in one hand, mice with extra copies of the plp gene ( plptg mice) have neurological symptoms and cns pathology similar to those found in pmd patients while mice with plp gene inactivation (plp null mice) share similarities with spg patients. then, these two mouse models represent very useful tools to evaluate the efficacy of a therapeutic strategy for the severe and the mild form of the plp related disorders on the neuropathological and behavioral aspects. olesoxime (tro ), a cholesterol-like small molecule, has been shown to rescue motor neurons from cell death and to promote axonal regeneration both in vitro and in vivo. more recently, olesoxime has also been shown to promote central remyelination by accelerating oligodendrocyte maturation both in vitro and in vivo. altogether, these results strongly suggest that olesoxime may be a potential drug candidate for the treatment of white matter neurodegenerative disorders and notably to plp related disorders. here we treated plptg mice with olesoxime for months starting at birth, as well as plp null mice from to months of age (presymptomatic treatment) or from to months of age (symptomatic treatment). behavioral and neuropathological analysis revealed that olesoxime failed to correct the development of symptoms in mice modeling the severe form of plp related disorders. on the contrary, treatment of plp null mice with olesoxime resulted in a -month delay in the onset of abnormal behaviors related to anxiety and in the slowing of central nerve conduction. these beneficial effects are observed only when the drug is administered before disease onset. treatment with olesoxime also reduces the effect of aging on rotarod performance decline in both wt and plp null old mice. altogether these results suggest that olesoxime could be of interest as a treatment to delay the onset of symptoms in plp related disorders provided that patients suffer from a mild form of the disease and are treated before the establishment of symptoms. objectives: glial cells play an important role in amyloid-beta (aß) clearance, however little is known about the cellular routes involved in internalization of different aß aggregation forms by adult human microglia and astrocytes. therefore, we evaluated the uptake mechanism of aß - by primary human adult microglia and astrocytes isolated from brain tissue of non-demented control and ad cases. methods: cells were preincubated for hour with different inhibitors, before exposure to fluorescence labelled (fam) oligomeric and fibrillar forms of synthetic aß. the number of amyloid positive cells was quantified by flow cytometry. inhibitors of various pathways tested, include cytochalasin b (inhibits actin polymerization; general endocytosis inhibitor), nocadozole (inhibits tubulin depolymerization; general endocytosis inhibitor) and fucoidan (scavenger receptor inhibitor). results: cytochalasin b inhibited aß fibril uptake ( % reduction, p < . ) and to a lesser extent oligomer ( % reduction, p < . ) uptake in microglia, whereas no effect was seen on aß uptake by astrocytes. in contrast, nocodazole was found to inhibit aß fibril uptake ( % reduction, p < . ) and oligomer ( % reduction, p < . ) uptake in astrocytes, whereas no effect was seen on aß uptake by the microglia. interestingly, fucoidan prevented aß uptake in both astrocytes (oligomers: % (p < . ); fibrils: % (p . ) and microglia (oligomers: % (p < . ); fibrils: % (p < . )). conclusions: these data indicate that both human astrocytes and microglia use scavenger receptors to internalize aß oligomers and fibrils. whether different types of scavenger receptors are involved in aß uptake by astrocytes and microglia will be further investigated. tel-aviv university, tel aviv, israel alzheimer's disease (ad) accounts for the majority of cases of dementia worldwide, affecting over % of the population over . the disease is characterized by a progressive memory loss, cognitive deterioration, behavioral disorders, deposit of beta-amyloid (ab) peptides, neurofibrillary tangles, reactive astrocytosis and activation of microglia cells. mutations in the genes that encode app (amyloid precursor protein) and components of the proteases that generate amyloid beta cause familial forms of ad. microglia, the resident immune cells of the central nervous system (cns) were suggested to play both beneficial and harmful roles in the course of the disease. therefore, modulating the microglial response is being considered as a promising approach for the treatment of this disease. we have recently shown using in vitro and in vivo systems that the ectoenzyme cd , regulates microglial activation. in view of the significant role of activated microglia in ad and the important role played by cd in microglial activation, we hypothesized that cd deficiency would affect the course of the disease. in order to test this hypothesis, we implicated the app swe ps de transgenic mouse model for ad, and compared the cognitive performance (as evident by behavioral studies) of aged app swe ps de mice, to app swe ps de cd -/mice. in addition, abload, accumulation of microglia and astrocytes was assessed in the brains of these mice. our preliminary results show that cd deficiency has a neuroprotective role in this ad mouse model as indicated by improvement in cognitive performance and dramatic reduction in ab load. oxidative stress induced by reactive oxygen species (ros) is associated with various pathological conditions, including aging, neurodegenerative disorders, traumatic and ischemic insults. the mechanism by which the gap junction protein connexin (cx ) contributes to oxidative stress-induced cell death or the "rescue" of the dying cells is still unclear. cx is highly expressed in astrocytes. we have previously shown that astrocytic cx is critical for neuroprotection during ischemic insults in vivo. to investigate the protective effect of cx in oxidative stress pathways, we induced oxidative stress in wild-type and cx knockout astrocytes using hydrogen peroxide (h o ). cx knockout astrocytes exhibited increased h o -induced cell death as measured by the loss of membrane integrity using the dye rhodamine b dextran, supporting a cell protective effect of cx ; this effect was not observed in wild-type astrocytes. to examine the involvement of cx in h o -induced cell death, we studied the expression and distribution of cx in response to h o in wild-type astrocytes. h o treatment altered the expression level and phosphorylation of cx . this was accompanied with changes in cx distribution and gap junction plaque formation. hemichannel activity was measured using dye uptake. h o ( . mm) increased dye uptake, an effect that was blocked by gap (syntethic mimetic peptide, mm) or carbenoxolone ( mm), two connexin hemichannels blockers. however, h o treatment did not result in any measureable gap junction activity in cx knockout cells as measured by a scrape-loading dye transfer assay. these findings suggests that gap junction activity contributes to cx -mediated protection in response to ros. one of the receptors found on microglia is the triggering receptor expressed on myeloid cells (trem ), which signals intracellularly via an adapter molecule referred to as tyro protein tyrosine kinase-binding protein (tyrobp, known also as dap ). in humans, loss of function of either trem or tyrobp leads to nasu-hakola disease, which is characterized by neuroinflammation and bone cysts. by using a trem knock-out (ko) mouse line, we provided further insights on the trem function in neurodegenerative diseases. first, we observed mild signs of dopaminergic neurodegeneration and a slight increase in microglial iba -immunoreactivity in different brain regions of months trem ko mice compared with the wild type (wt) control animals. however, the transcription levels of inflammatory cytokines (tnfa, il b) or inducible nitric oxide synthase (inos) were unaffected in mice of different ages ( , or months). to shorten the time required for neurodegeneration to occur, we have injected the mice intraperitoneally with lipopolysaccharides (lps) on four consecutive days and analyzed the dopaminergic neurodegeneration in the substantia nigra after a three weeks period. while the quantification of dopaminergic neurons in the substantia nigra showed a slight decrease in number for wt animals, a higher loss was recorded in the trem ko mice after systemic lps challenge. concomitantly, significant increase of microglia activation was detected in the lpstreated trem ko mice compared with lps-treated wt animals. our results are pointing towards a neuroprotective effect of the microglial trem receptor under chronic and systemic inflammatory conditions. increasing evidence suggest that brain energy metabolism is severely altered in multiple sclerosis patients, which may contribute to the process of neurodegeneration. imaging studies reveal a disturbed cerebral perfusion and glucose uptake and metabolism in ms patients. moreover mitochondrial dysfunction is recognized to play a crucial role in ms pathology. to date little is known about the distribution of glucose transporters (gluts), key molecules for the cellular uptake of glucose, in human brain and their role in the pathogenesis of ms. therefore, the aim of this study was to provide a complete overview of the distribution and expression levels of glucose transporters in control and ms brain. our data so far show that mrna levels of glut , and as well as their regulators hypoxia inducible factor (hif)- a, hif- a and peroxisome proliferator-activated receptor gamma coactivator (pgc)- a, are increased in normal appearing white matter (nawm) of ms patients compared to controls. immunohistochemical analysis revealed that brain endothelial cells not only express glut but also glut and glut . glut , and are present in resting microglia and highly expressed in activated microglia and macrophages in active ms lesions. interestingly reactive astrocytes expressed increased levels of glut and glut compared to control, in addition glut positive astrocyes were observed in ms brain. axons expressed high levels of glut in active ms lesions, which was evidently reduced in chronic lesions. strikingly, all glucose transporters studied were down regulated in chronic ms lesions. together, these data emphasize the extent to which glucose metabolism appears to be affected in all stages of ms. a better understanding of these metabolic changes, especially in the nawm and chronic stage of the disease, is essential to develop strategies to improve neuronal survival and to gain more insight in the mechanisms underlying lesion formation. pericytes are vascular mural cells which are enriched within the capillary basement membranes of the brain. recent studies have provided evidence that pericytes regulate the blood-brain barrier (bbb) and modulate cerebral blood flow. neurovascular dysfunction and bbb damage are hallmarks of alzheimer's disease (ad), but the role of pericytes in ad pathogenesis is still unclear. we have examined post-mortem samples of middle frontal gyrus in collaboration with the university of cambridge after ethical approval by the local irb. ten cases with neither history of neurological disorder nor evidence of neuropathology were compared with ad cases provided by the neurological foundation of the new zealand human brain bank. we performed immunostainings for laminin and plateletderived growth factor receptor (pdgfr)-b which mark vessels and pericytes, respectively. we also stained for ab - for evidence of amyloid pathology. quantification of pericyte and vessel density was performed using stereological methods, which are considered as gold standard in morphometric quantification. the spaceballs method was used to measure the density of capillaries. pericyte density was assessed using the optical fractionator. the age of the subjects did not differ between the control and ad groups ( , , vs. , , years). the cortex of ad cases contained more capillary vessels than controls (mean sd: vs. mm/mm ; p < , ; fig. a ). the capillary density increased in correlation to the total plaque load (r , ; p < , ). in contrast, we could not observe a significant difference in the number of pericytes per mm capillary length between control and ad cases ( , , vs. , , cells/mm; p , ; fig. b ). relative to tissue volume, the cortex of ad individuals showed a tendency to contain more pericytes than controls ( vs. cells/mm ; p , ; fig. c ). this is the first study using state-of-the-art techniques to investigate the relationship between capillaries and pericytes in ad brains. impaired clearance of b amyloid across the pericyte-regulated bbb and deficient microvascular regulation of blood flow may contribute causally to ad pathogenesis. in consequence, loss of pericytes could contribute to disease progression. however, our results show that ad cortical pathology is characterized by an increase in the density of capillary vessels without deficit in pericyte coverage. thus, they do not substantiate the notion that pericyte loss is a significative feature of ad. m. noack, j. leyk, c. richter-landsberg carl von ossietzky universit€ at oldenburg, oldenburg, germany histone deacetylase (hdac ), a member of the class ii hdacs, is a unique cytoplasmic a-tubulin deacetylase that interacts with the microtubule (mt)-associated protein tau. in healthy human brain six tau isoforms are expressed generated by alternative mrna splicing. these contain either three ( r-tau) or four ( r-tau) microtubule binding repeats regulating mt assembly and stability. tau hyperphosphorylation impairs its mt-binding activity. tau deposits in nerve cells and glia are the characteristic hallmark of a number of neurodegenerative diseases termed tauopathies. in progressive supranuclear palsy and corticobasal degeneration, pathogenic glial cell inclusions are observable in oligodendrocytes, the myelin forming cells of the cns. hdac plays an important role in the degradation and accumulation of misfolded proteins by promoting transport of aggregated proteins to the aggresome, localized at the microtubule-organizing center where protein aggregates are deposited and processed by autophagy. the present study was undertaken to elucidate the functional significance of hdac in oligodendrocytes and its role in pathological protein aggregate formation. towards this cultured rat brain oligodendrocytes and oln-t cells, an oligodendroglial cell line expressing the longest human tau isoform, were used. treatment of primary oligodendrocytes with tubastatin a (tst), a selective inhibitor of the tubulin deacetylation activity of hdac , caused a significant increase in the level of acetylated tubulin, as determined by indirect immunofluorescence and immunoblot procedure. mts appeared more bundled and stabilized. inhibition of hdac attenuated the phosphorylation of tau at the phf- -epitope (serine / ) and its enhancement at the e -epitope (serine , located within the microtubule binding repeat region ). furthermore, altered protein levels of tau isoforms containing either three or four mt binding repeats were observed. while r-isoforms were reduced, r-isoforms were increased in primary oligodendrocytes suggesting an impact on microtubule binding ability. hdac knockdown in oln-t cells revealed no alteration in tau phosphorylation at phf- -epitope, but also augmented phosphorylation at the e -epitope. cells were morphologically damaged and mt organization was disturbed. the data indicate that hdca modulates tau expression and phosphorylation, its inhibition leads to the accumulation of r-tau which is also prominent in tauopathies with oligodendroglial pathology. to test the hypothesis, that these cells are part of a possible compensatory mechanism to cope with the -ohda-induced loss of dopaminergic neurons, we studied the expression of tyrosine hydroxylase (th), the rate-limiting enzyme in the catecholamine synthesis pathway, in the cortex of -ohda-lesioned animals. performing immuncytochemistry at d after the lesion we demonstrated a -fold increase in the number of th-positive (th somata in rat cortex following -ohda injection) compared to sham-lesioned animals. th somata in the parietal cortex were restricted to the layers ii-v with most of the cells being bipolar. combining th immuncytochemistry with classical nissl stain yielded complete congruency. a small fraction of th cells co-expressed calretinin thus pointing to an interneuron affiliation. virtually none of the th cells expressed other markers of interneurons (calcium binding proteins, neuropeptides) or the glial markers gfap and nestin. in contrast, we found a co-localization of th with markers of glial progenitor cells (sox and s b) and with psa-ncam, which has been shown to be expressed in immature, but not recently generated cortical neurons in cortical layer ii of the cat (varea et al.; front. neurosci. : ). taken together, these findings indicate that -ohda lesions might induce a glial de-differentiation followed by a fate re-direction towards neuronal committed cells. future studies have to be performed to confirm this and to find out if the th cells are capable of dopamine synthesis. objective: using the mouse optic nerve (mon) wm model, we tested whether hydrogen in drinking water reduced functional wm ischemic injury, and if it reduced cellular lipid peroxidation and mitochondrial dysfunction including mitochondrial and nuclear dna oxidation. methods: functional integrity of mon was determined by quantitatively monitoring the area of mon compound action potential (cap) before, during and after a standardized min period of oxygen and glucose deprivation (ogd), the ischemia equivalent ex vivo. nuclear -oxoguanine (nu -oxog) was used as a marker of oxidative dna damage. results: a min period of ogd caused prompt loss of the cap, followed by an average % recovery. in mice that had received hydrogen-containing drinking water for - days, the cap area did not disappear during ischemia and recovered to a significantly greater extent during reperfusion (normal oxygen and glucose levels). immunostaining of axonal neurofilament by smi- showed significant protection in mons from mice drinking hydrogen-water. accumulation of nu -oxog was observed mainly in oligodendrocytes after ogd. the levels of -oxog after ogd were significantly reduced in optic nerves from hydrogen-water drinking mice. the 'protection' induced by - days of hydrogen-water drinking lasted several days. conclusions: our results show that several days of hydrogen exposure reduced the extent of cns wm irreversible injury associated with ogd. the importance of these observations is that oligodendrocytes may play an important role in the protective effect against ischemic injury. these observations raise intriguing therapeutic options. amyloid-b (ab) deposits in the brain extracellular space (ecs) and neurofibrillary tangles are typical features of alzheimer's disease (ad). both affections are expressed in triple transgenic ( xtg-ad) mice, making them a useful model for studying the pathophysiology of ad. in these mice, significant changes in astroglial morphology appear in the limbic brain structures [ , ] , which may affect the diffusion of neuroactive substances through the ecs. using the real-time iontophoretic method, we determined the ecs volume fraction a (a ecs volume / total tissue volume) and the geometrical factor tortuosity k (k free / apparent diffusion coefficient) in the ca region of the hippocampus, dentate gyrus (dg) and prelimbic cortex (plc) in brain slices obtained from -month-old ( m) and month-old ( m) xtg-ad mice and age-matched wild-type (wt) controls. to study potential diffusion anisotropy, the measurements were performed along orthogonal axes: medio-lateral, rostro-caudal and ventro-dorsal. astroglial morphology and the presence of ab were assessed using immunohistochemical staining for gfap and ab. we found no anisotropy in the ca region, dg, or plc, thus the data for each structure along the three orthogonal axes were pooled. the ecs diffusion parameters measured in the ca are shown in table . in the ca of m mice, there was no significant difference in the ecs diffusion parameters between wt and xtg-ad mice. in m mice, a decreased by % in wt but increased by % in xtg-ad mice in comparison with the values found in the younger animals. during aging, there was also a significant decrease in k in wt but not in the xtg-ad mice. a similar pattern of changes in the ecs diffusion parameters during aging was also observed in the dg and plc. unlike in the ca , we found significant differences in young animals: a was lower in the dg and k was higher in the dg and plc of xtg-ad mice than in wt mice. in m xtg-ad mice, ab deposits accompanied with astroglial atrophy or hypertrophy were observed. we suggest that the amyloid load in xtg-ad mice and the subsequent prevailing astroglial atrophy affect the normal aging process by inducing an increase in the ecs volume during aging instead of the normal decrease. this leads to a larger ecs space in aged xtg-ad mice than in age-matched wt mice, which may alter the efficacy of extrasynaptic as well as synaptic transmission and contribute to the impaired cognitive functions in ad. the study was supported by the grants: gacr p / / , gacr p / /g . significant differences between m and m mice of the same group are marked with *, differences between wt and xtg-ad mice of the same age are marked with . sequence or any treatment. however growing evidences are in favor of the involvement, besides neurons, of several partners such as glia and muscles. to better characterize the time course of pathological events in an animal model that recapitulates human als symptoms, we investigated functional and cellular characteristics of hsod g a mice. we have evaluated locomotor function of hsod g a mice through dynamic walking patterns and spontaneous motor activity analysis. we detected early functional deficits that redefine symptoms onset at days of age, i.e. days earlier than previously described. moreover, sequential combination of these approaches allows monitoring of motor activity up to disease end stage. to tentatively correlate early functional deficit with cellular alterations we have used flow cytometry and immunohistochemistry approaches to characterize neuromuscular junctions, astrocytes and microglia. we show that ( ) decrease in neuromuscular junction's number correlates with motor impairment, ( ) astrocytes number is not altered at pre-and early-symptomatic ages but intraspinal repartition is modified at symptoms onset, and ( ) microglia modifications precede disease onset. at pre-symptomatic age, we show a decrease in microglia number whereas at onset of the disease two distinct microglia sub-populations emerge. we are now establishing the transcriptomic profile of microglia over the course of the disease. in conclusion, precise motor analysis updates the onset of the disease in hsod g a mice and allows locomotor monitoring until the end stage of the disease. early functional deficits coincide with alterations of neuromuscular junctions. importantly, we identify different sets of changes in microglia before disease onset as well as at early-symptomatic stage. this finding not only brings a new sequence of cellular events in the natural history of the disease, but it may also provide clues in the search for biomarkers of the disease, and potential therapeutic targets. in order to gain more knowledge about the disease pathophysiology, two mouse models for vwm are developed in our lab. co-cultures between astrocytes (wt and vwm) and wt oligodendrocyte progenitor cells (opcs) are done to investigate the effect of astrocytes on opc maturation. furthermore, induced pluripotent stem cells (ipscs) from both vwm and wt mice are compared in their glial differentiation efficiency in vitro and in vivo. the astrocyte-opc co-cultures show that vwm astrocytes inhibit opc maturation. this suggest that astrocytes might have a crucial role in the development of the oligodendrocyte and myelin pathology in vwm. currently our research focuses on rescuing the maturation inhibition in vitro, which can aid the development of stem cell therapy for vwm. particularly in response to b-amyloid. using the appps alzheimer's disease mouse model, we observed the production of the common interleukin (il) and il- subunit p by microglia. genetic ablation of various il- /il- signaling molecules, of which deficiency in p had the strongest effect, resulted in a drastic decrease in cerebral amyloid burden. although deletion of il- /il- signaling from the radiation-resistant glial compartment of the brain was most efficient in mitigating cerebral amyloidosis, peripheral administration of a neutralizing p -specific antibody likewise resulted in reduction of cerebral b-amyloid in appps mice. furthermore, intracerebroventricular delivery of antibodies to p significantly reduced soluble ab species and reversed cognitive deficits in aged appps mice. our results suggest that inhibition of il- /il- signaling reduces cerebral amyloidosis and cognitive dysfunction, and may pose a novel potential pharmacological target to combat alzheimer's disease. charit e -universit€ atsmedizin berlin, berlin, germany question: microglia are attracted to and surround b-amyloid deposits, the main hallmark of alzheimer's disease (ad), suggesting a role for these cells in disease pathogenesis. however, recent in vivo data using the cd b-hsvtk system for microglial depletion suggests that resident microglia are not sufficiently capable of restricting and clearing bamyloid plaques. to underpin the cd b-hsvtk system and shed more light into the biological mechanistics of microglial depletion mediated by ganciclovir (gcv), we aim to intravitally monitor the depletion process. methods: we are using cd b-hsvtk transgenic mice crossed to fractalkine-gfp /mice harboring green fluorescent microglia. a chronic cranial window is implanted onto the head of the offsprings followed by depletion of microglia and subsequent in vivo two-photon ( p) imaging. as p imaging is able to visualize the upper mm of the cortex, a new topical depletion approach of cd b-hsvtk microglia was established. here, gcv is applied directly onto the brain surface in the area of the cranial window through a catheter. results: manipulation of the brain skull by installation of the cranial window and placing a catheter for topic gcv application resulted in strong depletion of microglia up to % in fractalkine-gfp /mice crossed to cd b-hsvtk mice. conclusions: taken together, we established a minimal invasive technique for visualization of the microglia depletion process in cd b-hsvtk mice using intravital p microscopy. these tools will allow the underpinning of the cd b-hsvtk system as well as the study of relevant biological questions involving resident and peripheral derived microglia in ad. (icrea) , barcelona, spain x-linked adrenoleukodystrophy (x-ald) is a metabolic genetic disorder of the central nervous system characterized by demyelination in brain and/or axonopathy in the spinal cords, adrenal insufficiency and accumulation of very long-chain fatty acids (vlcfa) in plasma and tissues. the disease is caused by inactivation of the abcd transporter with the consequence production of oxidative stress mediators and damage. here we aimed to determine: i) the existence of endoplasmic reticulum (er) stress and the ensuing unfolded-protein response (upr) in brains and fibroblasts from x-ald patients, and in the x-ald mouse model (the abcd null mouse); and ii) whether the early and rampant oxidative stress formerly identified accounts for the er stress. indeed, here we report signs of er stress and upr response in human and mouse tissues. a common feature is the activation of atf and lower amounts of ire , two er transducers constituting the core signature of upr in x-ald. chaperone grp and grp expression, by contrast, differ between variants and stages of the disease. we highlight how chaperones are down-regulated in x-ald mice at months of age, pointing to selective depletion of upr-related factors overtime. treatment of the mouse model with a combination of antioxidants reversed the initial activation of transducers and chaperones, indicating that oxidative stress caused er stress. altogether, we postulate that oxidative-stress elicited er stress occurs in x-ald, and that a defective upr may contribute to axonopathy progression. thus, potentiation of the upr may be a therapeutic avenue in x-ald. representing opc-like cells with the potential to differentiate upon growth factor withdrawal and addition of triiodothyronine (t ) into premyelinating oligodendrocytes. upon differentiation, significant fewer cg _wt-a-syn cells express the myelin basic protein (mbp; see figure a) as a marker for terminal differentiation and additionally, mrna levels were remarkably reduced compared to control cells (see figure b ). as mrna levels of the myelin protein proteolipid protein (plp ) as well as the major myelin-gene regulatory factor (mrf) are also reduced in differentiated cg _wt-a-syn cells (see figure b) we hypothesized that the process of differentiation is delayed upon a-syn expression. using various differentiation-promoting agents targeting distinct maturation-associated pathways we aimed to dissect the a-synmediated effect on differentiation in more detail and evaluate potential disease-modifying approaches for this devastating neurodegenerative disorder. methods: two nutritionally distinct groups of male newborn ratswell-nourished (w; n ) and malnourished (m; n ) were treated from the th to the th day of postnatal life with daily intraperitoneal injections of saline (sal) or cona (sigma-aldrich) at doses of mg/kg (group w-l and m-l ) or mg/kg (groups w-l and m-l ). two groups of "naive" (non-injected) pups were used as additional controls. when the pups reached adult age ( - days), under ip anesthesia ( g/kg urethane mg/kg chloralose) they were submitted to the csd recording (ecog and slow potential change) in two points of the parietal cortical surface. csd was triggered every minutes by applying % kcl for min at a point in the frontal cortex. this study was approved by the ethics committee of our university, case no.: . / - . results: compared to w-sal controls (mean sd velocity in mm/ min . . ), m-sal rats presented significantly higher velocities ( . . ; p < . ). in both nutritional conditions l and l treated groups presented significantly lower csd velocities ( . . and . . for the w-l and w-l , respectively, and . . and . . for the m-l and m-l ). this effect was dose-dependent, and was greater in the m-condition. the na€ ıve-and salgroups had similar csd velocities. conclusions: the lectin cona administered early in life to rats decelerated csd in a dose-dependent manner at adulthood, suggesting a long-lasting effect. the largest relative reduction was observed in the m group, suggesting modulation of the effect by the nutritional status. the involvement of glial cells in this effect is discussed. the endocannabinoid system is of growing interest as a therapeutic target in neurological diseases. the two major endocannabinoids arachidonoylglycerol ( -ag) and n-arachidonoyl ethanolamine (anandamide, aea), are the endogenous ligands for the cannabinoid receptors (cb ) and (cb ). cb is mainly expressed in the brain and associated with neuroprotective effects, whereas cb is primarily found in hematopoietic cells and associated with anti-inflammatory effects. in neurodegenerative diseases, e. g. amyotrophic lateral sclerosis (als) and parkinson's disease (pd), an increased cb expression on microglia has been reported and is therefore an interesting therapeutic target. furthermore, an increase of endocannabinoid levels occurs in the affected neuronal tissues. stimulating this disease-related increase in endocannabinoids might thus be of therapeutic interest. in our project, we evaluated the novel, highly selective inhibitor kml of an enzyme central to the degradation of the endocannabinoid -ag, the monoacylglycerol lipase [ ] . an in vitro monoacylglycerol lipase inhibitor screening assay for the comparison of the utilized inhibitor with others was performed. additionally, behavioral experiments including body temperature measurement, pain threshold measurement and motor activity analysis in healthy control mice were performed. furthermore, endocannabinoid system baseline levels of healthy control mice vs. sod (superoxide dismutase ) mice as an als mouse model were compared at the gene expression and protein level. in vitro inhibition of the monoacylglycerol lipase by the recently published compound kml resulted in lower ic values and therefore indicated a higher potency of kml when compared to the other monoacylglycerol lipase inhibitors jzl and cpc applied in the assay. in vivo administration of the reported monoacylglycerol lipase inhibitor kml resulted in significant decrease in body temperature, increase in pain threshold in b sjlf /j mice as well as in impairments in the nocturnal motor activity of c bl/ j mice when compared to vehicle-treated mice. in summary, our data show a comparatively high potency in vitro and significant dose-dependent effects on physiological parameters in vivo of the monoacylglycerol lipase inhibitor kml and therefore lay the foundation for a future therapeutic study targeting neuroinflammation in mouse models of als and pd. glaz/apod protects against oxidative stress and promotes axon regeneration after injury, but its mechanism of action is unknown. we hypothesize that glaz/apod modulates membrane dynamics and oxidation. to contrast this hypothesis we study glaz protective role on the polyq-based spinocerebellar ataxia type i (sca ) model in drosophila. human polyq-ataxin expression in fly photoreceptors triggers glaz up-regulation. overexpressing glaz in photoreceptors or in retinal support cells rescues neurodegeneration. when expressed in retinal support cells, the glaz rescue is dependent on lipocalin-receptor expression by photoreceptor neurons, and the glaz-gfp fusion protein co-localizes with photoreceptor markers, suggesting the existence of receptor-mediated endocytosis of glaz. upon neurodegeneration, oxidative stress and induction of autophagy coexist. to test whether the glaz beneficial effects are mediated by the modulation of autophagy we quantified atg a expression and monitored the accumulation of ubiquitinated proteins and p . overexpression of glaz reduces atg a induction, but concurrently reduces the accumulation of ubiquitinated proteins and p . upon autophagy stimulation by rapamycin treatment, the levels of sca -dependent accumulation of ubiquitinated proteins and p are further reduced by glaz. in addition, glaz decreases the sca -dependent induction of gsts , a sca genetic modifier contributing to the clearance of lipid peroxides. our data support that glaz enters the degenerating neurons by a receptor-mediated mechanism and promotes the resolution of autophagy, increasing its flow and helping to clear polyq-induced protein aggregates. moreover, the beneficial effects of glaz are linked to lipid peroxide clearance, either directly or through receptor-mediated modulation of protective gene networks. micinn(bfu - ); jcyl(va a - ); fund. rodr ıguez-pascual. we investigated the effect of three different intensities of running exercises on the survival of sod g a mice. at the early-symptomatic stage (p ), males were isolated and randomly assigned to conditions: sedentary groups ("sedentary" and "sedentary treadmill" placed on the inert treadmill), and different training intensity groups ( cm/s, cm/s and cm/s; min/day, days/week). we first demonstrated that an appropriate "control" of the environment is of the utmost importance since comparison of the two sedentary groups evidenced an . % increase in survival in the "sedentary treadmill" group. moreover, we showed by immunohistochemistry that this increased lifespan is accompanied with motoneurons survival and increased glial reactivity in the spinal cord. in a second step, we showed that when compared with the proper control, all three runningbased training did not modify lifespan of the animals, but resulted in glial and motoneuronal changes. conclusions/significance: we demonstrate that increase in survival induced by a slight daily modification of the environment is associated with motoneurons preservation and strong glial modifications in the lumbar spinal cord of sod g a . using the appropriate control, we then demonstrate that all running intensities have no effect on the survival of als mice but induce cellular modifications. our results highlight the critical importance of the control of the environment in als studies and may explain discrepancy in the literature regarding the effect of exercise in als. alzheimer's disease is the most prevalent neurodegenerative disorder, characterized by occurrence of senile plaques, neurofibrillary tangles and aberrant function of classical neurotransmitters and peptide messengers, such as neuropeptides and growth factors. in the last years a role of astrocytes in mediating and amplifying the progression of neurodegenerative disorders has been proposed. moreover, a growing body of evidence has shown that astrocytes can release non-peptide and peptide transmitters to influence neuronal development, function and plasticity. here we analyzed the impact of the main component of senile plaques, the amyloid-b (ab), on two key components of peptide vesicles, carboxypeptidase e (cpe) and secretogranin iii (sgiii), in astrocytes in vitro and in vivo. we consistently located cpe and sgiii proteins in cultured and in situ astrocytes. traffic and secretion of astroglial cpe and sgiii were analyzed under basal and stimulated conditions in primary cultures. we found that exposure of astrocytes to ab - markedly impairs expression, traffic and secretion of glial cpe and sgiii. protein levels were decreased in the media, but increased into the cells, by ab - incubations. the effect of ab on cpe and sgiii in glial cells was also investigated in vivo using the amyloid-forming transgenic mice appswe/ps de . an aberrant accumulation of cpe and sgiii was found in numerous activated-astrocytes surrounding senile plaques through all the cns of aged transgenic mice. taken together, the present study shows that ab dramatically alters expression, traffic and secretion of cpe and sgiii. because cpe and sgiii are essential in the process and targeting of neuropeptides and growth factors, an involvement of the astroglial secretory pathway in the pathology of alzheimer's disease is suggested. glia cytokine that is produced subsequent to h-i, that collaborates with other cytokines to stimulate the production of astrocytes from subventricular zone glial progenitors. the specific goal of this study was to evaluate gliogenesis after h-i when the tgfb receptor alk is antagonized in the vanucci p h-i rat model. by q-pcr, the relative amount of tgfb mrna peaked days after h-i. therefore, sb- , and alk antagonist, was given intraperitoneally (i.p.) days after the injury to a group of rats while another group received pbs. animals were sacrificed at different time points and brain samples processed for western blot analysis. validating the importance of alk- signaling, sb reduced the levels of phosphorylated smad / that increased after h-i. in another study, sb- was administered i.p. from p to p and animals sacrificed at p for immunohistochemistry for gfap, iba- , gstpi and mbp. gfap and iba- positive cells were dramatically increased in the injured striatum and corpus callosum after h-i and mbp staining was decreased. by contrast, both the extent of injury and the degree of reactive gliosis was decreased by sb- treatment. altogether, our results indicate that sb- inhibits alk signaling in the damaged brain. furthermore, sb- administration decreases the extent of microgliosis and astrogliosis while preserving myelination in the damaged brain after neonatal h-i. supported by nih r hd and a grant from the leducq foundation awarded to swl. [ ] . despite efforts to improve air quality, dep persists as a problem and the share of diesel engined passenger cars in western europe is increasing steadily. honey bees are an important pollinator of food crops and wild flowering plants. they not only contribute to food security by providing pollination services of an enormous economic value but also play an important ecological role. over the last five years, bee keepers worldwide have reported a decline in honey bee populations. the reasons for this decline remain unknown, but it is thought to be multi-factorial [ ] . honey bee colonies are confronted with a number of stressors, for example infection with the mite varroa destructor or exposure to pesticides. to forage successfully honey bees rely on their learning and memory abilities. our research aims to establish whether dep might be a factor contributing to declines in honey bee populations through impairment of healthy cns function. methods: we have investigated the impact of dep exposure on the learning abilities of the honey bee using the proboscis extension reflex and classical conditioning. western blotting and histology were used to determine regional expression levels of stress proteins in the brain in response to an acute diesel exhaust exposure. we are examining the morphological appearance of glial cells to investigate the impact of dep in the honey bee cns of dep exposed, and control, forager bees. results and conclusions: we hypothesize that dep acts as a stressor in the brain of the honey bee causing changes in glial cells that are detectable using morphological and molecular techniques -similar to microglial activation or immunological responses of astroglia in the mammalian brain. this work will further our understanding of how dep acts on the brain and how it might impact on the health of the animal. current results indicate that diesel exhaust pollution impacts on the neurobiology of the honey bee and this may contribute to decline by impairing cns functions. hepatic encephalopathy (he) is a serious neurological disorder caused by liver failure. the prime candidate responsible for he pathology is an increased ammonium concentration; and following acute liver failure, ammonium can reach levels of up to mm in the brain. astrocytes are a major target of ammonium toxicity and earlier studies suggested that this toxicity includes a disturbance in intracellular calcium signaling. in the present study, we analyzed the effect of acute hyperammonia on the intracellular calcium concentration of astrocytes in tissue slices of mouse brain, using quantitative intracellular calcium imaging with the indicator dye fura- . astrocytes were identified by staining with the vital dye sr ; cerebellar bergmann glial cells were identified based on their morphology. in all brain regions studied, the hippocampal ca area, somatosensoric cortex, and cerebellar cortex, bath perfusion with mm ammonium for min induced a small, but persistent elevation in intracellular calcium, averaging about nm. in addition, a fast and transient increase by on average nm that lasted about minutes was seen in a small percentage of astrocytes in hippocampal and cortical slices at the onset of ammonium perfusion. this transient increase was never observed in cerebellar bergmann glial cells. both the transient, as well as the plateau phase of ammonium-induced calcium changes were unaltered in the presence of ttx, indicating that they are independent from action potential generation by neurons. furthermore, ammonium-induced calcium increases largely persisted upon removal of extracellular calcium, indicating that they are caused by release from intracellular stores. taken together, our experiments demonstrate that ammonium evokes complex changes in the intracellular calcium concentration of astrocytes in the intact tissue, which differ both between cells in one preparation as well as between different brain regions. because of the central role of astrocyte calcium in gliotransmission, the observed ammonium-induced calcium dysbalance might result in disturbed neuronglia interaction and contribute to the pathology of he. it has been shown in a number of animal models that microglia in the degenerating brain are primed to show exaggerated cytokine responses to subsequent stimulation with toll-like receptors agonists such as lps and poly i:c. it is not clear whether the degenerating brain shows similarly exaggerated responses to pro-inflammatory cytokines. in the current study we hypothesised that glial cells in the hippocampus of animals with chronic neurodegenerative disease (me prion disease) would display abnormal responses to central cytokine challenges. in normal animals it has previously been established that intracerebral cytokine challenges elicit specific pathways, i.e. il- b a cxcl- a neutrophil recruitment or tnfa a ccl- a monocyte recruitment. unilateral ll doses of tnfa ( ng/ll), il- b ( ng/ll) or saline were administered intrahippocampally via pulled glass microcapillary, in normal (nbh) and me mice. these animals were terminally perfused for formalin fixation and paraffin embedding at , and hours post challenge. at hours post challenge me microglia produced il- b following either il- b or tnfa challenge while nbh microglia did not. furthermore, there was very robust nuclear localisation of the nfkb subunit p in the astrocyte population and this was associated with very marked astrocytic synthesis of the chemokines cxcl- and ccl- in response to both cytokine challenges in me animals. conversely, very limited expression of these chemokines was apparent in nbh animals similarly challenged. thus astrocytes are the primary chemokine synthesizing brain cell and in the prion-diseased brain are they primed to produce exaggerated chemokine responses to acute stimulation with pro-inflammatory cytokines. this abnormal pattern of chemokine expression is predicted to have consequences for leukocyte infiltration and the ramifications of an altered cellular infiltration pathway for the degenerating brain will be discussed. these findings suggested that the presence of mutated htt in astrocytes alters glial glutamate transport capacity early in the disease process and may contribute to excitotoxicity. to decipher whether astrocytic mutated htt expression was directly associated with msns dysfunction, we investigated the functional properties of individual msns in proximity of mutated htt expressing astrocytes in acute slices. results from whole-cell patch clamp technique showed that msns surrounded ( - microns distance) by astrocytes expressing mutated htt vs. astrocytes expressing normal htt did not differ with regard to passive membrane properties (input resistance, resting membrane potential), action potential firing rates nor spontaneous excitatory postsynaptic current properties (averaged amplitude and frequency). thus, astrocytic expression of mutated htt does not lead to readily apparent functional consequences in msns in these conditions. our data show that month old tg mice displayed a significant increase of ab-plaques in the brain, compared to wt mice. furthermore, we show that the a c subunit colocalizes with % of the abpositive plaques. the cellular expression of a c was predominantly found in activated gfap astroglia around plaques. qpcr profiles of the hippocampus revealed expression of the majority of calcium channel subunits, which was stable during aging. the auxiliary beta subunit was expressed in these mice but did not co-localize with a c astroglia around plaques. in cultures of primary astrocytes, ab( ) slightly enhanced the a c mrna expression after days. we are currently underway to characterize this expression in more detail. in summary, our data provide evidence for a largely stable expression of most ltcc subunits in alzheimer tg mice during aging. finally, the expression of a c but not beta is upregulated in activated astrocytes located around ab-plaques and ab( ) may directly induce astroglial ca v . calcium channels. this study was supported by the sonderforschungsbereich sfb f -b and f -b of the austrian science funds. increasing evidence indicate that both physical and cognitive stimulation induce plastic changes in the brain, such as increase in synaptic and spine densities or neurogenesis, and counteract b amyloid (ab) pathology recovering cognitive function. in the present study we, for the first time, demonstrate the effects of psychostimulation on glial fibrillary acid protein (gfap) architecture in the dentate gyrus (dg) of xtg-ad, a well-established mouse model of ad. starting from months of age, xtg-ad and their corresponding controls (non-tg) were housed either in the presence of a running wheel (run) or in the enriched environment (enr) for months. as reported previously (olabarria et al., ), in standard housing, xtg-ad animals showed marked atrophy of gfap-positive profiles, evidenced by significant reduction in gfap surface area, by %, and volume, by %, compared with non-tg mice. however, physical and cognitive stimulation affected astrocyte morphology by increasing their cytoskeleton surface area and volume, both in non-tg and in xtg-ad mice. the surface area of gfap-ir astrocytes increased by % and % in xtg-ad mice housed in run and enr, respectively, compared with transgenic mice kept in standard housing. in addition, the cell volume of gfap-ir astrocytes was higher by % and % in xtg-ad mice housed in run and enr, respectively. the hypertrophy was also evidenced by an increase in the surface area and the volume of astroglial somata and processes in both non-tg and xtg-ad mice. further correlation analysis between xtg-ad and their corresponding control mice housed under the same conditions, revealed that run and enr not only reversed the genotype-induced astrocytic atrophy, but even induced a hypertrophy in xtg-ad mice, with gfap positive profiles rising to the levels of non-tg mice. thus our study indicates that long-term exercise and enriched environment restore and improve astroglial plasticity in the context of adlike pathology, which may recover cognitive functions by compensating disease-associated degeneration of neuronal-glial network. methods: intraperitoneal administration of wa at a dosage of mg/ kg of body weight was initiated from postnatal day (p ) till end stage in sod g a mice, from months till end stage in sod g r mice and from months of age in tdp a t mice. results: wa was able to improve the survival by more than a week in sod g a and by two weeks in sod g r familial mouse model. in addition wa administration also conferred significant neuroprotection in tdp a t transgenic mice model. beneficial effects of wa in sod g a mice model was accompanied by reduction in loss of motor neurons and also by reduction in level of misfolded sod protein in the spinal cord as detected by immunoprecipitation with antibody specific to misfolded sod . wa was found to be an inducer of heat shock protein (hsp- ), which could possibly explain reduced level of misfolded sod and increased neuroprotection in wa treated mice. moreover real-time imaging with the use of biophotonic sod g a transgenic mice carrying luc (luciferase) and gfp (green fluorescent protein) reporter genes under the control of the murine gap- promoter revealed that wa was able to reduce neuronal stress at post symptomatic stage. conclusion: taken together, our results suggest that wa may represent a promising therapeutic drug for treatment of als. the temporal lobe epilepsy (tle) is the most common form of epilepsy that originates from the hippocampus and then propagates to other limbic areas such as the amygdala and entorhinal cortex. the pathological feature associated with tle is hippocampal sclerosis which is characterized by atrophy, induration, gliosis and loss of neurons in ca , ca and the dentate hilar regions. some animal models, albeit do not exactly match the complex etiologies identified in humans, are found to recapitulate most of the pathological features observed in tle. there is evidence that administration of kainic acid can cause seizures in the ca region of the hippocampus that can lead to loss of neurons and astrogliosis characteristic of tle, but the underlying mechanisms associated with the degeneration of neurons remain unclear. since lysosomal enzymes, cathepsins b and d, can have important roles in the loss of neurons in a variety of experimental conditions, we evaluated their potential roles along with the insulin-like growth factor-ii (igf-ii) receptor, which is involved in the intracellular transport of these enzymes, in the kainic acid treated rats. our results clearly showed that systemic administration of kainic acid evoked severe loss of neurons along with hypertrophy of astrocytes and microglia in the hippocampal region of the adult rat brain. the levels and expression of cathepsins b and d as well as igf-ii receptor increased progressively with time in the hippocampus of kainic acid treated rats compared to control rats. the activity of both cathepsins b and d was also found to be enhanced in the hippocampus of treated rats. our double labelling studies revealed that expression of both cathepsins were initially increased in the pyramidal neurons and then decreased with time. this was accompanied by the expression of immunoreactive igf-ii receptors as well as cathepsins b and d in a subset of gfap-labelled activated astrocytes and iba -labelled microglia in kainic acid treated rats. these results, taken together, suggest that enhanced levels/expression and activity of lysosomal enzymes may have a role in the loss of neurons observed in kainic acid treated rats. in addition, increased ng glia immunoreactivity and myelin loss were found specifically in the gray matter of patients' motor cortex and spinal cord ventral horn. furthermore, oligodendroglial specific monocarboxylate transporter expression is decreased not only in als mouse models but also in patients. given that oligodendrocytes not only facilitate saltatory conduction of action potentials by myelinating axons, but also support neuronal functions metabolically through monocarboxylate transporter (mct ), the injury to oligodendroglia in als may have pathogenic consequences. methods. to further investigate whether oligodendrocytes play a role in als development, we selectively removed mutant human sod (g r) from postnatal ng cells in pdgfar-creer;loxsod (g r) mice. results. the administration of -hydorxytamoxifen ( ht) caused significant decrease in the expression of mutant human sod transgene in ng glia as determined by quantitative pcr analysis. we found that excision of mutant hsod from ng glia dramatically delayed disease onset and early disease, and significantly prolonged animal survival. in addition, at disease onset stage, activated astroglial and microglial responses were delayed in the animals received ht treatment. moreover, the removal of mutant sod helped to preserve mct expression at disease onset. conclusions. these data indicate that expression of mutant sod in ng cells and their oligodendrocyte progeny has a deleterious effect on motor neuron survival, and suggest that a key negative consequence of mutant sod expression in oligodendrocytes is to diminish their capacity to provide metabolic support to neurons. recent studies suggest that innate immunity might be also involved in the pathogenesis of neurodegenerative diseases, cerebral ischemia and brain injury. although the recent works demonstrated that adaptive immune system is involved in the motor neuron disease process, the role of innate immune system in motor neuron disease was not fully investigated. to assess the contribution of innate immunity in the pathogenesis of amyotrophic lateral sclerosis (als), the gene expression profile of lumbar spinal cord from symptomatic mutant sod mice was obtained by microarray approach and subsequent pathway analysis indicated the involvement of innate immune pathway. next, to test the role of innate immunity in the pathogenesis of als, sod g a als model mice were mated with myd and trif (tir domain-containing adaptor inducing ifnb) deficient mice. myd and trif are the essential adaptor proteins for toll-like receptor mediated signaling pathway. as compared with sod g a mice, myd /trif double-deficient and trif-deficient sod g a mice exhibited the substantially shorter survival times with accelerated disease progression. the disease duration was shortened by % in trif-deficient sod g a mice. in contrast, elimination of myd in sod g a mice showed marginal effect in survival time. in addition, the expression levels of pro-inflammatory chemokines, ccl and cxcl- were significantly suppressed in the spinal cord of trifdeficient sod g a mice, as compared with sod g a mice. to determine the cell type in which trif-dependent pathway contributes to the production of these chemokines, we examined the expression of poster abstracts s glia chemokines in lps-stimulated primary microglia or astrocyte derived from trif-deficient or myd -deficient mice. trif-dependent induction of these chemokines was observed only in lps-stimulated microglia. moreover, we found that infiltration of t-lymphocytes and other immune cells were significantly decreased in the spinal cord of symptomatic trif-deficient sod g a mice. these results suggest that the basal level of trif-dependent innate immune activation of microglia is beneficial to slow disease progression of als models through the maintenance of pro-inflammatory chemokines and the infiltration of the immune cells to the spinal cords. the detailed analyses to clarify the role of these infiltrating immune cells are underway. alzheimer's disease (ad), the most common age-dependent neurodegenerative disorder, causes a chronically progressive decline in cognitive functions. there is growing evidence that glial changes are early involved in this pathology. pdapp mouse, a well-defined model of ad, accumulates toxic soluble and deposited ab, derived from proteolytic processing of the amyloid precursor protein (app) and develops adlike synaptic deficits and cognitive impairment. on the other hand, autophagy has been associated to the neurodegenerative process, in particular with the clearance of aggregation-prone proteins like ab. the amyloid plaques, mainly located in cortex and hippocampus, are closely surrounded by reactive and hypertrophic gfap astrocytes. the aim of this work was to evaluate the potential astrocyte autophagic activity during the progression of ad in pdapp mice from to months (m) of age. lc ii/i ratio was studied by western blot. amyloid deposit load, stained with congo red, exhibited a continuing rise according to age in transgenic mice. the markers gfap and lc were analyzed by immunofluorescence and confocal microscopy on hippocampal sections showing an increasing colocalization that reached the top at m, where . . % of plaque associated gfap cells were lc . at m, this proportion was lower. conversely, the subpopulation of astrocytes located far from ab deposits were gfap /lc -, besides a decreased cell volume compared with control mice astrocytes. additionally, the density of astroglial cells in the stratum radiatum diminished with aging ( compared to m, total gfap cells, p < . ) along with higher plaque load, in a more prominently manner than in control mice. our results clearly show that ) astroglia is strongly affected during ad progression: two different morphologic subpopulations -close to or far from deposits-are distinguished in the hippocampus. aging correlates with a glial density reduction; ) a gradual increase of autophagic activity in plaque associated-astrocytes is verified by the first time, suggesting a contribution to ab clearance until m in pdapp mice, with a significant decay after this age. it also shows widespread neuron loss and gliosis in the brain. interestingly, in cstb-/-mice pronounced microglial activation has been detected in selected brain areas already in presymptomatic mice, preceding astrocytosis and neuronal death. in this study, we examine the microglial contribution to the neuronal dysfunction and death in epm . we aim to characterize the functional properties of cstb-/-microglia and their effects on the survival of cstb-/-neurons. our results show that the cstb mrna level in cultured wild type mouse microglia analyzed by real time quantitative pcr is high in relation to primary astrocytes or neurons. a gene-expression profiling of microglia from cstb-/-mice and from wild type mice has been obtained by using the affymetrix mouse exon . st array and reveals a down-regulation of interferon-regulated pathways in cstb-/-microglia. the stimulation of primary wild type mouse microglia with the endotoxin lipopolysaccharide (lps) up-regulates cstb mrna expression measured by real time quantitative pcr. cstb-/-microglia show an altered inflammatory response to the stimulation with lps by increased secretion of chemokines demonstrated by cytokine array analyses. moreover, the release of nitric oxide from cstb-/-microglia measured by griess assay is elevated. our data indicate an altered response of primary cstb-/-microglia to inflammatory stimuli, which may contribute to neurodegeneration in epm . the data provide a basis for further detailed studies on the pathophysiology and therapy of this devastating disease. activation of astrocytes and microglia surrounding amyloid extracellular plaques in the brain is a hallmark of alzheimer's disease (ad). increasing evidence suggests that chronic neuroinflammation and elevated levels of several cytokines play important roles in ad development. beside astro-and microgliosis, demyelination and oligodendrogenesis can be observed in human patients as well as in murine models of ad. oligodendrocyte progenitor cells, also termed ng cells because they express the neuron/glial antigen (ng ) proteoglycan, comprise about % of total cell number of the adult brain and are the main proliferating cells present. ng cells receive gabaergic and glutamatergic synaptic input and can secrete pro-and antiinflammatory substances upon activation by microglia-derived cytokines. overall, ng cells are highly reactive cells and one of the first cells which respond to changes in the cns. however, their role during ad pathology is still uncompletely characterized. using immunohistochemistry, we have analyzed their expression pattern in a transgenic murine model of ad (coexpressing a mutated form of the amyloid precursor protein (app) and the presenilin (ps ) gene). we focused on the spatial arrangement of ng cells in the hippocampus of transgenic mice at , and months of age and age-matched controls (n / time point). our goal is to provide an anatomical and structural basis for elucidating the role of ng cells in ad. the first results indicate an uniform distribution of ng cells in the hippocampus of app/ps mice, with an accumulation at the sites of plaque deposits in close association with microglia. ng cells are normaly absent from the pyramidal and granular cell layers. however, when plaques deposits were found at these principal cell layers, ng cells could be seen surrounding them, suggesting an active migration. changes in ng cell phenotype were noted from months of age on. like microglia, ng cells assume an activated morphology, as in a more "bushy" appearance. in numerous plaques, ng immunoreactivity was strongly increased, mostly in the intermingled processes that encircle the core of the plaques. whether these activated ng cells proliferate and differentiate into oligodendrocytes or communicate with activated microglia in the plaque microenvironment remains to be investigated. further, levels of the glutamatergic ampa receptor expression will be evaluated. ultimately we aim at understanding how ng cells and microglia interact in ad, which could be usefull in the development of novel therapies. accumulating evidence supports an increasing role of astrocytes in the initiation or progression of a variety of neuropathological conditions. in alzheimer's disease (ad), numerous data indicate that astrocyte properties are modified with potential deleterious effects on neurons. accordingly, we have described changes in the expression of astroglial connexins, the gap junction channel and hemichannel forming proteins, in the vicinity of amyloid-b (ab) plaques in brains from ad patients and murine models of ad. also ab peptide was shown to trigger astroglial cx hemichannel activation in culture and in acute slices leading to neuronal degeneration. hence, we have investigated hemichannel function of astroglial connexins in - months old app swe /ps de mice that exhibit ab plaques in the cortex and hippocampus. ethidium bromide (etbr) uptake performed in acute brain hemisphere slices was used as an index of hemichannel activation and was quantified in astrocytes identified by gfap immunostaining. in app swe /ps de mice, compared to wildtype mice of the same age, an increased uptake of etbr was detected in the overall population of astrocytes. this uptake was blocked by carbenoxolone and lanthanum ions, indicating connexin hemichannel involvement. such activation may be linked to the increase in resting astroglial [ca ] i described in this mouse model. interestingly, etbr uptake was higher in reactive astrocytes contacting ab plaques than in non-reactive astrocytes located far from (! mm) these deposits. we are currently investigating their respective pharmacological profiles. moreover, in app swe /ps de mice knock-out for cx generated in our facility, etbr uptake was comparable to that observed in app swe /ps de mice, suggesting that cx is the major contributor to the hemichannel activity observed. altogether, these results indicate that neuroglial interactions could be affected by astroglial hemichannel activation and could account for neuronal alterations or death observed in neurodegenerative diseases. since it is well established that amyloid plaques are associated with activated astrocytes we asked whether concentrations of the astrocyte-derived proteins glial fibrillary acidic protein (gfap) and s b in human csf might serve as additional biomarkers. to functionally link the role of astrocytes to mechanisms involved in memory formation, we asked whether the amount of gfap-positive astrocytes correlates to the level of ltp in the hippocampus of aged ad transgenic mice. we used elisa kits for the quantitative analysis of gfap (ibl-international, hamburg, germany) and s b (ibl). in a mixed disease cohort (n diseased patients, n controls), we correlated standard biomarkers (abeta and tau-protein) and gliaderived proteins. furthermore we compared mean levels between groups of ad patients (n ) and healthy control patients (n ) and correlated levels of astroglial proteins in the csf and cognitive performance (mmst values). in transgenic (app/ps transgenic mice), aged ( - months) mice (n ), we performed ltp experiments at the schaffer collateral synapses in the ca region of the hippocampus. the number of gfap-positive astrocytes was quantified contralaterally. results: in the mixed cohort, we found a significant correlation of t-tau and gfap levels in csf (r . , pbetween the s b levels and the patients cognitive performance as measured by the mmst values (r - , ) as well as between the gfap levels and the mmst values (r - , ). when correlating the number of astrocytes in the ca region to the level of ltp, min after induction, we found a significant correlation of the number of astrocytes per mm to the level of ltp (r , , p conclusions: the astrocyte-derived proteins gfap and s b can be reliably detected in human csf. measurement of astroglial biomarkers might allow an improved pathobiological staging of ad, also since astrocytes appear to play a functional role in memory formation in the context of an ad-like pathology in mice. retinitis pigmentosa is a group of inherited disorders affecting photoreceptors or retinal pigment epithelium (rpe) that leads to progressive loss of vision. the pde b rd mouse model is a very well-known animal model for retinal degeneration, in which rods carry a mutation in b subunit of rod cgmp-phosphodiesterease. glial cell line-derived neurotrophic factor (gdnf) has already been shown to rescue morphology as well as function of rod cells in pde b rd mouse. this effect was indirect, through stimulation of m€ uller glial cells (rmg). to better understand the neuroprotective effect of gdnf, primary retinal rmg were stimulated in vitro with gdnf and the secreted proteome was analyzed by proteome profiler arrays. among others, cyr /ccn , a member of ccn family, was found strongly induced upon rmg stimulation with gdnf. when applied directly to medium, cyr significantly reduced photoreceptor death in organotypic ex vivo cultures of pde b rd retinas. in order to identify the target cells of cyr we treated, arpe and mio-m cell lines with cyr , and observed an increase in phosphorylation of akt and erk / signaling molecules. these results suggest that stimulation of rpe and rmg cells may have a protective influence on photoreceptor survival in organotypic ex vivo conditions. we postulate cyr as a novel potential candidate for future therapeutic approaches in neurodegenerative retinal disorders. since astrocytes react to multiple physiological and pathological stimuli in the microenvironment of the brain, they could be mediators for gene x environment interactions in the pathogenesis of psychiatric disorders. altered gene/protein expression and regressive changes have been reported for astrocytes in post-mortem studies of psychiatric disorders, i.e. schizophrenia (scz), bipolar disorder (bp) and autism spectrum disorders (asd). by contrast, no genetic link to astrocytes has emerged from the extensive genomic analysis of psychiatric disorders. genetic findings mostly relate to neurons for disorders rooted in neurodevelopment (asd, scz). it is timely to perform a formal analysis of genomic information emerging for psychiatric disorders for a contribution of genes expressed by astrocytes. methods: we generated a meta-list of genes highly expressed in rodent astrocytes using published gene lists and literature mining. datasets were prepared for candidate genes and genes from gwas in attention deficit/hyperactivity disorder (adhd), asd, bp and scz, by using literature and databases (szgene, sfari base, dbgap). datasets for copy number variations (cnvs) associated with neurodevelopmental delay were also assembled. then the overlap between the meta-list of genes highly expressed in astrocytes and gene datasets for psychiatric disease was determined. overlapping genes were pooled and subjected to david bioinformatics analysis. results: the meta-list of genes highly expressed in astrocytes contained n , genes ( . % of the genome). datasets for risk genes in psychiatric disorders showed random overlap with the meta-list (adhd %; asd %; bp %; scz %). cnvs related to neurodevelopment were not enriched for astrocytic genes ( %). when the overlapping genes were pooled from all disorders, a small set of shared astrocytic genes emerged (n ). cntnap , nrxn and sdc were linked by david analysis (p . ). conclusions: this correlative analysis makes it unlikely that genes highly expressed in astrocytes make a major contribution to the genetic risk in four major psychiatric disorders. no genetic link was found between astrocytes and neurodevelopmental delay. changes in gene/ protein expression and pathology in astrocytes in the adult brain in scz and bp may be explained by chronic neuronal dysfunction, chronic medication or acute changes. the neuronal ceroid lipofuscinoses (ncls, batten disease) are a group of autosomal recessively inherited lysosomal storage disorders affecting children and young adults, each of which is caused by a mutation in a different gene. a common feature across all ncls is the early, localised glial activation that occurs long before the onset of neuron loss. this glial activation appears to be an accurate predictor of which neuron populations are vulnerable and will subsequently die. both astrocytes and microglia express the ncl gene products and it is likely that normal glial cell function may be compromised. given the close functional relationship between neurons and glial cells it is possible that glial activation and/or dysfunction may affect neuronal health. we have begun to explore the biology of astrocytes and microglia in the juvenile form of ncl using primary cultures from cln -/mice. defects in both astrocytes and microglia, including altered protein secretion profiles and impaired morphological and proliferative responses were apparent. co-culturing with mutant microglia and astrocytes influenced the survival and morphology of wild type neurons, with more profound effects upon cln -/neurons. intriguingly, these effects were largely reversed by substituting wild type glia, which rescue mutant neurons. a pilot study has provided similar evidence for glial dysfunction in infantile ncl, including and altered protein secretion and response to stimulation. these changes appear to be different from those observed in juvenile ncl and we are in the process of exploring whether the glial cell phenotypes discovered so far, and their impact upon neurons are specific to the different forms of the disease. the accumulation of aggregated proteins in nerve cells and glia underlie the pathogenesis of many neurodegenerative diseases. glial pathology is characteristically observed in tauopathies, such as corticobasal degeneration and progressive supranuclear palsy, and alexander disease, a primary disease of astrocytes caused by mutations in the gfap (glial fibrillary acidic protein) gene. irreversibly damaged proteins can be degraded by the proteasomal system. for proteasomal degradation they are covalently linked to ubiquitin in a three-step enzymatic pathway (e , ubiquitin-activating enzyme; e , ubiquitin-conjugating enzyme; and e ubiquitin ligases). the polyubiquitin chain needs to be removed before the translocation of the substrates to the lumen of the proteasome. this is achieved by deubiquitinating enzymes (dubs), which oppose the functions of e -ligases and assist in targeting substrates to specific pathways. to assess whether impairment of dubs may contribute to age-related neurodegenerative diseases and the regulation of cell death and survival, we have subjected primary cultures of rat brain astrocytes to pr- , a dub inhibitor with broad specificity. immunoblot analysis demonstrates that after a h treatment, pr- caused the induction of heat shock proteins, hsp and hsp , and an increase in p , which is considered a cargo receptor for ubiquitinated proteins and a common constituent of ubiquitinated protein inclusions. as analyzed by indirect immunofluorescence, protein aggregates positively stained by antibodies against ubiquitin and p assembled in the perinuclear region at the microtubule organizing center (mtoc). these aggregates were surrounded by a cage-like network of gfap intermediate filaments, thus resembling aggresomes. using mitotracker and lysotracker staining procedures, the fluorescent images demonstrate that after dub inhibition lysosomes and mitochondria are recruited to the mtoc. this process was dependent on an intact microtubule network, since it was interrupted after treatment with nocodazole, a microtubule destabilization drug. furthermore, pr- caused mitochondrial fragmentation which may be a stress response to enable the removal of damaged mitochondria by mitophagy. in conclusion, dub inhibition impairs the cellular architecture, leads to protein aggregate formation and mitochondrial alterations in astrocytes. our data sustain the hypothesis that an imbalance in dub activities may contribute to the pathogenesis of neurodegenerative diseases. shown that the collapsin response mediator protein (crmp- ), which play a significant physiological role in neuronal cell bodies and axons within the cns, is phosphorylated during the neurodegenerative phase of the inflammatory disease. methods: we investigated the limitation of axonal degeneration by transducing retinal ganglion neurons with a phosphorylation mutant of crmp- utilising an intraocular adeno-associated virus (aav ) delivery system in eae-induced mice. the other group of mice injected with aav consisting of the green flourescent protein reporter only (aav -gfp) with eae was used as a control (n per each group). results: we showed substantial preservation of axons of the optic nerve in the eae-induced mice injected with the aav carrying the crmp- phospho-mutant compared ($ -fold difference) with those mice injected with aav -gfp. the aav -gfp transduced axons showed significant degeneration during the peak stage of eae. conclusion: our data suggest that phosphorylation of crmp- may be a central mechanism that governs axonal degeneration during inflammatory demyelination of the cns (as occurs in ms) and inhibition of phosphorylation of crmp- may be of therapeutic potential for the progressive phase of the disease in ms patients. y. shinozaki, s. koizumi univ. of yamanashi, yamanashi, japan atp, a major gliotransmitter integrating neuron-glia networks, is known to be released or leaked from injured cells. in the cns, atp dramatically changes glial phenotypesand could affect pathogenesis of brain disorders. however, whether such glial responses facilitate or rather inhibit the diseases is still under debate. to address this issue,we studied the neuroprotective role of atp/purinergic signaling using in vivo stab injury model on mouse cerebral cortex. without injury or the contralateral side of the injured brain exhibited no fluoro-jade(fj)-positive neuronal death, cd infiltrating leukocytes or gfap reactive astrocytes. three days after injury, fj signals, cd cells and reactive astrocytes were increased in the ipsilateral side of the brain. the cd cells were enclosed by reactive astrocytes-formed glial scar. administration of an atp-degrading enzyme apyrase, a broad p receptor antagonist ppads, and a p y receptor antagonist mrs significantly reduced the stabincreased fj signals. immunohistochemical analysis revealed p y receptor was expressed in gfap astrocytes. p y receptor knockout (p y ko) mice also exhibited reduced fj signals and infiltration of cd cells. they exhibited higher level of gfap expression, more remarkable hypertrophy of astrocytes and tighter glial scar formation than wild type mice did. the accelerated glial scar formation reduced cd cell number and inhibition of astrocytes by fluorocitrate increased cd cell number. although glial scar formation was accelerated, no enhanced proliferation was observed. we then analyzed the mechanism of enhanced glial scar formation and neuroprotection using in vitro scratch-wound model. in cultured astrocytes, the scratch induced a transient increase in extracellular atp, which was followed by decrease in extracellular atp mainly due to an increase in ectoatpase activity. suppression of purinergic signaling by either apyrase, ppads or mrs enhanced scratchevoked astrocytic migration rather than proliferation. neither activation nor inhibition of p y receptors in cultured cortical neurons affected the scratch-induced damages. our data suggest that the decrease in atp/ p y receptor-mediated signal should be a trigger that transforms astrocytes into migratory phenotype, which forms tighter glial scar structure thereby suppressing neuronal death. background: oligodendrocyte damage and loss are key features of multiple sclerosis (ms) pathology and oligodendrocytes appear to be particularly vulnerable to ros. in vitro studies showed that ros induce cell death and prevent the differentiation of oligodendrocyte precursor cells (opcs) into mature myelin-producing oligodendrocytes. hence, a potential therapeutic strategy to protect these cells from ros-mediated damage is urgently needed. here we investigated the efficacy of several compounds that are able to boost antioxidant enzyme production, including monomethyl fumarate (mmf), tert-butylhydroquinone (tbhq), sulforaphane (sfn) and protandim. these compounds are thought to exert their protective function via activation of the nuclear-factor-e -related factor- (nrf ) transcriptional pathway, which is involved in the production of antioxidant enzymes necessary for oxidative stress defense. methods: primary rat oligodendrocytes were treated with different concentrations of mmf, tbhq, sfn and protandim. expression of antioxidant enzymes were analyzed by pcr and western blot analyses. to study the beneficial effects of the different nrf activators, oligodendrocytes were first incubated with nrf activators and subsequently exposed to various concentrations of hydrogen peroxide. oligodendrocyte cell survival was measured by a live/dead cell viability assay. results: sfn, mmf and protandim are well-tolerated and induce nrf driven antioxidant enzyme production in oligodendrocytes. protandim was the most potent compound with regard to antioxidant enzyme induction and protected oligodendrocytes against ros-induced cytotoxicity. conclusions: our findings indicate that several nrf activators are able to induce antioxidant enzyme production in oligodendrocytes. interestingly, protandim, a dietary supplement consisting of herbal ingredients, was the most potent protector of primary rat oligodendrocytes. in future experiments we will determine whether protandim can also promote the differentiation of opcs under oxidative stress and test the clinical efficacy of protandim in an experimental animal model for ms. the arising of the "tripartite synapse" concept and the discovery of the astrocytic excitability have been highlighting astrocytes as active elements concerning information flow in the brain. however, the impact of such remarkable features in complex brain function is still under-explored mostly due to the difficulty of studying neuron-astrocyte interactions in vivo. the aim of this work was to use an in vivo model of astrocytic dysfunction and investigate the impact of this treatment in complex cognitive functions. the rationale consisted in studying behavioral performance that relies on the prefrontal cortex, such as behavioral flexibility and working memory in an animal model of astrocytic dysfunction. for that purpose, we used a pharmacological model in which wistar-han rats were subjected to bilateral intracranial injections of aminoadipate in the prelimbic portion of the medial prefrontal cortex to cause astrocyte depletion specifically in this region, mimicking pathological states such as depression, in which marked decreases of gfappositive cells are observed. this animal model was tested for its cognitive abilities by the attentional set-shifting task (asst) and water maze-based tests. a clear impairment of cognitive function was observed in the animals treated with aminoadipate. a detailed morphological and histological analysis showed that along with the astrocytic lesion, neurons were also affected at the site of lesion. this seems to contribute to the cognitive decline observed in this model and may explain similar observations under pathological states. a. sternotte, e. hermans universit e catholique de louvain, brussels, belgium amyotrophic lateral sclerosis (als) is an adult disease characterized by a selective loss of motor neurons, resulting in progressive paralysis and death within - years. in several inherited cases, the disease is caused by point mutations in the gene encoding for superoxide dismutase (sod ) which promotes its aggregation, leading to biochemical damages, including mitochondrial dysfunction. alteration of mitochondrial membrane potential and/or respiratory chain leads to atp depletion and these metabolic dysfunctions could contribute to motor neuron death in patients and animal models of als. in motor-neurons, the combination of altered axonal transport with mitochondrial dysfunction likely leads to considerable decrease in atp levels at distant neuromuscular junctions. commonly know as the fuel gauge of mammalian cells, amp-activated protein kinase (ampk) is a key enzyme in the control of cellular atp and energy homeostasis. in this study, the implication of ampk in the progression of als was specifically investigated by measuring the expression and activity of this enzyme in the spinal cord of mice overexpressing the mutated human sod (hsod g a ), a commonly used experimental model of als. no consistent modification of the enzyme was detected in spinal cord samples throughout the progression of als. indeed, such experiment did not discriminate between cell types present in the tissue. hence, a more detailed characterization of ampk in cultured astrocytes derived from als animals revealed a robust induction of the enzyme activity, which correlates with decreased atp levels in these cells. in a second part of our study, we have investigated the consequences of ablating ampk in the mouse model of als by breeding ampk knock out mice with hsod g a mice. while we did not detect any difference in the lifespan of ampk(-/-)/hsod g a mice as compared to hsod g a mice, we observed substantial alterations in the progression of the disease in selected behavioural tests. in particular, analysis of the gait (catwalk) which enables early detection of muscle weakness revealed that the onset was delayed by up to days while the progression of the disease after onset was accelerated. further studies will be conducted to establish the importance of atp-in the disease, and to validate this enzyme as a putative pharmacological target in als and in other neurodegenerative diseases involving mitochondrial dysfunction. institute of neuroscience, sibs, cas, shanghai, china ng glia is the fourth type of neuroglia in vertebrate nervous system, which also known as oligodendrocyte progenitor cell (opc) in a developmental point of view. in adult brain, ng glia has a small cell body with highly branched extensions and represents a new type of glial cell differing from other glial cell types such as astrocyte, microglia and mature oligodendrocytes. published studies by others have shown that ng glia is able to promptly respond to brain injury and activated in several neurodegenerative disease animal models including -ohdainduced rat parkinson's disease model. however, the pattern and function of these activated ng glia remain largely unknown. here, we performed a time-course study of ng glia activation in mptp mouse model of parkinson's disease using immunohistochemistry. it was found that ng glia was activated as early as hours after the last mptp injection in the substantia nigra (sn) of mptp-treated mice. this temporal character is very similar to those of microglia, indicating ng glia respond to mptp challenges very quickly. the activation of ng glia became stronger over time and reached the peak at about days after the final mptp injection. then, the extent of ng glia activation declined gradually and diminished days after mptp injection. interestingly, we found that prominent ng glia activation could only be observed in the substantia nigra. the dorsolateral striatum which is the projection target of nigral dopaminergic neurons, however, was devoid of ng glia activation in all the time-points examined. in summary, we characterized the ng glia activation in mptp mouse model of parkinson's disease. these data suggest that activated ng glia may play a role in the degenerative process of dopaminergic neurons in pd. hallmarks of cns inflammation, including microglial and astrocyte activation, are a feature of post-mortem tissue from amyotrophic lateral sclerosis (als) patients and in transgenic mice overexpressing mutant superoxide dismutase- (sod g a ). administration of glucocorticoids does not significantly alter disease progression, but this may reflect poor cns delivery. here, we sought to discover whether cns-targeted liposomally-packaged glucocorticoid would inhibit the cns inflammatory response and reduce motor neuron loss. sod g a mice were treated with saline, free methylprednisolone (mp, mg/kg/week) or glutathione pegylated liposomal mp ( b - , mg/kg/week) and compared to saline treated wild-type animals. animals were treated weekly with intravenous injections for weeks from days of age. animal weights and motor behaviour were monitored during this period. at the end of the experimental paradigm ( days) mice were imaged using t -weighted mri for brainstem pathology, and brain and spinal cord tissue was collected for histological analysis. all sod g a animals showed a significant decrease in motor performance compared to baseline from $ days. sod g a animals showed a significant increase in signal intensity on t weighted mr images, which may reflect the combination of neuronal vacuolation and glial activation in these motor nuclei. treatment with b - , but not free mp, significantly reduced t hyperintensity, which correlated with significantly reduced histopathological manifestations in the brainstem motor nuclei, but not the spinal cord. interestingly, there was a significant reduction in astrogliosis but not microglial activation following treatment with b - . the results of this work indicated that the cns-targeted anti-inflammatory agent b - has therapeutic potential in als. the toxic effects of new antiretroviral drugs on the central nervous system (cns) are unclear. because these drugs penetrate the brain even at low concentrations, it becomes crucial to determine the doses which can be toxic for the cns resident cells. moreover, after the recent introduction into clinical practice, it is unclear whether the efficacy of the antiretroviral drugs of new generation may also derive from their ability to exert extravirological effects on factors responsible for the development of hiv brain injury, e.g. matrix metalloproteinases (mmps). objective: to investigate on the toxicity of four different antiretroviral drugs and their ability to modulate the expression of gelatinase b (mmp- ) in astrocyte cultures. methods: primary cultures of rat astrocytes were activated by exposure to mg/ml lipopolysaccaride (lps) (positive control) and simultaneously treated for h with increasing doses ( - - - . mm) of: efavirenz (efv); darunavir (drv); maraviroc (mvc) or raltegravir (ral). mmp- mrna expression was assessed by rt-pcr. quantitative determination of mmp- expression was done by computerized scanning densitometry. single drug toxicity was assessed by the mtt test. each drug was considered toxic at the concentration able to induce a percentage of cell survival above %. results: the treatment with antiretroviral drugs inhibited mmp- mrna expression in lps-activated astrocytes in a dose-dependent manner. in particular, a statistically significant inhibition of mmp- expression was observed when astrocytes were treated with mm efv ( % of inhibition) or with mm ral ( % of inhibition). as assessed by the mtt test, the toxicity of the antiretrovirals ranges from and mm. in particular, efv was toxic for astrocytes at the concentration of mm, mvc at mm, while drv and ral were toxic at the concentration of mm. conclusions: the present results indicate that efv and ral directly inhibit mmp- expression in lps-activated astrocytes with mechanisms that are independent from their antiviral activity. the toxic doses of antiretrovirals are much higher than those found in the csf of hiv-positive patients. our results highlight some beneficial/deleterious extra-viral effects of the antiretroviral drugs that may be useful to improve the development of new therapeutic strategies for the management of hiv infection. we used a parahippocampal kainate injection mouse model to induce mtle-hs and to study early expression patterns of kir . and apq after se. mice were sacrificed h, h and d after kainate injection. immunhistochemistry of the hippocampus was performed to assess kir . and aqp expression, and gfap staining was used for identification of astrocytes. gfap expression was increased compared to saline injected controls, suggesting that kainate injection induces astrogliosis. comparison of kir . and aqp staining showed a similar change in the expression pattern. our data indicate a drop in kir . and aqp expression within hrs after se followed by an upregulation within the first week. altered expression of kir . and aqp could contribute to dysregulation of potassium and water homeostasis and play a role in the early phase of epileptogenesis. c. l€ o€ ov, a. erlandsson uppsala university, neuroscience/neurosurgery, uppsala, sweden we have previously shown that astrocytes effectively engulf dead cells after trauma both in vitro and in vivo, but that they store the ingested material rather than degrade it. compared to macrophages, which degrade engulfed, dead cells within hours, our data show that astrocytes store the ingested material for weeks before the degradation is completed. to further study the routes of degradation and the possibilities to speed up this process we have used a cell culture model where uvtreated, dead cells are added to stem cell-derived astrocytes. by labeling the dead cells with the ph-sensitive dye phrodo prior to the engulfment, we demonstrate that the ph in the astrocytic phago-lysosomes is higher than in professional phagocytes. interestingly, lamp and lamp , which are involved in the phago-lysosome fusion, are both highly expressed in the astrocytes, particularly around the ingested material. dendritic cells are known to express the inhibitory protein rab a that slow down the degradation in order to preserve antigen for presentation. rab a prolongs the actin coating around the phagosomes, which physically inhibit the phago-lysosome fusion, and interacts with nox- , which leads to an increased consumption of protons. western blot analysis shows a high expression of rab a in our cell cultures which may explain the slow degradation. moreover, dead cells in astrocytes are surrounded by actin rings for a long period of time after the ingestion. to counteract this, the astrocytes were treated with . or mm of the actin inhibitor latrunculin b. the number of actin rings were significantly lower in the cultures treated with mm of latrunculin b compared to controls, but the intensity of phrodo was unaltered indicating that the prolonged degradation may be due to a higher ph in the lysosomes rather than a delayed actin coating. next we will investigate the effect of rab a expression on the degradation process by performing sirna experiments. one of the promising strategies for the treatment of spinal cord injury is the transplantation of glial cells obtained from the olfactory system; olfactory ensheathing cells (oecs). effective proliferation and migration of oecs are essential for optimizing clinical applications and there is a need for identification of small molecules that regulate glial cell biology. curcumin is a natural polyphenol compound found in the spice turmeric, which is known for its neuro-protective properties and has been reported to have an effect on neurogenesis and nerve regeneration. however, the effect of curcumin on oecs has not been determined. we have examined the effect of curcumin on oecs at the cellular level using fluorescence and timelapse microscopy. oecs were purified from s b-dsred transgenic mice in which oecs express the fluorescent protein dsred. different treatments: (i) control medium, (ii) curcumin ( . to mm), (iii) g commercial growth factor, or (iv) a combination of g and curcumin were used to determine the effect of curcumin on oecs proliferation and migration. cell proliferation was quantified at two different times by cell counting and mts proliferation assay. timelapse microscopy was used to visualize changes in cell morphology and cell migration. cell branching, number and area of lamellipodia and speed of migration per cell were measured for each treatment. we found that lower concentrations of curcumin ( . and mm) increase oec proliferation. as well, combination of g and curcumin showed the highest proliferative effect on oecs suggesting a possible synergistic relation between g and curcumin. live cell imaging analysis showed that curcumin increased the number and area of lamellipodia resulting in faster migration of oecs. these results suggest that curcumin can regulate the proliferation, morphology and migration of oecs which could improve the therapeutic use of oecs for spinal cord injury repair. methods: the dams were divided in four groups and had received ml of nonalcoholic or alcoholic beer solution -control, vehicle (nonalcoholic solution), etoh % (nonalcoholic solution %v.v ethanol) or etoh % (nonalcoholic solution %v.v ethanol) during gestational day up to weaning (postnatal day ). male offspring ( - days old) were sacrificed and hippocampal acute slices were prepared. we analyzed gfap content, s b and glutamate uptake as astrocyte parameters. we tested the animals in the plus-maze discriminative avoidance task to check anxiety and memory. results: gfap content decreased after pnee for etoh % and etoh % treatment (f , . , p < . ). interestingly, we found that only the etoh % treatment showed an increase of hippocampal s b content (t . , p . ) and in the csf (f , . , p . ), but no difference in secretion. glutamate uptake was significantly decreased for etoh % group (f , . , p . ). to perform the plus-maze discriminative avoidance task we selected pups from control, vehicle and etoh % treatment on pnd . two-way anova revealed significant effects of treatment (f , . , p . ), arm type (f , . , p < . ) and treatment x arm type interaction (f , . , p < . ). in the test session, only a significant prenatal treatment group effect (f , . p < . ). bonferroni's post-hoc test revealed that only control group presented significantly less time in the enclosed aversive arm than nonaversive one. conclusions: in this study we showed that pnee with moderate doses could alter the structure and functional astrocytes balance. thus, high levels of s b are indicated in some neurodegenerative disorders. taken this data together we could demonstrate that moderate ethanol exposure can be harmful to fetal brain. aging has been associated to neuroinflammation in the central nervous system, however it is not known whether microglial changes induced by aging are affected by early effects, such as litter size and sedentary life style. in addition, the lateral septum has been recognized by its complementary role in the memory processing for tasks of object recognition for identity and spatial location. in the present report, we investigated whether aging cognitive decline and microglial morphological changes in the lateral septal region are influenced by changing litter size early in life and by a sedentary life style. to assess these questions, wistar rats suckled in litters of either six or pups per dam were raised sedentarily in groups of up to , from the st post-natal day onwards. at (young adult) or (aged) months-old, half of the sedentary rats underwent progressive daily treadmill exercise for five weeks, while the others remained sedentary. after performing the tests of recognition for spatial localization and object identity all of the animals were sacrificed and their brains were processed for selective microglia/macrophages immunolabeling with anti-iba- antibodies. a representative sample of the immunolabeled cells in the lateral septum was analyzed after three-dimensional reconstruction with neurolucida software (microbright field inc.) and morphological features of each cell were quantified by neuroexplorer (microbright field inc.). it was found that sedentary life style of wistar rats maintained in standard laboratory cages is associated with spatial memory deficits in both mature and aged subjects no matter the litter size, and that exercise decreased these effects in aged subjects raised in small but not in large litters. on the other hand, all sedentary aged rats, despite of the litter size, presented impairment of object recognition memory for identity, and exercise decreased this effect in animals from both large and small litter size groups. microglial morphological analysis revealed that cell soma area, perimeter and branches volume seem to be more intensely affected by aging and that these changes are mainly associated with animals raised in large litters. furthermore, it was observed important shrinkage and thickening of the microglial branches in aged individuals on a higher proportion in the sedentary group suckled in large litters. taken together the results suggest that litter size and sedentary life style may affect object recognition and spatial memories in both adult and aged rats and that exercise minimizes these effects on animals suckled in small but not larger litters. in addition, we suggest that these changes are related to distinct effects on the soma and branching patterns of septal microglia from young and aged subjects. huntington disease (hd) is a dominant inherited neurodegenerative disorder caused by an unstable expansion of a cag repeat within the gene encoding for huntingtin protein (htt). this mutation induces formation of a poly-glutamine tract in the mutant htt (mhtt) protein that prones its aggregation into the cells. hd is characterized by an initial and massive degeneration of the medium spiny neurons in the striatum with later neuronal loss in cortex, globus pallidus, and other structures leading to motor and cognitive symptoms. alterations of energy metabolism contribute to hd pathogenesis but the mechanisms responsible for these alterations are not well understood. a recent pet study provided evidence for a selective impairment of striatal glycolytic and not oxidative metabolism of pre-symptomatic hd patients. in the brain, glycolysis is predominantly an astrocytic metabolic process, whereas oxidative metabolism is primarily neuronal raising the question that metabolic defects in hd could originates from astrocytes. the purpose of our study was to characterize ( ) the metabolic alterations in vivo in a transgenic mouse model of hd (bachd mice) and ( ) the respective roles of astrocytes and neurons in metabolic defects occurring in hd using an in vitro strategy. bachd mice express the human full length htt protein with glutamine repetitions under the human htt promoter into a bacterial artificial chromosome. bachd mice present a slow disease progression with motor deficits occurring at months and progressive neuronal aggregates from months, reflecting human pathology. first, we performed [ c]- -deoxyglucose autoradiography experiments in vivo to assess energy metabolism in months bachd (n ) and wt (n ) mice. we performed a d analysis of the whole brain glucose uptake without any regional a priori. using statistical parametric mapping (spm), a voxel-wise statistical analysis approach, we showed that compared to age-matched wt mice, months bachd mice present hypometabolism in the striatum, like pre symptomatic hd patients, and also in the hippocampus. hypermetabolism was also detected in the hypothalamus. second, we performed an in vitro study to dissect out the cellular mechanisms responsible for these metabolic changes. by using cell insert, we realized neurons/astrocytes co-culture without any physical contact. this culture system allowed us to grow neurons in presence of wt or bachd astrocytes. we showed that both bachd and wt neurons have a decreased [ c]- -deoxyglucose uptake when cultured in presence of bachd astrocytes but not wt astrocytes. these results collectively suggest that energy defects in bachd mice are due to noncell autonomous mechanisms by which astrocytes expressing mhtt induce neuronal metabolic dysfunction. naag and naa are abundant metabolites in the brain which can be utilised by oligodendrocytes to make myelin. but elevated levels of naag and naa are associated with myelin loss in the leukodystophies canavan and pelizaeus-merzbacher-like disease, whereas lower levels of naa are observed in brain tissue of ms patients. currently, the physiological function of these two metabolites as well as their role in white matter pathology is unknown. naag and naa can also act as signalling molecules as they can affect glutamate receptors. oligodendrocyte precursor cells (opcs) express nmda receptors and activation of these receptors may be important for their differentiation and efficient myelination. in disease opcs are recruited to demyelinating lesions where they proliferate and differentiate into new myelinating oligodendrocytes. we therefore investigated the effects of both physiological and pathological concentrations of naa and naag on opc biology in vitro. we used calcium imaging to see whether naag or naa can induce intracellular calcium changes in opcs as they are known to act on neuronal nmda receptors. mm naa or mm naag evoked intracellular calcium changes in opcs (df[ / ] . . and . respectively), but only one third of the cells responded which corresponds to the proportion of nmda responsive cells in the cultures. however, not all opcs that responded to nmda did also respond to naag or naa. therefore, it seems that the naa and naag evoked calcium raise is via an nmda receptor independent pathway. naag and naa evoked a small but significant increase in intracellular calcium in opcs, which even after prolonged application of pathological levels of naa and naag did not affect the cells' viability as propidium iodide staining of opcs showed no significant difference in viability. over % of the opcs survived hrs application of mm naa and mm naag. naag and naa are produced and released by neurons but it is not known how opcs take them up from the environment and how it affects them. we therefore addressed which possible transporters opcs express and the effect naa and naag have on opcs' proliferation and differentiation. it is unlikely that naa and naag even at high concentrations have a detrimental effect on opcs. perhaps these high concentrations of naa and naag found in canavan and pelizaeus-merzbacher-like disease indicate rather opcs' deficiency to transport and utilise naa and naag for energy and myelin formation. vanishing white matter (vwm) disease is a severe leukoencephalopathy, classically starting in childhood. the disease is characterized by neurological symptoms, including uncoordinated movements and balance disturbances which are slowly progressive. episodes of rapid deterioration are triggered by febrile infections and minor head trauma, which can lead to coma. a treatment is unavailable and eventually all patients die within a few years upon diagnosis. the disease is caused by mutations in any of the five genes encoding the subunits of the eukaryotic translation initiation factor b (eif b). although this protein has an important function in all cells, mainly the cells in the brain white matter (astrocytes and oligodendrocytes) are affected by these mutations. pathologically the brain shows cystic degeneration with meager gliosis, dysmorphic astrocytes, lack of myelin and an increased number of oligodendrocyte and astrocyte progenitor cells. we have developed two mouse models for vwm to further unravel the disease mechanisms at molecular level and to develop and test possible treatments. the b mouse line is homozygous for a mutation in the eif b gene, encoding the eif bd subunit, and the b mouse line is homozygous for a mutation in the eif b gene, encoding the eif be subunit. both mutations have been described in human patients and are associated with a severe form of vwm. preliminary data show that these mice models recapitulate the clinical and neuropathological phenotype observed in vwm patients. pathologically, the central nervous system white matter is vacuolated and shows lack of myelin while astrocytes are dysmorphic and immature. these transgenic mice appear to be a representative model for vwm. microglial cell lines were differentiated from ips cells and represent in vitro models for human microglia. rt-pcr and flow cytometry analysis of ipsdm showed gene transcription and protein expression of cd respectively. the protein expression was increased by breaking the cis-interacting sialic acids on the microglial glycocalyx using sialidases. cd -fc fusion protein was designed and purified via the fc tag to study binding patterns of cd to various cell types expressing different glycocalyx structures. additionally, ipsdm exhibiting either an over expression or knockdown of cd will be used to determine protein functions. furthermore, preliminary immunohistochemical analysis of control brain tissue compared to ad patients showed increased cd protein expression in cd positive cells, in the latter. in summary, data show that cd is expressed by human microglial cell line enabling further characteristic and functional analysis of the protein. furthermore, preliminary analysis on primary brain tissue corroborates the association of cd -expressed on microglia -to lateonset ad. the neurotrophins are essential for peripheral nervous system development and myelination. we have previously demonstrated that the neurotrophin bdnf exerts contrasting influences upon myelinationacting through neuronal p ntr to enhance myelination, but inhibiting it via neuronal trkb. recently we have generated a small peptide called cyclo-dpakkr that structurally mimics the region of bdnf that binds p ntr . here we aim to investigate whether utilising cyclo-dpakkr to selectively target p ntr is an approach that could exert a unified promyelinating response. like bdnf, cyclo-dpakkr promoted myelination of ngf-dependent neurons in vitro, an effect dependent on the neuronal expression of p ntr . importantly, cyclo-dpakkr significantly enhanced the myelination of bdnf-dependent neurons in vitro, whereas bdnf inhibited it. local injection of cyclo-dpakkr adjacent to the neonatal sciatic nerve in vivo significantly enhanced myelin protein expression and increased the number of myelinated axons. we found that injection of cyclo-dpakkr also significantly upregulated the expression of neuregulin type-iii, a key factor required to induce peripheral myelination. thus, our data demonstrate that cyclo-dpakkr promotes peripheral myelination in vitro and in vivo. to investigate whether cyclo-dpakkr promoted the remyelination of peripheral neurons, we utilized the ean, a rodent model of peripheral demyelinating neuropathy. our data show that administration of cyclo-dpakkr significantly delayed the disease onset and reduced clinical severity in ean. this improved disease phenotype is supported by reduced demyelination, as cyclo-dpakkr substantially reduced the extent of myelin damage in myelinated peripheral nerves subjected to ean. collectively, our data demonstrate that using cyclo-dpakkr to selectively target p ntr promotes peripheral myelin development, and is effective at ameliorating ean disease and protecting against myelin damage. our findings suggest that selective targeting of p ntr is a strategy worthy of further investigation for the treatment of peripheral demyelinating diseases introduction huntington's disease (hd) is a neurodegenerative disease characterised by huntingtin protein misfolding and the intra-cellular accumulation of mutant huntingtin (mhtt). accumulation of mhtt is most pronounced in neurons and is thought to underpin neuronal dysfunction. oligodendrocytes have been shown to accumulate mhtt aggregates, which may contribute to cellular dysfunction in these cells. evidence from diffusion tensor mri (dt-mri) studies shows that white-matter changes are amongst the earliest pre-symptomatic changes observed in hd. these findings have been taken to implicate axonal and or glial aberrations prior to disease onset. methods we have used a variety of techniques, which include, emulsion in situ hybridisation, whole brain sub-fractionation, immunohistochemistry, meso scale cytokine assay, and western blots. results our investigations using the r / mouse model show that mhtt aggregates result in a specific down-regulation of the small heat shock protein (shsp) hspb (alpha-b-crystallin). localisation of hspb by whole brain sub-fractionation shows that hspb is enriched in the myelin fraction. emulsion in situ hybridisation further shows localises hspb in oligodendrocytes. detergent extraction of myelin fractions show differential hspb partitioning between soluble and insoluble fractions. cumulatively, these findings suggest that hspb is constitutively expressed in oligodendrocytes and is present in two distinct pools. recent studies implicate hspb as being a negative downregulator of inflammation. as such down-regulation of the shsp in r / mice may promote increased oligodendrocyte vulnerability to mhtt cytotoxicity. to investigate the role of hspb as a negative regulator of inflammation, we obtained transgenic hspb knockout mice. the knockout mice are viable and show very little phenotypic difference against littermate controls. we have analysed the inflammatory profiles of these mice using the meso scale. to validate our findings in the r / mice, we are currently investigating human tissue from distinct vonsattel stages of disease, to see if hspb downregulation is also observed in the human disease. conclusion as hspb has several critical cellular roles, its downregulation may compromise oligodendrocyte structure and function, which ultimately has negative consequences for neurons. it is therefore worthwhile to investigate it's specific role in the cns. the myelin sheath is attached to the axon through multiprotein complexes that form the axon-glial interactions. they consist of cell adhesion molecules and voltage gated ion channels and divide the axon into distinct domains: the node of ranvier, the paranode, the juxtaparanode and the internode. this molecular organization is crucial since recent studies identified distinct perinodal proteins as autoantigens in ms patients: nodal and paranodal neurofascin isoforms and juxtaparanodal tag- /contactin- . tag- is a cell adhesion molecule expressed both by axons and glial cells. in the adult nervous system, tag- organizes the juxtaparanodal domain of the fiber, where it interacts with caspr and the potassium channels (vgkcs). question: in this study we have analyzed the alterations of the juxtaparanodal proteins caspr , tag- and vgkcs in relation to paranodal caspr and nodal sodium channels upon the onset and progression of experimental autoimmune encephalomyelitis (eae) and in the cuprizone model of toxic demyelination. methods: we immunohistochemically and biochemically analyzed both models of cns demyelination. results-conclusions: our results show a higher paranodal susceptibility to disruption compared to juxtaparanodes in eae. as eae progresses there is subsequent compensatory efforts for myelinated fiber restoration that include paranodal protein re-clustering and increased heminodal formation, without subsequent juxtaparanodal protein aggregation. in contrast, juxtaparanodal organization was observed only when remyelination occured in the cuprizone model of toxic demyelination. supported by: imbb-forth scholarship, manassaki scholarship (university of crete), the project "myelintag" ( ) is implemented under the "aristeia" action of the "operational programme edu-cation and lifelong learning" and is co-funded by the european social fund (esf) and national resources. multiple sclerosis (ms) is a chronic disease of the human central nervous system that is characterized by focal lesions with inflammation, infiltration of immune cells, demyelination and axonal damage. activation of cannabinoid cb /cb receptors is considered a potential therapeutic strategy for the treatment of ms, based on the evidence that exogenous cannabinoid agonists exert neuroprotective and immunosuppressive effects in experimental models of the disease. nevertheless, the therapeutic use of synthetic and/or plant-derived agonists acting on brain cannabinoid receptors is limited by the possible adverse responses related to memory and learning impairment. an alternative approach that could avoid this limitation consists of enhancing the concentration of the main endocannabinoids (aea, -ag) by increasing their synthesis or decreasing their degradation. the main objective of this study was to analyze the effects of jzl , a selective inhibitor of -ag hydrolyzing enzyme monoacylglycerol lipase (magl), in the chronic eae model of ms. mice were treated daily with jzl ( mg/ kg and mg/kg) or vehicle from onset of the motor symptoms to the end of the experiment. comparison of the motor score curves indicated that both doses of jzl ameliorated the deficits observed in vehicletreated mice during the disease course. nevertheless, the beneficial effect of the mg/kg dose was no longer evident in mice scored at dpi. chronic treatment with mg/kg jzl reduced the conduction latency of the corticospinal tract measured at the end of the experiment, as well as the number and size of inflammatory lesions in spinal cord, whereas chronic administration of the high dose of the magl inhibitor had no effect on these parameters. importantly, treatment with the mg/kg dose of jzl reduced the coupling ability of cannabinoid receptors to g i/o proteins, measured by [ s]gtpcs autoradiography, in all the brain areas analyzed. finally, incubation with jzl prevented cytotoxicity by activation of ampa-type glutamate receptors in oligodendrocyte precursors in vitro. this protective effect of the jzl was blocked by the cb receptor antagonist am . our findings point to the involvement of cb receptors in the in vivo therapeutic effects of jzl , and suggest that chronic administration of magl inhibitors may be a promising strategy for the treatment demyelinating disorders in which oligodendrocyte excitotoxicity plays an important role as mechanism of white matter damage. under disease conditions, connexins can also release these signaling molecules into the extracellular milieu through hemichannels. since astrocytes are involved in the disease progression of als, we are exploring if abnormal gj activity can serve as a potential mechanism for the reported astrocyte-mediated toxicity. we used the sod g a mutation as a als mouse model to address the role of connexins in als. we observed that at the end stage of the disease, spinal cords of sod g a mice exhibited a significant increase in expression of connexin protein compared to their spinal cords of control mice. this increase in cx co-localizes primarily to astrocytes in the gray matter of the spinal cord. importantly, human post-mortem tissue from als patients compared to control patients exhibited a significant increase in cx rna and protein levels in the motor cortex and a trend for increase in cx in the cervical spinal cord. this is an important finding in pursuing cx as a potential candidate contributing to astrocyte-mediated toxicity in als. in addition, when astrocytes were isolated from wt and sod g a mice, endogenous levels of cx rna and protein were elevated in sod g a mice compared to the wt derived astrocytes. we are currently investigating if functional properties of astrocytes from sod g a mice are altered in addition to biochemical changes. in vitro studies using cx specific blockers to assess neuroprotection are also being conducted in co-cultures of neurons and astrocytes. these findings will be novel and an important step towards harnessing therapeutic strategies for als. the pdz protein omi/htra is localized in the intermembrane space of mitochondria. it is involved in mitochondrial homeostasis and may act as a chaperone. mutations in the serine protease domain of omi/ htra (mnd mice), leads to massive loss of striatal neurons and neuromuscular disorders in mice confirming the protective function within mitochondria. upon cellular stress, omi/htra is translocated from mitochondria into the cytosol when the mitochondrial outer membrane is permeabilised. in the cytosol, omi/htra blocks the action of the inhibitors of apoptosis proteins (iaps) by competitive binding or by degradation via the protease activity. this leads to caspase activation and apoptosis induction. omi/htra also regulates apoptosis in a caspaseindependent manner via its protease activity; recent studies have defined multiple substrates of the serine protease. in a yeast twohybrid screen we identified the ng proteoglycan as a binding partner of omi/htra . ng is expressed by oligodendrocyte progenitor cells (opc). in the presence of hydrogen peroxide, which is a model of cellular oxidative stress, omi/htra is released from mitochondria and can bind to ng in opcs. binding of ng to omi/htra via the pdz domain may thus be a way to sequester or regulate the serine protease. oligodendrocyte-lineage cells are known to be particularly sensitive to cellular stress and white matter diseases of new borns is a major pathology in situations where premature infants are exposed to unphysiological oxygen concentrations. it has been recently shown that glioma cells often express the ng protein which may play a protective role. our results indicate that the interaction between omi and ng may help protect opcs from cellular stress. microglia are the immunocompetent cells of the cns that continuously survey the extracellular milieu for foreign antigens and play an instrumental role in brain inflammation. in contrast, astrocytes extend highly ramified processes between neurons and synapses and play a vital role in regulating brain homeostasis, synaptic function and plasticity. interestingly, both microglia and astrocytes adopt a specialized 'activated' or 'reactive' state when exposed to adverse brain conditions. the changes in their molecular profiles include the release a diversity of proteins such as pro-and anti-inflammatory cytokines and extracellular matrix molecules that could impact each other's behavior and regulate the properties of nearby neurons. in alzheimer's disease (ad), activated microglia and reactive astrocytes are detected around plaques in mouse models of ad and human ad tissues. however, exactly how these glial types contribute to the onset or progression of ad still remains an open question. to gain a better understanding of the bidirectional communication and response of microglia and astrocytes in the development of ad-like symptomatology, we investigated the properties of these cells types in the crnd transgenic ad mouse model by applying confocal imaging and western blot analysis. crnd is an early-onset model of ad-like symptomatology showing ab deposits present at months of age and neuritic pathology by months. our results show complex spatio-temporal changes in the interaction of microglia and astrocytes around ab plaques during the progression of the disease in both cortex and hippocampus. we found that subtle rearrangements of microglial morphology and astrocyte reactivity were detected as early as month when ab plaque deposition is absent. by - months, low numbers of activated microglia and reactive astrocytes begin to surround small ab deposits. the establishment of complex 'reactive glial domains (rgds)' can be observed at months, when amoeboid microglia fully encompass large ab plaques and become surrounded by reactive astrocytes forming an elaborate outer shell-like structure. intriguingly, after months, the domains become progressively disrupted due to the reorganization and recruitment of additional microglia and astrocytes. we also observed gradated inflammatory phenotypes of glial cells. these are first restricted to rgds in the early and middle stages of ad but spread locally in late stages when disorganized rgds are observed. interestingly, neuronal dystrophy was correlated with the severity of the inflammation. we conclude that communication between microglia and astrocytes may be a key process in the ability of the brain to surmount a reparative response to early neurodegenerative conditions but may be disrupted or become overwhelmed in later stages of ad. supported by alzheimer society of canada (db) and by cihr (kkm and rq). poster topic extracellular matrix and cell adhesion molecules opcs exist in mature rodent and human central nervous system (cns; around - % of the total cells) and constitute an interesting source regenerative therapies in demyelinating diseases, like multiple sclerosis (ms). different molecules are involved in opc morphofunctional development during embryogenesis and postnatal stages, and some of them are upregulated in ms lesions, suggesting their involvement in the pathogenesis of the disease. this is de case of anosmin- , an extracellular matrix glycoprotein coded by the kal gene and responsible for the x-linked form of kallmann syndrome. the best known mechanism of action of anosmin- seems to be mediated through the interaction of this protein with fibroblast growth factor receptor (fgfr ) and the modulation of the activation of this receptor by fgf . this protein participates in the adhesion, migration and differentiation of various cell types in the cns; among others, anosmin- promotes the adhesion of neurons, neurite outgrowth, axonal guidance and branching of different cns projection neurons, as well as having a role in the migration of different types of neuronal precursors, immortalized gnrh-producing neurons and embryonic opcs. in addition, previous results of our group also suggest a role of anosmin- in demyelinated lesions from ms patients and point to the feasible pharmacological and genetic manipulation of the fgf /fgfr- /anosmin- system on endogenous and/or exogenous opcs in demyelinating lesions. in the present work we studied the functional implications of anosmin- and fgf- on neurobiology (cell death, proliferation, motility, migration) of postnatal/adult murine opcs isolated from cerebral cortex (p , p , p ) and of opcs isolated from adult human biopsies, using both in vivo (immunohistochemistry) and in vitro (chemotaxis chambers, migration, brdu uptake, tunel, immunocytochemistry) techniques. these results would be useful for the design of effective neuroreparative therapies in ms and other demyelinating diseases. funded we generated ipsc lines from fibroblasts isolated from pitx -gfp knock-in mice which allowed facs-purification of mature midbrain da neurons upon in vitro differentiation based on the expression of the mature da marker pitx . subsequently, mouse pitx -gfp knock-in ips cells were transduced with lentiviral vectors containing genes encoding for hl and for stx, the enzyme that add psa onto ncam. next they were differentiated in vitro into da neurons. the effects of hl and psa-ncam on differentiation, survival and outgrowth of ipsc-derived da neuron were analyzed in vitro and in vivo. indeed, we were able to obtain a pure suspension of mature ipscderived da neurons upon in vitro differentiation and fac sorting. transfection of pitx -gfp knock-in ips cells with hl or stx did not affect the pluripotent potential of these cells and did not interfere with the process of dopaminergic differentiation itself. we continued to establish the beneficial effect of l and psa-ncam expression on survival and outgrowth of ipsc-derived da neurons in vitro and after grafting in the striatum of parkinsonian rats. vascular endothelial growth factor (vegf) is a major regulator of neurovascular remodeling following brain injury. while much have been learnt about vegf functions on target endothelial cells, little is known about its intracellular trafficking, mode of secretion and matrix interactions in "source" cells, i.e. mainly reactive astrocytes. here, we generated a vegf::gfp fusion protein to follow the distribution of vegf during its trafficking in primary astrocytes and cos cells. we found that vegf::gfp forms dimers and gets glycosylated similarly to wild type, and as expected, the molecule follows the endoplasmic reticulum-golgi pathway. however, its post-golgi trafficking suggests a unique route with features that do not conform the classical constitutive secretory pathway: the secretion of vegf occurs even at c and shows a ca -and pkc-induced increase. we investigated the distribution of vegf in polarized primary astrocytes in an in vitro scratch wound assay. in these cells, vegf::gfp appeared to follow a vectorial distribution and accumulated behind the leading edge. the accumulation appeared to be on the extracellular surface, moreover, ultrastructural immunogold labeling revealed that extracellular vegf::gfp remains associated with discrete areas of the cell membrane and is accumulated in caveolae as well as in microvesicular shedding elements. this particular localization corresponds to fibrillar adhesions (fb), where vegf::gfp is co-localized with fibronectin. we also observed that vegf::gfp is targeted to adherens junctions between astrocytes, however, at these sites vegf::gfp was not co-localized with fibronectin. finally, we show in frap experiments that integrin turnover is decreased in vegf associated fbs, which raises the possibility of an autocrine/paracrine regulation of astrocytic functions. together, these findings have strong implications for understanding focal coordination of angiogenesis and vascular remodeling by astroglia derived vegf. after invading the embryonic cns, microglia migrate extensively to occupy their final positions. however, the cellular and molecular mechanisms of microglial migration are unknown. therefore, this study aims to elucidate the microglial migration behavior, cellular interaction partners and ecm-integrin interactions essential for migration in the developing neocortex. methods: immunohistochemical stainings and immunogold labeling for transmission electron microscopy were carried out on fixed brain tissue of c bl/ cx cr /egfp mice embryos (embryonic day (e) . - . ). multicolor facs analyses were performed on homogenized age-pooled cx cr /egfp embryonic cortici. microglial migration was recorded in cx cr /egfp -hgfap-cfp acute brain slices using multiphoton time-lapse excitation and analyzed using mtrackj in imagej. results: laminin (lm) is expressed as punctuate dots near the pia and in the ventricular zone at e . and disappears at e . to remain only in blood vessels and the pia. fibronectin (fn) is present as aggregates from e . until e . . both ecm proteins follow the course of radial cells. preliminary data indicate a transient upregulation of the lm receptor (lmr) on e . and e . . in addition, the percentage of microglia expressing the fn receptor (fnr) tends to decrease during development. ex vivo time-lapse recordings show that embryonic microglia are dynamic cells that migrate in random patterns. these cells seem to make brief contacts with the processes of radial glia. conclusions: embryonic microglia express laminin and fibronectin receptors and possibly change the expression level during development. they are dynamic cells that migrate in random patterns, and possibly make occasional contact with radial glia. knowledge about ecm-integrin interactions during microglial migration could reveal targets for intervention in pathological settings, such as multiple sclerosis and alzheimer's disease. multiple sclerosis (ms) is a chronic demyelinating disease of the central nervous system in which astroglial cells become hypertrophic, produce various extracellular matrix (ecm) proteins which become aggregated when secreted. these aggregated ecm proteins contribute to a non-permissive environment for repair. tissue transglutaminase (tg ) expression is enhanced in astrocytes in ms lesions. this enzyme is well-known for protein cross-linking capacities. moreover, tg can act as a co-receptor for b-integrins to bind to ecm proteins, thereby mediating cell adhesion processes. in this study we questioned whether astrocyte-derived tg affects ecm protein production and/or aggregation, and if astrocyte-derived tg mediates cell adhesion onto various ecm proteins. primary rat astrocytes were transduced with an empty lentiviral vector (mock) or with a lentiviral vector expressing human wildtype tg . alternatively, primary rat astrocytes were transduced with a lentiviral vector expressing scrambled shrna or tg shrna to knockdown endogenous rat tg . the rat primary astrocytes overexpressing human tg showed an increased production of fibronectin and collagen v. conversely, knocking-down tg showed a decrease in fibronectin and collagen production. interestingly, overexpression of tg resulted in enhanced aggregation of extracellular laminin. also, human astrocytoma cells (u ) were transduced with an empty lentiviral vector (mock) or with a lentiviral vector expressing human wildtype tg and allowed to adhere onto ecmcoated wells. astrocytoma cells overexpressing human tg showed increased adhesion onto laminin-, and fibronectin-coated wells. we conclude that astrocyte-derived tg affects production/aggregation of fibronectin and laminin. moreover, the astrocyte-derived tg mediated elevated production/aggregation of fibronectin and laminin coincides with more adherence of astrocytes onto these ecm proteins. we hypothesize that tg -mediated enhanced production/aggregation of fibronectin and laminin is related to increased astrocyte adhesion which together could contribute to the non-permissive environment for repair in the central nervous system of ms patients. a. tripathi, z. parikh, p. pillai the m.s. university of baroda, vadodara, india background: oligodendrocytes originate as progenitor cells (opcs) in discrete areas of the developing brain which undergoes a complex and precisely timed program of proliferation, migration, differentiation, and myelination in cns. during migration it interacts with its surrounding environment through integrins. purpose: to evaluate the interactions between integrins and growth factors (gfrs) that promote the molecular events such as proliferation, cytoskeletal reorganization and migration in oligodendrocytes leading to oligodendrocyte mediated myelination. methods: cortical cells were prepared from p rat cerebral cortex following standard protocols. immunocytochemistry (icc), western blot (wb), immunoprecipitation (ip), migration assay and real-time rt-pcr analysis were performed. for dissecting out the roles of different intracellular signalling proteins in the regulation of events downstream of receptor activation, use of pharmacological inhibitors was carried out. results: tnfa, pdgfa and estradiol significantly increased cell proliferation and cell processes. data also suggest differential effects of extracellular matrix (ecm) on signalling component activation. significant increase in erk expression was found following gfrs-integrin interactions. ecm proteins bind to integrin family members and mediate bidirectional signalling between the cellular interior and the external environment through the activation of focal adhesion complexes. conclusion: integrin switching, oligodendrocyte proliferation, cytoskeletal reorganization and differentiation profile is highly influenced by ecm and gfrs. integrin governs the signalling cascades underlying oligodendrocyte differentiation and myelination. this study was performed in strict accordance with the recommendations for the care and use of laboratory animals. all the protocols were duly approved by the institutional ethical committee, the m.s. university of baroda. topography, roughness and mechanical properties of micro environment are crucial parameters influencing cell adhesion/motility, morphology and mechanics as well as the development of cells e.g. stem/progenitor cells, and neuronal cells [ ] [ ] [ ] [ ] [ ] . atomic force microscopy is a powerful tool not only to study the morphology in terms of high resolution imaging and roughness measurements, but also to map mechanical and adhesive properties of the sample/cells and tissues. combining these remarkable abilities with advanced optical microscopy allows for extensive characterization of biomaterials and tissue slices [ ] [ ] [ ] . however, commercially existing technical solutions are either time consuming, or only limited practicable for soft, sticky, or fragile samples. therefore, we developed a new force curve based afm mode -quantitative imaging (qi tm ). the novel qi tip movement algorithm prevents lateral forces and controls the vertical forces for nondestructive imaging. to demonstrate the performance and flexibility qi tm mode we investigated topography, adhesion properties, and young's modulus local distribution of living dorsal root ganglion cells. a specialized platform -cellhesion v r -has been developed to run single cell force spectroscopy (scfs) with a need for long-range cellsurface and cell-cell binding experiments with up to microns pulling length. using single cell force spectroscopy (scfc), cell-cell adhesion can be quantified [e.g. ], and the contribution of different components e.g. from the extra cellular matrix, can be assessed [ ] . a new technical solution will be demonstrated if a cantilever attached cell can be transferred through the liquid-air interface. this approach allows to measure the adhesion of the same individual single cell on different materials within different dishes as it will presented for cho cell adhesion on plastic as well as albumin coated surfaces. we want to present the progress in automatic data processing and display to investigate topography, young's modulus and local adhesion phenomena on transparent and non-transparent biomaterials like titanium, peptide functionalized surface or cell layers, and tissue slices. l. vargova , m. cicanic , , e. sykova , charles university, nd faculty of medicine, prague, czech republic institute of experimental medicine, v.v.i., department of neuroscience, prague, czech republic bral- is a link protein that is localized in distinct brain regions, where it stabilizes the perineuronal nets of the extracellular matrix (ecm). our previous studies showed that qualitative and quantitative ecm changes, might evoke changes in the diffusion properties of the extracellular space (ecs), thus affecting the movement of neuroactive substances in the cns. in the current study, we determined by the real-time iontophoretic method the extracellular space volume fraction a (a ecs volume / total tissue volume) and the geometrical factor tortuosity k (k free / apparent diffusion coefficient) in coronal brain slices obtained from the sensorimotor cortex and the ventral posteromedial thalamic nucleus of sixteen-month-old bral- deficient mice (bral- -/-), age-matched bral- / controls and young adult ( -month-old) bral- / controls. the diffusion parameters in the cortex of young adult bral- / mice were: a . . (mean s. e. m.) and k . . , n , n (n -number of slices, n -number of animals). in the thalamus, there was a smaller a and a larger k ( . . and . . , respectively, n , n ). in young adult mice, we found no significant differences in the ecs diffusion parameters between bral positive and negative mice in either the cortex or the thalamus. in the cortex of aged bral- / mice we found a smaller a ( . . , n , n ) than in the cortex of young adult mice. during aging, there was no significant difference in the thalamic diffusion properties in wild-type animals, but in the thalamus of aged bral- -/-mice, a significantly decreased ( . . , n , n ) in comparison to the younger bral -/-animals as well as to age-matched controls. immunohistochemical analysis of the ecm in the ventral posteromedial thalamic nucleus revealed no positivity for bral protein in either group of bral -/-mice. in the youger mice, bral deficiency was associated with a small attenuation in the positivity of brevican and another link protein, ctrl ; however, in aged bral -/-animals, brevican and ctrl positivity was increased in comparison with the age-matched controls. in contrast to the cortex, we found no decrease in a in the thalamus of aged wt animals, suggesting that the process of aging may differ between the cortex and the thalamus. a decrease in the ecs volume fraction in the thalamus of bral- -/-aged mice may result from a disruption of the perineuronal nets and rearrangements of the molecular assembly, which seem to differ in young adult and aged animals. here we analyzed the global transcriptome of cultured astroglial cells incubated with activators of camp pathways. a bulk of astroglial transcripts were differentially regulated by camp signaling. camp analogs strongly upregulated genes involved in typical functions of mature astrocytes, such as homeostatic control, metabolic and structural support to neurons, antioxidant defense and communication, whereas they downregulated a considerable number of proliferating and immaturity-related transcripts. moreover, genes typically activated in reactive cells, such as immunological mediators and scar components, were repressed by camp. gene set enrichment analysis and evaluation in situ of gene expression in astrocytes in different states showed that camp signaling conferred a mature and in vivo-like transcriptional profile to cultured astrocytes. these results indicate that camp signaling is a key pathway restricting developmental and activation features of astrocytes and promoting their maturation. a positive modulation of camp signaling is suggested to suppress the mechanisms of activation driven by pathological situations and to promote the physiologically normal state of differentiated astrocytes. question: microglia, the innate immune system of the central nervous system, are crucial to maintain homeostasis by cleaning debris and attacking stressors. in the ageing brain, increased numbers of microglia with ameboid morphology and increased inflammatory cytokine levels are reported. the altered microglial phenotype in the ageing brain is characterized by hyperresponsive to immune stimuli, which is called immune priming. immune priming could be a potential cause of age-associated neurodegeneration. the aim of this study is to characterize the immune pathways and biological processes that are altered in ageing-induced immune primed microglia and to find genes potential drivers of this process. design/methods: the effect of ageing on microglia has been addressed in naturally aged mice ( months old) and in ercc transgenic mouse model in which accelerated ageing is induced by dna damage accumulation. from each of these models a pure population of microglia was isolated. microarray technology was used to obtain information about the genome wide gene expression profile. weighted gene co-expression network analysis (wgcna), uses correlational structures to find clusters of genes to make optimal use of a dataset in biological interpretation. results: using weighted gene co-expression network analysis, a very robust microglial immune priming gene module was discovered. in this module, there is a significant enrichment of the complement system, antigen presentation, interferon type- signaling and phagocytosis-machinery. the average expression of this module is stronger up-regulated in ercc transgenic than in normally physiologically aged mice, supporting the notion that ercc is characterized by excessive immune priming. conclusion: microglial priming which occurs in normal ageing or in accelerated ageing models is marked by increased expression of several immune signaling pathways. methods: we investigated three single nucleotide polymorphisms (snps) (rs (arg gly), rs (gln gly) and rs ) and five haplotypes in adrb to explore whether these polymorphisms were associated with ms progression in well phenotyped ms patients. genotyping was done using illumina bead microarray and missing genotypes were imputed using beagle. results: a trend towards a decreased frequency of rs c allele ( gln) was in people with progressive ms than in people with relapsing remitting ms. this was seen in both sp-ms (p (uncorrected) . ) and in pp-ms (v . , p (uncorrected) . ). however, those p values did not reach statistical significance after correction for multiple testing. neither haplotypes nor snps were significantly associated with altered msss, age of onset, time between fist and second relapse, processing speed (sdt) or brain atrophy. conclusion: we found no evidence that polymorphisms in adrb influence severity in multiple sclerosis. here we examine the expression of these brain-specific pgc- a isoforms in neurons and glia cells. we characterize the abundance of the different isoforms of pgc- a in primary murine cell cultures of neurons, glia cells, including oligodendrocytes, astrocytes and microglia under control conditions and metabolic stress. we show that pgc- a is differentially expressed in the different cell types, e.g. neurons and oligodendrocytes express much higher levels of the novel brain-specific isoforms of pgc- a. the different expression levels might reflect the different metabolic needs of the different cell types. s. marques, g. castelo-branco karolinska institutet, stockholm, sweden oligodendrocytes (ol) are neuroepithelium-derived cells that insulate neuronal axons through myelin-containing membranes, essential for axonal integrity and impulse transmission. defects in this process are present in several diseases, including the highly debilitating multiple sclerosis (ms). the process of remyelination can be promoted by ol precursor cells (opc) endogenously and occurs during a short window of time at earlier stages of ms, ultimately failing as the disease evolve. the understanding of development and differentiation regulation of ol is essential to clarify the reasons behind the defective myelination during pathologies and to open the pave to new remyelinating cell therapies. opcs start to be specified early during embryogenesis but regardless of the origin, their terminal differentiation and functional maturation occurs only at post-natal stages. in this project, we are characterizing the transcriptome of opcs during development, which will allow us to better characterize the heterogeneity of these cells and point out what might be the key factors involved in transition between states. recent studies suggest that non-coding rnas can interact with proteins complexes containing chromatin modifying enzymes and transcription factors, regulating their activity, acting as scaffolds and/or recruiting them to specific loci in the genome. therefore, we are investigating their role in opcs by rna interference and how they might modulate the function of key transcription factors. neural stem cells (nscs) represent a potentially valuable resource when considering repair strategies for cns damage. unlike their embryonic counterparts, adult neural stem cells (anscs) in the two neurogenic niches have an astrocyte-like identity, raising the question of what regulates this ansc state. some early postnatal astrocytes in the parenchyma also retain some nsc-like characteristics and in vitro display self-renewal and multipotency, though these properties are lost following in vivo maturation. interestingly, following injury, adult astrocytes can become reactive and reacquire nsc-like properties, whilst others can generate new neurons through forced expression of specific transcription factors. we are aiming to elucidate the gene regulatory networks underlying specific nsc and astrocyte states and transitions between them using transcriptomic and epigenomic tools alongside computational methods in different nsc and astrocyte populations. we will describe our latest findings from an in vitro system that aims to model immature versus mature astrocytes derived from a common progenitor. from microarray data, we have identified candidate genes and pathways involved in regulating astrocyte potential versus differentiation, including a role for specific pro-inflammatory signalling. we will also describe the associated epigenetic signatures that accompany cell state and discuss the implications of our latest findings. microglia are the cns immune-related cells and associated with the pathogenesis of several types of neurological diseases. following peripheral nerve injury (pni), spinal microglia become activated and have crucial roles in generating neuropathic pain. we have previously shown that interferon regulatory factor- (irf ), upregulated in spinal microglia after pni, regulates gene expressions that switch to a reactive phenotype. here we identified irf as a crucial factor of reactive microglia that works cooperatively with irf . we found that pni increased expression of irf in the spinal cord, the expression of which was restricted to microglia. forced expression of irf in cultured microglia markedly increased expression of irf in a manner that depended on its ability to bind dna. furthermore, irf -deficient mice failed to increase the expression of irf in spinal microglia after pni, indicating irf -dependent expression of irf in microglia in vivo. interestingly, upregulation of p x receptor (p x r; an atp-gated channel crucial for producing neuropathic pain) by forced expression of irf in cultured microglia was markedly suppressed by knockdown of irf expression. in a model of neuropathic pain, suppressing upregulated irf protein by spinal administration of sirna targeting irf alleviated pain hypersensitivity. altogether, the irf -irf axis drives a program of gene expression that promotes p x r hi microglia gating neuropathic pain. astrocytes are the most abundant glial cell type. they express a range of neurotransmitter receptors localized along their processes that cover synapses and constitute "microdomains" for signalling pathways. to investigate astroglial purinergic p y and glutamatergic nmda receptors we generated knockout mice in which gene recombination of "floxed" receptors was controlled by an astrocyte-specific and tamoxifen-inducible cre recombinase (glast-creert ). here, we present data on the dna recombination efficiencies of glast-creert x floxed p y mice and glast-creert x floxed glun mice in different brain regions (cerebellum, hippocampus, brainstem, optic nerve and cortex). recombination is determined by quantitative real-time pcr of genomic dna using primers across the loxp sequences flanking exon of the p ry gene and exon - of the grin gene, respectively. dna recombination was induced in young adult mice by intraperitoneal tamoxifen injection for three consecutive days and analyzed three weeks later. primers were designed such that reduction of the pcr signal reveals increasing recombination (i.e. gene excision and knockout) compared to control. the degree of reduction in the investigated brain regions is expected to be similar in both mouse lines; allowing conclusions ( ) about the reliability of the glast-creert /loxp system in general (i.e. recombination efficiency at different chromosomal locations), and ( ) gives a percentage of glastpositive cells (predominantly astrocytes) in the given brain area. first data demonstrate % recombination in the cortex, which is well in line with the percentage of astrocytes in this brain region. these results could be confirmed by using a reporter mouse line expressing the red fluorescent protein tdtomato (r -tdtomato) and using immunohistochemistry with astrocyte markers. in conclusion, the combination of quantitative real-time pcr analysis of gene recombination and cell-specific cre mouse lines represents a reliable approach to reveal the percentage of a given cell type in a given tissue region. the transcription factor creb is protective in injury and neurodegeneration, but the contribution of neuronal vs astrocytes is unknown. we have generated a mouse with targeted over-expression of a constitutively active creb (vp -creb) in astrocytes by crossing teto-vp and tta-gfap mice. vp -creb/gfap mice show expression of vp in astrocytes -as detected by immunohistochemistry -restricted to cerebellum, hippocampus and outer layers of cortex. we determined the role of astrocytic creb in the outcome of brain injury by subjecting wild type (wt) and vp -creb/gfap (tg) mice to a focal cryolesion (c) in the frontal cortex. at days post-lesion, wt mice presented a necrotic region filled with macrophages (labeled with lectin) surrounded by a rim of tissue with robust gliosis (labeled with gfap). tg mice showed reduced macrophage infiltration as compared with wt mice. a dna array analysis of the damaged zone (agilent) has revealed differentially expressed genes in paired comparisons. wtc/wt: , ; wtc/tgc: , ; tgc-t: ; tg/wt: (statistical linear model in limma., p values computed by empirical bayes moderated t-statistics at . ). there were significantly enriched kegg pathways regulated by the cryolesion, according to gsea and hypergeometric tests set at p < . . vp -creb had an effect on of the pathways. upon increasing the analysis stringency by setting the cut-off at p < . , the number of significantly enriched kegg pathways regulated by vp -creb was reduced to . we selected of these pathways related to functions: energy metabolism, inflammation, structural reorganization, genomic stability and apoptosis, which we define as the core signature of the vp -creb-elicited change. the differential expression of representative genes related to this signature was validated by real-time pcr. thus, vp -creb rescued the decreased expression of enzymes regulating energy metabolism, while decreasing the expression of pathways related to apoptosis, extracellular matrix and inflammation. the latter is consistent with the decreased macrophage infiltration detected by immunohistochemistry in tg mice. we posit that vp -creb protects the brain against bionergetic failure and secondary damage due to macrophage infiltration, while rendering the tissue more conducive to neurite regeneration by reducing extracellular matrix components. the study supports the notion of astrocytes as therapeutic targets in brain injury. furthermore, ca /cn/nfat dependent mmp expression was confirmed in pure astrocyte cultures derived from neural stem cells (ast-nsc), demonstrating that the induced mmp expression occurs in astrocytes, and not microglial cells. in an in vivo stab-wound model of brain injury, mmp expression was detected in nfatc -positive scarforming astrocytes. since [ca ] i increase is an early event in most brain injuries, these data support an important role for ca /cn/ nfat-induced astrocyte mmp expression in the early neuroinflammatory response. understanding the molecular pathways involved in this regulation could provide novel therapeutic targets and approaches to promoting recovery of the injured brain. the posterior hypothalamus (ph) is crucial for maintaining wakefulness whereas the anterior hypothalamus (ah) constitutes a sleep-promoting region. consequently, the energy needs of ah and ph should probably change throughout the sleep-wake cycle. the glycogen mobilization altogether with the astrocyte-neuron lactate shuttle (anls) likely constitutes two major mechanisms by which astrocytes ensure the neurometabolic coupling. therefore, we hypothesized that astrocytes of ah and ph may display differences in genes expression involved in these mechanisms during the sleep-wake cycle. to investigate this, we measured the gene expression levels in astrocytes obtained from transgenic mice expressing the fluorescent protein egfp under the control of the astrocyte-specific gene gfap promoter. mice were sacrificed at four time, zt , zt , zt and zt (zt corresponding to light-on). after brain dissection, "punches" of ah and ph were micro-dissected from slices. cell suspensions were obtained from a pool of samples by enzymatic digestion and cell trituration. then, gfp-positive cells, which were highly enriched in astrocytes, were sorted by facs. total rna was extracted from these cells and gene expression levels were assayed by qrt-pcr and normalized by cyclophylin mrna levels. seven genes related to anls were tested: the alpha sub-unit of the na-k atpase (atp a ), the two sub-unit of the lactate dehydrogenase (ldha and ldhb), the glutamate transporters (glast and glt ) and two monocarboxylate transporters (mct and mct ). levels of mrna encoding genes related to glycogen metabolism such as ptg, the glycogen synthase (gs) and the glycogen phosphorylase (gphos) were also measured results showed that the levels of mrna encoding ldha, mct and mct expressed significant circadian variations which can reach % for mct at zt , compared to zt levels in ah. the glt mrna levels as well as mct and mct mrna levels also displayed significant circadian variations in ph. levels of expression of genes encoding the gphos, gs and ptg did not display major regional circadian variation. this study shows that astrocytes displayed some transcriptional regulation in hypothalamic areas involved in the sleep-wake cycle. the difference in the pattern of expression of anls related genes in ah and ph, such as glt , suggests that neuro-metabolic coupling capacity of each structure might be different and might play a functional role in the sleep-wake regulation. , are a heterogeneous cell population that is despite its functional importance still not well defined. while astrocytes are no longer just considered the supporting cells in the cns and are recognized to play a significant functional role in e.g. synapse formation, regulation of the blood brain barrier and synaptic transmission, little is known whether specific subtypes of astrocytes fulfill these functional tasks differently. studies in our laboratory described brain region specific differences in the expression of astrocytic glutamate transporter (glt- ) with significantly increased levels of glt- in the spinal cord compared to brain. the mechanisms of glutamate transporter regulation are not well defined, and to date little is known about the factors that are responsible for regulating protein expression and transporter activity and whether this regulation is specific for certain subtypes of astrocytes. to study glt- regulation in more detail, we generated a family of transgenic mice using increasing lengths of the non-coding region of the glt- gene controlling expression of tdtomato. these mice were crossed with full length bac-glt- -egfp mice to compare astrocytic expression of the reporter genes. interestingly, only a subpopulation of gfp-positive astrocytes was found to have strong tdtomato fluorescence signal, when we crossed an . kb eaat promoter fragment mouse with the bac-glt- mouse, suggesting the existence of astroglial subtypes that are differentially regulated for eaat expression. such differences might account for variance in local glutamate-dependent excitotoxicity to motor neurons. to this end, we purified both tdtomato /gfp and tdtomato-/gfp astrocytes from multiple cns regions by fluorescence-assisted cell sorting (facs) and analyzed their transcriptomic profiles using microarray analysis. a list of candidate genes, mainly transcription factors and cell membrane molecules, was complied from this analysis and validated by both quantitative rt-pcr and immunofluorescence. pax and kir . were found to be highly expressed in astrocytes, consistent with previous findings. more importantly, kir . mrna was noted to be selectively enriched in cortical tdtomato /gfp cells, compared to gfp only cells. it suggests kir . may serve as an astroglial subtype marker. . we have hypothesized that microglial c/ebpb is a potential target to attenuate the neurotoxic effects of neuroinflammation. in order to test this hypothesis in vivo, c/ebpb knockout mice are not suitable because they show multiple phenotypic alterations as a result of the widespread expression of c/ ebpb. mice with specific c/ebpb deletion in microglia would be a better model. unlike gfap promoter for astrocytes, there is not a gold standard promoter for cre expression in microglia. cd b, cx cr or lysozime m (lysm), among other promoters, have been used to this end. we have generated lysm-cre/c/ebpb fl/fl mice with two main aims: ) to validate the use of lysm as a suitable promoter for microglial gene deletion by cre-lox recombination ) to analyze the effects of the absence of microglial c/ebpb in vitro and in vivo lysm-cre/c/ebpb fl/fl mice which were fertile and viable and did not show the female infertility and high perinatal mortality described in c/ ebpb knockout mice. western blot experiments revealed the presence of c/ebpb in protein extracts from wild-type but not from lysm-cre/c/ ebpb fl/fl microglial cultures. immunocytochemistry experiments confirmed this observation. thus, c/ebpb immunoreactivity was present in all cells in wild-type microglial cultures and it was absent in all microglial cells in lysm-cre/c/ebpb fl/fl microglial cultures. the microglial specificity of lysm-cre recombination was demonstrated in primary mixed glial cultures. whereas in wild-type primary mixed glial cultures c/ebpb immunoreactivity was observed both in astrocytes and microglia, in lysm-cre/c/ebpb fl/fl mixed glial cultures it was detected in astrocytes, but never in microglia,. to assess the functional outcome of microglial c/ebpb deficiency, we analyzed lps ifncinduced no production and we observed a marked decrease in lysm-cre/c/ebpb fl/fl microglial cultures. this decrease was also observed in mixed glial cultures in fitting with the microglial origin of no production in this model. in summary, these findings show that lysm-cre promoter causes recombination in % of microglial cells in primary culture and it is therefore suitable for microglial-specific gene deletion in vitro. experiments are in progress to determine the degree and specifity of microglial c/ebpb deletion in vivo in these mice. supported by grants pi / and pi / , instituto de salud carlos iii, spain. to study the functional role of cpeb , transgenic mice overexpressing cpeb in astrocytes were generated. cpeb overexpression without the endogenous untranslated region (utr) led to the downregulation of astrocytic connexins (cx & cx ). consistently, in the hippocampus of cpeb overexpressing mice, intercellular gap junction coupling was strongly impaired. cpeb overexpression also led to downregulation of glutamate transporter (glt- ) and glutamine synthetase (gs), key players involved in extracellular glutamate clearance. we have now raised mice overexpressing cpeb with the endogenous utr. in these mice, we observed the downregulation of cx , cx , glt- and gs. since interastrocytic coupling and astrocytic glt- and gs activities are downregulated in epilepsy patients with hippocampal sclerosis, we hypothesize that upregulation of cpebs in astrocytes may contribute to the pathogenesis of epilepsy by causing astrocyte dysfunction. to study the influence of the p gene in the structural and quantitative characteristics of astrocytes in the mice retina methods: adult mice of the c bl/ strain ( months old) were distributed into two groups: ) mice with two extra copies of p ("super p "; n ) and ) wild-type p age-matched control, as the control group (wt; n ). retinas were immunohistochemically processed with gfap. gfap astrocytes were quantified. results: in comparison con wt: i) retinal-astrocyte distribution followed established patterns; however, morphological changes were seen through the retinas in relation to p availability: astrocytes were more robust and secondary processes were more evident; ii) astroglial density was significantly higher in the sp retinas, both in the whole-retina (p < , student's t-test) and in the intermediate and peripheral concentric areas of the retina (p < . student's t-test). conclusion: the astrocyte changes in the sp retinas might improve the resistance of the retinal cells against oxidative stress and its downstream signalling pathways. however, neuronal damage caused due to excessive microglial activation is a well known phenomenon in neurodegenerative diseases. mirnas are novel regulators of gene function, controlling several biological processes. recent reports show altered mirna expression in immune-mediated pathologies, suggesting that mirnas have roles in modulating immune responses. thus, the present study was initiated to identify micrornas and their target mrnas which can modulate microglial inflammatory responses. a pilot study was designed to understand the role of mirna- b in modulating microglia-mediated immune response as this was found to be localized in the microglia. recently, mirna b has been shown to target several proteins including c-jun, the substrate of jnk mapk (mitogen-activated protein kinase) which mediates the release of proinflammatory cytokines in activated microglia. qrt-pcr revealed the decrease of mir- b expression level in activated bv microglia. loss-of-function and gain-of-function studies confirmed c-jun to be the target of mir- b in microglia. overexpression of mir- b in activated microglia resulted in a decrease in c-jun and jnk expression and activity, thereby dysregulating the mapk-jnk pathway and proinflammatory cytokines. further, treatment of neuronal cells, mn d with conditioned medium obtained from activated microglial cells, resulted in increased inflammatorymediated cell death upon knockdown of mir- b. overexpression of mirna- b also reduced the phagocytic ability in activated microglia. taken together, these results demonstrate the important role of mir- b in modulating the mapk pathway via c-jun which in turn affects different aspects of the inflammatory process accompanying microglia activation including cytokine response, no production, phagocytosis and neuronal cell death. thus, mir- b may prove to be a useful target for developing therapeutic strategies to control microglial mediated inflammation in neurodegenerative diseases. further in order to understand the global mirna changes contributing to microglial activation, a global mirna microarray was carried out using control, lps-activated and amyloid b-activated primary microglia. this screen identified several families of mirnas differentially expressed between the different treatment groups enabling us to analyze the mirna signature in activated microglia. national university of singapore, singapore, singapore an inflammatory process in the brain as a result of an injury or infection is mediated by the microglia, the prime immune effector cells of the central nervous system (cns). microglia have also been shown to display chronic inflammatory responses leading to neurodegenerative disorders. small ubiquitin-like modifier- (sumo- ) is a post-translational protein modifier, which has been shown to be associated with nuclear factor kappa b (nfjb)-mediated inflammatory pathway. in this study, we showed the expression of sumo- in microglia and characterized the roles of sumo- in activated microglia. we found that sumo- is specifically expressed in the amoeboid microglial cells of the early postnatal rat brain. secondly, lipopolysaccharide-mediated activation of microglia led to a significant increase in the expression as well the nuclear translocation of sumo- in microglia in vitro. in order to establish a function for sumo- in activated microglia, we sought to silence its expression using sirna-mediated gene knockdown. sumo- knockdown in lps-treated bv- microglia in vitro subsequently showed the decreased nuclear translocation of nfjb and expression of tumour necrosis factor-alpha (tnf-a) level. further, we performed an in silico screening to identify the targets of sumo- in the nfjb pathway. our study thus, suggests that sumo- regulates nfjb translocation into the nucleus for the transcription of pro-inflammatory genes including tnf-a in activated microglia. microglia are the main resident immunological cells the cns. in the healthy brain, microglial cells are in a surveillance state whereas upon rupture of cns homeostasis they enter activated states. in ischemic stroke, activated microglia is one of the most important cellular components of post-stroke neuroinflammation, which mainly occurs in and around the area of the infarct. microglia activation is characterized by morphological alterations, functional and transcriptional remodeling, which account for the acquisition of immune phenotypes. purinergic receptors are known to play important roles in microglia activation, and p x r have been suggested as potential therapeutic target to limit microglia-mediated inflammatory responses associated with brain diseases. in the present study, we have developed an experimental approach based on laser micro-capture to isolate microglia from the penumbra area at different post-lesion times (from h to days post-lesion). the repertoire of genes expressed by microglial cells was determined through cdna microarray analysis. with this approach we have been able to determine clusters of genes that are co-regulated thus revealing the time course of microglia activation in this model. in addition, we have compared the transcriptional remodeling of microglia in wild-type and p x -deficient mice. our results suggest that wild-type and p x r ko mice present specific transcriptional profiles, which only partially overlapped. thus our data suggest that p x r play significant roles in the regulation of microglial cells functions. z. chen , m. ghosh , r. sattler , m. robinson , j. rothstein johns hopkins university, baltimore, united states university of pennsylvania, philadelphia, united states question: astrocytes, the most abundant cell type in the central nervous system (cns), are a heterogeneous cell population that is despite its functional importance still not well defined. while astrocytes are no longer just considered the supporting cells in the cns and are recognized to play a significant functional role in e.g. synapse formation, regulation of the blood brain barrier and synaptic transmission, little is known whether specific subtypes of astrocytes fulfill these functional tasks differently. studies in our laboratory described brain region specific differences in the expression of astrocytic glutamate transporter (glt- ) with significantly increased levels of glt- in the spinal cord compared to brain. the mechanisms of glutamate transporter regulation are not well defined, and to date little is known about the factors that are responsible for regulating protein expression and transporter activity and whether this regulation is specific for certain subtypes of astrocytes. methods: to study glt- regulation in more detail, we generated a family of transgenic mice using increasing lengths of the non-coding region of the glt- gene controlling expression of tdtomato. these mice were crossed with full length bac-glt- -egfp mice to compare astrocytic expression of the reporter genes. results: interestingly, only a subpopulation of gfp-positive astrocytes was found to have strong tdtomato fluorescence signal, when we crossed an . kb eaat promoter fragment mouse with the bac-glt- mouse, suggesting the existence of astroglial subtypes that are differentially regulated for eaat expression. such differences might account for variance in local glutamate-dependent excitotoxicity to motor neurons. to this end, we purified both tdtomato /gfp and tdtomato-/gfp astrocytes from multiple cns regions by fluorescence-assisted cell sorting (facs) and analyzed their transcriptomic profiles using microarray analysis. a list of candidate genes, mainly transcription factors and cell membrane molecules, was complied from this analysis and validated by both quantitative rt-pcr and immunofluorescence. pax and kir . were found to be highly expressed in astrocytes, consistent with previous findings. more importantly, kir . mrna was noted to be selectively enriched in cortical tdtomato / gfp cells, compared to gfp only cells. it suggests kir . may serve as an astroglial subtype marker. conclusions: our data clearly suggest the existence of molecular subtypes of astrocytes. factor expressed by activated astrocytes and microglia. studies with non-glial cells have shown that c/ebpd is able to regulate the expression of proinflammatory genes. however, the role of c/ebpd in neuroinflammation has not been established. we have here analyzed the effects of c/ebpd absence on glial activation in vitro and in vivo. in primary mixed glial and microglial-enriched cultures the expression of the pro-inflammatory genes nos- , cox- and il- induced by lps ifnc, but not by lps alone, was attenuated in the absence of c/ebpd. chip experiments revealed binding of c/ebpd to the promoters of these genes in activated, but not in control, mixed glial cultures. in neuronal-microglial co-cultures the neurotoxicity elicited by lps ifnc-activated microglia was completely abolished when microglial cells were devoid of c/ebpd suggesting that this transcription factor plays a key regulatory role of the expression of potentially detrimental proinflammatory genes by activated microglia. to analyze the role of c/ebpd in neuroinflammation in vivo mice were treated systemically with lps ( ug/mouse; i.p.). this treatment induced a marked increase in c/ebpd mrna and protein brain levels. double immunofluorescence experiments showed the presence of c/ebpd in astrocytes and microglia in lps-treated mouse brain. in c/ ebpd -/-mice, systemic lps-induced brain expression of nos- , tnfa, il- b and il- was attenuated. finally, in mouse experimental autoimmune encephalitis (eae) c/ebpd was upregulated in the spinal cord and the severity of eae symptoms was reduced in c/ebpd -/-mice. altogether these findings demonstrate that c/ebpd plays a major role in the regulation of proinflammatory gene expression in neuroinflammation and point to microglial c/ebpd inhibition as a novel strategy to reduce the detrimental effects of neuroinflammation. supported by pi / and pi / from instituto de salud carlos iii, spain gene targeting strategies have become a powerful technology for elucidating mammalian gene function. the recently generated knockout (ko)-first strategy produces a knockout at the rna processing level and also allows for the generation of conditional ko alleles by combining flp/frt and cre/loxp systems, thereby providing high flexibility in gene manipulation. however, this multipurpose ko-first cassette might produce hypomorphic rather than complete knockouts if the rna processing module is bypassed. moreover, the generation of a conditional phenotype is also dependent on specific activity of cre recombinase. here, we report the use of an efficient molecular biological approach to test pannexin (panx ) mrna expression in global and conditional panx ko mice derived from the ko-first mouse line, panx tm a(komp)wtsi . using qrt-pcr, we demonstrate that tissues from wild-type mice show a range of panx mrna expression levels, with highest expression in trigeminal ganglia, bladder and spleen. unexpectedly, we found that in mice homozygous for the ko-first allele, panx mrna expression is not abolished but reduced by % compared to that of wild-type tissues. thus, panx ko-first mice present a hypomorphic phenotype. crosses of panx ko-first with flp deleter mice generated panx f/f mice. further crosses of the later mice with mgfap-cre or nfh-cre mice were used to generate astrocyte-and neuronal-specific panx deletions, respectively. a high incidence of ectopic cre expression was found in offspring of both types of conditional panx ko mice. our study demonstrates that panx expression levels in the global and conditional panx ko mice derived from ko-first mouse lines must be carefully characterized to ensure modulation of panx gene expression. the precise quantitation of panx expression and its relation to function is expected to provide a foundation for future efforts aimed at deciphering the role of panx under physiological and pathological conditions. agarwal, e. hughes, d. bergles johns hopkins university, baltimore, united states astrocytes elevate intracellular ca in response to stimulation of metabotropic receptors. these ca transients are linked to processes as diverse as synaptic plasticity and functional hyperemia, but the relevance of this signaling remains uncertain. although most studies of ca signaling in astrocytes have focused on somatic events, anatomical and functional studies indicate that ca signaling in astrocytes may be compartmentalized into functionally isolated "microdomains." to facilitate analysis of ca signaling, we generated transgenic mice in which the cytosolic and membrane anchored variant of genetically encoded calcium indicator gcamp , referred as cgcamp and mgcamp respectively, can be expressed conditionally in a cell specific manner. a ubiquitous cag promoter is used to control cgcamp and mgcamp expression, and a "stopper" sequence flanked by loxp sites was placed upstream of the coding sequence, preventing expression until cre excises this dna. these constructs were targeted to rosa locus to ensure widespread expression. to evaluate whether cgcamp and mgcamp were expressed at levels sufficient to resolve ca transients, we bred r -lsl-cgcamp and r -lsl-mgcamp mice to glast-creer mice. double transgenic offspring were injected with tamoxifen at weeks of age and analyzed - weeks later. histology revealed that cgcamp and mgcamp were expressed in astrocytes throughout the brain. astrocytes in acute cortical slices exhibited large amplitude, spontaneous increases in both cgcamp and mgcamp fluorescence. the signal to noise ratio of the fluorescence intensity was greatly improved in astyrocytes expressing mgcamp , partially due to the ability of mgcamp to better resolve near membrane ca flux. ca transients were typically restricted to small regions of an individual astrocyte, consistent with ca elevations within microdomains. spontaneous ca transients were occasionally observed in the soma of astrocytes expressing cgcamp but not mgcamp , and were not coincident with activity in the processes, suggesting that these regions are functionally uncoupled. to determine if gcamp expression is sufficient to visualize ca signaling in vivo, we made cranial windows over the somatosensory cortex and performed -photon imaging. microdomain ca transients were prominent in cortical astrocytes, indicating that these mice provide new opportunities for understanding the significance of this form of signaling. in the trigeminal ganglion (tg), satellite glial cells (sgcs) surround neurons and are able to release substances that may affect sensory transmission through the tg and contribute to mechanisms underlying craniofacial pain. the aims of this study were ) to investigate whether sgcs could be provoked to release glu in vitro and ) to study the in vivo response properties of trigeminal afferent fibres upon artificial elevation of glu concentration in the tg. methods: confocal microscopy was used to assess glu content in and the expression of excitatory amino acid transporter (eaat ) and eaat by sgcs of the tg. glu release was investigated in vitro, where sgcs were isolated from the tg of adult male sprague-dawley (sd) rats and treated with control medium or medium containing mm kcl together with , . , , mm of the eaat / inhibitor tfb-tboa. in vivo electrophysiology experiments were conducted in additional anaesthetized adult male sd rats to determine the effect of repeated intraganglionic injections of glu ( mm, ml , min apart) on the response properties of tg neurons that innervated either the temporalis or masseter muscle. cumulative afferent discharge was calculated as the sum of action potentials (ap) min post injection subtracted from the sum of ap min prior to injection. an electronic von-frey hair was used to measure afferent mechanical threshold (mt) at min intervals for min prior to the first and again min after the second microinjection of glu. results: most sgcs were found to be glu positive and express eaat and eaat . treatment with mm kcl resulted in a $ -fold increase in glu concentration, compared to control ( . . vs. . . mm, respectively; p . ). treatment with mm tfb-tboa significantly reduced the glu concentration to . . mm (p . ), which was further lowered to . . mm when mm was applied (p . ). cumulative afferent discharge evoked in tg neurons by the second injection of glu ( ap) was not significantly different from that evoked by the initial injection ( ap; p . ). glu injections significantly reduced muscle afferent mt (baseline: g) by % (p . ). these results indicate that glu can be released from sgcs through eaats, and that increased glu concentrations in the tg excite neurons and induce a state of peripheral sensitization. this may hypothetically be a mechanism, whereby activated tg sgcs could contribute to e.g. migraine headache. in an accompanying poster, we present data demonstrating that in cultured neurons l-lactate induces plasticity-related genes expression through a nmda-dependent mechanism (i.e. arc, zif and c-fos). here, we describe the effect of l-lactate on neuronal excitability by performing electrophysiological recordings of cultured cortical neurons. we observed that application of l-lactate ( mm) triggers an inward current with an amplitude of . /- . na and slow kinetics, with a peak reached at /- s (called ilac). the membrane current decreases gradually to reach a plateau, with a mean inward current of . /- . na persisting up to s ( minutes) after addition of l-lactate (called ipyr). in order to assess l-lactate specificity on the generation of these currents l-pyruvate, another monocarboxylate, as well as d-lactate, the non-metabolized enantiomer of l-lactate) were tested. in contrast to l-lactate, l-pyruvate ( mm) induced a low-amplitude sustained current ( . /- . na) similar to the late-phase slow current evoked by l-lactate (i.e ipyr), but no current similar to ilac. dlactate on its side did not elicit any specific current. pharmacological characterization of ilac and ipyr currents demonstrate that ilac is generated by nmda receptors (as it is blocked by mk ) and relies on active nmda receptors (as it is blocked by either glutamate or glycine binding sites inhibitors). in contrast, generation of ipyr by both l-lactate and lpyruvate is prevented by diazoxide, a katp opener, whereas ilac was unaffected by the treatment. in order to determine the importance of katp for plasticity-related gene expression, the katp closer glibenclamide ( um) was applied to neurons and mrna gene expression of arc and zif quantified. results obtained demonstrate that arc and zif mrna expression are unaffected by glibenclamide. as a whole this set of data demonstrates that l-lactate generates two types of inward currents in neurons i.e. nmda-dependent (ilac) or katp-dependent (ipyr). moreover, they highlight the nmda-dependent ilac current as the key mediator, in contrast to katp, of l-lactate-induced plasticity gene expression. this is further sustained by the observation that l-pyruvate does not reproduce the effect of l-lactate on gene expression (see accompanying poster). this set of results provides novel insights into the mechanisms of action of l-lactate as an inducer of plasticity gene expression, and an important signaling molecule for neuronal plasticity. the reliance on drg neuron co-culture for sclcs fate commitment stands as a major hurdle for the therapeutic application of this finding. thus, we aim to unravel the mechanisms underlying sclcs fate commitment in order to develop a drg neuron-free condition for generating fate committed schwann cells. we hypothesised that the switch is brought about by the membrane bound neuregulin (nrg), nrg type iii, but not the other soluble isoforms of neuregulin. as our results show that sclcs treated with soluble neuregulin did not show significant changes in morphology nor marker expression when compared with untreated sclcs. nrg type iii expression was observed on purified drg neurons by immunocytochemistry, western blot analysis as well as reverse transcription pcr for the mrna. mammalian expression constructs for nrg type iii were made by transfection into human embryonic kidney cells, hek t, and mouse embryonic fibroblasts (mef) with the aim of generating surrogate cell types for co-culture with sclcs to pursue cell-specific effects of nrg type iii on differentiation of sclcs. the findings promise a way for generating fate committed schwann cells for autologous transplantation for recovery after nerve injury. ( ) the co /hco buffer system. the latter being an open buffer system, because biomembranes are usually permeable to co , and, in addition, most cells express hco transporting membrane proteins. the enzyme family of carbonic anhydrases (cas) catalyses the reversible reaction from co and h o to hco and h , and thereby modulates the intracellular h buffer dynamics.we have performed calibrated in situ live-cell imaging with the proton-sensitive fluophore bcecf in glial cells and neurons of acute cerebellar slices of wild-type and knockout mice. the studies were used to quantify the intracellular buffer capacity and to dissect the contribution of the intracellular caii and the extracelluar caiv by comparing intracellular h shifts in glial cells and neurons from wild-type mice and mice deficient in their caii or caiv gene. we found that only one proton in is unbound and thereby chemically active, and that about % of this buffer capacity is mediated by the co /hco buffer system and the other % by intrinsic buffers. the rate of co -induced change in intracellular h concentration was increased by intracellular caii in both glial cells and neurons. the extracellular caiv on the other hand affected primarily neurons, but also showed unexpected intracellular activity (schneider et al. , pnas ). the rates of acidification and alkalinisation in wild-type and knockout mice were significantly reduced in the presence of the ca blocker -ethoxy- -benzothiazolsulfonamid ( mm). we confirmed ca protein expression by western blot and could also show that the expression of the remaining ca is not upregulated. these results provide new insights into cellular proton-coupled processes in acute neural tissue, which shows some new complex roles of carbonic anhydrases in shaping proton dynamics in cells and tissues. objective: it is becoming increasingly evident that inflammatory mechanisms promote neuronal hyper-synchronization and epileptogenesis following brain insults. our recent studies identified serum albumin as a key player in the inflammatory cascade leading to epilepsy, at least partially through the activation of tgf-b signaling pathway in astrocytes. in this study we set out to explore the mechanisms by which tgf-b induces glial activation and epileptogenesis. methods: primary cultures of microglia and astrocytes were treated with tgf-b and gene expression analysis along with protein quantification were performed by quantitative pcr and elisa. electrophysiological response to il- was obtained in acute brain slices (field potential recordings) and in-vivo (sub-dural corticoencephalography) following to a continuous administration of il- into the lateral ventricle for days. results: in glia cultures tgf-b induced early and rapid up-regulation of il- at both mrna and protein levels whereas the upregulation of other pro-inflammatory cytokines such as il- b and tnf-a was milder and at a later time point. notably, smad / -dependent tgf-b signaling induced the expression of il- primarily in astrocytes and to a significantly lesser extent in microglia. il- was sufficient to induce epileptiform activity in acute brain slices and recurrent seizures within - days a following continuous intracerebroventricular injection of il- . conclusions: we thus suggest astrocytic release of il- as a potential key mechanism in epileptogenesis. okayama univ., dept. of brain science, okayama, japan dopamine transporter (dat) is expressed not only in catecholaminergic neurons but also in astrocytes. we previously showed that repeated l-dopa treatment markedly induced expression of dat and apparent dopamine (da)-immunoreactivity in the reactive astrocytes in the striatum of animal models of parkinson's disease. it is thought that astrocytes can uptake both l-dopa and da via neutral amino acid transporter lat and dat, respectively. therefore, uptake and metabolism of l-dopa and da in the striatal astrocytes may influence their availability in dopaminergic system of parkinsonian patients. to clarify uptake and metabolism of l-dopa and da in striatal astrocytes, the contents of l-dopa, da and their metabolites were measured after the l-dopa/da treatment using primary cultured astrocytes. first, we revealed the expression of neutral amino acid transporter lat and aromatic amino acid decarboxylase (aadc) in the striatal astrocytes. the level of l-dopa in astrocytes was markedly increased after -hr l-dopa exposure, but da was not detected in the astrocytes or hr after the treatment. on the other hand, the da treatment for hr increased levels of da and its metabolites, especially dopac. these results indicate that uptaken da into astrocytes is rapidly metabolized and that uptaken l-dopa never be converted to da in astrocytes. furthermore, level of uptaken l-dopa in cultured striatal astrocytes was rapidly decreased after removing extracellular l-dopa. taken together, the present results suggest that striatal astrocytes act as a reservoir of l-dopa to uptake or release l-dopa depending on the extracellular l-dopa concentration, but have less ability to convert l-dopa to dopamine. s. limmer, c. kl€ ambt universit€ at m€ unster, m€ unster, germany neuronal function consumes a large amount of energy and thus the brain requires a constant but regulated supply of metabolites. in invertebrates, such as drosophila, the nervous system is floating in the hemolymph and all metabolites that enter the brain have to be transported across the blood brain barrier (bbb). as in primitive vertebrates, the drosophila bbb is generated by glia. a layer of so-called subperineurial glial cells completely encapsulates the nervous system and thus, all metabolites reach the neurons en route through glia. the main energy supply of the inner organs of drosophila is provided by trehalose that is found in high concentrations in the hemolymph. trehalose is a nonreducing disaccharide, in which two dglucose units are linked via a a,a- , -glycosidic bond. the uptake of trehalose is mediated by two trehalose transporters, which are members of the slc a gene family. the subperineurial glial cells then either secrete c sugars to the neurons -or alternatively supply energy to neurons by secreting c metabolites as described for the bee retina. to discriminate between these possibilities we have followed a number of different approaches. the relevance of trehalose transporters in the different cell types has been determined by mutant analysis and rnai studies. furthermore, we have studied the role of all other putative sugar and monocarboxylate transporters in glial cells by rnai knockdown. to determine whether c or c carbohydrates are secreted by glial cells we suppressed the expression of core enzymes regulating glycolysis or components of the respiratory chain in either glial cells or neurons. moreover, we have ablated mitochondria specifically in glia or in neurons through expression of a restriction enzyme targeted to mitochondria. we will summarize our results and discuss approaches towards identifying neuronal signals that control glial carbohydrate secretion during energy homeostasis of the brain. microglia have long been known to respond to virtually any insult to the central nervous system. they quickly migrate to the site of the insult, and play many important roles, such as barricading the injury site, phagocytosing debris, and releasing cytokines. the role of microglia in the normal brain, however, has only recently begun to be appreciated. with the advent of in vivo imaging, microglia have been shown to be constantly surveying their environment with rapid extensions and retractions of their processes. subsequent studies have shown that these 'resting' microglia (now known as 'surveying microglia') are indeed active throughout development and adulthood. during development, microglia have been shown to be involved in synaptic monitoring and pruning as well as synaptic stripping after injury. thus far, many studies have focused on the role of microglia at the synapse. however, we report here, for the first time, that in the cortex of normal adult rodents, a small percentage of microglia are specifically associated with the axon initial segment (ais). the ais is characterized by a high density of voltage-gated ion channels and plays an important role in initiation of the action potential and maintenance of neuronal polarity. this interaction seems to be limited to the 'surveying' phenotype of microglia and much less frequent in 'activated' microglia. this overlap of processes appears early in development and continues throughout adulthood although the function of microglia at the ais is still currently unknown. x. liu , j.-m. petit , , p.j. magistretti , , c. giaume cirb, collège de france, paris, france lndc, brain mind institute, epfl, lausanne, switzerland centre de neurosciences psychiatriques, chuv, prilly, switzerland astrocytes act as modulators of neuronal activity through mechanisms such as neurotransmitter uptake, 'gliotransmitters' release, and metabolic supply. recently, astrocytes were shown to modulate sleep by vesicular release of atp. besides the cellular mechanism, we are interested in the network behavior of astrocytes in sleep-wake cycle. indeed, astrocytes form networks of communicating cells via gap junction channels constituted by connexins (cxs). recently, we have demonstrated that astroglial networking is regulated by neuronal activity and that neuronal activity is affected by deletion of the two main astroglial cxs (cx , cx ). to investigate how astroglial networks behave in sleep-wake cycle, we used pharmacological treatments and sleep deprivation to manipulate sleep-wake status of mice and determined whether astroglial networks would change in response. astroglial networks in acute cortical slices were revealed by "dye coupling", i.e. by loading of a patch-clamped astrocyte with sulforhodamine b that diffuses into neighboring astrocytes through gap junction channels. also, cxs expression at mrna and protein levels was measured as an index of connections among astrocytes. the results are as follows. first, i.p. injection of modafinil, a potent wakefulness-promoting drug, increased both mrna and protein expression of cx in the mouse cortex. superfusion of modafinil also enhanced dye coupling among astrocytes in acute coronal slices of the mouse somatosensory cortex. this effect was abolished by ttx treatment, which demonstrates neuronal activity is necessary for the effect of modafinil. in contrast, c-hydroxybutyric acid (ghb), a sleep-promoting agent, and propofol, a general anesthetic, decreased astroglial coupling in cortical slices. interestingly, the effect of ghb was not affected by ttx, suggesting a different pathway of action from modafinil. next, we used a "gentle" sleep deprivation model to sleep deprive mice for hours from the onset of the light period. as a result, mrna of cx , but not cx , was increased in both the cortex and hippocampus. finally, dye coupling in cortical astrocytes was enhanced in mice after sleep deprivation compared to mice after normal sleep. this increase in dye coupling, however, was not observed in slices from cx knock out mice after the same sleep deprivation treatment. altogether these results indicate that astroglial networks are bidirectionally regulated by perturbations in sleep-wake cycle and that cx is the major cx sensitive to such perturbations. in neurons, synaptic and extrasynaptic gaba a receptors (gaba a rs) differ in their subunit composition, conferring them distinct functional and pharmacological properties. oligodendrocyte precursor cells, also called ng cells, are contacted by bona fide neuronal gabaergic synapses. however, we recently showed a synaptic to extrasynaptic switch in the mode of transmission between gabaergic interneurons and ng cells during postnatal development of the somatosensory cortex. we therefore hypothesized that the postnatal switch of gabaergic transmission in ng cells is accompanied by changes in the expression of gaba a r subunits. to test for this hypothesis, we stimulated neuronal fibers to evoke gaba a r-mediated responses in ng cells recorded in acute slices of the somatosensory cortex of ng -dsred transgenic mice. the effect of zolpidem and a ia on evoked gabaergic responses reveals the predominance of functional a -and a -containing gaba a receptors, respectively, at interneuron-ng cell synapses early in development. however, the expression level of a decreases when responses rely exclusively in extrasynaptic transmission at more mature developmental stages. more importantly, specific pharmacology for the c subunit, a crucial molecular component for the clustering of gaba a receptors at postsynaptic sites, demonstrated a down-regulation of this subunit in ng cells prior to the complete loss of gabaergic synaptic activity. in keeping with the synaptic nature of the c subunit in neurons, this molecular change in ng cells correlates with the switch from synaptic to extrasynaptic transmission. to corroborate these functional data, we performed single cell multiplex rt-pcr of a - , b - , c - and d subunit mrnas in ng cells of the second and fourth postnatal weeks. despite the large heterogeneity of subunit mrna expression at both developmental stages, we found that the number of cells expressing c mrnas dramatically decreases in the fourth postnatal week. in conclusion, the expression loss of the c subunit is an important molecular determinant impacting the change of transmission modes between interneurons and ng cells during cortical development. financial support: frc, anr blanche, arsep, dfg (sfb/tr ) and eu (fp - neuroglia). university of rochester, medical center, rochester, united states synaptic plasticity is a critical process throughout the lifespan for achieving and maintaining normal and efficient operation of the nervous system. while much is known about the functional and structural changes that occur at the synapse during synaptic plasticity, the mechanisms implementing these changes are poorly understood. despite being classically characterized as immune cells, microglia have recently been shown to play a role in normal brain function by restructuring and removing synapses. given the novelty of this role, few details are known about how microglia behave to implement such a role during periods of plasticity. to characterize the changes in microglial behavior during ocular dominance plasticity in the developing visual cortex, we examined microglial morphology in fixed brain sections as well as in vivo using two-photon microscopy. for analysis of microglia in fixed sections, c /bl mice were monocularly deprived for either hours, , , , or days during the visual critical period (p -p ). fixed coronal sections were stained with iba , a specific marker for microglia, imaged on a confocal microscope and analyzed using imagej. microglial density decreased rapidly (within hours) and remained low for the day deprivation period. characterization of microglial morphology revealed an increase in the complexity of the microglial process arbor after hours, which steadily decreased back to baseline by days. this suggests that microglia do undergo a rapid change in morphology during plastic events that is reminiscent but distinct from the traditional activation pattern observed during pathological events. for analysis of microglia in vivo, heterozygous cx cr /gfp (jung ) mice were monocularly deprived directly preceding the first imaging session, the binocular segment of primary visual cortex was imaged, and the same cortical area was re-imaged and days later. while no change in microglial density was observed, analysis of microglial process motility showed a decrease in motility after periods of monocular deprivation. these results, combined with the observed morphological changes, show that microglia undergo behavioral changes after a period of monocular deprivation that are inconsistent with pathological activation, suggesting that microglia take on different roles and activities during normal brain function. g. baltazar, j. oliveira, t. roxo, c. fonseca faculty of health sciences, cics-ubi, covilhã, portugal neuroinflammation is a pathological hallmark in patients and experimental models of parkinson's disease. both present the classical features of inflammation, with evidence of an uncontrolled process. moreover, microglia may become activated early in the disease process and remain primed, responding strongly to subsequent stimuli, and thereby enhancing inflammation-induced oxidative stress and cytokinedependent toxicity in vulnerable neuronal populations. we have previously demonstrated that glial cell line-derived neurotrophic factor (gdnf), released by ventral midbrain astrocyte cultures, potently prevents microglial activation induced by a pro-inflammatory agent. therefore, gdnf is an important mediator in the astrocytemicroglia crosstalk keeping microglia in a resting state. however, in the brain, microglia and astrocytes are not alone and other cell types, e.g. neurons, may interfere and influence this astrocytic control of inflammation. therefore, it is important to investigate if the presence of neurons alters gdnf-mediated astrocytic control of microglial activity. in this work we aimed at investigating whether the presence of neurons, injured or not, changes the ability of astrocyte-derived gdnf to prevent microglial activation. for that purpose, we used primary cultures of ventral midbrain astrocytes and microglia, as well as neuronastrocyte mixed cultures from the same brain region. neuron-astrocyte cocultures were exposed to vehicle (control) or to the da neurotoxin mpp . the media conditioned by control and mpp -challenged neuron-astrocyte mixed cultures, or by astrocyte cultures, was collected and transferred to ventral midbrain microglia cultures, which were then exposed to the pro-inflammatory agent lipopolysaccharide. the effect of conditioned media obtained from the different cell culture systems on microglial activity was determined by analyzing the production of nitric oxide by the griess reaction and of the phagocytic activity by determining the engulfment of fluorescent microspheres by fluorescence microscopy. university of lausanne, lausanne, switzerland lactate is produced by astrocytes and used by neurons as metabolic substrate during activity. recent evidence indicates that lactate may not only serve as energy substrate but also as modulator of glutamatergic or gabaergic neurotransmission. we tested this hypothesis using primary cultures of cortical neurons obtained from wild type and gad -gfp knock-in mice (c bl/ ). neuronal excitability was monitored by electrophysiological recordings and calcium imaging using the fluorescent probe fluo- am. spontaneous action potentials and rhythmic calcium transients were present in more than % of cultured neurons, which were either gabaergic or glutamatergic cells. in the presence of mm glucose, l-lactate application reversibly diminished calcium transient frequency in a concentration-dependent manner in both cell types (glutamatergic neurons ic . . mm and gabaergic neurons ic . . mm). to test whether lactate effects were dependent on metabolism, we applied the closely related substrate pyruvate ( mm) or switched to different glucose concentrations ( . or mm). none of these conditions altered the calcium transient frequency. in contrast, the application of d-lactate, thought not to be metabolized by neurons, decreased the spiking activity in the same concentration range as l-lactate (ic . . mm). we determined that d-lactate was taken up by neurons, however more than two-fold less efficiently than l-lactate. these results suggest that the mechanisms of lactate sensitivity are not purely metabolic. since d-lactate has the same potency as l-lactate but it is less internalized, it is likely that lactate acts as an extracellular ligand that triggers the reduction of neuronal activity. this hypothesis is currently under investigation. this modulating effect of lactate represents a novel role for the compound that has to be taken into account in the context of the interactions between astrocytes and neurons. ben-gurion university, ber-sheva, israel microglia integrate within the neural tissue with a distinct ramified morphology through which they scan the surrounding neuronal network, a process which appears to contribute to the integrity, maintenance and functioning of the brain. since microglia are long-lived, they are subjected to senescence processes which may severely compromise their function with age. here we used a digital tool for the quantitative morphometric characterization of fine cortical microglia structures in mice. we thus followed the morphological changes microglia underwent with aging and with the progression of alzheimer's-like disease. compared with microglia in young mice, microglia in old mice are less ramified and possess less branches and fine processes, a phenomenon which was associated with increased expression of pro-inflammatory genes. notably, a similar microglial pathology appeared - months earlier in mouse models of alzheimer's disease (ad). we thus demonstrate that in addition to promoting neurotoxic inflammation, amyloid plaques attract the microglia and modify their structure. this, in turn, causes a severe microglial process deficiency, which possibly results in compromised neuronal function and repair. blood-borne glucose is the main energy substrate for the brain under physiological conditions. however, the question remains unresolved if the two main cell types in the brain -neurons and astrocytes -consume glucose upon their individual needs or if glucose is mainly taken up by one cell group and glycolytically degraded to energy-rich substrates (e.g. lactate), which are then shuttled to the other cell group. a novel genetically encoded fret glucose sensor (bittner et al., , ) together with two-photon microscopy now provides for the first time the opportunity to determine intracellular glucose concentrations in single cells in the intact organism. cortical microinjections of an adenoviral vector (aav and aav ) coding for the sensor flii pglu md with the promoter sgfap for astrocytes and synapsin for neurons were performed in mice and chronic windows were implanted above the somatosensory cortex. after two weeks, specific expression in astrocytes and neurons was observed. the ratiometric fret signal in both cell types increased after intraperitoneal glucose injection and decreased after insulin application demonstrating the functionality of the construct in vivo. the astrocytic sensor displayed a large linear range at blood glucose levels ranging from to mmol/l. in a second set of experiments, we examined the dynamic behavior of cellular glucose concentrations upon increased brain activity. during electrical hindpaw stimulation % of examined astrocytes showed a . % decrease of the fret signal, whereas neuronal glucose levels did not exhibit activity-dependent fluctuations. to be able to draw conclusions about metabolic rates of substrate oxidation we recently started with experiments where cellular glucose uptake was transiently inhibited using different blockers of glucose transport. drugs were applied systemically and intracortically. we could demonstrate this principle on first measurements on astrocytes. glucose fret sensors allow measurements of the dynamics of glucose concentrations of single neurons and astrocytes in the intact organism. measurements of cellular glucose transients during transport blockade hold the potential to compare glycolytic rates of neurons and astrocytes. this tool may substantially contribute to uncover the complex organization of brain energy metabolism. neuron-glial communication in the cns is fundamentally important for many brain processes including synaptic transmission and plasticity. perisynaptic astrocytic processes (pap) contact excitatory synapses, forming tripartite structures with neurons. in the hippocampus, the morphology of pap has been shown to remodel rapidly and continuously but the mechanisms and roles of this form of structural plasticity remain unknown. this study investigated the physiological mechanisms driving pap movements and their role during long-term potentiation (ltp). pap and spines contacts were labeled by viral gene delivery of farnesylated fluorescent proteins in hippocampal slice cultures and imaged by confocal microscopy. electron-microscopy of infected slices confirmed that pap-synapse morphology is preserved in organotypic cultures. pap movements adjacent to dendritic spines were evaluated with an index of motility (mi). increasing neuronal activity by schaffer collaterals (sc) stimulation elevated pap mi and this was prevented by both ttx and mglur inhibition, suggesting that neuronal activity modulates pap movements. we next investigated whether intracellular calcium (ca i ) in astrocytes influenced pap motility. bapta-am bulk-loading specifically chelated ca i in astrocytes and reduced pap movements. when exogenous gq-coupled receptors mrga or mrgc were specifically targeted to astrocytes, their agonist fmrfa induced ca i increases and accelerated pap motility. moreover, fmrfa delivery at the synaptic level by two-photons flash photolysis was sufficient to elevate pap mi. we then investigated pap dynamics in relation to ltp. application of theta-burst sc stimulation initially increased pap motility, but then resulted min later in a decrease of pap motility. interestingly, the reduction of pap movements correlated with both spine enlargement and increased pap's spine coverage. to assess the consequences of these changes, we then specifically triggered elevation of pap movements at the synaptic level by flash photolysis. spine stability analysis hour after uncaging revealed that spines in contact with activated paps were more stable than others. this study suggests that excitatory synapses control the motility of surrounding paps through the triggering of neurotransmitter-evoked astrocytic ca i elevations. increased pap motility seems to be necessary to elevate their coverage of the synapse during ltp, leading to higher synapse stability. astrocyte reactivity occurs in response to many pathological brain situations. reactive astrocytes display morphological and functional changes that could result in concentration variations of some brain metabolites localized both in astrocytes and in neurons. such changes could be detected by magnetic resonance spectroscopy (mrs), allowing non-invasive monitoring of astrocyte reactivity and thereby neuronal dysfunction in vivo. one of the major peak detected by mrs in the brain corresponds to neuronal metabolite n-acetyl-aspartate (naa). although its function is unclear, it is considered as a biomarker for neurodegeneration and a decrease in its concentration is interpreted as neuronal death or dysfunction. however, there is not clear evidence that other cell types, specifically astroglia, could not contribute to change in naa levels in the brain. in this study, we used a rat model of astrocyte activation by stereotaxic injections of lentiviral vectors encoding for either the cytokine ciliary neurotrophic factor (lenti-cntf) into the right striatum or betagalactosidase (lenti-lacz) into the left striatum, as a control. mrs data were acquired on a t magnet from two voxels placed over each injected striatum. a diffusion-weighted laser sequence with echo time te ms was used. concentrations were measured using lcmodel software for total n-acetyl-aspartate (naa naag), myo-inositol (ins), total choline (cho), glutamate and taurine relative to total creatine. lenti-cntf injection promotes a sustained, extensive and selective activation of astrocytes, as evidenced by overexpression of gfap and vimentin and cellular hypertrophy (fig. ) . importantly, neurons displayed unaltered morphological, molecular and electrophysiological features, as described previously (escartin et al., ; beurrier et al., ). mrs evidences changes in metabolite concentration: in the lenti-cntf injected striatum, levels of the astrocyte metabolites ins and cho were increased, whereas levels of the neuronal metabolite naa and glutamate were decreased. increased levels of ins and cho are generally found associated with astrocyte reactivity, however, the decrease in naa is unexpected given that neurons are not dysfunctional with cntf. to understand how astrocyte reactivity can influence naa levels, we are currently characterizing the molecular changes occurring in the naa metabolism using quantitative pcr, biochemistry, histology and hplc. our data suggest that reactive astrocytes alone result in alterations of metabolite concentrations detectable by nmr. although the mechanisms by which activated astrocytes regulate neuronal metabolism remain to be elucidated, our work challenges the mrs dogma that decreased neuronal metabolites can be used as a biomarker for neurodegeneration. microglial phagocytosis is a vital phenomenon for the clearance of damaged and death cells or infectious agents in a context of brain injury or infection, respectively. in addition, microglia can boost synaptic plasticity by the phagocytosis of axon terminals and dendritic spines. therefore, it is crucial to better understand the mechanisms involved in microglia clearance in order to devise new strategies to promote an efficient brain repair. recently, we showed that histamine modulates microglia motility and cytokines release. in this work, we aimed to investigate the role of this molecule and its receptors in microgliainduced inflammation by evaluating microglial phagocytic activity and reactive oxygen species (ros) production. for that purpose, an iggopsonized latex bead assay was performed in n murine microglial cell lines exposed to lipopolysaccharide (lps) or histamine , and mm. we showed that histamine significantly stimulated phagocytosis of opsonized latex beads via h receptor activation. this effect was accompanied by the rearrangement of cytoskeleton analyzed by phaloidin and acetylated tubulin expression and cellular distribution. the phagocytic activity was significantly reduced after pretreatment with apocynin, a putative napdh oxidase (nox) inhibitor. all nox isoforms were expressed by microglial cells, but only the expression of nox was increased by treatment of histamine. rac , an important subunit for the activation of nox , was also activated by histamine. in addition, histamine induced ros production via h and h receptor activation. in conclusion, histamine plays an important role in the regulation of microglial phagocytosis, which is mediated by nox and rac activation. multiple sclerosis (ms) is a chronic inflammatory disease of the central nervous system characterized by demyelination and axonal damage, involving both white (wm) and gray matter (gm) areas. furthermore, cortical pathology correlates with ms progression and cognitive dysfunction. we previously found altered expression of gap junction (gj) proteins connexin (cx ) and cx in oligodendrocytes and cx in astrocytes in wm lesions and normal appearing wm (nawm) in brain samples from ms patients, and in a mouse model of experimental autoimmune encephalomyelitis (eae), implicating uncoupling of myelinating cells as an important aspect of ms pathology. here we studied cx and cx and their astrocytic partners cx and cx in and around cortical lesions and the normal appearing cortex (nacx) in ms samples and non-ms controls. we examined their expression by real-time pcr, quantitative immunoblot and immunohistochemichal analysis. compared to non-ms controls, expression levels of cx and cx mrna were reduced within lesions whereas cx and cx were increased. in nacx, all four connexins were upregulated with cx being significantly increased. immunoblot analysis of ms cortex revealed reduced cx and increased cx levels, while cx and cx showed no significant change. immunohistochemical analysis showed severely reduced cx gj plaques within lesions with gradual increase toward nacx. in contrast to nawm, cx gj plaques were not increased in nacx. both astrocytic gj proteins were increased in ms, but in distinct patterns: cx gj plaque counts were higher within lesions and perilesional areas. cx was increased in ms cortex compared to non-ms controls, but in contrast to nawm, cx gj numbers were higher in nacx than in lesions. similar alterations were found in the eae mice, in which cx and cx gj counts were reduced whereas cx and cx gjs were increased. our results indicate that oligodentrocytic as well as astrocytic gjs are affected not only in ms wm but also in the gm. loss of oligodendrocyte gjs and in parallel increase of astrocytic gjs reflecting astrogliosis occurs throughout the ms brain and may negatively affect the prospects of repair and remyelination, worsening disease progression. acknowledgements: this project was funded by the cyprus research promotion foundation (grants access/ / and health/bios/ / to kak) and by cyprus telethon ( - grant to kak). post-mortem human brain tissues were kindly provided by the uk multiple sclerosis society tissue bank, imperial college london, funded by the uk multiple sclerosis society. the mainly astroglialocated enzyme glutamine synthetase is required to synthesize glutamine from the re-uptake of either glutamate or gaba. thereby, this enzyme significantly contributes to the control of brain glutamate and gaba levels. there is good evidence that both neurotransmitter systems are dysregulated in depression. hence, we decided to study the cellular expression of this enzyme in subjects with major depression and bipolar disorder. material and methods: we investigated the numerical density of glutamine synthetase immunoreactive astroglial cells in different brain areas (prefrontal cortex, anterior insula) of subjects with major depression, subjects with bipolar disorder and matched control cases. results and discussion: we found that compared with controls in cases with major depression there was a significant reduction of the density of immunostained glial cells in all brain regions studied, whereas bipolar cases showed a normal expression of the enzyme. our findings point to a profound disturbance of the glutamateglutamine-gaba cycle in major depression, which is in good agreement with neuroimaging observations and molecular biologic data of others. m.-c. marx, d. billups, b. billups university of cambridge, cambridge, united kingdom perisynaptic astrocytes are thought to play a key role in the recycling pathway of glutamate by supplying the precursor glutamine to the presynaptic terminal. using fluorescent ph i measurements of astrocytes and direct patch-clamp recordings from the calyx of held synapse in rat brain slices, we have investigated the transport mechanisms that may mediate this release of glutamine from astrocytes in situ and assessed the role of glutamine in maintaining glutamatergic neurotransmission. glutamine transporter currents were elicited by puff application of mm glutamine from a nearby pipette. for imaging astrocytic ph i , hpts was included in the patch pipette. system a (sa) transporter function was probed using the substrate inhibitors meaib, alanine and serine, while system n (sn) function was isolated using its unique ability to transport li in place of na . histidine was used as a substrate inhibitor of both sa and sn. glutamine-induced membrane currents i gln ( . . pa, n ) were blocked by bath applied alanine ( mm) and meaib ( mm), as well as substitution of external na with li . fluorescent ph i measurement revealed an alkalinisation during puff application of glutamine. while bath application of histidine ( mm) abolished the ph change, meaib and serine ( mm) only attenuated it. most of the ph change ( %; n ) was insensitive to the substitution of na with li . in presynaptic terminals, puff application of glutamate did not induce a membrane current indicating a lack of direct glutamate uptake; however, puff application of glutamine resulted in a i gln which was eliminated by the removal of external na and by meaib. miniature epscs recorded from postsynaptic neurons ( . . pa) were inhibited by meaib ( . . pa; n ) following turnover of the presynaptic glutamate pool. single epscs were unaffected by meaib indicating no direct effect on postsynaptic ampa receptors. together, these data identify two glutamine transport systems within astrocytes. properties of the glial i gln indicate expression of sa while ph imaging reveals a non-electrogenic glutamine transporter with properties identifying it as sn. sn is known to be readily reversible under physiological conditions and we propose that this mediates the astrocytic glutamine release mechanism within the glutamate-glutamine cycle. furthermore, system a mediates glutamine transport in glutamatergic nerve terminals and is important in maintaining the supply of glutamate for excitatory neurotransmission during periods of high frequency stimulation. a. briens, m. schwalm, f. docagne, d. vivien inserm u , caen, france astrocytes are key players in the brain, cooperating with neurons to modulate crucial processes. in particular, they are able to uptake and release neurotransmitters and neuromodulators to control their synaptic level. in our study, we hypothesize that astrocytes can regulate some effects of the serine protease plasmin in the cns through a mechanism of uptake. conversion of the zymogen plasminogen to the serine protease plasmin by the activators (tpa or upa) is the basis of fibrinolysis in the vasculature. but cerebral functions for the plasminogen activator/plasmin system have recently arisen. it has been shown that plasmin was essential to activate the brain derived neurotrophic factor (bdnf) in its mature form, promoting long-term hippocampal plasticity. in alzheimer's disease, plasmin plays a protective role by participating in beta-amyloid clearance. plasmin can also lead to neuronal injury by degrading extra-cellular matrix proteins. in animal models of multiple sclerosis, plasmin can also help removing intraparenchymal deposits of fibrin, thus limiting axonal damage. these data suggest that a system of regulation of plasmin effects in the brain is necessary. in this study, we showed that astrocytes can actively and specifically uptake plasminogen and plasmin in a time-dependent and a dose-dependent manner. this mechanism involves clathrin pits formation in astrocytes and the lysine-binding site of plasminogen/plasmin. plasminogen and plasmin, following endocytosis are found in early endosomes, late endosomes, lysosomes and recycling endosomes. finally, we noticed that astrocytes uptake plasmin more efficiently than plasminogen. this study brings out a mechanism of glial uptake of plasminogen and plasmin. this could control their extra-cellular levels and regulate their effects within the brain. further investigations should determine whether this mechanism can modulate plasminogen activation and synaptic plasmin activity. besides, plasminogen activators (tpa) and cerebral substrates of plasmin (pro-bdnf) are also taken up by astrocytes. a link could thus exist between these processes to regulate the amount of synaptic mature bdnf. this would reveal a cross-talk between neurons, releasing precursors in the synapse (pro-bdnf and plasminogen) and astrocytes, regulating their activation and their clearance. understanding and modulating plasmin uptake could be very important in pathological conditions where the plasminogen/plasmin system is involved, such as alzheimer's disease, multiple sclerosis or stroke. healthy brain aging is characterized by neuronal loss and cognitive decline being inflammation a major causative factor. microglial activation is considered to be a major driver for the neuropathological findings in alzheimer's disease (ad). we evaluated whether young vs. aged microglia responded differently to ab soluble oligomers (abo) and to conditioned media from abo-treated hippocampal neurons. microglia from day cd pups were incubated for h at (young) and (old) days in vitro (div) with abo (ab - at and nm). in parallel, e mice hippocampal neurons were treated with abo at (young) and (old) div for h, and the incubation media used as conditioned media to treat microglia, at respective div, for further h. cell migration (boyden chamber), extracellular content in glutamate (commercial kit), and activity of matrix metalloproteinases (mmp) and (gelatin zymography) were assessed. an increased migration towards abo and atp was observed in young microglia but almost undetectable in aged cells (p < . ). interestingly, microglia exposure to conditioned media from nm abotreated neurons markedly decreased both young and old microglia migration, while treatment with media from nm abo-treated neurons enhanced migration, mainly in the young microglia (p < . ). concerning glutamate, aged cells released less than young ones upon abo treatment (p < . ). in addition, when exposed to neuronal conditioned media, only the young microglia were able to exert a neuroprotective role by reducing the media glutamate content. curiously, while abo promoted a microglia release of mmp- , independently of cell age, it only induced mmp- secretion in old cells in an abo concentrationdependent manner (p < . for nm abo). in contrast, conditioned media from abo-treated neurons elicited mmp- release only from young microglia. together, our data point to a loss of microglia function towards abo with ageing and are unique in suggesting that senescent microglia may release high levels of mmp- in ad. in addition, our results indicate that abo-treated neurons enhance the release of mmp- even by young microglia. whether this finding may contribute to enhance inflammatory stress and the onset of ad will be the aim of further studies. human immunodefeciency virus- (hiv- ) productively infects microglia within the central nervous system (cns). viral replication within microglia and the resulting immune response and neurotoxicity leads to significant cognitive impairments not limited to dementia, neurocognitive abnormalities, and memory deficits. combination antiretroviral therapy reduces the cognitive impairments associated with hiv- , but the regulatory hiv- tat protein is still produced in the cns during treatment, even in the absence of productive infection. our laboratory analyzes the effect of tat on the murine cns by stereotactically injected into cortex, which mimics in part the neuroinflammation and neurodegeneration characteristic of hiv- associated neurocognitive disorders (hands). additionally, in vitro tat-treated bv- microglial cells exhibit activation associated with increased cytokine expression and phagocytosis. mixed lineage kinase (mlk ) has recently been implicated in the tat-mediated activation of microglia. mlk activity is increased by the presence of tat protein, which results in microglial activation and increased production of inflammatory cytokines. conversely, the brain penetrable, small molecule, new chemical entity urmc- inhibits the mlk pathway. we hypothesize that urmc- attenuates tat-induced microglial activation as measured by microglia process length, phagocytosis assay and cytokine expression. in vitro application of urmc- decreased tatinduced production of ccl and il- , microglia process length, and phagocytosis of charged and uncharged beads in bv- microglia cells. these data, in aggregate, strongly support a role for the mlk pathway in neuroinflammation and suggest that inhibition of this pathway with urmc- may have significant anti-inflammatory effects in an inflammatory cns milieu. in conclusion, this work will advance our understanding of mlk activity and may provide a viable drug therapy for hands. the contributions of glial cells to the modulation of adult synaptic plasticity are becoming increasingly more appreciated. astrocytes in the supraoptic nucleus (son) of the hypothalamus demonstrate prominent temporary structural changes during physiological events such as dehydration and lactation. such structural remodeling is characterized by a reduction in the astrocytic coverage of oxytocin neurons and their synapses. these astrocyte morphological changes also contribute to the observed functional changes in synaptic activity during dehydration and lactation. in order to elucidate the underlying mechanisms regulating the astrocytic contributions to synaptic plasticity we have established an astrocyte protein expression profile for structural changes in the son during lactation and hyperosmotic challenge. for this, son from virgin, lactating, and hyperosmotic rats were collected from acute hypothalamic slices and analyzed by mass spectrometry to identify proteins differentially regulated by the changes in synaptic structure induced by lactation and hyper-osmolarity. our mass spectrometry data analysis has placed particular focus on regulation of astrocyte-specific proteins and the roles of these proteins in molecular pathways known to be involved in son plasticity, such as cytoskeleton remodeling, cell adhesion, and cell signaling. gene ontology analysis revealed over representation of pathways known to be highly active in astrocytes including metabolism and antioxidant activity. promising target proteins for future functional studies will be discussed. here we elucidate a novel merlin isoform (merlin-iso )-specific function in maintaining axonal integrity and propose that reduced axonal nf gene dosage leads to nf -associated polyneuropathy. we identify a merlin-iso -dependent complex corresponding to activation of the gtpase rhoa, enabling downstream rho-associated kinase to promote neurofilament heavy chain phosphorylation. in vivo, specifically merlin-iso knockout mice exhibit impaired locomotor capacities, delayed sensory reactions, and electrophysiological signs of axonal neuropathy. sciatic nerves from these mice and sural nerve biopsies from nf patients show reduced nf-h phosphorylation, decreased interfilament spacings and irregular-shaped axons. a. catenaccio, p. silva, f. court pontificia universidad cat olica de chile, santiago, chile axonal degeneration is an active process involved in a variety of neurodegenerative conditions triggered by diverse stimuli. this degenerative process can cause permanent loss of function, so it represents a focus for neuroprotective strategies. we have recently shown that axonal degeneration triggered by distinct mechanical and toxic damage is dependent on the activation of the mitochondrial permeability transition pore (mptp), which probably represents a central execution program of axonal degeneration, upon which several pathways converge (barrientos et. al., journal of neuroscience ). nevertheless, the participation of other cellular types in this degenerative process has been largely overlooked. after axonal damage in the peripheral nervous system (pns), schwann cells enveloping axons have been regarded as responsible for clearing degenerating axonal debris and to mount an inflammatory response for tissue clearance by invading macrophages. nevertheless, a possible function of glial cells in modulating axonal degeneration has not been addressed so far. here we explored the possibility that glial cells actively participates not only in the clearance of degenerating axons, but directly in the axonal degeneration program during wallerian degeneration. we found that axonal injury activates an early response of schwann cells that results in the fragmentation of their associated axons by actin-rich cytoplasmic domains of schwann cells known as schmidt-lanterman incisures (sli). this first step of schwann cell fragmentation of axons is dependent on the erk signaling pathway, wich control the dedifferentiation program initiated in schwann cells after nerve injury (napoli et. al., neuron ). in the axonal compartment, injury triggers a parallel cell autonomous degenerating program dependent on mitochondrial mptp activation, which is cell non-autonomously assisted by schwann cells through the activation of a cell extrinsic pathway of axonal destruction by tnf-alpha secretion. we showed for first that time that axonal degeneration in the pns proceeds by axonal autonomous mechanisms, as well as cell non-autonomous ones dependent on scs. therefore, effective targets for neuroprotection should consider not only the axonal compartment, but also the associated glial cell. glutamate is the major excitatory neurotransmitter in the brain. its concentration is the synaptic area is highly regulated in order to maintain point-to-point transmission and to prevent excitotoxicity associated with chronic activation of glutamate receptors. as there is no endogenous extracellular enzyme to degrade glutamate, the clearance of glutamate is ensured by transporters located on the surface of astrocytes. among these surface transporters glt- ensures almost % of glutamate uptake at synapses. it is widely accepted that many receptors and transporters on the surface of neurons and glia display surface trafficking properties and are not simply static proteins. the basic surface trafficking properties of glutamate receptors at the synapse have been studied in depth and have allowed us to understand many important trafficking events that occur during synaptic plasticity. the action of glt- is fundamentally important in synaptic communication. surface trafficking of this transporter may play a pivotal role in the spatial and temporal properties of glutamate diffusion at the synapse. however, it is still unknown whether glt- is dynamically regulated on the surface of astrocytes. thus we set out to uncover the surface trafficking of glt- in astrocytes by using a combination of high-resolution imaging, such as single particle (quantum dot [qd]) tracking, molecular biology and electrophysiological approaches. here we have demonstrated that glt- is indeed subject to surface trafficking on astrocytes. using pharmacological approaches we have shown that this diffusion is subject to regulation by the activity of the transporter itself as well as neuronal activity. furthermore, we have demonstrated that the speed of glt- diffusion is greatly reduced at excitatory synapses. this leads us to the conclusion that glt- surface diffusion is highly regulated at the synapse where it can effectively carry out its role as the principal glutamate transporter at the synapse. finally, to elucidate the physiological role of glt- surface diffusion, we have carried out electrophysiological experiments in which glt- trafficking is effectively reduced using a cross-link (x-link) protocol. we observed that a reduction in the surface trafficking of glt- resulted in an increase of neuronal excitability. thus glt- surface trafficking on astrocytes has an implicit role in regulating basal neuronal activity through glutamate uptake at the synapse. o. chever, e. dossi, n. rouach collège de france, paris, france astrocytes play crucial roles in brain physiology by dynamic interactions with neurons. they form plastic and extensive networks mediated by gap junction channels. it has recently been shown that astroglial networks limit neuronal network activity. however, it is currently unclear how astroglial networks influence neuronal excitability and population activity. to investigate how astroglial assembly regulates neuronal network activity, we performed electrophysiological recordings in hippocampal slices (ca and ca areas) from mice with disconnected astrocytes, in which both astroglial gap junction forming proteins, connexin (cx ) and connexin (cx ), are knocked out (gfap-cre cx -/-cx fl/fl , dko). synchronized excitatory discharges were recorded in an acute pharmacological model of epileptic-like activity. in this model, spontaneous bursting discharges are characterized by a sharp and large amplitude shift in field potential, generated by a major depolarization and firing of all putative neurons. pyramidal cells depolarized up to mv during seconds and such depolarization is followed by a long-lasting undershoot of the membrane potential. we found that the frequency of bursting activity in dko hippocampal slices is drastically increased. however, the duration of neuronal depolarizing bursts is severely reduced. furthermore, pyramidal neurons are more depolarized due to an increase of synaptic bombardment. this suggests that increased synaptic background promoting resting membrane potential depolarization facilitates triggering of neuronal bursts, but compromises the strength of synchronized events. to investigate local dynamics of bursts generation, we performed multielectrodes arrays recordings in the ca area of the hippocampus. in slices from dko mice, bursting activity in the ca region is generated within a more restricted area, indicating that the number of neurons to be recruited is highly decreased. altogether, these results indicate that gap junction-mediated astroglial networks strengthen the coordination of neurons during synchronized events. acutely loaded hypoxia increases ventilation, and increased ventilation does not immediately return to the pre-hypoxic level, but persists for a while after resuming room air inhalation. this phenomenon is referred to as short term potentiation or plasticity of breathing, and is thought to contribute to the stabilization of ventilation. although extensive studies have been conducted to clarify the cellular mechanism of respiratory plasticity focusing on neurons and synapses, its precise mechanism remains unknown. on the basis of the recent discovery that astrocytes are actively involved in neural plasticity in various brain regions, we hypothesized that the post-hypoxic potentiation of breathing is mediated by astrocytes. we used unanaesthetized unrestrained adult mice. ventilatory parameters (respiratory frequency, tidal volume and minute ventilation) were measured by whole body plethysmography. to test the hypoxic response, mice breathed room air, hypoxic gas ( % o , % n ), and again room air. we also analyzed the hypercapnic response that was conducted under a hyperoxic condition to eliminate the influence of the carotid body. to test the hypercapnic response, mice breathed pure o , hyperoxic hypercapnic gas ( % o , % co ), and again pure o . after recording of the hypoxic and hypercapnic responses with their recovery time courses, arundic acid (ono- ) was injected intraperitoneally. arundic acid is a compound that suppresses the function of astrocytes through the inhibition of s b production in astrocytes. arundic acid did not affect ventilatory augmentation induced by either hypoxia or hypercapnia. however, arundic acid suppressed the short term potentiation induced by hypoxia (but not by hypercapnia) in a dose-dependent fashion. high dose arundic acid completely eliminated hypoxia-induced short term potentiation. these results suggest that the post-hypoxic potentiation of breathing is mediated by astrocytes with the involvement of s b. the mechanism of hypercapnia-induced potentiation is different from that of hypoxia. we propose a model to explain the cellular mechanism of post-hypoxic potentiation: hypoxia stimulates the respiratory neuronal network in the brainstem through the excitation of the carotid body. neurotransmitters spilled from excited neuronal synapses activate nearby astrocytes. the activation of astrocytes is sustained, and these astrocytes persistently excite respiratory neuronal network by releasing gliotransmitters. monosynaptic c-fibre evoked excitatory postsynaptic currents (epscs) were recorded in lamina i neurons using whole-cell patchclamp in rat transverse spinal cord dorsal-root slice preparations. spontaneous epscs were recorded simultaneously. in all experiments bicuculline and strychnine were added to the bath solution to block gaba a -and glycine-mediated synaptic currents. the superfusion of spinal cord slices in vitro with fkn results in a rapid facilitation of evoked epscs in spinal lamina i neurons receiving monosynaptic input from primary afferent c-fibres. both the microglial cell inhibitor minocycline and a fkn neutralising antibody, completely prevented fkn-induced changes in synaptic transmission. in addition, the inclusion of the ca chelator bapta or the nmda receptor antagonist mk in the pipette solution completely prevented fkn-induced enhancement of synaptic strength, suggesting a post-synaptic ca -dependent mechanism of ltp induction. analysis of paired-pulse ratio (ppr) indicated that fkn-induced facilitation of synaptic strength is accompanied by a decrease in ppr suggesting a pre-synaptic mechanism of ltp expression. in support of this finding, fkn increases the number of spontaneous epscs recorded from lamina i neurons. these data indicate that stimulation of microglial cells in order to induce a pro-nociceptive activity state is sufficient for facilitation of synaptic strength within the dorsal horn. both pre-and post-synaptic mechanisms contribute to fkn-induced facilitation of synaptic strength. this work was funded by a wellcome trust flexible travel fellowship to akc. the organization and connectivity of the developing neocortex remains largely unresolved. the establishment of neuronal microcircuits is a complex process that partly depends on the diversity of neuronal cell types and their ultimate role in the mature brain. this complexity is increased by the existence of bona fide synaptic connections between neurons and oligodendrocyte precursor cells, also called ng cells, which properties and function remains largely unknown. to unravel the physiological characteristics of gabaergic synapses between interneurons and ng cells, we performed paired recordings in the developing somatosensory cortex. experiments were carried out during the second postnatal week in vgat-venus;ng -dsred double transgenic mice that allowed us to identify interneurons (venus ) and opcs (dsred ) in acute slices. our results demonstrated that ng cells are synaptically contacted by fast-spiking (fsi) and non-fast-spiking (nfsi) interneurons. however, a predominant input was received by fsi, a class of interneuron that constitutes only a minor population in the second postnatal week. on the contrary, only a small proportion of the abundant nfsi were connected to ng cells. all the connections showed paired-pulse depression, low probability of response, and preliminary analyses suggest that each interneuron form a single synaptic contact onto ng cells. paired recordings, extensively used to examine the synaptic properties of neuronal microcircuits, may open a new perspective to understand in detail the mechanisms of gaba release and signaling between interneurons and ng cells in the healthy and pathological developing brain. first, we characterized a microglial cell line obtained from cd mouse cortex (n ), in order to clarify the mechanisms involved in the activation pattern of these cells. for that, we incubated n microglia with ng/ml of lipopolysaccharide (lps) for h. morphology (anti-iba staining), phagocytic ability (fluorescent latex beads) and chemotaxis to atp (boyden chamber) were evaluated. we observed that n microglia is ramified in control conditions and after lps treatment they change to an amoeboid morphology, which is accompanied by increased phagocytosis and decreased migratory ability, indicating that lps induces activation of n cell line. next, we investigated the role of the released factors from a mn-like cell line expressing human sod with g a mutation (nsc- / hsod g a) on microglia functions. nsc- expressing human sod wt were used as control. thus, n microglia were incubated for and h with nsc- conditioned media that had been collected at and days of differentiation (div) and reactivity tests were performed as above. although after h of incubation no significant changes were observed, data evidenced that incubation for h with nsc- / hsod g a conditioned media collected at div changed the bipolar morphology of the n microglia into an active amoeboid state, decreased phagocytic ability ( %, p < . ) and induced apoptosis ( %) (fig. ) . moreover, microglia have shown to not be attracted by the released factors from degenerating mn, as determined by the chemotaxis assay. together, our results evidence that released factors from nsc- / hsod g a degenerating cells although inducing microglia morphological activation trigger a reduction in cell phagocytic ability and loss of viability, suggesting a role for microglia along als progression. organotypic explants culture has a pivotal role in studying the complex structure of neuronal tissue. although embryonic tissue explants and slices are used to obtain long term culture, most applications such as drug testing require adult tissue for reliable results. in this study, we show that nanostructured metal-oxide arrays can be employed to yield long-term organotypic culture of adult neuronal tissue explants as demonstrated for the adult guinea pig retina and adult murine brain slices. even after days of culture, the thickness and the layered structure of the retina were well maintained. quantitatively, the number of nuclei within the inner and outer nuclear layers was comparable to freshly isolated retinae and no significant change in the densities of the nuclei within the layers was observed. the outer plexiform layer, as the site of synaptic connection between photoreceptors and neurons, was still well preserved. moreover, the thickness of the photoreceptor layer was conserved, indicating that the inner and the outer segments of the photoreceptors survived well during two weeks culture. this is hardly observed in long-term cultures since the outer segments were cultured without retinal pigment epithelium. concerning the adult murine brain slices from neo-cortex, after days of culture, the neurofilaments and cell nuclei were still well preserved and the neuronal network displayed the typical arrangement of long axons. however, preservation of the tissue depends strongly on the geometry of the nanostructured array surface such as roughness and spacing, whereby organotypic brain slice culture requires different geometries than retina culture. since organotypic culture of adult tissue could only be obtained for about days with conventional techniques, our new substrate material could be the breakthrough for in vitro studies on tissue regeneration, neuroplasticity, as well as drug testing. the intrinsic optical signal (ios) is widely used for mapping neuronal activity in afferent activated brain areas. ios evoked by afferent stimulation in vitro is generally attributed to glial cell swelling via activation of na /k /clcotransporter that follows postsynaptic activation. despite the phenomenon is known for decades, the relative contribution of different cell types and the underlying molecular mechanisms have not been disclosed in detail. our goal was to explore the glial and neuronal correlates underlying in vitro ios genesis. we characterized ios initiated by schaffer collateral stimulation in the rat hippocampal slice using simultaneous high-frequency imaging and field potential recordings. we used a -element photodiode-array device (pda) that enables ios detection with . ms time resolution, making it achievable to align optical and electrophysiological signals. ios was primarily observed in the proximal region of the stratum radiatum and stratum pyramidale of the hippocampus. ios was decreased by blockade of neuronal activity by voltage-gated na channel inhibitor tetrodotoxin and was significantly enhanced by suppressing inhibitory signaling with the gaba a antagonist picrotoxin. we found that ios was predominantly initiated by postsynaptic glutamate receptor activation and progressed by the activation of astroglial glutamate transporters and mg -independent astroglial nmda receptors. in agreement with previous studies, furosemide, the blocker of both the neuronal k / clcotransporter kcc and the glial na /k /clcotransporter nkcc decreased the ios amplitude. to decide which cotransporter is responsible for this effect of furosemide, we applied selective blockers of nkcc and kcc , bumetanide and ( )[r]-dioa, respectively. we evidenced, that nkcc did not contribute to the ios generation, in contrast to previous suggestions. enhancement and slight inhibition of ios through anion and volume-regulated anion channels, respectively, were also depicted. major players of ios mechanisms disclosed by high-frequency ios imaging imply that spatiotemporal ios reflects glutamatergic neuronal activation and astroglial response, as observed within the hippocampus. our model may help to better interpret in vivo ios and support diagnosis in the future. question: a poor x polyunsaturated fatty acids (x pufa) status, favored by the low x /x ratio in western diets, seems to contribute to cognitive decline in the elderly, but mechanistic evidence is lacking. we therefore explored the impact of x status on the evolution of glutamatergic transmission, astroglial regulation and neurogenesis in the hippocampus during ageing in rats. these processes are involved in memory formation and their dysregulation participates to the agerelated brain damage leading to cognitive decline. methods: we have compared groups of rats aged to months fed x -deficient, x /x -balanced, or x (fish oil) supplemented diets: young x balanced (yb), deficient (yd) or supplemented (ys), and old x balanced (ob), deficient (od) or supplemented (os) rats. we have evaluated synaptic efficacy and plasticity (electrophysiological recording), astroglial regulations (glutamate uptake and gfap expression), neuronal markers (glutamate transporters and receptors), neurogenesis (proliferation of neuronal precursors in the sub granular zone), and analyzed brain fatty acids composition. results: dietary modulation of x intakes efficiently modified the incorporation of docosahexaenoic acid (dha, the main x in cell membranes) in brain ( % deficient vs balanced, % supplemented vs balanced). ageing induced a % reduction of synaptic efficacy due to decreased pre-synaptic glutamate release, and a % decrease in the astroglial glutamate uptake associated to a marked astrogliosis ( % gfap). x deficiency further decreased these hallmarks of ageing (od vs ob rats: % synaptic efficacy, % glutamate uptake, % gfap). on the opposite, x supplementation increased synaptic efficacy ( % os vs od) and seems to abolish astrogliosis (os vs ys : no change in gfap). neurogenesis was altered by x deficiency but not by supplementation. conclusion: our results characterize some specific age-related alterations of the glutamatergic synapse in the hippocampus that are aggravated by a dietary deficit in x and attenuated by x supplementation. methods: sgcs were isolated from the trigeminal ganglion (tg) of adult male wistar rats (n ). animals were deeply anaesthetized and both tg were extracted and digested first in mg/ml collagenase for min and then in . % trypsin containing dnase for min. in initial experiments, isolated sgcs were treated with control medium or medium containing . , , or mm cis for h. in the subsequent experiments, sgcs were pretreated for h with control medium or medium containing mm ibu or mm skf. afterwards, sgcs were stimulated for hours with medium containing mm cis. pge concentration was determined by elisa. results: cis caused a concentration-dependent increase in pge release from sgcs. treatment with mm cis significantly increased pge concentration to . . pg/ml, compared with control . . pg/ml (p . ). pge concentration was further increased to . . pg/ml (p . ) following mm cis. pretreatment with ibu significantly lowered the pge concentration compared with cis-stimulated sgcs ( . . pg/ml vs. . . pg/ml, respectively; p . ). a more pronounced inhibitory effect was observed following skf pretreatment, where it reduced the pge concentration to . . pg/ml (p . , compared with cis-stimulated sgcs). conclusion: these findings suggest that cis contributes to an inflammatory response in the tg by provoking release of pge from sgcs, which may lead to development or maintenance of peripheral sensitization involved in cipn. the data also suggest that treatment with agents that target pge release cascade may prove beneficial for preventing development or maintenance of cipn. functional alterations in synaptic contacts have often been described as a hallmark of major depressive disorder (mdd). antidepressants (ads) have been shown to restore neuronal circuits through an enhancement of synaptogenesis in some regions of the brain. nevertheless, the underlying mechanisms are still unclear. glia cells have been since long acknowledged as active partners of neurons in orchestrating molecular signals crucial for the proper arrangement of neuronal circuits in the developing and adult brain. therefore, understanding how both neurons and astrocytes respond to antidepressants (ad) is of high interest. using rat c glioma cells and primary cultures of astrocytes and neurons, we showed a time-dependent cell-autonomous modulation of the erk/mapk signalling pathway after acute treatment with various classes of ads in both cell types. specifically, glia cells responded to ads with the simultaneous and fast activation (after min treatment) of both erk / , in contrast to treatment with antipsychotics or mood stabilizers. this activation induced hrs later an increased release of gdnf, a factor involved in synapse formation and axonal wiring, which was mapk-dependent. on the contrary, hippocampal neurons showed a reduction in erk activity that correlated with neuronal activity inhibition after short term ( min) ads administration, as demonstrated by quantification of c-fos expression after kcl stimulation. interestingly, this inhibitory effect was reversible after long term ( hrs) ads treatment. to further identify whether gdnf released from astrocytes treated with ads might be responsible for long term changes in neuronal synapses, we examined how ads influenced synaptic densities in neurons alone or co-cultured with astrocytes. strikingly, we found that the number of synapses was reduced at hrs after ad treatments, but only in the presence of intact astrocytes and not in cultures of neurons alone or neurons treated with astrocyte-conditioned media. this effect was reversed after hrs treatment and rescued by the soluble form of the specific gdnf receptor gfralpha , but not by gdnf alone. moreover, live imaging of astrocytes showed dramatic morphologic changes of their processes in response to ads that might underlie the observed synaptic remodelling. our analysis of changes occurring at the neuronglia interface upon ad treatments might elucidate novel mechanisms of ads action that may open the avenue for unravelling the role of astrocytes in psychiatric diseases and implement pharmacologic treatment regimens for mdd patients. here we extend these findings showing that the neurotrophin nt- , which also belongs to the putative factors secreted from glial cells after t -stimulation, also increases the navd in neuron enriched cultures. the effects of nt- and fgf- are, however, not additive. antibodies against nt- potentiated the effects of fgf- , suggesting that by converging signal cascades nt- could reduce the effect of fgf- . in neuron-glia mixed cultures antibodies against nt- enhanced the t -induced up-regulation of navd, suggesting, that a co-secretion of nt- from glial cells could limit the effects of fgf- . in a second series of experiments we investigated, whether the well-known effect of t on the expression of na /k -atpases, thought to contribute to the effect of thyroid hormone on metabolism, is also influenced by glial cells. we found, that the effect of t on [h]ouabain-binding and on the expression of na / k -atpase alpha subunits detected by western-blots was highest in neuron-mixed cultures, suggesting that a neuron-glia interaction is also involved in the t effects on na /k -atpase expression. in the healthy brain microglia engage in bi-directional interactions with neurons and other brain cells, which is crucial for maintaining normal physiology and cognitive function of the brain. disturbances in homeostasis and cell-cell communication may predispose to age-related dysfunction and neurodegenerative disease. for decades it has been speculated that microglia exhibit phenotypic diversity allowing specialisation to a variety of brain microenvironments. previous observations of regional variations in microglial densities and morphologies support the existence of microglial heterogeneity. however it is clear that a more comprehensive understanding, in particular of the molecular basis of microglial phenotypic and functional diversity on a global scale, is required. the limitation of recent gene expression studies is the use of mixed whole brain or dissected mixed cell extracts which preclude the detection of cell-specific gene expression profiles. thus, our aim was to validate an efficient extraction process for microglia, ideally minimising the impact on the resting state. the method developed is based on existing procedures and was refined to ensure consistency and to maximise cell yield and purity. the extraction of highly pure microglia as a single cell type was achieved by a two-step isolation technique using density-gradient and magnetic bead separation. purity between - % was verified by flow cytometry and immunohistochemistry. quantitative pcr also demonstrated expression of specific microglial genes indicating that isolated cells are microglia. cytokine assays suggest that no or only minimal activation of cells was induced by the isolation. hence, the microglia retained their ability to respond to immunogenic stimuli and produce il- b as a key inflammatory cytokine. overall, the purification procedure was shown to achieve consistent high purities and retain crucial functional properties. thus, the extracted microglia are likely to adequately represent the real in vivo state and enable the study of microglial phenotypes and functionality in the healthy and ageing whole brain and accordingly brain regions. early diversity experiments showed region-specific microglia phenotypes in the brain of healthy and immune challenged animals as well as differences between unchallenged and challenged animals. question: activation of nmda receptors increase the respiratory frequency in mammals. astrocytes are in intimate contact with neurons, specially in glutamatergic synapses, and are able to sense neuronal activity, react to, and even influence nearby neurons. furthermore, they can sense changes in h or pco , and in response to these stimuli, they can release molecules like atp and d-serine, which will activate neuronal circuits. then d-serine, an agonist of the glycine site of the nmda receptor, can affect the respiratory rhythm during hypercapnia. our aim was to study the probable role of d-serine in the modulation of the respiratory rhythm in mice neonates. methods: fictive respiration was recorded with suction electrodes from c -c ventral roots in "en bloc" (brainstem-spinal cord) preparations from p -p cf mice. superfusion was done with artificial cerebrospinal fluid equilibrated with o :co %: %, (ph . , c) containing different d-serine concentrations ( . - mm) in presence or absence of d-amino acid oxidase (daao), which degrades d-serine. in addition, hypercarbic acidosis (switching equilibration from to % co , changing ph from . to . ) was done in absence or presence of daao. results: application of d-serine increased the frequency of the respiratory rhythm in a concentration-dependent way. application of dao attenuated the effects of d-serine application. likewise, daao reduced slightly the effect of hypercarbia on the respiratory rhythm. conclusions: our preliminary experiments indicate that d-serine is a potent agent increasing the respiratory rhythm frequency in in vitro neonatal preparations, and likely, in association with atp, is mediating the respiratory response to hypercarbia. there, we showed that mac- positive microglia accumulate within the motoneuron area and phagocyted apoptotic bodies at the onset of motoneuron developmental cell death (from embryonic day . -e . ). motoneuron developmental cell death and microglia accumulation in the motoneuron area increased at e . , decreased at e . and disappeared at e . . since it was supposed that microglia promote developmental cell death in the developing central nervous system, at least in vitro, we determined to what extent it was also the case in the embryonic spinal cord in vivo. to address this issue, we analysed in vivo the progression of developmental neuronal death in the spinal cord of transgenic mouse embryos lacking microglia (pu. -ko mice). as opposed to what is observed in the sc of wild type mouse embryo, we found that activated caspase- staining was detectable before e . in pu. -ko mouse embryos. in pu. -ko mouse embryos, activated caspase- staining was not restricted to the motoneuron area, as several positive cells are also present in the dorsal region of the spinal cord and in the progenitor zone. at e . , activated caspase- staining was times greater in the spinal cord ventral area of pu. -ko mouse embryos when compared to wild type littermates. moreover, and opposed to what occurs in wild type animals, caspase- staining in the motoneuron area of pu. -ko mouse embryos increased at e . and persisted at e . , being likely partly due to the accumulation of apoptotic bodies in the absence of microglia. our results suggest that, in addition to their phagocytic role, embryonic microglia are likely to protect neuron from death at the onset of developmental cell death and to promote survival of progenitor-like cells in the embryonic sc in vivo. accordingly, microglia could have an opposite effect on cell survival in the embryonic sc when compared to postnatal snc developmental stages and pathological conditions in the adult. we recently demonstrated a key role of the a-secretase tace, also known as adam , in peripheral nervous system (pns) myelination by tace-mediated cleavage and subsequent inhibition of neuregulin (nrg ) type iii activity. unlike the pns in which nrg type iii is an essential instructive signal for myelination, oligodendrocyte (ol) development and myelination in the central nervous system (cns) are likely controlled by several growth factors some of which undergo cleavage by secretases. to study the role of tace on opc development, immunopanned a b opcs were cultured in proliferating or differentiating medium and treated with a soluble form of tace (rhtace). when ols were scored according to their morphology, we observed enhanced opc differentiation upon addition of rhtace to proliferating opcs. addition of rhtace to differentiating opcs also increased the number of ols with a complex morphology and already presenting membrane sheets, suggesting that tace might promote opc differentiation. to further investigate the role of neuronal tace in cns, we used an in vitro ol myelinating coculture system. when cocultured with tace null drg neurons, rat wild type opcs never myelinate, suggesting that neuronal tace might control opc differentiation. in agreement, preliminary ultrastructural analyses of transgenic mice lacking tace in cns neurons showed hypomyelination and signs of myelin degeneration. accordingly, conditional transgenic mice lacking tace in ol are normally myelinated. these studies suggest that in the cns the a-secretase tace promotes opc differentiation and might regulate cns myelination. a better understanding of the role of tace will provide novel insights into the mechanism regulating cns myelination. in the excitatory network glutamate is released in the synaptic cleft during synaptic activity. glutamate clearance by astrocytes is mainly performed by na -dependent glutamate transporters. the na load during glutamate uptake evokes an activation of the na /k atpase, which dramatically increases energy demands in astrocytes. astrocytes are also involved in the regulation of extracellular potassium (k e ) which significantly increases during the repolarization of excitatory neurons. in this study we evaluated how these fundamental functions of astrocytes coexist and if they interact. we investigated the impact of altering k e concentrations on glutamate transporter activity measuring intracellular sodium (na i ) using the na sensitive fluorescent dye asante sodium green loaded in cultured astrocytes. we observed that glutamate uptake caused a reversible rise in na i , which was tightly modulated in amplitude, slope and recovery rate by k e levels. in order to evaluate the impact of physiologically relevant k e fluctuations on the kinetics of the glutamate transporter the na /k atpase was inhibited. therefore we found that low k e evoked a significantly faster influx of na , whereas high k e markedly slowed down glutamate capture, indicating that k e directly modulates the kinetics of glutamate transporters. we then investigated the energy demands of astrocytes caused by k e alterations. atp hydrolysis was indirectly measured through the changes in intracellular free mg using the mg sensitive fluorescent probe magnesium green. we found that low k e led to higher energy demands of astrocytes during glutamate uptake. overall these results indicate that k e acts as a negative feedback on glutamate uptake in astrocytes. the physiological consequences of these findings have to be considered in the context of k buffering and of maintenance of neurotransmitter and energy homeostasis. recovery was significantly improved by nf fractions, as well as tubulin (tub), added after lpc removal: compared to cultures treated with lpc alone the number of olp (a b cells), and their proliferation increased significantly, as well as the number of differentiated (cnp ) and mature (mbp ) ol. moreover, nf and tub protected ol from lpc toxicity when added at the time of lpc treatment: they increased olp proliferation, as well as the number of cnp and mbp ol significantly, compared to cultures treated only with lpc. on the contrary irrelevant proteins (actin, and skin proteins) were ineffective, demonstrating the specificity of the cytoskeleton proteins effects. importantly, nf and tub increase olp survival and proliferation when challenged with lpc, without delaying differentiation and maturation. we hypothesize that release of nf and tub following axonal damage in ms could participate in the regulation of remyelination through this process. this putative phenomenon might be complexified by alterations of axon protein expression, or of proteolysis, known to occur in ms models. purpose: microglial proliferation is commonly accepted as a hallmark of glial activation. an automatic method that allows assessing the number of microglial cells to analyze the proliferative microglial behavior in a laser-induced ocular hypertension (oht) model has been developed. methods: adult albino swiss mice were organized in two groups: naive (age-matched control; n ) and lasered (n ). retinal wholemounts were immunostained with anti-iba and images of iba- microglias were recorded with a fluorescence microscope. new algorithms of segmentation and control of distances were developed in matlab to obtain the number of iba- microglial cells. automatic results were compared with those obtain by direct human observation of the samples. results: the automatic method detected the number of iba- cells in the inner and outer plexiform layers of the retina in both, na€ ıve and oht-retinas. the number of cells present in the samples was not an obstacle for the program to run properly. the time required for counting iba- cells decreased from the human guide to the program based counting method from days to one hour. results show a strong correlation between both automatic and manual method (pearson correlation test, r . ; p . and r . ; p . for outer and inner plexiform layer respectively) indicating the consistency of the automatic counting. conclusions: a new, reliable and quick algorithm was developed with matlab to obtain the number of iba- microglial cells and also cellular density maps through retinal wholemounts in both na€ ıve and other proliferative conditions (i.e. glaucoma). due to this new automatic method, a bigger set of images or samples could be included in future studies to analyze the behavior of microglial cells. we are interested to understand whether scs in the peripheral nerves of mammals express functional ionotropic glutamate receptors, and whether scs use these receptors for communication with axons. to start addressing this question, we established a preparation of mouse live sciatic nerve slices employing "know-how" of live brain slices technique. using this preparation, we performed whole-cell patch-clamp recordings of scs at different stages of development (embryonic and early postnatal). we included a fluorescent dye into the pipette solution in order to perform post-recording morphological analysis of single scs. first, we recorded current responses of scs to a series of depolarizing voltage steps, aiming to test whether all scs in the nerve are electrophysiologically akin. we found that mouse scs differ in their passive membrane properties and expression of voltage-gated k channels: depending on the age, to cell types could be distinguished. post-recording morphological characterization of the recorded cells showed that scs in the mouse sciatic nerve differ in their size, as well as in the number and length of their processes. next, we used fast pressureinduced application of glutamate to investigate whether scs possess ionotropic glutamate receptors, and whether electrophysiologically distinct sc types differ in their expression of glutamate receptors. application of mm glutamate caused an inward current in at least two types of scs, and preliminary analysis revealed a peak current amplitude in the range of - pa (n ). glutamate-induced currents in scs were blocked by gyki , indicating that mouse scs carry ionotropic glutamate receptors of ampa/kainate type. currently we are studying which subunits of ampa receptors are present in scs, and how these receptors get activated in situ. although cx is expressed by myelinating schwann cells in the peripheral nerves, patients with cmt x develop relatively early axonal degeneration, which correlates best with chronic disability. we found in previous studies of gjb -null mice, a model of cmt x, that the diameter of myelinated axons was progressively reduced at the age of months compared to wild type (wt) littermates, resulting from progressive dephosphorylation of axonal neurofilaments and increased packing density. fast axonal transport was slower in distal axons of mutant compared to wt animals, with impaired mobility of synaptic vesicleassociated proteins and accumulation of beta-amyloid precursor protein and tau. how cx -formed gjs are involved in the regulation of axonal cytoskeleton dynamics remains unclear. methods-here we investigated the signaling mechanisms regulating axonal cytoskeleton focusing on major kinases and phosphatases involved in neurofilament phosphorylation by immunohistochemistry and immunoblot. in addition we examined the localization of axonal mitochondrial, which also depend on axonal transport. we focused on nerve pathology in month-old gjb -null mice, when demyelination and inflammation is minimal, in order to identify the direct effects of gj loss on the axon. results-our results show that erk / kinase and pp a phosphatase were localized in non-compact myelin areas, including the schmidt-lantermann incisures and paranodes, where gjs are normally formed by cx . another kinase, cdk , was more diffusely localized along the axon. biochemical analysis showed altered amounts of erk / , pp a and cdk as well as their upstream activators in gjb -null nerves. the pp a inhibitor phapi was also localized in non-compact myelin areas and significantly increased in gjb -null nerves. assessment of axonal mitochondria by porin immunostaining showed significantly increased density in gjb -null nerves, consistent with slower axonal transport. conclusion-our findings clarify some mechanisms of early axonal pathology that are independent of demyelination in this mouse model of cmt x. several components of the signaling pathway regulating axonal cytoskeleton and axonal transport appear to be functionally related to gjs at the non-compact myelin sheath. loss of myelin gjs results in impaired regulation of axonal cytoskeleton, energy supply, and axonal transport. we have recently discovered that oligodendrocytes secrete exosomes containing a distinct set of proteins as well as mrna and microrna. intriguingly, oligodendroglial exosome release is stimulated by the neurotransmitter glutamate indicating that neuronal electrical activity controls glial exosome release. in this study we examined the role of exosomes in neuron-glia communication and its implications in glial support. results: to analyze the transfer of oligodendroglial exosomes to neurons, we exposed cultured cortical neurons to fluorescently labeled oligodendroglial exosomes. indeed, cultured cortical neurons internalized and accumulated oligodendroglial exosomes in the neuronal cell soma in a time-dependent manner. addition of endocytosis inhibitors or expression of dominant negative dynamin interfered with neuronal exosome internalization indicating that exosome uptake is mediated by clathrin-dependent endocytosis. furthermore, neuronal internalization of exosomes resulted in functional retrieval of exosomal cargo in vitro and in vivo upon stereotactic injection of exosomes. to investigate the influence of oligodendroglial exosomes on neuronal gene expression we performed a microarray screen of neuronal mrnas after exosome exposure. interesting candidates were further validated by qrt-pcr. conclusion: taken together, our results represent a proof of principle of exosome transmission from oligodendrocytes to neurons suggesting a new route of horizontal transfer in the cns. astrocytes are essential for the regulation of neuronal excitability, synaptic plasticity, and the vascular coupling of brain metabolism. unlike neurons, they are electrically silent, but produce a complex repertoire of ca events that coordinate their major functions. however, the canonical cellular mechanism for ca integration in astrocytes is unknown. using cultured astocytes and astrocytes in brain slices expressing ca sensor, gcamp , we report a candidate principle for ca integration in single astrocytes. analysis of the spatiotemporal dynamics of ca signaling in cultured astrocytes demonstrated that ca event distribution can be described by a power law. we show that metabotropic glutamate receptor activation or changes in extracellular ca regulate the power law exponent. these findings demonstrate scale-free ca dynamics in astrocytes that can provide a potential computational theory for brain operation and metabolism. the biogenesis of the axon-myelin unit is controlled by reciprocal interactions between oligodendrocytes and neurons, which continue throughout life. oligodendrocytes secrete endosome-derived microvesicles termed exosomes, implicated in intercellular communication. these exosomes carry a specific set of proteins as well as rna species. here, we show that the neurotransmitter glutamate stimulates exosome secretion from oligodendrocytes by provoking ca entry through oligodendroglial nmda and ampa receptors. furthermore, active neurons evoke exosome release from oligodendrocytes, indicating that neuronal activity controls oligodendroglial exosome release. in turn, neurons internalize oligodendroglial exosomes and functionally retrieve the exosomal cargo. thus, oligodendrocytes may influence the neuronal metabolism by an exosome-dependent transfer of bioactive substances to neurons. axonal degeneration resulting from lack of glial support occurs in plp and cnp deficient mice. intriguingly, both proteins are components of oligodendroglial exosomes. a proteomic approach revealed that amount and composition of exosomes derived from plp -/and cnp -/oligodendrocytes are altered, supporting the hypothesis that disturbed intercellular transfer of substances by exosomes may contribute to axonal degeneration in these mouse models. a. shahraz, j. kopatz, h. neumann university of bonn, bonn, germany microglial cells are the resident macrophages of the brain, which are derived from the embryonic haematopoiesis of the yolk sac. recently, we established a protocol for in vitro differentiation of pluripotent stem cells into microglial precursors by mimicking the embryonic haematopoiesis in a neural microenvironment. we now succeeded to produce microglia from human induced pluripotent stem (ips) cells. these human microglial cells were responsive to amyloid-b by increasing reactive oxygen species (ros) production. in addition, we produced primitive neural stem cells (pnsc) from ips cells that differentiated with suitable growth factors towards neurons. in human microglia-neuron co-culture, amyloid-b treatment resulted in shorter neural branches length compare to the control group. furthermore, results showed that the inhibitory human lineage specific receptor sialic acid binding immunoglobulin-like lectin- (siglec- ) was expressed on human microglial lines. we are now studying the neuroprotective role of siglec- in the amyloid-b-mediated microglia-neuron co-culture model. by expressing the human-lineage specific receptors, these microglial cells will be a suitable model to investigate these receptors in a human microglianeurons co-culture system.x purpose: organotypic retinal culture is a useful tool to perform preclinical drug testing, in which cellular interactions as well as tissue threedimensionality are preserved. the present study aimed to provide a detailed characterization of the morphologic changes of macro and microglial cells in this culture system. methods: retinas were isolated from days old crl: cd(sd) rats with the retinal pigment epithelium attached as described previously (arango-gonzalez et al. ). explants were cultured for , and days (div , div and div ). glial cell populations were characterized by immunostaining cryosections and wholemounts of age-matched control and cultured retinas using specific antibodies against gfap, vimentin and cd -b. results: in div and div cultures, gfap-positive astrocytes were more robust and not homogeneously distributed as observed in vivo: in some retinal areas the astrocytic network was thicker and in other regions astrocytes were sparsely distributed. at div only few thinned gfap-positive astrocytes remained. gfap immunoreactivity of m€ uller cells was up-regulated in culture. however, no major difference was observed in vimentin staining between both, in vivo and in vitro retinas. m€ uller cells extended processes from the inner to the outer limiting membrane were observed for all examined retinas. microglial cells stained with cd -b at div and div had somas more robust than in vivo and cell processes were thicker and retracted. same cells at div exhibited mainly a rounded morphology. conclusions: progressive morphological changes were observed in glial cell populations in retina explants. these changes include reactive macrogliosis, rearranged astrocytic distribution and microglial activation. since glial response is a hallmark of several retinal diseases, our organotypic retinal culture is a valuable resource for future investigations in retinal degenerative processes and therapy. it is widely recognized that grouped housing in enriched environment (enr) impacts the animals' brain structure and function. for instance, rats reared in enr exhibit enhanced neurogenesis in the dentate gyrus, improved hippocampus-dependent spatial memory task performance, and spine density increases of ca and ca pyramidal neurons. we found that that enr housing for four weeks after weaning induces an enhancement in gamma band local field potential oscillation power and an increase in spine density in the right ca stratum radiatum of long-evans rats. the mean spine size, as assessed by serial measurements of postsynaptic density areas, did not change substantially by laterality or rearing condition. one standing question is how glial processes are organized in enr treated rats. unlike cerebellar parallel fiber synapses, the astrocytic coverage of a hippocampal synapse varies from synapse to synapse. our preliminary electron microscopic observation suggests that right ca str. radiatum excitatory synapses tend to have larger degrees of astrocytic coverage in enr treated rats than singly caged rats.we are currently making morphometric measurements to quantitatively assess the peri-synaptic glial changes. it is becoming more and more evident that proper functioning of the neuron-astrocyte-microglia triad is fundamental for the functional organization of the brain, and thus it is of the utmost importance to better characterize their interactions in physiological and pathological processes. we recently demonstrated, in rat models of normal brain aging and lps-induced acute inflammation, that astrocytes and microglia actively collaborate in the clearance of apoptotic neurons and neuronal debris associated with programmed cell death. here we studied the interactions between neurons, microglia and astrocytes within the ca region of the hippocampus after bilateral common carotid artery occlusion (bccao) in the rat, a valid model of chronic cerebral hypoperfusion which leads to persistent ischemic conditions and ultimately to neuronal death. male wistar rats were subjected to permanent bccao. a group of rats was infused into the jugular vein with dipyridamole ( days, mg/kg/day by an osmotic minipump). sham-operated animals were used as controls. three months after bccao, immunohistochemical studies were performed on brain coronal slices, focussing on the hippocampal ca region. using an antibody against glial fibrillary acidic protein (gfap) no astrogliosis was detected. we found a significant increase in the number of total microglia, visualized using the iba antibody, in bccao-treated rats in comparison to the sham group ( %, p < . , one way anova and newman-keuls) and this effect was completely reverted by dipyridamole (p < . , one way anova and newman-keuls). as exemplified in figure , in the ca and str. radiatum of bccao-treated rats, many neurons (anti-neun antibody, red) showing signs of degeneration were closely apposed to and infiltrated by astrocyte branches (anti-gfap antibody, green) which appeared to be bisecting the cell body into cellular debris, and microglia cells (anti-iba , antibody, blue) were actively phagocytosing the damaged neurons. this finding is consistent with the scavenging activity of microglia upon dying neurons or debris, a possible mechanism that prevents further injury to neighboring neurons. it will be interesting to investigate which intercellular communication mechanisms allow the recruitment and activation of different glial cells in a well-organized reciprocal interaction to scavenge the damaged neurons and to verify the mechanisms of the protective effects of dipyridamole found in this chronic cerebral ischemic model. astrocytes are glial cells which provide important metabolic support to neurons, actively tune synaptic activity and influence brain microcirculation [ ] . one of the key processes which sustain astrocyte communication with neighbouring cells is regulated exocytosis mediating the release of gliotransmitters (peptides, amino acids, atp), delivery of membrane transporters, channels and other molecules to the plasma membrane. the exocytic gliotransmitter release is thought to be associated with snare complexes. these consist of four a-helices where one of these is contributed by syntaxin- , one by synaptobrevin- (sb ) and two by snap- in astrocytes. out of these, sb is a trans-membrane protein present on the secretory vesicle [ ] . however, the subvesicular nano-architecture and the number of sb molecules per vesicle are unclear. moreover, it is also unknown how many sb molecules are involved in the fusion between the vesicle and the plasma membrane in astrocytes. to study single vesicles and their association with sb in live astrocytes, we generated a ph-sensitive indicator yellow synapto-phluorin (ysph) as a marker for sb and as a functional readout for monitoring the properties of fusion pores, which are formed following the merger between the vesicle and plasma membranes. in an acidic environment ysph is non-fluorescent and becomes fluorescent upon alkalinisation, permitting the study of fusion of vesicles with the membrane during gliotransmitter release [ ] . our preliminary results, obtained by confocal fluorescence microscopy (with the resolution limit of about nm) and by a super-resolution microscopic technique structured illumination microscopy (sim; with the resolution limit of about nm) show that the new probe (i.e. ysph) efficiently reports the fusion pore establishment in astrocytes and the configuration of sb molecules in a vesicle. the number of schwann cells is fitted to the axonal length in the peripheral nerves. this setting is lost when tumorigenic stimuli induce uncontrolled schwann cell proliferation, generating tumours such us neurofibromas and schwannomas. schwann cells proliferate as well during wallerian degeneration. in both cases proliferation is finally arrested. we show that in neurofibroma, the induction of jmjd removes trimethyl groups on lysine- of histone-h and epigenetically activates the ink a/arf-locus, forcing schwann cells towards replicative senescence. remarkably, loss of function of this mechanism allows unrestricted proliferation, inducing malignant transformation of neurofibromas. interestingly, our data suggest that in injured nerves, schwann cells epigenetically activate the same locus and go as well into the senescence program. indeed, when this pathway is genetically blocked, cell proliferation results increased after nerve injury. we postulate that the ink a/arf-locus is expressed as part of a physiological response that prevents uncontrolled proliferation of the de-differentiated schwann cells generated during nerve regeneration, a response that is also activated to avoid overproliferation after tumorigenic stimuli in the peripheral nervous system. university of rochester medical center, rochester, united states synaptic plasticity is critical for normal neurodevelopment and proper circuit function in the adult nervous system. recent evidence suggests that microglia, classically studied in neuroinflammation, play critical roles in neurodevelopment and synaptic plasticity. however, the mechanisms driving these roles are not well understood. to start elucidating the molecular players involved in microglial communication with neurons during plasticity, we decided to first explore the role of purinergic signaling in this process. while purinergic signaling has been implicated in microglial behaviors, studies have primarily focused on neuroinflammatory roles. however, noninflamed microglia highly express the purinergic receptor, p y , which has known functions in microglial chemotaxis in early inflammation and can stimulate the release of cytokines implicated in synaptic plasticity. thus, we posited that purinergic signaling could contribute to the microglial motility that underlies synapse surveillance in the non-inflamed brain. this in turn may be critical for microglial roles in synaptic refinement during development. to explore this possibility, we took advantage of the p y knock-out (ko) mouse and examined the morphology and motility of microglia in the visual cortex as compared to microglia in wildtype mice. first, we used immunohistochemistry for the microglia specific ionized calcium-binding adapter (iba- ) protein to stain microglia in fixed sections. we then used confocal imaging and scholl analysis to investigate the complexity of the microglial processes. we also used -photon microscopy to monitor microglial motility in p y ko mice by crossing them with cx cr -gfp knock-in mice that allow visualization of microglia including fine processes in vivo. our preliminary evidence suggests that p y disruption alters basal microglial morphology and behavior making microglia less complex and altering their motility patterns. we are now investigating whether microglia-synapse interactions, as well as functional plasticity elicited by visual experience are altered in p y ko mice. our studies will expand our understanding of emerging roles for microglia in neurodevelopment and will pioneer new non-pathological roles for microglial purinergic signaling. a. dvorzhak, a. wojtowicz, r. grantyn charit e -university medicine, berlin, germany in neurodegenerative diseases, the afflicted brain is both an important object of study and an opportunity to characterize a given cellular interaction from a pathophysiological perspective. this dual approach is particularly advantageous when human disease is based on a monogenetic defect and an appropriate animal model becomes available for detailed investigation, as in case of z_q _ki (q ), a new knock-in mouse expressing a mutant form of murine huntingtin. electrophysiological recordings of gabaergic unitary ipscs from striatal output neurons (sons) in sagittal slices from wild-type and homozygous q challenged the current viewpoint that gabaergic transmission is enhanced in the hd striatum. quantal analysis in combination with high frequency stimulation and paired pulse tests revealed that synaptic gaba release is in fact tonically suppressed resulting in disinhibition of striatal output activity (dvorzhak et al j physiol ). the underlying mechanism involves a retrograde endocannabinoid signalling pathway linking postsynaptic mglur with presynaptic cb and gaba release. in addition to this deficit, z-q _ki homozygotes exhibited a significant reduction of tonic inhibition via extrasynaptic gaba(a) receptors. using pharmacological tools to alter the ratio of ambient glutamate and gaba concentrations made clear that the hd-related depression of synaptic and extrasynaptic gabaergic actions depends on the state of both the astrocytic glutamate transporter glt- and the gaba transporter gat- . in support of h eja and colleagues, our imaging experiments suggest that in normal striatal astrocytes gat- is in a releasing mode and driven by the activity of glt- . however, the latter activity is much lower in the hd striatum. sbfi recordings of glt- -related na transients and patch clamp recordings of glt- transporter currents revealed a marked reduction (less than % of wt level) in sr-labelled astrocytes from symptomatic hd mice. together, our results suggest that the deficiency of astrocytic glutamate uptake might represent a pathophysiological key mechanism underlying the observed disinhibition in the striatum of mice affected by huntington's disease. nodes of ranvier are highly enriched in voltage-gated sodium channels (nav), allowing rapid action potential propagation on myelinated axons. cell adhesion molecules (neurofascin- (nfasc ) and contactin) and scaffold proteins (ankyring and bivspectrin), which are also enriched at the nodes, have a critical role in assembly and/or stabilization of na v clusters. oligodendrocytes have been described to induce nav channel clustering at nodes in the central nervous system (cns); however, the nature of the signal(s) involved in nodal formation remains mostly unknown in the cns. to gain insight into cns node assembly, we developed hippocampal neuronal cultures from e rat embryos. during the first week, as expected, na v channel accumulation was detected at the axon initial segment (ais), co-localized with ankyring and nfasc , whereas expression along the axon was diffuse and hardly visible. in contrast, at days in vitro (div), regularly spaced clusters of na v channels, ankyring and nfasc were detected along the axon. the percentage of hippocampal axons with clusters increased from . . % at div, to . . % and . . % at and div, respectively (mean sd). importantly, these clusters were formed in the absence of myelin and no accumulation of the paranodal protein caspr was detected at this stage. to study the role of extrinsic versus intrinsic pro-aggregating factors, we analyzed nearly pure hippocampal neuronal cultures. these purified neurons still formed ais, while only few axons formed na v clusters. interestingly, when these purified neurons were co-cultured with oligodendrocytes or with conditioned medium from oligodendrocytes, the percentage of neurons with nascent nodes was greatly enhanced. taken together, these results confirm that some glial soluble factor(s) might be involved in na v channels clustering, as previously shown for retinal neurons (kaplan et al., ). furthermore, nascent nodes were detected on gabaergic neurons, but not on glutamatergic hippocampal neurons, thus arguing also for the role of intrinsic neuronal factors in their establishment. using hippocampal sections from gad -gfp mice, we showed that some gabaergic interneurons were myelinated in vivo. moreover, we observed that nascent node formation prior to myelination also takes place in vivo, strengthening physiological relevance. the electrophysiological properties of neurons with nascent nodes will be studied. microglia in the degenerating or aging brain are primed by aspects of pathology to produce exaggerated pro-inflammatory responses to subsequent central and systemic inflammatory insults. however, the mechanisms by which microglia become primed, rather than adopting an m phenotype, are not clear. triggers for priming may include altered expression of complement, recognition of amyloid or neuronal/synaptic debris for phagocytosis, decreased engagement of microglial regulatory molecules such as cd r, trem or fractalkine receptor. there is also evidence that loss of basal neurotransmitter tone may contribute to this state. microglia are known to express the nicotinic cholinergic a receptor, which has been shown to influence macrophage and microglial reactivity. in the current study we performed limited lesions of the basal forebrain cholinergic system using the murine-p -saporin immunotoxin (mu-p -sap), to investigate the degree to which loss of cholinergic tone predisposes the microglia to subsequent inflammatory stimulation and to assess the consequences of this for cognitive function in the vulnerable brain. intracerebroventricular injection of mu-p -sap ( . lg) depleted cholinergic neurons in the basal forebrain and decreased cholinergic innervation of the hippocampus, but left performance on hippocampal-dependent reference and working memory tasks relatively intact. however, decreased cholinergic innervation of the hippocampus conferred increased susceptibility to cognitive deficits induced by systemic lps ( lg/kg) days after lesioning, but microglia were not primed days after % lesions. to investigate the regional, temporal and neurochemical basis of this we induced more severe lesions of the basal forebrain (using . lg mu-p- sap) and assessed microglial priming at or days post-lesion. these data demonstrate that microglia are primed by low dose challenge at but not days and, when more robust lesions are induced, priming remains even at days. these responses are observed at the site of injury (medial septum) but more profoundly at the site of denervation, the hippocampus. indicating that neuronal injury and, particularly, loss of cholinergic tone have a profound effect on the reactivity of the microglia to subsequent inflammatory challenge. these data expand our knowledge of microglial priming and have clear implications for the interaction of cholinergic tone and inflammation in cognitive dysfunction such as dementia and delirium in which cholinergic dysfunction is implicated. ). however, glucose may also be metabolized to glycogen or oxidized to co , so it is not obvious that the stimulation of glut and hexokinase leads to lactate release. the objective of this work was to investigate whether lactate may be released by cultured mouse astrocytes within the time frame of the interstitial lactate rise observed in vivo. a first approach with an enzymatic assay showed a significant increase in extracellular lactate after minute of exposure to mm k or lm glutamate. to obtain better time resolution we developed a "lactate sniffer", a hek cell that expresses the fret lactate nanosensor laconic (san mart ın et al., plos one ). seeded on top of an astrocytic monolayer, the sniffer cell responded within seconds of exposure to elevated k mm and a similar response was elicited by mm ba . the response to k and ba supports a role for the na /bicarbonate co-transporter nbce , previously shown to mediate the stimulation of astrocytic glycolysis (ruminot et al., j. neurosci., ). the sniffer cell detected an equally fast increase in extracellular lactate when the culture was exposed to lm glutamate. previous work had shown that glutamate does not stimulate glucose consumption in the short-term (bittner et al., j. neurosci. ), and therefore the rapid release of lactate came as a surprise. this phenomenon may relate to glycogen mobilization or perhaps to inhibition of astrocytic oxygen consumption (azarias et al., j. neurosci. ). k , ba and glutamate did not produce detectable effects on the sniffer cell in the absence of astrocytes. the rapid release of lactate by astrocytes exposed to k and glutamate supports a role for these cells in the fast increase in interstitial lactate concentration that accompanies synaptic activity. ] i ) are regularly occurring as a result of uptake of glutamate from the synaptic space. glutamate uptake occurs via the glutamate/na co-transporters glast and glt- , where one glutamate is accompanied by na and h in exchange for k . the salt pump, na,k-atpase (nka), is responsible for the maintenance of the trans-membrane na gradient by exporting na and importing k for each atp. nka is dose-dependently inhibited by ouabain. astrocytes express two isoforms of the catalytic nka a subunit, the ubiquitous a and in the cns the glia-specific a . the isoforms differ with regard to na affinity, which is lower for a and with regard to ouabain affinity, which is higher for a than for a . although a mutations give rise to neurological symptoms -familial hemiplegic migraine type , the relative roles of the a and a isoforms in astrocytes are still incompletely understood. we hypothesized that the low na affinity of a makes it more suitable to cope with transient large increases in na i . to test this, we compared the effect of mm glutamate for min on real time changes of [na ] i in primary astrocytes, which, following transient transfection, predominantly expressed either the a or the a isoform. in a cells glutamate caused a significantly larger increase in [na ] i and longer recovery time to base line [na ] i than in cells expressing a . glutamate uptake dependence on either a isoform was determined by aspartate uptake in astrocytes expressing endogenous a and a . in the presence of ouabain concentrations that selectively inhibit a , we found a modest ( . mm) increase in [na ] i , accompanied by a disproportionately large decrease ( %) in glutamate uptake. it has been suggested that astrocyte glutamate transporters are clustered in microdomains where they may interact with nka. in gst pull down assays on rat brain we found that both glt- and glast interacted with the st intracellular loop of both a and a , but the interaction was significantly stronger for the a isoform. no interaction was found with other segments of the a molecule. the data indicate that the nka a isoform, which has a more restricted expression than a , plays a specific role for the interaction with the glutamate transporters and for sodium homeostasis in astrocytes. this specificity may be attributed to low sodium affinity of a and to the relatively high capacity of a to interact with the astrocyte glutamate transporters. ozone, a major component of air pollution, has considerable impact on public health. besides its well described inflammatory and dysfunction effects on the respiratory tract, there is accumulating evidence indicating that ozone exposure also affects brain functions. however, the mechanisms through which ozone exerts toxic effects on the cns remain poorly understood. we previously showed that in addition to lung inflammation,ozone exposure caused a neuronal activation in the dorsolateral regions of the rat nucleus tractus solitarius (nts, a sensory nucleus involved in visceral information processing) overlapping terminal fields of lung primary afferent running in the vagus nerves to investigate this hypothesis, we used electron microscopy and immunoblot techniques. in ozone-exposed animals, the astrocytic coverage of nts glutamatergic synapses is increased while the astrocyte volume fraction and the astrocyte membrane densities are not significantly increased. moreover, the expression of specific astrocyte markers gfap, s beta and ezrin did not change in ozone-exposed animals. altogether, our results indicate that ozone inhalation induces glial plasticity, which is restricted to the peri-synaptic coverage. . we recorded pp-gc synaptic activity after eae induction via adoptive transfert (at-eae) and found that mepsc frequency was increased compared to control animals. moreover synaptic alteration in at-eae mice depended on astrocytic tnfr because the effect was absent in tnfr -/-mice but reappeared upon tnfr re-expression in astrocytes. nr b receptors are also necessary because in vivo ifenprodil injection prevented synaptic alteration. overall, our study reveals a specific mechanism responsible for hippocampal synaptic dysfunction possibly relevant for cognitive impairment in ms. research supported by snsf grant a- and nccr "synapsy" to av. we patched ng cells in the hippocampal ca region of ng -dsred mice ( - days old) and investigated in current clamp mode how sodium and potassium channels affected synaptic depolarizations. epsps and ipsps were evoked from a resting membrane potential of mv by injecting mock epsc or ipsc waveforms derived from miniature currents. epsps which depolarized ng cells to more than mv were increasingly shortened by vgcs. at mv, corresponding to a quantal content of , the half width of epsps was shortened to % while the amplitude was hardly altered. application of ttx, -ap and tea showed that most of the observed shaping of epsps was primarily due to activation of a-type k current rectifying the depolarizing input. na currents only slightly ($ %) amplified epsp input, but this effect was outcompeted by the dampening effect contributed by a-type current ($ %). tea was almost without effect on epsps. due to their slower time course ipsps were very differently shaped by vgcs: while recruitment of vgcs by mock ipsps started at a similar threshold voltage, the maximal suppression of ipsp amplitude was much stronger. when depolarizing ng cells to mv, corresponding to a quantal content of , ipsp amplitudes were suppressed to % and the duration of ipsps was not shortened but increased to %. a-type k current was the primary contributor for rectifying as well. interestingly, if a-type k current was blocked, ipsps with a quantal content of unmasked a small ttx-sensitive spikelike depolarization of mv (duration: ms). for comparison, while synaptic responses to simultaneous release of glutamatergic vesicles were hardly altered by vgcs, the synaptic response of gabaergic vesicles was substantially low-pass filtered by a-type potassium currents. altogether, our results suggest that vgcs in ng cells normalize the synaptic strength of excitatory and inhibitory inputs and support their differentiation by ng cells through further emphasizing their pre-existing kinetic differences. to explore the involvement of this pathway in pain and analyze its impact separately in sensory neurons and sgcs, we tested pharmacological inhibitors and transgenic mice in an orofacial pain model. transient inflammation in the submandibular region of mice was induced using complete freund's adjuvant (cfa) and tactile sensitivity was quantified using von frey filaments. although inflammation was resolved by days after injection, tactile hypersensitivity persisted in wildtype mice for at least days, consistent with chronic post-inflammatory pain. tactile hypersensitivity in mice was reversed by systemic injection of the panx /gap junction blockers mefloquine or carbenoxolone at days (peak inflammation) and at days after cfa-injection, suggesting the contribution of these channels to tactile hypersensitivity. in dissociated trigeminal ganglia (tg), which innervate the submandibular skin, bzatp-induced yopro uptake into neurons and glia was prevented by mefloquine, demonstrating the functional presence of the p x r-panx complex in tg. moreover, tg of cfa-injected mice showed more atp release and immunostaining of tg revealed higher expression of panx at week after cfa injection compared to controls. development of hypersensitivity was prevented in p x r-null and in panx -null mice, and was attenuated in mice with glia-specific deletion of panx (gfapcre-panx f/f) but not with neuron-specific deletion (mnfhcre-panx f/f), emphasizing the importance of glial panx signaling in pain. because p x r and panx both have a key role in the immune response by activating the inflammasome, we are currently dissecting the relative importance of neuron-glial communication compared to inflammatory responses in the development and maintenance of orofacial pain. overall, our results show that the p x r-panx complex likely plays a major role in signaling events contributing to tactile hypersensitivity. synaptic plasticity is central to the process of learning and memory and is accompanied by strengthening of existing synapses and/or formation of new synapses. numerous studies have investigated the molecular mechanisms of learning and memory with neurons as the primary interest. given the tight coupling between synaptic activity and brain metabolism, it seems reasonable to assume that synaptic plasticity changes occurring at the synaptic level may also occur at the metabolic level in order to keep up with the augmented demand at the synapse. in this study we investigated the importance of genes involved in brain energy metabolism, and in particular those involved in neuronglia metabolic coupling during fear-motivated inhibitory avoidance learning. using ( c) -deoxyglucose ( -dg) technique we first defined the brain areas that are involved in this learning process. context-dependent avoidance behavior was tested in c bl/ mice using the step-through inhibitory avoidance paradigm (ia). regional brain metabolic activity, as measured by the dg uptake, was quantified in several brain regions. brain metabolic mapping revealed increased glucose utilization in hippocampus, amygdala [(basolateral complex (bla) and the central nucleus (cea)], and anterior cingulate cortex and mammillary bodies. quantitative mrna levels were assessed in dorsal hippocampal tissue at different time points following ia learning. results obtained demonstrate that learning modulates the expression pattern of genes involved in brain energy metabolism, i.e. glycogen synthesis and degradation, pyruvate metabolism, the pentose phosphate shunt and the astrocyte-neuron lactate shuttle (anls) in a time dependent manner. we found a late-phase of enhanced dorsal hippocampal gene expression, particularly for anls related genes, including monocarboxylate transporter (mct ). furthermore, we found that mice deficient in mct transporter display impaired long term memory in the inhibitory avoidance and spatial learning in morris water maze. these observations indicate that metabolic adaptation shown by neuron-glia metabolic coupling might be a relevant phenomenon following learning in conjunction with synaptic plasticity. in order to systematically as well as functionally analyse rmg reactivity to retinal degeneration and thus explore the rmg-derived signalling molecules in depth, we aim at comprehensively profiling proteomewide cellular responses to stimuli. we thus developed a proteomics approach screening specifically for cell surface and membrane proteins as well as secreted protein expression profiles and in addition monitor quantitative changes induced by prototype inflammatory inducer lipopolysaccharide lps. this workflow utilises sugar residues of cell surface proteins on intact rmg which are mildly oxidized and then covalently coupled to biotin. biotinylated proteins are affinity purified and glycosylated peptides are specifically released by pngasef followed by protein identification and quantification by label-free lc-msms. this workflow resulted in the identification of more than proteins on cell surfaces complemented by more than proteins identified in the rmg secretome. bioinformatic analysis allocates % of the cell surface proteins to be truly membrane or extracellular matrix proteins. this comprehensive cell surfaceome includes transmembrane receptors, transporters, adhesion molecules, signalling molecules and proteases, including cd markers, integrins, solute carriers, two ephrins, five ephrin receptors and six plexins. treatment of cells with lps results in a highly reproducible significant shift of cell surface proteome with upregulation of proteins and downregulation of proteins. among those lps-induced cell surface expression changes are proteins that suggest an active role of these glial cells in inflammatory processes. the specific changes will be discussed in detail. cell surface biotinylation on glycosyl-residues in combination with label-free lc-msms is a sensitive and reproducible method to profile cell surface proteomics and sheds light on the biological properties of cells. background: neuronal activity alters calcium ion (ca ) dynamics in astrocytes, but the physiologic relevance of these changes is controversial. to examine this issue further, we generated an inducible transgenic mouse model in which the expression of an inositol , , -trisphosphate absorbent, "ip sponge", attenuates astrocytic ca signaling. results: attenuated ca activity correlated with reduced astrocytic coverage of asymmetric synapses in the hippocampal ca region in these animals. the decreased astrocytic 'protection' of the synapses facilitated glutamate 'spillover', which was reflected by prolonged glutamate transporter currents in stratum radiatum astrocytes and enhanced n-methyl-d-aspartate receptor currents in ca pyramidal neurons in response to burst stimulation. these mice also exhibited behavioral impairments in spatial reference memory and remote contextual fear memory, in which hippocampal circuits are involved. conclusions: our findings suggest that ip -mediated astrocytic ca signaling correlates with the formation of functional tripartite synapses in the hippocampus. they are the only non-neuronal cell type in the brain that receives synaptic input from neurons. ng -cells exhibit a variety of ca -signalling pathways, which might be triggered by pre-synaptic neuronal activity. however, no global increase in the intracellular ca -concentration ([ca ] i ) was detected in ng -cells employing the minimal stimulation technique at neuron-glial synapses. therefore we assume that post-synaptic ca -microdomains might be activated under physiological conditions. these ca -signals are locally restricted to the plasma membrane. membrane bound ca -sensors with a high signal-to-noise ratio, such as lck-gcamp , are best suited for optimal visualisation of ca -microdomains. here, we set out to express lck-gcamp in ng cells together with the cytosolic reporter dye dsred. we generated transgenic mice which express a bidirectional promoter encoding both proteins under the control of a tet-off system. this strategy allows an exclusive expression of the transgenes only in the presence of a tet-responsive transcriptional activator (tta). the construct was successfully cloned and expressed in hek cells. as expected, lck-gcamp was located only at the inner plasma membrane while dsred was expressed all-over the cytosol. functionality of the ca -sensor was tested in vitro. elevation of [ca ] i reliably increased the fluorescence intensity of lck-gcamp . after zygote injection seven positive founder animals could be identified and will be crossbred with a ng -tta mouse line. taken together, the presented construct appears suitable for functional imaging of ca -microdomains in post-synaptic ng -cells. according to the "lactate shuttle" hypothesis, glycogen stored in astrocytes can be converted to l-lactate and exported to neurones as preferred energy substrate. we use optogenetics to investigate signalling between astrocytes and noradrenergic (naergic) neurones of the locus coeruleus (lc). to selectively activate g s -protein-mediated signalling in astrocytes, we generated a viral vector to express a chimera of rhodopsin and b -adrenoceptor (optob ar). we confirmed that this construct activates camp-mediated signalling in astrocytes. in cultured astrocytes, excitation of optob ar resulted in acidification as detected using snarf- indicator, and this acidification could be blocked by pre-incubation with an inhibitor of glycogen breakdown , -dideoxy- , -imino-d-arabinitol (dab), suggesting that the acidification was due to build-up of l-lactate. fast scan cyclic voltammetry (fcv) was used to measure noradrenaline (na) release in organotypic slices cut at the level of the lc in which astrocytes were expressing optob ar. light stimulation of astrocytes powerfully induced release of na which could be prevented by pre-incubation with dab or application of d-lactate. bath application of l-lactate (! mm) in absence of optogenetic stimulation triggered the release of na. in patch clamp experiments, l-lactate depolarised lc neurones and evoked vigorous firing of action potentials. these experiments suggest that activation of g s -protein-coupled receptors in astrocytes could potentiate the release of na from lc neurones via l-lactate. the cellular and molecular mechanisms of this signalling mechanism are currently under investigation. supported by the british heart foundation. rgcs. in addition to this, the organization of astrocytes in the retina of a rat model of glaucoma was found to be significantly different to those of a healthy retina. these alterations in glial cell morphology may reflect changes in their relationship with rgcs and could signify profound changes in their secretome, which may influence rgc survival. in this study we investigated labeling of astrocytes by sr in acute slices from the ventro-lateral medulla and the hippocampus of transgenic mice expressing egfp under the control of an astrocyte-specific promoter. while sr efficiently labeled egfp-expressing astrocytes in hippocampus, we found that sr -staining was very weak in the ventro-lateral medulla and rather unspecific. thus, sr is not a reliable marker for brainstem astrocytes. although carbenoxolone decreased the labeling of astrocytes in the hippocampus significantly, mefloquine which blocks pannexin and connexin hemichannels, was unable to prevent sr uptake in hippocampal astrocytes. time-lapse -photon imaging revealed that in hippocampus both astrocytes and neurons showed temporary sr -loading. in astrocytes the rise of sr fluorescence was slower as compared to egfp-negative cells and sr was quickly removed from non-astrocytic cells during washout, while it was retained in astrocytes. in brainstem astrocytes, however, only a very weak and transient sr -labeling was observed. to test if sr is actively removed from astrocytes in the brainstem, we applied mk- to block the multi-drug resistance transporters mrp- . we did not observe any increase of sr -labeling in brainstem astrocytes. in contrast, astrocytic sr labeling was significantly reduced by substrates of organic anion transport, probenecid, estron- sulfate and dehydroepiandrosterone sulfate suggesting that sr is actively transported into hippocampal astrocytes by an organic anion transporting polypeptide (oatp). additionally, the data suggest that astrocytes modulate extracellular concentration of neurosteroids in the hippocampus. as an alternative approach we have used scanning electron microscopy (sem) to obtain high-resolution back scattered images (bsi) of serial ultrathin sections over a wide-area. in tem the size of the specimen is limited to less than mm , whereas in sem it can be expanded to - mm , allowing us to mount and examine more than serial ultrathin sections on the same stage, collecting d information about many nodal structures simultaneously. here we demonstrate a variety of nodal architectures assembled from bsi-sem (su ,hitachi). in our preliminary study using this technique, the pattern of cellular processes approaching the perinodal axolemma is quite different among axons even in only four nodes examined in optic nerve. in the present study, we analyzed perinodal elements at whole nodes in rat optic nerves and reconfirmed this variety of perinodal glial elements include the followings: ) perinodal space with extracellular matrix ( - %); ) closely abutting astrocytic processes ( - %) ; ) unidentified glial cell, presumable ng glial processes ( - %), but could not find ) pre-synapse-like pseudo-neuronal processes, which was observed at the perinode in the cns grey matter (cerebral cortex) in the previous study. additionally, we found unexpected structures, small teardrop-like protrusion(s) of axolemma at out of nodes examined ( %). in nodes out of these nodes ( . %), glial elements encircled or densely contacted with the teardrop protrusions. a typical case of d reconstructed image is shown in the figure. nodal axon (ax, red), unidentified glial process (yellow; ug, presumable ng cell) and teardrop-like protrusion (asterisks) are demonstrated. outer part of the protrusion from the position indicated two arrows is encircled by the process of ug cell. based on these observations, we propose that some glial cells, including astrocytes and/or ng cells, might contribute to remove wasted membranous elements from the axon at the node of ranvier, which is a new type of neuron-glial interaction. to understand what makes this ssc network more synchronous and excitable, we performed a morphological study of neurons and astrocytes by estimating densities of neun and s -positive cells respectively. although we found similar densities of neurons and astrocytes between gaers and non-epileptic controls, the thickness of the ssc was % smaller in gaers. western blotting quantification of gfap, another astrocytic marker, was significantly higher at p -p , as compared with non-epileptic control. moreover, gfap-immunolabeling suggested that these astrocytes were reactive. we hypothesized that these modifications are linked with an alteration of astrocytic and/or neuronal physiological calcium excitability that could lead to the occurrence of epileptic seizures. to better evaluate the role of astrocytes and neurons networks during the development of ssc between p and p , we used two-photon microscopy imaging coupled with eeg recording in animal under anesthesia and neuroleptanalgesia. we first analyzed neuropile, representing ascendant projection of deeper cortical layers, astrocytes and neurons calcium activities. preliminary results obtained from animals under isoflurane anesthesia showed similar neuropile activities. astrocytic and neuronal activities were too weak to highlight differences between gaers and non-epileptic control. this is likely due to the mode of anesthesia known to strongly decrease astrocyte calcium signaling and neuronal synchronization. current experiments are therefore performed using neuroleptanalgesia know to allow the occurrence of swd. the expected data should lead to a better understanding of neuron-astrocyte interactions during brain development and the mechanism underlying epileptogenesis in a genetic model of epilepsy. purpose: to study the effects of laser-induced ocular hypertension (oht) in the astrocytes of contralateral eyes two weeks after lasering. methods: adult swiss mice were divided into two groups: na€ ıve (n ) and lasered (n ). retinal whole-mounts were immunostained with antibodies against gfap. the gfap-labelled retinal area (gfap-ra) and the number of astrocytes were quantified. results: in comparison with na€ ıve: i) astrocytes were more robust in contralateral eyes. in oht-eyes, the astrocyte population was not homogeneous, given that astrocytes displaying only primary processes coexisted with astrocytes in which primary and secondary processes could be recognized, the former having less intense gfap-ir (p< . ). the mean percentage of astrocytes in which only primary processes could be detected was . %; ii) gfap-ra was increased in contralateral (p< . ) and decreased in oht-eyes (p < . ); iii) the mean intensity of gfap-ir was higher in oht-eyes (p< . ), and the percentage of the retinal area occupied by gfap cells with higher intensity levels was increased in contralateral (p . ) and in oht-eyes (p< . ); iv) the astrocyte number did not differ significantly among the eyes analyzed. however, in oht-eyes the number of astrocytes in which primary and secondary processes could be observed were decreased (p< . ). conclusions: two weeks of laser-induced oht caused changes in the gfap-labelled retinal area but not in astrocyte number in both, contralateral and oht-eyes. on the basis of the astroglial changes detected in the present work, the use of the contralateral eye as an internal control in experimental model unilateral oht should be reconsidered. astrocytes show a high level of functional and morphological heterogeneity and are involved in many aspects of neural function, e.g., formation of the blood-brain barrier, regulation of ion homeostasis, synaptogenesis, and synaptic plasticity. despite the diversity of this cell type, the various astrocyte subpopulations are not well characterized, which is mainly due to the lack of markers that would allow for classification of astrocyte subsets. our study aimed at phenotyping of astrocyte subpopulations based on the expression of cell surface markers, which are associated with certain functions. we used two novel monoclonal antibodies, acsa- and acsa- (acsa: astrocyte cell surface antigen), directed against extracellular epitopes of astrocyte-specific cell surface markers to identify astrocyte subtypes. the acsa- antibody was generated by immunization of glast knockout mice and specifically detects the astrocyte-specific l-glutamate/l-aspartate transporter glast (eaat , slc a ), whereas the acsa- antibody results from an immunization of rats with astrocytes isolated from gfap-egfp transgenic mice. a mass spectrometric approach for the ligand-based identification of the acsa- antigen pointed to a number of candidates, which have to be validated by further experiments. the antibodies acsa- and acsa- were carefully analyzed by costaining experiments with commonly used intracellular astrocyte markers. we found that both antibodies specifically detect astrocytes in the developing and adult central nervous system. in contrast to antibodies against intracellular markers, such as gfap, s ß, or glutamine synthetase, acsa- and acsa- can be used to detect and isolate living astrocytes, which enables further analysis and culture of the separated cells. flow cytometric analysis of acsa- and acsa- antigen expression on cells from different brain regions, as well as immunohistochemical analysis, revealed differences in the expression patterns. this allowed us to define different astrocyte subtypes especially in the cerebellum and olfactory bulb of the neonatal mouse brain. future work will focus on the identification of additional astrocytespecific cell surface proteins for a comprehensive classification of astrocyte subtypes based on cell surface marker expression or expression patterns. the reciprocal communication between neuronal and glial cells represents the key component of the immunosurveillance system of the brain. it has been proposed that the neuro-glial interaction is impaired in human alzheimer's disease. in this study we focus on neuronal "on" and "off" signalling molecules in the transgenic rat model for alzheimer's disease. using enzyme-linked immunosorbent assay we determined the level of cd , cx cl ("off" signalling molecules) and mmp , trem ("on" signalling molecules) in brain homogenates. our results demonstrated significantly up-regulated level of cd (p < , ) in our ad rat transgenic animal model. on the other hand, quantification of mmp level revealed significant decrease of mmp expression (p < , ) in tested transgenic animals. previous studies showed that cd and mmp are predominantly expressed on neuronal cells in cns where cd functions normally as a marker of "self" to protect intact body component or "don't eat me" molecule which protect cd -expresing cells from phagocytosis. our results indicate that overexpression of cd on neuronal cells expressing pathologically modified truncated tau protein can be used as a neuroprotective mechanism potentiating spread and accumulation of pathological modified tau protein in neurons affected by ad pathology which in final stage support progression of the developing disease. in conclusion, we showed, that pathologically modified tau protein can modulate specific "on and off" signalling molecules and thus affects neuron-glia interaction in ad. field-excitatory post-synaptic potentials (fepsp) were recorded from the ca area of hippocampal slices prepared from wistar rats ( - weeks old) as before (fontinha et al., ) . after obtaining a stable value of the fepsp slope for at least min, ltp was induced by delivery of trains of pulses at hz, each train being separated by ms. ltp magnitude (% increase of fepsp) was evaluated - min after induction. ltp magnitude in control conditions was . %, whereas in a second independent pathway of the same slices but in the presence of bdnf ( ng/ml) it was . % (n ; p < . ). ltp was completely abolished when hippocampal slices were superfused with the gliotoxin fluorocitrate (fc, mm), which selectively reduces the astrocytic metabolism decreasing intracellular ca signalling and the release of gliotransmitters. interestingly, in presence of fc, the facilitatory action bdnf ( ng/ml) upon ltp was completely prevented. noteworthy, when fc ( mm) treated slices were superfused with the selective adenosine a a receptor agonist, cgs ( nm), the facilitatory action of bdnf ( ng/ml) upon ltp was rescued (n ; p < . ), so that dltp caused by bdnf (ltp in the presence of bdnf -ltp in the absence of bdnf in two independent pathways of the same slices) in fc cgs treated slices ( %) was similar to that observed in control slices ( % in the absence of fc and cgs ). the results suggest that the facilitatory action of bdnf upon ltp is controlled by astrocytes, and that this role of astrocytes results from their contribution to the extracellular accumulation of adenosine allowing a a receptor activation to gate plasticity actions of bdnf. several studies demonstrated the ability of astrocytes to sense, respond to and regulate neuronal function. among the many functions of glial proteins, glutamate (glu) and gaba transporters play important roles in balancing excitatory and inhibitory signals in the brain. here we show that astrocytes regulate the tonic inhibition of neurons by the concerted action of glu and gaba transporters, thereby protecting both neurons and glial cells from overactivation. we demonstrate that the uptake of glutamate triggers the reverse function of glial gat- / transporters by elevating the intracellular na concentration in astrocytes. the released gaba significantly contributes to the tonic inhibition of neurons in a network activity-dependent manner. we also describe the source of the releasing gaba that is synthesized by an alternative pathway from polyamines. moreover, in the low-[mg ] model of epilepsy, we show that blockade of the glial glu/gaba exchange mechanism increases the duration of seizure-like events and also results in increased activity of astrocytes, demonstrating the neuroprotective impact of the mechanism. finally, we show that the released glial gaba modulates the power of gamma range oscillation in vivo, suggesting that the glu/gaba exchange mechanism is also functioning in the intact hippocampus under physiological conditions. revealing this novel molecular mechanism by which astrocytes provide an adjustable, in situ negative feedback on the excitability of neurons is expected to broaden our understanding about the regulation of neuronal activity by astrocytes and may open up new targets for the treatments of pathological conditions, such as epilepsy or ischemia. chronic stress is well recognised to decrease the number of gfap astrocytes within the prefrontal cortex (pfc). recent research, however, has suggested that our understanding of how stress alters astrocytes may be incomplete. specifically, chronic stress has been shown to induce a unique form of microglial remodelling, but it is not yet clear whether astrocytes also undergo similar structural modifications. such alterations may be significant given the role of astrocytes in modulating synaptic function. accordingly, in the current study we have examined changes in astrocyte morphology following exposure to chronic stress in adult rats, using three-dimensional digital reconstructions of astrocytes. our analysis indicated that chronic stress produced profound atrophy of astrocyte process length, branching and volume. we additionally examined changes in astrocyte-specific s b, which is both a putative astrocyte marker, and a protein whose expression is associated with astrocyte distress. while we found that s b levels were increased by stress, this increase was not correlated with atrophy. we further established that while chronic stress was associated with a decrease in astrocyte numbers when gfap labelling was used as a marker, we could find no evidence of a decrease in the total number of cells, based on nissl staining, or in the number of s b cells. this finding suggests that chronic stress may not actually reduce astrocyte numbers and may instead selectively decrease gfap expression. together, these results provide a significantly more elaborate picture of how chronic stress alters the pfc. the drosophila nervous system is ensheathed by several types of glial cells, one of which, namely the subperineurial glia, forms pleated septate junctions (psj) to build a tight blood-brain barrier (bbb). hence, nutrients from the surrounding hemolymph must cross these glial cells to reach the neurons. the amount of nutrients entering the nervous system is likely to be tightly controlled through neuron-glia communication to ensure optimal metabolic supply while avoiding disturbance of the extracellular homeostasis. we aim to elucidate signals involved in this regulatory interaction. to this end, an rnai-based screen in glia of adult flies is performed using the gal /gal ts system. we analyze the intake of blue-dyed food by photometric measurement. since impairment of the metabolic supply of the brain should severely affect the organism, compensatory changes in feeding behavior are expected. for instance, knockdown of nutrient transporters specifically in glia would then induce excessive feeding in normally nurtured individuals. the first set of rnai lines screened includes genes that have previously been found to be essential in glia for survival or locomotion of the animal. furthermore it comprises metabolic enzymes, all carbohydrate transporters, neuropeptides and their corresponding receptors. candidate genes will be further characterized regarding their role in controlling the energy homeostasis of the nervous system. the discovery that activation of non-neuronal cns microglia plays a causal role in spinal processing of nociceptive signaling has shed new light on the processes underlying neuropathic pain facilitation. however, there remains much uncertainty as to the necessary contribution of microglia to enhanced pain states. we aim to define the particular role of microglia for the initiation of neuropathic pain and by answering this question also learn if the function of peripheral myeloid cells is distinct or redundant in this process. methods: to specifically investigate spinal microglia and peripheral macrophages in the pathogenesis of neuropathic pain, we model chronic pain by performing partial ligature of the sciatic nerve in cd b-hsvtk mice engrafted with gfp bone marrow (gfp>cd b-hsvtk). cd b-hsvtk /mice allow the exchange with peripherally-derived, gfp macrophages upon central depletion of endogenous cd b microglia. following this depletion/ repopulation paradigm, behavioral analyses of mechanical and thermal allodynia are conducted. results: we established a selective tool to exchange cns parenchymal microglia with peripheral gfp myeloid cells. in chronic pain tests for mechanical and thermal hyperalgesia, gfp>cd b-hsvtk /mice show considerable decreases in paw withdrawal thresholds in response to mechanical, but not thermal stimuli ipsilateral to the injury, indicating distinct roles of microglia and macrophages in the facilitation of thermal hyperalgesia. conclusions: we identified a differential contribution of resident spinal microglia vs. peripheral myeloid cells in the development of neuropathic pain in gfp>cd b-hsvtk chimeras. future studies aim to examine the exact mechanisms underlying the distinction between these two populations. a deeper understanding of the processes involved in the compartmentation of brain energy metabolism is of major interest. not only to enwrap pathomechanisms of brain diseases associated with metabolic deficits but also for a more accurate interpretation of functional neuroimaging signals which often use metabolic signals as surrogate marker for brain activity (e.g. fdg pet). large amount of knowledge in the field has been obtained from small animal neuroimaging studies. but due to their invasiveness they often need anesthesia. anesthetics heavily alter physiological read-outs. the aim of this study was .) to test if metabolic imaging using two-photon microscopy ( pm) and genetically encoded glucose sensors is feasible in the awake, head-fixed mouse and .) to examine the effects of isoflurane anesthesia on the signals. in two mice, a recombinant adeno-associated virus as a carrier for the genetically encoded glucose sensor flii pglu md was injected into the whisker barrel cortex. by using an astrocyte-specific promoter (short gfap) astrocytic expression was ensured. in addition, a chronic window and a head post were implanted. behavioral training started a few days after surgery. animals had to learn to tolerate head fixation without spontaneous motor activity. within a single trial animals were trained not to move for seconds (imaging period) to receive a water reward. animals were subjected to water restriction during the behavioral training. animals were trained for three weeks before pm imaging started. animals learned to tolerate head fixation up to minutes allowing acquisition of up to trials per session. upon oral glucose administration (per single water reward $ mg glucose was administered) fret signal started to increase within minutes and went up to a maximum of %. surprisingly, isoflurane anesthesia led to an even higher increase of the signal (mean increase of %) compared to the awake state. in some experiments the barrel cortex was activated by vibrotactile single whisker deflections generated by a piezo bending actuator. fret signal did not show a difference between baseline and stimulated condition ( . . vs. . . ). in conclusion, the presented experiments demonstrate for the first time the feasibility of awake pm imaging for metabolic measurements on a single cell level. the signal increase upon peroral glucose administration unambiguously supports the functionality of the sensor. isoflurane exhibits a large effect on intracellular astrocytic glucose concentration distinctly revealing the need for experiments in awake animals for unbiased results. future experiments will now be expanded to neuronal glucose sensors to enable comparisons between astrocytes and neurons. our previous studies indicate that neuronal electrical activity controls glial exosome release resulting in subsequent neuronal uptake and functional retrieval of the exosomal content. proteomic analysis of oligodendroglial exosomes revealed a list of candidates with potential neurotrophic action in neurons. to elucidate the impact of oligodendroglial exosomes on the neuronal metabolism, we analyzed the metabolic activity of neurons after co-culture with oligodendrocytes or direct treatment with exosomes. methods: neurons were subjected to different stress paradigms such as oxidative stress, hypoxia, and nutrient deprivation. neuronal vitality was assessed by mtt assay and the mitochondrial membrane potential was visualized by staining with mitocapture. results: neurons grown under optimal conditions were not affected by the presence of exosomes. intriguingly, when neurons were subjected to stress (oxidative stress, nutrient deprivation, oxygen-glucose deprivation) their metabolic activity was significantly increased in the presence of exosomes. when challenged with oxidative stress prior to exosome treatment, neurons were not able to recover. mitocapture staining demonstrated that oligodendroglial exosomes prevent the breakdown of the mitochondrial membrane in nutrient-deprived neurons. conclusions: our results indicate that exosome supply is protective for neurons but is unlikely to support their recovery. we suggest that oligodendroglial exosomes carry neuroprotective substances, which protect neurons from stress. interestingly, this effect of lactate on synaptic plasticity mechanisms was not fully mimicked by glucose, suggesting that the effect was not only related to supporting the potential increase in energy demands related to plasticity, but that l-lactate could act as a signaling molecule for the regulation of expression of plasticity-related genes. we have followed up on these observations and show that l-lactate significantly stimulates mrna expression of key immediate early genes (iegs), in a time-and concentration-dependent manner, in cultured neurons. following one hour of treatment with mm l-lactate, arc, zif and c-fos mrna levels were increased by . , . and . folds, respectively. in addition, the increased iegs mrna expression levels induced by l-lactate are correlated at the protein level with respectively . , . and . fold increase compared to control values at the same time point. these effects are specific for llactate, since d-lactate (non-metabolized enantiomer of l-lactate), l-pyruvate and d-glucose (at equicaloric concentrations) have no effect on gene expression. interestingly, we observed that, in similar culture conditions, l-lactate-induced iegs expression is only observed in neurons but not in astrocytes, implying a cell specific effect. characterization of the underlying molecular mechanisms of l-lactate action on iegs expression demonstrate the involvement of nmda receptors. finally, we obtained evidence that such regulatory mechanisms of ieg expression also operate in vivo as direct intracortical injections of l-lactate ( mm) into the somatosensory-motor cortex areas resulted, within hour of application, in a significant stimulation of arc, zif and c-fos expression (by %, % and %, respectively) as compared to d-lactate ( mm)-injected contralaterally areas. taken together these findings demonstrate that l-lactate acts as a direct signaling molecule which regulates neuronal plasticity-related iegs expression both in vitro and in vivo. interestingly, the underlying mechanism of action of l-lactate involves nmda receptors.this set of observations therefore reveals a novel signaling role of lactate which may represent a likely mechanism to account for the role of astrocyticderived l-lactate on ltm formation. neuronal activity in the brain is associated with a transient increase in the extracellular k concentration. the excess k is removed from the extracellular space, primarily by the surrounding glial cells, leading to an intracellular accumulation of k via mechanisms not fully identified and/or quantified. post-stimulus recovery of [k ] o has been proposed to be dependent on kir . -mediated spatial buffering and/or to na /k -atpase activity. to resolve the molecular mechanisms involved in k clearance, we initially determined the contribution from the different k -transporting mechanisms present in primary culture of rat astrocytes and their k . for k . the na /k / clcotransporter, nkcc , increased its activity within a physiological concentration range of k , while the na / k -atpase saturated at much lower concentrations. consequently, a concentration-dependent switch occurs between the two mechanisms of k uptake in cultured astrocytes. in addition, nkcc was capable of mediating robust k -induced astrocytic cell swelling. thus, nkcc could potentially act as a molecular mechanism responsible for clearance of the stimulus-evoked rise in [k ] o and the associated shrinkage of the extracellular space. to determine the contribution of each of the three molecular mechanisms to k -clearance in native brain tissue, we used rat hippocampal brain slices and employed ion-sensitive microelectrodes in association with high-frequency electrical stimulation as well as focal appliances of k by ionophoresis. inhibition of kir . ( um bacl ) or nkcc ( um bumetanide) failed to show a significant effect on the rate of k removal from the extracellular space. in contrast, inhibition of the a and a isoforms of the na /k -atpase significantly delayed post-stimulus recovery of [k ] o and this delay was further potentiated by additional inhibition of the a isoform. the na /k atpase emerged as the primary factor responsible for stimulus-evoked k -clearance with no evidence in favor of kir . and nkcc involvement. further characterization of the different na /k -atpase isoforms, by heterologous expression in xenopus laevis oocytes, revealed that the a isoform reached its maximal activity already at resting k concentrations, hinting at a "housekeeping" function. the a subunit displayed voltage-sensitivity and increased turnover rate along physiologically relevant increments in extracellular k concentration. these a -related features may render this astrocyte-specific subunit variant specifically geared for post-stimulus recovery of [ as a model of ischemic injury, and sham-operated mice were used as controls. we isolated gfp cells from the dorsal part of the lv of sham-operated-and post-ischemic brains and employed a neurosphereforming assay in order to estimate their ability to proliferate and selfrenew. furthermore, we followed their immunocytochemical and electrophysiological properties during in vitro differentiation and compared the differentiation potential of gfp cells isolated from the controls to those isolated from post-ischemic brains. the gfp cells isolated from the dorsal part of the lv of controls formed neurospheres and differentiated only into a glial phenotype. the gfp cells isolated from the dorsal part of the lv of post-ischemic brains were able to form neurospheres as well; however, besides their differentiation into a glial phenotype, they also gave rise to cells with the properties of neuronal precursors. we also performed in situ immunohistochemical/electrophysiological analyses of gfp cells in the adult brain of controls and those after mcao. in situ analyses revealed that gfp cells expressed the phenotype of adult nscs or neuroblasts in controls or following ischemia and that their number was increased in both hemispheres following mcao. compared to controls, we found that the number of gfp/doublecortin-positive cells significantly increased in the dorsal part of the lv as well as the number of gfp cells in the olfactory bulb, where they probably differentiated into calretinin interneurons. our results indicate that gfp cells with an active mdach gene in the dorsal part of the lv play an important role in the increased production of neuroblasts after injury and that this process is also enhanced in the contralateral hemisphere. collectively, our results reveal the involvement of the mdach gene in adult neurogenesis. cells expressing this gene exhibit the properties of adult nscs or neuroblasts and respond to mcao by enhanced neurogenesis. experimental cerebral ischemia leads to activation of microglia and astrocytes, which subsequently release pro-and anti-inflammatory factors. furthermore, during the earliest periods of neuronal damage, large quantities of dopamine are released, which may exacerbate the vulnerability of the lesioned tissue. on the other hand, delayed treatment with levodopa has been proven beneficial in stroke patients, suggesting a more complex effect of dopamine. recent studies on astrocytic involvement in neuroinflammation have shown a remarkable modulation of the inflammatory response over the dopamine d receptor (d r). after stroke, reactive astrocytes in the astroglial scar were found to be immunopositive for d r. in this study, we report the expression of d r in activated microglia, both after experimental stroke in vivo and after oxygen-glucose deprivation (ogd), an in vitro model of ischemia. using immunohistological stains, we analyzed the expression of d r in iba positive cells , , , , and days after middle cerebral artery occlusion (mcao; n - mice/time point). for each animal, > pictures were taken from the ischemic core as well as from the contralateral, non-lesioned cortex from sections. a total of > (control) and > (ischemic core) iba cells were analyzed for each time point. in the ischemic core, $ % of cells immunopositive for iba expressed d r in contrast to < % in control areas. a strong depletion of d r immunoreactivity was observed in the ischemic striatum, accompanied by an increase in d r expression in the adjoining cortex and an elevated presence and activation of microglia. for in vitro experiments, we used fluorescence activated cell sorting to isolate cd low /cd microglial cells. no significant d r expression at mrna level could be detected by rt-pcr in this population, while we were able to detect d r in non-microglial cells. interestingly, cultured primary mouse microglia expressed low quantities of d r, as revealed by western blotting and rt-pcr, suggesting that culturing is sufficient to induce d r in microglial cells. upon in vitro activation of primary microglia through minutes of ogd, we found a . fold increase in d r expression after hours (n ). furthermore, treatment of primary microglia with the selective d r agonist pramipexole showed a dose-dependent tendency to increase lipopolysaccharideinduced tnfa release (n ). our studies indicate that activated microglia express a functional d r, which may contribute to the pro-inflammatory reaction of microglia after ischemia. white matter (wm) is injured in most strokes and axonal injury and dysfunction contribute to disability associated with clinical deficits. in young wm, the damage from ischemic injury involves the sequence of energy depletion (ionic pathway), excessive glutamate release (excitotoxicity), generation of reactive oxygen species and oxidative stress (oxidative pathway). in older wm the injury is mediated by ca -independent excitotoxicity due to an earlier and more robust glutamate release. because excitotoxicity leads to oxidative stress in wm we investigated whether blocking nitric oxide synthase (nos) activity before or after a period of oxygen glucose deprivation (ogd) promoted axon function in an age-dependent manner. acutely isolated optic nerves from young and old ( and month) swiss webster (sw) or c bl/ (bl ) mice were used to ascertain quantitative measurements of wm function and structure. to support a biological basis for nos inhibitor nitro-l-arginine methyl ester (l-name) action in themonpreparation, we evaluated the expression and localization of brain nos (bnos) using immunohistochemistry in conjunction with confocal imaging. the expression of bnos co-localized with gfap ( ) astrocyte nuclei, cytoplasm, end-feet as well as nf- ( ) axons. the pattern of bnos expression paralleled astrocyte morphology with age and became more punctate in appearance. evoked compound action potentials (caps) recovered to . . % (n ) after min of oxygen glucose deprivation (ogd) in young bl mons. pretreatment of mons with l-name ( mm) promotedcaprecovery to . . % (n ) compared to ogd while caps recovered to . . % (n ) when l-name was applied after the end of ogd. pretreatment of mons from or month old sw with l-name improvedcaprecovery to . . % (n , vs ogd . . %, n ) or to . . % (n , vs ogd . . %, n ) respectively. l-name application after the end of ogd failed to promote aging axon function ( . . %, n ). changes in nos activity help unveil agedependent oxidative injury mechanisms in ischemic white matter. question: cerebral endothelial cells have been reported to exert a protective effect against brain damage. does transplantation of healthy endothelial cells have a beneficial effect on the outcome of ischemic brain damage? methods: we injected endothelin- (et- ) into the rat internal capsule to induce lacunar infarction. seven days after et- injection, microvascular endothelial cells (mvecs) prepared from adult rat cerebral cortices were transplanted into the internal capsule. meningeal cells prepared from adult rat cerebra or . % bovine serum albumin-hank's balanced salt solution were injected as controls. two weeks later, the footprint test and histochemical analysis were performed. results: we found that mvec transplantation improved the behavioral outcome based on recovery of hind-limb rotation angle (p < . ) and induced remyelination (p< . ) compared with the control groups. also the inflammatory response was repressed by mvec transplantation, judging from fewer ed- -positive activated microglial cells in the mvec-transplanted group than in the other groups. conclusions: elucidation of the mechanisms by which mvecs ameliorate ischemic damage of the white matter may provide important information for the development of effective therapies for white matter ischemia. objective: though lactate is a known neuroprotective agent, its mechanism of action is not known. we hypothesized that lactate can provide neuroprotection by activating trek channels. the effect of lactate on the electrophysiology of trek channels was studied both in an in vitro brain slice model, after blocking the activity of other ion channels and in a heterologous expression system. results: whole cell patch clamp experiments, on ca stratum radiatum astrocytes, in acute hippocampal slices, significantly increased trek channel activity by . . % and hyperpolarized their resting membrane potential by . mv, on bath application of mm lactate. comparison of rectification index at negative potentials between control and mm lactate treatment showed no significant difference, suggesting that the conductance activated by lactate is outward rectifying. lactate-evoked increase in trek channel activity was reversibly inhibited by mm quinine, a potent inhibitor of trek channels. further, lactate was unable to increase trek channel activity after disruption of intracellular lactate uptake with mm a-cyano- -hydroxy cinnamate, a blocker of monocarboxylate transporter. this was confirmed by inside-out patch clamp experiments on human trek channel expressed in hek cells. conclusion: the experimental observations suggest uptake of lactate by astrocytes to activate trek channels intracellularly. regenerative responses occurring after different types of postnatal brain injury often involve expansion of an endogenous neural progenitor cell pool, but the molecular mechanisms underlying this process are still poorly understood. we studied expansion of the glial progenitor cell pool in a mouse model of neonatal hypoxia (hx) that reproduces the hallmarks of perinatal brain injury in infants, including diffused white matter injury (dwmi). we have previously shown that hx causes proliferation of wm oligodendrocyte progenitor cells (opcs), and that this process is regulated by the cyclin-dependent kinase (cdk ) pathway. our analysis in wm in vivo and in cultured cells demonstrated: i) higher expression of cdk , cycline, prb / and e f in wm opcs after hx; ii) activation of the cdk pathway after hx, and iii) reduced hx-induced opc proliferation in cdk -null mutant mice. here, we studied the upstream molecular mechanisms that regulate activity of the cdk signaling pathway resulting in opc proliferation after hx. we show that sirtuin (sirt ) histone deacetylase activity is a major regulator of cdk signaling and of the regenerative response of opcs after hx. under hypoxic conditions, sirt is upregulated in wm, and interacts with both rb and cdk . patterns of posttranslational modifications of cdk or sirt proteins after silencing either of these genes in cultured cells from normoxic and hypoxic wm indicate that cdk is a substrate for deacetylation by sirt , and that sirt is phosphorylated by cdk . also, sirt causes rb deacetylation resulting in dissociation of e f from the rb/e f complex, which ultimately leads to higher opc proliferation. knockdown of sirt in hypoxic wm cell cultures suppresses opc proliferation. finally, inhibition of sirt activity by sirtinol accelerates opc differentiation to mature oligodendrocytes, and reduces the number of opcs. we are currently analyzing the effects of hx on opc proliferation and differentiation in the wm of sirt -null mice. our results provide evidence that sirt is an essential regulator of the cdk -dependent regenerative response observed in wm opcs after hx, and that inhibition of sirt activity facilitates oligodendrocyte regeneration from opcs, which might ultimately promote wm recovery after hx. supported by nih r ns , p ns and iddrc p hd . gonadal steroid hormones reveal a potential neuroprotective role in acute brain ischemia. in rat in vivo studies using the transient occlusion of the middle cerebral artery as ischemic stroke model, we have shown that ß-estradiol and progesterone reduce the infarct area by more than % given h after the onset of stroke, prevent neuronal death and behavioral deficits. an intriguing aspect of this study was that the application of steroid hormones reduced the number of microglia and microglia-related markers in the penumbra during the first h after the onset of stroke. besides microglia, penumbral astroglia coevally appeared as cellular target for steroid hormones by reducing the expression of proinflammatory-and molecules necessary for the attraction and activation of microglia and lymphocytes. this suggests that protective steroid hormones influence glia cell cross-talk and activity during early damaging conditions in the lesioned brain site. by using an in vitro hypoxic approach, we have now demonstrated that microglia express steroid hormone receptors and directly respond to ischemic conditions and steroid hormone treatment by changing expression and secretion of inflammatory compounds, trans-signaling factors and by modulating phagocytotic activity. this clearly shows that microglia is an important direct target for steroid hormones but also indirectly influenced via cross-talk by adjacent astrocytes at the damaged brain site. this generally points at an extensive bidirectional crosstalk between both glial cell types to convey steroid-mediated neuroprotection in ischemic brain tissue. in summary, the balance of inflammatory astrogliamicroglia interactions within the injured brain tissue during an early phase of ischemic destruction is a pivotal step for tissue repair. cannabinoids are considered as key regulators in the pathology of various diseases, including ischemic stroke. we previously demonstrated that post-ischemic treatment of two naturally occurring phenylpropanoids, trans-and cis-hinokiresinols (hrs) significantly reduced ischemic injury in rats subjected to middle cerebral artery occlusion (mcao) . in studies to screen their cellular targets, we found that hrs selectively bind to both type and type cannabinoid receptors (cb r and cb r). in the cb r reporter gene assay, both hrs demonstrated antagonistic activity. in cb r-overexpressing u os cell lines, both hrs increased forskolininduced camp accumulation, suggesting their inverse agonism. since camp induces expression of heme oxyagenase- (ho- ), a well-known antioxidant stress protein, we further investigated the effect of hinokiresinols on ho- expression in mixed cortical neuron/glia cultures. trans-hr increased astroglial expression of ho- greater than cis-hr did. a selective ho- inhibitor, tin protoporphyrin ix significantly reduces the neuroprotective effect of trans-hr in cortical cultures exposed to oxygenglucose deprivation. in addition, both hrs reduced the number of ed- immunopositive cells (i.e., active microglia and macrophages) in ischemic lesions. also, both hrs significantly reduced microglial migration induced by an endocannabinoid, -arachidonoylglycerol. the present study suggests that hrs reduce ischemic injury via regulation of glial cbrs. , the main anaplerotic enzyme in the brain, is predominantly located in astrocytes. in the adult brain, ischemia is known to reduce pyruvate carboxylation. moreover, reperfusion after ischemia leads to production of reactive oxygen species (ros). the pentose phosphate pathway (ppp) is, by making nadph necessary for the regeneration of reduced glutathione, a major pathway in the protection against ros. however, astrocytic and neuronal metabolic function in the neonatal brain after hypoxic-ischemic brain injury (hi) remains to be explored. methods: hi was induced in -day-old rats by unilateral severing of the carotid artery followed by hours recovery and subsequent exposure to hypoxia ( %o ) for min. min after end of hypoxia (the early reperfusion phase), rats were injected with [ , - c]glucose and decapitated min later. one group was sham-operated, and not exposed to hypoxia, thus reflecting normal metabolism in the neonate. extracts of ipsilateral hemispheres from hi and sham animals were analysed with h-and c-nmr spectroscopy. results: the sham animals had similar glutamine content, but lower amounts of c labelled glutamine compared to corresponding values from adult rats, reflecting a generally lower rate of glucose metabolism in the neonatal astrocytes. however, approximately equal amounts of c labelled isotopomers of glutamine were derived from pc and pyruvate dehydrogenase (pdh), resulting in a relatively high pc/pdh-ratio compared the same ratio in adults. following hi and reperfusion, a reduction in glucose metabolism and mitochondrial metabolism was seen by increased glucose and reduced incorporation of c labelling in glutamate, glutamine and aspartate via pc and pdh. however, the pc/pdh-ratio in glutamine was similar in hi and sham. total mean values of glutamate, glutamine and aspartate were decreased, but only the latter to a significant degree. labelling in lactate via the ppp was reduced following hi. conclusion: the low labelling of lactate via the ppp indicates that the flux through the ppp was in fact reduced in the early reperfusion phase after hi. moreover, mitochondrial metabolism of pyruvate from glucose was reduced in both astrocytes and neurons. impaired anaplerosis in astrocytes was confirmed by lower amounts of aspartate. however, the proportionally similar decrease in c labelling in glutamate and glutamine via pc, and the maintained pc/pdh-ratio implies that astrocytes continue to provide metabolic support for the neurons during the early reperfusion phase. ret receptor tyrosine kinase is the signaling component of the receptor complex for the family ligands of the glial cell line-derived neurotrophic factor (gdnf). ret is involved in the development of enteric nervous system, of sympathetic, parasympathetic, motor and sensory neurons and it is necessary for the postnatal maintenance of dopaminergic neurons. ret expression has been as well demonstrated on microglia and several evidence indicate that gdnf regulates not only neuronal survival and maturation but also certain functions of microglia in the brain. here we demonstrated that isolectin ib , commonly used as a microglial marker in the brain, binds to the glycosylated extracellular domain of ret both on the surface of living nih t fibroblasts cells stably transfected with ret than in adult rat brain sections as revealed by immunoblotting. further, confocal immunofluorescence analysis demonstrated a clear overlap staining between pret and ib labeling in primary microglia cultures as well as in adult rat sections obtained from control or postischemic brain after permanent middle artery occlusion (pmcao). interestingly, ib staining identified activated or amoeboid retexpressing microglia under ischemic conditions. collectively, our data indicate ret receptor as one of the ib -reactive glycoconjugate accounting for the ib stain in microglia under physiological and ischemic conditions. astrocytes can adapt to lower oxygen tensions by enhancing their glycolytic rate of energy production. in this case, adequate expression of monocarboxylate transporters (mcts) may be essential to sustain prominent lactate efflux and prevent glycolysis inhibition. here we show that oxygen level is an important factor controlling mct expression in primary cultures of mouse cortical astrocytes. indeed, while mct is clearly expressed by astrocytes in vivo, its basal in vitro expression in primary cultures of mouse cortical astrocytes is undetectable under classical culture conditions ( % air, %co ). exposing astrocytes to more physiological brain oxygen tensions (hypoxic chamber flushed with %o / %co / %n , leading to a concentration of dissolved oxygen in the culture medium of - mmhg) restored mct expression. mct mrna expression became detectable after hours under lower oxygen tension and was still present after days with a maximum at hours of incubation. this effect was paralleled by the appearance of the mct protein which reached a plateau between hours and hours, with a further enhancement at and days of incubation. this induction was specific for mct since mct expression was not altered. lactate release was significantly enhanced after hours of incubation and further increased up to days compared to astrocytes maintained under atmospheric conditions. treatment with dimethyloxalylglycine (dmog), a compound that mimics hypoxia by stabilizing the hypoxia-inducible factor alpha (hif- a) , produced a similar effect. both mct mrna and protein induction reached their maxima when astrocytes were treated with a concentration of dmog ranging between to . mm, which is also the range for greatest hif- a expression. again, no effect was detectable on mct expression. transfecting astrocyte cultures with a sirna against hif- a significantly reduced the effect of lower oxygen tension on mct expression. our results suggest that ) under physiological conditions, changes in local oxygenation might regulate the capacity of astrocytes to supply lactate to neighboring cells via notably alteration in mct expression ) under pathological conditions, mct may be upregulated as a consequence of hypoxia and contribute to neuroprotection by favouring the release and accumulation of lactate in the extracellular space. indeed, this process might give rise to the beneficial effect of lactate for neurons observed during the recovery from hypoxic/ischemic conditions in vitro and in vivo. early onset dementia and. the loss-of-function of trem or its coreceptor dap is responsible for the recessively inherited nasu-hakola disease (also known as plosl). the brains of plosl affected patients show strong microglial activation in the cerebral white matter. reports demonstrate that trem -mediated phagocytic function of microglia is required for debris clearance and cns tissue homeostasis. perinatal brain injury is the underlying etiology for a host of developmental disabilities that includes spastic motor deficits and cognitive, behavioral and learning difficulties. hence, our aim is to characterize the expression of trem in the control of neuroinflammation following hypoxia/ ischemia (hi) in the newborn brain. we performed hi brain damage in postnatal day (p ) c /bl mice by permanent left carotid occlusion and litters were exposed to % of oxygen balanced with nitrogen for minutes in a hypoxic chamber with controlled humidity and temperature maintained at c. pups were then returned to their dam until sacrifice. the age-matched controls and samples from hours to days after hypoxia were collected and processed for immunohistochemistry. in control animals, trem expression was observed in the corpus callosum and in the subventricular zone at p that almost disappeared at p . following hi, an increase in trem staining was observed in the corpus callosum, hippocampus, caudate-putamen, fimbria, cortex and thalamus in the ipsilateral (il) hemisphere, following a similar regional pattern of microglia activation. the increased expression of trem was observed from hours to days post-hypoxia. trem colocalized mainly with microglia markers, such as iba- and cd . oligodendrocyte expression of trem was also analyzed. in conclusion, hi produced an increase in trem expression on the il damaged regions. these results suggest that the modulation of trem might be a possible target for damage control in neonatal brain. increasing evidence shows that astrocytes play a critical role in neuronal protection during an ischemic insult. in the vertebrate retina, most of astrocytic functions are executed by m€ uller glial cells, the major macroglial cell type in this tissue. the aim of this study was to evaluate the role of m€ uller glia in the onset and progression of retinal ganglion cell (rgc) degeneration after acute ischemic injury in the mouse eye in vivo. here, we used the selective gliotoxin fluorocitrate (fc) in order to transiently impair m€ uller glial metabolism at different time points during and after induction of an acute retinal ischemia/reperfusion by means of elevated intraocular pressure (iop). the effect of fc ( mm) on metabolism was determined by measuring retinal glutamine levels, aconitase activity and mitochondrial membrane depolarization after intravitreal injection of the gliotoxin. retinal ischemia was unilaterally induced by elevating iop for min. fc was intravitreally injected h before onset of ischemia and after , or h of reperfusion. saline was injected in the contralateral eye and served as control. surviving rgcs were quantified by means of confocal scanning microscopy and automated cell counting-based analysis on retinal wholemounts days after lesion. fc treatment reduced glutamine levels to - % as compared to control - h after injection. inhibition was completely reversed h after injection. aconitase activity was reduced to $ % from control levels h after fc injection. mitochondrial membrane potential was reduced by - % as indicated by reduced intensity of the specific stain mitotracker red cmxros. one week after ischemia, only % of rgcs survived as compared to unlesioned controls. impairment of m€ uller glial metabolism during ischemia further reduced rgcs by % as compared to ischemia alone. fc treatment did not significantly modify rgcs survival and h after lesion. results support the notion for a critical neuroprotective role of m€ uller glial cells during ischemia. in the acute phase following ischemia, however, m€ uller glia inhibition does not further affect rgcs survival. further research is required to establish the time point for this switch in m€ uller glial function and the underlying mechanisms. extract has been widely investigated in animal models and clinical studies for various diseases including ischemic stroke. its major metabolite, cordycepin, has been reported to act as a selective a adenosine receptor agonist and to protect neurons against ischemic injury. however, their underlying mechanisms remain unclear. in the present study, we report that the standardized c. militaris extract, wib- c, which contains cordycepin % of total dry weight of the extract, markedly reduced ischemic injury by inhibiting post-ischemic inflammatory responses. postischemic treatment with wib- c (orally administered twice at and h after onset of mcao) significantly reduced infarct size and edema in rats subjected to transient middle cerebral artery occlusion (mcao, . h) and subsequent reperfusion ( h). wib- c also significantly improved integrity of glial cells in ischemic lesions, as evidenced by reduced white matter degeneration and loss of blood-brain barrier. importantly, wib- c significantly ameliorated neurological deficits in not only transient but also in permanent stroke models. moreover, wib- csignificantly improved long-term survival of mcao rats over days. as we previously reported with selective a receptor agonists introduction: cd & cd r are immune inhibitory molecules that have been shown to be involved in inducing immune tolerance and in contributing to immune privileged status of the cns. the developing brain exhibits distinct morphological as well as physiological characteristics determining a peculiar response to injury showing an aggravated susceptibility to excitotoxicity and pro-inflammatory cytokines, along with an exacerbated inflammatory response. previous studies from our group have described the expression of cd -cd r in brain during development showing a distinct pattern of expression in the cortex and the hippocampus. hence, the aim of this study is phenotypic characterization of cd r microglia/macrophages and cd neuronal cells following hypoxia/ischemia (h/i) in neonatal mice brain. wild-type c /bl mice postnatal (p) day , , , , , , and adult were used for developmental studies. p mice was used for h/i injury that was administered using vannucci model modified for neonatal mice ( % o , min) and samples were collected h, h, h, h, h & days after hypoxia for immunofluorescence staining. results: phenotypic characterization of cd r microglia/macrophages showed them to express markers of pro-or anti-inflammatory phenotype in a temporal fashion post lesion. they expressed m phenotype as observed by colocalization with cd cells throughout the time points studied but a subpopulation of cd r cells exhibited m phenotype as exhibited by mhcii , cd cells from - hrs post lesion. calretinin (cr) used for the characterization of cd neurons showed that most cr neurons were cd especially the interneurons in the innermolecular layer of hippocampus, layer i of the neocortex and the hilus at most age groups studied. after h/i, cd immunolabeling was increased in the hippocampal fissure until days post-lesion. this followed a change in cd r microglial cells described above. to conclude, a balance between m /m phenotype of cd r microglia/macrophages and expression of cd by cr cells are involved in evolution of h/i induced brain injury in neonatal mice. objective: the cytokine interleukin- (il- ) and its naturally occurring receptor antagonist (il- ra) play a key role in determining neuronal cell death and survival in focal cerebral ischemia. the objective of this study was to determine the cellular production of il- /il- ra, and to test the neuroprotective potential of post-surgically injected il- ra overexpressing bone marrow (bm) cells in a mouse model of focal cerebral ischemia. materials and methods: c bl/ mice were injected i.v. with bm cells isolated from sil- ra overexpressing mice, min after permanent middle cerebral artery occlusion (pmcao). physiological parameters and behaviour were recorded post-surgically, infarct sizes were estimated, and microglial-leukocyte expression of il- /il- ra was analyzed by flowcytometry, in situ hybridization and immunohistochemistry. results: we identify microglia, and not recruited leukocytes as the major producers of il- ra after pmcao in mice, and we show by using il- ra knock out mice that microglial-produced il- ra is neuroprotective. we report that the sil- ra produced by the post-surgically injected bm cells, potentiates the neuroprotective effect of microglialderived il- ra, at both h and days, which is consistent with behavioural improvement at days, and detection of recruited bm cells in the ischemic area . h after pmcao. interestingly, we also find that the sil- ra overexpressing bm cells stimulate microglial production of il- ra h after pmcao, at which time both il- a and il- b is upregulated. conclusion: our results provide proof of principle that increasing the production of il- ra by recruited bm cells can counteract the effect of il- a/b, increase neuronal survival and improve motor function alone or through induction of microglial-produced il- ra after pmcao in mice. bogomoletz institute of physiology, kyiv, ukraine short-term oxygen-glucose deprivation (ogd) is known to activate excitatory glutamatergic synapses, induce long-term potentiation and result in neuronal and synaptical ultrastructural remodelling in organitypic cultured hippocampal slices (ochs). in this work we studied changes in glial synaptic coverage following min ogd episode using d reconstruction of ochs confocal and electron microscopic images. propidium iodide (pi) and mitotracker orange cmtmros (mto) were used to estimate cell viability and their mitochondrial potential. astroglial and microglial cells were identified using immunohistochemical staining with anti-gfap and anti-iba- antibodies. the study demonstrated that astroglial and microglial cells retain viability (there was no significant increase in the amount of pi-labbelled cells). d reconstruction revealed significant increase in excitatory synapses glial coverage as early as hour following ogd accompanied by a significant increase in number of astroglial and microglial processes. the deprivation also resulted in gradual increase of mitochondrial potential from hr to hr in both astroglial and microglial cells. it was previously shown that pyramidal neurons under the same conditions lose mitochondrial potential and became pi-positive after and reoxygenetion, indicating damage of cytoplasmic membrane, the effect prevented by application of nmda receptor antagonist d-ap . thus, unlike neurons astroglial and microglial cells are ogd-resistant and demonstrate marked mitochondrial activation -a process that might be involved in maintaining neuronal function during oxygen-glucose deficiency. using a microfluidic high throughput qpcr platform, we measured the expression of selected genes encoding astrocytic and polydendrocytic markers, ion channels, transporters and receptors that participate in maintaining k and glutamate homeostasis in each of individual gfap/egfp-positive glial cells. in this study we show how the expression of these selected genes changes during development (postnatal days , , and ) as well as , and days after middle cerebral artery occlusion (mcao). self-organizing maps and principal component analyses divided the cells according to their similarity in gene expression into three subpopulations of astrocytes present within the first - days of postnatal development (p -p ). the first subpopulation comprises immature glia mainly from p , characterized by the high transcriptional activity of all of the studied genes, including polydendrocytic markers. the second subpopulation is dominated by cells from p and displayed low transcript levels of all of the studied genes. the third subpopulation represents mature astrocytes mainly from p and p . three, seven and fourteen days after ischemia (d , d , d ), additional astrocytic subpopulations appear: resting astroglia (mainly from p and d ), transcriptionally active early reactive astroglia (mainly from d ) expressing the mrna of polydendrocytic markers, and, presumably, permanent reactive astroglia (solely from d ). following focal cerebral ischemia, reactive astrocytes undergo pronounced changes in their expression of aquaporins, nonspecific cationic and potassium channels, glutamate receptors and markers of reactive astrocytes and polydendrocytes. from the data obtained from single cell pcr analysis, we calculated the spearman correlation coefficients between pairs of genes, which revealed interesting positive gene expression correlations with polydendrocytic markers (gria - , grik - , grin a, kcnj , kcnk and kcnk genes). this study was further supplemented by an immunohistochemical analysis of nmda receptor subunits and pdgfar, which confirmed that changes in gene expressions correlate with immunodetected proteins. ga cr - s, astf - , gauk . reactive astrogliosis is often regarded as universal type of astroglial response to various forms of injuries. among its most characteristic features one may include: glial fibrillary acidic protein (gfap) up-regulation, cellular hypertrophy and proliferation and gliotic scar formation. an important element of glial cells response is their ability of de-differentiation, which is related with their re-gaining of immunological and molecular properties characteristic for earlier developmental forms. the origin of cells proliferating in response to various types of brain injury is still a subject of debate. among others it is related to the pathomechanism of lesion, affected brain structure and developmental stage of the nervous system. aim: in our study we aimed to assess the differences of de-differentiation and proliferation potential of astroglia localized in the cerebral cortex and striatum to the transient focal brain ischemia. material and methods: transient focal brain ischemia was evoked in adult male wistar rats by placement of the monofilament surgical thread into the internal carotid artery and occlusion of the middle cerebral artery for h. the postoperative survival period was up to weeks. immunocytochemical double-staining for astrocytic marker (gfap), developmental (pax ) and proliferative (ki- ) markers was performed and subsequent qualitative and quantitative study was done by means of confocal microscopy. results: double-labeled gfap/pax and gfap/ki reactive astroglial cells were revealed in the cerebral cortex and striatum of the ischemic region, starting from h after initiating the ischemia. the apparent difference in the intensity of morphological changes, quantity of de-differentiating and proliferating astroglial cells was observed between ischemic cerebral cortex and striatum. intensity of astroglial response and proliferative potential were much higher in the striatum than in the cerebral cortex. summary and conclusion: apparent difference in proliferative response between the cerebral cortex and striatum may be consequence of metabolic and molecular differences between astroglial cells populating two mentioned above structures. it may be also consequence of uneven decrease of cerebral blood flow and resulting different metabolic conditions influencing astroglial function in various fragments of ischemic area. reassuming, the differentiated potential of astroglial proliferative response to ischemic changes in different brain regions must be taken into account while analyzing clinical consequences of cerebral infarcts of different localization. triggering receptor expressed on myeloid cells- (trem ) is a microglial surface receptor involved in phagocytosis. clearance of apoptotic debris after stroke represents an important mechanism to re-attain tissue homeostasis and thereby ensure functional recovery. the role of trem following stroke is currently unclear. as an experimental stroke model, the middle cerebral artery of mice was occluded for minutes with a range of reperfusion times (duration of reperfusion: h/ h/ h/ d/ d/ d). quantitative pcr (qpcr) revealed a greatly increased transcription of trem after stroke ($ fold). we subsequently analyzed the expression of proinflammatory cytokines, chemokines and their receptors in trem knockout (trem -ko) mice via qpcr. microglial activation (cd , iba ) and cd -positive t-cell invasion were analyzed via qpcr and immunohistochemistry. functional consequences of trem knockout were assessed by infarct volumetry. the acute inflammatory response ( h reperfusion) was very similar between trem -ko mice and their littermate controls. however, in the sub-acute phase ( d reperfusion) following stroke, trem -ko mice showed a decreased transcription of pro-inflammatory cytokines tnfa, il- a and il- b, associated with a reduced microglial activity (cd , iba ). furthermore, trem -ko mice showed a reduced transcription of chemokines ccl (mcp ), ccl (mip a) and the chemokine receptor cx cr , followed by a diminished invasion of cd -positive t-cells. no effect on the lesion size was observed. conclusions although we initially expected an exaggerated pro-inflammatory response following ablation of trem , our data support a contradictory scenario that the sub-acute inflammatory reaction after stroke is attenuated in trem -ko mice. we therefore conclude that trem appears to sustain a distinct inflammatory response after stroke. metabotropic glutamate receptors (mglurs) are seven transmembrane domain g-protein-coupled receptors for l-glutamate, the main excitatory neurotransmitter in the mammalian brain. in addition, glutamatemediated excitotoxicity plays a central role in mediating neuron and oligodendrocyte death during ischemic episodes. in vitro, developing oligodendrocytes (ols) in particular have been shown to be highly vulnerable to excitotoxicity and oxidative stress. notably, activation of group mglurs has been shown to attenuate ol excitotoxicity in vitro and in brain slices has been shown to reduce ischemia-mediated impairment of astrocytes. here, we have examined the functional expression of mglurs and their role in acute ischemic injury in developing cns white matter of the mouse optic nerve. calcium imaging experiments were performed in optic nerves from p - wild-type mice. optic nerves were isolated intact, loaded with fluo- am and visualized using a zeiss lsm pascal confocal microscope; images were collected in optical z-sections and changes in fluorescence intensity were measured. the application of mglur agonists acpd and dhpg showed that group i mglur mediate a rise in glial [ca ] i . the action of the group i mglur antagonist aida versus acpd indicated that the rise in [ca ] i also involves group ii (and possibly group iii) mglur. immunohistochemistry confirmed glial expression of group i subunit mglur and group ii subunits mglur and mglur in the optic nerve, although further analysis of cellular localization is required. to examine the role of mglurs in white matter ischemia, optic nerves from p - mice in which fluorescent reporters are driven by astrocyte (gfap-egfp) or oligodendrocyte (sox -egfp) genes were isolated intact into artificial cerebrospinal fluid (acsf) and subjected to oxygen glucose deprivation (ogd) for hour. activation of group i or group ii mglur with the agonists acpd, dhpg or ly protected astrocytes and oligodendrocytes from ischemic cell death. further experiments are required to determine the mechanisms involved, but these findings demonstrate functional expression of mglur in optic nerve glia and indicate that activation of mglur is a possible therapeutic strategy to protect against glial damage in response to ischemia. the cerebral cortex is one of the most often affected areas after stroke, but there are no studies comparing the outcome in different cortical regions after focal ischemia. the aim of this investigation was to evaluate the patterns of glial activation, tissue degeneration and neuronal loss in different survival times following cortical ischemia. focal ischemia was induced by stereotaxic microinjections of endothelin- (et- ) into the somatosensory, motor and association cortices of adult rats (n ). the control animals were injected with the same volume of sterile saline (n ). the animals were perfused at , and days after ischemia. gross histopathology was evaluated using cresyl violet staining. lm sections were submitted to immunohistochemistry for astrocytes (anti-gfap), activated microglia/macrophages (anti-ed ) and microglia (anti-iba and ed ). tissue loss and glial activation were more intense in the somatosensory cortex than in other cortical areas. the motor cortex was the second more affected area. the association cortex was the less damaged area, which was confirmed by quantitative analysis (anova-tukey). the results suggest that an ischemic lesion of the same intensity induces a differential pattern of tissue loss and neuroinflammation, depending on the cortical area, and that the primary sensory and motor areas are more susceptible to ischemia than association areas. tlr is required for ipc-induced neuroprotection. in order to elucidate the mechanisms by which microglial tlr contributes to ipc, we carried out cell-targeted genomic analyses specifically on microglia exposed to either ischemic conditions in vitro or ipc in vivo. for our in vitro model, we exposed wt and tlr -/mouse primary microglia to hypoxic/hypoglycemic conditions for hours. for our in vivo model, we carried out transient middle cerebral artery occlusion (mcao) or sham surgery as our ipc pulse on wt and tlr -/adult male mice. three days later mice were sacrificed and microglia acutely isolated from ipsilateral cortex using magnetic affinity chromatography cell separation and ex vivo flow cytometry. microarray analysis was carried out on rna from both in vitro and in vivo microglia. results from both datasets identified robust expression of type and/or type interferon (ifn)-stimulated genes (isgs) as the predominant transcriptomal feature of ischemia-exposed microglia. in vitro, we found that out of the ( %) individual genes that were significantly up-regulated > fold by hypoxia/hypoglycemia in wt, but not tlr -/-, microglia were isgs including mx , oas , irf and ccl . promoter analysis demonstrated that the top four most active transcription factors were isgf , irf , irf and irf ; all ifn regulatory factors (irfs). a similar pattern emerged from the in vivo dataset with multiple irfs again among the most activated transcription factors in sorted microglia from ipcexposed mice. however, unlike the isg response in vitro, the isg response from the in vivo dataset was not tlr -dependent. the ifn family of cytokines is recognized as a key component of the innate immune response to infection and other types of injury. the role of microglial ifn-signaling in ipc is unknown. these results suggest that broad activation of isgs may be a key feature of the microglial response to ischemic conditions. astrocytes respond to central nervous system (cns) injury by the formation of a glial scar that develops within few days after insult and is characterized by astrocyte proliferation and cellular hypertrophy. reactive astrocytes change their immunohistochemical profile, such as increasing expression of gfap, nestin and vimentin; however, they also markedly alter the expression of ion channels, receptors and transporters, which participate in the maintenance of ionic-, water-and neurotransmitter homeostasis, such as k channels, aquaporins or glutamate transporters. here, we aimed to characterize the gene expression profile and functional properties of reactive astrocytes weeks after global cerebral ischemia (gci) or focal cerebral ischemia (fci) with a specific focus on k channels or non-specific cationic channels. gci was induced in adult rats by bilateral, -minute common carotid artery occlusion combined with low oxygen, while fci was induced in adult egfp/gfap mice by permanent middle cerebral artery occlusion. using the patch-clamp, we investigated the membrane properties of astrocytes in situ weeks after gci in the rat hippocampal ca region and weeks after fci in the mouse cortex. regardless of the type of ischemic injury, astrocytes from both cns regions depolarized their membrane potential by $ mv weeks after ischemia. when compared to astrocytes from the non-injured ca region or the cortex, the post-ischemic astrocytes displayed large hyperpolarizationactivated time-and voltage-dependent, non-inactivating inward currents, the current density of which increased -fold in response to gci or fci. in addition, these non-specific cationic channels were sensitive to zd and to a low extracellular na concentration, suggesting that they may belong to the family of hyperpolarization-activated cyclic nucleotide-gated (hcn) channels. we have taken advantage of fluorescently labeled astrocytes in egfp/gfap mice and isolated egfp cells by facs from non-injured as well as ischemic regions and performed gene expression profiling using single-cell rt-qpcr. our data revealed that cortical astrocytes after fci express, besides the increased expression of gfap, gfapd, aqp and , extremely high levels of hcn - transcripts that encode hcn - channels. moreover, our immunohistochemical analyses confirmed the presence of hcn - in reactive astrocytes, especially hcn , and channels. in summary, our results show that reactive astrocytes from post-ischemic tissue express hcn channels and that their activity might significantly contribute to na influx, possibly maintaining atpase function and/or contributing to intracellular na -dependent events, such as glutamate transporter or na /ca exchanger reversal. ga cr - s, p / /g and gauk . the optimal operation of electrogenic astrocytic transporters and exchangers for some well-defined astrocyte brain homeostatic functions depends on the presence of k channels in the cell membranes and the hyperpolarized membrane potential. our previous study showed that astrocytes functionally express two-pore domain k channel trek- , which helps to set the negative resting membrane potential. however, the roles of trek- on astrocytic function under normal and ischemic conditions remain unclear. in this study, we investigated the effects of trek- activity on astrocytic function and neuronal death under ischemic conditions. in an in vitro hypoxia model with astrocytes culture, trek- immunoreactivity was up-regulated after hypoxia. suppression of trek- activity inhibited the glutamate clearance capability, enhanced the inflammatory secretion of astrocytes derived s b and led to increased neuronal apoptosis after ischemic insult. after middle cerebral artery occlusion induced focal ischemia in rats, the trek- expression was significantly increased which correlated with reactive astrogliosis. application of lineonic acid, a trek- agonist which could potentiate the astrocytic conductance and hyperpolarization membrane potential, up-regulated the expression of astrocytic glutamate transporter glt- , inhibited the micro-glial inflammatory response and attenuated neuronal damage. our results suggest that trek- activity is involved in astrocytic function and neuronal survival. this would provide evidence showing astrocytic trek- involvement in ischemia pathology which may serve as a potential therapeutic target in stroke. oligodendrocytes are the myelinating cells of the central nervous system (cns). they are responsible for ensheathing myelin layers around axons, which contributes to improve conduction of neuronal impulses and additionally provides axonal support. oligodendrocytes are generated by oligodendrocyte precursor cells (opcs), a self-renewing and proliferating adult stem/precursor cell population in the cns. this process occurs not only during developmental myelination but also mediates cns remyelination, a regenerative process that restores myelin sheaths to demyelinated axons. although energy metabolism in the oligodendrocyte lineage is thought to be very active to support the lipid production specific to this cell type, it is still poorly characterized. as a first step towards a detailed characterization of the energy metabolism associated with specific lineage stages we investigated how glucose -the main cerebral energy substrate -is metabolized at an immature opc stage and in differentiated oligodendrocytes. primary cultures of opcs and oligodendrocytes were incubated with medium containing [ , - c]glucose for h and metabolites in both cell extracts and culture medium were collected and analysed for excess % c enrichment using gas-chromatography-mass spectrometry (gc-ms) as well as for total amounts using hplc. the significant c-enrichment in citrate, malate, and aspartate in cell extracts indicate that oligodendrocytes oxidize glucose-derived metabolites extensively in the tricarboxylic acid (tca) cycle. moreover, they release c-labelled aspartate and glutamate to the extracellular medium, although a significant consumption of aspartate, glutamate and glutamine from the medium was also observed. enrichment of extracellular lactate and increased alanine synthesis in oligodendrocytes as compared to opcs suggests that the rate of aerobic glycolysis is increased in immature as compared to differentiated cells. in conclusion, these data confirm that both opcs and oligodendrocytes are highly metabolically active and, in addition to extensively oxidizing glucose, synthesize and release distinct metabolites. we anticipate that further studies on this subject will contribute to a better understanding of the physiological role of oligodendrocytes in the cns and the role of glycolysis and mitochondrial metabolism during their maturation. schwann cells in the peripheral and oligodendrocytes in the central nervous system require a tremendous amount of lipids to be capable of producing all cell membranes forming the myelin structure. furthermore, when compared to other cells, myelin membrane presents an extremely high lipid content ($ % of dry weight) coupled to an unique lipid composition. the importance of myelin lipid composition towards myelin formation and maintenance is highlighted by the frequent appearance of myelin defects in lipid-metabolism human disorders. one of the main represented class of lipids in myelin are phospholipids, of which fatty acids (fas) represents the building blocks. most cells preferentially uptake circulating free fas. however, cells under increased membrane production, e.g. highly proliferating cells, can endogenously produce them through the enzyme fatty acid synthase (fasn), which synthesize palmitate. in these cells, fasn is transcriptionally augmented via the mtorc pathway. thus, we hypothesized that due to their increased lipid demand myelinating cells would require fasn activity towards myelin production. the functional role of fasn in the metabolic control of myelin membrane lipid composition and myelination is unidentified. thus, we addressed it experimentally by conditionally deleting fasn in myelinating cells in vivo. we show that endogenous fas biosynthesis in myelinating cells is essential to maintain myelin membrane lipid homeostasis. furthermore, maintaining a correct lipid homeostasis is critical for myelination. thus, we analyzed whether fasn is required to allow palmitoylation of myelin proteins and their targeting to the myelin membrane. in addition, we show that interfering with myelin fas homeostasis results in the activation of systemic compensating metabolic loops promoting fas uptake, although not sufficient to preserve efficient myelination. taken together our data demonstrate that in myelinating cells fas homeostasis is under the synergistic control of endogenous metabolic fas production via fasn and systemic functional regulation of free fas uptake. most importantly, maintaining a correct homeostasis of fa-derived lipids in myelin is critical for myelination. homeostasis and signaling, respectively ( ). in mammals, two cytosolic and two mainly mitochondrial located glutaredoxins have been identified ( ). we already described an essential role for the formation of a functional neuronal network during embryonic development of one of these proteins, the vertebrate specific glutaredoxin ( ). here, we will focus on the contribution of glutaredoxin on basic cellular functions of oligodendrocytes, the myelinating cells of the central nervous system, under physiological and pathophysiological situations, e.g. multiple sclerosis, which is characterized by inflammatory axonal demyelination ( ). oligodendrocyte progenitors and mature oligodendrocytes are very sensitive against oxidative stress induced by release of nitric oxide by activated microglia ( ) . using a b positive glia restricted progenitor cells as well as organotypic cerebellar slice cultures, we found that increased levels of glutaredoxin protect against cell death induced by either activated microglia or s-nitrosoglutathione, a physiological nitric oxide donor. under physiological conditions elevated levels of glutaredoxin increased migration of a b positive cells nearly three fold. western blot analyses, quantitative real time pcr, as well as immunocytochemistry revealed that glutaredoxin inhibited further differentiation of ng positive oligodendrocyte progenitor cells. in summary, our data indicate the importance of specific thiol redox signaling during cellular protection and function in this type of glia cells. current multiple sclerosis therapeutics act as immunomodulators and/ or immunosuppressors. this strategy is largely ineffective at preventing progression of the disease. significant benefits to multiple sclerosis patients might be seen with a therapeutic agent which induces remyelination, however, to date no such agents have been developed. using in vitro and in vivo models we have identified nefiracetam as a potential remyelinating therapeutic. to study remyelination in vitro, hippocampal organotypic cultures were exposed to lysophospatidylcholine (lpc) for hrs to induce demyelination before being allowed to recover. cultures were treated with nefiracetam for hrs of this recovery period. in all cases immunofluorescent labelling of myelin basic protein (mbp) was used as a measure of myelin. nefiracetam was shown to increase mbp expression, relative to untreated controls, accelerating recovery from lpc. two different models were used to investigate the efficacy of nefiracetam in vivo; an inflammatory model, experimental autoimmune encephalomyelitis (eae), and a toxin induced model of demyelination, achieved through the use of cuprizone chow. nefiracetam treatment did not alleviate the symptoms of eae. however, nefiracetam did restore the amount of myelin in cuprizone exposed animals, compared to saline-treated controls. in combination, these observations suggest that nefiracetam is capable of accelerating remyelination through a mechanism of action which is independent of immunomodulation. we conclude that nefiracetam encourages remyelination both in vitro and in vivo. these pro-myelin properties identify nefiracetam as a potential therapeutic for demyelinating disorders. university of naples "federico", neuroscience, naples, italy changes in intracellular [ca ] i levels have been shown to influence the developmental processes that accompany the transition of human oligodendrocyte precursor cells (opcs) into mature myelinating oligodendrocytes and are required for the initiation of myelination and remyelination processes. in the present study, we explored whether calcium signals mediated by the selective sodium calcium exchanger (ncx) family members ncx , ncx , and ncx , play a role in oligodendrocyte maturation. functional studies, as well as mrna and protein expression analyses, revealed that ncx and ncx , but not ncx , were divergently modulated during opc differentiation into oligodendrocyte phenotype. in fact, while ncx was down-regulated, ncx was strongly up-regulated during the oligodendrocyte development. the importance of calcium signaling mediated by ncx during oligodendrocyte maturation was supported by several findings. indeed, whereas the knocking down of the ncx isoform in opcs prevented the up-regulation of the myelin protein markers cnpase and mbp, its overexpression induced an up-regulation of cnpase and mbp. furthermore, ncx knock-out mice exhibited not only a reduced size of spinal cord but also a marked hypomyelination, as revealed by the decrease in mbp expression and by the accompanying increase in opcs number. collectively, our findings indicate that calcium signaling mediated by ncx plays a crucial role in oligodendrocyte maturation and myelin formation. to explore the role of kif b in myelination in vivo, we generated conditional knock-out mice with kif b specific ablation in either schwann cells or oligodendrocytes. we found that kif bfl/fl p cre mouse nerves are hypomyelinated with reduced myelin thickness, thus confirming in vitro data. in the pns, axonal neuregulin (nrg) type iii is one of the main signaling promoting myelination and remyelination after injury. nrg type iii binds to erbb /erbb receptors in schwann cells, which signal through a plethora of downstream signaling pathways also including the pi k/akt complex. the pi k/akt signaling pathway is attenuated by the pten phosphatase, which dephosphorylates ptdins( , , )p (s. goebbels et al., ). dlg is believed to interact with pten and potentiate its ability to dephosphorylate ptdins( , , )p , thus negatively modulating the akt-mtor pathway (l. cotter et al, ). as kif b interacts with dlg in schwann cells, we investigated the pi k/akt pathway in kif b-null nerves and surprisingly, we found that dlg expression levels are increased whereas akt phosphorylation is decreased in mutant nerves. our findings suggest that kif b negatively regulates dlg activity and acts as a promoter of myelination in the pns. on the contrary, when kif b is lost specifically in oligodendrocytes, myelin sheaths are thicker. at the molecular level, the hypermyelination is consistent with increased akt phosphorylation. we are currently investigating the molecular mechanisms at the basis of the kif b/dlg interaction in both schwann cells and oligodendrocytes. we have identified the nootropic nefiracetam as a potential novel remyelinating therapeutic using in vivo and in vitro models of demyelination. in this study we investigate the mechanism of action of nefiracetam in the context of remyelination as regulated by wnt/b-catenin signalling and spontaneous activity. to investigate whether nefiracetam acts by modulating wnt signalling, organotypic hippocampal cultures were exposed to nefiracetam or the wnt/b-catenin inhibitor cardamonin. both increased immunofluorescent staining for the opc marker ng . myelin formation, as assessed by mbp staining, was enhanced by nefiracetam but was not affected by cardamonin. thus, modifying wnt signalling appears to impact opc development without effecting differentiation, suggesting the pro-myelinating effects of nefiracetam involve an additional mechanism. we next investigated the effect of nefircatam on opc differentiation using purified opc cultures. nefiracetam increased the ol:opc ratio, as assessed by immunofluorescent staining for mbp and ng respectively, suggesting nefiracetam is capable of promoting ol maturation through a direct action on opcs. finally, we investigated the effect of nefiracetam on spontaneous activity. organotypic hippocampal cultures were exposed to lysophospatidylcholine (lpc) for hrs to induce demyelination before being allowed to recover. cultures were treated with nefiracetam for hrs of the recovery period. spontaneous calcium activity was then measured using fluo -am. cultures not exposed to lpc displayed low levels of activity which were unaffected by nefiracetam. lpc exposure caused an increase in activity which was enhanced by nefiracetam. additionally, in these cultures, lpc decreased mbp staining compared to control while nefiracetam post-lpc caused a return to control levels. these data suggest increased activity may be an adaptive response related to myelin repair which is enhanced by nefiracetam. together, these findings suggest a complex mechanism of action for nefiracetam as a remyelinating agent involving modulation of wnt signalling, spontaneous activity and opc differentiation. in an assay in which cortical oligodendrocytes ensheath dorsal root ganglion cells we found that nrg and nmda receptors interact to regulate myelination. without nrg, blocking nmda receptors had no effect on myelination. adding nrg increased myelination at all timepoints analysed ( - weeks), suggesting that nrg did not merely accelerate myelination. strikingly, nrg led to the majority of myelination becoming dependent on activation of nmda receptors and action potential activity. nrg's effect was associated with a -fold increase in nmda receptor current in oligodendrocyte lineage cells. thus, nrg switches myelination from a default programme, which is independent of neuronal activity, to a mechanism that is regulated by glutamate released from active axons. the effects of nrg were associated with an increase in the phosphorylation of akt and the transcription factor creb, but not associated with changes in the phosphorylation of erk. these data reveal a function for oligodendrocyte nmda receptors. the absence of nrg in multiple sclerosis lesions, and enhanced remyelination by added nrg, suggest a role for neuregulin/nmda receptor dependent remyelination after pathology. low-density lipoprotein receptor-related proteins (lrps) are endocytic receptors that internalize several ligands and are involved in lipoprotein homeostasis in adult brain. lrp- , also known as megalin, is involved in the development of the central nervous system (cns) and in the transport of molecules across the blood brain barrier (bbb). in a previous work, our group revealed that megalin is selectively involved in sonic hedgehog-mediated effects on oligodendrocyte precursor cell migration and proliferation during the development of the cns. although there are some reports describing the presence of lrps in acute lesions of multiple sclerosis (ms) patients and pointing to an important role of shh in the pathogenesis of this disease, there are not data about the expression of megalin in ms tissue. in the present study, we analyse the distribution and the cellular characterisation of megalin in samples of human brain from ms patients to elucidate the role of this receptor in the pathogenesis of demyelination. changes in megalin distribution parallel the different ms histopathological lesions: we detected megalin in active demyelinating lesions and in the periplaque of chronic-active lesions, areas where remyelination spontaneously occurs. on the contrary, megalin was absent in the demyelinated region of chronic lesions. in addition, we observed the up-regulation of megalin in a subpopulation of perivascular astrocytes within the normal appearing grey matter (nagm) of ms patients. moreover, megalin was up-regulated in blood vessels within active lesions, in the periplaque of chronic-active lesions and in the nagm. in parallel, we also analysed the bbb profile to determine structural and functional alterations. altogether, we suggest that megalin is an important receptor expressed in diverse areas of the cns of ms patients representing different aspects of ms development: i) a potential target to improve remyelination; ii) an indicator of a functional rather than a structural disruption of the bbb. therapeutic strategies to modulate specific lrps would be useful to effectively treat ms. bdnf is known to promote central nervous system myelination both in vitro and in vivo. adopting in vitro myelination assays, we identified that bdnf acts through oligodendroglial-expressed trkb receptors to promote myelination. this was verified in vivo, as deletion of trkb in mature oligodendrocytes (in trkb fl/fl mbp-cre mice) resulted in a hypomyelinating phenotype during development. question: here we investigate the consequence of trkb deletion in oligodendrocyte progenitor cells (in trkb fl/fl cnpase-cre mice) and the signalling pathways downstream of trkb that promote myelination. methods and results: trkb fl/fl cnpase-cre mice were born in mendelian ratios and were phenotypically indistinguishable from littermate control mice. surprisingly, analysis of myelinated axonal tracts in the spinal cord and optic nerve revealed normal myelin development. in addition, the number of cells in the oligodendroglial lineage was the same as control mice during postnatal development. these data indicate that the timing of trkb deletion in the oligodendroglial lineage is critical, as deletion in opcs results in no significant phenotype, whereas deletion in mature oligodendrocytes results in a hypomyelinating phenotype. these data suggest that opcs are able to compensate for the loss of trkb and myelinate normally in the absence of bdnf signalling. our in vitro data have shown that the promyelinating influence of bdnf strongly correlates with erk / activation. here we show that overexpression of erk in opcs exerts a more significant promyelinating effect than erk . as erk / are known to exert some of their effects through direct transcription factor phosphorylation, we have undertaken an in silico analysis of myelin-specific transcription factors and identified several that contain potential erk / binding domains and phosphorylation sites. co-immunoprecipitation experiments show an interaction between erk / and these transcription factors. investigation into the nature of these interactions and their functional consequences are ongoing. conclusions: this work suggests a novel role for erk / signalling within oligodendrocytes that regulates cns myelination, possibly via activation of myelin-specific transcription factors. as erk / is activated by multiple receptors, other receptors could potentially compensate for the loss of trkb in opcs, resulting in a normally myelinated cns in vivo. myelin is vulnerable to impairment by genetic, toxic, and immunological triggers, but the molecular basis for many aspects of myelin pathology has remained unknown. to identify molecules required for the structural integrity of central nervous system myelin we have systematically analyzed its protein composition by quantitative proteome analysis. based on the high abundance of filament-forming septins in myelin, we hypothesized that septin filaments may constitute a cytoskeletal membrane cortex in myelin. we find that septin filaments localize to the non-compact adaxonal myelin compartment, which is considered relevant for the metabolic maintenance and the turnover of myelin. for functional analysis, we generated mice lacking the most abundant septin of myelin, sept , from myelinating glia. our results indicate that sept , sept , sept and sept multimerize with sept and that this interaction is essential for their incorporation into myelin. myelin lacking septin filaments display pathogenic outfoldings emerging from adaxonal myelin. we propose that this phenotype reflects that myelin septins provide long-term structural integrity to the adaxonal compartment of cns myelin. here, we used primary opc cultures to investigate the mechanisms underlying gpr endogenous regulation. this is quite important since gpr is upregulated at injury sites in both a demyelinating in vivo model (boda et al., glia, ) and in multiple sclerosis (ms) patients (chen et al., nat neurosci ), which, could, in turn, cause defective myelination. in line with these findings, we have recently shown that gpr forced over-expression at late differentiation stages, obtained by transfecting cultured opcs with a gfp-gpr fusion vector, indeed impairs terminal cell maturation. this suggests that the receptor needs to be down-regulated/ desensitized to allow opc terminal maturation. physiologically, gpr downregulation may occur through agonist-induced receptor phosphorylation via g-protein coupled receptor kinases (grks) (daniele et al., j pharm exp ther, ; frantangeli et al., j biol chem, ), which are, in turn, controlled by mtor kinases, and are altered in ms. our in vitro data show that rapamycin, an inhibitor of mtor kinase, reduces grk levels, with parallel increases in gpr expression and strong impairment of opc maturation. globally, these data suggest that dysregulation of these interconnected pathways leading to aberrant gpr overexpression may prematurely block opc maturation. analysis of gpr in vivo in ms models will confirm if the receptor is dysregulated during disease development and will help finding ways to re-establish myelin repair in demyelinating diseases. loss of central nervous system myelin, and the failure of remyelination by oligodendrocytes, contributes to the functional impairment that characterizes neurodegenerative disorders and demyelinating diseases such as multiple sclerosis (ms). since incomplete remyelination will irreversibly damage axonal connections, treatments effectively promoting (re)myelination are pivotal in precluding progression of disease. although the reasons for remyelination failure are likely multifactorial, one of the major impediments to successful remyelination is the altered lesion microenvironment. our previous findings suggest that fibronectin aggregates, as an environmental factor, contribute to remyelination failure. here, we aim at elucidating whether exogenous gangliosides, i.e., cell surface lipids with a potential to modulate fibronectin-cell surface interactions, could overcome fibronectin-mediated inhibition of myelination. of the gangliosides tested, only addition of gd a, perturbed adherence of oligodendrocyte progenitor cells to fibronectin, but not to poly-l-lysine, without affecting cell viability. gd a slightly increased growth factor-mediated proliferation on fibronectin, but in the presence of the lipid migration of oligodendrocyte progenitor cells on fibronectin was reduced. furthermore, addition of gd a increased myelin-like membrane formation on (aggregated) fibronectin, whereas gm reduced oligodendrocyte differentiation, as reflected by a decrease in the number of mbp positive cells. most interestingly, gd a counteracted the myelination inhibiting effect of aggregated fibronectin in co-cultures of dorsal root ganglion neurons and oligodendrocytes. also, gd a enhanced remyelination in demyelinated cerebellar slice cultures that contained fibronectin aggregates. taken together, gd a is able to overcome the inhibiting effect of aggregated fibronectin in remyelination. given the persistent presence of these aggregates in ms lesions, gd a might therefore act as a potentially novel molecular tool to selectively modulate the impeding signaling environment, apparently detrimental to remyelination. its mechanism of action and exploration of gd a's potential in the treatment of ms, are currently investigated. in the peripheral nervous system (pns) it is secreted by schwann cells and fibroblasts and its expression is strongly induced upon injury. we are interested in the function of apod in the molecular events that take place after a pns injury. we have studied in vivo the process of wallerian degeneration following sciatic nerve crush and we have assayed ex vivo the phagocytosis of labelled myelin from wt and apod-ko mice by flow cytometry. the analysis of cellular processes and protein or mrna expression of a group of signalling molecules induced by injury shows that the lack of apod results in an exacerbated mcp- and tnfa-dependent macrophage recruitment. at the injury site, free aa decreases in the wt. lack of apod results in higher basal levels and a stronger injurytriggered depletion of aa. control by apod of the availability of aa to produce the lipid mediators involved in macrophage recruitment is therefore a key mechanism that conditions both wallerian degeneration and injury resolution later on. on the other hand, the phagocytosis of apod-ko cns myelin is less efficient than the phagocytosis of wt myelin. these results indicate that there are also genotype-dependent differences in myelin composition and/or in the interaction between myelin and macrophages. an electron microscopy analysis of crude myelin preparations reveals that the cns myelin from apod-ko mice has abnormal periodicity and shows defective myelin compaction. lipid analysis of apod-ko myelin shows altered phospholipid composition, particularly in phosphoinositid species. a study of the function of apod in the myelination process is currently underway, our results demonstrate that apod function is relevant for both, myelin membrane properties that influence myelin-macrophage interactions, and the control of the lipid-mediated signalling events controlling the extent of macrophage recruitment. adhesion g protein-coupled receptors (ad-gpcrs) are a subgroup of gpcrs that facilitate cell-cell and cell-matrix interaction. structurally, they differ from other gpcrs by the presence of an extremely larger extracellular n-terminal region that is separated from the -transmembrane region by a gpcr autoproteolysis-inducing (gain) domain that in most ad-gpcrs facilitates an auto-catalytic process to produce an nand c-terminal fragments during the maturation process. gpr is a member of the ad-gpcr family and its mutations are responsible for a human brain malformation known as bilateral frontoparietal polymicrogyria (bfpp). individuals with bfpp present with changes in the white matter tracts in the form of hypomyelination, in addition to cortical malformation. here, we show that gpr knockout mice and zebrafish exhibit a reduction of myelin-specific protein expression, fewer myelinated axons and decreased number of mature oligodendrocytes in the central nervous system (cns). during cortical development, gpr functions together with its ligand collagen iii to regulate neuronal migration and cortical lamination. however, it is not clear whether collagen iii is also the ligand of gpr during cns myelination. gpr has a very long and poorly characterized n-terminal fragment, allowing for the possibility of multiple binding partners that mediate cell-extracellular matrix interactions. indeed, gpr also binds to tissue transglutaminase (tg ) in melanoma cells. we have begun to study the possible role of collagen iii and tg in oligodendrocyte development and cns myelination. the likelihood of these two proteins as being the ligand(s) of gpr during oligodendroglial development will be discussed as will our ongoing work to define the role of gpr in cns myelination. proper control of cell cycle, proliferation and differentiation is fundamental to regulate oligodendrocyte (ol) development and recruitment of oligodendrocyte precursors (opc) after brain damage. failure in this process results in severe dysmyelinating disorders and defective brain repair. the molecular machinery that couples differentiation to proliferation withdrawal plays therefore a critical role in brain development and repair and is regulated by multiple extracellular cues including extracellular matrix (ecm) and growth factor derived signals. however, how these signals are integrated into ol to control cell cycle progression and differentiation is only partially understood. we are investigating the role of jun activating binding protein (jab ), an intracellular signalling molecule, shuttling between nucleus and cytoplasm, involved in the control of cell proliferation, survival and gene transcription. recent evidence showed that jab function is regulated by ecm and growth factors. we demonstrated that jab is expressed in glial cells in peripheral and central nervous system (cns), and conditional inactivation of jab in schwann cells results in dysmyelinating neuropathy. here we present preliminary data showing that jab plays a role in cns development. mice with conditional inactivation of jab in ol, by cnp-cre tansgene, show cns hypomyelination, including corpus callosum, optic nerve and spinal cord. concomitant fiber degeneration was observed. our preliminary data suggest that jab expression in ol plays a role in cns myelination and possibility axonal survival. c. gonsior, j. trotter university of mainz, mainz, germany myelin basic protein (mbp), a major component of central nervous system (cns) myelin, is essential for oligodendroglial function and myelination in the cns and its expression is tightly regulated in time and space. the mrna of mbp is sorted to cytoplasmic rna granules and transported up to the distal processes of oligodendrocytes in a translationally silenced state. we and others could show that, dependent on axonal signals, activation of the non-receptor tyrosine kinase fyn leads to a release of translational inhibition allowing synthesis of mbp protein at the axon-glial contact site. a key factor in this pathway is the fyn target and rna-binding protein heterogeneous nuclear ribonucleoprotein (hnrnp) a , that recognizes the a response element (a re) in the mbp utr and orchestrates the dynamics of the mbp mrna granule. moreover, we identified hnrnp f as an additional target of fyn kinase present in these ribonucleoprotein complexes and contributing to posttranscriptional regulation of mbp. cell stress conditions, as present in various disease states such as multiple sclerosis, may result in the formation of cytoplasmic rna-containing stress granules (sgs), potentially influencing the fate of myelin transcripts including their translation.mbp mrna was shown to be sorted to these cytoplasmic foci. we here identified the hnrnps a and f to be associated with oligodendroglial sgs. de-regulation of such factors involved in mbp mrna metabolism could affect stress dependent synthesis of mbp and thus (re-)myelination and myelin maintenance. we performed the experiments in biozzi abh mice known to develop a chronic relapsing-remitting form of eae following immunization with myelin oligodendrocyte glycoprotein. our findings suggest that in demyelinating lesions, myelin layers are pulled off from the myelin sheath and form bulb-like structures that we call "myelinosomes". we can detect the formation of such structures in acute and in chronic stages in the animal model, as well in biopsies from multiple sclerosis patients. we are currently investigating the molecular interactions that underlie the formation of myelinosomes to gather further insight into the mechanisms that underlie immue-mediated demyelination. adenosine is a potent neuromodulator which can be released from most cells and its extracellular concentration increases dramatically after brain injury. it acts on four g protein-coupled receptors, a , a a , a b and a , and their activation is involved in myelination as well as in apoptosis linked to neurodegenerative diseases. in this study, we initially found that rat oligodendrocytes in vitro express all four adenosine receptor subtypes, adenosine transporters ent and ent and the adenosine degrading enzymes adenosine deaminase and adenosine kinase. activation of adenosine receptors caused mitochondrial dysfunction and oligodendroglial apoptosis. notably, selective activation of adenosine receptors revealed that a receptors are the main subtype mediating oligodendrocyte death. thus, a receptor agonist -ci-ib-meca caused a robust increase in ros levels, mitochondrial membrane depolarization and caspase-dependent apoptosis. in turn, selective a receptor activation induced mapk modifications compatible with cellular damage, which was prevented by the selective antagonist mrs . the modulation of g protein and adenylate cyclase suggested that -ci-ib-meca acts via camp-pka. moreover, selective activation of a receptor triggered intracellular calcium mobilization via plc pathway. finally, we used cerebellar organotypic slices and optic nerve ex vivo to investigate adenosinemediated oligodendroglial death in more integral preparations. consistent with data in dissociated cultures, a receptor activation induced a significant damage in the optic nerve as well as in the cerebellum white matter, an effect that was prevented by caffeine, an adenosine receptor blocker. together, these results indicate that adenosine can trigger oligodendrocyte death via activation of a receptors, and suggest that this mechanism may contribute to the etiology of demyelinating diseases. question-one of the commonest forms of inherited neuropathy, xlinked charcot marie-tooth disease (cmt x) leads to progressive distal muscle weakness and sensory loss. the disease is caused by a large number of mutations in the gjb gene encoding the gap junction protein connexin (cx ). cx is expressed by schwann cells in peripheral nerves and forms important channels of communication between the layers of the non-compact myelin sheath and is necessary for the homeostasis and survival of the myelinating cells and the axon. transgenic disease models generated in our lab as well as clinical-genetic analysis of large number of patients have demonstrated that most cmt x mutations result in loss of cx function and failure to form gap junctions. cx ko mice offer a well characterized and relevant model of the disease to study future therapies for cmt x, for which gene replacement may be an effective treatment strategy. methods-for this purpose, we have generated a novel rd generation lentiviral vector to allow gene delivery to peripheral nerves. in this vector, the cx open reading frame is placed under the control of cell-specific promoter for schwann cells, the rat mpz/p promoter. the expression cassette also includes a green fluorescent protein (egfp) as a marker. we have successfully produced lentiviral vector particles and delivered them to the sciatic nerves of -month old wild type (wt) and cx ko mice, immediately distal to the sciatic notch. the expression of egfp on wt background and of cx on ko background was determined by western blot analysis and immunohistochemistry of teased nerve fibers at different time points after injection. results-our initial studies in wt mice (n ) show that more than % of schwann cells from different nerve fascicles expressed egfp as far as mm distal to the injection site. expression started a week after the intraneural injection and remained stable for up to months. when the lentiviral construct was injected into the sciatic nerves of cx ko mice (n ), virally delivered cx was expressed and was properly localized at the non-compact myelin areas including the paranodal regions and incisures of myelinated fibers, where gap junctions are normally formed. poster abstracts s glia conclusions-thus, we have generated lentiviral vector that leads to efficient gene expression specifically in schwann cells. these results indicate that gene replacement therapy is feasible and may lead to correction of the gap junction loss in myelinating cells of the pns. perinatal inflammation is intensively discussed to have a long-term impact on various cell populations and on the development of autoimmune diseases such as multiple sclerosis (ms) in adulthood. in order to determine long-term effects of perinatal inflammation on the course of demyelination in adulthood we mimic a bacterial inflammation by exposure to lipopolysaccharide (lps) of either pregnant or newborn mice. demyelination as a "second hit" was induced by feeding adult mice with the oligodendrocyte toxin cuprizone (bis-cyclohexanone oxaldihydrazone). a single prenatal lps injection at e . did not affect the course of demyelination in adulthood. in contrast, serial postnatal lps injections lead to a delayed demyelination and a reduced number of activated microglia in the corpus callosum, suggesting a long-lasting effect of perinatal inflammation on the function of microglia. surprisingly, mice exposed to lps after birth showed an enhancement of early remyelination accompanied by an increased number of newly differentiated oligodendrocytes. furthermore, independent of cuprizone administration the perinatal lps injections seem to induce a long-term impact on the blood-brain barrier (bbb) by decreasing the number of claudin- positive vessels. in summary, perinatal inflammation mimicked by the administration of lps has long-lasting effects on the bbb, microglia activation, and remyelination. question: epigenetic control is crucial for the differentiation of a variety of cells including oligodendrocytes, the myelinating glial cells of the central nervous system. however, studies about the implication of epigenetic factors in peripheral nervous system maturation are just emerging and we were wondering whether methylation of histones is involved in this process. methods: we describe the function of ezh in primary schwann cells and drg cocultures using immunohistochemistry, transfection and lentiviral transduction, shrna, qrt-pcr analysis, morphological analysis, western blotting and chromatin immunoprecipitation. results: here, we demonstrate for the first time the impact of a histone methyltransferase, encoded by the enhancer of zeste homolog (ezh ) gene, on schwann cell differentiation. in sciatic nerves, ezh expression was found in schwann cells and to peak perinatally. suppression of ezh expression in cultured primary rat schwann cells reduced the length of cell processes. these morphological changes were accompanied by widespread alterations in the gene expression pattern, including downregulation of myelin genes and induction of p kip , which we have recently identified as an intrinsic inhibitory regulator of schwann cell maturation. in addition, we show that ezh suppression in dorsal root ganglion cocultures interferes with in vitro myelination. chromatin immunoprecipitation analysis revealed binding of ezh at the p kip promoter and reduction of histone h k trimethylation upon gene suppression. ezh suppression-dependent effects on morphology and myelin genes could be reversed by concomitant suppression of p kip , indicating that p kip is a downstream effector of ezh . furthermore, we describe hes as transcriptional repressor of myelin genes in schwann cells, which was induced upon ezh suppression and downregulated in p kip -suppressed schwann cells. conclusions: we have identified a molecular link between histone methylation and control of schwann cell differentiation and demonstrate that this epigenetic mechanism is crucial for glial differentiation to proceed. previous studies, performed primarily in cell culture, suggest that electrical activity may regulate multiple stages of oligodendrocyte development including proliferation, migration, initial wrapping and myelination. in this study, we have examined the requirement for electrical activity in oligodendrocyte development in vivo using zebrafish as a model system. results: we find that electrical activity is not required for proliferation, migration, or the initiation of axon wrapping. rather, our data indicate a specific role for electrical activity in oligodendrocyte differentiation by regulating a subset of myelin genes, including myelin basic protein (mbp). silencing electrical activity with tetrodotoxin reduced mrna expression of mbp, as assessed by rna in situ hybridization and quantitative rt-pcr. in contrast, myelin protein zero, claudin k, claudin a, ugt , and k levels were not regulated by electrical activity. ongoing experiments are aimed to uncover the mechanism of activity-dependent mbp mrna expression during oligodendrocyte differentiation. conclusions: taken together, these data indicate that electrical activity can modulate myelin gene expression in vivo but do not support the notion that electrical activity drives key axon-glia messengers that are required for the initial stages of myelination. (fancy et al., ) . eight-week-old lgals -/-and wild type (wt) mice were fed a diet containing . % cpz w/w during weeks, after which cpz was withdrawn in order to evaluate remyelination weeks after. according to our results, cpz-induced demyelination in lgals -/mice showed an exacerbated astrocytic and microglial response as compared to wt littermates. electron microscopy showed a significant lack of myelinated axons in lgals -/-mice as compared to controls. furthermore, the few myelinated axons present were nearly % less myelinated than those of controls and were found to be collapsed. remarkably, the remyelination process seemed to be faster in lgals -/-mice than in wt. remyelinated lgals -/-mice showed a higher myelin basic protein (mbp) recovery rate as compared to their controls. flow cytometry assays showed a sharper microglial response in lgals -/-mice, which was supported by an exacerbated number of cd b and cd cells. however, electron microscopy images from remyelinated lgals -/-animals showed, again, collapsed axons with a defective myelin wrap, as compared to wt mice showing normal axons without any relevant myelin wrap disruption. behavioral performance observed during cpz treatment recovery correlates with alterations in the morphological studies, which show that neither lgals -/-nor wt mice reach basal myelination levels. we have shown that new oligodendrocytes (ols) continue to be generated in healthy adult mice after months of age, even in the optic nerves, which are considered to be completely myelinated already [ ] . this raises the question of whether the adult-born ols and myelin are engaged in remodeling existing myelin (e.g. intercalating among the existing myelin sheaths), or replacing ols that die in use. to ask whether ols die and are replaced during healthy adulthood we generated transgenic mice that express icreer t under the transcriptional control of the myelin basic protein promoter (mbp -icreer t ). the $ kb mbp upstream fragment that we used is reported to direct transcription to ols but not to either ol precursors (ops) or schwann cells [ ] . we confirmed that our mbp transgene targets cre recombination specifically to differentiated ols by crossing to ros -yfp conditional reporters; when tamoxifen was administered to double-transgenic offspring, essentially all yfp cells in the cns were also cc . when mbp -icreer t is crossed to tau-mgfp conditional reporter mice (mgfp is membrane-tethered) we can visualize differentiated ols and their myelin internodes in white and grey matter, allowing us to follow the fates of myelinating ols for extended periods of time after tamoxifen labeling. following tamoxifen administration to mbp -icre t : tau-mgfp mice on postnatal day (p ) or p , a large proportion of ols became gfp-labelled in the corpus callosum, optic nerve and spinal cord. examining these mice at increasing times post-tamoxifen will reveal if and how the number and fraction of mgfp-labeled ols/ internodes changes with age -whether there is net loss or gain of ols or whether new ol synthesis is balanced by the rate of ol death (i.e. there is ol turnover). our preliminary results indicate that new ol synthesis exceeds ol death, so the function of adult ol genesis is not simply to replace dying ols. this study aims to identify protein networks that participate in peripheral myelin formation and maintenance. we use a cell culture system, where c bl/ j mouse embryos (e . ) are used for the isolation of dorsal root ganglia neuron explants. explants are cultured in matrigel, and endogenous schwann cells appear in the culture within a few days. myelin formation is induced by adding myelination-promoting substances to the culture medium. immunocytochemistry is used to evaluate the myelination efficiency and the developmental timetable of myelination in culture. electron microscopy is used to study structural features of myelin in culture and to compare its ultrastructural features with those of myelin in situ. rna microarray analysis is performed to study gene expression changes during myelin development in culture. the rna expression data indicates the changes in the rna expression levels, but it does not give information about the amount of final protein product, their posttranslational modifications or stability. we combine the rna expression analysis with a proteomics approach. proteins are extracted for -dimensional electrophoresis analysis at the same time points, and protein spots exhibiting time-dependent changes in their expression levels are analyzed by mass spectrometry. we show that mouse dorsal root ganglion explant cultures produce substantial amounts of mature myelin in weeks after the induction of myelination. ultrastructural analysis shows that myelin in our cell culture system has the same structural characteristics as those observed in peripheral nerves in situ. rna microarray analysis reveals changes in myelin-, nervous system-and schwann cell-related genes. the most significant changes in gene expression occur at the induction of myelination. at the protein level, peaks of specific proteins can be observed at the first and the last time points, which complement the results obtained using microarray data. we have developed a reproducible and efficient method to study molecular changes in myelination at the proteomic and genomic levels. this approach will be exploited further for the characterization of the molecular networks involved in the myelination process. results: our findings show that primary rat schwann cells respond to ivig stimulation with altered cell morphologies accompanied by an accelerated growth of cellular protrusions in early stages of the differentiation process. stimulation with ivig was also found to reduce cellular proliferation rates without affecting cell survival. furthermore, we observed that ivig treatment transiently boosts myelin gene expression of immature cells, whereas myelin gene expression of differentiating schwann cells is promoted on a long-term scale. importantly, myelin gene expression responses cannot be detected upon application of igg control antibodies (herceptin, synagis, avastin). in addition, we could demonstrate that primary rat schwann cells express the cd fc receptor and that in differentiating schwann cells cd levels were significantly increased. on the other hand, we could also reveal a specific binding of the human ivig on the schwann cell surface. conclusions: we therefore conclude that schwann cells are not only able to respond to but also specifically bind immunoglobulins and that ivig stimulation can promote their differentiation. here, we demonstrate that cnp deficient mice exhibit alterations in mechanical and thermal sensation. histological analyses of cnp deficient mutants reveal loss of sensory axons when quantified in the dorsal root and the predominantly sensory saphenous nerve. in contrast ventral roots remain unaltered. moreover, electron microscopical assessments of saphenous nerves display a hypermyelination of small to mid-sized caliber axons and an overrepresentation of the smallest fibers. the myelin pathology of sensory axons is reflected by alterations of sensory nerve conduction velocities. thus, our data demonstrate that, contrary to findings in the central nervous system, cnp has an impact on myelination and is especially important for the integrity of small to mid-sized caliber axons within the sensory peripheral nervous system. to unravel the pathogenetic mechanism of the s del mutation, we generated transgenic mice that express p s del protein and manifest a demyelinating neuropathy similar to cmt b. p s del is a misfolded protein retained in the er, where it activates a chronic unfolded protein response (upr). the upr is a cellular response to er stress, aimed to reduce the load of abnormal proteins by attenuating protein translation, increasing the er folding capacity and stimulating protein degradation. among the degradation strategies upregulated by the upr is the er-associated-degradation (erad) pathway. in erad, proteins destined for degradation are retrotranslocated from the er to the cytosol, ubiquitylated and degraded by the proteasome. microarray analysis of s del nerves revealed that several erad genes, such as derlins, are strongly upregulated and derlin- expression returns towards normal in association with the amelioration of demyelination produced by ablation of chop, indicating a possible involvement of this pathway in cmt b neuropathy. derlins elicit particular interest for exerting a protective role in several er stress-mediated diseases. in s del nerves, derlins co-immunoprecipitate with p s del protein and, finally, derlin- overexpression reduces the level of er stress in cos cells expressing p s del protein, suggesting a positive role in the modulation of p s del protein retrotranslocation. these observations indicate that derlins may be part of the schwann cell adaptive response that attempts to cope with chronic er stress by modulating p s del clearance. we are now in the process of investigating the role of derlins, in the pathophysiology of pns myelination using in vitro, ex vivo and in vivo approaches. during the myelination process, the oligodendrocyte extends processes to and wraps around multiple axons of different diameter, keeping the number of wraps proportional to the axon diameter. this is one unique example of how cells in the central nervous system establish asymmetry. transport of mrna and local regulation of protein synthesis represent one mechanism by which such cellular asymmetry can be generated and the different requirements for myelin sheath at each axo-glia interaction can be controlled. the mrna of a key myelin sheath protein, myelin basic protein (mbp), is known to be transported into the oligodendrocyte processes, and a tight temporal and spatial control of mbp mrna translation is thought to be required for normal myelination. the molecular basis for the tight control of mrna transport and translation is the assembly of large mrna-protein complexes. prior work has identified a number of proteins that interact with the mbp mrna, including hnrnp-a , hnrnp-k and hnrnp-e . however, it is currently unknown how these protein functions together to prevent translation during transport. it is also unknown how targeting of the mbp mrna to the myelin sheets occurs. to delineate the precise role of the individual binding protein in regulating mbp mrna translation, we have identified regulatory elements within the utr of the mbp mrna involved in translational inhibition, and we have identified binding sites in the mrna for hnrnp-k and hnrnp-e . furthermore, we have analyzed the effect of sirna-mediated knockdown and overexpression of these proteins in primary oligodendrocytes. together, our results allow us to propose a model, in which the individual mrna binding proteins are assigned distinct roles for correct spatial and temporal expression of mbp. remarkably, this manipulation was sufficient to achieve both the expansion of oligodendrocyte precursor cells (opc) and the de novo myelination of a large fraction of enlarged parallel fiber axons in the cerebellar molecular layer, independent of axonal neuregulin- expression. by laser-guided microdissection and gene expression profiling of cerebellar gc, we identified neuronal factors that promote opc proliferation and myelin synthesis in vitro. these findings suggest that oligodendrocytes and their precursors respond to multiple 'instructive' signals expressed by cns neurons in vivo, but only on axons with diameters > . lm that became 'permissive' for myelination. l. marziali, p. franco, j. pasquini university of buenos aires, caba, argentina promyelinating effects of both apo-transferrin (atf) and thyroid hormone (th) on rat brain are well documented. th effects are mediated by nuclear receptors which act as transcription factors and regulate different cell processes. previous results from our laboratory showed that th promotes the commitment of oligodendrocyte progenitors (opcs) to mature myelinating oligodendrocytes (olg). on the other hand, atf is able to promote the commitment of neural stem cells (nsc) to cells of the oligodendroglial linage and favors olg maturation after an intracranial injection. our previous demonstration that th administration is able to up-regulate tf mrna expression leads us to hypothesize that both factors converge in the control of oligodendrogenesis. to test if the combined effects of both atf and th are required for proper myelination in the rat brain, studies were done at p and p for each experimental condition. a hyperthyroid state was rendered by daily subcutaneous th administration from the day of birth (hyper). hypothyroidism was achieved by giving propylthiouracil to the mother from gestational day to the end of the experiment (hypo). half of the hypo pups received an intracranial injection of atf at postnatal day (hypo atf). a set of euthyroid animals received an intracranial injection of atf at postnatal day (atf). control groups received an intracranial injection or subcutaneous saline solution (c). at p , hyper animals showed an up-regulation of % in tf mrna expression and % in protein levels as compared to controls. hypo showed a decrease of % in tf mrna levels. this effect was not affected by exogenous administration of atf. at p , hyper showed no differences in the expression of tf mrna or protein levels as compared to controls. hypo showed a decrease of % in tf mrna and a % decrease in protein levels. this effect was not affected by exogenous administration of atf. no differences were observed in the expression of insulin growth factor i (igf-i) or igf-i receptor between groups at p or p . immunohistochemical analyses were performed at p in all groups usingspecific markers anti caii, anti mbp, anti rip and pdgfra. our conclusion is that, in a hypothyroid state, tf is not able to produce olg and myelin maturation at the corpus callosum, which indicates that th is necessary for atf action. however, the finding that hyperthyroid animals showed a significant increase in tf mrna strongly suggests that tf could be involved in th effects. new experiments are carrying out in order to knock down the tf gen to probe this last piece of evidence. h. yigit, l. meyer, c. gonsior, p. hoch-kraft, j. trotter johannes gutenberg university, mainz, germany myelination is the prerequisite for fast saltatory conductance of action potentials. in the central nervous system, the myelin sheath is produced by oligodendrocytes which extend processes and wrap the axons. this structure is subsequently compacted by the help of certain proteins. of major importance here is the myelin basic protein (mbp) which interacts with the cytoplasmic leaflet of the plasma membrane. mbp is a highly basic protein and binds to the negatively charged plasma membrane with high affinity. it has isoforms in mice produced by alternative splicing. the mostly studied isoforms are kd, kd, , kd and , kd. interestingly, exon-ii containing kd and , kd isoforms are actively transported into the nucleus while kd and , kd isoforms are rather localized to the distal part of the plasma membrane. transport to the nucleus depends on temperature and energy. recently, it was reported that exon-ii contains a non-canonical py nuclear localization signal. as a cytoplasmic protein, mbp is essential for proper myelination. furthemore mbp is supposed to have multiple additional functions, e.g. in calcium homeostasis and cytoskeleton dynamics. it was also shown that the expression level of mbp exon-ii containing is changed in schizophrenia patients. selectively transport of exon-ii containing isoforms to the nucleus raises the question of its nuclear functions which have not been elucidated yet. this study aims to unravel the role of exon-ii containing mbps in the nucleus. gene expression profiling of the microdissected svz regions revealed that wnt-responsive genes were enriched in the postnatal dorsal svz compared to the lateral svz. we performed inhibition of gsk b by intraventricular infusion of ara to initiate wnt-signalling. several approaches confirmed activation of the wnt-signalling pathway in the dorsal svz as well as within ops. thus, gene expression profiles of wnt-target genes of the dorsal svz indicated that axin , lef , fzdl and tcf , but not those other pathways, were dramatically up-regulated. furthermore, nuclear b-catenin was transiently up-regulated in the dorsal svz including in a large population of sox -egfp expressing cells following pharmacological wnt-activation. gene expression profiling and immunostainings revealed that the densities of nscs, cycling progenitors and subsequent op differentiation in the dorsal svz were augmented. in parallel, we genetically manipulated wnt-signalling in stem and progenitor cells of the dorsal wall of the lateral ventricle. targeted dorsal svz electroporation of cre-gfp plasmids in floxed stabilised b-catenin (gain-of-function) and floxed b-catenin (loss-of-function) transgenic mouse lines were performed and resulted in a phenocopy of the results observed following ara-intraventricular infusion. in summary, our findings illustrate that wnt-signalling endogenously persists in the dorsal svz after birth. pharmacological as well as genetic interventions reveal an important role of this signalling pathway in the regulation of op genesis during the period of myelination. in the cerebellum the generation of neurons and glia and the relationships between these two lineages are poorly understood. all the cerebellar neuronal phenotypes derive from two distinct embryonic germinal neuroepithelia that are restricted regarding the neuronal cell fate: the rombic lip (rl) gives rise to the glutamatergic neurons, whereas the ventricular zone (vz) generates all gabaergic neurons. in particular, different types of inhibitory interneurons are generated during the postnatal life from a common pool of progenitors expressing the transcription factor pax . on the other hand, the glial development is relatively unexplored. part of the oligodendrocytes derives from extracerebellar sources, whereas some of them derive from endogenous precursors. astrocytes, instead, may derive from progenitors that delaminate from the vz and continue to divide in the prospective white matter (pwm) up to postnatal life. given the vn origin of both astrocytes and interneurons, we wondered whether a lineage relationship exists between these two cell populations and whether they share the same bipotent progenitor. in order to address these issues, we performed a genetic fate mapping study of glastcreert mice. we showed that both cerebellar gabaergic interneurons and astrocytes derive from proliferating glast precursors. in particular, we found that these progenitors are able to produce interneurons at earlier developmental phases, whereas after p they appear restricted to the glial lineage. proliferating glast cells that may produce interneurons comprise bergmann glia and pwm progenitors. however, when recombination was specifically induced in bergmann glia, we found that these cells were not neurogenic. therefore, we concluded that glast progenitors reside in the pwm. to get further insight in the behaviour of these precursors, we injected in the pwm a gfp-containing lentiviral vector with a specific tropism for astroglial cells. some days after, infected cells generated pax -interneurons that were able to mature into interneurons of different cortical layers. moreover, in vitro and in vivo clonal analysis of pwm derivatives indicates that the pwm is neurogenic and both astrocytes and interneurons may derive from a common progenitor. evidence is accumulating that neurogenesis in the subventricular zone (svz) is boosted after trauma or ischemia, also through the interaction with surrounding parenchyma or niche cells. nevertheless, the number of newborn neurons that survive and integrate in the damaged areas is negligible, suggesting a non-permissive environment. thus, understanding the complex signaling network guiding neuroblast generation/survival could help identifying strategies to limit negative inputs and promote regeneration. extracellular nucleotides (ents) are among the hypothetical modulators of svz cell functions, especially under pathological conditions where their concentrations raise several folds and they contribute to reactive astrogliosis. few literature data have recently pointed for a role of the p y receptor subtype (one of the known g protein-coupled nucleotide receptors) in controlling the proliferation and differentiative potential of svz cells. thus, we tested the ability of adpbs, a stable p y agonist, to modulate stem cell properties in the adult brain, with a focus on the possible modulatory effects exerted by reactive astrocytes. the administration of adpbs in the lateral ventricle of adult mice led to reactive astrogliosis in the surrounding brain parenchima, and to a massive reaction of gfapexpressing precursors and astrocytes in the svz. also proliferation was increased, paralleled by a significant expansion of the population of mash transit-amplifying cells and of doublecortin neuroblasts. thanks to the conditional glast::creert yfp mouse model, we also demonstrated that adpbs promoted the proliferation of glast-expressing progenitors in the neurogenic niche, and sustained their progression towards the generation of rapidly dividing transit-amplifying cells. in vitro the nucleotide analog increased the proliferation of svz cells grown as neurospheres, and their differentiation towards neurons, fully confirming in vivo data. interestingly, a significant enhancement in neurosphere generation was detected when svz cells were initially grown in the supernatant of astrocytes exposed to adpbs, and then shifted to normal medium. this suggests that adpbs stimulates the release of yet-to-be identified astrocytic mediator(s) whose removal from the culture medium boosted proliferation of svz cells. our results further strengthen the notion that the purinergic system is a key regulator of the neurogenic potential of svz cells, both directly and through the involvement of reactive astrocytes. cambridge stem cell institute, cambridge, united kingdom oligodendrocytes, one of the principal glial cell-types of the central nervous system, are critical for neuronal function and represent the primary target of many neurological diseases, including multiple sclerosis and leukodystrophies. despite the recent progress in stem cell research and the possibility to generate various neural cell types in vitro, the derivation of oligodendrocytes from human pluripotent stem cells remains inefficient and unreliable. we employed a new experimental approach termed "direct cellular reprogrammng" which was already succesfully applied for the generation of different types of neurons and neural stem cells. aided by bioinformatic filtering we have generated a pool of seventeen candidate reprogramming factors that could potentially convert fibroblasts directly into oligodendrocyte lineage cells. combinatorial testing of the candidate factors in overexpression experiments has revealed a combination of just two transcription factors that was sufficient to convert mouse embryonic fibroblasts directly into o -expressing oligodendrocyte precursors. supplementation of additional factors and multicistronic gene delivery strategies render the reprogramming process more efficient and widen the applicability of this protocol. cellular reprogramming offers a new tool for the generation of oligodendrocyte precursors. this approach, that circumvents the protracted specification and differentiation process inherent to the oligodendrocyte lineage will foster the quest for a culture system of human oligodendrocyte lineage cells. transplanted are derived from progenitors within the intestine. to gain further insight in possible stem cell niches the compelling evidence that mouse enteric glia can also be neuronal precursors was related to human tissue. human colon from infants with hirschsprung's disease was investigated with a panel of glial and stem cell markers. tissue samples from infants with hirschsprung's disease were investigated with glial and stem cell markers along the gut axis. the tissue samples from ganglionic, aganglionic and transient segments were immuonstained either for s /nestin, s /p , gfap/nestin and gfap/p . in all samples investigated, nestin positive ganglia could be found, even in the distal parts with a severe hypo-or aganglionosis. beside nestin positive cells in all segments, there were also different expression pattern glial markers within the ganglia, indicating that distinct phenotypes of glia cells could be found. neural and glial precursor cells are present in the ganglionic as well as in the hypoganglionic segments of hirschsprung's colon, suggesting that these cells might be suitable for ncsc generation and further transplantation. the subventricular zone (svz) of the lateral ventricle, the largest adult stem cell niche, has a striking pinwheel organization specific to regions of adult neurogenesis. the pinwheel's core contains the apical endings of b cells and in its periphery two types of ependymal cells: multiciliated (e ) and biciliated (e ) cells. previous studies showed that svz cells are reactivated in response to demyelination. however, the mechanisms inducing svz reactivation in response to inflammatory demyelination such as in multiple sclerosis (ms) are poorly understood. we hypothesize that b and e, which are in direct contact with csf through cilia, may play a crucial role in initiation of svz reactivation. in the present study, we examined the svz reactivation in mice in which we targeted an ms-like pathology to the forebrain white matter (t-eae). animals were immunized with subclinical doses of myelin oligodendrocyte protein and after days, received a stereotaxic injection of a cytokine cocktail consisting of tumour necrosis factor (tnfa) and interferon (ifn c) into the corpus callosum. to obtain an en face view of the ventricular surface, we dissected whole mounts of lateral ventricle of control and eae animals at , , and days post-eae induction (dpi). we investigated by immunohistochemistry whether and how the pinwheel architecture of the svz is modified and ependymal cells are reactivated during disease course of eae. in svz niche of t-eae animals we found that the number of b cells increased and peaked at dpi compared to control. the number of e cells and pinwheels decreased and then reached control level at dpi. while the number of e cells remained steady, their surface area increased at dpi and then returned to normal size over time. in addition, the number of their ciliary basal bodies increased during eae time courses, and e cells highly expressed gfap in eae animals compared to controls. thus, the findings of our ongoing research suggest that neuroinflammation induces some changes in cell number and morphology of svz niche as well as reactivation of ependymal cells, which altogether may contribute to svz reactivation mechanisms. further study will provide detailed information on the homotypic and heterotypic interactions between pinwheel cells in response to t-eae. to test whether hypothalamic tanycytes act as bona fide neural stem/ progenitor cells in vivo, we analysed their immumoprofile and proliferative potential, and lineage-traced them in vivo. for this, we exploited our earlier findings that in the adult hypothalamus, fibroblast growth factor (fgf ) is expressed exclusively by tanycytes (hajihosseini et al. mcn : - ) , a radial glial-cell like population that lines the floor and ventral walls of the third ventricle. using a line of fgf -lacz reporter mice, we found that fgf beta tanycytes express a panel of neural/stem progenitor markers such as nestin, musashi , blbp and sox . fgf tanycytes also incorporated brdu under cumulative brdu-labelling paradigms, and formed neurospheres in vitro. to directly test the potential of fgf ve tanycytes, we lineage-traced them at p and p by activating the constitutive expression of the marker gene, tomato-dsred, in these cells and their descendants. this was achieved by applying tamoxifen to fgf -creer-t ::rosa r-tomato double transgenic mice. after short survival time-points, tomato was detected exclusively in tanycytes, whilst with longer survival time-point, tomato cells emerged in the neighbouring parenhcyma. the majority of these were neun neurons with fine arborizations. we noted that the newly-generated neurons become associated mainly with hypothalamic nuclei that regulate appetite and energy-balance. moreover, they respond to appetite/energy balance regulating signals such as acute food deprivation and leptin administration. our findings establish fgf beta-tanycytes as a putative population of neural stem/ progenitor cells that generate appetite/energy balance regulating neurons in the postnatal and adult hypothalamus. we used an inducible transgenic mouse line (hgfap-creert ) to conditionally label and fate map putative gfap( ) nscs populations in the adult svz and spinal cord (sc), and compare their self-renewal and multipotential properties. we evidence that a population of gfap cells that share the morphology and antigenic properties of svz-nscs reside in the dorsal and ventral aspects of the central canal (cc) throughout the spinal cord. these cells are non-proliferative in the intact spinal cord, but incorporate edu, an s-phase marker following spinal cord injury. multipotent yfp-expressing neurospheres (i.e. which derive from recombined gfap-expressing cells) were successfully obtained from both the intact and injured spinal cord, confirming their early gfap origin. notably, only spheres isolated from the injured spinal cord revealed long-term self-renewal properties resembling those of svz neurospheres. altogether, this work highlights discrepancies in the identity of nscs that reside in distinct regions of the adult cns. our observations however reconcile divergent views on the location and identity of spinal cord-nscs by showing a population of multipotent gfap progenitors with limited self-renewal properties co-existing alongside with multipotent, self-renewing ependymal cells within the spinal cord central canal. during development neural precursors (nps) both divide, to expand the cell population, and produce many different kinds of neurons and glia. this balance appears to be regulated by par complex proteins, which polarize neural precursors and can thereby direct daughter cells for different fates. how par complex proteins are regulated to appropriately polarize nps remains unknown. in recent years, regulation of gene function by micrornas has emerged as an important mechanism during development. using bioinformatics we identified the polarity gene pard as a candidate target for microrna (mir- ). mir- is specifically expressed in the developing central nervous system (cns) of vertebrates and mir- -deficient zebrafish embryos have a deficit of oligodendrocytes, the myelinating glial cells of the cns. because a disruption in polarity could affect the types of cell divisions that nps undergo, thus altering the balance of cell types that arise, we hypothesize that neural precursor maintenance is regulated by modulation of polarity cues through mir- . by using an in vitro reporter assay we found that mir- can downregulate expression of a luciferase gene fused to the pard utr. reduction of pard function in zebrafish embryos suppressed the oligodendrocyte phenotype resulting from loss of mir- function and injection of a morpholino oligonucleotide designed to block binding of mir- to its pard target sequence phenocopied mir- knock down. together, these data provide strong evidence that pard is a functionally relevant in vivo target of mir- . to further investigate the role of mir- function we tested expression of np, radial glial and neuronal markers in mir- -deficient embryos. the number of cells expressing sox , a marker of nps and neural stem cells, was significantly increased upon mir- knockdown. concomitantly, mir- -deficient embryos had a dramatic deficit of radial glia and late born neurons. these data provide evidence for a new mechanism of np regulation, in which mir- regulates pard levels, thereby regulating the transition of dividing neural precursors to differentiated neurons and glia. in demyelinating diseases such as multiple sclerosis myelin repair activities based on recruitment, activation and differentiation of resident progenitor and stem cells can be observed. however, the overall degree of successful remyelination is limited. it is therefore of considerable interest to understand oligodendroglial precursor cell (opc) homeostasis and maturation processes in order to develop remyelination therapies. mesenchymal stem cells (msc) were shown to exert positive immunomodulatory effects, to reduce demyelination, to increase neuroprotection and to promote adult neural stem cell differentiation towards the oligodendroglial lineage. we here addressed whether msc secreted factors can influence primary opcs in a myelin non-permissive environment. methods: to this end we analyzed cellular morphologies, expression and regulation of key genes/proteins involved in oligodendroglial cell fate and maturation upon incubation with mesenchymal stem cell conditioned medium. results: this demonstrated that msc derived soluble factors promote and accelerate oligodendroglial differentiation, even under astrocytic endorsing conditions. accelerated maturation featured elevated levels of , -cyclic nucleotide -phosphodiesterase and myelin basic protein expression, reduced glial fibrillary acidic protein expression and was accompanied by downregulation of prominent inhibitory differentiation factors such as id and id . conclusions: we thus conclude that besides the previously established immunomodulatory and neuroprotective roles of mscs these cells can also positively influence oligodendrogenesis in the adult central nervous system. in this study, we evaluated the effects of epo and the epo splicing variant (vepo) on murine neurogenesis ex vivo. for this purpose, we analyzed the survival of cultured neural stem cells (nscs) either exposed transiently to recombinant protein, or transduced with lentiviral vectors to achieve stable protein expression. in comparison to non-exposed controls, recombinant epo and vepo enhanced survival of nscs ex vivo (epo: %; vepo: % survival). nscs transduced with pcl -mscv-epo-and pcl -mscv-vepo showed also higher viability ex vivo compared to nontransduced nscs (epo: % vepo: % survival). furthermore, pcl -mscv-epo, but not pcl -mscv-vepo, increased the proportion of differentiated neurons when compared to non-transduced controls. thus, stable expressed vepo may have rather an effect on nsc survival than on nsc differentiation. our results demonstrate that vepos are neuroprotective in vitro. vepo may serve as therapeutic protein for treatment of neurodegenerative disease associated with impaired neurogenesis such as huntington disease. in the subependymal zone (sez) cytogenic niche of the adult mouse brain neural stem cells drive the continuous generation of new cells, mostly of olfactory bulb interneurons, but also of cells of the oligodendroglial lineage. previous reports have shown that newly-born cells exit the sez and migrate to the adjacent corpus callosum (cc) where they differentiate into oligodendroglial cells. however, the real contribution of these niche-derived oligodendrocyte progenitor cells and oligodendrocytes (nopcs and noligos) to the oligodendroglial population of the cc has not been assessed so far. in this study we used the hgfap-cre ert rosa -eyfp double transgenic mice in order to label specifically the progeny of sez-located adult neural stem cells. we found that cells expressing markers of opcs, such as pdgfra and olig , are constantly generated in the sez and migrate to the proximal fraction of the cc. interestingly though, these cells do not remain in the cc for more than days with their contribution to the total population of oligodedroglial cells remaining stably below % even in the ageing brain. moreover, immunostaining for markers of cell cycle revealed that a higher fraction of nopcs undergoes mitosis, when compared with local opcs; hence, they constitute almost % of proliferating opcs of the cc. notably, during their transient presence in the cc, nopcs express markers of maturing oligodendrocytes and are incorporated in established local cell structures. when the cc is challenged with a focal demyelinating insult the local and the niche-derived oligodendroglial machineries exhibit differential cell kinetics, nopcs increase their contribution to the total pool of oligodendroglial cells; however, their presence remains transient. in the human and mouse retina m€ uller glia (mg) are well known to undergo gliosis in all major types of retinal diseases -which sometimes may even lead to scar formation due to proliferative gliosis. some studies suggest that in the mouse retina mg derived neuronal regeneration can be stimulated, but only to a very limited extent. here, we started to find out, if conditional immortalization might stimulate mg derived proliferative gliosis and /or neuronal regeneration. others and we reported that in the juvenile mouse retina, after retinogenesis is finished, some m€ uller glia shift from a quiescent differentiated state into a proliferative state upon damage of retinal explants ex vivo. we now observed that this process is differentially inducible by mitogens like egf (epidermal growth factor). edu (s-phase marker) pulse chase experiments revealed a tremendous increase in mg proliferation within the first two days, a peak of edu positive cells at day ( sem, n ) and a massive decrease until day ( . sem, n ) per mm of a central retina section. next, we used transgenic mice with tightly and temporally controlled expression of the proto-oncogene sv large t-antigen (csv lt). it is well reported that csv lt binds several proteins including the tumor suppressor p and retinoblastoma and bypasses cell cycle checkpoints. induction of the csv lt for days ex vivo led to an overall increase in proliferation compared to control. the number of edu cells was . fold increased (csv lt: sem, n ; control: . sem, n ; p our results so far suggest that induction of csv lt not only overcomes the proliferative restriction of m€ uller glia but also maintains its progeny in the cell cycle over extended period of time. surprisingly, major parts of the generated cell progeny formed gliotic cell clusters, which were all located within the boundaries of the retina. further, we present data of successful m€ uller glia isolation at high purity by fac-sorting. in our current and future work we study the m€ uller glia and its derived progeny to find out the underlying mechanisms that enable neuronal regeneration and prevent gliotic scar formation. in mammals, regeneration capacity of the central nervous system (cns) is considered too low to recover the neurological functions after traumatic injury. in fishes and amphibians, however, the spontaneous regeneration is vigorous enough to complete the anatomical reconstruction and functional recovery even after severe damage of cns, in which the ependymal cells play the central role to exhibit proliferation, epithelial-mesodermal transition, cell migration, support of regenerating axons and neurogenesis to contribute to recovery from injury. these positive roles of the ependymal cells observed in fish and amphibian damaged cns may be good candidates for treating mammalian cns injury. in this study, we attempted to control the activity of the ependymal cells in the adult rodent spinal cord by virus-mediated gene transfer for imitating the reactions that are observed in the ependymal cells of fish and amphibian damaged cns. we introduced the gene known to be expressed in both the ependymal cells and neural stem cells (nscs) and found the proliferation of ependymal cells. in this case, the ependymal cells expressed glial fibrillary acific protein suggesting that this gene regulates proliferation and differentiation into astrocytes. in addition, introduction of another gene, also known to be expressed in the ependymal cells and nscs, caused cell proliferation without differentiation tendency. these findings indicate that the cell fate of the ependymal cells in the adult rodent spinal cord can be modulated by artificial intervention. future studies will clarify whether cell fate modification in the ependymal cells can contribute to anatomical reconstruction and functional recovery in the adult mammalian damaged spinal cord. methods neural stem cells are isolated from different parts of the human gastrointestinal tract, and also from different compartments (muscle, submucous and mucous layer) using enzymatical digestion approaches. generated neurospheres were screened for neuronal, glial, neural stem cell and pluripotency markers, prior and after differentiation. additional experiments were performed where neurospheres were either co-cultured with tissue blocks from different parts of the brain, or transplanted into brain slices from the rat. results neurospheres could be generated from all areas investigated and differentiated on glas coverslips. nestin and musashi- could be found in all neurospheres. moreover sox- , nanog and oct- could be found in the differentiating cultures. after differentiation tubulin and pgp . positive neurons could be found. transplantation experiments showed that cells from the transplanted spheres migrated into the brain slices and differentiated into neurons or glial cells. neurospheres co-cultured with tissue blocks from cortex, cerebellum or hippocampus showed a significant higher outgrowth than neurospheres cultured without any brain tissue. conclusions in general, the ens is a suitable tissue for the isolation of neural stem cells in the individual patient and might so be an appropriate source for autologous neural stem cells. neurogenesis. at the end of embryogenesis, oligodendrocytes and astrocytes appear during the process of gliogenesis. although in the last decades diverse extrinsic and intrinsic factors involved in these developmental processes were identified, our understanding of detailed mechanisms is still comparatively low. in order to increase the knowledge about the cellular and molecular mechanisms that underlie central nervous system development, transfection of nscs showed to be a promising method to understand the function of specific genes and proteins. to overcome the well-known difficulties to transfect cells of the central nervous system, we improved the electroporation conditions to achieve high efficiency transfection and survival rates for embryonic and adult nscs isolated from mouse forebrain structures. one day after electroporation transfection and viability rates of around % were achieved in murine nscs (bertram et al. j neurosci meth ). due to our interest in differentiation and maturation of oligodendrocytes we are currently improving the transfection conditions of oligodendrocyte precursor cells (opcs), since a sufficient protocol for the transfection of opcs derived from neonatal rat cortices is currently not established. the opcs are isolated from rat cortical tissue by a shaking protocol and transfected with the d-nucleofector (lonza). when we used the same pulse protocol that successfully works on nscs, oligodendrocytes were sensitive to the transfection process, and many cells died or did not undergo normal differentiation. this demonstrates that other pulses are needed to electroporate opcs. in cooperation with lonza we tested several recommended pulses to increase the transfection and survival rates of opcs. a dramatic increase of the viability rate has already been achieved. in further experiments the pulse protocol will be improved to obtain transfection rates above %. by establishing this transfection protocol we want to accomplish transfection and viability rates comparable to murine nscs. this improved protocol for the transfection of sensitive opcs will allow for reliable studies of gene function by over-expression or knockdown experiments, and will enhance our understanding of cell biological mechanisms important for oligodendrocyte development. recent studies demonstrate that astroglial cells isolated from non-neurogenic brain regions have the potential to be reprogrammed into functional neurons through forced expression of transcription factors known to instruct neurogenesis. based on our previous studies on the potential of the neurogenic gene cend in directing neural stem/precursor cells (nsc) to exit the cell cycle and acquire a neuronal phenotype, in parallel with evidence demonstrating activation of cend expression by genes of the neurogenin family, we explored the combined effect of cend and neurogenin- on their reprogramming potential on postnatal cortical astrocytes. to this end, forced expression of either cend , neurogenin- or both, resulted in an important increase of two morphologically distinct subpopulations of gfapcells with elongated morphology, that strongly expressed the radial glial marker glutamate transporter glt- . further characterization revealed that a subpopulation of these cells differentiates towards the neuronal lineage, as they were exhibiting a differentiated neuronal morphology and expressed b-iii tubulin, as well as neuronal subtype-specific markers, including gaba and th. further analysis using long time live cell imaging and brdu cumulative labeling revealed that the majority of cend -transduced astrocytes undergo - cell divisions before differentiating to gabaergic neurons, whereas most ngn astrocytes directly transdifferentiate giving rise to th neurons. additionally, only in the doubletransduced cultures, cend /ngn astrocytes seemed to take a step back forming colonies of small round glast /nestin cells, detected h following transduction. a day later, these colonies grew as three-dimensional spheres of high proliferative potential attached to the culture dish. when 'astrospheres' were isolated and cultured under nsc conditions, they grew as neurospheres and were propagated for over ten passages. importantly, as soon as 'astrospheres' were cultured in the absence of growth factors, they differentiated into neurons, astrocytes and oligodendrocytes, implying that they possess neural stem cell properties. at the moment we are performing electrophysiology recordings to assess the functionality of cend derived neurons in vitro, while studies are in progress to explore the potential role of cend and ngn on astrocytic reprogramming in vivo following cortical brain injury. average membrane potential (v m ) was mv and input resistance (ir) was mx, while dcx or map cells expressing fast activating outwardly rectifying k currents (ka), and delayed outwardly rectifying k currents (kdr) were defined by a v m of mv and high values of ir ( mx). ng cells with a complex current pattern expressed inwardly rectifying k (kir) currents, in addition to kdr and ka currents, and their v m was mv and ir was mx. the electrophysiological analysis revealed that the inhibition of the wnt signaling pathway significantly increased the incidence of cells with a complex current pattern, while the number of cells with the marked expression of outwardly rectifying k currents declined. wnt signaling inhibition also resulted in increased kir and decreased ka current amplitudes. oht-treated cells were also hyperpolarized when compared to controls. immunocytochemistry using antibodies against map and dcx showed that neuronal progenitors in oht-treated cultures were less developed, having fewer and less branched processes. in summary, our data imply that the canonical wnt pathway in the neonatal svz promotes nsc differentiation into cells with neuronal characteristics. gacr p / / ; p / /g . adult neurogenesis plays a critical role in the overall health of, and repair processes occurring within the nervous system. while a great deal of information has been gained about the elements of the neurogenic niche and the factors that mediate neurogenesis, many unanswered questions remain. specifically, studies suggest that microglia have a significant impact on adult neurogenesis, but no unifying theory exists to explain their exact role in this process. in order to determine if microglia play a pivotal role in adult hippocampal neurogenesis and to examine the mechanisms through which microglia exert their effects on the hippocampal neurogenic niche, we employed a transgenic mouse model that allows for the selective ablation of microglia in the central nervous system (cns), the cd b-herpes simplex virus thymidine kinase (cd b-hsvtk) mouse. using this system, we removed microglia from the adult hippocampal neurogenic niche and evaluated the effects of this manipulation on neurogenesis and hippocampal homeostasis, including immunohistochemical analyses of stem cell survival and proliferation, as well as analyses of neuroblast development, migration and electrophysiological activity. taken together, these studies help to provide a better understanding of the factors governing adult neurogenesis and further our knowledge about the increasingly diverse role of microglia in the cns. the current assumption is that ependymal damage, caused by viral infections may result in reactive gliosis in the subventricular zone (svz), leading to sgns. however, as the svz is currently recognized as one of the neurogenic niches in the adult human brain, and svz astrocytes have been identified as neural stem cells (nscs), the assumption of sgns being reactive astrocytes has to be reconsidered. therefore, we have immunophenotyped the cell types present in these structures in the brains of individuals of various ages. in addition, we assessed nodule incidence in the svz in cases infected with hiv, where cells in the svz are likely to be to be affected. we found sgns in almost % of all cases, independent of age or hiv infection. we determined that cells in the sgns express adult nsc markers, rather than markers for reactive astrocytes. we hypothesized that direct contact of the nscs with cerebrospinal fluid (csf) may induce nodule formation. indeed, we could show that human ventricular csf stimulated the proliferation of human nscs in culture. from our data, we conclude that sgns most likely are formed upon ependymal damage in response to exposure to csf and represent local expansions of the svz. . in experimental murine model of demyelination, the repair capacity is robust and sufficient, even if it might decrease with aging. one major therapeutic goal in ms is to favour endogenous myelin repair, to prevent neurodegeneration and disability progression. using a transcriptomic approach on pure populations of opcs, we try to identify mechanisms specifically involved in opcs activation. we set up a method to isolate a purified population of opcs, by fluorescent-activated cell sorting from pdgfar::gfp mouse brains. we created the gene expression profile of neonatal opcs, adult opcs and adult oligodendrocytes (ols) in control conditions and of adult opcs in cuprizone-induced demyelinating conditions. we analyzed the transcriptomic profile of adult opcs, neo-natal opcs and ols, and identified more than differentially expressed genes. after demyelination, we identified more than adult-opc-specific genes that are either up-or down-regulated compared to control conditions. preliminary analysis suggests that adult opcs are more "mature" than neonatal opcs and partially revert to a more immature profile after demyelination. bio-informatic analysis is ongoing, with the perspective of identifying key candidates for myelin repair capacity, then develop functional experiments to validate their involvement in remyelination. in parallel, we plan to compare the transcriptomic profile of adult opcs from aged versus young mice, to get insight into age-related decrease of repair efficacy. question: after damage, astrocytes are activated and exert specific roles for central nervous system (cns) repair. among these, astrocytes within the injury site may represent a novel source of cells with stem cell potential. in this study, we used sedimentation field-flow fractionation (sdfff) method to rapidly sort well preserved astrocyte subpopulations and we identified stem cell properties within one of these subpopulations. methods: cells were collected from newborn rat cortex, separated mechanically and subjected to sdfff. cell fractions obtained were analyzed by immunocytofluorescence using antibodies against specific epitopes: glial fibrillary acidic protein (gfap), o , b iii tubulin, and cd , labelling respectively astrocytes, oligodendrocytes, neurons, and microglial cells. then, proteomic analysis was performed on astrocyte subpopulations and their behaviour in culture was analyzed, particularly under culture conditions usually allowing neurosphere development. results: three main fractions (f , f , and f ) were isolated and compared with the total eluted population (tp). after one week in vitro, tp and f derived cell cultures contained respectively . % and . % of gfap expressing astrocytes while this percentage was extremely high in f and f derived cell cultures ( . % and . % respectively). f derived cells displayed higher migratory capacities than those of f . proteomic analysis of f and f derived cells indicated a high expression of vimentin in f derived cells. in the presence of epidermal growth factor (egf), f derived cells formed neurospheres containing cells expressing gfap and nestin (figure) that, when placed in appropriate conditions, were able to generate the three major cell types of the cns (neurons, astrocytes and oligendrocytes). moreover, colony-forming cell assay in a collagen-containing semi-solid matrix allowed in the presence of egf the formation of large colonies. in addition, days after cortical lesion in adult rats, cells presenting similar stem properties were obtained after sdfff cell sorting. conclusions. our study demonstrates that sdfff enables the isolation of almost pure astrocyte subpopulations after one week in vitro. in addition, after sdfff cell sorting, we isolated an astrocyte subpopulation expressing vimentin and displaying the self-renewal and multipotency properties of neural stem cells. our data support that sdfff is an efficient tool to explore the different astrocyte subpopulations of the cns, particularly those involved in cns repair. experimental studies have shown that loss of myelin results in axonal loss and disability. so, finding of an expandable and autologous source of myelin-forming cells to enhance remyelination is required. induced pluripotent stem cell-derived neural progenitor cells (ipsc-npcs) have been developed recently. the remyelination potential and safety of these cells still remained to be well-addressed. the main goal of this study is to characterize mouse ips-npcs in vitro and in vivo after transplantation. we used embryonic mouse neural progenitor cells (mnpcs) as control and characterized mouse ips-npcs for their expression of major markers of immature or mature cells by immunostaining. rt-pcr was performed to analyze expression of nestin, olig , b -tubulin, olig , sox , and nkx . mrnas. furthermore, to investigate the fate of these cells in vivo, we injected the lysolecithin to induce focal demyelination in the spinal cord of adult nude or shiverer mice. we transplanted cells in the lesion site days after demyelination. animals were sacrificed days, , , and weeks post transplantation to assess survival and differentiation of the grafted cells. our preliminary results showed that mips-npcs similar to mnpcs were nestin , ki , olig , b -tubulin but o -and map -. mips-npcs, were more proliferative than mnpc. moreover, mips-npc expressed all mrna transcripts except sox . a first line of mips-npcs, was transplanted in the newborn shiverer:rag forebrain or the demyelinated spinal cord of immunosuppressed shiverer mice with a constant failure in terms of cell survival beyond days of transplantation, highlighting the fragility of some of the reprogrammed cell lines. cells of a second line of mips-npcs were transplanted in nude mice to be sacrificed at , and weeks. an additional serie of transplantation was performed in the demyelinated shi:rag spinal cord. data at and weeks indicate excellent survival and integration of the mips-npc at both time points. some of the grafted cells expressed gfap, olig and ki (few only) and so far, no tumors were observed at these timepoints. immunohistochemistry with other markers and analysis of weeks post transplantation animals, are in progress. our preliminary results show that although mips-npcs have very similar immature phenotype of mnpcs in vitro, they may have different survival capacities in vivo. future studies will reveal whether transplanted cells which showed short-term survival after grafting are capable of differentiation into myelin-forming cells. . however these putative stem cells have only been partly characterized in vitro and their therapeutic potential for cns repair has not been addressed. we cultured adult mouse drg cells in sphere-forming conditions. in parallel, we derived spheres from adult spinal cord of the same animals and compared their properties. this last tissue contains well-described stem/progenitor cells whose repair capacities have already been proven for spinal cord injury but not for primary demyelination. in vitro, regardless of their origins, all tissues generated self-renewable spheres up to passages. rt-pcr and immunocytochemistry revealed that sphere-forming cells actively proliferate and express neural stem cells markers. drg spheres were multipotent: as they differentiated into schwann cells, neurons and myofibroblasts, whereas spinal cord spheres differentiated into astrocytes, neurons and oligodendrocytes. we then compared the integration, differentiation potential and remyelination capacity of primary sphereforming cells isolated from actin-gfp transgenic mice after their engraftment in two distinct environments: the adult nude lpc-demyelinated spinal cord and the developing newborn shiverer forebrain. both cell types survived but with distinct integration and differentiation patterns. in the mbp deficient mutant, spinal cord cells exhibit a substantial tropism toward white matter tracts and differentiated mainly into myelinating oligodendrocytes while drg cells were largely distributed in the parenchyma in close association with blood vessels and differentiated in vascular smooth muscle cells. surprisingly, after focal demyelination, in the adult spinal cord, drg cells migrated extensively towards the lesion and differentiated into remyelinating schwann cells whereas adult spinal cord cells migrated less efficiently, and gave rise to oligodendrocytes and a large population of astrocytes. our study reveals that both cell types present different but promising potential for cns remyelination. funding by ela foudation, dim nerf, arsep foundation. ischemia-induced progenitor cell proliferation is a prominent example of the adult mammalian brain's ability to regenerate injured tissue resulting from pathophysiological processes. a key locus of regeneration is the neurogenic niche located in the subgranular zone of the dentate gyrus (sgz). within the sgz, radial glia-like progenitor cells generate neural precursor cells which migrate into the granule cell layer (gcl) of the dentate gyrus, mature, and integrate into the hippocampal synaptic network. in order to better understand and ultimately exploit the cell signaling mechanisms that regulate ischemia-induced proliferation in the sgz, we tested the role of the p / mitogen-activated protein kinase (mapk) cascade effector ribosomal s kinase (rsk) in this process. using the endothelin- ischemia model in c bl/ mice, we report that intrahippocampal cerebral ischemia triggered rsk activation in sgz progenitor cells. further, microinjection of a rsk inhibitor significantly reduced ischemia-induced sgz progenitor cell proliferation. using the neurosphere assay, we also show that sgz-and subventricular zone (svz)-derived adult neural stem cells (nsc) exhibit a significant reduction in proliferation in the presence of rsk and mapk inhibitors. taken together, these data indicate that rsk functions as a cell-autonomous regulator of ischemia-induced progenitor cell proliferation. here we demonstrated that the inpcs not only possess npc-specific marker genes, but also have qualitites of primary brain-derived npcs (wt-npcs), including tripotent differentiation potential, mature neuron differentiation capability and synapse formation. moreover, the mature neurons derived from inpcs exhibit significant physiological properties, such as potassium channel activity and generation of action potential-like spikes. importantly, besides survival, the transplanted inpcs exhibit the migratory property responding to inflammatory stimulation in vivo. these results suggest that directly reprogrammed inpcs closely resemble wt-npcs, which may suggest an alternative strategy to overcome the restricted proliferative and lineage potential of induced neurons (incs) and broaden applications of cell therapy in the treatment of neurodegenerative disorders. the pathogenesis of neurodegenerative disorders, including human immunodeficiency virus (hiv) associated neurocognitive disorders (hand), is exacerbated by an imbalance between metalloproteinases (mmps) and their inhibitors, tissue inhibitors of metalloproteinases (timps). as the timps exhibit diverse non-classical functions including anti-apoptotic effects, the induction of timp- in neuroinflammatory conditions likely serves multiple roles in addition to modulating mmp activity. our work demonstrates differential timp- expression in acute versus chronic activation of astrocytes and hiv- associated dementia (had) brain tissues. the timp- promoter harbors five consensus ccaat boxes. ccaat enhancer binding protein (c/ebp)b levels were elevated in brain specimens from hiv- patients and this transcription factor regulated astrocyte timp- expression. hiv-relevant stimuli increased c/ebpb expression in human astrocytes and localized the factor to the nucleus. overexpressing c/ebpb in astrocytes increased timp- promoter activity, mrna and protein expression, while knockdown of c/ebpb decreased timp- mrna and protein expression. erk / activation is critical for il- b-mediated astrocyte timp- expression and p k activation contributes to il- b-induced astrocyte timp- and c/ebpb expression. these data suggest that erk / signals downstream of c/ ebpb to facilitate il- b-induced astrocyte timp- expression. the role of astrocyte-timp- as a neurotrophic factor was examined. interestingly, cotreatment with timp- protected neurons from apoptosis and reversed neuronal morphological changes induced by staurosporine (sts) and hiv- ada virus. further, the anti-apoptotic effect was specific to timp- and partially independent of mmp-inhibition. additionally, timp- modulated the bcl- family of proteins and inhibited opening of mitochondrial permeability transition pores induced by hiv- or sts. together, these findings describe a novel function, regulatory mechanism and direct role of astrocyte-timp- in neuroprotection suggesting its therapeutic potential in had and possibly in other neurodegenerative diseases. mounting evidence and our pervious study has also confirmed that the axon outgrowth inhibition receptor ngr was also expressed on microglia, and regulated cell adhesion and migration behavior in vitro. however, microglia has been proposed to play a pivotal role in the neuroinflammation through expressing a range of proinflammatory enzymes and proinflammatory cytokines under pathological stimulus. these findings initiate the possibility of nogo/ngr signal on mediating neural inflammation processes during cns injury beside neurite outgrowth inhibition. in the present study, we found that nogo-p , a -aa core inhibitory peptide sequence of nogo- , was able to induce microglia expressing cox- , inos, as well as releasing proinflammatory cytokines including il- b, no and pge , which could been reversed by nep - or pi-plc treatment. microglia stimulated with nogo-p increased the phosphorylation lever of signal transducer and activator of transcription (stat ). the activation of stat further mediated the promotion of nogo-p on microglia expression proinflammatory factors. furthermore, administration nep - was capable to attenuate the inflammation response in a mouse spinal cord compression injury model by reducing the expression of several proinflammatory mediators, as well as attenuating the activation of stat . taken together, our results demonstrated, for the first time, that nogo- may directly take part in cns inflammation process by influencing microglia expressing proinflammatory factors, which was related to ngr /stat signal pathway. these results imply that the interaction of nogo- with ngr expressed on microglia may participate in diverse cns diseases related to neural inflammation, including trauma, stroke, and neurodegeneration diseases, etc. our results indicate that ccl- is one of the key molecules in pathogenesis and ccl- /ccr- signaling system can be a potential target for drug development in the treatment for neuropathic pain. demyelination plays a central role in the pathophysiology of multiple sclerosis (ms) not only because it disrupts nerve conduction, but also because of effects that compromise axonal survival. there is a growing consensus that any effective treatment for ms must include a component that restores or enhances remyelination by endogenous oligodendrocyte progenitor cells (opc). however achieving this goal requires a far better understanding of why remyelination fails in the first place. we now report that fgf is selectively up regulated in demyelinating ms lesions and acts to inhibit (re)myelination in vitro. to explore the mechanism involved we compared the activity of fgf to that of fgf , another member of the fgf family with well characterised effects on opc differentiation and myelination. fgf acts directly on opc to stimulate proliferation, while at the same restricting their differentiation into mature oligodendrocytes. in contrast, fgf is neither an opc mitogen, nor is it able to inhibit opc differentiation directly. however, opc differentiation and myelination in myelinating cultures were inhibited by supernatants harvested from fgf treated astrocytes, even in the presence of an excess of fgf neutralising antibody. microarray studies were performed to identify astrocyte derived factors responsible for this inhibitory effect. this approach identified several potential candidates that were significantly up regulated by fgf in astrocytes. these included lif, a member of the gp /il cytokine family that is generally described as having pro-myelinating effects, although our own studies indicate when present in excess relative to other gp /il family members it will inhibit myelination in vitro. our experiments demonstrate that fgf -mediated signal transduction in astrocytes disrupts their ability to maintain a growth factor milieu that can support myelination. been shown that inflammation is primarily mediated by macrophages that are recruited to the tissue. similarly, a recent study demonstrated reactive microgliosis in the hypothalamus of mice fed a high-fat diet. in the cns, however, the relevance of microglial activation in response to a high fat diet remains unclear. we aim to determine if microglia activation occurring in response to overnutrition is a cause or consequence of dysregulation of energy homeostasis and obesity. to determine if the absence of microglia (and therefore microglia-mediated inflammation) may influence the metabolic response to a high fat diet, we used a transgenic mouse model, the cd b-hsvtk mouse (tk), which allows for an inducible, specific ablation of microglia in response to treatment with the nucleoside analog, gancilovir (gcv). mice were treated with gcv for weeks during which time they were maintained on a diet high in fat ( %) or a respective control diet. body weight, food intake, locomotor activity and energy expenditure were measured and compared to wildtype mice. analyses revealed alterations in the metabolic phenotype of mice fed both hfd and control diet in the absence of microglia cells. our findings point towards a physiological role of microglia in energy homeostasis, the study of which will be the focus of further experiments. interleukin- (il- ), a cytokine classically related with anti-inflammatory and protective functions in the central nervous system (cns), has been reported to be produced by astrocytes and microglia in different neurodegenerative and neuroinflammatory conditions. in order to study the specific role of il- in the cns, we have created a new transgenic mouse with astrocyte-targeted production of il- (gfap-il tg). the aim of this study was to evaluate if production of this cytokine in the cns has any effect on the phenotype of glial cells. for this purpose, coronal cryostat free-floating sections from the brain of both adult transgenic mice and their corresponding wild-type (wt) littermates, were processed for the study of astrocytes using gfap immunohistochemistry and microglia using antibodies against iba and several markers commonly related to the activated phenotype of these microglial cells, such as cd / (fc receptor), f / , cd b, cd , cd and mhc-ii. our results showed that astrocyte-targeted production of il- induces an increase in the area covered by gfap immunolabelling. microglial cells in these gfap-il tg animals displayed also morphological changes in specific areas of the brain, including the hippocampus, cortex and cerebellum, characterized by coarser processes and a notable enlargement of the cell body. distinctively, in the hippocampus, microglial cells adopted an elongated morphology parallel to the dendrites of pyramidal neurons. in addition, in the aforementioned areas, microglia exhibited a statistically significant increase in iba , cd / , f / and cd b markers and "de novo" expression of cd , whereas no detectable levels of either cd or mhc-ii was found. in conclusion, these results indicate that in specific areas, glial cells are influenced by the presence of astrocyte-targeted il- . further studies are necessary to evaluate whether after injury these transgenic animals will present changes in the microglial and astrocyte activation that can influence the evolution of the lesion. networks. brain-resident macrophages, microglial cells were considered ''resting,'' but it has recently become evident that they actively sense neuronal microenvironment with their motile and ramified processes. they develop specific activities (cytotoxic, neuroprotective or phagocytic) depending on brain molecular context. for example, they contribute directly to the development of neuronal networks by eliminating or maintaining synapses in an activity-dependent manner. morphofunctional plasticity of microglial cells is finely tuned in order to keep brain homeostasis and to ensure such developmental activity. like macrophages at the periphery, microglial cells indeed adopt m or m phenotypes, distinguishable through their pattern of cytokine expression and of specific cell surface markers, a m phenotype being more pro-inflammatory while a m phenotype is more related to phagocytosis. linoleic acid (la, : n- ) and alpha-linolenic acid (ala, : n- ) are essential fatty acids that cannot be synthesized de novo by mammals and have to be provided through the diet. they are precursors of arachidonic acid (aa; : n- ) and docosahexaenoic acid (dha; : n- ) respectively which are key structural components of brain cell membrane phospholipids and precursors of bioactive lipid messengers involved in the regulation of inflammation and microglial activity. most aa and dha accumulate in the brain during the perinatal period via placenta and milk. we previously demonstrated that depleting the diet in n- pufas from the first day of gestation alters some neuronal functions of the offspring at adulthood. we here hypothesized that this is due to an alteration of microglial mrophofunctional activity in the developing brain. to test this hypothesis, mice were submitted to a diet deficient or not in ala throughout gestation and lactation. microglial morphology, phenotypes and functions were determined in the brain of pups at post-natal day (pnd ). we show that dha levels is decreased in membrane phospholipids of microglia. in addition, microglial cells from mice fed with an ala deficient diet present a drastic increase of the cd marker expression combined to a dysregulated cytokine/chemokine expression profile and an altered phagocytic activity. all together, our results show that dietary n- pufas deficiency is likely to impair brain innate immune system activity at pnd . the outcome of such microglial impairment on brain function is discussed. in recent years, one of the most promising therapeutic means for ad was thought to be immunization with specific antibodies against ab. several therapeutic antibodies were developed, however, most of the clinical trials were canceled due to unexpected deaths and neuroinflammatory responses in some patients. the causes and mechanisms of such responses are currently only partially understood. we have recently raised monoclonal antibodies against oligomeric forms of ab and were investigating whether these antibodies would prevent the neurotoxicity of ab oligomers in primary neuronal-glial cerebellar granule cell (cgc) cultures. antibodies were not directly toxic to cgcs when applied at concentrations relevant to concentrations reported to be in blood serum. however, surprisingly, the antibodies when in complexes with ab oligomers or firbrils dramatically increased the neurotoxicity of ab. similar effects were also observed with antibodies to other oligomeric proteins: hamster polyomavirus major capsid protein vp , human metapneumovirus nucleocapsid protein and measles virus nucleocapsid protein strongly potentiated the neurotoxicity of their antigens. the neurotoxicity of antibody-oligomeric antigen complexes was abolished by removal of the fc region from the monoclonal antibodies or by the removal of microglia from cultures, and was accompanied by inflammatory activation and proliferation of the microglia in culture. in conclusion, we find that immune complexes formed by ab oligomers or other oligomeric antigens and their specific monoclonal antibodies can cause neuronal death in primary neuronal-glial cultures via fc-dependent microglial activation. the results suggest that therapies resulting in antibodies to oligomeric ab or oligomeric brain virus proteins should be used with caution or with suppression of microglial activation. additionally, if endogenous antibodies contribute to neuronal loss in ad or viral encephalitis, suppression of microglial activation may be therapeutic. antidepressants have often been recommended for potential treatment of chronic pain, especially for neuropathic pain caused by nerve damage. it is known that nerve injury-induced activation of microglia and astroglia can be a cause for the development of neuropathic pain. recent data indicate that abnormal neuronal activity mediated by glia activation may influence the level of neuroimmune signaling molecules in chronic pain. however, it remains unclear whether the mechanism underlying antidepressant effects is related to glia activation. this study investigated the potential pain-relieving properties of milnacipran and venlafaxine, selective serotonin/noradrenaline reuptake inhibitors (snri), in neuropathic pain and attempted to resolve how these antidepressants affect the factors synthesized by the activated glia in neuropathy. chronic constriction injury (cci) was performed in wistar rats by loose ligation of the sciatic nerve. after a single intraperitoneal (i.p.) injection of antidepressants, the responses to mechanical and thermal stimuli in neuropathic rats were evaluated days after cci by the von frey test and the cold plate test, respectively. to confirm the influence of antidepressants on microglia and astroglia, western blot analysis of iba- (microglia marker) and gfap (astroglia marker) in the spinal cord and dorsal root ganglions (drg) was performed. the experiments were carried out according to the iasp and local bioethics committee rules. our findings confirmed that the administration of milnacipran ( mg/kg) and venlafaxine ( mg/kg) elicited antiallodynic and antihyperalgesic effects in cci-exposed rats. western blot analysis showed that milnacipran elevated the level of iba- protein in the spinal cord (but not in drg) in cci-exposed rats. venlafaxine decreased the cci-induced iba- protein level in the spinal cord and in drg. we observed no changes in gfap in the spinal cord and drg after milnacipran and venlafaxine administration. these results provide a substantial evidence that both used antidepressants belonging to snri attenuated allodynia and hyperalgesia in an animal model of neuropathic pain, although their effect on microglial cells and astrocytes was different, namely, milnacipran increased the microglia activation, in contrast to venlafaxine. this effect can be explained by comparing the effects of these drugs during their repeated application, and relevant experiments are currently in progress. myeloid cells can respond to intra-and extracellular danger/stress signals by inflammasome-mediated activation. this process consists of two steps: toll-like receptor-induced production of pro-il- b followed by inflammasome-mediated cleavage and secretion of bioactive il- b. inflammasome-mediated activation is strictly regulated by expression of regulatory proteins and by inhibitory processes like autophagy. recent studies describe that microglia are markedly different from other myeloid cells. they originate from a separate progenitor, are chronically exposed to the neural microenvironment and their activation may be regulated differently. the objective of this study was to determine the expression profile of inflammasome components in primary microglia and to compare inflammasome-mediated microglial activation and its regulation with other myeloid cells. primary microglia, bone marrow-and blood-derived macrophages (bmdms) of adult rhesus macaques of the same donor were profiled for all nod-like receptors (nlrs), inflammasome-associated adaptor proteins, caspases, and regulatory proteins by qpcr. inflammasome function and kinetics were assessed by exposing lps-primed microglia to atp, silica or msu (danger-associated molecular patterns: damps) followed by measuring the transcription of pro-il- b-encoding mrna and the secretion of il- b. primary microglia expressed nlrs (nalp - , nod / / , aim , ipaf, naip), adaptor proteins (asc), caspases ( / - / / ), and regulatory proteins (a ), and the expression profile closely resembled that of bmdms. priming of microglia with lps induced high levels of pro-il- b-encoding mrna, but il- b protein was only secreted in response to subsequent stimulation with various damps. interestingly, in lpsprimed bmdms the window for inflammasome-mediated activation was - hours, while lps-primed microglia remained sensitive for inflammasome-mediated activation for at least hours. in conclusion, primary microglia express multiple inflammasome components closely resembling the expression profile of bmdms and they can be induced to form functional inflammasomes. importantly, primed microglia remain sensitive to inflammasome-mediated activation for much longer than other macrophages. we will present data on whether this reflects a difference in negative regulation of the inflammasome, or differences in autophagy or apoptosis sensitivity. objective: the susceptibility of the aging brain to neurodegenerative diseases may in part be attributed to the intrinsic senescence of the microglia. cellular aging or senescence is linked to telomere shortening/dysfunction. evidence is accumulating that aging microglia show morphological changes, referred to as microglial dystrophy, which may be accompanied by accumulation of the iron-binding protein, ferritin. here, we investigated the effect of telomere shortening on microglial morphology, numbers, and activation state in telomerase deficient (terc -/ -) mice, a model of premature aging due to shortened telomeres. methods: male terc / mice and rd generation terc -/mice, which show a clear aging phenotype, were perfusion-fixed with % paraformaldehyde. brains were processed into lm thick horizontal sections. every fourth section was used for stereological estimation of cd b microglia in the molecular layer of the dentate gyrus by the optical fractionator method. sections were also stained for the microglial markers iba- to monitor microglial morphology, cd and cd to study microglial activation, and ferritin to study microglial aging. further sections were stained for the apoptosis marker cleaved poly (adp-ribose) polymerase (cparp). results: unlike resting microglia in littermate terc / mice, which display thin, finely branched processes, microglia in terc -/mice exhibited partial retraction and hypertrophy of processes. microglial cd and cd immunoreactivity were similar in terc / and terc -/mice. absolute numbers of mac- microglia were significantly reduced in young and old terc -/versus terc / mice. an increased microglial density in old terc -/versus terc / mice could be attributed to a volume reduction of the dentate gyrus. we also observed an increased number of cparp cells in old terc -/versus terc / mice. this increase was most prominent in the sub-granular zone, an active site of neurogenesis, but also occurred in the molecular layer of the dentate gyrus and might reflect microglial apoptosis. we observed no difference in ferritin staining in terc -/versus terc / mice. conclusion: our findings suggest that telomere shortening impairs microglial capacity for self-renewal. surprisingly, changes in microglial morphology in terc -/mice occurred without differences in cd and cd expression. ongoing studies will show if increased numbers of microglia in terc -/mice co-express cparp or ferritin as evidence of increased microglial aging. tetrahydrocannabivarin (d thcv) and cannabidivarin (cbdv). we have developed a series of new cannabinoid quinones, among them the cbg quinone (vce- ) that shows pparg and cb receptors agonism. in addition we have found that vce- activates the nrf /are pathway in neuronal cell lines. in the present study, we investigated the therapeutic potential of vce- in the experimental autoimmune encephalomyelitis (eae) model of multiple sclerosis (ms) by immunization con mog - . vce- ( mg/kg ip, daily) was administered to susceptible c /bl mice at the onset of symptomatology. clinical score and weights of mice were daily recorded until the day of sacrifice ( days post-immunization). vce- treatment delayed the onset of disease and ameliorated the symptomatology. histological analysis of spinal cord of eae mice treated with vce- showed decreased microglia reactivity and reduced cellular infiltrates, in particular cd t lymphocytes. double labeling with neurofilament and the myelin protein, rip indicated that vce- diminished the axonal damage. demyelination was evaluated by luxol fast blue labeling. changes in the expression of several cytokines, adhesion molecules and nrf -dependent genes were determined by qrt-pcr. the implication of pparg and cb receptors in the beneficial effects of vce- in the eae model of ms is being investigated by using specific receptors antagonists. taken together our results support the potential of vce- for the treatment of ms and other chronic inflammatory diseases. we found that the lipid molecule lactosylceramide (laccer), is upregulated in ms patients. to better understand the role of laccer in ms, we used an animal model of spms, and found that laccer synthase (b galt ) is upregulated during disease progression by astrocytes, and that its inhibition halts the progressive phase of the disease, attenuating cytokine and chemokine production by the astrocytes, and reducing the recruitment of inflammatory ly c high monocytes to the cns. moreover, it also skewed the microglia and monocytes towards a m phenotype, and shifted the astrocyte phenotype to support re-myelination and axonal growth. in vitro experiments, using primary cells demonstrated that the inhibition of laccer synthesis directly inhibits cytokines and chemokines production in astrocytes. importantly, similar results were also obtained with human astrocytes, reinforcing the biological importance of our findings. in summary, we identified a novel lipid-signaling pathway that promotes the activation of local cns immunity and neurodegeneration in experimental spms and is a potential therapeutic target for spms. converging clinical studies report an increased prevalence of comorbid neuropsychiatric symptoms, particularly major depressive disorders, in a number of conditions (e.g., aging, obesity) and diseases (e.g., atherosclerosis, congestive heart failure, rheumatoid arthritis) sharing inflammation as a common denominator. by using an experimental approach in mice exposed to innate immune system (iss) stimulation, we demonstrated that induction of depressive-like behavior is mediated by cytokine-induced brain activation of a tryptophan-catabolizing enzyme, the indoleamine , -dioxygenase (ido). in the metabolic syndrome (mets), a widely accepted concept that identifies a cluster of individual risk factors for type diabetes and cardiovascular disease (including obesity, metabolic dysregulations and basal low-grade inflammation), neuropsychiatric symptoms emerge as significant factors for aggravation of the disease and related outcomes. recently, we demonstrated in a model of mets, the diabetic and obese db/db mice that cognitive alterations and increased anxiety-like behavior are related to hippocampal inflammation. on the other hand, depressive-like behavior is not affected by basal inflammation displayed by db/db mice. given the data linking increased depressive-like behavior and ido activation by cytokines in conditions of iss stimulation, the question arises as to whether depressive-like behavior is increased in those conditions in db/db mice. to answer this question, we measured in db/db mice and in their healthy db/ littermates the effect of a lipopolysaccharide (lps) challenge ( mg/mouse, ip) on behavioral reactivity in a depression test (the forced swim test, fst), plasma levels and hippocampus expression of inflammatory cytokines and related neuronal targets, and brain ido activity. plasma levels and hippocampus expression of il- b, il- , tnfa and il- are similarly increased h after lps in both db/ and db/db mice. as expected, brain ido activity and duration of immobility in the fst are increased in lps-treated db/ mice h after lps. on the contrary, induction of brain ido activity is significantly blunted in db/ db mice compared to their db/ counterparts and no increase of depressive-like behavior is observed. moreover, some data suggest an impairment of the neuroimmune interactions in db/db mice. we need now to understand how obesity and related neurobiological alterations impair ido activation by cytokines and their consequences in terms of vulnerability to infections in mets. in this study, we tested it in a collagenase-induced mouse intracerebral hemorrhage (ich) model using tlr knock-out (ko) mice. to induce ich, collagenase or blood was injected into the right caudate putamen in - week old male mice. tlr expression was upregulated in the ipsilateral hemorrhagic tissues of the collagenase-injected mice. brain injury volume and neurological deficits following ich were reduced in the tlr ko mice as compared to the wild type (wt) mice. heterologous blood-transfer experiments show that tlr signaling in the brain-resident cells, but not leukocytes, contributes to the injury. in our study to elucidate underlying mechanisms, we found that damage in the blood-brain barrier (bbb) integrity following the ich was attenuated in the tlr ko mice compared to the wt mice, which may be due to reduced mmp- activation in brain astrocyte. the reduced bbb damages accompanies with reduced neutrophil infiltration and proinflammatory gene expression the injured brain parenchyma, which may account for the attenuated brain damage in the tlr ko mice after ich. conclusively, these data demonstrate that tlr contributes to brain injury following ich by compromising bbb through activating mmp- in brain. the pathological relevance of this autoantibody response is unknown, but it is generally assumed that it exacerbates demyelination via activation of complement and/or cell meditated effector mechanisms. however in a majority of cases the mog-specific autoantibody titre is far lower than that required to induce widespread demyelination and exacerbate disease severity in animal models of ms. to explore the possibility mog-specific antibodies may play other more subtle roles in disease pathogenesis we investigated possible effects in myelinating cultures derived from embryonic rat spinal cord; a model system that allows us to explore antibody-dependent effects in the absence of exogenous complement and effector cells. myelinating cultures were treated continuously with monoclonal antibodies specific for either mog (clone z , igg a), sulphatide (clone o , igm), proteolipid protein (plp) (clone o , igm), or an appropriate isotype control from days in vitro onwards. in the absence of an exogenous source of complement none of these antibodies induced demyelination, but by div had all had a significant inhibitory effect on myelination compared to cultures treated with appropriate isotype control antibodies. to investigate possible mechanisms contributing to this inhibitory effect qpcr arrays were used to determine if this complement-independent effect on myelination was associated changes in expression of immune mediators. unexpectedly we found recognition of antigens exposed at the surface of the myelin sheath induced a rapid increase in expression of three chemokines known to be involved in recruitment of effector t cells into the cns. within hours of adding antibody to the cultures expression of ccl , ccl and cxcl increased by at least three orders of magnitude and then declined to baseline over the following five days despite continuous presence of antibody. this transient increase in mrna transcripts for these cytokines resulted in sustained protein synthesis and secretion of biological active products as demonstrated by analysis of culture supernatants. these results challenge the traditional view that myelin-specific autoantibodies contribute to the pathogenesis of diseases such as ms and adem by virtue of their ability to initiate immune-mediated demyelination. we now demonstrate that even in the absence of recruited immune effector mechanisms myelin-specific autoantibodies not only inhibit myelination but trigger secretion of chemokines predicted to trigger or exacerbate inflammation within the cns. the blood-brain barrier (bbb), comprising specialized brain endothelial cells, is crucial in maintaining a controlled environment within the central nervous system (cns) to safeguard normal neuronal function. in multiple sclerosis (ms) the function of the bbb is disturbed, leading to uncontrolled influx of metabolites and immune cells and neuroinflammation. therefore, restoring the function of the bbb may be a novel therapeutic tool to halt disease progression or new lesion formation. we previously showed the importance of the vitamin a metabolite retinoic acid (ra) in bbb function during cns development , where fetal astrocyte-derived ra is needed to induce bbb function during cns embryogenesis. considering the possible involvement of developmental pathways that could regenerate the disrupted bbb, we investigated the possible role for ra in the protection or reinstatement of disrupted bbb function. we show that ra counteracts the deleterious effects of inflammatory cytokines such as tumour necrosis factor (tnf)-a and interferon (ifn)c on bbb function in vitro. moreover, ra diminishes the induction of inflammation-related genes in human brain endothelial cells, and induces general immune quiescence in resting brain endothelium. our preliminary data as well as a recently described study in the cuprizone animal model for demyelination suggest that ra synthesis in the cns is increased under inflammatory conditions. the impact of cns-derived ra on the inflamed bbb, as well as the cellular source is currently under investigation. insight into the ra-synthetic pathway and its protective effect on the bbb may lead to the development of novel therapies which are aimed to restore bbb function and reduce the inflammatory cascade in ms lesions. tissue transglutaminase (tg ) is a multifunctional enzyme whose expression and activity is enhanced during inflammatory processes. we previously observed that the expression of tg is increased in monocytes in lesions in post-mortem material of ms patients. moreover, tg activity and expression is enhanced in chronic-relapsing experimental autoimmune encephalomyelitis (cr-eae) in rats. in this same ms model, pharmacological inhibition of tg activity dramatically reduced clinical symptoms and attenuated the influx of monocytes. in the present study we question whether tg plays a role in mouse eae, as transgenic mice have to be used for subsequent in vivo imaging of leukocytes/monocytes during eae. methods and results: eae was induced in tg -/mice and littermate wildtype mice (c bl/ ) using mog - . during the early phase of disease (day - ), the clinical symptoms observed were significantly less severe in tg -/compared to the wildtype mice. moreover, the maximal clinical score was significantly lower in the knockout mice. there was no difference in the day of onset of disease symptoms between the two groups. secondly, we aimed at visualizing leukocytes/monocytes in the spinal cord of lysm-gfp and cd c-yfp mice suffering from mog - induced eae using intravital video microscopy. a permanent window was fixed on top of the spinal cord of mice to allow reimaging of the same field of view in the same mouse over the disease course. in a pilot experiment we observed numerous gfp positive cells in the white matter around the imaged blood vessel in the spinal cord of a lysm-gfp eae mouse. although equally present, this was less prominent in a cd c-yfp mouse suffering from eae. subsequent immunohistochemical analysis revealed that various cell types had infiltrated the spinal of cord of both mice. outlook: to further address the role of tg in the infiltration of leukocytes/monocytes into the spinal cord of eae mice in vivo, specific inhibitors for tg activity will be administered to the transgenic mice and leukocytes/monocytes will be visualized using intravital video microscopy. z. muneer , c. wiesinger , g. regelsberger , j. berger , s. forss-petter medical universty of vienna, center for brain research, vienna, austria medical university of vienna, institute of neurology, vienna, austria x-linked adrenoleukodystrophy (x-ald) is the most frequently occurring peroxisomal neurodegenerative disorder and is often associated with cerebral demyelination and inflammation. x-ald is caused by mutations in the atp-binding cassette sub-family d member (abcd ) gene encoding adrenoleukodystrophy protein (aldp), which is a transporter for coa esters of very long-chain fatty acids (vlcfa) across the peroxisomal membrane. adrenoleukodystrophy related protein (aldrp), another member of the abcd sub-family, encoded by abcd , is the closest homologue of abcd . upon over-expression, abcd has been shown to compensate for abcd deficiency in vitro and in vivo. several lines of evidence imply an important role of microglia/macrophages in the disease progression. here we used mouse peritoneal macrophages (mpmu) to correlate the gene expression levels of candidate modifiers with x-ald associated defects, like accumulation of vlcfa and decreased peroxisomal b-oxidation, in abcd and abcd single ko mutants as well as in abcd /abcd double deficient (doko) mice. by quantitative rt-pcr analysis, in mpmu from wild type mice the abcd mrna was present at about half the level of the abcd mrna. abcd ko mpmu showed elevated accumulation of vlcfa (c : ) compared to the mpmu from wild type mice as measured by gc-ms. the vlcfa levels of abcd ko mpmu were similar to wild type amounts. interestingly, there was much higher accumulation of vlcfa in the mpmu from doko mice. there were no differences in the mrna expression level of the elovl gene, encoding the enzyme catalyzing the elongation step of vlcfa biosynthesis. peroxisomal boxidation activity was decreased in abcd ko mpmu compared to wild type level. this defect was strongly enhanced in mpmu from abcd / abcd doko mice. these results show that the increased accumulation of vlcfa in mpmu from doko mice as compared to abcd ko mice was due to a more severe defect in peroxisomal b-oxidation in doko mpmu. we conclude that a substantial expression of abcd mrna prevents a more severe metabolic phenotype in abcd deficient mouse peritoneal macrophages. this study also supports the validity of abcd up-regulation as a potential therapeutic target in x-ald. j. claude, b. linnartz-gerlach, h. neumann reconstructive neurobiology, bonn, germany microglia have innate immune receptors recognizing pathogens and disease-associated molecular patterns but also molecules that could sense the intact tissue. a subfamily of these receptors is the inhibitory signaling sialic acid-binding immunoglobulin-like lectin (siglec) group including siglec-e that has an immunoreceptor tyrosine-based inhibitory motif (itim) in the cytoplasmic tail to suppress activatory microglial signals. in this study, we used primary and stem cell-derived microglia that were modified by lentiviral vectors. here we show that siglec-e is expressed on microglia and is up-regulated following interferon-c (ifnc) treatment. we performed lentiviral knock-down and overexpression of siglec-e. lentiviral overexpression of siglec-e decreased, while knock-down increased the phagocytosis of neural debris and its associated reactive oxygen burst. the extracellular domain of siglec-e linked to a fc-part of immunoglobulin bound to the sialic acid residues of the neuronal glycocalyx. therefore, we co-cultured these modified microglia with primary hippocampal neurons. overexpression and knock-down of siglec-e showed an increase and decrease in relative neurite-length, respectively. the neuroprotective effect of siglec-e was abrogated after removal of the sialic acid residues on the neuronal glycocalyx. treatment with the anti-oxidant trolox abolished the neurotoxic effect of the siglec-e knock-down on neurite length. in summary, our data suggests an immunomodulatory function of siglec-e on microglia which leads to a neuroprotective phenotype by decreasing the production of reactive oxygen species and a reduced phagocytosis rate of neural debris. a. nadjar, c. madore, a. sere, a. aubert, s. lay e university of bordeaux , bordeaux, france many epidemiological studies have linked maternal exposure to infections during pregnancy to later development of cognitive disorders in the descendants. these alterations are likely to originate from a generalized neuroinflammation in the fetal brain following activation of the maternal immune system. this neuroinflammatory response relies on microglial cells activation. these latter normally contribute to the development of neuronal networks especially by eliminating/maintaining synapses, a phenomenon also known as synaptic stripping, in optimal conditions of brain development. neonatal inflammation may alter the synaptic stripping capacity of microglial cells. thus, targeting this persistent inflammatory microglial activation could represent an original and promising strategy to improve cognitive performances in the descendants. omega- are known as immunomodulators and target microglial cells. we thus hypothesized that enriching the diet with omega- from the first day of gestation may impair the development of neurological deficits at adulthood, through limitation of microglial inflammatory response in favor of its synaptic stripping activity. to characterize the beneficial effects of omega- on microglial activity, pregnant mice were fed with an omega- -deficient diet, or a balanced omega- / omega- diet or supplemented with omega- and received a peripheral injection of either lps or saline at e of gestation. using the golgi method, we first evaluated the morphology of dendrites and quantified the dendritic spines density as a measure of neuronal networks maturation in the hippocampus. we found that a prenatal exposure to lps increased the number of immature spines and this was reversed by an omega- supplemented diet. to correlate these data with alterations of microglia-neurons interactions, we took advantage of the cx cr -gfp mice and injected them with a neuronspecific lentivirus containing dsred protein, in the hippocampus, days prior to two-photon experiments. we focused on microglia general dynamic and microglia-neuron physical interactions and found an alteration in microglial behavior and microglia-neurons interactions in pups from lps-treated females. these alterations were reversed by an omega- supplemented diet. overall, our data show that alterations of microglia-neurons interactions in the developing brain may explain the neurological disorders observed in the descendants of females with prenatal inflammation. these deleterious effects may be prevented by nutritional strategies. , which is characterized by a relapsing phase with inflammatory cell infiltrates and a remitting period, where patients partially recover. among the different cell types involved in the necessary immunomodulation to allow the relapsing-to-remitting transition, the role of myeloid-derived suppressor cells (mdscs) gains importance. mdscs form a heterogenic population of immature myeloid cells that is able to suppress the inflammatory response. this cells act, among other mechanisms, through arginase-i (arg-i) activity on tcells. a previous study of our group showed that arg-i -mdscs transiently enter the spinal cord of eae mice and takes part in the immune response control by inducing t cell apoptosis around the peak of the clinical score. therefore, changes in the mdsc population during eae should modify the evolution of the disease. the retinoid acid family molecules are used for the treatment of different leukaemia due to their role as mdsc differentiation factors into diverse cell populations, abolishing t cell immunosuppression. am , a synthetic analogue of the retinoic acid with a higher bioavailability and less side effects than the natural ones, has controversial actions on eae depending on its administration period. in this work, we administered am specifically in the critical moment for immune modulation (around the maximum clinical score). drug administration affected mdsc population and clearly worsened the eae course. our results point to endogenous strengthening of mdsc population as a new and promising therapeutic strategy to treat ms by speeding up the transition from the relapsing to the remitting period. sildenafil induces cgmp accumulation by pde inhibition. we have demonstrated that sildenafil decreases microgliosis and astrogliosis and proinflammatory cytokines expression in a demyelination model. however, little is known about mechanisms of sildenafil neuroprotection. since nfjb plays an important role in the regulation of glial activation, we examine the hypothesis that nfkb is part of the mechanism underlying the sildenafil neuroprotective effects. five male mice (c bl/ ), weeks-old, were used per group. the groups received for four weeks: . % cuprizone (cpz) mixed into the chow; cpz into the chow plus sildenafil (viagra v r , pfizer, mg/kg) in the drinking water, starting concomitantly (sild-t ) or fifteen days (sild-t ) after initiation of cpz; controls received pure chow/water. cerebella were processed for western blotting and immunofluorescence (if). results showed that cpz increased gfap expression compared to control (astrocytes activation). sild-t (but not sild-t ) significantly decreased gfap compared to cpz group. in controls, microglia showed resting phenotype, with thin and branched processes positive to iba /nfjbp (inactive fraction) double labeling. cpz treatment decreased inactive-nfjb expression, indicating that this transcription factor was activated. in agreement, ikba (nfjb inactivating protein) was also decreased. if showed increase of iba- expression, indicating microglia activation. sild-t strongly increased the nfjbp and its inhibitory protein, ikba, suggesting inactivation of nfjb. if showed decreasing in iba- labeling, suggesting inactive microglia. the delayed treatment (sild-t ) did not decrease iba- or increase inactive-nfjb and ikba expression. in conclusion, treatment with sildenafil concomitant with cpz exposure prevents micro-and astrogliosis in mice, possibly through ikba-nfjb signaling pathway. sildenafil may be suitable to improve neuroinflammatory/neurodegenerative diseases treatment. financial support: cnpq, capes, facepe, fapesp. mt- & levels have been found to be increased in several human neurodegenerative diseases including alzheimer's disease (ad). moreover, mt are also upregulated in different ad mouse models, for example tg mice, which show a significant up-regulation of these proteins in the vicinity of the amyloid plaques. in the present study we generated a double transgenic mouse line that develops ad-like pathology in addition to having an overexpresion of mt , in order to determine the role of mts in different aspects of ad pathology. the results show that the overexpression of mt does affect the mortality rate of the tg and control mice in a gender-dependent manner and partially reverses the behavioural phenotype of young ( - months) tg mice, reducing the exploratory activity and improving the learning process; in contrast, mt does not cause relevant changes in deambulations and anxiety. on the other hand, the amyloid cascade and neuroinflammation is increased in the hippocampus of old ( - months) apptgmt mice, but the lower level of gliosis in the hippocampus of young male apptgmt mice suggests that the overexpression of mt is capable of reducing inflamatory response well before amyloid plaques are formed but not afterwards. further molecular and immunohistochemistry analyses are underway to give further insight into the role of mts in amyloidosis and neuroinflammation in this ad mouse line. exposure to prenatal inflammation is a risk factor for neurodevelopmental and neurobehavioural abnormalities that manifest in later life in disorders such as autism, schizophrenia and seizure development. the amygdaloid complex is composed of more than nuclei with subdivisions that have different cytoarchitectonic, chemoarchitectonic, and projection characteristics and that are responsible for the processing of higher functions such as memory consolidation and emotional conditioning. it is also known that the basolateral nucleus of the amygdala is implicated in seizure propagation and initiation. seizure onset later in life may be linked to abnormal development of the amygdalaoid complex. neuropeptide y (npy) and its receptors are known to be involved in a number of important functions such as feeding behaviours, anxiety and seizure modulation. npy y and y receptors are the most abundant in the brain and are expressed at different stages of development. during foetal development different brain regions are populated by neurones and glia at different times and the interface between mother and foetus plays a crucial role in the development process. lipopolysaccharide (lps) induced maternal inflammation is a known animal model for studying the inflammatory effects on the developing foetus. in this study we are investigating the effects of an acute prenatal systemic insult using the lps model on amygdala morphology and structure. embryonic day (e ) c bl j pregnant mice receive an intraperionteal injection of lps ( mg/kg) or . % sterile saline. embryos at e , e , e and pups at p , p p and p are taken and used to study the developing hippocampus and amygdala. for each age and treatment group foetal brains, maternal brains, maternal serum and placentas are collected. incidence of preterm birth, resorption rate, litter size and weights are also assessed. immunohistochemistry, western blot and rt-pcr are then used to assess the expression of neuronal and glial markers in the amygdala and surrounding limbic structures. specifically alterations in expression, activation and distribution of npy and its receptors, microglia and astrocytes in the developing amygdala and hippocampus at different stages following lps treatment are being investigated. it is hoped that this study will further enhance our understanding of how the maternal environment influences brain development and, if disturbed at a critical time in the development of the amygdala, may predispose individuals to amygdala related disorders in later life. in vivo imaging of transgenic fluorescent mice in the cns by twophoton laser-scanning microscopy ( p-lsm) has become a powerful tool in neuroscience. for in vivo imaging of the cortex a craniotomy of the skull has to be made. in addition to anaesthesia and analgesia treatment, we occasionally apply anti-inflammatory drugs to prevent immunological activity that can obscure the images. as anti-inflammatory drug we used carprofen ((rs) -( -chloro- h-carbazol- -yl)propanoic acid), a known cyclooxygenase- (cox- ) inhibitor. adult cx cr egfp mice in which microglia are labeled by expression of the green fluorescent protein egfp were treated with a single dose of carprofen or with vehicle hours before the craniotomy. in all mice we exposed the right somato-sensory cortex, and quantified the microglial response to a laser-induced micro-lesion. this micro-lesion was caused by increasing the power of the laser for second in the middle of the region of interest. results -conclusions unexpectedly, we observed that carprofen reduced the microglial process motility significantly. the speed with which the processes approached the lesion dropped from . mm per minute in the untreated mice to . mm per minute in the treated mice. a molecular understanding of microglia response in inflammatory processes and how anti-inflammatory drugs modify normal microglia response will provide a strong impact in developing treatment strategies for diseases with strong inflammatory components. resting and activated (lipopolysaccharide, lps) cultured cstb-/-and control microglia were analyzed for cytokine production, chemotaxis and phagocytosis. microglia extracted directly from the mouse brains were studied for expression of mhc ii and m -m polarization using flow cytometry. mouse brain cortex (p ) was analysed for m -m microglial phenotypes and the presence of other immunological cells from the periphery. moreover, we have checked myeloid cell population in bone marrow and spleen of p animals as well as cytokines' level in blood serum. our results from cultured microglia show that secretion of proinflammatory chemokines is elevated by cstb-/-microglia. also, cstb-/microglia are chemotactically more active compared to the controls whereas their phagocytic activity is decreased. cstb-deficient microglia show decreased expression of mhc ii on the cell surface indicating reduced antigen presentation. activated cstb-/-microglia directly extracted from the brain has predominantly proinflammatory m phenotype. cstb-/-mice display inflammatory changes in the peripheral tissues at their early postnatal period. we have registered high concentrations of proinflammatory chemokines and cytokines in blood serum of p cstb-/-pups, but no difference in amount of neutrophils and lymphocytes between brains of wild-type and knockout mice. also, in bone marrow and spleen amount of granulocytes is enhanced in cstb-/mice during early postnatal period as well as level of granulocyte macrophage colony-stimulating factor in their blood serum. our results suggest a role for cystatin b in regulation of immune response and that cstb-deficiency is associated with early inflammatory processes both in the brain and the peripheral tissues. therefore, we consider that epm should be treated as a disorder combining neuropathological and immunological features. the contribution of glial cells in the pathophysiology of epilepsy is increasingly valued. furthermore, clinical and experimental evidence suggests a direct relationship between epileptic activity and cns inflammation, which is characterized by accumulation, activation and proliferation of microglia and astrocytes. concomitant glia-mediated mechanisms of action of several aeds have been proposed. however, their direct effects on glial cells, especially microglia, have jet been hardly investigated. we aimed to investigate the influence of commonly used aeds on the glial viability and microglial in-/ activation state in a physiological and inflammatory modified in-vitro astroglia/ microglia coculture model. methods:primary astrocytic cultures were prepared from brains of postnatal (p -p ) wistar rats and cocultured with a physiological amount of % (m ), as well as % (m ) microglia in order to mimic inflammatory conditions. cocultures were treated for hours with valproic acid (vpa), carbamazepine (cbz), phenytoin (phe) and gabapentine (gbt) with a concentration of , , and mg/ml. viability and proliferation was measured using the tetrazolium (mtt) assay. the microglial activation state was determined by immunocytochemical labeling using a monoclonal antibody to the ed marker. results:m and m cocultures showed a dose-dependent, significant reduction in glial viability after incubation with phe and cbz. furthermore, low doses of vpa led to highly significant microglial activation in m cocultures. however, cbz significantly reduced the amount of activated microglial cells and increased the total number of inactivated microglia in the inflammatory modified m cocultures. conclusion: cns inflammation is characterized by a disturbance of glial cell functions. strong microglial activation, a typical hallmark of inflammation, was induced by vpa in m and continued in m cocultures. with regard to the direct relation between cns inflammation and seizures, vpa seems to be unsuitable to reduce inflammatory condition. the reverse effect was achieved after cbz. we noticed significant microglial inactivation, after incubation of the m cocultures. as it has been demonstrated for levetiracetam before, we assume a beneficial therapeutic effect of anti-epileptic drugs (aed) with an antiinflammatory glial potential in epileptic patients with persistent inflammation. microglial cell activation due to homeostatic imbalances of external and/or internal etiology implies, among others, secretion of proinflammatory cytokines. the fact that microglial activation, inflammation and neurodegeneration often coexist, suggests that proinflammatory cytokines might induce and/or enhance neuronal damage. we investigated the neurotoxic impact of microglial activation in organotypic hippocampal slice cultures by exposing them to the toll-like receptor ligand, lipopolysaccharide (lps), for hours. microglial activation was characterized by unbiased estimation of their number (stereology) and quantification of soma and branching morphology (neurolucida v r -based cell reconstructions). moreover, proinflammatory cytokine and nitrite levels in the culture supernatant were estimated by elisa and griess. the neurotoxic impact was assessed by morphology (nissl staining, fluoro-jade v r b) and extracellular electrophysiological recordings of spontaneous and evoked neuronal activity in the hippocampal ca subregion. lps exposure induced microglial population expansion, morphological changes, such as process thickening and somatic enlargement, as well as substantial secretion of proinflammatory cytokines (tnfa, il ) and nitrite accumulation in the culture medium. however, these changes did not coincide with neurodegeneration and were associated with only minor effects on neuronal function, as demonstrated by the amplitude of evoked neuronal responses and short-term plasticity properties. we conclude that, in contrast to what has been observed in vivo, chronic microglial activation by lps is not sufficient to drive neuronal death and dysfunction in organotypic hippocampal slice cultures. the neonatal brain is particularly susceptible to oxidative stress. our group has previously shown that following hypoxic-ischemic injury, hydrogen peroxide (h o ) levels rise significantly particularly in the neonatal brain and are sustained for up to hours. this rapidly accumulated h o is detrimental in the iron-rich immature brain as it can lead to the generation of dangerous free radicals that can cause extensive injury. to date, there is limited literature on the effects of increased h o levels, particularly on microglial cells, which have been extensively indicated in the mediation of ensuing injury. here we describe the effects of a continuous exposure of microglia to h o , as generated using the glucose oxidase-catalase (gox-cat) system. this system allows us to generate and continuously maintain for up to h pathophysiological levels of h o > um. microglial cultures were derived from the p mouse brain and exposed to either bolus concentrations of h o [ - mm] or varying concentrations of gox-cat for varying lengths of time. conditioned medium was collected from cells at , and h of treatment and analyzed for secreted cytokine levels using the -plex cytokine microbead array kit. treated cell extracts were processed for protein and fixed cells were labeled with m and m a phenotype markers. continuous exposure to very low levels of h o can produce - -fold higher (over control) pro-inflammatory cytokine protein levels of il , il , il p , il p , il , il and ifng and at least a -fold higher level of il a, il b, il and tnfa by h. anti-inflammatory cytokine (il , il and il ) and chemokine (rantes, g-csf and gm-csf) protein levels were also increased by h. interestingly, no prominent cytokine responses were seen with bolus treatment at any of the time points studied. low, continuous h o promoted a predominantly m a microglial phenotype by h. we conclude that studying continuous exposure effects will help delineate the various specific effects h o can have on microglial cells. these specific effects can then be used to clarify when and how microglial cell responses can be beneficial or detrimental following injury in the immature brain. we have previously shown that natural ( -deoxy-d , prostaglandin j , d) and synthetic (pioglitazone) agonists of peroxisome proliferator activated receptor-c (ppar) potentiate intrinsic cellular mechanisms protecting oligodendrocyte (ol) progenitors (ops) from oxidative insults and promote their differentiation to ols. in addition, ppar-c agonists potentiate mitochondrial activities, as the mitochondrial respiratory chain activity and the regulation of cytoplasmic ca waves, which are known to be crucial for ol differentiation. in the present study we sought to investigate whether ppar-c agonists can protect ol cultures from conditions causing mitochondrial stress. first, to specifically induce a mitochondrial impairment, we used the complex i inhibitor rotenone. as expected rotenone, at concentration not affecting cell viability, significantly inhibited ol differentiation, as indicated by the reduced number of cells expressing specific markers of differentiation (o and o ). in ppar-c agonist-treated ols the inhibitory effects of rotenone were significantly attenuated, suggesting a protective effect of the agonists against the mitochondrial toxin. we next examined a condition mimicking inflammatory stress by challenging op cultures with tnf-a, an inflammatory cytokine known to retard the differentiating program of ops. in parallel with the expected reduction of the percentage of o and o positive cells, the cytokine induced a significant reduction of mitochondrial membrane potential (mmp), suggesting an impairment of the mitochondrial functions. the simultaneous treatment with tnf-a and ppar-c agonists ( d or pioglitazone) significantly reverted both tnf-a dependent reduction of ol differentiation and mmp. at the molecular level, we found that in op cultures, ppar-c agonists increased the expression of the uncoupling protein- (ucp- ), a mitochondrial protein known to contribute to the protection of mitochondria against oxidative stress. these findings suggest that ppar-c agonists protect ols and promote myelination through several mechanisms, including those involving mitochondrial functions. our studies support the therapeutic potential of ppar-c agonists in brain diseases in which mitochondrial alteration, oxidative stress and demyelination occur and point to the need to better understand the role of ppar-c and its agonists in ol biology. it is characterized by lesions of demyelination and inflammation, which can be classified according to the activation state of the microglia/macrophages. well before any myelin and blood-brain barrier damage clusters of activated microglia are noticeable. these clusters are considered to be the first stage of lesion formation and therefore called 'pre-active lesions. however, they do not always develop into demyelinating lesions. here we postulate that stressed oligodendrocytes play an crucial role in pre-active lesion formation, by producing factors that will attract and activate microglia to either a more pro-inflammatory (m ) or antiinflammatory (m ) activation status. this can contribute to the amount of damage to the environment and further lesion formation. this study sets out to identify the best method to mimic this early lesion formation in vitro. first, human primary isolated microglia are stimulated to either a m or m phenotype and characterized by marker expression and cytokine profile. oligodendrocytes are isolated out of the human brain. medium of (stressed) oligodendrocytes will be used as attractant as well as activator for microglia. by immunohistochemical staining the activation status of microglia in pre-active lesions will be determined. preliminary data reveal that human microglia are able to be skewed towards a pro-and anti-inflammatory phenotype. first experiments of primary human oligodendrocyte cultures, showed pure oligodendrocytes. thus far our data support our hypothesis, however further research is required to make our conclusions more robust. question: although cell transplantation is increasingly suggested to be beneficial for the treatment of various neurodegenerative diseases, the therapeutic application of such intervention is currently hindered by the limited knowledge regarding central nervous system (cns) transplantation immunology. in this study, we aimed to investigate the early post transplantation innate immune events following grafting of autologous mesenchymal stromal cells (msc) in the cns of immune competent mice. methods and results: first, the survival of grafted luciferase/egfpexpressing msc (msc-luc/egfp) was demonstrated to be stable from on day post implantation using in vivo bioluminescence imaging (bli), which was further confirmed by quantitative histological analysis of msc-luc/egfp graft survival. additional histological analyses at week and week post grafting revealed the appearance of (i) graftsurrounding/-invading iba microglia and (ii) graft-surrounding gfap astrocytes, as compared to day post grafting. while the density of graft-surrounding astrocytes and microglia did not change between week and week post grafting, the density of graft-invading microglia significantly decreased between week and week post implantation. however, despite the observed decrease in microglial density within the graft site, additional phenotypic analysis of graftinvading microglia, based on cd b-and mhcii-expression, revealed > % of graft-invading microglia at week post implantation to display an activated status. although microglial expression of cd b and mhcii is already suggestive for a pro-inflammatory m -oriented phenotype, the latter was further confirmed by: (i) the expression of nos by microglia within the graft site, and (ii) the absence of arginase -expression, an enzyme known to suppress no activity in m oriented microglia, on graft-surrounding and -invading microglia. conclusions: in summary, we here provide a detailed phenotypic analysis of post transplantation innate immune events in the cns of mice, and warrant that such intervention is associated with an m -oriented microglia response and severe astrogliosis. it is well known that cell surface immune receptors play a critical role in regulating immune and inflammatory processes in the central nervous system (cns). cd f is a novel immunoreceptor that dampens inflammatory reactions in experimental autoimmune encephalitis (eae) and diverse allergy models. we have analyzed the function of cd f immunoreceptor in an nmda excitotoxicity model and in a traumatic brain injury model (tbi). for this purpose, we determined the pattern of expression of both cd f and its putative ligand(s) in the cns, as well as the neuroprotective role of cd f after both acute brain injuries. first, we used a human and rat cd f-ig soluble protein to show by confocal microscopy the presence of endogenous ligand(s) for this receptor mainly in cns white matter oligodendrocytes and on the surface of oligodendrocytes, fibrous astrocytes and neurons in vitro. interestingly, when we analyzed the in vitro expression pattern of rcd f in brain cells by q-pcr and immunohistochemistry, in addition to the expected expression in microglial cells, we detected expression of cd f in oligodendrocytes and neurons. in vivo, after a tbi, cd f is expressed at early timepoints ( d) in small round macrophages, and at later time-points ( d) in neurons of the penumbra. q-pcr studies showed significant upregulation of the mrna for rcd f at and d after tbi. interestingly, a delayed ( hours after the lesion) intraparenchymal injections of a non-viral modular recombinant gene therapy vector termed nlsct overexpressing hcd f induced a decrease in the lesion volume d after nmda injection or tbi when compared to control gfp over-expression. in addition, the over-expression of hcd f decreased the long-term sensitive functional deficits observed by the "sticky tape" test. in order to validate these results with the endogenous molecule, we cloned the rat ortholog of cd f protein. on-going positron emission tomography (pet) studies using -[ f]-fluoro- -desoxiglucosa ( f-fdg) are being used to evaluate the long-term functional recovery after tbi. the overexpression of rcd f receptor had a comparable neuroprotective effect as the human molecule after nmda injection. these data suggest that cd f may be important in neuron-glia inflammatory and trophic interactions. in addition, our results suggest that delayed cd f overexpression by means of a modular recombinant gene therapy may constitute an interesting therapeutic strategy for acute brain injuries. depending on the type of signals produced after an inflammatory event, microglia develops specific activities, including cytotoxic, neuroprotective or phagocytic activities in order to come back to brain homeostasis. as described for macrophages, microglia can adopt m (proinflammatory) or m (phagocytic) phenotypes distinguishable through their pattern of factors expression and specific cell surface markers. interestingly, polyunsaturated fatty acids (pufas) are precursors of bioactive lipid messengers involved in the regulation of inflammation and in particular, docosahexanoic acid (dha, : n- ) an n- pufa, presents anti-inflammatory properties. the aim of our study was thus to investigate the effect of an increase of n- pufas on the inflammatory response and cognitive abilities after a peripheral immune challenge. to increase n- pufas, we took advantage of transgenic mice carrying the fat- gene from the roundworm caenorhabditis elegans. this fat- transgenic mouse is capable of producing n- fatty acids from the n- type endogenously, eliminating confounding factors of the diet. this conversion leads to abundant n- fatty acids with reduced levels of n- fatty acids in tissues, including the brain. to induce a neuroinflammation, we injected mice intraperitoneally with lipopolysaccharide (lps), components of gram negative bacteria walls. we first studied the microglial phenotype h after lps injection and found that microglia in fat- mice presented a significant increase of m phenotype markers. we then measured cytokine expression in the hippocampus after a lps challenge and found a significant decrease in il- b mrna in fat- mice compared to wildtype littermates. in terms of hippocampal memories abilities, only control mice presented a deficit in the y-maze task whereas fat- mice were able to perform the test correctly. our results indicate that h after a lps injection, fat- mice presented a decreased of the inflammatory response and normal hippocampal memory abilities compared to wild-type littermates. glia integrity and homeostasis as they have prominent role in blood brain barrier formation and maintenance, as well as in active regulation of an immune response. however, astrocytes are affected in neuroinflammation, when their protective roles might be suppressed and when they might be shifted towards pro-inflammatory phenotype. experimental autoimmune encephalomyelitis (eae) is a widely used model of autoimmune neuroinflammation. chemokine cxcl produced by astrocytes and endothelial cells has profound anti-inflammatory and anti-encephalitogenic role in eae. nitric oxide (no) produced by inducible no synthase (inos) within the cns is considered as disease promoting, while interleukin (il)- as protective in eae. the aim of this work was to investigate effects of no and il- on cxcl gene expression in astrocytes. methods: astrocytes were stimulated with supernatant collected from concanavalin a-stimulated splenocytes (conasn) and simultaneously exposed to no donor, sodium-nitroprusside (snp) or anti-il- antibody. also, astrocytes were stimulated with ifn-c il- in the absence or presence of il- . peritoneal macrophages isolated from healthy rats were co-cultivated with astrocytes. after h incubation period cxcl gene expression in astrocytes was measured by rt-qpcr. phosphorylation of p mapk and nf-jb was assessed by immunoblot in astrocytes exposed to snp. results: we found that no released from snp or macrophages significantly reduced cxcl gene expression in astrocytes. this reduction was mediated by inhibition of p mapk activation, but not of nf-jb signaling. on the contrary, il- stimulated and anti-il- antibody suppressed cxcl gene expression in astrocytes. conclusions: these results imply that expression of cxcl in astrocytes is inversely regulated by no and il- in the central nervous system affected by inflammation. having in mind, profound regulatory role of cxcl in neuroinflammation, these data contribute to our understanding of pro-and anti-inflammatory nature of no and il- , respectively. in the presence of pathological insults, they acquire reactive phenotypes aimed at re-establishing brain homeostasis and minimizing neuronal damage. however, reactive microglia produce several factors, typical of an inflammatory response, with a potential neurotoxic effect. consequently, the progression and resolution of microglial activation have to be tightly controlled to avoid detrimental secondary effects. signals arising from neuronal cells play an important role in the activation state of microglial cells. among them, inhibitory mechanisms, such as neuronal cd and microglial cd r interaction, keep the proinflammatory phenotype of microglia under control. alterations in the expression of cd and cd r have been described in pathological conditions, and the modulation of cd -cd r signalling could be an interesting target to be considered in therapeutic approaches against neuroinflammation occurring in neurodegenerative diseases. little is known on the molecular mechanisms involved in the regulation of cd and cd r expression. the aim of the present work is to study the possible modulation of cd and cd r by ppar-c agonists, and the involvement of cd -cd r in the anti-inflammatory and neuroprotective effects of ppar-c activation. we have used mouse microglial, mixed glial and neuronal cell cultures, as well as neuron-microglia co-cultures. cd r expression was detected in microglial cells, and it was decreased in response to pro-inflammatory stimuli such as lps/ifn-c. the ppar-c agonist -deoxy-d , -prostaglandin j ( d-pgj ) abrogated the inflammatory response in lps/ ifn-c-treated microglial cells and prevented cd r expression inhibition. cd expression was detected in neuronal cultures and, at a lesser extent, in astrocytes in mixed glial cultures. lps/ifn-c-treatment did not modify cd expression in neuronal cultures, but it induced an increase in mixed glial cultures. this increase was inhibited by d-pgj pre-treatment. d-pgj also protected against lps/ifnc-induced neurotoxicity in neuron-microglia co-cultures, but this effect was abolished when cd -cd r interaction was interrupted using an anti-cd r blocking antibody. these results show that ppar-c agonists modulate cd and cd r expression in reactive glial cells, and that cd -cd r interaction is necessary for the neuroprotective effect of ppar-c agonists. to gain insight into the contribution of the cwbc pathway to remyelination in ms, we analyzed the expression pattern of tcf l , a downstream target of the cwbc pathway and coactivatior of b-catenin in ms lesions. tcf l was expressed in ol and astrocytes in a subset of early ms lesions, but was also observed in tissue samples from patients with inflammatory, non-demyelinating diseases. in contrast, in chronic lesions, no expression of tcf l was detected. by wb we found slightly increased b-catenin levels in ms lesions compared to normal appearing white matter. aspirin (ass), a non-steroidal anti-inflammatory drug, attenuates bcatenin signaling activity by inhibition of protein phosphatase a (pp a) via increased phosphorylation. therefore, we determined the effect of ass on ol differentiation and myelin gene expression (mge). as a control for the cwbc pathway activation or inhibition the selective cwbc pathway inhibitor (icg- ) was chosen. icg- binds specifically to cbp and causes a disruption of the b-catenin/cbp interaction. exposure of ol to . mm icg- for hours increased mge and had a positive effect on ol differentiation. in contrast, ass had no positive effect on process formation and did not promote mge in primary murine ol. our results suggest that icg- , but not ass increases the expression of myelin genes. further experiments are required to determine the functional role of the cwbc pathway for ol differentiation and remyelination in ol and ms. (braak et al., ) . furthermore, neuroinflammation, i.e. activation of microglial cells in the substantia nigra (sn), has been widely implicated in pd progression (mcgeer et al., ) . in the present study, we question whether microglial activation occurs in brain regions beside the sn which are affected in pd patients. methods: to this end, we studied microglial activation and protein pathology in the ob and hc of clinically and neuropathologically verified pd patients (braak - ) and control subjects (braak ). human post-mortem formaldehyde-fixed material of pd patients included sn (n ), ob (n ) and hc (n ), and material from age-matched control subjects without neurological deficits included sn (n ),ob(n ), and hc (n ). results: the presence of a-syn pathology concentrated in the anterior olfactory nucleus of the ob and in the ca - region of the hc. furthermore, using cd as a microglial marker, we observed a significant increase in the number of immunopositive microglial cells, with an activated, amoeboid morphology, in the sn as well as in the ob and hc of pd patients compared to control subjects. co-localization studies indicated that these activated microglia were found in the proximity of a-syn inclusion bodies and neurites, but did not co-localize suggesting that the microglial cells do not actively phagocytose a-syn. conclusion: we conclude that microglial activation occurs in other brain regions beside the substantia nigra of pd patients. the functional role of the microglial cells in those brain regions and their contribution to pd pathology remains to be established. in the present study, we question whether ccl and cx cl and its receptors are present in hippocampal lesions of ms patients. to this end, semi-quantitative rt-pcr was performed on cdna transcribed from rna isolated from hippocampi of ms patients and control subjects. moreover, immunohistochemical analysis of ccl , cx cl and its receptors ccr and cx cr was performed on post-mortem, formalin-fixed hippocampal lesions (plp -) from ms patients and on hippocampal material (plp ) from control subjects. ccl and ccr mrna was significantly enhanced in demyelinated ms hippocampal homogenates compared to non-demyelinated and control hippocampal homogenates. cx cr mrna was significantly increased in demyelinated ms hippocampal homogenates compared tot non-demyelinated hippocampal homogenates, but cx cl mrna levels showed no difference between groups. ccl , ccr , cx cl and cx cr immunoreactivity was present mainly in white matter of control and ms hippocampi and was clearly enhanced in hippocampal grey (cornu amonis) and white (stratum lacunosum, stratum radiatum, alveus) matter lesions. the intensity of the ccl and cx cl immunoreactivity was most pronounced when active lesions were present. co-localization studies indicated that ccl and cx cl are present in astrocytes, whereas cx cr and ccr are present in or on microglial cells. we conclude that the chemokines ccl and cx cl and their respective receptors are enhanced in hippocampal ms lesions. as no clear influx of leukocytes is observed in the grey matter lesions, we propose that astrocyte-derived ccl and cx cl , via interaction with their receptors, affect microglial activation and/or function thereby contributing to neuronal dysfunction in the hippocampus as seen in ms patients. , is a kda cytokine-inducible protein, produced by activated macrophages during chronic transplant rejection and in inflammatory reactions. in central nervous system (cns), iba is a sensitive marker associated to activated microglia and is upregulated following neuronal death or brain lesions. iba -like factors have been described in several metazoan and share a well conserved amino acid primary structure throughout evolution suggesting a common, functional role. the medicinal leech hirudo medicinalis is able to regenerate its cns after injury, leading to a complete functional repair. similarly to vertebrates, leech neuroinflammatory processes are linked to microglia activation and recruitment at the lesion site. we investigated the expression of hirudo iba to track the activation state of leech microglial cells involved in nerve repair events. results: we recently identified a gene, named hmiba , coding a . kda protein showing high similarity with vertebrate iba factor. quantitative rt-pcr analyses showed that hmiba is constitutively expressed in cultured nerve chains. a weak down regulation was observed in the days following experimental injury. gene transcripts rise back to basal level one week later. cultured nerve chains stimulated with atp shows a significant increase of hmiba transcript hours after treatment. immunoblot analysis, performed with anti-hmiba polyclonal antibodies, revealed an immunopositive band at the predicted size. the presence of hmiba protein in na€ ıve and experimentally challenged tissues was evaluated by immunohistochemistry. the protein is constitutively present in spread, stellar shaped microglial cells, distributed in connective fibers and in segmental ganglia. a few hours after experimental injury of cns, the amount of immunopositive microglial cells increases at the lesion site and at the cut end of nerve fibers until. the amount of hmiba cells in connectives rapidly increases in atp treated nerve chains. this augmentation is visible in ganglia microglia and in connective fibers, where cells located between axon fibers display an elongated and stretched shape. conclusion: hmiba is a good marker of activated microglia. like in vertebrates, the atp induces its expression in leech cns. also if the functional role of hmiba has to be further elucidated, this molecule appears as a good activation marker of microglia and an interesting tool to study and follow the activity of such cells during nerve repair in leech. hypertension is the single most important risk factor for cardiovascular disease. despite significant advancements, - % of all hypertensives remain resistant to all current available pharmacotherapy. these patients exhibit elevated sympathetic drive, increased norepinephrine spillover, and dampened parasympathetic drive. the dysfunctional autonomic nervous system of these resistant patients indicates a neurogenic origin for their pharmacotherapy resistance. previous studies have proposed that neuroinflammation in the autonomic brain regions plays an important role in neurogenic hypertension. this coupled with the emerging interest in microglia led us to hypothesize that activation of microglial cells in the brain could be a critical event in the initiation and establishment of hypertension pathophysiology. we have previously established that chronic low dose angiotensin ii (ang ii) induced hypertension involves activation of microglia, and increase in proinflammatory cytokines and reactive oxygen species (ros) in cardioregulatory brain areas, such as the paraventricular nucleus of the hypothalamus (pvn). intracerebroventricular (icv) delivery of minocycline, an anti-inflammatory antibiotic that inhibits microglia activation, decreased activated microglia, inhibited increase in cytokines and ros, and attenuated hypertension. in addition, inhibition of brain mitochondrial ros attenuated hypertension, microglial activation, and the overexpression of brain pro-inflammatory cytokines. a time-course experiment demonstrated that there was a significant increase in activated microglia before the increase in blood pressure was detectable by radiotelemetry. furthermore, the origin of microglia in established hypertension appears to be both from resident and bone marrow derivation. these observations indicate that the activation of microglia is a critical player in the development of neurogenic hypertension. they suggest that activation of microglia in cardioregulatory regions of the brain is an early occurrence that may initiates a cascade of signaling events leading to increase sympathetic nerve activity and initiation of hypertension. this research is supported by nih grant (r hl to mkr). our results demonstrate significant inflammatory activation of the neurons and their sgc not only in drg associated but also non-associated with injured nerve. a distinct expression of cytokines and their receptors was identified in sgc surrounding largesized drg neurons. significant inflammatory reactions of sgc were found in all drg of pac-treated rats. moreover, inflammatory activation sgc was also observed in drg of sham-operated rats indicating other kinds of trigger than traumatic nerve injury. the nerve injury and pac-treatment also triggered socs expression in sgc to control stat activation. inflammatory activation of sgc significantly contributes to ectopic activation of the drg neurons not only associated with injured nerve, and is involved in npp induction. aging is associated with reduced function and degenerative changes of the central nervous system (cns). increasing evidence suggests that changes in microglia cells, i.e. the resident macrophages of the cns, contribute to the age-related deterioration of the cns. the most prominent age-related change of microglia concerns enhanced sensitivity to proinflammatory stimuli of microglia in mice, rats and primates, called priming. we have addressed this issue in ercc mutant mice, ercc d/mice, a progeroid, dna repair deficient mouse model that displays features of accelerated aging in multiple tissues including the cns. in aged ercc d/mice, microglia showed increased proliferation and enhanced immune function including expression of cytokines and antigen presentation molecules, increased phagocytosis and production of reactive oxygen species (ros). this microglial functionality was found to be surprisingly hyper-responsive to immune stimuli indicative of microglia priming. transcriptome analysis revealed an expression pattern that characterizes microglia priming, featuring genes associated with increased antigen pattern recognition and antigen presentation and phagocytic-, adhesion-and chemotactic ability. in mice where the ercc related dna repair deficit was targeted to forebrain neurons, microglia priming was restricted to forebrain areas, suggesting that the dna damage-induced changes in neurons provided the signals leading to microglial immune priming. acidosis is a clinical consequence of many major diseases. non-infectious diseases are increasing in prevalence and many have been shown to have an inflammatory component, which in the absence of infection, will be sterile. the nlrp inflammasome, a multimeric protein complex which induces processing of il- b into its active form, is a well established mediator of sterile inflammation and is known to drive the worsening of brain injury in numerous experimental disease paradigms. we sought to investigate whether acidic conditions, typical of a disease environment, affected il- b processing in primary mouse glial cells. mixed glial cultures were grown from wild type and nlrp knockout mice. culture media was reduced to ph . and il- b release was measured following addition of activators of the nlrp inflammasome (calcium pyrophosphate dihydrate crystals, monosodium urate crystals, atp) with or without pre-treatment with caspase- or cathepsin d inhibitors (yvad-cho or pepstatin a respectively). subsequently, il- b release was measured following addition of lactic acid with or without pre-treatment with the above inhibitors. at ph . , activators of the nlrp inflammasome (calcium pyrophosphate dihydrate crystals, monosodium urate crystals, atp) induced the release of il- b from mixed glial cultures. the il- b released at this low ph was kda in size in addition to the mature kda il- b. this kda il- b release was maintained in nlrp deficient cells and was not significantly altered by pre-treatment with the caspase- inhibitor yvad-cho. lactic acid, itself released during disease and a common cause of acidosis, also induced the release of kda il- b from mouse glial cultures. as with the nlrp activators under acidic conditions, the lactic acid-induced kda il- b release was maintained in nlrp knock-out cultures and with pre-treatment with yvad-cho. pre-treatment with the cathepsin d inhibitor pepstatin a, however, significantly reduced the kda il- b released with the nlrp activators and lactic acid. here we show that under disease relevant conditions (low ph), kda il- b is released from mouse glial cultures and this kda il- b is independent of the classical nlrp inflammasome/caspase- pathway and likely mediated by cathepsin d. further investigation of this caspase- -independent il- b pathway may in future provide novel targets for the treatment of inflammatory disease. the phoneutria nigriventer (ctenidae-araneaeomorpha) spider venom (pnv) causes reactive gliosis, neuroinflammation and blood-brain barrier (bbb) impairment. nitric oxide(no)-soluble guanylate cyclase(sgc)-cgmp has been implicated in pnv-induced cavernosal relaxation. we investigate whether no-sgc-cgmp signaling acts on astrocytes/microglia activation and neuroinflammation induced by pnv. male wistar rats ( - -week-old) were pre-treated (i.p.) with sgc inhibitor (odq, mg/kg), nnos inhibitor ( -nitroindazole- ni, mg/kg), no donor (nitroprussiate-ntp, mg/kg) or pde inhibitor (sildenafil, mg/kg). after minutes, the venom ( . mg/kg) was injected in the tail vein (i.v.). saline (i.v.), pnv alone or dmso (i.p) followed by pnv were used as controls. one hour after injection, cerebella were processed for immunofluorescence or western blotting. pnv increased gfap, iba- , ifn-c and sgc, compared to saline control, indicating astrocytes and microglia activation, neuroinflammation and sgc-cgmp involvement. the sgc inhibition by odq intensified the pnv effects, increasing even more gfap, iba- and ifn-c levels. this indicates that sgc-cgmp production can be inhibited by venom. in agreement, the cgmp accumulation by sildenafil inhibited the pnv effects, decreasing gfap, iba- , ifn-c and sgc. the increase of sgc by venom could result from a feedback mechanism, when sgc activity is inhibited and its expression is increased. ni and ntp pre-treatment did not show difference compared to venom alone, suggesting that no per se is not involved in the inflammatory effects of venom. however, it is not discarded no and sgs-cgmp coupling in the mediation of pnv effects. we suggest that pnv induces glial reaction and neuroinflammation by sgc-cgmp inhibition. the study gives evidence that sgc-cgmp, coupled or not to no signaling, mediates pnv cerebellar effects including the bbb impairment. pnv can be a useful tool for studies on the mechanisms involved in glial regulation. cnpq/fapesp/faepex support. j. engele, m. puchert, v. € odemis university of leipzig, leipzig, germany it is currently believed that cxcl predominantly signals through cxcr . in addition, it is assumed that the alternate cxcl receptor, cxcr , represents a non-classical g protein-coupled receptor which primarily acts as a modulator of the function of cxcr . discrepant from this view, we demonstrated recently that in primary rodent astrocytes and human glioma cell lines, sdf- exclusively signals through cxcr by a g protein-dependent mechanism. we now provide evidence that cxcr is essential for cxcl /i-tac signalling in primary rodent astrocytes and some human glioma cells. treatment of cultured rat astrocytes with cxcl for min resulted in the dose-dependent activation (phosphorylation) of erk / and akt with maximum activation in the presence of ng/ml of the chemokine. cxcl- -dependent activation of both signalling molecules persisted in astrocytes in which expression of the established receptors for cxcl , cxcr and cxcr , were inhibited by rnai. however, cxcl -dependent activation of erk and akt was abrogated following sirna-mediated inhibition of cxcr . likewise, cxcl failed to activate erk and akt in cortical astrocytes cultured from a cxcr -/-transgenic mouse line. moreover, similar to primary astrocytes cxcl activated erk and akt in the human glioma cell line, a . again activation of both signalling molecules was abrogated following rnai-mediated inhibition of cxcr . cxcl -dependent activation of erk and akt further remained undetectable in a non cxcr -expressing subpopulation of a cells, previously isolated by flow cytometry. together, these findings unravel a unique processing of cxcl and cxcl signalling in primary astrocytes which is preserved at least in some malignant astroglial cells. long-lasting activation of gfap-positive astrocytes occurs in brain tissue exposed to injuries that promote epilepsy development. studies in experimental models of epilepsy showed that activated astrocytes release various molecules, such as proinflammatory cytokines and danger signals, that play a role in seizures generation and recurrence. these activated cells also loose their ability to buffer extracellular k and glutamate. this set of evidence suggests, therefore, that astrocytes may contribute to epileptogenesis (i.e. the post-injury phase prodromal to generation of spontaneous seizures), thus representing a putative biomarker of epilepsy. to test this hypothesis, we set up an in vivo longitudinal study using h-magnetic resonance spectroscopy (mrs) to measure the hippocampal levels of metabolites that could reflect the extent and the duration of astrocytes activation after an epileptogenic brain injury. status epilepticus (se), which provokes epilepsy, was induced by pilocarpine in adult male rats. h-mrs measurements were done in the hippocampus every h for d post-se, thus encompassing the epileptogenesis phase, and in chronic epileptic rats using a tesla bruker biospec. spectra were processed and analysed using jmrui and tarquin freeware softwares. we studied changes in myo-inositol (mins) and glutathione (gsh), which reflect astrocytes activation. we found a progressive ( -fold, pwhich was maintained in epileptic rats. immunohistochemical analysis (ihc) of s ß and gfap confirmed the concomitant activation of astrocytes in separate timematched groups of rats. gsh levels during epileptogenesis showed a negative correlation with the frequency of spontaneous seizures which developed after se. a negative correlation was also found between gsh and mins levels during epileptogenesis and the extent of neurodegeneration in hippocampus of epileptic rats. ihc done in epileptic rats at the end of the mrs experiments, showed that hippocampal s ß levels positively correlated with spontaneous seizure frequency. since this is a soluble protein, further investigations of its csf and blood levels are warrented. our mrs findings show that gsh levels during epileptogenesis could serve as a predictive biomarker of the ensuing seizure frequency (thus of epilepsy severity) and, together with mins levels, also predict the extent of neuronal cell loss which develops following se in epileptic tissue. notably, ihc-detected s ß levels in the epileptic tissue also reflect seizure frequency. these findings highlight the potential use of serial h-mrs analysis of astrocyte activation for predicting the severity of epilepsy and the extent of neuropathology in the clinical setting. interleukin- (il- ) is a highly plurifunctional cytokine, with many pleitropic actions, considered one of the main cytokines controlling the immune system and coordinating it with the nervous and endocrine systems. il- is produced in multiple cell types in the cns, and in turn many cells do respond to it. it is therefore important to ascertain which the contribution of each cell type is in the overall role of il- during both physiological and pathological conditions. astrocytes are major responders to il- as well as one of the main cns producers of il- . for this work we used astrocytary il- ko (ast-il ko) mice, which we already proved to have an important role in physiological conditions (like body weight control and exploratory/ locomotion behavior), in order to test astrocytary il- role during a neuroinflammation situation. for this purpose, we induced either an extensively used animal model of multiple sclerosis, experimental autoimmune encephalomyelitis (eae), or a traumatic brain injury (cryolesion) in our mice. regarding eae, results indicate that lack of astrocytary il- delays clinical course of eae and ameliorate eae symptomatology in ast-il ko respect littermate controls in a gender-dependent way. further immunohistochemistry analyses confirm a decreased number of cellular and lymphocytes infiltrates and lesser demyelination, angiogenesis and gliosis in spinal cord of ast-il ko animals. regarding traumatic injury, astrocytary lack of il- facilitates microgliosis, lymphocytes infiltration and a faster decrease of the injured area. all these results are likely to bring some answers to astrocytesecreted il- involvement in neuroinflammation pathways and eae pathogenesis. interleukin- (il- ) is a counterregulatory cytokine that plays an important role in controlling inflammatory and immune reactions. in the central nervous system (cns), production of il- has been demonstrated in activated astrocytes and microglia after different types of injuries. the specific role played by il- inmodulating glial responses is however still unclear. hence, the objective of this study was to evaluate the effects of local astrocyte-targeted production of il- on glial reactivity using the axonal anterograde degeneration paradigm. for this purpose, unilateral perforant pathway transection (ppt) was performed on adult gfap-il transgenic (tg) animals and their corresponding wild types (wt) littermates. at and days post-lesion (dpl), animals were intracardially perfused with % of paraformaldehyde, brains frozen and parallel free-floating sections processed for glial analysis using immunohistochemistry for iba- (microglia) and gfap (astrocytes). our results showed that in comparison with wt animals, microglial cells in the dennervated dentate gyrus molecular layer of gfap-il tg showed differential reactivity changes. after lesion, astrocytic reactivity presented a higher hypertrophy in gfap-il tg animals when compared with their corresponding wt littermates. in conclusion, this study demonstrated that local production of il- inthe cns can modify the glial response associated with ppt. further studies are warranted to evaluate if these alterations in microglial and astrocytic reactivity have any repercussions for the evolution of the lesion and the associated axonal sprouting. astrocyte and microglia become reactive under many brain pathological conditions, making this process of neuroinflammation a surrogate marker of neuronal dysfunction. several studies have reported that reactive microglia overexpress the translocator protein kda (tspo, formerly known as peripheral benzodiazepine receptor). positron emission tomography (pet) using radioligands of tspo is thus considered as a potent technique to detect reactive microglia in situ. however, it is still controversial whether tspo pet imaging is also able to monitor reactive astrocytes in situ. this is an important question as reactive astrocytes and reactive microglia play very different roles in brain physiology and could impact disease progression in opposite ways. to address this question, we used a model of selective astrocyte activation through unilateral lentiviral gene transfer of the cytokine ciliary neurotrophic factor (lenti-cntf) in the rat striatum. cntf induced an extensive activation of astrocytes, which overexpressed gfap and became hypertrophic. microglia, on the contrary, displayed a minimal increase in the expression of markers of reactivity. cntf-activated astrocytes overexpressed tspo at the mrna and protein levels. pet imaging experiments demonstrated a significant and specific binding of two tspo radioligands [ f]dpa- and [ c]ssr in the lenti-cntf-injected striatum. we show that reactive astrocytes can be monitored by tspo-pet imaging in the rat brain. this technique is thus well suited to monitor reactive microglia as well as reactive astrocytes, the two cell types involved in neuroinflammation, but would not allow their discrimination in situ. chronicity might establish a neuroinflammatory ganglion profile with inflammatory cells contributing to the hypersensitive phenotype. we first investigated whether, in trigeminal sensory ganglia, cytokines such as tnfa might contribute to a local inflammatory phenotype of a transgenic mouse model of familial hemiplegic migraine type- (fhm- , cacna a r q knock-in mice). with respect to wild-type, r q ki trigeminal ganglia were enriched in activated macrophages and expressed higher mrna levels of il b, il , il and tnfa cytokines and the mcp- . functional consequences of crosstalk between macrophages and sensory neurons were studied in primary ganglia cultures, where larger release of soluble factors and larger currents mediated by pain-transducing atp-gated p x receptors were found. consistently, we observed that, following lps injection, tnfa expression and macrophage occurrence were significantly higher in r q knock-in ganglia with respect to wild-type ganglia. our data suggest that, complex cellular and molecular environment of sensory ganglia could support a new tissue phenotype compatible with a neuroinflammatory profile. we propose that, in selected patients, this condition might contribute to pain pathophysiology through release of soluble mediators, including tnfa and atp that may modulate the crosstalk between sensory neurons and resident glia, underlying the sensitisation process. cultures of astrocyte/microglia are stimulated with ifn-gamma and then infected with tachyzoites of neospora caninum they release nitric oxide (no) that controls parasite proliferation. in order of elucidating the immune response of cns against this parasite, this study investigated the participation of inducible nitric oxide synthase (inos) in the control of its proliferation in co-cultures of neuron-glia obtained from rat brain. the cells were stimulated with ifn-gamma ( iu/ml- h) then supplemented with l-ng-nitroarginine methyl ester/l-name, an inhibitor of inos ( . mm/ml - min) before infection with tachyzoites of n. caninum ( : parasite:cell). after h, in the cultures supplemented with l-name, it was verified, a reduction of tachyzoites number in . % (control cells) and . % (ifn-y stimulated cells). however, in infected co-cultures, it was not observed any release of no, even when cells where modulated by ifn-g. additionally, in cultures infected and treated with l-name, the levels of il- were increased in six fold (non stimulated) and nine fold (ifn-g stimulated). these data suggest that no should not participate as a mediator of n.caninum control in neuron/glia co-culture but, the regulatory pattern of immune response must play a role in its down modulation regulating the inflammatory mediators released during the cns infection, perhaps to protect neurons. methods: adult albino swiss mice were organized in two groups: naive (age-matched control; n ) and lasered (n ). anti-iba was used in retinal whole-mounts immunolabelled to analyze the distribution, morphology, density and arbor area of iba microglial cells. results: in na€ ıve, contralateral and lasered eyes, iba microglias were distributed throughout the retina in the: subretinal space (ss), outer plexiform layer (opl), inner plexiform layer (ipl), nerve-fibre layer (nfl) and ganglion-cell layer (gcl). in na€ ıve eyes, iba microglias: i) had rounded bodies with fewer and shorter processes in the ss; ii) exhibited a branched morphology emanating from small cell bodies in the opl and ipl; iii) were less ramified and were related with the retinal vessels in the nfl and in the gcl. by contrast, the morphological features exhibited by iba cells in contralateral and oht-eyes differed from na€ ıve: i) somas were more robust; ii) processes were thicker and more branched in contralateral eyes and thicker and retracted in oht-eyes. in oht-eyes and contralateral untreated eyes the iba microglial number was increased in comparison with na€ ıve (p < . and p < . respectively, t-test). in comparison to na€ ıve retinas the iba- cell arbor in the opl and ipl decreased in both oht-eyes (p < . and p < . respectively, t-test) and in the contralateral untreated eyes (p < . and p < . respectively, t-test). conclusions: two weeks of laser induced-oht triggered microglial retinal changes suggestive of activation in the contralateral untreated and oht-eyes. the microglial activation in contralateral eyes could be related to the immune response. this behaviour in contralateral untreated eyes, lead us to suggest that the use of contralateral eyes as internal controls in experimental unilateral oht, should be reconsidered. interleukin- (il- ) is a pleiotropic cytokine involved in inflammatory and non-inflammatory responses. among the latter, it participates in the regulation of body weight and metabolism. il- -deficient mice develop mature onset obesity and have higher blood glucose, as well as impaired glucose tolerance and elevated blood leptin levels, suggesting that il- participates in suppressing adiposity in mice. moreover, transgenic mice with astrocyte-targeted production of il- challenged with a high-fat diet are resistant to high-fat diet-induced obesity, highlighting the role of centrally produced il- in the regulation of body weight. in this context, we hypothesize that mice lacking il- produced by astrocytes (ast-il ko) will be more prone to develop obesity. we therefore challenged ast-il ko mice (obtained with the cre-lox technology) with a high fat diet ( % kcal from fat) for weeks and compared their body weight and food intake with those of mice fed a standard diet ( % kcal from fat). on weeks and , the metabolic status was evaluated by an insulin tolerance test (itt) and an oral glucose tolerance test (ogtt), respectively. regarding body weight gain, we see a clear increase in ast-il ko females on a high-fat diet in comparison to their controls with no apparent difference in food intake, which is also already apparent in the control diet. in males a similar tendency is observed. part of this difference can be attributed to the heavier subcutaneous white adipose tissue depots (relative to body weight) in ast-il ko mice (fig ) . insulin and oral glucose tolerance are compromised in mice fed a high-fat diet, but no differences between genotypes are observed. taken together, these results indicate that centrally produced il- has indeed a major role in the regulation of body weight, affecting subcutaneous white adipose tissue, but without significantly altering peripheral glucose metabolism. we are currently working on assessing hypothalamic neuropeptides with in situ hybridization to study the effects of astrocytic il- deficiency at the central level. minocycline is an agent with pleiotropic properties that targets multiple proteins and cellular processes associated with development of neuropathic pain, including inhibition of the injury-induced glia activation. the aim of our study was to examine the effects of minocycline on the injury-induced changes in immune factors and on morphine effectiveness in a rat model of neuropathic pain. chronic constriction injury (cci) to the sciatic nerve in rats was performed according to bennett and xie ( ) and behavioral tests were conducted to measure allodynia (von frey test) and hyperalgesia (cold plate test). minocycline was administered intraperitoneally h and h before cci and then twice daily for days. the studies were performed using competitive rt-pcr in the spinal cord and drg from control, cci-exposed and minocycline-treated cciexposed rats. morphine was administrated i.t. (chronic catheterization according to yaksh and rudy, ) and ip h after the last minocycline injection. the experiments were carried out according to iasp rules (zimmermann, ) . repeated administration of minocycline attenuated allodynia and hyperalgesia when measured days after cci. minocycline downregulated the spinal and drg level of several immune factors: c q, mmp- , mmp- , il- beta, il- , il- , nos and nos which were increased in consequence of the injury. moreover, chronic administration of minocycline improved the response to i.t. and i.p. morphine injections. our results demonstrate that minocycline reduces the level of pro-inflammatory factors and increases the effectiveness of morphine, which may have clinical significance for enhancing analgesic effects of opioids in neuropathic pain. to date, there is no standardized simple model available to investigate the biology of human microglia. the aim of this study was to establish a new in vitro microglia model using blood-derived precursor cells. for that purpose, human peripheral blood monocytes were cultured in serum free medium in the presence of a mixture of cytokines and chemokines (m-csf, gm-csf, ngf and ccl ) to generate monocyte-derived microglia (m-mg). monocyte-derived dendritic cells (m-dc) were also generated as a control population using gm-csf and il- . the human microglia cell line hmc was used as control. m-mg were clearly different in morphology, phenotype and function from m-dc, but shared many properties with hmc cells. m-mg acquired a ramified morphology with primary and secondary processes, comparable to hmc . they expressed very low levels of cd , cd and hla-dr, cd b and cd c; but a distinct pattern of chemokine receptors, including ccr , ccr , ccr , ccr , ccr , cxcr , cxcr , cx cr . similar to hmc , under non-activated condition, the m-mg secreted of il- and il- . in comparison with m-dc, m-mg displayed lower t-lymphocyte stimulatory capacity, as well as lower phagocytosis activity. in summary, we have established a new protocol for the generation of human monocyte-derived microglia, which is is feasible, well standardized and reliable, as it uses well defined culture medium and recombinant cytokines, but no serum or conditioned medium. this model will certainly be very helpful for future studies investigating the biology and pathology of human microglia. w. schaafsma university of groningen, neuroscience, section medical physiology, groningen, netherlands background: microglia are the innate immune cells of the cns. like other tissue macrophages, they can activate and commit to distinct reactive phenotypes in response to tissue damage and infections. however, the microglial response to tissue damage may change over age and by past experience. these changes in activation patterns may be caused by epigenetic modifications which in turn can result in changes in gene expression of, for example inflammatory cytokines. a well studied condition with an altered responsiveness of innate immune cells is 'endotoxin tolerance' (et). innate immune cells that are pre-exposed to endotoxins are dampened in their inflammatory response upon re-exposure to these endotoxins. while et is well described for peripheral macrophages/monocytes, very little is known about et in relation to microglia. objective: in our experiments we set out to investigate the presence of et in microglia and if an 'epigenetic' memory is involved. design/methods: in our in vitro experiments, microglia cells were stimulated with lps ( ng/ml). naive microglia only received one lps stimulation at the end of culture, pre-stimulated microglia received an h pre-stimulation of lps ( ng/ml) and a second stimulation days later. in vivo, male c bl/ mice received an i.p. injection with either pbs or lps ( mg/kg) and weeks later again an i.p. injection with pbs or lps. transcription levels and secretion of il -b and tnf-a were assessed by use of quantitative pcr and elisa. in order to answer if there is a long lasting epigenetic memory involved in this tolerance we used chromatin immune-precipitation (chip), to investigate changes in covalent histon tail modifications associated with either permissive or repressed chromatin. results : in vitro, we show a 'tolerant' phenotype in microglia which receive a second lps stimulation after days. a reduced expression level of pro-inflammatory genes il -b and tnf-a and reduced secretion of tnf-a were observed in response to a second lps challenge. furthermore, with in vivo experiments we showed that mice which received a second lps injection after weeks, showed a dampened response in their microglia inflammatory reaction at the level of il -b and tnf-a gene transcription. in both in vitro and in vivo experiments we showed a corresponding pattern for histon tail modifications in the enrichment of activation marks h k me and ach at the il -b and tnf-a promotors. we are the first to show a long lasting 'tolerant' phenotype in mouse microglia in vitro and in vivo and the involvement of epigenetic changes at the level of histon modifications. morphological criteria and dual immunofluorescent labelling confirmed expression of er stress protein in neurons, astrocytes, microglia or oligodendrocytes. an intriguing finding was localisation of crt to the rim of oro-positive myelin fragments and the 'patchy' nature of crt staining seen when tissue was dual-labelled with crt and gfap or iba . these results are the first demonstration of significantly higher levels of crt in rodent eae. chop and p-eif a data has also not been reported in rat eae. this study highlights the potential importance of er stress in inflammatory demyelination. the authors acknowledge support from the irish research council and from the foundation office of nui, galway. ( ). considering the responsible signaling pathways regulating adult neurogenesis ( ), we observed differential regulation of wnt members in the hippocampus on transcriptional level, e.g. wnt dependent transcription factors (axin , tcf ) and ligands (wnt , a, b and ), as well as on translational level represented by the wnt signaling cascade (active-bcatenin, phospo-glycogen synthase kinase b) during acute and chronic states of disease. by using in vitro studies in primary hippocampal cultures, we further show the role of transforming growth factorbb (tgfb ), a key cytokine early involved during eae ( ), in regulation of wnt ligand expression and wnt signaling activity. furthermore we are able to visualize the connection between tgfb and wnt signaling by using the axin -lacz mouse, a wnt reporter mouse, for functional and histological investigation of the hippocampus. taken together our results suggest a cross talk of inflammation and wnt signaling for dysregulated hippocampal neurogenesis. interleukin- (il- ) is a cytokine with major regulating effects of the inflammatory response. moreover, il- is a neuropoietin that has neurotrophic effects related to neuronal survival and protection. to establish the importance of il- produced only in the central nervous system, we have generated mice producing il- essentially only in the brain by crossing gfap-il mice (transgenic mice with astrocyte-targeted production of il- ) with il- ko mice (il- -deficient). we studied the inflammatory response in a traumatic brain injury model, cryolesion, after and days post-lesion gfap-il -il ko mice, comparing them with appropriate controls. in basal conditions wt and il- ko mice showed a similar phenotype. this was also the case for gfap-il and gfap-il -il- ko mice, which showed prominent astrogliosis, microgliosis, increased recruitment of t lymphocytes and vascularisation compared with the other two groups. in response to cryolesion of the cortex an increased astrogliosis, microgliosis, recruitment of t lymphocytes and vascularisation was observed. il- deficiency produced an altered inflammatory response, in a time-and gender-dependent manner. this study with il- ko and gfap-il transgenic mice indicates that during an acute neuropathological insult such as traumatic brain injury il- , from either the brain or the periphery, has an important role on the inflammatory response. increasing evidence suggests that iron accumulation in the brain might contribute to neurodegeneration. iron is a potential source of free radicals as it can catalyze the production of hydroxyl radicals under oxidative conditions. this can lead to amplification of tissue injury caused by oxidative damage. we thus characterized iron storage within the central nervous system (cns) of animal models of different neurodegenerative diseases. we examined animals with acute inflammation mediated by cd or cd positive t cells and animals suffering from t cell and antibody mediated chronic inflammation due to active immunization. further, we studied lps induced lesions which represent cns disease caused by the innate immune system. similarly, we characterized oxidative damage in the different models for inflammation. we did not find evidence for the presence of oxidized phospholipids or oxidized dna in the experimental lesions, which is in contrast to our results of ms lesions. none of these models showed iron accumulation in glial cells comparable to what is seen in ms tissue. however, some iron positive microglia and perivascular macrophages were observed in acute and chronic models. in preliminary experiments we found evidence that the presence of iron exacerbates h o induced cell death in purified glial cells in vitro. as a next step, we wanted to study a possible amplification of oxidative damage and neurodegeneration by iron in a more elaborate system. for this purpose we treated myelinating spinal cord cultures with iron chloride (fecl ) or ferritin. we observed iron loading in microglia in both experimental setups. moreover we found a selective decrease of microglia in iron chloride treated cultures but not after ferritin application. we did not find iron loaded oligodendrocytes in this in vitro model. iron accumulation is only minimal compared to the human brain in all the tested animal models. these observations necessitate the search for additional animal models mimicking the human situation more closely. objectives: microglia are a brain resident population of immune cells, involved in homeostatic surveillance of the brain parenchyma, with high importance in the regulation of inflammatory processes after injury. there are unresolved questions regarding microglia origin, and their similarities to peripheral macrophage populations. our objective was to compare the capacity of microglia and bone marrow derived macrophages (bmdms) to adopt different phenotypes, and how this influenced cell death after brain injury. methods: we compared the phenotype of bmdms and microglia (both bv microglial cell line and primary microglia) after treatment with well known polarising agents. lipopolysaccharide (lps) was used as an inducer of the classical m phenotype and il- was used to induce an alternative m phenotype. microglia or bmdms, of different phenotypes, were added to hippocampal organotypic slices (hosc) subjected to oxygen glucose deprivation (ogd) as an in vitro model of brain injury. results: bmdms and microglia (bv and primary microglia) both adopt m or m phenotypes after treatment with lps or il- respectively. however, addition of polarised microglia or bmdms onto hosc resulted in different outcomes. addition of activated bmdms, of either m or m phenotypes, led to cell death in control hosc and increased death when combined with ogd. on the other hand, addition of bv -microglia did not induce any cell death in control hosc, and were protective after ogd, except for m microglia which were toxic. endogenous microglia within the hosc were also able to adopt different phenotypes and exert neuroprotection after il- treatment. these ex vivo data correlated with the in vivo situation where we observed increases in microglial activation early after experimental stroke, although the vast majority of these activated cells did not express markers of the m phenotype. conclusions: this study highlights functional differences between macrophages and microglia, especially in response to brain injury. although microglia, like peripheral macrophages, can adopt different demyelination, thereby creating an unfavorable environment for remyelination. exploring efficient ways to prevent or overcome tlr-induced fibronectin aggregation by astrocytes might pave the way for new approaches in remyelination-targeted therapeutic strategies in ms. to study the tlr response in-vivo, sod g a mice were crossed with the tlr -luc-gfp reporter mice, a transgenic mouse bearing the dual reporter system luciferase and green fluorescent protein under the transcriptional control of the murine tlr promoter. double transgenic mice and littermates controls were monitored longitudinally using in-vivo biophotonic/bioluminescent imaging to measure microglial activation over the course of the disease. surprisingly, this analysis did not reveal an increase of activation in sod g a mice as the disease progressed, with only weak signal coming from olfactory bulb and brain area. however, quantification of this signal suggested a lower level of tlr activation in the sod g a mice compared to wild-type littermates. to further study the microglial impairment observed in the sod g a mice, these transgenic mice were given lps with i.p. injections at mg/kg and were followed for h by bioluminescence imaging, both at a pre-symptomatic age ( days) and closer to paralysis ( days). the level of tlr activation was lower in the sod g a mice than the wild-type littermates, both at days and days. immunostaining of olfactory bulbs reveal the same phenomenon, which is less iba and less tlr -positive cells in the sod g a tissue. mrna analysis by in-situ hybridization confirms a similar pattern of microglial response. in a parallel study, primary microglial cell culture, derived from adult mice ( days) were similarly stimulated with lps to further understand the altered sod mutant microglia response. here again, sod cells appears less responsive to lps stimulation as observed in our mice studies. these combined results suggest that sod g a microglial cells might have a pre-symptomatic suboptimal immune response. the role of reactive glia in the etiology and progress of neurological diseases is still unknown. reactive glia produce several factors, typical of an inflammatory response, with a potential neurotoxic effect, highlighting the relevance of a strict control of the progression and resolution of glial activation. under some circumstances glial activation does not resolve, but it exacerbates or becomes chronic resulting in detrimental secondary effects. it is necessary to develop strategies to halt the negative outcome of glial activation in these situations, by controlling the pro-inflammatory phenotype of reactive glial cells and potentiating their beneficial effects. we studied the temporal pattern of inflammatory reaction in spinal cord and brain regions in the experimental autoimmune encephalomyelitis (eae) model of multiple sclerosis. special attention was paid to the involvement of members of the c/ebp family of transcription factors, which regulate the expression of pro-inflammatory genes in reactive glia, and cd , cd r and trem- , membrane-associated inhibitors of the pro-inflammatory response in resting/surveillant microglia. mice were scored for signs of eae for up to days postimmunization (dpi). eae symptoms were present from dpi, reaching score peak at dpi. in the spinal cord, we observed a sustained increase in c/ebpa and a transient increase in c/ebpb and c/ebpd expression peaking at dpi. cd expression decreased at dpi, remaining below control values at dpi. cd r and trem- expression increased at dpi, thereafter this effect progressively attenuated. no alterations were observed in the brain. an acute increase in the expression of pro-inflammatory genes (il- b, il- , tnf-a, inos, cox ) occurred in the spinal cord at dpi. our results suggest that cd expression decreases before onset of eae symptoms resulting in downregulation of the microglia inhibitory signal mediated by cd -cd r , which would facilitate the proinflammatory response observed after onset of the eae symptoms. the increase in cd r expression observed after the onset of eae symptoms suggests a concomitant intend of microglia/macrophages to resolve the inflammatory response. the transient increase in c/ebpb and c/ebpd expression could be related to the acute increase in proinflammatory gene expression, while the sustained increases of c/ebpa expression and trem- could be involved in the resolution of the inflammatory response. recently, we showed a beneficial effect of myelin reactive t cells on oligodendrocyte precursor cell differentiation in zones of axonal degeneration in the hippocampal dentate gyrus, which mirrors grey matter injury in multiple sclerosis. this effect was associated with a marked expression of t-cell cytokines, such as interferon-c and interleukin- , enhanced microglial clearance of myelin debris, and an enhanced sprouting of calretinergic axons. to gain better insight in which cytokines and signaling pathways are elevated in response to the t cell enhanced regenerative responses, we performed a mrna microarray study, in which the transcript profile in hippocampi from mice with adoptively transferred proteolipid protein specific t cells combined with an axonal lesion were compared to the transcript profile from mice with axonal lesion. thereby we identified transcripts, which were differently expressed. we identified the interleukin- (il- ) signaling pathway as a potential target by performing pathway analysis using david and amigo databases. we discovered that il- a, il- b and il- receptor antagonist (ra) mrna as well as the il- signaling pathway were highly upregulated in response to myelin reactive t cells. in order to determine if il- b mrna was translated into protein, we performed immunohistochemical stainings on tissues from mice with days post lesion survival. expression of il- b was observed in the molecular layer of t cell infiltrated, but not in t cell na€ ıve mice with axonal lesion, suggesting that myelin reactive t cells stimulate il- b protein expression. ongoing studies will determine the expression of il- a and il- ra protein and determine the cellular expression of the il- signaling pathway in t-cell infiltrated versus non-t cell infiltrated mice with axonal lesion. understanding the regulation of the expression of il- a, il- b and il- ra in t cell compared to non-t cell infiltrated cns may lead to a better understanding of the functional consequences of inflammatory responses in the cns. when io were challenged with kainate in the presence of conditioned medium (cm) from astrocytes, susceptibility to kainate-induced cell death was reduced, but cm from l-ap -treated astrocytes reverted kainate toxicity. in contrast, treatment with cm from control or lpsactivated microglia, either untreated or pre-exposed to l-ap , was not able to affect kainate-induced io cell death. to establish whether mglur activation could modulate the antigen presenting activity of microglia, the bv microglia cell line was used. treatment with l-ap ( lm/ h) did not affect changes in morphology induced by activation with lps ( . lg/ml/ h), but significantly reduced the percentage of cells expressing mhc class ii, as detected by flow cytometry. these data suggest that during neuroinflammation, activation of mglur directly on io or through astrocytes and microglia may contribute to a protective effect resulting in survival of the oligodendrocyte lineage. mirnas are small non-coding rna molecules that modulate gene expression at a post-transcriptional level and their role in the regulation of biological processes makes them an emerging class of therapeutic targets. dysregulation of mirna networks has been linked to neurodegeneration and immune dysfunctions and has become a research focus in the context of alzheimer's disease (ad). in this work, we demonstrate that mir- expression is increased in the brain of trg ad transgenic mice, prior to senile plaque formation, and co-localized with intraneuronal app accumulation in the cortex and hippocampus. in view of the pro-inflammatory functions of mir- and aiming at clarifying its role in ad, we evaluated the levels of mir- in ab-stressed astrocytes and microglia cultures, two brain-related cell types involved in neuroinflammation. we show that mir- expression levels are increased in astrocytes and microglia following activation with ab fibrils but not with ab oligomers. these findings correlate with the observed decrease in socs- expression, a mir- validated target, and with the increase in the production of tnf-a, il- b and il- by these cells. importantly, we show that mir- expression is regulated by c-jun, since silencing of this transcription factor led to the reduction of mir- levels following astrocyte and microglia exposure to ab fibrils or lps. c-jun was also found to be upregulated in the trg ad transgenic model since early ages, which can help to explain the observed early increase in mir- expression. overall, our results demonstrate the important role of mir- in ad and show that silencing of c-jun in glial cells may constitute an interesting and promising anti-inflammatory therapeutic strategy towards this disease, by targeting mir- -mediated inflammatory responses. here we demonstrate that tgfb is an important endogenous factor promoting quiescence of microglia in vitro. inhibition of microglial tgfb signalling resulted in a microglia polarisation towards the classical activation state. moreover, tgfb is able to significantly decrease ifnc-induced classical activation of primary microglia and to enhance il -induced alternative microglia activation. we further provide evidence that il -mediated alternative microglia activation is dependent on active tgfb signalling. these results demonstrate the essential functions of tgfb as an important regulator of microglia activation states and further suggest that tgfb might be an interesting therapeutical molecule to modulate microglial functions during development and progression of neurodegenerative diseases. we found that young mice lost weight only in the first h after infection, whereas aged mice exhibited a biphasic pattern of weight loss, with the second phase of weight loss beginning week after infection and continuing for approximately = weeks. aged mice also demonstrated a co-ordination and balance deficit in the static rod test week after infection compared to uninfected or week post infection aged mice, whereas young mice were unimpaired at any timepoint. s. typhimurium infection caused a significant, progressive reduction in open field rearing activity at week and weeks after infection in both young and aged mice. a similar but not significant trend was apparent in novel object performance. forced swim test data showed a trend towards depressive behaviour at week after infection, but not at weeks. analysis of pro-inflammatory mediators in the hippocampus did not indicate significantly upregulated il- b, tnf-a, cox- or cox- transcript at any timepoint, suggesting that microglia might not play a significant role in mediating these behavioural effects, or that the changes in these molecules were too subtle to reliably detect using qpcr in the hippocampus. the effects of ageing on s. typhimurium induced weight and co-ordination changes may be a result of regional differences in the sensitivity of microglia to peripheral infection within the cns or increased sensitivity to activation of neuronal circuits involved in weight loss or co-ordination with age. we have studied microglial cells with anti-iba and anti-cd immunohistochemistry in the brains of subjects with pretangle neuropathology ranging from to years of age. regions examined included the locus coeruleus and surrounding brainstem areas, entorhinal cortex, hippocampal formation, and other parts of the mesial temporal lobe. ten of the subjects (median age ) showed severe neuroinflammation secondary to infectious disease (hiv, sepsis, toxoplasmosis) and cns trauma, while subjects (median age . ) died from non-infectious conditions and lacked neuroinflammatory changes (controls). we also examined two -year-old subjects with down syndrome both of which showed braak stage vi neurofibrillary degeneration, where one had comorbid sepsis and the other one did not. pretangle neuropathology was staged a and b in all subjects, i.e. they exhibited primarily subcortical tau pathology in the locus coeruleus and only sporadic lesions in the transentorhinal cortex. our findings show that rampant microglial activation in the ten subjects with infections and trauma was coincident with the same level of tau pathology as seen in the control subjects, demonstrating that neuroinflammation does not initiate or exacerbate neurofibrillary degeneration. in addition, our findings show that the extent of neurofibrillary pathology seen in down syndrome is unrelated to neuroinflammation since the down subject without comorbid sepsis revealed a complete absence of microglial activation. overall we conclude that neuroinflammation is not causally involved in the development of neurofibrillary degeneration which is most characteristically associated with alzheimer's disease dementia. it has been suggested that peripheral infection/inflammation may play a role in the onset or development of not only neurodegenerative diseases but also depression, autism and chronic fatigue syndrome through inflammatory responses in the brain. to investigate the role of microglia in this hypothesis we used a model of neuroinflammation induced by an intraperitoneal (i.p.) injection of the toll-like receptor (tlr ) agonist, polyinosinic:polycytidylic acid (poly i:c) in rats. as we reported previously an injection of poly i:c (i.p) decreased the daily amounts of spontaneous running wheel activity to $ % of the preinjection levels until day . microglia were morphologically activated in the prefrontal cortex (pfc) until hrs after the injection of poly i:c. pretreatment with minocycline for the consecutive days ( mg/kg/ day) blocked the poly i:c-induced decrease in the running wheel activity as well as microglial activation. quantitative analysis of mrna levels demonstrated that interferon-a (ifn-a) increased in the pfc on both days and . we found in the present study that poly i:c injection induced increases in mrnas relevant to tlr-signaling such as tlr , interferon regulatory factor (irf ) and irf in the pfc. furthermore, direct application of poly i:c to the primary cultured microglia also induced an enhanced expression of ifn-a, tlr , irf and irf . it has been reported that permeability of blood-brain barrier is increased after poly i:c injection (wang et al., ) . therefore, it is possible that the peripheral poly i:c enters into the brain to induce neuroinflammation by activating microglia. interestingly, intracerebroventricular (i.c.v.) injection of primary cultured microglia activated by poly i:c, but not by lipopolysaccharide, effectively produced a significant decrease in spontaneous activity in rats. taken together, these findings suggest that microglial activation is important for poly i:cinduced neuroinflammation through inducing tlr -related genes. roles of each gene in the behavioral changes will be further studied. results: microglia produced nanogram levels of pgrn ( fold higher in microglia than in astrocytes), and pgrn release from microglia was suppressed by the tlr ligands or il- /ifnc, but increased by il- or il- . unexpectedly, while astrocytes stimulated with proinflammatory factors released large amounts of slpi, none were detected in microglial cultures. we also identified mmp- as a pgrn proteolytic enzyme, and slpi as an inhibitor of mmp- -induced pgrn proteolysis in human microglia. conclusions: our results establish microglia as a significant source of pgrn. mmp- and slpi are modulators of microglial pgrn proteolysis. negative and positive regulation of microglial pgrn release by the proinflammatory/th and the th stimuli, respectively, suggests a fundamentally different aspect of pgrn regulation compared to other known microglial activation products. microglial pgrn appears to function as an endogenous modulator of innate immune responses. at pathological condition such as injury or ischemia to the central nervous system (cns), microglial cells and astrocytes become activate and inflammation occurs like proliferate, migrate and secrete some proinflammatory cytokines, il- ß, tnf-a or il- . however, it is unclear that the reason why inflammation is induced in the cns, and whether or not it is necessary for the brain during recovery from pathological situation. to know the molecular mechanisms for inflammation occurs in activated glial cells, we made a stab wound mouse model to the brain. we performed microarray analysis for rna extracted from the brain tissue around stab wound site compared among day after , , and . it revealed that most of the genes from top genes at high expression level around lesion site were concerned in immunological or inflammatory functions. we successfully identified and focused on to osteopontin (opn), which is an inducer of pro-inflammatory cytokine production expressed not only in reactive microglial cells but also in reactive astrocytes around the injured cns. furthermore, we also found receptors against opn functioning in the injured brain. however, functional role of opn in reactive astrocytes is not yet clarified. to analyze the central role of opn in reactive astrocytes, we examined primary culture of astrocytes from opn-deficient (opn/ko) mice and found that the morphology of these cells was unusual. by the stimulation with lipopolysaccharide to the primary culture of astrocytes from opn/ko mice, some of the pro-inflammatory cytokine expression was altered. moreover, reactivity of astrocytes caused by stab wound to the brain was examined using analogue of nucleic acid, bromo-deoxy-uridine (brdu), and clarified that it was decreased in opn/ko mice. from these results, we conclude that opn might play a major role in the activation of astrocytes under inflammation occurred to the cns. here we determined the influence of a hfd on eae. we show that feeding mice with a hfd exacerbates eae severity through the induction of the brain renin angiotensin system (ras), resulting in an increased immune cell infiltration into the cns, oxidative stress and th cytokines. moreover, peripheral inflammation was boosted upon a hfd due to a ras independent mechanism, indicating that a hfd exacerbates neuroinflammation in various manners. these data suggest that nutritional modulation may have an important influence on the course of ms and other neuroinflammatory conditions. the brain's immune privilege has been attributed to a lack of dendritic cells (dc) within its parenchyma and the adjacent meninges implying maintenance of antigens rather than their presentation in lymphoid organs (lo). using cd c-gfp mice, we have recently reported the existence of a cd c/iba- /cd b -population in the juxtavascular parenchyma which extent their processes into the glia limitans, an ideal position for antigen-presentation. therefore, we phenotypically compared them to the cd c /cd population (all isolated with the same protocol used for brain cd c cells) from lung, liver and spleen in healthy mice using -color flow cytometry. we found unique, sitespecific expression patterns of f / , cd , cd , cx cr , ccr , flt and mhc-ii. as described before, two different cd -populations (cd high and cd int ) in the brain can be separated, whereas liver, lung and spleen exhibit a more homogeneous cd high population. a higher percentage of the brain's cd /cd c -cells were f / -positive compared to spleen, liver and lung, but expressed it at lower levels. within the cd int /cd c -population most cells expressed the microglia marker cx cr and ccr low (marker for inflammatory, motile cells). most importantly, compared to spleen and liver, cd int /cd c -cells from the brain almost completely lacked mhc-ii expression and cd high /cd c -cells from the brain have a lower percentage of mhc-ii -cells. since the cd int cells are widely regarded as the resident, intraparenchymal microglial population and cd high as dcs and perivascular macrophages, our data confirm the view that a small subpopulation of microglia (cx cr high , f / ,ccr low ) share established immune phenotypical characteristics of dcs (cd c ). additionally we showed that intraparenchymal cd c microglia are unique in their low expression of mhc-ii. their weak expression of cd is in line with a tolerogenic phenotype. methods: we performed a series of experiments, where we examined the expression of pro-inflammatory factors in resident microglia and infiltrating macrophages after fluorescence-activated cell sorting (facs) at different eae stages. in order to properly discriminate the two cell types we used a set of markers where resident microglia were defined as cd b cd int ly c -, and infiltrating macrophages cd b cd hi ly c . using this protocol we have analyzed the rna expression levels of mhcii (cd , h -aa), co-stimulatory molecules (cd , cd , cd , cd ), pro-inflammatory genes (il- b, tnf-a) and inflammasome-regulated cytokines in these immune cells with quantitative pcr. followed by analysis of mhcii, cd and cd at the protein level with flow cytometry. results: our results indicate that infiltrating macrophages have a pronounced activated (cd and h -aa) and pro-inflammatory phenotype (il- b and tnf-a) at rna level. this is supported with an increase of the expression of mhcii and the key co-stimulatory molecules cd and cd at rna as well as protein level. in contrast, microglia display a decrease in the expression of the co-stimulatory molecules cd and cd at the rna level and do not up-regulate expression of il- b and tnf-a during the progression of eae. conclusion: our data suggest that upon eae, infiltrating macrophages display an inflammatory profile whereas microglia are only mildly immune activated. background: age-related hearing loss (presbycusis) affects half of people by the age of . it has a large impact on quality of life and is associated with accelerated cognitive decline. however, presbycusis is not an inevitable process of aging, suggesting that its progression could be minimised. hearing relies on sound vibrations from the middle ear to elicit movement of the basilar membrane in the cochlea, resulting in excitation of hair cells. spiral ganglion neurons relay the inner hair cell signals to the central auditory system via the vestibulocochlear nerve. sounds are integrated in the central auditory pathway and then processed by cognitive areas of the brain. degeneration of cochlear structures, including hair cells and spiral ganglion cells occurs in presbycusis. in the central auditory system there are alterations in neuronal plasticity and neurodegeneration may occur. chronic inflammation has previously been correlated with agerelated hearing loss in humans, implicating immune cells in the progression of presbycusis. microglia within the central nervous system are susceptible to priming by neurodegeneration or aging, both factors in presbycusis. it is feasible that microglia in the cochlea, an organ with similar immune privilege as the brain, could also become primed. priming increases the chance of microglia becoming activated by subsequent inflammatory events, such as 'flu, which may cause them to contribute to neurodegeneration in the cochlea and central auditory system. hypothesis: our working hypothesis is that as age-related hearing loss progresses, microglia in the cochlea and central auditory pathway will become primed. we predict that systemic inflammation will cause primed microglia to be activated to a pro-inflammatory damaging phenotype, which will increase the rate of cochlea and auditory pathway degeneration and hence hearing loss progression. methods: c bl/ j mice are genetically predisposed to develop age-related hearing loss. young and middle-aged mice will be challenged with lps or saline (control) and microglial phenotype assessed in the cochlea and central auditory system. neurodegeneration will be evaluated at corresponding points in the cochlea and auditory pathway using molecular techniques. conclusion: if our hypothesis is proved this would suggest that rigorous control of infection in older individuals would reduce progression of hearing loss by minimising degeneration in the auditory system. this receptor triggers the response via both myd -and trif-dependent signalling pathways, leading to production of cyto-and chemokines and thereby alarming and attracting the immune cells of the periphery to invade the brain. however, tlr is only fully functional in complex with several co/receptors, such as cd . even though cd has been traditionally considered simply as a lps affinity provider, recent data indicate that cd is also necessary for the endocytosis of tlr , which links tlr to intracellular signalling via the adapter protein trif. our latest data reveal the critical role of this receptor for the profile as well as the magnitude of microglial cyto/chemokine production in response to diverse lps variants. cd increases the sensitivity towards lps in a cell type-specific manner, making microglia far more sensitive to lps than bone marrow and peritoneal macrophages. while striatal applications of low lps doses reveal less incoming neutrophils into a brain of cd ko mice, as compared to wildtypes, high doses lps lead to an excessive neutrophil infiltration. this phenomenon is well correlating with the observations in vitro, where cd absence in microglia stimulated with high doses lps causes an excessive production of selected chemokines, with the highest impact on the neutrophil chemoatractant cxcl . these regulatory activities of cd require its membrane insertion and prolonged functionality, pointing to an involvement of signalling. in order to reveal candidates for such signalling mechanisms, we tested the role of syk and plc. even though these enzymes were described to play a role in cd -dependent signalling in dendritic cells, they have no contribution in microglia, further pointing to a cell typespecific organization of the cd /tlr complex in microglia. importantly, we identified receptor systems that impose influences on cd expression itself, which would thereby determine the extent and impact of a cd -mediated regulation of tlr functions. supported by the dfg (for ). in neurodegenerative disease misfolded proteins and cues released by dying cells can attract immune cells by chemotaxis and alter their phenotype. to better understand and differentiate between the effect of these cues, we aim to elucidate the spatiotemporal immunological events in the brain in vivo. zebrafish are an excellent model system to study leukocytes in vivo, and we previously showed by live imaging how engulfment occurs in the developing zebrafish brain (van ham et al., curr biol ). here we use genetically targeted cell ablation to specifically induce cell death of target brain cells. we subsequently track dying cells and immune cells by single and multiphoton d imaging using fluorescent reporter genes. to analyse immunological infiltrate and resident cells in an unbiased fashion, we use large-scale electron microscopy (em) of complete zebrafish cross-sections in parallel as a complementary approach. we find that within a day after onset of cell death multiple types of phagocytic cells, including microglia, accumulate in areas where cell death occurs, engulfing dying cells. granulocytes, do not migrate towards these dying cells and are not found inside the brain at these stages. we also find total numbers of immune cells, microglia in particular, are increased. in addition to microglia, we find phagocytic peripheral cells to be involved in clearance of dying cells. our findings provide an initial in vivo characterization of the immune response to programmed cell death in the brain, indicating involvement of resident and peripheral immune cells. we show spatiotemporal recruitment of these different immune cell types, and proliferation of microglia. the latter together with the recruitment of peripheral leukocytes are likely triggered to increase capacity to dispose of dying cells efficiently. our results indicate zebrafish provides a model to tease apart basic dynamic properties of immune maintenance of the diseased brain in vivo. our current studies focus on identifying the sequence of immunological events in resolving brain injury in vivo and how this is controlled, to ultimately help identify which of these aspects are harmful during brain damage or promote tissue recovery. in humans, lif and osm both signal through the lif receptor (lifr), however osm can also activate its specific receptor, osmr. lifr signaling promotes the survival of glial cells and neurons, and is thought to limit the development of pathogenic t helper cells while promoting differentiation of protective regulatory t cells. osmr signaling is also neuroprotective, however the effects on immune cells are not yet elucidated. in this study, the immunomodulatory effects of lifr and osmr signaling in humans are investigated. we determined which immune cells express the receptors for lif and osm in healthy donors and compared this to the expression levels in ms patients. we found that in blood of healthy donors approximately half of the monocytes express the lifr and osmr, while - % of the t cells and b cells express the receptors. more importantly, in untreated ms patients higher numbers of t cells and b cells express both the lifr and osmr as compared to healthy controls and treated ms patients. available treatments suppress the immune system, resulting in less activated t and b cells, which could explain the lower receptor expression. indeed, we measured the receptor expression on t cells and b cells after activation and demonstrated this strongly induces expression of both receptors. currently, we are investigating the effect of lif and osm on proliferation, cytokine production and antigen presentation of the different immune cell subsets. as blood circulating immune subsets of ms patients have a higher expression of lif and osm receptors, patients show increased susceptibility for modulation by these cytokines. thus, their immunoregulating properties together with the protection of glial cells and neurons indicates that these cytokines are promising candidates for the treatment of ms and other neuroinflammatory diseases. interleukin- (il- ) and interleukin- (il- ) are key cytokines with an important role in the regulation of the inflammatory and immune responses. in the central nervous system (cns), increased expression of both il- and il- occurs in a wide range of pathological conditions. meanwhile il- is recognized as a cytokine with a dual role acting as a pro-inflammatory or anti-inflammatory signal inducing glial activation, il- is well known by its ability to counteract the inflammatory and immune reactions. the objective of the present study was to evaluate the effects of local production of either il- or il- on the microglial response and the neuronal degeneration induced by facial nerve axotomy, a sterile neuronal injury model. to accomplish that, we used two transgenic mice, gfap-il tg and gfap-il tg, which express either il- or il- under the gfap promoter on astrocytes, i.e. a local production within the cns. unilateral facial nerve mm resection was performed, one set of transgenic and wild-type (wt) axotomized animals were sacrificed at , , , and days post injury (dpi) and cryostat free-floating sections were processed for immunohistochemical analysis. another set of animals were sacrificed at dpi to study neuronal survival. our results showed that, at days after facial nerve axotomy, in comparison with the usual neuronal death observed in wt, selective cns il- production had a detrimental effect on neuronal survival, whereas, il- production was able to reduce neuronal death. these effects on neuronal survival correlated with changes in the expression of different molecules associated with microglial activation such as iba , cd b, cd / , mhc class ii, and some integrins like osteopontin and its receptors (cd and a ), along the different time points after injury. remarkably, when compared with their wt littermates, cd b expression was higher on gfap-il tg mice, whereas remains lower on gfap-il tg animals along all the time-points analyzed. similarly, cd expression, which increases after axotomy in wt mice, was higher on gfap-il tg mice at dpi and dpi, whereas no induction of this molecule was detected on the gfap-il tg axotomized animals. in conclusion, our results indicate that astrocyte-targeted il- and il- production has a direct impact on neuronal survival, glial activation and integrin mediated signaling, producing a specific outcome of facial nerve axotomy. methods: we used bv- cells, a murine microglial cell line. in a first experiment aimed at determining the time course of the induction of expression of the lipoxygenases and receptors, they were incubated with lps. in a second experiment aimed at determining the effects of the resolvins and lipoxin on the production of il- , il- b, il- and tnfalpha, they were firstly incubated with lxa , rvd and rve and then incubated with lps. results: our results indicated that lps only enhanced the expression of alx, the receptor for rve and lxa ( i ) (p < . ). the expression of the -lipoxygenase was also affected by lps (p < . ) and varied during the time course, reaching a maximum after h of incubation (p < . ). the production of the proinflammatory cytokines decreased after h of incubation: il- b with rvd ( nm, p < . ) and rve ( nm, p < . ); il- with rve ( and nm, p < . and p < . ); tnfalpha with rve ( and nm, p < . and p < . ). the production of the anti-inflammatory cytokine il- increased only when cells were incubated with lxa after h ( and nm, p < . ). conclusions: these results suggested a potential role of pufa-derivates in the resolution of inflammation. methods: the expression of a panel of m and m markers on human monocyte derived m and m macrophages was analyzed using flow cytometry. this revealed that cd and mannose receptor (mr) were the most distinctive markers for m and m macrophages, respectively. we next examined the activation status of macrophages/microglia in ms and control tissue using a panel of m and m markers. results: our data show that m markers, were abundantly expressed by microglia in normal appearing white matter and by activated microglia/macrophages throughout active demyelinating ms lesions. m markers, mr and cd , were expressed by myelin-laden macrophages in active lesions and perivascular macrophages. double stainings using anti-cd and anti-mr revealed that % of the results and conclusions: the aim of the present study was to evaluate the specific binding of [ h]pk , a compound targeting tspo, to human tissue sections from patients with ms, and to correlate these findings with presence of tspo microglia in different types of lesions. specific binding of [ h]pk was evaluated using autoradiography, and histological analysis was performed on tissue sections to evaluate lesion type, tspo expression and presence of microglia. there was a clear correlation between level of specific binding of [ h]pk and lesion type, such that there was an increased binding to tissue with active or chronic-active lesions compared to control tissue or tissue with chronic lesions. this was concomitant with an increased tspo expression and increased presence of microglia in tissue sections with active or chronic-active lesions. in conclusion, specific binding of [ h]pk is correlated with lesion type, tspo expression and presence of microglia in tissue sections from ms patients. being astrocytes key regulators of microglial cell activation. during inflammatory response, glial cells secrete cytokines such as tnfa, il b and tgfb, as well as nitric oxide (no), and activate phagocytosis, chemotaxis, increased expression of receptors that bind cytokines and inflammatory ligands. among ligand receptors are induced pattern recognition receptors such as scavenger receptor (srs), including class a receptor (sra), expressed by astrocytes and microglia. we will evaluate if sra has active roles in ab clearance and inflammatory activation of glia. methods: we evaluated the role played by sra in glial cell activation, and the regulatory capacity of astrocytes upon microglia in vitro, assessing production of no griess assay and cytokines by elisa; phagocytosis and activation of activity identity markers of signaling pathways by western blot. results: in primary glial cultures from wild type (wt) and knockout mice (ko) for sra, it was found that lps-stimulated sra ko astrocytes release % more no than sra wt astrocytes. these differences were associated with an extended activation of erk signaling pathway. in contrast, evaluation of lps-induced cytokines production, only wt astrocytes released il b, whereas production of tnfa and tgfb reached similar levels in wt and sra ko animals. the abolition of il b release by sra ko astrocytes appeared to be associated to the inhibition of activation of jnk and p signaling, and to the delayed activation of ijb/nfjb pathway in response to lps. microglia from sra ko mice also showed several differences in response to lps stimulation compared with their wt counterparts. they failed to show activation-associated morphological changes and were unable to secrete il b, although they showed an increased release of no. in terms of activation pattern, sra ko microglia showed increased expression of mhc-ii, indicating that sra could be involved in a mechanism inhibiting mhc-ii expression that has not been previously documented. conclusions: our results suggest that sra participates in the activation of specific inflammatory responses by astrocytes and microglia, and the activation of signaling pathways involved in the production of soluble molecules, being also needed for astrocytes in order to be able to modulate microglia activation. microglia are the resident macrophages of the brain and the first responders to disturbances of brain homeostasis. they exist in one of two broadly defined states, a rather inaptly named "resting state" in which they exhibit a ramified morphology and, upon tissue disturbance, an "activated state" in which they exhibit an amoeboid morphology. the characterization as "resting" is in stark contrast with the frantic morphological changes that ramified microglia undergo in brain slices and in vivo, constantly extending and retracting processes. while the purinergic receptor p y has been identified as a key receptor via which microglial processes are guided to a source of atp or to the site of an injury, the signals and pathways guiding microglial processes in the healthy tissue remain elusive. here, by imaging microglia in acute hippocampal brain slices, we assessed the baseline and targeted motility of microglial processes in the presence of different pharmacological agents targeting purinergic signaling. we found that while bath application of a p y specific blocker alone (psb- at nm) or of a p y specific blocker alone (mrs at mm) did not affect the baseline motility of microglial processes, bath application of a p y / / blocker (mrs at mm) strongly reduced the baseline motility of microglia, leading them to retract most of their processes. moreover, a strong rebound of motility was observed after washing out mrs that did not lead to microglial activation within the time window of the experiment (up to minutes). in experiments measuring the chemotactic response of microglia to a pipette containing mm of atp inserted into the slice, we found that chemotaxis was completely abolished by psb- (bath applied at mm) but not by mrs ( mm). finally, no chemotaxis was observed in response to a pipette containing udp-glucose ( mm), an agonist of the p y receptor. these results confirm that p y alone controls the targeted chemotactic response of microglial processes to atp, with no discernable involvement of p y , or , but suggest that p y , and might collectively play a role in controlling the baseline motility of microglial processes in the healthy tissue. how the different p y receptors interact with each other to control microglial movements remains to be determined. supported by an eu marie curie fellowship, the wellcome trust and the erc. the ubiquitin-proteasome-system (ups) plays a central role in degradation and clearance of short lived regulatory as well as misfolded or damaged proteins. upon stimulation by interferons (ifn) the catalytic subunits b , b and b of the s proteasomes (s-proteasomes) can be replaced with b i/lmp , b i/mecl- and b i/lmp subunits, giving rise to the catalytically active immuno-proteasomes (i-proteasomes), respectively. since i-proteasomes play an important role in clearance of oxidant-damaged proteins that preferentially accumulate upon inflammatory stimulation, and many neurodegenerative diseases, including alzheimer's disease (ad) and parkinson's disease (pd) are associated with a chronic inflammatory reaction, we tested the impact of i-proteasome deficiency on the pathogenesis and progression of ad and pd. while appps mice, a model for ad associated extracellular plaque pathology, exhibited increased levels of i-proteasome subunits in the brain, genetic ablation of the b i/lmp subunit, which significantly impairs i-proteasome function, resulted in a reduction of cerebral plaque burden, indicating a disease exacerbating role of iproteasomes in ad. in contrast, transgenic alpha-synuclein (tgasn) mice, a mouse model for intracellular alpha-synuclein pathology associated with pd, crossed to b i/lmp subunit deficient mice, highlight an increase of aggregated alpha-synuclein accompanied by worsening of pathology in tgasn mice lacking functional i-proteasomes, suggesting a protective role of i-proteasomes for intracellular amyloid pathologies. future studies will aim at resolving the mechanistic underpinnings of the seemingly opposing effects of i-proteasome deficiency on the pathogenesis and progression of extracellular and intracellular protein aggregation to aid better understanding and guide novel therapeutic opportunities for neurodegenerative diseases. in the most severe form x-linked adrenoleukodystrophy (x-ald) is a fatal neurodegenerative disorder with inflammatory demyelination of the brain caused by a deficiency of the adrenoleukodystrophy protein (aldp), encoded by the abcd gene. aldp, a member of the abc transporter subfamily d, is located in the peroxisomal membrane and transports very long-chain fatty acids (vlcfa) as coa-esters for degradation into the peroxisome. currently, the only curative therapies are allogeneic hematopoietic cell transplantation (hct) or genetically corrected autologous hematopoietic cd stem cell therapy (hsct). the success probably relies on the settlement of microglia and perivascular macrophages derived from the myelo-monocytic donor cells in the brain of the patient. therefore, we explored the phenotypes of the major immune cell types derived from the cd stem cell. immune cells from peripheral blood of controls and x-ald patients were isolated by magnetic activated cell sorting (macs). purity of cell isolations was verified by flow cytometry. in purified cells qrt-pcr analysis was used to determine mrna levels of the abcd transporters and gc-ms was used for the quantification of vlcfa levels, a diagnostic marker. the three peroxisomal abc transporters were differentially expressed in cd derived cell types of healthy controls: abcd and abcd mrna were inversely expressed in all cell types, whereas abcd was equally distributed. abcd , the closest homolog of abcd could be expected to compensate for the loss of abcd function. however, the expression pattern of abcd was unchanged in x-ald patients; accordingly, cell types lacking abcd mrna (e.g. monocytes) displayed the most severe biochemical phenotype concerning vlcfa catabolism, whereas cells with substantial abcd expression (e.g. t-lymphocytes) were barely affected. thus, not all investigated immune cells present an intrinsic metabolic defect. therefore, we propose that the beneficial effect of hct and hsct may rely on the replacement of those cells lacking sufficient abcd expression in abcd deficiency. in addition, these findings support the concept that abcd is a target gene for pharmacological induction, as an alternative treatment strategy, to rescue vlcfa metabolism and possibly halt the inflammation in x-ald patients. interleukin- (il- ) is a cytokine that has important functions in inflammatory and autoimmune diseases. little is known, however, about il- in the brain. we analyzed expression of il- in the mouse brain during embryonic and postnatal development. being undetected until late embryogenesis, il- was highly expressed during the first two weeks of postnatal life, after which expression gradually decreased and was then absent from the normal adult brain. astrocytes and oligodendrocyte precursors expressed il- , but not neural progenitors or neurons. the vast majority of il- positive cells displayed nuclear staining. apart from its function as a pro-inflammatory cytokine, a role for nuclear il- , potentially in transcriptional regulation, is also emerging. the important role of neuro-inflammation in traumatic brain injury is being increasingly recognized, and local cytokine production can mediate the inflammatory response. because of its potential for attracting inflammatory cells we analyzed il- expression in a cortical contusion infarction model (cci). we found that il- expression was induced by injury, with a peak of expression three days after cci, and at six days the levels declined. il- positive cells showed an overlap with astrocytic and oligodendrocytic lineage markers, but not with neurons, neural progenitors, endothelial cells or microglia/macrophages. a similar time course for il- expression in human brain trauma was found, using cerebral microdialysis, which allows continuous sampling of the parenchymal concentrations of molecules over several days in vivo. the peak of released il- for the patient analyzed was days post trauma. il- binds to a heterodimeric receptor composed of st and il- racp. following injury of st knockout mice we found a general reduction of inflammatory cells at the site of injury, compared to wild type animals, and there were fewer microglia at the trauma site of mice lacking il- receptors. our data show that il- expression is under tight regulation in the normal brain, but is induced by traumatic injury where is important for attracting inflammatory cells. the small heat shock protein alpha b-crystallin (cryab) is expressed by oligodendrocytes (ol) and astrocytes at several stages of multiple sclerosis (ms) lesions. cryab has been shown to be protective and therapeutic in the eae model of ms, possibly due to its anti-apoptotic functions and regulation of inflammatory pathways. in this study, we further investigated the role of cryab in de-and remyelination in vivo, applying the cuprizone model of demyelination to cryab -/mice. surprisingly, we found that cuprizone-induced lesions are significantly smaller, less severe and less inflammatory in cryab -/mice, compared to wild type (wt) controls. staining for microglia and astrocytes revealed that astrocytes are the main inflammatory cell type that is affected by cryab expression: lesions in cryab -/mice display less reactive astrogliosis than wt controls. conversely, although less severe, lesions in cryab -/mice also contain fewer oligodendrocyte progenitor cells (opc) than in wt mice. in addition, our data suggest that remyelination is less efficient in cryab -/mice than in wt controls and that remyelinated areas in cryab -/mice contain fewer ol than remyelinated areas in wt mice. together, we hypothesize that cryab, due to its anti-apoptotic actions, mediates protection in both astrocytes and ol, enabling on one hand reactive astrogliosis, which contributes to demyelination, and on the other hand ol survival, facilitating remyelination. additional in vitro experiments support this hypothesis, revealing that cryab -/astrocytes are less reactive than wt astrocytes and that sirna-mediated knockdown of cryab affects ol survival. interestingly, analyzing phosphorylation patterns of cryab in cuprizone lesions revealed that cryab is differentially phosphorylated in astrocytes and ol. furthermore, we found that cryab is differentially phosphorylated in astrocytes in active demyelinating ms lesions, indicating that phosphorylation of the protein might underlie its (pathogenic) role in active demyelination. these paradoxical findings not only suggest a role for cryab as a potential target in a regenerative approach for ms treatment, but also point to a possible pivotal role for reactive astrogliosis in cuprizoneinduced demyelination and, more importantly, in early ms pathology. in a comprehensive immunohistochemical survey, we compared cortical ms lesions to those of inflammatory (tuberculous meningitis, rasmussen's encephalitis, b-cell lymphoma, and meningitis) and neurodegenerative (alzheimer's disease) diseases. although complex and fulminant immune responses were seen in many disease cases, primary demyelination was only detected in ms. in search of ms-specific molecular mechanisms, we performed whole-genome microarrays on micro-dissected archival formalin-fixed paraffin-embedded material. apart from cortical ms lesions, we also included brain tissue from patients suffering from tuberculous meningitis (inflammatory control) and alzheimer's disease (neurodegenerative control). additionally, control cases without brain pathology were included. using a restrictive cut-off, we identified genes differentially expressed in ms lesions. more than % of these candidate genes could be assigned to t-cell mediated inflammation, microglia activation, oxidative stress, tissue injury, dna damage/repair as well as remyelination and regenerative processes. subsequent neuropathological analysis confirmed that significantly more oxidatively damaged neurons (stained positive for oxidized phospholipids) were present in cortical ms lesions than in any other examined disease. tunel stainings for the detection of dna strand breaks showed that neurons and oligodendrocytes with tunel-positive nuclei were most abundant in ms lesions containing zones of active demyelination and tissue injury. interestingly, immunohistochemical stainings for radical producing enzymes revealed that oxidative stress-mediated tissue damage in cortical ms lesions seems to be driven by nadph oxidases rather than by nitric oxide synthases. taken together, we were able to show that ms-specific primary demyelination is driven by mechanisms of tissue injury that differ from any other investigated inflammatory and neurodegenerative disease. among these mechanisms, oxidative tissue injury seems to play a major role. f. labombarda , s. gonzalez , i. jure , a. de nicola ibyme/conicet/uba, buenos aires, argentina ibyme/coni-cet, buenos aires, argentina reactive gliosis and inflammatory mediators are implicated in demyelination and secondary damage after spinal cord injury (sci). we have previously reported that after sci, short-term progesterone treatment ( days) stimulates oligodendrocyte precursor cells proliferation and decreases reactive gliosis, whereas chronic treatment ( days) differenciates oligodendrocytes precursor cells into mature oligodendrocytes and enhances remyelination. presently, we further studied whether progesterone was able to modulate the inflammatory reaction and cytokine production by astrocytes and microglial cells. thus, the timecourse mrna expression of pro-inflammatory cytokines (il b, tnfa and il- ) and pro-inflammatory enzymes (cox- and inos) was studied by pcr in real time. results showed that the highest increase in cytokine and pro-inflammatory enzymes mrna production occurred in rat spinal cord h after sci. progesterone treatment significantly decreased the early rise of proinflammatory mediators mrnas at h. as progesterone action in spinal cord involves multiple mechanisms, the role played by the classical progesterone receptor (pr) on the progesterone inhibitory effects on cytokines, was assessed in prko mice. in agreement with data obtained in the rat model, sci strongly stimulated tnfa, il b and il- mrna levels in spinal cord of both wild type and prko mice. however, whereas progesterone treatment inhibited the mrnas of cytokines in wild type mice h after sci, it was ineffective in prko mice, involving pr in the inhibition of cytokines production. finally, we measured astrocyte and microglial cells density by immunohistochemsitry after h of progesterone treatment. the steroid administration significantly decreases gfap and ox- cells, which correlated with cytokine and pro-inflammatory enzymes inhibition. we conclude that progesterone attenuates reactive gliosis and inflammatory reaction, probably reducing the secondary spinal cord damage and favouring remyelination. in the steady state they are rarely observed in the cns parenchyma. however, during cns inflammation they cross the blood-brain barrier where together with microglia (cns-resident apc) they can present antigen to infiltrating t cells. the goal of this study was to compare two subpopulations of microglia: cd c-and cd c with brain dc in terms of promoting different t cell responses. two subpopulations of microglia: (cd c-cd dim cd b ) and (cd c cd dim cd b ) as well as (cd c cd hi) bdc have been sorted from the cns of c bl/ mice subjected to eae. sorted cells were used either for ex vivo proliferation and cytokine assays or for qrt-pcr. our results show that bdc and cd c microglia are similar with regard to ability to induce proliferative t cell response from both primed and na€ ıve t cells. nevertheless, they differ in their cytokine profile and ability to promote cytokine release by t cells. our findings suggest that different subtypes of apc in the cns promote different immune responses. this work has been supported by lundbeckfonden. melanocortin receptor (mc r) is predominantly expressed in the brain and it is the only mcr expressed in astrocytes. our previous results showed that a-melanocyte-stimulating hormone (a-msh) the anti-inflammatory action is mediated by mc r in astrocytes and in the hypothalamus of male rats and that these effects may lead to neuroprotection. we have already demonstrated that ndp-msh (an a-msh analogue) increased brain-derived neurotrophic factor (bdnf) expression through the camp-pka-creb pathway in astrocytes. in the present study we examined the participation of mitogen activated protein kinases (mapk) and phosphatidylinosotol- kinase (pi k)-akt pathways in mc r signaling in astrocytes. we also investigated the effect of a-msh on bdnf expression in vivo in brains of male rats.we preincubated cultured rat primary astrocytes with mapk (p , jnk and erk) and pi k-pdk -akt inhibitors min before addition of mm ndp-msh for h. we found that ndp-msh-stimulating effect on bdnf expression assayed by qrt-pcr was abolished only in the presence of erk and pi k inhibitors. accordingly, levels of phospho-erk / determined by western blot were increased by ndp-msh whereas phospho-akt levels were not modified at min. ndp-mshinduced erk / activation was decreased by adenylate cyclase and pi k inhibitors but not by a pka inhibitor, suggesting a camp and pi k involvement in this effect. we also investigated if a-msh was able to induced bdnf expression in vivo. for that purpose we injected male rats with a-msh ( . mg/kg, ip) or vehicle (saline) once and sacrificed them after h. rats were also injected twice daily and for two days and killed h after the first injection. we observed that bdnf mrna levels increased after a-msh treatment at h in the hypothalamus whereas in the cortex bdnf expression was not modified. at h bdnf expression was not modified by a-msh. we showed by immunohistochemistry that mc r and bdnf co-localizes with neurons and astrocytes in the brain. since melanocortins have anti-inflammatory and neuroprotective effects in the brain, the mechanisms described in astroglia help understand mc r action. our results indicate that melanocortins induce bdnf expression in astrocytes through erk and pi k, suggesting that this effect could be involved in the neuroprotective actions of melanocortins. the fact that bdnf expression also increases in vivo in the hypothalamus reinforce this idea. methods: we analyzed microglia activation and the role of nmda receptors in this process using the microglia cell line bv and cortical slice cultures. the in vivo analysis was performed in two models: first, in odtr mice that express the diphtheria toxin receptor specifically in oligodendrocytes (odcs) which allows induced killing of odcs (locatelli et al., ) and second in mice that were immunized with mog/ cfa to induce eae. results: the proinflammatory cytokine il- a plays a pivotal role in the pathogenesis of ms and eae. we showed il- a activates microglia in vitro. therefore, we activated the microglia cell line bv and cortical slice cultures with il- a to and subsequently treated the cultures with the nmda receptor antagonists mk and ap to block nmda receptor signaling. this treatment inhibited il- a-mediated activation of microglia and in particular ros production, proliferation and migration. additionally, we detected increased secretion of il- and g-csf. furthermore, il- a enhanced activation of the nmda receptor through phosphorylation leading to higher influx of calcium upon ligand binding. to further analyze nmda receptor-depended microglia activation we used two different mouse models that provoke different mechanisms of microglia activation. whereas in eae a strong inflammatory process activates microglia, in odtr mice, activation occurs due to induced odc death. interestingly, in the context of eae microglia activation was clearly diminished when mice were treated with mk whereas inhibition of the nmda receptor had no effect on microglia activation in the odtr model. in line with the in vivo data we show that inhibition of microglia activation takes place only when the cells were stimulated with il- a and not upon stimulation with lps. conclusions: our findings show that nmda receptors are not involved in general microglia activation but rather play a role in microglia activation in an inflammatory context with specific cytokines such as il- a. aims of the study: to investigate whether cx cl -cx cr signaling contributes to microglia migratio ctivation and subsequent astrogliosis following msc transplantation in the cns. methods: first, in order to allow localization of grafted msc in vivo, wt c bl/ msc were transduced with a lentiviral vector encoding the blue fluorescent protein (bfp). next, bfp msc were injected into the cns (striatum) of cx cr /-(n ) and cx cr -/-(n ) transgenic mice. these mice have respectively one or both copies of the cx cr gene replaced by the egfp reporter gene. at days posttransplantation, histological analyses were performed to determine iba microglia and s b astrocytes within and surrounding the bfp msc graft site. astrogliosis was determined based on staining for gfap. cx cr expression was evaluated based on egfp expression. all histological evaluations were quantified using tissuequest and/or imagej cell analysis software. results: (i) bfp msc graft survival is observed in both cx cr -/and cx cr /mice; (ii) microglia migration towards grafted msc occurs independent of functional cx cr -cx cl signaling; (iii) down-regulation of egfp gene-expression (and thus also cx cr gene expression) is observed in both cx cr -/and cx cr /mice on msc graft-invading microglia, but not on msc graft-surrounding microglia; (iv) the absence of functional cx cr -cx cl signaling in cx cr -/mice results in significantly less astrogliosis around the msc graft site, as compared to the msc graft site in cx cr /mice. conclusions: here we identified cx cr -cx cl signaling as a potential target to modulate and/or control astroglial scarring following (stem) cell grafting in the cns. the latter is of significant importance as grafted cells can only functionally interact with (injured) brain tissue in the absence of a graft-surrounding astroglial scar. this accumulation is known to be essential for the usual sprouting of injured axons and leads to a functional nerve repair. because of the insignificant infiltration of macrophages in the injured leech cns, we investigate the chemotactic mechanisms of the recruitment of only resident microglial cells in order to explore in fine the crosstalk between damaged neurons and activated microglia leading to the leech cns repair. methods: in vitro and ex vivo chemotactic assays were performed by using recombinant form of hmc q on microglial cells which were pre-incubated or not with anti-cc qr or anti-gc qr antibodies. then, affinity purification analyses using biotinylated c q were performed from leech microglial cell protein extracts. finally, immunohistochemistry analyses were located the production of newly characterized c q receptors in microglial cells. results: we demonstrated that rhmc q contributes to the recruitment of some leech microglial cells. chemotaxis assays showed that the blocking of respective receptors, homologous to human gc qr and cc qr, inhibits the hmc q-dependent recruitment. importantly, affinity purification analyses demonstrated the interaction between both receptors and hmc q. finally, those receptors were located on distinct microglial subsets. conclusion: this work is used to specify the activation processes of only resident microglial cells. the results show the importance of hmc q in the microglial accumulation leading to the nerve repair. in h. medicinalis, hmc q activity is driven through two different receptors which are not present on the same cells suggesting that hmc q activates two distinct microglial cell subpopulations. the question whether blood-borne immune cells are infiltrating brain areas afflicted with neurodegeneration in ad and other tauopathies yielded contradictory results. some authors showed that the chronic neuroinflammation in ad was provided almost exclusively by resident cns cells without any apparent influx of leukocytes from the blood while others reported that hematopoietic cells can enter the brain in alzheimer's disease and may contribute to an increased inflammatory burden. in order to address this issue we have used two independent transgenic lines expressing human misfolded truncated tau in two different genetic background (wistar and shr). we have shown that tau neurodegeneration can induce inflammatory responses mediated by reactive microglia in both transgenic lines, however the microglial responses showed a striking difference between the lines. we have identified two significantly different inflammatory responses to the same inducer. moreover, we found that the genetic background significantly modified the molecular cascade influencing leukocyte influx into the brain. in both transgenic lines, the majority of the blood cells entering the brain belonged to antigen presenting cell family -monocytes and dendritic cells. at the proteomic level we found striking differences between the lines. while in the wistar transgenic line we observed significant increase of cytokine-induced neutrophil chemoattractant- , in shr transgenic line, tissue metalloproteinase was significantly reduced. these findings suggest that blood cell infiltration of the brain affected by tau neurodegeneration is modified by genetic background. this inter-individual variability should be taken into the consideration in the development of novel drugs with anti-inflammatory properties. acknowledgement: this work was supported by axon neuroscience and research grants vega / / , / / , / / , apvv - , apvv - and structural fund . charit e -universit€ atsmedizin, berlin, germany toll-like receptors (tlrs) are key molecules of the innate and adaptive immune response in vertebrates. the original protein toll in drosophila melanogaster regulates both host defense and morphogenesis during development. tlrs recognize host-and pathogen-derived stimuli. single-stranded rna is sensed by tlr localized to the endolysosomal compartment of immune cells. we found that both extracellular viral rna and host-derived micro-rnas induce cell-autonomous and microglia-mediated neuronal cell death through the endosomal receptor tlr in vitro and in vivo. however, the exact intracellular signalling cascade and the cellular mechanisms through which tlr leads to tissue injury in this context remained unclear. unc b is a molecule specifically involved in trafficking of nucleotide-sensing tlrs, such as tlr , in immune cells of both humans and mice. unc b physically interacts with this receptor in the endoplasmic reticulum (er), and the function of the membrane protein unc b is to deliver the nucleotidesensing receptors from the er to endolysosomes. making use of real-time pcr, immunocytochemistry, and histochemistry we systematically examined the expression of unc b in the murine cns. whereas a distinct expression for this molecule was observed in microglia and astrocytes, expression of unc b in cultured neurons was negligible. however, expression of this molecule was detected in cortical neurons of brain sections. moreover, unc b was strongly regulated during different embryonic, postnatal, and adult stages of the developing mouse brain. neurons of various brain regions were identified as the main cell type expressing unc b in the developing brain. taken together, our data reveal a specific expression pattern of unc b in the cns, in particular in a developmental context, and lay foundation for further investigation of the pathophysiological significance of this molecule for both injurious and developmental processes in the central nervous system of vertebrates. has benefited from a number of studies that precisely depicted different types of plaques in white or grey matter areas. also, both inflammation and diffuse axonal loss in the normal appearing white matter (nawm) were extensively described. however, little attention was given to the so-called periplaque, which is usually defined as a partially demyelinated ribbon of tissue surrounding the plaque. whether periplaques correspond to expanding lesions, sites of ongoing remyelination or areas of tract degeneration remains uncertain. methods: in this context, our study aimed to bring quantitative insights to the neuropathology of ms periplaques in the spinal cord of primary or secondary progressive ms patients. a neuropathological quantitative assessment of inflammation, axonal loss and myelin loss was performed concurrently in the periplaques, plaques and normal-appearing white matter (nawm) of cervical spinal cord samples from patients with progressive ms. results: periplaques formed large areas of incomplete myelin loss that extended distant away from the border of plaques. axonal loss was quantitatively similar in plaques and periplaques but signs of axonal dystrophy were predominantly observed in plaques. surprisingly, axons that remained myelinated in periplaques presented a thicker myelin sheath than myelinated axons of the nawm. inflammation in the periplaque was mainly characterized by an accumulation of macrophages/microglia that were closely apposed to myelin sheaths but exerted poor phagocytic activity. finally, we found that neuropathological features of periplaques were overall disconnected from that of plaques with regard to size, shape, inflammation and axonal integrity. conclusions: our work indicates that in ms spinal cords, periplaques correspond to demyelinating rather than remyelinating lesions. it further suggests that in progressive forms of ms, periplaques are likely to impact significantly on neurological disability. finally, we propose that tract degeneration might not be the only cause of periplaque extension and that slowly-expanding demyelination, temporally remote from plaque formation, might occur in periplaques. molecular results will be presented that support these hypotheses. cell-adhesion between endothelial cells at the blood-brain barrier (bbb) makes the endothelium impermeable to blood-derivatives and immune cells. to establish and maintain this barrier during development, adulthood, and during disease, brain endo-thelial cells must develop and sustain these strong adhesive contacts, through expression of tight junction molecules. however, we do not know whether netrin supports inter-endothelial cell adhesion at the blood-brain barrier. given this, we hypothesize that netrin tightens the bbb during development, adulthood, and protects it during disease. methods: to test this, we used both human adult primary brainderived endothelial cells and newborn netrin- knockout mice and evaluated netrin's effect on inter-endothelial cell adhesion and barrier permeability. we also assessed netrins' therapeutic potential to maintain the barrier and limit immune cell infiltration into the central nervous system (cns) during experimental autoimmune encephalomyelitis (eae). results: our results demonstrate that brain endothelial cells express netrins. they help to form a tighter bbb during development. they also maintain and protect the adult barrier by increasing the expression of endothelial junction molecules, thus promoting inter-endothelial adhesion and reducing protein leakage across the barrier. netrins also reduce bbb breakdown and diminish initial myeloid cell infiltration into the brain and spinal cord during eae, which delays disease onset and ameliorates disease severity. discussion: we conclude that netrins enhance bbb stability, and protects the bbb during neuroinflammatory disease. microglial cells were shown to play an important role in many diseases of the central nervous system, not least due to their capability to become activated upon signs of brain injury. they migrate to lesion sites, proliferate, release cytokines and chemokines, and phagocytose cells or cellular debris. therefore, the microglial cytoskeleton has to be highly rearrangeable. one major element of the contractile system in nonmuscle cells is nonmuscle myosin ii (nm ii). as the role of nm ii in glial cells is poorly characterized, it is an aim of this project to study nm ii-dependent functions in microglia. we found that inhibition of nm ii by blebbistatin, which is a highly selective inhibitor of nm ii adenosine phosphatase, prevents any morphological shaping in freshly plated microglia and leads to functional deficits during migration and phagocytosis of fluorescence-labeled beads. using confocal microscopy, we could find diffuse expression of nm ii in resting microglia in vitro, whereas activation with bacterial lipopolysaccharide (lps) led to perinuclear accumulation of nm ii protein. in an activated state, microglial cells release pro-inflammatory cytokines and reactive oxygen species; interestingly, in the presence of blebbistatin the release of nitric oxide (no) as well as rearrangement of nm ii was prevented. taken together, these results illustrate a key role for nm ii in microglial shaping and functioning. ongoing studies will improve our knowledge on spatial and functional aspects of the actomyosin complex during demyelinating processes and open targets for development of therapeutic strategies towards remyelination in the cns. prolonged activation of the nucleus basalis of meynert (nbm), the primary source of cholinergic projection to the cerebral cortex has been reported to cause significant increases in cerebral cortical blood flow (cbf) in rodents. while the nbm also gives rise to gabaergic and glutamatergic projections to the cerebral cortex, the nbm-driven increase of cbf has been described to be dependent in part on muscarinic acetylcholine receptors. lately, several groups reported that astrocytes modulate local cbf via intracellular ca signaling. considering that cortical astrocytes express machrs and in vivo activation of the nbm leads to machr-dependent ca surges in astrocytes, cholinergic modulation of cbf via astrocytic ca surges is conceivable. we report here that a single train stimulation of the nbm (stnbm; hz, . s, ma) induces a biphasic (a rapid increase, followed by an overshooting slower decrease that goes back to baseline within a minute) laser doppler flowmetry (ldf) response in the somatosensory cortex. moreover, the stnbm-induced ldf response was sensitive to the machr antagonist atropine. stnbm with a weaker stimulation intensity ( ma) induces a transient decrease. surprisingly, we find that ip r knockout mice, which lack cytosolic ca surges in astrocytes, show similar ldf responses to stnbm. a.-c. boulay, c. giaume, m. cohen-salmon cirb, paris, france astrocytic endfeet are specialized processes, which enwrap blood vessels and express a large molecular repertoire dedicated to the physiology of the vascular system. one of the most striking properties of astrocyte endfeet is their enrichment in the gap junction proteins connexin (cx) and cx allowing for direct intercellular trafficking of ions and small signaling molecules through perivascular astroglial networks. however, the role of these proteins at the gliovascular interface is not fully understood. we have recently demonstrated that astroglial perivascular cxs expression starts after birth during bloodbrain barrier maturation and controls its integrity (ezan et al. cx d/d purified vessels showed essentially a strong upregulation (fold change > ) of sgcg. this gene encodes gamma-sarcoglycan, a membrane glycoprotein associated with the dystrophin-dystroglycan complex which is essential for muscle integrity and is impaired in duchenne muscular dystrophy (petrof et al., ) . at the gliovascular interface, the dystrophin-dystroglycan complex anchors astrocyte endfeet to the basal lamina (nico et al., ) , and is crucial to the bbb integrity. however, expression of sgcg and its role in the brain has until now only been poorly documented. moreover, we found an upregulation of s a and s a (fold change , and , , respectively) encoding the calgranulin a and b, two proteins present in neutrophils and monocytes, and involved in their migration to inflammatory sites, attachment to endothelial cells and extravasation (ryckman et al., ) . altogether, our results suggest that cx might modify the dystrophin-dystroglycan complex thereby influencing blood-brain barrier properties, and control the attachement as well as the transmigration of leukocytes through the endothelium, thus contributing to the regulation of inflammatory processes in the brain. the blood-brain barrier is supported by the perivascular glia. a crucial event during the gliogenesis is the replacement of radial glia by astrocytes. immunohistochemical staining of nestin visualized the first gliovascular connections after e , and by e the radial glial endfeet cover the vessels completely. by e numerous nestin immunoreactive astrocyte-like cells participated in it. in parallel, nestin immunoreactivity also appeared in the wall of the vessels. by about p both radial glia and nestin immunopositive astrocytes disappeared and gave place to astrocytes immunoreactive to s protein and glutamine synthetase, markers of mature astrocytes. in the radial glia these markers were not found, and they colocalized with nestin only scarcely. the s and glutamine synthetase immunoreactive cells appeared at first in the middle zone of the pallium about p , and their population gradually extended to both the pial surface and the deep cortical zones, colonizing the whole thickness by p , in parallel with the disappearance of nestin immunoreactive glial elements, either radial or astrocytic ones. at p gfap immunoreactive cells were still confined to the most superficial and deepest cortical zones, so showed no colocalization with s and glutamine synthetase, unlike that found after p . whether this glial 'takeover' results in a transitory increase of leakage of the blood-brain barrier, it remains to be answered. it seems however, to underly that cerebrovascular fibronectin and laminin immunoreactivities disappear at first from the middle zone of the developing cortex and only later from the superficial and deep ones, between p and p . according to krum and rosenstein ( ) disappearance of these immunoreactivities refers to the fusion of the glial and vascular basal laminae, the maturation of the definite gliovascular connections. surrounded by astroglial scars that inhibit such growth. little is known about how astrocytes assemble to form this scar, and a deeper understanding of the cellular and molecular mechanisms that control this process is needed if this goal is to be achieved. in vitro data suggest that certain bioactive molecules can alter astrocyte morphology into a more growth supporting state. other molecules have been shown to promote axon growth. clinical application of many of these molecules would require neurosurgical delivery directly into the spinal cord. injectable biomaterials have the potential to serve as depots and scaffolds for in vivo delivery of bioactive molecules. we are developing di-block co-polypeptide hydrogels (dch) as fully synthetic biomaterials that could safely and easily be injected into specific sites after sci to deliver molecules in order (i) to understand better, and (ii) to manipulate, the mechanisms that control glial scar formation. we have previously shown that dch are biocompatible after injection into brain or spinal cord and selfassemble into structures with finely controllable properties. our current work shows that dch depots can deliver diffusible bioactive growth factors that influence local neurons or astrocytes in predictable ways and form gradients that are effective up to several mm away from depots. we are now testing the ability of dch depots to deliver specific growth factors that will manipulate scar-forming cells when dch are injected at clinically realistic sub-acute times after sci. to investigate the molecular phenotype of esdm, we performed a whole transcriptome analysis of esdm in comparison to primary cultured microglia, flow cytometry-sorted microglia, different myeloid cells, t cells, astrocyte-and neuron-enriched cultures. while being distinct from t cells, neurons and astrocytes, esdm were closest related to primary cultured microglia followed by flow cytometry-sorted microglia and other myeloid cells. esdm and primary cultured microglia showed a strong overlap in their transcriptome by only having gene transcripts differentially expressed. most of these differentially expressed genes were immune-related genes that were up-regulated in primary cultured microglia. this indicates that esdm were in a less activated state rather reflecting the in vivo biology of these cells. flow cytometry analysis of cell surface markers selected from the microarray confirmed the close relationship between esdm and primary cultured microglia. esdm resemble primary microglia not only in respect to surface marker expression and functional properties, but also display a molecular phenotype similar to primary microglia. our data suggest that esdm are a reliable tool for the replacement of primary microglia. spinal cord injury (sci) is a devastating condition which usually leads to paralysis. this outcome of injury in most cases is permanent due to the cascade of immunological factors that create an anti-repair environment around the site of injury and due to the limited capacity of central nervous system (cns) axons to regenerate, creates a daunting challenge to establish an effective therapy. from many years of research it is apparent that a combination of therapies would be the best course of action for treatment of sci; this however greatly increase the animals cohorts and time to establish the most effective course of treatment. for this reason we have developed an in vitro model of spinal cord injury as a moderate throughput screen, which is in accordance with the r's. this culture consist of dissociated mixed rat embryonic spinal cord cells plated onto a monolayer of astrocytes this is essential for the spinal cord cells, obtained from rats of embryonic day , which then develop into a carpet of neurites that over time become myelinated forming internodes of myelin interspaced with nodes of ranvier. this mixed cns culture was utilized using a scalpel blade to axotomise the axons and a persistently neurite-free area is created which is accompanied by many features of sci, including demyelination and reduced neurite density adjacent to the lesion and the presence of and reactive astrocytes. these finding are congruent with changes seen in vivo after spinal injury. data will be presented regarding the effects from proven and novel pharmacological agents on neurite outgrowth and myelination looking to understand the mechanism in which they act. as well as modifications of the culture system to incorporate methods to align axons and isolate cell bodies from their processes for a more detailed analysis of regeneration. the role of increased activity of astrocyte networks in structural synaptic plasticity following remote axonal injury is unexplored. we set out to investigate the efficiency of synaptic rearrangements in models where astrocyte reactivity is selectively diminished by the inhibition of the main cytokine pathway in transgenic mice (tg) in comparison to that observed in wild type mice (wt). first, synapse densities were compared in the facial nuclei two weeks after unilateral facial nerve transection by synaptotagmin- /psd- immunolabelling and ultrastructural analysis. we found that the recovery of synapse density was decreased by % in tg mice compared to wt mice, and that the reduction in synaptic density correlates with a relative loss of neuronal coverage by reactive astrocyte end-feet in the same conditions. specifically, we observed a % reduction in excitatory synapse density in the tg mice. second, we developed an organotypic entorinho-hippocampal slice culture system in which astrocyte activation and synaptic plasticity could be more closely monitored in real time. this was assisted by two-photon microscopy, using glial ca -imaging and dendritic spine motility analysis after perforant pathway transection. in summary, we demonstrated that full astrocyte activation is necessary for partial structural recovery of synapses after axonal injury. our work, using simplified models, may help experimental approaches aimed at optimizing adaptive plasticity in cns injuries. pathological loss of myelin in diseases like multiple sclerosis (ms) is usually followed by a phenomenon of remyelination, in which oligodendrocytes synthesize new myelin sheaths to envelope exposed around axons in the adult central nervous system (cns). the importance of remyelination arises from the fact that this process not only restores saltatory conduction, but also protects axons from different insults and thus limits clinical disability in demyelinating diseases. in this scenario, the mammalian subventricular zone (svz) has garnered attention as a potential source of replacement cells after injury. this zone harbours stem cells and supports long-distance migration, and is activated in ms patients to promote gliogenesis. although ng precursor cells are the first to react to demyelination and show the highest proliferation rate in comparison with other cells, the relative contribution of the svz with respect to the inflammatory demyelination induced by the theiler's virus infection and oligodendrogenesis has never been addressed. in the present study, we have investigated the behavior of the svz in theiler's murine encephalomyelitis virus -induced demyelinating disease (tmev-idd). we report that in this viral model for ms, there is a preclinical phase of the disease with demyelination in the corpus callosum that is followed later by an attempt of remyelination. this phase is accompanied by an activation of the svz with no apparent activation of ng precursors, and a strong and clear stain- galectins (gals) are a family of soluble lectins that, when binding to b-galactosides, form multivalent complexes with glycoconjugates of the cellular surface and induce a modulation of intracellular signaling pathways for differentiation and survival. recently, galectin- has been described as a microglial deactivator which prevents neurodegeneration and promotes neuroprotection in an eae model. however, its action at neuronal level is poorly understood. spinal cord injury (sci) also remains a major challenge to neurological research, as several factors such as extracellular matrix molecules inhibit axonal regeneration. among them, semaphorine a (sema a) contributes to the inhibition of axonal regeneration by acting on microtubules and actin cytoskeleton. neuropilin- (nrp- ) is a neuronal receptor whose binding of sema a generates repulsion of axonal growth. in addition, nrp- has been described as a target of gal- in non-neuronal systems. the aim of this work was to study the role of gal- intraumatic sci, considering that sema a expression is "turned on" in sci and prevents axonal regeneration via nrp- . gal- knockout mice (lgals -/-) were submitted to a full traumatic sci at thoracic level (t -t ) and treated with different concentrations of recombinant gal- . we demonstrated that lgals -/mice treated with gal- have a dose-dependent functional recovery of motility as early as days post-treatment, which is structurally associated to neuronal repopulation and high axonal regeneration. these effects were only observed in animals treated with gal- with dimerizing capacity, but not in mice treated with a mutant gal- , only present in a monomeric form. in addition, we observed that microglial response was "turned off" in gal- -treated mice. moreover, exogenous dimeric gal- bound to the surface of injured motoneurons and promoted the spread of nrp- . d confocal microscopy reconstruction showed gal- and nrp- spatial proximity. to confirm this result, we carried out immunoprecipitation assays, which allowed us to determine that only the dimeric form of gal- interacted with nrp- and induced a decrease in the levels of sema- a bound to nrp- . our results show that gal- -based treatments combine its neuronal effect on nrp- with its deactivating effect on microglia, which leads to a better restorative process after medullar lesion. the findings presented here support the potential of dimeric gal- as a therapeutic agent for human sci patients. theophylline and caffeine. it depresses activation of microglial cells and astrocytes which is associated with neuronal damage during inflammation and hipoxia. it is largely known that ethidium bromide (eb) injection into the cns induces local oligodendroglial and astrocytic death, resulting in primary demyelination, blood-brain barrier disruption and schwann cell invasion, and thus serving as an experimental demyelinating model in animals. the aim of this study was to evaluate if prop had the capacity of affecting glial cell behaviour during the process of demyelination and remyelination following gliotoxic injury with eb. wistar rats were divided into groups: i-rats injected with microlitres of . % eb into the cisterna pontina and treated with prop; ii-rats injected with eb and not treated with the xanthine. prop (agener) treatment was done using . mg/kg/ day by intraperitonial route during all experimental period. the rats were euthanized from to days after eb injection and brainstem sections were collected and processed for light and transmission electron microscopy studies. results from both groups were compared by using a semi-quantitative method developed for documenting in semithin sections the extent and nature of remyelination in gliotoxic lesions. in general terms, by - days following gliotoxic lesion, the center of the lesion was expanded and filled with huge amounts of myelin debris among foamy macrophages and demyelinated axons. no astrocytic processes were found in this site and some lymphocytes were seen in the neuropil and around blood vessels. the most prominent feature of the th day was the initial association at peripheral locations between naked axons and remyelinating cells. schwann cells were associated with one or multiple demyelinated axons or already forming thin myelin lamellae around single axons in astrocytic-free areas. oligodendrocytes began to form thin myelin sheaths in areas completely or partially filled with astrocytic prolongations. by - days, it became evident that rats treated with prop presented a small improve in the repair process when compared to untreated animals, the latter presenting greater amounts of myelin-derived membranes in the central area and a lesser extension of oligodendrocyte remyelination, but without any apparent difference regarding to schwann cells. by days results showed that prop administration following eb injection seemed to stimulate oligodendroglial remyelination (mean remyelination scores of . peripheral axotomy results in a disconnection of the neuronal cell body from the target organ. a complex rearrangement of the nerve microenvironment, the so called wallerian degeneration, takes place distally to the lesion and involves schwann cell proliferation as well as macrophage infiltration. the process results in the formation of the bands of b€ ungner which guide the axonal sprouts throughout the nerve distal stump. although this process has been extensively studied, a thorough understanding of the three-dimensional nerve tissue reorganization is not completely known. such knowledge may in turn lead to more effective strategies aiming at the repair of peripheral nerves following transection or crushing. in this sense, the tetrahydrofuran (thf)-based tissue-clearing technique has been adapted for sciatic nerves of mice and used for subsequent observation to p-lsm. transgenic mice that express fluorescent proteins in neurons, astrocytes, schwann cells and microglia/macrophages as well as double and triple hybrids were used. additionally, by immunohistochemical analysis, it was possible to observe the structure of the nerve after injury by the expression of neurofilament and s b, in non fluorescent mice. also, increased non neural cell responsiveness after injury was studied with anti-p ntr immunostaining. transmission electron microscopy allowed the evaluation of the ultrastructure of the nerve microenvironment, as well as the regenerative process by , and days after injury. microscopic examination of cleared tissues of transgenic mice through p-lsm consisted in comparing the different cell type interaction before and after injury, with particular interest on the schwann cells, macrophages and growing axons. question: mass spectrometry (ms) has become nowadays an essential tool for the detection and identification of biomolecules in various contexts of biological problematic. ms can be performed from all types of biological materials ranging from biological fluids to tissues. end of 's it was shown that with certain instrumentation such as those equipped with a maldi ion source it was possible to record ms spectra directly in situ from tissue sections. more, point to point automated acquisition of maldi ms spectra from a whole tissue section was shown to allow for reconstruction of the d images of biomolecules contained in the section distribution through ion density maps (caprioli rm et al., anal. chem. ). methods: maldi ms imaging (maldi msi) is a molecular imaging modality that presents several advantages including possible imaging of hundreds of biomolecules in one step acquisition and the untargeted character of the method allowing to study molecules without any prerequisite knowledge or use of probes. results: over the past decade maldi msi has demonstrated its potentiality in terms of applications for biology and clinics (franck j et al., mol. cell. proteomics. ). maldi msi can be use to study both endogenous molecules namely metabolites, lipids, peptides and proteins or exogenous ones such as drugs or xeniobiotics and was applied to various problematic. understanding of physiological or physiopathological processes requires knowledge on the identity and localization of molecules within a tissue. maldi msi reseals a clear potential to answer this objective and give a unique contribution. indeed, more recently maldi msi has gained new strategies for identification of molecules in situ on tissues. in particular, many efforts were given to develop confident identification of proteins from tissue sections. conclusion: here we will review this novel technology and discuss some of its current and potential contributions for neurosciences application (wisztorski m et al., dev. neurobiol. ). this will be illustrated by presentation of the investigation of invertebrate neuroimmune response as well as vertebrate neuropathological disorders using either fresh frozen or formalin fixed paraffin embedded tissues. the sphingosine -phosphate (s p) signaling pathway is known to influence astroglial reactivity in experimental autoimmune encephalomyelitis and the synthetic s p analog fty has been shown to provide neuroprotection in experimental models of acute stroke. however, the effects of a manipulation of s p-s p signalling at later time points after experimental stroke have not yet been investigated. we examined whether a relatively late initiation of a fty treatment has a positive effect on long-term neurological outcome with a focus on reactive astrogliosis, synapses and neurotrophic factors. we induced photothrombotic stroke (pt) in adult c bl/ j mice and allowed them to recover for three days. starting on post-stroke day , mice were treated with fty ( mg/kg b.i.d.) for days. behavioral outcome was observed until day after photothrombosis and periinfarct cortical tissue was analyzed using tandem mass-spectrometry, taqman v r analysis and immunofluorescence with especial emphasis on markers of astroglial reactivity. fty treatment results in a significantly better functional outcome persisting up to day after pt. this is accompanied by a significant decrease in gfap-immunoreactivity (-ir) and an increase in psdsize. however no change in gfap-mrna, gs-ir or cs -ir was observed. while fty -treatment leads to a decrease in s p concentration, it increases vegf-mrna in the periinfarct cortex. the initiation of fty treatment in the convalescence period has a positive impact on long-term functional outcome, probably mediated through reduced astrogliosis, a modulation in synaptic morphology and an increased expression of neurotrophic factors. due to the restrictions in regenerative capacity of the brain the tissue most severely damaged after traumatic brain injury (tbi) will end up as a fluid filled cavity. presently, it is an impossibility to effectively restore lost brain tissue, but ongoing research on the stimulation of endogenous neural stem cells has increased the hope to viably and functionally repopulate the injured parenchyma. however, it is crucial that possible therapies can show a long-term effect on both regeneration and functional recovery to be of clinical interest. in this study the enhanced induction of neurogenesis in rats after experimental tbi was evaluated three or six weeks after injury. severe focal tbi was performed using the controlled cortical impact model after which a combination therapy of intracerebroventricular administration of epidermal growth factor (egf) for seven days followed by deposition of extracellular matrix scaffold, containing vascular endothelial growth factor, into the cortical cavity. the treatment was devised to accomplish an optimal effect on the stem cell regeneration. the animals with six weeks survival time were functionally evaluated using the morris water maze (mwm). before sacrifice the animals were injected with bromodeoxyuridine (brdu) to identify newly generate cells. sections were made and immunohistochemically double stained for brdu and cell type markers for neurons, astrocytes and blood vessels. the sections were micrographed and analyzed for hemispheric tissue loss as well as number of new astrocytes, neurons and blood vessels. three weeks after injury there was a significant treatment effect as shown by an increase in neuronal and astrocytic regeneration. however, after six weeks there was no difference in the number of newly generated neurons and astrocytes. evaluation of tissue loss and spatial learning in the mwm corroborated that the treatment had no effect at the later time point. the results clearly show the importance of longterm studies in rodents to ensure that a promising effect on tissue regeneration and functional outcome is not merely temporary. astrocyte activation to extracellular matrix (ecm) -producing cells is inhibitory to regeneration in cns injury or disease. here we show that the cleaved neurotrophin receptor p ntr controls transforming growth factor (tgf)-beta-mediated astrocyte activation via regulation of nucleocytoplasmic shuttling of smad . loss of p ntr completely rescues tgf-beta-induced hydrocephaly, astrocyte activation, and ecm production in gfap-tgf-beta transgenic mice. nuclear fractionation of tgfbeta-treated p ntr knock out astrocytes revealed reduced nuclear phosphorylated smad than their wt counterparts. in accordance to p ntr depletion, inhibition of tgf-beta-induced gamma-secretasemediated p ntr cleavage also reduces nuclear accumulation of smad and the secretion of proteoglycans that inhibit neurite outgrowth. taken together, our results identify regulated intramembrane cleavage of p ntr as a mechanism for astrocyte activation resulting in the regulation of tgf-beta signaling and scar formation in the diseased brain. the reaction of glial cells toward brain injury comprises a proliferative response with some reactive astrocytes even reacquiring stem cell potential as indicated by forming long-term self-renewing and mutlipotent neurospheres (for review: robel et al., ). here we addressed first the question to which extent the type of injury selectively affects the proliferative response of ng glia and astrocytes and to which extent the proliferative reaction of astrocytes may be linked to the stem cell response. interestingly, while both astrocytes and ng glia mounted a profound proliferative response after acute invasive injury, such as stab wound and mcao, non-invasive injury models, such as amyloid plaque deposition or widespread neuronal death could not activate the proliferation of these macroglial cells, while microglia proliferated actively in all these lesion models. intriguingly, the proliferative response of astrocytes correlated to the activation of their potential to form self-renewing, multipotent neurospheres with a steep decline with age. we then set-out to determine the signals regulation reactive astrocyte proliferation and neurosphere formation after invasive injury and demonstrate that invasive lesions result in entrance of sonic hedgehog (shh) into the brain from extraneural sources, such as the cerebro-spinal fluid, and activate astrocyte proliferation and neurosphere formation. this response can be blocked by systemic injection of the shh-signaling antagonist cyclopamine as well as inducible astrocyte-specific deletion of the receptor smoothened by glast creert . interestingly, shh-agonists can boost the proliferative response of astrocytes in vivo and their subsequent neurosphere-forming capacity, thereby providing a new approach how to activate this response in conditions where it is limited, as e.g. in age or noninvasive injury conditions. taken together, our work highlighted for the first time differences in the proliferative and stem cell response of reactive astrocytes in different injury paradigms and identified the shh signaling pathway as a key signal in this response. tumor necrosis factor (tnf) is a pleotrophic cytokine involved in normal brain function and inflammation, apoptosis, and other responses that occur following injury and disease. there is a considerable amount of confusion as to whether or not tnf activation is beneficial or detrimental following injury to the cns. genetic studies in mice have suggested that inflammation in disease models involves soluble tnf (soltnf) and that maintenance of innate immune function involves transmembrane tnf (tmtnf). these findings suggest that the outcome of selective pharmacologic inhibition of tnf may depend on whether the pharmacologic intervention is targeted towards soltnf or tmtnf. therefore, we took advantage of a dominant-negative inhibitor of soltnf, xpro , that selectively inhibits soltnf and compared the effect of this inhibitor to a more widely used igg fc-tnf receptor (tnfr) fusion protein etanercept, an inhibitor of both soltnf and tmtnf, that has been a successful treatment strategy for several autoimmune diseases. however, etanercept has been shown to display severe side effects by increasing the risk of infections. female c bl/ mice were subjected to a spinal cord injury at t level. mice were divided into three groups, receiving etanercept, xpro , or saline. the drug was delivered directly onto the lesion site using alzet mini osmotic pumps for days starting immediately after introduction of the lesion. mice were allowed to survive days. functional evaluation was performed using the basso mouse scale, rung walk, open field test and hargreaves test. in contrast to etanercept, xpro significantly ameliorated recovery of hind limb function (evaluated by motor recovery score) compared to saline when administered continuously to the lesioned cord, whereas s.c. injections every days for weeks following sci had no effect. our study suggests that selective inhibition of soltnf is beneficial following sci when administered directly to the contused spinal cord and raises the possibility that selective inhibition of soltnf may represent a new therapeutic strategy for the treatment of sci without compromising the innate immune response. here, we show that expression of oncostatin m (osm) is upregulated after spinal cord injury (sci). to reveal the relevance of increased osm signaling in the pathophysiology of sci, osm was applied locally after hemisection. acute osm-treatment significantly improved locomotor recovery after mild and severe sci. delayed osm-treatment in the chronic phase was ineffective. improved recovery in osm-treated mice was associated with a reduced lesion size, less astrogliosis and diminished neuroinflammation. the underlying mechanism involves neuroprotective effects of osm. furthermore, osm promoted nerve fiber regeneration both in vitro and in vivo. thus, local production of osm after sci is an endogenous response that limits cns lesion propagation and promotes recovery. the university of western australia, crawley, australia following neurotrauma, cells beyond the initial trauma site undergo secondary degeneration, with excess ca a likely trigger for loss of neurons, compact myelin and function. treatment of secondary degeneration by limiting excess ca entry into cells using inhibitors of specific ca channels showed promise in preclinical studies, but clinical trials were disappointing and combinatorial approaches are acknowledged as necessary. we assessed efficacy of every possible combination of three ca channel inhibitors at reducing secondary degeneration months after partial optic nerve (on) transection in rat. we used lomerizine to inhibit voltage gated ca channels (for months); oxidised adenosine-triphosphate (oxatp) to inhibit purinergic p x receptors and/ or -[ -( h-imidazol- -yl) -nitro- , -dioxo- , , , -tetrahydro quinoxalin- -yl]acetic acid (inq) to inhibit ca permeable a-amino- hydroxy- -methyl- -isoxazolepropionic acid (ampa) receptors (both for the first weeks after injury). only the three ca channel inhibitors delivered in combination completely preserved visual function to levels not different from normal animals. in order to determine the mechanism/s by which the three inhibitors delivered in combination preserved function, we assessed neuroprotection, nerve swelling, myelin compaction and node/paranode complexes. preservation of retinal ganglion cell (rgc) somata and axons is unlikely to have accounted for the full recovery of function as only partial neuroprotection of rgcs vulnerable to secondary degeneration was achieved. a range of the ca channel inhibitor combinations prevented swelling of optic nerve vulnerable to secondary degeneration, with no one agent selectively effective. each of the treatments involving lomerizine significantly increased the proportion of axons with normal compact myelin and decreased the proportion of axons with partially decompacted myelin, indicating the importance of sustained control of ca flux via voltage gated ca channels for prevention of chronic myelin decompaction. nevertheless, limiting decompaction of myelin or formation of atypical node/ paranode complexes was not sufficient for preservation of function in our model. it is likely that the additional prevention of the lengthening of the paranodal gap that was only achieved by treatment with the three ca channel inhibitors in combination was an important effect that contributed to the associated preservation of visual function by this combinatorial treatment strategy. to generate myelinating oligodendrocytes opcs undergo a distinct series of morphological and molecular changes. these are regulated by a combination of extrinsic and intrinsic factors, which have only been partially been revealed so far. in the present study we identify a novel signaling mechanism as potent negative regulator of opc maturation. ephrin b , a member of the b-class family of ephrins, inhibits opc process formation and blocks the formation of mature oligodendrocytes in vitro. the presence of ephrinb results in activation rhoa and pkca signalling and deactivation of fak kinase in opcs in vitro. moreover, epha rtk, a receptor responsive to ephrinb in opcs, is also expressed by opcs in demyelinating lesions. infusion of recombinant ephrin b into demyelinating lesions results in a failure of remyelination caused by an inhibition of opc maturation. in contrast, antibody-mediated masking of ephrin b epitopes in vitro as well as in demyelinating lesions of aged rats promotes opc differentiation and accelerates myelin regeneration. our results demonstrate an important role of ephrin b for the process of remyelination. after peripheral nerve injury, axons rapidly degenerate and start regrowing only after a few days. full functional recovery, however, is rare. a progressive loss of intact axons also determines the clinical phenotype of chronic demyelinating peripheral neuropathies, such as charcot marie tooth disease type a (cmt a). cmt a is the commonest inherited neuropathy, caused by a duplication of the peripheral myelin protein kda (pmp ) gene. we demonstrate that the transgenic neuronal overexpression of the growth factor neuregulin- type i enhances axonal regeneration and functional recovery after experimental acute peripheral nerve injury. similarly, neuregulin- type i overexpression preserves axonal loss and improves nerve function in a mouse model for cmt a. importantly, neuregulin- type i induced axonal preservation in cmt a mice was independent of myelination, as myelin sheath thickness was not altered. we conclude that axonal recovery and preservation relies on common supportive factors and that paracrine neuregulin- type i constitutes a beneficial signal in peripheral nerve disorders. g. szab o university of tuebingen, tuebingen, germany question: the enormous capacity for repair of the cns after lesion is one of the most amazing property of lower vertebrates. in the mammalian central nervous system (cns) the astrocytes reveal the so-called orthogonal arrays of particles (oaps); the clusterization of the aqp molecules at the astrocytic endfeet. in lower vertebrates these structures are lacking. in the case of the amphibia -anolis carolinensis-the caudal spinal cord has the capability for regeneration after loss of the tail, an ability which might be in relation with the presence of tight junctions (tj) between the glial cells like the olfactory ensheating cells act in mammalian olfactory system through the lifespan. the growing axons of the fish optic nerve are embraced by astrocytes which are also interconnected by tjs. tj connected glial cells could contribute to a growth-supportive microenvironment which might reinforce our hypothesis namely the mutual exclusion of aqp occurrence in astrocytes and regenerative growth of nerve fiber. methods: we performed immunocyto-and immunohystochemical staining, western blot, freeze-fracture electron microscopy and pcr on cultured aqp knockout and wildtype astrocytes and also on formalin fixed paraffin embedded tissue sections regarding junctional proteins, such as occludin, claudin , , . result: in the light of our experiments, there is no mutual exclusiveness between the presence of aqp and tight junctions in astrocytes, at least in mammals. further investigations need to be completed. glutamate and electrical activity influence opc differentiation and myelination in normal development. opcs receive synaptic input from unmyelinated axons, possibly to initiate myelination. both opcs and mature oligodendrocytes respond to glutamate via ampa and nmda receptors. activation of glutamate receptors in vitro increase opc migration but decreases proliferation. here we examine the role of glutamate signalling in remyelination following experimental demyelination in the adult rodent cns. we voltage-clamped opcs in brain-slices of adult rat cerebellar peduncle containing focal areas of primary demyelination and post-identified these by ng -immunolabelling. recruited opcs mainly expressed ampa receptors at the peak of the opcs proliferation ( - days postlesion), as over % of the glutamate evoked current was blocked with nbqx (ampa/kainate receptor antagonist) and unaffected by ap (nmda receptor antagonist). the demyelinated axons continued to propagate action potentials with latencies similar to those in unmyelinated axons during development. critically, the demyelinated axons established synapses with opcs expressing voltage-gated sodium currents. these synaptic inputs had identical decay times to those recorded during developmental myelination. pharmacological inhibition of neuronal activity or glutamate signalling decreased remyelination efficiency by impairing differentiation. these results indicate that ) glutamate currents are mainly mediated via ampa and kainate receptors during the opc recruitment ) opcs establish synaptic contact with demyelinated axons and ) the neuronal activity is essential for remyelination. together, these data suggest that demyelinated axons remain active and communicate with opcs through glutamate release during the regenerative process to guide their remyelination. there has been much recent interest in the use of intraocular stem cell transplantation to slow or reverse visual loss in glaucoma. however, migration and integration of stem cells into the retina is limited by reactive gliosis, a major barrier to retinal engraftment. our aim was to identify glial modulators of low toxicity and measure their potential to facilitate stem cell entry into the host retina. an explant organotypic culture system was used as a stem cell transplantation model and screening tool for candidate drugs. gfp mouse mesenchymal stem cells (mmscs) were co-cultured on the surface of the explants for days.the microglial contribution to mscinduced reactive gliosis was investigated by assessing microglial activation and proliferation using immunohistochemistry for f / and edu. distribution of chondroitin sulphate proteoglycans (cspgs) and their role in the barrier was also explored by immunostaining for chondroitin sulphate (cs- ) and by chondroitinase abc(chabc) treatment. stat inhibitor vi and jak inhibitor were used to suppress glial fibrillary acidic protein (gfap) expression. the efficacy of each drug to overcome the barrier was evaluated by immunohistochemistry for the glial markers gfap. mmsc integration was assessed by simultaneous immunostaining for gfp. cell viability after drug treatment was investigated by measuring the preservation of retinal thickness and by immunostaining for the neuronal marker neun and for caspase . co-culture of retinal explants with mmscs caused a . . fold increase in gfap expression. microglial activation and proliferation was not markedly affected by the presence of grafted mmscs on retinal surface. cspgs, highly expressed in the outer retina but only moderately in the inner retina, do not contribute substantially to the barrierto inner retinal engraftment. in contrast, interfering with the jak/stat pathway successfully reduced gfap expression by % % compared to control and facilitated mmscs integration into the host tissue. no toxic effect on cell viability was detected. on the contrary, over the days ex-vivo culture, stat inhibitor vi treatment resulted in a neuroprotective effect on the outer nuclear layer, whose thickness ( . . mm) is better preserved compared to control ( . . mm) the results show that microglia and cspgs do not play a dominant role in the barrier to retinal engraftment, but the jak/stat pathway represents a promising pharmacological target.targeting modulators of glia reactivity to promote stem cell integration in the host tissue may facilitate stem cell therapy in glaucoma and other neurodegenerative diseases. oecs possess many characteristics favorable for the repair of sci including the secretion of growth and survival factors, the expression of cell adhesion molecules and the ability to integrate with astrocytes. oecs have also been reported to promote exceptional outgrowth of severed axons across the inhibitory environment of the lesion scar. the unique combination of regenerative properties suggests oecs would be beneficial for the repair of ventral spinal root avulsion injuries. after ventral root avulsion anterior horn neurons often fail to regenerate axons back into the surgically re-implanted nerve root resulting in poor functional recovery. to evaluate the efficacy of oec transplants for the repair of ventral root avulsions, the ability of oecs to promote neurite outgrowth from spinal cord neurons was investigated in a spinal cord organotypic co-culture. the integration of oecs with the spinal cord tissue was also examined by confocal fluorescence microscopy. oecs were harvested from adult sprauge dawley (sd) rats, cultured for days and transfected with gfp. slices of cervical spinal cord were prepared from p sd pups and cultured on collagen coated membrane inserts. each slice was surrounded by a collagen gel with or without the addition of gfp oecs and glial derived neurotrophic factor (gdnf). our results indicate that oecs alone and in combination with gdnf have a positive effect on neurite outgrowth and morphology. the aim of this study was to obtain an hydrogel for the controlled delivery of vascular endothelial growth factor (vegf-a ). hydrogels are a class of liquid-gel biomaterials with high water content, typically composed of cross-linked, three-dimensional hydrophilic water-soluble polymer networks. the advantages of injectable hydrogel systems are: biocompatibility, biodegradability, adjustable shape, low cost and similarity to the extracellular matrix; moreover, they can be used as growth factor and cell delivery systems for tissue engineering applications. agarose/gelatin (a/gl) hydrogel was cross-linked with genepin. rheological measurements show that a/gl hydrogel can be injected through a syringe into the inner cavity of a nerve guide. in vitro assays performed to evaluate adhesion, proliferation, viability and migrationusing a fibroblast cell line (nih t ), neonatal olfactory bulb ensheathing cells (nobec) and a schwann cell line (rt -d p t)show that hydrogel allows cell adhesion, proliferation, viability and migration. vegf-a is a potent angiogenic factor and angiogenesis has long been recognized as an important and necessary step during tissue repair. vegf is known to have a positive effect on schwann cell proliferation and migration, neuron survival and outgrowth of regenerating nerve fibers. the hydrogel preparation protocol was optimized to be functionally efficient at physiological conditions, allowing the loading of vegf without the risk of its denaturation. different amounts of vegf ( - - ng/ml) were incorporated in the a/gl hydrogel and the releasing rate in vitro up to days was quantified by elisa . released vegf bioactivity was validated in human umbilical vein endothelial cells (huvec) and in nobec by evaluating phosphorylation of vegf-receptor (vegfr ), akt and erk - . moreover, dorsal root ganglia explants were cultured on hydrogel containing different amounts of vegf, and an increased neurite outgrowth was quantitatively assessed. these results demonstrate that vegf can be successfully incorporated and bioactive released from a/gl hydrogel inducing vegfr activation and neurite outgrowth. in vivo test are ongoing on adult rats: one centimetre lesions on medial nerve were performed, followed by implantation of porous polye-caprolactone tubes filled with a/gl hydrogel containing vegf-a . functional tests and histological analysis will be carried out to evaluate peripheral nerve regeneration. .spinal cord injury (sci) often leads to death of oligodendrocytes and considerable demyelination. demyelination of axons compromises signal conduction, and contributes to the functional deficits following sci. enhancing remyelination may serve to restore conduction and likely prevents axonal degeneration. here we focussed on a potential therapeutic intervention to promote remyelination, the transplantation of human oligodendrocytes precursor cells (opc)s capable of differentiating into mature oligodendrocytes and remyelinating denuded axons. human platelet derived growth factor (pdgf)-responsive neural precursor (prp) cells were isolated from the fetal forebrain and had been previously shown to differentiate into mature oligodendrocytes with a higher efficiency in vitro than traditional egf/fgf responsive neural stem cells (chojnacki and weiss ; deleyrolle et al. ). therefore, we tested the potential of human prps to differentiate into new oligodendrocytes and remyelinate axons in a rodent thoracic contusion spinal cord injury model. one week following injury, human prps were transplanted rostral and caudal to the lesion site. subsequent histological examination at two, seventeen and forty-two days post-transplantation demonstrated that human prps survived and integrated well into host tissue, particularly when nk cells were depleted. transplanted human prp's labelled with the mature oligodendrocyte marker apc, and % of the cells colabelled with the oligodendroglial marker olig . astrocyte differentiation was also observed, suggesting human prps function as a multipotent glial progenitor following sci. human-specific immunoreactive processes were seen in close association with axons expressing the myelin protein mbp, providing evidence of remyelination by the transplanted cells. overall, these findings indicate that human prps are a source of new oligodendrocytes capable of producing myelin in response to spinal cord injury. remyelination is a regenerative process during which demyelinated axons are enwrapped by newly formed myelin sheaths. in the central nervous system (cns), this process takes place in two stages: first, during the recruitment stage, oligodendrocyte precursor cells (opcs) divide, migrate and engage the demyelinated axons. then, during the differentiation stage, opcs differentiate to myelin-forming oligodendrocytes. under pathological conditions such as multiple sclerosis (ms), remyelination often fails and as a consequence chronic demyelinated areas accumulate in ms. post mortem evidence suggests that opc differentiation is often impaired in ms patients. however, the reason why opc differentiation fails in ms is not completely understood. it has been proposed that ms lesions contain factors that act as specific inhibitors of opc differentiation. amongst them, myelin associated inhibitors play an important role. we have demonstrated that the inhibitory effect of myelin protein extracts (mpe) is mediated by activation of signalling cascades including pkca-marcks in vitro. furthermore, modulation of pkca has been shown to rescue opc differentiation in the presence of inhibitory mpe. here we demonstrate that tamoxifen, a drug that is used in various clinical settings because of its pkc-inhibitory activity, is able to promote opc differentiation in vitro. in a series of in vivo experiments, we tested the hypothesis that inhibition of pkca by tamoxifen promotes cns remyelination. demyelination was induced in the caudal cerebellar peduncle (ccp) of young-adult sprague-dawley rats by the administration of ethidium bromide. animals received daily doses of tamoxifen. although tamoxifen treatment did not induce changes in the number of opcs expressing immature markers (ng and pdgfr-a), a significant increase in the number of cells expressing mature marker of oligodendrocytes (cc /olig and plp) was detected. furthermore, analysis of resin-embedded tissue demonstrated that tamoxifen significantly promotes remyelination in the cns when compared to controls (mann-whitney's test, p < . ). although further investigations are required to elucidate the mechanisms by which tamoxifen exerts cns remyelination, our results demonstrate that administration of tamoxifen represents a potent and clinically accessible strategy to promote endogenous myelin regeneration. induced pluripotent stem (ips) cell technology now allows development of autologous oligodendrocyte precursor cells for putative remyelination cell therapy for multiple sclerosis (ms). we have successfully developed mouse and human ips cells and converted these into oligodendrocyte precursor cells (opcs) that can differentiate into fully mature oligodendrocytes. furthermore, we have shown that (intracerebral) implantation of ips-derived opcs in mouse ms models enhances remyelination.before implantation-induced remyelination therapy with autologous ips-derived opc can proceed to a clinical trial for remyelination cell therapy in ms patients, we have to provide solid evidence for long term efficacy and to establish the safety thus ultimately excluding contamination of teratogenic (pluripotent) stem cells. these issues are best examined in a nonhuman primate model. the biomedical primate research center (bprc), rijswijk has developed a welldocumented experimental autoimmune encephalomyelitis (eae) model in marmoset monkeys that most closely approximates the pathology of ms in humans. in this animal model we will verify that intrathecally administered ips-derived opcs migrate to and remyelinate eae lesions and determine their long-term fate.we have developed an efficient protocol to induce marmoset ips cells from skin fibroblasts. for that we have used a lentiviral polycistronic construct, containing the four (human) reprogramming genes oct , klf , sox and cmyc, that is excisable using cre-recombinase. the pluripotent nature of the marmoset ips cells was established based on the endogenous expression of pluripotency transcription factors and the ability to differentiate in-vitro (via embryoid bodies) and in-vivo (subcutaneous injection for teratoma formation) into all the different tissues of the three germ layers. presently we are developing protocols for the differentiation of these marmoset ips cells into an oligodendrocyte cell lineage. published protocols have been modified for production of opcs for research purposes, and produced cells are used for characterization of opcs. ultimately the aim is to define an opc population with optimal myelination capacity. results and discussion: currently, human oligodendrocytes and opcs can be produced from pluripotent stem cells. these cells are routinely characterized in protein expression level, and methods for more comprehensive characterization are under development. produced opcs can be purified with fluorescence-activated cell sorting based on ng protein expression in order to study if this cell population is heterogeneous in terms of myelination capacity. conclusions: oligodendrocytes and opcs can be produced from human pluripotent stem cells. studies are ongoing to determine the molecular characteristics and myelination capacity of the differentiated and purified opc-population. remyelination, the regenerative process in which myelin is restored to demyelinated axons, is carried by oligodendrocyte precursor cells (opcs). in a previous study we have shown that oligodendrocyte precursor cells are multipotent and they can differentiate into oligodendrocytes as well as to schwann cells and occasionally astrocytes in the remyelinating lesions. we observed that schwann cells derived from opcs in the central nervous system occupied almost exclusively tissue around blood vessels in astrocyte deficient areas. therefore, we postulated the occurrence of specific niches or microenvironments that modulate opc fate. the aim of present study was to identify the factors and their downstream effectors that significantly discriminate vascular and nonvascular niche and determine the fate of oligodendrocyte precursor cells. we microdissected tissue from vascular and non-vascular regions of lesion areas at , and days after bilateral stereotaxic injection of ethidium bromide into the brain white matter of adult rats and performed microarrays to profile the whole transcriptome of both niches separately. this approach allowed us to distinguish between mrnas that are enriched within two separate environmental niches within the same lesion area. the study identified differentially expressed transcripts. comparative bioinformatic analysis of global gene expression combined with signalling transduction pathway structure and genes function analysis revealed specific candidate signaling pathways differentially expressed in local peri-vascular niche compared to nonvascular one. results of rt-pcr-based validation of microarray data have proven that expression gradient of bmp , bmp , their antagonist sostdc , as well as wnt b, wnt , dhh and scube act as the major regulators of opcs fate in the vascular niche. moreover, we observed that blood vessels undergo substantial regeneration in the course of remyelination and postulated the important role of blood vessels reconstruction in creating the specific microenvironment of neurovascular niche during tissue regeneration. our analysis shown that unique properties of tissue around blood vessels differ from the rest of the lesion which can be one of the factors favoring opcs alternative differentiation in this area of lesion. . however, key aspects of dynamic behavior, such as cell migration or process orientation and motility, can only be monitored by live imaging. to elucidate these key aspects of opc behavior after injury, we used repetitive in vivo two-photon laser scanning microscopy ( plsm) to follow ng -cells after smaller and larger stab wound injuries in the mouse somatosensory cortex. we therefore used the bac transgenic mouse line sox icreert (simon et al., ) crossed to the inducible reporter line cag-gfp (nakamura et al., ) selectively labeling cells of the oligodendrocyte lineage. gfp opcs were imaged through a cranial window at the day of injury and at different time points thereafter. identification of the same cells was possible based on blood vessels labeled by rhodamine dextran as stable landmarks. live imaging revealed that the majority of opcs reacts within days after injury with hypertrophy (enlarged soma and processes), polarization (elongation and concentration of processes towards the injury site), directed migration towards the injury site (defined as movement of the cell body for at least mm) and proliferation (defined by a single cell being replaced by or even more daughter cells). only a small proportion of cells within $ mm of the injury did not react in any detectable manner. we noted that polarization and hypertrophy occur rather fast after inflicting the injury, while proliferation peaks days after injury. taken together, the live observation of opcs reacting to stab wound injury supports the concept of opc heterogeneity and reveals new insights into the functional role of these cells after injury: the fast process orientation towards the injury site implies a contribution to wound closure and their substantial proliferation sometimes for several rounds amplifies the number of ng -cells surrounding the injury site with implications for scar formation. the aim of this study was to create a non-invasive tool for cell visualization and cell population tracking that can be used in long term studies. methods: this study was performed using human pluripotent stem cell lines derived in institute of biomedical technology (former regea), university of tampere, finland. cells were differentiated to neural cells using neural differentiation protocol described earlier by lappalainen and colleagues. fluorescent probes used in this study were ) chloromethylfluorescein diacetate (celltracker green cmfda, ct) and ) , -dioctadecyl- , , , -tetramethylindodicarbocyanine perchlorate (did). cell viability and proliferation of the labeled populations was briefly studied. also, the labeled cells were characterized using immunocytochemistry and neuronal functionality was studied using micro electrode array (mea). results: during labeling optimization a wide range of different labeling parameters was tested for both probes to obtain long term labeling. most suitable parameters for human neural cells were mm ct with hours incubation time and mm did with hours incubation time. with these concentrations the labeling was visible up to weeks and had no statistical effect on cell viability. ct labeling was not affecting to cell proliferation but with did slight decrease in proliferation was seen in long term culture. labeled populations contained both map- positive neurons and gfap-positive astrocytes. also, the ct or did labeled population and their mixed-cultures expressed normal spontaneous network activity when cultured on mea-platform. conclusions: this study indicated that fluorescent probes ct and did are suitable for long-term labeling of human derived neural cultures in vitro. the labeling had no negative effects on cell behavior with the exception of did labeled cells having slight decrease on cell proliferation. importantly, the labeled neural networks were electrically functional. axonal regeneration after spinal cord injury (sci) is limited mainly due to the presence of an inhibitory microenvironment, especially reactive astrogliosis and glial scar formation. overcoming this inhibitory barrier of reactive astrocytes and inhibitory substances making the microenvironment of the damaged neurons permissive for axonal regrowth might be crucial for cns repair. single-dose irradiation has been proved to be neuroprotective in cns trauma through elimination of reactive astrocytes, however, the irradiation conditions with singledose protocols are not in clinical use. in the present study, we aimed to investigate whether low-dose-fractionated irradiation, which is currently being used widely for clinical treatment of several tumor types, could regulate cell cycle elements of reactive astrocytes just like its use in cancers, inhibit glial scar formation, attenuate astrogliosis-mediated inhibition of axonal regeneration and facilitate recovery of motor function after spinal cord hemi-section in beagle dogs. as expected, our results demonstrated that low-dose-fractionated irradiation reduced reactive astrogliosis, attenuated astrogliosis-associated neural injuries, ameliorated microenvironment of axonal regeneration, and benefited recovery of the animals subjected to spinal cord trauma. accordingly, irradiation might be a promising therapeutic strategy targeting at excessive astrogliosis for sci in the near future. mesenchymal stem cell transplantation has been proven to have beneficial effects in various degenerative diseases, including demyelinating models. both in our lab as well as others have shown that they express a number of trophic factors that are capable of inducing remyelination, mainly by activating nearby oligodendrocyte progenitors towards mature oligodendrocytes that in turn remyelinate the damaged area. however, this effect was only observed locally, in the area surrounding the graft, thus in order to achieve general remyelination in various brain structures simultaneously, we decided to perform bone marrow-derived mesenchymal stem cell injections into the lateral ventricles. in this manner, the cells may attach to various areas such as the hippocampus, corpus callosum and fornix, among others, all of which are demyelinated. previous to the graft, the cells were incubated with iron nanoparticles. this way, it is possible to track in vivo the grafted cells by magnetic resonance imaging (mri). as a result, the cells were observed at different time points ( - - - - days) by mri. also, the demyelinated areas can also be visualized and myelin quantified using image analysis software. this allows the quantification myelin density in the same individual mice at different moments before and after transplantation. j. muenzel , , c. becker , t. becker , a. williams university of edinburgh, centre for regenerative medicine, edinburgh, united kingdom university of edinburgh, centre for neuroregeneration, edinburgh, united kingdom central nervous system (cns) remyelination is important for the restoration and protection of proper nervous system function in demyelinating diseases such as multiple sclerosis but is poorly understood, and inefficient in humans. in contrast, zebrafish are able to regenerate many tissues very efficiently. as zebrafish are being increasingly used to study myelination, we aimed to develop and characterise an adult zebrafish model of cns de-and remyelination, to answer the question if remyelination is also very efficient, to describe the biology and to identify similarities and differences with mammalian remyelination. this then may allow us to better understand why zebrafish have a high regenerative capacity compared to rodents and humans. previous studies in teleost fishes have used optic nerve crush as a paradigm of myelin injury to investigate myelin repair. this, however, involves the de novo myelination of regenerated axons, rather than deand remyelination of the same axons. lysophosphatidylcholine (lpc) is a detergent-like toxin, which is widely used in rodent models to demyelinate axons. we administer lpc to the optic nerve of adult zebrafish and observe less myelin by both immunoreactivity and electron microscopy at the lesion site at days post lesion (dpl) and microglia activation along the optic pathway. immunoreactivity and electron microscopy suggest complete regeneration of myelin at dpl. we cannot identify any axonal injury in this model. in young zebrafish (aged - months), the myelin thickness of remyelinated fibres shows no difference to the pre-lesion state, which is different to mammals, where the myelin thickness is reduced. however, in old fish (aged months), although the axon diameter is the same as pre-lesion and in young animals, , remyelinated fibres have thinner myelin, suggesting that the regenerative capacity of zebrafish declines with age. we believe that this new zebrafish model of cns remyelination can be added to the suite of current models to better understand the remyelination process, why it fails, and to test for compounds to improve it, with the added benefit that zebrafish are rapid breeders, transgenesis is easy and there is a high potential for live imaging. although often described as a hard-wired component of the vertebrate body, the nervous system is a plastic and considerably fluid organ system that reacts to external stimuli in a consistent, stereotyped manner, while maintaining incredible flexibility and plasticity of its core components. unlike the cns, the pns is capable of significant repair, but we have only just begun to understand the cellular and molecular mechanisms that underlie this phenomenon. peripheral nerves are composed of axons surrounded by layers of glia and connective tissue. they are ensheathed by myelinating or non-myelintaing schwann cell glia, which are in turn wrapped into a fascicle by a cellular sheath called the perineurium. this structure forms from centrally-derived glial cells and serves as a protective barrier that is essential for nerve function. following an injury, adult peripheral nerves have the remarkable capacity to remove damaged axonal debris, regenerate and re-innervate targets. schwann cells have been shown to play an important role in this process by trans-differentiating, proliferating, clearing debris, and guiding re-growing axons, but less is known about the potential role of perineurial glia. to investigate the role of perineurial cells in pns regeneration, we have developed an injury response assay that uses a micropoint laser to create injuries along the motor nerves in live transgenic zebrafish. time-lapse imaging of injured nerves reveals that perineurial glia rapidly respond to nerve injury and extend processes toward the injury zone. this is in contrast to schwann cells, which we observe orienting towards the distal stump where they engulf and clear axonal debris. these data demonstrate that perineurial glia respond immediately to motor nerve injuries in a manner distinct from schwann cells, and future work is aimed at defining the molecular mechanisms that mediate the cellular responses of perineurial glia and schwann cells, as well as determining if developmental paradigms are recapitulated in these glial populations during the regenerative process. we have previously shown that in schwann cells the transcription factor c-jun acts as a master regulator of wallerian degeneration and is required for successful repair following nerve injury (arthur-farraj et al., methods. we here investigate on the release of the gliotransmitter glutamate from superfused isolated purified glial processes (gliosomes) obtained from adult rat cerebellum. intracellular ca levels were measured by a microfluorimetric technique. immunocytochemical confocal and western blot analysis were also carried on cerebellar gliosomes. results. confocal and western blot analysis confirmed that cerebellar gfap-positive gliosomes are a purified preparation of glia processes. more then % of gliosomes were positive for brain-specific lipid binding protein (blbp), indicating that they derived from bergmann cells. by measuring the release of glutamate we obtained evidence for the presence of glutamate release-facilitatory ampa receptors on the bergmann glial processes. in fact, ampa evoked [ h]d-aspartate or endogenous glutamate release which was abolished in ca -free medium; effectiveness of the selective inhibitor naphthylacetyl spermine indicated the involvement of ca -permeable, glua -lacking ampa receptors. ca imaging confirmed the presence of ca -permeable, glua lacking ampa receptors on bergmann glia processes. activation of the glua -lacking ampa receptors triggered vesicular exocytotic glutamate release: inhibition of vesicular loading by the vesicular glutamate transporter (vglut) inhibitors rose bengal or trypan blue, or by the h -atpase inhibitor bafilomycin a prevented the ampa-evoked glutamate release (see figure) . confocal analysis confirmed that blbpand gfap-positive processes expressed vglut and vglut . conclusions. we here report evidence that: . functional processes of bergmann cells are recovered in purified preparation of adult rat cerebellar gliosomes; . ampa receptors located on bergmann glia processes show features of ca permeable, glua -lacking ampa receptors; . activation of the ampa receptors evokes ca entry in isolated bergmann glia processes and ca -dependent vesicular glutamate release from the processes. activation of ca -permeable, glua lacking ampa receptors coupled to vesicular glutamate release might represent a mechanism by which bergmann glia processes modulate synaptic transmission at parallel fibers/purkinje cell synapses. the formation of brain edema, which accompanies ischemic or traumatic brain injuries, originates from a disruption of ionic/neurotransmitter homeostasis that leads to extracellular k elevation and neurotransmitter accumulation in the extracellular space. an increased uptake of these osmotically active substances, predominantly provided by astrocytes, is accompanied by intracellular water accumulation via aquaporin- (aqp ). previously, it has been shown that the removal of perivascular aqp via the deletion of alpha-syntrophin, a protein responsible for anchoring aqp on the astrocytic membrane, delays edema formation and k clearance (amiry-moghaddam et al., , pnas ; ( ): - ). therefore, we aimed to elucidate the impact of alpha-syntrophin deletion on astrocyte volume changes in the cortex during pathological states, such as hypoosmotic stress or oxygen-glucose deprivation (ogd), using three-dimensional ( d) confocal morphometry in situ. in addition, single cell rt-qpcr profiling was carried out to reveal possible differences in the expression profiles of ion channels/transporters that participate in maintaining ionic/neurotransmitter homeostasis. in order to visualize individual astrocytes that lack alpha-syntrophin, double transgenic mice were generated by crossbreeding gfap/egfp mice ( . d-confocal morphometry revealed that alpha-syntrophin deletion results in significantly smaller/slower astrocyte swelling when induced by min hypoosmotic stress ( mosm), min ogd or by high extracellular k ( mm), while alphasyntrophin deletion had no effect on the astrocytic shrinkage evoked by hyperosmotic stress ( mosm). the volume recovery of cortical astrocytes from gfap/egfp/alpha-syntrophin knockout mice was significantly slower following their exposure to hypo-or hyperosmotic stress, whereas no differences were found in astrocyte volume recovery following ogd or after their exposure to high extracellular k . compared to the cortical astrocytes of gfap/egfp mice, single cell rt-qpcr analyses revealed that astrocytes from gfap/egfp/alpha-syntrophin knockout mice express higher mrna levels for two-pore domain k channels (twik- , task- , task- ), inwardly or outwardly rectifying k channels (kir . , kir . , kv . , kv . ) and chloride channels (clc ,clc ), while mrna expression for the glutamate transporter glt- is lower. in summary, the deletion of alpha-syntrophin slowed down astrocyte swelling during hypoosmotic stress, ogd or high k ; however, it also resulted in alterations in astrocytic gene expression profiles. supported by grants ga cr - s and p / /g from the gacr. microglia undergo a process of activation in any type of pathology which is controlled by many factors such as cytokines, chemokines or growth factors, but also by neurotransmitters. we found that a subpopulation ( %) of freshly isolated microglial cells from the adult brain respond to the muscarinic acetylcholine receptor agonist carbachol with a ca response, indicating the expression of functional receptors. the carbachol-sensitive population increased in microglia/ brain macrophages isolated from the tissue of mouse models for stroke ( %) and alzheimer's disease ( %), but not for glioma and multiple sclerosis. microglia cultured from adult and neonatal brain contained a similar carbachol-sensitive sub-population ( % and %) which was increased by treatment with interferon-c to % within hours, and was sensitive to blockers of protein synthesis. carbachol was a chemoattractant for microglia and decreased phagocytosis activity, indicating that microglia are a functionally heterogeneous population. t - a astrocytes and s p receptors the family of sphingosine -phosphate receptors (s prs) are g protein-coupled comprising five subtypes (s p r-s p r). these receptors are expressed in cells of the immune, cardiovascular, and central nervous systems (cns), in addition to others. s prs play important roles in celular proliferation, differentiation, survival and migration. recently, the immunomodulatory drug, gilenya v r has been approved as the first oral therapy for multiple sclerosis (ms), after proving efficacious in clinical trials. the active ingredient of gilenya v r is the phosphorylated compound fty (pfty ), which is a potent agonist on all s prs, except s p rs. pfty has been suggested to work as a 'functional antagonist' causing s p r internalisation in lymphocytes, thus limiting t cell auto-immunity. in addition to regulating the immune system, the lipophilic nature of the pro-drug fty allows it to readily cross the blood-brain-barrier (bbb) where it may also activate s prs expressed on both neurons and glia. here, the role of s prs in the cns was investigated, by specifically investigating their role in astrocyte function. a range of methods were used to examine the effects of s pr activation and their roles in astrocytes, including: (i) s p r subtype trafficking, (ii) transient and continued intracelular signalling, (iii) astrocyte cell migration, (iv) release of pro-inflammatory cytokines, (v) oligodendrocyte cell differentiation and survival and (vi) myelination state in brain slice cultures. the data showed that activation of the s p r subtype leads to (i) its internalisation and (ii) continued signalling in astrocytes, and that s p rs were found to (iii) promote astrocyte migration, (iv) limit release of pro-inflammatory cytokines, (v) promote oligodendrocyte survival and (vi) limit demyelination. these studies demonstrate a role for s p rs in regulating astrocyte function and suggest their use a drug targets for neuroinflammtory and neurodegenerative disorders. in particular, both astrocytes and oligodendrocytes express kir . , which are essential for setting their strongly negative rmp, as well as the maintenance of [k ] o following neuronal activity. notably, strong expression of the kir . subtype was determined during a whole genome microarray analysis of the mouse optic nerve, a typical cns white matter that contains mainly astrocytes and oligodendrocytes. the kir . subunit is known to be involved in potassium transport in epithelial cells and has been identified in cerebellar purkinje neurons, but has not been reported in glia. here, we examined functional expression of kir . in mouse brain and optic nerve. rt-pcr and western blot confirmed expression of kir . mrna and protein expression in the brain and optic nerve. positive kir . immunostaining was observed in astrocytes and oligodendrocytes in brain sections and in optic nerve explant cultures and the results indicated a developmental increase in kir . expression. in astrocytes, kir . was localised to perivascular end-feet, supporting a potential role in k regulation. in oligodendrocytes, kir . were localised to cell somata, suggesting a developmental role in setting their rmp. in addition, oxygen and glucose deprivation (ogd) experiments were performed on isolated intact optic nerves from mice aged p and examined for the effects of the kir . channel blocker vu . after min ogd, blockade of kir . resulted in increased cell death of optic nerve glia, measured using propidium iodide (pi) labelling, from in controls to in vu (n nerves per group, fov per nerve; p < . , anova). our results demonstrate expression of kir . in glial cells, and indicate they are important in protecting glial cells from ischemic damage. is an eight transmembrane domain protein highly expressed in brain. human mutations in mlc lead to the rare genetic disorder "megalencephalic leukoencephalopathy with subcortical cyst" (mlc). characteristic for the disease are the onset of macrocephaly within the first year of life, and a slowly progressive loss of motor skills with epilepsy and cognitive decline. at the cellular level, countless fluid-filled vacuoles occur within myelin sheaths surrounding axons and in astrocytic endfeet. in cell culture models, mlc mutations are associated with defects in chloride currents and cell volume regulation. thus far, the expression and function of mlc in intact murine and human brain are still controversial. to understand the role and developmental expression of mcl in the brain, we have developed a mlc mutant null mouse model carrying an egfp reporter gene under the mlc promotor. we now show the exclusive expression of mlc in astrocytes throughout the brain with specifically higher expression around blood vessels, at the sub-ventricular zone and at the glia limitans. the functional loss of mlc in mutant mice recapitulates in part the human mlc disease. ko mice show macrocephaly with high brain wet weight. water filled vacuoles develop in the white matter of the cerebrum and in large fiber tracts of the brainstem. by contrast, the heterozygous loss of mlc has no consequences on myelin structural integrity. both heterozygous and homozygous mlc ko mice have dysmorphic peri-vascular and peri-ventricular astrocytes. in contrast to humans, ko mice have no motor deficits, but are hyperactive and show an anxiety-like behavior. in the mlc ko mouse, a dysfunction of an astrocytic protein causes loss of myelin structural integrity leading to vacuolating myelinopathy. therefore, the mlc mutant mouse could be a key model to study the astrocytic involvement in brain water and ion homeostasis. gaba activated slowly desensitizing responses in ng cells which were mimicked by muscimol and inhibited by bicuculline. to elucidate the subunit composition of the receptors we tested its pharmacological properties. co-application of pentobarbital, benzodiazipines and zolpidem all significantly increased the gaba responses. the presence of small tonic currents indicated the presence of extrasynaptic gaba a receptors. to further analyze the subunit expression, single cell transcript analysis was performed subsequent to functional characterization of ng cells. the subunits a , a , b , c and c were most abundantly expressed, matching properties resulting from pharmacological characterization. importantly, lack of the c subunit conferred a high zn sensitivity to the gaba a receptors of ng cells. to determine the effect of gaba a receptor activation on membrane potential, perforated patch recordings were performed. in the current-clamp mode, gaba depolarized the cells to about mv, indicating a higher intracellular clconcentration (about mm) than previously reported. gaba-induced depolarization in ng cells might trigger ca influx through voltage-activated ca channels. schwann cells (sc) play important roles in the development and regeneration of the peripheral nervous system (pns) following injury. several molecules such as neurosteroids and neurotransmitters have been suggested as potential pharmacological targets in regulating sc physiology and regenerative potential. nevertheless, the slow growth rate and difficulties in harvesting limit sc applications in regenerative medicine. adipose-derived stem cells (asc) can be differentiated into a sc-like phenotype (dasc) sharing morphological and functional properties in the present study, we analysed changes in glutamate transporter expression and function following a mechanical lesion in organotypic slice cultures of the mouse hippocampus using immunohistochemistry, western blots and dynamic imaging. the lesion was positioned perpendicular to the stratum pyramidale in the ca area and comprised the entire hippocampus proper. after three-six days, a glial scar had formed along the lesion site. activated astrocytes in close proximity ( - mm) to the lesion ("scar cells") directed long, palisading gfap-positive processes towards the lesion, had significantly swollen somata and lost their ability to take up sr . furthermore, some exhibited distinct clustering of glast and glt- immunoreactivity. scar cells showed greatly diminished increases in intracellular sodium in response to application of d-aspartate, an agonist for glutamate transporters. astrocytes in the periphery to the lesion, in contrast, maintained their ability to take up sr and showed only slight upregulation of gfap, as well as less swollen cell bodies. cells in the periphery displayed only marginal changes in glutamate transporter immunoreactivity and unaltered amplitudes of sodium changes in response to d-aspartate. taken together, our data show that mild astrogliosis in the periphery of a mechanical lesion is not accompanied by a significant change in glial glutamate uptake capacity. at the scar itself, a strong clustering of glutamate transporters is observed that apparently goes along with a severe functional reduction in glutamate uptake. transport of base equivalent (hco -) across the membrane is a crucial process to maintain the intracellular and extracellular proton concentrations within a narrow physiological range. the electrogenic sodium-bicarbonate cotransporter (nbce , slc a ) is a major base transporter in eukaryotes and expressed in many tissues, especially in epithelial cells. nbce may transport significant amounts of bicarbonate into and out of the cell. in the brain, nbce is predominantly expressed in glial cells and operates with a transport stoichiometry of na: hco -. we have studied the bicarbonate sensitivity of nbce , in primary cultured mouse cortical astrocytes (c bl /n) using live cell fluorescence imaging with confocal microscope and bcecf-am as a proton sensitive fluorescence probe. we have also used the primary cortical astrocyte culture from nbce -ko mouse to compare the nbce -mediated processes in wt astrocyte culture. bicarbonate dose response protocols suggest that nbce has a very high affinity for bicarbonate (k m < mm). due to this high affinity for bicarbonate, nbce can sensitize even residual bicarbonate concentrations of - lm present in nominally bicarbonate-free extracellular solutions (buffered with hepes). the removal of residual bicarbonate by saturating extracellular buffer (hepes) with % o reduced these nbce -mediated intracellular proton changes significantly. in astrocytes of nbce -ko mice, cytosolic h changes could not be detected at hco concentrations lower than mm. it has been reported that astrocytic nbce activity mediates the activation of astrocytic glycolysis by a mechanism dependent of intracellular bicarbonate and ph (ruminot et al j neurosci : - ). using a genetically encoded fret-based glucose nanosensor, we show that glucose metabolism can be activated at low millimolar bicarbonate concentration, which was largely absent in astrocytes from nbce -ko. our results demonstrate the highest bicarbonate sensitivity yet described in animal cells, mediated by nbce in cortical astrocytes. nbce may function as a bicarbonate sensor in these cells, e.g. for modulating energy metabolism. supported by the deutsche forschungsgemeinschaft de / - . inward currents which were significantly blocked by the l-type calcium channel antagonist nimodipine. to better investigate the role of l-type calcium channels in ng glia at different developmental stages of the cns, we take advantage of the inducible cre-lox system by breeding homozygous cav . floxed mice and cav . flexed mice with ng -creert knock-in mice. we are currently investigating how the removal of cav . / . alters proliferation, migration and survival of ng glia. a particular focus lies on the analysis of behavioral abnormalities generated by cav . / . -deficient ng glia. chronic neuropathic pain is a frequent consequence of spinal cord injury (sci). yet despite recent advances, upstream releasing mechanisms and effective therapeutic options remain elusive. previous studies have demonstrated that sci results in excessive atp release to the peritraumatic regions and that purinergic signaling, among glial cells, likely plays an essential role in facilitating inflammatory responses and nociceptive sensitization. we sought to assess the role of connexin (cx ) as a mediator of cns inflammation and chronic pain. to determine the extent of cx involvement in chronic pain, a weight-drop sci was performed on transgenic mice with cx /cx deletions. sci induced robust and persistent neuropathic pain including heat hyperalgesia and mechanical allodynia in wild-type control mice, which developed after weeks and was maintained after weeks. notably, sciinduced heat hyperalgesia and mechanical allodynia were prevented in transgenic mice with cx /cx deletions, but fully developed in transgenic mice with only cx deletion. sci-induced gliosis, detected as upregulation of glial fibrillary acidic protein in the spinal cord astrocytes at different stages of the injury, was also reduced in the knockout mice with cx /cx deletions, when compared with littermate controls. in comparison, a standard regimen of post-sci treatment of minocycline attenuated neuropathic pain to a significantly lesser degree than cx deletion. these findings suggest cx is critically linked to the development of central neuropathic pain following acute sci. since cx /cx is expressed by astrocytes, these findings also support an important role of astrocytes in the development of chronic pain. mast cells (mcs) are immune cells that reside in normal brain tissue. they store in granules an array of pro-inflammatory mediators, which are rapidly released to the extracellular milieu upon activation through an extracellular ca -dependent mechanism. neurotoxic amyloid peptides are known to induce mcs degranulation but the membrane transduction mechanism remains unknown. here, the possible role of pannexin hemichannel (panx hc) known to be permeable to ca was studied. objective: since ca influx is essential for mcs degranulation, we evaluated if the neurotoxic amyloid fragment ab - peptide promotes mcs degranulation via activation of panx hcs. methods: primary na€ ıve mcs were differentiated from bone marrow precursors of wild type (wt) and panx knock out (panx -/-) c bl/ mice using il- present in wehi conditioned medium. the presence of panxs , and mrnas presence was evaluated by rt-pcr analyses. the hc activity was assessed using the ', -diamidino- -phenylindole (dapi) uptake assays and measurements of membrane current using whole cell patch clamp while intracellular ca signal was evaluated using fura- am. degranulation was assessed by quantification of extracellular histamine as well as release of toluidine blue (tb) from tb preloaded mcs from wt and panx -/mice. results: only panx mrna was detected in wt mcs. stimulation with ab - but not ab - peptide induced histamine and tb release. it also enhanced dapi uptake and total membrane current in wt mcs. these responses were drastically reduced in wt mcs pretreated with mm carbenoxolone and mm panx , blockers of panx hcs and were absent in panx -/-mcs. in wt mcs treated with ab - increase the intracellular ca signal and was drastically prevented by panx , absence of extracellular divalent cations and in panx -/-mcs. conclusion: these findings indicate that mcs express functional panx hcs, which are essential for ca influx required for the degranulation response induced by ab - . thus, inhibition of panx hcs might avoid spreading of mc-dependent inflammatory mediators released during alzheimer's disease progression. gliomas are the most frequent primitive cns tumors and are thought to derive from astrocytes or from neural progenitors/stem cells. however, the precise identity of the cells at the origin of gliomas remains a matter of debate because no pre-neoplastic state has been yet identified. tgf alpha is frequently over-expressed in the early stages of glioma progression. sharif & al (oncogene. ( ): - ) previously demonstrated that prolonged exposure of normal astrocytes to tgf alpha is sufficient to trigger their reversion to a neural progenitor-like state. when astrocytes de-differentiated with tgf alpha were submitted to oncogenic stress using gamma irradiation, they acquired cancerous properties: they were immortalized, showed cytogenomic abnormalities, and formed high-grade glioma-like tumors after brain grafting. our study aims to identify and characterize the protein signature of those in vitro transformed cells in an attempt to understand their neoplastic behavior and the effect of transformation on metabolic processes. this involves a global proteomic analysis using the d-dige methodology and a study of the metabolism of these cells (carbon source, lactate, glucose and glutamine use, ros metabolism…) by hand c-nmr nmr quantification of metabolites. such approaches are expected to provide information allowing understanding the metabolic reprogrammation that occurred during transformation. the comparative d-dige proteomic analysis of normal and transformed astrocytes shows that during transformation, the cells increase their expression of glycolytic enzymes, thus acquiring the ability to use aerobic glycolysis (warburg effect). moreover, the transformed cells reduce their capacity for tricarboxylic acid oxidation and for neurotransmitters (glutamate and gaba) metabolism. ingenuity pathway analysis indicates major effects on carbohydrates, amino acids and nucleotides metabolic components. using enzymatic activity measurements and the detection of protein isoforms by d-western blot and zymography, we document a change in expression and activity of various isoenzymes that may be responsible for those metabolic reprogrammations. r.e. k€ alin, a. jarczewski, f. apel, r. monk, s. kraft, j. radke, f.l. heppner charit e -universit€ atsmedizin berlin, neuropathology, berlin, germany question: glioma are the most frequent malignant primary tumors of the brain. glioma survival and growth is determined by the interaction with brain parenchyma, which includes intense tumor angiogenesis. this process is supported by glioma-invading myeloid (gim) cells, namely brain resident microglia or brain-invading macrophages. depletion of gim cells leads to a significant decrease in glioma volume. in a previous study, we established the novel neuroendocrine hormone apelin as an angiogenic factor and described its upregulation in human glioblastoma multiforme. our aim is thus to study the role of apelin signaling in tumor angiogenesis but also its possible effect on brain monocytes for the regulation of glioma growth. methods: to investigate apelin signaling during gliomagenesis we took advantage of an orthotopic mouse model for tumor formation. intracerebral xenotransplantation of the human glioma cell line u mg, expressing lentiviral control or apelin shrna, allows us to study the specific contribution of glioma-derived apelin to gliomagenesis. to further dissect a putative immune function of apelin, we used the isogenic mouse glioma cell line gl for intracerebral implantation into immunocompetent mice lacking (apelin-ko) or overexpressing apelin (apelin-tg). results: loss-of-apelin expression in u mg cells resulted in a reduced glioma volume and attenuated the formation of the xenograft vasculature. gl implants in apelin-ko mice produced a similar phenotype. in addition, invasion of glm cells was significantly reduced. interestingly, expression analysis showed an upregulation of the apelin receptor apj in brain myeloid cells making them competent to respond to glioma-derived apelin. conclusions: our findings demonstrate that apelin plays an important role in tumor angiogenesis and glioma growth. we show here that both, glioma cell-derived apelin and apelin expressed by the tumor neovasculature are contributing to tumor angiogenesis, gim invasion and glioma growth. we anticipate that our study will provide insights whether apelin signaling may serve as a future novel target for antiangiogenic tumor therapy. tumor infiltrating microglia/macrophages (tims) constitute the largest population of infiltrating cells in glioblastoma (gbm), the most aggressive brain tumor. data from the clinic and from experimental work performed in murine and human models indicate that tims play a significant role in gbm biology as they support proliferation, migration and invasion of tumor cells. evidence is amounting that tumor cells actually polarize tims towards this m -like, tumor-supportive phenotype. we showed that toll-like receptor ligand reverses this m -into a m -like phenotype. pre-activated, m -like polarized tims incubated with spheroids of gbm cells reduced migration, killed and phagocytosed tumor cells over a day-period, indicating a sustained m activation of tims in absence of exogenously added stimuli. in order to analyse in more detail tims-gbm cells interactions, we have undertaken two approaches. we use spheroids of cells (tumor with/without tims) embedded in collagen matrix, in which tims can be implanted, as an experimental model. this three-dimensional in-vitro system is well suited for a qualitative and quantitative monitoring of cell proliferation, death, migration and invasion. data experimentally generated are then implemented in a mathematical model that is, to our knowledge, the first one proposed to take into account tumor cells and tims in order to simulate gbm progressive behaviour. in a first step, the capacity of spheroids made of murine glioma cells to invade collagen matrices was evaluated in absence and presence of microglia. invasion was monitored by photography of the spheroids and images were processed with photoshop cs software. in order to achieve a precise determination of invasion, we chose to measure the diameter of the core plus the invasive rim as a read out of expansion rate. untreated microglia promoted growth and invasion of tumor cells. data generated through the proposed in-vitro system were comparable to and reproduced the insilico simulations obtained with the mathematical model, hence validating our mathematical and experimental approaches. we currently evaluate the rate of proliferation and death of tumor cells in various settings that modulate tims polarization, using flow cytometry and confocal (live) imaging. data contributed by these two approaches should facilitate the delineation of a predictive model for tumor progression in a tims-enriched microenvironment with possible therapeutic fallouts. sialic-acid-binding immunoglobulin-like lectins (siglecs) are type membrane proteins displaying an amino-terminal immunoglobulin-like variable (v-set ig-like) domain that binds sialic acid and variable numbers of immunoglobulin-like constant region type (c -set ig-like) domains. siglec-h is a recently identified mouse-specific cd -related siglec that signals via the itam linked adaptor protein dap . so far the function of siglec-h and its ligand are unknown. in this project, we demonstrate gene transcription and protein expression of siglec-h by mouse microglia after interferon-c (ifn-c) treatment or polarization into a m -subtype. targeting of beads to siglec-h by specific antibodies triggered the phagocytic uptake of the beads by the microglia. a siglec-h fc fusion protein generated by our laboratory selectively bound to cells with an altered glycocalyx, particularly glioma cells, but not to normal control cells. m -polarized microglia phagocytosed glioma cells and also limited their proliferation in a co-culture system. interestingly, no signs of apoptosis were observed before phagocytosis of the glioma cells by siglec-h expressing microglia. phagocytosis of glioma cells was reduced after shrna mediated siglec-h knock down in microglia. furthermore, microglial clearance of glioma in vitro was dependent on the interaction of siglec-h with its corresponding adaptor protein dap . our data show that m -polarized microglial cells can engulf glioma cells via a dap -mediated and siglec-h dependent uptake. glioblastomas (gbm) are the most common brain tumors in humans. although advances in the chemotherapeutic management of glioblastoma have been made, almost % of the patients die within the first years after diagnosis. on the basis of these observations, the identification of new molecules that can counteract the gbm growth and invasiveness appears relevant. previously, we have demostrated that m muscarinic receptor agonist arecaidine inhibited cell proliferation in a time and dose-dependent manner and induced a severe apoptosis both in two glioma stable cell lines (u and u ) and in primary cultures derived from human biopsies. in order to clarify the mechanisms causing the decreased cell proliferation, we have evaluated the ability of m receptor activation to counteract the notch- and egfr pathways. the analysis by real time pcr and western blot have demonstrated that, in both cell lines, m receptor caused a decreased expression of egfr and notch- and its ligands (e.g. delta- , jagged- and ), suggesting that the decreased cell proliferation may dependent on altered expression and activity of these pathways. although facs analysis have showed that arecaidine induced an arrest of cell cycle progression, we observed, in both cell lines, a significant increase of dividing cells already after hours of treatment. in particular, the number of metaphases increased significantly, while the percentage of anaphases diminished. furthermore we observed in both cell lines, a dis-regulation of mitotic spindle assembly, misalignment of chromosomes and the presence of multipolar spindles. moreover, due to prolonged activation of the promethaphase/methaphase mitotic checkpoint, chromosomes appeared more condensed in cells treated with arecaidine. facs analysis and immunocitochemistry using anti-histone c-h ax, a marker of double strand breaks, have also demonstrated that arecaidine treatment induced dna damage. on the other hand, m agonist induced also oxidative stress, followed by an increased expression of the enzyme superoxide dismutase and sirtuins (sirt and sirt ). in conclusion, the data obtained suggest that the activation of m receptor induces cytostatic and cytotoxic effects in glioblastoma cell lines, opening new therapeutic perspectives for this receptor in glioblastoma therapy. univdersidade federal do rio de janeiro, rio de janeiro, brazil glioblastoma (gbm) is one of the most aggressive human cancers. despite current advances in multimodality therapies, such as surgery, radiotherapy and chemotherapy, the outcome for patients with high grade glioma remains fatal. there is now a growing awareness that the main limitations in understanding and successfully treating gbm might be bypassed by the identification of a distinct cell type that has defining properties of somatic stem cells, as well as cancer-initiating capacitybrain tumor stem cells, which could represent a therapeutic target. in addition, experimental studies have demonstrated that the combination of antiangiogenic therapy, based on the disruption of tumor blood vessels, with conventional chemotherapy generates encouraging results. emerging reports have also shown that microglial cells can be used as therapeutic vectors to transport genes and/or substances to the tumor site, which opens up new perspectives for the development of gbm therapies targeting microglial cells. finally, recent studies have shown that natural toxins can be conjugated to drugs that bind to overexpressed receptors in cancer cells, generating targeted-toxins to selectively kill cancer cells. further, extensive effort is being dedicated to characterize the molecular basis of gbm resistance to chemotherapy and to explore novel therapeutic procedures that may improve overall survival. we show that a non-cytotoxic concentration of equinatoxin ii (eqtx-ii), a pore-forming toxin from the sea anemone actinia equina, potentiates the cytotoxicity induced by temozolomide (tmz), a first-line gbm treatment, and to etoposide (vp- ), a second-or third-line gbm treatment. we also suggest that this effect is selective to gbm cells and occurs via pi k/akt pathway inhibition. finally, magnetic resonance imaging (mri) revealed that a non-cytotoxic concentration of eqtx-ii potentiates the vp- -induced inhibition of gbm growth in vivo. these combination therapies constitute a new and potentially valuable tool for gbm treatment, leading to the requirement of lower concentrations of chemotherapeutic drugs and possibly reducing, therefore, the adverse effects of chemotherapy. a growing body of evidence indicates that glioblastoma stem-like cells (gscs) play a central role in glioblastoma development and resistance to current therapies. we recently described a cluster of micro-rnas, the mir- - , which induces the exit of gsc from their stem state and suppresses their tumorigenic properties in an irreversible manner (fareh et al, ). we postulated that such a drastic change in the cell phenotype should be accompanied with metabolic alterations that could be instrumental in the loss of functional properties of gscs induced by the micro-rna cluster. metabolome profiling by mass-spectrometry of gscs and gsc-mir- - pointed to changes in the gaba synthesis pathway (see abstract el-habr et al). remarkably, exposure of na€ ıve-gsc to metabolites of the gaba synthesis pathway found to be overproduced in gsc-mir- - reproduced at least in part the effects of mir- - , including inhibition of clonality, down-regulated expression of selfrenewal markers, and loss of self-renewal properties. the metabolites studied acted by interfering with progression of the cell cycle and the nuclear localization of transcription factors crucial for maintenance of gsc stem-like properties. studies assaying the effects of the metabolites on gsc tumor-initiating properties are under way. these results demonstrate that metabolic regulations can participate in the control of gsc properties, and opens novel paths in therapeutic targeting of glioblastoma. *lgd and eae contributed equally. glioblastomas are the most frequent and aggressive form of adult primary brain tumors. glioblastoma stem cells (gscs) are thought to play key roles in the development and resistance of the tumor to existing radio-and chemotherapies. our previous results showed that the cluster of micrornas, mirna- - , induces loss of gsc stem-like and tumor-inducing properties (fareh et al. ). to further understand the molecular pathways controlling the peculiar properties of gscs, we sought for metabolic changes likely to accompany the drastic change in cell phenotype induced by mir- - expression. we measured the intracellular and secreted levels of metabolites by mass spectrometry. reconstitution of the metabolomes revealed significant and coordinated changes in components of the krebs cycle, and the glutamine/glutamate neuropeptide metabolic pathway that suggested altered turnover of the gaba synthesis pathway. exon array hybridization and western blot analysis, revealed changes in expression of several enzymes of the gaba synthesis pathway in mir- - -gscs. of note, none of the analyzed enzyme transcripts appeared as a direct target of mir- - . our results could be integrated in a complex multistep schema compatible with enhanced turnover of the gaba synthesis pathways, resulting in decreased cell gaba levels and increased levels of gaba by-products, as observed in mir- - gscs. further studies showed that these metabolic changes participate in the induced loss of gsc properties (see abstract by dubois et al). * eae and lgd contributed equally colonization of the brain by carcinoma cells is barely understood, in particular when considering interactions with the host tissue. we recently demonstrated that microglia assist carcinoma cell invasion. current findings indicate that this is part of a danger response of the entire brain tissue. in the brain slice coculture model, contact with both benign and malignant epithelial cells induced a response by microglia as well as astrocytes comparable to that seen at the interface of human cerebral carcinoma metastases. this response tries to protect the brain from intrusion of benign epithelial cells by inducing apoptosis. however, this is ineffective against malignant cells which did not undergo apoptosis and actually exploited this reaction to invade instead. gene expression and functional analyses revealed that c-x-c chemokine receptor type (cxcr ) and wnt signaling are involved in this process. furthermore, cxcr regulates the recruitment of microglia towards brain injury in a zebrafish model and was expressed in human stroke patients, suggesting a conserved role of cxcr in various danger reactions. we propose that glia-assisted malignant invasion takes advantage of the physiological two-stage glial defense reaction. carcinoma cells escape the second step of cell killing and misuse the damage response to colonize the brain. university of tuebingen, tuebingen, germany aquaporin- (aqp ), the main water channel of the brain, is highly expressed in animal glioma and human glioblastoma in situ. in contrast, most cultivated glioma cell lines do not express aqp , and primary cell cultures of human glioblastoma lose it during the first passages. accordingly, in two glioma cell lines of the rat (c cells and rg cells) and in sma mouse glioma cell lines we found no aqp expression. this let us consider the possibility that aqp expression depends on brain microenvironment. aqp negative rat glioma cells were implanted into rat brain. within two weeks, a tumor developed. aqp staining of the tumor cells was positive. however, if the identical cells were implanted into the rat's flank, they did not express aqp . in contrast to the normal brain, where aqp staining is polarized in the astrocytic endfoot membranes, the aqp staining in c and rg tumors was distributed over the whole glioma cell as in human glioblastoma. we conclude that the micro-environment is crucial for aqp expression in brain and brain-tumor. glioblastomas (gbm) are infiltrative and highly vascularised brain tumors containing a subpopulation of multipotent stem-like cells. here we questioned whether notch pathway activation in these cells influenced the extent of tumoral vascularisation and dissemination. we used two cd sox gbm stem-like cultures generating highly infiltrative but poorly vascularised tumors upon intracranial grafts. the use of a hes promoter reporter construct indicated that the notch pathway was only active in a small fraction of cells. sustained notch activation in these cultures, using an activated form of the notch- receptor (nicd), induced a profound alteration of their morphology, phenotype and properties. a severe decline of their migration and proliferation rates was observed in vitro and in vivo. intracranial and subcutaneous transplantation revealed that nicd triggered an extensive vascularisation of tumoral cells indicated by the presence of wellformed vessels with large lumens. close interactions between gbm cells and host endothelial cells were readily observed but no evidence for endothelial transdifferentiation was found either in vitro or in vivo. microarray and proteomic analyses showed that nicd induced the expression of vascular adhesion proteins (icam- , vcam- , itga ), proangiogenic cytokines (plgf, il- , hb-egf), metalloproteinase (mmp- ), a-smooth muscle actin and dll , a notch ligand essential for vascularisation. this was associated with a remarkable transcription factor switch whereby cells became klf snai while ascl , olig , and sox were reduced or lost. thus in addition to its well-described role in vascular development and remodelling, the notch pathway is also central in controlling proliferation, migration and vascularisation of gbm-stem like cells. t - b extracellular space diffusion parameters in the mouse thalamus in bral knock-out mice retinal wholemounts were immunostained with antibodies against gfap, iba- and mhc-ii. results: in the na€ ıve retinas, weak constitutive mhc-ii expression was scarcely found in some iba- microglial cells and rarely in gfap astrocytes. only a small dendritiform subpopulation of iba- cells, located in the juxtapapillary area and in the marginal region of the retina, had a strong mhc-ii immunoreaction. in comparison with na€ ıve both, in contralateral and in oht-eyes: i) gfap was upregulated in m€ uller cells and microglia was activated; ii) mhc-ii was upregulated on macroglia and microglia. in microglia, it was similarly expressed in contralateral and ohteyes. by contrast, in macroglia, mhc-ii upregulation was observed mainly in astrocytes in contralateral eyes and in m€ uller cells in oht-eyes conclusions: both, contralateral and oht-eyes had macro-and microglial retinal changes in mhc-ii expression after two weeks of laser-induced oht approximately % of the nmo patients harbor antibodies against the water-channel aquaporin- , aqp -igg (seropositive nmo), expressed mainly by perivascular astrocytes. however, some patients diagnosed with nmo don't have aqp -igg (seronegative nmo). the aim of this work is to compare in vitro the neuroinflammatory profile of igg from seropositive (igg-nmo ) and seronegative (igg-nmo-) nmo patients. pool of purified igg from igg-nmo and igg-nmo-patients fulfilling diagnostic criteria of , and from multiple sclerosis (igg-ms) patients and healthy controls (igg-hc) were include in the study. mixed glial cell cultures of astrocytes ( %), oligodendrocytes ( %) and microglia ( %) were obtained from spinal cord of postnatal c bl /j mice. after days in vitro, cells were treated with the igg pools for hours for rt-pcr, and hours for western blot and immunocytofluorescence (if) igg-nmo caused a stronger upregulation of c/ebpb mrna and nos protein than igg-ms and igg-nmo-. all these data show that igg-nmo and igg-nmo-induce a different proinflammatory glial activation. the proinflammatory pattern of igg-nmo-is quite similar to that induced by igg-ms patients. these findings raise the question if igg-nmo-could be a different disease. such as gdnf, artemin, bdnf and sonic hedgehog, adhesion molecules including p ntr, n-cadherin and n-cam and the transcription factor olig . c-jun also controls the structure of the regeneration tracks. in schwann cell c-jun null mice, the initial rate of axonal regeneration after injury is impeded and long term recovery of function measured by footprint analysis, toe pinching , von frey hairs, and the hargreaves test is not seen weeks after injury. there is widespread death of both large and small drg neurons and although spinal cord motor neurons survive there is widespread failure of both sensory and motor neurons to reinnervate target muscles united states mutations in the neurofibromatosis (nf ) gene cause neurofibromatosis type (nf ) characterized by formation of multiple schwannomas and meningiomas. nf encodes a tumor suppressor protein called merlin. loss of function of merlin is associated with an increased level of active rac and p -activated kinases (pak). the lim domain kinases (limk and ) are rac-pak substrates and modulate actin dynamics and cytoskeletal organization by phosphorylating cofilin, an actin severing and depolymerizing agent acknowledgements: this work was supported by grant ncn / /b/ nz / and statutory funds. phenotypes, their functions are fundamentally different in a model of brain injury. these data provide new insights into the protective role of microglia after brain injury. . although the pathogenesis is still not clear, activation of the immune system at some point of the disease process plays a crucial role. it is known that myelin-specific autoreactive t-cells and monocytes cross the blood-brain barrier (bbb) and migrate into the cns. within the cns, these cells secrete proinflammatory cytokines which stimulate local glial cells to produce inflammatory factors, including reactive oxygen species that contribute to demyelination and ultimately axonal loss. the destruction of the bbb and the influx of monocytes and t-cells from the circulation are key events in the progression of ms pathology. tissue transglutaminase (tg ) is a multifunctional enzyme that has been shown to play a role in monocyte/macrophage adhesion and migration onto extra cellular matrix proteins in vitro. this binding occurs via interaction with bintegrins. previously, we observed the appearance of tg in monocytes in active human post-mortem ms lesions in the cns.in the present study we question whether tg expression is regulated in primary human monocytes by inflammatory mediators and whether it is modulated in ms patients. methods: to this end, tg in monocytes was detected by semiquantitative rt-pcr.results and conclusions: our preliminary results show that activation of primary human monocytes with lps or lps ifnc results in a time-dependent increase in tg mrna whereas the expression of factor xiiia (another member of the transglutaminase family) remains stable over time. moreover, the tg mrna level is higher in unstimulated monocytes derived from ms patients compared to control subjects while factor xiiia mrna level remains unaffected.these initial data are promising with respect to a possible role for monocyte-derived tg being involved in adhesion to and migration across the bbb under inflammatory conditions (e.g. multiple sclerosis). this hypothesis has not been proven yet. in addition, by increasing the number of patients, a consistent difference in tg expression in monocytes between ms patients and control subjects may point to tg as a novel biomarker for disease. an important aspect of chronic neurodegenerative diseases, such as alzheimer's, parkinson's, huntington's and prion disease, is the generation of an innate inflammatory response within the central nervous system (cns). microglial and astroglial cells play a key role in the development and maintenance of this inflammatory response, showing enhanced proliferation and morphological activation. using a laboratory model of chronic neurodegeneration (me murine model of prion disease), we studied the time-course and regulation of microglial proliferation. our results show that resident microglial cells have an increased proliferation rate during the development of the disease, leading to a significant increase in the population, without a contribution from circulating cells. microglial proliferation is differentially regulated in diverse regions of the cns, pointing to a heterogeneous development of the pathology. we have identified novel molecular regulators of the proliferative response, and addressed the significance of the contribution of microglial cells to the pathological course of the disease by modifying their proliferation. we also found a correlation of our results with the scenario present in chronic human neurodegenerative conditions variant creutzfeldt-jakob disease (vcjd) and alzheimer's disease. our results demonstrate that microglial proliferation is an important feature of the evolution of chronic neurodegenerative disease, with direct implications for understanding the contribution of the cns innate immune response to disease progression. here, we examined the underlying mechanism of fibronectin aggregation and address whether inflammatory mediators, such as toll like receptor (tlr) agonists or pro-inflammatory cytokines induce fibronectin aggregation by astrocytes. our findings reveal that only tlr and agonists, including the endogenous tlr agonist stathmin, increased stable fibronectin aggregation. interestingly, only white matter-derived astrocytes displayed fibronectin aggregation whereas in cortical astrocyte cultures fibronectin remained unaffected, suggesting regional differences in the functional response of astrocytes. furthermore, in rat cerebellar slice cultures, the tlr agonists only induce aggregation of fibronectin after lysolecitin-induced demyelination. in this manner, efficient remyelination is impeded and, consequentially, progressive neuronal decline occurs.taken together, tlr and agonists promote fibronectin aggregation by primary rat astrocytes. since tlr on astrocytes and stathmin are both upregulated in ms lesions, stathmin, as an endogenous tlr agonist, might play a role in inducing fibronectin aggregation after c-jun reprograms schwann cells of injured nerves to generate a repair cell essential for regeneration. neuron. ( ): - ). in this study we identify a novel role for c-jun in the activation of notch signalling in the denervated schwann cell. we find that c-jun is required to activate notch signalling, leading to upregulation of the bhlh protein hes . hes then plays two functions in the denervated cell, promoting myelin breakdown and acting as part of a negative feedback loop to reduce c-jun levels. as a result of this, ablating notch signalling specifically in schwann cells acts to increase c-jun levels. we show that this upregulation of schwann cell c-jun accelerates axon outgrowth, target re-innervation and remyelination by generating a cell, which promotes faster than normal functional recovery. these results identify novel functional links between the c-jun and notch signalling pathways. they also show that not only is schwann cell c-jun necessary for successful nerve regeneration, but that nerve repair can be improved by enhancing normal c-jun signalling. it is well known that cell surface immune receptors play a critical role in regulating immune and inflammatory processes in the central nervous system (cns). after injury of the peripheral nervous system (pns), schwann cells and macrophages phagocyte myelin debris in wallerian degeneration in order to regenerate axons to distal targets. the immunoreceptor cd f is normally expressed in the myeloid line and in nervous system in microglia, oligodendrocytes and neurons under certain conditions. however, little is known about the cd f ligands. by using a cd f-fc fusion protein we have analyzed the expression of the cd f ligands in the pns. moreover, we have also analyzed the possible role of the cd f immunoreceptor in peripheral nerve regeneration by blocking the interaction between cd f and its ligands with the same fusion protein. thy -yfp-h mice sciatic nerves were injected with cd f-fc, control migg a or pbs immediately before a crush injury. the results show that cd f ligand is expressed in schwann cells. moreover, after a sciatic nerve injury, animals injected with the cd f-fc protein show a lower number of yfp-positive fibers growing into the tibial nerve after days post-lesion (dpl) than control groups. moreover, the cd f-fc group shows a higher number of macrophages and cd -positive cells at dpl when compared to control groups. we do not see these differences in axon regeneration and macrophage infiltration after dpl. we have also evaluated the mrna expression of the pro-inflammatory cytokine il- b at hours after crush and injection of the fusion protein. q-pcr shows an up-regulation of the mrna in control groups and a lower mrna expression level in the cd f-fc protein group. together these results show that the pair cd f receptor and ligand is implicated in some aspects of wallerian degeneration and nerve regeneration such as the modulation of both the influx and phenotype of macrophages. in response to the central nervous system (cns) injury, hematopoetic cells migrate to the lesion where they can potentially contribute to tissue regeneration. following cns injury, these cells can secrete a plethora of immunomodulating cytokines and/or pro-regenerative growth factors as well as phagocyte proinflammatory tissue debris. nevertheless, the role of primitive hematopoetic stem/progenitor cells (hspc) -derived from bone marrow -to support cns regeneration has not been studied in detail. therefore, the aim of the present study was to examine characteristics of hspc in vitro as well as to test proregenerative potential of these cells to support cns repair in vivo.highly pure population of primitive hematopoetic stem/progenitor cells was isolated from the bone marrow and the cns with fluorescence activated cell sorting (facs). the number of hspc in the cns lesion was significantly increased compared to intact cns tissue. sorted-bone marrow hspc were co-cultured with primary astrocytes to examine their phenotype according to the presence of specific antigens and gene expression as well as to test their phagocytosis potential. in the presence of astrocytes, bone marrow-derived hspc differentiated in vitro into microglia-like cells expressing specific myeloid/microglia markers and showing high ability to ingest fluorescent microbeads. the in vivo potential of hpsc for supporting regeneration was examined by their transplantation into the cns lesion.results of our study demonstrated that primitive hematopoetic stem/progenitor cells are the source of microglia-like cells which can support regeneration in the central nervous system. when the brain or spinal cord is injured, glial cells in the damaged area undergo complex changes resulting in the formation of the glial scar. whether the scar is beneficial and detrimental to recovery remains controversial. the signals that initiate the formation of the glial scar are unknown. because both canonical and non-canonical wnts are increased after spinal cord injury (sci), we examined the role of canonical wnt signaling in the glial reactions to cns injury. to disrupt b-catenin-dependent wnt signaling specifically in opcs, we created transgenic mice carrying an opc-specific conditionally deleted bcatenin gene. after moderate contusion injuries to the thoracic spinal cord of control mice, opcs proliferate and accumulate in the penumbra region surrounding the injury epicenter. in the b-catenin-depleted mice, there is reduced proliferation and accumulation of opcs after sci, reduced accumulation of activated microglia/macrophages and reduced astrocyte hypertrophy. using a crushed optic nerve model, we show that these reduced glial reactions create an environment that is permissive for axonal regeneration. these results suggest that canonical wnt signaling in glia after cns injury is necessary for the formation of the glial scar and they identify wnt signaling as a new therapeutic target for promoting axon regeneration. the crucial role of central nervous system (cns) glial cells in the integrity and physiology of neuronal networks, is well known. however, little is known about glial cells in the peripheral nervous system (pns). one of the main glial cell types in the pns is the perineuronal satellite glial cells (sgcs), that surround drg neurons in an envelope-like fashion. despite their abundance in peripheral nerves, having stem cells properties and playing a role in neuropathic pain, there is limited information on their physiology. although sgcs have similarities to astrocytes in terms of purinergic-and gap junction-mediated signaling, their membrane properties and ionotrophic glutamate receptor expression are more or less unknown. our aim was therefore to characterize the membrane properties and glutamatergic ion channel expression of sgcs. we performed patchclamp experiments on sgcs wrapped around sensory neurons in the rat dorsal root ganglion (drg) in vitro. whole-cell recordings revealed that sgcs are slightly hyperpolarized in comparison to the neurons they ensheath, with a resting membrane potential of approximately $ mv and a high input resistance (> gx). moreover, upon membrane depolarization no detectable voltage-gated na , ca or k currents were detected. extracellular application of glutamate agonist, kainic acid, n-methyl-d-aspartic acid (nmda) and -amino- -( hydroxy- -methyl-isoxazol- -yl) propanoic acid (ampa) during the recording, did not evoke any response from the cells, indicating their lack of ionotropic glutamatergic receptor (iglur) expression. double immunocytochemistry on lucifer yellow (ly) filled scgs revealed that they express the scip/oct transcription factor, which is also expressed by promyelinating schwann cells.drg ganglion sgcs differ from cns perineuronal cells as they lack glutamatergic receptors, but are similar to astrocytes as they have no voltage-gated ion channels, are gap-junctionally coupled and express glutamine synthetase. thus, we are currently addressing if sgcs respond to neuronal activity in a similar manner to astrocytes in the cns with the use of calcium imaging. these studies will provide further insights into the physiological role of sgcs in the pns. multiple sclerosis (ms), a chronic neuroinflammatory disease with presumed autoimmune etiology, is the most common demyelinating disorder of the central nervous system among young adults. progressive neurological impairment of the patients are associated with accumulation of characteristic brain lesions characterized by inflammation, demyelination, gliosis and axonal damage. demyelination and loss of oligodendrocytes contributes to axonal loss and permanent neurological deficits. remyelination occurs but is limited; one contributing factor is the incapability of oligodendrocyte precursor cells (opc) to differentiate and form new myelin sheaths. k channels are not only known to predominantly set and maintain the membrane potential but also to be important regulators of various cell functions like proliferation and maturation. specific regulation of these cellular functions is based on high channel diversity and their ability to form heteromers as well as alternate mrna splicing and posttranslational modifications. so far the expression and function of potassium channels in cells of the oligodendroglial lineage has not been clearly identified. previous studies indicate that an unspecific blockade of k channels in opc suppresses maturation as well as proliferation. moreover, for one distinct channel (k ir . ) a functional relevance has been shown, as deficiency of kir . leads to altered opc maturation and hypomyelination in mice. here, we elucidate the expression and functional relevance of k channels in oligodendrocytes. our first electrophysiological recordings from primary murine opcs revealed the presence of different k channels with both inward and outward rectification. in future experiments, we aim at identifying k channel subtypes that specifically regulate opc cell functions and might influence cell maturation under neuroinflammatory conditions. thereby, we want to offer new insights in the functional role of k channels in opcs/oligodendrocytes and contribute to novel therapeutic strategies for the treatment of ms. aqp ) is the predominant water channel in the mammalian brain and is mainly expressed in the perivascular glial endfeet at the brain-blood interface. aqp has been described as an important entry and exit site for water during formation of brain edema and regulation of aqp is therefore of broad interest. phosphorylation of some aquaporins has been proposed to regulate their water permeability via gating of the channel itself. protein kinase (pk)-dependent phosphorylation of ser has been reported to increase the water permeability of aqp expressed in an astrocytic cell line. this possibility was, however, questioned based on the crystal structure of the human aqp . our study aimed to resolve if ser was indeed a site involved in phosphorylation-mediated gating of aqp . the water permeability of aqp -expressing xenopus oocytes was not altered by a range of activators and inhibitors of pkg and pka. mutation of ser to alanine or aspartate (to prevent or mimic phosphorylation) did not change the water permeability of aqp . pkg activation had no effect on the water permeability of aqp in primary cultures of rat astrocytes. molecular dynamics simulations of a phosphorylation of aqp .ser recorded no phosphorylation-induced change in water permeability. a phospho-specific antibody, exclusively recognizing aqp when phosphorylated on ser , failed to detect phosphorylation in cell lysate of rat brain stimulated by conditions proposed to induce phosphorylation of this residue. thus, our data indicate a lack of phosphorylation of ser and of phosphorylation-dependent gating of aqp . undocked connexons may form open hemichannels in the plasma membrane when exposed to specific stimuli, e.g. reduced extracellular concentration of divalent cations, and allow passage of fluorescent molecules with masses lower than kda. a range of physiologically relevant molecules, smaller than the assumed molecular cut-off of kda, have therefore been proposed to permeate connexin (cx ) in its hemichannel configuration. however, the permeability profile of cx hemichannels remains unresolved as does the molecular substrate for hemichannel activity in astrocytes. exposure of cx -expressing xenopus laevis oocytes to divalent cation free solution induced a gadolinium-sensitive uptake of the fluorescent dye ethidium. in spite thereof, a range of smaller biological molecules, such as water, glutamate, lactate, glucose, and atp, did not gain detectable access through the pore. in contrast, permeability of glutamate, glucose and atp was observed in oocytes expressing the other major astrocytic connexin, cx . exposure to low divalent cation solutions also induced a robust membrane conductance in cx -expressing oocytes but none in cx -expressing oocytes. expression of cx in c glioma cells failed to induce hemichannel activity. our results thus call for caution when assigning molecular identity to astrocytic channel activity and when interpreting hemichannel-mediated dye-uptake as equal to permeability of physiologically relevant molecules. atp-gated p x receptor channels expressed in spinal microglia actively participate in central sensitization, making their functional regulation a key process in chronic pain pathologies. p y metabotropic g q -coupled receptors, also expressed in microglia, are involved in the initial response to nerve injury, triggering phagocytosis upon activation by udp. it has been reported recently that expression of both p x and p y is upregulated in activated microglia following nerve injury. we show here, in resting as well as lps-activated primary microglia, that p y decreases p x -mediated calcium entry and inhibits the dilation of p x channels into a large-conductance pore measured with a yo-pro- uptake assay. furthermore, p y activation modulates the atp-dependent migration of microglia, a process likely involved in their shift from migratory to phagocytic phenotype.reconstituting the p x -p y interaction in recombinant systems shows that p y activation decreases p x current amplitude, activation and desensitization rates, and reduces p x channel permeability to the large cation nmdg . phospholipase c-mediated hydrolysis of the phosphoinositide pi( , )p , a necessary cofactor for p x channel function, underlies this inhibitory crosstalk. as extracellular levels of both atp and udp are increased in the spinal cord following nerve injury, the control of p x activity by p y might play a critical role in regulating neuropathic pain-inducing microglial responses. with sc, thus representing a valid sc alternative. we have previously shown that dasc express c-aminobutyric-acid (gaba) receptors (i.e. gaba-a and gaba-b receptors), which modulate their proliferation and neurotrophic potential, although little is known about the role of other neurotransmitter systems in asc.in this study we investigated the expression of purinergic receptors in dasc. using rt-pcr, western blot analyses and immunohistochemistry we have demonstrated that asc express p x , p x and p x purinoceptors. interestingly, differentiation of asc towards glial phenotype was accompanied by up-regulation of p x and p x receptors. using ca imaging techniques, we have shown that stimulation of purinoceptors with adenosine- -triphosphate (atp) results in intracellular ca signals, indication functional activity of these receptors.. moreover, we have shown that the increase of intracellular ca leads to sc death, an effect that can be prevented using specific p x or p x antagonists.altogether, these results show, for the first time, the presence of functional purinergic receptors in sc-like derived from asc and their link with critical physiological processes such as cellular death and survival. the presence of these novel pharmacological targets in dasc might open new opportunities for the management of cell survival and neurotrophic potential in tissue engineering approaches using dasc for peripheral nerve repair. university of freiburg, freiburg, germany intracellular ph homeostasis is a vital function shared by all cells and is mainly regulated by the coordinated action of several acid-base transporters. in the brain, ph homeostasis is of particular importance since changes of extracellular ph (pho) are events associated with both physiological conditions and pathological states in brain function. transient pho changes usually accompany physiological processes, including neuronal activity, and astrocytes respond to a rise in extracellular k with plasma membrane depolarization and intracellular alkalinization. on the other hand, loss of brain ph homeostasis can lead to severe pathological conditions and significant changes of pho have been observed during seizures and spreading depression.in the present study, we sought to investigate regulation mechanisms of the electrogenic sodium/bicarbonate cotranspoter , nbce , and of the vacuolar h -atpase (v-atpase) in primary hippocampal astrocytes following extracellular acid-base changes, following neuronal activity, and using the -aminopyridine ( -ap) model of epilepsy in vitro.we show that extracellular acidosis, but not extracellular alkalosis, increased the number of activated astrocytes. our data also show differential regulation of acid-base transporters in the hippocampal glial cells during extracellular acid-base changes. intracellular ph measurements revealed a crucial role for v-atpase in regulation of intracellular ph, a process accompanied by increased membrane expression of the transporter, as assessed by immunofluorescence and surface biotinylation. nbce -b is the nh -terminal variant of nbce predominantly expressed in glial cells and is transcriptionally regulated followed neuronal activity or treatment of the cells with -ap.these data propose transcriptional and post-translational modification of acid-base transporters as putative regulatory mechanisms in astrocytes to cope with changes of extracellular ph serving the maintenance of intracellular ph homeostasis. these astroglial networks fulfil a variety of functions in the brain, e.g. potassium buffering and metabolite transport. in this study we compared glial networks in different brain regions to investigate site specific effects on network formation. we combined electrophysiology and immunohistochemstry with semi-quantitative rt-pcr and western blot analysis to investigate connexin expression, gap junction coupling and antigen profiles. experiments were performed in wild type and transgenic mice with glia specific fluorescence labelling as well as in cx ko mice. astrocytes were investigated between postnatal days - . gap junction networks in the ca region of the hippocampus and the ventrobasal thalamus show abundant coupling in hgfap-egfp mice. intriguingly, we found significant coupling between oligodendrocytes and astrocytes in the thalamus, while in the hippocampus panglial coupling was less abundant. other glial cells did not participate in the networks. we also found that a fluorescent glucose analog, -nbdg, propagates through the thalamic panglial network. the function of these panglial networks remains largely unclear.in heterozygous cx -ecfpki mice, deletion of one allele of the major hippocampal cx significantly reduced the number of coupled astrocytes only in the hippocampus, while the thalamic networks remained unchanged. sr labelling of astrocytes and subsequent p microscopy identified a significant subset of thalamic sr cells lacking cx -ecfp expression. sr did not label oligodendrocytes as analysed in plp-gfp mice. semi-quantitative rt-pcr and western blot analysis revealed stronger expression of cx in thalamic nuclei while cx levels were higher in the hippocampus. this indicates a minor role for cx in gap junction coupling of astrocytes in the thalamus. consistent with these findings, the thalamus of cx ko mice displayed a strong decrease in astrocytic cell coupling compared to wild type littermates.together, these results indicate that thalamic astrocytes differ in various aspects from their counterpart in other brain regions and support the emerging concept of astrocyte heterogeneity. the mechanism of secondary damage spread after brain trauma remains unresolved. in this work, we redirected the attention to astrocytic communication pathways. using an in vitro trauma model that consists of a scratch injury applied to a rat astrocyte monolayer, we found a significant induction of connexin hemichannel activity, demonstrated by ethidium uptake, in regions distal from the injury ($ mm away and maximal $ h after injury). this response was abolished by two connexin hemichannel blockers, la and the peptide gap . in addition, the trauma-induced increase in hemichannel activity was prevented by inhibition of purinergic p receptors. the scratch-induced increase in hemichannel activity was absent in astrocytes from cx knockout mice. this activity took place with a singular spatial distribution, since cells located at $ mm away from the scratch gained connexin hemichannel activity. however, the functional state of gap junction channels (dye coupling) was not significantly affected in the same locations. the connexin hemichannel activity was also enhanced by the acute extracellular application of mm k . the increase in hemichannel activity was correlated with an increment in apoptotic cells, measured by immunofluorescence to annexin v at h post-trauma, which was totally prevented by gap peptide. these findings could open a new approach to prevent or reduce the secondary cell damage due to brain trauma. l. schlosser, a. scheller, f. kirchhoff institute of physiology, saarland university, homburg, germany current research suggests that astrocytes represent heterogeneous neuroglial populations in different regions of the brain. this is particularly evident when the expression pattern of various transmitter receptors is analyzed. hippocampal astrocytes, for example, do not express ampa receptors, while cerebellar bergmann glia is loaded. functional analysis of glial receptors requires dedicated efforts for each distinct brain region and developmental age. unfortunately, the molecular identification using antibody approaches is often hampered by either poor antibody quality or abundant receptor expression on adjacent neuronal membranes. therefore, we are using cre/lox-mediated gene ablation for proper functional analysis.here, we are focusing on the characterization of astrocyte-specific gene deletion of the gaba b receptor. metabotropic gaba b receptors consist of two subunits gaba b and gaba b forming a heteromeric receptor complex of which gaba b is essential. in contrast to neurons, activation of astroglial gaba b receptors leads to transient rises of intracellular ca . the functional impact of these glial gaba b -receptors is largely unknown.to address the gaba b receptor function in astrocytes we took advantage of the cre/lox system and crossbred glast-creert knockin mice with floxed gaba b receptor mice. first immunohistochemical analysis reveals a selective deletion of gaba b on a majority of astroglial processes. functional studies addressing the role of these receptors in astroglial ca signaling are in progress. purinergic signaling is the most diverse system in astrocytes to communicate with other glial cells and neurons. astrocytes express a variety of p x (ionotropic) and p y (metabotropic) receptors. especially in case of brain injury, atp levels and receptor expression on astrocytes are increased. atp is immediately degraded to adp, amp and adenosine. these nucleotides/nucleosides act back on the preferred purinoreceptor expressed on glial and neuronal cells. in the last decade researchers tried to evaluate the functional impact of astrocytic purinoreceptors on the complex information flux at the tripartite synapse. a prominent role has been suggested for the p y receptor subtype. we generated conditional mouse mutants to investigate the function of p y receptors in astrocytes in vivo and crossbred mice carrying the floxed p y gene with mice that express the inducible dna recombinase (creert ) under the control of the glast (l-glutamate/ l-aspartate transporter) locus. ablation of the p y receptor is induced in development and adulthood by intraperitoneal tamoxifen injections. successful recombination of the targeted p y receptor is evaluated by qrt-pcr on genomic dna and mrna usually days after tamoxifen application. at the genomic level we find $ % and $ % recombination in astrocytes of the cerebellum and hippocampus, respectively. similar recombination frequencies are observed in young (p ) or adult mice ( - weeks). when looking at the transcript level, the mrna is reduced to % in the cerebellum of adult mice and to % in the hippocampus. in young mice we determined a reduction to % and % mrna expression in the cerebellum and hippocampus, respectively. given the high level of p y expression on other cells of the brain these findings indicate a successful astrocyte-specific knockout. further fluorescence-activated cell sorting (facs) of recombined cells expressing the red fluorescent protein td-tomato will be used to verify the knockout. the potential function of these astroglial receptors in neuronal networks will be as well investigated using histological (em and light microscopy), twophoton ca imaging in situ and in vivo and behavioral approaches. we recently showed that they invade the cortex at . days of embryonic age (e . ). they first accumulate at the pial surface and within the lateral ventricles, after which they spread throughout the cortical wall, avoiding the cortical plate region in later embryonic ages. the absence of the expression of mac- and mhc ii suggest these cells have a na€ ıve/quiscent phenotype during embryonic development of the cortex. besides immunohistochemical markers is the presence of different k channels on microglia also an indication of their activation stage. however most studies have been conducted on postnatal and adult microglial cells. therefore we aimed at determining the electrophysiological activation phenotype of these embryonic microglia.at the age of e . and e . , microglial cells display a small inward rectifying k current and this independent of their location in the embryonic cerebral cortex and their cell morphology. these cells also express functional p x receptors, which based on the profile of the response are most probably p x receptors. time-lapse analysis showed that embryonic microglia are highly dynamic cells. suggesting that these cells are already very active during fetal development. rapid extracellular removal of glutamate, the major excitatory neurotransmitter in the cns, is essential for normal brain function. this task is primarily accomplished by the action of the sodium-dependent, high-affinity transporters glast and glt- (rodent analogous of eaat and eaat ), which are mainly expressed by astrocytes. impairment or failure of glast and glt- plays an important role in many pathological conditions. question: pelizaeus-merzbacher-like disease (pmld) is a hypomyelinating leukodystrophy caused by mutations in the gjc gene encoding the gap junction (gj) protein connexin (cx ). cx is expressed in oligodendrocytes and forms most gj channels to astrocytes and to other oligodendrocytes. since pmld-associated gjc mutations cause loss of cx function, gene replacement strategies may be promising for developing future treatments. a mouse model for plmd, the cx / cx double knockout (ko), is characterized by early onset of severe leukodystrophy at weeks of age leading to death by weeks, offering the possibility to test therapies. the aim of the present study was to generate a lentiviral vector to allow gene delivery specifically to oligodendrocytes, and to examine the transduction efficacy, distribution, duration and levels of egfp reporter gene and cx expression throughout the cns, in order to establish a method for oligodendrocyte-targeted gene therapy.methods: the expression cassette with the cx gene along with the ires-egfp as a reporter gene under the control of oligodendrocyte-specific cnp promoter was cloned into the lentiviral vector pcclsin.ppt.hpgk.gfp.pre. mock vectors lacking the cx gene were also generated as controls. viral particles were produced to high titers and purified, before injection. lentiviral vectors were delivered into the brain of wild type (wt) and cx ko mice at postnatal day (p ), intraventricularly and in the stratum radiatum of the dorsal hippocampus. egfp and cx expression was assessed using immunochemistry and immunoblot analysis at different time points post injection.results: we found widespread expression of virally delivered egfp in different brain regions colocalizing with oligodendrocyte markers (cc and olig ). the expression of egfp was detected from p until months post-injection. on average . . % of oligodendrocytes were egfp positive, with highest rates in the subventricular zone ( . . %, n ) and olfactory bulb ( . . %, n ) and lower rates in the cortex ( . . %, n ) and corpus callosum ( . . %, n ). in cx ko brain expression of virally delivered cx was also found after p with formation of gj plaques in a subpopulation of oligodendrocytes.conclusions: our results show that neonatal lentiviral gene delivery may result in stable and widespread cns expression targeted to oligodendrocytes by using cell specific promoters. thus, gene therapy approaches using lentiviral vectors may be feasible for future treatment of leukodystrophy and should be further studied. recent reports identified mrna coding for different cav subtypes like . and . in hippocampal ng glia. using acute brain slices prepared from developing and mature ng -eyfp mice, we also found that all ng glia upon depolarization in different brain regions elicited a. grimaldi, g. d'alessandro, c. lauro, m. catalano, c. limatola sapienza university of rome, rome, italy glioblastoma (gbm) is one of the most aggressive tumor of the central nervous system, being characterized by a great invasiveness and a very low survival rate of patients. this work wants to better define the role of tumor microenvironment in the modulation of tumor invasiveness. it is known that tumor migration and invasiveness can be modulated by the chemokine cxcl and its receptor cxcr . the expression of both these proteins has been demonstrated in various human gbm cell lines and it has been shown that, blocking this pathway, tumor cells greatly reduce their invasive ability. we have recently demonstrated that an important molecule directly implicated in tumor migration is the intermediate conductance calcium-activated potassium channel (kca . ). given the expression of these channels also in other cell types infiltrating the tumor mass, like microglia, we investigated the effect of kca . inhibition on microglia-glioblastoma interaction. preliminary data indicate that kca . activity on microglia is involved in the movement of these cells toward the tumor mass. a growing body of evidence indicates that glioblastoma stem-like cells (gscs) play a central role in human glioblastoma development and resistance to current therapies. we recently described a cluster of micro-rnas, the mir- - , which induces the exit of gscs from their stem state and suppresses their tumorigenic properties in an irreversible manner (fareh et al, ) . to elucidate the gene networks that govern gsc exit from their stem state, we used chip-seq (chromatin immunoprecipitation followed by next generation dna sequencing), to identify gene loci showing enrichment of the active (h k me ) and repressive (h k me ) epigenetic marks in gsc-mir- - as compared to na€ ıve gscs. functional significance of the chip-seq results was evaluated with confrontation of the chip data with the transcriptomes of the corresponding cells derived from exon array hybridization. western blot analysis showed that global h k me and h k me expression levels were similar in gsc and gsc-mir- - . chip-seq analysis revealed similar gross number of gene loci associated with h k me or h k me ( and genes, respectively) in either cell type. interestingly, % ( genes) of the analyzed genes exhibited a change in either h k me or h k me , or both in mir- - -gscs as compared to na€ ıve gscs. these changes resulted into a transcript variation in % of the cases ( / ) considering as significant an increase or a decrease of at least -fold and the % of these genes translated into congruent alterations in the corresponding transcript levels.analysis of the functional significance of the changes observed using functional annotation databases identified novel gene networks, likely to participate in the regulation of gsc properties. studies under way focus on members of these networks specifically activated in mir- - gscs that might alter gsc dialog with their microenvironment. malignant gliomas are the most frequent primary tumors of the brain with poor clinical prognosis. infiltrating peripheral macrophages and resident microglia contribute significantly to the tumor mass. we have previously shown that microglia as the intrinsic immune competent brain cells promote glioma expansion by up-regulating metalloprotease mt -mmp through toll-like receptor (tlr) and its adaptor protein myd . in this study we identified tlr , as the main tlr controlling mt -mmp expression and pro-tumorigenic signaling in microglia. glioma-derived soluble factors and synthetic tlr specific ligands induced mt -mmp expression in microglia from wt mice but not tlr -/-mice. by using the organotypic brain slice model, we found that tumor expansion depended on both parenchymal tlr expression and the presence of microglia. tlr is also highly expressed in human gliomas and inversely correlates with patient survival. in search for an endogenous tlr ligand released from glioma cells, we screened glioma conditioned medium (gcm) by mass spectrometry for endogenous tlr agonists produced by glioma cells and found versican, an extracellular matrix proteoglycan and a reported ligand of tlr . to examine if versican is the factor mediating the glioma-microglia interaction, we silenced versican expression in gl cells. primary microglia were then stimulated with gcm from versican sirna and non-target sirna transfected gl cells and microglial mt -mmp expression was analyzed by real-time pcr. an almost -fold up-regulation in microglial mt -mmp expression was observed using gcm from non-target transfectants while the expression was reduced with gcm from versican knock-down gl transfectants. our results show that tlr activation is an essential part of the signaling cascade employed by glioma cells to convert microglia into a pro-tumorigenic phenotype and tlr thus may be a novel target for glioma therapies. two ipsdms were used to determine their responses in the presence of glioblastoma cells. using lc-ms/ms method, proteomic profiles were generated and compared between ipsdms exposed to human glioblastoma cells and ipsdms exposed to normal human astrocytes as control.results: immunolabeling of the two ipsdm cell lines showed specific expression of microglial markers such as iba- , cd b, and cd . the comparative proteomic analyses indicated that microglia exposed to glioblastoma cells showed differential protein expressions relevant for cytoskeletal activity, energy production, and cell survival compared to control.conclusion: this is the first study showing the proteomes of human induced pluripotent stem cell-derived microglia exposed to glioblastoma cells, and the findings could provide further insight of the interaction between microglia and glioblastoma. key: cord- -rz r sj authors: nan title: abstracts for the th annual meeting of the japan neuroscience society (neuroscience ) date: - - journal: neuroscience research doi: . /j.neures. . . sha: doc_id: cord_uid: rz r sj nan the brain basis of conscious experience is one of the great unsolved mysteries of science. how can a material object became aware of the world around it and of its very own awareness? many scholars think this question is unanswerable. new approaches to this age-old mind-body problem have recently been encouraged by the development of pet and mri and the powerful tools of modern neuroscience. we are now witnessing the explosive growth of a new field, called cognitive neuroscience which focuses on behavior and uses classical reaction-time paradigms together with new computer-based technology. a parallel approach is to cross-correlate formal aspects of conscious experience as the brain spontaneously pursues its regular trajectory through the objectively defined states of waking nrem and rem sleep. at the level of the brain it is possible to record pet and mri and by using an animal model to analyse cellular and molecular mechanisms. the advantage of this approach, which i call the conscious state paradigm, is that it, is quantitative, integrative, and holistic (in the rigorous sense of that word). the brain basis of activation (a) input-output gaining (i) and modulation (m) can now be described in relation to the changes in consciousness associated with them. the data can be used to create a three-dimensional model (aim) which describes the brain-mind trajectory in its state space. neural circuits in many parts of the developing nervous system exhibit highly rhythmic episodes of electrical activity. such activity has generally been considered to fine tune connections that are initially made by axons responding to a complex array of molecular guidance cues. however, chick and mouse spinal cords exhibit activity at early stages as motoneurons are still migrating and beginning to extend their axons. modest alterations in the frequency of such episodes produced changes in the expression of several guidance/recognition molecules and caused motor axon pathfinding errors. interestingly the type of pathfinding decision, the binary dorsal-ventral choice or the subsequent pool specific fasciculation and projection of axons to specific muscles, was differentially affected by decreasing or increasing the frequency respectively. thus, activity generated by developing circuits interacts at early stages with the molecular signaling pathways that govern the formation of precise circuits and any drugs that alter this activity could adversely affect circuit formation. supported by nih grant ns . sl - - frontal cortex and higher-order motor control: preferential use of multiple premotor and prefrontal areas dependent on behavioral context jun tanji brain science research center, tamagawa university, machida, tokyo, japan in the cerebral cortex of primates, there exist a number of motor areas rostral to the primary motor area and caudal to the prefrontal cortex. although each area has been defined based on anatomical and physiological criteria, functional roles played by each of them have been the matter of much debate. in fact, in a recent trend of research reports, it is popular to stress commonalities rather than specificities in the use of multiple cortical areas in motor control. for instance, the involvement of the primary motor cortex in cognitive aspects of motor behavior has been an eye-catching subject of recent reports. the involvement of the prefrontal cortex in movement planning has also been inferred. up to a certain extent, it is true that the use of different areas have some factors in common. however, it is a mistake to ignore profound differences in the use of each area, depending on specific aspects of motor behavior. in this lecture, i will describe such differences that could be clarified only when neural activities are examined properly, by studies designed to reveal individual aspects of functional significance of each area. sl - - neuronal functions and molecular motor, kinesin superfamily proteins, kifs: from transport of synaptic proteins and mrnas, to brain wiring, neuronal survival and higher brain function nobutaka hirokawa department of cell biology and anatomy, graduate school of medicine, university of tokyo, japan the intracellular transports are fundamental for neuronal morphogenesis, functioning and survival. to elucidate this mechanism we have identified and characterized kinesin superfamily proteins, kifs, using molecular cell biology, molecular genetics, biophysics, and structural biology. kifs play essential roles on neuronal function and survival by transporting synaptic vesicle precursors (kif a/kif bbeta), nmda type(kif ) and ampa type(kif s) glutamate receptors and mrnas(kif s) such as camkii  mrna and arc mrna with a large rna-transporting protein complex containing at least rna related proteins such as hn rnp-u, staufen, and fmrps. kif a is fundamental for correct brain wiring by suppressing elongation of axon collaterals through depolymerizing microtubules in growth cones. kif suppresses tumorigenesis by transporting ncadherin/beta catenin containing vesicles from golgi to plasma membrane in the neuroepithelium. kif controls the activity-dependent survival of postmitotic neurons by regulating parp- activity in brain development. thus, kifs play significant roles not only on various neuronal functions but also on brain wiring, development and higher brain functions such as memory and learning. tsukahara award - what we can learn from the functional recovery after brain injury tadashi isa national institute for physiological sciences, okazaki, japan it is believed that when a part of a neuronal system is damaged, some of the lost functions can be taken over by residual systems through training. such concept is considered as the basis of neurorehabilitation, however, the mechanism of the "take-over" is not well understood. in this talk, i will present our recent progress in two lines of studies using non-human primate models related to this issue. the first topic is on the recovery of dexterous finger movements after lesion of the lateral corticospinal tract at the cervical spinal cord. after the virtually complete lesion, relatively independent finger movements can be recovered in - months. we have found that bilateral primary motor and ventral premotor cortices are involved at various stages of the recovery process by combining the pet imaging and reversible local inactivation technique. in the second, i will talk about the visuo-motor processing in monkeys with unilateral lesion of the primary visual cortex (v ). after complete ablation of v , the monkeys recover performance of visually guided saccades toward the blind field in months. saccades to the blind field have low sensitivity and less accurate. however, the monkeys can perform surprisingly cognitively demanding tasks in the blind field. i will discuss on our hypothesis on the bottom-up and top-down control of learning during recovery. takuji iwasato riken brain science center (bsi), wako-shi, saitama, japan in the rodent primary somatosensory (barrel) cortex, the configuration of the whiskers on the face is topographically represented as "barrels", discrete modules of layer iv neurons and thalamocortical afferent (tca) terminals. while barrel formation is an important model of the establishment of patterned topographic connections between the sensory periphery and the brain, the molecular mechanisms underlying this process are poorly understood. we developed and applied mouse reverse genetic technologies to examine these molecular mechanisms. we have focused on the nmda-type glutamate receptor (nmdar) and calcium-stimulated adenylyl cyclases, as nmdar-and camp-cascades are central to various types of neuronal plasticity, both in adulthood and during development. series of global and region-specific knockout mice have revealed the roles of these molecules in the patterning of barrel cortex and differentiated the specific mechanisms at presynaptic tca terminals compared to those at postsynaptic cortical neurons. research funds: presto (jst), kakenhi , kakenhi sy - - - distinct roles of two -pass transmembrane cadherins in neurite growth control tadashi uemura , , yasuyuki shima , , keisuke sehara , manabu nakayama , shinya kawaguchi , mikio hoshino , yoichi nabeshima , tomoo hirano , graduate school of biostudies, kyoto university, kyoto, japan; school of science, japan; kazusa dna research center at chiba, japan; graduate school of science, japan; graduate school of medicine, japan; crest, jst, japan drosophila flamingo (fmi) and mammalian celsr - , which are pass transmembrane cadherins, have been considered to mediate the regulation of neuron contact-dependent neurite growth. we show that mammalian -pass transmembrane cadherins celsr and celsr are activated by their homophilic interactions and regulate neurite growth in a distinct manner. both gene-silencing and co-culture assay showed that celsr enhanced neurite growth whereas celsr suppressed it. our result suggested that celsr had a stronger activity as g-protein coupled receptor than celsr did, most likely due to a difference of a single amino acid residue in the transmembrane domain, and this functional difference resulted in distinct effects in neurite growth regulation. thus, neuron-neuron interactions modulate neurite growth differentially through this couple of -pass transmembrane cadherins. masatoshi takeichi riken center for developmental biology, kobe, japan for synapse formation, axons need to recognize their specific partners, and subsequently stabilize their contacts. while a number of cell surface molecules should be involved in such processes, cadherin adhesion molecules play a role. when cadherin activities are blocked, synaptic contacts become destabilized in cultured neurons. this is also the case in vivo; e.g., in the neural retina whose cadherin activities are impaired without perturbing their overall architecture, a certain class of synaptic contacts does not normally form. another series of our study demonstrate that the cadherins cooperate with nectins, a subfamily of ig-domain molecules, for establishment of axon-dendritic contacts: in early hippocampal pyramidal neurons, nectin- is preferentially localized in axons; and nectin- , in both axons and dendrites. we present evidence that the heterophilic binding between axonal nectin- and dendritic nectin- is important for facilitating the axon-dendritic attachment; and cadherins seem to be required to stabilize the nectin-initiated contacts. thus, multiple classes of adhesion molecules work together to ensure the correct linking between axons and dendrites. hitoshi sakano department of biophysics and biochemistry, university of tokyo, tokyo, japan we have studied how the olfactory sensory neurons (osns) expressing the same odorant receptor (or) gene converge their axons to a specific set of glomeruli in the olfactory bulb (ob). retrogradestaining of osn axons indicated that the dorsal/ventral (d/v) arrangement of glomeruli in the ob is correlated with the expression areas of corresponding ors along the dorsomedial/ventrolateral axis in the oe. in contrast, the anterior/posterior (a/p) arrangement of glomeruli appears to be independent of the epithelial locations of osns and more dependent on the expressed ors. it was found that g proteinmediated camp signals regulate the positioning of glomeruli along the a/p axis in the ob. we also found that multiple sets of cell adhesion molecules, e.g., ephrin-as and eph-as, are expressed in a complementary manner, whose transcription levels are uniquely correlated with the expressing or species. we propose that differential levels of repulsive/adhesive interactions of axon termini may regulate the sorting of like-axons during the process of osn projection. research funds: crest, jst, kakenhi sy - - - lamina-restricted guidance of hippocampal mossy fibers hajime fujisawa , fumikazu suto nagoya university, nagoya, japan; institute of genetics, mishima, japan axons from different sources terminate at particular dendritic segments of target neurons in a laminal fashion. one important issue to be addressed is how individual axons are instructed to invade and arborize in particular laminae. projection of hippocampal mossy fibers is one of good experimental models to analyze molecular mechanisms that govern lamina-restricted termination of axons, because the fibers project to the proximal dendritic segment of ca pyramidal cells. we here report the following three mechanisms that provide lamina-restricted projection of mossy fibers. first, a neural repellent sema a is expressed in ca pyramidal cells and principally suppresses invasion of mossy fibers to ca . second, the repulsive signal of sema a is mediated by plexin-a expressing in mossy fibers. third, the repulsive activities of sema a are attenuated by plexin-a in the proximal dendritic segments of ca pyramidal cells, resulting in the segments permissive for mossy fibers to invade. over the course of development, children become increasingly able to control their thoughts and actions (i.e., cognitive control). the term cognitive control is an umbrella term for a set of putative control processes. these control processes may reach adult levels at different rates, depending on the rate of functional development of the specific brain structures involved. the structure most closely associated with cognitive control is prefrontal cortex (pfc). pfc is composed of what are believed to be functionally distinct subregions, including ventrolateral, dorsolateral, rostrolateral, and medial pfc. i will discuss the control processes associated with each of these regions, and how the functionality of these regions differs between school-aged children, adolescents, and young adults. three fmri studies will be presented, focusing on ( ) working memory maintenance and manipulation, ( ) rule representation and task-switching, and ( ) relational reasoning. based on these data, i will discuss some general points about neurodevelopment changes in cognitive control, and outline the approach that our laboratory has taken in our developmental cognitive neuroscientific research. sy - - - towards manipulative neuroscience based on non-invasive brain decoding in atr computational neuroscience laboratories, we proposed several computational models such as cerebellar internal models, mosaic, modular and hierarchical reinforcement-learning model. some of these models can quantitatively reproduce subject behaviors given sensory inputs and reward and action sequences which subjects received and generated. these computational models possess putative information representation such as error signals for internal models, action stimulus dependent reward prediction, and they can be used as explanatory variables in neuroimaging and neurophysiology experiments. we named this approach as computationalmodel-based neuroimaging, as well as computational-model-based neurophysiology. this new approach is very attractive and appealing since this is probably the only method with which we can explore neural representations remote from either sensory or motor interfaces. but, sometimes limitation of mere temporal correlation between the theory and data became so apparent, and we started to develop a new paradigm 'manipulative neuroscience' where physical causality is guaranteed. research funds: nict, karc sy - - - neural mechanisms in williams syndrome-insights from neuroimaging andreas meyer-lindenberg nimh, bethesda, md, usa williams syndrome (ws), a rare disorder caused by hemizygous microdeletion of − genes on chromosome q . , has long intrigued neuroscientists with its unique profile of striking behavioural abnormalities, such as hypersociability, combined with a differential impact on cognitive functions, with some types of abilities only mildly affected while others are severely impaired. ws, thus, raises fundamental questions about the neural mechanisms of social behaviour, the modularity of mind, and brain plasticity in development, and provides a privileged setting to understand genetic influences on complex brain function in a bottom-uph way. recent months have seen dramatic advances in uncovering the functional and structural neural substrates of ws and a beginning understanding of how these are related to dissociable genetic contributions characterized both in special participant populations and animal models. we will review neuroimaging work indicating abnormal function and structure in subsystems of visual processing, long term memory, and emotional regulation and social cognition, and discuss advances in relating them to the underlying molecular biology of this unique syndrome. daisuke yamamoto tohoku university graduate school of life sciences, japan the fruitless locus of drosophila was originally recognized by its mutants, the males of which preferentially court males rather than females. the fruitless primary transcript is subject to sexually dimorphic splicing mediated by transformer, and encodes a group of proteins that are putative transcription factors of the btb-zn finger protein family. the male-specific fruitless protein is expressed in small groups of cns neurons of males, but not of females. fruitless masculinizes these neurons thereby establishing the neural substrates for male-typical behavior. by experiments that label individually the neurons that express fruitless, we have identified a subset of brain interneurons that display marked sexual dimorphism in their number and projection pattern. fruitless supports the development of those neurons with male-specific dendritic fields, which are programmed to die during development in females as a result of the absence of fruitless. thus, the fruitless protein expression can produce a male-specific neural circuit likely used for heterosexual courtship by preventing cell death in identifiable neurons. hitoshi okamoto riken brain science institute, wako, saitama, japan the emotional behavior depends on the evolutionarily most conserved neural circuits. especially the fear behaviors involve the basal telencephalic nuclei such as the amygdala and the nucleus accumbens. thanks to the progress in understanding of the telencephalic development among different species, we can determine the correspondence of the parts between the teleost and mammalian telencephalons. with these in mind, we initiated the characterization of the emotional neural circuits in the zebrafish brain which are amenable to various modern technology. we already reported the asymmetric axonal projection from the left and right habenulae which act as the relay station to conduct the emotional information from the telencephalon to the monoaminergic neurons in the midbrain and the hindbrain. to investigate whether such asymmetric neural circuits cause the laterality for emotional behaviors, we are now in the process of establishing the paradigms for combining the behavioral assay with genetic manipulations to control the activity of the emotional neural circuit in zebrafish. research funds: kakenhi ( ) sy - - - a molecular biological approach for songbirds to study learned vocal communication kazuhiro wada, erich jarvis duke university, usa songbirds possess one of the most accessible neural systems for the study of brain mechanisms of behavior, particularly that for learned vocal communication. however, neuroethological studies in songbirds have been limited by the lack of high-throughput molecular resources and gene manipulation tools. to overcome this limitation, we generated a resource of full-length cdnas for gene expression analyses and functional gene manipulation in songbirds. we constructed total full-length cdna libraries from brains in different behavioral and developmental conditions. with these cdnas, we created a novel database and k songbird cdna array. we used the arrays to reveal a set of genes regulated by singing behavior. their molecular functions spanned most cellular and molecular categories, including signal transduction, structural, and synaptically released molecules. with the full-length cdnas, we were able to express proteins of singing-regulated genes in targeted brain area, using a lentiviral system. this resource now opens to more thoroughly study molecular neuroethological mechanisms of behavior. research funds: uehara memorial fellowship to k.w. and nih grant to e.j. - - - stepping pattern learning using mice: histochemical identification of activated neuronal circuits takashi kitsukawa graduate school of frontier biosciences, osaka university, osaka, japan identifying brain areas and neuronal circuits activated in a behavior is a critical step in understanding how the brain works in that behavior. also, identifying neuronal types involved in a behavior is a key step toward connecting behavioral approaches with molecular and genetical approaches. an efficient method of clarifying neuronal types activated by behavior is histochemical identification of neuronal types combined with c-fos staining. i would like to introduce our work as an example of using this method. in order to understand the neural processing involved in sequential motor skill learning we built a wheel running system in which a mouse learns sequential stepping patterns. we double-stained brain sections from mice which performed this task with c-fos and a neuronal marker such as enkephalin, substance p or nitric oxide synthase, each of which denotes a particular neuronal type. our results indicate that particular types of stiriatal neurons are activated during this learning, suggesting that cortico-striatal circuits are involved. synaptic plasticity that is dependent on precise timing of spikes between pre-and postsynaptic neurons plays important role in development and plasticity of brain functions. such spike-timingdependent plasticity (stdp) has attracted wide attentions because of its high computational power and physiologically plausible induction. we previously demonstrated that long-term potentiation was closely associated with structural plasticity of dendritic spines. however, how stdp is associated with structural changes has not been elucidated. we here report that paired two-photon uncaging of a caged-glutamate compound at a single spine and postsynaptic spike of whole-cell clamped neuron rapidly induced long-lasting bidirectional structural plasticity of spines in hippocampal ca pyramidal neurons. our results indicate that stdp is intimately associated with bidirectional structural plasticity at the level of single spines. research funds: kakenhi sy - - - role of camkii as a structural protein that stabilizes actin cytoskeleton in dendritic spines kenichi okamoto, radhakrishnan narayanan, yasunori hayashi massachusetts institute of technology, usa actin serves as a major cytoskeleton which maintains spine structure and exists in a equilibrium between f-actin and g-actin. tetanic stimulation causes a persistent shift of actin equilibrium towards f-actin which enlarges dendritic spines. but the mechanisms which maintain these changes remain elusive. we propose that camkii  acts as an actin stabilizing molecule to maintain spine structure. camkii  is not only an abundant f-actin binding protein, it can also make oligomers. we found that camkii  oligomer crosslinks f-actin and stabilizes actin depolymerization kinetics. in spines, camkii  oligomer slows down actin dynamics and camkii  is enriched in spines by actin polymerization. the suppression of endogenous camkii  alters spine shape to filopodia-like structures. these experiments suggest that camkii  plays a role as a major stabilizer of the actin cytoskeleton to maintain spine structure. we also found that camkii  detaches from f-actin in an activity dependent manner. we will discuss how camkii  maintains actin equilibrium in activity dependent dendritic plasticity. ryohei yasuda duke university medical center, usa the small gtpase protein ras plays central roles in calcium signaling important for many forms of synaptic plasticity and regulation of neuronal excitability. using -photon fluorescence lifetime imaging microscopy in combination with a fret-based ras activity sensor, we visualized the activity of ras signaling with high spatiotemporal resolution. our studies indicate that calcium entry due to action potentials causes ras to activate in a supra-linear manner (yasuda et al., ) . furthermore, in response to single spine stimulation using -photon glutamate uncaging, ras activation initially occurs at the stimulated spine, subsequently spreading into its parent dendrites and nearby spines. these results suggest that nonlinear filtering by ras regulators as well as the spatial spreading of ras and ras regulators shape spatiotemporal patterns of ras signaling. hiroshi shibasaki takeda general hospital, kyoto, japan involuntary movements are unintended, generalized or focal, movements of abnormal nature, and include tremor, myoclonus, dystonia, chorea/ballism, athetosis and dyskinesia. myoclonus is characterized by abrupt, shock-like movements caused by brief muscle contraction (positive myoclonus) or abrupt cessation of on-going muscle contraction (negative myoclonus), or their combination. depending on the estimated origin, it is classified into cortical, brain stem, and spinal myoclonus. cortical myoclonus is short in duration ( ms). by back averaging eeg or meg time-locked to spontaneous myoclonus, a cortical activity is demonstrated in the corresponding area of the contralateral primary motor cortex immediately preceding the myoclonus (by ms for hand). it is mediated by fact-conducting corticospinal pathway. cortical myoclonus is often stimulus-sensitive (cortical reflex myoclonus), showing extremely enhanced cortical responses to somatosensory or visual stimulus, and enhanced longloop transcortical reflexes. these findings, together with transcranial magnetic stimulation, suggest increased excitability of sensorimotor cortex in cortical myoclonus. mark hallett ninds, bethesda, md, usa there have been many recent advances in the understanding of the physiology of focal dystonia. three main avenues of research have shown abnormalities in cortical inhibition, sensory processing including sensorimotor integration, and plasticity. this lecture will emphasize the abnormal inhibition. abnormal inhibition appears to be the most direct cause of unwanted muscle contractions that make up both the involuntary spasms and the overflow movements also seen in this condition. a loss of inhibition is seen in spinal and brainstem reflexes, but these changes are likely secondary to cortical abnormalities. cortical inhibition is also diminished as demonstrated most clearly with transcranial magnetic stimulation. gaba content may be decreased as shown with magnetic resonance spectroscopy. a particular type of defective inhibition is surround inhibition, the inhibition that normally operates to sharpen fine skilled movements. studies are now in progress to determine the synaptic mechanisms of surround inhibition and how this becomes abnormal in dystonia. understanding about inhibition in dystonia has led to some new treatments including some non-invasive cortical stimulation methods. research funds: nih intramural program sy - - - basal ganglia-cortical systems reinforcing tonic motor activity in health and disease peter brown sobell department, institute of neurology, uk the synchronisation of neuronal activity in the beta frequency (∼ hz) band has been noted in healthy primates, including humans, at both striatal, putamenal and cortical levels. it is most obvious in the motor cortex during tonic motor activity and is suppressed by voluntary movement. in this talk i will develop the idea that beta band synchronisation in the basal ganglia-cortical loop promotes tonic/postural contraction at the expense of new movements. thus, spontaneous phasic increases in beta activity in healthy subjects can be shown to be associated with a slowing of voluntary movements and a reinforcement of transcortical stretch reflexes. beta synchrony is also greatly exaggerated in untreated parkinson disease, where it may bias against new movement and contribute to bradykinesia and rigidity. excessive dopaminergic stimulation, either during treatment for parkinson disease, or in conditions such as dystonia, may overly suppress beta activity in bg-cortical loops leading to excessive movement. recordings of local field potentials in the basal ganglia of patients with movement disorders will be described that support this schema. research funds: mrc sy - - - coding of reward value of actions and valuebased action selection in the basal ganglia a damage of the nigrostriate dopamine system results in severe impairments of voluntary movements as well as involuntary behavioral states like rigidity, akinesia and tremor as typically observed in parkinson disease. recent studies revealed that long-term potentiation of corticostriatal synaptic transmission occurs dopaminedependent manner, and that neuronal firing related to external stimuli and body movements are modulated by whether the stimuli and movements are associated with reward or not. we recorded striatal neurons of monkeys who chose between left-and right-handle-turns based on the estimated reward probabilities of the actions. during a delay period before the choices, activity of more than one-third of striatal projection neurons was selective to the values of one of the two actions. during handle-turns, another subset of neurons was activated. these results suggest representation of action values in the striatum, which can guide action selection in the basal ganglia circuit. roles of the basal ganglia circuit in voluntary and involuntary action selection will be discussed. in vivo reporter gene imaging is expected to be a powerful tool in gene and cell therapy monitoring. we designed a new pet reporter gene system with f- fluoroestradiol (fes) and human estrogen receptor ligand binding domain (herl), which would work in various tissues with little physiological effect. we have been evaluating its potential in gene therapy monitoring constructing a plasmid co-expressing thymidine phosphorylase (htp), a factor works for revascularization, as therapeutic gene and herl. cos cells transfected with the plasmid expressed the both proteins and, when the plasmid was in vivo electroporated into mouse calf muscle, the electroporated muscle accumulated significantly higher amount of fes that the control side. this system was successfully applied to es cell transplantation monitoring also. inducible herl expression system was stably transfected into mouse es cells and viable es cells could be detected in vivo using fes. these data support the prospect that our in vivo reporter gene system would be useful in gene/cell transplantation therapy monitoring. tetsuya suhara molecular imaging center, national institute of radiological sciences, chiba, japan the molecular imaging using positron emission tomography enables to visualize various brain molecules with radio labeled ligand. neurons and glias express various receptors and transporters and those can be a specific target of the imaging. the functions of those molecules can be examined in various types of pharmacologically or genetically modified animal models. amyloid precursor protein transgenic mice provide the target of in vivo imaging of amyloid protein and glial reaction. because pronounced neuronal death is frequently heralded by microgliosis, in vivo analysis of glial activation in a quantitative manner could be a powerful means for assessing neuroglial degeneration. on the other hand, clinical finding of molecular imaging can also provide important cues for the basic research targets. since there is no ideal animal model for psychiatric disorders, the abnormal dopamine d receptor found in clinical research indicates a possible therapeutic target of negative symptoms of schizophrenia. the bidirectional interaction between basic research and clinical research using the molecular imaging technique can expand our knowledge in brain disease. sumiko mochida department of physiology, tokyo medical university, tokyo, japan in presynaptic terminals, packages of neurotransmitters, synaptic vesicles (svs), are localized at specific sites in different stages by regulation of proteins complexes. the recent outstanding studies have revealed molecular mechanisms of presynaptic structure and function. for svs, new proteins were found and the anatomy of vesicle structure was clarified. readiness for transmitter release, svs are docked and primed at the cytomatrix at the active zone where proteins complex formation is regulated by phoshorylation. new kinase sad- found at the sv and active zone or pka phosphorylates specific proteins. sv exocytosis is triggered by conformational changes in the fusion proteins complex when ca + sensing protein was activated. synchronies of sv fusion are maintained by a ca + sensing protein, synaptotagmin i. after transmitter release svs are recycled: surprisingly, recycled svs are shared between synaptic boutons regulated by cytoskeletal and motor proteins. these new findings suggest fine mechanisms in presynatic terminals that regulates transmitter release. shigeo takamori st century coe program, department of neuorology and neurological science, tokyo medical and dental university, tokyo, japan synaptic vesicles are storage organelles for neurotransmitters that recycle in the presynaptic terminals. to achieve their functions, i.e. neurotransmitter uptake and membrane fusion, they have to be equipped with specified proteins that play essential roles for each process. since decades ago, we have witnessed major advances in our knowledge about the molecular constituents on synaptic vesicles and we've functionally characterized many key players on membrane fusion machinery such as snare proteins and the rab gtpases and on neurotransmitter uptake such as vesicular transporters and vacuolar h + -atpase. however, a detailed picture of a vesicle membrane with all of its constituents is not yet available. in the present study, we have applied a combination of biochemical and biophysical approaches on purified synaptic vesicles from rat brains in order to arrive at a comprehensive and quantitative description of synaptic vesicles. in particular, with a newly developed counting method for synaptic vesicles in solution, we estimated the copy number of each molecule in a single synaptic vesicle. sy - - - sad: a novel kinase implicated in phosphoproteome at the presynaptic active zone toshihisa ohtsuka department of clinical and molecular pathology, faculty of medicine/graduate school of medicine, university of toyama, toyama, japan sad is a serine/threonine kianse, which has been shown to regulate various neuronal functions during development, including clustering synaptic vesicles, maturation of synapses, and axon/dendrite polarization: these have recently been revealed by genetic studies in c. elegans and mice. to test if sad is also involved in synaptic functions at mature neurons such as neurotransmitter release, we have recently isolated and characterized human orthologues of sad. interestingly, sad localizes both on synaptic vesicles and at the presynaptic active zones. moreover, sad, together with prominent active zone proteins cast and bassoon, is tightly associated with the active zone cytomatrix. in accord with its unique localization at the nerve terminals, sad appears to be involved in a late step of neurotransmitter release, via direct phosphorylation of another active zone protein rim . thus, these results suggest that sad regulates not only neural polarization during development but also neurotransmitter release at mature synapses. toshiaki sakisaka , takeshi baba , sumiko mochida , yoshimi takai department of molecular biology and biochemistry, osaka university graduate school of medicine, osaka, japan; depatment of physiology, tokyo medical university, tokyo, japan two types of factors are involved in ca + -dependent neurotransmitter release: the snare system and its regulators. the regulators of the snare system include many factors, such as the rab system, munc- , munc- , doc- , and tomosyn. we have previously reported that tomosyn, a syntaxin- -binding protein, works as a molecular clamp that controls free syntaxin- availability for the formation of the snare complex and thereby regulates synaptic vesicle exocytosis. here we show that pka-catalyzed phosphorylation of tomosyn decreases its binding to syntaxin- , resulting in enhanced the formation of the snare complex. conversely, rock phosphorylates syntaxin- , which increases the affinity of syntaxin- for tomosyn and forms a stable complex with tomosyn, resulting in inhibition of the formation of the snare complex. thus, tomosyn is likely to be an upstream regulator of the snare system, whose activity is regulated via well known signal transduction pathways. tei-ichi nishiki department of physiology, okayama university, okayama, japan a synaptic vesicle membrane protein, synaptotagmin i is thought to be a ca + sensor for neurotransmitter release. however, the physiological contributions of its ca + -binding domains (c a and c b) are still unclear. we have studied the roles of aspartate (asp) residues in the c b ca + -binding sites. in synaptotagmin i deficient neurons, although synchronous release was abolished as previously reported (cell , p. ) , asynchronous release was significantly increased. this defect was completely rescued by expressing wild-type synaptotagmin i, indicating the crucial roles of synaptotagmin i for triggering synchronous release and suppressing asynchronous release. synaptotagmin i with mutations in the second or third asp inhibited synchronous release, but still could suppress asynchronous release. thus, we conclude that synaptotagmin i maintains the synchrony of transmitter release in two ways. ca + binding to the c b is essential for synchronizing release. suppressing of asynchronous release seems not to require ca + binding to the c b because mutation in the second asp inhibits ca + binding, yet still allows the protein to suppress asynchronous release. yukiko goda, kevin j. darcy, kevin staras, lucy m. collinson mrc cell biology unit and lmcb, university college london, uk it has been assumed that vesicle replenishment at central synapses operates autonomously at individual presynaptic terminals. we tested the classical model of a compartmentalized synaptic vesicle cycle using fluorescent styryl dyes in combination with methods of fluorescence recovery after photobleaching and correlative light and electron microscopy in cultured hippocampal neurons. we found that endocytosed, recycling synaptic vesicles travel along axons and incorporate into non-native synapses by an actin-dependent mechanism. these newly-incorporated vesicles underwent exocytosis upon stimulation, demonstrating that they form part of the functional recycling pool at their host synapses. our findings indicate that synaptic vesicle recycling is not confined to individual presynaptic terminals but rather a substantial proportion of synaptic vesicles are shared constitutively between synapses. research funds: mrc, nih and narsad hiroshi kunugi department of mental disorder research, national institute of neuroscience, ncnp, japan neurotrophins such as brain-derived neurotrophic factor (bdnf) and neurotrophin- (nt- ) have been implicated in the phathogenesis of several neuropsychiatric diseases including schizophrenia, mood disorders, and neurodegenerative diseases. in the past decade, we have systematically screened bdnf, nt- and its low-affinity receptor p genes for polymorphisms and their possible association with neuropsychiatric diseases. as a result, three polymorphisms of the bdnf gene (c t in the noncoding region, bdnf-linked polymorphic region, and val met), three polymorphisms of the nt- gene (g- a, microsatellite in intron , and gly- glu) and a missene polymorphism of the p gene (ser leu) have been found to be associated with susceptibility to schizophrenia, bipolar disorder, depressive disorder, or alzheimer's disease, although some contradictive negative results have also been reported. here i summarize these findings, review the relevant literature, and discuss future directions of the promising role of the genetic variations of neurotrophins and p in neuropsychiatric diseases. recently, a single nucleotide polymorphism (val met) in the bdnf gene resulting in a prodomain substitution at position from a valine (val) to methionine (met) has been shown to lead in humans to altered hippocampal size and function, and susceptibility to neuropsychiatric disorders. we have recently determined in vitro that bdnf met aberrantly engages a specific vps protein, sortilin, that is part of a highly specialized sorting machinery that regulate bdnf trafficking to secretory pathways. in order to determine whether these trafficking defects are responsible for impaired hippocampal functioning, we have developed a transgenic knock-in mice containing the genetic variant bdnf (bdnf met/met ). we have determined that there is a regulated secretion defect for bdnf met , as well as altered hippocampal structure and function in these bdnf met mice, in a manner similar to that reported in humans with this variant bdnf. thus, this bdnf met/met mouse may provide an in vivo model system to inform human studies focused on associations of this variant bdnf with clinical disorders. research funds: nih grant# ns sy - - - processing of bdnf and brain disorders masami kojima , aist, osaka, japan; sorst, jst, saitama, japan the fact that pro-and mature neurotrophins elicit opposite effects through p neurotrophin receptor (p ntr) and trks, respectively suggests that proteolytic cleavage of proneurotrophins is an important mechanism that controls the direction of neurotrophin actions. here we examined the effects of two rare single nucleotide polymorphisms (snps); (t/g) and (t/g) of the human bdnf gene, causing amino acid substitution (r m and r l) near the cleavage site. western blot analysis and two-side elisa demonstrated that these snps prevented the cleavage, resulting in secretion of probdnf, but not mature bdnf. these snps did not affect intracellular distribution or mode of secretion of the protein. application of the uncleavable probdnf (probdnfml) elicited apoptosis of cerebellar granule neurons, but inhibited dendritic growth of basal forebrain cholinergic neurons. together, these results reveal structural determinants for the cleavage of probdnf, and demonstrate distinct functions of probdnf for different populations of neurons. we have now analyzed the brain functions of the mice expressing this form of bdnf. sy - - - pleiotropic effects of gdnf in regulation of enteric nervous system development hideki enomoto laboratory for neuronal differentiation and regeneration, riken center for developmental biology, kobe, japan formation of the enteric nervous system (ens) is governed by multiple extracellular signals at a given time during development. inactivation of the gene encoding gdnf, ret or gfr␣ leads to nearly complete absence of enteric neurons during early development. although this finding establishes gdnf as an essential extracellular signal acting at the initial stage in ens development, little is known about whether and how enccs continue to depend on gdnf later in development. we have generated mice in which function of gfr␣ , the high affinity receptor for gdnf, is conditionally inactivated in a time-specific fashion. we will show how gdnf signal influences cell migration, differentiation, proliferation and survival of developing enteric neurons, and discuss the biological significance of the findings in development and regeneration of the nervous system in general. bone marrow stromal cells (mscs) including the primitive pluriopotent mesenchymal stem cells and the multipotent adult progenitor cells, are attractive targets for cell and gene therapy for the range of central nervous system disorders. we present using replicationincompetent hsv- vector that msc population can be efficiently engineered to secrete a series of various cytokines in the large quantities and in long term in vivo to be able to treat the ischemic stroke of the brain potentially. three kinds of gene-transferred mscs, hgf, il ss+fgf- , and vegf were prepared and directly transplanted into the lesioned brain of rat transient middle cerebral artery occlusion model. each growth factor gene-transferred mscs achieved the remarkable amelioration of neurological symptoms and apparent decreasing of infarct volume comparison with native research funds: kakenhi ( ) sy - - - hgf gene therapy for the treatment of spinal cord injury masaya nakamura , akio iwanami , kazuya kitamura , , yoshiaki toyama , hideyuki okano department of ophthalmology surgery, keio university, tokyo, japan; department of physiology, keio university, japan hepatocyte growth factor (hgf) has recently been reported to exhibit neurotrophic activity and to play a role in angiogenesis. in this study, we demonstrated the validity of hgf for treatment of spinal cord injury (sci) in adult rats. first, we analyzed temporal expression of hgf and c-met in the injured spinal cord. hgf-mrna expression was relatively low in the acute phase of sci compared with c-met mrna expression. hypothesizing that hgf is insufficient immediately after sci, we induced sci at th level in adult rat days after injecting herpes vector-mediated gene transfer of hgf (hgf group) or lacz (control) into spinal cord. motor function was evaluated by bbb score. or weeks after injury, histological analyses were performed. there were significant decreases in apoptotic cell number and significant enhancements of angiogenesis and gap +fibers in hgf group compared to the control group. animals of the hgf group showed better functional recovery than the controls. these findings suggest that hgf could have therapeutic effects for sci. hiroshi funakoshi, toshikazu nakamura division of molecular regenerative medicine, osaka university graduate school of medicine, osaka, japan hgf was initially identified and molecularly cloned as a mitogen for primary hepatocytes (nakamura et al., ) . recently, hgf is found to be a novel neurotrophic factor for various types of neurons, such as hippocampal, cerebral cortex, cerebral granular, motor, and sensory neurons. mutant c-met/hgf receptor knock-in mouse reveals that hgf decreases neuronal survival and axonal elongation of several types neurons, including motor, sympathetic and cerebral granular neurons, during development (maina et al., ) . therefore, it is possible to speculate that hgf could play an important role in the retardation or regeneration of neurons in neurodegenerative diseases. here we show the examples of beneficial effects of hgf on model animals of different neural and neurodegenerative diseases, using several delivery methods for hgf including gene therapy approach. we also present the possible application of hgf in modifying the neurogenesis for the disease. references maina et al., . nature neuroscience. nakamura et al., . nature. hiroyuki kato center for clinical medicine and research, international university of health and welfare, nasushiobara, japan we examined stroke patients using fmri at acute/subacute and chronic stages, and visualized areas of brain activation during paretic hand movements. normal hand movement activated the contralateral primary sensorimotor cortex, supplementary motor areas, and ipsilateral cerebellum. at the acute/subacute stages, we observed reductions of these activations and/or addition of activation in ipsilateral cortex or contralateral adjacent cortex during paretic hand movements. at the chronic stages, recovery of activation and/or persistent addition of activation were observed. thus, motor functional recovery was accompanied by restoration of brain activation and/or appearance of additional activation within the motor network of the brain. the findings suggest that cortical motor reorganization as well as recovery from reversible injury plays a role in the restoration of motor function. interestingly, the time period during which reorganization occurred was limited to first - months after stroke, suggesting the presence of a critical period. in the cat, illert et al. ( ) first demonstrated that disynaptic pyramidal excitation in forelimb motoneurons can be mediated via propriospinal neurons located in the c -c segments. in contrast, recently it has been shown that polysynaptic pyramidal epsps are only rarely observed in forelimb motoneurons of macaque monkeys and humans. we reexamined the indirect corticomotoneuronal inputs in the primates, and obtained the following evidence for the pathway. ( ) in the macaque, recordings from forelimb motoneurons showed polysynaptic pyramidal epsps after blockade of glycinergic inhibition by strychnine. moreover, we recently identified c -c propriospinal neurons, which receive pyramidal inputs and project to forelimb motor nuclei. ( ) in human arm motor units, magnetic stimulation of the motor cortex produced multiple peaks at short latency in the post-stimulus time histogram, whose total duration was longer than the corresponding value of a finger muscle. stimulation of the pyramidal tract in the medulla could also produce multiple peaks, though in a lower frequency. functions of the pathway both in physiological and pathological conditions will be discussed. bisphenol-a (bpa) has been extensively evaluated for toxicity in a variety of tests as the most common environmental endocrine disruptors. we previously reported that prenatal and neonatal exposure to bpa potentiated central dopaminergic neurotransmission, resulting in supersensitivity to psychostimulant-induced pharmacological actions. many recent findings have supported the idea that astrocytes, which are a subpopulation of glial cells, play a critical role in neuronal transmission in the central nervous system. we found that in vitro treatment with bpa caused the activation of astrocytes, as detected by a stellate morphology and an increase in levels of gfap. a low concentration of bpa significantly enhanced the ca + responses to dopamine in both neurons and astrocytes. these findings provide evidence that bpa induces dopaminergic changes in neurons and astrocytes. this phenomenon may, at least in part, contribute to the enhancement by bpa of the development of psychological dependence on drugs of abuse. mami yamasaki, yonehiro kanemura the department of neurosurgery, clinical research institute, osaka national hospital, national hospital organization, osaka, japan l cam(l ) is a member of the immunoglobulin superfamily of cell adhesion molecules. x-linked hydrocephalus, masa syndrome and certain forms of x-linked spastic paraplegia are now known to be due to mutations in the gene for l . therefore, these syndromes have been reclassified as l syndrome. we performed a nation-wide l gene analysis and identified li gene mutations in families with l syndrome. all the patients showed developmental delay in various degree. we discussed genotype and phenotype correlations, a striking correlation between the mutation class and the severity of symptoms and molecular basis of severity of developmental delay. the loss of extracellular domain functions like l -mediated cell adhesion and cell migration is considered to be responsible for molecular genesis of ventricular dilatation and disturbance of the functions of cytoplasmic domain would cause symptoms related axon growth in l syndrome. research funds: kakenhi ( ), ( ) sy - - - rett syndrome and developing brain yoshiko nomura segawa neurological clinic for children, japan rett syndrome (rtt) is a neurodevelopmental disorder with mental retardation, autistic feature, and stereotyped hand movements. hypofunction of the brainstem monoaminergic neurons is suggested. pathology showed no degeneration. methyl-cpg-binding protein gene (mecp ) located at xq is the causative gene. types of mutation at different functional domains are correlated to clinical severities. x-inactivation also influences phenotypic variability. mecp was thought as a global transcriptional repressor, but finding of bdnf as a target gene suggest its role in the neuronal activity-dependent gene regulation. genetic heterogeneities have been suggested and the mutation of cyclin dependent kinase-like gene (cdkl ) manifest as atypical rtt. the mutations of mecp are found in other clinical conditions, such as x-linked mental retardation, angelman syndrome, autism, and severe neonatal encephalopathy. thus, the evaluation of rtt gives the clue to study the clinical, pathophysiological, biological and molecular correlation of not only rtt but also other neurodevelopmental disorders. in our previous studies, we have proposed that ros and/or ros-mediated signal play(s) an essential role in -ohda-induced, caspase-dependent apoptosis. in contrast, mpp+-mediated death is not blocked by caspase inhibition and is accompanied by an increase in intracellular free calcium. subsequently, we have demonstrated that mpp+ induces release of cytochrome c but not activation of caspase and proposed that depletion of atp and/or calcium-activated calpain-mediated degradation of procaspase- are responsible for the absence of subsequent activation of caspases. furthermore, we have identified that degradation of several important proteins by activated calpain and proteasome system is linked to mpp+-mediated dopaminergic neuronal death. as such, we have found that one of onconeural proteins seems to play a role as a potential survival factor, degradation of which is involved in mpp+-induced cell death. taken together, we reason that distinct set of proteases activation is involved in experimental models of pd. therefore, novel strategies interfering activation of these proteases may contribute to prevention of dopaminergic neuronal death. satoshi ogawa department of neuroanatimy, kanazawa university of medical school, ishikawa, japan we discuss the role of er-stress in neuronal cell death in snpc by introducing two models. upregulation of pael-receptor in the substantia nigra pars (snpc) of mice induces endoplasmic reticulum (er) stress leading to a decrease in tyrosine hydroxylase and death of dopaminergic neurons. the role of er stress in dopaminergic neuronal vulnerability was highlighted by their enhanced death in mice deficient in the ubiquitin-protein ligase parkin and the er chaperone orp , suggesting parkin dysfunction result in er-stress mediated neuronal cell death. conversely, transgenic rats overexpressing megsin (tg meg), a newly identified serine protease inhibitor (serpin), demonstrated intraneuronal periodic-acid schiff (pas) positive inclusions, which distributed throughout the deeper layers of cerebral cortex, hippocampal ca , and substantia nigrta. enhanced er stress was observed in dopamine neurons in snpc, accompanied with loss of neuronal viability and motor coordination. in both subregions, pas-positive inclusions were also positive with megsin. these data suggest that enhanced er stress causes selective vulnerability in a set of neuronal populations. noradrenaline (na) transmission modulates synaptic excitability and plasticity through distinct receptor subtypes. accumulating evidence has suggested that the central na system modulates consolidation and reconsolidation of long-term emotional memory. here we show that the na system is particularly important for retrieval of reconsolidated emotional memory. the mutation of the gene encoding tyrosine hydroxylase causes a deficit in conditioned taste memory after its reactivation. this memory deficit is restored by pharmacological stimulation of na activity before the test and is also restored by intra-amygdala na stimulation through ␣ or -adrenergic receptors. moreover, intra-amygdala na stimulation in the wild type animals increases their susceptibility to recall reconsolidated memory. our findings indicate that the amygdalar na system, primarily through ␣ and -adrenergic receptors, acts to improve the retrieval of reconsolidated memory trace. shigeru morinobu, shigeto yamamoto, shigeto yamawaki departmnet of psychiatry and neurosciences, hiroshima university, hiroshima, japan as psychophysiological reactivity on exposure to cues resembling an aspect of the trauma is the major symptom in ptsd, it is hypothesized that impaired extinction may be involved in ptsd. rats subjected to single prolonged stress (sps) exhibit the enhanced negative feedback of the hpa axis, exaggerated startle response, and analgesia. thus, sps is a good model of ptsd. we examined whether extinction of fear memory was impaired in sps rats, using the contextual fear conditioning. sps rats exhibited the significant longer freezing during re-exposure to the context - days after the conditioning. furthermore, repeated administration of d-cycloserine markedly inhibited the development of enhanced freezing in sps rats. we measured the levels of nmda receptor subunits (nr , nr a, b, c), glycine transporter , and eaac , by real-time pcr. no significant changes were found in the hippocampus. based on these findings, it is speculated that the increase in other types of glutamate transporters or nmda receptor modification may play a role in impaired extinction in sps rats. ichiro masai masai initiative research unit, riken, wako, japan in human, there are hereditary retinal diseases such as retinitis pigmentosa. to understand these molecular mechanisms, we performed a large-scale mutagenesis using zebrafish as an animal model. here we report two zebrafish mutants, twilight (tli) and eclipse (els), both of which show no normal erg and okr response. in the tli mutant, photoreceptors initially differentiate but degenerate later. electron-microscopic analyses revealed that photoreceptive membranes are severely disorganized in the tli mutants, suggesting that tli is required for the formation of photoreceptive membranes. in the els mutant, photoreceptors seem normal in morphology, suggesting that phototransduction is compromised. we found that the els gene encodes cgmp phosphodiesterase ␣ -subunit (pde c), a component of cone-type pde. since genetic mutations of pde c have not been reported in human, the els mutant provides a good model for studying roles of cone-pde in visual functions. shinichi nakagawa , masatoshi takeichi , , fumi kubo , nakagawa initiative research unit, riken, wako, japan; riken cdb, kobe, japan; department of biostudies, kyoto university, kyoto, japan the marginal region of the optic vesicle contains retinal stem cells that remain undifferentiated and proliferate for a much longer period compared to other progenitor cells in the central retina. we have previously shown that wnt b, a signaling molecule expressed in a region neighboring the stem cell area, functions as a putative stem cell factor that endows undifferentiated retinal cells with the characteristics of the stem cells. interestingly, wnt b inhibits cellular differentiation in the absence of notch activity, a well-known signaling receptor that inhibits neuronal differentiation. wnt b antagonizes proneural gene functions independent of the notch signaling pathway, presumably through unidentified transcriptional repressors. we have isolated several candidate genes that are upregulated upon an activation of the wnt signaling pathway, and some of them are expressed in the stem cell containing region. physiological roles of those genes will be discussed. research funds: kakenhi ( ) sy - - - identification of cell lineage of retinal progenitor cells by cell surface markers sumiko watanabe, hideto koso, shinya satoh department of molecular and developmental biology, university of tokyo, tokyo, japan i would like to discuss about early cellular developmental stages of retina, which we identified by examination of the expression pattern of cell surface markers. we found c-kit and ssea- to be spatiotemporal markers of distinct populations of retinal progenitor cells, and these cells dramatically changed their expression profiles of c-kit and ssea- during development. c-kit-positive cells expressed various immature retina specific genes; and later onset of rhodopsin expression and stronger proliferation activities were observed. c-kit/ssea- double-positive cells showed stronger proliferation activities than ckit single-positive ones. although the number of ssea- -positive cells was augmented by beta-catenin signal, c-kit-positive cells were positively regulated by notch signaling, suggesting that c-kit and ssea- have intrinsically distinct characters. prolonged expression of c-kit by a retrovirus resulted in promotion of proliferation and the appearance of nestin-positive cells in response to scf, suggesting a role for c-kit in retinal development. the retinal photoreceptor cells play a primal and central role in the phototransduction system. they are susceptible to deterioration in human retinal diseases, which lead to severe visual impairment. we have been demonstrated that transcription factors, otx and crx play critical roles in retinal photoreceptor development. while otx is a key molecule for retinal photoreceptor cell fate determination, crx is essential for the terminal differentiation and maturation of photoreceptors. meanwhile, the photoreceptor cell is a highly polarized neuron and also has epithelial characteristics such as adherens junctions. our investigation of a role of apkc, which has been proposed to play a critical role in the establishment of epithelial and neuronal polarity, in differentiating photoreceptors has shown that apkc is required for the formation of outer & inner segments and ribbon synapse. in addition, we also found that photoreceptor polarity formation has important roles in proper retinal lamination. we would like to present our recent analysis of photoreceptor development. research funds: kakenhi ( , , ) raj ladher riken center for developmental biology, kobe, japan the inner ear translates mechanical energy into neural signals that the appropriate centers of the brain can decode into balance or sound information. the inner ear forms from bilateral thickened discs of ectoderm located on either side of the hindbrain, early during development. induction of the inner ear is mediated by localized signals emanating from the paraxial mesoderm. in the chick, the inner ear is induced by localized fgf found in the mesoderm. we find that although fgf can induce the inner ear, it is unable to support differentiation of the inner ear. differentiation, that is the development of the chick inner ear hair cells, is triggered by another family member fgf and is actually inhibited by fgf . for full functionality, the inner ear needs to be integrated into the larger auditory complex, made up of the middle ear, the external ear and the auditory centers in the hindbrain. these components develop from diverse origins but are intimately linked during development. we have been trying to understand how integration occurs and present one model by which this could occur. research funds: center support grant, mext leading projects grant sy - - - how is olfactory receptor-dependent axonal wiring conducted? shou serizawa, kazunari miyamichi, haruki takeuchi, yuya yamagishi, tokiko tsubokawa, hitoshi sakano department of biophysics and biochemistry, crest jst, university of tokyo, tokyo, japan in the olfactory system, termini of primary axon segregate depending on the type of olfactory receptor (or) expressed, forming the olfactory sensory map. to study how the or-dependent axonal wiring is conducted, we analyzed the gene expression profile in the olfactory epithelium of the transgenic mouse in which the majority of olfactory sensory neurons (osns) express a particular or gene. we found that the expression of the immunoglobulin superfamily gene kirrel , encoding homophillic adhesion molecule, is down-regulated in the transgenic mouse compared to the wild type control. the expression level of kirrel in each osn is found to be correlated with the type of or species expressed in the osn. moreover, kirrel promoted fasciculation of osn axon termini in the mosaic gain-of-function experiment. here, we propose that the information of which type of or is expressed in the osn is converted to the expression level of kirrel which determines the adhesiveness of axon termini, contributing to or-dependent segregation of osn axons. in spite of its morphological similarity to the other species in the melanogaster species subgroup, drosophila sechellia has evolved distinctive physiological and behavioral characters adapting to its host plant morinda citrifolia, known as the tahitian noni fruit. the ripe fruit of m. citriforia contains hexianoic acid and octanoic acid, the main components of the odor from the fruit. d. sechellia is attracted to these two fatty acids, while the other species are repelled by them. using inter-species hybrid between d. melanogaster deficiency mutants and d. sechellia, odorant binding protein e was identified as the gene responsible for this behavioral difference among the species. obp e forms a gene cluster with obp d, and these two genes are expressed in the same cells associated with the chemosensory organ. the history of dynamic obp d/e-cluster evolution was revealed by comparison of the genomic sequences of the obp d/e region obtained from species phylogenetically located between d. melanogaster and d. pseudoobscula. sy - - - an approach of dissociating complex traits into fine genetic elements using consomic strains of mouse aki takahashi , , akinori nishi , , toshihiko shiroishi , , tsuyoshi koide , sokendai, kanagawa, japan; national institute of genetics, shizuoka, japan much of the genetic variation that underlies most behavioral traits is complex and is regulated by loci that have quantitative effect on the phenotype. we have previously shown that laboratory strain c bl/ (b ) and wild-derived strain msm/ms have great differences in many behavioral traits. consomic strains were established by natural mating between b and msm, and those strains have the same genetic background as b except for one chromosome from msm. by examining bunch of consomic strains on many behavioral trait, such as spontaneous activity, anxiety-like behavior, pain sensitivity, and social behavior, we were able to map which chromosome have a locus or loci affecting those phenotype. one strain b - msm, which have msm chromosome , showed increased fear responses and riskassessment behavior, and thus it is thought that there is a locus/loci related to the emotionality. to identify the gene in the loci, we have made congenic strains, and successfully narrowed the locus down in the telomeric region. research funds: kakenhi ( . ) sy - - - cloning of the major quantitative trait locus underlying capsaicin resistance in mice capsaicin is the main compound of hot chili peppers, and induces sensations of heat and pain. however, sensitivity to capsaicin differs among individuals. a genetic approach using a mouse model reveals some quantitative trait loci for this sensitivity. capsaicin resistance linked on chromosome (capsq ) is the major locus affecting reduced taste sensation in kjr mice. here we show that intracellular recycling of capsaicin receptor (trpv ) was impaired in kjr neurons in contrast to that of c bl/ j mice. by searching the candidate genes, eh domain-containing four (ehd ), a trp-binding scaffold protein encoding gene was found. ehd binds to c-terminal of trpv . three mutations were found in ehd of kjr, which remarkably diminished the binding, leading to changes in the intracellular distribution of trpv . this study is the first genetic dissection associated with capsaicin/heat resistance in a nature strain and shows a novel binding protein to trpvs. sy - - - comprehensive behavioral analysis of genetically-engineered mice tsuyoshi miyakawa hmro, kyoto university, kyoto, japan one of the major challenges in the life sciences of the post-genome era is to elucidate the functions of the genes at the level of individual animals. final output level of the functions of the genes expressed in the brain is behavior, indicating a need for systematic investigations of the behavioral significance of the genes. in our laboratory, we use a "comprehensive behavioral test battery" for genetically-engineered mice to reveal causal relationships between genes and behaviors. the battery covers broad areas of behaviors, from simple reflexes to highly cognitive functions. so far, we have assessed more than different strains of mutant mice with the battery. surprisingly, more than % of the strains showed at least one significant behavioral phenotype, suggesting that a large part of the genes expressed in the brain may have some functions. representative results for a few strains of mutant mice and the meta-analytic results of the combined data will be presented. also, a potential impact of our approach to "large-scale neuroscience" will be discussed. research funds: kakenhi ( , , , ) , jst bird hiroshi takashima department of neurology and geriatrics, kagoshima university, kagoshima, japan inherited neuropathies are clinically and genetically heterogeneous. at least genes and loci have been associated with charcot-marie-tooth disease (cmt) and related inherited neuropathies. most causes of inherited neuropathy have been discovered by positional cloning technique and in the past two years, the pace of cmt gene discovery has accelerated. these recently discovered cmt causing genes/proteins include those which, although showing unpredictable correlations with the peripheral nervous system, are definitely important for the peripheral nerve. their discovery should pave the way for dramatic progress in the understanding of peripheral nerve biology. on the other hand, genotype-phenotype correlations of these genes are also important in order to understand the pathomechanisms of inherited neuropathy since, based on mutation studies, a large number of genes associated with both the demyelinating and axonal forms of cmt have been identified. to clarify the specific features and molecular mechanisms, we reviewed recent progress in cmt research, especially cmt f caused by prx, and scan caused by tdp . sy - - - gangliosides are important for the maintenance of the nodes of ranvier nobuhiro yuki, keiichiro susuki department of neurology, dokkyo university school of medicine, tochigi, japan gangliosides are abundant in vertebrate nervous system, but the function has yet to be elucidated. some patients with guillain-barre syndrome have autoantibodies to gangliosides such as gm , who show failure of peripheral motor nerve conduction. sensitization of rabbits with gm can produce the disease model. in ventral roots from the paralyzed rabbits, igg and complements deposited on the nodes of ranvier, and sodium channel clusters were disrupted. in ganglioside-deficient mice with disrupted gm /gd synthase gene, motor nerve conduction velocities were reduced in the sciatic nerves. some myelin loops failed to contact the paranodal axolemma, and potassium channels were aberrantly localized at the paranodes. the abnormality became prominent with age. these findings using different models showed that gangliosides are important for the maintenance of the node of ranvier and saltatory conduction along the myelinated nerve fibers. hiroshi ueda division of molecular pharmacology and neuroscience, nagasaki university graduate school of biomedical sciences, nagasaki, japan neuropathic pain caused following nerve injury is one of important issues in neuroscience as well as clinics, since its pain pathway is apparently distinct from that in healthy humans and naive experimental animals. this is clearly evidenced by the finding that the tactile information is converted to noxious one in allodynia characterized in neuropathic pain. in our recent paper (nature medicine, ) , we firstly demonstrated that lysophosphatidic acid (lpa) and its receptor (lpa ) activation initiate the neuropathic pain. in this and following studies we proposed that the demyelination of nociceptive fibers reorganizes the nociceptive spinal inputs through sprouting and electrical synapses (ephapses). i will discuss four issues, lpa-induced demyelination of dorsal root fibers using in vivo and ex vivo culture models, the signal transduction of underlying lpa-mediated downregulation of myelin proteins, evidence for sprouting and ephapses following demyelination and the origin of injury-specific lpa production in terms of demyelination and allodynia. research funds: kakenhi ( ) toshihide yamashita department of neurobiology, graduate school of medicine, chiba university, chiba, japan axons of adult central nervous system are capable of only a limited amount of regrowth after injury, and an unfavorable environment plays major roles in the lack of regeneration. some of the axon growth inhibitory effects are associated with myelin. three myelin-derived proteins have been identified to inhibit neurite outgrowth in vitro. these proteins induce activation of rho in some neruons. inhibition of rho or rho-kinase promotes axon regeneration in vivo. these findings establish rho and rho-kinase as key players in inhibiting regeneration of the central nervous system. i will review recent findings regarding the signaling mechanism of axon growth inhibitors. our experiments suggest that several new candidate proteins may be axon growth inhibitors. these proteins activate not only rho/rhokinase but also other signals to inhibit neurite outgrowth from some neurons in vitro. these findings suggest that agents that block the multiple signals elicited by these axon growth inhibitors may provide efficient tools that produce functional regeneration following injuries to the central nervous system. sha mi biogen idec, usa lingo- is a cns-specific protein expressed in both neurons and oligodendrocytes. in neurons, lingo- mediates the inhibition of axonal growth as a component of the ngr /p /lingo- and ngr /troy/lingo- signaling complex. inhibition of endogenous lingo- by soluble lingo- or dominant negative lingo- can reverse the inhibition of neurite outgrowth by myelin components. soluble lingo- treatment significantly improves functional recovery of spinal cord injured rats as determined by bbb scores. soluble lingo- treatment promotes axonal regeneration and reduced axon dieback in the corticospinal tract, rubrospinal tract, and optic nerve. in oligodendrocytes, lingo- mediates the inhibition of differentiation and myelination. loss of lingo- function using dominant negative lingo- , lingo- rnai, or soluble lingo- or lingo- knockout increased oligodendrocyte differentiation and myelination, whereas over-expression of lingo- led to inhibition of oligodendrocyte differentiation and myelination, in vitro and in vivo. the discovery of a significant role for lingo- in neurons and oligodendrocyte biology are an invaluable step for understanding cns axon regeneration and myelination. alex reyes new york university, usa neurons in the auditory cortex exhibit a wide range of firing patterns. to elucidate the cellular properties and circuitry that give rise to these responses, a d sheet of excitatory and inhibitory neurons were reconstructed in vitro using an iteratively-constructed network (icn) modified to contain both feedback and feedforward circuits. a disc of neurons was stimulated and the resultant firing pattern and spread was documented. simultaneous whole-cell recordings were performed from pyramidal and interneurons in a slice preparation of the mouse auditory cortex. a computer simulated the activities of thalamic neurons and calculated the net synaptic conductance that would be generated by their firing. this waveform was converted to current, injected into the recorded neurons via a dynamic clamp circuit, and the resultant firing documented. using the icn method, we reproduced the firing of a realistic network of excitatory and inhibitory neurons. we replicated many of the responses recorded in vivo. morever, the firing patterns of neurons depend substantially on their distance from the stimulus center and on the identity of the local interneurons. research funds: nih dc - a sy - - - neuronal avalanches reveal neuronal wirings of layer / cell assemblies jun-nosuke teramae, tomoki fukai brain science institute, riken, japan how cortical neurons process information crucially depends on how their local circuits are organized. spontaneous synchronous neuronal activity propagating through neocortical slices displays highly diverse, yet repeatable, activity patterns called 'neuronal avalanches'. they obey power-law distributions of the event sizes and lifetimes, presumably reflecting the structure of local cortical networks. however, the explicit network structure underlying the power-law statistics remains unclear. here, we present a neuronal network model of pyramidal and inhibitory neurons that enables stable propagation of avalanche-like spiking activity. we demonstrate a neuronal wiring rule that governs the formation of mutually overlapping cell assemblies during the development of this network. the resultant network comprises a mixture of feedforward chains and recurrent circuits, in which neuronal avalanches are stable if the former structure is predominant. we investigate how the resultant power laws depend on the details of the cell-assembly formation as well as on the inhibitory feedback. research funds: kakenhi ( ) sy - - - spike-timing dependent and homeostatic plasticity from an optimality viewpoint taro toyoizumi , jean-pascal pfister , kazuyuki aihara , , wulfram gerstner department of complexity science and engineering, university of tokyo, japan; school of computer and communication science & bmi, epfl, japan; aihara complexity modelling project, erato, jst, japan maximization of information transmission by a spiking neuron model predicts changes of synaptic connections that depend on timing of pre-and postsynaptic spikes as well as on the postsynaptic membrane potential. under the assumption of poisson firing statistics, the synaptic update rule exhibits all the features of the bienenstock-cooper-munro rule, in particular regimes of synaptic potentiation and depression separated by a sliding threshold. the learning rule is found by maximizing the mutual information between presynaptic and postsynaptic spike trains under the constraint that the postsynaptic firing rate stays close to some target firing rate. an interpretation of the synaptic update rule in terms of homeostatic synaptic processes and spike-timing dependent plasticity is discussed. research funds: grant-in-aid for jsps fellows j and sci. res. on priority areas from mext of japan, and swiss natl. sci. found. - / sy - - - timing computations in the auditory brain stem john rinzel center for neural science and courant institute of mathematical sciences, new york university, usa sound localization involves precise temporal processing by neurons in the auditory brain stem. the first neurons in the auditory pathway to receive input from both ears can distinguish interaural time differences (itds) in the sub-millisecond range. these cells in the mammalian medial superior olive have specialized biophysical features: two dendrites, each receiving input from only one side; very short membrane time constant; specialized ionic channel properties, including a low-voltage activated k+ current, i-klt. this i-klt contributes to phasic firing (one spike in response to a step of current), precise phase-locking, and extremely timing-sensitive coincidence detection. we will describe the temporal feature-selecting properties of mso cells based on biophysical (hh-like) modeling, in vitro electrophysiology and application of concepts from dynamical systems theory and coding theory. neuronal information is often inferred by counting spike numbers over tens to hundreds of milliseconds. however, if relative spike timings at the scale of milliseconds would carry information, neuronal circuits could have large information capacity. in response to various visual inputs, the retina fires spike bursts separated by hundreds of milliseconds of silent periods. onsets and spike numbers of these bursts are highly reproducible. we asked if spike patterns, i.e., combinations of interspike intervals within single bursts, carry information. using the retinas of salamanders and mice, we found that bursts have various spike patterns, which are unique to the preceding inputs. differences in spike patterns at the scale of milliseconds encode differences in the input as long as - ms. when single bursts contain three or more spikes, the multiple interspike intervals combinatorially encode multiple features of the input. this suggests the spike patters are not determined sorely by slowly modulating instantaneous firing rates. we propose that the retina encodes multiple features in hundreds of milliseconds of input into burst spike patterns at the scale of milliseconds. accumulating evidence reveals that the generalized seizure activity can produce regenerative, in addition to degenerative, structural changes in the hippocampus, including the enhancement of progenitor cell division of dentate granule cells. although the regulatory mechanisms underlying such neurogenesis are unknown, we hypothesized that newly generated granule cells may contribute to the reorganization of the hippocampal formation in the early course of seizures, constituting a possible substrate for epileptogenicity. to address this issue, we examined the division of dentate granule cell progenitors in rats after kainic acid administration, or perforant path kindling. the results indicate that initial limbic seizures trigger the enhancement of dentate progenitor cell division, but progenitor cells may become unreactive to prolonged generalized seizures. the degenerative process is not necessary for triggering the upregulation. it is also suggested that newly generated granule cells may play a role in the network reorganization that occurs during epileptogenesis. the molecular basis underlying such neurogenesis will be discussed. keiichi itoi , ikue otaki , saya suzuki , yasunobu yasoshima , kazuto kobayashi laboratory of informational biology, graduate school of informational science, tohoku university, sendai, japan; institute of biomedical science, fukushima medical university, japan in order to examine functional roles of the noradrenergic (na) neurons in the locus coeruleus (lc) we developed a novel method to ablate specifically the na neurons in the lc, and examined the behavioral and stress responses using the animal model. a transgenic mouse line was used in which human interleukin- receptor ␣ subunit (hil- r␣) was expressed under the control of dopamine -hydroxylase gene promoter. anti-hil- r␣ antibody fused to pseudomonas exotoxin was microinjected into bilateral lc of a transgenic mouse stereotaxically to destroy specifically the na neurons. as behavioral paradigms, elevated plus maze and open field test were used. plasma adrenocorticotropin levels were measured following lipopolysaccharide injection intraperitoneally, as an immune stress. thus, the effect of lc ablation how it affects the behavioral and stress responses will be elucidated. - - - integrated circuits controlling the stress response james p. herman department of psychiatry, university of cincinnati, oh, usa the hypothalamo-pituitary-adrenocortical (hpa) axis is a primary stress-response system in all vertebrates. the end-product of hpa activation, glucocorticoids, serve the general function of redirecting bodily resources to meet a real or perceived challenge. however, prolonged glucocorticoid secretion has deleterious effects on metabolism, immune function and behavior, making control of hpa activity a priority for the organism. this control is exerted in large part by limbic structures in the brain. our studies indicate that the amygdala, hippocampus and prefrontal cortex play major roles hpa axis regulation. the amygdala is primarily stress excitatory, whereas the hippocampus has an inhibitory influence on hpa activity. the role of the prefrontal cortex is considerably more complex; its prelimbic region is primarily stress inhibitory, whereas the infralimbic region may participate in stress activation. all of these regions exert their influence via subcortical relays to hypothalamic paraventricular nucleus (pvn) neurons controlling the hpa response, allowing convergence of information from multiple limbic sources prior to the pvn. sy - - - molecular mechanism for the inverse incidence of parkinson's disease and cancer: synuclein as stimulator of tumour differentiation makoto hashimoto department of chemistry and metabolism, tokyo metropolitan institute for neuroscience, tokyo, japan neurodegenerative disease and cancer are major age-associated disorders. however, the pathogenesis of these diseases may be in sharp contrast, since the former is featured by cell death, whereas, the latter is associated with immortalization. in parkinson's disease (pd) research, smoking, the risk factor for a variety of cancers, had been known to reduce the risk of pd. furthermore, epidemiological studies described that the incidence of cancer was reduced in pd patients. recent study provides evidences of the inverse relationship of pd and some cancers at the molecular level. for example, loss of neuroprotection of dj- is causative for familial pd, while increased expression of this molecule stimulates oncogenesis. in this context, we show that proteasomal inhibition by ␣-synuclein, which has been thought as one major pathogenic mechanism for pd, may induce differentiation of cancer cells. thus, unifying approach on the basis of the opposite pathogenic mechanism to neurodegenerative disease and cancer might uncover unexpected findings in both fields. kiyomitsu oyanagi department of neuropathology, tokyo metropolitan institute for neuroscience, tokyo, japan neurodegenerative diseases and malignant tumors develop symptoms usually at middle or old-age in humans. however, it is well known that critical periods of some malignancies are in fetal period, which are ( ) leukemia in patients exposed with atomic bomb during the iind world war, and ( ) brain tumors in rats with ethylnitrosourea administration. as to neurodegenerative diseases, ( ) many genetic/familial diseases show clinical symptoms at the middle or old age. ( ) epidemiological study revealed that emigrants from guam to the main land of usa show relatively high incidence of amyotrophic lateral sclerosis, and the critical period of exposure to some environmental noxiousness was considered to be childhood/adolescence. ( ) relating to parkinson disease, low magnesium intake over generations induced selective degeneration of the dopaminergic neurons in the substantia nigra in rats [oyanagi et al., in press] . these findings indicate that not only certain malignant tumors but also some sporadic neurodegenerative diseases may be induced originally by the insults in embryonic stage/childhood. to understand the role of synuclein, the major component of pathological inclusions, we examine the expression of synuclein in the embryonic mouse cerebral cortex. we found that a-synuclein and b-synuclein were predominantly detected in the subplate neurons, which are known to enter programmed cell death at a postnatal stage. in another line of inquiry, we are interested in a zinc finger protein containing poz domain, rp , which functions as a sequence specific transcriptional repressor and involved in cortical layer formation. when the rp gene is disrupted, apoptosis is enhanced, and a-synuclein, but not b-synclein, is upregulated in the mutant cortex, suggesting that a-synuclein is involved in the cell death. interestingly, in the mutant cortex the expression of s-phase marker, pcna increased, suggesting that rp mutant mice are useful to analyze the relation among neurodegeration, synuclein and cell cycle. minoru saitoe, junjiro horiuchi, daisuke yamazaki tokyo metropolitan institute for neuroscience, tokyo, japan age-related memory impairment (ami) is a striking feature of ageassociated neuronal dysfunction. to identify gene mutations that affect ami, we screened ∼ drosophila lines and found that heterozygous mutants for the pka catalytic subunit (dc /+) exhibit robust suppression of ami without affecting memory at young ages. this result suggests a causal relationship between pka and ami. of particular interest, igf/pi k/akt signaling, which results in decreased gsk activity, has also been shown to ameliorate ami. both pka and gsk phosphorylate the microtubule-associated protein tau, causing tau aggregation and neurodegeneration. while igf signaling suppresses activity of gsk at young ages, declining igf levels during aging may increase gsk activity in aged animals. in support of this idea, we found suppression of ami in flies fed gsk inhibitors. we hypothesize that similar to the mechanisms occurring in neurodegenerative diseases, tau phosphorylation by pka and gsk causes neuronal dysfunction during normal aging. research funds: kakenhi sy - - - molecular mechanism of cancer progression by gamma-synuclein koji okamoto radiobiology division, national cancer center research institute, tokyo, japan synucleins, a family of small proteins consisting of three known members, are implicated in both neurodegenerative disorder and tumorigenesis. ␣synuclein is involved in the formation of pathologically insoluble deposits characteristic of neurodegenerative diseases such as alzheimer disease and parkinson disease, whereas overexpression of ␥synuclein is associated with progression of breast and ovarian cancer. however, the normal cellular function of synucleins remains largely unknown. in order to get an insight into biological function of synucleins, we focus on cancer progression induced by ␥synuclein. we introduced ␥synuclein into breast cancer cells in order to recapitulate malignant transformation of breast cancer. using such cells, the attempt to elucidate the biochemical function of ␥synuclein is underway. the impact of synuclein over-expression, especially on known tumor suppressor pathways such as the p pathway, will be discussed. research funds: kakenhi ryuichi sakai growth factor division, national cancer center research institute, - - tsukiji, chuo-ku, tokyo - , japan numbers of growth factors and their membrane receptors which possess tyrosine kinase activity are involved in proliferation and differentiation of the neural system. shc family docking molecules conduct signals directly downstream of various growth factor receptors as substrates and binding partners of these tyrosine kinases. in the neural systems, two unique shc family molecules, shcb and shcc, are found to be specifically expressed and analysis of mice lacking these proteins revealed that they have redundant functions during mammalian neural development as mediators of ngf/trka signaling. it was recently found that tyrosine phosphorylation of shcc is frequently detected in majority of neuroblastoma cell lines. we showed that hyperphosphorylated shcc detected in some of neuroblastoma cell lines is associated with constitutively activated anaplastic lymphoma kinase (alk) caused by the gene amplification. identification of binding partners of shcc and expression of mutant shcc in several cancer cell lines revealed novel roles of shcc as a regulator of differentiation and proliferation of neuroblastic tumors. research funds: kakenhi sy - - - identification of estrogen receptor target genes and role of their gene products in cancer and nervous system satoshi inoue , department of geriatric medicine, university of tokyo hospital, tokyo, japan; research center for genomic medicine, saitama medical school, saitama, japan estrogen has crucial roles in the cancer growth and in the neural function. here, we have isolated and characterized novel estrogenresponsive genes to clarify the molecular mechanism of the estrogen action in target cells using genomic binding-site cloning (gbsc) method. one of the first identified genes is the estrogen-responsive ring finger protein (efp). efp expression was observed in uterus, mammary gland and certain regions of the brain where er is also expressed and positively regulated by estrogen. we revealed that efp targets proteolysis of - - sigma, a negative cell cycle regulator that causes g arrest and that efp is an essential oncogenic factor in breast cancer growth. on the other hand, another gene identified by gbsc is nr d, an nmda receptor. this gene was regulated by estrogen in the hypothalamus, together with er, pr and efp. these estrogen responsive genes could mediate roles of estrogen action in specific organs, utilizing differential mechanisms as well as sharing common mechanisms. keiji tanaka , hossein esteky , kiani roozbeh , tadashi sugihara , gang wang riken brain science institute, wako, saitama, japan; institute for studies in theoretical physics and mathematics, tehran, iran; kagoshima university, kagoshima, japan individual cells in the monkey inferotemporal cortex, which is the final unimodal stage along the ventral visual pathway, respond to moderately complex features, but not to objects nor to object categories. then, questions arise where and how view-general objects and object categories are represented. a possibility is the representation by a population of inferotemporal cells. to examine it, we recorded responses of inferotemporal cells to object images in a fixation task. we also conducted psychophysical experiments with monkeys to determine conditions for view-invariant object recognition. the results suggest that a population of inferotemporal cells represent object categories and their relational structure, and that the representation is common to nearby views of objects with up to • rotation. research funds: kakenhi alexander thiele , gene stoner , louise s. delicato , mark roberts university of newcastle upon tyne, uk; the salk institute, japan a variety of different roles of synchronized activity for sensation and perception have been proposed, ranging from object binding, through attentional enhancement, to mechanisms of learning. we have employed different paradigms to investigate the role of neural synchrony in visual perception and attentional selection in the awake macaque monkey. using two different tasks and stimulus conditions, well suited to probe the role of feature binding in the motion domain, we found no support for the idea that neuronal synchrony in macaque area mt underlies the binding of an object's component features. recent reports have focused on the role of synchrony in the mediation of attention. we will discuss the role of synchronized activity in primate v while attentional load was varied, and how it could be mediated by cholinergic mechanisms. research funds: hfsp, wellcome trust, bbsrc sy - - - context-dependent changes in noise correlation in mt william newsome, marlene r. cohen stanford university and howard hughes medical institute, usa changes in the correlated firing of a pair of neurons may provide a metric of changes in functional circuitry within the nervous system during ongoing behavior. we studied dynamic changes in functional circuitry by analyzing the noise correlations of simultaneously recorded mt neurons in two behavioral contexts: one that promotes cooperative interactions between the two neurons and another that promotes competitive interactions. we created cooperative or competitive contexts by changing the axis of motion of the discrimination task from trial to trial. we found that identical visual stimuli indeed give rise to differences in noise correlation in the two behavioral contexts. specifically, noise correlations were higher in the cooperative than in the competitive context. this result suggests that mt neurons receive inputs of central origin whose strength changes with the task structure. the changes in correlation appear to reflect differences in how mt neurons are pooled for the purpose of perceptual discrimination, and may derive from higher-level cognitive processes such as feature-based attention. research funds: howard hughes medical institute sy - - - effects of task demands on color processing in area te of the monkey hidehiko komatsu , , kowa koida national institute for physiological sciences, okazaki, japan; sokendai, okazaki, japan color vision has two different functions, namely, categorization and discrimination, and the same color stimulus can be processed according to these two functions depending on task demands. lesion studies suggested that inferior temporal (it) cortex of the monkey plays a key role in color vision, and we have recently found that color selective neurons are concentrated in a small region in area te of it cortex. to study how the color selective responses in this region are affected by the task demands, we trained monkeys a color categorization task and a color discrimination task using the same set of color stimuli, and analyzed how the responses are affected. we found response magnitudes of many neurons changed between two tasks while the color tuning is well reserved. in several extreme cases, large gain change almost completely eliminated the responses in one task. these results suggest that color signals are gated by the top-down signal representing task demands in area te and color channels specific to different tasks are formed at this level of the visual cortex. yoichi sugita neuroscience research institute, aist, tsukuba, japan early visual experience is indispensable to shape the maturation of cortical circuits during development . monocular deprivation in infancy, for instance, leads to an irreversible reduction of visually driven activity in the visual cortex through the deprived eye and a loss of binocular depth perception - . here, i show exposure only to monochromatic illumination in infancy results in the disruption of color perception. infant monkeys were reared for nearly a year in a room where the illumination came from only monochromatic lights. after extensive training, they were able to perform color matching. but, their judgment of color similarity was quite different from that of normal animals. furthermore, they had deficits in color constancy; they could not compensate for the changes in wavelength composition. these results indicate that early visual experience is also indispensable for color perception. research funds: crest sy - - - dendritic growth, spinogenesis and synaptogenesis in response to neurosteroids in the developing purkinje cell kazuyoshi tsutsui , hirotaka sakamoto , katsunori sasahara , hanako shikimi , kazuyoshi ukena , mitsuhiro kawata laboratory of brain science, faculty of integrated arts and sciences, hiroshima university, higashi-hiroshima, japan; department of anatomy and neurobiology, kyoto prefectural university of medicine, kyoto, japan new findings over the past decade have established that the brain synthesizes steroids de novo from cholesterol. such steroids synthesized de novo in the brain are called neurosteroids. recently we have identified the purkinje cell as a major site for neurosteroid formation in the brain. this is the first demonstration of de novo neuronal neurosteroidogenesis in the brain. in mammals, the purkinje cell actively synthesizes progesterone and estradiol de novo from cholesterol during neonatal life, when cerebellar cortical formation occurs. subsequently, our recent studies on mammals using the purkinje cell have demonstrated organizing actions of neurosteroids. both progesterone and estradiol promote dendritic growth, spinogenesis and synaptogenesis via each cognate nuclear receptor in purkinje cells. research funds: kakenhi ( and to kt) sy - - - roles of estrogen receptors in the regulation of socio-sexual and emotional behaviors-studies with knockout mice and rnai sonoko ogawa kansei, behavioral and brain sciences, university of tsukuba, tsukuba, japan the gonadal steroid estrogen plays a major role in the regulation not only of female reproductive behavior but also an array of social and emotional behaviors in both sexes, by acting through intracellular estrogen receptors (ers), ligand dependent transcription factors. a series of studies using single and double knockout mice for er-␣ and/or er- genes have revealed that activation of er-␣ and er- differentially regulate a number of behaviors as well as neuroendocrine functions. our studies have suggested a unique role of activation of er- in the hypothalamic and limbic brain areas, dorsal raphe nuclei and locus coeruleus in the regulation of socio-sexual and emotional behaviors. in this talk, our findings from behavioral studies using er-␣ and er- knockout mice along with possible brain mechanisms underlying the behavioral effects will be first overviewed. our most recent studies on brain site-specific manipulation of er gene expression with the use of small interference rna combined with adeno-associated virus will then be presented. research funds: kakenhi ( , ) sy - - - sex steroid receptor function in sexual behavior shigeaki kato , , takashi sato , takahiro matsumoto , imcb, university of tokyo, tokyo, japan; erato, jst, saitama, japan androgen actions are believed to mediate nuclear androgen receptor (ar)-mediated gene regulations. ar is a member of nuclear receptor, and acts as a hormone-induced transcription factor to control of target genes through chromatin remodeling/histone modification. we generated the floxed ar mice to avoid testicular feminization mutant (tfm) abnormalities with infertility, and then crossed with female ar(−/+) heterozygoutes expressing cre to generate ar(−/−) female mice. the ar(−/y) ko males grew healthy with typical features of tfm abnormalities, and genital organs were atrophic with a marked decrease in the serum testosterone level, but with normal estrogen level (kawano et al., ) . no sexual behaviors and reduced aggressive behaviors were seen in ar(−/y) male mice (sato et al., ) . female ar ko mice were normal in sexual behavior but exhibited premature ovarian phenotype (shiina et al., ) . together with these results, the ar function will be discussed in terms of ar function as a transcription factor. references kawano, h., et al., . pnas usa , . sato, t., et al., . pnas, usa , . shiina, h., et al., research funds: probrain sy - - - annexin : a mediator of cell-cell communication in the neuroendocrine system julia buckingham , helen christian , john morris imperial college london, uk; department of human anatomy and genetics, university of oxford, uk annexin (anxa ) plays an important part in mediating the regulatory effects of glucocorticoids (gcs) on neuroendocrine function, particularly within the hpa axis. it is expressed by folliculostellate (fs) cells in the pituitary gland and by ependymal cells and activated glia in the hypothalamus but not by classical secretory cells. gcs act on cells expressing anxa to cause the translocation of the protein to the plasma membrane at points with particular accumulation at points where the cells make contact with endocrine cells. this process is effected via a non-genomic mechanism and is dependent upon phosphorylation, lipidation and a transport protein, possibly abca . the released protein then acts, via cell surface receptors on the endocrine cells to suppress stimulus-evoked peptide release. the nature of the anxa receptor is unclear but, increasing data suggest that members of the formal peptide receptor family may be important in this regard. katsuhiko nishimori , yuki takayanagi , masahide yoshida , yoshiyuki kasahara , masaki kawamata graduate school of agricultural science, tohoku university, sendai, japan; department of physiology, jichi medical university, minamikawachi-machi, japan we examined the behaviors of mice lacking oxtr gene and discovered that oxtr null females displayed impaired nurturing behavior, and their pups showed defect in ultrasonic vocalization, instead, increased locomotor activity by social isolation test. those are implying impaired mother-infant relationship. oxtr null males also showed more aggressive and having social amnesia as well as the phenotype of oxt null mice. in addition, oxtr null mice failed to maintain their body temperature after acute cold exposure. their rectal temperature rapidly dropped in comparison of that of wildtype animals at • c ambient temperature. our studies demonstrate that oxtr plays a critical role in regulating several aspects of social behavior and the other physiological function, and may have important implications for developmental psychiatric disorders, such as autism. research funds: grant-in-aid for scientific research (b) ( ) sy - - - cortical mechanisms mediating visuomotor control of primate grasp roger n. lemon ucl institute of neurology, uk primates demonstrate an exquisite ability to precisely shape their hand when grasping an object. a network of parietal and frontal motor areas is thought to play a key role in this behaviour. our work shows that: hand shape can be unambiguously determined from emg activity of hand and digit muscles. information about grasp is represented by neuronal populations in the ventral premotor cortex (area f ); f activity shows graspspecific discharge soon after an object becomes visible, well in advance of activity in primary motor cortex (m ). local field potential activity in f and m is also tuned to grasp, and there is strong beta coherence between f and m , indicating reciprocal transmission of information. this is also seen in synaptic responses of m neurones to stimulation of f (and vice-versa). single pulse stimulation in f strongly modulates corticospinal outputs from m through corticocortical pathways between these two areas. paired-pulse tms can probe the excitability of these pathways in humans. facilitation of meps is both object and muscle specific and indicates that activity in these pathways is selectively enhanced during object grasp. research funds: wellcome trust, bbsrc sy - - - where tactile signals are ordered in time shigeru kitazawa , department of neurophysiology, juntendo university graduate school of medicine, tokyo, japan; crest, japan science and technology agency, saitama, japan how does the brain order successive events? it is generally accepted that the brain can resolve the order of two stimuli that are separated in time by ms. this applies to temporal order judgment of two tactile stimuli, delivered one to each hand, as long as the arms are uncrossed. however, crossing the arms caused misreporting (that is, inverting) of the temporal order. the reversal was not due to simple confusion of hands, because correct judgment was recovered at longer intervals (e.g., . s). when the stimuli were delivered to the tips of sticks held in each hand, the judgment was altered by crossing the sticks without changing the spatial locations of the hands. we recently found that temporal order judgments of tactile stimuli are strongly affected by visual distractors and/or eye movements. the results suggest that tactile stimuli are ordered in time only after they are referred to relevant locations in space, where multiple modalities of sensory signals converge. results from functional imaging support this idea. sy - - - decision making and underlying neural mechanisms-auditory-visual ambiguity solving and preference shinsuke shimojo , biology/cns, california institute of technology, pasadena, ca, usa; jst.erato shimojo implicit brain function project, atsugi, japan we explore mechanisms underlying crossmodal ambiguity solving (passive decision), and preference (active decision). we've employed the illusory flash effect, where a single flash appears to be doubled when accompanied by two sounds. -channel meg was employed, while the observer reported number of flashes. partial directed coherence was applied to see if there was a causal influence by the auditory on the visual cortices. the results indicate a strong causal influence in a-v direction in alpha ( ) ( ) ( ) ( ) ( ) and ranges only in the illusion-reported trials, while stimulus parameters were identical. no such difference was found in v-a direction. for preference, the observer's gaze shifted towards the to-be-chosen stimulus (face) before conscious decision. our fmri study shows activity specific to preference task in the ventral amygdala and the ventromedial prefrontal. while such results enable the same causality analysis, it also raises a question as to what determines active/passive nature of decision. research funds: jst.erato, hfsp sy - - - why look there? insights from spatial neglect and the medial frontal cortex masud husain ucl institute of neurology, uk why do we look where we do? studies in humans show that when we look at a scene, our initial fixation patterns can be predicted to a high degree of accuracy. our eyes go to the most salient locations where local feature contrast is greatest. these findings have led to the concept of a salience map which directs attention and gaze bottomup. in humans, damage to the right posterior parietal cortex often leads to dramatic neglect of the left side of space. recent research has begun to unravel the components of this syndrome, demonstrating several underlying mechanisms. these include a disturbance of the salience representation, a failure to keep track of spatial locations across saccades and difficulty in sustaining attention over time. gaze is directed not only bottom-up by but also top-down by voluntary mechanisms. our recent investigations of human medial frontal regions reveal important roles for the supplementary eye field and the pre-supplementary motor areas in the control of competing eye movement plans and deciding where to look. parietal and medial frontal gaze regions appear to play different, complementary roles in controlling why we look where we do. research funds: wellcome trust ( ) sy - - - recognizing self actions through externalized eyes atsushi iriki , symbolic cognitive development, riken brain science institute, saitama, japan; cognitive neurobiology, tokyo medical and dental university, tokyo, japan we can recognize ourselves and our own actions through the mirror or video images. thus, human can use such apparatus as externalized eyes (sensory tools), while non-human animals can normally use tools as extension of their effectors (motor tools) at most. human fmri studies revealed that the right temporo-parietal junction region and the mesial superior frontal gyrus are involved in perceiving and manipulating the representation of the self actions under different third person perspectives. japanese monkeys could be trained to use a hand-held video camera as a manipulable extension of their eyes only when their own vision was gradually transferred to the distant cues via motor-tools to extend their body images. the emergence of novel cortico-cortical projections between temporo-parietal junction and the intra-parietal cortex was described in monkeys that were trained to use motor tools, therefore, integrate the tool in their own body image. thus, presence of a self-objectification mechanism is suggested for acquisition of sensory tools as externalized eyes to recognize self actions. yoshiyuki kubota division of cerebral circuitry, nips, okazaki, japan gabaergic nonpyramidal cells in the neocortex are composed of several different subtypes. we found that most of gabaergic cell types, including fs basket and somatostatin martinotti cells, that innervate dendritic spines in addition to the somata and dendritic shafts. most postsynaptic spines also received an asymmetrical input, called double innervated (di) spines. to better characterize the other asymmetrical input on the di spines, excitatory presynaptic terminals were stained by immunohistochemistry for two types of vesicular glutamate transporters (vgluts): vglut , existing mostly in cortical pyramidal cells, and vglut , found in thalamocortical fibers. gabaergic inputs were rarely observed in spines innervated by vglut -expressing terminals (n = ), but were found in- % of spines innervated also by vglut -expressing terminals (n = ). symmetrical synapses on di spines were positive for gaba, as shown by postembedding immunohistochemistry. these results indicated that some thalamocortical inputs are likely selectively inhibited at the spine level by gabaergic synapses from cortical nonpyramidal cells. research funds: kakenhi sy - - - gabaergic recruitment of excitation by cortical axo-axonic cells gabor tamas, csaba varga, gabor molnar, szabolcs olah, pal barzo, janos szabadics university of szeged, hungary the axon has the lowest threshold for action potential generation and axons in the cerebral cortex receive input only at the axon initial segment exclusively from axo-axonic cells (aacs), which use the dominant inhibitory neurotransmitter, gamma-aminobutyric acid (gaba). thus, aacs are considered as strategically placed inhibitory neurons controlling cortical information flow. we applied multiple patch clamp recordings in slices of rat and human neocortex and found that single spikes in aacs can trigger action potentials in pyramidal cells and initiate stereotyped series of multiple synaptic events in the cortical network. the excitatory action of aacs is based on a depolarized reversal potential for axonal relative to perisomatic gabaergic inputs as determined in paired perforated patch recordings. powerful axo-axonic depolarization from the resting membrane potential is supported by a ∼ -fold decrease in the potassium-chloride co-transporter (kcc ) expression from somatic to axon initial segment membranes detected by quantitative immunogold labeling. in my talk i will describe the integrative and plasticity properties of thin basal dendrites of cortical pyramidal neurons. these dendrites receive the majority of the cells' synaptic inputs, so determining their integrative and plasticity properties is of prime importance. previous studies have most often reported global linear or sublinear summation in these dendrites. using confocal imaging and dual-site focal synaptic stimulation of identified thin dendrites in rat neocortical pyramidal neurons we show that thin dendrites provide a layer of independent computational "subunits" that sigmoidally modulate their inputs prior to global summation. next i will describe the plasticity rules used by these fine basal dendrites putting a special emphasis on the role of nmda-spike in local synaptic plasticity processes. yumiko yoshimura department of visual neuroscience, research institute environmental medicine, nagoya university, nagoya, japan neocortical circuits contain fine-scale networks of excitatory neurons interconnected precisely. we previously showed that layer / pyramidal cells in visual cortex share common excitatory inputs from layer and from within layer / , when they are directly connected. here, we tested whether inhibitory cells are incorporated into the fine-scale specificity of excitatory connections. we recorded photostimulation-evoked synaptic currents from pairs of layer / cells, consisting of one inhibitory cell and one pyramidal cell in rat visual cortex slices, and measured the extent of common inputs to the pairs based on cross-correlation analysis. fast spiking inhibitory cells shared extensive common excitatory inputs with neighboring pyramids only when the pairs of cells were reciprocally connected. adapting inhibitory cells shared little or no common input with neighboring pyramids, regardless of their direct connectivity. therefore, fine-scale specificity depends on the type of inhibitory cell and on the direct connectivity between neighboring pyramidal-inhibitory cell pairs. research funds: kakenhi ( , ) sy - - - local circuitry of cortical inhibitory neurons edward callaway, takuma mori, xiangmin xu the salk institute, usa we used laser scanning photostimulation to map local input to inhibitory neurons in layer of rat visual cortex and layer / of mouse barrel cortex. mouse studies used transgenic animals with gfp expressed in subsets of inhibitory neurons. in layer , axondescending cells receive excitatory input predominantly from layer / while neurogliaform cells receive stronger input from deeper layers. layer / neurons also receive inputs that vary systematically by cell type. two subtypes of martinotti cells, distinguished by calretinin (cr) expression, also differ in morphology and intrinsic physiology. cr+ martinotti cells receive excitatory input predominantly from layer / , while the cr− martinotti cells also receive strong excitation from layer . irregular-spiking basket cells also receive strong excitatory input from layers / and , but they often have a gap at the top of layer , with little or no input. fast-spiking basket cells and pyramidal cells in mouse barrel cortex receive input indistinguishable from cells in rat visual cortex, with strong input from layers / and , and only weak input from deeper layers. research funds: nih sy - - - physiological genomics of cortical microcircuits sacha b. nelson brandeis university, czech republic cortical microcircuits are comprised of highly diverse neuronal cell types that differ in their morphology, synaptic connectivity and intrinsic electrophysiology. presumably, these phenotypic differences are orchestrated and maintained by unique transcriptional programs. in order to begin to reveal those programs we have recently developed methods for measuring genome-wide gene expression from small numbers ( - ) of fluorescently labeled, hand-sorted neurons. subsets of pyramidal neurons and gabaergic interneurons were labeled genetically with gfp or anatomically with fluorescent microspheres. labeled neurons were characterized electrophysiologically and sorted for expression analysis. the resulting expression profiles revealed highly diverse expression of molecules involved in cell-cell signaling and cell-cell adhesion, as well as transcription factors. based on this diversity of expression we constructed a taxonomic tree using an unsupervised clustering algorithm, that correctly reflects known relationships between cortical cell types. research funds: r ey , mcknight neuroscience of brain disorders award sy - - - axon guidance mediated by localized ca + signals in the growth cone hiroyuki kamiguchi laboratory for neuronal growth mechanisms, riken brain science institute, wako, japan axonal growth cones migrate along the correct paths, not only directed by guidance cues but also contacted by local environment via cell adhesion molecules (cams). many guidance cues attract or repel the growth cone via asymmetric ca + signals. its turning direction depends on the occurrence of ca + -induced ca + release (cicr) through the ryanodine receptor type (ryr ). the activity of ryr is controlled by cams via camp and pka. in this way, axon-guiding and cam-derived signals are integrated by ryr , that serves as a key regulator of axon guidance. attractive ca + signals facilitate intracellular membrane transport to the leading front and subsequent vamp -mediated exocytosis on the side with elevated ca + . in contrast, repulsive ca + signals do not trigger such membrane dynamics. growth cone attraction, but not repulsion, is prevented by inhibition of vamp -mediated exocytosis. the results indicate that growth cone attraction is driven by asymmetric membrane dynamics and that growth cone repulsion is driven by different mechanisms, not simply by changing the left/right polarity of the same molecular machinery. sy - - - molecular mechanisms for signaling through plexin-a hitoshi kikutani, atsushi kumanogoh, toshihiko toyofuku research institute for microbial diseases, osaka university, suita, japan sema a acts as a guidance cue for axons through a receptor complex comprising neuropilin- as the ligand-binding subunit and plexin-a as the signal-transducing subunit. the ferm domain-containing gef, farp , associates directly with plexin-a . sema a induces the dissociation of farp from plexin-a , resulting in activation of farp 's rac gef activity, rnd recruitment to plexin-a and down regulation of r-ras. simultaneously, the ferm domain of farp sequesters pipki␥ from talin, thereby inhibiting its kinase activity. these activities are necessary for sema a-mediated repulsion of outgrowing axons. plexin-a also functions as a ligand binding receptor of a transmembrane semaphorin, sema d and contributes to cardiac morphogenesis. sema d exerts a migration-inhibitory activity on cells from the ventricle and a migration-promoting activity on those from the conotruncal segment. plexin-a forms a receptor complex with off-track in the ventricle or with vegf receptor type in the conotruncal segment, which are responsible for the effects of sema d on the respective regions. research funds: crest sy - - - axonal transport elicited by axon guidance molecules yoshio goshima department of molecular pharmacology & neurobiology, yokohama city university graduate school of medicine, yokohama, japan for the wiring of individual neurons together into an orderly network, control by axon guidance molecules of navigation to their targets is a critical event, and molecular components destined for specific subcellular domains of neuron must be targeted correctly. we previously reported that semaphorins a (sema a), a repulsive axon guidance cue, increases the rate of bi-directional axonal transport in dorsal root ganglia (drg) . to address if the individual molecules rides on the sema a-facilitated cargo, we used time-lapse imaging of several egfp-fusion proteins expressed in drg. sema a stimulated the transport of neuropilin- ::egfp, plexin-a ::egfp, and fyn::egfp, which are the components of sema a signaling, but not neuropilin- ::egfp. interestingly, sema a accelerated the anterograde transport of trka::egfp. these facts suggest that sema a selectively facilitates the transport of its own signaling components and that sema a may modulate ngf-sensitivity of neurites by accelerating the transport of trka. kozo kaibuchi department of cell pharmacology, nagoya university graduate school of medicine, japan neurons are one of the most highly polarized cells, comprised of two structurally and functionally distinct parts, axon and dendrites. however, it remains largely unknown how neuronal polarity is established. we previously showed that collapsin response mediator protein- (crmp- ) is enriched in growing axon, and play a crucial role in axon specification. crmp- interacts with tubulin dimers to promote microtubule-assembly, and binds to sra- , an effector of rac to regulate wave-dependent reorganization of actin filaments. crmp- links kinesin- to tubulin dimmers and sra- , and participates in the kinesin- -dependent transport of tubulin dimmers and the sra- /wave complex to growing axons. we also found that the par- /par- complex and the ras/pi -kinase/akt/gsk- b pathway are involved in neuronal polarization. akt appears to phosphorylate gsk- b and inactivates its kinase activity downstream of ras/pi -kinase, thereby increasing non-phosphorylated active crmp- which promotes axon growth. this time, i summarize and discuss functions of these polarity molecules in regulation of neuronal polarity. research funds: grant-in-aid for creative scientific research sy - - - regulation of actin dynamics during neurite initiation and axon guidance frank gertler, adam kwiatkowski, doug rubinson, erik dent, leslie mebane mit, usa nervous system development requires extensive cell migration and elaboration of neurites that become axons and dendrites. axons are guided to their targets by motile growth cones. both whole cell and growth cone migration involve dynamic remodeling of the actin and microtubule cytoskeleton in response to environmental cues. the ena/vasp protein family regulates cell migration and axon guidance. ena/vasp proteins modulate actin remodeling and promote the formation of long, sparsely branched actin networks, such as those found in filopodia. results of recent work on phenotypes arising in mice lacking all three ena/vasp proteins (mena, vasp, evl) will be presented. such animals exhibit a "cobblestone cortex" in which groups of neurons migrate beyond the pial membrane. the mutants also contain little if any cortical axonal fiber tracts. analysis of primary cells from the mutants indicates ena/vasp function is required for neurite initiation. mutant neurons express differentiation markers but form few, if any filopodia and exhibit alterations in actin-microtubule interactions. kimitaka anami department of psychiatry, national center hospital for mental, nervous and muscular disorders, ncnp, tokyo, japan recent years, studies using eeg and fmri in simultaneous manner has become flourished. this is because the feasibility that any eeg events is, in principle, able to be mapped on the mri tomographic view has attracted many researchers. applications of this methodology are to basic eeg researches including event-related potentials and background activities, and as clinical aspect, localization of epileptic foci. and applications of this methodology is not matured yet but still developing. in this presentation, we will introduce our study using this method to epilepsy and to other eeg events. masaya misaki , , takashi abe , , , shigeyuki kan , , satoru miyauchi , national institute of information and communications technology, kobe, japan; japan society for the promotion of science, tokyo, japan; graduate school of biosphere sciences, hiroshima university, higashi-hiroshim, japan; kyushu institute of technology, kitakyushu, japan; crest, japan science and technology agency, tokyo, japan recording fmri and an eeg simultaneously is effective for studying spontaneous brain activities. we used this method to examine the relationship between an eeg rhythm and a bold signal. some studies have hypothesized that the hemodynamic response for a change in power of certain eeg frequency bands, such as alpha waves, is canonical in shape. however, the appropriate response shape for a change in the rhythmic eeg has not yet been determined. we measured the eeg and fmri simultaneously while subjects were in a resting or sleeping state. we applied nonlinear regression analysis using an artificial neural network to detect correlations between the changes in rhythmic eeg waves and fmri signals without a priori hypothesis of the response shape. research funds: crest of jst and grant-in-aid for jsps fellows norihiro sadato, hiroshi toyoda department of cerebral research, national institute for physiological sciences, okazaki, japan previous studies have demonstrated a nonlinear relationship between blood oxygenation level dependent (bold) response and stimulus parameters. however the origin of this nonlinearity still remains unclear. to investigate the origin for the nonlinearity of bold response, we underwent simultaneous measurement of fmri and near infrared spectroscopy (nirs) . temporal dynamics of the responses in oxy-, deoxy-and total hemoglobin concentrations as well as bold signal were simultaneously measured during a visual stimulation with various durations. to quantify the degree of the nonlinearity of responses, we introduced a model using an impulse response function modified with additional nonlinearity scaling. this model was applied to the nirs measures as well as bold responses. the nonlinearity of the response in oxygen extraction fraction (oef) was also estimated from nirs measures. the non-linearity of bold was almost identical to oef across the wide range of nonlinearity of the neuronal responses. and hence we conclude that the non-linearity of bold responses to the neural activity is mainly due to the nonlinear response of oef. the bold-fmri signal is ambiguous regarding the underlying neurophysiology. in our work we attempt a) to better understand the neurophysiological basis of fmri and b) to improve on the information obtained by functional brain imaging by adding additional information, e.g. obtained by electrophysiological measurements. in one series of experiments, we combined transcranial magnetic stimulation with near-infrared imaging in order to clarify how changes in deoxy-hb concentration (the inverse of bold) is related to neuronal inhibition. in another series of experiments, we combine eeg with fmri in order to identify bold correlates of neuronal background rhythms such as alpha rhythm, mu rhythm, etc. in a third series of experiments, we combine fmri with the measurement of high-frequency oscillations in eeg. the latter is an expression of evoked spike burst in the somatosensory cortex, i.e. this kind of measurements adds the information about action potentials to fmri haruhiko akiyama tokyo institute of psychiatry, japan activation of microglia is a part of host defense mechanisms in the brain. microglia remove invading microorganisms as well as cell debris that contains hazardous substances such as lysosomal proteases. brain is particularly vulnerable to the immune and inflammatory attacks and, therefore, has multiple mechanisms that regulate the immune and inflammatory responses more strictly than other organs. nevertheless, many neurodegenerative lesions are associated with activated microglia and low-grade, but sustained, inflammation. neuroinflammation is a term that refers to such conditions. in alzheimer's disease (ad), microglia play a central role for phagocytic removal of amyloid beta-protein (abeta) from the brain. the process is enhanced by complement activation. however, these cellular and humoral responses to abeta may be toxic to neurons in ad. neuroinflammation could be a double-edged sword in the brain. in patients with neurodegenerative diseases, complication of systemic inflammatory diseases, depletion of some neurotransmitters such as catecholamines, and the presence of brain lesions may adjunctly upregulate neuroinflammation, which further accelerates neuronal degeneration. makoto sawada department of brain life science, riem, nagoya university, japan microglia, macrophage-like cells in the cns, are multi-functional cells; they play an important role in removal of dead cells or their remnants by phagocytosis in the cns degeneration as well as are one of important cells in the cns cytokine network. they are thought to be originated from mesoderm, and to be similar cells to other tissue-resident macrophages. activated microglia have been shown to remove potentially deleterious debris and promote tissue repair by secreting neurotrophic factors at the neuronal injury sites, however, they can release potentially cytotoxic substances in vitro, and at least so-called fully activated form of microglia which are observed at the injury site in aids dementia is completely neurotoxic. these suggest that some factor(s) may contribute to change microglial phenotype from protective to toxic, but the detail is not clear. recently we generated hiv-derived nef protein tranduced microglia. they are found to increase both the potential to produce o -and mpo-like peroxidase activity resulting in the neurotoxicity. therefore, the target protein(s) of nef might to be involved in the control of microglial neurotoxicity. there is abundant evidence that extracellular atp have an important role in pain signaling. the focus of attention now is on the possibility that atp receptor of microglia might be involved in neuropathic pain. neuropathic pain is often a consequence of nerve injury through surgery, bone compression, diabetes or infection. this type of pain state is generally resistant to currently available treatments. we recently reported that the expression of p x receptors in the spinal cord is enhanced in spinal microglia after peripheral nerve injury, and blocking pharmacologically and suppressing molecularly p x receptors produce a reduction of the neuropathic pain behaviour ( . nature , - ) . more recently, we have reported that brain-derived neurotrophic factor (bdnf) released from microglia by the stimulation of p x causes the depolarizing shift in reversal potential of anion in li neurons of rats with nerve injury ( ( . nature , ( - . understanding the key roles of these atp receptors may lead to new strategies for the management of neuropathic pain. research funds: kakenhi ( ) sy - - - pet imaging of microglia using peripheral benzodiazepine ligands richard b. banati university of sydney, australia brain disease often results in significant changes in the functional state of microglia, the brain's resident tissue macrophages. the response is thought to be an important step in the pathophysiology of traumatic, inflammatory, neoplastic and degenerative brain disease. part of the structural and functional plasticity of microglia is the de novo expression of the kda transposor protein or "peripheral benzodiazepine binding site" (pbbs). the pbbs is bound by the isoquinoline pk , which labeled with carbon- can be used for positron emission tomography (pet). using c-(r)-pk pet in inflammatory and neurodegenerative brain disease as well as receptor autoradiography, we have shown that distributed regional pbbs up-regulation correlates with clinical deficit and mirrors the histologically described activation of microglia in the penumbra of focal lesions, but importantly also in the distant, anterograde and retrograde projection areas of the lesioned neural pathway. sy - - - application of simulation of light propagation in tissue to nirs imaging of brain function eiji okada department of electronics and electrical engineering, keio university, japan in nirs imaging, the functional image is obtained from the variation in intensity of detected light caused by concentration change in haemoglobin in cortical tissue. the serious problem of nirs imaging is ambiguity in light propagation in the head caused by the scattering of tissue. this poses results in poor spatial resolution and contrast in the image. the development of simulation model to calculate light propagation in the head to deduce the path length in the brain and the spatial sensitivity profile is very important to improve the nirs imaging. in this study, simulation of light propagation in the head model for nirs imaging is briefly reviewed. the heterogeneity of the tissues in the head, especially low-scattering cerebrospinal fluid (csf), has a strong effect on the light propagation in the brain. the sensitivity to concentration change in haemoglobin in the cortical tissue is improved by the effect of the csf. the simulation of nirs imaging indicates that the intensity and size of activated region in the functional image depend on the relative position of activated region to fibre pairs. yoko hoshi integrated neuroscience research team, tokyo institute of psychiatry, tokyo, japan quantification of near-infrared spectroscopy (nirs) data has been a central issue in the nirs field. over the past years, many approaches to quantification have been tried, and in the case that hb concentration changes are global within the tissue, the quantitative accuracy of time-resolved spectroscopy (trs) and phase-resolved spectroscopy (prs) has been established. when the changes are localized, however, as with functional brain activation, the difficulty of quantification has not yet been fully overcome because elimination of the effects of hemodynamic changes in the extracerebral tissue is also challenging. the temporal profile of detected light intensity measured by trs carries information about depth-dependent attenuation, because light that penetrated into deeper positions in the head is detected later. thus, several time-domain methods to determine absorption changes with depth resolution have been proposed. diffuse optical tomography (dot) is also a potential technique for quantitative detection of focal changes in cerebral hemodynamics. in this symposium, i will outline these approaches. sy - - - brain functional imaging in cerebral ischemic disorders: comparison of nirs and fmri kaoru sakatani department of neurological surgery, nihon university school of medicine, tokyo, japan we compared the evoked cerebral blood oxygenation (cbo) responses measured by nirs and activation maps of bold-fmri in stroke patients with mild and severe (misery perfusion) cerebral ischemia (ci). in the age-matched controls, deoxyhemoglobin concentrations decreased with increases in oxyhemoglobin and total hemoglobin in the primary sensorimotor cortex (psmc) during contralateral motor tasks. the psmc on the non-lesion side exhibits normal cbo response pattern. on the lesion side, the mild ci did not affect the cbo response pattern, but the severe ci caused an increase of deoxyhemoglobin during the task associated with increases of oxyhemoglobin and total hemoglobin. the mild ci caused only slight reduction of the activation volumes of bold imaging on the lesion side; however, the severe ci, caused markedly reduction of the activation volumes on the lesion side. misery perfusion caused marked reductions of activation volumes of bold imaging associated with an increase of deoxyhemoglobin during activation. bold-fmri should be performed, giving consideration to the baseline circulatory conditions. masato fukuda, toru uehara, masahiko mikuni department of psychiatry and human behavior, gunma university graduate school of medicine, gunma, japan near-infrared spectroscopy (nirs) has been increasingly employed in psychiatry researches such as personality ( . neuropsychobiology , ), aging ( . neuroimage , , and psychiatric disorders ("progress in schizophrenia research", nova science publishers, ) . frontal lobe reactivity was investigated using multichannel nirs machines in unipolar depression, bipolar depression, and schizophrenia ( . biol. psychiatry , ; . neuroimage , ) by monitoring changes of oxy-hemoglobin concentration ([oxy-hb]) every . s during a verbal fluency task. the unipolar depression was characterized by smaller [oxy-hb] increase, the bipolar depression by comparable but delayed [oxy-hb] increase, and the schizophrenia by reduced [oxy-hb] increase during the task period followed by [oxy-hb] re-increase during the post-task period, suggesting reduced, preserved but delayed, and inefficient frontal lobe reactivity, respectively. collaborators: itsuro ida, akihiko oshima, makoto ito, tomohiro suto, masaki kameyama, yutaka yamagishi, toshimasa sato, masashi suda sy - - - clinical application of nirs to neurorehabilitation optical imaging using near-infrared spectroscopy (nirs) is suitable for assessing cortical activation during human gait because of its flexibility and portability. in healthy subjects, walking induced increase of oxygenated hemoglobin levels that centered in the medial sensorimotor cortex and supplementary motor area. in hemiparetic patients with stroke, cortical activation was characterized by asymmetrical activation in the sensorimotor cortex and recruitment of the premotor and prefrontal cortex. a longitudinal study revealed that locomotor recovery was associated with improvement of asymmetrical activation in the sensorimotor cortex as well as enhanced activation in the premotor cortex. sensorimotor stimulation by facilitation technique induced enhanced activation in the motor related areas, particularly in the premotor cortex. partial body weight support and speed-dependent exercise decreased sensorimotor activation, suggesting relative shift of locomotor control to the hierarchically lower structures including the spinal cord. thus nirs may contribute to establishing brain-based strategies for neurorehabilitation. research funds: funds for comprehensive research of aging and health sy - - - measurement of language related brain activities during recovery from aphasia eiju watanabe , yumiko muroi , chizuru nakajima department of neurosurgery, jichi medical university, tochigi, japan; department of neurosurgery, tokyo metropolitan police hospital, tokyo mechanism which supports the recovery of language after aphasia is not well understood. it has long been discussed that language related areas including the regions surrounding the language area and compatible cortex on non-dominant side could be candidates which support the recovery. several studies suggest the compensation by these areas using fmri and pet. we used optical topography (ot) to know the participation of these areas during the recovery from the aphasia. we measured aphasics who showed recovery from the aphasia after apoplexy. word generation task was used. in seven cases ot was measured more that twice. seven cases showed the activity on the non-dominant frontal lobe. they all showed activities on the dominant frontal lobe in the follow-up measurements along with the deactivation of non-dominant side. these findings showed dynamic participation of non-dominant frontal lobe during the recovery phase suggesting that the rehabilitation protocol should be changed according to the area activated in each phase. tamami fukushi research institute of science and technology for society (ristex), japan science and technology agency (jst), tokyo, japan recent development of neuroscience has provided remarkable scientific discoveries, as well many newer philosophical, ethical, legal and social issues. for example, the consequences of the progress of non-invasive neuroimaging technologies, such as functional magnetic resonance imaging (fmri) and near infrared spectroscopy (nirs) show the possibility to read the mind of others, which may lead the criminal and commercial applications. brain-machine interface (bmi) technology and pharmacological manipulation of the human brain can cause the unpredictable enhancement of human ability. in advance to the expansion of "applied neuroscience", neuroscientists should consider "what the ethical problem is in the current neuroscience" and "how we learn and interact with the ethics". in this symposium, the panels will talk about the history of neuroethics then provide the ethical aspects of basic research. we will also discuss the future perspective of the neuroethics in japan and world in terms of sharing the problems across neuroscientists, ethicists, mass media, and public. judy illes stanford university, usa akin to genetic testing in the s, the translation of neuroimaging capabilities from the laboratory to the clinical setting has raised ethical questions about how new diagnostic and predictive information will be managed in the absence of effective treatments for certain diseases, about the timing of technology transfer and handling of technology that falls in the regulatory gray zone between research and clinical use, and what impact increasing opportunities for selfreferral to health care will have on patient-physician relationships, medicine, and society overall. potential off-label uses of advanced neuroimaging outside the health care setting -in law, education, employment and even for national security -are already being tested and debated. we will discuss how these issues converge in st century neuroethics, the presence of neuroethics in the international domain, and the critical role of ethics in neuroscience in the future. sy - - - neuroethics from primate's eyes naotaka fujii bsi, laboratory for symbolic cognitive development, japan neurophysiologists working on monkeys have been trying to understanding how their brains are working. the aim of the studies was not merely revealing functions of primate's brain but also trying to extrapolate the findings in primates onto human brain functions. this was true but not really true due to technical limitations which prevented us from expanding the findings in primates directly to the human brain function. however, recent advancement of technology has changed the world. findings in primates can be directly applied to human studies with or without researcher's intention. technologies invented in primate physiology are now readily transferred into human without much discussion. brain machine interface is one example. now, monkey's brains are forcing us to think about social impact of our research from ethical view, which we have not discussed before. as an experienced primate neurophysiologist but with little ethical training, i will discuss 'what is ethically correct primate research' and 'how our scientific contribution has to be made' from very practical and ground level of neuroethics. sy - - - neuroethics of nurturing the brain takao k. hensch critical period mechanisms group, riken brain science institute, japan at no time in life is the brain so easily shaped by experience than in infancy and in early childhood. it is during these critical periods that neural circuits acquire language and other basic brain functions. unraveling mechanisms that limit such dramatic plasticity to early life would pave the way for novel paradigms or therapeutic agents for rehabilitation, recovery from injury or improved learning in adulthood. conversely, a deeper insight into early postnatal brain development will provide valuable inspiration for effective brain-based education methods for our children-perhaps the greatest potential contribution of neuroscience to society. this raises urgent and important ethical questions for our consideration: is there an "ideal" brain we should be nurturing? to what extent can/should drugs be used not merely to correct developmental disorders but also to enhance performance? how do we determine what is good or bad for the maturing brain? research funds: riken bsi sy - - - neuroethics beyond laboratories and across cultures daofen chen national institute of neurological disorders and stroke, usa recent progress in systems and cognitive neuroscience poses new ethical challenges to both investigators and to the funding agencies that support scientific investigations. potential uses of many of these recent advances go beyond their intended medical applications. a growing array of neurotechnologies capable of monitoring or even intervening in human cognition makes it imperative to consider the social, ethical, and legal implications of these scientific advances. while it once might had been possible to conduct research with naive ignorance of its societal implications, this is no longer the case. moreover, we need to be cognizant that modern brain science is profoundly influenced by the distinct cultural and social values held by different sectors of the world population. we will discuss what can be done from the perspective of funding agencies to facilitate intercultural dialogue, foster mutual understanding, and develop a framework and strategies to address emerging neuroethical issues and prioritize our future efforts in neuroscience research. sy - - - bridging neuroscience and public: neuroethics in cultural contexts osamu sakura , interfaculty initiative in information studies, university of tokyo, tokyo, japan; research institute of science and technology for society (ristex), jst, japan to bridge between neurosciences and public society-it should be one of the important aims of neuroethics. for this purpose we need to draw the outline of neuroscience within the cultural context. the method and the result of natural sciences are universal, of course, but its social consequences are highly variable among cultures. although the systematic comparative researches are open to the future, we should discuss how the neurosciences could create the healthy relationship between the public society, especially focusing on the method for public participation and on the previous successful cases. mitsuru kawamura , , akira midorikawa , yoshiki kaneoke , shinichi koyama , masato taira , argye hillis showa university school of medicine, japan; crest, jst, saitama, japan; national institute of neuroscience, ncnp, tokyo, japan; national institute for physiological sciences, okazaki, japan; nihon university, tokyo, japan; johns hopkins university, baltimore, usa this symposium aims to provide an opportunity to talk between clinical neuropsychologists and neuroscientists. focusing on the visual system, we will discuss up-to-date studies from neuropsychological and neuroscientific viewpoints. the topics include motion perception in brain-damaged patients, neuroimaging of motion perception, surface and depth perception in brain-damaged patients, and neuroimaging of surface and depth perception, and neuropsychological and neuroimaging studies of visual attention. we will discuss consistency and inconsistency of our findings, and discuss what to do in order to produce synergy between clinical neuropsychology and neuroscience. research funds: kakenhi ( ), crest sy - - - impairment of surface perception in patients with ventral occipital damages shinichi koyama , mitsuru kawamura , akira midorikawa , yoshiki kaneoke , masato taira , argye hillis showa university, tokyo, japan; national institute of neuroscience, ncnp, tokyo, japan; national institute for physiological sciences, okazaki, japan; nihon university, tokyo, japan; johns hopkins university, baltimore, usa we examined the perception of faces and objects in two patients with ventral occipital damages, using psychophysical techniques. patient was a -year-old woman with bilateral damage in the fusiform and parahippocampal gyri. although she could recognize pictures of famous people, she frequently failed to decide the races of unfamiliar faces and occasionally failed to see the hollow-face illusion (gregory ) . in addition, her performance in object identification task became poorer when the objects were presented in inverted (negative) pictures. patient was a -year-old men with bilateral damage in the lingual and fusiform gyri. his recognition of faces and objects became poorer when they were presented in inverted pictures. based on the above results, the role of the ventral visual cortex in the perception of faces and objects will be discussed. research funds: grant-in-aid for jsps fellows sy - - - how do pictorial cues influence d information processing in the parietal association cortex? masato taira , arish, nihon university, tokyo, japan; department of applied system neuroscience, nihon university graduate school of medical science, tokyo, japan pictorial cues are one of the most influenced cues for d perception. basically, it is thought that the parietal association cortex processes d visual information by binocular disparity cues and the temporal association cortex processes that by pictorial cues in the concept of two visual information processing systems in the brain. however, recent studies have suggested that there are many crosstalk of d information between these association areas. our recent studies have shown that a group of neurons in the parietal cortex (area cip) is involved in perception of d surface orientation and used both disparity and pictorial cues. functional mri data in human also suggest that pictorial cues, such as attached and cast shadow, are processed in the parietal cortex in d perception. furthermore, human psychophysical data gives us some insights of how the pictorial cues influence d information processing in the parietal association cortex. research funds: kakenhi sy - - - case report of a patient with posterior cortial atrophy who relatively preserved perception of moving objects akira midorikawa national center of neuroscience, national center of neurology and psychiatry, tokyo, japan it has been presented that severe type of bálint syndrome behaves like a blind person; however, it also reported that there are some cases who behave like a blind person but could walk without collision. up to today several cases have been reported, but the underlying mechanism of the phenomenon has not been mentioned. in this report, i will talk about a patient with bálint syndrome due to degenerative disease known as posterior cortical atrophy (pca), who could not only walk around without collision but also play catch very well, nevertheless having blind like behavior. the crucial visual information underlying these phenomenons was assumed to be motion parallax induced by not only objects movement but also self movement. in addition, discrepancy between the patients who could walk and not walk will be discussed. research funds: crest, japan science and technology agency sy - - - neural mechanism underlying visual perception of motion as revealed by non-invasive human study yoshiki kaneoke department of integrative physiology, national institute for physiological sciences magnetoencephalography (meg) measures the neural activity representing the synchronized inputs to the millions of pyramidal neurons in the localized cerebral cortex. thus, it will show us another aspect of the neural activity related to the specific brain function that would not be revealed by the recording of single neuronal activity. our meg studies have revealed several important findings in the human visual motion detection system. first, the response properties for the apparent motions indicate the importance of the human mt/v + for the perception and the existence of the parallel processing for the motion and light blinking. second, the existence of the spatiotemporal filtering mechanism for the perception of motion speed is shown by the various motion stimuli. third, we present the evidence that the spatial integration of the speed without direction information occurs in our visual system. based on the results, scalar fields theory for the spatial integration of motion is proposed to explain various complex motion perception. research funds: kakenhi ( ) sy - - - neural correlates of visual attention argye hillis , mitsuru kawamura , akira midorikawa , yoshiki kaneoke , shinichi koyama , masato taira johns hopkins university, usa; showa university, tokyo, japan; national institute of neuroscience, ncnp, tokyo, japan; national institute for physiological sciences, okazaki, japan; nihon university, tokyo, japan in this paper i report a series of studies of unilateral spatial neglect (usn) in acute stroke, demonstrating a frequent double dissociation between stimulus-centered neglect and viewer-centered neglect, and showing that these types of neglect can be modality-specific. other data reveal that different types of usn are observed when there is hypoperfusion of temporal cortex versus parietal cortex. still other data provide evidence that severity of neglect depends on the volume of hypoperfused tissue in acute stroke, and that reperfusion results in early recovery of neglect. finally, i will review new evidence that right usn is common after left cortical infarcts or hypoperfusion in acute stroke, but the distribution of types of usn is very different from the distribution of types of usn after right hemisphere stroke. takeo kubota, takae hirasawa, kaoru nagai department of epigenetic medicine, university of yamanashi faculty of medicine, chuo, yamanashi, japan how are brains controlled molecularly? this is one of fundamental questions in neuroscience. several lines of evidences have suggested that genes are more strictly controlled in the brain than in other organs. epigenetics is one of such systems to control expression of the genes not based on dna sequence, but based on 'beyond the dna sequence' (chromatin modifications and small rnas). the failure of this system is known to result in neurodevelopmental diseases, such as an autistic rett syndrome. it has recently been demonstrated that the epigenetic system is affected by an environmental stress after birth, and that the system is associated with neural differentiation and cell fate determination and human brain diversity. these findings imply that epigenetics is an important research field to understand mechanisms of neural development and mental diseases. topics from update epigenetic researches in neuroscience will be discussed in this symposium. as one of epigenetic disorders, we introduce studies of rett syndrome (rtt) and its model mouse. rtt is a neurodevelopmental disorder, characterized by mental retardation and peculiar behavior. mutations of the mecp gene, encoding methyl-cpg binding protein , cause rtt. mecp acts as a transcriptional silencer. abnormal expression of some genes due to mecp dysfunction is thought to be the first step of rtt pathophysiology. to understand how mecp mutation makes rtt, we have two approaches that are morphological investigation of brain and discovery of mecp -downstream genes. using mecp -null mice (mecp −/y ), we revealed small number of dendritic spines and premature postsynaptic density at presymptomatic period. premature synaptogenesis may be the initial neuronal changes of rtt. we also found that mecp directly regulates expression of insulin-like growth factor binding protein (igfbp ) gene in human and mouse brains. pathological features of mecp −/y have the similarity of igfbp transgenic mice, which show reduction of early postnatal brain growth. over-expression of igfbp due to lack of mecp may lead to delayed brain maturation. growing evidence suggests that alterations in the epigenetic status such as dna methylation and histone modifications in brain are involved in the behavioral response to environmental factors and the pathogenesis of psychiatric diseases. however, in contrast to mrna profiling, there are few established methods for profiling the genomewide epigenetic status to date. we developed a method for profiling the genome-wide dna methylation pattern using tiling arrays, and focused our analysis on human brain samples derived from psychiatric patients and control subjects. in this symposium, general picture of the genes that are heavily methylated or non-methylated in human brain, and the relationship between dna methylation and mrna expression patterns will be presented. understanding what produces neuronal diversification has been a longstanding challenge for neuroscientists. the recent finding that long interspersed nucleotide elements- or l (line- ) retroelements are active in somatic neuronal progenitor cells provided an additional mechanism for neuronal diversification. together with their mutated relatives, retroelements sequences constitute % of the mammalian genome with l elements alone representing %. the fact that l can retrotranspose in a defined window of neuronal differentiation, changing the genetic information in single neurons in an arbitrary fashion, allows the brain to develop in distinctly different ways. this characteristic of variety and flexibility may contribute to the uniqueness of an individual brain. however, the extent of the impact of l on the neuronal genome is unknown. the characterization of somatic neuronal diversification will not only be relevant for the understanding of brain complexity and neuronal organization in mammals but may also shed light on the differences in cognitive abilities, personality traits and many psychiatric conditions observed in humans. sy - - - notch-induced acquisition of astrocyte differentiation potential of neural stem cells kinichi nakashima , jun kohyma , tetsuya taga , masakazu namihira grad. sch. biol. sci., naist, ikoma, japan; inst. mol. embryol. genet., kumamoto univ., kumamoto, japan neurons and astrocytes are generated from common neural stem/precursor cells, however, neurogenesis precedes astrocytogenesis during brain development. we have previously reported that gfap-positive astrocyte differentiation is dependent on the transcriptional activity of stat . a cpg dinucleotide in the stat recognition sequence within the gfap gene promoter is highly methylated at midgestation which becomes demethylated as the brain develops, enabling stat to induce gfap expression. thus, it is conceivable that the epigenetic modification such as dna methylation of cell type-specific gene promoter controls the switch from neurogenesis to astrocytogenesis in the developing telencephalon. here we report that neurons, which are generated earlier than astrocytes can potentiate neural precursors at midgenstation to differentiate into astrocyte through the activation of notch-signaling. the activated notch-signaling accelerates demethylation of stat binding element in the gfap gene promoter. sy - - - neurogenesis and stem cell supply as therapeutic approach to overcome ischemic stroke masayasu matsumoto department of clinical neuroscience and therapeutics, hiroshima university graduate school of biomedical sciences, japan in order to overcome the brain damage caused by ischemic stroke, several strategies have been so far applied. in the present symposium, i will address the following points to be considered prior to clinical application of neurogenesis and/or stem cell supply to repair the damaged brain function. ( ) which type of brain infarction will be a future target of this therapeutic approach? ( ) which phase of brain infarction (i.e., acute or chronic phase) will be selected as a future timing of therapeutic intervention? ( ) which will be the best way to be applied in the clinical settings, neurogenesis control, stem cell supply or both? the present research status and future directions will be presented and fully discussed. research funds: kakenhi sy - - - gene therapy for cerebral ischemia setsuro ibayashi, hiroaki ooboshi department of medicine and clinical science, graduate school of medical sciences, kyushu university, fukuoka, japan cerebrovascular disease is the leading cause of the disabled people in japan and western countries. gene transfer technique may be applicable to the treatment of serious types of cerebrovascular disease. cerebral blood vessels have been targeted by gene transfer with intravascular or perivascular approaches. several experimental studies have revealed potential usefulness of gene therapy for prevention of vasospasm after subarachnoid hemorrhage. as for cerebral infarction, studies using various brain ischemia models have shown effectiveness of gene transfer in reduction of infarct size and functional recovery. our recent studies of post-ischemic gene transfer have provided promising results in attenuation of ischemic damages by inhibiting apoptosis, inflammation and vascular permeability. approaches to cerebral ischemia using gene transfer for angiogenesis and neurogenesis appear to be novel and promising strategies. thus, gene therapy has a potential for the future therapy against cerebral ischemia. isao date department of neurological surgery, okayama university, okayama, japan cerebral ischemia is one of the neurological disorders that cell transplantation is expected to be applied. in this presentation, the author will summarize our recent basic research and clinical application reported in the literature. it is now possible to make several types of neurotrophic factor secreting cell line by genetic manipulation. in order to prevent immunological reaction and tumor formation, we have been using encapsulated cell grafting technique. we transplanted several types of neurotrophic factor secreting cell line into the middle cerebral artery occlusion model and could confirm the histological and behavioral efficacy. we have also been using adultderived neural stem cells as donor cells because they have merits to make autografitng possible. as donor tissue, neural protection can be expected similar to fetus-derived neural stem cells. the effect of neural protection increases when neurotrophic factor secreting genes such as gdnf were inserted into neural stem cells. cell transplantation is considered a new therapeutic approach for cerebral ischemia and clinical application is expected. we now know that ( ) motor function may recover after minor injury to the primary motor cortex, ( ) this recovery is, at least in part, associated with reorganization of cortical motor representation, ( ) the molecular mechanism for synaptic plasticity and axonal regrowth is being elucidated, and ( ) recent clinical experience revealed that the motor function in patients with spinal cord injury is improved after transplantation of her/his own olfactory mucosa. furthermore, recent neuroimaging techniques can display the cortical functions as we as the specific fiber connections in individual brain. virtually any part of the brain can be approached with the accuracy of millimeters by the current image-guided neurosurgery. those theoretical and technical backgrounds suggest we might be ready for the reconstruction of brain function. nobuyuki nukina laboratory for structural neuropathology, riken brain science institute, japan a major hallmark of the polyglutamine (pq) diseases is the formation of pq inclusions. recently, misfolding has come to be considered one of the primary factors for pq protein aggregation, although, the nature of misfolding is not yet well known. the protein misfolding induced by pq expansion was investigated with our molecular model system using mutant myoglobin which is inserted different size of pq. expanded polyglutamine stretches form intramolecular and intermolecular beta sheets and amyloid fibrils. the surface of the mutant myoglobin with expanded pq was partially unfolded and destabilized. we also investigated the early phase of fibrillization by small-angle x-ray scattering and electron microscopic studies, revealing that the expansion of pq to repeats induced the formation of quasi-aggregate in the earliest stage of the protein fibrillization. this structure could be closely involved in recruitment of various functional proteins into aggregates, leading to the cellular dysfunction that causes pq diseases. furthermore using cellular model system we also studied the aggregates interacting proteins (aips) by analyzing the purified polyglutamine inclusions and the lists of aip including chaperones, proteasome subunits, ubiquitin interacting proteins and others suggest the pathological role of aips in the disease cascades. sy - - - neuronal dysfunctions in dentatorubralpallidoluysian atrophy (drpla) shoji tsuji , toshiya sato , mitsunori yamada department of neurology, the university of tokyo, tokyo, japan; center for bioresource-based researches, japan; department of pathology, brain research institute, niigata university, niigata, japan to investigate molecular mechanisms of neurodegeneration in drpla, a polyglutamine disease caused by expansions of cag repeats of drpla gene, we have established transgenic mice harboring a single copy of the full-length human mutant drpla gene with cag repeats. the q mice exhibited neurological phenotypes similar to juvenile type of drpla characterized by ataxia, myoclonus and epilepsy. electrophysiological studies disclosed age-dependent abnormalities in the globus pallidus and cerebellum. neuropathological studies revealed progressive brain atrophy without obvious neuronal loss and an age-dependent increase in neuronal intranuclear accumulation of mutant proteins with the regional distribution vulnerable to drpla. expression profiling analyses revealed down-regulated genes including camp responsive genes. these results suggest that "neuronal dysfunction", but not the "neuronal cell death", is the essential mechanism of neurodegeneration in drpla. huntington's disease (hd) is caused by an expansion of a cag repeat encoding polyglutamine in the huntingtin protein and involves progressive motor, cognitive and psychiatric symptoms. using a transgenic mouse model of hd, we have shown that environmental factors can dramatically modify the disease process and delay the onset and progression of motor and cognitive symptoms. further, we have attempted to correlate these behavioural findings with changes in gene expression, neuronal morphology, neurogenesis, and cortical plasticity, in an attempt to elucidate cellular and molecular mediators in hd, and understand how gene-environment interactions can modulate these pathogenic pathways. our findings indicate that the modulatory effects of environmental manipulations are mediated by amelioration of specific molecular and cellular deficits, and provide experimental paradigms for the identification of novel therapeutic targets for hd and related brain disorders. sy - - - control of neural organization in the developing cerebral cortex yasuto tanabe mitsubishi kagaku institute of life sciences, tokyo, japan in the developing cerebral cortex, the generation of neurons with distinct identities and patterns of connectivity is controlled by a hierarchical series of cellular interactions that culminate in the laminar organization of distinct cortical areas. over the past three years we have begun to examine cerebral cortical development by focusing on three distinct major neuronal subtypes, namely, cajal-retzius cells, cortical projection neurons, and cortical interneurons. the analyses of these distinct neuronal subtypes allowed us to identify several candidate molecules and cellular interactions that might contribute to the laminar and areal organization of the cerebral cortex. in the first part of my talk, i would like to deal with the issue of ontogeny of cajal-retzius cells, and present the way cajal-retzius cells are generated, migrate and finally distribute in the developing cerebral cortex. then, i would like focus my talk on the issue of the way the acquisition of radial migration and axonal trajectory patterns of distinct cortical projection neurons is controlled during the development of the cerebral cortex. research funds: kakenhi , sy - - - mechanisms of the regulation of neuronal migration and corticogenesis kazunori nakajima , dept. of anat., keio univ. sch. of med., tokyo, japan; inst. of dna med., jikei univ. sch. of med., tokyo, japan mammalian cerebral cortex has a six-layered structure where the neurons are aligned depending on their birth-date. to determine whether the migration from the ventricular zone (vz) to beneath the marginal zone (mz) is essential for neuronal segregation into layers, we investigated whether migrating neurons have different cell aggregation properties in vitro depending on their birth-dates, even before they arrive beneath the mz. we analyzed vz cells and cells from the intermediate zone (imz) mainly composed of migrating cells, and found that the cells had acquired a birth-date-dependent preferential segregation mechanism in a reelin-independent manner. these findings suggest that cortical neurons acquire a birth-date-dependent segregation property (or fate) before their somas reach the mz. in silico experiments of the reaggregation culture supported that this mechanism might indeed contribute to the layer formation in the developing cerebral cortex in concert with other mechanisms such as reelin signaling. kenji shimamura division of morphogenesis, institute of molecular embryology and genetics, kumamoto university, japan neurons of the thalamus originate in restricted regions of the proliferative zone of the diencephalic compartment before settling in their final locations in the nuclei. to investigate cellular and molecular mechanisms underlying nucleus formation, we analyzed the sequence and pattern of expression of specific markers that distinguish the subsets of neuronal precursors during development of the thalamus. we found that a morphogen-like activity of sonic hedgehog (shh) precisely defines positions of neurons with distinct properties, and that some gabaergic interneurons migrate from their birth place to distant nuclei in a highly organized manner. we also provide evidence that shh produced by the zona limitans intrathalamica (zli), which abuts the prethalamus and thalamus, is likely to be a cue for this directed migration. our results suggest that local production of prespecified neurons coupled with distinct migration properties and local guidance cues such as compartment boundaries could be principle elements for the nucleus formation. layers and nuclei are important functional units in the vertebrate cns. neurons in these structures have common physiological and anatomical features. despite their importance, mechanisms for nucleogenesis are poorly understood. we focused on the lower rhombic lip (lrl)-derived precerebellar neurons, and utilized exo utero electroporation with an enhanced yellow fluorescent protein (eyfp) gene, to study the process of nucleogenesis. after the unilateral transfer of eyfp to the lrl of embryonic day . mice, eyfp-labelled neurons migrate tangentially from the lrl in two distinct streams, one toward the ventral metencephalon and the other toward the ventral myelencephalon. the former formed the pontine grey nucleus and reticulotegmental nucleus and the latter the external cuneate nucleus and lateral reticular nucleus. before forming the clusters, the labelled neurons begin to migrate toward the ventricle along the radial fibres, and aggregate as they detach from the fibres. perturbation experiments such as introduction of dominant negative constructs and sirna suggested involvement of several molecules in the migration of these neurons. the brains of fetal alcohol syndrome patients exhibit impaired neuronal migration, but little is known about the mechanisms underlying this abnormality. here we show that ca + signaling and cyclic nucleotide signaling are the central targets of alcohol action in neuronal cell migration. an acute administration of ethanol reduced the frequency of transient ca + elevations in migrating neurons and cgmp levels, and increased camp levels. experimental manipulations of these second messenger pathways, through stimulating ca + and cgmp signaling or inhibiting camp signaling, completely reversed the action of ethanol on neuronal migration in vitro as well as in vivo. each second-messenger has multiple but distinct downstream targets, including camkii, calcineurin, pp , rho gtpase, mapk and pi k. these results demonstrate that the aberrant migration of immature neurons in the fetal brain caused by maternal alcohol consumption may be corrected by controlling the activity of these second-messenger pathways. sy - - - membranes, water and diffusion denis j. le bihan shfj/cea, france among einstein papers is one which unexpectedly gave birth to a powerful method to explore the brain. molecular diffusion was explained by einstein on the basis of the thermal random translational motion of molecules. in the mid s it was shown that water diffusion in the brain could be imaged using mri. a dramatic application of diffusion mri has been brain ischemia, following the discovery that water diffusion drops immediately after the onset of an ischemic event, when brain cells undergo swelling through cytotoxic edema. also, water diffusion is anisotropic in white matter, because axon membranes limit molecular movement perpendicularly to the fibers. this feature can be exploited to map out the orientation in space of the white matter tracks and image brain connections. more recently, it was discovered that diffusion mri could detect transient swelling of activated cortical cells. this represents a significant breakthrough, allowing non invasive access to a fast and direct physiological marker of brain activation. this approach will bridge the gap between invasive optical imaging techniques and current functional neuroimaging approaches in humans, which are based on indirect and remote blood flow changes. sy - - - diffusion tensor fiber tractography using a tesla mr system yukio miki department of diagnostic imaging and nuclear medicine, kyoto university, kyoto, japan diffusion tensor imaging (dti) is an mr imaging technique that is sensitive to orientation of mobility in water molecules. dti reveals two specific characteristics: diffusion anisotropy; and directional distribution of water diffusivity. white matter shows high diffusion anisotropy, because diffusion is faster in parallel to fiber direction than in other directions. dti of the brain can be reconstructed to display d macroscopic fiber tract architecture, in a process known as fiber tractography. with recent advances in actively shielded -t magnets and parallel imaging techniques, high-field mr imaging has become practical in clinical settings. we have demonstrated that depiction of most fiber tracts was improved on -t tractography compared to . t. we have also established an integration of tractography and intraoperative subcortical motor-evoked potential, and demonstrated that diffusion tensor tractography of the corticospinal tract using -t mr was able to provide interactive information on fiber tracts, depicting the course of eloquent fiber tracts during an operation. to test whether mr tractography is reproducible and reliable, we used this technique to assess acute tiny infarcts located in the supratentorial brain. we analyzed the data of patients who presented to our institute with sensorimotor symptoms. there was an excellent correlation between the location of the infarct as assessed by tractography and clinical symptoms. next, we applied the technique to patients with evolving symptoms after admission to hospital. we specifically assessed the change in the tract-infarct relationship over time. the data showed that, in most cases when there was symptomatic progression, the distance between the tract and the infarct border depicted on dwi diminished. finally, we studied whether the use of tractography could help predict a patient's prognosis. to simplify the analysis, we specifically focused on patients with lenticulostriate artery (lsa) infarcts. we analyzed the correlation between the extent of cst involvement within the infarcts and the severity of motor deficits. the data indicated that the tractographic technique could be useful to predict a patient's outcome. sy - - - anatomical and functional tractography: a combined approach with diffusion tractography and corticocortical evoked potential riki matsumoto department of neurology, kyoto university graduate school of medicine, japan recent advances in diffusion-weighted imaging have raised the possibility of in vivo investigations of brain circuitry in humans. the probabilistic tractography provides estimates of the likelihood of a pathway between two brain regions without tensor estimation and thus could trace the fiber pathways beyond regions of low diffusion anisotrophy into the grey matter. however, the results depend merely on anisotropic movement of water molecules and need validation. for presurgical evaluation of epilepsy patients, we developed an in vivo tracking method, cortico-cortical evoked potential, to electrically track the cortico-cortical connections by stimulating a part of the brain through epicortical electrodes and recording the cortical evoked potentials that emanate from a distant region of the cortex via projections. combined with preoperative diffusion analysis, this invasive evaluation provides a unique opportunity to study the cortico-cortical connectivity both functionally and anatomically. results of the combined approach will be presented. parkinson disease (pd) is the second commonest neurodegenerative disorder after alzheimer disease characterized by tremor, rigidity, bradykinesia, and postural instability. pathologically, the most outstanding change is the neurodegeneration of the nigral dopaminergic neurons. although familial forms of pd can be encountered up to % of the patients, the remaining cases are sporadic. it has been postulated that nigral neurodegeneration in pd is induced by the interaction of genetic risk factors and environmental factors. epidemiological studies revealed numbers of environmental factors that are positively correlated with increased risk of pd; such factors include pesticide, herbicides, rural living, well water drinking, metals such as manganese and iron, fuel oil, industrial chemicals, and hydrocarbon solvents. in addition, certain employments were reported to be associated with increased risk of pd; these include steel/alloy industry, wood/pulp plant, farming, carpentry, cleaning, orchard, mining, and welding. these studies suggest importance of environmental factors in the pathogenesis of pd. recent progress in these areas will be discussed. masami ishido national institute for environmental studies, tsukuba, japan there are getting much public concerns about children health since environmental factors such as industrial chemicals cause deficit in developing brains. it has been suggested that they may be incident of attention deficit hyperactivity disorder or autism. epidemiologic studies also suggested that parkinson's disease was found in the peoples who were exposed to pesticides in their childhood. thus, we examined the effects of industrial chemicals, called endocrine disruptors, on rat neurodevelopment. oral administration of an endocrine disruptor ( - mg/kg) into male wistar rats (from days to weeks of age) significantly caused hyperactivity at - weeks old. immunohistochemical analyses of the brain tissues at weeks of age revealed a large reduction of immunoreactivity for tyrosine hydroxylase, but not for glutamic acid decarboxylase, both of which are localized in the substantia nigra, suggesting the specific degeneration by the chemical of dopaminergic neurons. tunel-positive cells were seen in the substantia nigra. thus, environmental insults in early life may be of particular etiologic importance. sy - - - non-thermal effects of mobile phones upon the rat brain leif g. salford, b. persson, j. eberhardt, g. grafstrom, l. malmgren, a. brun dept of neurosurgery and the rausing laboratory, lund university, sweden we have shown that rf electromagnetic fields can cause significant leakage of albumin through the bbb of exposed rats as compared to non-exposed animals. one remarkable observation is that sar values around mw/kg give rise to a more pronounced albumin leakage than higher sar values -all at non-thermal levels. if the reversed situation were at hand, we feel that the risk of cellular telephones, base-stations and other rf emitting sources could be managed by reduction of their emitted energy. the sar value of around mw/kg is exists at a distance of more than one meter away from the mobile phone antenna and at a distance of about - meters from a base station. another remarkable observation in our studies is the fact that a significant (p < . ) neuronal damage is seen in rat brains days after a hour exposure to gsm at sar values , and mw/kg. we have followed up this observation in a study where animals were sacrificed and days respectively after an exposure for hours to gsm mobile phone electromagnetic fields at sar values , , , . and (controls) mw/kg. significant neuronal damage is seen after days and albumin leakage after . our findings may support the hypothesis that albumin leakage into the brain is the cause for the neuronal damage observed after and days. sy - - - the mitochondrial toxin -nitropropionic acid: an environmental toxin to study striatal degeneration in huntington disease emmanuel brouillet neuronal death laboratory, ura cea-cnrs , france huntington disease is a neurodegenerative disorder caused by a mutation in the gene encoding huntingtin. the mechanisms underlying the preferential degeneration of the striatum, the most striking neuropathological change in huntington disease, are unknown. the behavioral and anatomical similarities found between huntington disease and animal models of striatal degeneration using the environmental toxin -nitropropionic acid ( np) support the hypothesis that mitochondrial defects could play a role in huntington disease. we will discuss the mechanisms of np toxicity and show that np and mutated huntingtin have certain mechanisms of toxicity in common. in particular, we show that mutated huntingtin can alter the expression of mitochondrial complex ii, the respiratory chain enzyme specifically inhibited by np. in summary, the np story is a good example showing how the study of environmental toxins can greatly help to elucidate the complex mechanisms underlying chronic neurodegenerative disorders. we recently demonstrated that rats received intrecisternal injection of -ohda or environmental chemicals, such as bisphenol a, nonylphenol, p-octylphenol, diethylhexylphthalate or dibutylphthalate, at days of age showed behavioral hyperactivity at - weeks of age. immunohistochemical studies revealed a deficit in the development of dopamine (da) neurons. adult rats received these chemicals showed degeneration in nigro-striatal da neurons similarly to parkinson's disease. in this study, we investigated the mechanism of -ohda-induced neurotoxicity, using pc cells as an in vitro model system. we observed the generation of reactive oxygen species (ros) and p-quinone via auto-oxidation of -ohda. we also characterized the oxidation of cellular proteins by -ohda and the protective effect of antioxidants such as catalase, glutathione, and n-acetylcysteine with different manner. we will discuss about apoptotic cell death pathway including cytochrome c release and caspase activation induced by -ohda, ros and p-quinone. sy - - - environmental factors in the pathogenesis of alzheimer's disease joanna l. jankowsky california institute of technology, usa epidemiological studies indicate that environmental factors significantly influence the risk of developing alzheimer's dementia. foremost among those factors are education, occupation, and leisure activities. although not universal, most studies have found that individuals with greater education, more challenging occupation, or active leisure hobbies show relative protection against dementia. animal models for alzheimer's disease have recently been used to explore the mechanism of this effect. transgenic mice designed to recapitulate alzheimer's amyloid pathology are protected from functional decline by enriched housing designed to provide cognitive stimulation. both enriched housing and exercise modify the level of amyloid-beta in the brains of transgenic mice, demonstrating that environmental factors can significantly influence brain biochemistry. intriguingly, traditional environmental enrichment can improve cognitive behavior while paradoxically elevating amyloid-beta levels in transgenic mice, suggesting that environmental stimulation may alter amyloid metabolism and cognitive function by competing mechanisms. the efficacy of synaptic inhibition depends on the number of gaba a receptors expressed on the neuron surfaces. in the present study, we have elucidated the role of prip (plc-related inactive protein) in trafficking of the receptors by analyzing prip knockout (ko) mice; the sensitivity to diazepam was reduced as assessed by biochemical, electrophysiological and behavioral analyses of ko mice, suggesting the dysfunction of the ␥ subunit-containing receptors. we then examined the mechanisms by which prip molecule regulates cellsurface expression of ␥ subunit-containing receptors. disruption of the direct interaction between prip and the  subunit of receptors by prip-binding peptide inhibited cell-surface expression of ␥ subunit-containing receptors in gh and hek cells. constitutive internalization of the receptors was also modified by the peptide. collectively, prip molecules are involved in trafficking of ␥ subunit containing gaba a receptors to/from cell-surface membrane. research funds: kakenhi ( ) sy - - - involvement of bdnf in the induction of ltp at visual cortical inhibitory synapses yukio komatsu dept. visual neurosci., res. inst. environ. med., nagoya univ., nagoya, japan high-frequency stimulation (hfs) induces long-term potentiation (ltp) at inhibitory synapses of layer pyramidal cells in rat visual cortex. this ltp requires a postsynaptic ca + rise for induction and spike firing of presynaptic cells for maintenance, although the necessary frequency is low, suggesting that ltp is expressed presynaptically and some information must be sent backwards from the post-to presynaptic cells during induction. in this study, we investigated whether bdnf could act as such retrograde messengers. ltp did not occur when hfs was applied in the presence of k a at nm, inhibiting trk receptor tyrosine kinases selectively at that dose. hfs induced ltp when k a application was started soon after hfs or when k a was loaded into postsynaptic cells. ltp did not occur in the presence of trkb-igg or anti-bdnf antibodies. in cells loaded with bapta, the addition of bdnf to the medium enabled hfs to induce ltp without affecting basal synaptic transmission. these results suggest that bdnf released from postsynaptic cells activates presynaptic trkb, enabling the induction of ltp. research funds: kakenhi ( ) sy - - - autocrine mglur activation in cerebellar purkinje cells regulates gaba-mediated synaptic inhibition trevor smart, ian c. duguid university college london, uk in the cerebellum, retrograde signalling is important for the induction of short-and long-term changes to synaptic inhibition at interneuron-purkinje cell (in-pc) synapses. endocannabinoids, via cb receptors, mediate a short-term decrease in synaptic efficacy, while glutamate, via presynaptic nmda receptors, induces a sustained increase in gaba release. we now report that dendritically released glutamate also acts as an autocrine messenger, activating mglur on pcs to enhance synaptic inhibition via the release of endocannabinoids. this process was triggered by repetitive pc stimulation and blocked by uncoupling the mglur -gq/ transduction pathway as well as being initiated by direct mglur activation during pc depolarisation. glutamate uptake by excitatory amino acid transporters controlled the extent of autocrine mglur activation, whilst basal glutamate levels were unable to enhance endocannabinoid release. our study suggests that autocrine mglur activation provides a powerful homeostatic mechanism to dynamically regulate inhibitory synaptic transmission. sy - - - regulatory mechanism of inhibitory synaptic transmission in the cerebellum shin-ya kawaguchi , , tomoo hirano , dept. biophys., grad. sch. sci., kyoto univ., kyoto, japan; crest, jst, kawaguchi, japan at the gabaergic synapses between inhibitory interneurons and a purkinje neuron in the cerebellum, postsynaptic depolarization induces long-term potentiation of transmission efficacy mediated by gaba a receptors (rebound potentiation: rp). the signaling cascades regulating the induction of rp has been clarified. the balance of activities of protein kinases and phosphatases determines whether rp is induced or not. here we show another molecular mechanism involved in the rp induction. using both electrophysiological experiments and computational kinetic simulation of biochemical reactions, we demonstrate how the long-term potentiation of gaba a receptormediated responses is brought about. rp induction was impaired by inhibition of ca + -activated protease calpain or by disturbance of association of gaba a receptor ␥ subunit with gabarap (gaba a receptor associated protein). binding of gabarap to microtubule was also involved in the regulation of rp. our results suggest that structural alteration of gabarap caused by calpain activity is critical for establishment of rp. sy - - - contribution of hebb's "organization of behavior" to the development of brain science masataka watanabe tokyo metropolitan institute for neuroscience, tokyo metropolitan organization for medical research, tokyo, japan more than years have passed since the publication of "organization of behavior". this book has been one of the most influential books in neuroscience. around the time of the th anniversary of this book, special issues and articles concerned with this book appeared in several journals. his idea, which is a general framework for relating behavior to synaptic organization through the dynamics of neural networks, has stimulated variety of neuroscience researches in relation to, for example, environmental effects on development, naturenurture interaction, memory consolidation and sensory deprivation. however, he also made some mistakes, for example he advocated frontal lobotomy. in this symposium, i will briefly review how influential this book has been on basic and practical neurosciences, and will re-consider the importance and limitation of studying mental processes, such as emotion, memory and thought by exploring brain mechanisms, in reference to the idea of cell assembly, phase sequence and hebb synapse. sy - - - detection of cell assembly in neuroscience experiments and brain-machine interfaces yoshio sakurai , , susumu takahashi department of psychology, kyoto university, kyoto, japan; crest, japan science & technology agency, japan the reality of cell-assembly coding in the working brain depends on how we could detect specific properties of cell assembly from multi-neuronal activities in behaving animals. first in the present paper, we show experimental results indicating some of the properties, i.e., functional overlapping of individual neurons and connection dynamics among multiple neurons, that depend on tasks and events being processed and on the distance among the neurons. second, we demonstrate a newly developed method, brain-machine interface (bmi), to test the reality of cell assembly as neural information and the plastic formation of cell assemblies during learning processes. we introduce our recent bmi system with independent component analysis (ica) and specific multi-electrodes and show some neuronal and behavioral data obtained by the bmi system. hebb postulated that coincident activities of pre-and postsynaptic neurons trigger input-specific plasticity. how relevant is it in protein synthesis-dependent late-phase plasticity (lp)? synaptic tagging hypothesis explains how new proteins reach the activated synapses to establish input-specific lp. using live-imaging techniques, we measured entry of vesl- s-egfp into dendritic spines (ve trapping) of rat hippocampal neurons in culture, and found that ve trapping activity serves as synaptic tag in many criteria. ve trapping conforms to the hebb's rule in a sense that it required both presynaptic activity and postsynaptic no-pkg pathway, but their coincident time window was far wider (∼h) than that of early-phase plasticity, suggesting an involvement of persistently synchronized rather than transiently coincident activity. no spreading from the activated synapses may persistently prime the postsynaptic tag components at the surrounding synapses, during which brief inputs to these synapses will establish associative and heterosynaptic tags. thus, tagging one synapse would lead surrounding synapses to multiple metaplastic states. tomoki fukai laboratory for neural circuit theory, riken brain science institute, saitama, japan in the cell-assembly hypothesis, cortical neurons are considered to form functional subnetworks depending on a particular demand of information processing. such cell assemblies may be organized through synchronous firing of the constituent neurons and synapses modifiable by hebbian learning. in this talk, i will overview recent in vivo and in vitro experimental findings that provide new evidence for synchronous or precisely timed neuronal activity. i propose a hardwired structure of local cortical networks, "entangled synfire chains", on the basis of the experimental observations of cortical activity. in this model, multiple cell assemblies can be defined by the pattern of neuronal wiring. however, the same experimental findings can lead us to a different type of cortical network models. in this type of models, cortical networks may self-organize to develop a critical dynamical state, which may be useful for realizing a hypothetical "liquid-state machine". i will discuss the characteristic properties of both types of models and the possible implications in cortical computations. research funds: kakenhi ( ) sy - - - impact of hebbian hypothesis on neuroscience keisuke toyama shimadzu institute of basic technology, seikacho, hikaridai, kyoto - , japan hebb has seeded two major concepts in the modern neuroscience, i.e., cell assembly hypothesis for perception, and hebbian synapses to construct that cell assembly. the former concept stimulated extensive searches for the response selectivity extending from the primary visual cortex to the inferotemporal cortex and even to the hippocampal cortex, while the later concept triggered neuroscience studies of the learning and memory in the developing and adult brains. recently, these concepts refreshed the impact with new dressing of 'dynamics'. cell assembly that was originally assumed to be static, became dynamic and opened a new possibility for the neural computation, combined with dynamic hebbian synapses conceptualized as the spike-timing dependent plasticity (stdp). i would like to discuss about speaker's talks in this context. sy - - - tonic gaba a receptor mediated conductances: properties, functions and plasticity alexey semyanov riken brain science institute (bsi), japan communications mediated by non-synaptic receptors are important for information processing in the brain. high affinity extrasynaptic gaba a receptors mediate a persistent "tonic conductance" which reflects their activation by ambient concentrations of gaba. this phenomenon is found in different brain regions, shows cell-type specific differences in magnitude and pharmacology, and changes during brain development. our findings have revealed functional significance of gaba a receptors mediated tonic conductance in the hippocampus. we have shown that it modulates rate-coded information processing by individual neurons, and acts in a cell-type specific manner to regulate the excitability of the local neuronal circuit. the magnitude of the conductance is regulated by efficiency of gaba uptake and membrane potential. gaba a receptor mediated tonic conductance undergoes adaptive plasticity. it is up-regulated in hippocampal pyramidal cells in a model of pilocarpine status epileptics in rats. in mice lacking gad the amount of the tonic inhibition is reduced in ca hippocampal interneurons, while unchanged in pyramidal cells. sy - - - modulatory effects of peri-interneuronal glial cells on neuronal activities in hippocampal ca region yoshihiko yamazaki , yasukazu hozumi , kenya kaneko , satoshi fujii , hiroshi kato dept. of neurophysiol., yamagata univ. sch. of med., yamagata, japan; dept. of anat. & cell biol., yamagata univ. sch. of med., yamagata, japan glial cells, in addition to their supportive roles in the nervous system, make up a functional unit with neurons and have been suggested to play novel roles in neuronal activities. we focused on interneuron/peri-interneuronal glial cell (pg) pairs in the hippocampal ca region and performed dual whole-cell recordings to investigate the modulatory effect of glial cells on neuronal activities. direct depolarization of pg suppressed the excitatory postsynaptic currents in an adjacent interneuron. this suppression was inhibited by adenosine a receptor antagonist. moreover, pg activation modulated the firing pattern of the interneuron. since interneurons in the hippocampus are mainly inhibitory and the terminals of a single interneuron make a large number of synapses on a group of pyramidal cells, direct inhibitory regulation via pg would have marked effects on the information processing of neurons in the ca region. research funds: kakenhi ( ) sy - - - carbachol-induced beta oscillations in rat hippocampal slices kiyohisa natsume, jun arai graduate school of life science and systems engineering, kyushu institute of technology, fukuoka, japan rat hippocampus has the cholinergic input from medial septum and diagonal band in vivo. the input involves the generation of hippocampal rhythm, theta, gamma rhythm. to mimic the system, we applied carbachol, a cholinergic agent, to rat hippocampal slices. carbachol can induce beta oscillation as well while carbachol can induce theta, and gamma oscillations in the slices. in the present paper, we introduce the beta oscillations. the application of m carbachol induces beta oscillations which occur intermittently with the interval of - s. during the intervals, gamma oscillations are induced. the mean frequency of the oscillations is . ± . hz (mean ± s.e.m.). the oscillations are induced via muscarinic m , , receptors. the frequencies of them are significantly decreased by the application of bicuculline, a gaba a antagonist. they are sensitive to bicuculline, while theta oscillations are not. it is indicated that the character of beta oscillations are different from those of theta oscillations. the neocortex and the hippocampus are connected by way of the entorhinal cortex and the subiculum. to examine ongoing network interactions among these distinct cortices during neocortical slow oscillations ( - hz), we recorded intracellular potentials in single neocortical, entorhinal, subicular, and hippocampal neurons, together with hippocampal field and multi-unit activities in adult anesthetized rats. we have found that ( ) most entorhinal and subicular neurons displayed bimodal active (up) and quiet (down) states of membrane potential, in synchrony with neocortical slow oscillations, ( ) no bimodal up-down transition was present in hippocampal neurons. hippocampal granule cells were directly driven by entorhinal up-state activity, while ca and ca neurons discharged during both up and down states, ( ) gamma and fast (ripple) oscillations were observed in hippocampal ca area irrespective of up-down transition. these observations suggest that entorhinal and subicular regions are "neocortex-like" and hippocampal networks can generate self-organized activity independent of neocortical slow oscillations. the cholinergic neurons in the mesopontine reticular formation (mprf) seem to control sleep-wake cycle and hippocampal activity, because stimulation of the mprf elicits rem sleep and hippocampal theta wave. in this study, we recorded neuronal activity in the mprf and pontine and hippocampal eeg during rem sleep and investigated time-relationship between them. our results are summarized as follows: ( ) most of the mprf neurons were active during rem sleep; ( ) the mprf activity increased over ten seconds before transition from nrem to rem sleep, i.e. from non-theta to theta period; ( ) the theta wave was instantaneously accelerated concomitant with activation of the mprf neurons. these results suggest that cholinergic neuron in the mprf is important in generation and maintenance of rem sleep and theta wave. because hippocampal theta waves are involved in memory consolidation during rem sleep, our findings might help to clarify this mechanism. research funds: kakenhi ( ) sy - - - clock mechanisms of the scn involving in the entrainment to the morning and evening light sato honma, natsuko inagaki, nobuko tokumaru, ken-ichi honma dept. physiology, hokkaido univ. grad. sch. med., sapporo, japan the circadian clock, by entraining to the light-dark cycle of different day length, controls seasonality in biological functions. the mechanism is currently explained by morning and evening oscillators which change their coupling intensity depending on the day-length. by using clock gene expression as a marker of clock functions, we examined the localization and molecular bases of the two oscillators. rats and mice were housed under light-dark (ld) : h and ld : h. clock gene expression patterns in the entire scn were examined by in situ hybridization on the first day of constant darkness. the phase relations of per and per rhythms suggest that light-on resets per rhythms in both light conditions, while per rhythm also relates to the light-off. in cultured scn of transgenic mice expressing luciferase under the control of per promoter, we observed two bioluminescent peaks a day only in the anterior scn from the mice kept in ld : . the finding suggests that two distinct oscillators, which respond to the day-length, reside in the anterior scn. the suprachiasmatic nucleus (scn) is the center of the mammalian circadian clock. tissue transplantation of the scn restores the behavioral circadian rhythm in scn-lesioned mice in spite of the impaired neural connection with the host brain. we have investigated whether grafted scn regulates the circadian oscillator in peripheral organs using the scn-transplanted mice that have a limited time information transmission paths. as a result, the grafted scn restored not only circadian behavior rhythm but also the circadian rhythms of peripheral organs. many of clock genes showed dynamic oscillations with identical phase relationship as shown in intact animals, however, per and per showed low amplitude of oscillation. the findings suggest that diffusible signal molecules released from the transplanted scn entrain the circadian clock in peripheral organs and that they differentially modulate the expression of clock genes. sy - - - genome-wide analysis of adrenal-dependent and independent circadian regulation of mouse hepatic genes norio ishida clock cell biology group, national institute of advanced industrial science and technology (aist), ibaraki, japan recent progress in genome-wide expression analysis has identified hundreds of circadian regulated genes in the suprachiasmatic nucleus as well as in peripheral tissues of mammals. adrenal gland is important for circadian regulation for mammalian peripheral clocks. to identify circadian expressed genes regulated by adrenal glands pathways, we performed dna microarray analysis using hepatic rna from adrenalectomized (adx) and sham-operated mice. we identified genes that fluctuated between day and night in the livers, lost circadian rhythmicity in adx mice. these included the genes for key enzymes of liver metabolic functions such as glucokinase, hmg-coa reductase, and glucose- -phosphatase. the present study showed that the circadian expression of mouse liver genes is governed by core components of the circadian clock such as clock, and the other genes depend on adrenal glands pathway such as glucocorticoids. hitoshi okamura kobe university graduate school of medicine, japan light is a powerful synchronizer of the circadian rhythms, and bright light therapy is known to improve metabolic and hormonal status of circadian rhythm sleep disorders, although its mechanism is poorly understood. in the present study, we revealed that light induces gene expression in the adrenal gland via the suprachiasmatic nucleus (scn)-sympathetic nervous system. moreover, this gene expression accompanies the surge of plasma and brain corticosterone levels without accompanying activation of the hypothalamoadenohypophysial axis. the abolishment after scn-lesioning, and the day-night difference of light-induced adrenal gene expression and corticosterone release, clearly indicate that this phenomenon is closely linked to the circadian clock. the surge of plasma corticosterone after light exposure indicates that environmental light signals are instantly converted to glucocorticoid signals in the blood and csf. the light-induced clock-dependent secretion of glucocorticoids adjusts cellular metabolisms to the new light-on environment. sy - - - neuronal and hormonal control of peripheral clock function through suprachiasmatic nucleus shigenobu shibata, naomi hayasaka, takashi kudo, tsuyoshi yaita department of pharmacology, school of science and engineering, waseda university, tokyo, japan the clock genes are expressed not only in the suprachiasmatic nucleus (scn) of the hypothalamus where the master clock exists, but also in other brain regions and various peripheral tissues. in the liver and lung, clock genes are abundantly expressed and show clear circadian rhythm. although oscillation of clock genes in the liver and lung is controlled under the circadian clock mechanism in the scn, we do not know the resetting signals on peripheral clock function. communication between the scn and peripheral tissues occurs through various systems involving the sympathetic, nicotinic and glucocorticoide functions. this symposium mainly describes both anatomical and physiological experiments to reveal the sympathetic and glucocorticoid control over peripheral clock function. sy - - - a to z of gene transfer with adenoviral vector-application to neuronal birth date-specific gene transfer using replication-defective adenoviral vectors, we successfully performed 'pulse gene transfer' into progenitor cells in a neuronal birth date-specific manner. when adenoviral vectors were injected into the midbrain ventricle of mouse embryos between embryonic days (e) . to e . , the adenoviral vectors introduced a foreign gene into a specific cohort of birth date-related progenitor cells. this technique allows us to distinguish a cohort of birth date-related progenitor cells from other progenitor cells with different birth dates and to introduce a foreign gene into specific subsets of neurons by performing adenoviral injection at specific times. this adenovirus-meditated gene transfer technique will enable us to examine the properties of each subset of progenitor cells that share the same neuronal birth date. i will explain directions how to use an adenoviral vector and application of an adenoviral vector in my talk. research funds: crest sy - - - live imaging in the specific neuronal cells by the combination of transgenic mice and viral vectors kaori kashiwagi, naoaki saito lab. mol. pharmacol. biosig. res. ctr, kobe univ, kobe, japan live imaging analysis has revealed that each protein kinase c (pkc) subtype shows spatio-temporally distinct targeting in response to various stimuli. we demonstrated that the trans-synaptic stimulation induced translocation of ␥pkc-gfp in cerebellar slices from bitransgenic mice (nse-tta/tetop-␥pkc-gfp) which express ␥pkc-gfp in time and region-specific manner. this translocation was not restricted, but propagated from the distal to the proximal dendrites close to the soma of purkinje cells. in order to gain further insight in to the molecular mechanisms of pkc translocation, we introduced viral vectors to primary cultured purkinje cells. the propagative ␥pkc-gfp translocation was also observed in cultured purkinje cells derived from nse-tta mice. the molecular mechanisms of pkc translocation in purkinje cells were analyzed by live imaging with various kinds of viral vectors. the combination of tg mice and viral vectors is useful to understand the physiological role of pkc in the specific neuronal cells. research funds: kakenhi ( ) sy - - - visualization and manipulation of the signaling systems in the cns using sindbis viral vectors sho kakizawa dept. of pharmacol., grad. sch. of med., the university of tokyo, tokyo, japan virus vectors can efficiently deliver genes to neurons and other cells in the nervous systems in vitro and in vivo. because many viral vectors are in common use, it is important to select the best viral vector for each specific application, and a number of factors must be considered when making a decision. sindbis virus is an enveloped plus-strand rna virus belonging to the alphavirus genus of the togaviridae family. the sindbis viral vector is characterized by its effective, rapid, high-level and preferential transduction of neurons. these facts indicate that the vector is a powerful tool for the robust expression of target genes in the specific population of neurons. in this symposium, we will introduce our recent topics on the synaptic functions in the cerebellar systems revealed by visualization and manipulation of signaling molecules, such as nitric oxide and inositol , , -trisphosphate, in the cerebellar purkinje cells. research funds: grant-in-aid for scientific research on priority areas-molecular brain science-from the ministry of education, culture, sports, science and technology of japan sy - - - rescue of phenotypes of null-mutant mice by virus vector-mediated gene transfer kazuhisa kohda, wataru kakegawa, kyoichi emi, michisuke yuzaki dept. of physiol., keio univ. sch. of med., tokyo, japan even after the completion of genome project in major species, functions of many molecules remain uncharacterized. a transgene-based rescue approach is one of the powerful methods to decipher the mechanisms of actions of an orphan receptor; however, it is quite labor intensive and time consuming. here, we have developed a virus vector-based rescue approach and applied to investigate the mechanisms of action of the orphan glutamate receptor ␦ (glur␦ ) in the cerebellum. by introducing a sindbis virus carrying a wild-type glur␦ into glur␦ -null cerebellum in vivo, we could rescue abnormal phenotypes, such as impaired long-term depression at parallel fiber-purkinje cell synapses. by examining whether a mutant glur␦ lacking a specific domain could similarly rescue the phenotypes, we could evaluate functional importance of the domain. alternatively, by introducing a partial sequence of the gene of interest into wild-type brain and examining its dominant-negative effect, we will be able to identify the region of the gene product that is functionally important. research funds: kakenhi sy - - - gene transfer into in vivo cerebellar purkinje cells by hiv-derived lentiviral vectors hirokazu hirai advanced science research center, kanazawa university, ishikawa, japan cerebellar purkinje cells are key elements regulating motor coordination and motor learning. gene transfer into purkinje cells is an effective approach for the study of cerebellar function and treatment against cerebellar disorders. although adenoviral vectors or sindbis vectors are frequently used for gene delivery into neurons, the former has extremely low affinity for purkinje cells, while the latter causes substantial damage to the infected cells. to achieve effective gene transfer into purkinje cells, we used human immunodeficiency virus (hiv)-derived lentiviral vectors. purkinje cells were efficiently transduced without significant influence on the cell viability and synaptic functions. gene expression was also detected, though less efficiently, in other cortical cells, whereas no transduced cells were observed outside of the cerebellar cortex. these results suggest that hiv-derived lentiviral vectors are useful for the study of gene function in purkinje cells as well as for application as a gene therapy tool for the treatment of diseases that affect purkinje cells. research funds: jst/presto, kakenhi ( ) sy - - - physiological basis for stereotaxic surgery in basal ganglia atsushi nambu division of system neurophysiology, national institute for physiological sciences, and school of life science, the graduate university for advanced studies, okazaki, japan stereotaxic surgery in movement disorders such as parkinson's disease dramatically improves the symptoms of such diseases. i assume the physiological basis for the treatment is to disrupt abnormally increased information flow through the basal ganglia. i will discuss the pathophysiology of basal ganglia disorders and the effect of stereotaxic surgery in light of the three major pathways in the cortico-basal ganglia loop, i.e., hyperdirect (cortico-stn-gpi), direct (cortico-striato-gpi) and indirect (cortico-striato-gpe-stn-gpi) pathways, that dynamically control the activity of the thalamus and cortex to perform correct motor programs in correct timing. research funds: kakenhi ( ) sy - - - deep brain stimulation of subthalamic nucleus on parkinson's disease fusako yokochi department of neurology, tokyo metropolitan neurological hospital, tokyo, japan parkinson's disease is a progressive and degenerative disease caused by dopamine deficiency attributed to the degeneration of neurons in the substansia nigra. consequently, various symptoms appear, such as cardinal symptoms, those in the advanced stage and those as the side effects of long-term levodopa therapy. many antiparkinsonian drugs have been developed, but all of the symptoms are not improved by these drugs. stereotaxic surgery was started for treating severe tremor and rigidity in the mid- th century. stimulation by chronically implanted electrodes in the brain, that is, deep brain stimulation (dbs), has recently been applied and has been shown to have marked effects on the symptoms resisted to conventional treatments. in particular, dbs of the subthalamic nucleus (stn) is an effective treatment for the symptoms. much basic research on the function of stn has been reported, but stn function is still unclear. clinical outcomes including the side effects of stn dbs, the neural activities recorded from stn and the localization of clinical effects are reported in this paper. sy - - - firing patterns of basal ganglia neurons and effects of deep brain stimulation in parkinson's disease takao hashimoto center for neurological diseases, aizawa hospital, matsumoto, japan high-frequency electrical stimulation of the subthalamic nucleus (stn), internal segment of the globus pallidus (gpi) and thalamus can improve motor signs in patients with parkinson's disease, however, its mechanism of action remains unclear. a leading hypothesis regarding the development of movement disorders of basal ganglia origin suggests that hyperkinetic and hypokinetic disorders occur as a result of changes in the mean discharge rate in the gpi and substantia nigra, which in turn suppress thalamocortical output. on the other hand, altered firing patterns in the basal ganglia have been reported in mptp-induced parkinsonian animals: increases in bursting activity and periodic oscillatory activity in the gpi and stn, and synchronization of gpi, or gpi and striatal neurons. synchronous oscillation in the basal ganglia may break down independent processing in the motor circuit and disrupt signal processing at the cortical level. kaoru takakusaki department of physiology, asahikawa medical college, asahikawa, japan locomotion is composed of volitional and automatic processes. particular attention is required to perform volitional processes such as avoiding obstacles and accurate limb placements during locomotion. however, we are largely unaware of the automatic control processes of rhythmic limb movements, muscle tone and postural reflexes that accompany locomotion. because each process is seriously impaired in parkinsonian patients, the basal ganglia must play a crucial role in integrating the volitional and automatic processes of locomotion. the basal ganglia contribute to the planning and execution of voluntary movements via basal ganglia thalamocortical loops. on the other hand, recent our findings suggest the importance of the direct basal ganglia outflow to the brainstem where fundamental neuronal networks for controlling postural muscle tone and locomotion are located. in this presentation we discuss the role of the basal ganglia in the integration of volitional and automatic movements during locomotion, which has been less understood aspect of gait control. research funds: grant-in-aid for scientific research (c) and priority area (area no. ) sy - - - characteristics of neuronal activity within the globus pallidus interna (gpi) in patients with dystonia yoichi katayama , department of neurological surgery, nihon university school of medicine, tokyo, japan; division of applied system neuroscience, nihon university graduate school of medical science, tokyo, japan dystonia represents disordered muscular tonicity of the trunk and/or extremities, and is often dramatically controlled by chronic gpistimulation in humans, indicating that dystonia is attributable to certain abnormal activity of gpi neurons. little is yet known, however, regarding characteristics of neuronal activity within the gpi underlying dystonia. we analyzed activity of gpi-neurons in patients with dystonia or parkinson's disease, which were recorded during surgery for chronic gpi-stimulation. as compared to gpi neurons in patients with parkinson's disease, gpi neurons in patients with dystonia were distinctive with following three characteristics; firing rate ( . ± . hz, n = ) was low, firing pattern was often composed of irregular pauses and bursts, and many were responsive to body movement with wide receptive fields. these findings suggest that dystonia may be related to unstable movement-related sensory processing within the gpi. it has been considered that dystonia, which is generally characterized by postural abnormalities and involuntary movements including torsion, is caused by dysfunction of the basal ganglia. the purpose of the present work is to clarify the neural mechanisms underlying the onset of dystonia by analyzing the pathophysiology of a model for torsion dystonia, wriggle mouse sagami (wms). the genomic mutation of wms occurs in the gene encoding plasma membrane ca + -atpase isoform that is located on the th chromosome. recent immunohistochemical and electrophysiological investigations on wms have shown that ( ) d -type dopamine receptors are downregulated presynaptically in the striatum, and ( ) a large number of purkinje cells in the cerebellum express tyrosine hydroxylase (the synthesizing enzyme for dopamine) and their excitability is greatly reduced. in this symposium, the possible correlation between these data and dystonic phenotypes will be discussed. kei-ichiro maeda reproductive science, graduate school of bioagricultural sciences, nagoya university, nagoya, japan gnrh has been well established to regulate reproduction through two modes of secretion: surge and pulse. the surge mode is female-specific and induces lh surge and then ovulation through positive feedback action of estrogen. the pulse mode tonically activates gonads in both sexes with being negatively regulated by gonadal steroids. environmental cues, such as photoperiod or nutrition, are considered to affect reproductive activity through altering pulse mode of gnrh release. pulse mode seems much more robust than surge, because estrogen can induce a surge under a certain condition when the pulse is suppressed. the following four papers aim to unveil the physiological mechanism underlying two modes of gnrh secretion in various experimental models. sy - - - metastin: a neuropeptide playing a central role in the regulation of ovulatory cycle hiroko tsukamura, kei-ichiro maeda graduate school of bioagricultural sciences, nagoya university, japan estrous cyclicity is regulated by a sequence of neuroendocrine events consisting of hypothalamus-pituitary-gonadal axis. gonadotropinreleasing hormone (gnrh)/luteinizing hormone (lh) surge is induced by positive feedback action of estrogen secreted by mature ovarian follicles. the central mechanism of positive feedback action of estrogen on gnrh/lh secretion, however, is not fully understood yet. metastin was first isolated as a natural ligand for a g-proteincoupled receptor, gpr ( . nature , ) . recent studies reported that a genetic alteration leading to homozygous loss of function of gpr impairs pubertal development in mice and human. we have first demonstrated that endogenous metastin plays a physiological role in inducing ovulation through stimulating gnrh/lh surges to control estrous cyclicity in the female rat ( . endocrinology , ) . the present paper focuses on the role of metastin in regulating gnrh/lh surge based on our recent study in rats and discusses possible mechanism underlying positive feedback action of estrogen. suzanne moenter, catherine christian university of virginia, usa a surge in gonadotropin-releasing hormone (gnrh) secretion is the cns signal that triggers the luteinizing hormone (lh) surge, which causes ovulation. the gnrh surge depends on a switch in estradiol (e) feedback from negative to positive and in rodents on time of day, occurring in the pm. treating ovariectomized (ovx) mice with a constant release e capsule (ovx+e) elicits daily pm lh surges; there is no diurnal change in ovx controls. likewise, extracellular recordings of firing activity of gfp-identified gnrh neurons showed no diurnal changes in cells from ovx mice. in contrast, e increased firing in the pm compared to am. gabaergic neurons form a major input to gnrh neurons, and activation of the gabaa receptor can be excitatory in these cells. whole-cell patch-clamp recordings of synaptic activation of gabaa receptors on gnrh neurons revealed e-dependent decreases in transmission during the am (negative feedback) and increases in transmission near surge onset that persisted for some populations of afferents thru the surge peak. together these data indicate one mechanism by which e induces the gnrh surge is by altering gaba transmission to gnrh neurons. yoshitaka oka grad. sch. sci., univ. of tokyo, tokyo, japan the gonadotropin-releasing hormone (gnrh) peptidergic neuronal systems consist of hypothalamic neuroendocrine and extrahypothalamic neuromodulatory gnrh neurons. here, i introduce our recent studies on the physiological properties of neuroendocrine preoptic (poa) gnrh neurons in comparison with the neuromodulatory terminal nerve (tn) gnrh neurons. to study the both types of gnrh neurons, we use a fish model system in which we can easily identify both of them in intact brain preparations in vitro, which is a great advantage over most other vertebrates. the poa-gnrh neurons had quite different basic electrophysiological membrane properties from those of tn-gnrh neurons and showed alternating active and silent phases of firing activities, in contrast to the regular pacemaker activities of tn-gnrh neurons. now that we have various experimental approaches (electrochemical measurement of gnrh release, ca + imaging after single-cell electroporation, single-cell rt-pcr, double patch clamp recording, etc.) in hand, simultaneous multidisciplinary approaches should be possible to study the physiology of gnrh neurons. research funds: kakenhi sy - - - metabolic regulation of puberty onset using the prepubertal rat model helen i'anson biology department and neuroscience, washington and lee university, usa we hypothesize that glucose availability determines the timing of puberty onset in rats. abdominal fat stores are low in dieted, prepubertal female rats with delayed puberty, suggesting that such rats may be particularly sensitive to dietary fuel changes. such rats are fed a single daily meal and demonstrate decreased blood glucose levels between meals. we hypothesized that such daily hypoglycemic bouts delay onset of puberty during dieting. when glucose supplement was given to prepubertal dieted rats, and they exhibited first estrus with similar timing to previously dieted re-fed rats. conversely, when dieted rats were re-fed ad libitum and simultaneously glucose-deprived, first estrus was delayed. blood glucose levels during glucose-supplementation which induced first estrus, and during glucoprivation and re-feeding which delayed first estrus, were similarly elevated, suggesting that central, rather than peripheral, glucose levels are monitored in the prepubertal animal. in summary, central glucose availability may be an important signal timing puberty onset. research funds: jeffress memorial trust and washington and lee university sy - - - molecular mechanisms of thyroid hormone action in developing brain toshiharu iwasaki, noriyuki koibuchi department of integrative physiology, gunma university graduate school of medicine, maebashi, japan thyroid hormone (th) plays an important role in the developing brain. the action is mainly exerted by controlling gene expression through binding to its specific receptor, th receptors (trs). trs are ligand-regulated transcription factors that are expressed in many organs, including brain. trs bind to target dna sequences known as th-response element (tre). coactivators and corepressors are involved in the tr-mediated gene regulation through histone modification. we characterized the coactivator and the corepressor that were expressed in embryo brain. although precise mechanism of the th action for brain development has not been fully clarified, these cofactors may be involved in these actions. th affects brain development only during a limited period, which is referred as the critical period of th action. recently, it has been speculated that environmental chemicals may cause the abnormal brain development. possible mechanism of action of such chemicals on tr system will be discussed. research funds: kakenhi , sy - - - thyroid hormone and organic anion transporters in brain hiroyuki kusuhara, yuichi sugiyama graduate school of pharmaceutical sciences, the university of tokyo, tokyo, japan organic anion transporting polypeptides (oatps/slco) currently consist of isoforms in human and rodents. they are initially identified as part of detoxification system in the body, and involved in the tissue uptake of xenobiotics, especially amphipathic organic anions. some members accept a variety of structurally unrelated compounds as substrate. oatp c is characterized by its unique substrate specificity, highly selective for thyroid hormones, particularly for t and reverse t , but the transport activity of t is quite low. it is predominantly expressed in the brain capillaries and choroid plexus, acting as barrier of central nervous system, where oatp a , the transport activities of t and t are similar, is also expressed. the uptake of t and t by the brain determined using in situ brain perfusion technique was saturable and inhibited by oatps substrates and inhibitors. these two transporters may play a role in regulation of brain levels of t and t . research funds: the advanced and innovational research program in life sciences from the ministry of education, culture, science and technology sy - - - alterations of gene expression profiles in the developing brain by chemicals disrupting thyroid hormonedependent signals takayuki negishi , masaki takahashi , yasuhiro yoshikawa , tomoko tashiro department of chemistry and biological science, aoyama gakuin university, kanagawa, japan; department of biomedical science, the university of tokyo, tokyo, japan there is increasing concern about the possibility that environmental chemicals such as polychlorinated biphenyl (pcb) and its hydroxylated metabolites interfere with normal brain development through acting as thyroid hormone disrupting agents. in this presentation, based on comprehensive dna microarray analysis, we demonstrate alterations in gene expression in the brain of neonatal rats perinatally exposed to -hydroxy- , , , , pentachlorobiphenyl (anti-thyroid hormone-like), as well as in primary cultured rat hippocampal neurons exposed to -hydroxy- , , , , , , -heptachlorobiphenyl (thyroid hormone-like). among genes whose mrna expression levels were affected by these compounds, a number of genes essential for the establishment of synaptic networks were detected, suggesting that long-lasting effects on higher brain functions may result from exposure of the developing brain to these compounds. hydroxylated metabolites of pcbs (oh-pcbs) have chemical structures similar to thyroid hormones (ths). we reported that low doses of two types of oh-pcb inhibited th-dependent extension of purkinje cell dendrites ( . dev. brain res. , ) . koibuchi et al. ( ) clarified that they interfere with th-dependent gene expressions in reporter gene assays. further, takasuga et al. ( ) detected some pcb and oh-pcb congeners in human csf, of which oh-cb is the highest concentration. to determine its effects on developing neurons, we examined oh-cb in rodent cerebellar culture cells. interestingly, oh-cb promoted dendritic development of purkinje cells in the absence of th and increased significantly their survival numbers. these results indicate that oh-pcb congeners may disrupt normal brain development by different mechanisms depending on their chemical structures. we have reported that rat pups exposed to an antithyroid agent, propylthiouraci l (ptu), via maternal milk exhibit hyperactivity, impairment in spatial learning and social interaction, and audiogenic hypersensitivity extending to audiogenic seizures, thus this ptu rat can be a possible candidate of animal model for autism. in ptu rats, the delay in migration of the extragranular cells of cerebellum, and in innervation from inferior olive nuclei to purkinje cells were shown. these neurons transiently expressed serotonin-ir, therefore we treated ssri or -htp to examine the relevance of serotoninergic function. the treatment of -htp but not ssri recovered the delay of cell migration. these effects of serotonin manipulation in ptu rats on the behavioral impairment and the development of cns will be discussed. it is hoped that embryonic stem (es) cells will be used in transplantation therapy for neurological diseases. however, because grafts of neural stem cells derived from es cells may contain residual undifferentiated cells, there would be a risk for teratomas. to reduce this risk, we applied to es cells herpes simplex virus thymidine kinase (hsv-tk) gene and ganciclovir (gcv) treatment. stable mouse and cynomolgus monkey es cell lines expressing hsv-tk were obtained. gcv sensitivity was higher in undifferentiated es cells than in es cell-derived neural stem cells. es cell-derived neurons were resistant to gcv treatment. nude mice with transplants of undifferentiated es cells expressing hsv-tk formed teratomas, but the tumor growth was suppressed after the gcv treatment. suicide gene delivery might increase the safety of the use of es cells in cell replacement therapy. enzymatic degradation of chondroitin sulfate is known to promote axonal regeneration in the central nervous system. the physiological role of chondroitin sulfate up-regulated after injury was examined in the nigrostriatal dopaminergic system which was unilaterally transected and treated with chondroitinase abc. in transected mice, dopaminergic axons did not extend across the lesion. chondroitin sulfate was up-regulated around the lesion and a fibrotic scar containing type iv collagen deposits were developed in the lesion center. in chondroitinase abc-treated mice, numerous dopaminergic axons were regenerated across the lesion. in these animals, chondroitin sulfate immunoreactivity was remarkably decreased and the formation of a fibrotic scar was unexpectedly prevented. these results support our previous supposition that chondroitin sulfate does not act as an obstacle to regenerating axons, but involved in the repair process of the brain injury including the formation of the fibrotic scar (kawano et al., ) . reference kawano et al., . j. neurosci. res. , - . research funds: kakenhi os a- - neurotransmitters that maintain and suppress the tonic firing of the serotonergic neurons in the dorsal raphe during sleep waking cycles yoshimasa koyama , kazumi takahashi , yukihiko kayama cluster of science and technology, fukushima university, fukushima, japan; department of physiology, fukushima medical university, fukushima, japan the present experiment was done to examine, under unanesthetized natural sleep-waking condition, which neural systems were involved to regulate the firing of the serotonergic ( ht) neurons in the dorsal raphe (dr) during sleep waking cycles. using head restrained, unanesthetized rats, single neuronal activity was recorded and each drug was applied iontophoretically or by pressure close to the recording neurons. spontaneous firing of the ht neurons in dr were excited by glutamate and orexin a or b. they were inhibited by noradrenaline. an ␣ receptor agonist (phenylephrine or methoxamine) increased the firing rate during sws or ps, but had no effect when applied during w. in ps-off type dr neurons, cessation of firing during ps was recovered by bicuculline, however in the dr neurons that did not stop firing during ps, bicuculline had almost no effect. os a- - correlation between regional grey matter volume and proficiency increase in second language: a vbm study arihito nauchi , , kazuyoshi hirano , , yukimasa muraishi , , kuniyoshi l. sakai , department of basic science, university of tokyo, tokyo, japan; crest, jst, japan; secondary education school, university of tokyo, japan although neuroimaging studies have contributed to clarify the brain function, the neural basis of individual variation in cognitive abilities such as language still remains unknown. in the present study using voxel-based morphometry (vbm), a whole-brain unbiased technique for detecting regional differences in mr images, we examined the relationship between the proficiency increase in second language (l ) and grey matter volume among students aged - , who received special classroom training in the use of english verbs. in specific regions including the left lateral premotor cortex and the left inferior frontal gyrus (the grammar center), we found a positive correlation between the regional grey matter volume and improved performance for the grammaticality of english sentences. these results suggest an anatomical basis for the language faculty, such that the capacity of a specific region is related to proficiency increases in l . os a- - grammar center activation in honorification judgment of japanese sentences kanako momo , , kuniyoshi l. sakai , department of basic science, university of tokyo, tokyo, japan; crest, jst, japan one linguistic theory proposes that japanese honorification is a syntactic feature, because syntactic agreement is required between subject/object and honorific forms. to investigate whether such syntactic processing is actually realized in the brain, we examined cortical activation using fmri under several types of normal/anomalous judgment for japanese sentences including honorification. when activation in a honorification task was contrasted with that in a spelling task, we observed significant increase in the left including grammar center (ifg) as well as the bilateral cerebellum for all tested participants. moreover, the lower performance group showed greater activation in the left f op/f t and the bilateral cerebellum. these results suggest that syntactic process is required for japanese honorification and that activation in these regions shows modulation according to performance level, even in native language. research funds: crest, jst os a- - top-down modulation for melody-related activity in the right auditory areas: an meg study takuya yasui , , , kimitaka kaga , kuniyoshi l. sakai , dept. of basic science, univ. of tokyo, tokyo, japan; dept. of otolaryngology, univ. of tokyo school of medicine, japan; crest, jst, japan we previously reported right-hemisphere dominance for melody error-induced fields (m ) (neurosci. res. , s ). in a subsequent study, we confirmed that m was independent from mismatch negativity usually induced by oddballs. in the present study, we examined whether m was induced by deviation from a memorized melody. we used four pairs of unfamiliar songs, each pair consisting of an original song and a modified song in which the third note deviated from that of the original. subjects learned these songs and judged whether there were one or two deviations in notes. there was no significant difference in dipole amplitudes between m elicited by the original songs and that by the modified ones. however, while m without the deviation showed no significant effect of lateralization, m with the deviation resulted in significant enhancement in the right hemisphere. these results suggest the existence of memory-induced, i.e., top-down modulation for melody-related activity in the right auditory areas. research funds: crest, jst os a- - cortical plasticity in adulthood for learning phonics rules for english orthography and phonology makiko muto , , kuniyoshi l. sakai , department of basic science, university of tokyo, tokyo, japan; crest, jst, tokyo, japan although matching english orthography with correct pronunciation is difficult for second language learners, learning phonics rules may rapidly improve their performance. in the present fmri study, we tested an english matching task during the course of phonics training in sessions, in which infrequent words were visually shown, while matched/unmatched speech sounds were simultaneously presented. comparing the first half of the sessions with the latter half, the left posterior inferior temporal gyrus (the letter center) and a part of the left lateral premotor cortex (the grammar center) showed activation decreases, when the performance was significantly improved. these results suggest that the plasticity of functional systems involving these critical regions is essential for establishing phonics rules and for forming a new link between orthography and phonology. research funds: crest, jst os a- - hierarchical syntactic processing in the left frontal region: an meg study kazuki iijima , , naoki fukui , kuniyoshi l. sakai , dept. of basic science, univ. of tokyo, komaba, japan; crest, jst; dept. of linguistics, sophia univ., yotsuya, japan previous erp studies have shown word-related activation based on semantic association or context. however, it remains unclear how syntactic information of preceding words is integrated into the ongoing sentence processing. in the present meg study, we measured brain activity during each of four tasks: a syntactic task, a semantic task, a memory task, and an evaluation task. sentence stimuli consisted of one noun phrase and one verb, where the noun phrase had either an objective or nominative case particle. the first peak of the activity for a verb presentation was observed at the left frontal region as early as ms after the onset. in the objective-case condition, this activity was enhanced only for the syntactic task, while in the nominative-case condition no such task-selectivity was observed. these results are consistent with the current linguistic theory (the minimalist program), which holds that a noun phrase with an objective case particle is directly merged with a verb, to form a new hierarchical level. research funds: crest, jst os a- - individual difference of brain activity in medial prefrontal cortex and superior temporal sulcus during social cognition koji jimura, seiki konishi, tomoki asari, junichi chikazoe, yasushi miyashita dept. physiol. univ. tokyo sch. med., japan previous neuroimaging studies have reported brain activity in the medial prefrontal cortex (mpfc) and the superior temporal sulcus (sts) during performance of theory of mind tasks. the present fmri study explored individual difference of the mpfc and sts activity by employing false belief paradigms. the task consists of two sessions, study and test. during the study session, subject studied a brief story in which two characters have false beliefs. then, the subject answered questions about the false belief and the fact that constitutes the false belief during the test session. consistent with previous studies, significant activity was observed in the mpfc and the sts during representing the false belief. the individual differences of the mpfc and the sts activity were correlated with psychodiagnostic indices that represent controlled and automatic idealization, respectively. these results suggest that the two indices represent distinct neural mechanisms participating in social cognition. research funds: grant-in-aid from mext ( ), jsps research fellowship ( ) os a- - brain activity of happy facial recognition in mother-daughter relationship jun shinozaki , nobukatsu sawamoto , toshiya murai , takashi hanakawa , hidenao fukuyama hbrc, kyoto univ. grad. sch. of med. kyoto, japan; neuropsychiatry, kyoto univ. grad. sch. of med. kyoto, japan relationship between parents and children is special, and affective facial recognition between them should evoke specific neural activity not shared by other personal relationships. eleven healthy females participated in this fmri experiment. the subjects saw happy and neutral faces of their own mothers, and newly learned other subjects' mothers during the scan. when happy face recognition was compared with neutral face recognition, the mother-daughter combination induced greater activity than the non-familial combination in the following areas; the lateral prefrontal cortex, anterior cingulate cortex, middle temporal cortex, striatum, and anterior insula. it has been shown that the lateral prefrontal and anterior cingulate cortices are associated with familial facial recognition, whereas the middle temporal cortex is related to happy facial recognition. the activity in the striatum and anterior insula might be related to positive affection and empathy, respectively. os a- - anatomical connections among functionally identified brain regions for sentence processing yukari yamamoto , , atsushi maki , , kuniyoshi l. sakai , advanced research laboratory, hitachi, ltd., tokyo, japan; crest, japan science and technology agency, saitama, japan; dept. of basic science, univ. of tokyo, tokyo, japan we have functionally identified the left dorsal inferior frontal gyrus (ifg), the left lateral premotor cortex, and the triangular/orbital part of the left ifg (f t/f o) as regions associated with sentence and discourse level processing. in the present study, we examined whether there are direct anatomical connections among these regions by using diffusion tensor tractography. f t and f o of the left ifg were chosen as seed areas for fiber tracking. fiber bundles that went through two spherical regions were extracted from the tracking data. the central coordinates of these regions were (− , , ) , (− , , ) , and (− , , − ) in the standard brain, which are associated with syntactic processing (the first and second coordinates) or sentence comprehension (the third coordinate). direct connections among these regions were consistently observed among the subjects. this result suggests a critical network among multiple regions that are associated with sentence processing. os a- - effect of the incongruity controlled by semantic distance on visually evoked magnetic fields nobuyoshi harada , sunao iwaki , mitsuo tonoike aist, osaka, japan; chiba university, chiba, japan visual incongruities of heads changed on animal pictures, which were controlled by the semantic distance of the word of the animal, were investigated on visually evoked magnetic fields. the semantic distance was decided by the numbers of links of the semantic network of the taxonomic layer in a japanese thesaurus. the words for mammalians were grouped into five semantic categories in the thesaurus. the heads of the animals were changed with those from another semantic category (deviant, d = ), and with those from an inner semantic category (middle, d = ), while others were not changed (normal, d = ). peak amplitudes of waveforms of the root mean square values on the components of ms (f ( / ) = . , p = . ) and ms (f ( / ) = . , p = . ) were significantly decreased with increments of the semantic distance in left occipital sensors. the gradient of the decreasing line of the amplitudes of the and ms components indicated the capability of extracting the structure of a typical prototype of the form of the animal. we call this capability, the structural sensitivity for prototype (ssp). ryohei yasuda duke university medical center, usa calcium signaling in dendritic spines is important for many forms of synaptic plasticity. however, the quantitative mechanisms of how calcium elevations are translated into spatial and temporal patterns of biochemical reactions leading to modifications of synaptic strength are unclear. identifying and following the spatiotemporal activation of molecules necessary for synaptic plasticity is crucial for a better understanding of this complex process. to visualize the activity of signaling pathways in neurons deep in brain tissues, we have combined fluorescence lifetime measurements and two-photon microscopy. this technique allowed us to measure spatiotemporal aspects of the activity of signaling proteins including ras gtpase proteins in response to physiologically relevant stimuli with single spine resolution. research funds: burroughs wellcome fund, dana foundation os p- - mechanisms of p y purinoceptor-mediated long-term enhancement of inhibitory transmission examined by multiple-probability fluctuation analysis at cerebellar gabaergic synapses yumie ono , xiaoming zhu , takashi tominaga , fumihito saitow , shiro konishi , waseda-olympus bioscience research institute, singapore, singapore; department of neurophysiology, tokushima bunri university, kagawa, japan; department of pharmacology, nippon medical school, tokyo, japan postsynaptic p y receptor activation by atp enhances ipscs at cerebellar interneuron-purkinje cell (pc) synapses. to investigate the underlying mechanisms, we here employed the non-stationary fluctuation analysis to estimate the number (n) and single channel conductance (i) of gaba a receptors in pcs using whole-cell recordings of evoked ipscs in pcs of rat cerebellar slices before and - min after application of atp. the atp-induced enhancement of the ipsc amplitude was associated with a significant increase in the single channel conductance, but not the number, of gaba a receptors in pcs: i and n after atp treatment were ± . % and ± . % of the controls, respectively. pretreatment with the protein kinase a inhibitor h- , but not the calmodulin kinase ii inhibitor kn- , completely abolished the atp-induced ipsc enhancement. os p- - activin induces long-lasting nmda receptor activation via scaffolding pdz protein arip isao inoue, akira kurisaki, hiromu sugino institute for enzyme research, tokushima university, tokushima, japan calcium entry into the postsynaptic neuron through nmda type glutamate receptors (nmdars) triggers the induction of long-term potentiation (ltp). the ca + permeability of nmdar is regulated by phosphorylation of its tyrosine residues. we report here that activin, a member of the transforming growth factor-b (tgf-b) superfamily, and one of proteins synthesised after ltp, promotes phosphorylation of nmdars and increases the ca + influx through those receptors in primary cultured rat hippocampal neurons. this signal transduction occurs in a functional complex of activin receptors, nmdars, and src family tyrosine kinase, fyn formed on a multimer of postsynaptic scaffolding pdz protein, activin receptor interacting protein (arip ). activin-induced nmdar activation persists more than h, which is complimentary to the transient activation of nmdars by brain derived neurotropic factor (bdnf). our results show that activin is a long-lasting potentiator involved in synaptic plasticity regulatory mechanisms. os p- - roles of cam kinase i in the hippocampal longterm potentiation kohji fukunaga, takashi komori, shigeki moriguchi department of pharmacology, graduate school of pharmaceutical sciences, tohoku university, sendai, japan cam kinase i (camki) family members are highly expressed in the adult rat hippocampus and camki-alpha is predominantly localized in the cytosol. camki activation requires phosphorylation of thr by camkk as an upstream kinase. we here documented a marked increase in camki-alpha-thr phosphorylation following ltp induction in rat hippocampal ca region. like camkii activation following ltp (fukunaga et al., ) , the increased camki-thr phosphorylation remained elevated at least for min after ltp induction. the increased camki-thr phosphorylation was closely associated with prolonged increases in phosphorylation of creb and myosin light chains in the ca region. this is in contrast with transient increases in camkiv and erk phosphorylation. treatment with camkk inhibitor, sto- significantly inhibited both creb and mlc phosphorylation with concomitant reduction of ltp in the ca region. taken together, camki likely mediates the late phase of creb phosphorylation and an increased mlc phosphorylation in the hippocampal ltp. to investigate how the excitatory postsynaptic inputs of the proximal dendrite effect the information processing of synaptic inputs at the distal dendrite, stimulation was applied to induce bap and epsp at the alveus and the proximal dendrite, respectively. the resulting coincidence of magnitude of bap and epsp at the distal dendrite was enhanced when the bap was delivered at a timing ( ms) to induce ltp. furthermore, the magnitude of bap at the distal dendrite was attenuated by the input from the proximal dendrite at a timing ( ms) to induce ltd. these results suggest that the magnitude of bap delivered to the distal dendrite can be amplified or attenuated depending on the relative timing between proximal input and bap. this may be due to an effect on the coding process at the distal dendrite and could support the basis for a novel learning rule in the brain. research funds: kakenhi ( ) os p- - mouse brains deficient in neuronal pdgf receptor- develop normally but are vulnerable to injury yoko ishii, takeshi oya, lianshun zheng, masakiyo sasahara department of pathology, faculty of medicine, university of toyama, japan the platelet-derived growth factors (pdgfs) and pdgf receptors (pdgfrs) are widely expressed in the mammalian cns. here, we developed novel mutant mice in which pdgfr- subunit gene was genetically deleted in the neurons of cns to elucidate the role of pdgfr-. our mutant mice reached adulthood without apparent anatomical defects. the cerebral damage after cryogenic injury was severely exacerbated in the mutants compared with the controls. furthermore, this exacerbated lesion formation was suggested to be, at least partly, due to the enhanced excitotoxicity after injury, because nmda-induced lesion formation was also extensively enhanced in the cerebral cortex of the mutants without altered nmda receptor expression. this is the first known report to address the postnatal function of pdgfr- expressed in cns neurons, using genetically engineered mutant. it was clearly demonstrated that pdgfr- expressed in neuron protects cns neurons from cryogenic injury and nmda-induced excitotoxicity. early postnatal days (especially the first three weeks in the rat) are the critical period for newborn hippocampal granule cells (gcs) to dynamically migrate from the dentate hilus and form the gc layer. to investigate the mechanism that regulates newborn gc migration, we developed a new slice coculture system. the hilar parts of entorhino-hippocampal slices prepared from postnatal six-day-old (p ) rats that had received a single brdu injection at p were substituted with the corresponding region of entorhino-hippocampal slices from p rats. after five days in vitro, newborn gcs, detected by brdu and prox , migrated out of the hilar graft and reached the host gc layer. chronic application of picrotoxin, a gaba a receptor antagonist, facilitated the migration of newborn gcs into the gc layer. these results indicate that gaba a receptors regulate the migration of newborn gcs in early postnatal days. os p- - cdk is required for neuroblast migration in the adult mouse brain yuki hirota , , , toshio ohshima , takuji iwasato , ashok b. kulkarni , hideyuki okano , , kazunobu sawamoto , , bridgestone lab. keio univ., tokyo, japan; dept. physiol., keio univ., tokyo, japan; dev. neurobiol. riken, tokyo, japan; behavioral gen. riken, tokyo, japan; nih, bethesda, usa; sorst, jst, saitama, japan neuroblasts generated in the subventricular zone (svz) of the lateral ventricles migrate into the olfactory bulb (ob) through the pathway called rostral migratory stream (rms). molecular mechanisms regulating the directional long-distance migration remain largely unknown. here we studied adult function of cyclin-dependent kinase (cdk ) that has been revealed to play a role in neuronal migration in the embryonic brain. crossing the floxed-cdk mice to emx cre mice resulted in decreased size of ob and abnormal distribution of neuroblasts. svz explants from these mice cultured in matrigel showed decreased migration distance. leading process of neuroblasts infected with cre-encoding retrovirus were found in random orientations and frequently failed to migrate out of the svz compared to control cells. these results indicate that cdk has a cell autonomous function in neuroblast migration in the adult brain. os p- - colocaliztion of neuron markers and glial markers in gabaergic neuron progenitors as revealed by singlecell microarray analysis shigeyuki esumi , wu sheng-xi , yuchio yanagawa , , kunihiko obata , nobuaki tamamaki kumamoto univ., kumamoto, japan; fourth military medical univ., xi'an, people's republic of china; gunma univ., maebashi, japan; sokendai, hayama, japan; riken, wako, japan gabaergic neurons and oligodendroglia share many characters in the murine forebrain. both of the cell types has been reported to originate in the medial ganglionic eminence and migrate to the neocortex. in addition, it is reported that they share several glial markers, such as ng , plp, and cnp at their prematured stages. in order to investigate its nature, we have established a single-cell microarray analysis method. single gfp-positive gabaergic neuron progenitors were corrected from the subventricular zone of the gad -gfp knock-in mouse neocortex at e -p by dissociation and picking. complemental dna from the single cells was amplified by universal pcr amplification and converted into biotin-labeled crna using t rna polymerase. after these procedures, crna sufficient for a microarray analysis was obtained. as the result we found, mbp and s - expression in the gabaergic neuron progenitors. os p- - role of -catenin signaling in regulating proliferation of transit-amplifying cells in the adult mouse subventricular zone kazuhide adachi , , , masanori sakaguchi , toru yamashita , , , yuko fujita , yukiko gotoh , arturo alvarez-buylla , takeshi kawase , hideyuki okano , kazunobu sawamoto , neurosurgery, keio univ. sch. med., tokyo, japan; bridgestone lab. dev. regenerative neurobiol., keio univ. sch. med., tokyo, japan; physiol., keio univ. sch. med., tokyo, japan; inst. mol. cell biosciences, univ. tokyo, tokyo, japan; neurosurgery, ucsf, san francisco, usa; neurol, okayama univ, med, dentistry and pharmaceutical sci, okayama, japan the subventricular zone (svz) continuously produces olfactory bulb neurons in the adult rodent brain. neural stem cells generate migratory neuroblasts via highly proliferative transit-amplifying cells in this region. here, we studied the role of -catenin signaling in the adult mouse svz. -catenin accumulated in the nucleus of only the transitamplifying cells in the svz. activated -catenin signaling promoted the proliferation of transit-amplifying cells, resulting in an increased number of new neurons in the olfactory bulbs. these results suggest that -catenin signaling plays a role in the proliferation of transitamplifying cells in the adult mouse svz. the ciliary marginal zone (cmz) is a region between the neural retina and ciliary epithelium, and contains retinal progenitor cells that give rise to neuron and glia. wnt b is expressed in cmz, and has been shown to control the differentiation of the retinal progenitor cells. we have isolated a novel bmp antagonist, chick tsukushi (c-tsk), which belongs to the small leucine-rich proteoglycan family. in the eye, the expression of c-tsk is observed in the cmz which is similar with that of wnt b. to examine the molecular interactions between c-tsk and wnt b, we co-electroporated them into the optic vesicle at stage - chick embryo and observed the proliferation of the retinal progenitor cells. we found that c-tsk inhibited the wnt b activity that sustains prolonged proliferation of retinal progenitor cells. our result suggests that c-tsk controls the proliferation of retinal progenitor cells interacting with wnt b. to reveal the role of epigenetic gene regulation in neuronal differentiation, we studied subcellular distributions of histone deacetylase (hdac) in developing cortical neurons. an expression vector of gfp-tagged hdac was transfected to dissociated cortical cell cultures as well as cortical neurons in vivo. hdac was primarily localized in nuclear until week in vitro, but was translocated to cytoplasm in the later stages. such translocation was found in a similar time course after birth in vivo. to examine a possibility that neural activity is involved in the translocation, firing activity of cultured neurons was examined using multi-electrode dishes. as a result, spontaneous firing activity was prominent in the late stages when cytoplasmic translocation occurred. however, ttx addition to the culture medium produced the inverse translocation. these results suggest that activity-dependent intracellular localization of hdac contributes to neuronal differentiation in cortical development. research funds: kakenhi ( ) dept. of physiology, fujita health univ. sch. of med., japan we described electrical synapses in alpha retinal ganglion cells (␣-gcs). precise temporal synchronization of spikes is generated from ␣-gcs (hidaka et al., ) . the fraction of open channels in gap junctions were evaluated with techniques of dual patch-clamp, connexin immunocytochemistry, and high-voltage electron microscopy. junction conductance (maximum . ns) was measured. in high-voltage electron microscopy (hitachi m, nips, , gap junctions (average size . m long) were present in contacts. in confocal laser-scanning imaging, connexin localization at contacts counted gap junctions (seven sites in a pair on average). assuming that the density of connexons would be /m and a single channel conductance is ps, the conductance of each junction would be ns. the presence of seven junctions between a pair will lead to estimate a total junction of ns. the measured conductance could allow to estimate a fraction of open channels as . %. the open fraction is small, when we consider whether electronic transmission acts to synchronize the spikes in the intercellular network. the visual system separates different types of information into parallel, anatomically distinct processing streams. despite their significance for visual processing, the molecular mechanism underlying the physiological stream formation is largely unknown, partially because these physiological streams have not been reported in mice. to identify molecular correlates of functionally distinct streams, we fabricated a custom cdna microarray of higher mammal ferrets. we successfully identified molecules whose unique distribution and developmental profiles define the lgn itself, its constituent layers, or identify cells comprising one of the physiological streams in the lgn. using these molecules as temporal and spatial markers, we investigated mechanisms of the physiological stream formation in the ferret lgn. research funds: kakenhi ( ), coe research akira muto, herwig baier department of physiology, university of california san francisco (ucsf), usa the visual system operates over a broad range of luminances. this is accomplished by adjustment of photosensitivity, called light adaptation. to study the molecular mechanisms of light adaptation, we screened for zebrafish mutants that showed compromised optokinetic responses (reflexive eye movements to large field motion) after an abrupt dark-to-light transition. in this experimental paradigm, wildtype fish larvae recover their full optokinetic response within about two minutes after being brought back to light. in a screen of almost genomes, we identified five mutants all of which showed substantially delayed recovery of the okr. positional cloning of one of the loci revealed a mutation in the dna-binding domain of glucocorticoid receptor (gr). gr is known for its role in the stress response, but its function in the visual system is unexplored. we propose that gr is regulating genes essential for light adaptation in the retina. os p- - multisite recording of the signal propagation pattern in the visual cortex makoto osanai, yusuke takeno, satoshi tanaka, tetsuya yagi graduate school of engineering, osaka university, suita, japan recently, the visual prosthesis systems with implanted stimulus electrode in the visual cortex are developed. but the signal propagation pattern induced by electrical stimuli in the visual cortex is not fully investigated. therefore, we studied the signal propagation pattern induced by electrical stimuli in the mouse visual cortex slice, using a channel multielectrode array and a calcium imaging system. in the electrophysiological study, the responses conducted vertically against the layer of the cortex with layer stimuli and propagated horizontally in the layer / . in the calcium imaging study, the area of the higher calcium concentration region spread vertically with layer stimuli. signal propagation was restricted within several tens m around the stimulus electrode by ap + cnqx administration and was completely blocked by ttx administration. administration of bicuculline increased the area of the signal propagation in a dose-dependent manner. we concluded that these restricted patterns of the signal propagation in the visual cortex were due to the inhibitory system. os p- - presence of two phases in the sensitive period of orientation plasticity shigeru tanaka , toshiki tani , kazunori o'hashi , , jerome ribot brain science institute, riken, saitama, japan; graduate school of life science and systems engineering, kyushu institute of technology, kita-kyushu, japan recently we have revealed that orientation-restricted visual experience induces drastic reorganization of orientation maps in the cat visual cortex. in this study, we examined the effect of release from single orientation exposure on once reorganized orientation maps during the sensitive period using intrinsic signal optical imaging. when kittens were returned to the normal visual environment by removing the goggles after weeks of goggle rearing starting around the age of weeks, the over-representation of the exposed orientation was preserved. on the contrary, when the goggle rearing started around the age of weeks and then the animals were returned to the normal visual environment, orientation maps rapidly changed to represent orientations equally. these findings indicate that the sensitive period of orientation plasticity consists of two phases: orientation map reorganization is irreversible in an early phase and reversible in a late phase. os p- - residual visuomotor processing in the animal model of blindsight: comparison with normal, near-threshold vision masatoshi yoshida , , , tadashi isa , , dept. dev. physiol., nat'l inst. physiol. sci., okazaki, japan; sch. life sci., grad. univ. adv. stud., hayama, japan; crest, jst, kawaguchi, japan in two macaque monkeys with unilateral v lesion performing a visually guided saccade task, saccadic parameters were compared between the saccades to the affected hemifield and those to the intact hemifield. the luminance contrast of the target presented in the intact hemifield was reduced so that the detectability was comparable to that in the affected hemifield ( - % correct). in the saccades to the affected hemifield, the curvature of the trajectories was smaller and the deviation of the saccadic end points from the target was larger than those to the intact hemifield. these results suggest that without geniculo-striate pathway, online compensation for the variation of the initial saccadic command is not fully functional, thus leading to inaccurate saccades. we propose that the residual visuomotor processing of monkeys with v lesion is unlike normal, near-threshold vision. research funds: kakenhi , kakenhi and crest, jst os p- - comparison of the angle representation in macaque visual areas v and v minami ito , , hidehiko komatsu , national institute for physiological sciences, okazaki, japan; the graduate university for advanced studies, hayama, japan previously, we have reported that fairly large number of area v neurons has angle selectivity. here, we studied the angle selectivity of area v , which is the major source of inputs to area v . we conducted single-unit recordings from the superficial layer of area v , while animals performed a fixation task. for comparison, we used a similar stimulus set. the stimuli were much larger than the size of the classical receptive fields. area v neurons responded mainly to sharp angles ( • ), straight lines ( • ) or right corners ( • ), but not to intermediate angles ( • or • angle width). this contrasted with area v , where neurons showed a variety of the optimal angle width including intermediate angles. we also observed several v neurons showed fine orientation tuning to short line segments, while weak or no responses were induced by a set of large angle stimuli. we suggest that area v neurons largely contribute to representing line components (lines and line-ends) and to sending such information to area v . os p- - firing rates and dynamic spatiotemporal patterns of ganglion cells both contribute to retinal information processing xin jin, ying-ying zhang, xue liu, hai-qing gong, pei-ji liang department of biomedical engineering, shanghai jiao tong university, shanghai, china population activities of retinal ganglion cells (rgcs) were recorded using a multi-electrode recording system. single unit analysis showed that firing rate of individual neuron was strongly dependent on the luminance intensity of stimulation. however, population activity of ganglion cells usually showed particular spatiotemporal pattern, in response to a specific velocity of the moving bar. differing from single direction-selective ganglion cell (dsgc), which responds to its preferred direction of movement by firing at its maximal rate, population activity of non-direction-selective ganglion cells may encode the motion information in a temporally ranked manner, independent to their individual firing rates. these results suggest that an efficient and economical coding mechanism may be employed by the retina, where the firing rate of individual neurons and spatiotemporal pattern of population neuronal responses could act in parallel to encode different aspects of visual information. yasuto tanaka, satoru miyauchi, masaya misaki brain information group, nict, kobe, japan visual long-range interaction was reported to be limited in space. here, we show the evidence of long-range interaction extending to an order magnitude larger using the right-left symmetrical configuration. two horizontally collinear gabor signals, one defined as probe and the other cue, were presented at the left and right side of the visual field at mirror symmetrical regions. detection threshold of gs probe reduced with cue-probe separations up to • . the facilitation was highly tuned to the symmetrical locus. furthermore, the facilitation was substantially longer at upper visual field than the lower visual field. the reduction was specific to orientation, phase, and horizontal direction, the results indicate long-range mirror symmetrical interaction across vertical meridian, suggesting symmetrical neuronal communication between early visual cortices. the anisotropy between left-right hemifield (symmetry) and upper-lower hemifield (upper-field advantage) signifies hemifield inhomogenity in human vision. os p- - integrity of visuospatial attention in a split brain patient noudoost behrad, seyed reza afraz, maryam vaziri, hossein esteky school of cognitive sciences (scs), iran transfer of visual information between hemispheres is severely impaired following transection of posterior part of the corpus callosum. we investigated whether attentive visual object tracking across vertical meridian of the visual field is possible for a posterior callosotomized patient (md). we asked md to track one bouncing ball among four identical distracters while fixating at the center of the screen. target crossed the vertical midline in half of the trials. her performance in crossed conditions was significantly above chance level. also, we asked her to make decision about horizontal alignment of two balls presented simultaneously in one of three conditions: both in right or left hemifield, or each in one hemifield. in this alignment task md was able to compare location of the two bilaterally presented stimuli well above chance level. our data suggest that inter-hemispheric transfer of position information required for spatial attention is preserved without posterior corpus callosum. pei sun, justin l. gardner, mauro costagli, kenichi ueno, r. allen waggoner, keiji tanaka, kang cheng laboratory for cognitive brain mapping, riken brain science institute, wako-shi, japan although the preference for stimulus orientations in human visual cortex has been inferred indirectly in a few studies using fmri, tuning to particular stimulus orientations has not been directly demonstrated using this technique. in an effort towards revealing orientation selectivity and its spatial arrangement in human v , we have conducted an fmri study with a novel stimulation paradigm and a differential mapping method. we found that responses of the majority of activated voxels were modulated by the grating orientation and individual voxels were sharply tuned to particular orientations. our results provide the first demonstration that orientation selectivity in humans can be directly studied using fmri. os p- - probing the spatial scale of classifier performance with high spatial resolution fmri justin l. gardner , , pei sun , keiji tanaka , david j. heeger , kang cheng department of psychology and center for neural science, new york university, usa; laboratory for cognitive brain mapping, riken brain science institute, japan recently, classifier analysis with conventional resolution fmri has been used to decode the orientation of a grating stimulus from the fmri responses of early visual cortex. it has been proposed that classifier analysis exploits small but robust orientation biases in voxels that are created by local inhomogeneities in the columnar organization. we have examined this proposal by using classifier analysis to decode stimulus orientation using high spatial resolution fmri ( . mm × . mm × mm voxels) in human v . we found that many voxels that are weighted heavily in the classifier analysis and carry similar orientation biases closely follow draining veins that are visible on t *-weighted venograms. we suggest that large draining veins with orientation specific responses, rather than local inhomogeneities in orientation maps, may provide a basis for classifier performance using large voxels. research funds: nrsa fellowship from the nih ( f ey - ) os p- - relationship between horizontal connections and functional structure in macaque anterior inferotemporal cortex (area te) hisashi tanigawa, kathleen s. rockland, manabu tanifuji riken brain science institute, wako, japan we have studied the relationship between horizontal connections and functional structure in te using a combination of optical imaging, unit recording, and anatomical tracing. intrinsic signal imaging was performed in exposed te, under anesthesia, during presentations of visual object stimuli. this resulted in multiple optical spots evoked by each stimulus. in some animals, subsequently, unit recording was carried out at multiple sites within the imaged region. then, an anterograde tracer was injected into one of the spots. both optical imaging and unit recording revealed regions with stimulus preference similar to that at the injection site. however, these regions and the injection site were not always connected by horizontal axons. some regions sharing a preference to particular stimuli were connected, even though they showed different preferences to the other stimuli. these results suggest that horizontal axons can connect regions with different stimulus preferences in te, in contrast to like-to-like connectivity, as understood in early visual cortices. we recorded single cell responses from the inferotemporal cortex of a fixating monkey while visual stimuli with various durations ( - ms; isi = s) were presented. presentation of visual stimuli at all of the tested durations resulted in prolonged responses. the brief presentations evoked multiple phasic responses while the long presentations evoked sustained activities. there was a significant difference in average firing rate of late phase ( - ms) of response to optimal stimulus across presentation durations. but no such differences were found for the first phase ( - ms). in addition, the optimal stimulus evoked significantly different response magnitudes in the first and second phase particularly in the short presentation durations. but the suboptimal stimulus (∼ % of max response) evoked similar response magnitudes in the first and second phase. these results suggest that stimulus selectivity of inferotemporal cells depends on the stimulus presentation duration and the time window that is used to measure the firing rate. os p- - the perceptual learning effect in myopes by the lateral masking procedure keiko mizobe , kazuto terai , osamu hieda , shigeru kinoshita dept. of ophthal., kyoto second red cross hospital, kyoto, japan; dept. of ophthal., kyoto pref. univ. of med., kyoto, japan; baptist eye clinic hospital, kyoto, japan purpose: the study of the visual cortex revealed the lateral masking collinear configuration modulated the neuronal responses and psychophysical studies also showed perceptual learning improved the visual detection. we asked whether perceptual learning could improve the myopic blurred vision, using the new instrument of the lateral masking technology, neurovision. method: nine low myopes were studied. non-corrected digital visual acuities (va) ranged from . to . . the logmar average was . . eight sessions of neurovision treatment were performed to each individual. the estimation was done by comparison of va before and after the treatment. results: four eyes showed more than one octave improvement of va. the logmar average of the four was . , improved from . . the residual five eyes showed less or no improvement. the change of logmar average was from . to . . conclusion: some myopes showed the perceptual learning effects by new treatment, using the lateral masking technology. os p- - the hindbrain neuroepithelial cells exclude the migrating facial motor neurons by expression of planar cell polarity (pcp) genes hironori wada , hideomi tanaka , , satomi nakayama , miki iwasaki , , hitoshi okamoto , laboratory for developmental gene regulation, bsi, riken, japan; crest, jst, japan many neurons migrate tangentially through one cell layer at a specific depth within the brain. in the developing zebrafish hindbrain, the facial (nvii) motor neurons originate in rhombomere (r) and migrate tangentially to r near the pial surface of the hindbrain. in this study, we demonstrate that expression of the planar cell polarity (pcp) genes celsr and frizzled a in neuroepithelial cells maintain the nvii motor neurons near the pial surface during their caudal migration in the zebrafish hindbrain. mosaic analyses show that expression of the frizzled a gene in the surrounding neuroepithelial cells prevented the entry of the nvii motor neurons in the neuroepithelial layer. the demonstration of a role for neuroepithelial cells in excluding differentiated neurons from the neuroepithelial layer may provide new insights into the general mechanisms underlying formation of the layered structures in the mammalian brain, such as in the cerebral cortex. os p- - disrupted-in-schizophrenia (disc ) regulates the transport of the nudel/lis complex to axons via direct interaction with kinesin- shinichiro taya, kozo kaibuchi department of cell pharmacology, nagoya university, nagoya, japan disc is a candidate gene for susceptibility to schizophrenia. in a scottish family, the chromosome translocation interrupts the coding sequence of disc . disc is reported to interact with nudel, which forms a complex with lis . although the functional significance of this complex in axon growth and neuronal development has been reported, the transport mechanism of the complex into axons and the functions of disc remain largely unknown. here we report that disc interacted with kinesin- , a motor protein of anterograde axonal transport. kinesin- interacted with the nudel/lis complex through disc , and these molecules accumulated at the distal part of axons. the knockdown of disc by rnai of disc induced the delocalization of nudel and lis from the axons and reduced axonal growth. the knockdown of kinesin- induced the delocalization of disc from the axons. taken together, these results indicate that disc links kinesin- to the nudel/lis complex and regulates its transport as a cargo receptor for axon elongation. research funds: mext os p- - role of a novel collapsin response mediator protein- interacting molecule, synaptotagmin-like protein in hippocampal neuron nariko arimura, saeko kawabata, atsushi hattori, kozo kaibuchi department of cell pharmacology, graduate school of medicine, nagoya university, nagoya, japan during the development, neurons recognize the extracellular signals and extend the axons to proper directions. certain kinds of receptors are transported from the nerve cell body to the axon terminal, and participate in the recognition of extracellular environments. however, the mechanism of controlled recruitment of receptors remains unsolved. here, we report that synaptotagmin-like protein (slp ) can mediate the vesicle transport. slp is known to associate with rab . we found that slp associates with collapsin response mediator protein- (crmp- ), which is a key regulator of axon formation. slp could form the trimeric complex with rab b and crmp- , and also associate with kinesin- through crmp- . slp is accumulated on microtubules at the axonal growth cones, and is co-localized with a receptor of growth factor. these findings suggest that slp functions as a mediator of recruitment of certain receptor depending on crmp- and kinesin- . os p- - absolute quantification of mdr a, mrp , mrp and bcrp proteins at the mouse brain blood barrier by lc-ms/ms junichi kamiie , , yuki katsukura , , sumio ohtsuki , , xiao-kun cai , , tetsuya terasaki , graduate school of pharmaceutical sciences, tohoku university, sendai, japan; sorst, jst, japan the abc transporter proteins are thought to limit permeability across the blood-brain barrier as the efflux transporters. however, contribution of each transporter to the bbb function is not clarified. the purpose of this study was to clarify the protein amounts of mdr a, mrp , mrp , and bcrp in the brain capillaries of mouse by newly developed membrane protein quantification method using lc-ms/ms. by this method, the standard curve showed linearity between and fmol, and amino acid sequence of the detected fragment was confirmed by ms/ms spectrum. in the brain capillaries, the protein amounts of mdr a, mrp , bcrp were . , . and . fmol/g, respectively, while it of mrp was under detection limit of standard curve. this quantitative profile suggests that mrp and bcrp function as the efflux transporter at mouse blood-brain barrier as well as mdr a. os p- - dominant expression of claudin- in highly purified brain capillary endothelial cells sumio ohtsuki , , hirofumi yamaguchi , saori sato , tmoko asashima , , tetsuya terasaki , graduate school of pharmaceutical sciences, tohoku university, sendai, japan; sorst, jst, japan claudins are major constituents of tight junctions (tjs). the purpose of this study was to clarify the expression levels of each claudin subtype in brain capillary endothelial cells (bcecs), which form the blood-brain barrier. mouse bcecs were highly purified using endothelial surface antigen (pecam- ) and magnetic cell sorting. mrna expression of caludin- - was measured by real-time rt-pcr. claudin- showed the highest mrna expression in the purified mouse bcecs. mrna levels of claudin- and - were . % and . % of that of claudin- . claudin- mrna was concentrated in the purified bcecs, while claudin- and - mrna in the purified bcecs were lower than that in the whole brain. rat claudin- mrna was also concentrated in rat brain capillary fraction, but claudin- mrna did not. these results suggest that claudin- is a dominant tjs protein in bcecs, and expression of claudin- and - , which was reported as tj protein in bcecs, are not restricted in bcecs. os p- - effects of hydrogen peroxide towards gap junction communication in astrocytes and permeability of blood brain barrier f. ahmad , a. pauzi m. yusof , p.d. mourad , m. bainbridge , s. ab ghani universiti sains malaysia; university of washington, seattle, usa; brody school of medicine, east carolina university, nc, usa h o is the main peroxides produced in mammalian cells that consume o . the main source of h o in the brain, produced in large amount, was from the superoxide dismutase catalyzed reaction in mitochondria. therefore, we look into the effects of h o towards the gap junction communication in astrocytes and permeability of blood brain barrier. in this study, by using a h o microsensor, we investigated the level of h o in the brain that altered the permeability of bbb. the microsensor was implanted in the rat's brain and operated amperometrically. we measured h o level from the current generated by the electron transfer at the electrode. we observed a change in permeability when external h o was injected into the brain. fatality occurs when the injected h o exceeds m. these finding showed that the altered paracellular permeability in the presence of h o is caused by a series of events that happen one after another. research funds: short term grants pkimia and pfar-masi os p- - somato-ovarian sympathetic reflex discharges in anesthetized rats sae uchida, fusako kagitani, harumi hotta dept. auton. nerv. syst., tokyo metropol. inst. gerontol., tokyo, japan ovarian sympathetic efferent reflex discharges caused by single electrical shock stimulation of spinal (t - ) afferent nerves or limb (tibial) afferent nerves were studied in urethane anesthetized rats. in central nervous system (cns) intact rats, stimulation of the t - spinal afferent nerve produced early and late a-reflex discharges, and a late c-reflex discharge. after spinalization at the third thoracic level, stimulation of the same spinal afferent nerve produced an a-reflex with the same latency as the early a-reflex in cns-intact rats and an early c-reflex discharge with the similar latency as the late a-reflex in cns-intact rats. on the other hand, stimulation of the tibial afferent nerve produced late a-reflex and c-reflex discharges in cns intact rats, those were not observed after spinalization. it was concluded that ovarian sympathetic aand c-reflex discharges evoked by stimulation of a segmental spinal afferent nerve in cns-intact rats are of spinal and supraspinal origin, and those evoked by tibial nerve stimulation are of supraspinal origin. os p- - responses of renal sympathetic nerve activity and sodium excretion to days sodium loading in rats misa yoshimoto, nozomi iinuma, rie itokawa, eri hayashi, kenju miki integrative physiol. grad. sch. humanities and sci. nara-women's univ., nara, japan in the present study, a month recording of renal sympathetic nerve activity (rsna) in freely moving rats was made to explore the long-term regulation of rsna and sodium excretion. wistar male rats were instrumented chronically with electrodes for the measurements of rsna and electrocardiogram. after the days recovery period, rsna, heart rate and sodium balance were measured over three weeks. animals were allowed to drink four different concentration of sodium chloride solutions ( , , , meq./l nacl) over days. the sodium loading with meq./l nacl suppressed rsna significantly and then it gradually recovered while either meq./l nacl or meq./l nacl loading had no effects on rsna. sodium excretion changed significantly in proportion to the each sodium loading levels. these results indicated that the changes in rsna were not always correlated with the changes in sodium excretion in rats. os p- - cross correlation analysis of respiratoryrelated optical imaging signals yoshitaka oku , haruko masumiya , yasumasa okada dept. physiol., hyogo col. med., nishinomiya, japan; dept. med. keio univ. tsukigase rehab. ctr. shizuoka, japan we aimed to establish an objective method to identify the distribution of respiratory-related regions and the timing when these regions are activated relative to the inspiratory activity from optical imaging signals. optical signals were recorded from the ventral medullary surface of neonatal rats in vitro using a voltage-sensitive dye. cross correlation between integrated c ventral root (c vr) activity and each pixel was calculated after cycle-triggered averaging and detrending. the maximum of cross correlation coefficients and the lag at which the cross correlation became maximal (lagmax) were displayed as d pseudocolor maps. in all preparations, two respiratory-related regions were consistently identified: ( ) a continuous column extending from the para-facial region to the pre-bötzinger complex, and ( ) a region corresponding to the ventral horn. pixels where lagmax were negative (meaning that the activity preceded the c vr activity) tended to be distributed in the para-facial region, and this tendency was more evident when superfusate ph was lowered. os p- - slow afterhyperpolarization determines the firing pattern of action potentials in rat gnrh neurons masakatsu kato, yasuo sakuma department of physiology, nippon medical school, tokyo, japan gonadotropin-releasing hormone (gnrh) neurons play a pivotal role in the hypothalamo-pituitary-gonadal axis. gnrh neurons must be able to continuously fire in response to depolarizing stimuli. for this type of firing, gnrh neurons may have a certain intrinsic property. to address this issue, we investigated the ca + -activated voltage-independent k + currents underlying afterhyperpolarization. dispersed gnrh neurons from adult gnrh-egfp transgenic rats were cultured overnight and used for an electrophysiological experiment with perforated patch clamp configuration. the gnrh neurons showed a slow afterhyperpolarization current (i sahp ). in contrast to previous reports, the i sahp observed in rat gnrh neurons was potently blocked by an sk channel blocker apamin. in current clamp condition, gnrh neurons evoked a train of action potentials to depolarizing current pulse. apamin increased the susceptibility to spike failure. the results indicate that rat gnrh neurons exhibit an apaminsensitive i sahp , which regulates the firing pattern. research funds: kakenhi , os p- - the effect of music to sex hormones of elderly person hajime fukui , kumiko toyoshima , kiyoto kuda , katsuhiko iguchi nara univ. of edu., nara, japan; grad. school of human sciences, osaka univ. japan; nara city medical clinic, japan it has been known that testosterone or estrogen protects nervous system and regulates cell death in a brain. also, it is pointed out that the decline of t and est accelerates depression. therefore the treatment such as hormone replacement therapy (hrt) has been tried to cure depression and alzheimer's disease. however, it has been pointed out that hrt has serious side effects. on the other hand, there are reports that music influences on a steroid hormone. in addition, it is known that music has certain therapeutic gain toward ad and dementia. in this study, from a point of view of the prevention of ad and dementia, we examined the effect of music to sex hormones of normal elderly person. four males and females participated music session and t and est were evaluated. as a result, in female, in the high hormone group, the values decreased after the session, and in the low hormone group, the values increased. from above, there might be possibility that through a steroid hormone music participates in protection and improvement of function on brain. tuberculous meningitis (tbm) is the most common form of chronic infection of the central nervous system. despite the magnitude of the problem, the general diagnostic outlook is discouraging. this study identifies a specific protein marker in csf, which will be useful in early diagnosis of tbm. we have demonstrated the presence of a -kda protein band in csf of % (n = ) of confirmed and % (n = ) of suspected tbm patients out of tbm patients. the -kda protein band was analyzed by lc-ms/ms analysis. in the present study we have identified two mycobacterial proteins rv c (ag a) and rv c (ag b) and one host derived protein as the components of the tbm specific -kda protein. involvement of mitochondrial extrinsic and intrinsic apoptotic pathways in dopaminergic neurodegeneration was tested in rotenone-and mpp + -induced rat models of parkinsonǐs disease (pd). hplc-ec, patch clamp, fluorimetry, immunoblot and rt-pcr were used for measuring neurotransmitters/free radicals, membrane currents, caspases activities, levels of proteins and mrna of mitochondria-linked signaling in brain. we report here a retrograde mode of neuronal death via mitochondrial intrinsic pathway in mpp + -, but an extrinsic mode of cell death in rotenone-induced model. drug screening in these models (l-deprenyl as positive control) indicated that quercetin, coenzyme q , vitamin d and melatonin act via interfering the signaling events in neurons. loss of complex-i and -iv activities and changes in some of the protein subunits in pd postmortem brains were confirmed in pd and control cybrids. results from the present study provide evidences for a direct involvement of mitochondria and are suggestive of existence of both intrinsic and extrinsic apoptotic pathways in dopaminergic neuronal death. os p- - involvement of thioredoxin on the neuroprotective effect of (−)-deprenyl tsugunobu andoh , boon chock , dennis l. murphy , chuang c. chiueh dept. applied pharmacol., univ. toayama, toyama, japan; lab. bioch., nhlbi, nih, md, usa; lab. clin. sci., nimh, nih, md, usa; cent, brain diseases and aging, taipei med. univ., taipei, taiwan the present study investigated whether the induction of thioredoxin (trx) involves in the cytoprotective mechanisms of (−)-deprenyl which is known as the inhibitor of mao-b. after confirming (−)-deprenyl protects against mpp + -induced cytotoxicity in human sh-sy y cells, we observed further that (−)-deprenyl induced trx for protection against oxidative injury caused by mpp+. the induction of trx was blocked by pka inhibitor through a pka-sensitive phosphoactivation of map kinase erk / and the transcription factor c-myc. (−)-deprenyl-induced trx and associated neuroprotection were concomitantly blocked by the antisense against trx mrna in human sh-sy y cells. consistently, trx increased the expression of mnsod and bcl- supporting cell survival. in conclusion, (−)-deprenyl augments the gene induction of trx leading to elevated expression of antioxidative mnsod and antiapoptotic bcl- proteins for protecting against mpp + -induced neurotoxicity. os p- - pgd induces neuronal apoptosis via d- , -pgj tatsurou yagami , noboru okamura , toshiyuki sakaeda facul. health care sci., himeji dokkyo univ., himeji, japan; kobe univ. grad. sch. med., japan prostaglandin d (pgd ) is abundant in the brain, but its neuropathologic role has been unclear. here, we found that pgd induced neuronal apoptosis in rat cortical cultures. however, a pgd receptor blocker did not suppress neurotoxicity of pgd . little pgd receptor was detected, suggesting an involvement of pgd metabolites in the apoptosis. among pgd metabolites, -deoxy- , -prostaglandin j ( d- , -pgj ) caused neuronal apoptosis most potently and rapidly. although d- , -pgj is an endogenous ligand for peroxysome proliferator-activated receptor ␥ (ppar␥), ppar␥ activators did not kill neurons, suggesting that d- , -pgj induces apoptosis independently of ppar␥ activation. we found specific binding sites of [ h] d- , -pgj (jbs) in plasma membranes. there was a close correlation between the neurotoxicity of various eicosanoids and their affinity for jbs. in conclusion, we demonstrated that pgd induced apoptosis via d- , -pgj in rat cortical neurons, and suggested that jbs in the plasma membrane was involved in the d- , -pgj -induced apoptosis. yoshiki iwamoto, daisuke umetsu, shigeru ozaki, naohito terui department of physiology, university of tsukuba, tsukuba, japan stability of a driver's head is crucial for clear vision and consistent, smooth operation of a vehicle. we reported last year that bilateral sternocleidomastoid muscles (scm) of drivers showed a symmetrical increase in activity during forward acceleration of a vehicle. in the present study, we analyzed the relationship between scm activity and vehicle acceleration. emgs of the right and left scm of drivers were recorded during rapid forward acceleration. the time course of the rectified, smoothed emgs did not match that of vehicle acceleration. for a given acceleration, emg was larger when acceleration was increasing than when it was decreasing. we compared emgs and a linear sum of acceleration and its time derivative, jerk. with optimal weights for the two variables and a proper time lag, the linear sum reproduced the emg profile. the optimal weight and lag varied across subjects and vehicles. we suggest that the jerk-related muscle activity may be necessary to quickly restore proper head position after sudden acceleration. grasping is a highly developed movement in primate including human. in contrast to the well-known involvement of cerebral cortex, role of spinal neurons in controlling this behavior has never been examined. here, we show the first direct evidence suggesting the significant contribution of spinal neurons. we trained japanese monkeys to perform the precision grip task, pinching the two springloaded levers with their index finger and thumb, and recorded neural activities through an oval recording chamber implanted over the cervical spinal cord (c to t ). majority of the recorded neurons showed movement-related modulation of firing rate, and the modulation sometimes started before movement onset. spike-triggered averaging of muscle activities revealed some neurons had post-spike effects to hand muscles, suggesting that spinal neurons were capable to generate and modulate muscle force during precision grip. we suggest that primate spinal neurons have a significant role in preparation and execution of grasping movement. research funds: kakenhi os p- - compartmentalization of the cerebellar nuclei: aldolase c expression and the olivonuclear projection pattern izumi sugihara, yoshikazu shinoda dept. systems neurophysiol., tokyo med. & dental univ., tokyo, japan the cerebellar cortex is compartmentalized into more than longitudinal stripes by the aldolase c (=zebrin) expression pattern, which is tightly correlated with the topographic olivocortical projection. however, no equivalent compartmentalization has been known in the cerebellar nuclei. we mapped aldolase c labeling of terminals of purkinje cell axons and anterograde labeling of collaterals of olivocerebellar axons in the rat cerebellar nuclei. the cerebellar nuclei were divided into the caudoventral aldolase c-positive and rostrodorsal negative parts, indicating purkinje cells in the positive and negative stripes in the cortex project to the caudoventral and rostrodorsal parts in the nuclei, respectively. olivonuclear projections showed clear topography within these parts, which was completely congruent with the olivocortical topography. these results clarified the compartmentalization of the cerebellar nuclei and supported that the aldolase c expression is tightly related with the functional organization of the cerebellum. we examined a context dependency of neuronal activity of the pedunculopontine tegmental nucleus (pptn) in monkeys during visually guided saccade tasks. about half of movement-related activities occurred for only the saccades to the saccade target in the task, but they did not occur for the saccades outside the task. on the other hand, for the other half of neurons, movement-related activities occurred for every saccade regardless of the task condition. for visual responses, some neurons responded either the initial fixation point or saccade target, and others responded equally to both stimuli. we further analyzed mutual relationship among modulation timing, preferred direction, effect of reward expectation and this context dependency of the activities, and discussed the visuo-motor processing of pptn. in the reinforcement learning theory, the midbrain dopamine (da) neurons send reward prediction error signal to the striatum. the cholinergic pedunculopontine tegmental nucleus (pptn) is one of the strongest excitatory input sources to da neurons. we hypothesized that pptn may play an important role for relaying necessary components of reward prediction error signals to da neurons. during recording of pptn neurons, we utilized reward predictable visually guided saccade tasks where a shape of fixation point indicated a reward volume. for more than half of the neurons, which showed cue related responses, the cue responses were dependent on association of cue feature and reward size. from another population, we recorded reward related activity. in conclusion, pptn neurons may relay both reward and reward prediction signals, sufficient for computation of reward prediction error. research funds: kakenhi ( ) os p- - timing activity in supplementary eye field during a saccadic eye movement task shogo ohmae , xiaofeng lu , , yusuke uchida , toshimitsu takahashi , , shigeru kitazawa , dept. of neurophysiol., juntendo univ. grad. sch. of med., tokyo, japan; crest, jst, tokyo, japan to act properly in our daily life, the ability to detect and predict timing of events is always required. how do we deal with timing in the brain? to address this question, we trained two japanese monkeys to perform a visually guided saccadic eye movement task in which the monkeys made saccades to each of targets following a gosignal given at a random timing between and ms after the appearance of the target. we recorded neuronal activity from the supplementary eye field (sef) during the task. we found a group of cells that showed activity related to the length of the delay period from target-on to the go-signal. these cells were classified into two types: ( ) those that showed buildup activity during the delay period until the go-signal, and ( ) those that displayed changed activity after the go-signal in relation to the length of the delay period. the results suggest that sef is involved in timing the onset of the go-signal during the saccadic eye movement task. in reaching, a spatial visuomotor transformation should occur in our brain. we can make the transformation not only when the relationship between visual and motor coordinates is default, but also when a gain for the relationship is changed, for example, in a microsurgery. we trained monkeys to make reaching movements when visuospatially identical targets were presented on a computer display by aligning a cursor that indicated their hand position, while the gain was systematically changed. we recorded and analyzed movement-related neuronal activity in the ventral premotor cortex (pmv) and the primary motor cortex (mi) during reaction time. it was revealed that a majority of the mi neurons and a part of the pmv neurons showed activity changes depending on executed movement direction, amplitude, and velocity, whereas a number of the pmv neurons exhibited activity consistent to the visual location of the targets, but not to motor parameters such as amplitude and velocity. the results indicate that the pmv contributes to gain control of reaching during visuomotor transformation. local oscillatory changes in the human sensorimotor cortex induced by simple motor tasks were investigated using supragyral and intrasulcal surface electrodes which was temporarily implanted for the treatment of intractable deafferentation pain. time frequency spectrogram and coherence between electrodes revealed that, before and after several hundred milliseconds of the motor execution, the coherence in the premotor cortex increased cooperatively between neighboring electrodes but that the coherence in the intrasulcal primary sensorimotor cortex decreased exclusively. this result reflects that the premotor cortex plays a role in motor planning with diffuse network while the primary motor cortex plays a role in selective motor execution with local motor output unit. the human sensorimotor processing may be hierarchical and similar to an artificial neural computer. we have shown that the trigeminal oral nucleus (vor) neurons with the receptive field in the intraoral structures project bilaterally to either the jaw-closing (jc) or jaw-opening (jo) motor nucleus in the cat. it is known that neurons in the somatosensory cortex project to the trigeminal sensory nuclei in the rat. thus, we conducted this study to reveal whether there are vor neurons that receive cortical projections and project to the jc or jo nucleus in the rat. we injected a retrograde tracer, fluorogold (fg), in the vor, and found many retrogradely labeled neurons in the contralateral rostral primary somatosensory cortex (si). thus, we injected an anterograde tracer, biotinylated dextranamine (bda), in the rostral si, and also fg in the jc or jo nucleus in the same animals. we found a considerable number of fg-labeled vor neurons made contact with bda-labeled axon terminals. these results suggest that si neurons control jawreflexes through vor neurons. tsunehiko kohashi, yoichi oda grad. sch. science, nagoya univ., nagoya, japan the mauthner (m) cells, paired large reticulospinal neurons in teleost hindbrain, are known to initiate fast escape from sudden aversive stimuli. to investigate how the fast escape is established during early developmental stages, we examined motor performance of the escape in zebrafish embryos or larvae, and the contribution of mcell activity on the behavior. the rostral portion of the zebrafish, - h post fertilization (hpf), was embedded in agar and the tail flip in response to water pulse applied to the head was examined. thirty hpf embryos, in which m-cell has already received trigeminal nerve innervation and is still extending its axon in the spinal cord, showed tail flips contralateral to the stimulated side with longer latency (> ms) than larvae (> hpf, ms). m-cell activity monitored with confocal ca + imaging during the tail flip (> hpf) tightly correlated with the initiation of fast escape, whereas delayed escapes without m-cell firing appeared in some cases (< %) after hpf. thus, the development of the escape behavior coincided with that of m-cell circuit. junctophilins (jps) expressed in the er/sr interacts with plasma membrane thereby constructing junctional membrane complexes (jmc). we here report that lacking neural jps subtypes exhibit an irregular hindlimb reflex and impaired memory. to define neural mechanism of memory deficit in jp-dko mice, we performed whole-cell patch clamp recording of hippocampal neurons. in wild mice, an obvious afterhyperpolarization (ahp) was observed and its ahp was totally blocked by apamin. by contrast, ahp was absent in the jp-dko mice and was insensitive to apamin treatment. the er ca + release through ryanodine receptors, triggered by glutamate receptor-mediated ca + influx, is essential for the activation of sk channels toward ahp generation in the hippocampal neurons. therefore, jp-mediated jmc formation likely plays an essential role in neural excitability underlying neural plasticity and memory. os p- - distribution of voltage-gated calcium channel ␣ ␦- mrna in mouse central nervous system takeshi houtani, satoru sakuma, masahiko kase, tetsuo sugimoto department of anatomy and brain science, kansai medical university, moriguchi, osaka - , japan the ␣ ␦ subunits are the auxiliary subunit of voltage-gated calcium channels and modulate the biophysical properties of the pore-forming ␣ subunits. these auxiliary subunits are composed of four genetically different molecules, ␣ ␦- to ␣ ␦- . the distributions of ␣ ␦- , - , - mrna have been intensively investigated in the rat central nervous system by in situ hybridization, but that of ␣ ␦- remains to be determined. we cloned ␣ ␦- cdna fragment from mouse brain by rt-pcr and examined the distribution of ␣ ␦- mrna-expressing cells in the mouse central nervous system by in situ hybridization using digoxigenin-labeled crna probe. while the ␣ ␦- mrna was found to be broadly expressed, some neuronal types or sites such as piriform cortex, hippocampal pyramidal cells, paraventricular hypothalamic nucleus, facial nucleus and motor neurons of the ventral horn had intense mrna expression. our results suggest that ␣ ␦- subunit may play an important role in learning and memory, neuroendocrine secretion and somatic motor control. the mushroom bodies of insect brains are essential in associative olfactory learning. here we show that the drosophila larval mushroom body calyx, the dendritic region, is organized in about glomeruli, which we have mapped. individual glomeruli receive specific innervation from second order olfactory neurons. by contrast, they contain dendrites from hundreds of mushroom body neurons (kenyon cells), which show low specificity for individual glomeruli. glomeruli therefore potentially transmit specific sensory inputs to a large fraction of kenyon cells. quantitative analysis of dendritic termini of single larval-born kenyon cells suggests that they arborize in about glomeruli in an apparently random manner. this pattern of connectivity is consistent with a model in which kenyon cell dendrites process olfactory input by a combinatorial mechanism that allows the discrimination of a large number of odors. withdrawn os p- - hypothalamic defense reaction involves purkinje cells in the flocculus folium p via orexin and gaba in anesthetized rabbits, electric stimulation in the hypothalamic defense area either excited or inhibited "simple spike" discharges in purkinje cells located in folium p of the flocculus. iontophoretic application of an orexin antagonist (sb ) depressed the excitation, while bicuculline depressed the inhibition. h or h histamine antagonist had no effect. labeling orexin fibers by immunocytochemistry showed that they were most numerous in folium p as compared with other folia of the flocculus. stimulation of the hypothalamic defense area produced little field potentials in the folium p unlike those evoked by mossy fibers. these observations suggest that the excitation and inhibition are mediated by orexin-containing fibers, which contact purkinje cells directly and also indirectly via other gabaergic neurons. os a- - activity-dependent development of corticispinal synapse in mouse slice co-culture takae ohno, masaki sakurai dept. physiol., teikyo univ. sch. med., tokyo, japan we showed nmda-dependent synapse elimination of corticospinal (cs) tract in vitro in rat. in order to use the genetically modified mice to study the underlying molecular mechanisms of this developmental plasticity, we studied development of cs synapses in c bl/ mice. by recording field epsp (fepsp) along m interval lattice in the spinal gray matter in response to the stimulation of deep cortical layer, we evaluated spatial distribution of synapse formation quantitatively. fepsps were recorded diffusely throughout the spinal gray matter at - div, then the amplitudes of fepsps in the ventral side began to decrease at - div, and dominated in the dorsal area at div. cs axon terminals labeled anterogradely with biocytin distributed diffusely throughout the spinal gray matter at - div but the axons terminals in the ventral area were eliminated until div. this synapse elimination from the ventral side was blocked by apv application from div, indicating that this process is also nmda-dependent. in slice coculture study, we showed that corticospinal (cs) axons grow rapidly and reach the ventral spinal gray until div. the number of those ventral axons is reduced before div. to study the behavior of the cs axons at the single axonal level, we transfected a small number of cortical neurons with eyfp expression vector pcag-eyfp by way of electroporation to visualize them and took the time-lapse images of their axons under the confocal microscope equipped with an on-stage co incubator. some axons showed rapid growth, reaching the ventral most part of the spinal gray matter already at div. some axons had collaterals at the dorsal part and retracted the ventral branch while extending the dorsal branch during - div. some ventral axons showed a fragmented tip during retraction, which was indicative of axonal pruning. these observations provide direct evidence that there are early cs axons that once reach the ventral spinal gray and then retract to stay dorsally. we identified click-iii/camki␥ as a novel brain-enriched isoform of the camk-i family that was lipid-anchored by multiple lipid modifications, prenylation and palmitoylation, resulting in enrichment of click-iii into lipid rafts fractions. in situ hybridization revealed the abundant presence of click-iii transcript throughout the central nervous system in mouse embryos. to test the role of click-iii during early neuritogenesis, a shrna vector specific for click-iii was delivered into dissociated cortical culture. we found that knock-down of click-iii resulted in significant decrease in the number and total length of dendrites. results from introduction of click-iii into a click-iii-null context confirmed this finding. surprisingly, lipid modifications of click-iii seemed to contribute to fully elicit such an effect. we thus uncovered a novel signaling mechanism by which lipid raft insertion and local activation of a camk can be efficiently coupled to actin cytoskeletal signaling during dendritogenesis. os a- - transcription factor control of dendrite arbor ultrastructure adrian moore, reiko amikura, shiho nakao, andrew liu, emi kinameri riken brain science institute, japan the different functions of neurons in a complex nervous system are reflected in a large diversity of dendrite arbor morphologies. the drosophila larva dendritic arbourization (da) neurons consist of four classes (i-iv) with increasing levels of arbor complexity. these diverse arbor shapes develop due to class specific mechanisms of dendrite branching and outgrowth. here we show that these class specific differences in dendrite arbor morphology are controlled by a combinatorial code of transcription factors. we have developed a system to label individual dendrite arbors then subsequently identify them in electron microscopic sections. using this method we illustrate that the dendrites of class i neurons, with a simple arbor, contain a high density parallel array of microtubules; on the other hand class iv neurons, with a complex arbor, contain a low density meshwork of microtubules. we are presently investigating how these differences in ultrastructure are controlled by the transcription factors making up the combinatorial code. os a- - segmental and hox related cues are involved in the establishment of the somatotopy yasunori murakami igbmc, strasbourg, france in the rodent, trigeminal sensory inputs are topographically relayed, and mapped in the somatosensory cortex. little is known about the mechanism underlying the development of the somatotopic organization. by fate mapping of specific rhombomeres (r), we found that principal sensory (prv) neurons derived from r receive predominantly inputs from the maxillary branch of the trigeminal nerve and uniquely contribute to the whisker map. by conditional inactivation, we found that early expression of hoxa in r is required for pathfinding and positioning of trigeminal nerve afferents. at later stages, hoxa expression in prv neurons provides instructive cues for topographic arborization of maxillary axons. moreover, while prv neurons appeared normally specified, loss of hoxa function resulted in selective loss of eph expression, and altered axonal projections from prv to the ventral posterior medial (vpm) nucleus of the thalamus, and absence of a postnatal whisker map at any level of the neuraxis. thus, hoxa dependent cues are required to determine the territory for whisker representation in r and the assembly of a somatosensory circuit. os a- - the second wave of corticospinal innervation after synapse elimination of the first wave tsutomu kamiyama, masaki sakurai dept. physiol, teikyo univ. sch. med., tokyo, japan in the previous study we showed that the rat corticospinal (cs) terminals and synapses were widely distributed at p and those in the venrtolateral (vl) area were eliminated from p to p and that the number of terminals in the dorsomedial (dm) and vl area began to increase again from p and further increased thereafter. in the present study we further studied the subsequent developmental time course of cs terminal distribution. cs axons were anterogradely labeled by injection of biotin dextrane (bda) into the sensorimotor cortex. the number of the terminals began to increase from p , reaching peak around the third postnatal week. labeling of single or a few by microinjection axons revealed that at p some additional cs axon branches appeared within the dorsal column of the target spinal segment and further ramified after entering the gray matter. however, the number of axons did not increase in the brainstem and the upper cervical cord. these suggest that the second wave of innervation is explained mainly by branching of cs axons just before and after entering the spinal gray matter. os a- - proteomics of the growth cone: i. protein profiling of the growth cone the growth cone is a motile tip formed at the developing neuronal processes, and functions for the accurate determination of the axon pathway and the synaptogenesis. in higher organisms, however, the molecular basis of the growth cone is poorly understood for the present, since the information on the protein localization there is insufficient to explain the growth cone functions. proteomics is a powerful strategy for identifying the protein composition in a given cell or a subcellular compartment, and the application of this method to the growth cone should help us solve the above question. we obtained the whole growth cone (gcp) obtained from neonatal rat forebrain and the membrane subfraction of the gcp (gcm), and then those fractions were analyzed using proteomics. we have identified several hundreds of the distinct proteins of these fractions. here, we show the profiling of gcp and gcm, and will discuss the overview of these protein profiles in relation to the growth cone functions. axonal branching is thought to be regulated by not only genetically specified molecules but also neuronal activity. however, the interplay between these two mechanisms remains largely unknown. to study this issue, we analyzed the role of electrical activity in layer-specific thalamocortical (tc) axon branching by using organotypic cocultures. during the second week in vitro, yellow fluorescent protein-labeled tc axons formed branches primarily in the target layer. spontaneous firing was found to increase when branches were formed abundantly. pharmacological blockade of synaptic transmission diminished layer-specific branching considerably. moreover, time-lapse imaging showed that branching was generated dynamically by elimination as well as addition in the target layer and that blockade of synaptic activity reduced this remodeling. these findings suggest that synaptic activity modifies layer-specific tc axon branching by regulating the remodeling process with molecular cues expressed in the target layer. research funds: kakenhi ( ), kakenhi ( ) os a- - the application of navigation-guided repetitive transcranial magnetic stimulation for intractable deafferentation pain naoki tani, yoichi saitoh, haruhiko k., satoru oshino, masayuki hirata, amami katoh, toshiki yoshimine department of physiology, university of osaka, osaka, japan repetitive transcranial magnetic stimulation (rtms) has been applied to control intractable deafferentation pain (dp). but nobody has investigated which cortical area is the most effective target for pain relief. therefore, we stimulated m , s , sma, premotor accurately with a navigation-guided rtms and compared their effects of pain relief. at the same time, rtms ( , , hz, stimulations) was compared in dp patients. the pain relief was evaluated with visual analogue scale. high frequency ( , hz) rtms of m was the only effective stimulation for treating intractable pain in of patients ( %). the pain relief continued for h significantly. we would like to discuss the mechanism of pain relief with high frequency rtms of m . os a- - involvement of atp on nociceptive modulation in rat model of masseter muscle pain yasuo sugiura, noriyuki ozaki, masamichi shinoda department of functional anatomy and neuroscience, nagoya university graduate school of medicine, nagoya, japan we determined the role of p x r on pressure pain and mechanical hyperalgesia in a newly developed rat model of pain in masseter muscle (mm) . the pain in the mm was assessed by the pressure pain threshold (ppt) defined as the amount of pressure required to induce head flinching. the mm injection of ␣,-meatp (p x , , / rspecific agonist) significantly enhanced the behavioral response to the pressure. this enhanced response was completely blocked by the co-application of ␣,-meatp with ppads (p x , , , , / , / r-specific antagonist). excessive muscular contraction of mm produced by the electrical stimulation significantly decreased the ppt indicating mechanical hyperalgesia of the mm. administration of ppads to the exerted mm produced a complete recovery of decreased ppt. p x rpositive neurons innervating the exerted mm increased in trigeminal ganglia. our results suggest that p x r plays an important role in pressure pain, and mechanical hyperalgesia caused by excessive muscular contraction of mm. the present study was undertaken to investigate the change in the activation of the nociceptive neuronal circuit under a neuropathic pain-like state. here we found sciatic nerve ligation (snl) produced a marked increase in the number of c-fos-positive cells in the periaqueductal gray (pag). using the fluoro-gold (fg) microinjection into the pag, numerous fg-labeled cells were detected in the hypothalamus. in the arcuate nucleus (arc) of the hypothalamus, the immunoreactivity (ir) for an excitatory neuronal maker, fosb was increased, whereas the -endorphin (-ep)-ir was decreased days after snl. furthermore, the subpopulations of -ep-positive cells were co-labeled with fosb in the arc. the present data suggest that the hypothalamus can be received by snl-induced concomitant nociceptive signals, leading to continuous activation of neurons projecting to the pag. this phenomenon, in turn, indirectly controls pain transmission in the dorsal horn through the descending antinociceptive pathway. os a- - the cantor-like patterns in rat hippocampal ca pyramidal neurons tsuda and kuroda proposed a mathematical model for the cantor coding in the hippocampal ca . this prediction includes an attractor dynamics expected in the associative network, which was proposed by many authors, since marr's theory of simple memory in the hippocampus. however, our mathematical model is too abstract to describe physiological feature of neurons. then, we have tried to find cantor-like patterns experimentally from the ca pyramidal neurons. temporally associated and non-associated electrical stimulations were delivered to schaffer collaterals, and membrane potentials were recorded by patch-clamp recording method. in our results, cantor-like patterns were observed in hippocampal ca pyramidal neurons. young songbirds shape their songs using memorized tutor songs and auditory-vocal feedback. we prevented zebra finches from hearing their own vocalizations by exposure to loud noise after days of age, before which they had been reared with song tutors from birth. when the noise stopped at - days of age, the birds sang unstable and noisy song syllables that did not resemble the tutor syllables. the similarity to the tutor syllables steadily increased until the time of song crystallization ( days later). these findings show that the memory of tutor syllables still exists well beyond the normal age of song crystallization (d of age) and that zebra finches can develop songs using the memory well after the normal period of song development. the temporal order of syllables resembled the tutor model only in birds released from the noise before days of age. thus, different schedules and processes may govern the learning of syllable phonology and syntax. in addition to well-characterized areas, a novel adult neurogenic region; the temporal germinal layer (tgl) was identified in rats (takemura, ) . a tracer study revealed that there is an interconnection between the dorsal part of the tgl and the lateral nucleus of the amygdala, suggesting a functional implementation of tgl neurogenesis in amygdala-dependent emotional memory processing. to investigate this possibility, we performed a tgl region-specific low-dose irradiation, which can selectively kill proliferating cells and hence can reduce neurogenesis, using a gamma knife. the tgl-irradiated rats expressed a significantly increased tone-related long-term fear memory, indicating a functional significance of the tgl neurogenesis for aversive memory reduction. we (tsukada and pan, ) systematically examine the functional difference between spatio-temporal learning rule (stlr) proposed by tsukada ( ) and hebbian learning rules in a single-layered neural network, computing their ability to differentiate spatiotemporal sequence. in this paper, we tested physiologically the cooperative plasticity without a postsynaptic spike in the ca hippocampal network. tsuda and kuroda proposed a mathematical model for the cantor coding in the hippocampal ca . they also predicted chaotically transitory dynamic behavior called chaotic itinerancy in the hippocampal ca . this prediction includes an attractor dynamics expected in the associative network, which was proposed by marr and others. the time series of events, which could be output from ca , may be encoded in ca in an efficient way. the proposed cantor coding is effective, because the topology of time series is naturally measured on the cantor set since each element of cantor set represents a single time series. however, our mathematical model is too abstract to describe physiological feature of neurons. then, we have tried to make more realistic model of ca , using -compartment model of neuron, and we found the cantor coding of information of time series in the model ca . it is known that neurons can propagate action potentials with high temporal precision. however, it is unclear how precisely closely neighbouring neurons synchronize and whether they can code information. here we show that sub-millisecond synchronization can code information as well as the discharge rate modulation. we found that closely neighbouring pyramidal neurons in the ca region of the hippocampus synchronize with sub-millisecond precision. the optimal frequency bands for transmitting these synchronizations matched the beta, gamma and fast-ripple oscillations. moreover, we found that the synchronizations were commonly coupled with rate modulations in relation to both internal (retention and comparison) and external (stimulus and motor) events. the synchronization often occurred in relation to stimulus inputs even when rate modulation was clearly absent. therefore, our results suggest that sub-millisecond synchronization plays an important role in propagating information in the hippocampus. the alterations of cerebral motor function by chronic ischemia are poorly understood, since no motor symptoms are noticeable in most of the cases. we evaluated spatial distribution and intensity of eventrelated desynchronization of beta band (beta-erd) evoked in motor area using synthetic aperture magnetometry in patients with chronic ischemia due to diverse vascular occlusive diseases (n = ) and moyamoya disease (n = ). contrary to the normal motor activation, ipsilateral beta-erd was dominant during grasping task of affected hand in patients. this abnormal activation was obscured by self-paced finger tapping requiring more selective hand motor programming. and it was more frequently observed in the atherosclerotic hypoperfusion (with white matter change) than in other pathogenesis. ipsilateral beta-erd may be a new indicator of subclinical functional alteration in motor cortices caused by chronic ischemia. os a- - hypothermia protects against cerebral ischemia by suppressing ␦pkc activation takayoshi shimohata , , heng zhao , gary steinberg department of neurology, brain research institute, niigata university, niigata, japan; department of neurosurgery, stanford university, stanford, usa hypothermia protects the brain from ischemia, but the underlying mechanisms of this effect are not fully elucidated. ␦pkc is reported to induce apoptosis upon activation. its activity is modulated by phosphorylation, translocation and proteolytic cleavage. we investigated effects of hypothermia on ␦pkc activation using a rat permanent distal mca occlusion model. mild hypothermia ( • c) reduced infarct size by %. western blots indicated that ␦pkc cleavage increased markedly in ischemic core but moderately in penumbra after stroke, which is suppressed by hypothermia (p < . ). p-␦pkc (t ) dephosphorylated after stroke; this effect is blocked by hypothermia. full-length and cleaved form ␦pkc as well as p-␦pkc (s ) translocate from the cytoplasm to the mitochondria and nucleus, which is suppressed by hypothermia. ␦pkc activator suppressed the protective effect of hypothermia. taken together, hypothermia blocks ␦pkc activation after focal ischemia. this effect might contribute to hypothermic neuroprotection. calcium responses in situ following ischemia remain unclear. we sought to determine, in rats, the calcium changes following transient forebrain ischemia. in anesthetized adult rats, -vessle occlusion was induced. fluo- /am was microinjected, and the fiber-coupled confocal microscope [imaging fiber bundle coupled to the microlensattached nipkow-disk scanner (csu- , yokogawa, japan) equipped with × objective lens] was inserted into the brain. -vessle occlusion induced comparable ischemia in both hippocampus and frontal cortex. fluorescence intensity of fluo- increased up to %, and persistently increased up to % during -min reperfusion, indicating the long-lasting ca + increase in the ca region. in contrast, in the frontal cortex, -min ischemia increased fluorescence intensity during ischemia but not reperfusion. in the ca region but not in the frontal cortex, transient forebrain ischemia induces long-lasting increase in ca + in situ. research funds: kakenhi # , # os a- - reevalution of classical view on resident microglia: neutrophils may play more critical roles than resident microglia at acute phase of ischemic and traumatic brain insults hiroaki matsumoto , h. watanabe , y. kumon , t. ohnishi , chi ii , y. imai , j. tanaka dept. neurosurgery, ehime university, japan; dept. molecular and cellular physiology, ehime university, japan resident quiescent microglia (mg) are thought to respond quickly to a variety of pathologic events in the brain, by proliferating and producing a number of bioactive substances including proinflammatory cytokines and nitric oxide (no). in the present study, however, we found that the majority of resident mg died through apoptosis within h after the onset of ischemic and traumatic brain insults. we further noticed that traditional mg markers isolectin b and cd b recognized with ox antibody histochemically stained neutrophils, which were identified by neutrophil-specific elastase, rather than iba + mg or macrophages. accumulation of neutrophils was observed at the very early phase of the insults, while they expressed proinflammatory cytokines and inducible no synthase. iba + amoeboid-shaped mg started to accumulate days after the insults. the data prompted us to reevaluate the roles and the fate of resident mg in the brain. os a- - insulin regulates the hepatic clearance of amyloid  peptide tetsuya terasaki , , chihiro tamaki , sumio ohtsuki , graduate school of pharmaceutical sciences, tohoku university, sendai, japan; sorst, jst, japan the liver is the major organ that eliminates amyloid -peptide (a) from the circulation, and we have revealed that low-density lipoprotein receptor-related protein (lrp- ) is a molecule responsible for the hepatic clearance. since epidemiologic investigations suggest the high incidence of alzheimer's disease in diabetes mellitus, the purpose of this study was to clarify the effect of insulin on the hepatic clearance of a . insulin infusion into the rat portal vein increased lrp- expression in plasma membrane fraction of liver, but did not affect the expression in whole lysate. insulin treatment also increased the hepatic uptake of a( - ), which reached . -fold greater uptake than non-treated control after min treatment. increase of the hepatic uptake of a( - ) by insulin was concentration dependent (ec = pm), and was completely suppressed by rap ( m), an lrp inhibitor. these results suggest that insulin induces translocation of lrp- to the plasma membrane of hepatocytes, leading to increase of a hepatic clearance from the circulation. research funds: sorst, jst os a- - mr images of intra-arterially administered microglia surrounding -amyloid deposit in the rat brain the therapeutic use of microglial cells has recently received some attention for the treatment of alzheimer disease (ad), but few noninvasive techniques exist for monitoring cells. here we present a magnetic resonance imaging (mri) technology to track micrgolia cells injected intra-arterially in a rat model of ad. we labeled microglia expressing gfp with resovist using the hvj-e vector. we administered labeled microglia into the carotid artery of the rats. mri revealed clear signal changes attributable to resovist-containing microglia in a-injected areas. this study demonstrates the usefulness of mri for non-invasive monitoring of exogenous microglia, and suggests a promising future for microglia as therapeutic tools for ad. extravasation of protease-activated receptor (par) activators, such as thrombin, into brain parenchyma can occur after blood-brain barrier breakdown in a number of cns disorders, which causes pathophysiological changes in neurons and glial cells. to elucidate the mechanism of thrombin-induced activation of astroglial cells, we used n astrocytomas that show a characteristic retraction of bipolar protrusions after activation of pars with thrombin. the thrombin-induced morphological change of n cells was inhibited by an inhibitor of ip receptors, -aminoethoxydiphenyl borate ( -apb) or an endoplasmic reticulum ca + -atpase inhibitor, cyclopiazonic acid (cpa). in parallel, thrombin-induced mobilization of ca + was inhibited by -apb and cpa. moreover, removal of external ca + accelerated the reversal of thrombin effects. these results suggest that refilling of ca + store by ca + entry play an important role in the cytoskeletal dynamics of astroglial cells. to clarify the occurrence range of neurofibrillary tangles (nft), we reexamined an autopsied alzheimer patient with the onset at age and a -year-clinical course. the brain showed severe atrophy ( g). microscopic examination disclosed that all telencephalic neocortices had nft of more than and sp of more than . all limbic cortices and nuclei had nft of more than and sp of more than . although there was no sp, various numbers of nft were observed in the following structures: claustrum , caudate , globus pallidus , hypothalamus , meynert's nucleus , thalamus , substantia nigra , central gray , locus ceruleus , purkinje cells , posterior root ganglion , adrenal medulla . this study revealed that there exist nft-rich neurons and free neurons. the latter includes purkinje cells and posterior root ganglion cells. considering the pathogenesis of nft, it must be valuable to clarify qualitative/quntitative differences between nft-rich neurons and free neurons. os a- - transcriptional regulation of androgen receptor in aging mouse brain androgen receptor (ar) mediates action of androgen, which is involved in memory, behavior and other brain functions that deteriorate with advancing age. in aging mice brain, ar mrna expression was measured by rt-pcr, ar promoter methylation by southern hybridization, and proteins binding to promoter by emsa. ar mrna level was significantly higher in male than female, and it was downregulated by testosterone, but upregulated by estradiol in adult mice. female mice exhibited higher methylation of ar promoter than males. methylation was increased by testosterone, but decreased by estradiol. furthermore, dnasei accessibility to ar promoter was reduced in males, increased by gonadectomy but reduced by sex steroids in adult male. incubation of brain nuclear extract with plabeled ar promoter yielded three specific complexes. the intensity of these complexes varied with age and sex. these findings show that ar mrna expression and promoter methylation are inversely regulated by sex steroids in the adult mice cerebral cortex. such regulation of ar expression might influence androgen action and consequently brain function during aging. reliability of synaptic transmission depends on the efficiency of transmitter removal from the synaptic cleft, as well as on the release machinery and the postsynaptic response mechanism. it has been shown in various synapses that postsynaptic and glial excitatory amino acid transporters (eaats) contribute to glutamate removal. however, the role of presynaptic eaats remains unclear. using mouse retinal slices, we examined the contribution of eaats at the rod to rod bipolar cell (rbc) synapse. the kinetics of the rbc current evoked by electrical stimulation of rods was slowed by pharmacological blockade of eaats. recordings of the evoked rbc currents from eaat subtype-deficient mice and the eaat-coupled anion current revealed that functional eaats are localized to rod terminals but not to postsynaptic or glial cells. model simulations suggest that rod eaats are densely packed near the release site, and that rods are equipped with an almost self-sufficient glutamate recollecting system. trpv is a thermosensitive trp channel, and activated by body temperature. we found functional-trpv was expressed in soma, dendrites and synapses in the neurons. since trpv was firstly cloned as an osmotically activated channel, we hypothesized trpv might be involved in volume regulation of the spines. therefore, we quantified the spine volume changes by glutamate stimulation, and confirmed trpv expression related to the volume increase of spines. next, we compared the resting membrane potential (rmp) between wild type and trpv -deficient neurons at • c, and found rmp in wild type was more depolarized by approximately mv than rmp in trpv -deficient neurons. we also performed current-injection experiments in both neurons, and found that trpv -deficient neurons required much bigger currents to get their firing. thus, we conclude that trpv is involved in regulation of both neural activity and spine motility in hippocampus. os p- - a system for rapid uncaging in defined patterns and its application hiroshi kojima department of intelligent information systems, tamagawa university, tokyo, japan neurons integrate many sysnaptic signals at dendrite. understanding these information processes is a central topics in experimental and computational neuroscience. the use of focused laser beam for uncaging can provide fine spatial resolution to analysis of neural function. however, most experiments were carried out either at spatial locations or in a very simple scanning patterns. we developed a system for performing uncaging in arbitrary pattern in order to emulate realistic neural activity. our system is capable of patterned photorelease of caged neurotransmitters at locations per ms with submicron resolution. ultraviolet laser light is steered by galvano-mirrors and projected onto the surface of preparations for uncaging the caged chemicals. simultaneously, imaging of neurons are obtained by -photon microscopy and electrophysiological experiments can be done. we briefly report the present system for rapid uncaging and its application to neurophysiological research. os p- - d -like receptors selectively block p/q-type calcium channels to glutamate release onto cholinergic neurons in the rat basal forebrain a number of molecules have been identified in the sensory ganglia including those involved in the signal transmission to the brain. their functions, however, remain largely unknown. we tried to develop a method enabling to inhibit gene expression in the sensory ganglia in vivo by rnai and to evaluate its effect on the synaptic transmission in the brain slices. for this purpose, we selected the nodose ganglion (ng), in which the neurons sending glutamatergic projections to the nucleus tractus solitarii in the brainstem, are located. in anesthetized young wistar rats, synthetic sirna against the genes coding adenosine a receptors (adora ) was introduced to the ng by electroporation. one to five days after sirna delivery, the expression level of adora in the ng decreased by > % of that in the non-treated ng, being not accompanied by a change in mrna level for a a receptors. this technique might be promising in analyzing the function of specific molecules involved in transmitter release regulation at the brain synapses. nmda-receptors are specific constituents of glutamatergic system in brain responsible for molecular mechanisms of recognition and learning. activation of neurons by nmda results in intracellular generation of reactive oxygen species (ros) and reorganization of cell metabolism. exposure of rodent and human lymphocytes with nmda results in ros increase within the cells which is suppressed by nmda antagonists. moreover we have demonstrated by rt-pcr technique and by using anti-nmda-antibodies the expression of nmdareceptors on lymphocyte membranes. in addition, we shown that nmda receptor dependent signal from lymphocyte membrane is transformed into specific intracellular reactions controlling caspase- activity and interferon-␥ synthesis. in the presentation, properties of nmda-receptors and their functional role in immunnocompetent system are discussed. small molecule g-protein arf in combination with phospholipase d (pld) is essential for intracellular trafficking of the proteins from endoplasmic reticulum to golgi apparatus. however, it is recently reported that it also regulate ionic channel activity at the cytoplasmic membrane. to examine possible involvement of arf and subsequent pld in regulation of receptor-induced responses in neurons, we recorded k + -current response to dopamine (da) in the ganglion cells of aplysia under conventional two-electrode voltage clamp. intracellular application of arf blockers such as brefeldin a, exo , and arf n-terminal peptide, markedly suppressed the da-induced response. furthermore, intracellular application of ␣-synuclein, a specific blocker of pld, significantly depressed the k + -current response to da. these results suggest that arf and subsequent pld may regulate the k + -current response induced by da. os p- - p gap, a brain-enriched rhogap, is involved in the nmdar-mediated signaling takanobu nakazawa , toshihiko kuriu , ayako m. watabe , toshiya manabe , shigeo okabe , tadashi yamamoto div. of oncology, inst. med. sci., univ. of tokyo, tokyo, japan; dept. of cell biol., tokyo medical and dental univ., tokyo, japan; div. of neuronal network, inst. med. sci., univ. of tokyo, tokyo, japan nmdar regulates structural plasticity by modulating actin organization within spines. however, the signaling pathways that link nmdar activity to the postsynaptic actin cytoskeleton are poorly understood. we identified a brain-enriched rhogap, p gap, which interacts with the nr b subunit of nmdar. within neurons, p gap was highly concentrated in the postsynaptic density and co-localized with nr b and an actin-binding protein, cortactin. p gap promoted gtp hydrolysis of cdc and rhoa in vitro and in vivo. nmdar stimulation led to de-phosphorylation and redistribution of p gap. when over-expressed in dissociated neuron, p gap suppressed the activities of rho gtpases, which resulted in spine elongation. taken together, the results suggest that p gap is likely to be involved in nmdar activity-dependent actin re-organization in spines. os p- - non-static method to directly quantify the transfer of firing correlation from one neural population to another: fokker-planck method hideyuki cateau riken brain science institute, saitama, japan firings of only a few neurons are too weak to be transmitted safely, to activate other neurons to fire, or to contract muscles. therefore, we implicitly assume that brain function is exerted by macroscopic population of neurons. to characterize how a macroscopic neural population behave, the simulation method provide an indirect approach. many single neuron simulation runs need to be performed first before extracting macroscopic features by statistically averaging. unlike this method, the fokker-planck (fp) method directly evaluates the macroscopic features, thereby giving a clearer insight into function achievable with neuronal population. despite the lasting interests in firing correlation in coding and conveying information, theoretical studies on it have been largely confined to complicated simulation studies. here, we provide a first non-static fp analysis to directly calculate how correlation and population rates are transferred from one population to another and elaborate a dynamical interplay between these macroscopic quantities at work in time. os p- - spatial frequency tuning of disparity-selective neurons in macaque v hironori kumano , seiji tanabe , ichiro fujita grad. sch. of engineering science, osaka univ., osaka, japan; grad. sch. of frontier biosciences, osaka univ., osaka, japan to examine whether convergence across spatial frequency channels contribute to stereoscopic processing, we recorded single neuron activity from area v of awake, fixating monkeys. for each neuron tested, we first measured the spatial frequency tuning with sinusoidal gratings or two-dimensional ( -d) filtered noise images, and then examined the disparity tuning with both correlated and anticorrelated dynamic random-dot stereograms (rdss). neurons with broader spatial frequency tuning had more attenuated disparity tuning for anti-correlated rdss. in a subset of v neurons, we analyzed responses to various combinations of binocular disparity and spatial frequency by using -d filtered noise stereograms. the disparity tuning of most v neurons was consistent across a range of spatial frequencies to which they were sensitive. we suggest that v neurons pool disparity signals across spatial frequency channels to create an unambiguous representation of stereoscopic depth. os p- - predicting the monkey's behavioral choice in a stereoacuity task from neuronal responses in area v hiroshi shiozaki, seiji tanabe, ichiro fujita lab. cognitive neurosci., grad. sch. frontier biosciences, osaka univ., japan many neurons in visual area v of macaque monkeys are selective for binocular disparity. most disparity-selective neurons in v are sensitive to small changes in disparity near zero, suggesting that they might contribute to stereoacuity. however, the role of these neurons in stereoscopic depth discrimination has not been directly addressed. we recorded single unit activity from v while a monkey was engaged in a fine stereoscopic depth discrimination or stereoacuity task. the monkey was trained to report by saccadic eye movement whether the center region of a random-dot stereogram was nearer or farther than its immediate surround. trial-to-trial fluctuation of visual responses of v neurons was correlated with the monkey's subsequent behavioral choice. given the cell's disparity preference, an ideal observer can predict the monkey's upcoming behavioral response from the visual response of v neurons. the results suggest that v neurons are involved in mediating stereoacuity. os p- - the role of disparity energy and binocular matching processes in stereopsis takahiro doi, seiji tanabe, ichiro fujita lab. cognitive neurosci., osaka univ., japan the early visual system computes disparity energy of stereo images. some of the next stages retain this information, while other stages perform further computation to solve the stereo correspondence problem. we addressed how the energy and correspondence computations underlie stereopsis. we asked human subjects to discriminate depth of random-dot stereograms with various amounts of disparity. at each disparity level, we manipulated the proportion of dots with the same luminance contrast between the two eyes by reversing the contrast of some dots in one eye. at small disparities, the proportion of correct choices increased monotonically from chance to perfect as the proportion of the same-contrast dots was increased. at large disparities, the subjects perceived reversed depth when contrastreversed dots dominated, and the proportion of correct choices reached only chance level when the two types of dots were balanced. the results suggest that the correspondence and energy computations underlie fine and coarse stereopsis, respectively. we introduce a novel receptive field (rf) analysis, lsrc, which can reveal various aspects of visual receptive fields that were undetectable previously in a single measurement. the visual stimuli are standard wide-field -d ternary dynamic random noise, generally refreshed every - ms. unlike the conventional reverse correlation which computes a spike-triggered average (sta) of the stimuli themselves, lsrc computes the sta of the spectra of localized regions of the stimuli. both simulations and recordings from cat v /v neurons demonstrate that lsrc is capable of revealing details of complex cell rfs, cross-orientation suppression, variations of orientation tuning within rfs that might lead to shape selectivites. since the stimuli can cover a wide visual field area, and few assumptions are made regarding specific shapes or features in stimuli, lsrc is highly suitable for multi-neuron, multi-area studies spanning retina, v , and especially areas beyond. research funds: mext( ), jsps( ), coe os p- - analysis of center-surround organization of v neurons as a high-order receptive field hiroki tanaka, izumi ohzawa graduate school of frontier biosciences, osaka, japan responses of area (v ) neurons are influenced by stimuli not only in their classical receptive field (rf) center, but also in its surround. such a center-surround organization may be considered as a unified higher-order rf. we have sought to obtain detailed structures of such a rf by harmonic analyses of responses to drifting contrast-modulated sinusoidal gratings that cover both the center and surround regions. of cells analyzed, % showed spatial frequency tuning curves that were well fitted with gaussian. by taking the inverse fourier transform of these curves, spatial center-surround rf was obtained as gabor functions with spatial phases between ± degrees. highly asymmetric structures were observed for cells with strong surround suppression. estimated sizes of center and surround were well correlated with those from size tuning curves. moreover, there was no space-time tilt in the center-surround rf. the results suggest that neurons with surround suppression are capable of coding various spatial forms of higher-order features (figure-ground borders), but are insensitive to motion of such stimuli. os p- - spatial organization of receptive fields of complex cells in the early visual cortex kota sasaki , izumi ohzawa , grad. school of eng. sci., osaka univ., japan; grad. school of frontier biosci., osaka univ., japan little is known about the quantitative internal structure of the receptive fields (rf) of complex cells, although this is crucial for understanding how a complex cell acquires its function by collecting inputs from neurons in the preceding stage. therefore, we have analyzed the relationship between the spatial nd-order interaction kernels and the rf envelopes of complex cells. extracellular single unit recordings were performed in anesthetized and paralyzed adult cats. threevalued (i.e. gray, dark, and bright) dynamic white noise stimulus with × dots was presented over an area to times larger than the rf of a complex cell. for each dot location, a nd-order kernel and its envelope (by hilbert transform) were calculated. the rf envelope of the neuron was determined by summing the envelopes of nd-order kernels at all locations. nd-order kernels had roughly comparable extent as the rf, and contained . subregions on average (n = ). among complex cells, whose rf envelopes were elongated, cells exhibited the horizontal elongation. research funds: mext( ), jsps( ), coe os p- - orientation tuning of neuron in cat lateral geniculate nucleus tomoyuki naito , osamu sadakane , masahiro okamoto , hironobu osaki , hiromichi sato grad. sch. med., osaka univ., osaka, japan; grad. sch. front. biosci., osaka univ., osaka, japan; med. sch., osaka univ., osaka, japan we examined the orientation selectivity of lgn neurons of anesthetized cats and found that although about % lgn neurons showed significantly orientation-biased response to the grating with optimal size and spatial frequency (sf), and that % of lgn neurons exhibited significant orientation selectivity to gratings with diameter larger than its classical receptive field (crf) and sf higher than the optimal for crf response. two stimulus-size tuning curves measured for responses to stimulation with the optimally-or null-orientated grating exhibited profile similar to each other under the optimal sf condition. however, high sf grating caused stronger surround suppression for response to the orthogonally oriented stimulus than that to the optimally orientated stimulus. our results suggested that elliptic crf center produces orientation-biased response of lgn neurons. furthermore, surround suppression of lgn neurons tuned to particular stimulus orientations enhances orientation selectivity of lgn neurons. os p- - temporal dynamics of suppressive receptive field surround in cat v satoshi shimegi, hiroyuki kida, ayako ishikawa, hiroshi sakamoto, hiromichi sato graduate school of medicine, osaka university, toyonaka, japan in the primary visual cortex (v ), a neuronal response to stimulation of the classical receptive field (crf) is suppressively modulated by the stimulus presented at the receptive field surround (srf). using stationary flashes ( ms) of sinusoidal grating with optimal parameters and varying radii as stimuli, we examined the temporal dynamics of the surround suppression in v cells of anesthetized cats. stimulus slightly larger than the crf caused suppression in early response (< ms) but not in middle ( - ms) and late responses ( - ms). as stimulus size was further enlarged, the middle and late responses were remarkably suppressed while the early response was only moderately or weakly suppressed. radius of surround suppressive field progressively expanded in temporal sequence from . deg (early response) to deg (middle response) and . deg (late response). thus, modulation of early response seems to reflect whether stimulus is larger than crf size or not, and late response to reflect how wide area is stimulated. research funds: kakenhi ( ) os p- - spatial-frequency dependent surround suppression in cat v ayako ishikawa , satoshi shimegi , hiroyuki kida , hiromichi sato grad. sch. front. biosci., osaka univ., osaka, japan; grad. sch. med., osaka univ., japan; grad. sch. eng. sci., osaka univ., japan we examined the temporal dynamics of the surround suppression of visual response in terms of spatial-frequency (sf) tuning of neurons in cat v . we used a stationary flash (duration, ms) of a circular sinusoidal grating patch with optimal orientation and sf as crf stimulus, and that of an annulus ( ms) with optimal orientation but varying sf as srf stimulus. first, we stimulated crf and srf simultaneously (stimulus-onset-asynchrony (soa) = ) and analyzed time course of surround suppression. sf tuning of the surround suppression changed along time course of response, and effective sf of surround suppression shifted from the sf lower than that optimal for crf response (c-sf) to that near c-sf. next, changing soa, we examined surround suppression on different temporal phases of crf response. soa-dependency of surround suppression changed according to the temporal phase of response. these results suggest that multiple mechanisms with different sf-and temporal characteristics are involved in the surround suppression. os p- - contrast-dependency of spatial summation property in cat v and lgn masahiro okamoto , tomoyuki naito , osamu sadakane , hiromichi sato grad. sch. front. biosci., osaka univ., japan; grad. sch. med., osaka univ., toyonaka, japan we examined contrast-dependent change in a receptive field (rf) size and strength of surround suppression of neurons in the primary visual cortex (v ) and the lateral geniculate nucleus (lgn) of anesthetized cats. rf structure was modeled by spatial interactions of excitatory and inhibitory gaussians. both in v and lgn, ratio of gaussians (rog) model captured size-tuning curves of responses better than difference of gaussians (dog) model. under the high contrast stimulus condition, the peak of size tuning curve shrank by . and . times in v and lgn, respectively. in lgn, surround suppression was strengthened under high contrast stimulus condition, but in v , the strength of surround suppression did not affected by stimulus contrast on average. we conclude that ) rog model describes the surround suppression better than dog model both in v and lgn, ) under high contrast stimulus condition, there is a reduction of rf size with a shrinking of excitatory gaussian, which is confirmed with rog model. hiroyuki kida , satoshi shimegi , ayako ishikawa , hiroshi sakamoto , hiromichi sato grad. sch. eng. sci., osaka univ., japan; grad. sch. med. sci., osaka univ., japan; grad. sch. front. biosci., osaka univ., japan in the primary visual cortex (v ), neuronal responses to stimulation of the classical receptive field (crf) were suppressed by the presence of stimuli at surround receptive field (srf). we examined whether the suppression varied according to spatial configuration of srf stimuli in v neurons of anesthetized cat. the crf stimulus was a circular patch of sinusoidal grating with optimal stimulus parameter. srf was divided into flanks ( • step), and stationary stimulated with an annulus, oppositely-faced flanks ( -fk) or a flank ( -fk) stimulus. the durations of stimulus presentation were ms for crf and ms for srf stimulation. localized srf stimulation with either -fk or -fk exerted significant suppression on crf responses. according to the analysis of spatiotemporal change in srf effects, there was no particularly suppressive srf area for -fk stimulation throughout the crf response. however, -fk stimulation of end position to crf had strong and long-lasting suppression on responses during - ms after onset. os p- - temporal-frequency dependency of receptive field size and surround suppression in lgn and v osamu sadakane , tomoyuki naito , hironobu osaki , masahiro okamoto , hiromichi sato grad. sch. med., osaka univ., japan; med. sch., osaka univ., japan; grad. sch. front. biosci., osaka univ., osaka, japan spatial summation property of neurons in the primary visual cortex (v ) varies depending on stimulus parameters (e.g., stimulus contrast). in this study, we examined how temporal frequency (tf) of grating stimulus affects size-tuning properties of cat v neurons. our results showed that, when the tf was higher than the optimal, the strength of surround suppression became weak and receptive field size became larger, suggesting that v neurons change their spatial property according to tf in such a way that neurons integrate wide visual field for fast moving stimulus, whereas localized field for slow stimulus. we also tested the effect of changing stimulus size on tf tuning curve. consistent with above-mentioned results, large grating made the peak and the high cut-off of tf-tuning curve higher than those for small grating. in the lateral geniculate nucleus (lgn), we obtained basically similar results to those of v neurons, suggesting that the subcortical tf tuning property contributes to that in v . ryo sasaki, takanori uka department of physiology (i), juntendo university, tokyo, japan a few studies have shown that basic tuning functions in early visual cortex change during visual perceptual learning (schoups et al. ; yang and maunsell ) . the change in neuronal sensitivity in these studies, however, is small compared to the improvement in behavioral sensitivity. here we hypothesized that the read out of information from sensitive neurons was modified by learning. to test this hypothesis, we investigated whether learning modifies neuronal sensitivity or read out of middle temporal (mt) neurons during learning of a depth discrimination task. two monkeys were trained to report the depth of moving dots (near or far), and we recorded from isolated mt neurons during the course of training. the monkeys showed improvement in discrimination thresholds across daily sessions. in contrast, the sensitivity of mt neurons did not change, whereas the correlation between neuronal activity and the monkey's behavioral choice increased during the course of training. these results suggest that plasticity due to perceptual learning occurs within the neural pathway following area mt. we developed an in vivo method to localize the fine tip of a glassinsulated tungsten microelectrode for chronic recording using . t mri. the scan conditions were first optimized by imaging a microelectrode that was sunk into copper sulfate solution. the microelectrode tip was precisely localized up to a resolution of m under particular geometrical scan condition. we then examined the applicability of the method in vivo under this optimized scan condition in the temporal cortices of three monkeys. the microelectrode was penetrated into the dorsal or ventral bank of the superior temporal sulcus and the tip was localized by the high-resolution mri. the accuracy of this method was validated by comparing the localized positions of the microelectrode tips with the corresponding electrolytic lesion marks in histological sections. a transient signal change in diffusion-weighted image of the brain has been detected in human visual cortex. the time course of this signal was ahead of the bold signal and characterized by a steep onset. diffusion-mri thus represents a new exciting mechanism for fmri. in order to increase its efficiency we aimed at defining a diffusion response function (drf) as a counterpart of the hemodynamic response function (hrf). an volume of interest was defined using spm with a boxcar function. gamma-variate functions were used to model the steep onset. the parameters of the drf were estimated by fitting the time-course with the drf convolved with a boxcar. although the magnitude of the signal change (around %) was smaller than that of bold (> %), the temporal profile showed a constant precedence of the diffusion signal by . s. os p- - new insights on normal and pathological brain function from tomographic analysis of magnetoencephalographic signals laboratory for human brain dynamics, brain science institute (bsi), riken, wako-shi, japan tomographic analysis of magnetoencephalography (meg) data combines exceptional temporal resolution with accurate localization, at least for places a few centimeters away from the center of the head [moradi, et al., neuroimage; ioannides et al., cerebral cortex] . this unique capability of probing brain function across the entire cortex and deep brain structures from milliseconds to minutes in the same experiment has already provided new insights about normal [ioannides et al., cerebral cortex;ioannides et al., neuroimage] and pathological [ioannides et al., j. neurosc.] brain function. novel ways of analyzing meg data provide direct measures of regional brain activity over much longer timescales. these new methods are used in ongoing studies to probe the nature of global brain activity in different states of awareness (e.g. different stages of sleep) and explore the relationship between estimates of electrophysiological activity derived from meg with hemodynamic measures of brain activity. os p- - spatial registration of stand-alone fnirs data to mni space ippeita dan, archana singh, masako okamoto national food research institute, japan the registration of functional brain data to the common brain space offers great advantages for inter-modal data integration and sharing. however, this is difficult to achieve in functional near-infrared spectroscopy (fnirs) because fnirs data is primary obtained from the head surface and lacks structural information of the measured brain. therefore, we present a method for probabilistic registration of fnirs data to the standard montreal neurological institute (mni) template through international - system without using the subject's magnetic resonance image (mri). the standard deviation in probabilistic registration thus performed for given head surface points is approximately within cm. this means that if the spatial registration error is within an acceptable tolerance limit, it is possible to perform multisubject fnirs analysis to make inference at the population level and to provide information on positional variability in the population, even when subjects' mris are not available. stochastic perturbation in scale is a basic property of biological systems and generates scale-independent structuration and functional dynamics in spatial and temporal patterns, which can be characterized by fractal dimensionality. it allows a user-independent evaluation and does not rely on subjective evaluation in image assessment. we have used a box-counting algorithm in scale-space segmented images to determine the mass fractal dimension of ventricles in different neurological disorders. three groups of subjects [alzheimer disease (ad), obstructive hydrocephalus (oh) and normal controls] were examined. mass fractal dimension is high for ad ( . ), approaching unity (∼ . ) for oh, and in between for control ( . ). statistical analysis was performed and significant differences were observed for these groups (p < . ). the observations are accounted by a flow dynamics heterogeneity model. the implications are that stochastic structuration and fractal dimension may be useful to track temporal progression of disease and assess therapeutic management. thrombin, a serine protease essential for blood coagulation, also plays an important role in injury associated with intracerebral hemorrhage. in this study, we revealed that mitogen-activated protein kinase (mapk) pathways contribute to thrombin-induced brain injury in two experimental models. firstly, we employed organotypic cortico-striatal slice cultures. application of thrombin to slice cultures resulted in cortical neuronal injury and striatal shrinkage. the cortical neuronal injury was ameliorated by inhibition of extracellular-signal regulated kinase (erk) but not p mapk, while the striatal shrinkage was prevented by both of them. secondly, thrombin was injected into rat striatum. thrombin-induced brain injury determined by immunoreactivity of neuronal marker was reduced by inhibition of erk and p mapk. these results suggest that mapk pathways play important roles in thrombin-induced brain injury and they should be therapeutic targets against neurodegeneration associated with blood-brain barrier destruction. positron emission tomography was used to study brain activations during motor imagery of standing and during performance of standing posture in parkinson's disease (pd). eight pd patients performed mental and motor tasks: ( ) resting, ( ) staring at a standing human object, ( ) thinking of standing, ( ) standing with eyes open, ( ) standing with eyes closed. regional cbf data analyzed by spm were compared with normal counterparts. the cerebellar vermis was more activated during imagination of standing in the pd group than in healthy group. as seen in healthy subjects, standing also activated the primary sensorimotor foot area and cerebellar vermis in pd patients, but the between-group comparison generated greater activations in the vermis and prevuneus in pd. the cerebellar vermis engages in postural balance both in mind and reality, and the precuneus may play a more important role in postural control in pd. os p- - potentiation of nmda receptor-mediated current by metabolic failures through glycine release facilitation in the hypoglossal motoneurons of the rat yu kono , , eiji shigetomi , kiyoharu inoue , fusao kato dept. neurol., jikei univ., sch. med., tokyo, japan; lab. neurophysiol., jikei univ., sch. med., tokyo, japan to elucidate the mechanism underlying the selective vulnerability of motoneurons (mns) to metabolic failures (mfs), we compared the membrane current responses of mns and non-mns to mfs. experiments were performed on neurons in the hypoglossal nucleus (xii) and dorsal motor nucleus of the vagus nerve (dmx) in the young rat brainstem in the presence of ttx. mfs were induced by nacn or oxygen deprivation. in xii neurons, mfs induced large persistent inward currents accompanied by marked increase in strychnine-sensitive synaptic inputs, indicating facilitation of glycine release onto xii neurons. furthermore, nmda receptor-mediated current evoked by exogenous nmda was increased by nacn. in dmx neurons, mfs evoked outward currents without affecting synaptic inputs. these pre-and postsynaptic responses to mfs in mns might play a role in their selective vulnerability in various neurodegenerative diseases including the amyotrophic lateral sclerosis. os p- - effects of mdma on serotonergic neurons in rat organotypic mesencephalic slice culture including the raphe nuclei yuichi suzuki, megumi higuchi, takayuki nakagawa, shuji kaneko dept. mol. pharmacol., grad. sch. pharmaceu. sci., kyoto univ., kyoto, japan , -methylenedioxymethamphetamine (mdma) is a recreational drug of abused which has been shown to increase serotonin ( -ht) release and cause degeneration of -htergic nerve terminals via -ht transporter, although the mechanisms are unclear. in this study, we developed rat organotypic mesencephalic slice culture including the -htergic raphe nuclei, and examined the effects of mdma and methamphetamine (meth) on -ht release and -htergic neurotoxicity. immunohistochemical studies for tryptophan hydroxylase revealed abundant -htergic neurons around the raphe nuclei. treatment with a -htergic neurotoxin , -dihydroxytryptamine dramatically reduced the tissue contents of -ht and its metabolite, which was blocked by a selective -ht reuptake inhibitor. mdma and meth ( . - m) increased -ht release, and reduced the tissue contents of -ht and its metabolite at higher doses. the mesencephalic slice culture including the -htergic raphe nuclei may be useful to examine the mechanisms underlying -htergic neurotoxic effect of mdma in vitro. os p- - studies on drug dependence (rept. ): involvement of platelet-derived growth factor (pdgf) receptor in the morphine-induced rewarding effect masami suzuki, minoru narita, michiko narita, tomoko takeuchi, yasuyuki nagumo, keiichi niikura, tsutomu suzuki dept. of toxicol., hoshi univ. sch. pharm. pharmaceut. sci., tokyo, japan the present study was undertaken to investigate the involvement of platelet-derived growth factor (pdgf) receptor in the morphineinduced rewarding effect in rodents. extensive coexpression of tyrosine hydroxylase with pdgf receptor was apparently observed in the rat ventral tegmental area (vta). the levels of dopamine and its major metabolites in the nucleus accumbens (n.acc.) were markedly increased by the microinjection of pdgf into the rat vta. the morphine-induced rewarding effect was suppressed by intra-vta microinjection of pdgf receptor fc chimera. the increased level of dialysate dopamine produced by morphine in the rat n.acc. was significantly decreased by intra-vta injection of pdgf receptor fc chimera. these findings suggest that the stimulation of -opioid receptors in the vta by morphine leads to the activation of pdgf receptor, which may be directly responsible for the morphine-induced rewarding effect in rodents. os p- - prostaglandin d is a strong mediator of neuroinflammation in genetic demyelinating mouse model prostaglandin (pg) d , an inflammatory mediator, mainly produced by hematopoietic pgd synthase (hpgds). microglial activation and gliosis are commonly observed during the neuroinflammation. in twitcher (galct wi/twi ), a genetic demyelinating mouse model, we found that hpgds expression was upregulated in activated microglia accompanied by the dp receptor induction in hypertrophic astrocytes. using primary culture of glial cells, we demonstrated that activated microglia produced large amount of pgd by hpgds and that astrocytes expressed both dp and dp receptors and were activated by pgd . we found that gliosis and demyelination were well suppressed in hpgds-or dp -null twitcher and twitcher treated with an hpgds-inhibitor. these results suggest that pgd is a key molecule of neuroinflammation involved in the demyelination. research funds: , os p- - on a sodium channel distribution enabling high frequency signal processing go ashida , , kousuke abe , kazuo funabiki grad. sch. medicine, kyoto univ., kyoto, japan; grad. sch. informatics, kyoto univ., kyoto, japan some auditory neurons, such as the owl's nucleus laminaris (nl) cells, can sense very high frequency signals (up to khz). from the theoretical point of view, it seems exceptionally difficult to handle these high frequency signals because the membrane time constant is far longer. first, we discuss a biophysical mechanism of shifting the membrane time constant by connecting the large cell body (soma) with the small node of ranvier. next, we discuss the effect of sodium channel distribution on the impedance function of the membrane. sodium conductance in the soma amplifies low frequency signal components below khz, while that in the node does up to khz. last, as a typical example, we discuss the capability of high frequency signal processing in the owl's nl neuron. some biological evidences indicate that sodium channels in the nl neuron are distributed mainly in the nodes but less in the soma. by using an nl neuron model, we show that a neuron with low somatic sodium conductance and high nodal sodium conductance can achieve fine sensitivity to high frequency signals. interaural time difference (itd) is calulated using axonal delay lines and coincidence detector neurons (nucleus laminaris:nl). however, little is known about the cellular mechanisms of coincidence detection. here, we report the results of in vivo intracellular recordings from the barn owl's nl. we used coaxial glass electrodes in which one (microelectrode) was inserted into a patch-electrode type capillary. the inner sharp electrode was protected by the outer one during penetration of the cerebellum. we isolated nl cells from owls and achieved intracellular recordings in of them, as judged by a sudden dc potential drop and the resting membrane potential (mean rp = ± mv). nl neurons produced small spikes and oscillatory potentials whose waveform closely resembled the superposition of the tones delivered to the two ears (sound analogue psps:sap). the amplitude of saps varied as a function of itd. spike rates changed in linear proportion to the amplitude of sap. we evaluated sound localization ability of vision impaired and sighted persons by using a 'two-sound sources discrimination test' in a semianechoic darkroom. in total, vision impaired ( blind and low vision) and sighted persons participated. the stimuli were pure tone pulses. for each trial, the same single sound pulse was emitted consecutively from a pair of speakers with the same angle either left or right from the midline of the subject. localization ability was assessed whether the subjects are able to discriminate two sound sources or not in each trial. the discriminability of the blind subjects slightly exceeded that of sighted subjects but the difference was not significant. the discriminability of the low vision subjects, on the other hand, was significantly lower than that of blind or sighted. it was suggested that a peculiar 'object perception' of blind persons is not able to measure by means of 'two-sound sources discrimination test.' os p- - autocrine bmp signaling in astroglia sensitizes the glial scarring masahisa yamada , runa araya , naoto kitamura , yuji mishinsa yamada unit, riken bsi, saitama, japan; nihs, nieh, nc, usa bone morphogenetic proteins (bmps) affect growth of glial cells however, contribution of bmps during glial scar formation is unknown. to study the role of bmp signaling in vivo, we disrupted bmpr a, one of the type i receptors for bmps, in a telencephalic neuronal stem cell-specific manner. we found that aberrant architecture of microvessels that led to a failure in maintaining the blood-brainbarrier in the mutant mice. although mutant mice showed inflammation around the cortical microvessels, proliferation of hypertrophic reactive-astrocytes in the mutant mice was attenuated. disruption of astroglial bmpr a expression by cre-adenovirus recapitulates the same phenomena. bmps were upregulated in reactive astrocytes in after brain injury. knocking down of bmpr a by small interfering rna in primary astrocytic culture negatively affected their astrocytic growth injured by scratch, which reinforced the importance of autocrine bmp signaling in astrocytes. this result opens up the understanding of novel mechanisms underlying the autocrine bmp signaling on glial scarring after cns injury. the present study was undertaken to evaluate the functional role of the glial cells in the induction of stress. here, we found that aging mice promoted anxiety-like behaviors as characterized by both the light-dark and elevated plus-maze tests, and they exhibit an increase in astrocytes in the cingulate cortex. a robust increase in gfap-positive astrocytes was noted in the cingulate cortex of nerve-ligated mice that exhibited the anxiety-like behavior. in contrast, iba -positive microglial cells were dramatically increased as compared to that in control mice and some of them were co-localized with brdu-like immunoreactivity in the hippocampus of mice exposed to chronic psychological stress. our results indicate that the increase in astrocyte or microglia in the cingulate cortex or hippocampus may lead to emotional disorders including aggravated anxiety under aging, chronic pain-like state or exposure to chronic psychological stress. withdrawn os p- - fucosylation prevents overshooting of the migration by the vagus motor neuron precursors shigeharu kinoshita , , hideomi tanaka , , sachiko tsuruoka , hironori wada , , hitoshi okamoto , riken bsi, wako, japan; jst crest, kawaguchi, japan; riken rrc, wako, japan the vagus motor nuclei are important as the autonomic center for the maintenance of homeostasis. aberrant positioning of nuclei is implicated in the etiology of the sudden infant death syndrome (sids). therefore, control of precursor cell migration into the right position may be crucially important. the zebrafish embryo has two vagus motor nuclei, the dorso-laterally and medially located nuclei (dmx and mmx). the dmx precursors are born near the floor plate, migrate dorso-laterally and then are accumulated at the defined position. in the towhead mutant embryos, ectopic neurons are distributed between bilateral dmx where precursors aberrantly migrate in dorsal direction and fail to stop at the right position. positional cloning and mrna rescue analysis identified towhead as a gdp-mannose , dehydratase (gmds), a key enzyme for de novo synthesis of a gdp-fucose. as a result, the mutant embryos showed exclusive reduction of fucosylated glycans. our findings represent that fucosylation is responsible for maturation of these neurons. in development of the drosophila visual center, photoreceptor cells extend their axons (r axons) to the lamina ganglion layer and trigger proliferation and differentiation of synaptic partners (lamina neurons) by delivering the inductive signal, hedgehog (hh). this mechanism helps to establish an orderly arrangement of connections between the r axons and lamina neurons, termed a retinotopic map because it results in positioning the lamina neurons in close vicinity to the corresponding r axons. it is found that the bhlh-pas transcription factor single-minded (sim) is induced by hh in the lamina neurons and is required for the association of lamina neurons with r axons. in sim mutant brains, lamina neurons undergo the first step of differentiation but fail to associate with r axons. as a result, lamina neurons are set aside from r axons. the data reveal a novel mechanism for regulation of the interaction between axons and neuronal cell bodies that establishes precise neuronal networks. research funds: kakenhi ( ) os p- - initial molecular steps in synaptogenesis in vivo: trans-synaptic interaction of cell adhesion molecule is involved in postsynaptic assembly of psd -homolog dlg hiroshi kohsaka, etsuko takasu, akinao nose department of physics, university of tokyo, tokyo, japan trans-synaptic interaction via cell adhesion molecules (cam) is essential in constructing synapse structures. although this notion has been supported by various studies in vitro, evidence in vivo has been lacking. here we used live-imaging and genetic analysis to show that a drosophila cam fasciculin (fas ) mediates early interaction between pre-and postsynaptic cells in synaptogenesis in vivo. by visualizing gfp-tagged fas genetically expressed on a muscle, we found fas accumulated at postsynaptic site just after the contact between growth cones and its target muscle. genetic and deletion analysis implied that trans-synaptic interaction with presynaptic fas is crucial for the postsynaptic localization of fas . in addition, postsynaptic localization of a scaffolding protein dlg, psd -homolog, and glutamate receptors was impaired in fas mutants. these results provide the first in vivo evidence that trans-synaptic cell adhesion molecule has a role in inducing the assembly of synapses. gaudilliere brice harvard medical school, usa postsynaptic differentiation of dendrites is an essential step in synapse formation. we report here a requirement for the transcription factor myocyte enhancer factor a (mef a) in the morphogenesis of postsynaptic granule neuron dendritic claws in the cerebellar cortex. a transcriptional repressor form of mef a that is sumoylated at lys promoted dendritic claw differentiation. activity-dependent calcium signaling induced a calcineurin-mediated dephosphorylation of mef a at ser and thereby promoted a switch from sumoylation to acetylation at lys , leading to inhibition of dendritic claw differentiation. our findings define a mechanism underlying postsynaptic differentiation that may modulate activity-dependent synapse development and plasticity in the brain. research funds: ns , ag ps a-a characterization of mrna species that are associated with postsynaptic density fraction by gene chip microarray analysis we previously reported the partial identification by random sequencing of mrna species that are associated with the postsynaptic density (psd) fraction (tian et al., ) . we report here further characterization by gene chip analysis of the psd fraction-associated mrnas, which were prepared in the presence of rnase inhibitor. we confirmed that a large number of mrna species are associated with the psd fraction and found that mrnas encoding various postsynaptic proteins were highly concentrated in the psd fraction. we identified some mrna species that were highly concentrated in the psd fraction. we also constructed a cdna library using the psd fraction-associated mrnas as templates, and identified randomly selected clones by sequencing. our data suggested that the psd fraction-associated mrnas are a very useful resource, in which as yet uncharacterized genes are concentrated. tian et al., . mol. brain res., , - . research funds: kakenhi ( ) ps a-a the distribution of snap- protein is regulated in isofom-specific manner makoto itakura, saori yamamori, kouta takano, masami takahashi department of biochemistry, kitasato university school of medicine, sagamihara, japan two isoforms of snap- derived from exon splicing are expressed in brain. we generated two specific antibodies for snap- a and b, and studied the distribution in rodent brain. there was a sticking difference in expression of snap- a and b during early postnatal period. snap- b was low at the birth and increased remarkably thereafter. by the contrast, snap- a increased transiently and attained a maximum level around seven day after birth. furthermore, there seemed to be a difference in their distributions in plasma membrane, since a substantial amount of snap- b but not snap- a was recovered in raft-enriched fractions of triton x- -treated lp membrane after sucrose density gradient centrifugation. immunohistochemistry demonstrated that snap- b was widely distributed throughout brain, whereas, snap- a was restricted to some particular regions of brain. these results indicate that expression and distribution of snap- protein are regulated differently in isoformspecific manners, and snap- a and snap- b play different functional roles in brain. ps a-a erc(elks/rab ip /cast) regulates syaptic short-term plasticity by recruiting bmunc - to the active zone camkii in the postsynaptic sites is localized as a psd-anchored or a cytoplasmic form. camkii in the two sites is interchangeable by its translocation. translocation and targeting of this kinase to appropriate subcellular compartments are crucial for its physiological function. we have previously suggested that postsynaptic camkii is also localized in lipid raft microdomain ( . mol. brain res. , - ) . in this report, we proved the lipid raft localization of camkii by detergent-treatment and successive sucrose floatation assay of spm or cos cells expressing camkii, and by cholesterol depletion from membrane using mbcd. we also investigated the mechanism and properties of camkii targeting to lipid raft. camkii targeted to lipid raft microdomain possibly through protein-protein interaction. our data suggest that lipid raft microdomain is a major site of camkii distribution, as well as postsynaptic density and cytoplasmic region, at the postsynaptic site. glial glutamate transporters, glast and glt- , are co-localized in processes of bergmann glia wrapping excitatory synapses on purkinje cells (pcs). although glast is expressed six-fold more abundantly than glt- , the decay kinetics of climbing fiber (cf)mediated excitatory postsynaptic currents (cf-epscs) in pcs in glast(-/-) mice are not significantly different from those in wildtype mice. here we attempted to clarify the roles of glial glutamate transporters in cf-pc synapses using glast(-/-) and glt- (-/-) mice, and a novel antagonist of glial glutamate transporters, ( s, s)- -[ -( -methoxybenzoylamino)benzyloxy]aspartate. our results indicate that glial glutamate transporters can retain the fast decay kinetics of cf-epscs in the normal range if a small proportion (approximately %) of functional transporters, glast and/or glt- , is preserved. glutamate is well known as an essential neurotransmitter in nervous system. how glutamate-mediated synaptic transmission is controlled in neural circuit of live animal, however, remains to be poorly understood. we found that the loss-of-function mutations in vglut (vesicular glutamate transporter) encoded by eat- gene led to abnormal sensory behaviors including thermotaxis in c. elegans. thermotaxis defect of eat- mutant was caused by malfunction of both thermosensory neuron afd and its downstream interneuron ria, suggesting that thermal signals from afd or ria to their downstream neurons are transmitted by glutamate through eat- vglut. a mutation in avr- glutamate receptor also led to abnormal thermotaxis. we are trying to investigate whether avr- functions in the downstream neurons of afd or ria, and to identify other glutamate receptors involved in thermotaxis. through the analysis of thermotaxis neural circuit, we are hoping to reveal the mechanisms of glutamate-mediated synaptic transmission at neural circuit level. ps a-a biochemical characterisation of the vesicular glutamate transporter stephan schenck, shigeo takamori department of neurology and neurological science, st century coe program, tokyo medical & dental university, tokyo, japan vesicular glutamate transporters (vgluts) load synaptic vesicles with glutamate, the major excitatory transmitter in the brain, thus making these transporters of outstanding importance for the function of the central nervous system. the three known isoforms of these secondary active transporters have been characterised in terms of tissue distribution, developmental expression patterns and some pharmacological features. while the third isoform constitutes only a minor fraction, vglut and vglut are abundantly expressed in the brain with a complementary distribution pattern and divergences in the expression profile during ontogeny. so far, no clear difference in the function of vglut and vglut has been found. to further characterise the specific properties of the transporters we make use of the vglut -ko mouse which gives us the opportunity to investigate a brain devoid of vglutl. we focus on synaptic vesicle fractions from ko-mice to study the vglut-associated transport biochemically. in recent years, three isoforms of vesicular glutamate transporters (vgluts) have been molecularly identified in mammals. histological investigations have revealed that the distribution of three vglut isoforms in the cns is largely complementary with limited overlap, suggesting that differential expression of vglut isoforms may contribute to functional diversity in glutamatergic synapses. however, functional differences among the isoforms remained poorly understood. to get insights into their isoform-specific property, we searched for interacting protein(s) to the c-terminus of vglut by yeast two-hybrid screening and found endophilin a . as expected for the interacting molecule to vglut , endophilin a was typically localized to vglut -positive synaptic terminals in cultured hippocampal neurons. we are currently investigating physiological significance underlying their direct interaction and co-localization. the aim of this study is to investigate the molecular basis for lactate utilization. hippocampal neuronal culture was continuously superfused with glucose or lactate solution and spontaneous excitatory postsynaptic currents (sepscs) were recorded from a voltage-clamped pyramidal neuron. in lactate solution, amplitude of epscs was decreased in ∼ min, followed by spontaneously recovered after min, while epsc in glucose medium remained unchanged. application of apv+ni in lactate medium, spontaneous recovery was not observed. in neuron cultures, incorporation of c-lactate was gradually increased, which was suppressed by applications of inhibitors for calcium calcium channels or protein kinase c. in glial cell cultures, incorpotation of lactate was initially maintained. increased expression of monocarboxylate transporter (mct) was demonstrated in the lactate medium. results suggested that increased mct expression of neurons may lead to utilization of lactate to sustain synaptic function via calcium-dependent manner. yumei wu , kazuhito tomizawa , shuang liang , iori ohmori , teiichi nishiki , kohji takei , hideki matsui dept. of physiol., okayama univ., okayama, japan; dept. of neurosci., okayama univ., okayama, japan synaptic vesicle endocytosis is regulated by phosphorylation of endocytotic proteins, such as amphiphysin (amph) i and dynamin i. here, we show a novel type of regulation of vesicle endocytosis by proteolysis. in mouse hippocampal slices, amph i was found to be cleaved by a ca + -activated protease, calpain during prolonged depolarization or stimulus trains. the calpain-cleaved n-terminal amph i fragment lost its ability to bind dynamin and inhibited transferrin uptake as overexpressed in cos- cells, indicating that the calpain cleavage of amph i inhibits endocytosis. amph i in hippocampus was also cleaved by calpain in vivo after kainate seizure. although the second administration of kainate caused less severe seizure activity than the first one, this relieved second seizure was not observed in pre-treatment with a calpain inhibitor, allm during the first seizure. thus, the proteolytic activity of calpain could protect neurons from excitotoxicity by inhibiting vesicle recycling. synaptic vesicles (svs) are effectively recycled by endocytosis for continuous synaptic transmission. previously, we have suggested that a high level of synaptic transmission is maintained by recycling of svs through two types of endocytosis operating coordinately ( th this meeting). in the present study, we labeled endocytosed svs at nerve terminals of drosophila with fluorescence dyes, fm - and fm - , and also measured quantatively exocytosis and endocytosis of svs, using these dyes. egfp-labeled cacophony ca + channels and anti hrp stained the active zone and non-active zone at synapse, respectively. imaging analysis revealed that two distinct types of endocytosis of svs occurred at the active zone and the non-active zone of motor nerve terminals. we have previously shown that baclofen, a gaba b receptor agonist, inhibits exocytosis in synapses of mouse hippocampal neurons. syntaxin a is also known to modulate exocytosis. to characterize the molecular mechanisms involved, the inhibitory effects of baclofen in neurons transfected with antisense oligonucleotide to syntaxin a were investigated by patch-clamp recording and counting the number of release sites. transfected neurons showed higher frequency of miniature epscs and stronger inhibition by baclofen than controls, but no change in number of sites. increased exocytosis is thus induced by increases in transmitter release per site, rather than by more sites due to neurite sprouting. these results suggest that gaba b receptor shares part of the mechanism involved in modulation of exocytosis with syntaxin a in mouse hippocampal neurons. we have previously shown a transient localization of tubulin (tub) during synaptic vesicle (sv) cycling in drosophila nerve terminals. the tub localization is detected during sv recycling, while microtuble (mt)-loop is observed throughout sv cycle. in this study, we characterized the two distinct tub localizations and showed their relation with sv pool formation. axonal mts and mt-loops abounded in acetylated (acetyl) tub. the transient localization was either polymerized or depolymerized, and organized by non-acetyl tub. taxol decreased the non-acetyl tub localization but not mt-loops, and inhibited exo/endo cycling pool (ecp) formation. in boutons containing mt-loops, ecp formation was also inhibited. acetyl mt-loops tend to be stable whereas presynaptic non-acetyl tubs are either free dimers or dynamic mts. these results suggest that presynaptic dynamic tub, especially non-acetyl tub, controls ecp formation. presynaptic tub dynamics may regulate functional presynaptic plasticity by controlling sv pools. research funds: grant-in-aid for jsps fellows the mechanism by which pregnenolone sulfate (pregs) enhances synaptic transmission was studied at the rat calyx of held. pregs increased the amplitude of evoked epscs, without affecting that of spontaneous miniature epscs, indicating that the site of its action is presynaptic. pregs facilitated presynaptic voltage-gated ca + channel (vgcc) currents via accelerating their activation kinetics, but had no effect on k + currents, resting conductance, or action potential waveforms. in simultaneous pre-and postsynaptic recordings pregs did not change the relationship between presynaptic ca + influx and epscs, suggesting that exocytotic machinery downstream of ca + influx was not involved in the pregs effect. neither bapta nor gtp␥s loaded into presynaptic terminals blocked the effect of pregs. we conclude that pregs enhances transmitter release via facilitating vgccs by a novel mechanism, which is independent of intracellular ca + or g-proteins. ps a-b spine targeting of endocannabinoid synthesizing enzyme, diacylglycerol lipase-␣ in the cerebellum and hippocampus endocannabinoids are neuromodulator that is released from postsynaptic neurons, acts retrogradely on presynaptic cb cannabinoid receptor, and induce suppression of transmitter release. to understand the retrograde signaling mechanisms, we investigated subcellular localization of a major endocannabinoid biosynthetic enzyme, diacylglycerol lipase-␣ (dagl␣), in the mouse brain. in the cerebellum, dagl␣ was predominantly expressed in somatodendritic membrane of purkinje cells, and highly concentrated at the base of spine neck. however, dagl␣ was excluded from the main body of spine neck and head. in hippocampal pyramidal cells, dagl␣ was selective to spines, but widely distributed within spines. these results indicate that dagl␣ is essentially targeted to postsynaptic spines in cerebellar and hippocampal neurons, but its fine distribution within and around spines is differently regulated between the two cell types. synprint site of voltage-gated ca + channels interacts with synaptotagmin. however, its physiological role is not entirely clear. here we report that ap- subunit can directly bind with synprint site. this interaction was ca + -dependent, being weaker at concentrations higher than nm. in contrast, the interaction of synaptotagmin with synprint was optimal at m ca + , being weaker at lower or higher concentrations. the binding domain of synprint for ap- and synaptotagmin was indistinguishable, and these proteins competed with each other for the synprint site. to assess physiological role of these interactions, we made a peptide containing synprint site, and loaded it directly into the nerve terminal at the calyx of held. this peptide blocked endocytosis measured with capacitance, and gradually diminished exocytosis upon repetitive presynaptic activations. we conclude that ca + channel synprint site makes ca + -dependent interactions with ap- and synaptotagmin thereby contributing to vesicular endocytosis. ps a-b acl- , an evolutionarily conserved acyltransferase like gene is required for normal synaptic transmission in c. elegans naoko hara, takao inoue, yasukazu takanezawa, hiroyuki arai department of health chemistry, graduate school of pharmaceutical sciences, university of tokyo, tokyo, japan it is generally accepted that various phospholipid molecular species are formed by phospholipids acyltransferase reactions. however, the physiological significance and the molecular mechanism of the remodeling are largely unknown. to address these questions, we focused on evolutionarily conserved acyltransferase like genes in c.elegans acl- ˜ , and generated their deletion mutants. the mutants of acl- gene, which is predominantly expressed in neurons and muscles, showed no apparent phenotype. however, the mutants exhibited severe movement abnormalities in fat- mutant background in which long chain polyunsaturated fatty acids are depleted. pharmacological analysis revealed that these mutants showed presynaptic defects in synaptic transmission. these abnormalities were rescued by neuron specific acl- expression, suggesting that certain phospholipid species produced by acl- are involved in maintaining normal synaptic transmission and motility of c.elegans. daisaku yokomaku , hussam jourdi , akiyoshi kakita , tadasato nagano , hitoshi takahashi , nobuyuki takei , hiroyuki nawa dept. mol. neurobiol., brain res. inst., niigata univ., japan; brain resource center, brain res. inst., niigata univ., japan; dept. pathology, brain res. inst., niigata univ., japan scaffolding proteins containing pdz domains interact with synaptic receptors and cytoskeletal components and are therefore implicated in synaptic development and plasticity. little is known, however, about what regulates the expression of the pdz proteins and how the levels of these proteins influence synaptic development. here, we show that ligands for epidermal growth factor (egf) receptors (erbb ) decrease a particular set of pdz proteins and negatively influence synaptic formation or maturation. in neocortical cultures, egf decreased the expression of grip and sap . moreover, egf treatment resulted in a decrease in the frequency of pan-pdzimmunoreactive aggregates on dendritic processes. these findings revealed a novel negative effects of erbb receptor ligands that attenuates the expression of the pdz proteins and inhibits postsynaptic maturation in developing neocortex. takatoshi iijima , eriko miura , keiko matsuda , tetsuro kondo , , masahiko watanabe , michisuke yuzaki dept. physiol., sch. med., keio univ., tokyo, japan; dept. anatomy, hokkaido univ., sch. med., sapporo, japan; mol. neurophysiol., aist, tsukuba, japan cbln is a member of the c q and tumor necrosis factor families predominantly produced in cerebellar granule cells. recently, we have shown that cbln is secreted as a glycoprotein and plays crucial roles in synaptic plasticity and synaptic integrity of purkinje cells. although other members of the cbln family, cbln - , are known to be expressed in the brain, their precise expression patterns and biochemical properties remained unclear. here, we show that each cbln member is expressed in various regions of developing and mature brains. all cbln family members could form both homomeric and heteromeric complexes each other in heterologous cells. like cbln , cbln and cbln were secreted as glycoproteins, whereas cbln was retained in the endoplasmic reticulum. these results suggest that each cbln member is potentially involved in synapse development and plasticity in various brain regions. s-scam is a synaptic membrane-associated protein with pdz domains, a guanylate kinase domain and ww domains. it interacts with various synaptic components including nmda receptor subunits, psd- and neuroligin. as we previously reported, s-scam is recruited to excitatory synapses by -catenin. s-scam forms a ternary complex with neuroligin and psd- . more importantly, s-scam is involved in synaptic accumulation of neuroligin and subsequently affects the localization of psd- at excitatory synapses. in the course of these studies, we observed signals detected by anti-s-scam antibody at inhibitory synapses. we have here examined whether s-scam is indeed localized at inhibitory synapses in hippocampal neurons. we have raised questions which molecules s-scam interacts with at inhibitory synapses and which role s-scam plays in the assembly of inhibitory synapses. eriko fujita, yuko tanabe, takashi momoi division of differentiation and development, department of inherited metabolic disorder, national institute of neuroscience, ncnp, oawahigashi, tokyo, japan igsf /ra (ra ), which is a member of immunoglobulin superfamily having pdz binding domain at c-terminals, has ca +independent homophilic trans-cell adhesion activity. ra participates in synaptic junction and epithelial junctions in various tissues including testis. homozygous null (ra -/-) male is infertile and shows the defective elongating spermatids and fails to mature further. ra interacted with par- being involved in the polarity of epithelial cells via pdz binding domain at c-terminals. par- was colocalized in the cell adherent region of p embryonal teratocarcinoma cells during ra-induced differentiation into epithelial-like cells and mainly localized in the spermatid of ra +/+ testis, whereas it was undetectable in the spermatid of the ra −/− testis. ra and jam-c were localized around the head portion of spermatid and ra deficiency provided the abnormal polarization of the jam-c, which is necessary for the differentiation of round to elongated spermatid. jam-c inhibited the interaction between ra and par- . research funds: izumi kawabata, shigeo okabe department of cell biology, tokyo medical and dental university, tokyo, japan coordinated development of excitatory and inhibitory synapses is critical for both stability and temporal fidelity of neuron network in the hippocampus. however, there have been few analyses on postsynaptic molecular assembly in interneurons during development. to address this question, we examined dynamic properties of psd- clusters in cultured hippocampal interneurons. higher density of dendritic psd- clusters was observed in interneurons at div. at div, this difference was less prominent, mainly due to > -fold increase of psds in excitatory neurons. psd- -gfp imaging revealed lower rate of cluster appearance/disappearance in interneurons at div. the higher rate of cluster turnover in excitatory neurons, together with their higher rate of net cluster increase, may explain the delayed boost of cluster density. photobleaching of psd- -gfp revealed similar kinetics in two neuron types, suggesting additional determinants of cluster dynamics apart from the steady-state assembly rate. possible involvement of other postsynaptic molecules in interneuron psd dynamics is now being investigated. ps a-c two-photon imaging of immature dendritic protrusions and astroglial processes in hippocampal slice cultures hideko nishida, shigeo okabe department of cell biology, tokyo medical and dental university, tokyo, japan several lines of evidences indicate roles of astroglia in synaptogenesis, possibly mediated by either cell adhesion or diffusible factors. however, structural evidences supporting this claim are virtually lacking, mainly due to technical limitations in simultaneous imaging of neuronal and astroglial structures. here we visualized astroglia and pyramidal neurons in hippocampal slice cultures by combining adenovirus-mediated, cre-dependent expression of gfp with electroporation of rhodamine-dextran. two-photon time-lapse imaging of immature dendritic protrusions and astroglial processes in - div slice cultures revealed longer lifetime of dendritic protrusions having experienced astroglial contacts than those without contacts. dendritic protrusions with astroglial contacts also showed higher tendency to form spines. furthermore, expression of mutant rac in astroglial cells induced significantly longer, non-spiny protrusions than control. these findings suggest an involvement of direct astroglia-filopodia contacts in subsequent maturation of dendritic protrusions. taiko imura, fusao kato lab. neurophysiol., jikei univ. sch. med., tokyo, japan application of p x receptor agonists to the neurons in the nucleus of the solitary tract (nts) results in glutamate release facilitation (kato & shigetomi, ; shigetomi & kato, ) . recently accumulated evidence indicates that astrocytes affect the neuronal excitability by releasing gliotransmitters such as atp. this study was performed to determine whether such astrocyte-neuron interaction takes place in the nts. first, we analyzed the spatial localization of these cells by immunohistochemistry. a large number of gfap-positive cells with processes in the close apposition to the neun-positive neurons were found. second, we analyzed the effect on synaptic activity of localized application of atp using laser-based photolysis of caged atp in brainstem slices. uncaging of atp at neuronal dendrites ( s, -micrometer diameter) resulted in an immediate rise in mepsc frequency, in a manner sensitive to p x receptor antagonists. these results provide supports for the possible interaction between astrocytes and neuronal presynaptic terminals. research funds: kakenhi ( ) ps a-c ealy synapsin i accumulation in a granule cell axon at the filopodial attachment site of developing rodent purkinje cell dendrites in vitro isao nagata, junko kimura-kuroda department of brain structure, tokyo metropolitan institute for neuroscience, tokyo, japan synapse formation between the parallel fibers (pf) and dendrites of purkinje cells (pc) occurs at an early stage in the developing cerebellar cortex of the neonatal rodent. however, the precise spatio-temporal pf-pc interaction has not been elucidated. we have found that growth of pc dendrites was initiated by the attachment of axonal neurite bundles of granule cells orienting at right angles in several types of d-and d-cerebellar cultures. here, we investigated the expression of a synaptic vesicle marker, synapsin i, in granule cell axons by multiple immunofluorescence labelings in these cultures. synapsin i was first expressed at the filopodial attachment site of a pc dendrite as a cluster of faint punctate deposits in a long axon, then they appeared to gather into a slender and finally into a small round deposit. thus, the filopodial attachment of the juvenile pc dendrites to the axons of granule cells may induce rapid formation of presynaptic terminals via local clustering of synaptic vesicles. ps a-c integrative spike dynamics of rat ca neurons: an in situ multineuronal imaging study takuya sasaki, rie kimura, norio matsuki, yuji ikegaya department of pharmacology, university of tokyo, tokyo, japan the brain operates through a coordinated interplay of numerous neurons. our new technique with large-scale optical recordings reveals the diversity of synaptic integration in hundreds of neurons. in hippocampal slices bolus-loaded with calcium fluorophores, we stimulated the schaffer collaterals and monitored the bulk presynaptic activity from the stratum radiatum and individual postsynaptic spikes from the ca stratum pyramidale. single neurons responded to varying synaptic inputs with unreliable spikes, but at the population level, the networks output a linear sum of synaptic inputs. the network activity varied from trial to trial, even though given constant stimuli. this variation emerged through time-varying recruitment of different neuron subsets, which were shaped by correlated background noise. our imaging approach enables linking single-cell behaviors to their communal dynamics, and we discovered that, even in a relatively simple ca circuit, neurons could collectively be engaged in complex information processing. it is assumed based on previous in vitro experiments by other researchers that mglur connects with syntenin at the dendrites and mglur with pick at the axon terminal in on cone bipolar cells. to prove this possibility, we investigated wild-type mouse retinas immunohistochemically and confirmed their co-localized immunopositive labels at the respective places. next, we examined which scaffold protein would connect with mglur that was known to be ectopicly expressed in the dendrites of mglur -deficient on cone bipolar cells. we observed no pick but only syntenin at the mglur -deficient dendrites, and also the syntenin immunopositivity was co-localized with mglur immunopositivity. these findings suggest that mglur connects with syntenin in place of mglur that was knockout from the on cone bipolar dendrites. noriko trpv family is identified as thermosensitive, ca + -permeable channels. trpv , expressed in sensory neurons, is activated by noxious heat above • c, whereas trpv , expressed in keratinocytes, is sensitive to moderate temperatures (> • c). here we examined the role of trpv and in regulation of body temperature (bt) by using infrared laser as a heat stimulus. in wild type mouse, though the laser irradiation which caused the increase in skin temperature up to • c did not induce the change in bt, desensitization of trpv with capsaicin resulted in the increase in bt. on the other hand, in trpv -knockout mouse, moderate thermal radiation (> • c) caused the increase in the bt. the processing of noxious and moderate thermal radiation stimuli may depend on the trpv and respectively. research funds: kakenhi ( ) ps a-c generation and biochemical analysis of a glur␣ knockout mouse hirotsugu azechi , manabu abe , rie natsume , , kenji sakimura , department of cellular neurobiology, brain research institute, niigata university, niigata, japan; sorst/jst, saitama, japan glur␣ (glur ) is a key subunit of ampa receptors, since it is a critical determinant of their calcium permeability. to clarify the molecular function of glur␣ , we generated a conditional glur␣ knockout mouse using the cre/loxp recombination system. we first established a "floxed" mutant line gra f using c bl/ (b ) es cell line renka. the homozygous floxed mutants showed no significant abnormalities, thus our gra f was used as a target of glur␣ line. by crossing gra f and tlcn-cre that expressed cre in germ line cells, glur␣ null ko mice were produced, but most of them died within days after birth. to overcome the lethality, the glur␣ mutation was transferred onto b / or b /cd- genetic background. subcellular fractionation and quantitative immunoblot showed changes in the amount of ampa receptor subunits. these results indicated a significant role of glur␣ in the distribution of functional ampa receptors in vivo. ps a-c gisp: a novel brain specific protein that binds to the gaba b subunit and promotes its surface expression here we report the identification and characterisation of a novel brain specific kda protein, gaba b r interacting scaffolding protein (gisp), that interacts directly with the gaba b subunit via a coiledcoil domain. gisp coimmunoprecipitates with gaba b and gaba b from rat brain. in cultured hippocampal neurons gisp displays a punctate dendritic distribution and colocalises with gaba b receptors. when co-expressed with gaba b rs gisp increases the amount of gaba b protein and also promotes gaba b surface expression in the heterologous cells. furthermore, gisp increases surface expression of gaba b /gaba b complexes. these results suggest that gisp is involved in the forward trafficking and stabilisation of gaba b rs. thus gisp is an novel gaba b -binding protein potentially involved in the cell surface and/or synaptic targeting of the gaba b rs. three distinct isoforms of vesicular glutamate transporters (vglut - ) have been cloned and shown to exhibit differential distribution patterns in the brain. recent work shows the presence of vgluts in synaptic-like microvesicles (slmvs) of endocrine cells. mammalian pineal melatonin-secreting cells, pinealocytes, contain numerous slmvs which likely accumulate glutamate to inhibit melatonin synthesis. vglut and vglut seem to participate in this glutamate accumulation. in the present study, we found that vglut mrna is also expressed in the adult rat pinealocytes. vglut immunoreactivity (ir) was distributed throughout the pineal gland, and was co-localized with vglut -ir or vglut -ir in many, but not all, processes of pinealocyte. these data indicate that there are some subpopulations of slmvs which differ in the kind of vglut isoforms contained and/or in their combinations, suggesting vglut isoform-dependent sorting of slmvs to pinealocyte processes. kenzi saito , , , kenji nakamura , toshikazu kakizaki , , satoe ebihara , masakazu uematsu , shigeo takamori , minesuke yokoyama , shiro konishi , masayoshi mishina , , jun-ichi miyazaki , kunihiko obata , yuchio yanagawa , gunma univ., maebashi, japan; sokendai, hayama, japan; sorst, kawaguchi, japan; mitsubishi kagaku inst. life sci., machida, japan; kumamoto univ., kumamoto, japan; toyohashi univ. tech., toyohashi, japan; tokyo med. den. univ., tokyo, japan; univ. tokyo, tokyo, japan; osaka univ., suita, japan; riken, wako, japan the vesicular gaba transporter (vgat) loads gaba from neuronal cytoplasm into synaptic vesicles and is selectively expressed in inhibitory neurons containing gaba and/or glycine. to assess the functional role of vgat in development, we have disrupted the gene encoding vgat using cre-loxp system. western-blot analysis showed that vgat protein was absent in the homozygous embryos, indicating that the mutation had generated in a null allele. vgat knockout mice died around birth. all vgat knockout mice displayed cleft palate and omphalocele. our results suggest that vgat plays essential roles in both palate formation and ventral body wall development. research funds: kakenhi ( ) ps a-c postnatal changes in the colocalization of vglut and vglut immunoreactivities at single axon terminals of the mouse neocortex kouichi nakamura , , akiya watakabe , hiroyuki hioki , fumino fujiyama , yasuyo tanaka , tetsuo yamamori , takeshi kaneko , dept. morphol brain sci., grad. sch. med., kyoto univ., japan; crest, jst, japan; div. brain biol., nat. inst. basic biol., okazaki, japan vesicular glutamate transporter (vglut) and vglut accumulate transmitter glutamate into synaptic vesicles. the vgluts show a complementary expression pattern in the brain, but colocalize at single axon terminals in some synapses. here we quantitatively evaluated postnatal changes in the colocalization of vgluts at single axon terminals of the developing mouse neocortex by using a pixel-based correlation coefficient (cc) as an index of the colocalization. the cc was calculated from pixel values for vglut and vglut in each pixel of confocal micrographs of double immunofluorescence-labeled brain sections. in the barrels, the cc showed a prominent increase transiently around p . the cc was higher in area s than areas m and area v throughout postnatal development. our results indicate that the colocalization of vgluts in the neocortex is regulated in an age-, area-and layer-specific manner. gaba b receptors mediate slow and prolonged synaptic inhibition in the brain, and are members of the g protein-coupled receptors. here we have investigated the role of amp-activated protein kinase (ampk), as an endogenous regulator of gaba b receptor function. site-specific mutagenesis identified multiple phosphorylation sites for ampk within the cytoplasmic tails of both gaba b r and r . the activation of ampk regulated stability of gaba b receptors coupling with k + channels. together highlights a novel role for ampk in regulating the functional properties of gaba b receptors, by direct phosphorylation. given the role of ampk as a sensor of cellular stress this potential mechanism may be relevant in regulating the efficacy of synaptic inhibition under anoxic conditions and during periods of high synaptic activity. takao hirai, hiroaki nishio department of molecular pharmacology, faculty of pharmacy and pharmaceutical sciences, fukuyama university, hiroshima, japan serotonin ( -hydroxytryptamine, -ht) is a central neurotransmitter that is widely implicated in the regulation of mood and cognition, and is a peripheral signaling molecule that affects hemostasis, immune function, intestinal physiology, and other systems. there is increasing evidence for contribution of neuronal system to regulation of bone metabolism. this study was thus aimed at elucidation of possible functional expression of serotonergic system in mouse osteoblasts. rt-pcr analysis revealed constitutive expression of mrna for several -ht receptor subtypes, -ht transporter ( -htt) and vesicular monoamine transporter (vmat ) in primary cultured mouse osteoblasts and mc t -e osteoblastic cells. sustained exposure to fluoxetine, a selective -ht reuptake inhibitor, significantly prevented increase in alkaline phosphatase activities and mineralization in mc t -e . these results suggest that serotonergic system may be functionally expressed to regulate mechanisms underlying cellular differentiation and maturation in mouse osteoblasts. junko motohashi department of physiology, keio university school of medicine, tokyo, japan hotfoot mice are spontaneous mutants with ataxic phenotype. most hotfoot alleles identified so far have deletions of one or more exons coding for portions of the n-terminal domain of the ␦ glutamate receptor (glur␦ ). however, because only genomic dna was available for most hotfoot mutants, it was unclear whether truncated forms of glur␦ were actually translated and involved in the ataxic phenotype. here, we report that a newly identified hotfoot mutant, ho j, was caused by a new type of intragenic deletion of the grid gene, which was indeed translated as glur␦ lacking -amino acids in the n-terminus. mutant glur␦ proteins were retained in the soma of purkinje cells and degraded. as a result, ho j mice exhibited a severe motor discoordination on rotarod tests. furthermore, these mice exhibited sustained innervation of purkinje cells by multiple climbing fibers, and impaired long term depression, which is thought to underlie motor learning. these results indicate the importance of the n-terminal domain in glur␦ signaling and cerebellar functions. research funds: kakenhi ( ) ps a-d role of the dry motif in melanin-concentrating hormone receptor in signaling yumiko saito , yoshimi aizaki , mituse nakano , kei maruyama dept. pharamacol., saitama med. sch., saitama, japan; international university of health and welfare, tochigi, japan considerable attention has been focused on the functional importance of the highly conserved dry triplet in class a g protein-coupled receptors (gpcr). here we investigated the role of asp , arg and tyr in the dry of rat melanin-concentrating hormone receptor (mch r). in transfected cells, mutation of asp (d/a) resulted in nonfunctional receptor despite of showing moderate level of cell surface expression and an apparent affinity to mch. d/a mutation occurred with no increase in basal signaling pathway, suggesting no indication for constitutive activity. y/a mutation also yielded a loss of function phenotype that is similar to d/a mutation. mutation of the arg (r/a) showed higher ec value in signaling with a decrease in mch binding, while the level of cell surface expression exhibited only moderate decrease. these data suggest that a function for dry motif different from that widely accepted for class a gpcrs in regulating mch r-mediated signal pathway. in this study we confirmed functional heteromultimerization between a r and p y r electrophysiologically using xenopus oocyte expression system. when a r and p y r were coexpressed, application of non-hydrolyzable atp analogue induced g i/o response, showing formation of functional heteromultimers with a unique phenotype. it was also observed that the heteromultimers can activate g q/ pathway by atp analogue and also g i/o pathway by adenosine analogue, maintaining the features of the original subunits. ps a-d dual signaling via metabotropic glutamate receptor ␣ is regulated by a cytoskeletal protein . g michihiro tateyama , , yoshihiro kubo , department of biophysics and neurology, nips, aichi, japan; sorst, jst, saitama, japan the g protein-coupled metabotropic glutamate receptor ␣ (mglur ) is known to functionally couple to different types of g proteins. recently we have reported that the signaling pathways through mglur are differentially regulated by different types of ligands, glutamate and gd + . on the other hand, several cytoskeletal proteins have been reported to interact with the c-terminal cytoplasmic tail of mglur . these proteins, such as homer and . g, are also known to change the membrane expression of and modulate the function of mglur . here we investigated whether or not these cytoskeletal proteins regulate the multi path signaling of mglur . interestingly, the functional couplings of mglur to gq and gs pathways were altered by co-expression of . g, but not by homer. deletion of the c-terminal tail abolished the effect of . g, indicating that the interaction of . g with the c-terminal tail of mglur regulates the multi path signaling. ps a-d modulation of the eaac -mediated glutamate uptake by the addicsin mutant mitsushi j. ikemoto , , saori akiduki , age dimension research center, aist, ibaraki, japan; graduate school of science, toho university, chiba, japan addicsin is a murine homologue of rat glutamate-transporterassociated protein - (gtrap - ), an inhibitory modulator of neural glutamate-transporter excitatory amino acid carrier (eaac ). it contains two potential pkc phosphorylation motifs at positions - and - . however, its physiological function remains almost unknown. to clarify a significance of these pkc phosphorylation motifs, we investigated eaac -mediated glutamate transport activity in c bu- cells provided with a mifepristone-inducible expression of addicsin (wt), its mutants mutated at serine into alanine (s a) or at serine into alanine (s a). as compared with wt, s a had no inhibitory effect on glutamate transport activity under exposure to nm pma, and had increased glutamate transport activity under normal condition. by contrast, s a had the same glutamate transport activity as that of wt. thus, the eaac -mediated glutamate transport activity may be regulated by a pkc-dependent phosphorylation at serine in addicsin. kaori akashi , manabu abe , toshikazu kakizaki , rie natsume , , kenji sakimura , department of cellular neurobiology, brain research institute, niigata university, japan; sorst-jst, saitama, japan kainate type glutamate receptors are composed of various combinations of glur - (glur - ) and glur␥ - (ka - ) subunits. although their physiological functions and subunit compositions have been inferred from various studies, they are still not clear. to clarify the functions and subunit dynamics of kainate receptors, we generated glur ko mice from c bl/ es cell line. the glur ko mice were viable, fertile, and displayed no overt phenotype. on the other hand, the amounts of glur␥ and glur␥ proteins were significantly decreased in the crude fraction of ca region of glur ko. furthermore, subcellular localizations of both subunits were also changed in glur ko. these results suggested that native kainate receptors might function as heteromeric channels (glur/␥) and the glur subunit might determine subcellular localization of the glur␥ subunits, similar to the roles of nmda receptor glur subunits determining stability and distribution of the glur subunits. ps a-d sema d/plexin-b activates gsk-  via r-ras gap activity, inducing growth cone collapse yuri ito, izumi oinuma, hironori katoh, manabu negishi laboratory of molecular neurobiology, graduate school of biostudies, kyoto university, kyoto, japan plexins are receptors for repulsive axonal guidance molecules semaphorins. we have recently reported that semaphorin d (sema d) receptor plexin-b induces growth cone collapse by functioning as an r-ras gap. here we characterized the downstream signaling of plexin-b -mediated r-ras gap activity, leading to growth cone collapse. sema d suppressed the endogenous r-ras activity in hippocampal neurons, in parallel with dephosphorylation of akt and activation of gsk- . ectopic expression of the constitutively active mutant of akt, myr-akt, or treatment with gsk-  antagonist suppressed the sema d-induced growth cone collapse. the r-ras gap activity was necessary for plexin-b -induced dephosphorylation of akt and gsk- . plexin-a also induced dephosphorylation of akt and gsk-  through its r-ras gap activity. thus, we conclude that plexin-b dephosphorylates akt and gsk-  through r-rasgap activity, inducing growth cone collapse. to find proteins having relations in receptor trafficking, we searched human genome database and selected hepatocyte odd protein shuttling (hops) as a candidate gene. hops had three transmembrane domains, and expressed abundantly on a brain tissue. hops was detected in membranous regions from subcellular fractionation and immunohistochemistry. hops was recruited to membranous structures when overexpressed in cos cells. when expressed in hippocampal cultures, hops enhanced the amplitude of mepsc. from antibody feeding assay, we discovered that hops enhanced the recycling of glur . hops was co-immunoprecipitated with grip (glutamate receptor interacting protein ) when they were co-transfected to hek cells. thus, it was suggested that hops had roles in synaptic transmission enhancement by stabilization of surface glur via grip binding. serine must be taken up into neurons for their survival, because neurons lack serine biosynthetic enzyme. we have recently identified a serine transporter asc- . a neural amino acid transporter snat /ata also transports serine. we investigated their roles as serine transporters by comparing the localization of these serine transporters in the rat brain. the asc- immunoreactivity (asc- -ir) was detected in dendrites and somata of pyramidal neurons. the snat -ir was widely detected in neurons, whose intracellular localization was similar to that of asc- -ir. deferent from asc- -ir, snat -ir was also located in astrocytes and ependymal cells, especially around capillary blood vessels and ventricles. these results suggest the significant contribution of asc- and snat to the neuronal uptake of l-serine. snat might also accumulate l-serine in astrocytes from the extracellular spaces including blood and csf. ps a-d neuronal glutamate transporter eaat controls climbing fiber-mediated presynaptic inhibition of gabaergic transmission at cerebellar interneuron-purkinje cell synapses shin'ichiro satake , si-young song , shiro konishi , keiji imoto natl. inst. physiol. sci. (nips), okazaki, japan; mitsubishi kagaku inst life sci, tokyo, japan; tokushima bunri univ., sanuki, japan through extrasynaptic diffusion and activation of presynaptic ampa receptors in bc terminals. we here examined possible roles of glutamate transporters in this cf action. the eaat /glt- blocker threo- -methylglutamate, but not the glt- blocker dihydrokainate, augmented the cf-induced inhibition. cf stimulation obviously inhibited gabaergic transmission onto pcs in the lobule iii, where eaat expression was low, whereas the cf-induced inhibition was minimal in the lobule x, where eaat was abundant. the results suggest that eaat plays a major role in regulating the concentration of cf transmitters, possibly glutamate, in the route of its extrasynaptic diffusion, and determining the degree of cf-induced inhibition of gaba release from bcs depending on the regional difference of eaat expression in postsynaptic pcs. chitoshi takayama , yoshiro inoue department of molecular neuroanatomy, hokkaido university school of medicine, sapporo, japan gaba mediates inhibitory transmission in the adult central nervous system (cns). in contrast, gaba induces depolarization in the immature cns. this developmental shift from depolarization to hyperpolarization may be caused by decreasing of the intracellular chloride ion concentration regulated by two chloride ion co-transporters, na-k- cl co-transporter (nkcc ) and k-cl co-transporter (kcc ). in this study, we focused on kcc , which lowers the intracellular chloride ion concentration, and examined the developmental localization of the kcc with special reference to the neuronal development in the cerebellum. kcc was negative in the proliferating and migrating neurons. post-migratory neurons, which formed synapses, expressed the kcc . the kcc -protein was localized at the membrane of dendrites and cell bodies, whereas growth cones, axons and terminals were negative. these results suggested that formation of synapses might induce kcc -expression and localization, and gabaergic transmission might shift from excitation to inhibition after synapse formation. akinori nakajima , hisashi mori molecular neuroscience, university of toyama, toyama, japan the actions of many neurotransmitters are mediated by the members of a superfamily of receptors coupled to heterotrimeric guanine nucleotide binding proteins (g-proteins). the dopamine receptors are classified into two categories, d -like and d -like according to their pharmacological properties. the d -like receptors consist of d and d receptor, and are coupled to the adenylyl cyclase activating g proteins (gs). in the present study, we have generated a series of d receptor mutants and examined the effect on the gs coupled receptor signaling. we found that the expression of the third intracellular loop ( i loop) domain of d r fused with egfp effectively reduce camp production mediated by d and d receptors. interestingly, we also identified that the i loop domain of d r interfere with gs coupled beta adrenergic receptor signaling. these results suggest that the third intracellular loop of the d receptor is a primary determinant in its coupling to gs signaling. the activation of phosphatidylinositol-linked d -like dopamine receptor profoundly suppresses the exaitatory transmission in the developing hippocampus yoshinobu noriyama , yoichi ogawa , hiroki yoshino , masayuki yamashita , toshifumi kishimto dept. psychiatr.; dept. physiol i, nara med. univ., kashihara, japan we studied the effect of dopamine (da) on gabaergic and glutamatergic transmission in neonatal rat hippocampus from the early period of synapse formation by whole-cell patch-clamp recordings from ca pyramidal cells. da ( m) profoundly decreased gaba a receptor-mediated postsynaptic currents to % in the first postnatal week, when gaba provides excitatory drive. da also decreased ampa receptor-mediated excitatory post synaptic currents to % in the second postnatal week, when glutamate responses first appear. the da-induced inhibition declined after these periods. the receptor subtype involved in the da-induced inhibition was phosphatidylinositol (pi)-linked d -like receptor, since skf , a selective agonist for pi-linked d -like receptor, clearly mimicked the action of da. these results suggest that the activation of pi-linked d -like receptor profoundly suppresses the excitatory transmission during the early period of synapse formation in the developing hippocampus. ayuka ina , jinko konno , sachine yoshida , hideki ohmomo , hitoshi kawano , fumihiro shutoh , haruo nogami , setsuji hisano graduate sch., comprehensive human sci, univ. tsukuba, tsukuba, japan; tokyo metro inst neurosci, tokyo, japan supporting critical neurobiological roles of glutamate in mouse corticogenesis, we recently reported that cortical cells express vglut or - mrna at early fetal ages. to know roles of fetal vglut in cortical development, we studied expressions of vglut proteins in mouse fetuses by immunohistochemistry. on embryonic day (e ), vglut immunoreactivity (ir) was first detected in the marginal zone (mz), subplate (sp) and intermediate zone (imz). on e , vglut -ir was seen as puncta close to l -ir thalamocortical fiber tracts in the sp and also localized to fiber tracts expressing l or tag -ir in the imz, whereas vglut -ir was first observed in the sp and upper imz where l -ir existed. these results show that vglut ir corticofugal fibers appose to elongating vglut -ir thalamocortical fibers, suggesting that vglut may play a crucial role in glutamatemediated axon guidance to determine thalamic innervation patterns in the developing cortex. ps a-d developmental changes in the mechanism underlying activity-dependent swelling of the hippocampal ca regions michie kon , yoichi avil , hiroshi tsubokawa , dept. of information engineering, tohoku univ., sendai, japan; grad. schl. of information sciences, tohoku univ. to investigate the mechanisms underlying swelling of brain cells in association with neuronal activity, we analyzed interactions between changes in cell volume and synaptic activities in mouse hippocampal slices. swelling of several areas within the ca region were detected as increases in transmittance of near infrared light (irt). field epsps (feps) were recorded simultaneously from the stratum radiatum of ca region. in adult mice, repetitive stimulation of afferent fibers induced transient increases in irt at both somatic and dendritic regions in a frequency-dependent manner, which was temporally associated with feps. application of the bicuculline, a gaba-a receptor antagonist, reduced these optical signals. however, in mice under days old, the optical signals did not follow by high-frequency stimulation of inputs, and were not affected by an application of bicuculline. these results suggested that gaba-dependency in the mechanisms of cell volume regulation developmentally changes in the hippocampal ca region. in the present study, the effects of bilateral injections of glutamatergic agents into the hippocampal ca region on morphine-induced conditioned place preference (cpp) were investigated in rats. subcutaneous administration of different doses of morphine ( . - mg/kg) produced a dose-dependent cpp. using a -day schedule of conditioning, it was found that intra-ca administration of nmda receptor antagonist, mk- ( and g/rat) significantly attenuated the morphine ( . mg/kg)-induced cpp. moreover, nmda receptor agonist, nmda ( . , . and g/rat) significantly potentiated the morphine ( . mg/kg)-induced cpp. these results suggest that the development of morphine-induced cpp may be related to nmda and mk- receptors in that the glutamatergic system can modulate opiate reward. takahiro sonomura , kouichi nakamura , , hiroyuki hioki , masanori uemura , takeshi kaneko , dept. anatomy for oral sciences, grad. sch. med. and dent., kagoshima univ., kagoshima, japan; dept. morphological brain sciense, grad. sch. med., kyoto univ., kyoto, japan; crest, jst the majority of neostriatal neurons are medium-sized projection neurons with spiny dendrites and have so far been classified into three groups: striatonigral neurons producing ppd, and striatopallidal neurons producing ppe, and striatoinnominatal neurons producing pptb. these projection neurons are regulated in part by dopaminergic input from the substantia nigra pars compacta. it has been assumed that d receptor are expressed in striatonigral neurons and d receptor are expressed in striatopallidal neurons. in recent years, molecular cloning work has shown that there are at least five dopamine receptor genes (d , d , d , d , d ). in this study, the double-labeling method combining in situ hybridization and immunocytochemistry revealed how these five dopamine receptor subtypes are distributed among three projection neuron groups. the cerebellar tissue is good model system for the analysis of neuronal development, since dynamic neuronal development such as migration and axonal and dendritic outgrowth after birth. in the present study, we examined the localization of chondroitn sulfate proteoglycans (cspgs) by cspg-specific antibodies and lectins. cspgs are mainly observed at molecular layer in developing cerebellum (p - ) but they scarcely seen at external granular layer. electron microscopic observation demonstrated that phosphacan, one of cspgs, is localized at axonal membrane of parallel fibers. moreover, phosphacan inhibited adhesion and axonal extension of cerebellar granular neurons, while it promoted axonal fasciculation of their aggregated cultures. thus, cspgs, inhibitory molecules for axonal extension, are participated in axonal guidance cue in developing cerebellum. the vasopressin neurons are well knwon to show structural plasticity during chronic physiological stimulation such as salt loading. in the present study, salt loading significantly diminished the levels of chondroitin sulfate proteoglycans (cspgs) in vasopressin neurons. this downregulation is possibly due to proteolysis by tpa, since ( ) tpa immunoreactivity was observed at neurosecretory granules of vasopressin dendrites and terminals, ( ) salt loading increased protein and mrna levels of tpa in the somata and dendrites in the supraoptic nucleus but reduced protein levels of it in the terminals of the neurohypophysis, ( ) depolarizing agent released tpa from isolated neurosecretosomes, ( ) tpa knockout mice revealed lower ability of osmotic homeostasis and vasopressin release. thus, it is probable that tpa is participated in regulating structural plasticity of vasopressin neurons by degrading cspgs. chondroitin sulfate (cs) proteoglycans are essential for neuronal morphogenesis, including neural migration, survival and neurite formation in the developing brain. cs chains are modified by various sulfotransferases generating diverse sulfation patterns, which are assumed to be involved in the selective binding to various proteins such as growth factors. in this study, we analyzed the expression patterns of several cs sulfotransferases in the developing mouse cerebrum. using in situ hybridization analysis, it was revealed that cs sulfotransferase mrnas (u st, galnac - st, d st) were expressed in various types of cells, especially in the ventricular zone, and the cortical plate neurons just below the marginal zone. immunohistochemical analysis with anti-cs antibodies revealed that cs were highly expressed in the ventricular zone and the marginal zone. these results suggest that the cs structural domains generated by these cs sulfotransferases are involved in the regulation of the proliferation of neural progenitor cells and neuronal migration. nobuaki maeda , maki ishii , isao nagata , yumiko shimazaki dept. of dev. neurosci., tokyo metro. inst. for neurosci., tokyo, japan; dept. of brain structure, tokyo metro. inst. for neurosci. chondroitin sulfate (cs) is a long polysaccharide with enormous heterogeneity that binds with various proteins in a structure-dependent manner. previously, we revealed that cs is involved in the morphogenesis of the purkinje cell dendrites. in this study, we analyzed the expression of cs in the postnatally developing cerebellum using monoclonal antibodies that recognize specific structural motifs in cs. among the epitopes recognized by these antibodies, the expression of mo- epitopes, glca( s) - galnac( s) (d unit)containing structures, remarkably increased during development. detailed immunohistochemical analysis indicated that d unit-rich cs was deposited between purkinje cell surface and the processes of bergmann glia. furthermore, it was found that pleiotrophin bound to d unit-rich cs on phosphacan distributed around purkinje cells. these observations suggest that d-type structure in cs is important for the signaling of pleiotrophin, which play roles in purkinje cell-bergmann glia interaction. nobuna fukazawa , mineko kengaku , nobuaki maeda dept. of dev. neurosci., tokyo metro. inst. for neurosci., tokyo, japan; lab. for neuronal cell polarity, riken bsi, wako, japan ptp is a receptor-type protein tyrosine phosphatase, which is synthesized as a chondroitin sulfate proteoglycan that pleiotrophin-ptp signaling regulates the morphogenesis of purkinje cell (pc) dendrites. we previously revealed that ptp associated with delta/notchlike egf-related receptor (dner), which mediates the pc-bergmann glia (bg) interaction and regulates morphological differentiation of these cells. here, we found that ptp was expressed by both pcs and bgs and the expression by pc occurred at relatively late developmental stage. ptp showed patchy distribution in the dendritic shafts of pcs, which partially overlapped with the localization of dner. furthermore, we revealed that multiple tyrosine residues in the cytoplasmic domain of dner were phosphorylated and that these tyrosine phosphorylated residues were efficiently dephosphorylated by the ptp catalytic domain. these results suggested that ptp participate in the pc-bg interaction by regulating tyrosine phosphorylation level of dner. masahiko tanaka , tohru marunouchi division of cell biology, institute for comprehensive medical science, fujita health university, toyoake, aichi, japan cerebellar purkinje cells have the most elaborate dendritic trees among the neurons in the cns. to investigate the cellular and molecular mechanisms of dendrite development of purkinje cells, we cocultured purkinje cells on a coverslip with other cerebellar cells such as granule cells and astrocytes on the cell culture insert of m pore size. when purkinje cells were co-cultured with granule cells, dendrite development of purkinje cells was promoted in comparison with that in control conditions. this co-culture effect was abolished by addition of a glutamate antagonist in the cultures. in contrast, dendrite development of purkinje cells was inhibited when purkinje cells were co-cultured with astrocytes. we propose that (i) glutamate secreted by granule cells and diffused through the porous membrane of the cell culture insert promotes the dendrite development of purkinje cells and (ii) astrocytes inhibit the effect of glutamate through their glutamate transporting activity. heparan sulfate (hs) proteoglycans regulate neural development through the interaction with cell surface proteins and extracellular matrix molecules. an extracellular endosulfatase, sulffp , has been implicated in the regulation of growth factor/morphogen signaling through hs remodeling in vitro, but its physiological roles remain unknown. here we generated knockout mice lacking the sulffp gene, and examined the motor control. homozygotes appeared to be normal, showing no sign of ataxia. performances of the rotarod and beam-walking tests were normal compared with the control mice. both short-term and long-term adaptations in the optokinetic response were normal, while the gains in optokinetic response and vestibulocular reflex were significantly reduced. heparan sulfate (hs) proteoglycans regulate a number of developmental signaling through interactions with cell surface proteins and extracellular matrix molecules. these interactions are mediated by the specific sulfation patterns in hs, but the mechanism generating such modifications has not been fully elucidated. here we show that a new class of hs endosulfatases plays an important role in brain development. the mice deficient in either sulffp or sulffp appeared to be normal, while most of the double knockout mice died soon after birth. mutant brains had higher content of -o-sulfated disaccharide units in hs, suggesting a role of sulffps in heparan sulfate remodeling in vivo. the double mutant brains were smaller than the controls and showed some axon guidance defects. these data demonstrate that specific hs modification generated by sulffps is important for normal brain development. recent studies have suggested monoamine affects neural development, but it is unclear which receptor subtypes mediate actions of monoamine. here, we examined roles of -hydroxytryptoamine ( -ht), noradrenaline (na) and dopamine (da) in the formation of dendrites and synapses by dissociation culture. embryonic day or rat cerebral cortex was cultured in the presence of -ht, na or da. after days, we analyzed dendrite formation using anti-map antibody. after - days, we analyzed synaptogenesis with anti-psd- , anti-synaptophysin, and anti-map antibodies. the addition of -ht ( - nm), na ( - nm) or da ( - nm) increased dendritic length of pyramidal neurons. -ht ( - nm) also increased the synaptic density. by using receptor agonists and antagonists, it was suggested that dendritic outgrowth may be promoted by -ht a receptor, ␣ a receptor and d receptor, while inhibited by -ht a and receptors. in addition, synaptogenesis was promoted by -ht a and -ht c receptors, whereas inhibited by -ht a receptor. tatsuya mori, tomoe wada, takahiro suzuki, naoyuki inagaki department of cell biology, nara institute of science and technology, nara, japan most neurons have polarized shape consisting of a single long axon and multiple dendrites. several proteins have been implicated in the establishment of neuronal polarity; however, the mechanism for neuronal polarization is not well understood. in this study, with proteomic approach, we identified a novel protein, singar, as one of the proteins which are up-regulated during neuronal polarization of rat cultured hippocampal neuron. singar was expressed specifically in brain and developmentally up-regulated during neuronal polarization in vitro and in vivo. in t cell, singar associated with p and p , the subunits of pi kinase which is considered as one of the key molecules in neuronal polarization. moreover, inhibition of singar by rna interference induced the formation of multiple axon-like neurites. these data suggest that singar ensures the formation and maintenance of neuronal polarity by suppressing the formation of surplus axons. ps a-e lrfn , a neuronal leucine-rich repeatcontaining transmembrane protain can interact with psd- naoko morimura, takashi inoue, kei-ichi katayama, jun aruga laboratory for comparative neurogenesis, riken bsi, saitama, japan in a variety of organisms, proteins with leucine-rich repeat domain (lrr) function significantly in neural development. lrfn, a neuronal lrr transmembrane family, was expressed in the brain specifically. expression of lrfn was low in embryonic brain, and increased dramatically after birth. in the rat dissociated hippocampal neurons, lrfn protein was detected predominantly at mature dendrites, where it was accumulated at spines and colocalized with psd- , a postsynaptic scaffold protein. we examined the physical interaction between lrfn and psd- by immunocrecipitation and pull-down assay, since lrfn contains class i pdz domain-binding motif at its c-terminal tail. we revealed that lrfn associated with psd- /nmda receptor complex in the brain extracts and lrfn directly bound to psd- via its pdz domain-binding motif. in this study, we suggest that lrfn may play an important role in the regulation of synaptic functions. tsuya taneda, shingo miyata, hiroaki okuda, masaya tohyama department of anatomy and neuroscience, graduate school of medicine, osaka university, osaka, japan protein arginine methylation is a common post-translational modification catalyzed by a family of protein arginine n-methyltransferases (prmt - ). among the prmt proteins, the prmt has some characteristic motifs in the n-terminal tract which follows its active methyltransferase site. although little attention has been paid to protein methylation in the nervous system. first of all, we have examined the distribution of the prmt in the rat brain. the prmt was expressed in the cell bodies and dendrites in the hippocampal neurons. further, the ontogenetic analysis revealed the prmt expression increased from the perinatal stages to the adulthood. these findings suggest that the prmt relates to the neural function in the young and adult brain. furthermore in order to study the role of the prmt in the brain, we tried to identify novel interacting proteins with the prmt in rat hippocampal neurons using tandem affinity purification assay coupled with mass spectrometry. ps a-e proteomics of the growth cone: ii. the systematic immunostaining analysis of the growth cone proteins identified by the proteomic research motohiro nozumi , , michihiro igarashi , div mol cell biol, grad. sch. med dent sci; trans-diciplinary res program, niigata univ., niigata, japan proteomics is a powerful method to understand the molecular composition of a given cell or a compartment of the cell. in the accompanying paper, we applied this method to the growth cone from the rat forebrain, and we identified more than several hundred proteins there. although the proteins have been determined using the powerful methods, the localization of each protein in the neuron should be confirmed; thus, we checked the immunostaining in the cultured rat cortical neurons. currently, we have already performed the immunocytochemistry concerning more than identified proteins including cytoskeletal components, signaling molecules, receptors, and cell adhesion molecules. by quantitative analyzing the fluorescent intensity using the digital imaging, we classified the growth cone proteins into several groups. we have found more than twenty proteins specifically localized in the growth cone by this analysis. research funds: kakenhi ; project-promoting grant from niigata univ ps a-e netrin- is involved in the sensory axonal projection toward the spinal cord as a repulsive guidance cue tomoyuki masuda , keisuke watanabe , kazuhiro ikenaka , katsuhiko ono , hiroyuki yaginuma dept. anat., fukushima med univ. sch. of med., fukushima, japan; div neurobiol bioinfo, nat inst physiol sci, aichi, japan in higher vertebrate embryos, the ventral spinal cord exerts chemorepulsion for dorsal root ganglion (drg) axons to orient them toward their targets. netrin- is known to be a chemorepellent for a subset of axons, the role of netrin- for ventral spinal cord-derived repulsion is, however, unknown. by employing culture assays, we report here the involvement of netrin- in this repulsion. in the mouse embryo at e , netrin- is expressed in the floor plate and the dermamyotome, and the netrin- receptor unc c is expressed in drg neurons. we show that hek-cell aggregates secreting netrin- repelled chick e drg axons. moreover, using function-blocking antibody against netrin- , we revealed the fact that netrin- plays an important role in ventral spinal cord-derived repulsion. together, these findings suggest that the ventral spinal cord repels drg axons by secreting netrin- to shape the initial trajectories of drg axons. research funds: grants-in-aid on priority area (c) (mecst to t.m.) hitoshi maeda, masaki sakurai department of physiology, teikyo university school of medicine, tokyo, japan in the previous reports, we showed that in the early development, corticospinal synapses (cs) were formed widely in the spinal gray matter but those in the ventral side were eliminated later in an activity dependent manner. however, the property of postsynaptic cells to cs input is poorly understood. in the present study, we investigated the electrophysiological and morphological properties of the neurons that receive cs synapses in the acute spinal cord slices of neonatal rat. the postsynaptic neurons that were confirmed by the stimulation of the posterior funiculus, where the cs tract is located in rodents, were whole cell patch clamped and labeled by neurobiotin tm . responsive neurons are widely distributed in the p neonates, but the ventral neurons became unresponsive after p . the majority of the ventral neurons are of multipolar type with large somata showing "repetitive" or "phasic" firing patterns; on the other hand, most of dorsal neurons have smaller somata and multipolar branches with "single" or "phasic" patterns. keisuke watanabe , hirohide takebayashi , , kazuhiro ikenaka , katsuhiko ono div. neurobiol. bioinfo., natl. inst. physiol. sci., okazaki, japan; dev stem cell biol. program, ucsf, usa netrin- is a long-range diffusible factor that exerts chemoattractive or chemorepulsive effects on developing axons growing to or away from the neural midline. however, it is not known whether netrin- also exerts chemoattractive effect on ventral-ward migrating dorsal interneurons in the developing spinal cord. to test this hypothesis, we examined dorsal interneuron migration in netrin- −/− background, using olig -lacz knockin allele, which marks most of ventral-ward migrating dorsal interneurons. in the embryonic spinal cord of olig +/lacz ;netrin- −/− mice, ventral migration of olig cells was significantly impaired. furthermore, a netrin receptor, dcc was expressed in olig -positive cells. these results suggest that netrin- exerts chemoattractive effects on ventral-ward migrating dorsal interneurons in vivo. netrin-g and netrin-g are vertebrate-specific membrane-anchored members of the unc- /netrin family that have no affinity to classic netrin receptors and their function is unknown. here we show that netrin-g and netrin-g proteins are selectively distributed on axons of distinct pathways, and each interacts with a specific receptor on target dendrites. netrin-g and netrin-g differentially bind to lrrcontaining proteins, ngl- and a related molecule nag , in vitro. ngl- and nag in the mouse brain are concentrated in distinct dendritic segments, corresponding to lamina-specific termination of axons expressing netrin-g and netrin-g , respectively. furthermore, in netrin-g and netrin-g deficient mice, in which axonal pathfinding is normal, there is selective mislocation of individual receptors within dendrites. together, these results suggest that axonal netrin-g proteins transneuronally regulate the localization of distinct receptors on dendrites, and thereby determine the properties of subdendritic segments. jinhong huang , ryuichi sakai , teiichi furuichi lab. molecular neurogenesis, brain science institute of riken, saitama, japan; division of cell growth factor, national cancer center of japan, tokyo, japan cas is a tyrosine-phosphorylated docking protein that is indispensable for the regulation of actin cytoskeletal organization and cell migration in fibroblasts. the neuronal function of cas, however, is poorly understood. here we report that cas is dominantly enriched in the brain, especially the cerebellum, of postnatal mice. during cerebellar development, cas is highly tyrosine phosphorylated and is concentrated in the neurites and growth cones of granule cells. cas coimmunoprecipitates with src family protein tyrosine kinases, crk, and cell adhesion molecules. the axon extension of granule cells is inhibited by either rna interference knockdown of cas or overexpression of the cas mutant lacking the crk binding motifs. these results demonstrate that cas acts as a key scaffold to link the proteins associated with tyrosine phosphorylation signaling pathways to the granule cell axon elongation. research funds: huang was a postdoctoral fellowship recipient of jsps in in vitro cerebellum-pons-medulla block preparations isolated from neonatal rats on p -p , stimulation of parallel fibers produces excitation of purkinje cells lasting for - ms. this unusually prolonged response is observed in the lateral region of the cerebellum (paraflocculus/flocculus), where purkinje cells develop primary dendrites on p -p . since -agatoxin iva and -conotoxin mviic abolished the prolonged response, we suggest the involvement of p/q type ca channels. immunohistochemical labeling revealed that p/q type ca channels emerged in paraflocculus/flocculus and uvula/nodulus lobules on p and that they then locate in purkinje cells, in cell body on p and in primary dendrites on p -p . the parallel development of p/q type channels, primary dendrites, and the occurrence of prolonged parallel fiber-purkinje cell transmission suggests their causal relationships. naoya ichikawa, yasuo kitagawa, tatsuhiko kadowaki graduate school of bioagricultural sciences, nagoya university, aichi, japan we have recently identified a novel gene, mahya, which is specifically conserved between hymenoptera and deuterostome. mahya encodes a secretory protein with a follistatin-like domain, two immunoglobulin domains, and a c-terminal novel domain. mouse mahya genes (mmahya- and mmahya- ) are expressed in the olfactory bulb, hippocampus, and cerebellum of the adult brain. we have found that mmahya- protein is specifically synthesized in the pre-migratory granule cells and localized at the molecular layer of the postnatal cerebellum. these results suggest that mmahya- is involved in either the migration of granule cells or the dendritic maturation of purkinje cells. we will further report the analysis of the functions of mmahya- for the early cerebellum development. toshitaka morishima , erina fukushi , kazuto kobayashi , naohiro hozumi , sachiko yoshida toyohashi university of technology, toyohashi, japan; honda electronics co. ltd., toyohashi, japan in cerebellar development, granule cells migrate with elongation their axon, called parallel fibers, and form neuronal circuit in molecular layer. although density and thickness of parallel fibers are important information for cerebellar development, few were simple and useful methods. we have proposed a new method for two-dimensional acoustic impedance imaging for developing cerebellar slices. an acoustic impedance microscopy was obtained by mechanically scanning the transducer and the reflection intensity was interpreted into local acoustic impedance of no treated acute slices with no invasion. the developing parallel fibers were clearly observed as the contrast in acoustic impedance, whereas they were cloudy in immature egl from neonatal rat. the reflection from molecular layer enlarged and floated to deep layer, so that its spatial pattern was changed during cerebellar development. this imaging method is believed to be a powerful tool for observation of neuronal development, as neither fixation nor staining is required. tatsuro yamamoto , hideyuki dekimoto , tomiyoshi setsu , masahiko watanabe , mikio hoshino , yo-ichi nabeshima , toshio terashima dept. of anat., kobe univ. grad. sch. of med., kobe, japan; dept. of anat., hokkaido univ. grad. sch. of med., sapporo, japan; dept. of pathol and tumor biol, grad. sch. of med., kyoto univ., kyoto, japan a mutant mouse, cerebelles (cbll), lacks the entire cerebellar cortex but survives into the adult. the responsible gene for this mutation is ptf a, whose expression is lost in this mutant. in the present study, we examined cerebellar afferent and efferent systems of this mutant mouse by neural tracing methods with a combination of immunohistochemistry. the injection of fluoro-gold (fg) into the cbll thalamus resulted in retrograde labeling of neurons in the contralateral cerebellar nuclei. these fg-labeled neurons were glutaminase-positive. after the injection of bda into the cbll lumbar cord, spinocerebellar terminals projecting to the deep cerebellar nuclei were anterogradely labeled in spite of absence of the cerebellar cortex. these findings suggest that afferent and efferent systems of the cerebellar nuclei of the cbll are preserved in spite of absence of the cerebellar cortex. kumiko ishida , tomoko nishiyama , hitoshi tatsumi , masahiro sokabe , department of physiology, nagoya university graduate school of medicine, nagoya, japan; icorp, cell mechanosensing project, japan science and technology corporation sprouting and synaptic reorganization of the mossy fiber (mf) are commonly found in the hippocampus of temporal lobe epilepsy patients. as the muscarinic agonist, pilocarpine, can induce similar morphological changes, hippocampal slices treated with this drug have been widely used as a model of epilepsy. we found that pilocarpine induced a transient retraction and subsequent elongation of the neurites of granule cells in the slice cultures; the retraction was peaked approximately h and the elongation started at approximately h after the drug application. tetrodotoxin strongly inhibited both the retraction and elongation, while the bdnf sequestering protein, trkb/fc, retarded only the elongation. this result suggests that na + channel dependent neuronal excitation and following activitydependent bdnf releases are essential in the biphasic morphological changes induced by pilocarpine in hippocampal slices. rieko muramatsu, yuji ikegaya, maki k. yamada, norio matsuki, ryuta koyama laboratory of chemical pharmacology, graduate school of pharmaceutical sciences, the university of tokyo hippocampal granule cells extend their axons, i.e. the mossy fibers (mfs), from the dentate gyrus (dg) to the area ca . once this oneway projection is disrupted, the mfs retrogradely innervate granule cell dendrites and make excitatory synapses that induce epileptic neural activities in the dg. to clarify the mechanism that regulates normal, anterograde mf projections, we used a co-culture system of hippocampal slices. when a dg slice from a gfp(+) rat was juxtaposed to the ca region of a host hippocampal slice from a wild type rat, the gfp(+) mfs ran through the host ca toward the host dg but failed to invade it even after ten days in vitro. thus the dg seemed to serve as a barrier that blocks retrograde projections of mfs. however, the mfs extended into the dg when forskolin, an activator of adenylate cyclase, was chronically applied. these results suggest that the dg has a mechanism supporting anterograde mf projections to ca , which is regulated by the levels of adenylate cyclase activation. calcitonin gene-related peptide (cgrp) is a amino acid neuropeptide that is widely distributed in central and peripheral nervous systems. cgrp is expressed from early developmental stage in rat brain, suggesting that cgrp may be involved in not only neurotransmission but also neural development. but roles of cgrp in neuronal development of cerebral cortex and hippocampus remain unclear. in the present study, we made dissociation culture of cerebral cortex and hippocampus of embryonic day (e) or e rat. dendritic outgrowth of pyramidal neurons was analyzed after days using anti-map antibody. synapse formation was analyzed after - weeks, using anti-psd- and anti-synaptophysin antibodies. in the presence of cgrp ( - nm), both dendritic length and synaptic density were increased. however, the number of dendritic branching was not affected. these results suggest that cgrp promotes dendritic outgrowth and synapse formation. chisako kanamaru, kazunori suda, kouji senzaki, takashi shiga university of tsukuba, graduate school of comprehensive human sciences, tsukuba, japan recent studies have suggested monoamine affects neural development, but it is unclear which receptor subtypes mediate actions of monoamine. in this study, we examined roles of hydroxytryptoamine ( -ht) and noradrenaline (na) in the formation of dendrites and synapses using dissociation culture of rat hippocampus. embryonic day rat hippocampus was cultured in the presence of -ht or na. after days, we analyzed formation of dendrites using anti-map antibody. after days, we analyzed formation of synapses using anti-psd- , anti-synaptophysin, and anti-map antibodies. the addition of -ht ( nm) or na ( nm) increased dendritic length and number of branches of pyramidal neurons, whereas decreased number of primary dendrites -ht ( - nm) and na ( - nm) also increased the synaptic density. by using receptor agonists and antagonists, it was suggested that ␣ a receptor promotes dendritic outgrowth, while  receptor suppress dendritic outgrowth and branching. in addition, -ht a receptor and ␣ a receptor promote synapse formation. kenji amano down syndrome cell adhesion molecule (dscam) knock-out (ko) mouse died within h after the birth. to investigate possible etiology of the neonatal death, we examined the respiratory activity using whole body plethysmography and the c inspiratory activity using brainstem-spinal cord preparation. the respiratory activity of dscam-ko mice using plethysmography was irregular frequency and small amplitude accompanied with apnea. furthermore, c inspiratory activity also showed irregular frequency and narrow duration of the bursting. we then analyzed spatio-temporal pattern of the respiratory neuronal activity using combination of the voltage-sensitive dye (di- anepeq) and the imaging system (micam ). in dscam-ko mice, the optical signal which precedes c inspiratory activity was depressed. these results suggest that pre-inspiratory neuronal network, which determines respiratory rhythm, does not develop normally in dscam-ko mice and causes lethal respiratory dysfunction. ps a-f hippocampal cells cultured on d collagen substrate secrete a dense extracellular matrix, supporting neuritic outgrowth shantanu sur, thomas launey, masao ito brain sc. inst., riken, japan the brain extracellular matrix (ecm) influences neuronal migration and morphogenesis. we explored how hippocampal cells modify their extracellular environment when seeded onto collagen gel, a major component of the ecm. after weeks in vitro, neurons formed a dense layer, > . mm below the gel surface, with neurite outgrowth toward the surface, within the top gel layer (tgl). initially, we thought that hippocampal cells were penetrating the gel, following partial degradation of the collagen matrix. however, ( ) collagenasespecific inhibitor did not affect cell depth, ( ) limiting gliosis by antimitotics reduced the thickness of the tgl by %, ( ), neither glial nor neuronal cell body were found in the tgl by gfap/map detection, ( ) neurite outgrowth was observed only within this tgl, but not toward the bottom of the gel. to see whether the tgl is the remains of the initial collagen substrate, we embedded fluorescent beads in the collagen gel before cell seeding. the tgl was completely devoid of beads after weeks, suggesting that the tgl is newly formed by ecm material, largely secreted by glial cells. emi kumamaru, tadahiro numakawa, yuki yagasaki, hiroshi kunugi disorder research, national institute of neuroscience, ncnp, tokyo, japan the level of glucocorticoid is regulated through hpa axis, and glucocorticoid itself has a negative feedback effect on hpa axis. however, under the intense stress, the glucocorticoid level is increased, and the high level of it is suggested to induce neuronal damage and to cause the mood disorder. on the other hand, it is possible that the reduction of neuronal function mediated by bdnf is partly related to the cause of the disorder. therefore, in the present study, we investigated the effect of glucocorticoid (dexamethasone, dex) on synaptic maturation and function enhanced by bdnf in early developing hippocampal neurons. we found that bdnf increased the expression of synaptic proteins including glutamate receptor and presynaptic protein, however, pretreatment with dex significantly inhibited the up-regulation of these proteins by bdnf. further, increase in release of glutamate and in intracellular ca + by bdnf was suppressed after dex pretreatment, suggesting that dex inhibits the maturation of synaptic function mediated by bdnf. takashi ueyama , kazuto kujira , tetsuya kawabe , takao ito , yoshihiro tsuruo department of cell biology and anatomy, wakayama medical university, wakayama, japan; department of cardiovascular medicine, wakayama medical university, wakayama, japan in this study, we investigated the effect of castration on the emotional stress response in the brain by comparing the c-fos expression in response to immobilization stress (imo) between castrated rats (cast) and sham-operated rats (sham). increased c-fos immunoreactive cells in response to imo were observed in septum, thalamus, hypothalamus, midbrain, pons and medulla oblongata in accordance with previous findings. in cast compared with sham, the numbers of c-fos-ir cells were significantly lower in the medial parvocellular part of paraventricular hypothalamic nucleus, while they were significantly higher in the supraoptic nucleus and medial amygdaloid nucleus. these data suggest that neuronal activity in these areas is influenced by systemic androgen level. this may underlie the pathophysiology of partial androgen deficiency in aged men (padam). research funds: grant-in-aid for scientific research (c) ( ) ps a-f metabolic and glucagon response of a genetically heat-tolerant rat to ambient heat and cold fujiya furuyama , hitoo nishino , takehiro yahata nagoya city university graduate school of medical sciences, nagoya, japan; nayoro city college, nayoro, japan the inbred fok rat was developed by us using heat selection and inbreeding for generations. fok rats avoided serious multisystem disorders caused by heat stroke and by extreme dehydration. saliva spreads widely over the whole ventral body surface in fok rats. however, no strain difference was not found in vitro in the salivation rate, suggesting exsisting of a negative feedback loop between the central thermoregulation system and evaporation system. on the other hand, body temperature of the fok rats did not decreased in a extream cold environment as those in control rat strain. thermogenesis induced by cold in fok rats was larger than those in control rat strains. the larger increase in thermogenesis was partly attributable to glucagon-induced thermogenesis in brown adipose tissue. blood levels of triglryceride was lower, but polyunsaturated fatty acids were higher in fok rats than those in control rat strains. these changes can be considered to be results of genetically acquired heat-tolerance. oxidative stress is involved in the degeneration of nigrostriatal dopaminergic system in parkinson s disease (pd). vitamin e is a potent antioxidant, and its retention and secretion are regulated by alpha-tocopherol transfer protein (ttp) in brain. dysfunction of ttp has been shown to result in systemic deficiency of vitamin e in human and mice. in the present study, we using the ttp knockout mice, investigated the effect of vitamin e deficiency in pd development by generating mptp mouse model of pd. we confirmed that vitamin e depleted in the brain of ttp knockout mice completely. while the mptp treatment decreased striatal dopamine in the all three ttp genotypic groups, there were no significant differences among them. our results suggest that vitamin e does not play a major protective role in mptp-induced nigrostriatal dopaminergic neurodegeneration in the brain. priyanka dikshit , anand goswami , nobuyuki nukina , nihar ranjan jana national brain research centre, india; laboratory for structural neuropathology, riken brain science institute, - hirosawa, wakoshi, saitama - , japan a major pathological hallmark of the polyglutamine diseases is the formation of neuronal intranuclear inclusions (niis) of the disease proteins, often associated with various chaperones and proteasome components. but, how the polyglutamine proteins are ubiquitinated and degraded by the proteasome is not known. here, we demonstrate that the expanded polyglutamine proteins that are misfolded, become ubiquitinated. secondly, we identified chip ubiquitin ligase that is able to target polyglutamine expanded huntingtin and ataxin- for the misfolding-dependent ubiquitination and degradation by the proteasome. the over expression of chip reduces the aggregate formation and cell death mediated by expanded polyglutamine proteins and the suppressive effect is more prominent when chip is over expressed along with hsc . finally, we show that the expression of chip is increased in the expanded polyglutamine protein expressing cells. hypothalamic-pituitary-adrenal axis is central to the regulation of stress response. for the comprehensive detection of genes responsive to stress, we identified and catalogued the entire partial complementary dna sequences (expressed sequence tags (ests)) from rat hypothalamus. we have identified the total of , ests ( , non-redundant sequences). of them matched known genes of rodents in the genbank databases, but remained unknown. now we classified a full set of hypothalamic ests on the basis of their functional domains. complete profile of them will be presented in the meeting. these ests will also be applied to a cdna microarray for stress experiments. the present study will provide a refined genomic resource for molecular studies of animal models of stress-related disorder. research funds: grants-in-aid from the ministry of health, labor and welfare shinya yanagita, seiichiro amemiya, satoko suzuki, ichiro kita graduate school of science, tokyo metropolitan university, japan our previous study suggests that acute running is one stressor activating corticotropin-releasing hormone (crh) neurons in the hypothalamic paraventricular nucleus (pvn). many studies have reported that several weeks of voluntary running improved stress tolerance during non-exercise stress. it is, thus, possible that housing in cages attached running wheel can alter activation of stress-related neurons during acute running. in this study, we examine the effects of , , or weeks prior wheel running (i.e. housing in the cages attached running wheel) on activation of stress-related neurons, such as pvn, central nucleus of amygdala (cea), locus coeruleus, dorsal raphe, ventral tegmental area (vta), and prefrontal cortex during acute running using immunohistological methods in rats. prior wheel running altered activation of various stress-related neurons during acute running, especially markedly decreased activation of cea, and increased that of vta. these results suggest that prior wheel running influences stress-related neuronal activity during acute running. ps a-f transforming growth factor- in the brain regulates fat metabolism during exercise kazuo inoue, toma ishikawa, wataru mizunoya, tetsuro shibakusa, tohru fushiki division of food science and biotechnology, graduate school of agriculture, kyoto university, kyoto, japan we have previously reported that the concentration of transforming growth factor- (tgf-) increases in the cerebrospinal fluid of rats during exercise and that an increase in fat oxidation was observed following intracisternal administration of tgf-. these results led us to postulate that tgf- in the brain regulates the enhancement of fatty acid oxidation during exercise. to test this hypothesis, we carried out respiratory gas analysis during exercise while inhibiting the effect of tgf- in the brain using intracisternal administration of anti-tgf- antibody or sb- , an inhibitor of the type tgf- receptor (tr ). we found that each reagent blocked the increase in fatty acid oxidation. these results suggest that brain tgf- has a role in enhancing fatty acid oxidation in peripheral tissues during endurance exercise, and this regulation is executed at partly via the tr signal transduction system. yoshii takanobu it has been demonstrated that vasopressin (avp) might play a role in anxiety-related behavior. we hypothesized that traumatic stress changes avp activity and avp contribute to the symptom of ptsd. we carried out in situ hybridization (ish) for avp mrna expression and avp immunohistochemistry (ihc) with an experimental paradigm of single prolonged stress (sps) as ptsd model. sd male rats were exposed to sps ( h restraint; min forced-swimming; ether anesthesia) then they were put in untouchable situation for days. avp mrna expression significantly decreased in the son. ihc showed no significant change in avp-ir, but after additive stress (forced swimming min), avp-ir in the son was significantly diminished. we considered that the stress decrease avp synthesis, but has little effect to the storage of avp. mumeko tsuda, takaaki ozawa, aosa fukushi, sonoko ogawa kansei, behavioral and brain sciences, university of tsukuba, tsukuba, japan neonatal maternal separation (ms) is known to affect anxiety and fear responses in adult whereas its effect on socio-sexual behaviors is not fully understood. in the present study, we examined the effect of ms on an array of emotional and socio-sexual behaviors in both sexes of c bl/ j mice. pups were separated from mothers daily ( h) on postnatal days through . starting at weeks of age they were tested for ( ) emotionality and anxiety levels in open field (oft), light-dark transition (ldt), and elevated plus maze tests; ( ) responses to social stimuli in social investigation (sit) and social preference tests; and ( ) socio-sexual behaviors in aggressive and sexual behavior tests. overall, there was no apparent effect of ms on behaviors measured in the oft and ldt except for higher levels of exploration in the ms group compared to the non-stressed (ns) group in both sexes. during the sit, social investigation time and general activity in ms females were much lower than those in ns females suggesting ms females may be more fearful to social stimuli. in the present study, we investigated the effect of environmental stress applied during perinatal period on spatial learning activity of mouse evaluated by morris water maze test. mice were exposed to the noise of db (so), or were forced to swim (sw). these manipulations were performed for min once a day at weeks after birth (from postnatal days to ) or weeks after birth (from postnatal day to ). normal mice were left undisturbed (no). the spatial learning activity was tested at the age of weeks. it was found that the spatial learning activity of both so and sw mice manipulated weeks after birth was impaired as compared to no mice. so mice manipulated weeks after birth exhibited the same learning behavior as no mice, while that of sw mice manipulated weeks after birth was impaired. present results indicated that the effect of the environmental stress on the learning activity of the adolescent mice might be dependent on the period of the stress manipulation. kin-ya kubo , yukiko yamada , mitsuo iinuma , yasuo tamura , fumihiko iwaku , kazuko watanabe , minoru onozuka dept. oral anat., asahi univ. sch. dent., japan; dept. ped. dent., asahi univ. sch. dent.; dept. physiol., gifu univ. sch. med., japan; dept. physiol. and neurosci., kanagawa dent. coll., japan recent studies have suggested that occlusal disharmony is related to temporomandibular arthorosis and braxism, which may come from a hypothalamic-pituitary-adrenal (hpa) axis. in addition, aged mice with masticatory dysfunction show deficits in spatial memory, being due to various pathological changes in the hippocampus, suggesting the link between malocclusion induced by abnormal occlusion and hippocampal pathology. in this study, to prove this hypothesis, we examined the effect of this malocclusion on plasma corticosterone levels, the numbers of hippocampal neurons and spatial performance in water maze in samp mice. this treatment age-dependently advanced a decline in spatial memory, an increase in plasma corticosterone levels, and a decrease in neuron density in the hippocampal ca region. the results suggest that abnormal occlusion may progress hippocampal neuron loss via stress, thereby leading to senile deficits in memory. yurie nakamoto, go mugishima, mitsuko sato, masako miwa, mitsunobu yoshii division of psychobiology, tokyo institute of psychiatry, tokyo, japan it has been shown that pbr are increased after acute stress and decreased under chronic stressful conditions. in our previous studies, expression of pbr was significantly correlated with trait anxiety in normal human subjects, which might reflect polymorphism of the pbr gene. in addition, males appeared to have higher pbr densities than females in their prime lives. the present study was designed to analyze these sexual differences in rats. blood samples were obtained from adult male and female slc wistar rats immediately after acute random electrical footshock and also from these animals after chronic social isolation (for weeks after weaning). in naïve, male rats expressed higher densities of platelet pbr than females. chronic social isolation caused a marked increase in platelet pbr in male rats compared to female. the results indicate that pbr responses to environmentally induced stress are much less in female, probably under the influence of estrogen. kanako tambara , yayoi kitamura , junichi tanaka , yukio hattori , yasushi hayashi department of human nutrition, notre dame seishin university, okayama, japan; department of curriculum, teaching and memory, naruto university of education, tokushima, japan we investigated the effects of exogenous putrescine on stressinduced hyperthermia (sih) in male c bl/ j mice after systemic injection of putrescine to clarify the role of brain putrescine in stressful conditions. in addition, we examined the effects of spermidine, spermine, and the anxiolytic diazepam on sih. the rectal temperature of singly housed mice was measured twice at a -min interval, to measure the basal temperature (t ) and stress-enhanced temperature (t ), respectively. the difference ( t = t − t ) gives the sih. in control mice, t was approximately • c. pretreatment with diazepam caused dose-dependent inhibition of the sih. similarly, putrescine reduced t, although it caused a dose-dependent decrease in t . furthermore, spermidine and spermine also lowered t and t at doses lower than that of putrescine. these results suggest that endogenous brain putrescine and other polyamines have an anxiolytic-like effect in stressful conditions. eriko iguchi, yasuhiro tanaka, toshiyuki matsuoka, shuh narumiya department of pharmacology, kyoto university, kyoto, japan prostaglandins (pgs) are synthesized in many organs including the brain. of their synthesis, the rate limiting step depends on cyclooxygenase (cox), which has two subtypes, cox- and cox- . it has been known that, under some stressful conditions, cox- is induced in some neurons and increases pgs production. but the roles of the increased pgs under stress are not fully elucidated. in this study, we restrained mice in small tubes individually for h and subjected them to the elevated plus maze task h later. these mice showed more anxiety. immunohistochemistry showed significant induction of cox- by restraint in some parts of the brain, such as cerebral cortices and amygdala. next, we examined the effect of indomethacin on this stress-induced anxiety. indomethacin is expected to reduce pgs production. mice treated with indomethacin stayed on open arms longer than control mice. these data suggest that pgs synthesized during stress may have anxiety-increasing effect. ps a-g imaging brain and immune association accompanying cognitive appraisal of acute stressor to investigate association between brain and immune systems accompanying cognitive appraisal of an acute stressor, we recorded o-water positron emission tomography, cardiovascular, neuroendocrine, and immune indices, when male subjects conducted a mental arithmetic task in a high controllability (hc) condition and a low controllability (lc) condition. activation in the orbitofrontal (ofc) and medial prefrontal (mpfc) cortices was observed in the lc compared to the hc. furthermore, significant correlations between brain activation and hr, hrv, bp, and nk cells were found commonly in the ofc in the lc, but not in the hc. thus, the ofc is a pivotal region for top-down regulation over immune activity accompanying cognitive appraisal on a stressor. wei zhang , takesi sakurai , yasuitirou fukuda , tomoyuki kuwaki , dept. molec. integ. physiol., chiba univ., japan; dept. pharmacol., univ. tsukuba, japan; dept. autonom. physiol., chiba univ., japan we have previously proposed that orexin plays as a master switch to elicit multiple efferent pathways of the defense response. it is still open question, however, how information of stressor activates the orexinergic neurons. in this study, we examined possible afferent nuclei to activate orexinergic neurons. in urethane-anesthetized mice, a gaba-a receptor antagonist, bicuculline, was microinjected into the amygdala or the bed nucleus of stria terminalis (bnst), of which electrical stimulation induced simultaneous increases in blood pressure, heart rate, and respiration. bicuculline dose-dependently induced cardiorespiratory excitation in both orexin neuron-ablated and wild-type mice. however, dose-response curve was rightward shifted in the former. we conclude that the amygdala and bnst constitute one of the afferent pathways to the orexinergic neurons that involved in the defense response against stressor. in this study, developmental changes of anxiety behavior as well as myelin formation were investigated in male balb/c mice. the early-weaned mice had lower number of entries to the open arms of elevated plus maze at the age of - weeks, indicating persistent higher anxiety. high performance thin layer chromatography analysis was conducted for amygdaloid galactosyl ceramide, which is a typical lipid of myelin. the early-weaned mice had higher levels of galactosyl ceramide at the age of weeks, and an electron microscopic study suggested increased number of myelinated axon and reduced diameter of myelinated axon in the basolateral amyglaloid nucleus. these results suggest that the early weaning induces precocious myelin formation in the amygdale between and weeks of age, which would be related to higher anxiety state in the early-weaned mice. research funds: sasakawa sci. res. grant takefumi kikusui, yuji mori veterinary ethology, university of tokyo, tokyo, japan we previously reported that early-weaned mice developed persistent increase in anxiety as well as aggression. in this study, developmental changes of brain derived neurotrophic factor (bdnf) protein levels were investigated in early-weaned icr mice. the early-weaned male and female mice had lower number of entries to the open arms of elevated plus maze at the age of weeks, and this change was persistently observed in males. concurrently, the early-weaned males showed decrease of bdnf in the prefrontal cortex between and weeks of age, and in the hippocampus at the age of weeks. however, there was no difference of bdnf expression in females. in addition, the early-weaned males, but not females, showed reduced brdu immunoreactivity in the dentate gyrus. these results suggest that the deprivation of mother-infant interaction during the late lactating period augments the anxiety in the adulthood by decreasing the level of bdnf in the pre-limbic system, and that these stress responses are sexually dimorphic, i.e., male is more vulnerable to early weaning stress. research funds: kakenhi # ps a-g a systematic analysis of genetic factors associated with behavioral diversity between msm and c bl/ koide tsuyoshi , , aki takahashi , , toshihiko shiroishi , , akinori nishi mgrl, national institute of genetics, mishima, japan; sokendai, hayama, japan; mammalian genetics lab, nig, mishima, japan in the previous study conducting a multi-phenotype behavioral tests, we observed a great difference of the behavioral phenotype between mouse strains, msm and c bl/ . in order to elucidate a genetic factors underlying the behavioral difference, we analyzed a series of consomic strains which are made by replacing one of the chromosomes with that of msm strain. the behavioral data clearly indicated involvement of multiple genetic factors for each behavioral phenotype. one of the consomic strains, b - cmsm, which carries chromosome of msm, showed extreme behavioral differences from c bl/ . the strain showed lower activity in home cage and novel cage, and showed decreased number of transition in the light dark box test. by conducting analyses of composite interval mapping and a series of sub-consomic strains, we successfully identified genetic loci for the behavioral phenotype. tomoko soga , yu kajiyama , shigenobu shibata , hiroshi kunugi department of mental disorder research, national institute of neuroscience, center of neurology and psychiatry, tokyo, japan; department of electrical engineering and bioscience, waseda university the hypothalamus-pituitary-adrenal (hpa) axis plays an important role in the pathophysiology of depression. alterations of brain derived neuronal factors (bdnf) have been implicated in depression. we examined the effects of synthetic glucocorticoid (dexamethasone; dex) on emotional behavior and gene expression of hpa-related molecules and bdnf in mice. dex treatment for days after birth showed a significant decrease in locomotor activity and a significant rise in the time of immobility during forced swimming test. dex treatment to mature mice resulted in significant decrease in the number of entries into the open arm during elevated plus maze test. there was no change in gene expression of hpa-related molecules in dex-treated group. bdnf gene expression decreased significantly in dex-treated group, which showed behavioral abnormalities. our results lend further support for the involvement of glucocorticoid and bdnf in depression-related behavior. sachiko chikahisa, hiroyoshi sei, atsuko sano, kazuyoshi kitaoka, yusuke morita department of integrative physiology, the university of tokushima graduate school, tokushima, japan music is known to be able to elicit emotional changes including anxiolytic effect. the gonadal steroid hormone estrogen (e ) has been associated with anxiety levels. in this study, we examine whether the effect of music on anxiety is related with ovarian steroid in female mice. behavioral paradigms measuring anxiety (open field, elevated plus maze, dark-light transition and marble burying test) were tested in gonadally intact (sham-operated) and ovariectomized (ovx) female mice treated with placebo (ovx + placebo) or chronic estradiol (ovx + e ) replacement. in three behavioral tests except for open field, sham-operated mice exposed to music showed less anxiety than those exposed to white-noise and silence, while ovx + placebo mice did not show these effects at all. ovx + e mice showed the anxiolytic effect of music only in the marble burying test. these results suggest that exposure to music reduce anxiety levels, and ovarian steroids may be, at least partially, involved in the anxiolytic effects of music observed in female mice. tatsuhiro yasuda free, tokyo, japan strength and periodicity of periodical air pressure ascent around ones' ears induced by others' respiration may impact upon ones' awaken level, i.e. cognition. the air vibration acts upon tympanic membrane and then cochlear receptor stereocilia transforms it to neural signals which are sent via cochlear nucleus to inspiration nucleus in the medulla, and inspiration is induced. simultaneously afferent signals generated by external intercostals contraction are forward to medulla, thalamus and cortical areas. the stimuli with larger strength and periodicity compared to ones body size yields to auditory startle reflex. continuation of this may induce hyper ventilation or tension. if the input may be lasting with smaller strength and periodicity, insufficient diaphragm activity after hypoxia and gasping fade-out may induce afferent signal shortage that shrinks various cortical neural activity. lasting this situation may fall into depression. suitable timing of inspiration inducing may keep good mood, strong motivation and effective cognition. body system is suggested to own inherent observer that detects alerting or safe state so called homunculus. kenichi sasaguri , takero in general, it has been proposed that the mandibular retrusive position resulted from either malocclusion or inadequate occlusal reconstruction is one of the causes of indefinite complaint. we determined whether the malocclusion model influences brain activities by using fmri study. the results indicated that in some of volunteers, significantly bold signals in the hypothalamus and the amygdala, being associated with emotion and/or stress increased during clenching. it is, therefore, suggested that malocclusion influences the whole body through emotional system, thereby causing the indefinite complains. ps a-g synaptic organization between the amygdaloid axon terminals and the parvicellular reticular formationprojecting neurons in the retrorubral field of the rat toshiko tsumori, yi qin, shigefumi yokota, tatsuro oka, yukihiko yasui dept. anat. & morphol. neurosci., shimane univ. sch. med., izumo, japan the retrorubral field (rrf) is known as one of the areas containing numerous dopaminergic neurons in the midbrain. in the present study, we showed that the axon terminals from the central amygdaloid nucleus (ace) made synaptic contacts with non-dopaminergic rrf neurons sending their axons to the parvicellular reticular formation (rfp), where many premotor neurons projecting to the orofacial motor nuclei have been well known to exist. the ace axon terminals, which usually contain small pleomorphic vesicles and occasionally contain both small pleomorphic vesicles and large dense-cored vesicles, formed symmetrical synapses with cell bodies and dendrites of the rfp-projecting rrf neurons. moreover, most of these axon terminals showed glutamic acid decarboxylase immunoreactivity. the present study suggests that the ace exerts inhibitory influences upon the non-dopaminergic rfp-projecting rrf neurons to control orofacial movements closely related to emotional behavior. research funds: kakenhi ( ) ps a-g involvement of nr b tyrosine-phosphorylation in emotional responses mediated at the amygdala mina delawary , takanobu nakazawa , yuji kiyama , toshiya manabe , tadashi yamamoto div. of oncology, ims, univ. of tokyo, tokyo, japan; div. of neuronal network, ims, univ. of tokyo, tokyo, japan nr b is tyrosine-phosphorylated, with tyr- being its major phosphorylation site. to investigate the role of tyr- phosphorylation, we generated mice with a tyr phe knock-in mutation (yf/yf mice). in the elevated plus-maze test, time spent in open arm was reduced in yf/yf mice as compared to that in wild-type mice. similar phenotype was seen in the corticotropin-releasing factor (crf) overexpressing mice. this phenotype of yf/yf mice was canceled by the administration of crf receptor antagonist. as expected, in yf/yf mice, the expression level of crf in the amygdala was increased compared with that in wild-type mice. in the slice of amygdala from wild-type mice, nmda application induced de-phosphorylation of tyr- and up-regulation of crf mrna level. given that crf is important in emotional responses, these data strongly argue that phosphorylation of nr b is involved in the control of emotional responses by regulating crf content. ps a-g increase in anxiety in transgenic mice overexpressing camkii in forebrain previous studies have shown that ␣calcium/calmodulin dependent protein kinase ii (␣camkii) plays important roles in aggressive and fear response in mice. to understand roles of alpha camkii in emotional behaviors, we have generated transgenic mice overexpressing ␣camkii in forebrain. because these mutant mice showed increase in anxiety in open field and elevated zero maze tests, we here examined effects of administration of selective serotonin reuptake inhibitor (ssri) on anxiety-related behavior of these mutant mice. treatment with ssri suppressed anxiety-related behavior of camkii mutant mice, suggesting that camkii mutant mouse is a mouse model of anxiety disorder. to investigate the mechanisms for increase in anxiety led by overexpression of camkii, we next compared the expression profiles between wild and mutant mice using dna micro array. these mutant mice showed abnormal changes in expression levels of genes related to ca + signal transduction in hippocampus. yumiko ikeda, katsunori kobayashi, hidenori suzuki department of pharmacology, nippon medical school, tokyo, japan environment is known to influence behavior of animals. however, cellular and synaptic mechanisms underlying behavioral changes by environment remain largely unknown. we examined effects of changes in environment on locomotor activity and mossy fiber (mf) synaptic transmission in hippocampal slices. in mice housed in enriched condition for weeks, locomotor activity and longinterval ( , and ms) paired-pulse facilitation (ppf) at mf synapses were reduced. in contrast, in mice housed in isolated condition for weeks, there was no detectable change in either the total ambulation distance or the magnitude of ppf. we compared properties of the mf synaptic transmission with the locomotor activity in individual mice used in all experiments and found that the magnitude of synaptic potentiation induced by dopamine was negatively correlated with the ambulation distance. our results suggest that the modification of the hippocampal mossy fiber synaptic transmission could be involved in the environmental regulation of locomotor activity. yilong cui , , yosky kataoka , , yasuhisa tamura , yasuyoshi watanabe , , hisao yamada department of anatomy and cell science, kansai medical university, osaka, japan; department of physiology, osaka city university graduate school of medicine, osaka, japan; molecular imaging research program, riken frontier research system, saitama, japan during long-term intracranial self-stimulation (icss; electrical stimulations to the hemi-lateral medial forebrain bundle of rats by their lever pressing behavior at - times/min), inhibition periods (less than times/min) were often observed h after start of icss. we have been demonstrated that the inhibition was not induced by thermal effect on the neural function or by muscular fatigue. furthermore, the inhibition period was significantly decreased by pre-treatment with ns- , a selective cox- inhibitor. these observations indicate that the arachidonic acid cascade is involved in inhibition of long-term icss and would be in weariness or fatigue sensation. male bluegill, lepomis macrochirus, is known to display alternative reproductive tactics. "parental" males defend nests and provide parental care, and "satellites" or "sneakers" are non-nesting, attempting to achieve parasitic fertilizations via sperm competition. in teleost and other non-mammals, arginine vasotocin (avt), the homologue of mammalian avp, is known as an important hypothalamic peptide involved in the alteration of reproductive behavior. behavioral evaluation and immunohistochemical study in preoptic area (poa) were conducted in parental and satellite bluegills to clear the role of avt in teleost reproductive tactics. parentals displayed more aggressive and courtship behavior than satellites and satellite males had significantly more cells than parentals, while the size of avt cells showed no difference between the male morphs. these results suggested that hypothalamic avt might play some part in the central control of reproductive behavior in teleost. ps a-g impulsive choice in domestic chicks: context dependence and dissociation between delay and handling cost toshiya matsushima , naoya aoki , andras csillag biology, hokkaido univ., sapporo, japan; agriculture, nagoya univ., nagoya, japan; anatomy, semmelweis univ., budapest, hungary choice between small/immediate reward and large/delayed reward has been widely used as a behavioral measure of impulsiveness. to study how ecological factors shaped underlying neural processes, we examined week-old chicks in four different tasks with identical economical consequences. in task , chicks chose between small reward (one pellet) delivered immediately and large reward (six pellets) after a delay up to s. in task , chicks chose between small reward located at cm and large reward at − cm, where cues signaled the distance of invisible food. task was similar to the task , except that cues signaled the food quantity. in task , total handling time differed due to lowered food accessibility, while the delay was kept identical. lesion experiments revealed that ventral striatum was specifically involved in choices based on anticipated proximity (but not quantity), whereas arcopallium (association cortex analogue) in choices based on anticipated handling cost. research funds: kakenhi ( , ) ps a-g analysis of the brain regions associated with the dance language of the honeybees taketoshi kiya, takekazu kunieda, takeo kubo dep. biol. sci., univ. tokyo, tokyo, japan social animals have highly developed communicative abilities. the worker honeybees (apis mellifera l.) can transmit location of food sources by the dance language. in spite of the simple structure of the honeybee brain and the stereotyped dance behavior, its neural mechanisms remain totally unknown. previously, we found active brain regions in the dancing workers (dancers) by using a novel immediate early gene, kakusei, as a marker for neural activities and found its prominent expression in the small-type kenyon cells (skcs) of the mushroom bodies. here, we report that kakusei was similarly expressed in the skcs of the foraging workers (foragers), which do not always show the dance behavior. in contrast, the skcspreferential kakusei expression was not observed in the brains of the orienting workers, which were flying to learn the hive location. these results imply that the activities of the skcs in the dancer brain are neither due to dance presentation itself nor sensory inputs during foraging, but complex information processing accompanying the foraging behavior. c. elegans wild type animals are usually attracted to nacl, but show avoidance behaviors after being conditioned with nacl and starvation (food−/nacl+). this behavioral plasticity is not induced under the food−/nacl− or food+/nacl+ conditions. we isolated learning-defective mutants including pe , which had a missense mutation in the casy- gene. several casy- deletion mutants also showed learning defects. casy- has an extensive similarity to human calsyntenin/alcadein, which is a single-pass transmembrane protein with cadherin-like repeats localized to the postsynaptic membrane of cns synapses. alcadein forms a stable tripartite complex with app and x l/mint . however, after dissociation of x l, alcadein is susceptible to cleavage by protease(s). we found that casy- was expressed mainly in neurons and functioned at the adult stage. we are now investigating the localization pattern of the gfp-tagged protein, and whether casy- can also be proteolytically cleaved. ps a-g insulin-like signaling is required for association between temperature and feeding state in c. elegans eiji kodama , atsushi kuhara , akiko mohri , , kotaro kimura , , masatoshi okumura , masahiro tomioka , yuichi iino , ikue mori , div. of biol. sci., nagoya univ., japan; present address: univ. of texas, health sci. cent., usa; present address: natl. inst. of genet., japan; mol. genet. res. lab., univ. of tokyo, japan; inst. for advanced res., nagoya univ., japan c. elegans can associate cultivation temperature with feeding state. mutations in ins- encoding insulin homologue caused defective associative learning, mutations in daf- and age- encoding the homologues of insulin receptor and pi -kinase, respectively, suppressed the defect of ins- , and the mutation in daf- encoding forkhead transcriptional factor caused the learning defect. this suggests that ins- antagonizes daf- insulin-like signaling for associative learning. interestingly, age- animals associate their cultivation temperature with feeding-state quicker than wild type. this defect was rescued by expressing age- in some head interneurons. in addition, the activity of these interneurons were down-regulated by starvation through ins- . we suggest that insulin-like signaling modulates the neuronal activity of interneurons essential for associative learning. ps a-g analysis of ttx- : novel thermotaxis gene conserved among various organisms akiko miyara, akane ohta, yoshifumi okochi, masatoshi okumura, ikue mori laboratory of molecular neurobiology and institute for advanced research, nagoya university, nagoya, japan c. elegans can memorize the food condition in relation to the cultivation temperature and migrate to the cultivation temperature when looking for the food. this response to temperature is called thermotaxis. several neurons and genes required for thermotaxis have been identified, but molecular mechanism of thermotaxis is still poorly understood. the ttx- (nj ) and ttx- (nj ) mutants are obviously defective in thermotaxis and partially defective in chemotaxis. we revealed that ttx- encodes novel protein and is expressed in many neurons and functions in several neurons responsible for the thermotaxis behavior. the predicted protein structure of ttx- is similar to ric- , identified in c. elegans at first and conserved among several species (halevi et al., (halevi et al., , . ric- is thought to be required for the maturation of acetylcoline receptor (halevi et al., ) , so ttx- may play a similar role such as folding, assembly, transmission or anchoring of some kind of membrane protein. ps a-g analysis of aho- mutant that cannot associate cultivation temperature with feeding state in c. elegans nana nishio , akiko mohri , , eiji kodama , atsushi kuhara , mizuho koike , kotaro kimura , , ikue mori , div. of biol. sci., nagoya univ., japan; present address: univ. of texas, health sci. cent., usa; present address: natl. inst. of genet., japan; inst. for advanced res., nagoya univ., japan the nematode c. elegans can associate cultivation temperature with feeding state: well-fed animals migrate to and starved animals avoid from the cultivation temperature on a temperature gradient. to identify genes required for this associative learning, we screened mutants that are defective in starvation-induced cultivation temperature avoidance. we isolated aho- (nj ) mutants that were normal in thermotactic migration after cultivated well-fed state and normal in response to food in locomotion assay (sawin et al., ) , indicating that they are normal in temperature and food recognition and may be defective in the associative learning. aho- gene encoded a predicted hydrolase and the molecular properties have not been characterized yet, although aho- is a highly conserved protein throughout yeast to human. currently, we are trying to dissect the molecular and cellular analysis of aho- gene further. we have demonstrated aversive conditioning in lymnaea using mm sucrose presentation as the appetitive stimulus (cs) and mechanical tactile stimulation to the head as the noxious stimulus (ucs). we measured the feeding response before and after pairing with the aversive stimulus to determine whether learning alters the innate preference for sucrose. we also measured the neuronal activity of b , located in the buccal ganglion. an associative memory, lasting h, was produced with pairings of cs and ucs. the learning was characterized by a shift in the response to the ucs from a whole body withdrawal response to the cessation of feeding behavior. b neuron responded with repetitive impulse discharge regularly as fictive feeding patterns to a sucrose application in naive animals, on the other hand cs application failed to generate regular impulse activity rather it resulted in generation of epsps in the conditioned animal. this can interpret that the conditioning decreased the excitability of b neuron activity thus to decrease the fictive feeding behavior. yasutaka nomura , dai hatakeyama , tetsuro horikoshi , etsuro ito , manabu sakakibara lab. neurobiol. engr, sch. high-tech, tokai univ., numazu, japan; cris, hokkaido univ., sapporo, japan calexcitin, low molecular weight gtp-binding protein is found to be phosphorylated in the visuo-vestibular conditioned hermissenda at the type b photoreceptor. we found positively stained neurons to anti-calexcitin antibody (gift from dr. kuzirian) at the cerebral and pedal ganglion in the circumesophageal nervous system of conditioned lymnaea with two different ways. one was the same conditioning paradigm as hermissenda and the other was taste aversion conditioning. both of these conditioning response is the whole-body withdrawal. no positive neuron was found in naïve animal. neurons in cb cluster and pea cluster showed both positivity to calexcitin and serotonin. this suggested the functional role in conditioning. ken honjo, katsuo furukubo-tokunaga graduate school of life and environmental sciences, university of tsukuba, ibaraki, japan the fruit fly drosophila melanogaster has been utilized as a successful model to study underlying mechanisms of learning and memory. we have established a novel larval olfactory paradigm and found that appetitive and aversive memories are considerably different in their stability whereas both are localized to the mushroom bodies (mbs). we found that larval memory induced by sucrose lasts six times longer than that induced by quinine although the initial learning performances are comparable. by expressing shi ts in larval mbs, we demonstrate that disruption of neural output from mbs abolishes both appetitive and aversive memory indicating that both memories are stored before the mb output synapses. moreover, we show that disruption of either creb or amnesiac functions abolishes appetitive but not aversive memory. thus these data suggest that appetitive and aversive reinforcements stimulate different intracellular and/or intercellular signaling pathways generating distinct memory components in mbs. motomi matsuno , minoru saitoe , , tim tully tokyo metropolitan institute for neuroscience, tokyo, japan; department of biology, tokyo metropolitan university, japan; cold spring harbor laboratory, usa we identified ruslan as a novel memory mutant, and found that it encodes a cell adhesion molecule, klingon (klg). klg belongs to the immunoglobulin superfamily and was originally identified as an essential gene for the development of photoreceptor neurons. we report here that klg is necessary for long-term memory as well as early-phase memory. we show that klg expression is dependent on neural activity and functions as a downstream of both the transcription factor, creb and the cell surface receptor notch, both of which are well known to function in ltm formation. transgenic expression of klg improves memory of a klg mutant. since klg protein localizes along the surface between neuropil and neuropil glia, we propose that klg mediates an interaction between neurons and glia that is required for memory formation. we have investigated the ability of context-dependent olfactory learning in the cockroach, periplaneta americana. we trained one group of cockroaches to associate peppermint odor (conditioned stimulus, cs, p) with sucrose solution (appetitive unconditioned stimulus, us+), and vanilla odor (cs, v) with saline solution (aversive us, us−) under illumination (l), and to associate p with us− and v with us+ in the dark (d). another group received training with opposite stimulus setup (l: v+/p−, d: v−/p+). before training, cockroaches preferred v over p. day after training, the former group significantly preferred p over v under illumination but preferred v over p in the dark, and the latter group displayed the invert odor preference. result of the control experiment excluded the possibilities that conditioning hours of the day or its order was used as cues to disambiguate the meaning of css. thus cockroaches are capable of disambiguating the meaning of cs odors according to the visual context. hidehiro watanabe, makoto mizunami graduate school of life sciences, tohoku university, sendai, japan a century had passed since pavlov reported classical conditioning of salivation in dogs. however, the cellular mechanisms underlying this conditioning remain obscure. in insects, salivation is regulated by salivary neurons of the subesophageal ganglion which innervate the salivary grand. here, we established antennal classical conditioning of salivation and that of activities of salivary neurons in cockroaches, periplaneta americana. in insects, antennae are elaborate sense organ that processes many sensory modalities including odor and taste. we found that responses of salivary neurons to an odor was increased after repetitive pairing of the odor with sucrose or saline solution presented to an antenna, but those to an odor paired with water or tactile stimulus presented to an antenna did not changed. the level of salivation to sucrose-associated odor was significantly greater than that to non-associated odor. these results are the first to suggest the classical conditioning of salivation in non-mammalian species. these results are useful to study neural mechanisms underlying classical conditioning of salivation. research funds: kakenhi ke zhang , jian z. guo , ai k. guo , institute of neuroscience, chinese academy of sciences, china; institute of biophysics, chinese academy of sciences, beijing, china the cooperation of dopamine system and other brain cortices is essential for decision-making in mammal. drosophila can make clearout choice in visual flight simulator when facing conflicting visual cues based on the saliency of the cues previously trained to follow. here we show this behaviour is impaired when the transmission of dopaminergic neurons or mushroom bodies (mb), was genetically silenced by gal /uas-shi ts system, suggesting that this behaviour is mediated by dopaminergic system acting through mb, a structure shown to be densely innervated by dopaminergic fibers. however, the dopaminergic and mb synaptic activities were required only during the early choice period (< min), but not for the sustenance of the chosen flight path. thus the dopaminergic system and mb are specifically devoted to the cognitive function exemplified by the flyǐs choice behaviour and further studies of the circuit in drosophila may help to understand the neural basis of higher cognitive functions. sae unoki, yukihisa matsumoto, makoto mizunami graduate school of life sciences, tohoku university, sendai, japan in mammals, the dopaminergic reward system plays ubiquitous roles in reward learning. previous studies in insects suggested that octopamine (oa) and dopamine (da) mediate various kinds of reward and punishment signals in olfactory learning. however, whether such roles can be generalized to learning of sensory signals other than odors remained unknown. we pharmacologically studied the roles of oa and da in appetitive and aversive forms of visual pattern learning in crickets. crickets injected with oa receptor antagonists exhibited no significant levels of appetitive visual learning, but aversive one was unaffected. the opposite influences were observed by injection of da receptor antagonists. our finding that oa and da participate in reward and punishment conditioning in visual learning, together with results of previous studies in olfactory learning, suggests ubiquitous roles of the octopaminergic reward system and dopaminergic punishment system in insect learning. this suggests conserved roles of aminergic reinforcing systems among different phyla. aiko watanabe, neal a. hessler laboratory for vocal behavior mechanisms, riken brain science institute, saitama, japan in adult songbirds, neural turnover occurs in hvc, a forebrain motor control nucleus. cells labeled by bromodeoxyuridine (brdu), a cell birth marker, appear in the ventricular zone, migrate into hvc, and some of them mature into projection neuron. to assess the role of neurogenesis in adult song plasticity, we deafened adult bengalese finches, whose songs are disorganized and become plastic within the first month after deafening, and then stabilize. deafened birds had more brdu-labeled cells in hvc than control birds within the first month. more tunel-stained apoptotic cells also tended to be seen in hvc of deafened birds. however, number of the brdu-labeled cells decreased months after deafening, when the songs had stabilized. most of the brdu-labeled cells in hvc of deafened birds were immunoreactive for a neuron-specific marker, hu. additionally, amount of singing in deafened birds, which may affect amount of neurogenesis, did not significantly differ from that in control birds. these results suggest that the amount of neurogenesis is related to adult song plasticity. yasko tobari , , kazuo okanoya , , lab. for biolinguistics, riken-bsi, wako, japan; grad. sch. of sci. and tech., chiba university, chiba, japan; presto, jst. kawaguchi, japan a set of brain nuclei controls song production in songbirds. among these nuclei, the robust nucleus of arcopallium (ra) is the telencephalic site of direct projections onto vocal motor neurons and respiratory premotor neurons. the projections of ra to the mudulla included the tracheosyrigeal part of the hypoglossal nucleus (xiits), which innervates the syrinx, the birds , vocal organ, and respiratoryrelated nucleus, retroambigualis (ram) were present in bengalese finches. in this study, we have focused our attention on the descending projections of ra, with a view to the presence of contralateral projections to xiits and ram, using in vivo tract-tracing technique. the results indicated that ipsilateral and contralateral projections of ra to respiratory-vocal nuclei in the brainstem were defined in adult male bengalese finches. birdsong is composed of various song elements that have typical frequency modulation. each element is aligned in own sequential rule. especially in bengalese finches, the sequential rule obeys finite state grammar. it has been focused what neural mechanism enables such a complex sequential rule. in order to learn and maintain their own song, they have auditory neural representation of their own song in the forebrain area hvc. we collectively recorded the activities of hvc neurons driven by all possible element pair stimuli. the results show that most of neurons in hvc respond not only the sequence included in their own song but also the sequence not included. each neuron has typical response distribution toward the whole element sequence. in addition, the distribution property is different among neurons in same individual. taken together, information of the entire song element sequence would be stored in the neural ensemble of these neurons as a population coding. hironobu sakaguchi department of physiology and biological information, dokkyo university, school of medicine, japan avian vocal learning provides a good model for human speech learning. young male songbirds learn to imitate their tutor's song during a specific time in development, which is referred to as a sensitive period. many behavioral studies have shown that vocal learning is affected by a song template and social factors. if a young bird is raised without a tutor's song template (father) and/or social contacts with other birds, including its mother and siblings, it produces an abnormal isolated song, meaning that isolation delays the sensitive period for song learning. here, we investigated for the delayed song learning of socially isolated zebra finches from new tutors. consequently, isolated birds, exposed to new tutors from day , developed the zebra finch-typical song (song syntax), similar to song acquisition in young birds during the sensitive period of song learning. however, they were not able to imitate the syllable phonology from new tutors. the differences between two aspects of song organization suggest that the schedules and processes of the learning of phonology may be different from those of song syntax. ps a-h facilitatory effects of oxytocin on synaptic plasticity in the olfactory bulb and olfactory learning in young rats fumino okutani, jing-ji zhang, guang-zhe huang, hideto kaba department of integrative physiology, kochi medical school, nankoku, japan oxytocin (ot) within the olfactory bulb (ob) has been reported to be important for the induction of maternal behavior and recognition of offspring. the activity of mitral cells, olfactory relay neurons in the ob is inhibited by granule cells via reciprocal dendrodendritic synapses. electrophysiological studies have revealed that ot modulates mitral cell activity by acting on mitral and granule cells. in a classical conditioning paradigm, young rats show aversion to the odor that has been paired with foot shock. our studies have shown that plasticity in the ob is critical for this olfactory learning. pups that received ot infusion into the ob in the presence of citral odor developed an aversion to the odor without shock, suggesting that ot infusion has a facilitatory effect on olfactory learning. using ob slices, long-term potentiation (ltp) was induced in field epsps recorded in the granule cell layer. ot administration also facilitated ltp. these results demonstrate that ot is involved in olfactory learning in young rats. research funds: kakenhi ps a-h the gaba a receptors in the ventral pallidum are involved in the retrieval of conditioned taste aversion in rats tadashi inui, tsuyoshi shimura, takashi yamamoto div. behav. physiol., dept. behav. sci., grad. sch. human sci., osaka univ., japan we examined the effects of microinjections of gaba a receptors antagonist bicuculline into the ventral pallidum (vp) on the retrieval of conditioned taste aversion (cta). in experiment , rats received a pairing of saccharin or quinine hydrochloride (cs) with an i.p. injection of . m lithium chloride (us). after this conditioning, vehicle or bicuculline was bilaterally infused into the vp just before the re-exposure to the cs. the microinjections of bicuculline significantly increased the intake of saccharin cs, but not quinine hydrochloride. in experiment , rats were presented with saccharin as cs via an intraoral cannula. the microinjections of bicuculline significantly increased ingestive responses and decreased aversive responses. these results suggest that the gaba a receptors in the vp play an important role in the expression of ingestive and/or aversive responses to saccharin cs during the retrieval of cta so that the microinjection of bicuculline might increase the intake of saccharin cs. research funds: kakenhi ( ) ps a-h transient blockade, but not genetic deficiency, of c-fos gene expression impairs long-term memory in taste aversion learning roles of c-fos gene expression and its protein product, fos, in conditioned taste aversion (cta) learning were examined using the antisense oligodeoxynucleotide (odn) method in rats and in mice carrying c-fos gene deficiency. infusion of antisense odn (as-odn) directed against c-fos mrna into the parabrachial nucleus (pbn), but not into the amygdala or insular cortex (ic), impaired the acquisition, while infusion of randomized and inverted control odns had no effect. suppression of fos synthesis in the amygdala or ic impaired the retention. retrieval of an acquired cta was not impaired by as-odn infusion into the pbn or amygdala. in contrast, mice carrying c-fos gene deficiency showed normal acquisition and retention. the present results suggest that the fos-mediated signals in the pbn, amygdala or, ic plays key roles in the acquisition and/or consolidation, but not the retrieval, of long-term cta memory. ps a-h gaba receptors in the deep cerebellar nuclei are essential for mouse eyeblink conditioning classical eyeblink conditioning is a useful experimental system to analyze the neuronal substrate underlying learning and memory. the knowledge on the mouse eyeblink conditioning is far less compared with rabbit's. we examined the role of the deep cerebellar nuclei (dcn) during delay eyeblink conditioning in c bl/ mice by using gaba a receptors agonist and antagonist. in the acquisition tests, in which muscimol (msc) or picrotoxin (ptx) was injected from beginning of training, acsf-injected control mice learned this task, but both msc-and ptx-injected mice showed a significant impairment in acquisition of conditioned response (cr). in the retention tests, in which the drug was injected after acquisition of training, cr % in acsf-injected mice were kept over %, while those in the mscand ptx-injected mice decreased to %. these results revealed that gabaa receptors in the dcn play important roles in acquisition and retention of mouse eyeblink conditioning. various forms of synaptic plasticity are found in cerebellar circuits, but their significance in motor leaning remains unknown. in the cerebellum, delphilin is expressed selectively in purkinje cells (pcs) and localized exclusively at parallel fiber (pf) synapses, where it interacts with glutamate receptor ␦ that is essential for long-term depression (ltd) and motor learning. here, we showed that ablation of delphilin proteins facilitated ltd induction at pf-pc synapses and enhanced optokinetic response adaptation without affecting histology. this finding suggests that threshold regulation of ltd at pf-pc synapses is a limiting step for motor learning efficiency. ps a-h post-training cerebellar cortical activities are necessary for transfer of memory trace of motor learning from cortex to nuclei soichi nagao , , takehito okamoto , fumihiro shutoh , lab for motor learning control, riken bsi, saitama, japan; sorst, jst, saitama, japan; dept. anat., grad. univ. tsukuba, ibaraki, japan one-hour optokinetic training induces short-term adaptation of horizontal optokinetic response (hokr) gains in mice. succession of h daily training for week induces long-term adaptation. we recently reported that the memory trace of adaptation of hokr is initially acquired within the cerebellar flocculus through long-term depression (ltd), and later transferred to the vestibular nuclei for consolidation. in order to reveal the neural mechanisms underlying the memory transfer, we reversibly inactivated the neural activities of flocculus bilaterally by local application of muscimol immediately after the end of daily training. mice treated with muscimol showed depressed long-term adaptations, while the short-term adaptations were intact, suggesting that the neural activities of cerebellar cortex in a certain period after training are necessary for the transfer of memory trace from flocculus to vestibular nuclei. research funds: kakenhi ( ) ps a-h modification of gene expression in the cerebellar cortical neurons related with long-term motor learning yuji t. katagiri , , takehito okamoto , shin-ichi nishimura , fumihiro shutoh , , soichi nagao , lab. for motor learning control, riken bsi, saitama, japan; univ of the air, chiba, japan; dept. of anatomy, human comprehensive science, grad. univ. tsukba, japan; sorst, jst, japan we recently reported that the cerebellar ltd plays a crucial role for both acquisition and consolidation of memory trace of long-term motor learning using the adaptation paradigm of mouse horizontal optokinetic eye movements (shutoh et al., ) . in order to listup the molecules involved in the motor learning, we sampled total rna from the cerebellar flocculus of short-and long-term adapted mice, and quantified amounts of gene expression by the microarray methods. we found that the number of genes modulated by longterm motor learning much exceeded that modulated by short-term motor learning, and the number of down-regulated genes were larger than that of up-regulated genes. we furthermore examined the gene expression of purkinje cells by the laser micro-dissection and quantitative rt-pcr methods. ps a-h influence of spatial cues on hippocampal neuronal activity in spatial navigation tasks in mice hippocampal neurons were recorded while mice performing spatial tasks of searching for unpredictable and predictable rewards. the influence of spatial cues, including distal and proximal cues, on the response of hippocampal cells that exhibited place-related activity was examined. place cells predominantly shifted their fields accordingly by changes of visual and auditory distal cues, and fewer cells shifted their fields by changes of proximal cues. these results provide evidence that hippocampal neurons of mice can use flexibly information of spatial cues to represent the environment, and this ability is important for spatial learning. son ho , , t kobayashi , , e hori , , k umeno , , t ono , h nishijo , system emotional science, univ. of toyama, toyama, japan; crest, tokyo, japan we investigated a role of the hippocampal formation (hf) in encoding of a moving object in an open field. rats acquired icss rewards if they moved freely. then, a remote-controlled car was placed inside the open field. the rats could receive icss if it chased and approached the car. of a total of place cells recorded, activity of was significantly modulated by the car speed and/or distance between the car and rat; , and cells displayed distance-dependent, car speeddependent, and distance and car speed-dependent firing, respectively. furthermore, six cells, which did not show the place field in reference to rat position, but showed the place fields in reference to car position. in a control experiment, the same car was introduced, but the rats could receive icss rewards without relation to relative distance between the rat and car. so far, place cells were recorded in this experiment. of these, six and three place cells displayed distancedependent and car speed-dependent firing, respectively. the results suggest that hf encodes not only spatial information of own location, but also that of other moving object in an environment. hisae gemba, kazuko nakao, ryuiti matsuzaki, yusaku amaya department of physiology, kansai medical university, moriguchi, japan cortical field potentials were recorded by electrodes implanted on the surface and at a . - . mm depth in the cortex of monkeys in the process of learning somatosensory-initiated hand movements and then analyzed. it was found that an s-n, d-p potential, at about ms latency from stimulus, in the caudal bank of the left arcuate sulcus (homolog of broca's area) was related to recognition learning (association of stimulus with movement), and that an s-n, d-p potential in the motor and somatosensory cortices, and areas and , contralateral to the operating hand, was related to skill learning (making movements quicker and more appropriate). in visuo-initiated hand movements, the left prefrontal cortex was related to recognition learning; the motor and somatosensory cortices, and area to skill learning, as previously reported. this indicates that motor programming for somatosensory-initiated and visuo-initiated hand movement differs. computational studies of hippocampal function generally assume that ca performs a match-mismatch comparison of memory retrieval with sensory input. here we investigated this comparator model using an ensemble recording during task behaviors in the rat. we employed directional memory-guided alternation and visual cue discrimination tasks for the same animal. after training, the animals tended to predict a next direction according to the alternation paradigm even in the visual cue discrimination task. during this task, we found that some ca neurons showed specific bursts when a predicted event did not occur or along the trajectories of their corrective movements from a wrong cite to a correct cued one. these data suggest that ca plays an important role in the mismatch detecting and correcting process of behavior. n-methyl-d-aspartate (nmda) receptor has high permeability to ca + but is blocked by mg + in a voltage-dependent manner. this property is a molecular basis of nmda receptor-dependent long-term potentiation, which is thought to play a central role in learning and memory. we have generated the genetically engineered mice in which mutated nmda receptors defecting in mg + binding ability are expressed specifically in the granule cells of the dentate gyrus, the entry point to the hippocampal trisynaptic circuit. the mutant mice showed a variety of behavioral abnormalities including hyperactivity, impaired prepulse inhibition. to elucidate the effect of mutation on the information processing in the hippocampus, we recorded the place-related activity from hippocampal ca cells, the output stage of hippocampal circuit. the link between the behavioral anomaly and the hippocampal activity is discussed. mikako sakurai, ko zushida, masayuki sekiguchi, keiji wada department of neurodegenerative diseases, national institute of neuroscience, ncnp, tokyo, japan uch-l is a component of the ubiquitin system. uch-l is expressed at high levels in the hippocampal neurons. however, the functional role of uch-l in synaptic plasticity and behavior is not understood. we examined behavior and synaptic plasticity in gad mouse which is an autosomal recessive spontaneous mutant carrying an intragenic deletion in the gene encoding uchl . gad mice have significantly impaired performance in the open field and one-trial passive avoidance tests. theta burst stimulation (tbs) of shaffer collateral in hippocampal slices from gad mice elicited decremental long-term potentiation (ltp) in the area ca . in contrast, non-decremental ltp was induced in control wild-type mice. the maintenance of tbsinduced ltp in the wild-type mice was impaired by actinomycin d, an inhibitor of transcription, whereas tbs-induced ltp in gad mice was insensitive to actinomycin d. these results suggest that uch-l is a molecule participating in the synaptic plasticity elicited by tbs and the memory function. ps a-i learning stages in rat operant reversal task and cross-correlation between hippocampal and prefrontal local field potential powers yoshinori izaki, tatsuo akema department of physiology, st. marianna university school of medicine, japan to investigate whether the relationship between hippocampus (hip) and prefrontal cortex (pfc) spontaneous local field potentials changes with leaning stages, we analyzed cross-correlation (cc) of these local field potential powers during operant reversal training sessions. rats were trained with initial discrimination task until a stable discriminative performance was achieved (learning stage ). then the rats received the reversal training. learning stages examined were as following: the first training session (stage i), leaning stage for s+ (stage ii) and for s− (stage iii). different changes of the cc in some frequency-band powers with learning stages were observed. the cc in higher gamma-band ( - hz) was strong at stage and changed with leaning stages. particularly, the cc decreased to almost zero at stage ii. these results suggest that functional connection between hip and pfc is reflected in this frequency-band and changes with learning stages. ps a-i longitudinal fiber systems in the dentate gyrus of the rat norio ishizuka, yoshitomo umitsu department of brain structure, tokyo metropolitan institute for neuroscience, tokyo, japan longitudinal fiber systems in the dentate gyrus of the rat were investigated by anterograde labeling method of pha-l and retrograde labeling method with fluorescent dyes. the flattened hippocampal formation allowed sections to be cut perpendicular to the full septotemporal axis of the dentate gyrus. injection of pha-l into the hilar region elucidated that two longitudinal fiber systems existed in the dentate gyrus. the first fiber system gives rise to projections to the superficial portion of the dentate molecular layer, and the longitudinal axonal trajectory of this system ceased within the range of about . mm from the injection level. in the second fiber system, axonal terminations began to appear at the level of mm apart from the injection level and were distributed in further full septotemporal extent of the dentate molecular layer. the axonal arborizations of the second system were found in the deepest portion of the dentate molecular layer immediately above the granular cell layer. in the experiment of fluorescent dye injection, several kinds of cells in the hilus were retrogradely labeled. ryoichi moki, ryang kim, hisahiro umeeda, akinobu suzuki, satoshi kida department of bioscience, tokyo university of agriculture, tokyo, japan recent studies have shown that when conditioned fear memory is retrieved, fear memory becomes labile and requires gene expressiondependent reconsolidation for the re-storage. in addition, previous our study using conditional creb mutant mice indicated that creb is required for reconsolidation of conditioned fear memory. we also observed protein synthesis-dependent reconsolidation of spatial memory using morris water maze. in this study, to understand the mechanisms of reconsolidation of spatial memory, we examined a role of creb in reconsolidation of spatial memory. using conditional creb mutant mice that enable to induce the inhibition of creb activity in a tamoxifen-dependent manner, we found that inhibition of creb activity leads to disruption of spatial memory after the retrieval. this result indicates that creb is required for reconsolidation of spatial memory. shunsuke hasegawa, hirosi hosoda, satoshi kida department of bioscience, tokyo university of agriculture bhlh-pas transcription factor bmal ubiquitously expresses in brain. bmal functions by forming a heterodimer with either clock or npas , which has been known to play important roles in control of circadian rhythm and memory formation, respectively. to understand roles of bmal in forebrain function, we generated conditional mutant mice that enable to induce the inhibition of bmal function in a forebrain. using a dominant negative mutant of bmal (bmal r a) that forms a heterodimer with clock but loses the binding activity with e-box (hosoda et al., ), we generated transgenic mice expressing this mutant under the control of tetracycline-dependent promoter. these mutant mice were crossed to transgenic lines expressing tetracycline-dependent transcription factors (tta) under the control of alpha camkii promoter. we observed the expression of bmal r a in several double transgenic lines in a tta-dependent manner. behavioral analyses showed that these mutant mice showed an impairment of memory formation, indicating crucial roles of bmal in learning and memory. stress sometimes causes memory deficits. and chewing has been shown to reduce stress. however, the chewing-related mechanism in stress-induced memory deficits is unclear. we thus examined the effects of chewing on spatial memory using morris water maze and fos induction in the hippocampus and amygdala in stressed mice. when mice were exposed to restraint stress, reduction in learning ability and density of fos-positive cells in the dg and the bla was seen, but not in the mice chewing a thin wooden bar during stress exposure. the results suggest involvement of the amygdaloidmechanism by which chewing may prevent the stress-induced impairment of hippocampus-dependent memory. seiichiro amemiya, shinya yanagita, satoko suzuki, ichiro kita graduate school of science, tokyo metropolitan university, tokyo, japan we examined the effect of background noise (bgn) on spatial learning and its neuronal activity related to arousal and stress using maze task and c-fos immunostaining. rats performed maze task under different intensity of bgn ( , , or db; intermittent white noise). db bgn induced significant decreases in number of error and time to goal in maze compared with and db. although bgn increased fos positive acetylcholinergic neurons (fos-chat) in mesopontine tegmentum (mt) regardless of the intensity, fos-chat in basal forebrain (bf) increased intensity-dependently. in locus coeruleus (lc) and cortex, fos positive cell increased intensitydependently. furthermore, db bgn remarkably enhanced fos expression in stress-related nuclei, such as paraventricular nucleus and central nucleus of the amygdala. these results suggest that bgn improve spatial performance by enhancing arousal following activation of cholinergic neurons in mt and bf, and lc neurons. however, higher bgn intensity could evoke over-arousal and stress responses, thereby prevent the maze task. siriporn chattipakorn , , anucha pongpanparadorn , wasana pratchayasakul , anchalee pongchaidacha , nipon chattipakorn fac. dent. cmu, chiang mai, thailand; cert, cmu, chiang mai, thailand current pharmacotherapy of ad is the use of ache inhibitors. previous in vitro study showed that tde inhibited ache activity. this is the first study investigating the effects of tde on cortical ache activity and neuronal activity in in vivo. we used fos immunohistochemistry to determine the neuronal activity and the colorimetric method to investigate cortical ache activity following the single injection of various tde doses. mean fos-positive neurons in cortex were ± , ± and ± in the groups administered , and mg/kg tde, respectively. cortical fos-positive neurons in all three tde-treated groups were greater than those in the control group. percent inhibition of cortical ache activity was . ± . , . ± . and . ± . for , and mg/kg tde, respectively. these ache inhibitory effects were significantly different from the control. these findings suggest that tde could be beneficial as a possible novel therapeutic agent for ad. we showed the effects of a -h tde administration in animals on the inhibition of cortical ache activity and the enhancement of cortical neuronal activity. ache activity in circulation following a -h tde administration was not different compared to the control. this study investigated that the effects of tde on circulating ache activity (cache) in animal models was time-dependent. we used the colorimetric method to investigate cache activity in rats following the single administration of tde at various doses at different time courses ( , and min). percentage inhibition of cache activity following a single tde injection at doses and mg/kg significantly decreased at and min after tde injection, but not at min. cache inhibitory effects among two doses of tde administrated groups at various time courses were not significantly different. these findings suggest that tde may be a short-acting ache inhibitor. donepezil, galanthamine and tacrine are acetylcholinesterase (ache) inhibitors used for treatment of alzheimer's disease. we examined the neuroprotective mechanisms of ache inhibitors against apoptotic glutamate neurotoxicity using cortical neurons. we show that they protect neurons through mechanisms other than ache inhibition. the protective effects are mediated through nicotinic receptors (nachrs). donepezil and galanthamine protect neurons through ␣ and ␣ -nachr and kinases involved in pi k-akt pathway, and increase the levels of phosphorylated akt and bcl- . these results suggest that these ache inhibitors express their neuroprotective effects against glutamate neurotoxicity through nachrs and that donepezil and galanthamine protect neurons through pi k-akt pathway via ␣ and ␣ -nachrs. ps a-i arachidonic acid preserves hippocampal neuron membrane fluidity in senescent rats yasuto kashiyae , yoshiyuki ishikura , shigeaki fujikawa , yoshinobu kiso , manabu sakakibara lab. neurobiol. engr., tokai univ., numazu, japan; inst. health care sci. suntory, shimamoto, japan previous studies indicate that long-term dietary supplementation with arachidonic acid (aa) in -month-old rats (oa) effectively restores performance in a memory task and induction of long-term potentiation in the hippocampus to the level of young control animals (yc). this study examined fluorescent recovery after photobleaching (frap) in yc, old control (oc), and oa neurons in hippocampal slice preparations. three measures: mobile fraction (mf), diffusion constant (d), and time constant (τ), were estimated among yc, oc, and oa. each of these parameters was significantly different between oc and yc, suggesting that membrane fluidity is lower in oc than in yc. in contrast, d and τ were almost comparable in oa and yc, indicating that hippocampal neuronal membranes supplemented with aa were more fluid than those in oc, whereas the fraction of available molecules remained smaller than in yc. long-term administration of aa to senescent rats might help to preserve membrane fluidity and maintain hippocampal plasticity. ps a-i thimet oligopeptidase co-exists in gfap-and cd b-positive glia in rat pc/rsc treated with mk- takeshi kato , mohammad arif , michiyuki yamada , toshiyuki chikuma , md. mahiuddin ahmed lab. natural info. sci., grad. sch. integr. sci., yokohama city univ., yokohama, japan; grad. sch. integr. sci., yokohama city univ., yokohama, japan; dept. hygien. chem., showa pharmaceut. univ., machida, japan; dept. r&d, bioelectro. anal. sci., japan thimet oligopeptidase (ep . ) hydrolyzes not only neuropeptides but also the peptides generated by proteasomes. in the present immunohistochem study we found that mk- activated gfapand cd b-positive glia cells in rat posterior cingulate/retrosplenial cortex (pc/rsc) day after the treatment. mk- also increased ep . and prolyl oligopeptidase. immunohistochem data showed that ep . co-localized with gfap and cd b positive glial cells. since mk- causes schizophrenia-like psychosis and produces neurotoxicity in adult rodent brain, we further examined the pretreated effect of neuroleptics. clozapine co-administration suppressed the increased ep . in the pc/rsc. these data suggest that ep . in the astroglia and microglia cells of rodent brain might in part control positive and/or negative schizophrenia symptoms. ps a-i effect of age and sex steroids on the expression of alzheimer's disease presenilin (ps) and in the mouse brain soumi ghosh, m.k. thakur banaras hindu university, india alzheimerǐs disease is a neurodegenerative disorder characterized by the impairment of cognition and memory. these functions are improved by supplementation of sex steroids. the genes causing lateonset of ad, presenilin (ps) and , code for highly homologous integral membrane proteins. the proteolytic fragments of these proteins are main biological components. we have analysed the effect of age, sex and gonadal hormone supplementation on ps expression at protein level by western blotting. ps shows a significant decrease with aging in both males and females. however, there is no significant variation in expression of ps and ps with sex. gonadectomy also lowers the level of presenilin proteins in old age. ps protein shows increase in expression with gonadal hormone treatment in both ages, but estrogen supplementation to old mice lowers ps level. these modulatory effects of age, sex and gonadal hormones on ps proteins may explain the therapeutic interventions of hormone replacement therapy. research funds: ministry of science and technology, india ps a-i effects of the monomeric, oligomeric and fibrillar beta-amyloid peptides on the proliferation and differentiation of adult neural stem cells from svz dept. of pharmacol., seoul natl. univ., south korea the subventricular zone is the largest neurogenic area of the adult brain. in this region, neural stem cells (nsc) serve to produce newly generated neurons and glia cells throughout adulthood. however, the common neurogenesis of nsc cannot replace neuronal loss in alzheimerǐs disease (ad) induced by amyloid deposits composed mainly of amyloidproteins. in vitro, we examined the effects of various form of apeptide on the proliferation and differentiation of nsc from svz of -week-old adult mice. in this study, a peptide was prepared three forms of aggregating stage, monomeric, oligomeric and fibrillar a peptide. we found that treatment of nsc with oligomeric form of a peptides remarkably increased the number of neurospheres during proliferation and neurons during differentiation in-vitro. we also found that these neurogenesis was accompanied by morphological change of neuron. the number of secondary and tertiary neurites increased at submicromolar concentrations of oligomeric a peptide without shrinkage of axonal length. in alzheimer's disease (ad) brain, the formation of senile plaque with accumulated microglia is observed. although the role of microglia in ad is not clarified, their involvement in a clearance is noted. high mobility group box protein- (hmgb ) is a non-histone chromosomal protein. here, hmgb was associated with senile plaques and protein level was increased in ad brain. diffuse hmgb immunoreactivity was observed around dying neurons in the kainic acid-and a - (a )-injected rat hippocampi. hmgb was not co-localized with a in transgenic mice which show massive a production without neuronal loss. furthermore, co-injection of hmgb delayed the clearance of a and accelerated neurodegeneration in a -injected rats. these results suggest that hmgb released from dying neurons may inhibit microglial a clearance and enhance the neurotoxicity of a. perineuronal nets consisting of chondroitin sulfate proteoglycan (cspg) and hyaluronic acid (ha) are associated with distinct populations in mammalian brain. in the present study, we observed perineuronal net-like structure by rat cortical neurons in dissociated culture using wisteria floribunda lectin, ha binding proteins, and cspgspecific antibodies. this perineuronal net-like structure was observed often at parvalbumin-positive neurons, indicating gabaergic ones. it is well known that perineuornal nets-containing neurons are survive against alzheimer disease in human. to elucidate significance of perineuronal nets in alzheimer disease, we applied beta-amyloid peptide into cultured cortical neurons. perineuronal nets-containing neurons were resistant against beta-amyloid peptide, while negative neurons were often dead. these results indicate that perineuronal nets are participated in protecting neurons from cytotoxic substances such as beta-amyloid. ps a-i x -like protein regulates metabolism of app in the mouse brain yoshitake sano , , tadashi nakaya , shigeyoshi itohara , toshiharu suzuki riken bsi, saitama, japan; hokkaido university, neuroscience, sapporo, japan abnormal metabolism of amyloid beta precursor protein (app) results in the accumulation of beta amyloid (a) in the brain, and contributes to the pathogenesis of alzheimer's disease. app has a functional sequence in its cytoplasmic domain, the yenpty motif, which is involved in trafficking, internalization, and metabolism of app. x -like protein (x l) was identified as a molecule that interacts with the motif and regulates app metabolism in cultured cells ( . j. biol. chem. , . j. biol. chem. , ) . there is no evidence, however, that endogenous x l suppresses app metabolism and a generation in vivo. to examine the physiologic role of x l in app metabolism in the brain, we generated x l null mutant mice. the mutant mice developed normally without gross anatomic brain abnormalities. there were increased amounts of cterminal fractions cleaved at the -site and a, but the amount of total app was unaltered in the mutant mouse brain. these results suggest that x l suppresses the production of a by inhibiting secretase-induced proteolysis of app. it is still unknown how human's central nervous system (cns) controls its body system to keep the body balanced. this study aims to analyze the characteristics of spectral response of body sway in eyes open and in eyes closed during static upright stance based on a pid control model. in this model, body sway in medial-lateral direction is considered, and the body is simply modelled as a multi-link inverted pendulum system. spectral response analysis showed the gain varied with input frequency and time lag. peaks of the gain were intensively influenced by controller's parameters (kp, kd and ki). parameters identification showed that kd is decreased in eye-closed. by simulation, the spectral responses of the pid model quite agreed with the experimental data. the results proved that the spectral characteristics of body sway is determined by the dynamics of body system and its controller's parameters, suggest the balance-keeping control in cns can be modelled as a pid controller. nuclear dysfunction is a critical element of the pathology of polyglutamine (polyq) diseases. proteome analysis of soluble nuclear proteins in the nuclear matrix of neurons expressing normal or mutant huntingtin or ataxin- protein by d-electrophoresis and tof-mass delineate that mutant at and htt proteins similarly reduce transcriptional factor x and x . immunoprecipitation and pull-down assays support interaction between polyq and factor x and x . immunohistochemistry of hela cells and primary neurons reveal sequestration of factor x and x into inclusion bodies and reduction of them in the nuclear matrix. compensatory expression of factor x and x ameliorates poly-q pathology in htt-/at -expressing neurons and transgenic drosophila. these results suggest that factor x and x are critical regulators of polyglutamine disease pathology and could be a target for developing therapeutics. ps a-j ba - was reduced in rat brains fed with coconut juice n. radenahmad, p. subhadhirasakul psu, thailand young coconut juice (ycj), cocos nucifera (arecaceae), believed to contain phytoestrogen and other sex hormone-like substances, was investigated for its possible beneficial effects on halting dementia in ovariectomized (ovx) rats, a model system for the postmenopausal condition. sixty ovx rats were divided into six groups, rats/group (g). group received e at . g/kg per day; groups and received ycj at ml, and ml/kg day, respectively, once everyday. group received ycj ml/kg plus e at . g/kg day twice a week, all for weeks. the other two were ovx and sham-operated controls. using a chemiluminescent immunoassay, circulating e in group was insignificantly different from the control groups. after rats were sacrificed, brains were removed, fixed and paraffin embedded for ihc staining. using anti--amyloid - antibody, this alzheimer pathology was found in cytoplasm and dendrites, but not in nuclei or axons, of pyramidal cells both in hippocampus and in layer and layer of cerebral cortex. it was found that amyloid deposition in frontal, temporal and hippocampus of rat brains in group was lesser than ovx and control groups. amyloid deposition was correlated with e serum at r = − . . ps a-j correlation between semantic memory and regional gray matter volume of anterior aspect of right temporal lobe in normal elderly subjects. a voxel-based morphometry yasuyuki taki , shigeo kinomura , kazunori sato , shinya uchida , ryoi goto , kentaro inoue , ichiro tsuji , hiroyuki arai , ryuta kawashima , hiroshi fukuda department of radiology and nuclear medicine, institute of development, aging and cancer, tohoku university, sendai, japan; tohoku univ. grad. school of med., sendai, japan; niche, tohoku univ., sendai, japan the purpose of this study was to determine whether there is a correlation between semantic memory and regional gray matter volume in community-dwelling normal elderly people by voxel-based morphometry. we collected brain magnetic resonance images of community-dwelling normal elderly subjects. we performed multiple regression analysis of raw score in the wais-r information subtest, gender, and regional gray matter volume. the volumes of the right superior and middle temporal gyri showed significant positive correlations with raw score in the information subtest. our study indicated that normal elderly individuals show a significant correlation between regional gray matter volume and semantic memory. research funds: (h -kenko- ), (h -choju- , h -choju- ) ps a-j effects of fluoxetine on the cognition of patients with mild cognitive impairments arash mowla, azadeh pani shiraz university of medical sciences, iran recent researches suggest a role for monoaminergic hypofunction in age related cognitive decline. in several studies selective serotonin reuptake inhibitors demonstrated neurogenesis in hippocampus. we studied the effects of fluoxetine on cognition of patients with mild cognitive impairment (mci). fifty-two non-depressed patients with mci were randomly assigned to take fluoxetine or placebo. the patients were administered mini-mental status examination (mmse) and wechsler memory scale iii (wmsiii) pre intervention. twenty-six patients completed the weeks trial. treatment response was defined as a final mmse and wms-iii scores. the patients in the fluoxetine group showed improvement in mmse and immediate and delayed logical memory scores of wms-iii. the placebo group had not significant changes in the cognitive measurements. fluoxetine enhanced memory and cognition in the patients. this was consistent with pervious studies that emphasized on the role of fluoxetine in improving memory and promoting neurogenrsis in the hypocampus. however, this conclusion should be tempered by the small sample size. lisa l. cook , d.g. goodenowe , y. yamazaki , j. flax phenomenome discoveries inc., saskatoon, canada; precisionmed inc., san diego, ca, usa dementia affects about % of the population over the age of and can result from various neuropathological conditions. currently, there is no way to differentiate specific forms of dementia (alzheimer's disease (ad), vascular dementia, etc.) prior to autopsy. pdi has discovered an -metabolite biomarker panel within the serum of patients with ad, non-ad dementia and healthy non-demented controls that can simultaneously differentiate the type of dementia and identify cognitive impairment using a non-targeted metabolomics technology based a fourier transform ion cyclotron resonance mass spectrometry (fticr-ms). the accurate measurement of the metabolite mass is sufficient to elucidate its molecular formula, thereby leading to metabolite identification, explication of biological significance and efficient biomarker validation. the -metabolite biomarker panel could provide a non-invasive method to aid in the diagnosis of specific subtypes of dementia. the development of a high throughput assay for these markers will also be presented. neurons become photosensitive by genetically introducing one of green algae-derived protein, channelrhodopsin- (chr ). in this study, we quantitatively investigated the rapidness of the light-gated current of chr expressed in pc cells using blue led light. the light-gated current consists of two components, inactivating and noninactivating. the magnitude of inactivating component was almost linearly related to the light intensity. the non-inactivating component showed the tendency to saturate at high illumination. we also found that the activation rate is about -fold faster than the inactivating rate, but both are linearly dependent on the light intensity. since the photoactivated current was very rapid in both onset and offset, the neuronal firings were phase-locked to short light pulses in an acute slice of hippocampus. it is suggested that the genetic expression of chr is one of the most ideal photostimulation methods of a genetically identified neuron with defined activity patterns in intact nervous system. yujiro hattori , shigeki ohta , kenji hamada , naofumi yamada-okabe , yonehiro kanemura , hideyuki okano , yutaka kawakami , masahiro toda , neuroimmnology research group, keio univ., tokyo, japan; chugai co. ltd., kanagawa, japan; inst. cli. res., onh, japan; physiology, keio univ., tokyo, japan; cellular signaling, institute for advanced medical research, keio univ., tokyo, japan; neurosurgery, keio univ., tokyo, japan to identify neuron specific genes, we performed two gene profiling techniques, dna microarray and est analysis. in this study, we focused on genes expressed specifically in the normal brain tissues but not in glioma tissues and identified the human kiaa gene which was a homologue of rat synarfgef (po). rt-pcr analysis revealed that the human kiaa homologue was expressed only in adult brain tissue. western blot and immunocytochemical analyses showed the kiaa protein was expressed in adult brain tissues and differentiated neuronal cells but not in fetal brain tissues nor neural stem/progenitor cells. in conclusion, we identified an adult neural-specific gene using the combined gene profiling method and our results suggest the usefulness of this method to identify tissue specific genes. ritsuko the objective of this study was to find the proteins related to sexual differentiation and to elucidate its molecular mechanism. methods: developing hypothalamic and cortical cells from fetuses on embryonic day were dissociated. after the cells were treated with nm estradiol- beta (e ) or ethanol for days, proteins were extracted and labeled with cydyes. two-dimensional difference gel electrophoresis ( d dige) was then performed. the differential protein spots were analyzed by software analysis, subject to in-gel digestion, and identified by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (maldi-tof-ms). results: more than spots were detected from d dige. compared with ethanol treatment, e increased the expression of spots in the hypothalamic cells and spots in the cortical cells (p < . , difference > . ). proteomics analysis showed different effects of e for hypothalamic and cortical cells. in order to relate cellular brain structure to function, it is necessary to manipulate neural circuits at the level of individual cell types. genetic methods for neuronal inactivation combined with cell-typespecific promoters will achieve this goal. here, we have developed a genetic method for quickly and reversibly inactivating in vivo mammalian neurons using allatostatin receptor (alstr), which causes neuronal hyperpolarization when treated with peptide ligand allatostatin (al). in rat barrel cortex neurons expressing alstr, al reversibly inactivated neuronal activity evoked by electrical stimulation of the whisker pad. both inactivation and recovery were seen within several minutes. we also confirmed the effectiveness of the al/alstr system in ferret visual cortex, lateral geniculate nucleus (lgn), and monkey lgn. therefore, the al/alstr system will be a powerful tool to investigate neuronal circuits and function. prospective purification of neural stem cells (nsc) through the specific cell surface marker is crucial for functional recovery of the damaged brain. in the last meeting, we showed that living nsc were enriched from mouse whole brain as positive cells for erythro-phytohemagglutinin (e-pha), which binds to complex type asparagine-linked oligosaccharide (n-glycans). in this study, by using facs system, we found that high selective affinity of e-pha binding to the brain cells; e-pha negative cells were neurons, mid-positive cells were nsc, and highly positive cells were endothelial cells, respectively. ligand blot analysis revealed the existence of the e-pha binding proteins different from the known selective nsc markers in e days brain homogenate, suggesting that n-glycosylated proteins could be distinctive markers for nsc. masahiro waza, hiroaki adachi, masahisa katsuno, makoto minamiyama, fumiaki tanaka, manabu doyu, gen sobue department of neurology, nagoya university, nagoya, japan the pathogenic gene product of spinal and bulbar muscular atrophy (sbma) is polyglutamine (polyq)-expanded androgen receptor (ar), which belongs to hsp client protein family. -allylamino- -demethoxygeldanamycin ( -aag) is a new derivative of geldanamycin that shares its important biological activities but shows less toxicity. -aag is now in phase ii as a potential anti-cancer agent because of its ability to selectively degrade several cancer-related client proteins. we examined the efficacy and safety of -aag in a mouse model of sbma. administration of -aag significantly ameliorated polyq-mediated motor neuron degeneration by preferential proteasome degradation of mutant ar. the ability of -aag to preferentially degrade mutant protein would be directly applicable to sbma and other neurodegenerative diseases. modulation of hsp function by -aag has emerged as a candidate of molecular targeted therapy for neurodegenerative diseases. the avian embryo has long been a popular and an excellent model for studying vertebrate development because of its classical manipulative advantages. in the present study, we tried to develop regulated gene transfer by using the tet regulatory system in the chick. the reverse tetracycline-controlled transactivator was expressed under the control of the motor neuron (mn) specific hb promoter. tetracycline responsive elements were used for inducible gfp expression. after these constructs were introduced into neural tube by in ovo electroporation, gfp expression was induced in spinal mns in the presence of doxycycline. approximately - % of mn express gfp very intensely whereas the remaining mns never express gfp, suggesting that, although the transgene is induced in limited numbers of mns, once activated, cells express a large amount of the protein product of the experimental gene. thus, this strategy can be applicable for a variety of experiments that require specially and temporally regulated gene expression in the chicken embryo. ps a-k selective collection of catecholaminergic (ca) neurons in the brain and its application to gene expression analyses hiroaki nakamura , yoshiyuki ishii , kazuto kobayashi , yasufumi sato , keiichi itoi lab. info. biol., grad. sch. info. sci., tohoku univ., sendai, japan; dept. mol. genet., fukushima med. univ., japan; inst. develop., aging, cancer, tohoku univ., japan ca neurons are involved in a wide spectrum of physiological functions in the brain. most ca neurons are localized in the brainstem and hypothalamic regions and typically make clusters of cells, among which the noradrenergic (a , a and a ) and dopaminergic (a , a and a ) neurons predominate. in order to explore functional roles of these neurons, we collected ca neurons selectively using tyrosine-hydroxylase (th)-green fluorescent protein (gfp) transgenic mice in which gfp was expressed under the control of th gene promoter. in fetal mice, most gfp-positive neurons expressed th-immunoreactivity in limited brain regions including the locus coeruleus (lc). therefore, lc-containing region was dissected under fluorescent microscopy, and neurons were dispersed by treating with trypsin, then gfp-positive cells were sorted out by flow-cytometry (facs). rna was extracted from the gfp-positive (th) neurons, reverse-transcribed, and analyzed by pcr. atg b has been shown to play an important role in the processing of lc , a mammalian homologue of yeast atg , but the tissue distribution of atg b remains unknown. to understand the role of atg b in rat tissue cells, we prepared an antibody to atg b and pc cells in which atg b expression was knocked down by rnai. in rnaitreated pc cells where atg b expression was % of that in the wild-type pc cells, the expression of cytosolic lc -i was similar to that in wild-type cells. the knockdown cell lysates, however, suppressed cleavage of prolc to lc -i. moreover, the expression of atg b mrna was high in the cerebellum and olfactory bulb, while its protein was evenly distributed in the brain. immunostaining for atg b was intense in neurons, especially in the cerebellum. these results suggest that atg b plays a major role in the processing of lc , while autophagy is deeply associated with the metabolism in neurons, especially in the cerebellum. ps a-k spatial and time-dependent transneuronal propagation of swine coronavirus (hemagglutinating encephalomyelitis virus, hev) in the rat central nervous system after its hind footpad inoculation transneuronal propagation of hev n strain into the central nervous system was examined after its subcutaneous inoculation ( pfu) in the rat hind footpad. on day post-inoculation (p.i.), antigenpositive neurons were detected in cell groups of the ipsilateral spinal cord of lumber segments. on day p.i., they increased in number, and higher-order transneuronally infected neurons were observed in restricted brain areas that project to the spinal cord. on day p.i., the viral infection became more extensive and complex, and neurological signs appeared from this period. in this model the th day would be critical for the analysis of the long-distance connections. hev can be used as a novel tracing probe, being equivalent to other reported virus probes. hiroyuki hioki , hiroshi kameda , hisashi nakamura , taro okunomiya , koji ohira , kouichi nakamura , , takahiro furuta , takeshi kaneko , dept. of morphol. brain sci., grad. sch. of med., kyoto univ., kyoto, japan; crest, japan vesicular stomatitis virus g-protein (vsv-g) pseudotyped lentiviral vectors are useful vectors for gene transfer into the central nervous system. vsv-g achieves a broad transduction spectrum and lentiviral vectors provide an efficient vehicle to integrate transgenes into dividing and non-dividing cells. thus, vsv-g pseudotyped lentiviral vectors with ubiquitous promoters, such as human cytomegalovirus (hcmv) promoter, infect and express transgenes in neuronal and glial cells. the purpose in this study is to explore neuron-specific promoters and to quantitatively examine their characteristics. at first, we used five kinds of well-known neuron-specific promoters; hsyn , rta , mcamkii, rnse and hpdgf promoters. then, we developed new hybrid promoters by a combination sequence of hcmv enhancer and neuron-specific promoters listed above. all of the new hybrid promoters dramatically improved expression of reporter gene (gfp), but the specificity deteriorated in the rat striatum, thalamus and neocortex. although green fluorescent protein (gfp) is a useful tool to label living neurons, neuronal processes are not completely labeled with gfp. in the present study, we tried to develop dendritic membrane-targeted gfp using non-prolilferative lentivirus vector with human synapsin i promoter. palmitoylation site of gap n-terminal and myristoylation site of fyn n-terminal were first tested for membrane targeting of gfp. myristoylated gfp (myrgfp) was efficiently localized at the plasma membrane of infected neurons, but not palmitoylated gfp. since myrgfp was located at both dendritic and axonal membranes, we further added the putative dendrite-targeting or basolateral targeting signals, such as c-terminals of telencephalin (tlc), fc ␥ ii  receptor (fcr), polymeric immunoglobulin receptor (pigr), and low density lipoprotein receptor (ldlr), to c-terminal of myrgfp, and compared their efficiency on dendrite targeting. recently, we developed recombinant rabies virus vectors which were expected to act as a potential neurotracing tool. the vectors infected neurons specifically from axon terminals and were transported to the downstream neurons trans-synaptically. by using two different recombinant vectors, each of which expresses reporter protein of different kind, we attempted double labeling of a neuron. it was expected that we could detect and visualize the divergence or convergence of a neurocircuit. in the study, we could demonstrate the efficiency of double labeling in vivo. in the present study, we examined the potential of this technique particularly in terms of the quantitative detection of double labeled neurons in complicated neurocircuit. the experiments were performed in the hippocampus and the neighboring cortices of rats. we could show that this technique is also useful for the quantitative analysis of neurons which forms projections to different region of the brain. shuchen lee, lihao ge institute of neurobiology, institute of brain science, fudan university, shanghai, china glycine receptors on bullfrog retinal cone photoreceptors were characterized by immunocytochemical and whole-cell patch clamp techniques. cone terminals were both gly␣ and gly immunoreactive. in freshly dissociated cones, an inward current could be induced while glycine was focally applied to the terminal. the glycine-induced current was strychnine-sensitive and reversed in polarity at a membrane potential, close to the equilibrium potential of chloride ions. these results suggest that glycine, which may be released by glycinergic inplexiform cells, could modulate functions of cone photoreceptors. ␦-catenin has armadillo motifs and a carboxyl terminal type i pdz ligand. in neurons, ␦-catenin is enriched in the postsynaptic density, where it serves as a link between the adherens junction and the post-synaptic protein complex including the nmda and ampa receptors. electrophysiological recordings from ca hippocampal neurons overexpressing ␦-catenin demonstrated that ␦catenin increased the ampa receptor-mediated epsc but had no significant effect on the nmda receptor-medicated epsc. the effect of ␦-catenin on the ampar-epsc was medicated by its pdz ligand. in cos cells, co-transfection of ␦-catenin/grip showed that ␦-catenin regulated the membrane localization of grip through its pdz ligand. co-transfection of ␦-catenin/grip/gulr increased the surface expression of glur in cos cells compared with grip/glur or ␦-catenin/glur transfection. this study points to ␦-catenin as a regulator of glur receptor trafficking. inseon song, kunihiko obata, alexey semyanov bsi, riken, japan gaba a receptor mediated tonic conductance is a major component of membrane conductance which determines the way how neuron integrates incoming synaptic signals as well as input-output characteristics of the cell. we measured density of picrotoxin (gaba a receptor antagonist) sensitive holding current (which reflects gaba a receptor mediated conductance) in interneurons of hippocampal ca area in wild type (wt) and gad knockout (ko) mice. this parameter was twice lower in gad ko mice (wt: . ± . pa/pf, n = ; gad ko: . ± . pa/pf, n = ; p = . ). the total membrane conductance was similar in both types of animals suggesting adaptive compensation. application of gaba ( m) increased tonic current in both type of mice by the same amount. no significant difference in amplitude or frequency of spontaneous ipscs was detected, although their decay time was shorter in gad ko animals (wt: . ± . ms, n = ; gad ko: . ± . ms, n = ; p = . ). the changes in inhibition which we have found may explain previously reported behavioral abnormalities in gad ko. research funds: bsi, riken hiroki mutoh, thomas knopfel lab. for neuronal circuit dynamics, bsi, riken, wako, japan olfactory glomeruli constitute the first stage of central odor processing. yet, their role in integration of odor information is only partially understood. we previously discovered that hz olfactory nerve (on) stimulation induces long-term depression (ltd) in young (p to p ) mice. the present experiments were designed to understand in more detail the molecular mechanisms underlying on ltd. bath application of dhpg, a selective group i mglur agonist, induced on ltd and occluded subsequent hz stimulation-induced ltd. on ltd was not induced by activation of group ii or iii mglur agonists. the dhpg-induced on ltd was mediated by mglur but not by mglur because it was antagonized by the mglur antagonist ly but not by the mglur antagonist mpep. expression of dhpg-induced on ltd was accompanied by an increase in paired-pulse ratio suggesting that on ltd is caused by a decrease of release probability. we propose that mglur is expressed at the on. on ltd may be important for establishment and maintenance of odor maps in the olfactory bulb but may also involve in the regulation of the sensitivity for specific odorants. ps p-a involvement of dopamine system in long-term potentiation of thalamo-prefrontal cortex pathway masatoshi takita , michiko ohtomi cognition and action group, national institute of advanced industrial science and technology (aist), ibaraki, japan; department of biomolecular science, faculty of science, toho university, chiba, japan a mesocortical dopaminergic (da) input to prefrontal cortex (pfc) with the d receptor is necessary for long-term potentiation (ltp) to occur at hippocampal-pfc synapses, which is involved by working memory (wm) in rats. here the da system was investigated in another wm-involved pathway from mediodorsal nucleus of the thalamus (md) to pfc. preliminarily, local perfusion of the d antagonist sch into pfc by using a microdialysis method impaired md-pfc ltp but the d antagonist sulpiride did not. extracellular da levels in the pfc robustly increased after the tetanus of md (by - %). as a result both excitatory synaptic inputs to the pfc involved the wm-related da profile, implying da system enables a contrast-emphasis for cooperative crosstalk among several neuroplasticities in the pfc to selectively store intersectional information of multiple brain areas. neuronal activity is necessary for postnatal maturation of synaptic connections only grossly laid out in the neonatal brain. in sensory cortices, synaptic maturation involves strengthening of sensory-evoked responses and development of receptive field (rf) maps with defined rf size and shape. evoked activity is thought to shape synaptic maturation in sensory cortices by mechanisms of competitive hebbian plasticity. dendritic excitability, mediated by voltage-gated na + channels, is required for active backpropagation of axosomatic action potentials (aps) and initiation of dendritic spikes; backpropagating aps and dendritic spikes enable forms of synaptic hebbian plasticity, such as spike-timing dependent plasticity (stdp). here we examined the role of dendritic excitability in synaptic maturation of layer / pyramidal neurons in the rat somatosensory barrel cortex. in the present study we compared ltp induction in neocortex of captreated and normal rats in present of gaba antagonist, picrotoxin (ptx). the result of present experiment showed that ptx plays an important facilitatory role in the induction of ltp in both normal and cap-treated group. in cap-treated group, in present of ptx, the ltp responses significantly were higher than normal group. we conclude that the enhancement of ltp by ptx can be explained by product of competition between excitatory and inhibitory pathways or synapses. these results suggest that gabaergic system has an important role in synaptic plasticity. also, these results indicated that gabaergic inhibition has been increased in cap-treated group. tohru kurotani, komatsu yukio department of visual neuroscience, research institute of environmental medicine, nagoya university, nagoya - , japan we showed in previous study that somatic inhibitory synapses of neocortical layer pyramidal neurons undergo long-lasting depression and potentiation depending on the intrinsic firing pattern of the cell that mimics slow wave sleep (sws) and arousal states. in the present study, using a minimal stimulation method, we recorded somatic ipscs from layer pyramidal cells in visual cortical slices prepared from rats at sws like state under urethane anesthesia and in those prepared from rats at arousal state. the average amplitude of somatic ipscs recorded in slices from the former group was significantly larger than that recorded in slices from the latter group. the mean rise time, decay time constant of ipscs and the mean input resistance of the cells were not significantly different between these two groups. the present results further confirmed that the somatic inhibition in neocortical layer pyramidal neurons is bidirectionally modified in accordance with behavioral state. corticothalamic fibers (ct), originated from cerebral layer pyramidal cells, make excitatory synapses with both thalamic relay neurons and reticular neurons. since these pyramidal cells abundantly express kainate receptors (kars) mrna, we studied the effect of kainate on the presynaptic function of the two ct synapses in mouse thalamic vb nucleus. bath application of kainate ( nm) depressed ct-epscs and increased the paired pulse ratio in relay neurons. in contrast, kainate at the same concentration facilitated ct-epscs and decreased the paired pulse ratio in reticular neurons. these results suggested that kars differentially regulated release at the two ct synapses. furthermore, high frequency stimulation of ct depressed relay cell synapses but facilitated reticular cell synapses. blocking endogenous kars abolished these effects. because reticular cells are the main source of inhibitory input to relay neurons, we suggested that endogenous kars presynaptically regulate the balance of excitatory and inhibitory inputs to thalamic relay neurons. to examine the involvement of ntr in the regulatory mechanisms for ltp in the amygdala, we utilized ntr -knockout (ko) mice. we performed whole-cell patch-clamp recordings from the pyramidal neurons in the basolateral amygdala (bla), where da-nt neurons project. we found that the bla-ltp, induced by la stimulation, was significantly greater in ntr -ko mice than in wild-type mice. the bla-ltp in ntr -ko mice was attenuated by sulpiride, a d receptor antagonist. these results suggest that d -ntr interaction regulates the extent of ltp in the mouse la-bla synapses. ps p-a facilitation of axonal plasticity in recovery from traumatic brain injury and the role of tnf␣ in mouse model recent studies suggest axonal plasticity as possible mechanism of recovery from brain injury. apart from that, tnf␣, an inflammatory cytokine, has also been suggested to serve neuroprotective roles. the present study evaluated motor function recovery after controlled cortical impact (cci) brain injury, and also the facilitation of plasticity by biotin dextran amine (bda) axonal tracing in tnf␣ko mice and wild type (wt) mice. mice were subjected to left sided cci or served as sham controls, and were evaluated by composite neuroscore and rotarod over -day period. bda was injected in right cerebral cortex to observe new axonal connections. so far, we observed recovery of motor function in wt mice, whereas tnf␣ko mice showed continuous functional deficit. we also observed greater number of new axonal connections in wt mice. our results suggest that tnf␣ is necessary for functional recovery after brain injury, and axonal plasticity may be the mechanism involved. disuse of synaptic activity causes homeostatic adaptation presynaptically and/or postsynaptically. here we show that in hippocampal autaptic cultured neurons tetrodotoxin-induced chronic inactivity increases the fraction of high vesicular release probability pool with the entire readily releasable vesicle pool size remained intact. kinetics of short-term plasticity and unchanged apparent ca + sensitivity indicate that ttx-induced presynaptic modification is unlikely due to an increase in the fusion rate crucial for the ca + at the final fusion step. in addition, analysis of neurons genetically lacked the synaptic vesicle protein synaptotagmin- , and timing-dependent rescues using two different viruses provide a novel conception, namely, vesicle machinery requires prolonged period so that the fast burst vesicle pool orchestrates presynaptic homeostasis system underlying "vesicle mobilization". ps p-a inhibitory modulation of the hippocampal ca transmission and plasticity by glucagon-like peptide- jun-ichiro oka, takashi iwai lab. pharmacol., fac. pharm. sci., tokyo univ. sci., japan glucagon-like peptode- (glp- ) is a proglucagon-derived peptidehormone in the intestine and brain. we reported that glp- (i.c.v.) improved the concussive brain injury-or scopolamine-induced amnesia in mice. however, the mechanisms of glp- effects on hippocampal neurons are unclear. in this study, we investigated the effects of glp- on the synaptic function of neurons in the acute hippocampal slices. hippocampal slices ( m) were prepared from to days wistar rats of both sexes. patch-clamp recordings were made from pyramidal cells of the ca in the whole-cell mode using glass microelectrodes (resistance: - m ). in extracellular recordings, field excitatory postsynaptic potentials (fepsp) were evoked with a bipolar tungsten electrode, placed in the mossy fibers. glp- ( nm- m) inhibited spontaneous excitatory postsynaptic current. glp- ( nm) did not affect fepsp amplitude or the paired-pulse ratio, but attenuated the long-term potentiation. these results suggest that glp- may play an inhibitory role in the dg-ca transmission. ps p-b quantitative imaging of exo-endocytosis at mossy fiber presynaptic terminals of hippocampus by genetically expressed fluorescent probe takuya hkima, rikita araki, toru ishizuka, hiromu yawo dept. of dev. biol. and neurosci., tohoku univ. grad. sch. of life sci., japan both presynaptic and postsynaptic mechanisms are proposed for the synaptic plasticity. however, the presynaptic mechanisms have been analyzed indirectly on the postsynaptic responses. it has been difficult to quantify the exocytosis at the presynaptic terminals, particularly those in vivo or in acute slices. to measure exocytosis directly, we applied the synaptophluorin (sph) method to the individual presynaptic terminals in hippocampal slices of a mouse genetically expressing a conjugate protein of vamp- and superecliptic phluorin selectively in the mossy fiber terminals. the sph fluorescence at individual mossy fiber terminal was increased by nerve stimulation and was followed by its reduction which is blocked by bafilomycin a , a vesicular h+-atpase inhibitor. therefore, the rising phase of sph fluorescence corresponds to exocytosis whereas the decreasing phase to endocytosis and subsequent re-acidification of vesicles. this method would enable us to evaluate the presynaptic contribution to synaptic plasticity. jyoti parkash, gurcharan kaur gndu amritsar, india we have earlier reported that gnrh nerve terminals in the me continue to express high levels of polysialylated form of neural cell adhesion molecule (psa-ncam) in a cyclic fashion and psa-ncam covers both the gnrh axon surfaces and the associated glial cells in the proestrous phase rats indicating that psa plays important role in the neurosecretory activity in hypothalamus. to further establish the functional significance of psa-ncam molecule, we have studied the expression of psa-ncam on gnrh axon terminals and glial cells in the proestrous phase of cycling rats as well as gaba and pbz treated proestrous rats by using dual immunohistofluorescent staining. both gnrh and psa-ncam immunostaining was much higher in proestrous phase rats, whereas, gaba and pbz treatments significantly reduced their expression. the expression of pst has been studied within gnrh cell bodies as well as at their terminals by combining in situ hybridization with immunohistofluorescent in poa and me-arc regions of cycling female rats as well as in gaba and pbz treated proestrous rats. cortical plasticity has important roles in the development of neural circuits in sensory cortices. however, the roles and mechanisms for various types of ltp and ltd are not clear. we investigated supragranular ltp and two types of supragranular ltd in the slices obtained from the rat auditory cortex, and compared their properties. frontal cortical slices were prepared from male wister rats. supragranular field potentials elicited by the stimulation applied to layer vi were recorded. ltp was induced by tetanic stimulation (ts, hz for s) applied to layer vi. ltd was induced by low-frequency stimulation (lfs, hz for s) applied to layer vi. ltd was also induced by ts applied to supragranular layers near the recording site. lfs-induced ltd and ts-induced ltd were completely abolished in the presence of m apv, m bicuculline, but not m mcpg. lfs-induced ltd and ts-induced ltd occluded each other, suggesting that that both types of ltd share cellular and molecular mechanisms. kazuyoshi kawa department of neurophysiology, tohoku university, graduate school of medicine, sendai, japan using slice-patch techniques, synaptic transmission in neurons of the area postrema (ap) of the rat was studied. when mm kcl was applied from a "y tube" to ap neurons (whole-cell clamped at − mv), massive inhibitory postsynaptic currents (ipscs) were induced. most of the evoked ipscs were blocked by bicuculline confirming gabaergic identity. when nicotine ( - m) or capsaicin ( . - m) was applied to ap neurons, robust appearance of ipscs with gabaergic identity was induced. after blocking action potential generation in the slice with tetrodotoxin ( m), nicotine and capsaicin could still induce gabaergic ipscs. interestingly, responses to capsaicin of the synaptic facilitation showed marked desensitization even after min of rigorous washout. it is concluded that nicotinic receptors, as well as capsaicin receptors (presumably, trpv ), are expressed at gabaergic presynaptic terminals in area postrema neurons and play a distinctive role in controlling autonomic neural functions. research funds: grant from the smoking research foundation (japan) takako morimoto-tanifuji , akira komatu , akinao nose dept. phys., univ. tokyo, tokyo, japan; dept. physiol., sch. med., tokyo women's med. univ., tokyo, japan the molecular mechanisms that target neurotransmitter receptors to the postsynaptic membrane and keep them clustered remain unknown. we investigated how the localization of glutamate receptors (glurs) is regulated in neuromuscular junctions (nmjs) of drosophila rd instar larvae. there are mainly two classes of glurs, containing either gluriia or iib. gluriia has a sequence predicted as ca + -permeable site. when camkii was inhibited by the expression of inhibitory peptide, ala, the content of gluriia in synapses was dramatically increased and the mean amplitude of extrajunctional potential (ejp) was enhanced. the expression of constitutively active form of camkii (t d) resulted in decreased gluriia content and enhanced gluriib content. although miniature ejp amplitude was reduced, ejp amplitude was normal in t d expressing larvae, suggesting the existence of some homeostatic mechanisms. taken together, camkii regulates the localization of glurs in a subunitspecific manner and modulates synaptic function in nmjs. ) . notably, neuronal dnrs from dnr * flies did not show mg + blockade, and dnr * flies displayed significant impairment in transcription-dependent long-term memory (ltm) but not in transcription-independent acquisition and short-term memory. we identified salient increases in genes involved in l-ltp formation, e.g. homer, and activin, as well as the increase in genes involved in ltm, e.g. staufen, upon ltm formation. however, such increases were absent in dnr* flies. transcription for ltm is mediated, at least, by transcription factors such as creb, adf- , and notch. we examined how mg + blockade of dnr links to these transcription factors. research funds: kakenhi ps p-b response properties of wind-sensitive giant interneurons in the th-instar nymphs of the cricket tetsuya matsuura , masamichi kanou dept. of welfare eng., iwate univ., morioka, japan; dept. of biology, ehime univ., matsuyama, japan the response properties of four wind-sensitive giant interneurons (gis) - , - , - and - in the th-instar nymphs of the cricket gryllus bimaculatus were investigated. air current was presented to the animal from different directions in the horizontal plane. the intensity-response curves showed that the response magnitudes of gi - increased with stimulus velocity up to mm/s regardless of the stimulus direction. the response magnitudes of gi - reached a plateau at a stimulus velocity of mm/s in most stimulus directions. the response magnitudes of gis - and - increased with stimulus velocity up to mm/s regardless of the stimulus direction. the directional sensitivity curves revealed that the preferential directions of the gis in nymphs were the ipsilateral-side in gi - , the ipsilateralfront and contralateral-rear in gi - , the ipsilateral-rear in gi - and the ipsilateral-front in gi - , designated with respect to the side of the ventral nerve cord containing the axons, which were basically the same with those of adults. yasuyuki ishikawa, sadao shiosaka division of structural cellular biology, nara institute of science and technology, nara, japan long-term potentiation (ltp) is an enhancement of synaptic strength that may contribute to information storage in the mammalian brain. ltp expression can be regulated by previous synaptic activity, a process known as "metaplasticity." we report a novel form of cellwide metaplasticity in hippocampal area ca . serine protease, neuropsin, is involved in the regulation of synaptic plasticity. neuropsin increased the stability of ltp induced later at the same inputs via l-type vdcc. moreover, neuropsin-deficient mice impaired l-ltp induction by l-tbs. our findings have revealed the effects of neuropsin on the conversion of e-ltp to l-ltp. ptp, a form of presynaptic short-term plasticity, is mediated by a transient increase in transmitter release probability caused by tetanic stimulation. although it has been known that ptp is induced by the elevation of presynaptic ca + , the molecular mechanism of ptp is poorly understood. in order to elucidate the specific role of presynaptic trkb receptors in ptp, we analyzed ptp using hippocampal slices from conditionally gene-targeted mice in which the knockout of the trkb gene is restricted to presynaptic sites in the ca region. we found that ptp induced by the -hz tetanus was reduced in mutant mice, and that ptp in control mice was partially reduced by an n-type ca + channel blocker, while ptp in mutant mice was unaltered by the blocker. thus, these data suggest that the n-type ca + channel-dependent component of ptp requires trkb receptor activation. research funds: jsps and mext of japan kiyoshi ohnuma , kunihiko kaneko , , makoto asashima , grad. sch. of arts & sci., univ. of tokyo, tokyo, japan; jst, tokyo, japan measuring fluctuations or population distributions of a system can be used to understand the dynamics of the system. we have used this approach to study intercellular interaction between neuronal cells. here we show that the shape of the population distribution of intracellular ca + concentration ([ca + ] i ) may change because of nonsynaptic communication. we loaded pc cells with a ca + indicator, indo- am, and the [ca + ] i of more than , cells was measured using flowcytometry. the [ca + ] i distribution of unstimulated single cells had a long right tail, suggesting that [ca + ] i is usually low but sometimes becomes high. on the other hand, the distributions of cell clumps and depolarized single cells were bell shaped, suggesting that many ca + -related mechanisms such as channels and pumps were activated by nonsynaptic communication or by depolarization to change the shape into a normal distribution according to the central limit theorem. our results suggest that measuring the distributions is useful in researching intercellular interaction. na x is a sodium channel involved in sensing the sodium level of the body fluid. our recent studies showed that na x is specifically localized to perineuronal lamellate processes of specialized glial cells in the circumventricular organs, the cns organs involved in the sodium reception. however, molecular and cellular mechanisms underlying the sodium reception of the glial cells has not been elucidated. to address this issue, we developed a functional expression system of the channel protein in cultured glial cells, and found that na x enhances glucose uptake and lactate release in an extracellular sodium-dependent manner. these results suggest that na x alters the state of energy metabolism of the glial cells by sensing a physiological increase of the sodium level. the state of inexcitable glial cells thus play a key role for the control of excitable neural cells in the circumventricular organs. we have isolated spinesin/tmprss from human and mouse cns. in mouse cns, four isoforms (types - ) were expressed. subcellular localization analysis revealed that type (full length) spinesin was predominantly localized to the er, golgi apparatus and plasma membrane, whereas type variant was localized to the cytoplasm. furthermore, we performed expression analysis of m-spinesin in some cell lines. nsc and nb a derived from neuronal cell express only type , whereas os and kt- derived from astrocyte express both type and type . interestingly, it was observed that the level of spinesin mrna was increased by a dibutyryl cyclic amp treatment only in os and kt- . we analyzed promoter region of m-spinesin gene, and identified that -flanking region from − to − bp was essential for m-spinesin gene expression. however, this region did not involve camp-dependent regulation of m-spinesin expression. these results indicate that cell-specific expression and regulation of spinesin gene may play multifunctional roles in cns. it has recently been elucidated that l-serine (l-ser) is one of the glia-derived neurotrophic factors in the brain and its biosynthetic enzyme -phosphoglycerate dehydrogenase (phgdh), which is the first committed enzyme of l-ser biosynthesis in the phosphorylation pathway, is selectively expressed in glial cells, but not in neurons. since l-ser seems to be important in retinal functions as well, we investigated in the present study the cellular distribution of phgdh in the mouse retina. phgdh immunoreactivity was detected in müller cell soma in internal nuclear layer, being close to internal plexiform layer. immunopositive profiles were cellular processes surrounding rod spherules and retinal neurons in internal nuclear layer through nerve fiber layer. it was suggested that müller cells contribute in l-ser synthesis and its transportation to neurons in the retina. astrocytes frequently show spontaneous intracellular ca + signals, such as intra-and intercellular ca + waves; however, their physiological roles remain elusive. the overexpression of an ip -hydrolyzing enzyme, ip -phosphatase, suppressed the spontaneous ca + signals in rat hippocampal astrocytes in culture without noticeable effects on their viability. hippocampal neurons were cultured on a monolayer of astrocytes, and their neurite outgrowth was analyzed. the total neurite length and the number of proximal dendrites and branches decreased significantly when neurons were cultured on the monolayer of ca + -signal-deficient astrocytes. moreover, time-lapse imaging revealed that the extension speed of growing neurites was markedly reduced on ca + -signal-deficient astrocytes. these results indicate that spontaneous ca + signals in astrocytes are essential for glial cells to promote neurite outgrowth. katsuyasu sakurai , noriko osumi , tohoku univ. sch. med., japan; crest, jst, japan astrocytes are the most numerous cells in mammalian brain tissues, although factors regulating their structure and function are still poorly understood. we have previously reported that pax transcription factor is expressed in gfap positive cells in the rat hippocampus. in the present study, we first investigated the expression patterns of pax in postnatal mouse brain and found that pax was expressed in almost all astrocytes in the cerebral cortex. to address the role of pax in the astrocytes, we examined the morphology of the astrocytes in the wild type (wt) and pax heterozygote mutant (sey/+) mice at weeks. confocal imaging revealed that arborization and extension of the astrocytes were poor in sey/+ mice as compared with the wt. in primary culture, the astrocytes isolated from sey/sey cortex showed no morphological difference. however, and h after dibutyryl-camp treatment, the majority of the wt astrocytes had undergone the conversion from a polygonal to stellate shape, while sey/sey astrocytes rarely showed this response. these results suggest that pax regulates the morphology of astrocytes, thereby being involved in astroglial functions. we raised mouse monoclonal antibody (mab) dim to study its distribution and function in cell membrane and found not only its preferential reaction with ptdglc on tlc, but also its labeling in rodent cns (yamazaki et al., ) . we previously reported a unique expression of dim ag in developing mouse brain, especially in cell membranes of embryonic radial glia (kinoshita et al., ) . we show here that mab dim also recognizes adult neural stem cells and glial cells at postnatal period. dim , brdu and gfap co-expressed in cells of mouse neurogenic subventricular zone. we discuss a possibility that the dim ag may be expressed in the radial glia/astrocyte lineage cells. the bone morphogenetic protein (bmp) receptors are thought to have a role in neural patterning of early neuronal development. the bmp receptor is widely expressed throughout the central nervous system (cns) including cerebellum. however, the physiological roles of bmp signaling in mature brain remains obscure. to understand bmp function in cns, we generated a transgenic mouse line that conditionally overexpresses bmp signaling through the type i receptor alk (alternatively known as avcri) in a purkinje cell-specific manner using a cre-loxp system. we bred this mouse line with the cre transgenic mouse line of which expression was driven by l promoter. tissue specificity of cre recombination was monitored by a bicistronically expressed egfp following a constitutively active alk cdna. increased bmp signaling was confirmed by ectopic phosphorylation of smad / / (p-smads) in purkinje cells. we will discuss functional changes of the purkinje cells which receive excess amount of bmp signaling through alk . lipopolysaccharide component of the cell wall of certain bacteria is pyrogenic whose administration to spinal cord injured animals was found to inhibit glial scar formation. glial scar being considered as an impediment for axonal growth, it had been proposed in s and s that sub-febrile doses of pyrogen could be considered for spinal cord injury repair research. we tested this ignored hypothesis in paraplegic bonnet monkeys and found that such sub-febrile doses of bacterial pyrogen derived from salmonella typhi was indeed effective in preventing the glial scar formation in short-term and at least prolong the formation of such scar in long term. therefore, pyrogen therapy may be considered as an adjunct to other strategies such as transplantation approaches to treat spinal cord injury. kavita seth, r.k. chaturvedi, s. shukla, a.k. agrawal dev. tox. div., industrial toxicology reserch center, lucknow, india crosstalk between neurons and glial cells (astrocyte and microglia) in neurodegenerative conditions such as parkinson's disease has gained attention of more than supportive interaction. here contribution of glial cells in -ohda induced degeneration of dopaminergic neurons was investigated. glial cultures showed significant loss in cell viability after h ( and %) and h ( and %) exposure to − and − m -ohda respectively. it was accompanied by morphological changes and induction of gfap, s- and ox . -ohda ( − m, h) was found to cause a significant impairment in h glutamic acid uptake ( %) and gsh levels ( %). further neurons (in coculture with -ohda pre exposed glial cells) on exposure to -ohda ( − m), showed loss of th expression and significant neuronal cell death ( %). the results of the present study suggest that -ohda may impair glial cell functioning, which eventually affect neuronal fate making them more vulnerable toward toxic insults. nestin is an embryonic intermediate filament component, which is transiently expressed by the immediate precursors to neurons and glia during brain development. we studied nestin distribution in the olfactory system after injection of diethyldithiocarbamate in adult rats to cause reversible lesion of the olfactory epithelium (oe). the oe presented a near-complete destruction at day after injection, then started to repair at days and returned to the normal levels at weeks. nestin was expressed in olfactory ensheathing cells (oecs) of the olfactory mucosa at ∼ days, but not in those of the olfactory bulb (ob). simultaneously strong expression of nestin was detected in certain population of astrocytes in glomeruli. the reversion of astrocytes in glomeruli to immature phenotype may reflect their involvement in reinnervation of glomeruli. (ng ) is currently considered a marker of multipotent progenitor cells in the brain. in the present study, most iba + cells accumulated in stab wounds and ischemic lesions were found to express ng , of which molecular weight of its core protein was higher by kda than that of ng expressed in contralateral brain region. this was due to the lack of shedding of ng in the brain lesions. we found that iba + cells accumulated in stab wounds and ischemic core lesion, most of which were ng +/pdgfra+. furthermore, some of these cells expressed gfap, nestin, cd and von willebrand factor. ng + mg isolated from stab wounds often formed cell aggregates bearing alkaline phosphatase activity turned into cells with neuroectodermal phenotypes in serumfree culture medium. these variety of antigens expressed by ng + mg in brain lesions may be related to their multipotentiality to regenerate damaged brain tissue. saroj sharma, l.k. singh, b. ray, t.s. roy all india institute of medical sciences, india oculomotor nerve (on) supplies most of the extra-and intraocular muscles. it shows changes with normal ageing, metabolic and degenerative diseases. though there are various studies on the on, no definitive data regarding the morphometry and the fine structure is available. so, in the present study, neural and the connective tissue organization of the extradural part of the on from cadavers were studied. light microscopy revealed multi-fascicular nerve with myelinated fibers of various calibers. small sized myelinated fibers were noted at the junction of the central and the paracentral zone of most of the nerves. using unbiased stereology techniques the size of myelinated fiber axonal areas showed a multi-modal distribution and presented range from < to m . most of the fibers were myelinated and counts produced a mean of , ( , - , ). ultrastructurally, difference in the compactness of arrangement of connective tissue was observed with advancing age. the cell junctions of the perineurial cells and the endoneurial capillaries were observed. myelin thickness ranged from . to . m (from fetal age to years age). during the development of the drosophila visual system, retinal axons project to the optic lobe through the optic stalk. the optic stalk is composed of glial cells and adopts tube-like structure. fak is a non-receptor protein tyrosine kinase involved in many aspects of cell behavior including cell migration through the regulation of actin or microtubule dynamics. in drosophila fak (dfak) mutant animals, the optic stalk was abnormally broadened and retinal axons were defasciculated. cdgapr encodes one of gaps that regulate rho-family gtpases. putative cdgapr mutants showed dfak-like phenotype. since dfak and cdgapr interacted genetically, they are likely to act in the same signaling pathway to regulate cytoskeletal rearrangement via rho-gtpases. tissue specific rescue experiment showed that dfak autonomously acts in the glial cells. our results suggest that dfak and cdgapr regulate glial cell rearrangement to establish precise tubelike structure of the optic stalk and organized retinal axon projection. astrocytes are thought to be active participants in synaptic plasticity in the developing nervous system. spontaneous gabaergic postsynaptic activity is reported to be decreased in small neurons of the caudal nts at the end of the first postnatal week. to investigate whether astrocytes might be involved in this phenomenon, we examined developmental expression of gfap, an astrocytic marker. gfap began to be immunohistochemically expressed in the caudal nts at p - . costaining with calbindin, a marker for a certain type of small neurons, showed that gfap positive processes were thereafter closely apposed to soma of small calbindin neurons. electron microscopy showed that some astrocytic processes were interposed between orphan gabaergic varicosities and soma of small neurons at the specific developmental stages. these findings indicate that astrocytes may participate actively in regulating the postnatal differentiation of local neural network of the caudal nts. hitoshi ozawa, naoyuki yamamoto, nobuhiko sawai, hao-gang xue department of anatomy and neurobiology, nippon medical school, tokyo, japan it is well known that the hypothalamo-pituitary-adrenal (hpa) axis is an important system for responding and mediating the stress. in addition, hippocampus is also an important area for the stress response. in the hippocampus, the expression of glucocorticoid receptor (gr) has been reported in the ca , ca pyramidal neurons and the dentate gyrus neurons. on the other hand, while astroglia around the hippocampus also expresses gr, the morphological and functional changes under different corticosteroid condition have not been well elucidated. in the present study, we investigated morphological changes of astroglia around pyramidal neurons. under the lack of corticosteroids, astroglia showed well developed morphology with the spread fibrous processes, however the changes recovered to the control level with corticosterone replacement. these suggested that the astroglia were directly regulated by glucocorticoids as associated with the changes of hippocampal neurons. ps p-d impact of s b on hippocampal spontaneous activities in anesthetized and epileptic conditions seiichi sakatani , akiko seto-ohshima , shigeyoshi itohara , hajime hirase neuronal circuit mechanisms research group, japan; lab. for behavioral genetics, bsi, riken, wako, japan s b is a calcium binding protein mainly expressed in astrocytes and has a role in synaptic plasticity and learning. in order to assess the physiological roles of s b, we have recorded hippocampal spontaneous activities from urethane anesthetized s b ko and wt mice. typical eeg patterns including theta ( - hz) and sharp wave associated fast ripple ( - hz) oscillations were observed in both populations and these patterns were indistinguishable between the wt and ko. when epileptic activity was induced by kainic acid (i.p.), a difference appeared in ca radiatum, where ictal event was characterized by hyper-synchronous gamma band ( - hz) activity. while both populations developed ictal event within min, mean power during the development was significantly smaller in ko mice. our results suggest that deficiency of s b does not have a profound impact on neural activity in normal conditions. however, when neural activity was raised, activation of s b related pathways could potentially be activated. yoshiko takagishi , erina okabe , xiaoyang sun , sen-ichi oda , yoshiharu murata riem, nagoya univ, nagoya, japan; grad sch bio-agricult sci, nagoya univ, nagoya, japan shambling (shm) is a spontaneous mouse mutation that causes neurological and motor deficits, characterized by ataxia and the hind limb paralysis. we have recently identified the shm gene that encodes caspr, which constitutes paranodal junction of myelinated nerves. to determine whether the mutation alters the node of ranvier, we performed morphological analysis of myelinated nerves in shambling mice. by electron microscopy, we found that paranodal loops were disorganized and septate-like transverse bands were absent in mutant mice. immunohistochemistry revealed that caspr was diffusely located at the paranodal region, though the staining was extremely weak in mutant sciatic nerves. contactin, a component of the paranodal junction, was distributed similar pattern to that of capsr. further, k + channels were mislocalized to the paranode, while na + channels were normally restricted to the node. these findings suggest that the mutation disrupts the paranodal structure and may disturb salutatory conduction of myelinated nerves in shambling mice. ps p-d regulation of hippocampal neurocircuit activity by glutamate transporter glt- noriko koganezawa, shinsuke muraoka, ken-ichiro tsutsui, toshio iijima div. systems neurosci. tohoku univ. grad. school of life science, japan glial cells are now recognized as an essential functional element in synapses. in order to investigate their function, we focused on the activity of glt- , the glutamate transporter which is expressed in the astrocytes of hippocampus, in the rat brain slice preparations. response to an electrical stimulation of the schaffer collaterals was recorded using the optical imaging technique. by combining the application of the glutamate receptor blockers (nbqx, ap ) and the glt- blocker (dhk) with the signal subtraction, we could visualize the activity of glt- as a slow, tonic rise of the optic signal following electrical stimulation. then we evaluated the function of glt- by applying its blocker dhk. an obvious reduction of neural activity was observed in the hippocampal neurocircuit after application of dhk. furthermore, the blocking of glt- function in the ca region was elicited by much lower concentration of dhk than that in the ca region. ps p-d monoclonal antibody rip specifically recognizes , -cyclic nucleotide -phosphodiesterase in oligodendrocytes the antigen recognized with monoclonal antibody (mab)-rip has been used as marker for oligodendrocytes and myelin sheaths. however, the rip-antigen has been unknown yet. to identify the rip-antigen, we performed immunopurification with mab-rip using the differentiated cg- cells lysate. maldi-qit/tof ms n analyses revealed that one of molecules was , -cyclic nucleotide phosphodiesterase (cnp). immunocytochemical and immunohistochemical studies showed that rip-antigen colocalized with cnp in rat cerebellum, cultured rat oligodendrocytes and cg- cells. moreover, the same localization was also observed in rat cnp transfected hek t cells. overall we first demonstrated that the antigen labeled with mab-rip is cnp in oligodendrocytes. the expression of bdnf gene is regulated by four promoters (pi-piv), and is under activity-dependent control. until now, it has been established that bdnf pi is activated by ca + signal via cre. on the other hand, neuron-restrictive silencer cis-element (nrse), located in bdnf pii, represses bdnf gene expression through binding nrsf and recruiting hdac in non-neural cells. here, we found that nrse repressed the activity of bdnf pi in neuron. using rt-pcr and chip assay, the bdnf exon i expression and the histone acetylation of bdnf pi were increased by the administration of ca + signals or hdac inhibitor. in addition, nrsf bound to bdnf pii in neurons but was detached by ca + signals. these results suggest that bdnf pi activity is regulated by creb and nrsf through an alteration of chromatin structure. since creb and nrsf are playing an important role in neuronal differentiation, it is considered that the bdnf pi is deeply involved in the regulation of neurogenesis. singo suzuki , , hisatsugu koshimizu , megumi kashihara , tomoko hara , masami kojima , research institute for cell engineering; sorst, jst, japan brain-derived neurotrophic factor (bdnf) plays a crucial role in synapse development, especially, in the central nervous system (cns) . although this concept is now accepted extensively, the underlying molecular mechanisms are poorly understood. here we show that -day treatment with bdnf leads to a significant increase in cholesterol content in primary neuron. this change was in its dose-dependent manner and blocked by co-application of a cholesterol synthesis inhibitors. to understand the molecular relationship between cholesterol content and synapse development, we estimated the amount of cholesterol and sv proteins in lipid raft fractions prepared from cultured cortical neurons. the results indicated that bdnf treatment increased the amount of cholesterol and sv proteins in lipid rafts, but not in non-rafts fraction. these data suggested a possibility that bdnf regulated synapse development by increasing the amount of cholesterol and sv proteins in synaptic rafts. (p ) is known to be expressed in the cells of the central nervous system, and supposed to be involved in the control of cell proliferation, differentiation and survival. in this study, we found that p expressed in the neural progenitor cells at embryonic days (e ) mice brain. to ascertain the function of p on these cells, we treated the cultured e cells with the selective chemical inhibitor for p (sb ) for days, and determined the number of neural progenitor cells. the inhibitor specifically enhanced the number of neural progenitors compared to the control cells. this result suggests the involvement of p in the proliferation and/or survival of neural progenitor cells in developing mouse brain. hemragul sabit , takashi yamazaki , , takeshi oya , yoko ishii , masakiyo sasahara pathology ii, university of toyama, toyama, japan; oral and maxillofacial surgery, university of toyama, toyama, japan purpose: we had reported the increase of pdgf-b and active src in rat peripheral nerve regeneration. here examined activation of pdgf receptors (pdgfrs) and signals in the peripheral nerve regeneration. method and result: crushed sciatic nerve was removed on to days after injury, and activation of pdgfrs, mapks, akt and p were examined by phosphoprotein purification kit (qiagen) and western blot. expression of pdgfrs increased from to days after injury. p-tyr was highly detected from to days after injury, and activation of pdgfrs also increased during this period. activation of erk and jnk increased up to days after injury and then gradually decreased. activation of akt and p continuously increased from to day after injury. conclusion: pdgfrs and their signals were activated in rat peripheral nerve regeneration. autocrine signal of pdgf may contribute to the regenerative processes, such as proliferation and differentiation of schwann cells and axonal extension. cbln is a cerebellum-specific protein structurally related to the c q and tumor necrosis factor families. recently, we have shown that cbln is secreted from cerebellar granule cells (gc) and controls synaptic structure and plasticity of gc-purkinje cell (pc) synapses. however, because cbln was previously shown to serve as a precursor of a pc-specific peptide cerebellin, it remains unclear whether cbln needs to be processed before it trans-synaptically activates signaling pathways in pc. here, we show that purified recombinant cbln proteins, which formed a hexamer, preferentially bound to spines on pc dendrites. furthermore, cbln mutants that did not form a hexamer lost the binding affinity to pc spines. although cerebellin peptide may also contribute to different aspects of signaling, these results indicate that cbln released from gc directly bind to postsynaptic pc as a hexamer and activates signaling pathways in pc. activity-dependent gene expression in neurons contributes to expressing a variety of neuronal functions including a long-lasting neuronal plasticity. recently, we found that specific kinds of mrna can be stabilized in an activity-dependent manner. to elucidate the mechanisms for activity-dependent mrna stabilization, we have focused on bdnf, which is a member of neurotrophin family and plays an important role in exerting neuronal functions. we constructed firefly luciferase gene fused to -untranslated region (utr) of bdnf mrna to investigate the effect of the utr on the calcium signal-mediated mrna stabilization. in cultured neurons, we found that the degradation of firefly luciferase-bdnf utr mrna induced by the treatment with actinomycin d was prevented by calcium signals evoked via l-type voltage-dependent calcium channels (l-vdccs) and nmda receptors. we are now investigating to identify the cis-regulatory elements involved in the calcium signal-mediated stabilization of bdnf mrna using a series of mutant bdnf utr. recently, it has been established that bdnf and pacap regulate the expression of a group of genes which encode proteins involved in expressing neuronal functions. in this study, we found that the treatment of cultured rat cortical neurons with bdnf or pacap acutely induced the mrna expression of the activityregulated cytoskeleton-associated protein (arc) and homer a, whose products are necessary for the synaptic plasticity. bdnf induced arc mrna expression through the activation of trkb-erk/mapk pathway, whereas pacap induced it partly through the activation of nmda-receptors. using affymetrix genechips, we are now investigating a comprehensive profile of gene expression controlled by bdnf or pacap in cultured rat cortical neurons. ps p-e bmp expression in the adult rat brain bone morphogenetic protein- (bmp ) is a member of the transforming growth factor  (tgf-) superfamily and plays important roles in multiple biological event. although bmp expression has been well described in the early development of central nervous system (cns), little information is available for its expression in the adult cns. we, thus, investigated bmp expression in the adult rat cns using immunohistochemistry. bmp is intensely expressed in most neurons and their dendrites. in addition, intense bmp expression was also observed in the neuropil of the gray matters where high plasticity is reported, such as the molecular layer of the cerebellum and the superficial layer of the superior colliculus. furthermore, we found that astrocytes also express bmp protein. these data indicate that bmp is more widely expressed throughout the adult cns than previously reported, and its continued abundant expression in the adult brain strongly supports the idea that bmp plays pivotal roles also in the adult brain. ps p-e hgf as a target-derived trophic factor for rat nigro-striatal dopaminergic (da) system during post natal development wakana ooya, hiroshi funakoshi, toshikazu nakamura div. molecular regenerative medicine, osaka univ. grad. sch. med., osaka, japan hgf is a novel neurotrophic factor in vitro on da neurons. however, little is known about expression and biological activities of hgf in nigral-striatal system in vivo. here we show that hgf is a targetderived trophic factor for rat da system. real-time rt-pcr revealed that c-met mrna was expressed in substantia nigra (sn) and striatum (str), while hgf mrna was expressed in str but not in sn in programmed cell death period. hgf, c-met, phospho-c-met, th, da transporter immunostaining revealed the presence of concentration gradient of hgf from sn to str and c-met was phosphorylated in da nerve end during early postnatal development. phospho-c-metpositive da neurons decreased at later developmental stage, while it became prominent in oligodendrocytic leanage. hgf application into str increased da neuronal number and neurites and modified oligodendrocyte maturation, while opposite effects were observed by the application of blocking antibody for hgf. therefore, hgf may be a critical trophic factor for nigro-striatal da system development. the neuroprotective effects of g-csf were reported in neurological disease models. in the present study, we examined whether g-csf can protect dopaminergic neurons from mptp-induced cell death in a pd. the mice were intraperitoneal injected with mptp for five consecutive days, g-csf is intraperitoneal administered two days and one day before first mptp injection, and min before each mptp injection. in our results, g-csf significantly prevented mptpinduced loss of th-positive neurons, and increased bcl- protein, decreased bax protein expression. these findings suggest that g-csf has therapeutic potentiality to protect mptp-induced cell death through increasing the level of bcl- expression, decreasing the level of bax expression in c bl/ mice. kazue takahata has been shown to increase the expression of brain-derived neurotrophic factor and glial cell line-derived neurotrophic factor, and the activity of superoxide dismutase . here, we evaluated the effects of (−)-bpap on the phosphorylation of mitogen-activated protein kinase (mapk) and akt in slice cultures as well as in an in vivo model. (−)-bpap significantly increased phosphorylation levels of mapk, but not those of akt. (−)-bpap attenuated the decrease in nigrostriatal tyrosine hydroxylase immunoreactivity of -methyl- -phenyl- , , , tetrahydropyridine-treated mice. (−)-bpap may exert antiparkinsonian activity through neuroprotective effects on dopaminergic cells in addition to catecholaminergic enhancement in parkinsonian substantia nigra. shyuichi maeda , yoko tohyama , shinichi kohsaka , tadashi kurihara , kazuyuki nakajima , soka university, hachioji, tokyo, japan; dept. of neurochem., national institute of neuroscience, kodaira, tokyo, japan astrocytes (ast) are a cell type that supports cns not only nutritionally but also neurotrophically, by supplying neurotrophic factors (ntf) required in the neuronal survival, maturation and protection. however, the ability of ast to produce/secrete ntf has not been accurately known. thus, in the present study, we investigate the capacity of ast to produce/secrete various ntf in vitro. ast were prepared from the mother culture of neonatal rat brain. the ntf in the conditioned medium (cm) were detected by immunoblotting. the analysis of neurotrophins in the cm revealed that ast produce/secrete ngf, bdnf and nt- , and promote the production of them by stimulation with lipopolysaccharide (lps). furthermore, tgf among tgf family was detected in the cm of ast, and the production was enhanced by stimulation with lps. these profiles of ast were different from those of microglia, suggesting the differential regulation of ntf by glial cells. ritsuko katoh-semba , chiaki nakagawa , masako tsuzuki , motoko matsuda , satoshi ichisaka , yoshio hata inst. dev. res., aichi human ser. ctr., kasugai, japan; div. neurobiol., tottori univ., sch. med., yonago, japan; div. integrative biosci., tottori univ. grad. sch. med., yonago, japan the sleep-awake rhythm is formed during the early period after birth. the rhythm is very important for the functional development of brain. when the rhythm formation is disturbed, autism-like behaviors are often observed. brain-derived neurotrophic factor (bdnf) is known to be one of the factors forming circadian rhythms. we have found increases in levels of bdnf in the entorhinal cortex as well as the visual cortex from adult male rats h after beginning an -h phase advance of the light-dark cycle. here we planned to reveal the effects of the phase advance on neurotrophins in juvenile rats. we first examined circadian changes in the concentrations of bdnf and neurotrophin- (nt- ) in selected brain regions from -day-old male rats and compared to those from adults. the changes in levels of bdnf and nt- were observed in the neocortex and hippocampus. objective: this study was aimed to investigate the possible beneficial effects of granulocyte colony stimulating factor (g-csf) compared to methylprednisolone (mp) in experimental spinal cord injury (sci). materials and methods: (in vivo) adult female sprague-dawley rats had moderate sci ( kdyne, ih injury device) at t / and were assigned to three groups; a (placebo), b (mp treated; mg/kg i.v. immediately after injury), c (g-csf treated; g/kg i.v. for days after injury). animals were assessed with the bbb locomotor rating scale for w post injury and then killed for assessment of tissue sparing around the lesion. result: the behavioural recovery rates of the group c was as good as group b and significantly better than that of group a. morphological assessment showed better tissue sparing in group b and c compared to group a. these results suggest that g-csf is a possible neuroprotective agent in sci. research funds: kakenhi ( ) ps p-e glutamate signals enhance the expression of rnase-l in primary cultured cortical neurons of mice chie sugiyama, kiyokazu ogita dept. pharmacol., setsunan univ., osaka, japan mitochondrial dysfunction results from a decline in the mitochondrial rna (mtrna) transcripts and mitochondrial enzyme activity, as well as from mitochondrial dna (mtdna) damage. to evaluate involvement of mtdna expression in glutamate-induced neuronal death, in this study, we examined the effects of glutamate exposure on mtrna level in cultured cortical neurons of mice. cultured neurons ( div) exposed to glutamate for min at m. glutamate exposure led to a decrease in mrna of nd and nd , which are subunits of nadhubiquinone oxidoreductase, before cell death. since mtrnas level is regulated at least in part by rna degradation mediated by rnase-l, we next examined the effect of glutamate on expression of rnase-l. rt-pcr analysis revealed that glutamate was effective in increasing the level of rnase-l mrna at least - h after treatment. the increase in the expression of rnase-l was abolished by the nmda receptor antagonist mk- . these results suggest that the activation of nmda receptor by glutamate reduces mtrna level probably through enhanced expression of rnase-l in cultured cortical neurons. yuka gotoh, kiyokazu ogita dept. pharmacol, setsunan univ, osaka, japan expression of dj- is enhanced by oxidative stresses. although exact functional significance of dj- has still unknown, it is thus proposed that dj- is protective against neural damage under oxidative stresses. in this study, we tested expression of dj- in the hippocampus damaged by trimethyltin (tmt) treatment in mice. tmt was systemically injected into mice to cause neural damage in the dentate gyrus selectively. immunohistochemical analysis indicated that dj- was markedly increased in the molecular layer of the dentate gyrus on days and post tmt injection. on day post tmt injection, enhanced expression of dj- was observed in the stratum lucidum of the ca . in glutathione-depleted mice, tmt was more effective in enhancing expression of dj- , compared with that in untreated mice. furthermore, double staining of dj- and gfap demonstrated that most of cells highly immunoreactive to dj- were co-localized with gfap in the dentate gyrus of tmt-treated animals, but not of untreated animals. these results suggest that dj- is enhanced in the dentate astrocytes activated by tmt treatment. kei higuchi, kiyokazu ogita dept. pharmacol, setsunan univ., osaka, japan the systemic administration of trimethyltin (tmt) is known to induce granule cell death in the dentate gyrus of mice. we have previously shown that an injection of tmt ( . mg/kg, i.p.) led to significantly reduction of granule cells in the dentate gyrus days later, with visually apparent recovery of the granule cell layer days afterward. in this study, we examined the effects of glucocorticoids on tmt-induced damage in the dentate granule neurons of mice. tmtinduced neuronal cell damage was assessed by the immunohistochemical analysis using an antibody against single-stranded dna. the systemic injection of dexamethasone ( . - mg/kg) led to a significant reduction in neuronal damage induced by tmt in the dentate gyrus. the neuronal damage induced by tmt at the dose of . mg/kg was enhanced by adrenalectomy. dexamethasone was effective in completely preventing this neuronal damage in adrenalectomized animals. taken together, these results suggest that glucocorticoid released from adrenal cortex may be capable of protection against tmt-induced dentate granule cell death in mice. masami ishido national institute for environmental studies, tsukuba, japan melatonin, a secretory product of the pineal gland, has antitumor activities and is involved in the regulation of circadian, seasonal rhythms and in inducing osteoblast differentiation. furthermore, melatonin is reported to be a scavenger of a number of reactive oxygen and reactive nitrogen species both in vitro and in vivo. in this chapter, antioxidant nature of melatonin was demonstrated to prevent the cultured neural cells from apoptosis induced by endocrine disrupting chemicals, maneb. neurotoxicity of maneb ( g/ml) on the pc cells was elicited through apoptotic cell death. activation of caspase- / was associated with this process. a fluorescence rationing technique using mitochondrial dye revealed that maneb altered mitochondrial membrane potential of the neural cells. however, melatonin ( nm) could largely prevent the neural cells from the neural toxicant by inhibition of both caspase- / activation and disruption of the mitochondrial transmembrane potential. thus, melatonin could be a powerful free radical scavenger against manebcaused mitochondrial dysfunction in pc cells. ps p-e kinesin superfamily protein (kif ) regulates activity-dependent neuronal survival by suppressing parp- enzymatic activity ryosuke midorikawa, yosuke takei, nobutaka hirokawa department of cell biology and anatomy, university of tokyo, tokyo, japan in brain development, apoptosis is a physiological process that controls the final numbers of neurons. here we report that the activitydependent prevention of apoptosis in juvenile neurons is regulated by kinesin superfamily protein (kif ), a microtubule-based molecular motor. the c-terminal domain of kif is a module that suppresses the activity of poly (adp-ribose) polymerase- (parp- ), a nuclear enzyme known to maintain cell homeostasis by repairing dna and serving as a transcriptional regulator. when neurons are stimulated by membrane depolarization, calcium signaling mediated by camkii induces dissociation of kif from parp- , resulting in upregulation of parp- activity, which supports neuron survival. after dissociation from parp- , kif enters into the cytoplasm from the nucleus, and moves to the distal part of neurites in a microtubule-dependent manner. we suggested that kif controls the activity-dependent survival of postmitotic neurons by regulating parp- activity in brain development. research funds: ps p-e expression of hsp , apg- , and apg- in the hippocampal neural cells by trimethyltin masanari orita, kiyokazu ogita dept. pharmacol, setsunan univ, osaka., japan we tested changes in expression of high-molecular-weight heat shock proteins (hsps) in the hippocampal dentate gyrus in vivo and in the cultured cortical neurons in vitro after trimethyltin (tmt) treatment, which caused neuronal damage in the dentate gyrus and cultured hippocampal neurons. tmt ( . mg/kg) was systemically injected into mice, and then an immunohistchemical analysis was performed to identify cells immunoreactive to antibodies against hsps, neun, and gfap in coronal sections of hippocampus. tmt was effective in enhancing the expression of hsp , apg- , and apg- in the granule cell layer of the dentate gyrus, but not in ca -ca pyramidal cell layer, h to days later. double staining of neun and these hsps revealed that these hsps expressed by tmt almost co-localized with neun in granule cells of the dentate gyrus. whereas hsp highly expressed in survival neurons in the culture, apg- and apg- highly expressed in damaged neurons with nuclear condensation. taken together, the high-molecular-weight hsps may be involved in neuronal survival and damage caused by tmt. brain irradiation is often performed in patients with brain tumors. however, little has been known about radiosensitivity of neurons, especially in the developmental stages. in this study, we investigated the effect of irradiation on immature neurons with that on mature neurons. primary neuronal cultures were prepared from fetal rat hippocampi at embryonic day . thirty gray of x-irradiations were performed on the cultured cells at or days in vitro (div). then the cells were fixed at h after the irradiation with dapi. at -div, irradiation significantly increased the number of nuclear pyknosis of neurons. in contrast, radiation did not induce any nuclear pyknosis of neurons at -div. this indicates that the radiosensitivity of -div immature neurons is higher than that of -div mature neurons. glutamate receptors are believed to be involved in various neurological disorders via its excitotoxicity. ataxic mice lurcher (lc) are caused by a mutation in the ␦ glutamate receptor (glur␦ ), which shows constitutive channel activities in purkinje cells and leads to the cell death. thus, lc is the first example of neurodegeneration caused by chronic excitotoxicty. interestingly, glur␦ is also suggested to regulate autophagy via its association with beclin. however, it is unclear how excitation caused by constitutive channel activities is related to the autophagic pathway and cell death. here, using heterologous cells in vitro, we show that continuous influx of na + , but not ca + , was necessary and sufficient to induce autophagic cell death. in addition, we found that intracellular atp levels and subsequent activation of map kinase are involved in this process. junko taniguchi a major pathological hallmark of the polyglutamine diseases is the formation of neuronal intranuclear inclusions (niis) of the disease proteins that are ubiquitinated and often associated with various transcription factors, chaperones and proteasome components. but, how the expanded polyglutamine proteins or their aggregates elicit a complex pathogenic responses in the neuronal cells are not fully understood. here, we demonstrate that the expression of expanded polyglutamine proteins down-regulates the nf-kb-dependent transcriptional activity. expression of expanded polyglutamine proteins increases the stability and the levels of ikb-a and its phosphorylated form. we have also found that various nf-kb subunits and ikb-a aberrantly interacts with the expanded polyglutamine proteins and associates with their aggregates. finally, we have shown several nf-kb-dependent genes are down-regulated in the expanded polyglutamine protein expressing cells. molecular mechanisms for selective neuronal death in polyglutamine diseases remain to be clarified. by microarray analysis, we compared gene expression profiles in cerebellar granular cells under expression of normal and mutant ataxin- and found a novel gene down-regulated in response to mutant ataxin- in cerebellar granular neurons. we named the novel gene maxcell (mutant ataxin-affected gene in the cerebellum). nothern blot shows that maxcell mrna in human brain is expressed in cerebellum and cerebral cortex. immunohistochemistry with anti-maxcell antibody shows cytoplasmic stains of neurons but not glial cells in mouse brain. confocal microscopy shows that maxell-egfp is colocalized with ribosomal protein s as a ribosomal marker. we are analyzing the function of maxcell protein that might relate to sca molecular pathology. ps p-f permeability transition in mitochondria isolated from cold perfused brain and spinal cord-a detailed comparison of calcium sensitivity the purpose of this study was to compare the sensitivity of isolated brain and spinal cord mitochondria to ca + -induced permeability transition (mpt). the spinal cord is more traumatized than the brain during the extraction because it takes more time. in order to minimize confounding factors, we induced severe hypothermia in animals prior to removal of tissue and isolation of mitochondria. sensitivity to ca + -induced mpt was evaluated in brain and spinal mitochondria in energized and de-energized model of swelling with or without mpt inhibitor, cyclosporin a (csa). the present findings imply that the general features of mpt are similar in brain and spinal cord mitochondria and that mpt may be an important pharmacological target in disorders affecting the spinal cord. the role of cyclophosphamide monohydrate (cp), which is known as an immunosuppression drug, in the central nervous system (cns) has not been elucidated. in the present study, we found that treatment with cp prevented the cultured cortical neurons from cell death induced by serum deprivation. furthermore, cp exposure induced the activation of both the map kinase (mapk) and pi kinase (pi k) pathways. interestingly the up-regulation of bcl- , a survival promoting molecule was observed after cp treatment. these observations suggest that cp protects cns neurons from neuronal damage through intracellular signaling pathways. the research of cell death mechanisms has rapidly progressed. however, cell death is an inherently difficult process to measure. to investigate the roles of cell death in vivo, we introduced scat probe. scat is an indicator protein for caspase- activation that uses fret between two types of fluorescent protein, ecfp and venus, linked by a peptide containing the caspase- cleavage sequence. using this probe, we could monitor the activation of caspase- at the singleneuron level in culture. we will discuss about neural cell death through the detection of caspase activity. keiichi seko, koichi kawada, chie sugiyama, masanori yoneyama, kiyokazu ogita dept. pharmacol., setsunan univ., osaka, japan trimethyltin chloride (tmt) is a kind of organotin derivates that are known to induce neuronal damage in human and rodent. in this study, we examined tmt-induced neuronal death in mouse primary cultured cortical neurons in vitro and the frontal cortex in vivo of mice. in vivo analysis using mice revealed that injection of tmt ( . mg/kg, i.p.) led to an increase in single-stranded dna-positive cells, as well as in dnase ii-positive cells, in the frontal cortex days later. in cortical neurons, tmt exposure for h led to a marked decrease in the cell viability, as well as to an increase in nuclear condensation and ldh released. tmt exposure was effective in activating dnase ii in the nucleus. in addition, caspases and , but not caspase , were significantly activated by tmt treatment. cytochrome c release was not affected by tmt treatment. the caspase inhibitor zvad-fmk completely prevented tmt-induced neuronal death. these results suggest that tmt-induced neuronal death is involved in caspases and dnase ii activated by mitochondria-independent pathway in cortical neurons. we investigated the pattern of hippocampal damage and the levels of brain polyamines after systemic injections of trimethyltin (tmt) chloride ( . mg/kg, i.p.) in -( w) and -week-old ( w) icr mice. in addition, we measured the brain tin level following tmt injection. tmt induced marked, localized cell death in granule neurons of the dentate gyrus in w mice. by contrast, slight, diffuse neuronal damage was found in the ca and ca subfields and dentate gyrus of w mice. the hippocampal putrescine level was elevated markedly in w mice on tmt administration, whereas a minor putrescine increase was detected in w mice. there was no difference in the brain tin level between these two age groups. these results revealed the age-dependent vulnerability of mice hippocampal neurons to tmt administration, and suggest that massive activation of polyamine metabolism is associated with tmt-induced neurodegeneration. withdrawn ps p-f establishment of memory guided actions of taking food with tweezers in monkeys naoki hirai , toshinori hongo , kimisato naito , shigeto sasaki department of physiology, kyorin university, school of medicine, tokyo, japan; tokyo metropolitan institute for neuroscience, tokyo, japan monkeys learned a task of taking food with tweezers (twz) under the visual guidance. the task consisted of sequential actions of looking at twz, grasping it by hand simultaneously shifting gaze to food, bringing twz to food, and picking it up with twz. brief interruption of vision for . - . s during any actions by a liquid crystal shutter disrupted the ongoing actions, indicating that each action needed visual information as guidance. this contrasted with the task of taking directly with hand, which was done without vision. with repeated practice, they developed a mode of using more memory and somatosensory cue as guidance. they directed their gaze to invisible food in advance, and when vision of . s became available, they grasped twz, brought the twz to memorized location of food and grasped the food without vision. these results show that they acquired food taking actions using twz based on memory and somatosensory cues, the latter allowing monkeys to use twz as an extension of the hand. masahiko nishimura, yoshihiko yoshii university of ryukyus, okinawa, japan we have experienced that the patients with arm impairments by brain disorder were difficult to manipulate the tools with the paralyzed arm, and healthy arm. lt. parietal lobe and ifg are commonly recognized tools-semantic neuro-system. however, nobody knows a neural network contributed to suitability for a purpose of toolsmanipulation. we examined an fmri to evaluate the brain activation of tools-manipulation in volunteers. experiment was performed by three tasks, control task is a forearm rotation, task is simulation of tools-manipulation, task is execution of tools-manipulation. we found different brain regions by this experiment. task to investigate the role of synchronous firing in the prefrontal cortex (pfc), we performed cross-corelational analysis of the pfc neurons, while monkeys performed a path-planning task, which required multiple steps of actions to reach goals. first, we analyzed synchrony among pfc neurons during the execution period in comparison with that during the preparatory period. we found that neuronal synchrony was enhanced transiently for each step of movement during the execution period. next, we examined relationship between neuronal synchrony and task-related activities. we found that the relationship between neuronal synchrony and response selectivity of pfc neurons was more distinct during the preparatory period than during the execution period. we would discuss dynamical roles in neuronal synchrony in planning multiple steps of actions. the functional significance of primate medial prefrontal cortex in the selection of action has been unclear. we studied neuronal activity in this region while monkeys were performing a variant of conflict solving task in which visual cues instructed them to push either the left or right. the location of the cue was either compatible (congruent) or incompatible (incongruent) with the target's location. we found a focus of reaching-related neurons in the medial prefrontal cortex rostral to the pre-sma. the activity of neurons in this newly identified area was dependent on conflict. intracortical microstimulation in this area did not evoke eye movements, distinguishing this area from the sef. we found that the local field potential in this area, but not in other areas, differed when congruent and incongruent trials were intermixed, and when only the congruent trials were presented repeatedly, suggesting the involvement of this area in the selection of actions is dependent on the task demand. masaki maruyama, peter fenwick, andreas a. ioannides laboratory for human brain dynamics, riken-brain science institute, saitama, japan we used infrared corneal reflection, sampling at khz, to record simultaneously and independently the • horizontal saccades of each eye for subjects. two paradigms were used, in go-only sessions saccade direction with the cue to move, and in go/no go sessions, saccade execution, direction and move. mutual information (mi) analysis showed the two eyes were most consistently yoked for position than for velocity, but both provided adequate signals. mi showed coupling between the start and end of saccades and the importance of velocity signals in their ballistic nature. surprisingly leftward movement latency was longer to peak-velocity and showed more complex mi interactions. comparing go-only to go/no go saccades, significant differences were longer onset latencies and a higher eye velocity before the end of saccades. a recent meg study using this protocol, found just before and during the saccade the additional go/no go difficulty led to more interaction between left and right brainstem and cerebellum. these could be related to eye velocity changes with the higher cognitive loading. wriggle mouse sagami (wms) has been presented as a mouse model for dystonia, as it is characterized by postural and motor impairments, such as sever tremor, sustained muscle contractions of the limbs, and wriggling of the neck and trunk without coordination. by extracellular unit recordings under awake conditions, we analyzed neuronal activity in the basal ganglia and the cerebellum of this mutant mouse. in the basal ganglia (globus pallidus, entopeduncular nucleus, substantia nigra pars reticulata), neither rates nor patterns of spike discharges were significantly different as compared to normal mice. on the other hand, the discharge rate of cerebellar purkinje cells in wms was markedly decreased. these results suggest that the decreased activity of purkinje cells may be responsible for movement disorders in wms. hiromi hirata division of biological science, nagoya university, nagoya, japan wild-type zebrafish respond to mechanosensory stimulation with multiple fast alternating trunk contractions at day, whereas bandoneon (beo) mutants contract trunk muscles on both sides simultaneously. muscle voltage recordings confirmed that muscles on both sides of the trunk in beo are likely to receive simultaneous synaptic input from the cns. recordings from motor neurons revealed that glycinergic synaptic transmission was missing in beo mutants. furthermore, immunostaining with glyr antibody failed to show clusters in beo neurons. these data suggest that clustering defect of glyrs at synapse causes the impairment of glycinergic transmission and abnormal behavior in beo. indeed, mutations in the glycine receptor beta subunit were identified in beo. this is the first direct demonstration that glyr is essential for physiologically relevant clustering of glyrs in vivo. since glycine receptor mutations in humans lead to hyperekplexia, a motor disorder characterized by startle responses, zebrafish bandoneon mutant should be a useful animal model for this condition. medium-sized spiny projection neurons in the striatum receive inputs from gabaergic and cholinergic interneurons as well as from extrinsic sources, including the cerebral cortex. in the present study, the effect of gabaergic modulation on striatal projection neuron activity was investigated by infusion of the gaba a receptor blocker gabazine in the vicinity of the recorded neurons in monkeys who performed a memory-guided reaching task. the gabazine infusion enhanced the activity of striatal projection neurons in response to both cortical stimulation and task events, while the neuronal activity specific to the task events was decreased. these results suggest that local gabaergic input may play an important role in fine tuning of striatal projection neuron activity. research funds: kakenhi ( ) ps p-f dependence of synchrony in the subthalamic network on temporal characteristics of afferent inputs katsunori kitano, fumito kosuga department of human and computer intelligence, ritsumeikan university, japan the subthalamic nucleus (stn) and the external segment of global pallidus (gpe) constitute the indirect pathway of the basal ganglia and highly modulate the basal ganglia functions. the evidence that the emergence of synchronized oscillatory activity in the network of the two nuclei is relevant to movement disorders such as parkinson's disease shows temporal structures of the neuronal firings play an important role for the functions. among possible underlying mechanisms for the abnormal activity, the characteristic membrane properties of stn neurons is likely to be one of the most crucial origins. to clarify the detailed mechanism, we focus on and investigate the dynamical properties of the neuron theoretically and numerically with the model neuron. we apply the phase reduction method to the dynamics of the neuron to analyze the stability of synchronous activity. in particular, how the stability depends on temporal characteristics of afferent inputs to the neurons as well as the intrinsic membrane properties are investigated. during phasic voluntary movement, electromagnetic oscillatory activities of ∼ hz around the central sulcus show decrement and increment (erd/ers, respectively), that are assumed to reflect the cortical activation and inhibitory/recovery process respectively. we investigated the correlation between personality and these oscillatory changes. from healthy subjects, high and low scorers (n = each) of novelty seeking dimension on the psychometry were selected. magnetic fields were recorded while they performed selfpaced movements of their right index fingers, and frequency analysis was carried through the beta band ( - hz). high ns group showed less amount of erd in the left hemisphere, smaller magnitude, larger latency of ers in the right hemisphere and smaller amount of baseline activity in both hemispheres than low ns group. it was suggested that individuals with high ns trait may have less inhibition after the movement and higher readiness during resting state. despite the emerging methodology of combined fmri and tms, the quantitative relationship between tms intensity and bold signals is poorly understood. eight healthy subjects were scanned on a -t scanner, with an mri-compatible figure-of-eight tms coil attached for eliciting right hand movement. bold measurement was performed with the stepping stone sequence (tr = . s) with online monitoring of meps. the intensity of tms pulses was varied from % to % at a % step (frequency at ∼ . hz). bold signal changes were assessed in the primary motor cortex. a sharp increase in bold signals was observed above % stimulation. bold signals were weakly but significantly correlated with tms intensity adjusted by the resting motor threshold (r = . , p = . ). this finding gives a theoretical background for the application of fmri with tms to cognitive brain regions. ps p-f shift of activation areas induced by hand movement during recovery from post-stroke hemiparesis: an nirs study kotaro takeda , , yukihiro gomi , itsuki imai , nobuaki shimoda , , hiroyuki kato , international university of health and welfare, ohtawara, japan; crest, jst, kawaguchi, japan; nasu neurosurgical center, nasushiobara, japan we investigated the cerebral hemoglobin (hb) changes in hemiparetic stroke patients under voluntary hand grasping task from acute to chronic phases by using near infrared spectroscopy (nirs). fortyfour channels ( channels on each side) were placed on the scalp overlying both sensorimotor cortices, and the cerebral hb changes were observed during four to six cycles of s task and s resting periods while sitting on a chair. the amounts of oxy-hb change were significantly increased in the bilateral sensorimotor areas during hemiparetic hand grasping at the acute phase, though the significant increase was mainly observed in the contralateral sensorimotor area during hemiparetic hand grasping at the chronic phase and during normal hand grasping at all phases. this result suggests that the functional recovery from post-stroke hemiparesis may be attributed to neuronal reorganization of sensorimotor areas via recruiting ipsilateral cortex. research funds: crest, jst todd pataky , , rieko osu , hiroshi imamizu , mitsuo kawato , computational brain project, icorp, jst, japan; atr cns laboratories, japan movement direction encoding in primate single cortical cells has been widely documented. this study was designed to test whether this directional tuning is observable at the voxel level in human fmri. three subjects performed hz isometric force pulses to seven targets separated by • in the shoulder/elbow flexion-extension plane. while in mri, online force feedback was provided by a d strain gauge. twenty repetitions of each condition were performed in s blocks (tr = s). all subjects showed broad activation over the contralateral motor area, and from functional rois an average of voxel time series were extracted. many voxels exhibited continuous cosine-like tuning with movement direction. decoding using linear svm revealed that while correct classification rate was only . % (chance: . %), errors were distributed normally about the target such that . % (chance: . %) of the data was classified correctly to within • . these data demonstrate that non-invasive neuroimaging is sufficiently sensitive to study the problem of coordinate system representation. the behavioral thermoregulation is important for living in various temperature environments. however, the neural mechanism of behavioral thermoregulation is poorly understood. in this study, we aimed to establish a new model to analyze the neural mechanism of behavioral thermoregulation using zebrafish. we investigated whether zebrafish perform behavioral thermoregulation against heating. when water temperature was changed from • c, fish showed repetition of short time swimming in the range . - • c. the frequency of the heat induced escape behavior was increased with temperature dependant. these results suggest that the heat induced escape behavior is a part of behavioral thermoregulation. the heat induced escape behavior was observed stably in - days past fertilization, indicating that the neural mechanism which control behavioral thermoregulation is matured in days. in conclusion, we established an effective new model to analyze behavioral thermoregulation. ps p-f to learn with one limb or two: limited transfer between unimanual and bimanual skills recent studies on neural activity in primary motor cortex of nonhuman primates suggest that unimanual and bimanual movements are controlled by partially overlapping neural processes. here we demonstrate that unimanual and bimanual motor learning also reflect a partially overlapping process. first, motor adaptations to reach with a novel force field applied to a limb could not be fully transferred to the same limb across unimanual to bimanual conditions, and vice versa. second, learning acquired during unimanual reaching could not be fully eliminated by repeated bimanual reaching with no loads, and vice versa. rather, some learning remained intact (but invisible) until the original context was again performed. lastly, two conflicting force fields can be learned simultaneously if they are separately associated with unimanual and bimanual reaching. these results support the view of partially overlapping neuronal processes and illustrate the intimate relationship between neural control and motor learning. research funds: jsps and nserc rieko osu , ken-ich morishige , jun nakanishi , , hiroyuki miyamoto , mitsuo kawato atr computational neuroscience labs, kyoto, japan; kyushu institute of technology, japan; jst-icorp, japan human can execute multiple motor tasks by using the same limbs, which makes human different from industrial robots. recently the optimal feedback control hypothesis has given a significant impact on the motor control community because it produces an optimal behavior for a given task by avoiding offline computation of optimal desired trajectory that would result in suboptimal behavior in the presence of noise. it, however, requires considerable amount of resources and learning to realize multiple tasks on nonlinear system. on the other hand, a desired trajectory enables the brain to share resources with multiple tasks and save learning time by dividing a difficult problem into easier sub-problems of plan and execution. considering the modularity of the brain and viability for nonlinear system, the hierarchical implementation is a better solution for global optimality as a versatile creature. here, we experimentally demonstrate that the hand variance modulation during multiple via-point tasks supports the existence of a desired trajectory. the purpose of this study is to computationally predict arm-reaching movements and posture controls from neuronal activity of premotor (pm) and primary motor area (mi). the activity was collected with single-unit recording method during the animal performing a visually guided arm-reaching task. electromyograms (emgs) and kinematics were also measured. we reconstructed the emgs from the neuronal data using a linear regression model, and then we estimated the kinematics from the reconstructed emgs with an artificial neural network model and proportional derivative controller. as a result, these serial processes allowed us to accurately predict the kinematics during both moving and maintaining her posture from the activity. the advantage of our bmi system is to estimate not only the kinematics but also the muscle tension from the neuronal activity. we have recently reported essential role of the tongue in breastfeeding in the hypoglossal (xii) nerve-injured newborn rats. of particular interest were the findings that the rates of the amounts of milk intake in the unilateral xii nerve-injured p pups of the surviving cases increased greatly between p ( % of the control value) and p ( %), suggesting adaptive tongue movement during development. this study was undertaken to reveal underlying basic mechanisms for such adaptation focusing on neural plasticity allowing effective suckling. after resection of the ipsilateral xii nerve on p , dii, a postmortem neuronal tracer, was applied to the contralateral uninjured xii nerve of p and p pups. dii-labeled neuronal fibers were successfully traced within the tongue and were found to extend over the xii nerve-injured side with gradual increase from p to p . we show evidence for functional neural plasticity that allows effective suckling in the xii nerve-injured newborns with suckling disturbance. previously we reported that decorticated rats showed abnormal righting movements in the air when dropped from the supine position, while the air righting reflex (arr) could be evoked purposefully ( • turn around the body axis) in decerebrated ones. thus, the basal ganglia might send interference signals to the arr center via the midbrain tegmentum. to clarify its functional roles in arr control, we examined arr movements in rats with the midbrain lesioned. wistar, male rats were prepared; after the posterior cerebral cortex was removed by sucking, the superior colliculus and surrounding structures were ablated in various degrees. arr movements were examined post-operative , , and days. in rats with the superior colliculus lesioned extensively on both sides, arr onset were delayed and body turn around the longitudinal axis was weakened, so that either insufficient or no rotation occurred in the air. furthermore, coordination between the body and tail rotations was lost in many cases. the ablated region may relay cortical signals that give a top priority to the arr center. ps p-g role of plateau potentials in feeding system of aplysia kurodai aiko kinugawa , tatsumi nagahama dept. of life sci., grad. sch. of sci. & technol., kobe univ., kobe, japan; fac. phar. sci., toho univ., funabashi, japan rhythmic motor activities seen in the animal behaviors can be generated by specific neural circuits termed the central pattern generator (cpg). in the feeding system of aplysia kurodai, the le neuron we identified produces the long-lasting plateau potentials and may be a cpg element. during the feeding-like responses duration of the depolarization of the follower neurons was shortened by hyperpolarization of the le. in this study we found that the le plateau potentials had refractory periods and they were turned to activation periods by application of large depolarizing currents. and various depolarizing pulses tended to produce the stable plateau potentials with almost constant depolarizing size and duration, suggesting that the le can supply the constant long-lasting depolarizing outputs to the follower neurons even when it receives various length and intensity of excitatory inputs from the presynaptic neurons. the le may be an important cpg element to determine the size and duration of the basic depolarization of many buccal neurons. withdrawn ps p-g neural organizations for vocal control in a social rodent, the deguneural organizations for vocal control in a social rodent, the degu naoko tokimoto , sayaka hihara , kazuo okanoya , atsushi iriki lab for symbolic cognitive development, bsi, riken, japan; lab for biolinguisutics, bsi, riken, japan vocalizations of most animals are innate, the region for the direct control of such sound is known to be localized in the pag. on the other hand, a few animals with the cortico-medullary projection path can learn a new sound. in this research, we investigated about vocal control in pag of social rodent, the degu (octodon degu). it is known that degus have fifteen kinds of vocal repertoires, and that their courtship song has a complex structure. we verified the hypothesis by electrical stimulation of the pag that the neural mechanism of degus that enables complex vocalization differs from that of guinea pigs with simple vocalization. guinea pig is near relation with degu. as a result, in guinea pigs, each sound is controlled in different area of the pag. in degus, however, multiple sounds are controlled by the same area, and the different sound was occasionally evoked by the different kind of stimulation. the sound with the time-series specific to the spontaneous vocalization of degu was not emitted. the effects of a heat-and steam-generating sheet (hsg sheet) on autonomic nerve activity and bowel movement were examined in women suffering irregular defecation, the hsg sheet was applied to the lumbar or abdominal regions, causing the temperature between the sheet and skin to increase to about . • c. application of the hsg sheet to either the lumbar or abdominal region significantly increased the rate of miosis in the pupillary light reflex. as for changes in r-r, the hf increased after application, suggesting that the parasympathetic nerve system had become dominant. bowel movement assessed by electrogastrography increased in amplitude. based on the above findings, we concluded that the application of an hsg sheet to the lumbar or abdominal region may lead to dominant parasympathetic nerve activity and improve gastrointestinal motility. ps p-g prostaglandin e -induced thermogenesis involves a gaba-receptive mechanism in the preoptic area toshimasa osaka national institute of health and nutrition, - - toyama, shinjuku, - , japan unilateral microinjection of pge into the region around the rostroventral wall of the third ventricle (av v) elicited thermogenic, tachycardic, vasoconstrictive, and hyperthermic responses simultaneously in urethane-chloralose anesthetized rats. the magnitude of these responses increased dose-dependently in a range of - pg, except for the vasoconstrictive response. next, the effects of pretreatment with a gaba a receptor antagonist, bicuculline methiodide ( . mm, nl), microinjected into the preoptic area (poa) ipsilateral or contralateral to the pge injection site was examined. this treatment alone had no effect on the o consumption rate and temperatures of colon and skin but elicited a bradycardic response. however, all pge -induced responses were blocked min after the pretreatment with bicuculline, and recovered at ∼ min. pretreatment with vehicle saline had no effect on the pge -induced responses. these results suggest that the gaba-receptive mechanism in the poa is required for the pge -induced thermogenesis. tetsufumi ito , hiroyuki hioki , kouichi nakamura , takeshi kaneko , yoshiaki nojyo dept. anat., univ. of fukui, fukui, japan; dept. of morphological brain sci., kyoto univ., kyoto, japan although gaba-immunoreactive (ir) fibers in the rat superior cervical ganglion (scg) were thought to originate in small cells located in the cervical sympathetic trunk (cst), almost all gaba-ir axon terminals showed markers for sympathetic preganglionic neurons (spns) in our recent report. in this study, we performed series of experiments to confirm the origin of gaba-containing fibers. gad -ir fibers were not found in dorsal roots (drs), but in ventral roots (vrs), stellate ganglion, cst, and scg. gad -positive somata were not found in dr ganglia and cst, but in intermediolateral (iml) nucleus of thoracic spinal cord (tsc). after intraperitoneal injection of fluorogold (fg), to label the entire spns, some fg-ir neurons were also positive for gad . we injected sindbis virus, an anterograde tracer, in iml, and some labeled terminals in scg showed gad -ir. after cutting t -t vrs and drs, almost all gad -ir fibers were abolished in scg. these results indicate that gaba-containing fibers in scg originate from spns in iml of tsc. masato nagahama , ning ma , reiji semba , satoru naruse dept. of anat. ii, mie univ. school of med., tsu, japan; inst. for developmental research, kasugai, japan; dept. of int. med., nagoya univ. graduate school of med., nagoya, japan aquaporin (aqp ) is first found as a water-transporting protein and has been demonstrated in various organs and tissues. in the present study, we have demonstrated the presence of aqp immunoreactivity in a particular neuronal subtype in the enteric nervous system (ens) of the rat ileum. aqp -immunoreactive (ir) neurons simultaneously expressed a neuronal marker huc/d. moderate numbers of aqp -ir neuronal somata were found in the myenteric plexus, and a very few were found in the submucosal plexus. aqp -ir neurons can be classified as dogiel type i cells, which have several short processes and a single long process. many aqp -ir fibers were found both in the myenteric and submucosal plexi. many aqp -ir varicose fibers were closely associated with neuronal somata in the ganglia, whereas other aqp -ir fibers penetrated into the muscle layers. these results suggest that aqp -ir neurons probably play a significant role within the ens to control gut functions. research funds: kakenhi ( ) ps p-g abdominal expiratory nerve activity in the decerebrate neonatal rat center for medical sciences, ibaraki prefectural university of health sciences, ibaraki, japan the abdominal expiratory activity was recorded from the iliohypogastric nerve in the decerebrate, vagotomized, paralyzed, ventilated neonatal rat at postnatal days - . the increase in the volume and frequency setting of the artificial ventilator (fio = %, fico = %) failed to make the rat apnea. under this condition, the phrenic nerve showed unstable rhythmic inspiratory bursts, and the tail pinch increased the respiratory frequency. although the iliohypogastric nerve showed expiratory discharges, their amplitudes and shapes were not consistent. when fico was increased, the cycle period was prolonged and the abdominal expiratory activity was enhanced. in many rats, the iliohypogastric nerve showed biphasic discharges that consisted from the pre-and post-inspiratory discharges. the preinspiratory discharge has larger amplitude and shorter duration than the post-inspiratory discharge. since the post-inspiratory discharge was usually small or indistinguishable in the adult rat, the present results suggest that the pattern of abdominal expiratory activity will change during the postnatal development. we investigated the role of gabaergic neurons in the rostral ventrolateral medulla (rvm) in central respiratory control. we used gad -gfp knock-in mice in which we could identify gabaergic (i.e., gfp-positive) neurons in a living condition. we recorded gabaergic neuron activities (n = ) in medullary transverse slices. about % of gabaergic neurons were inspiratory, and all of the remaining neurons were non-respiratory. about % of gabaergic neurons recorded in the superficial rvm were co inhibitory, and all of the remaining neurons were co insensitive. we suggest that gabaergic inhibition in the rvm respiratory neuron network is mediated mainly by inspiratory neurons. gabaergic neurons are also involved in central chemosensitivity. we investigated two groups of people with a different initial level of an emotional tension before intellectual loading (il). first group had the initially increased emotional level, stressed group (sg), and the second group had not, calm group (cg). reaction time (rt) of simple visual sensorymotor reaction and asymmetry of skin potential level (spla) in two facial zones: forehead and nasal were measured. from research groups, subgroups with and mv spla were extracted. thus the distinction in the greater rt gain after il in subgroups with mv forehead spla, in comparison to subgroups mv forehead spla. such law was common for both for sg and for cg. in two subgroups of and mv nasal zone spla in sg the greater rt gain after il was in mv nasal spla subgroup, and for cg in mv subgroup. it was shown, that individual features of il performance are related to spl lateralization. but the low of the relationships of spl asymmetry in nasal zones depends on the level of emotional tension of investigated groups. mitsuko kanamaru, ikuo homma department of physiology, showa university school of medicine, tokyo, japan we have reported that serotonin ( ht) in the dorsomedial medulla oblongata in mice increases tidal volume and minute ventilation via ht receptors. peripheral administration of p-chlorophenylalanine (pcpa) reduces the whole brain ht level. the present study examined whether peripheral pcpa pretreatment affects hypercapnic ventilatory responses in mice. adult male mice (c bl/ n) were pretreated with pcpa ( . g/ ml/kg body weight) or saline intraperitoneally for consecutive days. on the next day, each mouse was placed into a double chamber plethysmograph to obtain respiratory flow curves. one hundred percent o inhalation was changed to stepwise , and % co in o inhalation every min. hypercapniainduced increases in tidal volume and minute ventilation during % co inhalation were reduced by pcpa pretreatment; these results suggest that ht may increase tidal volume in hypercapnic ventilation. saori nishijima, kimio sugaya, minoru miyazato division of urology, department of organ-oriented medicine, faculty of medicine, university of the ryukyus, okinawa, japan we investigated the effect of gosha-jinki-gan on bladder activity and the autonomic nervous system in rats. forty-two female rats were divided into a control diet group and a . % gosha-jinki-gan diet group. after weeks, continuous cystometry with physiological saline or . % acetic acid solution and biochemical analysis were done. the amplitude of bladder contraction with physiological saline was lower in the gosha-jinki-gan diet group than in the control diet group, and plasma dopamine and serotonin levels were also lower in the gosha-jinki-gan diet group. when cystometry was done with . % acetic acid, the interval between bladder contractions was shortened in the both groups. however, the interval and duration of bladder contractions were longer in the gosha-jinki-gan diet group than in the control diet group. therefore, it is suggested that gosha-jinki-gan inhibits bladder activity by maintaining the balance of the sympathetic nervous system and the parasympathetic nervous system at a low level. satoko suzuki, shinya yanagita, seiichiro amemiya, ichiro kita graduate school of science, tokyo metropolitan university, japan we examined the effects of negative air ions (nai) on physiological responses and neuronal activity with c-fos immunohistochemistry. in addition, we investigated the effect of vagotomy to reveal afferent pathways of nai stimulation. we analyzed neuronal activity of the paraventricular nucleus of hypothalamus (pvn), the locus ceruleus (lc), the nucleus ambiggus (na), and the nucleus of solitary tract (nts). nai significantly decreased blood pressure, heart rate, and respiratory rate, and increased hf component which is an index of parasympathetic nervous activity. nai decreased c-fos expression in the pvn and lc, and enhanced in na significantly. after vagotomy, the physiological responses and changes of c-fos expression in pvn, lc, and na was disappeared. furthermore, increase of c-fos expression in nts induced by nai was also disappeared. these results suggest that effect of nai on sympathetic and parasympathetic nervous activity was induced by reducing the activity of the pvn and lc, whereas enhancing the na activity, and that these effects of nai was caused through vagus nerve. yusuf o. cakmak, umit suleyman sehirli school of medicine, university of marmara, turkey previous assessments of the autonomic nerve supply of testis from vagus and brainstem nuclei were conflicting in the literature. we challenged this consensus by using neuronal tracer fluorogold in rats. fluorogold dye solutions was injected unilaterally under the capsule of rat testis. rats were sacrificed by transcardiac perfusion-fixation in the fifth and seventh days after injection. brainstem of the control group, subdiaphragmatic vagotomy group and main group rats were dissected. in the main group the fluoroscent-labelling were dense in area postrema. dorsal vagal nucleus, nucleus of solitary tract and nucleus ambiguous were also labelled. these preliminary data provide an evidence of testicular innervation by vagus nerve. taking into account that brainstem structures could be labelled from the testis, it can be assumed that the areas detected might be involved in the neural control of testicular functions. the results of this study cautioned that innervation of the testis may not be fully explained by innervation from pelvic and paraaortic ganglia. research funds: scientific researches comittee of medical school of marmara university ps p-g differential control of renal and lumbar sympathetic nerve activity during freezing behaviour in conscious rats yoshimi tahara, misa yoshimoto, keiko nagata, kenju miki integrative physiol. grad. sch. humanities and sci. nara-women's univ., nara, japan the present study was designed to examine sympathetic and hemodynamic responses to loud noise exposure, which induced freezing behaviour, in chronically instrumented rats. wistar male rats were instrumented with electrodes for measurements of renal (rsna) and lumbar (lsna) sympathetic nerve activity and catheters for measurements of systemic arterial and central venous pressure. rats were exposed to db white-noise for min. db noise exposure resulted in an immediate and significant increase in rsna while lsna did not change significantly during the exposure in sham-operated (so) rats. there was a significant difference in the response between rsna and lsna during the db noise exposure in so rats. sinoaortic denervation attenuated the magnitude of the increase in rsna while it had no influence on the changes in lsna observed in so rats. these data suggest that arterial baroreceptor significantly contribute to the differential control of rsna and lsna during freezing behaviour in conscious rats. here, we first demonstrated that in both the kolliker-fuse nucleus (kf) and the rostral ventral respiratory group (rvrg) region, phrenic nucleus (phn)-projecting neurons were embedded in the plexus of axons originating from the ventrolateral subnucleus of the nucleus of the solitary tract (vlnst) and that the vlnst axon terminals made synaptic contacts with somata and dendrites of the phnprojecting neurons, using a combined anterograde and retrograde tracing technique. secondly, we indicated that some of the vlnst neurons innervate both the kf and the rvrg by way of axon collaterals, using the double-labeling method. using retrograde tracing combined with in situ hybridization for mrna encoding glutamic acid decarboxylase (gad ), we finally showed that most of the kf/rvrg-projecting vlnst neurons expressed gad mrna. these results suggest that vlnst neurons may exert inhibitory influences upon the phn-projecting kf/rvrg neurons for inspiratory control. we have examined whether the neurons of the dmv have direct synaptic contacts on the myenteric ganglia using wga-hrp. the myenteric ganglia of the stomach were composed of four types of neurons. the average numbers of axosomatic terminals per profile were . on the small neurons, . on the medium-sized neurons, . on the large neurons, and . on the elongated neuron. most of the terminals contained round vesicles and formed asymmetric synaptic contacts on the small, medium-sized and large neurons. about % of the axosomatic terminals on the elongated neurons contained pleomorphic vesicles and formed asymmetric synaptic contacts. when wga-hrp was injected into the dmv, many anterogradely labeled terminals were found around the myenteric neurons. the labeled terminals were large ( . m), and contacted exclusively the somata. most of them contained round vesicles and formed asymmetric synaptic contacts. serial ultrathin sections revealed that almost all neurons in a ganglion received projections from the dmv. ps p-h neuronal mechanisms of respiratory rhythm modulation induced by external k + concentration change in the newborn rat brainstem-spinal cord preparation hiroshi onimaru, ikuo homma dept. physiol., showa univ. school of med., tokyo, japan it has been suggested that two distinct rhythm generators (pfrg-pre-i and pre-bötzinger insp) for respiration in the medulla possess different sensitivity to various neuromodulators. we hypothesize that the dominancy of these rhythm generators to determine basic respiratory rhythm depends on the back ground stimulation level. to verify this hypothesis, we studied neuronal mechanisms of respiratory rhythm modulation induced by external [k + ] change. we recorded membrane potential of pre-i neurons, c nerve and facial nerve activities. addition of mm k + to the standard superfusate decreased burst rate of c activity. addition of or mm k + caused initial inhibition of c burst and subsequent high frequency c burst. the facial nerve burst was depressed. pre-i neuron was depolarized strongly by application of high k + , and the burst activity was disturbed and action potentials were inactivated. results suggest that pfrg-pre-i or pre-bötzinger insp rhythm generator is dominant in low or high back ground stimulation level, respectively. research funds: kakenhi ( ) ps p-h regulation of synaptic transmission in the reticular formation of medulla oblongata by substance p we have examined the response of neurons in the reticular formation near the nucleus ambiguous (na) to the administration of substance p (sp). whole-cell recording was applied to the postsynaptic neurons in coronal slice preparations of medulla oblongata isolated from infant rat. bath application of sp ( m) increased or decreased the frequency of spontaneous activities. several neurons were clamped at − mv and recorded epscs evoked by electrical stimulation to dorsoventral adjacent area from recording neurons. in several neurons, evoked inward epscs were augmented by sp perfusion. i-v curve suggested that voltage dependent current was both augmented and not changed by sp. our previous studies have shown that administration of neurokinin receptor (nk r) antagonist near the na inhibited gastric and respiratory movement in anesthetized rat. these results indicated that sp affect to both post and presynaptic nk r and regulate the transmittance efficiency to generate the output signal of certain autonomic reactions. ps p-h effects of local warming in the back or abdominal region by means of a heat-and steam-generating sheets on physiological response in the low temperature room to investigate effects of local warming of the body on physiological functions as well as subjective feeling, eegs, ecg, respirometer, bis (bispectrum) index, blood pressure (bp), and local skin temperature of the body were monitored while a steaming heat pack was put on the lower lumber or abdominal region of the subjects for h in the cold room. in the control experiment without the heat pack, lf/hf of hr variability (lf/hf-hr) and lf of bp variability (lf-bp) increased, while hf of hr variability (hf-hr) and skin temperature decreased, suggesting elevation of sympathetic nervous activity. in the warming experiment with the heat pack, an increase in lf/hf-hr and/or lf-bp was suppressed and hf-hr increased. we will discuss these autonomic data in relation to subjective unpleasant or pleasant feeling, eeg and bis data. junichi arai, yasuhisa endo, ryouichi yoshimura, huan wang kyoto institute of technology, japan in the ventromedial hypothalamic-lesioned animals, the abnormal cell proliferation in liver and pancreas are thought to be due to the vagus hyperactivity and/or the sympathetic repression. we conducted the co-culture system of several cell lines and demonstrated that the proliferation of hepatocytes and min- cells (a cell line of pancreatic b cells) were stimulated by the administration of carbachol, when they were co-cultured with cell lines of endothelial cells or smooth muscle cells. these effects were also found in the filter-insert coculture system, but never seen in the culture using single cell line. we discuss the possible mechanism of their intracellular signal transduction. research funds: kakenhi to study the correlation between the trans-cranial oxy-and deoxyhemoglobin (hb) dynamics and sbp, we measured hb dynamics (f-nirs ® , omm- , shimadzu corp. japan) over the frontal area and sbp (finapres ® , bp monitor, ohmeda , usa) at the right middle finger from volunteers ( . ± . years). mild thermal stimuli ( ± • or ± • ) were administered every min alternatively to the left hand. some area showed positive correlation between the oxy-hb and sbp, the other showed negative correlation between them. hb dynamics over the frontal area have any correlation to sbp to some extent. so, trans-cranial nirs should be discussed carefully for neural activation. we thank shimadzu corp. for the use of omm- . we examined the effects of color environments on cognitive function in healthy subjects and patients after traumatic head injury using p components and loreta analyses. the examination was performed in color environments of red, green, or black using visual oddball tasks with photographs of a crying baby face as the target stimuli. the p latency in the red environment was significantly shorter in controls than in patients. the p amplitude in the red environment was significantly larger in controls than in patients. loreta analysis demonstrated that the neurological activities in the occipital lobes, left tonsillar nucleus, anterior cingulated gyrus, and brodmann area in the red environment were significantly higher in controls than in patients. hironori nakatani, cees van leeuwen riken brain science institute, saitama, japan some figures, such as rubin's vase/face and the necker cube, have two or more distinct interpretations and are, therefore, called 'ambiguous'. when an ambiguous figure is presented continuously for a period of time, we experience spontaneous switching between the alternative interpretations. as this occurs without any changes in the figures themselves, perceptual switching phenomena are eminently suitable to study how perceptual processes are influenced by the intrinsic dynamics of neural activity. we analyzed eye-movement and eeg during perceptual switching in the necker cube. blink probability showed a peak about ms before the button press responses. we found that only blinks that appeared around the peak time led to a characteristic spatiotemporal pattern of eeg. our results indicate that some, but not all, blinks play an active role in perceptual switching processes. ps p-h neural basis of social cognition investigated by functional near infrared spectroscopy and electroencephalograms recorded from the whole brain tsuneyuki kobayashi , , mikinobu takeuchi , , takahiro omote , naoyuki yosimura , etsuro hori , , kazuo sasaki , taketoshi ono , , hisao nishijo , system emotional science, univ. toyama, toyama, japan; crest, japan science and technology agency, japan; bio-information engineering, univ. toyama, toyama, japan; molcul. & integ. emotional neurosci., univ. toyama, toyama, japan neural basis of social cognition was investigated by functional near infrared spectroscopy (fnirs) and electroencephalograms (eegs). a head cap for recording fnirs and eegs was set on heads of subjects. the probes of the fnirs imaging systems ( channels) and/or electrodes of the eeg system were attached on the heads of the subjects. the subjects were required to perform social cognition tasks to discriminate ( ) human facial stimuli with different gaze directions and ( ) simple animation videos representing social interaction. whole brain hemodynamic images were superimposed on the d reconstructed mri images of the brains. now we are analyzing hemodynamic images and eeg data related to social cognition, and the results indicated some heterogeneity of the cortex in social cognition. hiroshige takeichi , sachiko koyama , ayumu matani , andrzej cichocki riken, wako, japan; hokkaido university, sapporo, japan; university of tokyo, kashiwa, japan to evaluate the level of spoken sentence comprehension objectively and quickly, electroencephalograms (eeg) were recorded from five japanese adults, while they were listening to fifty-second spoken sentences. natural japanese (native) and spanish (foreign) sentences were modulated in amplitude by an eleventh-order m-sequence at hz, and played twice: forward and backward. evoked responses to the modulation were analyzed as follows: ( ) circular cross correlation functions were calculated between the eeg data and the m-sequence for each subject. ( ) the functions were averaged across subjects. ( ) independent component analysis (ica) was applied to the averaged functions and independent eeg components were estimated for each stimulus for each subject. ( ) phase-locked component responses to the modulation were inspected. as a result, two components showed differential responses to the comprehensible forward japanese and the other incomprehensible stimuli. research funds: jst and kakenhi ( ) perceptual rivalry, such as ambiguous figure perception and binocular rivalry, reflects the flexibility of our brain, because it produces fluctuating perception though an unchanging stimulus. in this study, we carried on meg recordings of healthy subjects while they reported perceptual alternation of bistable apparent motion. we investigated power and phase synchronization analyses of meg signals and compared the spatiotemporal patterns during spontaneous perceptual alternation (rivalry condition) with the externally-triggered alternation (replay condition) to extract the inherent dynamics of perceptual alternation. as results, we detected transient anterior-posterior synchronizations in advance of subjects' reports of perceptual alternation in the rivalry condition. these results suggest that these synchronized activities are involved in a higher-order process inducing spontaneous alternations in perceptual rivalry. ps p-h the reflection of category perception of sound in the auditory evoked n m magnetic responses to periodic complex sounds with equivalent acoustic parameters except for different fundamental frequencies (f ) and different spectral envelopes of vocal, instrumental and linear shapes were recorded to clarify the cortical representation of timbre categorization. responses to vocal and instrumental (nonlinear) sounds were localized significantly anterior to linear sound responses. n m source strength for nonlinear sounds was significantly larger than that for linear sounds. n m peak latency only for vocal sounds was not affected by f . these results suggest that perceptual categorization was reflected in n m source strength and location (linear or nonlinear), and in n m latency (vocal or nonvocal). sunao iwaki , hiroko kou , kouichi sutani , mitsuo tonoike national institute of advanced industrial science and technology, osaka, japan; chiba univ., chiba, japan interactions between neural activities detected at multiple brain regions involved in the visual target detection processing were assessed using meg and the causal modeling. meg signals were measured during subjects performing a visual infrequent target detection task. distributed source model was used to infer the dynamic neural activities at the multiple regions and the structural equation modeling (sem) was then used to compare two possible causal models underlying the generation of major event-related components, namely p , related to the target detection. we used akaike information criteria (aic) and goodness-of-fit index (gfi) as measures of the goodness of the models. the results of the comparison of two possible sem models, whose major difference was on the contribution of the activities in the parieto-temporal region to the generation of p components, suggested the involvement of frontal and anterior cingulate cortex in the early p component (p a) and the contribution of the parietal and temporal regions to the later component (p b in our study, we investigate whether or not bilinguals use distinct neural substrates to recognize words in their first and second languages (l and l , respectively). we compared the brain activity of chinese learners of japanese as l with that of japanese natives studied in our previous study. we obtained written informed consent from each subjects. in data analysis, we used spm . while natives showed specifically greater activation in the left middle temporal gyrus than learners, learners showed specifically greater activation in the bilateral parieto-occipital and left occipito-temporal junction than natives. these results indicate that there are distinct neural substrates for word recognition of l and l . neural activations for lexical processes were measured using noun, vowel, and pseudo-character decision tasks with magnetoencephalography (meg) and functional magnetic resonance imaging (fmri) on ten right-handed subjects, and their time courses were analyzed with an fmri-constrained meg-multi-dipole method. the average activations rose at latencies around ms in the occipital gyrus or cuneus (og/cuneus) and ventral occipito-temporal areas (vot), and at latencies around ms in the posterior superior temporal and inferior parietal areas (pst/ipl), anterior temporal area (at), and posterior inferior frontal gyrus (pifg). the differences in activation between tasks are considered to reflect visual-form process in the og/cuneus and r.vot, phonological process in the l.pst/ipl and l.pifg, and semantic process in the l.at. the decay of activation for these areas was found to be well fitted to exponential functions with time constants around ms. the effectiveness of a habituation/dishabituation paradigm for determining the cerebral dominance for language was examined using a . t fmri. healthy right-handed adult volunteers with prior written informed consent were instructed to listen to analysis-synthesized words. after habituated to a single word presented repeatedly, the subject was presented with contrastive words which comprised comparison and habituation words in a pseudo-random order. the two blocks were repeated alternately for times. comparison words were phonemic or intonational derivative of the habituation word, and presented in respective sessions. the results showed that the left auditory cortex responded more to the phonemic contrast, and the right to the intonational contrast, which is in line with other paradigms/techniques for determining cerebral dominance, while the present paradigm demands little effort on the subject. the issue that whether meaning of kanji words is accessed from orthography, or from both orthography and phonology representations is still debated. the present fmri study investigated brain areas underlying the use of orthography and/or phonology in kanji reading by engaging subjects in semantic categorization task with homophone and orthographic similarity effects. fifteen native japanese volunteers participated. stimuli were pairs of definitions and their target words, including correct words and foils. the subjects were asked to decide the correct target words of definitions. the results showed that homophone versus non-homophone foils increased activation of the left fusiform and middle frontal gyri. orthographically similar versus dissimilar foils increased activation of the left middle and inferior frontal gyri. these findings reflected the roles of both orthography and phonology in kanji reading. moreover, homophone versus non-homophone minus orthographically similar versus dissimilar foils revealed activation of the left fusiform gyrus. this might suggest the role of this area in character-to-sound conversion of kanji words. chieko takamiya , mie matsui , , tsuneyuki kobayashi , , hisao nishijo , , michio suzuki , , yasuhiro kawasaki , , masayoshi kurachi , , jun nakazawa , kyo noguchi , hikaru seto neuropsychiatry, univ. toyama, toyama, japan; crest, japan science and technology corporation, japan; psychology, univ. toyama, toyama, japan; system emotional science, univ. toyama, toyama, japan; neuropsychiatry, univ. toyama, toyama, japan; developmental psychology, univ. chiba, chiba, japan; radiology, univ. toyama, toyama, japan an individual has a theory of mind (tom) if he imputes mental states to himself and others. this ability is necessary for our well-rounded social communication. we used functional magnetic resonance imaging (fmri) in ten healthy subjects to study the neural mechanisms underlying tom. we adopted the picture sequencing tasks which demanded inferring mental states to self and others as tom task. as a result, there were significant brain activations in the medial frontal cortex and middle frontal gyrus. these activations coverged with a part of results in previous neuro-imaging studies on tom and social cognitive functions. objective: the purpose of this study was to investigate the neural bases of evaluation of ambiguous facial expression using whole brain functional magnetic resonance imaging (fmri). methods: participants underwent fmri scanning during which they performed a task evaluating facial expression of human (happy or sad). the task consisted of three conditions: ambiguous, middle, and high intensity of facial expression. pictures were chosen from atr facial expression image database. results: subtraction between ambiguous and other conditions revealed the activation of anterior cingulate cortex and prefrontal cortex in evaluation of ambiguous expression. the present results suggest that these area may be involved in evaluation of ambiguously expressed emotions. motoaki sugiura , atsushi sekiguchi , keisuke wakusawa , , yuko sassa , , hyeonjeong jeong , , kaoru horie , , shigeru sato , , ryuta kawashima , miyagi university of education, sendai, japan; niche, tohoku univ., japan; dep. pediatrics, tohoku univ. school of medicine, japan; ristex, jst, japan; gsics, tohoku univ., japan; lbcrc, tohoku univ., japan using an fmri, we examined the cortical mechanisms for risk perception during observation of risky tool usage. normal subjects were presented with a picture of a naturalistic situation involving two actors, in which risks related to a tool and the direction of action were modulated in a two-factorial design. after the fmri, each subject self-evaluated the degree of risk in each picture. main effects of object-and direction-related risks were observed in the left ventromedial prefrontal cortex, and dorsolateral parieto-frontal network, respectively, suggesting that the object-and direction-related risk signals are separately processed in these networks. significant positive correlation between self-evaluated risk and cortical activation was observed in the anterior part of the left superior frontal sulcus, suggesting an involvement of this region in phenomenal risk-perception. in this fmri study, we identified cortical areas where activation during experience of risky situation is correlated with the harm avoidance (ha) scores, subscale of temperament and character inventory (tci). forty-six healthy subjects performed a rule speculation task in risky, normal, and safe situations in fmri. each situation was arranged for subjects to gain , , points or lose , , points, respectively. cortical activation induced by experience of risky situation was estimated. a significant positive correlation with the ha scores, was observed in activated areas in the right anterior insula in risky versus safe comparison. the results suggested that activation in this region predicts the individual difference in behavioral response to risky situation. this finding indicates that the right insula underlies individual difference in response to risky situation. ps p-h brain activation related to the evaluation of absolute and relative value of outcome juri fujiwara , masato taira , , toshio iijima , ken-ichiro tsutsui div. sys. neurosci., tohoku univ. grad. sch. life sci., sendai, japan; arish, nihon university, tokyo, japan; appl. sys. neurosci., nihon univ. grad. sch. med. sci., tokyo, japan one way to evaluate the behavioral outcome is in terms of absolute gain or loss (absolute value), but the evaluation can also be achieved by comparing the outcome with the possible outcomes of unchosen options (relative value). here we attempted to disentangle the brain processes involved in the absolute and relative value evaluation by using event-related fmri. subjects were instructed to compete with a computer to maximize the income in a task, in which they had to choose one option out of two, each of which were associated with either yen or a gain or loss of , , or yen. in each trial, a choice period was followed by a serial presentation of the outcomes of the chosen and unchosen options. we analyzed the brain activity during the presentation of each outcome. the activation changes related to the evaluation of absolute and relative value were observed mainly in the basal ganglia and in the cerebral cortex, respectively. ps p-i neural activation during experience-based reasoning chisato suzuki , , takashi tsukiura , hiroko mochizuki-kawai , yayoi shigemune , , toshio iijima neurosci. res. inst., aist, japan; div. systems neurosci., tohoku univ., japan the aim of this study is to investigate neural activations when reasoning future events based on experienced events. before fmri, subjects encoded two kinds of four-scene comics; the complete version with four scenes and incomplete one without the last scene. after encoding, subjects performed three tasks during fmri. in the first task, subjects chose a last scene associated with the first scene encoded in the incomplete version (memory-based reasoning: mr), whereas in the second task, subjects recognized a last scene encoded in the complete version (memory: m). in the third task, subjects chose a last scene appropriate to the first scene in the new comics (reasoning: r). activations specific to mr was found in a relatively anterior part of the left pfc and right pfc. the common activations between mr and m were identified in the right mtl, whereas a relatively posterior part of the left pfc was activated commonly between mr and r. the findings suggest that the network including bilateral pfc and right mtl may contribute to the experience-based reasoning of future events. to assess neural responses to reciprocal mindreading in socially strained human relationships, we performed an fmri study in healthy subjects who participated in the chicken game. statistical parametric mapping showed that the counterpart effect (human versus computer) activated the anterior paracingulate cortex (pcc) and the posterior superior temporal sulcus (sts). when we analyzed the data to evaluate whether the subjects made aggressive or reconciliatory choices, the posterior sts showed that the counterpart had a reliable effect regardless of risky or safe decisions. in contrast, a significant opponent x selection interaction was revealed in the anterior pcc. it could be inferred that the posterior sts and the anterior pcc play differential roles in mentalizing; the former serves as a general mechanism for mentalizing, while the latter is exclusively involved in socially risky decisions. creativity is the ability to generate new and original ideas. the most of studies of creativity used linguistic tasks which involve multiple aspects oflinguistic information processing in addition to creativity. we used new artistic creativity task such as designing new tools, in which we could quantitatively evaluated the creativity by the originality (os: originality score) of the products. using fmri, we observed bold signal change during designing task in art students (trained) and non-art students (untrained). we observed clear difference between two groups; in the trained highly creative group, the os is correlated with the interhemispheric difference of neural activities of the prefrontal cortex with right hemisphere dominance. in the untrained group we saw no such correlation. thus, our result supports the notion that both right prefrontal dominance and the increase of interhemispheric cooperativity could be the source of the artistic creativity. ps p-i the difference of brain activity elicited by different styles of art hiromi yamamura , yasuyuki kowatari , , shigeru yamane , miyuki yamamoto , comprehensive human sciences, university of tsukuba, tsukuba, japan; system brain science division, aist, tsukuba, japan artworks are categorized according to time and place where they were produced (cultural effects). surrealistic art is one of those categories and it gives uneasy impression to our mind. we investigated brain activity during viewing pictures of different art styles using functional magnetic resonance imaging (fmri). works of several artists who are well-known as representatives of renaissance, impressionism and surrealism were used as stimuli and results were analyzed by spm . while renaissance arts or impressionism arts elicited a similar activation pattern in the occipital and inferior temporal areas, surrealisms showed deactivation in parietal with the activation in the right dorsal prefrontal cortex (ba , ba ). these results suggest that a particular style of artwork may have commonly activated brain regions. research funds: coe(j- ) ps p-i effects of chewing on the activity of the prefrontal cortex in working memory processing: an fmri study in general, it has been proposed that chewing produces holding or enhancing effect on attention. furthermore, recent studies have shown that chewing causes activation of various brain regions, including prefrontal cortex. we therefore examined the influence of chewing on brain activities using fmri. the subjects used were - aged healthy adults, being conducted continuously to two-back task with intermittent gum-chewing. gum without odors and taste component was used to remove effects other than chewing. the results indicated that chewing tended to increase the bold signals in the prefrontal area including the dorsolateral prefrontal cortex during two-back task. this suggests the possibility that chewing may accelerate the process of working memory. research funds: kakenhi , ps p-i the tip-of-the-tongue with an emotional reaction caused by recall of celebrities' names hirohito m. kondo , michio nomura , jun kawaguchi ntt commun. sci. labs., ntt corp., atsugi, japan; dept. psychol., tokai women's univ., kakamigahara, japan; dept. psychol., grad. sch. environ. studies, nagoya univ., nagoya, japan the tip-of-the-tongue (tot) phenomenon is a mental state where you cannot recall something though you have every confidence that you know it. the tot state generates emotional reactions, but it is not clear what neural mechanisms are involved in the awareness of frustration. participants were instructed to recall the full names of celebrities when their faces were presented. event-related fmri analysis demonstrated that the anterior cingulate cortex (acc), anterior insular cortex (aic), inferior frontal cortex, intraparietal sulcus, and fusiform gyrus were activated during the tot state with frustration. activity of the acc and right aic was positively correlated with the degree of frustration in unsuccessful retrieval. roi analysis indicated that the acc and right aic were sensitive to retrieval demands and awareness of frustration, respectively. we suggest that the cinguloinsular circuit regulates the self-monitoring processes during the tot state. noriko kudo , , , yulri nonaka , katsumi mizuno , kazuo okanoya , riken, bsi, biolinguistics, saitama, japan; chiba university, chiba, japan; jsps, japan; department of pediatrics, showa university, tokyo, japan; presto, jst, japan segmentation of speech stream is a prerequisite for language acquisition. language learners use the transitional probability between vocal tokens to segment continuous auditory stream into distinctive words. we consider that the ability for statistical learning is not specific to language, but more general cognitive competence. and we ask whether this ability could be considered as innate. in this study, we measured erps for neonates within days, in order to examine whether neonates can learn transitional probabilities and statistically segment words. four three-tonal-words were presented in random order without intervals during recording of the eeg. as a result, only the first tone of each word evoked a significant positive component in the frontal area. since this potential is not evident during the first session, this is likely to be due to statistical learning. these results suggest that the ability to distinguish words based on statistical information is innately prepared in humans. using near-infrared spectroscopy (nirs), changes in concentration of oxygenated hemoglobin (oxy-hb) in the prefrontal cortex were evaluated while eleven human subjects performed the paintings appreciation task. in this task, subjects were required to appreciate abstract and representational paintings that appear one after another on a computer monitor. subjects were then required to judge the degrees of interest, beauty, and desirability immediately after the appreciation. it was shown that the peak of averaged oxy-hb change was higher while subjects appreciated abstract paintings. average differentiation for each oxy-hb change revealed that the changes while the appreciation of representational paintings were more accelerated than that while the appreciation of representational paintings. these results suggest the different cortical activity dependent on appreciation of abstract and representational paintings. we used meg to investigate the spatiotemporal cortical activities during mental calculations and their modulation by arithmetic complexity. eleven healthy subjects have participated in the study. three conditions were considered: easy: add three ( ) to a two-digits number without carry-over; difficult: stimuli were the same as easy, but with carry-over; nocalc: add zero to the two digits number. probe stimuli were presented s after the presentation of task stimuli (a pair of two-digit and one-digit number), and the subjects were required to respond by lifting the right index or middle finger. root-mean-square values for different meg sensor groups covering entire cortical area were calculated to evaluate local signal power in each condition. increased neural activities in the bilateral frontal/prefrontal and the parietal regions during both calculation conditions were observed in the latencies around - ms. the activities in the bilateral prefrontal and the left parietal areas in the same latencies were found to be complexity-dependent, i.e., increased activities in these regions were observed in difficult condition compared to easy condition. we investigated an effect of auditory feedback on self-produced speech in children with and without autism by measuring the lombard effect. ten children with autism ( : - : ) and agematched typically developing children ( : - : ) were instructed to name pictures of objects aloud in control and masking conditions. in masking, weighted-white noise was continuously delivered through a headset. the subjects' speech responses were recorded from a microphone. in typically developed children, the enhancement (masking/control) in masking was significantly greater (duration = . ± . , loudness = . ± . ) than in the children with autism (duration = . ± . , loudness = . ± . ) (p < . ). the present findings suggest that deficits in speech audio feedback in autistic children and this could be one of the reasons for their delay in speech development. since the mechanism underlying the effect of low power laser irradiation on the soft tissue is still unknown, we examined whether it can influence the muscle contraction as well as its fatigue in the frog (xenopus laevis) gastrocnemius or not. muscle tension continuously induced by a supramaximal stimulus to the sciatic nerve at . /s chronologically attenuated and showed a simple fatigue curve. direct irradiation of laser ( nm, mw) to the muscle surface ( . mm ) significantly delayed its attenuation (p < . ). when the rest period was set between stimulating sessions and the laser irradiation was applied during the rest period, averaged muscle tension during stimulating period for min decreased according to the session sequence. however, comparing with no or cooling application during the rest periods, such laser irradiation case significantly delayed the muscle fatigue (p < . ). it is suggested that laser irradiation has a potential to more activate atp synthesis during as well as after muscle contraction. ps p-i nedl , a novel e ubiquitin ligase for dishevelled- , targets mutant superoxide dismutase- and interacts with p yuanyuan li , , , kou miyazaki , toshinori ozaki , akira nakagawara division of biochemistry, chiba cancer center research institute, chiba, japan; production technology development center, the furukawa electric co., ltd., ichihara, japan; hisamitsu pharmaceutical co., ltd., tokyo, japan we have cloned a novel hect-type e ubiquitin ligase gene termed nedl . previous study has shown that nedl is exclusively expressed in neuronal tissues and its expression level is high in favorable neuroblastomas and undetectable in unfavorable ones. dishevelled- , a regulatory molecule in the wnt signaling pathway, was identified as the physiological target of nedl for uniquitination and proteasome-mediated degradation. on the other hand, nedl bound and ubiquitinated mutant (but not wild-type) sod in a mutant sod type-dependent manner, which is proportionally related with the fals severity. in the present study, we show that nedl physically bound p , and induced apoptosis in a p -dpendent manner. taken together, our results suggest that nedl may play a critical role in neuronal cell death occurring in fals through interacting with mutant sod and p . spinal and bulbar muscular atrophy (sbma) is an inherited motor neuron disease caused by the expansion of polyglutamine tract within the androgen receptor (ar). chip (carboxyl terminus of hsc interacting protein), u-box type e ubiquitin ligase, has been shown to interact with hsp or hsp and ubiquitylates unfolded proteins trapped by molecular chaperones and degrade them. we demonstrated in a neuronal cell model that transient over-expression of chip reduced the monomeric mutant ar more than the wild-type, suggesting that the mutant ar is more sensitive to chip than is the wild-type. we also demonstrated high expression of chip ameliorated motor impairments in the sbma transgenic mouse model. these findings suggest that chip over-expression ameliorates sbma phenotypes in mice by reducing nuclear-localized mutant ar, which probably due to enhanced mutant ar degradation. we performed an electrophysiological study demonstrating inhibition of spontaneous muscle action potentials within a co-culture of rat muscle and spinal cord by exposure to patients with guillain-barré syndrome (gbs) serum, as well as purified igg, from selected patients with gbs. using a whole-cell recording technique, we then investigated the effects of serum and purified igg from patients with gbs on voltage-dependent calcium currents (vdcc) in ngf-differentiated pc cells and cerebellar purkinje cells. serum from selected patients with gbs and purified igg from some serum of patients with gbs inhibited ca + current in both cells. these results suggest that muscle weakness in some patients with gbs might be induced by changes in p/q-type calcium channel function within motor nerve terminals. the aim of the present study was to explore the possible role of cox- inhibitor, rofecoxib in pentylenetetrazol (ptz, mg/kg, i.p.)induced kindling. rofecoxib was administered orally daily min before either ptz or vehicle. seizure severity was measured according to a prevalidated scoring scale. biochemical estimations were performed on the th day of ptz treatment. chronic treatment with rofecoxib ( . and . mg/kg, p.o.) for days showed significant decrease in ptz-induced kindling score. chronic treatment with ptz significantly increased lipid peroxidation, nitrite levels (no levels), and myeloperoxidase levels and decreased the reduced glutathione (gsh) levels in brain homogenate, which was reversed with rofecoxib treatment. research funds: university supportted study ashish dhir, shrinivas kulkarni uips, panjab university, chandigarh, india the objective of the present study was to elucidate the effect of cyclooxygenase inhibitors on pentylenetetrazol (ptz)-induced ( mg/kg) convulsions in mice with possible mechanism of action. various cox-inhibitors were administered min prior to the ptz administration. onset, duration of clonic convulsions and percentage mortality/recovery were recorded. pretreatment with cox-inhibitors aspirin ( and mg/kg, p.o.), naproxen ( and mg/kg, p.o.), nimesulide ( - mg/kg, p.o.) or rofecoxib ( - mg/kg, p.o.) dose dependently showed protection against ptz-induced convulsions. rofecoxib ( mg/kg) or nimesulide ( mg/kg) also enhanced the subprotective effect of diazepam or muscimol showing gabaergic modulation of cox- inhibitors. cox- inhibitors also antagonized the effect of flumazenil ( mg/kg) against ptz-induced convulsions further confirming the gabaergic mechanism. ps p-j cell proliferation after domoic acid-induced neuronal damage in adult rats domoic acid (da) is structurally related to kainic acid, which is a rigid analogue of the putative neurotransmitter l-glutamate that causes neuronal excitation. da-induced convulsions affects limbic structures such as hippocampus and entorhinal cortex. in this study we examined the neuronal damage after intraperitoneal da administration and cell proliferation in the adult rat brain. the most extensive neuronal cell damage was observed in ca subfield as evaluated by he staining, while tunel positive cells were mainly observed in the granular cells of cerebellum and dentate gyrus (dg) of the hippocampus. to elucidate the relations between damage and cell proliferation, we examined bromodeoxyuridine (brdu) labeled cells. brdu labeled cells were detected in dg and the granular cells of cerebellum. the cell proliferation was not associated with damage. ps p-j a-type potassium channel truncation mutation in temporal lobe epilepsy the role of voltage dependent calcium channels on the pentylenetetrazol (ptz) kindling induced learning deficits was investigated in rats. in this study animals were divided into three groups. in the test group verapamile were injected in the hippocampus ( mg/ min). after min kindling was established in rats with ptz. the control animals were the same age and undergone the same treatment in term of acsf injections and post-kindling waiting time as the kindled animals. and in sham group the animals received saline. one month after induction of kindling spatial learning and memory was tested by morris water maze. results showed that intra-hippocampal injection of verapamil significantly decreased spatial learning, suggesting that only working memory impaired but reference memory remain intact. the results with this study suggest that intera-hippcampal injection of verapamil significantly impaired spatial learning in rats. we showed that -oxoguanine ( -oxog) in mitochondrial (mt) dna and cellular rna increased significantly in the ca subregion of the mouse hippocampus after kainate administration. laser scanning confocal microscopy revealed that -oxog accumulated greatly in mtdna of the ca microglia. wild-type and mth -null mice, the latter lacking an ability to hydrolyze -oxo-dgtp and -oxo-gtp to the monophosphates to avoid their misincorporation into dna or rna, exhibited similar degree of the ca neuron loss after kainate administration, however, levels of -oxog accumulated in mtdna and cellular rna in the ca microglia were significantly increased in mth null mice in comparison to wild-type mice. we thus demonstrated that mth efficiently suppresses the accumulation of -oxog in both cellular dna and rna in the hippocampus, especially in microglia, caused by excitotoxicity. ps p-j transcription factor nrf regulates brain response to kainate-induced excitotoxicity yukihiko dan , kosuke kajitani , noriko yutsudo , ken itoh , masayuki yamamoto , yusaku nakabeppu kyushu univ., med. inst. bioreg., div. neurofunc. genomics, japan; univ. tsukuba, grad. sch. comp. hum. sci., japan nf-e related factor (nrf ) is the key transcription factor that serves to transmit the inducer signal to an antioxidant response element (are), a cis-acting element required for gene expression of a battery of proteins acting on anti-oxidative stress and detoxification of electrophiles. since loss of nrf has been reported to increase neuronal death under increased oxidative stress, nrf seems to play a role for neuroprotection. administration of kainite, a potent agonist of an excitatory neurotransmitter glutamate, to rodents produces epileptiform seizures followed by a delayed loss of pyramidal cells in the ca subregion of hippocampus. to unveil the functional significance of nrf in the brain, we compared seizure responses between wild-type and nrf -null mice after systemic kainate administration. we found that nrf -null mice exhibited an increased susceptibility to the kainate-induced seizure, and their loss of the pyramidal cells and gene expression profiles are now under investigation. ps p-j synaptic plasticity and -aminopyridineinduced epileptic discharges in rat hippocampal slices makoto otani, tetsuo furukawa, kiyohisa natsume department of brain science and engineering, kyushu institute of technology, kitakyushu, japan four-aminopyridine ( -ap) at the concentration below . mm suppresses k d channel and induces the epileptic discharges in rat hippocampal slices. in the present study, the involvement of the activation of nmda receptor on the ictogenesis of the -ap induced discharges in ca region was studied. ten m -ap induced the epileptic discharges with the frequency of . ± . hz (mean ± s.e.m.; n = ) and the amplitude of . ± . mv. when ap- , an nmda receptor blocker, was applied to the pre-established epileptic discharges, the frequency and the amplitude of the discharges did not change significantly. on the other hand, when ap- was applied from the ictogenesis period of the discharges, the discharges did not appear. these results suggest that the nmda receptor-dependent synaptic plasticity involves in the ictogenesis of -ap-induced epileptic discharges. chronic exposure of cultured astrocytes to morphine is reported to induce differentiation of the cells. using primary astrocyte cultures, we observed that under thyroid hormone (th) deficient conditions, morphine significantly decreased cell viability. further studies showed that the loss of cell viability was due to apoptosis of the cells. the effect is attenuated by th supplementation to the culture medium. the observed effect of morphine appears to be mediated through the opioid receptor since the opioid antagonist, naloxone, inhibited the decline in cell viability. ni, a specific inhibitor of nnos, completely blocked loss of cell viability suggesting that morphine induced intracellular no production, leads to cell death. studies suggested that no acts through a cgmp independent pathway. the involvement of no induced cgmp independent pathway in morphineinduced apoptosis during th deficiency has been investigated. collectively, the present study demonstrates that morphine mediated cytotoxicity of astrocyte is critically influence by the level of thyroid hormone in cultured medium. ps a-a influence of conductance-input signal and prior activation history on spike generation in rat somatosensory cortical neurons takashi tateno , hugh p.c. robinson engineering science, osaka university, osaka, japan; university of cambridge, uk in the cortex, a profusion of electrophysiological cell types, which form specific synaptic connections, is becoming apparent. a quantitative understanding of the dynamics of different cell types when responding to complex, natural inputs, is an important prerequisite for understanding the cortical network. neurons compute by transforming excitatory and inhibitory synaptic conductance inputs into a spike train output. we have examined the properties of synaptic conductance inputs which are most effective in evoking spikes, by injecting broad-band excitatory and inhibitory conductance inputs, and using spike-triggered reverse correlation and wiener-kernel estimation to calculate the average conductance input trajectory (acit) preceding spikes. the time course of the acit provides a general description of a neuron's response to dynamic conductance stimuli. our analysis showed that the acit reflects both previous stimulus history and previous discharge history, and that the relative influences of these two factors depend on the cell type. amyotrophic lateral sclerosis (als) is a rapidly progressive neuromuscular disease caused by the destruction of motor neurons. our study has investigated the effects of als-csf on voltage-gated calcium p/q-type channel (␣ a) expression in pre synaptic terminals of rat spinal motor neurons. csf from als and non-als (neurological patients) was injected into the -day-old rat pup spinal subarachnoid space at the rate of l/ . min. the rats were sacrificed h after csf injection and spinal cord sections were processed for immunocytochemistry with p/q-type channel ␣ a antibody and also for cytochrome oxidase labeling. als-csf significantly increased p/qtype channel expression compared to csf from non als patients. als-csf significantly decreased cytochrome oxidase activity in the rat spinal motor neurons, which may be a sign of degeneration. it is probable that, toxic factors present in the als patients csf might induce the expression of p/q-type channel observed in pre synaptic terminals synapsing on the spinal motor neurons. ps a-a on the membrane potential profile of ca pyramidal cells recorded with voltage sensitive dye imaging in rat hippocampal slices takashi tominaga , , yoko tominaga dept. neurophysiol., kagawa sch. pharmaceutical sci., tokushima-bunri univ., kagawa, japan; lab. for dynamics of emergent intelligence, riken bsi, hirosawa - , wako, saitama, japan integration of membrane potential response in a single neuron is a basis of neuronal calculation. we have been aiming to visualize this with voltage sensitive dye (vsd). hippocampal slices, with its unique laminar structure, allow us to assign optical signals to particular membrane fractions. but, it has not been clear whether the profile of optical signal could be a measure of membrane potential profile. to solve this, we visualized rather steady membrane potential change caused by perfusion of high potassium medium. a steep peak in optical signal was seen along stratum pyramidale. an application of ttx diminished this peak, and made the optical signal profile flat along the cell. thus, we concluded that the specificity of the vsd is small. with "neuron", by assuming a population nature to the optical signal, the membrane potential profile in a response to stimulation was successfully simulated. ps a-a overexpression of inwardly rectifying k + channel . in hippocampal slice culture masayoshi okada, hiroko matsuda department of physiology, kansai medical university, japan the expressions of mrnas for the inwardly rectifying k + channel (kir) . have been reported in mammalian central nervous system, but regulation of expression or its role in synaptic transmission remains unknown. in our rat hippocampal slice cultures, the endogenous kir current was hardly detected with whole cell recordings in the ca pyramidal neurons. then, egfp and kir . expressing virus vectors were constructed, and infected to the neurons in the slices. the vectors succeed to express the kir current, and the translocation of the fusion protein to the plasma membrane was also observed. furthermore, the overexpression significantly reduced the raise in whole-cell membrane potential evoked by depolarizing current injection, suggesting that kir plays a role of noise-filter for synaptic input in central neurons. takeshi otsuka, mieko morishima, yasuo kawaguchi div. cerebral circuitry & structure, nips, okazaki, japan layer pyramidal cells are heterogeneous in morphological and physiological properties, and project to multiple subcortical areas. although recent studies have addressed anatomical features of pyramidal cells identified projection regions, little is known about intrinsic membrane properties of these subtypes. here, we obtained whole cell recordings from rat frontal layer pyramidal cells that project to the striatum (ccs) or pontine nucleus (cpn), identified by injection of fluorescent retrograde tracer to these regions. firing properties of pyramidal cells had similarity depending on the projection regions. ccs cells showed strong adaptation of successive spike intervals in response to the depolarizing current injection. however, cpn cells exhibited very little spike frequency adaptation during current injection. we also examined synaptic inputs from layer / neurons to these subtypes by single cell stimulation, and detected excitatory inputs in both subtypes. our results suggest that physiological properties of layer pyramidal cells are correlated with their subcortical target. this study aimed to clarify expressional changes in types and of ryanodine receptors (ryr and ryr ) in the cerebellum of a ca + channel ␣ a subunit mutant, rolling mouse nagoya. semi-quantitative rt-pcr revealed altered mrna signal levels of ryr but not ryr in the rmn cerebellum: a less ryr mrna signals than in the control cerebellum. well consistent with the semi-quantitative rt-pcr results, ryr immunostaining in soma and primary dendrites of purkinje cells was less intense in rmn than in control mice. in contrast, ryr immunostaining was detected in cerebellar glomeruli but the staining intensity was not different between rmn and controls. the present study suggests that somatodendritic ryr expression in purkinje cells was decreased in the cerebellum of rmn. this may suffer ryr -mediated ca + release, contributing altered ca + homeostasis in the rmn purkinje cells. ps a-a dopamine-based modulation of lateral amygdala neuron excitability: a possible involvement of potassium current ryo yamamoto, yoshifumi ueta, noubuo kato integrative brain sci. med., kyoto univ., kyoto, japan the amygdala and dopaminergic innervation thereonto are considered to cooperatively regulate emotional states and behaviors. in the present slice experiments, we investigated the effects of dopamine (da) on lateral amygdala (la) neurons by whole cell recordings. application of da depolarized la neurons, reduced the action potential threshold, and induced slow afterdepolarization (sadp). this sadp was induced voltage dependently, and lasted for more than s. d receptor agonists induced the same sadp. previous reports have repeatedly suggested that sadp is triggered by calcium influx. consistently, calcium channel blockers or chelating intracellular calcium inhibited the present da-induced sadp. a membrane conductance decreased at the peak of sadp current (i sadp ). also, i sadp was suppressed by including cesium in the pipette solution. these results suggest that the present da-induced modulation of la neuron excitability may depend on a potassium current that can be masked by calcium influx. toru aonishi , , hiroyoshi miyakawa , masashi inoue , masato okada , tokyo institute of technology, japan; brain science institute, riken, japan; tokyo university of pharmacy and life science, japan; the university of tokyo, japan it has been reported that amplification of ap paired with epsp boosts the induction of ltp. there are two alternative hypotheses of such amplification mechanisms; one is activation of the na channel and other is inactivation of the a-type k channel. which is essential? in this talk, by mathematical analyses and the neuron simulator, we demonstrate that the balance of inward and outward currents, which can be controlled by down/up-regulation of the a-type k channel induces a divergence of the membrane input resistance, i.e. a singularity, and such super-sensitivity is the fundamental mechanism for boosting amplification of ap paired with epsp. the balance of na and a currents is essential for controlling dendritic integration manners. we also show that the down-regulation of the a-type k channel, which modifies the ratio between the inward and outward currents, leads to a drastic change from amplifying ap mode to shunting epsp mode. miharu komai , maya yamazaki , , mika tsujita , manabu abe , rie natsume , , kenji sakimura , department of cellular neurobiology, brain research institute, niigata university, niigata, japan; sorst/jst, saitama, japan we previously reported that stargazin family (␥ , ␥ , ␥ , and ␥ ) not only promoted ampa receptor surface expression but also modulated receptor activity and channel property (yamazaki et al., ) . therefore, we assumed these family proteins were auxiliary subunits of ampa receptors. to prove this hypothesis, we generated ␥ subunit knockout (ko) mice using the cre/loxp recombination system and analyzed their phenotypes. the ␥ subunit ko mice were viable, fertile, and displayed no overt phenotype. on the other hand, on western blot analysis, protein expression levels of ampa receptor subunits were reduced in ko mice compared with those in wild-type at postnatal day , while the reduction was not so significant in adult brain. these results suggested that ␥ might regulate dynamics of ampa receptor subunits during early development. in the cns, neural damages, such as hypoxia, ischemia and degenerating diseases, are often accompanied by disturbances in the ph environments. ambient ph plays as a significant signal for neural functions. microglia (brain phagocytes) express abundant voltagegated proton (hv) channels which have extremely high selectivity for h + and potent h + efflux ability. exposure to na-lactate (ph . ) induced cell acidosis and activation of the hv channels. the channel activation was characterized by increased conductance, facilitation of activation kinetics, prolongation of deactivation kinetics and a shift of the activation voltages to negative potentials. consequently, the hv channel could open more easily over a wide range of the membrane potential during lactic acidosis, and may contribute to a quick relief of the cell acidosis. mari sasaki, masahiro takagi, yasushi okamura okazaki institute for integrative bioscience, aichi, japan here we report a novel four transmembrane protein similar to the voltage sensor domain (vsd) of the voltage-gated channels that exhibits activities of a voltage-gated proton channel. voltage-gated proton channel currents have classically been described in snail neurons and recently in mammalian blood cells. however, the molecular basis underlying this channel has been elusive. here we identify a novel cdna clone named as mouse voltage-sensor domain only protein (mvsop ). cells overexpressing this protein showed depolarization-induced outward currents accompanied by tail currents during repolarization, which reversed at equilibrium potentials for protons. imaging analysis demonstrated that phin recovers rapidly after an acid load in mvsop -transfected cells. mvsop induced currents exhibited two key features of native voltage-gated proton channels: ph-dependent gating and zn + sensitivity. neutralization of a positive charge in the s -like segment caused shift of the voltage-conductance relationship, suggesting that it plays important role in gating. oscillatory extracellular electric fields have been observed in mammalian brains. the electric fields modulate neuronal excitability and synaptic events. to investigate the effect of the oscillatory electric fields on the ca pyramidal neuron, we applied sinusoidal electric fields to the rat hippocampal slice and recorded voltage responses with a voltage sensitive dye (rh ). application of sinusoidal electric fields induced transmembrane voltage oscillations in all the layers of the ca region. in the pyramidal layer, the amplitudes of the responses to the -hz field were the largest. the amplitudes were decreased monotonically when the frequency of the fields became higher. however, in the stratum radiatum, the amplitudes of the responses to the - -hz fields were larger than those to the other frequencies. the frequency preference in the dendritic region may be an underlying mechanism for the synchronization of the membrane potentials among large population of neurons within the theta frequency range. acid sensing ion channels (asic ) have proposed to constitute mechanoreceptors and nociceptors. we examined the localization and characterization of asic -expressing cells in rat central nervous system (e -p ) using immunohistochemical techniques. asic positive fiber first appeared in brain stem and spinal cord at e - stage. asic -expresseing cells appeared in white matter of brain stem and spinal cord at e stage. in early postnatal stages asic expressing cells appeared in corpus callosum, cerebellar medulla and dorsal horn of spinal cord at p stage. these cells were identified as an oligodendroglia by oligodendrocyte specific antibody and immunoelectron microscopy. these results are suggested the hypothesis that the function of asic mediate the myelin formation in the developmental stages of central and peripheral nervous system. masato shino, seiji ozawa, yasuhiko saito department of neurophysiology, gunma university graduate school of medicine, maebashi, gunma, japan nucleus prepositus hypoglossi (nph) is involved in horizontal eye movement. previously, we found nph neurons exhibiting a characteristic firing pattern in response to depolarizing current pulses (fil neurons). fil neurons exhibited a spike train with a long first interspike interval ( st isi) that is attributed to a large, slow hyperpolarization (ahp) after the first spike. in this study, we investigated ionic conductances underlying the long st isi by whole-cell recordings in rat slices. application of m apamin, an sk-type ca + -activated k + (kca) channel blocker, shortened the st isi and decreased the amplitude of the slow ahp. the shortening of the st isi was observed when membrane potentials were depolarized. moreover, application of m mibefradil, a t-type ca + channel blocker, shortened the st isi. these suggest that the firing pattern of fil neurons arises from activation of sk-type kca channels induced by ca + influx through t-type ca + channels. research funds: kakenhi (c) ( ) jafar vatanparast , , mahyar janahmadi , houri sepehri , ali haeri-rohani , ali reza asgari neuroscience research center, shaheed beheshti medical sciences university, tehran, iran; dept. of biology, university of tehran, tehran, iran the roles of the ionic channels and muscarinic receptors in paraoxon (px) induced burst firing in snail neurons were studied using current clamp method. px ( . m, within min) increased the frequency of spikes and shortened ahp. slow waves of depolarization with superimposed bursts were recorded within min. atropine blocked the depolarization shift but not the other effects of px. px was able to reversibly decrease the duration of calcium spikes elicited in a na + free ringer. this effect observed in the presence of atropine and was along with a decrease in the duration of ca + spike ahp and an increase in the spike frequency. the px blockade of ca + channels may decrease the activation of ca + dependent k + channels that underlies ahp. blockade of these channels possibly makes the neurons susceptible for burst induction, while activation of metabotropic muscarinic receptor by px underlies the depolarization shift with associated bursts. dendritic membrane properties are reported to be non-uniformly distributed in a single neuron and the non-uniformity could be important for synaptic integration. however their distribution is still unclear. we estimated distribution of membrane resistance by fitting a compartment model to voltage imaging data of membrane response in hippocampal ca slices to perturbation, such as propagating epsp induced by synaptic inputs and biphasic response to extracellular electric field. by numerical simulations, we found that these imaging data were consistently reproduced if we assume a step function as distribution of membrane resistance. this implies that a steep decrease of membrane resistance exists in distal dendrite of hippocampal ca pyramidal neuron. it is known that cooling-induced desensitization of cold receptors, however, its intracellular mechanism has remained unresolved. in this study, we analyzed molecular mechanism of desensitization of cold/menthol receptors (trpm ). repeated menthol application induced trpm desensitization. this desensitization was depended on extracellular ca + , indicating that involvement with ca + -dependent kinase. pkc activator (pma) desensitized trpm and go (pkc inhibitor) abolished pma-induced trpm desensitization. pma similarly desensitized wild type trpm and mutant trpm , in which serine or threonine residues in some putative pkc phosphorylation sites were replaced by alanine. pma treatment did not induce internalization of trpm . as the basis of cooling-induced desensitization of cold receptors, we conclude that cooling-activated trpm causes pkc to desensitize trpm itself. yosuke sawada , hiroshi hosokawa , kiyoshi matsumura , shigeo kobayashi dept. of int. sci. and tech., grad. sch. of info., kyoto univ., kyoto, japan; dept. of info. sci. and tech., osaka institute of technology, osaka, japan cooling below • c evokes cold pain sensation. however, the molecular basis of the cold pain sensation is still unknown. trpa is activated by pungent compounds stimuli. if trpa responded to cooling to noxious cold range, it could be candidate for evoking cold pain sensation. however, whether trpa is activated by cooling or not is still controversial. here, we investigated that trpa -expressing hek cells responded to noxious cold stimuli. whole-cell recording demonstrated that cooling below threshold evoked inward current. threshold temperature was . ± . • c. in inside-out singlechannel recording, cooling activated trpa directly. single channel conductance was . ± . ps. single channel currents showed inword rectification. in conclusion, trpa is the cooling activated cation channel. yoshiki matsuda, foong yen ang, jinsun yoon, noriko ebisu, satoshi takahashi, shinichi kogure dept. bioengin., soka university, tokyo, japan hyperplarization-activated and cyclic-nucleotide-gated nonselective cation channels (hcn - ) have been demonstrated in the cns. since they contribute to various physiological functions including neuronal pacemaking activity, setting of resting membrane potential and generation of paroxysmal discharge, we examined their expressions as well as functions in the pns using the frog (xenopus laevis) sciatic nerves. western blot analyses for hcn - demonstrated that samples from the nerve and the heart showed an hcn band whereas those from the dorsal part of skin and the gastrocnemius did not, and that immunoreactivities for hcn , hcn and hcn could not be found in those samples. when an hcn channel blocker, zd was applied on the stimulus portion of sciatic nerve and the nerve was elicited at . /s by a duration of ms pulse with supramaximal intensity, the generation of anode-break-excitation rather than cathode-makeexcitation was significantly blocked (p < . ). it is suggested that hcn channels exist in the pns and they contribute to the burst or recurrent discharges. ifenprodil, a clinically used cerebral vasodilator, interacts with several receptors, such as ␣ adrenergic, n-methyl-d-aspartate, serotonin and receptors. however, the molecular mechanisms underlying the various effects of ifenprodil remain to be clarified. here, we show that ifenprodil inhibited g protein-activated inwardly rectifying k + (girk; kir ) channels, which play an important role in the inhibitory regulation of neuronal excitability in most brain regions and the heart rate, expressed in xenopus oocytes. in contrast, kir . and kir . channels in other kir channel subfamilies were insensitive to ifenprodil. the girk currents induced by -opioid receptors or ethanol were also attenuated in the presence of ifenprodil. the inhibitory effects of ifenprodil were not observed when ifenprodil was applied intracellularly. our results suggest that inhibition of girk channels by ifenprodil, at submicromolar concentrations or more, may contribute to some of its therapeutic effects and adverse side effects. ps a-b proliferation of rat c glioma cells is controlled by the concentration-sensitive na + channel (na c ) shigeru yoshida, hiroyuki yamaguchi, takashi takeuchi, hokuto tanaka, yoshiyuki morimoto, teruki hagiwara department of life science, kinki university, higashi-osaka, japan the role of na + as a regulator of cell growth was studied using the tumor cell line (c ), which has a large quantity of concentrationsensitive na + channels (na c ; c = concentration). cell proliferation was suppressed when [na + ] o was raised from control ( mm) to or mm. an increase in [na + ] i was revealed by an image processor in c cells loaded with a na + indicator (sbfi), under high [na + ] o conditions. [na + ] i elevation was augmented by ouabain or by bumetanide (na + /k + /cl − cotransporter blocker), while it was decreased when na c expression was inhibited by rnai techniques. the real-time pcr method revealed that the expression level of the immediate early gene egr- , which is involved in cell growth, was concomitantly reduced. it is to be noted that similar alterations in cell growth, egr- level and [na + ] i were induced by a na + ionophore (monensin) without raising [na + ] o . these data indicate that na + enters through na c upon [na + ] o increase, and [na + ] i elevation itself is responsible for these phenomena. hiroshi kuba, takahiro ishii, harunori ohmori dept. physiol., univ. kyoto, kyoto, japan na + channels are concentrated in the axon to generate action potentials. however, little is known about how distribution of na + channels contributes to the activity and function of single neurons. in avian nucleus laminaris (nl), neurons act as coincidence detectors for sound source localization, and are tuned to both characteristic frequency (cf) and interaural time difference (itd) of sounds. we show here in the chick that nl neurons have distinct distribution of na + channels along the axon and optimize the itd sensitivity depending on their cf. neurons of high and middle cf (higher than khz) had small action potentials, and had no na + currents in the somatic membrane, but clustered only in the axon at some distance from the soma ( - m). while, neurons of low cf generated large overshooting spikes, and na + channels were clustered closer to the soma ( m) in the axon. thus, nl neurons had a spike generator on the axon, at a greater distance from the soma with the increase of cf. by computer simulation, these unique distributions of na + channels were found essential to enhance the coincidence detection. research funds: kakenhi ( ) il-sung jang , in-sun choi , eun-ju park , jin-wha cho , man-gee lee , byung-ju choi kyungpook national university, school of dentistry, south korea; kyungpook national university, school of medicine, south korea bisphenol a (bpa), an endocrine disrupter, is contained in cans, polycarbonate bottles and some dental sealants. here we report the effect of bpa on gaba a receptors using a conventional whole-cell patch clamp technique from acutely isolated rat ca pyramidal neurons. bpa itself elicited a postsynaptic current, which is highly sensitive to bicuculline, in a dose-dependent manner. bpa increased postsynaptic currents induced by gaba at lower concentrations (< m), but decreased those induced by gaba at higher concentrations (> m). in addition, bpa decreased both the current amplitude and decay time constant of gabaergic mipscs. finally, mechanisms underlying bpa-induced modulation of gaba a receptors will be discussed. we recently generated nav . -deficient mice and showed that these mutant mice developed epileptic seizures and died prematurely. we have now used these nav . -deficient mice as negative controls to examine nav . distribution in the mouse brain using rna in situ hybridization histochemistry and immunohistochemistry. at low magnification, nav . expression was higher in the thalamus, superior colliculus, inferior colliculus, pons, medulla and cerebellar nuclei relative to other brain regions. contrary to previous studies indicating a somato-dendritic nav . distribution, in the present study, higher magnification analysis revealed that nav . is predominantly distributed to axons in some brain parts. this apparent discrepancy may reflect the lack of specificity of anti-nav . antibodies used in these previous studies, none of which utilized nav . -deficient mice. based on our findings, we propose that nav . might be involved in propagating action potential to presynapses. keiji miura , , masato okada , , , shun-ichi amari department of physics, kyoto university, kyoto, japan; "intelligent cooperation and control", presto, jst, japan; department of complexity science and engineering, university of tokyo, chiba, japan; brain science institute, riken, saitama, japan we considered a gamma distribution of inter-spike intervals as a statistical model for neuronal spike generation. a gamma distribution is a natural extension of poisson process and it can generate spike trains with various irregularities. the model parameters consist of a time-dependent firing rate and a time-independent spiking irregularity. because the environment changes over time, the firing rate varies for each interspike interval. we used a novel method of information geometry to estimate the spiking irregularity whatever the functional form of the firing rate is. our estimator is simple and easily applicable to experimental data. the estimator is useful for characterizing spiking irregularity which varies among neuron types. it may be possible to classify neurons into functional groups according to their spiking irregularities. research funds: grant-in-aid for scientific research (nos. and ) mitsuyo watanabe, yuko ishimaru, taketo nakadai, tomoyuki kanamatsu graduate school of bioengineering, soka university, tokyo, japan we examined the effect of colchicine, inhibitor of axonal flow, on cerebral amino acid metabolism in the rat. the rats were injected with [ - c] glucose intravenously ( g/kg) or h after the intraventricular injection of colchicine ( g/ l) and the amino acid fractions were extracted from the brains at , or min after the glucose injection. the amount of [ - c] glucose in the cerebra was increased, however, the c incorporation into glutamine, glutamate, gaba and aspartate from [ - c] glucose were decreased. only glutamine concentration in all amino acids was increased in the cerebra of the colchicine group, compared to those values in the control group. the microdialysis analysis showed that the amount of gln in the dialysate was increased by three times in the colchicine group compared with the control group. these data may suggest that the glycolysis of glucose is decreased and that the influx of glutamine from blood to brain occurs with neuronal dysfunction induced by colchicine. these results indicate that a  alters the bhlh gene expression in neural stem cells toward cell death. ken kojima, akiko nishida, shinji takebayashi, jyuichi ito department of otolaryngology-head and neck surgery, graduate school of medicine, kyoto university, japan basic helix-loop-helix (bhlh) transcription factors play crucial roles in development of the central and peripheral nervous systems. to visualize expression of hes or hes gene, phes -and phes -egfp transgenic (tg) mice were generated (ohtsuka et al., ) . in each transgenic mouse, a promoter of hes or hes gene drives enhanced green fluorescent protein (egfp) gene. in the inner ear, it is suggested that hes or hes regulate cell division and differentiation of sensory and supporting cell progenitors via notch signaling pathway. by use of immunohistochemical technique, we examined distribution of gfp expressing cells in the inner ear of the transgenic mice from embryonic day (e ) to postnatal day (p ). in the phes -egfp tg mouse inner ear, gfp immunoreactive (gfp-ir) cells were detected from e to p . in the phes -egfp tg mouse inner ear, gfp-ir cells were observed from e . to p . gfp-ir cells in phes -gfp tg mouse are candidates of sensory cell progenitors in mature mammalian inner ear. ohtsuka et al., . mol. cell neurosci. ps a-c expression of zfh- in the developing mouse brain: mrna, antisense rna and protein expression yuriko komine , kenji nakamura , motoya katsuki , tetsuo yamamori national institute for basic biology, okazaki, japan; mitsubishi kagaku institute of life science, machida, japan zfh- is a transcription factor containing three homeodomains and zn fingers and expressed in differentiating neurons. we have reported that the level of zfh- mrna is negatively regulated by antisense transcripts of the zfh- gene. in several types of neurons, including pyramidal cells in the hippocampus and granule cells in the cerebellum, the zfh- antisense rna is expressed prior to the mrna; as the level of the antisense rna gradually decreases, zfh- mrna starts to be expressed. recently, we have raised an antibody against mouse zfh- and examined the expression profile of the zfh- protein. in the most regions of the brain, the protein expression pattern consisted with that of mrna. however, in the several types neurons mentioned above, zfh- protein was not detected even when the zfh- mrna was already expressed. this observation together with other data suggested that the zfh- protein level is regulated by several mechanisms including suppression by the antisense rna and translational control. takashi inoue , maya ota , katsuhiko mikoshiba , jun aruga laboratory for comparative neurogenesis, riken bsi, saitama, japan; laboratory for developmental neurobiology, riken bsi, saitama, japan zic family zinc-finger proteins play various roles in animal development. in mice, five zic genes (zic - ) have been reported. despite their partially overlapping expression profiles, mouse mutants for each zic gene show distinct phenotypes, suggesting the functional redundancy of zic proteins. it is expected that the common and specific roles of mouse zic proteins can be clarified by studying compound mutant mice. in the present study, we characterized zic /zic compound mutant mice. mice carrying homozygous zic mutant allele together with zic null allele showed defects in midline structures, including abnormalities in forebrain and thalamus. especially, the compound mutants showed severe anatomical abnormalities in the dorsal and ventral telencephalon and olfactory system, which are not obvious in either zic -or zic -single mutant. these observations indicate that zic , in cooperation with zic , have an essential role in controlling proliferation and differentiation of the neuronal projenitors in the medial telencephalon. chiaki maruyama, haruo okado department of molecular physiology, tokyo metropolitan institute for neuroscience, japan rp , a novel zinc finger protein containing a poz domain, functions as a sequence specific transcriptional repressor. rp gene disrupted mice show severe abnormalities in brain cortical layer formation, suggesting that rp has a crucial role in cerebral development. to understand the role of this protein in brain development, we examined rp gene expression in mouse embryo and adult brain by in situ hybridization. as a result, we found that rp transcripts are first detected at embryonic day in the neuroepithelium of the spinal cord and telencephalic vesicle. in the day - embryos, rp transcripts are predominantly observed in the preplate region but not in outside the nervous system. at e , rp transcripts were detected throughout the neocortex and hippocampus, but not in the thalamus and striatum. in the cortex, the transcripts were detected primarily in cortical neurons, but not in the marginal zones and ventricular zone. in adult mice, rp is expressed in neocortical and hippocampal neurons and granule cells in the cerebellar cortex toshiki kameyama, fumio matsushita, yuzo kadokawa, tohru marunouchi division of cell biology, fujita health university, toyoake, japan neural zinc finger (nzf) proteins are transcription factors with dnabinding domains of c hc-type zinc finger motifs. using p cells, we demonstrated that nzfs were expressed transiently during neuronal differentiation, and forced expression of nzf cdnas resulted in neuronal differentiation. these results suggest that nzf family have a function regulate neuronal differentiation. to elucidate in vivo functions of nzf family in detail, we generate knockout mice of nzf- and nzf- respectively. nzf- null mice are born alive, but die within min after birth with cyanosis. on the other hand, nzf- null mice are viable, fertile and appear normal. these mice look normal morphologically. then we generate double knockout mice of nzf- and nzf- by intercrossing. double knockout mice have a forelimb posture abnormalities like arthrogryposis multiplex congenita. and we find out that the spinal nerves projecting forelimb and trunk are decrease dramatically in the double knockout mice embryo. , ) . in the present study, to examine the role of runx in the development of drg in more detail, we examined the development of drg neurons in runx -deficient mice from the early embryonic stages to birth, using various markers for subpopulation of drg neurons. in newborn runx −/− mice, parvalbumin-positive drg neurons were greatly reduced in number, whereas calretinin-positive neurons were slightly decreased. similar decreases were observed in embryonic days . and . . shin hisahara , , susumu chiba , hiroyuki matsumoto , yoshiyuki horio department of pharmacology, sapporo medical university, sapporo, japan; department of neurology, sapporo medical university, sapporo, japan in mammalian cns, the function of histone deacetylase sirt is still unclear. recent studies indicated that sirt interacts with nuclear receptor co-repressor (n-cor) and n-cor represses intracellular domain of notch-icd activation of the hes promoter. we performed overexpression of sirt and n-cor in neurosphere by nucleofection, then induced differentiation. we found remarkable promotion for neural differentiation by overexpression of sirt and n-cor in the sirt with n-cor. sirt and n-cor suppressed hes transcription by notch-icd in the luciferase assay. hes transcription was suppressed in overexpression of sirt and n-cor, suggesting that interaction between sirt and n-cor represses hes transcription. consistent with this, chip assays revealed that not only n-cor but also sirt bind to the promoter of hes gene. taken together, these results indicate that sirt and n-cor accelerate neural differentiation of the undifferentiated cells via binding hes promoter site and repressing hes transcription. yasushi maruyama , mitsuhiko kurusu , masataka okabe , katsuo furukubo-tokunaga grad. school life and envir. sci., univ. tsukuba, japan; natl. inst. genetics, mishima, japan; inst. dna medicine, jikei univ. school of medicine, japan during brain development, a large number of neurons are generated by proliferation of neural stem cells. with a characteristic proliferation mode that persists through development, the neuroblasts of drosophila mushroom bodies (mb) provide an attractive model system to study mechanisms of neural stem cell proliferation. here we show that tailless (tll), a member of the orphan nuclear receptor super family, has a crucial function in maintaining cell cycle progression of the mb neuroblasts. mosaic analysis demonstrates that cell autonomous activity of tll is crucial for maintenance of the mb neuroblast cell cycles. moreover, gain-of-function analyses confirm instructive functions of tll in maintaining neuroblast activity. we propose that tll plays pivotal roles in proliferation of the mb neuroblasts and suggest a conserved mechanism of neural stem cell control with the tll/tlx homologs in both drosophila and vertebrate brains. kouji senzaki, masaaki yoshikawa, shigeru ozaki, takashi shiga graduate school of comprehensive human science, university of tsukuba, ibaraki, japan runx family transcription factor is an important component of tgf- and bmp signaling. we reported previously that runx mrna is expressed in the dorsal root ganglion (drg) from the early developmental stages, and that runx regulates axonal projection of trkcexpressing proprioceptive drg neurons (inoue et al., ) . furthermore, we announced previously that runx mrna is expressed in cranial ganglia of v, vii, viii, ix and x in mouse developmental stages. the expression was restricted to subset of neurons in each ganglion. to examine the influence of runx on the differentiation of trigeminal ganglion neurons, we investigated the expression of neurotrophin receptors, calcium binding proteins and neuropeptides in trigeminal ganglia of runx knockout mice using immunohisitochemical staining. we found the decrease of trkc-expressing neurons in trigeminal ganglia of neonatal runx knockout mice, however, we observe little change in the proportions of nuen-expressing neurons. kouko tatsumi , hirohide takebayashi , takayuki manabe , kazuhiro ikenaka , akio wanaka dept. anatomy, nara med. univ., kashihara, nara, japan; division of neurobiology and bioinformatics, nips, nins, okazaki, aichi, japan our previous study with double labeling of brdu and cell lineage markers suggested that a number of astrocytes were differentiated from resident oligodendrocyte progenitor (opcs)-like cells in the injured adult brain. and we found out that these opcs expressed ng proteoglycan and olig at early phase after injury. to directly trace the lineages of these opcs, we employed double transgenic mice that express tamoxifen-sensitive creer under the control of the olig promoter together with rosa-egfp reporter. the gfp positive cells were detected around the injured region, and the almost all of these cells co-expressed gfap at late phase after injury. furthermore, we confirmed that the morphological characteristics of these cells were those of the astrocyte by immunoelectron microscopy. our results clarified that dormant opcs in vivo differentiate into astrocytes in adult injured brain, and suggested that these cells participate in glia scar formation after brain injury. olig is a bhlh transcription factor, essential for oligodendrocytes (ols) and motoneurons differentiation in the spinal cord. however, differentiation of olig lineage cells in the forebrain is largely unknown. here we examined fates of olig expressing cells in the fetal forebrain by tamoxifen (tm)-inducible cre-loxp system. olig -creer knockin mice were mated with reporter mice, and tm was injected at embryonic day (e) . or . , when most of olig + cells are observed in the basal forebrain. the olig + cells at e . gave rise to more neuron than glia that included both ols and astrocytes. majority of neuronal olig lineage cells differentiated into gabaergic neurons, and a lesser number, into cholinergic neurons. the olig + cells at e . generated more glial cells than neurons. these results indicate that olig lineage cells generate three major types of neural cells with a stage dependent manner, and may have multiple functional roles on neural differentiation in the fetal forebrain. mana igarashi , , masato yano , , satoru hayashi , , hirotaka j. okano , , hideyuki okano , dept. physiol., keio univ. sch. med, tokyo, japan; sorst jst, japan the mammalian neuronal hu rna binding protein family is homolog of drosophila elav protein which is essential for differentiation and maintenance of the nervous system. in mammals, neuronal hu expresses in both early postmitotic and mature neurons and has ability to induce neuronal differentiation by binding to the utrs of specific target mrnas. to understand the molecular mechanism of hu induced neuronal differentiation, we purified hub associated complexes. among them, nf family, a double strand rna binding protein which is one of hu associated proteins, is known to bind to utrs of p , p and tau mrna known as hu targets. we generated rabbit polyclonal antibodies against nf and nf , binding partner of nf , respectively. in mouse embryonic brains, we found that nf / expressed highly in postmitotic neurons where neuronal hu proteins are highly distributed. moreover, we found that hu and nf / formed mrnp complexes in mouse brain extracts. we will discuss the role of hu binding partners in neuronal differentiation through post-transcriptional regulation. sachiko the pallium is specified as a homologous field in the vertebrate telencephalon. however, little is known about how species-specific pallial structures are generated during embryogenesis. to address this issue, we compared several neuronal subtypes and their migration patterns in the developing pallium of the mouse and quail. cell tracing analysis revealed that neurons born at the dorsal pallium tangentially migrated in the developing quail telencephalon, as in the mammalian cortex. next we investigated distribution of later-born neurons in the quail telencephalon using laminar specific genes (er and brn ) in the cerebral cortex. in situ hybridization and immunohisitochemical studies indicated that these neuronal markers were expressed in discrete regions of the developing quail telencephalon. our data suggest that early stages of cortex/pallium development are comparable between the mammalian and avian embryos, whereas neuronal specification in later stages is regulated by distinct mechanisms in each species. research funds: kakenhi ( ) ps a-d protein expression in hippocampal cells dissociated and re-cultured from organotypic slice cultures we established a re-cultivation technique of hippocampal cells dissociated from long-term cultured organotypic slices. protein phenotype of the cells was analyzed using immunocytochemical technique. antinestin immunoreactivity was observed in cells with short processes days in the re-cultivation. the anti-nestin immunoreactivity was progressively declined, whereas number of cells expressing anti-iii tubulin immunoreactivity increased through the re-cultivation for - weeks. presence of neurons, astrocytes and oligodenderocytes was examined using anti-iii tubulin, anti-glial acidic fibrillary protein and rip antibody, respectively. apart from the cells expressing one of the markers, the cells marked with multiple sets of antibodies were observed. these results suggest that protein expression was changed backward in normal differentiation course in hippocampal cells once matured in organotypic slices. we have shown that perineuronal ng + cells are major populations of proliferating cells in the cerebral cortex of rats. in the adult cortex, ng is known as a marker for oligodendrocyte progenitor cells (opcs) that retain ability to proliferate and differentiate into new oligodendrocytes. however, it is still unclear whether all ng + cells in the neocortex are the opcs. we investigated about subtypes of ng + cells found in the perineuronal regions of the cerebral cortex using cell markers. two subtypes of perineuronal ng + cells could be distinguished by the subcellular localization of gst-protein. one is nuclear type, the other is cytoplasmic type. only the nuclear gst-+ cells have the proliferative activity. these data suggest that the nuclear gst-+ /ng + cells in the perineuronal territory are progenitor cells engaging in reproduction of cortical cells. muguruma keiko, su hong-lin, matsuo-takasaki mami, watanabe kiichi, sasai yoshiki neurogenesis and organogenesis group, riken center for developmental biology, kobe, japan in this study, we report in vitro generation of math + cerebellar granule cell precursors and purkinje cells from es cells by using soluble patterning signals. when neural progenitors induced from es cells in a serum-free suspension culture are subsequently treated with bmp and wnt a, a significant proportion of these neural cells become math + . the induced math + cells mitotically active and express markers characteristic of granule cells precursors (pax , zic , and zipro ). after purification by facs and coculture with postnatal cerebellar neurons, es cell-derived math + cells exhibit typical features of neurons of the external granule cells layer, including extensive motility and a t-shaped morphology. interestingly, differentiation of l + /calbindin-d k + neurons (characteristic of purkinje cells) is induced under similar culture conditions but exhibits a higher degree of enhancement by fgf rather than by wnt a. this is the first report of in vitro recapitulation of cerebellar neurons by using the es cell system. sachiyo misumi, kim hye-jung, hideki hida, hitoo nishino department of neurophysiology and brain science, nagoya city university graduate school of medical science, nagoya, japan regulation of the cell cycle plays an important role in cell proliferation, differentiation, and apoptosis. we have shown that pretreatment with cell cycle blocker increase the number of neurons from neural stem or progenitor cells (npcs) without influencing apoptosis after differentiation. in this study, we investigate the molecular mechanism of neuronal differentiation by cell cycle arrest. in rt-pcr, the expression of p cip , p kip and p kip mrnas were elevated during differentiation to neuron from npcs. especially, prolonged enhancement of p kip mrna was shown. transfection of p kip into npcs induced activation of neurod promoter and increase of number of tublin iii-positive cells. treatment with deferoxamine to npcs from e . rat midbrain and hb .f cell line did not activate erk and akt phosphorylation during the treatment. date suggest that prolonged p kip elevation is related to enhanced production of neuron from npcs, and that cell cycle regulation in g /s phase did not activate mapk and pi -k signaling. yuichi tanaka , yusuke tozuka , dai muramatsu , kin-ichi nakashima , tatsuhiro hisatsune departement of integrated biosciences, graduate school of frontier sciences, university of tokyo, kashiwa, japan; graduate school of biological sciences, nara institute of science and technology, ikoma, japan we previously reported no definite evidence for in vivo neurogenesis in adult neocortex. however, we also confirmed dividing cells in this area. in this study, we analyzed the characteristics of adult cortical nestin+ cells. in vivo, they belonged to ng + and olig + cells, showed slowly proliferating ability compared to those in adult dentate gyrus. for in vitro analysis, we precisely isolated progenitor cells by percoll gradient procedure. they differentiated into tuj- + or map- + neuronal cells by adding retinoic acid or bdnf. more than % of newborn neurons expressed gabaergic neuronal markers, gaba, gad or calretinin. we also purified nestin-gfp+ cells from nestin-gfp transgenic mice using the facs system, and confirmed their neuronal potential. moreover, integration of a neural bhlh transcription factor neurod significantly promoted this neurogenesis. we demonstrated neurogenic potential of adult cortical nestin+ cells. mie gangi , michiko imanishi , teiko kuroda , masao tachibana , masahiko takada department of psychology, graduate school of humanities & sociology, university of tokyo, tokyo, japan; tokyo metropolitan institute for neuroscience, tokyo metropolitan organization for medical research, tokyo, japan a kv subfamily of voltage-gated k + channels is thought to play an important role in high-frequency repetitive firings. it is unknown which subtype of kv channels is expressed in the frog retina where ␥-range oscillatory spikes are evoked presynaptically by light stimulation. we found immunohistochemically that kv . b and kv . were expressed both in the mouse and frog retinas. however, a laminar pattern with two bands in the inner plexiform layer was displayed by kv . in the frog retina and by kv . b in the mouse retina. it has been shown that mammalian cholinergic amacrine cells express kv . b. thus, the differential expression of kv channels may reflect their functional diversity between the frog and mouse retinas. hiroshi jouhou , , kazunori yamamoto , masayuki hara , akinori homma , akimichi kaneko , masahiro yamada tokyo metropolitan univ., hino, tokyo, japan; astellas pharma. inc., osaka, japan; sch. rehabili., seijoh univ., aichi, japan in order to interpret the formation of receptive field surrounds in the retinal neurons, hirasawa and kaneko ( ) proposed a phmediated mechanism to substitute for the gaba-mediated feedback hypothesis from horizontal cells (hcs) to cone photoreceptors. to verify the idea that the depolarized hcs release protons we measured, by a fluorescent ratio imaging technique, the ph of the immediate external surface (ph o ) of hcs isolated from carp or goldfish retina. when hcs stained by -hexadecanoylaminofluorescein, a phsensitive lipophilic dye, were depolarized by application of kainate or by high extracellular k + , ph o acidified. the amount of ph o acidification was monotonically dependent on the amount of depolarization, as much as . ± . ph unit by mm k + . acidification of pho was suppressed by . m bafilomycin a , a specific inhibitor of v-atpase, suggesting that the hc depolarization enhanced an outward proton movement by the outward electrogenic h + pump. ps a-e analysis of spread of activity in the local circuit of superior colliculus by using multi-channel field potential recording system penphimon phongphanphanee, katsuyuki kaneda, tadashi isa national institute for physiological sciences, japan to study how the visual signal is processed in the local circuit of superior colliculus (sc) from the superficial layers (ssc) to the deeper layers (dsc), we analyzed the propagation of excitation following the electrical stimulation of the ssc by using a planar -channel field potential recording system in slice preparations obtained from to days old mice. stimulation at ssc induced negative field potential with short latency and short duration ( - ms) at the recording site in ssc adjacent to the stimulating site. after application of bath containing m bicuculline, the same stimulus induced a large negative field response with long duration ( - ms) that spreads laterally in ssc and ventrally to dsc. these responses disappeared after application of m apv, when only short latency response remained. the results suggest that when gaba a receptormediated inhibition is reduced, visual signal in the ssc propagates to the dsc as large response with long duration and nmda receptors contribute to propagation of the response. osamu hosoya , ken tsutsui , kimiko tsutsui dept. of neurobiol. and neuroanat., okayama univ. grad. sch. of med., dent., and pharm. sci., japan; dept. of genomics and proteomics, okayama univ. grad. sch. of med., dent., and pharm. sci., japan amphiphysin ir (amph ir) is alternatively spliced variants of amphiphysin i which is specifically expressed in retina. amph ir is composed of conserved domains including the n-terminal bar, the central clathrin/ap- binding, and the c-terminal sh domains and the variant specific two novel insertions (a and b). insert a may be a determinant for the retina-specific expression. insert b has no significant homology to known proteins and two shorter transcripts with -truncations in the insert were also expressed. recently, we found that a human retinal pigment epithelia cell line, arpe- , also expresses amph ir. arpe- thus can be a useful tool for investigating the cellular function of amph ir in retina. immunofluorescence analyses with arpe- cells revealed that amph ir occasionally colocalized with mitochondria, raising the possibility that amph ir may participate in structural or functional organization of mitochondria. further characterization of the variant is under investigation. hironori takamura , satoshi ichisaka , chihiro hayashi , hirotoshi maki , yoshio hata div. integrative biosci., tottori univ. grad. sch. med. sci., yonago, japan; div. neurobiol., sch. life sci., fac. med., tottori univ., yonago, japan monocular deprivation (md) induces significant plasticity in the primary visual cortex (v ) during critical period. it was reported that inhibition of extracellular signal-regulated kinase (erk) activity in the visual cortex suppressed the ocular dominance plasticity. if erk is involved in the mechanism of this plasticity, visual deprivation would change the activity of erk in v and such change might be induced only in the critical period. to test this possibility, we examined effects of md on the amount of phosphorylated (activated) erk (perk) in the rat visual cortex. by md, we found a significant decrease in the amount of perk in v receiving deprived eye inputs in both young and adult rats. as to the subcellular localization of erk, we found a significant increase of the nuclear perk only after md in young rats. these results suggest that erk signaling might be regulated by different mechanism between young and adult rats. research funds: kakenhi ( ) ps a-e rapid pre-synaptic weakening by experiencedependent competition in mouse visual cortex nobuko mataga, yoko mizuguchi, takao hensch neuronal circuit development, riken brain science institute, saitama, japan in the binocular zone (bz) of mouse visual cortex, critical period (cp) plasticity is accompanied by a transient loss of spines on pyramidal cell dendrites. to explore a correspondingly rapid and local pre-synaptic refinement by sensory deprivation, excitatory intracortical or thalamocortical axon terminals were visualized in the bz by vesicular glutamate transporters (vglut) and vglut , respectively. a complementary distribution of vglut and vglut was established by postnatal day (p) and both signal intensities increased further by p - (peak cp). the immunoreactivity for vglut decreased around layer iv after brief monocular deprivation ( dmd) during the cp. interestingly, both signals in all layers were lower in the bz contralateral to an eye injected with ttx than in the ipsilateral bz, consistent with the stronger functional plasticity and rapid dendritic refinement as compared to dmd. these results suggest that rapid and local weakening of excitatory inputs corresponds to dendritic spine pruning during experience-dependent competition. reiko meguro, masao norita department of sensory and integrative medicine, niigata university, graduate school of medical and dental sciences, niigata, japan we investigated how the geniculate and the extra-geniculate visual systems reorganize by monocular deprivation at birth. using anterograde/retrograde tracer, biotinylated dextran amine (bda), we made a small injection into the dorsal lateral geniculate nucleus (dlgn) or the lateral posterior nucleus (lp) of the degenerated side of the monocular deprived rat. the geniculate projection terminated mainly in layer iv of area , with a small projection to layer vi of areas and a. cells in layer vi of area projected to dlgn. in addition, cells in layer v of area projected to dlgn, which is not observed in normal rats. in area a, cells in layers v and vi projected to dlgn. the projection from lp terminated mainly in layer iv of a. cells in layers v and vi of area a projected to lp. smaller number of cells in layer v of area also projected to lp. these findings suggest that major parts of visual system developed normally, but some developed cross talk between geniculate and extra-geniculate systems. ps a-e activity dependent plasticity of feedback projection from the primary visual cortex to the dorsal lateral geniculate nucleus miho yoshida , takemasa satoh , yoshio hata div. integrative biosci., tottori univ. grad. sch. med. sci., yonago, japan; div. neurobiol., sch. life sci., fac. med., tottori univ., yonago, japan the projection from the lateral geniculate nucleus (lgn) to the primary visual cortex (v ) shows significant morphological plasticity responding to visual experiences in early life. such experiencedependent plasticity enables the geniculocortical projection to form functionally specific connections. it is not clear whether similar plasticity operates in other neural connections in the visual system. therefore, we tried to investigate the plasticity of feedback projection from v to the lgn. we focused on the density of type metabotropic glutamate receptor ␣ (mglur ␣) in the lgn which locate postsynaptically at synapses of feedback projection. immunohistochemical signal for mglur ␣ in the lgn decreased after elimination of v , showing that this signal reflects density of functional feedback synapses. to explore the activity dependent plasticity, we examined the effect of cortical activity blockade on the mglur ␣ signal in the lgn of young rats. yu morishima , hiroshi sakamoto , takahumi akasaki , yoshio hata div. integrative biosci., tottori univ. grad. sch. med. sci., japan; div. neurobiol., sch. life sci., fac. med., tottori univ., japan monocular deprivation (md) during postnatal development reduces cortical response to the deprived eye and input axons serving the deprived eye retract. however, when md is combined with continuous inactivation of the visual cortex by muscimol, cortical neurons lose their response to the open eye and the open eye axons retract. to clarify mechanisms underlying the two forms of ocular dominance (od) plasticity in different direction, we examined other characteristics of them, ( ) how rapidly the reverse od shift proceeds and ( ) whether the shift is induced only in young animals. we infused the cortex with muscimol in -week-old kittens and in adults. the reverse od shift was observed after days md, but not significant after days md. in adults, od distribution remained unchanged. morphological change of individual input axons was also examined after days md. the reverse od shift might reflect a mechanism of developmental plasticity that has a slower time course than the normal od shift. ps a-e experience-dependent plasticity in the absence of ampa receptor subunits in mouse visual cortex youichi iwai , nafiseh atapour , john renger , john roder , peter seeburg , takao hensch neuronal circuit dev., riken, bsi, wako, japan; mount sinai hospital, toronto, canada; max planck inst. med. res., heidelberg, germany two ampa receptor subunits (glur , ) play prominent roles in hippocampal models of homosynaptic plasticity (ltp/ltd). brief monocular deprivation ( d md) rapidly alters both the phosphorylation state and surface expression of glur in visual cortex. here, we addressed whether these coincidental events are essential for subsequent ocular dominance (od) plasticity. mice lacking glur (ko) displayed little ltd in visual cortical slices, while baseline transmission was normal. they also exhibited normal visual receptive field properties in vivo and shifted responsiveness toward the open eye after d md during a typical critical period. the rate of plasticity appeared somewhat slowed, as d md eventually led to full od shifts. in glur ko mice, even d md robustly activated od plasticity. thus, experience-dependent modification of ampa receptors is not essential for plasticity in vivo, although glur may contribute to the very earliest stages. shigeyoshi higo, nobuaki tamamaki department of morphological neural science, kumamoto university, kumamoto, japan virus-assisted transduction with reporter genes is a useful technique to investigate morphology of neurons in the central nervous system. however, the mechanisms to induce reporter expression in vivo often depend on gene-manipulated mice. since mice are not the best experimental animal for the study of mental disorder, we developed an adenovirus in which gfp expression is driven by dlx promotor and dlx / enhancer. this virus labels gabaergic neurons and oligodendrocyte in the wild-type mouse neocortex and allows us to trace gfp-labeled axons of gabaergic neurons in serial brain sections. we used this virus to investigate gabaergic neurons with long projecting axon branches beyond a functional area. the virus was injected into the stratum oriens of the mouse hippocampal field ca and revealed a nonpyramidal neuron projecting to ca and fimbria. further we shall introduce this virus to the cat brain and investigate axon branches of gabaergic projection neurons in the neocortex. akiko yamashita , takao oishi , motoharu hayashi div. appl. system neurosci., nihon univ. grad. sch. med. sci., tokyo, japan; dept. cell. and mol. biol., primate res. inst., kyoto univ., inuyama, japan gabaergic cells in the cerebral cortex are divided into subgroups: parvalbumin (pv)-, somatostatin (som)-, calretinin (cr)-, and calbindin-containing types. to clarify inhibitory system in primates, we determined coexistence of these molecules and proportions of these subtypes within gabaergic cells in the various cortical areas. pv, som or cr did not coexist with each other in primates as observed in rodents. more than % of gabaergic cells contained pv; showing that pv cells are more abundant in primates than in rodents. proportion of som cells in gabaergic cells was smaller in the primary visual area ( . %) than in other areas, such as the prefrontal ( . %), primary motor ( . %), somatosensory ( . %) and secondary visual areas ( . %), indicating cortical differentiation in gabaergic system of the primate cerebrum. our recent retrograde labeling studies in mice and cats showed that the neocortical areas are connected not only by excitatory neurons but also by gabaergic projection neurons. in order to address the importance of the gabaergic projection neurons in the neocortical information processing, we need to know the branching pattern and postsynaptic elements of the gabaergic projection axons. since more than % of the gabaergic projection neurons showed npy immunoreactivity, we used npy-cre transgenic mouse that express cre in npy neurons and adenovirus that encodes gfp in the downstream of floxed stop to label the gabeergic projection axons. after injection of the adenovirus into deep layers of the npy-cre mouse neocortex and immunoperoxidase staining of gfp in the brain section, we could reveal gabaergic neurons in a golgi-like image with their axons. also this method seemed to allow us to label gabaergic projection neurons retrogradely. koji ikezoe , guy n. elston , , tomofumi oga , hiroshi tamura , , ichiro fujita , osaka univ., japan; univ. queensland, australia; crest, jst, japan layer iii pyramidal cells in adult monkeys exhibit systematic differences in their dendritic morphology among cortical areas. basal dendrites of cells in visual association cortex such as inferior temporal area te spread more extensively and are more branched than those in the primary visual cortex (v ). pyramidal cells in prefrontal cortex, such as area , have even more dendritic branches than those in area te. here, we investigated whether a similar regional difference in the dendritic morphology was present in infant monkeys. we stained individual layer iii pyramidal cells in v (n = ), area te (n = ), and area (n = ) of a -week old monkey (macaca fascicularis) using intracellular dye-injection techniques in lightly fixed tissues. the number of branches and the tangential extent of dendrites was greatest in area , followed by area te, and v . thus, considerable heterogeneity in pyramidal cell structure already exists -weeks after birth. hiroaki matsushita , mahito ohkuma , masami watanabe , ei-ichi miyachi department of physiology, fujita health university, aichi, japan; department of perinatology, institute developmental research, aichi, japan acetylcholine (ach) receptors are believed to be expressed in developmental and regenerative process of retinal neurons. we performed the patch-clamp recording and fura- based calcium imaging in cat retinal ganglion cells (rgcs). under whole cell clamp conditions, transient sodium currents and action potentials were observed in all of normal or axonal regenerated rgcs. however, these currents and spikes were not observed in the % of axotomized rgcs. bath application of m carbachol, an ach receptor agonist, rose [ca + ] i in % of normal rgcs. although the % of rgcs responded to carbachol at days after axotomy, no responsive rgcs appeared during - days. ach responsiveness recovered in axonal regenerated rgcs ( %). since pycnotic cells were observed few days after axotomy, ach may modulate neurotrophic effect in survived rgcs. these results suggest that ach is an important marker for neuronal degeneration and regeneration in cat rgcs. research funds: kakenhi ( to em, to mw) kenichiro miura, masakatsu taki, hiromitsu tabata, kenji kawano dept. integrative brain sci., grad. schl. of med., kyoto univ., kyoto, japan the initiation of smooth pursuit eye movements is facilitated by the bottom-up attention to the target (hashimoto et al., ) . to study the effects of the attention on the processing of second-order motion stimuli, we recorded smooth pursuits in three humans with a dualpurkinje-image eye tracker. the pursuit target, presented on a crt monitor ( hz), was a gaussian patch of texture displayed on a neutral gray background. the gaussian envelope moved at deg/s, while the texture consisting of black and white random-noise pixel blocks remained stationary (drift-balanced stimulus). the number of the frames displaying the target before the motion onset was selected to manipulate the attention to the target, either eight frames ( ms) or only one frame ( ms). the initial tracking responses were larger when the target became visible eight frames before the motion onset. the result suggests that the second motion processing underlying the smooth pursuit initiation is facilitated by the attention to the target. ps a-e motion picture effects on eye movements and blood flow in the frontal area atsuhiko iijima , , tohru kiryu , kazuhiko ukai , takeshiko bando div. integrative physiol., grad. sch. med., niigata univ., niigata, japan; div. inform. sci., grad. sch. & tech., niigata univ., niigata, japan; dept. appl. phys., sch. sci. & tech., waseda univ., tokyo, japan motion pictures taken by rider's view of motocross bike elicited horizontal eye movements coherent to the motion vectors in some subjects, and not coherent in the other subjects, while those taken by passenger's view of roller coaster evoked similar eye movements in all of the subjects. subjects watched the two-dimensional motion pictures in random order. eye movements were measured by a binocular video oculography (newopto), and head movements were measured by a magnetic motion sensor (polhemus). blood flow in the frontal area was simultaneously monitored with a near infrared spectroscopy (hamamatsu). the patterns of eye movements and the blood flow variation during movie presentation changed in relation to motion components of the movie. possible mechanisms of the differences will be discussed. kiyoto matsuura , kenichiro miura , masakatsu taki , hiromitsu tabata , naoko inaba , kenji kawano , frederick a. miles grad. schl. med., kyoto univ., kyoto, japan; lab. sensorimotor res., nei, nih, bethesda, md, usa human ofrs show winner-take-all behavior when elicited by moving grating patterns composed of two sinusoids (sheliga et al., sfn ) . we recorded the ofrs to the motion of vertical grating patterns composed of two sinusoids of spatial frequency f and f, which created a repeating pattern with beat frequency, f, in two monkeys. motion consisted of successive steps ( hz), each one-fourth of the wavelength of the beat, so that with each step the two components shifted one-fourth of their wavelengths and had opposite directions, the f forwards and the f backwards. the contrast of the f was fixed at , , or %, while the contrast of the f was varied from one-fourth to four times the contrast of the f. when the contrast of the f ( f) was less than about half that of the f ( f), the f ( f) dominated initial ofr: winner-take-all. thus, the motion processing underlying the ofr in monkeys, like that in humans, includes nonlinear interactions. masazumi katayama, takahiro fujita department of human and artificial intelligence systems, faculty of engineering, university of fukui, fukuki, japan when executing prehension movement to grasp an object such as a tool, we plans the hand shape and grasping position to grasp a target object. while, goodale proposes the hypothesis that the roles of two visual streams (dorsal and ventral streams) are "vision for action" and "vision for perception", respectively. from the above points of view, we investigated independence of visual estimation and motor execution for grasping position of a target object. in this experiment, grasping positions were measured under the following four conditions: visual estimation without grasping, grasping without lift-up movement, grasping and lift-up movement and visual estimation without grasping. as a result, we found that grasping positions of visual estimation are significantly different from grasping positions of motor execution in the second and third conditions (p < . ). we concluded that grasping positions of visual estimation and motor execution are independent and these results support the goodale's hypothesis. ryuichi hishida, masaharu kudoh, katsuei shibuki dept. neurophysiol., brain res. inst. niigata univ., niigata, japan cortical sensory areas are divided into modality-specific domains such as the visual and auditory cortices, in which sensory neurons are driven by modality-specific inputs. recently, wallace et al. found that multimodal neurons clustered in deep layers are present near the borders between sensory cortices. multimodal properties of these neurons may be explained by three types of inputs: overlapped projections from the thalamus, projections from multi-modal sites, or overlapped horizontal projections from the modality-specific sensory cortices. in this study, we tested the third possibility. we prepared the mouse cortical slices including the visual and auditory cortices. the horizontal activity propagation elicited by local electrical stimulation were visualized using flavoprotein fluorescence imaging. these results indicate that cortical areas between the visual and auditory cortices receive horizontal projections originated in the visual and auditory cortices, suggesting that multimodal horizontal connections are important for the multimodal properties of sensory neurons. jumpei naito , yaoxing chen , yukiko tanada dept. animal sci., teikyo univ. sci. & tech., uenohara, japan; china agricul. univ., beijing, china twenty white-leghorn chicks (p - ) were perfused with % paraformaldehyde through the heart under deep anesthesia of nembutal ( mg/kg bw). two to three small crystals of dii were implanted into the optic tectum, thalamus, or hypothalamus under a dissecting microscope. a total of rgcs were classified into six groups according to the somal area and dendritic field (naito and chen, ). group ic projected dominantly to the tectum. group is and iiis showed high hypothalamic-and thalamic-dominance, respectively. group iic was non-specific in the central projections. group ivc was tectal-dominant. patterns of the dendritic stratification were counted to in tec-rgcs, in tha-rgcs, and in hyo-rgcs. of these stratification patterns, many patterns were common among tec-, tha-, and hyp-rgcs. in contrast, the rgcs that showed a same dendritic pattern were consisted of a single rgc in most of the non-common rgcs, and their dendrites extended mainly to the superficial inner plexiform layer (sublayers - ). yasuro atoji, shouichiro saito laboratory of veterinary anatomy, gifu university, gifu, japan the present study was examined afferent and efferent fiber connections of the intermediate part of the caudal nidopallium (nci) in the pigeon by a tract-tracing method. in the present study we define nci an area which is located lateral to the field l complex and ventral to ncl. following a ctb injection into nci, a large number of neurons was labeled in nci, the mesopallium, and intermediate arcopallium (ai) and in the thalamic posterior dorsointermediate and posterior dorsolateral nuclei. contralateral ai contained a small number of labeled neurons. a few labeled neurons were found in lst. few labeled cells were found in ncl, field l, piriform cortex, or hippocampal formation. following a bda injection into nci, a large number of labeled fibers extended in nci, mesopallium, and ai. lst contained a small number of labeled fibers. few labeled fibers were located in ncv and limbic regions. the diencephalon contained very few labeled fibers. in summary, nci has strong reciprocal connections within nci itself and with the mesopallium and ai, and little connections with the limbic system. hidenori horie , kenji yuda , eiichi okawa , katsuyoshi maruyama , hiroshi uozato , hiroko horie , satomi nakajima , kenkichi tanioka , yuji ohkawa , tomoki matsubara , wolfram tetzlaff advanced res. centr. biological sci., waseda univ., tokyo, japan; technomaster co. ltd., yokohama, japan; kikuna yuda eye clinic, yokohama, japan; healthcare business co., matsushita electric ind. co. ltd., yokohama, japan; dept. ophthalmol. & visual sci., kitasato univ., kanagawa, japan; nhk sci. technical res. labo., tokyo, japan; icord, univ. british columbia, vancouver, canada we describe here a highly effective method to improve visual acuity of children with myopia and adult with presbyopia by repeatedly offering a visual object at variable distances while keeping the apparent retinal projection size of the object constant using a novel electronic device. in our experiments on human subjects, we used an lcd screen that was rhythmically moved between and cm toward and away in a high speed (top speed: mm/s) from the subjects. the device significantly improved visual performances in over % of the school-aged children with myopia and % of adults with presbyopia. hiroyuki miyamoto , toral s. surti , takao k hensch laboratory for neuronal circuit development, riken brain science institute, wako, japan; san francisco, usa competitive plasticity of binocular response following monocular deprivation (md) is prominent in the primary visual cortex (v ) during an early critical period. recently, md has been shown to enhance head-tracking behavior induced by slow rotation of grating stimuli in adult mice and is critically dependent upon the integrity of v . here, we addressed to what extent these two types of plasticity induced by the same md share common mechanisms. adult mice lacking a gaba-synthetic enzyme (gad ko), which do not exhibit ocular dominance (od) plasticity by brief md during the critical period, showed normal optomotor acuity and enhancement with day md. od shifts did not correlate with optomotor enhancement in these mice. finally, early md spanning the entire critical period had no effect on optomotor acuity through the deprived-eye. these observations support the view that adult perceptual learning and classical od plasticity are independent. junya hirokawa , miquel bosch , shuzo sakata , yoshio sakurai , tetsuo yamamori division of brain biology, national institute for basic biology, okazaki, japan; mit, ma, usa; rutgers university, nj, usa; department of psychology, kyoto university, japan the brain is able to integrate information from different sensory sources to enhance behavioral responses. to identify the neuronal populations responsible for multisensory enhancement in rats, we have mapped the activation of neurons during an audiovisual integration paradigm (sakata, et al., ) by the expression of c-fos. a pronounced c-fos upregulation was found in superior colliculus and in layer iv and deep layer of latero-medial secondary visual area (v lm). local injection of gaba agonist muscimol into this region selectively suppressed the behavioral enhancement related to multisensory integration, while no suppression was found by the injection into primary auditory and visual areas. these results suggest a key role of v lm in integration of auditory and visual information to facilitate the behavioral reaction for bimodal stimuli. takashi shinozaki , youichi miyawaki , tsunehiro takeda department of complexity science and engineering, university of tokyo, chiba, japan; mathematical neuroscience, riken bsi, saitama, japan drifting grating patterns with different motion directions independently presented to the two eyes induce two sets of perceptual rivalries: interocular rivalry (left or right eye's image) and motiontype rivalry (pattern or component motion). we studied this double rivalry process based on psychophysical and magnetoencephalography (meg) measurements. pattern-motion percept exclusively arose and persisted for a long duration whereas component-motion percept was soon followed by percept of either of left or right eye's single motion direction. reaction time (rt) measurement showed that the pattern-motion was perceived faster than left or right eye's motion direction. we then compared meg signals among those perceptual conditions and found a meg response of interocular rivalry in the latency range expected from the result of rt measurement. these results suggest that the double rivalry process has a hierarchical structure in which motion-type rivalry is resolved before interocular rivalry. visual stimuli evoke several brain potentials with relatively precise time courses. the role of these brain potentials in visual object categorization is not clear. in this study we recorded event related brain potentials (erp) while subjects participated in a face/non-face categorization task. gray face and non-face natural object images were presented briefly ( ms) followed by a noise mask with pseudo randomly selected stimulus onset asynchrony (soa = - ms). subjects reported presentation of face or non-face images by pushing one of the two assigned keys. we found that the face category discrimination performance significantly declined only in short soa ( and ms) with a larger impact of masking on non-face discrimination. in erp, the peak amplitude and latency of p , n and area under curve of a late positive potential expanding from to ms were correlated with the subjects behavioral performance. the effect of backward masking on early erp components may be due to altering sensory processing of visual stimuli while the effect on late erp potential could be related to its impact on decision making processes. yasushi naruse , ayumu matani , , tomoe hayakawa , , norio fujimaki university of tokyo, kashiwa, japan; nict, kobe, japan; teikyo university, tokyo, japan to study the process of alpha rhythm resetting, we investigated the relationship between visual evoked potential and the seamlessness: how much the phase angle of prestimulus alpha rhythm and the backward-extrapolated phase angle from poststimulus alpha ringing synchronize. alpha ringing is an evoked potential in alpha frequency band around ms in latency. eight clinically normal adult volunteers participated in the experiment, in which the subjects passively viewed a series of flash stimuli with their eyelids closed throughout the experiment. eeg was simultaneously recorded during the experiment. we classified the trials into four subsets owing to the seamlessness, and then averaged the trials in each subset. the result showed that the larger the amount of the alpha rhythm resetting is, the larger the p amplitude becomes. this suggested that a factor of the variability of the p amplitude is the amount of the prestimulus alpha rhythm resetting. research funds: a grant-in-aid from the ministry of education, culture, sports, science and technology (no. ) hitoshi sasaki , takuya ishida , masayoshi todorokihara , junichiro miyachi , tahei kitamura , ryozo aoki dept. physiol. & biosignal., osaka univ. grad. sch. med., suita, japan; dept. phys. & elec., osaka pref. univ. grad. sch. eng., sakai, japan; dept. elec. eng. & elec., col. industri. tech., amagasaki, japan recently it has been shown that noise can improve detection of sensory stimuli in several modalities. here we investigated whether visual contrast detection sensitivity can be improved by adding a certain amount of noise. contrast detection thresholds of a light changing brightness periodically were measured with or without overlapping noise in five normal participants. the contrast detection threshold, measured by using the psychophysical method (up-anddown method), decreased at around the threshold level of the noise intensity. these findings are consistent with our previous findings obtained by using another psychophysical method and confirm that noise can improve signal detection in human visual perception. narumi katsuyama , nobuo usui , izuru nose , , masato taira , department of applied system neuroscience, nihon university school of medical science, tokyo; faculty of human science, bunkyo university, saitama, japan; arish, nihon university, tokyo, japan when an object is moving, perception of its d trajectory in depth can be strongly influenced by the trajectory of its cast shadow. for example, a ball moving in a diagonal trajectory can appear to rise in a frontal plane when the shadow moves along the horizontal trajectory (rising configuration) or to roll in depth when the shadow follows the same trajectory as the ball, while the trajectory of ball is identical. using fmri, we found that several visual areas, including human mt and the posterior sts and the posterior parietal cortex, are activated in the comparison between rising configuration and ball only condition. additional correlation analysis by modifying the slope of the shadow' s trajectory also showed activation in the posterior part of sts and the posterior parietal cortex, including precuneus. these results suggest that cortical areas in the temporal and parietal cortex might be involved in the processing of apparent motion of ball induced by the moving cast shadow. ps a-f local area network in the gerbil's auditory cortex: reversible focal inactivation with infrared laser irradiation akira yamamoto, hiroshi riquimaroux gratuate school of engineering, doshisha university, scnrl, japan this study investigated local area networks in the primary auditory cortex (a ) and the anterior auditory field (aaf) by blocking neural activities with the near-infrared laser irradiation (wave length = nm). in previous in vivo studies, the laser irradiation could focally inactivate neural activities in a few minutes after the irradiation started, while the activities recovered in a few minutes after its cessation. by using this technique, the present study examined corticocotical relationships in the auditory cortex of the mongolian gerbils (meriones unguiculatus). cf (constant frequency) and fm (frequency modulated) tones were presented to anesthetized animals, and neural responses were extracellularly recorded contralaterally to the ear of stimulation. when irradiated aaf area and recorded neural responses from ai, the irradiation changed phasic responses into tonic responses, and vice versa. these results indicate that there are functional connections within ai or aaf, and between ai and aaf. takashi doi, hiroshi riquimaroux department of knowledge and engineering and computer sciences, doshisha university, kyoto, japan in a previous behavioral study, ablation of right auditory cortex (ac) made the discrimination between ascending and descending frequency modulated (fm) tones by mongolian gerbil (meriones unguiculatus) difficult (wetzel et al., ) . this result indicates that some neurons in gerbil's right ac represent the directions of fm sweeps. actually, we could find direction-dependent neurons and these neurons were mainly in anterior auditory field (aaf). in aaf, bfs are gradually shifted along the rostrocaudal direction, and the same bfs are arranged in dorsoventral direction (thomas et al., ) . moreover, aaf has dense synaptic connections within the area (budinger et al., ) . we made network models based on this structure of aaf and could gain similar responses to the actual responses of directiondependent neurons. this result suggests that aaf in gerbil's ac has good structure to process fm tones. research funds: a grant to rcast at doshisha univ. from mext, innovative cluster creation project by mext it has been demonstrated that the auditory space, namely the direction of a sound source, is represented topographically in the mammalian superior colliculus (sc). however, it is unclear as to how this auditory space map of the mammalian sc is formed in the auditory pathway. the present study investigated the topographical representation of auditory space in the external nucleus of the inferior colliculus (icx) of anesthetized gerbils. the icx is the major auditory nucleus that has projections to the sc. the stimuli were -ms noise bursts whose azimuths varied on the horizontal plane in the virtual acoustic space. single-unit responses were recorded from the icx. the majority of units exhibited some degree of spatial selectivity and preference for the azimuth contralateral to the recorded side. for supra-threshold stimulus only, there were topographical gradients of preferred azimuths in the icx. however, the spatial tuning width and preferred azimuth of the units depended markedly on stimulus level. the results indicate that in mammals, the formation of a rigorous auditory space map is incomplete at the icx level. manabu toyoshima , yasuo takeda , yasushi shimoda , kazutada watanabe department of bioengineering, nagaoka university of technology, nagaoka, japan; department of clinical pharmacy and pharmacology, kagoshima university, kagoshima, japan nb- that we isolated and identified is a neural cell recognition molecule belonging to contactin subgroup. we reported previously that nb- expression is prominent in the auditory system. nb- knockout mice exhibit impaired neural function in the auditory system. these findings indicate that nb- is indispensable for the function of auditory system. here we report the detailed analysis of the nb- localization using anti-nb- monoclonal antibody that we produced recently. immunohistochemical analysis of the rat brain showed that nb- was detected not only in all brain regions of the auditory pathway, but also in substantia nigra (sn), caudate putamen (cpu) and fibers projecting from sn to cpu. the nb- immunopositive cells in sn are restricted to gabaergic neurons. since gabaergic neurons play essential roles in the development and function of the auditory system, it is highly likely that nb- regulates the development and/or function of gabaergic neuron in the auditory pathway. reiko nagashima, kiyokazu ogita department of pharmacology, setsunan university faculty of pharmaceutical sciences, osaka, japan sensorineural hearing loss can be caused by a variety of insults, including acoustic trauma. there is compelling evidence that reactive oxygen species (ros) are formed in the cochlea during acoustic stimulation. glutathione (gsh) protects against the hearing loss through scavenging ros generated by noise. in this study, we investigated the changes in expression of gamma-glutamylcysteine synthetase (gcs) gene, which is the rate-limiting enzyme in de novo gsh synthesis, in the cochlea following acoustic stimulation. nuclear extracts were prepared from the cochlea at various time points after acoustic stimulation ( khz octave band, db, h), and then subjected to electrophoretic mobility shift assay to determine activator protein- (ap- ) dna binding. ap- binding was increased - h after the exposure. rt-pcr and immunostaining revealed that noise exposure was effective in elevating the expression of gcs in the cochlea h later. taken together, ap- may participate in the expression of gcs gene in the cochlea after acoustic stimulation. masaharu kudoh, ryuichi hishida, katsuei shibuki department of neurophysiology, brain research institute, niigata university, niigata, japan multiple formants compose vowels. we have previously reported that bilateral lesions including in the auditory cortex (ac) of rats impaired discrimination learning between synthesized vowel-like sounds with multiple formants, while discrimination between stimuli of a single formant or pure tones was not significantly impaired. in the present study, we determined the responsible auditory fields, which were required for the discrimination leaning between vowel-like sounds. water-deprived rats were trained to discriminate between two sounds including four different formants. licking a spout during presentation of one sound was rewarded with water while the other was not. surprisingly, local lesions in the primary ac or the ventral association cortex had no clear effect on the discrimination learning. in contrast, the dorsal association areas impaired the discrimination learning. these findings indicate that the dorsal auditory association cortex plays a critical role in discrimination learning of vowel-like sounds with multiple formants. hiroaki tsukano, yamato kubota, manavu tohmi, masaharu kudoh, katsuei shibuki department of neurophysiol, brain research institute, niigata university, niigata, japan we used transcranial flavoprotein fluorescence imaging for visualizing cortical responses to missing fundamentals in mice. c bl/ mice were anesthetized with urethane. the skull on the auditory cortex was exposed and covered with liquid paraffin to keep the skull transparent. cortical images of green fluorescence in blue light were recorded by a cooled ccd camera. responses in the auditory cortex elicited by sound stimuli ( - khz for ms) exhibited mirrorsymmetrical tonotopic maps in the primary auditory cortex (ai) and anterior auditory field. the activity patterns in ai elicited by khz were different from those elicited by or khz. however, the areas activated by khz were also activated by the mixture of plus khz but not by that of plus khz, suggesting that cortical responses to missing fundamentals in ai were visualized using flavoprotein fluorescence imaging. hiroko kosaki national priting bureau, tokyo, japan we constructed a functional scheme of macaque auditory by distribution of calcium binding protein, parvalbumin (pv). auditory cortex is consisted of one core (primary cortex), and five surrounding rings, which correspond with secondary, tertiary, quaric, and quintic cortices. parvabumin showed a graduation, that is, inner core is most pv-rich, and outer rings showed the decrease of pv concentration. comparing with pv staining in visual cortex, these six-levels suggested similar hierarchic and reciprocal structure, which are proposed by deyoe and vanessen by analysis of feed-forward and feedback connections. akihisa kimura, tomohiro donishi, keiichiro okamoto, yasuhiko tamai department of physiology, wakayama medical university, wakayama, japan tonotopically comparable subfields of the primary and non-primary auditory areas in the rat cortex have similar topographies in the projection to the medial geniculate body but reverse topographies in the projection to the thalamic reticular nucleus (trn). in the present study, we determined how cortical and thalamic afferents intersect in the trn with regard to tonotopic organization. in light of the fact that a subset of auditory cells in the trn responds to visual or somatosensory stimulus, we also explored the potential sources of cortical and thalamic afferents that would set up polymodal sensory interaction in the trn. small injections of biocytin into the trn, which were made with guidance of electrophysiological recording of auditory response, resulted in retrograde labeling. retrogradely labeled cortical and thalamic cells exhibited distinctive patterns of distribution depending on the injection sites. the results indicate anatomical nodes in the auditory trn that would implement selective relay of auditory and/or polymodal sensory inputs. ps a-f functional connections between the core and belt fields of the guinea-pig auditory cortex observed by optical recording and partial cortical inhibition using muscimol junsei horikawa, daisuke uchiyama, tatsunori matsui, shunji sugimoto department of knowledge-based information engineering, toyohashi university of technology, toyohashi, japan guinea pigs were anesthetized with ketamine and responses to puretones in the auditory cortex stained with a voltage-sensitive dye rh were recorded with a photodiode array. after determining the core (ai and dc) and belt fields, ai or dc was inhibited by putting a muscimol-containing agar piece on each field. the inhibition of ai resulted in reduction of responses in the belt fields by - %, whereas the inhibition of dc resulted in reduction only by - %. the reduction by ai inhibition was larger in the anterior and ventral belt fields and that by dc inhibition was larger in the posterior belt fields than in the other fields. further inhibition of dc after the ai inhibition or vice versa resulted in suppression of the responses in all the fields. these results suggest that the responses of the belt fields are elicited mostly via connections from the core to the belt fields and the belt fields receive differential connections from ai and dc. masataka nishimura, hiroyuki kaizo, wen-jie song department of electrical, electronic, and information engineering, graduate school of engineering, osaka university, suita, japan the auditory cortex of many mammals has a core area and surrounding belt regions. in guinea pigs, the primary auditory area, the secondary auditory area, and many belt regions have been reported. however, the activity of the belt regions has not been fully examined. using a high-resolution optical imaging system, we examined cortical responses to tone stimulations in anesthetized guinea pigs. the auditory cortex of six guinea pigs was exposed to the ventral end and stained with the voltage-sensitive dye rh- . a novel field in the ventro-anterior region was identified based on its isolated responses to a pure tone stimulation and the relatively long latency of the responses. the field was located ventro-caudal to the primary auditory area, and was close to the ventral edge of the auditory cortex. we thus named the field as ventro-caudal field (vc). smooth frequency gradient was observed in vc in rostro-caudal direction, with the frequency axis in opposite direction to that of the primary auditory area. yoko kato , , kazuo okanoya laboratory for biolinguistics, riken bsi, wako, saitama; graduate school of humanities, university of chiba, chiba, japan bengalese finches sing complex courtship songs. to sing complex sequences, they require auditory feedback during singing. song nucleus nif has a projection from primary auditory area field l and then it projects to sensory/motor nucleus hvc. moreover, bilateral lesion of nif cause song deterioration on complex sequences (hosino and okanoya, ) . we recorded auditory responses by multiunit activity from nif and field l. auditory responses of nif showed selectivity to bird's own song (bos) than its reversed song (rev). comparing selectivity of nif and field l, nif showed stronger selectivity than field l. however, nif did not show sequence dependent selectivity. these results suggest that nif relays auditory information and enhances bos selectivity. however, we did not observe a direct evidence that nif related to generation of complex sequences. we started to record responses extracellularly from ac neurons of guinea pigs. in general, animals show a stereotyped pattern of behaviors; they have a quiet, almost-motionless period, usually for tens of min. during this period, animals do not appear to sleep but be sensitive to the environmental disturbance. thereafter they usually fall asleep with their eyes closed. during this presleeping period, the best frequency tone was repeatedly presented - times at a fixed interval, through a speaker at - db spl. responses to such repetitive tones are apparently irregular, with the occurrence of spikes in most trials but no spikes in some trials. however, if all the trials are accumulated, there was global phase alternation every a few to tens of seconds. one phase constitutes relatively high rates of spike occurrence, while the other very low rates of spike occurrence. we suppose that, unlike a machine, the brain has a unique mechanism that automatically turns on and off the cortical processing of the redundant sensory stimulus. masashi sakai, sohei chimoto, ling qin, yu sato department of physiology, university of yamanashi, japan a periodic click train produces a continuum of several perceptual qualities: (i) at low repetition rate (< hz), the individual clicks are clearly heard as discrete events so that the entire train produces "rhythm" percept, (ii) at high repetition rate (> hz), the entire train is heard as a single continuous event leading to a strong "pitch" percept, and (iii) in the transition range, the periodicity can still be detected as "roughness". we physiologically explored how those perceptual qualities are represented in the primary auditory cortex in awake cats. we found that distinct population of cells conducted two coding modes: (i) representing low-rate stimuli through stimuluslocking activity (i.e., temporal code) and high-rate stimuli as only onset responses or (ii) exhibiting sustained responses with generating larger amount of discharges at higher repetition rate (i.e., rate code). in addition, pure-tone stimuli elicited onset responses or sustained responses in each of these cell populations, respectively. we will discuss functional consequences and spike evocation mechanisms of each population. atsuhito toyomaki , , , , , hokkaido university, sapporo, japan; hokkaido university, sapporo, japan; sakushin gakuin university, utsunomiya, japan; kobe shoin women's university, kobe, japan; riken, wako, japan; sakushin gakuin university, utsunomiya, japan gaps in a continuous sound play important roles for perception of voiceless consonants (i.e./k/,/p/,/t/) and japanese special mora (sokuon). we recorded auditory evoked responses to short gaps and tones from children ( - years old, n = ) and adults ( - years old, n = ). there were six gap conditions with durations of , , , , and ms embedded in a continuous tone and six tone conditions with the same durations. the frequency of all the tone was hz. the responses elicited by the onset of gaps differed between the children and the adults: the responses in children were significantly larger and more sustained than those in adults for all the durations. in contrast, an n and p complex followed the onset of all the tones in all the subjects. thus development time course of neural process is conceivably different between gaps and tones. ps a-f an fmri study on pitch control of voice using transformed auditory feedback method akira toyomrua , tamaki miyamoto , atsushi terao , sachiko koyama , takashi omori , harumitsu murohashi , shinya kuriki jst, saitama, japan; hokkaido university, sapporo, japan auditory feedback plays an important role in natural speech production. in the present study, we conducted an fmri experiment while subjects performed a transformed auditory feedback (taf) task to delineate the neural mechanism for control of pitch. the subjects were required to vocalize a and to hold the pitch of a feedback voice constant. in taf condition, the pitch was altered suddenly two or three times, whereas in non-taf condition the pitch was not modulated. under the taf condition, auditory feedback control is selectively expected to work more strongly than the non-taf condition. thus, a comparison between these conditions could neatly extract brain regions involved in auditory feedback control of pitch. as a result, right supramarginal gyrus, right frontal lobe (ba ), right anterior insula, left premotor area and right superior temporal gyrus showed greater activation ( subjects, p < . corrected). this result suggests that auditory feedback of pitch is mainly controlled by the right hemisphere. sachiko koyama-takeichi , yuko toyosawa , fumiya takeuchi , michinao matsui , shinya kuriki research institute for elecronic science, hokkaido university, sappro, japan; jst, saitama, japan; hokkaido university, school of medicine, japan; kobe shoin institute for linguistic science, kobe, japan sounds with relatively long duration elicit a sustained component (slow field, sf). in the present study, we recorded cortical magnetic responses elicited by vowels and examined whether sf differs between native and non-native vowels (n = ). four synthesized vowels were used as stimuli (stimulus duration ms). two of the vowels (a, o) are native for japanese and the other vowels (ae, schwa) are not. two inter-stimulus intervals were used ( / ms). for the native vowels, an early sf ( - ms) was larger for the long than for the short interval session in both hemispheres. for the non-native vowels, the early sf was larger for the long than the short interval session only in the right hemisphere. neither an effect of interval nor hemisphere was significant for a late part of sf ( - ms) regardless of stimulus types. research funds: japan science and technology agent (brain sciences and education), kakenhi ( ) ps a-f spatio-temporal representation of frequencymodulated sounds in the auditory cortex revealed by optical imaging shunji sugimoto , , junsei horikawa department of knowledge-based information engineering, toyohashi university of technology, toyohashi, japan; riken brain science institute, wako, japan optical imaging (voltage-sensitive dye, rh ) showed spatiotemporal response patterns for frequency-modulated (fm) sounds in the multiple fields of the guinea pig auditory cortex. an fm sound evoked a strong onset response spreading widely over the cortex, which was followed by a later response moving across the iso-frequency contours in the core fields. the location of the later response was corresponding roughly to the instantaneous frequency input of each fm sweep. on the other hand, a pure tone evoked a wide-spreading onset response followed by strong and long-lasting inhibitory effects. the later response to an fm sound appeared clearly when the frequency of the fm sweep was modulated over a wider range of frequencies, while it was diminished when the sound frequency was less modulated. these results imply that the cortical representation of such a later response contributes to a detection of frequency modulations in sounds. yamato kubota, kuniyuki takahashi, ryuichi hishida, masaharu kudoh, katsuei shibuki department of neurophysiology, brain research institute, niigata university, niigata, japan mitochondrial flavoprotein fluorescence is intimately coupled with energy metabolism. if the flavoprotein fluorescence is photobleached, energy metabolisms and neural activities can be inactivated. we applied this photo-inactivation technique to demonstrate auditory signal transmission from the anterior auditory field (aaf) to the primary auditory cortex (ai). cortical responses in aaf and ai after sound stimuli ( - khz) were visualized using transcranial flavoprotein fluorescence imaging in mice anesthetized with urethane. after determination of tonotopic maps, the auditory cortex was irradiated with strong blue light derived from a xenon lamp for min, while the surface either aaf or ai was covered with a piece of carbon paper for preventing photo-inactivation. although photoinactivation of ai had almost no effect on the responses in aaf, photo-inactivation of aaf significantly reduced the responses in ai. these results suggest the presence of auditory signal transmission from aaf to ai. kousuke abe , go ashida , , kazuo funabiki graduate school of informatics, kyoto university, kyoto, japan; hmro, faculty of medicine, kyoto university, kyoto, japan sound signals are translated to dispersed sporadic firing of the auditory nerves, and are converged to the third auditory station called the nucleus laminaris (nl) in birds. in vivo intracellular recording from owl's nl cells revealed that sound waveforms are observed in the postsynaptic membrane potentials (sound analogue potential; sap). we simulated synaptic inputs to the owl's nl neurons by recruiting the convergence of phase-locked excitatory inputs. several parameters such as the degree of phase-locking, the number of convergence and the time course of a unitary synaptic input affected the amplitude of sap, the amplitude of dc depolarization and the spectral features of synaptic noise in a complex manner. biophysical mechanisms for recreating sound waveforms by synaptic potentials will be discussed. takashi nihashi , shigenori takebayashi , masahiko bundo , masazumi fujii , toshihiko wakabayashi , jun yoshida , hiroyuki fujisawa , kazunori ando , kazumasa hayasaka department of radiology, national hospital for geriatric medicine, obu, japan; department of neurosurgery, national hospital for geriatric medicine, obu, japan; department of neurosurgery, nagoya university school of medicine, nagoya, japan we identify si, using fmri in a routine scan for the patient who need a surgical approach. the activation of the brain with a tumor is complicated. we considered the pattern of the response. twelve patients were participated in this study. using . tesla mr imager, tactile stimulation was applied to bilateral palm, respectively. the statistical threshold was set for individual. contralateral activation on si was found in out of patients in the affected hemisphere. when region is near central sulcus, the multiple sites were activated. on the other hand, when the tumor is from central sulcus, the activation is simple: contralateral si. this method is useful to decide si in affected hemisphere in a short time. however, there are great inter-individual differences due to the locations of the tumor. takayuki iwano, shinya yamamoto national institute of advanced industrial science and technology (aist), neuroscience research institute, tsukuba, japan to examine how body surface with low spatial resolution is represented in the brain, we conducted a tactile identification task on toes. subjects (n = ) lay on their backs with their eyes closed, and one of their toes was touched with a toothpick. the subjects were required to identify the toe by verbal response. the subjects responded correctly when the great or fifth toe was touched (cf. fein, ) . surprisingly, subjects tended to misidentify the second toe as the third ( . %), and the third toe as the fourth ( . %), while the reverse misidentification rarely occurred (third as second, . %; fourth as third, . %). this unidirectionality suggests that misidentification arises not only from large overlapping receptive fields associated with the toes, but from some additional factors such as a lack of experience with visuotactile integration, which could be used to reshape the toe receptive fields. ps a-g effects of saccades on subjective temporal order of somatosensory signals toshimitsu takahashi , , shunjiro moizumi , ayami okuzumi , humine saito , shigeru kitazawa , department of neurophysiology, juntendo university graduate school of medicine, tokyo, japan; crest, jst, saitama, japan morrone et al. ( ) recently reported that subjective temporal order of two successive visual stimuli was reversed when the stimuli were delivered just prior to the onset of a saccade. in this study, we examined whether saccades affect temporal order judgments of tactile stimuli. right-handed subjects were required to make a visually guided rightward saccade ( • ), and to judge the order of successive tactile stimuli that were delivered one to each hand at various timing relative to the onset of the saccade. with a stimulation interval of ms, subjects generally judged the order correctly as long as the stimuli were delivered after the saccade. however, they often misreported (i.e., inverted) the order when the stimuli were delivered just prior to the onset of the saccade (within ms). the results show that the reversal effect of saccades is multimodal and further suggest that multimodal brain areas are involved in ordering sensory events in time. ps a-g function-directed organization of the postcentral somatosensory cortex representing oral structures takashi toda , , miki taoka , department neuroscience oral physiology, osaka university graduate school of dental sciences, suita, japan; secondrary cognitives neurobiology, tokyo medical & dental university, tokyo, japan; department physiology, toho university school of medicine, tokyo, japan the representation of oral structures in areas b and of four conscious macaque monkeys was studied by recording single-neuron activities. a total of electrode penetrations were made in areas b and . in penetrations, pairs of adjacent neurons along the track had receptive fields (rfs) on continuous oral portions with or without overlapping, or otherwise on the same portion. in the remaining penetrations, however, % of adjacent pairs ( / ) had rfs on discrete but functionally-related sets of oral portions, e.g., the lip and tongue tip, the cheek mucosa and lateral margin of the tongue, the corresponding portions of the upper and lower lips, the corresponding portions of the palate and tongue, etc. we speculated that such an organization in areas b and might be responsible for forming composite rfs of area neurons. those composite rfs often covered discrete but functionally-related oral portions as reported earlier. research funds: kakenhi ( , ) miki taoka , michio tanaka , hisayuki ojima , atsushi iriki secondrary cognitives neurobiology, tokyo medical and dental university, tokyo, japan; laboratory of symbolic cognitive development, riken brain science institute, wako, japan we previously reported neuronal projections from the hand region of the second somatosensroy cortex (sii) to higher motor cortices (vetral premotor cortex etc.) suggesting that sii may be related to motor control of the hand movement. in the present study, we investigated the activities of sii hand neurons during voluntary movements. we recorded neurons from two animals that were active when animals took small pieces of food by hands and put them into the mouth. among them ( % contra-, % bi-and % ipsilateral hand movements), we could determine receptive fields for only neurons ( %). most of activities ( neurons) were related to a certain phase of movements such as reaching, pinching a food piece, and putting it into the mouth. we found neurons showing phasic activities just before/after a certain phase, for example, just before pinching the object, or just after putting it into the mouth. those results suggest that sii hand neurons code the start or end of a certain act of hand. takahiro furuta , , kouichi nakamura , takeshi kaneko department of morphological brain science, graduate school of medicine, kyoto university, kyoto, japan; crulrg, laval university, canada we investigated response properties of whisker-responsive neurons in the nucleus interpolaris (spvi) combining juxtacellular recording and a piezo-stimurator. the spvi is one of the first relay stations in the vibrissal system. rostral part of the spvi sends axons to the posterior thalamic nuclear group, whereas the caudal part of the spvi projects to the ventral lateral part of the ventral posterior medial nucleus. in the rostral part of the spvi, the vast majority of recorded neurons were multi-whisker responsive neurons, which are considered as projection neurons. in the caudal part of the spvi, about a half of neurons were mono-whisker-responsive neurons, which are thought as local circuit neurons. almost all neurons had angular tunings to upward deflection of whiskers in the rostral part of the spvi, while neurons in the caudal part of the spvi exhibited various angular tunings to all directions. these results indicate that the spvi could be divided into two sectors by response properties. seiji komagata , , shanlin chen , hiroki kitaura , masaharu kudoh , minoru shibata , katsuei shibuki department of neurophysiology, brain research institute, niigata university; department plastic surgery, school of medicine, niigata university, japan we visualized neural responses in the mouse somatosensory cortex using transcranial flavoprotein fluorescence imaging. mice were anaesthetized with urethane ( . g/kg, i.p.), and somatosensory responses were elicited by vibratory brush stimulation ( hz for s) applied to the left plantar forepaw. changes in green fluorescence in blue light were observed in the right somatosensory cortex. immediately after cutting the left median and ulnar nerves, the somatosensory responses were almost completely abolished. however, the responses appeared again within a few hours after the partial denervation, and almost complete recovery was observed a few weeks after the partial denervation. the recovered responses were eliminated by cutting the remaining radial and muscle cutaneous nerves. the rapid recovery of the responses observed in the present study may explain the mechanisms for allodynia and cortical plasticity in the somatotopic maps. shin-ya nakamura, takaaki narumi, ken-ichiro tsutsui, toshio iijima division system neuroscience, tohoku university graduate school of life science, sendai, japan in the rat somatosensory pathway, information received with a whisker is conveyed to the barrel cortex via trigeminal and thalamic nucleus mainly by two parallel pathways, the lemniscal and paralemniscal. the former includes the nucleus of trigeminal prv and thalamic vpm, which are known to contain neurons selective to the direction of whisker stimulation, and the latter includes trigeminal spvi and thalamic pom. in this study, we examined the specific involvement of the lemniscal pathway to the discriminative perception of whisker stimulus direction. rats were trained to perform a single-whisker directional discrimination task, and the task performance was evaluated before and after the selective electrolytic lesion or muscimol inactivation of each trigeminal and thalamic nucleus. the lesion or inactivation of prv or vpm significantly impaired the task performance, whereas those of spvi or pom did not. this result suggests the specific involvement of the lemniscal pathway in the single-whisker directional discrimination. kumiko yokouchi , nanae fukushima , tetsuhiro fukuyama , akira kakegawa , tetsuji moriizumi department of anatomy, shinshu university school of medicine, matsumoto, japan; department of pediatrics, shinshu university school of medicine, matsumoto, japan to know sensory cues for suckling behavior, rat pups of the suckling period received unilateral or bilateral resection of the infraorbital (io), mental (m) or lingual (l) nerves responsible for sensation of the upper and lower lips, and the tongue. for comparison, unilateral or bilateral removal of the olfactory bulb was done in these pups. the control, unilaterally io or m nerve-injured, and unilaterally bulbectomized pups showed successful suckling by their access to the mother's nipple, oral contact to it and long-lasting sucking. the bilaterally bulbectomized pups could not have access to the nipple. the bilaterally io nerve-injured pups could have access to the nipple with no oral contact, while the bilaterally m nerve-injured pups showed successful suckling. suckling behavior of the bilaterally l nerve-injured pups was characterized by frequent oral contacts for a very short duration, resulting in ineffective milk-intake. the results show fundamental roles of olfaction and sensation of the upper lip and the tongue in suckling. takahiro kawashima , takeshi kawano , hidekuni takao , , , kazuaki sawada , , , hidekazu kaneko , , makoto ishida , , department electrical & electronic engineering, toyohashi university of technology, aichi, japan; issrc, toyohashi university of technology, aichi, japan; aist, hsbe, ibaraki, japan; jst, crest, japan a si microprobe array (probe: au-coated recording site at the tip, m in diameter, m in length; array: -m pitch) to record neuronal activities has been developed by using selective si probe growth technique. to examine electrical properties of the array, single motor unit action potentials evoked by the electrical stimulation of the sciatic nerve of a rat were recorded in the left tibialis anterior muscle. signal-to-noise ratio of observed signals decreased with probe impedance, suggesting that lower impedance is better for recording small action potentials. however, lower impedance makes more difficult to record local signals, because signals observed at probes with low impedance were highly correlated (r = . ). to record local signals, it is necessary to decrease the area of the recording site of each probe at the expense of an increase in the impedance. research funds: kakenhi ( ), the st century coe program "intelligent human sensing" ps a-g a microelectrode positioning system for longterm extracellular recording of multiple neuronal activities hidekazu kaneko , hiroshi tamura , shinya s. suzuki , takahiro kawashima aist, hsbe, ibaraki, japan; laboratory of cognitive neuroscience, graduate school of frontal bioscience, osaka university, osaka, japan; department electrical & electronic engineering, toyohashi university of technology, aichi, japan a novel microelectrode-positioning system was devised that tracks a target neuron by countermoving a microelectrode against uncontrollable movements of brain tissue. the system automatically adjusted the position of a seven-core microelectrode such that the amplitude of each spike of a target neuron at the tip was the same as that at a lateral recording site (differential mode). the differential mode was compared with a conventional (peak-search) mode in which the spike amplitude of a target neuron at the tip was continually maximized. the differential mode was more stable to forced electrode movements and more sensitive to small displacements than the peak-search mode. furthermore, the differential mode enabled stable recording of not only spikes of a target neuron but also those of non-target neurons for over h. seiji matsuda , takehiro terashita , tetsuya shimokawa , kyoujy miyawaki , yuji miguchi , takuya doihara , jie chen , shuang-yan gao , chun-yu li , min wang , zhong wang , bing xue , naoto kobayashi , , kazuhiro shigemoto department anatomy, ehime university of medicine, ehime, japan; medical education c, ehime university of medicine, ehime, japan; department of hygiene, ehime university of medicine, ehime, japan this study shows the phylogenetic development of cajal's initial glomeruli (ig) and dogiel's pericellular nests (pcns) in the dorsal root ganglion of the healthy adult frog, chick, rat, and rabbit. the three-dimensional architecture of the neurons was observed in ganglia by scanning electron microscopy, after removal of the connective tissue. the proportion of neurons having ig or pcns increased with increasing phylogenetic complexity in the species examined here. in the chicks, the stem processes were longer and sometimes tortuous. typical ig were observed not in frogs or chicks, but in rats and rabbits. dogiel's pcns also have been observed in the drg of rats and rabbits. the nerve fibers in the pcns were less than . m in diameter and had some varicosities. some pcns contain tyrosine hydroxylase-positive nerve fibers and varicosities. masayo okumura, eiji kondo matsumoto dental university, institute for oral science, shiojiri, japan we established a rat nerve injury model using axotomy of the inferior alveolar nerve (ian), and investigated its effect on gene expression in the trigeminal ganglion. microarray analysis three days after surgery showed the up-regulation of some genes which are regulated by transcription factor stat , whereas other genes known to be regulated by stat were not detected. stat is expressed in many tissues and plays various roles. however, there have been few reports about the role of stat in the peripheral nervous system, despite its welldocumented activation in the central nervous system after injury or stress. the aim of this study is to elucidate the role of stat in gene expression in the trigeminal ganglion after ian injury. at various time points, we analyzed and investigated changes of gene expression which are known to be influenced by stat and stat phosphorylation, which indicates transcriptional activity, as well as cell types in which the genes and stat are expressed. these results should help us understand injury-induced change mechanisms of the peripheral nerve. hirofumi hashimoto , susumu hyodo , makoto kawasaki , minori shibata , takeshi saito , hiroaki fujihara , takashi higuchi , yoshio takei , yoichi ueta department of physiology, school of medicine, university of occupational and environmental health, kitakyushu, japan; laboratory of physiology, department of marine bioscience, ocean research institute, university of tokyo, japan; department of integrative physiology, university of fukui, japan adrenomedullin (am ) (identical to intermedin) belongs to the super family of am. centrally administered am and am activated oxytocin (oxt)-secreting neurons and increased plasma oxt level in rats. in the present study, we examined the effects of central administration of am on oxt-secreting neurons and sympathetic outflow in comparison with that of am in conscious rats. effects of central administration of am was stronger than those of am and the effects of am on oxt secreting neurons could not be blocked completely by pretreatment with cgrp or/and am receptor antagonists. these data suggested that am would have unknown receptor except cgrp and am receptor. arata oh-nishi , makoto saji , taku uchida , sen-ichi furudate , nobuyuki suzuki division of brain science, kitasato university, graduate school of medical science, kanagawa, japan; division of reproduction and fetal development, kitasato university, graduate school of medical science, kanagawa, japan the mechanism whereby neonatal hypothyroidism impairs cognitive function has not been well studied. in this respect, nmda receptors are thought to be crucially involved in cognitive and memory function. we have examined the effect of neonatal hypothyroidism and hyperthyroidism on the nmda receptor function, using rats treated with methylmercaptoimidazole (mmi), which specifically blocks the biosynthesis of thyroid hormone and mmi-treated rats injected with thyroxine, respectively. dose-response curves indicated that the sensitivity to nmda of the nmda receptors was significantly reduced in the hippocampus of the hyperthyroid rats, compared to that of normal and the hypothyroid rats. concomitant with this observation, western blot analysis showed that the nmda receptor subunit nr expression significantly decreased in the hippocampus of the hyperthyroid rats, compared to that of normal and the hypothyroid rats. our lab demonstrated that estradiol is endogenously synthesized within hippocampal neurons in the adult male rat (pnas, ) . here we report that the density and morphology of spines of pyramidal neurons in ca region are rapidly altered by treatments with nm estradiol and bisphenol a (xenoestrogen). hippocampal slices are incubated with estradiol or bisphenol a for h, and then neurons were injected iontophoretically with lucifer yellow. three-dimensional imaging of neurons is performed by confocal laser microscopy, and the analysis of individual spines is performed by neurolucida software. the results showed that in ca , both estradiol and bisphenol a induce a significant increase in the total spine density, especially the density of thin spine. synaptic plasticity of hippocampal neurons is demonstrated to be rapidly modulated by estrogen and xenoestrogen. we investigated the effects of stress on enhanced green fluorescent protein (egfp) expression in the arginine vasopressin (avp)-egfp transgenic (tg) rats. after bilateral adrenalectomy and intraperitoneal administration of lipopolysaccharide egfp fluorescences were increased in the parvocellular division of the paraventricular nucleus and the external layer of the median eminence. this tg rat is a convenient tool to study dynamic changes of avp expression in the hypothalamus under stressful condition. chitose orikasa, yasuhiko kondo, yasuo sakuma department of physiology, nippon medical school, tokyo, japan we report here a sex difference expression of somatostain mrna within the sexually dimorphic nucleus of the preoptic area (sdn-poa), the volume of which depends on gonadal hormones during the ontogeny. in infant rats aged day - , the volume of somatostain mrna-positive region within the poa was significantly larger in males than in females and overlapped the sdn-poa in both sexes. the sdn-poa visualized by nissl staining in adjacent sections agreed precisely with the extent of somatostain mrna-positive cellular distribution. orchidectomy of males neonates and estrogen treatment of female pups reverse brain phenotypes when examines on day . the staining of somatostain mrna in individual neurons was diminished when examined on day or , albeit that the sex difference of the volume of somatostain mrna-positive region persisted thoughtout the observed period. somatostatin may play a role in the establishment of the sdn-poa, which lacks classic nuclear receptor for estrogen. ps a-g short chain sugar acid, -buten- -olide, activates oxytocin-secreting neurons in the hypothalamus of rats makoto kawasaki , tatsushi onaka , hirofumi hashimoto , hiroaki fujihara , yoichi ueta department of physiology, school of medicine, university of occupational and environmental health, kitakyushu, japan; department of physiology, jichi medical school, tochigi, japan -buten- -olide ( -b o), an endogenous sugar acid, which may be involved in the regulation of feeding. we examined the effects of -b o on the hypothalamo-neurohypophyseal system in rats. the plasma oxytocin (oxt) levels were significantly increased at - min after intraperitoneal (i.p.) administration of -b o ( mg/kg), whereas plasma arginine vasopressin (avp) levels did not change. dual immunostaining revealed that fos-like immunoreactivity (li) was predominantly observed in oxt-secreting neurons in the paraventricular and the supraoptic nuclei min after i.p. administration of -b o. in addition, many fos-li neurons were also observed in the nucleus of the tractus solitarius (nts) after i.p. administration of -b o. these results suggest that peripherally administered high dose of -b o activates oxt-secreting neurons in the hypothalamus through the activation of the nts neurons. ps a-g estrogen receptor ␣ gene promoter activity is a marker for the sexually dimorphic nucleus of the preoptic area tomohiro hamada, yasuo sakuma department of physiology, nippon medical school, tokyo, japan the volume of the sexually dimorphic nucleus in the preoptic area (sdn-poa) is two to four times larger in male rat than in female, however function of this nucleus has not well known. in contrast, estrogen causes the sexually dimorphism by acting in perinatal periods. recently, transgenic rats expressing enhanced green fluorescent protein (egfp) under the control of an estrogen receptor (er) ␣ promoter were generated to tag er␣-positive neurons in the brain. in the present study, we examined gfp expression could be used a marker for the sdn-poa. gfp labeled cells were distributed in the core of sdn-poa of male and female transgenic rats and in the majority of these cells included er␣, immunohistochemically. both area and number of gfp expressed cells in the sdn-poa were larger in male than in female, however, female gfp cells in the sdn-poa showed concentrated distribution than male. these results suggest that gfp labeled cells in sdn-poa could be useful marker to make clear the function of the sdn-poa. recent studies on gonadal steroids imply that testosterone and estradiol are involved in learning and memory with modification of excitatory synapses in the hippocampus. although previous in vivo studies have demonstrated that these steroids increase the number of dendritic spines in neurons, it is still unclear whether each steroid has a direct effect on the modulation of the spatio-temporal patterns of dendritic morphogenesis. in the present study, we investigated steroid-induced morphological changes using cultured hippocampal neurons derived from neonatal or embryonic mice. the neurons were transfected with venus-actin. time-lapse images were taken by laser scanning confocal microscope during steroid treatment. testosterone but not estradiol increased the number of spines/filopodia of the dendrites within h. these results obtained from in vitro studies suggested that testosterone affects dendritic morphogenesis of hippocampal neurons in short term. tetsuya kimoto , , shinpei higo , , yasushi hojo , , kouhei nakajima , , hironori nakanishi , , hirotaka ishii , , suguru kawato , department of biophysics & life science, university of tokyo, tokyo, japan; crest, jst, japan hippocampus is one of the main target of sex steroids (androgen and estrogen) and stress steroids (corticosteroids). neuronal signal transmission in the hippocampus is modulated acutely by these steroids, and we recently demonstrated that the hippocampus of the adult male rat contained enzymes required for the synthesis of these steroids. however, the full diagram of hippocampal neurosteroid synthesis has not been obtained yet. in the present study, we therefore investigated the synthesis of sex steroids and corticosteroids in the hippocampus of adult male rats, by monitoring the metabolism of tritiated steroids with hplc system. ps a-g gaba depolarizes gnrh neurons isolated from adult gnrh-egfp transgenic rats chengzhu yin, nobuyuki tanaka, masakatsu kato, yasuo sakuma nippon medical school, department of physiology, tokyo, japan gnrh neurons are essential in the reproductive neuroendocrine system. in regulation of gnrh neurons, gaba may be one of the major players, especially in relation to gnrh/lh surge. we, therefore, performed a cell-physiological analysis of gaba action on rat gnrh neurons. cells were dispersed from adult gnrh-egfp transgenic rats and cultured overnight. gnrh neurons, were applied to the perforated patch-clamp configuration with gramicidin d. gaba evoked cl − conductance, which was almost completely blocked by either picrotoxin or biccuculin. the reversal potential of the response was ranged from − to - mv in identified gnrh neurons in both sexes. there was no difference in the reversal potential among the stages of estrous cycle. in unidentified neurons, however, the reversal potential was more negative than - mv and most of them were ∼− mv. in conclusion, gnrh neurons isolated from adult rats express gabaa receptor and its reversal potential is more positive than the resting potential. although the neural activation in the subfornical organ (sfo) by angiotensin ii (angii) is widely regarded for the increments of angii-induced water intake and vasopressin release, galanin (gal) have been reported to inhibit them. therefore, gal may inhibit neural activity of angii-sensitive sfo neurons. rt-pcr analysis demonstrated existences of all mrnas of gal receptor subtypes, galr , galr and galr , in the sfo. in extracellular recording on sfo slice preparation, gal dose-dependently led to inhibition of neural activity. all gal sensitive neurons showed excitatory response by angii. galr selective agonist m induced inhibitory responses, as well as gal. in patch-clamp recordings, gal induced outward current in some neurons. these results suggest that gal inhibits neural activity in sfo neurons through, at least partially, outward current following activation of galr . hiroaki fujihara , tomoki fujio , david murphy , yoichi ueta department of physiology, school of medicine, university of occupational and environmental health, kitakyushu, japan; molecular neuroendocrinology research group, the henry wellcome laboratories for integrative neuroscience and endocrinology, university of bristol, bristol, uk we have generated transgenic (tg) rats expressing an arginine vasopressin (avp)-enhanced green fluorescent protein (egfp) fusion gene. in this study, we investigated the amount of drinking and food intake, the urinary output, the urine osmotic pressure, the urine sodium concentration and body weight after drinking % saline for days in , , and months old tg rats. in and months, there were no difference between tg rats and tg(−) rats about the amount of drinking and food intake, the urinary output, the urine osmotic pressure, the urine sodium concentration and body weight under normal condition and salt loading. in aged tg rats ( and months old), there were no obvious changes in water balance. these results suggest that the expression of avp-egfp transgene does not disturb body fluid homeostasis in tg rats. ps a-h prolactin-releasing peptide is a potent mediator of stress response in the brain through the hypothalamic paraventricular nucleus takashi mera , hiroaki fujihara , hirofumi hashimoto , makoto kawasaki , tatsushi onaka , takakazu oka , sadatoshi tsuji , yoichi ueta department of neurology (division of psychosomatic medicine), school of medicine, university of occupational and environmental health, japan; department of physiology, school of medicine, university of occupational and environmental health, japan; department of physiology, jichi medical school we examined the effects of restraint stress (rts), nociceptive stimulus and acute inflammatory stress on the prolactin-releasing peptide (prrp) gene expression in the hypothalamus and brainstem. moreover, we examined the effects of pretreatment with an anti-prrp antibody on nociceptive stimulus-induced c-fos gene expression in the hypothalamic paraventricular nucleus (pvn). rts, nociceptive stimulus and acute inflammatory stress upregulated the prrp gene expression in the brainstem. pretreatment with anti-prrp antibody significantly attenuated nociceptive stimulus-induced c-fos gene expression in the pvn. these results suggested that prrp is a potent and important mediator of stress response in the brain through the hypothalamic pvn. taieb bousejin , afsaneh eliassi , nasser naghdi , ali ghanbari ghsemi; pastor institute, tehran, iran the purpose of this study was to consider the role of the ventromedial hypothalamus (vmh) d receptors on histamine-induced gastric acid secretion (gas). the animals were anasthetized and guide cannulas were implanted unilaterally above ( . mm) vmh. animals were anasthetized and two polyethylene tubes were introduced into the stomach through esophagus and pylorododenal junction. iv infusion of histamine in sham grup induced marked increase in gas with a peak response that started from min up to the end of experiments ( min). at the peak acid response, the vmh microinjection skf ( . , ) significantly reduced the amount of gas (p < . ). there was no any effect by microinjected sch ( . ) into the vmh. injecting skf into the vmh, min after sch , had no effect on gas in compare with control. but, the acid suppressant effect of skf was completely removed by peripheral injection of sch (p < . ). our results show that the vmh d dopamine receptors have regulatory mechanisms of gas by interaction with h receptors through an inhibitory neural pathways. zhilin song, celia d. sladek department of physiology, uchsc, aurora, co, usa although prior studies demonstrated expression of p x purinoceptors in supraoptic neurons (son) and indicated their importance in atp stimulated vasopressin release, in studies monitoring the effect of atp on intracellular ca ++ ([ca ++ ] i ), we have obtained evidence that p y purinoceptors (p y r) are important in the response to atp. atp stimulated [ca ++ ] i increase was maintained in ca ++ -free medium and reduced by pretreatment with thapsigargin to deplete [ca ++ ] i stores. p y r agonists increased [ca ++ ] i in son, with p y r agonist being the most effective. the possibility that p y r mediates atp induced [ca ++ ] i increase in son was further evaluated using a p y r antagonist, mrs . atp stimulated increase in [ca ++ ] i was greatly attenuated by mrs ( m) in mm ca ++ medium. in ca ++ -free medium, there was no significant response to atp in the presence of mrs . furthermore, combined treatment with mrs and ppads ( m, a p x r antagonist) also abolished the [ca ++ ] i response to atp. these results demonstrated that p y r mediates a large portion of the [ca ++ ] i response to atp challenge in son. ps a-h analysis of the ontogenic expression of enzymes for brain neurosteroids in the male rat hippocampus hirotaka ishii , , yasuhiro sonoki , , aizo furukawa , , yasushi hojo , tetsuya kimoto , , suguru kawato , department of biophysics and life science, university of tokyo, tokyo, japan; crest, jst, japan; kurihama national hospital, japan brain neurosteroids are steroids synthesized endogenously in the brain. our recent studies have demonstrated that the adult male rat hippocampus is equipped with a complete machinery for the synthesis of androgen and estrogen. to define the physiological role of brain neurosteroids in the hippocampal development and function, detailed information about the expression profiles of enzymes for brain neurosteroids in the hippocampus is essential. this study have comprehensively investigated the temporal patterns of enzymes for brain neurosteroids in the male rat hippocampus from postnatal day (pd ) to the adult stage using rt-pcr/southern blotting. enzymes required for the synthesis of estradiol from cholesterol were expressed form pd to pd with a higher level than in the adulthood. these results indicate that the rat hippocampus synthesizes estradiol more vigorously during the postnatal stage than in the adulthood, which may play an important role in the hippocampal development and function. hideo mukai , , gen murakami , shirou kominami , john h morrison , william g.m janssen , tetsuya kimoto , , suguru kawato , department of biophysics and life sciences, graduate school of arts and sciences, the university of tokyo, meguro, tokyo - , japan; crest project, jst, japan; faculty of integrated arts and sciences, hiroshima university, higashi-hiroshima , japan; kastor neurobiology of aging laboratories, fishberg research center for neurobiology, usa estrogens elicit rapid non-genomic effects on the synaptic transmission, and spinogenesis in the hippocampus. however, the existence of estrogen receptor alpha (er␣) still remains elusive. with highly purified antibody rc- , mass spectrometric analysis identified er␣ in the hippocampus and immunohistochemistry showed er␣ localization in principal neurons of ca , ca , and granule cells in dentate gyrus. further, western blot revealed that er␣ is contained in psd fraction, confirming the observation with immunoelectron microscopy. these results imply that the synaptic er␣ mediates the effects of estrogen in hippocampal neurons. ken takumi gnrh neuron is the key modulator of reproductive systems, directly regulating the synthesis and secretion of gonadotropins from anterior pituitary gland. gnrh neurons have been reported to be contacted by various neuronal systems, suggesting that the biosynthesis and release of gnrh is controlled by a complex of excitatory and inhibitory inputs. however, anatomical studies which quantified the direct input on gnrh neuron are few. in this study, we quantitatively analysed glutamatergic and gabaergic input onto gnrh neurons of the rhesus monkey by immunofluorescence method and confocal laser scanning microscopy; the close appositions between gnrh neuron and axon terminals immunoreactive for either vgluts or vgat were counted and the densities of the appositions on the dendrites and soma were calculated. sabine gouraud , song t. yao , jing qiu , julian fr paton , david murphy university of bristol, hw-line, uk; department of physiology, bristol heart institute, university of bristol, united kingdom the neuropeptide hormone vasopressin (vp) is produced in the magnocellular neurons of the hypothalamic supraoptic (son) and paraventricular (pvn) nuclei and stored in the posterior pituitary (pp). dehydration evokes an increased expression of the vp gene in magnocellular neurons and a massive release of vp from the pp in the circulation to promote the water conservation at the kidney level. in parallel, a functional remodelling of the hypothalamo-neurohypophyseal system (hns) is observed but poorly understood. we investigated this activity dependent plasticity of the hns using proteomic ( d fluorescence difference gel electrophoresis (dige)) combined with maldi mass spectrometry approaches to identify proteins that change in abundance in the son and the pp from days dehydrated rats. a truncated form of prosaas, a granin-like neuroendocrine peptide precursor known as a potent inhibitor of the prohormone convertase , has been found decreased in the pp and increased in the son. ichiro nishimura, masakatsu kato, yasuo sakuma department of physiology, nippon medical school, tokyo, japan function of gonadotropin-releasing hormone (gnrh) neurons is regulated by gonadal steroid estrogen. however, the precise mechanism of estrogen action upon these cells has not been clarified. we investigated a direct action of estrogen on the regulation of potassium current in gnrh neuronal cell line gt - . delayed rectifier potassium current (i k ) and large-conductance calcium-activated potassium (bk) current were recorded by patch clamp configuration in gt - cells cultured in dmem supplemented with % fbs for days. bk current was increased by addition of -estradiol (e ) in culture medium in a physiological concentration range. this action of e was blocked by ici- , , a potent estrogen receptor (er) antagonist. we further examined whether e acted through er ␣ or er  by using selective agonists ppt and dpn, respectively. the dpn augmented the bk currents similar to the effect of e but ppt had no effect. e had no effect on the i k . these results indicate that e increases the bk current by activating er without affecting the i k . research funds: kakenhi , ps a-h myelin protein zero is one of the components of the detergent-resistant membrane microdomain fraction derived from rat pituitary katsutoshi taguchi , haruko kumanogoh , shun nakamura , seiji miyata , shohei maekawa department of biosystems science, kobe-university, kobe, japan; division of biochemistry and cellular biology, national institute of neuroscience, ncnp, tokyo, japan; department of applied biology, kyoto institute of technology, kyoto, japan a membrane microdomain enriched in cholesterol and glycosphingolipids was found to contain many signal transducing and cell adhesion molecules. here, we studied the components of the membrane microdomain fraction derived from rat pituitary, and found specific enrichment of several proteins in this fraction. one of them, kda protein, was identified as myelin protein zero (p ) from mass analysis and this result was confirmed by western blotting that a specific antibody to kda band reacted to an authentic p prepared from rat sciatic nerve myelin. p is a type i transmembrane glycoprotein and a member of the immunoglobulin superfamily. the expression of p has been believed to be restricted to the peripheral myelin in mammals. our result, however, indicates that p expresses more widely and participates in cell communications. mari ogiue-ikeda, norio takata, suguru kawato department of biophysics and life sciences, graduate school of arts and sciences, university of tokyo, tokyo, japan -estradiol (e ) has a rapid effect on synaptic transmission. recently, we found that hippocampal neurons synthesize e (hojo et al., ) , and express estrogen receptor ␣ (er␣) at synapses. endocrine disrupters are representative estrogenic industrial compounds. while their disrupting effects on reproductive organs are well documented, their effects in the central nervous system are almost unknown. in this study, we investigated the effects of e and endocrine disrupters (des, bpa, np, op and tbt) on nmda-induced ltd in the rat hippocampal ca , ca and dg with a custom made multi-electrode measuring system (med ). ltd was enhanced by e dose-dependently in ca , ca and dg. des, bpa, np, op and tbt had similar or different effects on ltd dose-dependently. our results suggest that estradiol and endocrine disrupters rapidly modulate synaptic plasticity in the hippocampus and that the action of endocrine disrupters can be quantitatively analyzed by measuring the modulation of ltd of the hippocampal neurons. we examined the effects of chronic salt loading on the hypothalamic expressions of the green fluorescent protein (gfp), arginine vasopressin (avp) and oxytocin (oxt) genes and body fluid balance in avp-enhanced (e) gfp transgenic rats. chronic salt loading caused marked increase of the egfp fluorescence in the hypothalamoneurohypophyseal system in transgenic rats. there were no differences of the avp and oxt gene expressions in the hypothalamus, plasma avp and oxt levels and water balance between nontransgenic and transgenic rats under normal condition and after salt loading. humoral responses to chronic salt loading were maintained in avp-egfp transgenic rats. takeshi saito , takushi x. watanabe , tomoko urabe , hirofumi hashimoto , hiroaki fujihara , yukio hirata , yoichi ueta department of physiology, school of medicine, university of occupational and environmental health, kitakyushu, japan; peptide institute, inc., osaka, japan; department of clinical and molecular endocrinology, tokyo medical and dental university, tokyo, japan salusin- was newly discovered as a bioactive endogenous peptide. low concentration of salusin- stimulates the secretion of arginine vasopressin (avp) from perifused rat hypophysis. salusin- coexists with avp, but not oxytocin, in the rat magnocellular supraoptic (son) and paraventricular nuclei (pvn). to further investigate the physiological role of salusin- in body fluid homeostasis, we examined the effects of salt loding for days on salusin--like immunoreactivity (li) in the son and pvn of rats by immunohistochemistry. the marked increase of salusin--li in the son and pvn were observed in the salt loaded rats. the result suggests that salusin- may play a role of body fluid balance by regulating avp release. naoyuki yamamoto , hao-gang xue , yuji ishikawa , yoshitaka oka , hitoshi ozawa department of anatomy and neurobiology, nippon medical school, tokyo, japan; national institute of radiological sciences, chiba, japan; department of biological sciences, graduate school of science, the university of tokyo, tokyo, japan the terminal nerve gnrh (gonadotropin-releasing hormone) system, an extrahypothalamic peptidergic system, is thought to modulate neural circuitries involved in the control of motivational status for certain behaviors in teleosts. the major afferent source to the gnrh neurons is a midbrain nucleus, the nucleus tegmento-terminalis in percomorph teleosts, while a comparable nucleus appears to be missing in cyprinids teleosts. here, we examined the presence of such an afferent pathway in medaka oryzias latipes. injections of a dii crystal into the cluster of gnrh neurons resulted in labeled cells in the midbrain tegmentum, and injections to the midbrain tegmentum resulted in labeled terminals close to the gnrh neurons. these results suggest that the afferent pathway to the gnrh neurons is a character shared by "advanced" teleosts like medaka and percomorphs. takeshi yamazaki , eiji munetsuna , asuka kamogawa , suguru kawato , shiro kominami graduate school of integrated arts science, hiroshima university of higashihiroshima, japan; graduate school of arts and science, tokyou university of tokyo, japan tributyltin, tbt, an endocrine disruptor, induced increases in estradiol content in rat hippocampal slice culture. to analyze molecular mechanism of stimulation of estrogen synthesis, we determined mrna contents of estrogen biosynthetic enzymes and activity of p arom in the hippocampus. method: the cultured hippocampus slices from days male rat were treated with - nm of tbt for h. after the treatment, total rna was extracted and the levels of mrna of estrogen synthetic enzymes were quantified by real-time rt-pcr. activity of p arom was determined by quantification of [ h]estradiol from [ h]testosterone. result: forty-eight hours treatment of hippocampal slices with nm tbt induced increases in mrna contents of p arom, and with nm tbt induced that of -hsd. estradiol content was increased by the treatment with nm tbt, but not affected by nm tbt. tbt may modulate estradiol synthesis by alteration of expression of p arom. the medial preoptic area (mpoa) is an important neural site for regulation and maintenance of sleep. studies have indicated that gabaergic neurons and terminals at the mpoa are active during sleep. present study was carried out to elucidate the contribution of gaba-a receptor at the mpoa in sleep-wakefulness (sw) in male wistar rats. the sw was assessed by chronically implanted electrodes for eeg, eog, and emg. a bilateral guide cannula was also implanted for drug injection into the mpoa. after recovery, three baseline sleep recordings were taken for h on different days. bicuculline methoiodide (gaba-a receptor antagonist) at a dose of , , and ng in nl was injected bilaterally into the mpoa in different groups of rats and their sw was studied for subsequent h. the ng dose of bicuculline methoiodide had minimum effect whereas and ng produced arousal. maximal wakefulness was observed at dose of ng with no further increase in wakefulness at higher dose of ng. the results suggest the involvement of gaba-a receptors at the mpoa in sw. yoshiaki isobe , hiroyuki tsuda department of neuro-physiology and brain science, nagoya city university, graduate school of medical sciences, nagoya, japan; department of molecular toxicology, nagoya city university graduate school of medical sciences, japan locomotor activity in rodents shows free-running circadian rhythms even under the constant light. constant light exerts a promoting effect on hepatic carcinogenesis. after the partial hepatectomy, hepatic cell proliferation is regulated by circadian rhythm information (via wee ). to know the relation of proliferating factor (cell cycle) with circadian rhythmicity, locomotor activity against a diethylnitrosamine (den), widely used to initiate the hepatic neoplastic foci, is analyzed in preliminary. den was injected (i.p., mg/kg) on rats during the free-running condition under the constant dim light (dd) and constant light (ll). the effects of den were gentle under the dd. however, under the ll, phase delay accompanying the elongation of circadian period () was observed. decrement of an amount of activity in h after the den administration was obvious under the ll compared with that under the dd. this study was designed to investigate the central regulating system of hypothermia during maintenance phase of hibernation. although intracerebroventricular (icv) injection of naloxone (non-selective opioid receptor antagonist) and naloxonazine, ( antagonist) were effective, naltrindole (␦ antagonist) and nor-bni ( antagonist) did not interrupt the hibernation. the increment of c-fos expression was observed in arcuate nucleus (arc) at h after from hibernation onset compared with before hibernation. in addition, a localized -endorphin-like immunoreactivity (-end ir) was observed in neuronal perikarya in arc at h after from hibernation onset. although -end ir in arc got weak, the -end ir of nerve fibers in preoptic nucleus (pon) got strong with progression of hibernation. these results suggest that the -endorphin was transported to pon from arc by axonal flow and then played an important role in maintenance of hypothermia via -opioid receptors in hibernation. wei-min qu, zhi-li huang, naomi eguchi, yoshihiro urade, osamu hayaishi department of molecular and behavioural biology, osaka bioscience institute, osaka, japan prostaglandin (pg) d is a potent somnogenic substance, and isomerized from pgh through the action of pgd synthase (pgds). pgds has two distinct types, the lipocalin-type pgds (l-pgds) and hematopoietic pgds (h-pgds). selenium compounds have been reported to decrease sleep by inhibiting pgds in rats. to clarify what type of pgds inhibition is involved in sleep reduction by selenium or whether selenium intoxication decreases sleep, we intraperitoneally injected secl into l-pgds and h-pgds knockout (ko), and their wild-type (wt) mice. in wt mice, secl decreased rapid eye movement (rem) and non-rem sleep for h after injection and, concomitantly, increased wakefulness. similar results were observed in h-pgds ko mice. in contrast, l-pgds ko mice did not exhibit any significant changes in sleep-wake profiles after secl administrations. these findings indicate that pgd plays an essential role in the maintenance of the sleep state under physiological conditions, and l-pgds is a key enzyme for the production of pgd involved in sleep-wake regulation. under baseline conditions, h r ko mice showed essentially identical sleep-wakefulness cycles to those of wild-type (wt) mice but with fewer incidents of brief awakening (< s epoch), prolonged duration of non-rapid eye movement (nrem) sleep episodes, a decrease in the number of state transitions between nrem sleep and wakefulness, and a shorter latency for initiating nrem sleep after an intraperitoneal injection of saline. the h r antagonist pyrilamine mimicked these effects in wt mice. these results indicate that h r is involved in the regulation of behavioral state transitions from nrem sleep to wakefulness (huang et al., ) . ps a-h dissociation of responsibility in firing activity to dim light between the optic nerve and the suprachiasmatic nucleus neuron of mice koichi fujimura, ai fukushima, takahiro nakamura, toshihiro jogamoto, kazuyuki shinohara division of neurobiology & behaviour, nagasaki university, graduate school of biomedical science, nagasaki, japan the involvement of light response in the optic nerve to the firing activity of suprachiasmatic nucleus (scn) neuron was investigated by extracellular single unit recordings from the optic chiasma and the scn in mice. recordings were carried out during the early night in a light:dark cycle, and the illuminations were applied to a contralateral retina with a high-power led (λ = nm). the scn neurons responded to the light in intensities above photons/cm /s and were activated maximally at around photons/cm /s, they were about . log units less sensitive than optic fibers with high sensitivity. a sustained illumination in the intensity range between suprathreshold for the optic fibers and subthreshold for the scn neuron did not suppress the subsequent light response in the scn neurons, except in a few neurons. these results suggest that the most of the light responsive scn neurons are driven by any inputs independent of the high sensitive optic fibers. masayuki ikeda, tomoyoshi kojiya department of biology, faculty of science, toyama university, japan the hypothalamic suprachiasmatic nucleus (scn) has a pivotal role in the mammalian circadian clock. scn neurons generate circadian rhythms in action potential firings and neurotransmitter releases, and the core oscillation is thought to be driven by clock gene transcription-translation feedback loops. we have found robust circadian rhythms in the cytoplasmic concentration of ca + in scn neurons. since cytosolic ca + regulates diverse cellular systems, we have hypothesized that the cytosolic ca + rhythms may mediate the cellular output from the clock gene oscillations. here, to address the clock gene functions on the ca + rhythms, mouse bmal and its dominant negative sequence (mbmf r ) are transfected into the organotypic culture of scn with a yellow cameleon ca + sensor by the gene gun. the results demonstrated that over-expression of bmal or mbmf r significantly inhibited the circadian ca + rhythms and thus we concluded that the native bmal rhythm is essential for cellular output processes of the murine clock system. ps a-h the activation of ␣ adrenergic receptor increases the frequency of carbachol-induced  oscillation in rat hippocampal slices masafumi nakano, jun arai, kiyohisa natsume kyushu institute of technology, kyushu, japan recently it is found that locus ceruleus (lc) activation suppresses  rhythm in hippocampus in vivo. noradrenergic fibers derived from lc project to hippocampus. carbachol, a cholinergic agent, can induce  oscillation in rat hippocampal slices like  rhythm in vivo. in the present study, the effect of epinephrine on the generation of carbachol-induced  oscillation in ca region of rat hippocampal slices. carbachol ( m) induced  oscillation with the frequency and the amplitude of . ± . hz, and . ± . mv, respectively (mean ± s.e.m.; n = ). epinephrine ( m) significantly increased the frequency of . ± . hz (**p < . ), not change the amplitude. clonidine ( m), an ␣ receptor agonist, alone significantly increased the frequency at the concentration of m (*p < . ). yohimbine, an ␣ receptor antagonist, suppressed the oscillation. these results suggest that the application of adrenaline will increase the frequency of hippocampal  rhythm via ␣ receptor. attractor dynamics of recurrent neural network are believed to play an important role in information processing in the brain. we recorded transient activities of two neuron groups by two tetrodes apart . mm from each other in the hippocampal ca region in vitro and applied micro-iontophoresis of glutamic-acid near the tetrodes to activate the neurons selectively. it was found that number of spikes during twosite (pairing) stimuli is fewer than the total number of spikes during the single-site stimuli, suggesting synaptic interaction in the network. peri-stimulus time histogram (psth) of ensemble as well as individual neuronal activity in response to the single-site stimuli applied far from the recording site composed of transient (latency - ms), oscillatory ( - hz) and sustained responses. following the pairing stimuli, the psth showed change in transient response properties ( / slices). these results suggest the pairing stimuli would change attractor dynamics of the neural network in the ca region. ryozo aoki , hiroshi wake , hitoshi sasaki , kiyokazu agata dept. physiol. & biosignal. osaka univ. grad. sch. med., suita, japan; dept. elec. eng. & elec. col. industri. tech., amagasaki, japan; dept. biophys., kyoto univ. grad. sch. sci., kyoto, japan by insertion of a stainless-steel monopolar electrode to the head of planarian, continuous waveform of electrical potential could be first observed in microvolts. the frequency spectrum showed an almost monotonously decreasing distribution likely as /f, ranging from − to + hz. during the eeg recordings the planarian was kept still by cooling in several degrees. when it was cooled down to lower temperatures the amplitude of eeg was suppressed, and by warming again restored with spikes provably due to motions. this eeg active state continued beyond min after the electrode insertion but the amplitude gradually decreased, and became natural noise at the time up to min. by observing the sample it turns out the sticked head was degraded. strong photo stimulation suppressed this eeg signals and recovered after over min. however little response to light pulse stimuli was observed on the eeg spectrum. mariko uchida , hiroki sato , , naoki tanaka , atsushi maki , japan science and technology agency, crest, saitama, japan; advanced research laboratory, hitachi, ltd., saitama, japan previous studies about electroencephalography (eeg) described that alpha-wave power (the frequency band from to hz) decreases and the sleep spindle power (from to hz) increases in falling asleep. the purpose of this study is to analyze the crosscorrelation between the eeg power changes (eegpc) of each band and the cortical hemoglobin concentration changes (hbcc) during sleep. we measured optical topography (ot) and eeg simultaneously. the hbcc was measured at eighty-eight positions covering whole head of subject by ot probes. five females and eight males participated in this measurement. the results showed the high correlation between eegpc and hbcc at the location of dorsolateral prefrontal area, both in the period of (i) dominance of alpha-wave and (ii) dominance of sleep spindle. the time lag from eegpc to hbcc was from to s in (i), and from to s in (ii). we examine these differences between (i) and (ii) in detail. carnitine deficiency disturbs fatty acid oxidation under the fasting condition (fc). we show herein that nocturnal locomotor activity (la) was reduced under fc and recovered to normal by carnitine injection in jvs −/− mice, a model of systemic carnitine deficiency. as judged from eeg/emg profiles, jvs +/+ mice showed prolonged wakefulness under fc, but jvs −/− mice revealed disruption of the prolonged wakefulness with a high frequency of non-rem sleep. as the orexinergic arousal system plays an important role in la, we determined orexin neuronal activity in the fasted mice. fasted jvs −/− mice had fewer c-fos + orexin neurons in their lateral hypothalamus and a reduced orexin-a content in their csf, suggesting that the fasted jvs −/− mice exhibited reduced la and fragmented of wakefulness due to suppressed orexin neuronal activity. juhyon kim , kazuki nakajima , yutaka oomura , kazuo sasaki div. of bio-information eng., univ. of toyama, toyama, japan; dept. of integrat. physiol., kyushu univ., fukuoka, japan novel peptide, orexin, identified in the lateral hypothalamus (lh) participates in the regulation of sleep-wakefulness. orexin-containing neurons in the lh project to the pedunculopontine tegmental nucleus (ppt). the ppt is one of brain sites which control sleepwakefulness. thus, we examined effects of orexin on ppt neurons electrophysiologically using brain slice preparations in rats. applications of orexin depolarized the membrane potential of ppt neurons dose-dependently, and the depolarization was associated with the increase in membrane resistance. when extracellular k + concentration was increased, the magnitude of the depolarization significantly decreased. when extracellular na + was replaced by n-methyl-dglucamine, the magnitude of the depolarization also decreased significantly. these results suggest that the ionic mechanism for orexininduced depolarization includes k + channel, non-selective cation channel and/or na + /ca + exchanger, and that orexin participates in the regulation of sleep-wakefulness via the excitatory effect on ppt neurons. ben-shiang deng , wei zhang , akira nakamura , masashi yanagisawa , yasuichiro fukuda , tomoyuki kuwaki , dept. molec. integ. physiol., chiba univ., japan; dept. autonom. physiol., chiba univ., japan; dept. molec. genet., univ. texas, usa we examined whether the respiratory chemoreceptor reflex in prepro-orexin knockout mice (ko) was blunted or not, and if so, whether supplementation of orexin restored the abnormality. we also studied whether pharmacological blockade of orexin in the wildtype mice (wt) resulted in a similar abnormality. ventilation was recorded by whole body plethysmography before and after intracerebroventricular injection of orexin-a, -b, sb- (an orexin receptor antagonist), or vehicle. data were examined for only awake periods because sleeping distorts the chemoreflex. hypercapnic ventilatory responses but not hypoxic responses were attenuated in ko. similar abnormality was reproduced in wt treated with sb- . icv injection of orexin partially restored the hypercapnic chemoreflex in ko. our findings suggest that orexin plays a crucial role for co -sensitivity at least during waking periods. research funds: kakenhi , junko hara , taizo matsuki , katsutoshi goto , masashi yanagisawa , takeshi sakurai , department of pharmacology, basic medical science (coe), university of tsukuba, ibaraki, japan; yanagisawa orphan receptor project, erato, jst, tokyo, japan; howard hughes medical institute and department of molecular genetics, university of texas, dallas, texas, usa when the production of inflammatory cytokines is stimulated by acute inflammatory, the nonrem-sleep amount of animals increases. this is possibly due to changes in the biological activity of the tnfalpha system. besides their important function in sleep regulation during acute immune response, cytokines also seem to be involved in physiological sleep regulation. orexins (hypocretins) are recently identified neuropeptides that are derived from a common precursor peptide. recent studies suggest that specific degeneration of orexincontaining neurons occurs in brains of human narcolepsy patients, suggesting critical roles of these neurons in the regulation of vigilance states. here, we examined the effects of inflammatory cytokines on the activity of orexin neurons, by means of patch-clamp recording. these effects might also possibly be involved in the pathophysiology of narcolepsy. ps a-i prenatal exposure to bisphenol a enhances avoidance response to predator odor and impairs sexual differentiation of olfactory response of medial amygdala neurons tetsuya fujimoto , kazuhiko kubo , shuji aou dept. brain sci. eng., kyushu inst. technol., kitakyushu, japan; dept. otorhinolaryngol., chidoribashi hospital, fukuoka, japan prenatal exposure to bisphenol a (bpa) impairs the sexual differentiation of exploratory behavior and enhances depressive behavior (fujimoto et al. ) . in this study, the effects of bpa on general motor activity and avoidance response to predator odor and olfactory responses in medial amygdala neurons were examined. the smell of fox predominantly suppressed locomotor activity and enhanced avoidance response by bpa. in the electrophysiological study, male medial amygdala neurons showed selective excitatory responses to predator odors. this type of neurons did not respond to plant odors. in contrast female amygdala neurons did not show such selectivity. the sex difference in this neuronal response pattern was attenuated by bpa exposure. these findings suggest that bpa impairs sexual differentiation of medial amygdala neurons which affect emotional responses to the olfactory cues of predators. research funds: grants-in-aid for scientific research (no. , s.a.) shuji aou , tetsuya fujimoto , yumi ichihara , kimiya narikiyo , toru ishidao , hajime hori , yukiko fueta dept. brain sci. eng., kyushu inst. technol., kitakyushu, japan; dept. environm. manage., sch. health sci., univ. occup. environm. health, kitakyushu, japan -bromopropane ( -bp), an ozone-depleting substance replacement, has neurotoxicity and exhibited reproductive toxicity in adult animals. in this study, we investigated the effects of prenatal exposure to -bp on sexual differentiation of reproductive and non-reproductive behaviors. pregnant rats were exposed to ppm of -bp during prenatal period. the open-field test, lashley iii maze test and sexual behavior were evaluated at adult age. -bp significantly reduced the locomotor activity and the number of entries into the center area in female rats but not in males in the open-field test. in sexual behavior, the number of ear wiggles, an index of proceptive behavior, was decreased and the rejection score was increased in female rats. these results suggest that -bp is the potential candidate of endocrine disruptors which affect brain development. ( ) ps a-i changes in hippocampal excitability of rats prenatally exposed to -bromopropane yukiko fueta , toru ishidao , susumu ueno , yasuhiro yoshida , hajime hori department of environmental management, school of health sciences; department of pharmacology; department of immunology, school of medicine, university of occupational and environmental health, kitakyushu, japan inhalation exposure to -bromopropane ( -bp), a substitute for ozone depleting compounds, alters the function of gabaergic system in the hippocampus of adult male rats. but the neurotoxcitiy induced by prenatal exposure has not been well investigated. in this study pregnant rats were exposed to -bp ( ppm) during gestational day - ( h/day), and the hippocampal excitability in pregnant rats and their offspring was examined. basic excitability was enhanced and disinhibition was observed in the hippocampus of pregnant rats. offspring, however, exhibited an enhancement of averaged s/r curve of ps in the ca at the pnd - . conversely, s/r curves of fepsp as well as ps in the ca were inhibited at the age of - weeks. our results suggest that -bp causes hyperexcitability in pregnant rats, and disrupts basic excitability in the ca of the offspring during development. research funds: grant-in-aid for exploratory research ( ) ps a-i effects of endocrine disrupting chemical bisphenol a on the development of mouse cerebral cortex keiko nakamura , , kyoko itoh , takeshi yaoi , tohru sugimoto , shinji fushiki dept. pathol. appl. neurobiol., kyoto pref. univ. med, kyoto, japan; dept. pediatr., kyoto pref. univ. med, kyoto, japan bisphenol a (bpa), a widely distributed xenoestrogen, has been shown to disrupt thyroid hormone function. we have thus studied whether prenatal exposure to low-doses of bpa affects morphology and the expression of thyroid hormone-dependent genes in murine fetal neocortex. pregnant mice were injected subcutaneously g/kg of bpa daily from embryonic day (e ). control animals were injected vehicle alone. for evaluating cell proliferation, neuronal differentiation and migration, bromodeoxyuridine (brdu) was given to pregnant mice and processed for immunohistochemistry. the total rna was extracted from embryonic telencephalons at different embryonic period. brdu-labeled cells were decreased in the ventricular zone at e . and e . , whereas those cells increased in the cortical plate at e . , as compared with control mice. some of the genes associated with neurogenesis and thyroid hormone function were upregulated in bpa-treated group. research funds: jsps grant keiko ikemoto , teruko uwano , hisao nishijo , taketoshi ono , masayuki ito , ikuko nagatsu , katsuji nishi , shin-ichi niwa dept. neuropsychiat., fukushima med. univ. sch. med.; toyama med. pharm. univ.; faculty med., mie univ., mie, japan; fujita health univ. sch. med., toyoake, japan; dept. leg med., shiga univ. med. sci., japan we examined the effect of maternal repeated cold stress (rcs) on development of catecholamine neurons of offsprings using by tyrosine hydroxylase (th) immunohistochemistry. rcs was loaded to pregnant rats between day and after fertilization. pups were perfused at postnatal day . in the frontal cortex, the number of largesized (more than m in diameter) th-immunoreactive (-ir) varicosities was significantly smaller in prenatally rcs rats than controls. in the locus coeruleus of prenatally rcs rats, th immunoreactivity was less than that of controls. in the medullary c /a catecholaminergic field, the size of th-ir neurons was smaller and the quantity of thir fibers were less in prenatally rcs rats, although there were not significant differences. it was suggested that prenatal rcs impaired development of catecholaminergic neurons, especially noradrenergic neurons of neonates. ps a-i developmental exposure to pentachlorophenol affects thyroid hormone responsive gene in the brain but not stress response maiko kawaguchi , , , kaori morohoshi , , rie yanagisawa , erina saita , gen watanabe , , masatoshi morita , kazuyoshi taya , , hirohisa takano , , toshiyuki himi , , hideki imai , dept. toxicol and pharmacol., facul. pharmacy, musashino univ., tokyo, japan; res. inst. pharmaceut. sci., musashino univ., tokyo, japan; nation. inst. for environ. stud., ibaraki, japan; grad. sch. environ. sci., univ. tsukuba, ibaraki, japan; wildlife rescue veterinarian associ., tokyo, japan; facul. agriculture, tokyo univ. agriculture & technol., tokyo, japan; the united grad. sch. veterinary sci., gifu univ., gifu, japan; div. environ. health sci., dep. social med., facul. med., miyazaki univ., miyazaki, japan antiseptic pentachlorophenol (pcp) treatment to rats affects thyroid hormone (th) system, which is essential for normal development of central nervous system. in this study, we show the exposure to pcp during gestation and lactation suppressed plasma th level, and induces gene expression of neurogranin and th receptor , which play a role in neural formation. the present data suggest that pcp may affect central nervous system development, though stress response was not affected by pcp exposure. ps a-i the effect of psychological stress during pregnancy on the open-filed behavior, the forced swim test, the fos expression in the brain, and the level of plasma corticosterone in offspring rat hiroshi abe, noriko hidaka, kei odagiri, yuko watanabe, yasushi ishida dept. of psychiatry, miyazaki med. coll., univ. of miyazaki, japan one group (psy) was born from the dams which observed, during their pregnancy, that another rat was exposed to the foot-shock stress in a communication box. the other group (c) was born from the dams not exposed to such stress. psy, comparing to c, showed decreased activities in the open-field test and prolonged immobility time in the forced swim test. on the other hand, there were no significant differences between the number of fos immunopositive cells in various regions of the brain in two groups before and after the foot-shock. however, plasma corticosterone was elevated in psy compared with c. these results suggest that the prenatal psychological stress might enhance reactivity to novel environment and depressive behavior induced by forced swim, and chronically elevated level of corticosterone might be involved in this neurobiological substrate. akane nakasato, yasushi nakatani, yoshinari seki, hideho arita department of physiology, toho university school of medicine, tokyo, japan to evaluate roles of da and serotonergic ( -ht) systems in stressinduced anxiety, we measured brain da and -ht levels before, during and after a forced swimming test (fst) in autistic model of the rat. the model rat was made by exposing a pregnant rat to valproic acid (vpa). our previous study demonstrated that the autistic model exhibited abnormality of -ht system and behavioral impairments related to autism. in the present experiment, we gave a prolonged fst for min in the model rat, which frequently experienced to be drowned after the immobility time during fst. brain da and -ht levels were measured from samples collected from the prefrontal cortex (pfc). we found a gradual and steady increase in pfc da level during fst, although -ht level showed only transient augmentation. behavioral alteration after fst was characterized by an increased appetite during light phase (sleep) of circadian cycle. we suggest that the feeding abnormality may be caused by the stress-induced anxiety mediated by mesocortical da system. shigeo masaki, eiko aoki, satoshi yonezawa, atsuo nakayama dept. embryology, inst. developmental res., kasugai, aichi, japan neuroligin (nl - ) is a family of neuronal cell-surface proteins to be involved in intercellular junctional formation and signalling. recently, several studies have implicated nl and nl in autistic disorders. nls have a relative identical structure (∼ %); nl and nl localize in the glutamatergic excitatory, and inhibitory synapses, respectively, while nl seems express in the olfactory glia, but nl distribution is unknown. here we have generated antibody against human nl , and explored its distribution in the post mortem human tissues. in the central and peripheral nervous system, nl was expressed exclusively in the neurons, and was especially abundant in particular subsets of neurons, including neurons producing nonapeptides. nl was observed in paraneurons and some endocrine cells outside the cns. these results suggested that nl is important for neuroendocrine function. nl cdna was transfected to in neuroblastoma. formed spine-like structures on the cells expressing nl were rough and thicker than those of nl or nl transformants. it suggested the unique activity of nl for synapse formation. motopsin is a serine protease secreted from pyramidal neurons of cerebral cortex and hippocampus. recently, the truncation of motopsin gene has been reported to cause non-syndromic mental retardation. however, the underlying mechanisms are yet to be elucidated. we report here that the knockout (ko) mice deficient in the expression of motopsin exhibit morphological abnormality. golgi-cox staining revealed that spine density on both apical and basal dendrites of hippocampal ca pyramidal neurons in the ko mice significantly decreased than in the wild type mice. similarly, spine density tended to decrease at cingulate cortex of the ko mice than wild type. our results suggest that motopsin affects dendritic spine formation and/or stabilization. mental retardation is a frequent disorder affecting - % of the population. recently the truncation of motopsin/neurotrypsin gene has been identified in algerian family in which four out of eight children affected by a severe impairment of cognitive functions with an iq below . here we report that knockout (ko) mice lacking motopsin gene mildly impaired water maze performance and social behavior. the ko mice significantly delayed the latency to the platform area on a probe test of hidden version of morris water maze although they showed the similar performance to wild-type mice during training session. in a social memory test, the ko mice showed significant elongation of sniffing time to an intruder, despite of normal performance of social memory. our results suggest that the ko mice provide insights into the molecular mechanisms important for development of cognitive functions. natsue yoshimura , daisuke horiuchi , tomoyuki miyashita , minoru saitoe , hitoshi okazawa department of neuropathology, medical research institute, tokyo medical and dental university, tokyo, japan; tokyo metropolitan institute for neuroscience, tokyo, japan polyglutamine tract binding protein- (pqbp- ) was originally isolated as one of the candidates for polyglutamine disease related protein. recently, several groups has reported about pqbp- disease that pqbp- mutant causes x-linked mental retardation (xlmr). to investigate the function of pqbp- in xlmr pathology, we produced two kinds of flies, human pqbp- overexpression flies (hpqbp flies) and drosophila pqbp- knock-down flies (dpqi flies), and examined olfactory learning and memory to analyze their memory consolidation process from short-term memory (stm) to long-term memory (ltm). the hpqbp flies showed memory impairment in ltm. in current study, we analyze memory abilities of the dpqi flies to observe detailed function of pqbp- in memory formation. seiji hayashizaki, masahiko takada tokyo metropolitan institute for neuroscience, usa when two alternatives are available in instrumental behavior, animalǐs behavior is biased toward responding on one lever with which each behavioral response results in delayed large reward delivery, and against responding on the other lever with which each response results in immediate but small reward delivery. this has been used as an index of impulsive behavior and is known to be susceptible to lesions of brain structures such as the basolateral amygdala (bla) and the nucleus accumbens (na). it has been shown that the bla and na are involved in maintaining reward seeking behavior with a secondary reward when a secondary reinforcer is available. thus, a question arises as to how the behavioral response on the delayed lever is maintained through functions exerted by these structures when no secondary reinforcer is available. to this end, we implanted cannulae bilaterally and electrodes into the bla and na to identify neuronal substances and activities involved in the mediation of 'putative secondary reward' without secondary reinforcer. xue-zhi sun , sentaro takahashi , yoshihisa kubota , rui zhang , chun cui , yoshihiro fukui natl. inst. radiol. sci., chiba, japan; sch. med. tokushima univ., tokushima, japan heavy ion irradiation has the feature to administer a large radiation dose in the vicinity of the endpoint in the beam range, and its irradiation system and biophysical characteristics are different from ordinary irradiation instruments like x-or gamma-rays. using this special feature, heavy ion irradiation has been applied for cancer treatment. the safety and efficacy of heavy ion irradiator have been demonstrated to a great extent. for instance, brain tumors treated by heavy-ion beams became smaller or disappearance. however, fundamental research related to such clinical phenotypes and their underlying mechanisms are little known. in order to clarify characteristic effects of heavy ion irradiation on the brain, we developed an experimental system for irradiating a restricted region of the rat brain using heavy ion beams. the characteristics of the heavy ion beams, histological, behavioral and elemental changes were studied in the rat following heavy ion irradiation. yukio imamura department of psychiatry, university of ottawa, on, canada nmdars contain two nr subunits paired with two nr subunits. nr and nr (a-d) subunits harbor the glycine and glutamate binding sites, respectively. nmdars are localized in both synaptic and extra-synaptic areas, but they are found at higher density within the synapse. after the peak of synaptogenesis, the nr /nr a complex, characterized by rapid offset kinetics, dominates at the synapse, while the nr /nr b complex, characterized by slow kinetics, predominates in the extra-synaptic area. the activation of extrasynaptic nmdars by glutamate escaping from the synaptic cleft during episodes of high synaptic activity suggests that they may have a different role. using whole-cell voltage-clamp recordings from ca pyramidal neurons from mice (at weeks of age), we found that following induction of ischemia, ifenprodil, a selective nmdar-nr b antagonist, reduced the inward current of the isolated nmdar at extra-synaptic site while it had less effect at the synaptic nmdar. the molecular mechanisms involved are currently under investigation and these new data will be also presented at the meeting. in the present study, we observed expression and changes of mineralocorticoid receptor (mr) and glucocorticoid receptor (gr) in the gerbil hippocampal ca region after ischemia. in blood, corticosterone levels increased biphasically at min and h after ischemia, and thereafter its levels decreased. in the sham group, mr and gr immunoreactivities were weakly detected in the ca region. by days after ischemia, mr and gr were not significantly altered in the ca region. from days after ischemia, mr and gr immunoreactivities were detected in astrocytes and microglia in the ca region, and at days after ischemia. the specific distribution of corticosteroid receptors in glia may be associated with the differences of mr and gr functions against ischemic damage. the present study was investigated the effects of early treadmill training after cerebral infarction in rats. we determined whether treadmill exercise changes cellular expression of caspase- and midkine in the mca area. stroke was induced by a -min mca occlusion using an intraluminal filament. rats were exercised for min each every day on a treadmill. brain damage in ischemic rats was evaluated by infarct volume. exercised and non-exercised rat brains were processed for immunocytochemistry to quantify the areas of caspase-and mkimmunoreactive calls. no significant differences in infarct volume were found between rats trained with treadmill and non-exercised controls. cellular expressions of mk were significantly increased in striatum (glia) of the exercised rats. treadmill exercise was shown to suppress the decrease in caspase- expression in the penumbra. the present study showed the exercise after cerebral infarction might have important implication for post-ischemic recovery. ps a-j reversed astrocytic glutamate transporter glt- crucial to the ca + paradox-like insult-induced neuronal death in neuron/astrocyte co-cultures tatsuro kosugi, koichi kawahara, takeshi yamada, motoki tanaka lab. of cellular cybernetics, graduate school of information science and technology, hokkaido univ., sapporo, japan "ca + paradox" is the phenomenon whereby the intracellular concentration of ca + paradoxically increases during reperfusion with normal ca + -containing media after brief exposure to a low ca + solution. the present study aims to characterize the ca + paradoxinduced cell injury in neuron/astrocyte co-cultures. prior exposure of the cultures to a low ca + solution for min significantly injured only neurons after reperfusion with a normal ca + medium for h, but astrocytes remained intact. after the onset of reperfusion, the intracellular concentration of na + in astrocytes increased significantly during the reperfusion episode, resulting in a reversal of the operation of the astrocytic glt- . the present findings suggested that ca + paradox-induced accumulation of na + in astrocytes was involved in the reperfusion-induced excitotoxic neuronal injury resulting from the reversed operation of astrocytic glt- during the reperfusion episode. common genetic mutation in cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (cadasil) has been associated with missense mutations of notch concerning cysteine residues within the extracellular amino-terminal region. we report new mutations of two japanese cadasil families, which did not directly involve a cysteine residue. exons of the notch were amplified by pcr and subsequently analysed for dhplc and direct sequence. the first patient carried the missense mutation c t, which results in pro ser. the second patient carried the missense mutation c g, which results in arg pro. new mutations had not changed the number of cysteine residues, but coding the extracellular amino-terminal region of the notch receptor which may involve an alteration in the ligand binding or putative dimerisation properties. ps a-j mci - , a radical scavenger, protected cortical neurons from cell death through the activation of mitogen-activated protein kinase and phosphatidylinositol kinase madinyet niyaz , tadahiro numakawa , yoshinori matsuki , emi kumamaru , yuki yagazaki , harumi kitazawa , hiroshi kunugi , motoshige kudo pathology department of tokyo medical university, tokyo, japan; department of mental disorder research, national institute of neuroscience, ncnp, tokyo, japan the role of mci - , a radical scavenger, in the central nervous system (cns) has not been fully elucidated. in the present study, we found that treatment with mci - prevented the cultured cortical neurons from cell death induced by serum deprivation. furthermore, we found that mci - exposure induced the activation of both the map kinase (mapk) and pi kinase (pi k) pathways and that the mci - -dependent survival effect was blocked by the inhibitors, u (an mapk pathway inhibitor) or ly (a pi k pathway inhibitor). these results suggested that mci - exerts a protective effect on cns neurons via enhancing survival-signaling pathways in addition to a role such as a radical scavenger. osamu tokumaru , noriko yoshimura , tetsuro sakamoto , takaaki kitano , naoko nisimaru , isao yokoi dept. physiol., sch. med., oita univ., japan; med. edu. ctr., sch. med., oita. univ., japan protective effects of ethyl pyruvate (ep) on energy metabolism of rat brain exposed to ischemia were investigated by p-nuclear magnetic resonance ( • c). brain slices were incubated in standard artificial cerebrospinal fluid (acsf) with mm ep (ep- ), acsf replaced by acsf with mm ep after ischemia (ep- ), or acsf only (control). the brain slices were exposed to ischemia by stopping the perfusion for h. high-energy phosphate, creatine phosphate (pcr) and ␥-atp, levels were measured. decrease in pcr level was not different among the three groups when exposed to ischemia. but increase in pcr level after the reperfusion was significantly larger in ep- than in control (p < . ). these results indicate that ep is effective in the reperfusion period and is more protective when administered before ischemic exposure. the importance of timing of administration of ep in clinical use was suggested. research funds: grant-in-aid for scientific research (c) # from mext to t.k. hideaki tamai , kuniko shimazaki , norimasa seo department of anesthesiology and critical care medicine, jichi medical university, graduate school, tochigi, japan; department of physiology, jichi medical university, tochigi, japan we investigated the effects of acupuncture on cell proliferation in the dentate gyrus (dg) and the lateral ventricle (lv) of adult rats. in this study, acupuncture was performed at the acupoints neiguan (pc ), yintang (ex-hn ) and sanyinjiao (sp ), which have been used for the enhancement of conscious and functional recovery in stroke patients. eight weeks old male wistar rats were used in the experiment. through -bromo- ,-deoxyuridine (brdu) immunohistochemistry, a significant increase in cell proliferation in the dg of the acupunctured group was observed. however, the cell proliferation in the lv was not affected with the acupoints pc , ex-hn and sp . the present findings indicate that the sensitivity on cell proliferation in the dg by acupuncture stimulation is higher than in the lv. yukio ago , keiko takahashi , shigeo nakamura , akemichi baba , toshio matsuda laboratory of medicinal pharmacology, graduate school of pharmaceutical sciences, osaka university, osaka, japan; laboratory of molecular neuropharmacology, graduate school of pharmaceutical sciences, osaka university, osaka, japan this study examined the effect of isolation rearing on anxiety-related behavior of mice in the staircase test, an animal model of anxiety. the staircase test consisted of placing an experimentally naive mouse in an enclosed staircase with five steps. in group-reared mice, an anxiolytic diazepam increased the number of steps climbed to the top step of the staircase, but did not affect the frequency of rearing behavior. the anxiogenic drug -cca increased the number of rearing, but did not affect the number of steps climbed. on the other hand, methamphetamine increased the number of steps climbed to the second step. in these circumstances, isolation-reared mice showed an increase in the numbers of steps climbed to the top step and rearing in the staircase. these findings suggest that isolation rearing increases in exploratory and anxiety-like behaviors in mice. tomonori fujiwara , tatsuya mishima , takefumi kofuji , kimio akagawa department of cell physiology, kyorin university school of medicine, mitaka, tokyo, japan; radio isotope laboratory, kyorin university school of medincine, mitaka, tokyo, japan hpc- /syntaxin a is believed to regulate the exocytosis of synaptic vesicles. in order to examine the neurophysiological function in vivo, we have produced hpc- /syntaxin a knock-out mice. surprisingly, the null mutant mice revealed normal development and basal synaptic transmission in cultured hippocampal neurons appeared to be normal. however, in conditioned fear memory test, consolidation of the memory was impaired in homozygous mutant mice but not in heterozygote. however, once memory consolidation was acquired, the extinction process was disturbed in homozygote. we further examined latent inhibition of cued fear memory (li) to access behavioral property. interestingly, li was suppressed both in heterozygous and homozygous mutant mice unlike the case of conventional conditioned fear memory test. implication of these behavioral abnormalities in hpc- /syntaxin a knock-out mouse will be discussed. research funds: kakenhi ( ) ps a-k effects of local administration of the gaba agonists into the hippocampus ca area on active avoidance learning and serotonergic systems in the administration area in rats satoko hatakenaka , hiroko miyakubo , junichi tanaka , yasushi hayashi , yukio hattori , masahiko nomura department of curriculum, teaching and memory, naruto university of education, tokushima, japan; department of human nutrition, notre dame seishin university, okayama, japan; department of physiology, saitama medical school, saitama, japan in fischer male rats, bilateral injections of the ␥-aminobutyric acid (gaba) a agonist muscimol into the ca area slightly decreased the avoidance rate in an active avoidance task. similar injections of the gaba b agonist baclofen enhanced the avoidance rate. there are significant differences between the muscimol-and baclofen-treated groups in the avoidance rate, implying that gaba a and gaba b receptors have the opposite action on the performance of avoidance learning. perfusion with muscimol through the microdialysis probe decreased the serotonin metabolite -hydroxytryptamine ( -hiaa) concentration in the ca area, whereas baclofen perfusion had no effect, suggesting that the gabaergic system may exert to inhibit the serotonin release in the ca area through gaba a receptors. sawako arai, taku nagai, kenji takahashi, hiroyuki kamei, kazuhiro takuma, kiyofumi yamada lab. neuropsychopharmacol, kanazawa univ., kanazawa, japan we performed immunohistochemical c-fos mapping after a prepulse inhibition (ppi) test of the startle reflex in mice. startle stimulus increased the number of c-fos-positive cells in the somatosensory cortex, nucleus accumbens shell and the caudal pontine reticular nucleus (pnc), while prepulse trials without startle stimulus increased c-fos expression in the lateral globus pallidus (lgp). in mice subjected to startle stimulus with prepulses, most of the startle stimulus-induced c-fos expression was diminished but c-fos expression remained in the lgp. prepulse-induced c-fos expression in the lgp was colocalized with gad- . fluoro-gold infusion into the pnc and the pedunculopontine tegmental nucleus (pptg) retrogradely labeled neurons in the pptg and lgp, respectively. microinjections of phaclofen, but not picrotoxin, into the pptg impaired ppi of the startle reflex. these results suggest that gabaergic neurons in the lgp which project to the pptg play a crucial role through the activation of gaba b receptors in the ppi of the startle reflex. shiho kitaoka , sho koyasu , akinori nishi , tomoyuki furuyashiki , toshiyuki matsuoka , shuh narumiya department of pharmacology, university of kyoto, kyoto, japan; department of physiology, university of kurume, kurume, japan prostaglandins e (pge ) exert their actions in various organs through specific receptor, ep to . the previous study suggests that ep modulates da system. to investigate the roles of ep in da system, we examined ep ko mice with behavioral sensitization induced by cocaine. the administration of cocaine elevated da concentration in the nucleus accumbens up to ∼ % in both wild-type and ep ko mice. however, increase of locomotor activity in ep ko mice was significantly lower than that in wild-type mice. because locomotor activity is closely related to dopamine d receptor (d r) signaling, we tested the density of d r and d r signaling with phosphorylation of darpp- . there were no differences in d r binding. d r signaling was significantly attenuated in the striatal slices from ep ko mice. the effect of d r agonist on locomotor activity was also attenuated in ep ko mice. these results indicate that pge has enhancing effects on locomotor activity via ep by potentiating the d r signaling. central serotonin ( -ht) function has been implicated in impulsivity. the present study examined rats with -ht depletion by parachloroamphetamine (pca) in simple and reversal go/no-go visual discrimination tasks, and analyzed the relationships between learning performance and focal concentrations of -ht and its metabolites ( -hiaa) in the brain. for both tasks, significant negative correlations between learning performance and -ht and -hiaa concentrations were observed in the medial prefrontal cortex and nucleus accumbens. in contrast, for reversal task only, significant correlations between learning performance and -ht and -hiaa concentrations were observed in the orbitofrontal cortex and amygdala. these data suggest the regional difference of -ht roles on selective indices of impulsivity. yuki sato , , , tatsushi onaka , norimasa seo , eiji kobayashi dept. anesthesiol., jichi med. univ., tochigi, japan; dept. physiol., jichi med. univ., tochigi, japan; div. organ replacement research, center for mol. med., jichi med. univ., tochigi, japan cyclosporine is widely used for preventing allograft rejection. however, in a considerable number of transplant recipients, cyclosporine causes neuropsychological side effects such as confusion, depression, and anxiety. cyclosporine inhibits calcineurin activity and forebrain-specific calcineurin knockout mice exhibit deficits in social behaviour. it is thus possible that cyclosporine causes psychological side effects via disturbing social interactions. here, we examined effects of cyclosporine upon anxiety and social behaviour in mice. calcineurin did not significantly change percent entries into open arms and time spent on open arms in the elevated plus maze test. on the other hand, in the social interaction test in home cage, cyclosporine increased the number of particles in home cage, an index of social activity. all these data suggest that impaired social interaction is a cause of psychological side effects of cyclosporine. to investigate the distribution of functionally activated vestibularrelated brainstem neurons during postnatal development, ombined immuno-/hybridization histochemistry of c-fos expression was performed in sprague-dawley rats (p - ; adult). conscious animals were subjected to rotational or translational stimulus which activates hair cells of the horizontal semicircular canals or utricle, respectively. neuronal activation within brainstem nuclei was defined by the expression of c-fos. labyrinthectomized controls and normal stationary controls showed only a few sporadically scattered fos-expressing neurons. with rotational stimulation that comprised cycles of constant angular acceleration and deceleration, fos-labeled neurons were observed by p in the vestibular nucleus and downstream relay stations of vestibular pathways, such as the prepositus hypoglossal nucleus and inferior olive (subnuclei dmcc, ioa, ioc, iok). a later maturation time was evidenced for the utricular system. fos-labeled neurons were only identifiable in the vestibular nucleus by p ; in the prepositus hypoglossal nucleus and inferior olive (subnuclei dmcc and io) by p . within the vestibular nucleus of p - rats, neurons activated by canal or utricular inputs were intermingled throughout its rostro-caudal length. in p and adult rats, neurons activated by canal or utricular inputs were intermingled in localized regions of the medial and spinal vestibular nuclei. however, neurons in the rostral half of spinal vestibular nucleus were activated only by utricular inputs. taken together, we have demonstrated that canal-and otolithrelated brainstem neurons that encode rotational and translational movements in the horizontal plane are histologically segregated and exhibit different developmental time frame. to determine whether perineuronal nets (pn) within the vestibular nuclei contribute to plasticity of central connectivity, we studied the presentation of pn within the vestibular nuclei during development (rats, p to adult) and after unilateral labyrinthectomy (ul) in the adult. histochemistry with the lectin wisteria floribunda agglutinin was used to map pn about neun-immunopositive neurons within the vestibular nuclei. in normal postnatal rats, pn was detectable by p in the vestibular nucleus as fuzziness about neuronal cell bodies. from p onwards, the fuzzy pn progressively consolidated into a network organization. the fuzziness was no longer observable after p . during postnatal development, the number of neurons showing pn increased with age, reaching the adult level by p . with ul, the pn network on the lesioned side remained compact until days post-lesion when the fuzziness reminiscent of that in early postnatal rats became evident. by days after ul, the pn of some neurons resumed the network pattern as was observed in normal adult rats. this phenomenon was found in the pn of the remaining neurons by days after ul. the pn on the labyrinth-intact side showed the compact network of uninjured age-matched rats. taken together, our findings indicate pn changes that suggest possible correlation with vestibular nuclear neuronal function both during postnatal development of normal rats as well as in adult rats following destruction of the ipsilateral inner ear. minori ueda, takayuki suzuki, hiroyoshi miyakawa laboratory of cellular neurobiology, tokyo university of pharmacy and life science, tokyo, japan dynamics of transmitters in the synaptic cleft depends on many processes such as transmitter release, uptake and diffusion. to better understand these processes, we analyzed ampar-and nmdarmediated epscs and synaptically induced transporter currents (stcs) elicited with high-frequency stimuli. recordings were made from pyramidal cells and astrocytes in the ca region of rat hippocampal slices, hz/ pulse tetanic stimulations were delivered to schaffer-collaterals, and the evoked currents during the course of tetanic stimulation were isolated. the decay time course of the last isolated stc during the tetanic stimulation was not significantly different from that of the first. while the amplitude of the ampar-mediated epscs showed significant decay in the presence of cyclothiazide, there was no marked decay of the amplitude of the nmdar-mediated epscs. these findings imply that synaptic fatigue and saturation of glutamate transporters do not take place during the course of high-frequency stimulation at hz. ikuko yao , hiroshi takagi , hiroshi ageta , tomoaki kahyo , ken hatanaka , kaoru inokuchi , mitsutoshi setou , mitsubishi kagaku institute of life sciences, tokyo, japan; university of tokyo, tokyo, japan; okazaki institute for integrative bioscience, national institute for physiological sciences, okazaki, japan we identified and characterized a novel ubiquitin ligase named scrapper. scrapper is an f-box protein which has leucine rich repeat and c-terminal membrane localization sequence, highly expressed in neurons throughout the brain. to investigate the physiological role of scrapper in the neuron, we recorded mepscs from the neuron over-expressed the egfp-tagged full-length scrapper construct or truncated form of scrapper constructs. they exhibited a strong suppression or enhancement in the frequency of mepscs while showing a non-significant change in mepsc amplitude, rise, and decay time compared with neurons expressing egfp. the passive membrane properties of neurons such as membrane resistance (rm), series resistance (rs), and membrane capacitance (cm) were not statistically different from those of control. these data suggests a presynaptic effect of scrapper protein. ps p-a presynaptic membrane potential-dependent regulatory mechanism of transmitter release tetsuya hori, tomoyuki takahashi department of neurophysiology, university of tokyo graduate school of medicine, tokyo, japan in simultaneous pre-and postsynaptic recordings at the calyx of held, we addressed the mechanism underlying presynaptic membrane potential-dependent changes of transmitter release. a weak sustained depolarization (e.g., mv, s) of calyceal nerve terminal potentiated epscs despite that it diminished presynaptic action potential (a.p.) amplitude. as we further depolarized the terminal epscs became eventually depressed concomitantly with a marked reduction in the a.p. amplitude. when presynaptic ca + currents (i pca ), induced by an a.p.-waveform command pulse, were used to evoke epscs, a weak sustained depolarization enhanced i pca and epscs in parallel. this epsc facilitation was robust at the calyx of held both in rats and mice, but was almost absent in p/q-type ca + channel knockout mice. we conclude that the p/q-type specific ca + channel facilitation plays an essential role in the facilitation of transmitter release following presynaptic depolarization. hiroshi takagi , koji ikegami , ken hatanaka , , , yoko fujiwara-tsukamoto , mineo matsumoto , ikuko yao , mitsutoshi setou , , mitsubishi kagaku institute of life sciences, japan; presto, japan; school of pharmaceutical sciences, the university of tokyo, japan; okazaki institute for integrative bioscience, japan a variety of post-translational modifications to the exposed cterminal tails of tubulin, such as detyrosination/tyrosination, polyglycylation and polyglutamylation would play a crucial role in the neuron. however, evidence for the implication of these modifications in regulating the translocation of channels and receptors is currently unavailable. of the modifications, polyglutamylation is highly abundant in the mammalian brain, thus, this modification might account for the translocation of channels and receptors in the mammalian brain. in the rosa (−/−) mouse, which shows a gross loss of polyglutamylated ␣-tubulin, transient a-type currents were largely suppressed in hippocampal pyramidal neurons in vitro. we provide herein, using rosa mice, the evidence for the implication of ␣tubulin polyglutamylation in the regulation mediated a-type k current. satoshi kawasaki , shingo kimura , reiko fujita , shuji watanabe , kazuhiko sasaki dept. of physiol., sch. of med., iwate medical univ., morioka, japan; dept. of chem., sch. of lib. arts & sci., iwate medical univ., morioka, japan application of dopamine (da) induces a slow na + -current response in the identified neurons of aplysia ganglia under voltage clamp. this type of response is produced by the activation of trimeric g-protein sensitive to cholera toxin (ctx) as previously reported. the na +current response to da was gradually and irreversibly depressed after intracellular injection of clostridium difficile toxin b, which is known to inactivate all types of rho family g-proteins. intracellular application of clostridium botulinum exoenzyme c , a specific toxin to rhoa-c, also depressed the da-induced response irreversibly. furthermore, the da-induced current response was significantly depressed by gap domain of p rhogap applied intracellulary. in contrast, gef domain of rhogef dbs had a tendency to increase the response. these results suggest that the da-induced na + -current response may be regulated by the activation of rho family g-protein. the ␦ glutamate receptor (␦ r) plays a crucial role in cerebellar functions. although ␦ r has a putative channel pore domain, and ␦ r displayed ca + -permeable channel activities in lurcher mutant mice, it has been unclear whether wild-type ␦ r functions as a channel. here we introduced a ␦ r transgene, which had a mutation (gln arg) in the putative channel pore conserved in ca + -permeable glutamate receptors, into ␦ −/− mice. surprisingly, a mutant ␦ r transgene, as well as a wild-type transgene, rescued all abnormal phenotypes of ␦ −/− mice, such as ataxia and loss of long-term depression. these results indicate that ca + influx through ␦ r is not required for its function in the cerebellum in vivo, and that wild-type ␦ r may not function as a ca + -permeable ion channel. research funds: kakenhi ( ) and takeda science foundation ps p-a distribution of tarp - on hippocampal neurons and its key role in synaptic and extrasynaptic expression for ampa receptors masahiro fukaya , mika tsujita , maya yamazaki , etsuko kushiya , manabu abe , kaori akashi , masanobu kano , haruyuki kamiya , kenji sakimura , masahiko watanabe department of anatomy, hokkaido university school of medicine, sapporo, japan; department of cellular neurobiology, brain research institute, niigata, japan; department of cellular neuroscience, graduate school of medical science, osaka university, suita, japan; department of molecular anatomy, hokkaido university school of medicine, sapporo, japan the - is one of four transmembrane ampar regulatory proteins (tarps). pre-and post-embeding immunogold visualized - on excitatory synaptic and extrasynaptic membrane. in - -ko mice, ampars were reduced in hippocampal homogenates ( % of control) and psd fraction ( %). immunogold labeling also exhibited reduction of extrasynpatic ( %) and synaptic ( %) ampars in ca pyramidal cells. the reduction of extrasynaptic receptors was particularly severe on dendrites ( %) and spines ( %). ampar-mediated responses were reduced at ca synapses ( %). therefore, - is the major auxiliary subunit of hippocampal ampars. etsuko tarusawa , yugo fukazawa , elek molnar , masahiko watanabe , ryuichi shigemoto , div. cerebral structure, nips, okazaki, japan; mrc, univ. of bristol, bristol, uk; hokkaido univ., sapporo, japan; sorst, jst, kawaguchi, japan relay cells in the dorsal lateral geniculate nucleus receive two types of glutamatergic inputs; retinogeniculate (rg) and corticogeniculate (cg) synapses. it has been shown that the synaptic transmission at both rg and cg synapses is mediated via ampa and nmda receptors. however, how ampa and nmda receptors are expressed in these two types of synapses have not been elucidated. we examined the expression pattern of ampa and nmda receptors in rg and cg synapses using sds-digested freeze-fracture replica labeling (sds-frl). the sds-frl revealed that synaptic size of individual rg synapses was significantly smaller than that of cg synapses. rg synapses expressed . to times higher density of ampa receptors than cg synapses. on the other hand, cg synapses expressed . to times more nmda receptors than rg synapses. these results indicate differential effects on the relay cell by the retino-and cortico-geniculate inputs through ampa and nmda receptors. katsuyuki kaneda , , , hitoshi kita dept. of anat. & neurobiol., univ. of tennessee, memphis, tn, usa; japan society for promotion of science, tokyo, japan; dept. of developmental physiology, nips, okazaki, japan to investigate the properties of synaptically induced slow responses in globus pallidus (gp) neurons, whole-cell recordings were performed using rat brain slice preparations. repetitive stimulation of the gp and internal capsule induced mixed fast epsps/ipsps followed by a slow ipsp (sipsp), and a long-lasting slow depolarization (sdepo). bath application of nbqx, cpp, and gabazine blocked the mixed epsps/ipsps. the gaba b receptor antagonist cgp abolished the sipsp. an mglur antagonist, but not an mglur antagonist, partially blocked the sdepo. in addition, cgp enlarged the amplitude of fast ipscs, but not of epscs, that were evoked during the repetitive stimulation, suggesting an involvement of presynaptic gaba b receptors in gaba release. these results indicate that synaptically released gaba and glutamate can evoke gaba b receptor-and mglur -mediated responses in the gp. contribution of these responses to the control of gp activity will be discussed. research funds: nih and the jsps ps p-a essential contribution of glutamate to gaba depolarization involved in hippocampal seizure-like activity yoko tsukamoto , yoshikazu isomura , , michiko imanishi , tomoki fukai , masahiko takada system neurosci., tokyo met. inst. neurosci., tokyo, japan; neural circuit theory, riken bsi, saitama, japan we have previously shown that neuronal synchronization is achieved by excitatory gabaergic and glutamatergic inputs during a hippocampal seizure-like afterdischarge. however, it still remains unclear how the gaba response is converted from inhibitory to excitatory in the process of afterdischarge induction. here we traced the time-course of amplitude and reversal potential of gabaergic transmission in pyramidal cells and interneurons entraining the afterdischarge, and examined influence of glutamate on the conversion of gaba response. the gaba reversal potential in pyramidal cells rose to spike-threshold levels for > s after the induction. gaba amediated cl-influx lasted for . s, and then glutamate enhanced the conversion effectively in a gaba a -independent manner, which was dependent on an extracellular k increase. coapplication of gaba and glutamate caused a similar oscillatory activity. the results show gaba and glutamate may cooperatively induce as well as maintain seizure-like activity. michiko nakamura , yuko sekino , , toshiya manabe , division of neuronal network, department of basic medical sciences, institute of medical science, university of tokyo, tokyo, japan; crest, jst, japan profound activity-dependent facilitation of synaptic transmission at hippocampal mossy fiber synapses is a unique and functionally important property. in the present study, we found that this synaptic strengthening was partially mediated by presynaptic gaba a receptor activation during the developmental period (p < ), using electrophysiological methods and optical imaging. in immature animals (p ), fiber volley amplitudes were activity-dependently increased during short-train stimulation of mossy fibers. this fiber volley facilitation was significantly decreased by either inhibition of gaba a receptors or suppression of gaba release from interneurons. these results suggest that gaba released from inhibitory interneurons and gaba a receptors on mossy fibers contribute to activity-dependent facilitation of the excitatory synaptic transmission during development. takuya nishimaki, il-sung jang, jyunichi nabekura dep. dev. physiol., nips, sokendai, okazaki, crest, jst, japan lateral superior olive (lso) is the first auditory center. during the early postnatal period, the inhibitory synaptic inputs to lso neurons from medial nucleus of the trapezoid body (mntb) change from predominantly gabaergic to glycinergic. we focused on metabotropic gaba b receptor (gaba b r) as the key molecule of difference between gaba and glycine. in immature lso neurons postsynaptic gaba b r could activate k + channels, but this effect ceased by the third postnatal week. baclofen, a gaba b r agonist, reduced ipsc amplitude at mntb-lso synapses in neonate ( cadherin-related molecules and are encoded by three gene clusters (␣,  and ␥). the molecular features and synaptic localization of the clustered pcdhs have raised the possibility that they are synaptic recognition molecules. we have demonstrated that overexpressed pcdh␣ family proteins alone in several cell lines are rarely transported into the plasma membrane. furthermore, we found that a stretch of about fifty amino acids located at the c-terminus of pcdh␣s interfered the trafficking to the cell surface. in the present study, we compared the transport properties of a series of the cytoplasmic region truncation mutants and found that truncation mutants lacking or more c-terminal residues were detectable at the cellular surface suggesting a role for lysine-rich motif in the c-terminus of pcdh␣s in the intracellular retention. mdga is a novel cell surface glycoprotein similar to ig-containing cell adhesion molecules (igcams) with functions in migration and process outgrowth. mdga is expressed by layer / neurons throughout the neocortex at p mice, but is absent in adults. between e . and late p , stages that span the generation and radial migration of layer / neurons, mdga is expressed in patterns consistent with its expression by migrating layer / neurons, suggesting a role for mdga in controlling their migration and settling in the superficial cortical plate. we performed loss-of-function studies using rna interference (rnai) with in utero electroporation into the lateral ventricle at e . to transfect progenitors of superficial layer neurons. we found that an rnai suppressing mdga protein blocks proper migration of superficial layer neurons to the superficial cortical layer. we conclude that mdga acts cell autonomously to control the migration of superficial layer cortical neurons. in various pathological conditions, activated microglia mediate immune responses to injured cns neurons. however, it is not clear whether and how activated microglia affect neurons via direct contacts. this study aimed at examining whether direct contacts between microglia and hippocampal neurons increase following cns injury and whether telencephalin (tlcn), a dendrite specific adhesion molecule, which potentially binds to immune cells, mediates the direct contact. hippocampal neurons were damaged by local injection of excitotoxin, kainic acid (ka). compared to control animals, ka-injected mice showed higher density of contacts between activated microglia and dendrites of ca pyramidal neurons. contacts with longer interface appeared in ka-injected mice. these results suggest the importance of direct contacts for the immune response of microglia to injured neurons. similar contact formation was also observed in tlcn-deficient mice, indicating that the direct contacts are mediated by other molecules than tlcn. kilon is belonging to immunoglobulin superfamily of cell adhesion molecules and contains three igg-like domains. western analysis revealed that the expression levels of kilon is low at early neuronal culture and increased with progress of culture days. immunocytochemical observation showed that kilon was localized at elongating axon and growth cones but not at dendrites on days in vitro (div), while kilon was observed at synapses, mainly at presynaptic terminals on div. similar tendency was observed in kilon immunohistochemistry of brain sections in vivo. kilon was observed at axonal fibers of the cerebral cortex on postnatal day , but it was seen at synapses in adult brains. these results suggest that kilon is axonal cell adhesion molecule to control axonal guidance and/or extension. ps p-f analysis of mice that show abnormal expression of neuroglycan c, a central nervous system-specific transmembrane proteoglycan sachiko aono , yoshiyuki kuroda , fumiko matsui , yoshihito tokita , keiko nakanishi , michiru ida , masahito ikawa , masaru okabe , katsuhiko ono , atsuhiko oohira institute for developmental research, aichi human service ctr., kasugai, japan; research institute for microbial diseases, osaka university, suita, japan; national institute for physiological sciences, okazaki, japan neuroglycan c (ngc) is a membrane-spanning chondroitin sulfate proteoglycan that is exclusively expressed in the central nervous system. to study the role of ngc in the brain, we produced two strains of ngc-mutant mouse by gene-targeting; a mouse strain with no ngcexpression and a strain with low expression (knockdown mice). both mice were viable and fertile. they did not show obvious abnormalities in gross brain anatomy. to examine their behavioral phenotype precisely, the ngc-knockdown mice were subjected to several kinds of behavioral tests sequentially. they displayed obvious abnormalities in morris water maze and passive avoidance tests, suggesting that ngc is involved in learning and memory. we are now carrying out the same experiments using the ngc-knockout mice. research funds: kakenhi ( ) ps p-f phosphorylation of extracellular signal-regulated kinase in aged rats with acute face inflammation koichi iwata , tatsuhisa watanabe , ikuko suzuki , junichi kitagawa , akiko ogawa , kenro kanda , kazunao kuramoto dept. of physiol., sch. of dent., nihon univ., tokyo, japan; dept. of oral and maxillofacial surgery, sch. of dent., nihon univ., tokyo, japan; dept. of oral diagnosis, sch. of dent, nihon univ., tokyo, japan; shinjuku vocational school of acupuncture, moxibustion and judo therapy, tokyo, japan; division of research animal center, tokyo metropolitan institute of gerontology, tokyo, japan the capsaicin-induced perk expression was studied in the aged rats ( - months) following noxious face stimulation. a large number of perk-li cells were expressed in the superficial laminae of the trigminal spinal nucleus in adult and aged rats following subcutaneous capsainsin injection into the whisker pad region. the larger number of perk-li cells was expressed in adult rats than aged rats following intravenous administration of naloxone before capsaicin treatment. the present results suggest that the descending modulation system was impaired in the aged rats, resulting in the abnormal pain sensation advancing age. hirokazu katsura , koichi obata , masafumi sakagami , koichi noguchi department of anatomy and neuroscience, hyogo college of medicine, hyogo, japan; department of otorhinolaryngology, hyogo college of medicine, hyogo, japan recent studies demonstrated that the activation of extracellular signal-regulated protein kinase (erk) / and p mitogen-activated protein kinase (mapk) in dorsal root ganglion (drg) neurons contributes to the development of inflammatory and neuropathic pain. in the present study, we examined whether the newest member of the mapk family of proteins, erk (also known as big mapk or bmk ) is activated in the drg and participate in pain-related behaviors in the complete freund's adjuvant (cfa) model. peripheral inflammation induced an increase in the phosphorylation of erk , mainly in tyrosine kinase a-containing small-to-medium-diameter drg neurons at days and after cfa injection. furthermore, time course of phosphorylated-erk level in the drg matched the emergence of cfa-induced pain hypersensitivity. our data suggest that activation of erk in drg neurons may contribute to the development of inflammatory pain. ps p-f activation of erk in drg neurons contributes to acute pain toshiyuki mizushima , , koichi obata , takashi mashimo , koichi noguchi department of anatomy and neuroscience, hyogo college of medicine, hyogo, japan; department of anesthesiology, osaka univ. recently, we have reported that phosphorylation of extracellular signal-regulated protein kinase (erk) / and p mitogen-activated protein kinase (mapk) occurred in primary sensory neurons in response to natural noxious stimulation of the peripheral tissue, i.e., activity-dependent activation of erk and p in dorsal root ganglion (drg) neurons. however, there has been no study examining erk (also known as big mapk or bmk ) activation in drg neurons after noxious stimulation of normal tissue. here, we report intensity-dependent erk phosphorylation in drg neurons by painful stimulation. noxious stimulation induced phosphorylated-erk in small-to-medium diameter sensory neurons with a peak at min after stimulation. furthermore, we found a stimulus intensitydependent increase in the number of activated neurons. our data suggest that activation of erk in drg neurons may contribute to acute pain induced by noxious stimulation. koichi obata, koichi noguchi department of anatomy and neuroscience, hyogo college of medicine, japan there is compelling evidence indicating that the activation of extracellular signal-regulated protein kinase (erk) / and p mitogenactivated protein kinase (mapk) in the dorsal root ganglion (drg) and spinal cord contributes to the development of inflammatory and neuropathic pain. in the present study, we examined whether the newest member of the mapk family of proteins, erk (also known as big mapk or bmk ) is activated in the drg and spinal cord and participate in pain-related behaviors in the l spinal nerve ligation (snl) model. l snl induced an increase in the phosphorylation of erk not only in the injured l drg, but also in the spared l drg at day after surgery. furthermore, l snl induced a striking increase in erk phosphorylation in glial cells in the ipsilateral dorsal horn. our data suggest that activation of erk in the drg and spinal cord may contribute to the development of neuropathic pain. atsushi sakai , minoru asada , naoki seno , hidenori suzuki department of pharmacology, nippon medical school, tokyo, japan; pharmaceutical research center, kyowa hakko kogyo co., shizuoka, japan glial cell line-derived neurotrophic factor (gdnf) has been known to alleviate the neuropathic pain. however, the mechanisms of gdnfinduced analgesia remain almost unclear. gdnf binds to gfr␣- , which forms receptor complex and signals intracellularly through ret. recently, neural cell adhesion molecule (ncam) has been found to be an alternative signal-transducing receptor for gdnf. here, we report that ncam is involved in gdnf-induced analgesia in a rat model of the neuropathic pain. ncam mrna expression was decreased in the ipsilateral dorsal horn of the spinal cord after the nerve injury, but gdnf treatment returned its expression to the normal level. treatment with ncam antisense oligodeoxynucleotide blocked the analgesic effect of gdnf without affecting ret phosphorylation. these results suggest that activation of ncam signaling may provide a new strategy to relieve intractable chronic pain. ps p-f interleukin-  enhanced the excitability of trigeminal root ganglion neurons via activation of satellite glia following inflammation mamoru takeda , jun kadoi , msanori nasu , masayuki takahashi , shigeji matsumoto dep. physiol and res. cent. for odont. nippon dent. univ., japan the present study was investigated whether activation of satellite glial cells modulates the excitability of trigeminal root ganglion (trg) neuronal activity via the il-  paracrine mechanism following inflammation. two days after cfa into the whisker pad area, the mean number of trg neurons that were encircled by glial fibrillary acidic protein and il- -immunoreative satellite cells were significantly increased compared with those in the control. fg labeling was used to identify the trg neurons innervating the site of inflammation. in the fg-labeled small trg neurons, the occurrence of il-  induced depolarization in inflamed rats was larger than that in control rats. il-  application significantly increased the firing rate evoked by depolarizing pulses in the inflamed neurons compared with the control neurons. these results suggest that activation of trg satellite glial cells modulates the excitability of trg neuronal activity via the il-  paracrine mechanism following peripheral inflammation. junichi kitagawa , mamoru takeda , jun kadoi , yoshiyuki tsuboi , shigeji matsumoto , koichi iwata dept. of physiol., sch. of dent., nihon univ., tokyo, japan; dept. of physiol, sch. of dent. at tokyo, japan, nippon dental univ., tokyo, japan the present study was designed to elucidate an involvement of the primary afferent neurons in the trigeminal neuropathic pain using the rats model with chronic constriction nerve injury of the infraorbital nerve (ion-cci). the mechanical escape threshold was significantly lower in ion-cci rats at day after ion treatment and the threshold decrement was lasting more than day . single unit activities of ion were recorded from the ion-cci rats. the firing frequency was significantly higher in a␦ fibers in ion-cci rats as compared with naive at day - after ion-cci. whole cell patch clamp recording was performed from the middle trg neurons. ik and ia currents were significantly smaller and ih current was larger in ion-cci rats than that of naive rats. the present results suggest that ik, ia and ih currents are involved in abnormal firing of trg neurons in the rats with ion-cci, resulting in neuropathic pain in trigeminal region following peripheral nerve injury. hisako urai, munehiro uda, katsuya kami graduate schools of sport and exercise science, osaka university of health and sport sciences, osaka, japan mechanical hyperalgesia of skeletal muscles has been known to occur following intense eccentric contraction such as downhill running (dhr). the present study examined the number of c-fos-positive neurons in spinal dorsal horn to determine peak of mechanical hyperalgesia following dhr in rats. furthermore, we investigated whether glial cells are activated in dorsal horn with excitation of secondary afferent neurons (san). rats performed an intermittent bout of dhr for min. at , , , and h post-dhr, the rats were applied a weight on the right triceps surae muscle. immunohistochemical staining for c-fos and gfap on spinal cords was performed by freefloating abc method. the number of c-fos-positive neurons detected in superficial dorsal horn were increased at h, peaked at h and then decreased. intense gfap immunoreactivities were also detected at and h post-dhr. these results suggest that dhr generates mechanical hyperalgesia by increasing responsiveness of san, and moreover astrocytes may regulate excitability of san. katsuya kami, hisako urai, munehiro uda department of health science, osaka university of health and sport sciences, osaka, japan a production of inflammatory cytokines is increased in injured skeletal muscles. the present study examined relationship between production of inflammatory cytokines in skeletal muscles and fospositive neurons in spinal dorsal horn following downhill running in rats. the rats performed the downhill treadmill running for min at m/min. after the running, rats were applied the weight on the gastrocnemius muscles for min, and then spinal cord and soleus muscles were removed from the rats. productions of il- beta, il- and tnf-alpha in soleus muscles and expression of fos protein in dorsal horn were examined using immunohistochemical approach. at h post-running, number of fos-positive neurons was increased, peaked at h and then decreased to control level at h post-running. vigorous inflammatory reactions with necrotic myofibers in soleus muscles were observed at days post-running. these results indicated that increased numbers of fos-positive neurons in dorsal horn are induced prior to vigorous inflammation of skeletal muscles. shinichi sugiyo , yusuke sakai , aya masawaki , takashi shimoda , masayuki moritani , motohide takemura dept. oral anatomy and neurobiology, osaka university grad. sch. of dentistry, osaka, japan; dept. of fixed prosthodontics, osaka university grad. sch. of dentistry, osaka, japan; dept. of dental anesthesiology, osaka university grad. sch. of dentistry, osaka, japan diabetes mellitus is among the most common causes of painful peripheral neuropathy, worldwide. we examined if there exist the diabetic rat (dm)-specific difference in nociceptive behavioral and c-fos immunoreactivity (ir) by formalin test. injection of formalin into the upper lip weeks before streptozotocin injection induced biphasic specific pain related behavior (prb) for min. first phase was greater in dm than in the control rat (ctrl). in dm, second phase was much greater than ctrl. c-fos ir in the trigeminal caudal nucleus was also greater in dm than in ctrl. these results indicate that dm induced greater prb and c-fos expression following formalin injection into the rat upper lip. yasuko kozaki, satoshi hurune, fukushi kambe, hisao seo, kazue mizumura res. inst. environ. med., nagoya university, nagoya, japan we have reported that prostaglandin ep receptor (ep r) activation attenuates the desensitization of bradykinin (bk)-induced increase of intracellular calcium ([ca + ] i ) in a ptx-sensitive manner in cho cells expressing canine ep r and mouse bk b receptor (b r). in this study, we examined the involvement of protein kinase a (pka) in the desensitization of the bk response. when bk ( nm) was applied twice with a -min interval to the cells expressing b r, the second [ca + ] i increase by bk was markedly attenuated. however, the pretreatment with a specific inhibitor of pka, h- ( m) restored the second response. to further confirm camp increase by bk, the expression of a camp responsive reporter gene was examined. bk ( pm) treatment for h significantly increased the reporter gene expression. it is likely that bk increases the level of intracellular camp, and thus activates pka, resulting in the desensitization of the bk response. these results suggest that the desensitization of bkinduced increase in [ca + ] i was at least in part mediated by pka. ps p-f contribution of peripheral ht a or ht receptors to fos expression in the trigeminal spinal nucleus (vsp) produced by the masseter muscle injury of rats keiichiro okamoto, akihisa kimura, tomohiro donishi, yasuhiko tamai dept. physiology, wakayama med univ., japan we have recently reported that orofacial nocifensive behavior evoked by the masseter muscle (mm) injury is attenuated by blocking peripheral ht a or ht /r in male rats with tmj inflammation. here we tested if these two ht/r subtypes contribute to fos responses in vsp after mm injury. formalin injection into mm produced fos-like immunoreactivity (li) in several areas of vsp and c . fos-li was distributed mainly in the ventrolateral trigeminal subnucleus interpolaris/caudalis transition (vl-vi/vc) and vc/c transition regions. the number of fos-li induced by mm injury was increased in these areas in cfa-evoked tmj-inflamed rats for days compared to naive rats. we tested if local ht a or ht /r antagonist affects fos expression in both groups. the number of fos-li in the vc/c but not vl-vi/vc region was reduced when drugs were injected locally prior to formalin injection in tmj-inflamed rats. these data suggest that peripheral ht a and ht /rs play critical roles in mediating mm nociception during tmj inflammation. keiko abe, hidemasa furue, kohei kga, go kato, toshiharu yasaka, akihiro tamae, toshihiko katafuchi, megumu yoshimura department of integrative physiology, graduate school of medical sciences, kyushu university, japan we examined the postsynaptic effects of -ht on substantia gelatinosa (sg) neurons in slice preparations of rat spinal cord and their relationship to the morphological features. in ∼ % of sg neurons examined, -ht induced an outward current. the outward current was mimicked and suppressed by a -ht a agonist and -ht a antagonist, respectively. in ∼ % of sg neurons, -ht evoked an inward current which was mimicked by a -ht agonist. the outward current was observed mostly in excitatory neurons such as vertical cell, while the inward current was induced in an inhibitory neuron, islet cell. these findings suggest that -ht inhibits excitatory neurons and excites inhibitory neurons in the sg through activation of -ht a and -ht receptors, respectively. the reciprocal postsynaptic actions of -ht on sg neurons in addition to presynaptic inhibitory effects on primary afferents might play an important role in descending control of nociceptive transmission by -ht. we examined effects of levobupivacaine, ropivacaine, bupivacaine and r-bupivacaine on epscs in substantia gelatinosa (sg) neurons of the spinal dorsal horn evoked by dorsal root stimulation, and on action potentials in dorsal root ganglion neurons generated by the dorsal root stimulation. in sg neurons, levobupivacaine reversibly suppressed the amplitude of monosynaptic a␦ and c fiber-evoked epscs. however, a fiber-evoked epscs were slightly inhibited in amplitude. on the other hand, bupivacaine equally suppressed those three fiber-evoked epscs. in drg neurons, ic of bupivacaine and r-bupivacaine were almost equal on a, a␦ and c neurons. however, ic of levobupivacaine and ropivacaine on a␦ and c neurons were lower than that on a neurons. the present results suggest that pure s (−) enantiomers especially levobupivacaine effectively inhibits noxious transmission to the spinal dorsal horn by the blockade of ap conduction through a␦ and c fibers. ps p-g nitric oxide-dependent long-term potentiation revealed by real time imaging of nitric oxide production and neuronal excitation in spinal dorsal horn hiroshi ikeda, kei kusudo, kazuyuki murase dept. human & artificial intelligence systems, univ. fukui, fukui, japan no plays an important role in the induction of long-term potentiation (ltp) in spinal dorsal horn, which is believed to underlie hyperalgesia and allodynia. in this study, to elucidate the relationship of no to ltp, we measured the spatiotemporal distribution of no signal with the no-sensitive dye, and neuronal excitation with the voltagesensitive dye, in rat spinal cord slices. in superficial dorsal horn, neuronal excitation evoked by dorsal root stimulation was potentiated for more than h after low-frequency conditioning stimulation (lfs). in the same slices that exhibited ltp, no was produced and distributed in the superficial dorsal horn during lfs. ltp and production of no were inhibited in the presence of no synthase inhibitors and an inhibitor of heme oxygenase, the synthetic enzyme for carbon monoxide (co). research funds: kakenhi to hi ( ) and km ( ) and grants from novartis foundation and promotion of science and sumitomo foundation to hi tao liu, tsugumi fujita, akiko koga, masafumi kosugi, terumasa nakatsuka, eiichi kumamoto dept. physiol., facult. med., saga univ., saga, japan in order to know the effect of a pla activator melittin on inhibitory transmission in the substantia gelatinosa (sg; lamina ii of rexed), we applied the blind whole-cell patch-clamp technique to sg neurons in adult rat spinal cord slices. in about % of neurons examined, melittin ( m) superfused for min gradually increased the frequency and amplitude of spontaneous glycinergic inhibitory postsynaptic currents which were recorded at mv in the presence of bicuculline. this action was visible about min after the beginning of its superfusion and subsided within min after washout. these melittin actions were reduced in extent by a pla inhibitor -bromophenacryl bromide, while being unaffected by tetrodotoxin, and also by inhibitors of cyclooxygenase (cox) and lipooxygenase (lox). it is concluded that pla activation pre-and postsynaptically enhances glycinergic transmission in sg neurons, possibly not through metabolites of cox and lox; this action would contribute to a modulation of nociceptive transmission. research funds: kakenhi ( ) ps p-g presynaptic p y receptor-mediated enhancement of inhibitory synaptic transmission in the rat spinal dorsal horn terumasa nakatsuka, shugo koga, tsugumi fujita, tao liu, masafumi kosugi, eiichi kumamoto department of physiology, faculty of medicine, saga university, saga, japan using whole-cell patch-clamp recordings, we examined whether the activation of p y receptors can modulate synaptic transmission in dorsal horn (dh) neurons of adult rat spinal cord slices. bath applied -methylthio adp ( mesadp, m), a p y receptor agonist, did not change excitatory transmission, but clearly increased the frequency and amplitude of spontaneous inhibitory postsynaptic currents (ipscs) in about % of dh neurons recorded. miniature ipsc in the presence of ttx was increased in frequency by mesadp with no change in the amplitude. the mesadp-induced increase in miniature ipsc frequency was attenuated in extent by mrs ( m), a selective p y receptor antagonist. these results indicate that the activation of presynaptic p y receptors enhances inhibitory but not excitatory synaptic transmission in a subpopulation of dh neurons. thus, spinal p y receptors can be involved in an inhibitory effect on pain transmission. research funds: kakenhi ( ), the japanese health sciences foundation (kh ) ps p-g presynaptic enhancement by proteinaseactivated receptor- agonist peptide of glutamatergic excitatory transmission in rat substantia gelatinosa neurons tsugumi fujita, terumasa nakatsuka, akiko koga, tao liu, masafumi kosugi, eiichi kumamoto dept. physiol., facult. med., saga univ., saga, japan we have previously reported that proteinase-activated receptor (par)- but not par- agonist (each m) enhances glutamatergic excitatory transmission in substantia gelatinosa (sg) neurons. the present study examined a detail of the par- mediated enhancement by applying the whole-cell patch-clamp technique to sg neurons in adult rat spinal cord slices. par- agonist (sfllrn, m) reversibly increased the frequency of spontaneous epsc without a change in the amplitude and also in holding current at - mv. this facilitatory action was resistant to tetrodotoxin, and was not seen in the presence of par- antagonist (yfllrnp, m). these results indicate that the activation of par- s existing in nerve terminals in the sg results in an increase in the spontaneous release of l-glutamate from there. it is suggested that par- activation in glutamatergic neuron terminals in the sg may be involved in the modulation of nociceptive transmission from the periphery. research funds: kakenhi ( ) ps p-g effect of tramadol metabolite m on glutamatergic excitatory transmission in rat spinal dorsal horn neurons akiko koga, tsugumi fujita, tao liu, terumasa nakatsuka, eiichi kumamoto dept. physiol., facult. med., saga univ., saga, japan in order to know the antinociceptive effect of tramadol, we examined the effect of m , which is one of its metabolites, at mm on glutamatergic excitatory transmission in substantia gelatinosa (sg) neurons of an adult rat spinal cord slice by using the whole-cell patchclamp technique. bath-applied m reduced the frequency but not amplitude of spontaneous excitatory postsynaptic currents (epscs) at − mv. this action was not seen in the presence of a -opioid receptor antagonist ctap ( m). m also reduced the peak amplitudes of epscs which were monosynaptically evoked at − mv by stimulating primary-afferent a␦-and/or c-fibers in a spinal cord slice with an attached dorsal root. we conclude that m inhibits the quantal release of l-glutamate from nerve terminals in the sg through the activation of -opioid receptors; this action is not distinct in extent between primary-afferent a␦-fiber and c-fiber transmission. this effect of m would give a cellular basis for the antinociceptive effect of systemically-administered tramadol. narihito iwashita, natsu koyama department of physiology, shiga university of medical science, otsu, japan in our previous study, subcutaneous injection of glutamate into the human forearm evoked pain and produced skin temperature increase around the injection site. these results suggest peripheral glutamate receptors contribute to nociceptive signaling and neurogenic inflammation. in order to further investigate which subtype of glutamate receptors is involved in neurogenic inflammation, effect of nmda receptor antagonist mk- or non-nmda receptor antagonist cnqx was evaluated in hindpaws of pentobarbital-anesthetized rats. attenuation of skin temperature increase induced by simultaneous mk- injection with glutamate was larger than that of skin temperature increase induced by simultaneous cnqx injection with glutamate at the same concentration. on the other hand, inhibition of paw edema formation by cnqx was stronger than by mk- . these data demonstrate that peripheral nmda receptor predominantly contributes to vasodilatation, while peripheral ampa/ka receptor predominantly contributes to increase of vascular permeability in glutamate-induced neurogenic inflammation. ps p-g studies on pain control system (rept. ): changes in phosphorylation of nr b-contained nmda receptor in the spinal cord obtained from rats with painful neuropathy following chronic ethanol consumption kan miyoshi, minoru narita, michiko narita, tsutomu suzuki dept. toxicol., hoshi univ. sch. pharm. pharmaceut. sci., tokyo, japan chronic ethanol consumption produces a painful peripheral neuropathy. mechanical hyperalgesia was clearly observed during ethanol consumption and even after ethanol withdrawal in rats, and it lasted for weeks. under these conditions, the immunoreactivities of phosphorylated-ser- nr b (p-ser- nr b) subunit and phosphorylated-conventional protein kinase c (p-cpkc) were significantly increased in the spinal cord following chronic ethanol consumption, whereas p-tyr- nr b subunit immunoreactivity was not changed in this region. the hyperalgesia induced by chronic ethanol consumption was significantly attenuated by repeated i.p. injection of ifenprodil, a selective nr b-containing nmda receptor antagonist. these findings provide evidence for a substantial role of the phosphorylation of cpkc-dependent nr b-contained nmda receptor in the development or/and maintenance of ethanoldependent neuropathic pain-like state in rats. ps p-g prolonged depression of nociceptive response in the prefrontal cortex with high frequency stimulation of the amygdala yumi izawa, yoriko kawakami dept. physiol. tokyo women's medical university, tokyo, japan high frequency stimuli (hfs, hz, a, s) delivered to the basolateral nucleus of the amygdala (bl) induced prolonged depression of the nociceptive specific response in the prefrontal cortex (pfc). we examined the receptor mechanism underlying this depression of pfc neuron activity. extracellular neural activities, induced by nociceptive stimulation applied peripherally, were recorded in the rat pfc. inhibitory effects of hfs delivered to the bl on nociceptive responses were blocked by specific antagonists of a metabotropic glutamate receptor (mglur) or nmda receptor microinjected locally into the pfc. dopamine depletion, produced by -ohda injected into the substantia nigra, also reduced the inhibitory effects of hfs. the mglur and dopamine receptor mediated prolonged depressions of nociceptive responses were induced by hfs of the amygdala. our results suggest that emotional condition modulates pain sensation. ps p-g the nav . sodium channel pathologically reconfigures the thalamic pain amplifier-generator after spinal cord injury bryan c. hains, stephen g. waxman yale university school of medicine, usa spinal cord injury (sci) induces pain-related phenomena associated with the aberrant expression of nav . , a rapidly repriming voltage-gated sodium channel. in this study we hypothesized that, following sci, neurons in the thalamus undergo similar electrophysiological changes linked to nav . . four weeks post-sci, nav . protein was upregulated within thalamic neurons, where unit recordings revealed increased spontaneous discharge, afterdischarge, hyperresponsiveness to innocuous and noxious peripheral stimuli, expansion of peripheral rfs, and bursting. these properties persisted after interruption of ascending spinal barrage. lumbar intrathecal administration of specific antisense oligodeoxynucleotides against nav . caused a significant reduction in nav . expression and reversed electrophysiological alterations. these results show, for the first time, a change in sodium channel expression within neurons in the thalamus after injury to the spinal cord, and suggest that these changes contribute to altered processing of somatosensory information after sci. tomoki fukuda , hiroyuki ichikawa , ryuji terayama , tomosada sugimoto department of oral maxillofacial rehabilitation, okayama university, okayama, japan; department of oral function and anatomy, okayama university, okayama, japan ib -sap is a neurotoxin designed for targeting primary nociceptors with ib binding sites on the cell surface. however, the exact cell spectrum that is affected by the toxin has not been thoroughly investigated. we, therefore, unilaterally injected ib -sap ( . l of . % solution for each ganglion) directly into the th and th lumbar (l and ) dorsal root ganglia (drgs). three weeks later, the rats were killed and drg sections were stained for ib -binding. after counterstain, the cell body size of neurons were measured. ib -sap reduced the total number of drg neurons in l and ganglia combined by % ( ± on untreated side versus ± on treated side). small neurons (< m ) were reduced by % whereas large ones (≥ m ) were not affected. ib -binding neurons were mostly small (≥ %) and were reduced by %. the number of small neurons, that were not stained for ib -binding, increased by % ( ± versus ± ). schuichi koizumi , kaoru nasu-tada , makoto tsuda , emiko kunifusa , , kazuhide inoue div. pharmacol., natl. inst. hlth. sci., tokyo, japan; dept. mol. system pharmacol., grad. sch. pharmaceut. sci., kyushu univ., fukuoka, japan although microglial p x receptor, a key molecule for the mechanical allodynia, is increased after peripheral nerve injury, the molecular mechanisms underlying its upregulation remain unknown. here, we describe the influence of fibronectin on p x receptor expression in microglia. microglia that were cultured on fibronectin-coated dishes showed a marked increase in p x receptor expression. western blot examination of the spinal cord from rat with spinal nerve injury indicated that fibronectin was upregulated on the ipsilateral side. interestingly, intrathecal injection of atp-stimulated microglia revealed that microglia cultured on fibronectin-coated dishes was more effective in the induction of allodynia than microglia cultured on control dishes. taken together, our results suggest that spinal fibronectin is elevated after the peripheral nerve injury and it may be involved in the upregulation of the p x receptor in microglia, leading to neuropathic pain. research funds: mf , kakenhi ( ) ryousuke fujita, hiroshi ueda div. mol. pharmacol. & neurosci., nagasaki univ. grad. sch. of biomed. sci., nagasaki, japan we have reported that intrathecally administered lpa or endogenous lpa generated upon sciatic nerve injury causes demyelination of dorsal root (dr), which is supposed to be one of key molecular mechanisms underlying neuropathic pain (nat. med. ). however it remained whether lpa has direct actions on myelinated schwann cells (sc). in the present study we examined the direct effects of lpa on dr fibers in ex vivo culture system. scanning electron microscopy (sem) study revealed that lpa caused a swelling and disruption of myelinated fibers at h. in transmission em analysis, the addition of lpa caused a disruption of myelin sheath of a␦-and a-fibers. on the other hand, it was found that c-fibers were separated to each other by scs in naive fibers. following the addition of lpa, c-fibers showed direct contacts and some of them were uncovered. all these effects were also observed either with or without dr ganglion. thus, it is suggested that lpa has direct actions on myelinated and unmyelinated scs to cause demyelination of a-fibers and to uncover c-fibers. research funds: kakenhi ps p-g lysophosphatidic acid (lpa) down-regulates myelin associated proteins in cultured dorsal root fibers norikazu kiguchi, ryousuke fujita, hiroshi ueda div. mol. pharmacol. & neurosci., nagasaki univ. grad. sch. biomed. sci. nagasaki, japan we have reported that intrathecally administered lpa or endogenous lpa generated upon sciatic nerve injury causes demyelination of dorsal root, which is supposed to be one of key molecular mechanisms underlying neuropathic pain (nat. med. ). these treatments also caused a decrease in myelin protein and their gene expression levels. here we report the biochemical evidence underlying this demyelination in cultured fibers. the addition of lpa at mm decreased the protein levels of myelin basic protein (mbp) at h. this action was significantly inhibited by botulinum neurotoxin/c (bont/c ). on the other hand, lpa also caused a decrease in gene expression of various myelin proteins, such as mbp, pmp , mag, p in cultured fibers. the maximal decrease was observed all at as early as h after the addition of lpa. bont/c and y abolished the lpainduced down-regulation of mbp gene. all these findings suggest that the down-regulation of gene expression of myelin proteins is through rhoa-rock pathway underlying lpa-induced demyelination. neuropathic pain arise from peripheral never injury. the purpose of this study was to explore behavioral characteristics and investigate the involvement of nmda receptors and opioid receptors in the behavioural responses following spared nerve injury (sni). the hind paw withdrawal threshold to cold-and mechano allodynia and heatyperalgesia were tested at and , , , , days after operation. pre-emptive co-administration of mk- and morphine were tested. sni produces mechanical and cold allodynia and heat hyperalgesia. co-injection of morphine and mk- decreased cold-and mechanoallodynia, but had slightly effect on heat-hyperalgesia. the present data demonstrate that the sni procedure result in severe changes in behavioral responses in whether hyperalgesia or allodynia. coadministration of both drugs seems to be more effective to alleviate induced neuropathic pain. satoshi deyama , , naomi akiyama , mikie hirata , takayuki nakagawa , shuji kaneko , masabumi minami dept. of pharmacol., grad. sch. of pharm. sci., hokkaido univ., sapporo, japan; dept. of mol. pharmacol., grad. sch. of pharm. sci., kyoto univ., kyoto, japan the bed nucleus of the stria terminalis (bst) is involved in the regulation of negative affective states such as anxiety and fear. in this study, we examined the role of the noradrenergic (na) transmission within the bst in the negative affective component of pain in rats. we found that excitotoxic lesion of the bst attenuated intraperitoneal acetic acid-or intraplantar formalin-induced conditioned place aversion (cpa) without reducing nociceptive behaviors. we showed that na release within the bst was significantly elevated by these noxious stimuli. intra-bst injection of a -adrenoceptor antagonist timolol significantly suppressed these noxious stimuli-induced cpa without affecting nociceptive behaviors. these results suggest that visceral and somatic noxious stimuli-induced na release within the bst contributes to the negative affective, but not sensory, component of pain. noriyuki ozaki, mariko kawai, yasuo sugiura department of functional anatomy and neuroscience, nagoya university, graduate school of medicine, nagoya, japan neonatal maternal separation induces visceral hyperalgesia in colon. this study compares the effects of maternal separation on response sensitivity to gastric and colorectal distension in long-evans rats. maternal separation was performed for h per day between postnatal day and . visceral sensitivities were assessed in stomach and colon at weeks of age by visceromotor responses induced by either gastric or colorectal distension. somatic pain sensitivities were also assessed by von frey filaments and radiant heat. in contrast to the response to colorectal distension, maternal separation induced decreased response to gastric distension, especially in male rats. no difference was found between control and separated rats in somatic pain sensitivities. these results indicate that maternal separation differentially modulates visceral pain sensation in stomach and colon. research funds: grant-in-aid for scientific research ps p-g change by aging in muscular mechanical hyperalgesia after lenghtening contraction k. mizumura, t. taguchi, t. matsuda, t. nasu res. inst. environ. med., nagoya univ., nagoya, japan our previous experiments have shown that the mechanical threshold of the edl muscle underwent lengthening contraction (lec) lowered to days after exercise in rats ( w old). c-fos expression in the superficial dorsal horn increased in l spinal segment when the edl muscle was compressed days after exercise. from these results we have concluded that the muscle became hyperalgesic after lec. in the present experiment, we examined whether this hyperalgesia after lec changes along aging. male sd rats , ( - ) and ( - ) w old were used. the basal mechanical threshold (randall-selitto method) of edl muscle tended to be higher in w old rats, but not significant. after lec, the threshold started to decrease day after lec in all three age groups. it returned to the pre-lec level days after lec in and w old rats only. recovery of w old rats delayed up to days after lec. increased c-fos expression in the superficial dorsal horn was observed in l as well as in l in w old rats. these results suggest that hyperalgesia occurs in larger areas and lasts longer in aged animals. tong liu, hong p. wei, chun y. yuan, ai k. guo institute of neuroscience, chinese academy of sciences, china drosophila can display complex courtship behavior. male-male courtship behavior shown in some fly mutants, but here we report for the first time that the male-male courtship behavior can be induced by disturbance of dopamine level. to up-regulate dopamine level, uas-th/th-gal males were used, which showed high level of dopamine and performed active male-male courtship behavior. this behavior was attenuated by decreasing dopamine level either through drug breeding or genetic method. the increased courtship behavior in uas-th/th-gal males is specific to male partners, because the males courted females normally. to down-regulate dopamine level, pale ts , th temperature sensitive mutant was used. when raised at restrictive temperature, pale ts showed obvious attraction to wild type males. our study shows that the high level or low level of dopamine can induce male-male courtship behavior in active or passive manner. athushi yokoyama , masaharu akita kanagawa life-science res., japan; kamkura, kanagawa, japan we have developed the screening system for drug and chemical compounds of food by the used of ratwhole embryo culture. the advantages of whole embryo culture are to examine the direct effects of l-calnitin (lcal) on embryo and also to find the non-teratogenic agent (d-calnitin:dcal). as the testing agent, lcal was examined in this study using the rat embryo cultured from day to of gestation. in treated embryos of lcal, the embryonic heart beat, the crown-rump length, the embryo weight and the total embryonic somites were not decreased. on the other hand, the malformation (the defects of neural tube) and the short size of head length were observed in the embryos cultured with lcal. in treated embryos of d-calnitin (dcal), there parameter was not decreased. the observed malformation of lcal was not observed in the embryos cultured with dcal. these results might be due to the differences between lcal and dcal in the embryo toxicity. yoshihisa uenoyama, kenji takase, junya hirata, hiroko tsukamura, kei-ichiro maeda laboratory of reproductive science, nagoya university, nagoya, japan the mechanisms underlying the pubertal increase in gonadotropinreleasing hormone (gnrh) secretion are poorly understood. recently, metastin was found to stimulate gnrh secretion and mutations of its receptor are associated with lack of puberty. effect of immunoneutralization of endogenous metastin in the brain on the onset of puberty was examined to clarify the physiological significance of metastin in timing the puberty. when wistar-imamichi strain female rats received an infusion of anti-metastin antibody into the third ventricle during days - of age, they did not show the first estrus before days of age with mean age of . ± . day. in contrast, most of normal mouse igg-treated controls showed the first estrus by days of age with mean age of . ± . day. the age of vaginal opening was also delayed in the anti-metastin-treated rats. thus, the present study demonstrates that the puberty onset was delayed by immunoneutralizing central metastin. central metastin may be involved in timing the onset of puberty in female rats. kenji takase, yoshihisa uenoyama, shunji yamada, hiroko tsukamura, kei-ichiro maeda lab. of reproductive science, nagoya university, aichi, japan metastin has been considered to be involved in triggering pulsatile gonadotropin-releasing hormone (gnrh)/luteinizimg hormone (lh) secretion to time the onset of puberty. the present study aimed to determine expression of metastin, a novel kiss- gene product, in the rat brain during peripubertal period. wistar-imamichi strain female rat shows vaginal opening on around days of age (d ), and first estrus on around d . brain tissues were obtained on d , , , and . kiss- mrna expression in the arcuate nucleus-median eminence region (arc-me) and anteroventral periventricular nucleus (avpv) increased significantly from d to and was kept at a high level thereafter. gpr mrna expression in the medial preoptic area increased significantly from d to . metastin-immunoreactive cells were not found on d but were apparent in the arc-me on d onward. these results indicate that metastin expression increases in the arc-me and avpv before vaginal opening, suggesting that metastin triggers the onset of gnrh/lh secretion in female rats. toshiyuki saito , sei-etsu fujiwara , kenjiro konno , takashi yamaguchi , tetsu nemoto , etsuko kasuya , ryosuke sakumoto anim. neurophysiol. lab., natl. inst. agrobiol. sci., tsukuba, japan; inst. exp. anim. res., fac. med., gunma univ., maebashi, japan; grad. sch. sci. & eng., yamagata univ., yonezawa, japan; sch. health sci., fac. med., kanazawa univ., kanazawa, japan in the present study, we recorded and examined local field potentials (lfps) in the hippocampus of piglets performing an operant task by a radio-telemetry system. under halothane anesthesia, a pair of tungsten electrodes was implanted into the hippocampus and fixed on the surface of the skull with a transmitter using dental cement. after recovery from surgical procedures, the piglets were moved to a training cage. in the lfps, spike-shaped waves were frequently found just before the piglets pushed a switch with their noses. these waves may represent some of the hippocampal neural activities associated with switch manipulation for getting a food reward. yasuo osawa department of bioscience, tokyo university of agriculture, tokyo, japan memory extinction is an inhibitory learning rather than forgetting or erase of conditioned memory. from the view of treatment of phobia and post traumatic stress disease (ptsd) caused by fear memory, it is important to find the drugs to facilitate extinction of fear memory. importantly, previous studies using pavlovian fear conditioning have shown that d-cycloserine, a nmda receptor agonist, facilitates memory extinction. in this study, to examine whether d-cycloserine is applicable for the treatment with another type of fear memory, we investigated effects of d-cycloserine on extinction of aversive memory in mice. indeed, we performed conditioned taste aversion (cta) task, where the ingestion of a novel taste is paired with transient sickness. our results indicated the injection of d-cycloserine before but not after the re-exposure to cs facilitates extinction of cta. ps p-g hippocampal neural responses during a conditional delayed stimulus-response task in awake mice nobuhide kitabayashi , teruko uwano , , anh tran , , eturou hori , , taketoshi ono , , hisao nishijo system emotional science, univ. of toyama, japan; integrative neurosci, molecular and integrative emotional neurosci., univ. of toyama, japan; crest, japan to investigate a hippocampal (hf) involvement in the representation of temporal sequence in mice, neural responses were recorded during performance of a conditional delayed stimulus-response association task. a trial was initiated by one of two different conditioned tones. after a s delay, two serial reinforcements with an intervening delay was presented; aversive air puff-delay-tube protrusion to evoke licking sucrose solution and the opposite order of the same reinforcements. of hf neurons, responded to the tones, the reinforcements, and during the delay. some neurons responded to a presentation of a sensory stimulus, and other responded differentially during the delay depending on the reinforcement sequence. the results suggest a crucial role of the hf in representation of serial events in episodic memory in mice as well as in rats and primates. further studies will be conducted using genetic modified-mice to clarify the neural substrate in episodic memory. naoko inoue , atsu aiba , kaoru inokuchi mitsubishi kagaku inst. life sci. (mitils), tokyo, japan; grad. sch. med., kobe univ., kobe, japan vesl- s/homer- a and vesl- l/homer- c are splicing isoforms encoded by the vesl- gene. vesl proteins bind and regulate mglur / , ip receptor, ryanodine receptor, and trp channel at the postsynapse. the expression of vesl- s is upregulated by tetanic stimulation that elicits l-ltp. vesl- s is thought to play a critical role in the conversion from short-term to long-term memory (ltm). in this study we generated vesl- s gene-targeting mice (ko) and examined whether vesl- s plays a role in the ltm formation. analysis with the contextual fear conditioning revealed a defective in consolidation process, reconsolidation process, and remote memory formation in ko. ko further showed an enhanced freezing decrement within a test session, indicating faster within-session extinction. in contrast, consolidation process of the extinction was normal in ko. these results demonstrate that the vesl- s protein plays critical roles in various processes of the ltm formation. we now examine the signaling pathways important for ltm formation that are altered in ko. hiroshi ageta , r. migishima , s. kida , k. inokuchi , mitsubishi kagaku inst. life sci (mitils), japan, tokyo univ. agricul., japan; crest, jst, japan memory process consists of at least four distinct phases, acquisition, consolidation, maintenance, and retrieval. activin a mrna increases following l-ltp induction in the hippocampus, suggesting that activin plays a role in the memory formation. here, we generated activin and follistatin (antagonizes activin function) transgenic mice in which the transgene expression was tightly regulated by dox in a forebrain-specific manner (tet off system). transgene expression was turned off or on within d by (+/−) dox. contextual fear conditioning with these mice revealed that activin function is required during maintenance phase of fear memory for one week retention. furthermore, activin plays a role in the re-consolidation process. thus, fear memory that was once acquired and consolidated tightly could be "erased" by inhibiting the activin function during maintenance phase. these mice are useful for the study of ptsd. ps p-h sex differences in the effects of chronic estrogen treatment on fear conditioning in c bl/ j mice takaaki ozawa, mumeko tsuda, sonoko ogawa kansei, behavioral and brain sciences, university of tsukuba, tsukuba, japan it has been suggested that estrogen may play a role in the regulation of learning and cognitive functions. although most of previous studies have focused on elucidating facilitatory effects of estrogen on learning in females, estrogen is also known to affect various behaviors in males. in the present study, we investigated the effects of different doses of estrogen on fear conditioning (fc) learning in both sexes of mice. gonadectomized c bl/ j mice were implanted with a silastic capsule containing , , or g of estradiol benzoate. since it is possible that estrogen may indirectly modify learning by affecting general activity, emotionality and anxiety levels, we tested the mice in open-field and light dark transition paradigms prior to fc. mice were then conditioned for fear responses (freezing) to tone stimulus and tested for both contextual and cued fc responses. we found that estrogen facilitated both types of fc learning in females, whereas it inhibited them in males especially at a higher dose, with a small effect on emotional behaviors. ps p-h analysis of brain regions activated during memory consolidation in passive avoidance task zhang yue department of bioscience, tokyo university of agriculture, tokyo, japan short-term memory (stm) is labile. to generate long-term memory (ltm), stm is stabilized through a process known as memory consolidation. importantly, previous studies have shown that memory consolidation requires the function of transcription factor creb whose activation induces c-fos expression. in this study, we tried to understand molecular mechanisms of consolidation of passive avoidance memory that has been known to be amygdala and hipocampusdependent. indeed, we investigated brain regions that are activated following the learning by analyzing the expression level of c-fos using immunocytochemistry. consistent with previous study, we observed increase in c-fos expression in amygdala and hippocampus. more interestingly, we also found this increase in prefrontal cortex, indicating that prefrontal cortex plays critical roles in memory consolidation in light-dark passive avoidance task. hiroshi nomura, norio matsuki laboratory of chemical pharmacology, graduate school of pharmaceutical sciences, university of tokyo, tokyo, japan we have demonstrated the effect of ethanol on reactivated fear memory for the first time, using contextual fear conditioning. rats were conditioned with mild footshock, reexposed to the training context, immediately injected with ethanol or saline, and finally tested h after reexposure. ethanol-treated groups expressed longer freezing and the effect lasted for weeks. reactivation was necessary for the effect. the injection of ethanol itself did not induce a fearful response. as memory retrieval triggers memory extinction and reconsolidation, we investigated whether extinction process is involved in this ethanol effect. increasing retrieval time did not enhance freezing by ethanol, suggesting that ethanol had no effect on memory extinction. post-reactivation injections of anisomycin revealed that retrieval triggered reconsolidation. moreover, picrotoxin inhibited the memory enhancement by ethanol. these studies demonstrate that ethanol enhances reactivated contextual fear memories via activation of gaba a receptors. ps p-h analyses of brain regions activated in reconsolidation and extinction phases of contextual fear memory nori mamiya, akinobu suzuki, satoshi kida department of bioscience, tokyo university of agriculture, tokyo, japan the retrieval of conditioned fear memory by conditioned stimulus (cs) initiates two processes; reconsolidation or extinction. we previously found that the change in memory stability after retrieval (reconsolidation) associates with memory extinction. to understand the regulatory mechanisms of memory stability after the retrieval at the anatomical level, we here investigated the brain regions that are activated in reconsolidation and extinction phases. we measured the levels of phospho-creb inducing changes in neural plasticity following the re-exposure to cs. short re-exposure to cs inducing reconsolidation increased in phospho-creb in amygdala and hippocampus. in contrast, longer re-exposure inducing extinction increased in phospho-creb in amygdala and prefrontal cortex. these results indicate that distinct brain areas are activated in response to short or long re-exposure to cs and suggests that amygdala plays crucial roles in the interaction between reconsolidation and extinction. ps p-h analysis of molecular mechanism for the destabilization of retrieved contextual fear memory akinobu suzuki, satoshi kida department of bioscience, tokyo university of agriculture, tokyo, japan reconsolidation acts to stabilize, whereas extinction tends to weaken the expression of the original memory. to understand the mechanisms for the regulation of memory stability after the retrieval, we have investigated the relationship between reconsolidation and extinction using contextual fear conditioning. we previously found that memory extinction is associated with regulation of fear memory stability, indicating the interaction between memory reconsolidation and extinction phases. in this study, we compared molecular signatures of reconoslidation and extinction using mice. pharmacological experiments using antagonists for cannabinoid receptor (cb ) and l-type voltage-gated calcium channels (lvgccs) indicated that both cb and lvgccs are required for memory extinction but not consolidation and reconsolidation. more interestingly, blockade of either cb or lvgccs function prevents the disruption of the original memory by protein synthesis inhibition. these results suggest that cb and lvgccs are required for not only memory extinction but also the destabilization of reactivated memory. hotaka fukushima, akinobu suzuki, satoshi kida department of bioscience, tokyo university of agriculture, tokyo, japan previous our studies using contextual fear conditioning revealed three distinct time-dependent phases following memory retrieval: stable, reconsolidation, extinction phases. to understand the nature of memory processing following retrieval, we examined the effects of reexposure on memory reconsolidation and extinction using light-dark passive avoidance task. this task is thought to allow us to discriminate between reconsolidation and extinction phases at the time point when mice enter dark box from light box. brief re-exposure to light box did not affect the stability of fear memory (stable phase). further extending re-exposure to light box triggered the requirement of protein synthesis for re-storage of fear memory (reconsolidation phase). in contrast, entry from light into dark box initiated extinction of fear memory (extinction phase). additionally, using pharmacological blockade of cb and lvgccs, we also found that cb is required for only memory extinction but that lvgccs are required for memory extinction and reconsolidation. wakoto matsuda , takahiro furuta , kouichi nakamura , , takeshi kaneko , ps p-h difference in organization of corticostriatal and thalamostriatal synapses between patch and matrix compartments of rat neostriatum fumino fujiyama , tomo unzai , kouichi nakamura , , sakashi nomura , takeshi kaneko , department of morphological brain science, kyoto university, kyoto, japan; department of physical therapy, kyoto university, kyoto, japan; crest, japan the striatum, which has patch/matrix compartments, receives glutamatergic inputs from cortex and thalamus. in the present study, the differences in synaptology of these inputs between both compartments were examined. axon terminals positive for vesicular glutamate transporter (vglut) , thalamostriatal inputs, were less dense in patch region, whereas vglut -positive corticostriatal inputs were evenly distributed. quantitative analysis revealed % of vglut positive synapses in patch region were formed with spines, whereas % in matrix region were made with dendritic shafts. in contrast, the targets of vglut -positive inputs were mainly spines in both regions. moreover, vglut -positive axospinous synapses in patch region were larger than vglut -positive ones. the present observation suggests that thalamostriatal connection is more plastic in patch region. research funds: kakenhi ( , , , ( ), ) ps p-h single cell tracing of thalamostriatal projection neurons with reference to patch and matrix compartments of rat striatum tomo unzai , fumino fujiyama , takeshi kaneko , department of morphological brain science, university of kyoto, kyoto, japan; crest, japan the striatum consists of patch and matrix compartments, and receives glutamatergic inputs mainly from the cerebral cortex and thalamus. thalamic intralaminar nuclei are known to project exclusively to matrix compartment. on the other hand, it has not been clarified which thalamic nuclei project to patch compartment. in the present study, we combined single cell tracing with immunohistochemistry for mu opioid receptor, which is specifically expressed by patch neurons, to reveal the distribution of thalamostriatal axon terminals in relation to striatal compartments. recombinant sindbis virus expressing membrane-targeted green fluorescent protein (palgfp) was injected into the rat thalamus. a single neuron in the thalamic paraventricular nucleus extensively projected to the striatum and preferentially to patch compartment compared with matrix compartment. the axons were also distributed in the thalamic reticular nucleus, accumbens nucleus, amygdala, and cerebral cortex. research funds: kakenhi ( , , , ( ) , ) ps p-h lesion of the nucleus accumbens dopamine system shortens the lever pressing interresponse time and delays the response initiation in mice yuji tsutsui , kayo nishizawa , nobuyuki kai , kazuto kobayashi , dept. of psychology, fukushima univ., japan; dept. mol. genet., fukushima medi. univ., japan; crest, jst, kawaguchi, japan dopamine transmission is thought to be important for rodents to perform operant behaviors such as lever pressing. the lever pressing experiment was conducted to examine the effects of -ohda injections into the nucleus accumbens (acb) in c bl/ j mice. all mice were trained to press the lever for a food pellet using a fixed ratio (fr ) schedule. the mice were injected with ascorbate vehicle or -ohda into the acb, and then tested post-surgically using the fr schedule again. the -ohda-injected mice showed the acceleration of response speed, which was revealed by the shortening of interresponse time between each of the five lever pressings, and the suppression of the initiation of the response to the next step. this suppression of initiation was revealed by the increase of time from the last presentation of food to the next initiation. these results suggest that the acb dopamine system is important for the initiation and control of the operant behaviors in rodents. hideshi shibata laboratory of veterinary anatomy, tokyo university of agriculture and technology, fuchu, tokyo, japan retrosplenial area is one of the important structures for spatial memory and behavior in the rat. to understand more fully the functional roles played by area , it is essential to clarify the neural circuitry subserving these functions. in the present study, we analyzed the organization of frontal cortical projections to area in the rat, using retrograde transport of cholera toxin b subunit (ctb). ctb injections into area d retrogradely labeled cells in the orbital cortex and the caudal parts of the anterior cingulate and primary and secondary motor cortices. ctb injections into area c labeled cells in similar cortical regions, except for the orbital cortex. ctb injections involving areas a and b labeled cells in the caudal part of the anterior cingulate cortex. the results show that the orbital, anterior cingulate, and primary and secondary motor cortices have a different pattern of projections to each subdivision of area , suggesting different functional roles played by each subdivision of area in spatial memory and behavior. eiichi jodo , yoshiaki suzuki , tadahiro katayama , ken-yo hoshino , yukihiko kayama dep. of physiol., fukushima med. univ., fukushima, japan; dep. of neuropsy., fukushima med. univ., japan it has been shown previously that the dopaminergic neurons in the ventral tegmental area (vta) selectively respond to a stimulus repeatedly paired with reward stimuli in a classical conditioning paradigm. since the vta receives dense projection from the medial prefrontal cortex (mpfc), such response selectivity of vta neurons may in part be produced by inputs from the mpfc. however, few studies have compared the firing pattern between these two regions. our present experiment was designed to make such a comparison in freely moving rats. two different tones were sequentially presented, one of which (target, %) was paired with intracranial simulation of the reward area. the unit activity was recorded from the mpfc and/or the vta. pfc and vta neurons exhibited phasic excitation with the peak latency of about . s to both tones, while only the target tone induced sustained activation of firing activity lasting until presentation of the reward. masato inoue, akichika mikami primate research institute, kyoto university, inuyama, aichi, japan to investigate the neuronal mechanism in the ventrolateral prefrontal cortex (vlpfc) and inferotemporal cortex (it) for holding information for object and their order of presentation, we examined single neuronal activities in the vlpfc and it while monkeys were performing a serial probe reproduction task. in the task, two sequentially presented objects were memorized and then a target object was selected from memorized objects based on a color stimulus. in % out of vlpfc neurons, the delay-period activity showed objectselectivity and order-selectivity. in only % out of it neurons, the delay-period activity showed object-selectivity and order-selectivity. the starting time of the order-selective activity was earlier in the vlpfc. these results suggest that the vlpfc plays a role in holding information for object and their order of presentation and the it receives information for object and their order of presentation from the vlpfc. masao yukie, yasutaka oosawa department of behavioral physiology, tokyo metropolitan institute for neuroscience, fuchu, japan relational memory theory (eichenbaum et al., ) has been proposed from evidence that the hippocampal damage in rats impairs learning of transverse patterning task (a+ versus b−; b+ versus c−; c+ versus a−). very recent monkey study (alvarado et al., ) demonstrated that lesion of the hippocampus produced a significant impairment in that task and supported such a theory. in our study, however, ischemic damage of the hippocampus has not impaired learning of such a transverse patterning task (yukie et al., ) . in the present study, we examined effects of lesion of the monkey perirhinal cortex on transverse patterning task using two sets of d visual stimuli presented in a wgta. our three monkeys with perirhinal lesions failed to attain a learning criterion within a training limit of sessions in phase , although they learned easily the four problems in phases and . our results suggest that the perirhinal cortex, but not the hippocampus, is important for learning of transverse patterning task, that is, for formation of relational memory. yasuko sugase-miyamoto , noriyuki higo , munetaka shidara neuroscience research inst., aist, tsukuba, japan; grad. sch. of tsukuba univ., tsukuba, japan a recent dopamine d receptor study using antisense cdna showed that d receptor in rhinal cortex is crucial for learning associations between visual stimuli and reward schedules. neuronal responses in the perirhinal cortex differentiate the visual cues only when the cues are associated with the schedule states, while those in area te are related to physical attributes of the cue independently of the schedule states. to investigate the cellular substrate for d mediated associative learning, we examined monkey temporal lobe immunohistochemically with a d receptor antipeptide antiserum. d receptor immunoreactivity was observed in the pyramidal cells in layers ii-vi of the rhinal cortex and area te. the signal was mainly observed in cell bodies, and also in both apical and basal dendrites for some cells. the signal in layers v-vi was stronger in area of the perirhinal cortex than in area teav. the differential localization between area and te suggests the differential roles of the two areas in associative learning process. by using axonal transport of fast blue, diamidino yellow and tritiated amino acids, we determined the afferent and efferent connections of the retrosplenial cortex (rsp) in the macaque monkey. the rsp receives heavy projections from the subiculum, presubiculum and the caudal entorhinal areas (ec-ecl), and projects back to the presubiculum and the ec-ecl. the supracallosal portion of the rsp has connections primarily with the caudal half of the subiculum and presubiculum, as well as the lateral zone of the ec-ecl. the caudoventral portion of the rsp is, in contrast, mainly connected with the rostral half of the subiculum and presubiculum as well as the medial zone of the ec-ecl. the two portions of the rsp, thus, have access to different portions of the medial temporal lobe. these results indicate that there are two distinct neural systems in the retrosplenial-medial temporal network. hideko nakano , natsuko yoshida , kiyohisa natsume kyushu kyoritsu university, fukuoka, japan; kyushu institute of technology, fukuoka, japan; kyushu institute of technology, fukuoka, japan eeg activity was examined in english rhythm acquisition of japanese students who learn english as a foreign language (efl). we measured theta, alpha and beta rhythms of five subjects while they were reading aloud the materials and listening to the audio-recording, using eight electrodes attached to their skulls. the result shows that the increase of theta power at f and f was the highest and suggests that the theta rhythm at f and f may have a relationship to the process of english rhythm acquisition. moreover we found the highest increase in theta power when the subjects began to orally reproduce every line of the rhythm materials. this finding was observed in three right handlers except a left handler and a right handler who had just returned after -month english study experience in australia. these results suggest that the change of theta power at frontal areas may be more closely related to the japanese efl learners' english rhythm acquisition. research funds: kakenhi ( ) ps p-h neural correlates of music retrieval: an eventrelated fmri study using sparse temporal sampling takamitsu watanabe , sho yagishita , hideyuki kikyo , department of physiology, the university of tokyo school of medicine, tokyo, japan; department of molecular neuroimaging, national institute of radiological sciences, chiba, japan we investigated neural correlates of music memory using eventrelated functional magnetic resonance imaging and sparse temporal sampling technique with originally composed musical materials. written informed consent was obtained from all the subjects in accordance with the declaration of helsinki, and the experimental procedure was approved by the institutional review board of the university of tokyo school of medicine. a . t scanner system was used (te = ms; tr = s; acquisition time = . s). we demonstrated that the right hippocampus, bilateral lateral temporal cortices, left prefrontal cortex and left precuneus are involved in music retrieval. in addition, performance-based analysis suggested that the right hippocampus is associated with the accuracy of music memory. in this fmri study, we determined the neural correlates of the intellectual excitement. sentences describing facts in natural and human science were visually presented, and subjects judged whether they know the fact or not. after the fmri, each subject self-evaluates subjective "intellectual excitement" of each sentence. positive correlation with the self-evaluated intellectual excitement for known facts and novel facts were analyzed. significant correlation between cortical activation and self-evaluated intellectual excitement for novel facts was observed in the left and the right parahippocampal gyrus and for known facts was in the left orbital part of inferior frontal gyrus. it suggests the cortical areas related to self-evaluated intellectual excitement are different between getting of novel knowledge and recognition of existing knowledge. hyeonjeong jeong , motoaki sugiura , yuko sassa , keisuke wakusawa , , kaoru horie , , shigeru sato , , ryuta kawashima , gsics, tohoku university, sendai, japan; niche, tohoku university, japan; miyagi university of education, sendai, japan; department of pediatrics, school of medicine, tohoku university, japan; the lbc research center, tohoku university, japan a foreign language word is learned and retrieved either in daily situations (situation) or written text (text), and memory transfer is required when the learning and retrieval modes are different. in this experiment, normal japanese subjects learned korean words in the situation and text modes in video clips. during a subsequent fmri session, subjects were presented with the learned words in different movie clips; half of the learned words was presented in the same mode as in the learning session (match), and the rest was presented in a different mode (mismatch). comparison of the mismatch with match condition revealed significant activation in the orbital part of the left inferior frontal gyrus. the results suggest that this area plays a role in the memory transfer of foreign language words when the learning and retrieval modes are different. georgina e. cruz , christie l. sahley , kenneth j. muller physio. & biophys., univ. of miami, miami, fl, usa; biol. sci., purdue univ., west lafayette, in, usa in some animals much is known at the level of single synapses about mechanisms underlying behavioral sensitization, but in no system is the involvement of interactions at the network level well understood. the s-cell network of the medicinal leech is a chain of electrically coupled interneurons spanning the nerve cord with distributed sensory input and motor output and is crucial for sensitization of reflex shortening. its firing increases with sensitization although few additional s-cells initiate impulses during the reflex. we tested the hypothesis that the initial burst of impulses from the s-cell in the stimulated segment suppresses initiations in adjacent segments. hyperpolarizing the central s-cell to reduce its firing during skin stimulation markedly increased the number of initiations in adjacent s-cells, which corroborated the limited expansion of initiation sites seen in the behaving animal. a computational model of s-cell refractoriness further supported the idea of interaction among s-cells during sensitization. research funds: nih, u.s.a. ps p-h sensory/motor modules regulating the development of peer social relationship mamiko koshiba , , shun nakamura jst, crest, japan; ncnp social intelligence is indispensable for animal's survival and could have evolved to language capability. further, as a recent problem in japan, 'fewer children' supposedly causes the more tight interaction of child-parent, reciprocally the less between siblings or friends. in order to study the genetic and epigenetic development of peer social relationship after birth, we controlled peer interaction through limiting a particular sensory/motor modality as social deprivation and examined the effect on the active attachment behavior of domestic chick to conspecific mates. the chick has a merit of being precocial and unique in higher animal with no need of parent-care. comparing to the chicks reared as a group, the isolated chicks didn't develop their active attachment to peers. meanwhile, the behavior study with the chicks deprived not sensory/motor function itself, but only social interaction in auditory, visual, olfactory or tactile system, suggested that vocal communication at least must play a key role for the development. dna-chip study along the different social context brought candidates of social genes. shogo sakata , minoru hattori department of behavioral sciences, hiroshima university, higashi-hiroshima, japan; graduate school of biosphere science, hiroshima university, higashi-hiroshima, japan peak interval (pi) -s procedure is a very good method to investigate for timing. six male wistar rats were trained for five days a week in pi -s procedure over days. the -s bin of lever press responses on probe trials showed a clear peak point. the temporal distributions had the peak time of regression curve fitting with the gaussian function. the peak time corresponded to near the -s with reinforcement durations. then nicotine was administrated to the rat by intraperitoneal injection before daily pi -s session. results showed that the peak time in the nicotine administration was slightly leftward shift compared to the saline injection. however the pattern of temporal distribution of responses was not changed by the nicotine treatment as well as control condition. it suggests that the nicotine administration affects on the time perception that was reflected by the peak durations of responses. ps p-i the effect of random practice schedule on arbitrary stimulus-response association learning satoshi tanaka , , ritz oshio , , norihiro sadato , , manabu honda , nips, okazaki, japan; jsps, tokyo, japan; nagoya univ., nagoya, japan; ristex, jst, tokyo, japan; sorst, jst, tokyo, japan; ncnp, tokyo, japan previous studies suggest that randomly ordered practice facilitates retention and transfer of motor skills compared to blocked or regularly ordered practices. it remains unclear, however, whether the advantageous effects of random practice can be expended to cognitive skill learning in humans. we examined the simultaneous learning of multiple arbitrary stimulus-response (s-r) associations under three different practice schedules: blocked, random and regularly ordered. behavioral data indicate that subjects performing the random practice showed better performance of the retention and transfer of learning compared to those performing the blocked or regularly ordered practice. the present result indicates that random practice schedule is effective also for s-r association learning, which are considered as a bridge between motor control and cognitive control. ps p-i sports rats show increased level of bdnf in the cerebellum, possibly learning and memorizing well masaki morishima, sayuri hara, yutaka nakaya dept. nutrition and metabolism, univ. of tokushima, japan previously, we reported that the activation of hippocampal norepinephrine neurotransmission following a decrease in monoamine oxidase a was observed in sports, a novel hyper-running rat on wheel. this study assessed whether sports show increased bdnf levels and better learning and memory. compared to control, both protein and mrna levels of bdnf in cerebellum were significantly elevated in sports even without wheel running, and slightly increased in hippocampus. in the cerebellum of sports, trkb/pi k pathway was activated, whereas mapk pathway was activated in the hippocampus. locomotor activity assessed by the open field test showed that the sports were significantly more active in center coat than control. in the passive avoidance test, sports did not enter a dark area at next time indicating that sports showed better passive avoidance learning. these results suggest that bdnf signaling of sports were activated from trkb to mapk and pi k in the hippocampus and cerebellum, respectively, and that these signaling pathways might play an important role in learning and memory. research funds: kakenhi ( ) ps p-i selective manipulation of working memory through d and d receptors: computer simulation shoji tanaka, hiroki yata dept. of electrical & electronics eng., tokyo, japan though a number of experimental results suggest that working memory processes are controlled by the dopaminergic system, its mechanism is still unclear. to elucidate the mechanism, we have constructed a model of the prefrontal cortical neural circuit for working memory. the neurons in the model are leaky integrate-and-fire model with ampa, nmda, gaba, and leak conductances and have dopamine d and d receptors. the computer simulation with this model shows that d receptor activation mainly affect working memory activity itself, while d receptor activation affect the termination of working memory, being consistent with the experimental result. the simulation also mimics the hyper-and hypo-dopaminergic states. under such conditions, like schizophrenia, simulated pharmacological treatments using agonists and antagonists of d and d receptors indicate efficacies of some these treatments for the restoration of working memory. in conclusion, this kind of simulation shows how dopamine controls working memory by using the synergism of the actions of dopamine, glutamate, and gaba. kozo sugioka, tomiyoshi setsu, tatsuro yamamoto, toshio terashima div. anat. & dev. neurobiol., dept. neurosci., kobe univ. grad. sch. med., kobe, japan we examined activity and habituation in rats with experimentallyinduced abnormal morphogenesis of the hippocampus. pregnant rat (jcl:wistar) was injected with saline or mg/kg mam on the th day of gestation. the activity of male and female offspring was measured for each h light and dark period, and the habituation to the visual stimulation was observed by measuring the activity with every min interval for h under min dark/light alternative schedule during weaning and adult periods. activity was measured using infra-red sensor in a home-cage placed in the experimental room. the mam-treated rat showed hyperactivity for dark-period during both weaning and adult periods, and showed retarded habituation during weaning period. sex difference of behavioral alteration was evident during adult period in both groups. these behavioral disorders were discussed in relation to the mam-treated rat showing abnormal hippocampus (disruption of the ca pyramidal layer and ectopic neuron mass). ps p-i long-lasting tagging of functionally activated neurons in the mouse brain naoki matsuo, leon reijmers, mark mayford the scripps research institute, la jolla, ca, usa immediate-early genes (iegs) have been widely used as activity markers for mapping neurons involved in specific animal behaviors including learning and memory. however, conventional ieg approaches that use immunohistochemistry or in situ hybridization allow to detect neurons only shortly after their activation and does not enable genetic manipulations. here we have developed transgenic mice that allow selective and long-lasting tagging of neurons that were activated in a given brain region at a given time point. the mice consist of two components; c-fos promoter driven tetracycline-controlled transactivator (tta) and teto promoter regulated feedback loop. when strong neuronal activity occurs in the absence of tetracycline analogs such as doxycycline (dox), c-fos promoter driven tta initiates the teto-linked expression of mutant tta (tta*) that is not inhibited by dox. this teto-linked gene expression is then maintained indefinitely by feedback activation via the tta* even in the presence of dox. using this system, we have examined the expression of bicistronic teto promoter driven tau-lacz and egfp-glur . hamid gholamipour , shirin babri , khameneh saied department of physiology, university of tabriz, tabriz, iran; university of tabriz, iran; university of tabriz, iran diabetes mellitus is one of the most prevalent diseases in the world. because hippocampus is an important area for memory formation, the present study is scheduled to investigate the effect of insulin injection in ca region of hippocampus on memory formation. fifty male rats were divided into five groups. ( ) control ( ) sham operation ( ) test ( ) diabetic/saline ( ) diabetic/insulin. groups and were made diabetic by treatment with stz ( mg/kg, i.p.). in all but the control group, two canula were stereotaxically implanted in ca region of hippocampus. learning was tested and compared between groups through passive avoidance test. results showed that in the test group the latency increased as compared to control and sham groups (p < . ). compared to sham group diabetic/insulin group showed increased latency (p < . ) but no significant difference was found between diabetic/saline and diabetic/insulin groups. in conclusion, according to the results obtained in this study, insulin facilitates memory in intact rats but not in diabetic sex differences in hippocampus-dependent memory formation are well documented, but the mechanisms are poorly understood. the ca + /calmodulin (cam) kinase cascade regulates gene transcription in the hippocampus, which is required for long-term memory (ltm) formation. we hypothesized that sex differences in transcriptional regulation may account for the sexual dimorphisms in memory formation. we tested this idea by studying the role of cam kinase kinases (camkks). using mouse molecular genetics we found that camkk is required for spatial, but not contextual ltm. consistent with the impaired spatial memory formation, camkk null mutants lacked spatial training-induced creb activation and had impaired late ltp. in contrast to camkk, camkk␣ is required for contextual, but not for spatial ltm. furthermore, female camkk mutants had normal spatial and contextual ltm. thus, we show that there are malespecific mechanisms to regulate gene transcription that may explain sex differences in hippocampus-dependent memory formation. akshay anand, sudesh prabhakar, monika bhatia, c.p. das department of neurology, post graduate institute of medical education and research, chandigarh, india background: parkinson's disease has a prevalence rate of per , in india. we studied the park polymorphism in north indian population and parkin expression in early parkinson's disease (n = ) and sporadic parkinson's disease (n = ). methods: pcr, sscp, rflp and direct sequencing analysis were used to screen mutations. results: our results revealed homozygous exonic mutations in exon- , and in early pd and exon- and in sporadic pd, heterozygous mutations in exon and in five early pd and one sporadic pd patient. frequency of s/n polymorphism was significantly high suggesting that exchange of serine to asparagine at position of protein affects the secondary structure or hydrophobicity of the protein resulting in pathogenicity. our facs analysis of these samples indicates reduced parkin expression correlating with severity of mutations. conclusions: we conclude that high frequency of parkin mutations in pd population in india affect parkin expression resulting in pd. wanida tripanichkul , kittisak sripanichkulchai , david finkelstein faculty of medicine, srinakharinwirot university, thailand; faculty of medicine, khon kaen university, thailand; university of melbourne, australia emerging data suggests beneficial effect of estrogen for parkinson's disease (pd), yet the exact mechanisms implicated remain obscured. activated glia observed in mptp mouse model and in pd may participate in the cascade of deleterious events that ultimately leads to dopaminergic nigral neuronal death. estrogen can modify glial expression of inflammatory mediator, such as cytokines and chemokines implicated in neurodegeneration. to determine whether estrogen-elicited neuroprotection in pd is mediated through glia, adult male c bl/ mice were pretreated with beta-estradiol (e ), injected with mptp on the day and brains were collected on day . e pretreatment decreased nigral neuronal loss and diminished striatal fibers deficit induced by mptp. the neuroprotective effect of e was coincident with an attenuation of a glial response within the snpc and striatum. these findings propose that e neuroprotection in mptp mouse model may mediate through reactive glia inhibition. ps p-i effect of angiotensin-converting enzyme inhibitor perindopril in mptp-treated mice; immunohistochemistry and in vivo electron spin resonance (esr) study rumiko kurosaki , fumihiko yoshino , masaichi chang-il lee dept. of food sci. and nutrition, showa women's univ., tokyo, japan; clin. care and med. div. of pharmaco., kanagawa dental college, japan we investigated the effects of perindopril on the dopaminergic system and the oxidative stress in mice after mptp treatment. administration of perindopril showed dose-dependent neuroprotective effects against striatal dopamine and its metabolites depletion after mptp treatment. we have reported that th, gfap, pv, nnos and cu, zn sod positive cells in the substantia nigra was changed after mptp treatment in our immunohistochemical study. the administration of perindopril significantly attenuated mptp induced changes of these immunopositive nigral cells. we could measure increased oxidative stress in the brain of mptp and perindopril treatment mice using by in vivo esr technique. our results provide further evidence that the ace inhibitor perindopril may offer a novel therapeutic strategy for parkinsonǐs disease. research funds: kakenhi ( ) ps p-i recruitment of calbindin into substantia nigra dopamine neurons suppresses the onset of parkinsonian motor signs shigehiro miyachi , kaori sawada , haruo okado , atsushi nambu , masahiko takada tokyo met. inst. neurosci., fuchu, tokyo, japan; natl. inst. for physiological sci., okazaki, japan there is a consensus that dopaminergic neurons in the substantia nigra that express calbindin, a calcium-binding protein, are selectively invulnerable to parkinsonian insults. based on this notion, an attempt was made to test the hypothesis that parkinsonism may be suppressed by recruitment of calbindin into a subpopulation of nigral dopamine neurons that does not normally contain calbindin. an adenoviral vector expressing calbindin was injected unilaterally into the striatum of macaque monkeys, to let calbindin express in the dopaminergic neurons via retrograde transport. two to three weeks later, the parkinsonism-inducing drug mptp was systemically administered several times. parkinsonian motor signs, such as muscular rigidity and flexed posture, appeared only on the side ipsilateral to the calbindin recruitment when cumulative doses of mptp exceeded threshold for their bilateral onset. toru yasuda, hideki mochizuki, yoshikuni mizuno department of neurology, juntendo university school of medicine, tokyo, japan using the serotype- raav vectors, we have recently reported the protective effect of parkin on the ␣-synuclein (␣s)-induced nigral dopaminergic neurodegeneration in a rat model. here we investigated the neuronal specificity of ␣s toxicity and the effect of parkin co-expression in a primate model. another serotype (type- ) of raav (raav ) carrying αs cdna (raav -␣s), and a cocktail of raav -␣s and raav carrying parkin cdna were unilaterally injected into the striatum of macaque monkeys, resulting in protein expression in striatonigral gabaergic and nigrostriatal dopaminergic neurons. the injection of raav -␣s alone caused a decrease of th-immunoreactivity in the striatum, while there was no effect on gabaergic neurons. in the presence of overexpressed parkin, ␣s seemed to be less accumulated and/or phosphorylated at ser residue in gabaergic neurons. these suggest that the ␣s toxicity is not expressed in non-dopaminergic neurons but the ␣s-ablating effect of parkin is exerted in all neurons introduced in primates. tomokazu oshima, yohsuke narabayashi narabayashi memorial laboratory of neurology, neurological clinic, tokyo, japan rigidity in aged parkinsonians is often intractable against surgeries. we investigated how their rigidity scored by updrs was related with -band local field potentials (-waves) of the surgical targets in thalamic ventrolateral nucleus (vl) and posteroventral pallidal internal segment (pvp). forty patients aged - s gave informed consent for thalamotomy and/or pallidotomy. we divided the patients into groups with rigidity of . - (i), . - (ii), . - (iii), and . - (iv). the waves were rated with total periods (%) of - hz wavelets in -s sample records. rigidity was re-scored after the surgeries. the vl -waves were rated - % in groups i-iii with a slightly increasing tendency for increasing rigidity, but declined to about % in group iv. the pvp -waves were - %, but with a decreasing tendency for increasing rigidity. the surgeries alleviated rigidity in all the groups, but were least effective in group iv with least vl and pvp -waves. the results suggest that the pathology of aged parkinsonian rigidity develops beyond the pallido-thalamic pathway. yoshihisa tachibana , , hirokazu iwamuro , , masahiko takada , atsushi nambu , div. syst. neurophysiol., natl. inst. physiol. sci., okazaki, japan; sokendai; dept. neurosurg., univ. tokyo, tokyo, japan; dept. syst. neurosci., tokyo met. inst. neurosci., fuchu, tokyo, japan to approach a new therapy for parkinson's disease, extracellular unit recordings combined with microinjections of glutamate-related drugs were performed in the external and internal segments of the globus pallidus (gpe/gpi) of mptp-treated parkinsonian monkeys (macaca cyclopus). compared with the normal state, spontaneous oscillatory discharges were so often observed in the gpe/gpi and the subthalamic nucleus (stn) of the parkinsonian monkeys. microinjections of ionotropic glutamate receptor antagonists into the vicinity of recorded gpe/gpi neurons reduced their abnormal oscillations. these results suggest that glutamatergic excitatory input from the stn contributes to the oscillatory activity of gpe/gpi neurons, and that intrapallidal injections of ionotropic glutamate receptor antagonists may ameliorate some of parkinsonian symptoms. one of the pathological features of parkinsonǐs disease (pd) is loss of dopaminergic neurons in the substantia nigra pars comapacta (snpc). and it has been known that ␣-synuclein is involved in the neuronal loss. during the dopaminergic neuronal loss, activated microglia were centered in snpc. we hypothesize that ␣-synuclein may play a role in microglial activation to migrate to the pathological regions and to perform the neuronal cytotoxicity. we demonstrated that ␣-synuclein induced the cd expression on microglia and also enhanced the mt -mmp expression to shed off cd at the cell surface and degrade surrounding ecm to open the migratory way. a t mutant ␣-synuclein showed greater level of cd shedding and cell migration. extracellular treated ␣-synuclein also increased cd and mt -mmp expressions dose-dependently. among the multiple signaling pathways, erk pathway was involved in ␣-synuclein induced cell migration. these induced cell migration were also confirmed in human pd patients. research funds: national creative research initiative grant ( grant ( - ps p-j serotonergic fibers are involved in the conversion of l-dopa to dopamine in the striatum and the substantia nigra pars reticulata of parkinsonian model rats ryohachi arai , hiromasa yamada , yoshinari aimi , ikuko nagatsu department of anatomy, shiga university of medical science, otsu, japan; fujita health university school of medicine, japan dopaminergic neurons in the substantia nigra pars compacta (snc) project their axons to the striatum (st) and their dendrites to the substantia nigra pars reticulata (snr). dopamine released from these axons and dendrites is important in the regulation of motor activity. in parkinson's disease, dopaminergic neurons in the snc degenerate. l-dopa is the most effective drug for this disease. we hypothesize that, in parkinson's disease, a part of administered l-dopa is converted to dopamine in serotonergic fibers of the st and snr. here we produced parkinsonian model rats by the unilateral injection of hydroxydopamine into the snc, and found that serotonergic fibers in the st and snr were immunohistochemically positive for dopamine after l-dopa administration in the rats. therefore, it is possible that serotonergic neurons may be involved in the therapeutic effects of l-dopa for parkinson's disease. in mptp-induced pd monkey, reactive microglia are observed around neurons in nigra several years after mptp treatment and may be related to the progression of pd. to evaluate if reactive microglia in striatum and/or nigra of mptp-induced pd mice are present for a long time after mptp administration, like pd monkey. iba -and tb distribution in microglia were immunohistochemically investigated at h and days after twice mptp-treatments (one treatment comprised of intraperitoneal injections of mg/kg mptp at h interval) to c bl/ and balb/c at months (mo) interval. the recognizable change of iba -and tb -distibution in microglia of both mice strains was observed even mo after the first treatment. the twice mptp treatments tended to aggravate the symptoms in both mice strains, compared with once treatment. these results suggest that reactive microglia are present for a long time after the treatment by mptp and must play a role in the chronic progression of pd. ps p-j activated microglia affect the nigro-striatal dopamine neurons differently in neonatal and aged mice treated with mptp hirohide sawada , ryohei hishida , yoko hirata , kenji ono , hiromi suzuki , shin-ichi muramatsu , imaharu nakano , kunihiro tsuchida , toshiharu nagatsu , , makoto sawada school of medicine, fujita health university, aichi, japan; division of neurology, jichi medical university, japan; department of biomolecular science, gifu university, japan; research institute of environmental medicine, nagoya university, japan microglia play an important role in inflammatory process of parkinson's disease. we examined the effects between neonatal and aged microglia activated with lps on the nigro-striatal dopamine (da) neurons in mice treated with mptp. by mptp administration to neonatal mice, the number of da neurons in the substantia nigra was significantly decreased, whereas that in mice treated with lps and mptp was recovered. on the contrary, the number of da neurons of the week-old mice treated with mptp was significantly decreased with lps treatment. these results suggest that activated microglia in neonatal mice have neurotrophic potential, in contrast to the neurotoxic effect in aged mice. hyposmia is one of the most characteristic symptoms of parkinson's disease (pd). it may occur even before the motor symptoms start. in the olfactory bulb (ob), dopaminergic cells were present at glomerular layer. furthermore, it has been reported that ob contains neural stem cells. thus, ob has attracted attention because of its unique regenerative potential. in the present study, we established isolation of neurosphere forming cells (nsfcs) derived from adult mice ob, and examined proliferation potential in ob after dopaminergic neuronal loss induced by mptp, a selective toxin for dopaminergic neurons, utilized frequently as pd model. the number of neurospheres derived from adult ob was not decreased with mptp administration, rather significantly increased. we also evaluated nsfcs differentiation into neural subtypes. the isolation of neural stem cells has helped to establish the cellular basis of neurogenesis and the exciting potential for transplant-mediated treatment of degenerative cns disease like pd. ps p-j phosphorylation of erp in adult rat brain with neonatal -ohda treatment qinghua li, yasuyoshi watanabe department of physiology, osaka city university graduate school of medicine, osaka, japan dopaminergic neuron degeneration occurs in sporadic parkinson's disease (pd), but the mechanism of sporadic pd is not clarified. we prepared neonatal dopamine depleted rats, by i.c.v. injection of -ohda at (p ) and days after birth, to investigate the mechanism of dopaminergic neuron degeneration. at p , tyrosine hydroxylase (th) immunostaining cells were significantly reduced in the substantia nigra, and th immunostaining fibers were significantly reduced in the striatum, thus this model mimics the selective dopaminergic neuron degeneraion in sporadic pd. by two-dimensional electrophoresis we found that a certain protein was phosphorylated in the -ohda lesioned rats at p , and it was identified as disulfide-isomerase a precursor (erp ) a kind of molecular chaperone of the endoplasmic reticulum (er) by maldi-tof ms. the result suggests that the phosphorylation of erp may have the key function to induce dopaminergic neuron degeneration and somehow relates to the pathogenesis of sporadic pd. katsunori nishi department of neurology, tokyo metropolitan institute for neuroscience, tokyo, japan regrowth of survived dopaminergic (da) neurons after the administration of psi, a potent proteasome inhibitor, was examined in vitro. dissociated cell co-culture was prepared from embryonic rat mesencephalon and striatum. psi ( or nm, h) was applied to cultures at days in vitro and succeeding changes of da neurons were investigated up to days. more than % of da neurons reduced in number after the administration of psi, and a few truncated da neurons, devoid of neurites, being observed. non-da neurons were less severely affected at these concentrations of psi. regrowth of da neurites was observed approximately weeks after the administration of psi and continued during the observation period. in most of the regrowing da neurons, one of the processes extended far longer than the rest, suggesting that severely injured neurons retain the capacity to reextend axons. regrowth was less remarkable in mesencephalic culture lacking striatum indicating that target cells are necessary for this effect. in conclusion, psi-damaged da neuron has strong regrowth potential in vitro. ps p-j specific expression of proapoptotic factor pag on motor neurons in spinal cords of l-dopatreated parkinsonian models ikuko miyazaki, masako shimizu, francisco j. diaz-corrales, maria f. esraba-alba, masato asanuma dept. of brain sci., okayama univ. grad. sch. of med., dent. and pharmaceut. sci., japan we previously identified a proapoptotic gene, p -activated gene (pag ), as a dopa-induced gene in the striatum of l-dopa-treated parkinsonian models, which increased p expression to promote apoptosis by its nuclear translocation. last year, we also reported specific induction of pag in the internal capsule of l-dopatreated and constitutive expression in the smi- -immunopositive motor neurons in the pontine nucleus and motor nuclei of trigeminal nerve and facial nerve. in the present study, we examined distribution of pag in the spinal cords of l-dopa-injected parkinsonian rats by immunohistochemistry. l-dopa treatment showed inducing tendency of pag expression on the motor neurons in the anterior corn and lateral corticospinal tract of spinal cords. the expression of pag in the motor nuclei of cranial nerves and its induction in the spinal cords suggests its possible involvement in motor dysfunction such as dyskinesia. ps p-j an approach to the generation of ar-jp mouse model: crossbreeding of pael-r transgenic mice with parkin knockout mice hua-qin wang , , yuzuru imai , haruhisa inoue , , ayane kataoka , sachiko iita , nobuyuki nukina , ryosuke takahashi , neurology, university of kyoto, kyoto, japan; bsi, riken, saitama, japan since loss of parkin e activity appears to be causal of ar-jp, accumulation of potentially toxic parkin substrates should result in degeneration of da neurons. however, parkin knockout mice show no different da neuronal loss even at old ages, presumably due to relative short lifespan of mice. pael-r is one of the best characterized parkin substrates. we generated pael-r transgenic mice and crossbred it with parkin knockout mice. pael-r transgenic mice showed modest alterations in dopamine metabolism and behavioral deficits without displaying obvious dopaminergic neuronal loss at the age of one year. however, when pael-r transgenic mice were crossbred with parkin knockout mice, the da neuronal loss was induced in a pael-r gene dosage-dependant manner. these results strongly support that pael-r accumulation substantially contributes to dopaminergic neurodegeneration in ar-jp. parkinson's disease, a common motor disorder, is caused by a degeneration of dopaminergic neurons in the substantia nigra. after dopamine denervation, an over-activity of glutamatergic pathways has been found and that is implicated in the neuropathology of parkinson's disease. previous study (lai et al., ) have found that application of an antisense oligodeoxynucleotide specific for nr have successfully knockdown the expression of nr gene expression in the striatum of -hydroxydopamine-lesioned rats. in the present study, modulation of gene expression of nr was re-addressed using a small interfering rna (sirna) specific for nr . in pc cells, reductions of nr proteins after a single application of nr sirna were found by western blot experiments. and after one single application of nr sirna in the striatum of the lesioned rats, a significant reduction in apomorphine-induced rotation was found. slight reductions in the levels of nr immunofluorescence were found in the striatum after the sirna treatments. lai et al., . neurochem. int. , - . research funds: faculty research grant, frg/ - /ii- , hong kong baptist university ps p-j homocysteine and parkinson's disease: effects of acute intranigral administration on dopaminergic system g. chandra, k.p. mohanakumar indian institute of chemical biology, kolkata, india homocysteine (hcy) is implicated in a number of geriatric multisystem disorders and patients with hyperhomocysteinemia exhibit profound neuropsychological abnormalities. parkinsonǐs disease (pd) patients receiving long-term l-dopa therapy are reported to have elevated plasma hcy levels. we studied whether hcy is neurotoxic to the nigrostriatal dopamine (da)-ergic system in sd rats. animals infused unilaterally in substantia nigra pers compacta (snpc) with hcy ( . - mol in l) showed dose dependent loss of da and its metabolites, in the ipsilateral striatum on th day. animals with mol hcy exhibited significant motor disabilities and spontaneous and da-ergic drug-induced turning behaviors. in these animals a clear loss of neurons was visible in snpc, which were shown to be daergic by tyrosine hydroxylase immunoreactivity. intra-raphe infusion of hcy did not alter the neurotransmitter levels in the serotonergic perikarya or terminals. these results indicate the toxic potential of hcy to the da-ergic system and suggest that chronic l-dopa therapy in pd patients may further deteriorate the disease. ps p-j ubiquitin proteasome system was impaired by the aggregate formation of mutant ␥pkc found in sca takahiro seki , takayuki shimahara , naoko adachi , naoaki saito , norio sakai dept. mol. pharmacol. neurosci., grad. sch. biomed. sci., hiroshima univ., hiroshima, japan; lab. mol. pharmacol., biosig. res. ctr., kobe univ., kobe, japan we have previously demonstrated that several mutant protein kinase c gamma (␥pkc), found in several families of spinocerebellar ataxia type (sca ), are susceptible to cytoplasmic aggregation and cause cell death in cho cells, indicating that this property is involved in the etiology of sca . however, the relationship between the aggregate formation of mutant ␥pkc and cell death remains unclear. accumulating evidences indicate that the impairment of the ubiquitin proteasome system (ups) is related to the pathogenesis of many neurodegenerative disorders. therefore, we examined whether the aggregate formation of mutant ␥pkc affects ups function. the immunoreactivities for ubiquitin and proteasome were intensely accumulated in the aggregates of mutant ␥pkc. decreased proteasome activities were also observed in cells having aggregated mutant ␥pkc. these results indicate that the aggregation of mutant ␥pkc exert cytotoxic effect via the impairment of ups. it is well known that oxidant stress is involved in many pathologic conditions including brain ischemia and neurodegenerative diseases. recently, however, another type of stress, endoplasmic reticulum (er) stress has also been reported to be associated with such diseases. er stress is characterized by accumulation of unfolded proteins in the er that is caused by inhibition of protein modification, disturbance of ca + homeostasis or oxygen deprivation. we recently reported that targeting disruption of herp, a novel er stress-related gene, caused f cells vulnerable to er stress. using these cells, we developed a screening system for molecules that suppress er stress. approximately compounds have been screened, and we found some molecules that protect human neuroblastoma cells against er stress and oxidative stress. we speculate that this system could provide novel therapeutic targets to the er stress and oxidative stressrelated diseases. toshiyuki araki , yo sasaki department of peripheral nervous system research, national institute of neuroscience, ncnp, tokyo, japan; washington university school of medicine, st. louis, missouri, usa axonal degeneration which is observed in a variety of neuropathological conditions or physical damage to axons is a self-destructive program that is independent from programmed cell death. we previously reported that increased nicotinamide adenine dinucleotide (nad) production by the overexpression of nicotinamide mononucleotide adenylyltransferase (nmnat ) or exogenously applied nad can protect neurites from degeneration caused by mechanical or neurotoxic injury of neuronal cells. the mammalian nad biosynthesis is mediated by at least different kinds of enzymes and each enzyme converts different substrate to nad or its precursors. here we investigated whether overexpression of these enzymes or exogenous application of nad precursors protects neurites from degeneration through increased supply of nad. cocaine is considered to affect spine morphology and the composition of postsynaptic density (psd) of medium spiny neurons in nucleus accumbens (nac). we examined the accumulation of several proteins altered by cocaine challenge after withdrawal of repeated cocaine administration in psd fraction of rat nac at different time points. total psd protein yield was decreased at min, but next increased at h and returned to basal at h after cocaine challenge. actin showed a similar pattern but was maintained at high level at h. both psd- and glur were increased between h and h like actin. by contrast, some proteins such as drebrin were decreased after the peak at h. interestingly, the s proteasome subunit demonstrated a dramatic upregulation at h. these data suggest that the composition of psd proteins is regulated by proteasome activity as well as actin cycling. it is possible that some proteins may be removed from psd by proteasome following transient requirement for organizing psd in the nac of chronic cocaine-administrated animals. ps p-k effects of mdma and -meo-dipt on serotonin transporter and dopamine transporter yosuke yamauchi, takaya izumi, takayuki nakagawa, shuji kaneko dept. mol. pharmacol., grad. sch. pharm. sci., kyoto univ., kyoto, japan by two electrode voltage-clamp recordings from xenopus oocytes heterologously expressing serotonin transporter (sert) or dopamine transporter (dat), the effects of two addictive agents, , methylenedioxymethamphetamine (mdma) and -methoxy-n,ndiisopropyltryptamine ( -meo-dipt), on sert and dat were examined. as previously reported, mdma ( . - m) dose-dependently induced transport-associated, inward current response in the sertexpressing cells. interestingly, mdma-induced current response was also observed in dat-expressing cells. on the other hand, -meo-dipt ( . - nm) evoked an outward current response in sertexpressing cells similarly to that of selective -ht reuptake inhibitors. no current response was observed when -meo-dipt was applied to dat-expressing cells. these results suggest that mdma is transported not only by sert but also by dat, and that -meo-dipt suppresses the spontaneous transport activity of sert. junichi kitanaka , nobue kitanaka , tomohiro tatsuta , , yoshio morita , motohiko takemura department of pharmacology, hyogo college of medicine, nishinomiya, japan; department of neuropsychiatry, hyogo college of medicine, nishinomiya, japan we examined the effects of pretreatment with clorgyline on morphine-induced behavioral changes and antinociception. a single administration of morphine ( mg/kg, i.p.) to male icr mice induced a hyperlocomotion. the anova analysis revealed statistical significance of a morphine effect (hyperlocomotion) and of a clorgyline pretreatment x morphine interaction effect (inhibition), but not of an effect of clorgyline pretreatment. clorgyline pretreatment itself did not affect the spontaneous locomotion. clorgyline at a dose of . mg/kg but not other doses tested significantly potentiated morphine-induced antinociception evaluated by tail flick but not hot plate test. clorgyline at the doses of and mg/kg significantly inhibited dopamine and serotonin metabolism. these results suggest that clorgyline showed its inhibitory effect on morphine-induced hyperlocomotion, but not antinociception, through mao inhibition. recent studies in our laboratory have shown that methamphetamine (meth)-induced hyperlocomotion and behavioral sensitization in mice were inhibited by clorgyline, an irreversible monoamine oxidase inhibitor. in this presentation, the effect of clorgyline pretreatment on meth reward was assessed by conditioned place preference (cpp) paradigm, using an apparatus developed with supermex ® sensors. although intact male icr mice showed a significant cpp for meth ( . mg/kg, i.p.), pretreatment with subchronic clorgyline ( . - mg/kg, s.c.) did not affect the magnitude of cpp. pretreatment with clorgyline significantly decreased apparent dopamine and serotonin turnovers in the striatum in a dose-dependent manner. these results indicated that clorgyline pretreatment did not influence meth reward in mice. of lobeline pretreatment on methamphetamine-induced stereotypy and monoamine metabolism in mice motohiko takemura , nobue kitanaka , tomohiro tatsuta , , yoshio morita , junichi kitanaka department of pharmacology, hyogo college of medicine, nishinomiya, japan; department of neuropsychiatry, hyogo college of medicine, nishinomiya, japan the effects of lobeline, an alkaloid constituent of indian tobacco, on methamphetamine (meth)-induced stereotypy and monoamine metabolism were investigated in male icr mice. pretreatment with lobeline ( . - mg/kg, i.p.) min prior to drug challenge significantly decreased an intensity of stereotypies and increased its latency to onset in a dose-dependent manner. in saline challenge groups, doses of lobeline examined did not affect the spontaneous locomotion nor induce any stereotyped behaviors. the range of lobeline doses examined except mg/kg did not affect apparent monoamine turnovers in the brain regions including striatum min after drug challenge. these results suggested that the inhibitory effect of lobeline ( . - mg/kg) on meth-induced stereotypy did not attribute to the change in the brain monoamine metabolism. kazuto sakoori, niall murphy riken bsi, wako-shi, japan previously we showed that endogenous nociceptin suppresses drug reward. here, we examined the effect of blockade of nop receptors on methamphetamine (meth) induced behavioral sensitization in order to understand the role of endogenous nociceptin in the chronic response to addictive drugs. first, nop receptor ko and wt mice were treated with mg/kg meth and locomotor activity measured daily for days. wt mice showed gradually increasing sensitivity to meth with repeated treatment of meth, whereas nop receptor knockout mice did not. next, nmol ufp- (a nop receptor antagonist) and mg/kg meth were co-administrated to mice and locomotor activity measured daily for eight days. ufp- strongly suppressed locomotor activity. thus, it was unclear if ufp- suppressed behavioral sensitization to meth during chronic drug treatment. however, when challenged with meth after four or more days without treatment, ufp- co-administrated mice showed a lower locomotor response. these results suggest that endogenous nociceptin facilitates the plastic changes induced by chronic treatment with addictive drugs. the influence of olanzapine (a d dopamine receptor antagonist) on the morphine-induced conditioned place preference (cpp) in male and female mice was investigated in the present study. subcutaneous (s.c.) injection of morphine ( - mg/kg, three drug sessions) induced place preference both in male and female mice. intraperitoneal (i.p.) administration of olanzapine ( . - mg/kg) induced place aversion (cpa) in female mice but not in male mice. administration of olanzapine ( , . and mg/kg, i.p.) reduced both the acquisition and expression of morphine-induced cpp in male and female mice. however, olanzapine ( mg/kg, i.p.) caused more than % mortality in female but not male mice. the effects of olanzapine were reversed by l-arginine ( mg/kg, i.p.) pre-administration. in conclusion, it seems that olanzapine reduced morphine effects in part via a nitric oxide (no) mechanism. feed-forward associative learning (ffal) theory of cerebellar motor learning proposed by the author presumes that higher motor centers have place-coding systems and the same systems are shared by the cerebellum. when a new motor learning proceeds with respect to a certain movement, previous learning results of the movement will turn out to be modified or erased. ffal theory presumes that transferred memory from the cerebellar cortex to nucleus will serve as the maintenance of the previous learning. from this line, many aspects of saccadic adaptation are successfully demonstrated by computer simulation based on the theory. another theoretical issue is the credit assignment problem of motor error. a motor error is generally an integrated result of maladjusted multiple learning elements, and is to be decomposed to each element credit. this problem naively leads to an idea of a dual redundant system for movement, one for execution and the other for error decomposition. ffal theory naturally and simply resolves the credit assignment problem and demonstrates a computer simulation of motor learning of multi joint movement system, using the place-coding hypothesis. ps p-d regulation of camp responsive element binding protein to stress in rat amygdala and hippocampal formation the department of anatomy and histology, shanghai medical school, fudan university, china amygdala (am) and hippocampal formation (hf) are important structures relating with emotional learning and memory. transcription factor, camp-responsive element binding protein (creb) in am and hf plays important roles in memory modulating processes. creb is a nuclear protein and is wldely accepted as prototypical stimulusinducible transcription factor. creb is activated in response to a vast array of physiological stimuli and then becames phosphorylated creb (pcreb). neurophysiological and neuropharmacological studies said that creb may regulate gene transcription and protein synthesis to maintain the long term and sustaining changing of synaptic efficiency during the long-term process of synaptic plasticity. but we cannot tell exactly via what kind of neurons in am and hf creb regulate these processes. we used the animal model, forced swimming (fs) as emotional stimuli and the experiment methods such as, immunocytochemistry, western-blotting with anti-pcreb antibody. the distributing profiles and changing rules of pcreb immunoreactive nuclei in amygdala and hippocampal formation of both control and experiment groups were investigated. the neuronal types of pcreb immunoreactive nuclei were analyzed by double-labelling immunocytochemistry with anti-pcreb, anti-glu and anti-pv antibodies. the results were: ( ) the number of pcreb immunoreactive neuclei and total amount of pcreb in the subnuclei of rat amygdala, dentate gyrus (dg) and cornu ammonis (ca ) were increased after fs. the rule of this kind of changing was of region-and time-specific. ( ) pcreb immunoreactive neuclei were expressed in glutamate immunoreactive neurons and were devoid in interneurons. these results suggested that pcreb in limbic system regulated the fs process and the regulation was finished via exciting neurons, glutamate neurons. hideto takahashi , , tomoaki shirao dept of neurobiol & behav.; ercgsm, gunma univ. grad. sch. of med., maebashi, japan dendritic spines are developmentally-regulated and activitydependent polymorphic structures based on actin cytoskeleton. drebrin is a spine-rich actin-binding protein regulating spine morphogenesis during development. here we find that chronic blockade of ampa receptors (ampar) inhibits synaptic drebrin clustering during development of hippocampal neurons, but not that of nmdar. further, the analysis of fluorescence recovery after photobleaching for egfp-drebrin a reveals that only . ± . % of drebrin in the spine is stable, with a turnover time of . ± . min. blockade of ampar by m cnqx reduces the population of stable drebrin ( . ± . %), and has no effect on a turnover time. on the other hand, blockade of nmdar by m ap has no effect on the population of stable drebrin, whereas shortens a turnover time ( . ± . min). these data suggest that ampar activities increase the binding capacity of drebrin in spines, and therefore promote drebrin clustering at spine synapses. instead, nmdar activities regulate spine-shaft shuttling of drebrin. itsuko nihonmatsu , yoshito saitoh , kaoru inokuchi , mitsubishi kagaku inst. life sci. (mitils), tokyo, japan; crest, jst, tokyo, japan dendritic protein synthesis requires dendritic localization of mrnas in neurons. however, ultrastructural localization of these mrnas have not been well described. here we employed in situ electronmicroscopic technique to examine the precise localization of ␣camkii mrna in dendrites. ␣camkii mrna was located at the specific sites of dendritic shafts of pyramidal neurons, close to the spines, rather than in a diffused manner. we observed an increase in the ␣camkii mrna signals at the synaptic layer undergone l-ltp in the hippocampal dentate gyrus in unanesthetized freely moving rats. the increase was transient and returned to the basal level at h. the alteration in the ␣ camkii mrna localization in dendrites may reflect a functional change in the translational apparatus along with synaptic plasticity. reiko okubo-suzuki , , daisuke okada , kaoru inokuchi , , mitsubishi kagaku inst. life sci. (mitils), tokyo, japan; yokohama natl. univ. environment information sci., kanagawa, japan; crest, jst, japan late-phase long-term potentiation (l-ltp) depends on de novo protein synthesis. synaptopodin (synpo), an f-actin-associated protein, increases in the activated synapses following l-ltp induction. spine volume and f-actin content in the spines also increase during l-ltp. to reveal the roles synpo plays in the regulation of spine volume and f-actin content, we examined synpo-egfp (se) localization and spine volume in the hippocampal neurons using time-lapse confocal imaging techniques. se-overexpression did not alter spine volume, but the amount of se in spines positively correlated with the spine volume. pharmacological activation of the nmda receptors increased both spine volume and synpo content in spines. furthermore, experiments with ptk cells indicated that synpo stabilizes f-actin. these results suggest that synpo synthesized in soma and transported into the activated spines following l-ltp induction stabilizes spine f-actin that may lead to the maintenance of increased spine volume. mineo matsumoto , mitsutoshi setou , , kaoru inokuchi laboratory for molecular gerontology, mitils, japan; laboratory for nano-structure physiology, nips, japan subcellular localization of rna is an efficient way to localize proteins to a specific region of a cell. a requirement for dendritic rna localization and subsequent local translation has been demonstrated in several forms of experience-dependent synaptic plasticity. in spite of several attempts to identify these rnas, the population of rna species present in dendrites as a whole has not been well described. here we show the results of microarray analyses with rnas isolated from rna granule or synaptosome fractions prepared from the rat brain. these analyses revealed the complex nature of the dendritic rna population, which included rnas that were not expected to be in the dendrites. neural activity caused by an electroconvulsive shock triggered a redistribution of the dendritic transcriptome towards the synaptosome, a translationally active region. our results suggest that the redistribution of dendritic rnas is one of the mechanisms regulating local translation in response to synaptic inputs. ps a-a an activity that traps vesl- s protein into spines serves as synaptic tag synaptic tagging hypothesis explains how new proteins reach the activated synapses to establish input-specific late-phase plasticity, but it has not yet been substantiated. original idea of synaptic tagging is supposed to regulate protein entry into synaptic region including spines. using live-imaging techniques, we measured entry of vesl- s-egfp into spines (ve trapping) of rat hippocampal neurons in culture, and found that ve trapping activity serves as the synaptic tag in many criteria. ve trapping required synergistic activation of postsynaptic no-pkg pathway and an activity abolished by ttx at m, but not nm. because nm ttx is supposed to suppress na channels only postsynaptically, we concluded that ve trapping is a hebbian-like process that requires both pre-and postsynaptic activities. however, their coincidence time window was far wider (hrs) than that of early-phase plasticity, suggesting a requirement of persistently synchronized, rather than transiently coincident, activities, and a possibility of metaplastic states for late-phase plasticity. ps a-a acute effects of dehydroepiandrosterone sulfate (dheas) on the synaptic transmission and plasticity in rat hippocampal slices yuxia xu , ling chen , masahiro sokabe , , dept. physiol., nagoya univ., grad. sch. med., nagoya, japan; dept. physiol., nanjing med. univ., nanjing, china; sorst cell mechanosening, jst, nagoya, japan; dept. mol. physiol., nips, okazaki, japan the neurosteroid dehydroepiandrosterone sulfate (dheas) is known to improve memory and learning in mammals. recently we report that chronic administration of dheas facilitates the induction of ltp in the rat hippocampus. to elucidate the underlying synaptic mechanism of the dheas effects, we examined in this study the acute effects of dheas on the synaptic transmission and plasticity at the ca region in rat hippocampal slices. an application of . dheas for min to the slice augmented instantly the epsp, which was terminated within min. however, even h after the drug application, a subthreshold tetanus could induce ltp without alteration of ppf. this facilitating effect of dheas on ltp induction was blocked by a coapplication of a nmda receptor antagonist with dheas for min, suggesting that the dheas effect involves a sustained modulation of the postsynaptic signaling mediated by nmda receptor. xiaoniu dai , ling chen , masahiro sokabe , , dept. physiol., nagoya univ., grad. sch. med., nagoya, japan; dept. physiol., nanjing med. univ., nanjing, china; sorst cell mechanosensing, jst, nagoya, japan; dept. mol. physiol., nips, okazaki, japan to know whether -estradiol (e ) can protect ca neurons from functional deficit due to ischemia, adult male wistar rats were subjected four-vessel occlusion ( vo) for min, and the effect of e against this ischemic injury was examined. the electrophysiological properties of ca -ca synapses were examined by a real-time optical recording method days after ischemia. the ischemic brain showed a decreased synaptic transmission and an impairment of ltp induction but no alteration in paired-pulse facilitation. administration of e ( mg/kg) h before vo was able to protect ca neurons from these ischemic synaptic dysfunctions. the estrogen receptor-␣ selective agonist ppt ( mg/kg) produced a similar protective effect, but the estrogen receptor- agonist dpn ( mg/kg) did not. above results suggest that e can protect neurons not only from cell death but also from functional damages caused by cerebral ischemia. ps a-a non-genomic rapid effects of estradiol on hippocampal synapses: multi-electrode dish analysis kohei nakajima , mari ogiue-ikeda , yuki oishi , suguru kawato , department of biophysics and life sciences, graduate school of arts and sciences, university of tokyo at komaba, tokyo, japan; department of physics, university of tokyo, tokyo, japan estradiol has a non-genomic, rapid effect on synaptic transmission, which is manifested within seconds to minutes. recently, hippocampal neurons were shown to synthesize estradiol de novo, and to express estrogen receptor ␣ (er␣) at synapses. although these results imply that estradiol rapidly modulates synaptic plasticity through synaptic er␣, there are few electrophysiological evidence about it. here we investigated effects of estradiol on ltd by using wild type, er␣ hetero and er hetero mouse hippocampal slices with a multi-electrode dish (med, panasonic). med enabled us to measure epsps in ca , ca , and dentate gyrus simultaneously. hippocampal slices were perfused with estradiol before nmda-induced ltd. we found that estradiol enhanced ltd both in wild type and er hetero mouse, but not in er␣ hetero mouse. our data suggested non-genomic rapid action of estradiol through synaptic er␣. withdrawn ps a-a morphological changes of dendritic spines mediated by glucocorticoid receptor (gr) in rat hippocampus yoshimasa komatsuzaki , gen murakami , , tetsuya kimoto , , suguru kawato , college of humanities and sciences, nihon university, tokyo, japan; department of biophysics and life sciences, university of tokyo, tokyo, japan; crest, jst, japan modulation of hippocampal synaptic plasticity by glucocorticoids has been attracting much attention, due to its importance in stress responses. dendritic spines are essential for memory storage processes. here we investigated the effect of dexamethasone (dex), a specific agonist of glucocorticoid receptor (gr), on density and morphology of dendritic spines in adult male rat hippocampus by imaging of lucifer yellow-injected spines in slices. the application of nm dex induced rapid modulation of the density and morphology of dendritic spines in ca pyramidal neurons within h. the total spine density increased from . spines/m to . spines/m. dex significantly increased the density of thin and mushroom type spines, however only a slight increase was observed for stubby and filopodium type spines. because the presence of m cycloheximide, an inhibitor of protein synthesis, did not suppress the dex effect, these responses are probably non-genomic. hideki tamura , yuji ikegaya , sadao shiosaka division of structural cell biology, naist, nara, japan; laboratory of chemical pharmacology, university of tokyo, tokyo, japan the capacity of activity-dependent synaptic modification is essential in processing and storing information, yet little is known about how synaptic plasticity alters the input-output (i-o) conversion efficiency at the synapses. in the adult mouse hippocampus in vivo, we carefully compared the i-o relationship, in terms of presynaptic activity levels versus postsynaptic potentials, before and after the induction of synaptic plasticity and found that synaptic plasticity led synapses to respond more robustly to inputs, that is, synaptic gain was increased as a function of synaptic activity with an expansive, power-law nonlinearity, i.e., conforming to the so-called gamma curve. in extreme cases, long-term potentiation (ltp) and depression (ltd) coexist in the same synaptic pathway with ltp dominating over ltd at higher levels of presynaptic activity. these findings predict a novel function of synaptic plasticity, i.e., a contrast-enhancing filtering of neural information through a gamma correction-like process. research funds: st century coe research ps a-a actin organizations within single dendritic spines in ca pyramidal neurons studies with two-photon photoactivation naoki honkura, masanori matsuzaki, haruo kasai center for disease biology and integrative medicine, faculty of medicine, the university of tokyo, japan the major cytoskeleton of dendritic spines is filamentous actin (factin). we have here investigated sub-spine actin organizations using two-photon photoactivation of pa-gfp fused with -actin in rat ca pyramidal neurons. we found segregated and discontinuous organizations of two pools of f-actin, dynamic and stable pools, which turned over with time constants of . min and min, respectively. fractions of the stable f-actin pool were greater in larger spines, therefore, the entire f-actin pool was more stable in larger spines. we succeeded in visualizing a retrograde flow of f-actin in the dynamic pool from the apex to the base of spine, and found that both the speeds ( . - . um/min) and lengths ( . - . um) of the f-actin flow were greater in spines with larger head volumes. moreover, spine heads rapidly shrank when actin polymerization was blocked by latrunculin a, suggesting that the rate of actin polymerization in each spine actively and continuously determines the volume of spine head via the length of f-actin. tomoharu nakamori , katsushige sato , kohichi tanaka , hiroko hamazaki mol. neurosci., tmdu, tokyo, japan; physiology, tmdu, tokyo, japan the visual wulst (vw) in the thalamofugal pathway in chicks is known to have a critical role in the visual learning. to understand the function of the vw in the learning process of imprinting, we investigated the neuronal activity of vw region in chick brain. the slice stained with a voltage-sensitive dye was prepared for a multiple-site optical recording. when chicks were reared in quasi-dark condition, the extent and amplitude of response induced by electrical stimulation were different between at or days post-hatching (p or p ), and at p . this corresponds to behavioral data showing that chicks have high ability of visual learning in imprinting behavior until p , but they lose this ability at p . in addition, the light-exposed chick showed larger optical response than the dark-rearing one. the optical response in the vw was partly inhibited by the glutamate-and gaba-receptor antagonists. these results suggest that the glutamatergic as well as gabaergic neurons are active in the area including vw and that the neuronal activity of vw affects the learning ability for imprinting. withdrawn ps a-b effect of estrogen on hippocampus in male and female mice takanori sugawara , shinji hayashi , victoria luine graduated school of integrated sience, yokohama city university, yokohama, japan; department of psychology, hunter college, city university of new york, new york, usa we examined structural difference in the hippocampal neurons with golgi stain among the male, the female and the female treated with estrogen neonatally. the mice were gonadectomized and received -estradiol (e ) or oil-vehicle injections at adult before golgi impregnation. spine densities m of apical dendrites of the pyramidal neurons in the hippocampus ca region were calculated with categorization into three shapes, i.e., mushroom type with large head, thin type and filopodia-like type. as a result, only in the female not estrogen treated neonatally, the mushroom type and total spine densities were increased but the thin type spine density was decreased by e treatment in adult. the present results indicate that estrogen given at adult induces an enlargement of spine to mushroom type and generates new spines only in the female mice not treated with estrogen neonatally. thus, dendritic spine formation seems sexually dimorphic and depends on the sex steroid environment during the neonatal period. jun-ichi goto , , takafumi inoue , , akinori kuruma , katsuhiko mikoshiba , , lab. developmental neurobiology, brain science inst., riken, saitama, japan; div. molecular neurobiology, inst. medical science, univ. tokyo, tokyo, japan; calcium oscillation project, icorp-sorst, jst, tokyo, japan changes in synaptic efficacy at the parallel fiber (pf)-purkinje cell (pc) synapse are postulated to be a cellular basis for motor learning. although long-term efficacy changes lasting more than an hour at this synapse, i.e., long-term potentiation and depression, have been extensively studied, relatively short lasting synaptic efficacy changes, namely short-term potentiation (stp) lasting for tens of minutes, have not been discussed to date. here we report that this synapse shows an apparent stp reliably by a periodic burst pattern of homo synaptic stimulation. this stp is presynaptically expressed, since it accompanies with a reduced paired-pulse facilitation and is resistant to postsynaptic ca + reduction by bapta injection or in p/q-type ca channel knockout cerebella. this novel type of synaptic plasticity at the pf-pc synapse would be a clue for understanding the presynaptic mechanisms of plasticity at this synapse. aya ishida, wataru kakegawa, michisuke yuzaki department of physiology, keio university, tokyo, japan mitogen-activated protein kinase (mapk) cascade is thought to be essential for the synaptic plasticity and learning. in the hippocampus, three different mapk subfamilies, including extracellular signalregulated kinase (erk), p mapk and c-jun nh -terminal protein kinase (jnk), have been shown to selectively regulate different forms of synaptic plasticity -long-term potentiation (ltp), longterm depression (ltd), and depotentiation after ltp, respectively. although erk was previously shown to play a role in cerebellar ltd in cultured purkinje cells, the role of mapks has not been systemically studied. here, we examined the effect of specific inhibitors of three different mapks on ltd by patch-clamp recordings from cerebellar slices. we found that u , a specific inhibitor for erk activation, significantly inhibited ltd induction, whereas sb and sp , antagonists for p mapk and jnk, respectively, had no effect. therefore, unlike hippocampal ltd, cerebellar ltd was dependent on erk, suggesting involvement of different intracellular downstream pathways. ps a-b regulation of ampa receptor trafficking by aaa atpases in cerebellar purkinje cells: are nsf and vcp playing complementary or antagonistic roles? thomas launey , chou-chi li , yumiko motoyama , junko yamaoka , masao ito riken brain sci. inst., japan; national cancer institute, nih, ma, usa the number of postsynaptic ampa receptors (ampar) is regulated by interactions with multiple protein complexes, throughout its synthesis, maturation, transport, synaptic insertion and degradation. aaa atpases influence several of these stages, the most extensively studied being nsf's contribution to ampar trafficking. in cerebellar purkinje cell (pc), we show that valosin containing protein (vcp), an atpase with high homology to nsf, is bound to ampa receptors in pc's dendritic compartment. following glur co-ip from molecular layer, vcp was detected by ms/ms and by monoclonal anti-vcp. pull-down assay showed a direct interaction between vcp and glur c-term domain, requiring vcp n-term domain and both the nsf and pdz binding domains of glur . glur phospho-ser promotes vcp complex dissociation, suggesting a relation with synaptic plasticity. further, pep m-related peptides, thought to interfere specifically with nsf-regulated ampar trafficking, also blocked the glur -vcp interaction. yuichi kitagawa , , shin-ya kawaguchi , , tomoo hirano , dept. biophys., grad. sch. sci., kyoto univ., kyoto, japan; crest, jst, kawaguchi, japan at inhibitory synapses on a cerebellar purkinje neuron (pn), postsynaptic depolarization induces long-lasting potentiation of the gaba a receptor (gaba a r) responsiveness (rebound potentiation: rp). previous studies have clarified the molecular mechanism regulating rp induction. whether rp is induced or not is determined by the balance of activities of protein kinases (camkii and pka) and phosphatases (pp- and calcineurin). to understand the complex behavior of biochemical reactions systematically, a kinetic simulation model to analyze the behaviors of signaling network was developed. computer simulation reproduced the bistable states of gaba a r phospholyration according to stimulation patterns, which apparently corresponded to whether rp was induced or not. we further studied the systematic property of the molecular network, and obtained several experimental predictions. these possibilities were evaluated by experiments such as immunocytochemistry using cultured pns. ps a-b long-term depression of synaptic transmission in a songbird motor nucleus essential for song learning yuki haruta, yachun huang, neal hessler vocal behavior mechanisms riken brain science institute, japan in order to fully understand the neural basis of song learning, it is critical to characterize forms of synaptic plasticity that could be involved in this process. we previously reported that, in synapses of the song motor nucleus ra, participation of postsynaptic nmda receptor nr b subunits and presynaptic transmitter release both decrease from young birds to adults. here, we tested whether synaptic function could be modified in a similar way by acute stimulation. after pairing slight postsynaptic depolarization with presynaptic stimulation, ltd was reliably induced at both hvc and lman inputs in juvenile birds from to days old. this depression required activation of postsynaptic nmda receptors, and was expressed by decreased transmitter release, which required activation of cannabinoid receptors. no ltd could be induced in normal birds over days old, when song learning is nearly complete, but ltd remained possible in birds over days old who had been isolated from song tutors, and thus retained the capacity for learning. ps a-b involvement of ca + -permeable ampar in the repetitive-ltp induced synaptic enhancement (rise) yukiko ueno, keiko tominaga-yoshino, akihiko ogura graduate school of frontier biosciences, university of osaka, osaka, japan we showed previously that exposures to glu of cultured rat hippocampal slices at h intervals produced a long-lasting enhancement in synaptic strength accompanied by synaptogenesis (rise). we examined here whether the conversion of ampar subunits occurred during the development of rise. immunochemical staining for ampar subunits, glur and glur , showed that the number of glur -positive puncta increased transiently after the repeated glu exposures, whereas the number of glur -positive puncta increased gradually and persistently. jstx (a ca + -permeable ampar blocker) suppressed fepsp amplitude recorded at ca -ca synapses by - % in the period corresponding to the transient increase of glur -positive puncta. this transient increase should represent the delivery of ca + -permeable (glur -lacking/glur -including) ampar to synaptic sites. furthermore, jstx application at that period blocked the rise production. these results suggest that the transient delivery of ca + -permeable ampar to synaptic sites is involved in the rise production. yoshihiro egashira, tsunehiro tanaka, yuji kamikubo, yo shinoda, keiko tominaga-yoshino, akihiko ogura osaka univ. grad. sch. frontier biosciences, toyonaka - , japan long-lasting synaptic plasticity, the cellular basis of long-term memory, is assumed to be associated with protein synthesis. using cultured rat hippocampal slices, we previously found that a long-lasting synaptic enhancement coupled with an increase in the number of synaptic structures was established after inductions of ltp, not after its single induction. this synaptic enhancement required protein synthesis for its establishment. we recently found an apparently mirror-image phenomenon; inductions of ltd led to a long-lasting synaptic decrement coupled with a decrease in the numbers of synaptic structures. to know whether this synaptic decrement also requires protein synthesis, we induced ltd times ( h intervals) by applications of dhpg (a type i mglur agonist), during or after which anisomycin (a protein translation blocker) was applied. we found that anisomycin did not block the induction of ltd but blocked the establishment of the long-lasting synaptic decrement. haruo mizutani, tetsuya hori, tomoyuki takahashi department of neurophysiology, graduate school of medicine university of tokyo, tokyo, japan bath-application of -ht ( m) attenuated the amplitude of evoked epscs and facilitated paired-pulse ratio without affecting the miniature epsc amplitude, suggesting that its site of action is presynaptic. the -ht b receptor agonist cp mimicked the presynaptic inhibitory effect of -ht. -ht b receptor antagonist nas- reversed the -ht inhibitory effect, indicating that the -ht induced inhibitory effect occurs by mediating -ht b receptors. the presynaptic inhibitory effect of -ht became weaker as animals matured. in whole-cell recordings from calyceal presynaptic terminals, -ht attenuated voltage-dependent calcium currents, but had no effect on potassium currents. this -ht effect was characterized with a marked desensitization, but sustained under the fast calcium chelating agents, bapta. these results suggest that -ht, upon activating -ht b receptors, inhibits presynaptic calcium channels thereby inhibiting transmitter release and induces receptor desensitization by calcium influx at the immature calyceal synapse. takako ohno-shosaku , masato ano , yuki hashimotodani , tadasato nagano , masanobu kano dept. impair. study, grad. sch. med. sci., kanazawa univ., kanazawa, japan; dept. neurophysiol., grad. sch. med., osaka univ., osaka, japan; dept. cell. neurosci., grad. sch. med., osaka univ., osaka, japan retrograde endocannabinoid signal contributes to activitydependent modulation of synaptic transmissions in various brain regions. endocannabinoid release is triggered by depolarizationinduced elevation of intracellular calcium level or activation of gq-coupled receptors. here we report that nmda receptors can also contribute to generation of endocannabinoid signal. inhibitory postsynaptic currents (ipscs) were recorded in cultured hippocampal neurons prepared from newborn rats. application of nmda induced a transient suppression of cannabinoid-sensitive ipscs but not cannabinoid-insensitive ipscs. the nmda-induced suppression of ipsc was blocked by a cannabinoid receptor antagonist. these results indicate that activation of nmda receptors induces the endocannabinoid release, and suppresses the inhibitory synaptic transmission through activation of presynaptic cannabinoid receptors. the most caudal region of the rat spinal cord, the conus medullaris has a simple anatomical feature, which lacks ventral as well as dorsal root fibers and somatic motor neurons in the ventral horn. a small number of neurons distribute around the central canal, and some of them are nitric oxide synthase (nos) positive. a dense distribution of nerve fibers immunoreactive to cgrp, sp, and npy was found in dorsal part of the conus medullaris similarly to that of other spinal cord levels. in addition, enk-, -ht-, and th-immunoreactive varicose fibers were richly distributed throughout the sectional plane. to analyze this unique structure may provide valuable information on the basic neural cytoarchitecture and fiber connections of the spinal cord, particularly for the intraspinal circuitry. for this purpose, we made an electron microscopic study using nadph-diaphorase histochemistry combined with immunohistochemistry for neuronal markers. adenosine has been known to be a neuro-modulator in the nervous systems and four types of adenosine receptor are identified (a , a a, a b and a ). adenosine a and a receptors have been reported to inhibit high-threshold ca channel currents in neurons. to investigate the interaction between adenosine a and a receptors in rat striatum neurons in culture, l-type ca channel currents were recorded by whole-cell clamp method before and after administration of a agonist (cpa) and a agonist ( -cl-ib-meca). ca currents were decreased after administration of low concentration of cpa and -cl-ib-meca as reported previously. although ca currents were decreased by -cl-ib-meca in the presence of cpa, ca currents applied with cpa were not decreased on cells in the presence of -cl-ib-meca. at administration of cpa and -cl-ib-meca on cells simultaneously, ca currents were not decreased. these results suggested that adenosine a receptor may inhibit adenosine a receptor throughout a intracellular pathway in neurons. ps a-c influence of extracellular gaba and taurine to gaba a receptor-mediated actions in radially migrating cortical plate cells with identified by in utero electroporation t. furukawa , j. yamada , k. inoue , y. yanagawa , a. fukuda , dept. physiol., hamamatsu univ. sch. med., japan; dept. biol. info. process, grad. sch. elec. sci. & tech., shizuoka univ., hamamatsu, japan; dept. developmental and integrative neurosci., gunma univ. sch. med., gunma, japan it is well known that role of gaba a -r mediated actions is important for early cns development. the radially migrating cells may affected by the actions. gaba content in the brain of gad -gfp knock-in mouse decrease compared with the wild type mice. therefore, we investigate the influence of the circumferential gaba concentration to radially migrating cells. furthermore, as it was known that gaba a -r is affected by taurine, the influence of taurine to radially migrating cells was also investigated. there was no significant difference in distribution of radially migrating cells that was labeled by means of electroporation. evoked gaba a -r mediated currents of labeled cells had dose-dependent manner and had no differences among genotypes. therefore, we have examined the influence of circumferential taurine to gaba a -r mediate actions. takashi hayakawa , hiroyuki hioki , kouichi nakamura , , hisashi nakamura , takeshi kaneko , dept. morphol. brain sci., grad. sch. med., kyoto univ., kyoto, japan; crest, jst, kawaguchi, japan we previously reported that almost all vesicular glutamate transporter (vglut )-immunoreactive (ir) cells were also gabair in neocortex and choline acetyl transferase (chat)-ir in caudate-putamen in rat. although, in dorsal and median raphe nuclei, many vglut -positive cells showed immunoreactivity for -hydroxytryptamine ( ht), a significant proportion ( . %) of vglut -postive cells was ht-negative. in this study, triple immunofluorescence staining was performed for vglut , ht and one of the following proteins: neuronal nuclear antigen (neun), glial fibrillary acidic protein (gfap), glutamic acid decarboxylase (gad ) and tyrosine hydroxylase (th). our results showed that all of the vglut -positive/ ht-negative cells were immunoreactive for neun but not for gfap. furthermore, we found that these vglut positive/ ht-negative neurons didn't show any immunoreactivities for gad nor th, and thus it is indicated that there is a group of exclusively glutamatergic vglut -positive neurons in these nuclei. research funds: kakenhi , , ps a-c cortico-striatal and fast-spiking cell activity in the rat frontal cortex during cortical oscillations in vivo: modulation by serotonin m victoria puig , mika ushimaru , yoshiyuki kubota , akiya watakabe , tetsuo yamamori , yuchio yanagawa , yasuo kawaguchi div. cerebral circuitry, nips, okazaki, japan; div. brain biology, nibb, okazaki, japan; dept. genetic and behavioral neurosci., gunma univ. graduate school of med., japan we studied how cortico-striatal (cs) and fast-spiking (fs) cells are modulated by slow-wave-sleep (sws) oscillations and by serotonin ( -ht). cs and fs cells were recorded simultaneously with the electrocorticogram in the secondary motor area of anesthetized rats that expressed a gfp in gabaergic interneurons. fs displayed a highsuccess excitation to striatal stimulation, suggesting a control of cs over fs. during sws, both cs and fs fired during the up-states though with different patterns. the stimulation of the dorsal raphe promoted longer up-states. moreover, % of the cs were inhibited by -ht through -ht a r and % were excited through -ht a r. however, % of the fs cells were inhibited and % excited. these results show that cs cells are more inhibited by -ht than fs. the expression of -htr was confirmed by in situ hybridization. research funds: jsps pe and ryohei tomioka, kathleen rockland laboratory for cortical organization and systematics, riken brain science institute, saitama, japan in small mammals, gabaergic neurons have been shown to contribute to ipsi-and contralateral cortical projections. here, we report in monkey as well that some gabaergic neurons send long-distance projections. identification was partly based on golgi-like labeling of the dendritic tree, achieved by injecting adenovirus as a retrograde tracer in areas v , teo, or tep. aspiny or sparsely spinous nonpyramidal neurons were clearly visualized in the white matter or, less frequently, in cortical gray matter, in mainly layer but also in layer and/or . in each of the cases, about - gabaergiclike neurons were scored, with a preferential location anterior to the injection sites. in addition to their characteristic dendritic morphology, the neurons were identified as positive for gabaergic neuronal markers; namely, gad , somatostatin, or nos. thus, we conclude that gabaergic projection neurons are phylogenetically conserved; but more work is needed to determine ( ) their other features, ( ) possible species variability, ( ) their functional significance. supported by riken bsi. withdrawn ps a-c regional, cell type, and layer-specific differences in cholinergic modulation of neocortical neurons allan gulledge , , susanna b. park , greg j. stuart , yasuo kawaguchi national institute for physiological sciences, japan; div. neurosci., jcsmr, australian national university, canberra, australia we examined cholinergic modulation of pyramidal and nonpyramidal neurons in neocortical areas (prefrontal, somatosensory, and visual cortex). transient ach exposure ( m) inhibited layer pyramidal neurons in all areas via activation of an sk-type potassium conductance. pyramidal neurons in layers / were generally less responsive to ach, but ach inhibited layer cells in visual cortex. prefrontal layer pyramidal neurons were more responsive to ach than were layer cells in other areas of cortex. fast spiking (fs) nonpyramidal neurons were completely non-responsive to ach, even at very high concentrations ( mm). on the contrary, ach generated fast, nicotinic receptor-mediated responses in % of non-fs interneurons ( of cells). laminar or regional differences in ach responses were not observed in nonpyramidal neurons. these data suggest that ach may act to inhibit the output of cortical projection neurons while preserving information processing in superficial neurons. toshikazu kakizaki , , kenzi saito , , yuchio yanagawa , department of genetic and behavioral neuroscience, gunma university graduate school of medicine, maebashi, japan; sorst, jst, kawaguchi, japan; sokendai, hayama, japan a major inhibitory neurotransmitter gaba is synthesized by glutamate decarboxylase (gad), and is accumulated into synaptic vesicles by vesicular gaba transporter (vgat). another inhibitory neurotransmitter glycine could be transported into synaptic vesicles by vgat, and be co-released with gaba. several molecules related to gabaergic or glycinergic neurotransmission are expressed in nonneural tissues, suggesting that gabaergic and glycinergic systems exert their activities outside the cns. vgat-deficient mice die in the perinatal period, and display omphalocele, defect in ventral body wall closure, suggesting that gaba and/or glycine are involved in body wall formation. to further investigate whether gaba is essential for the ventral body wall formation or not, we have been examining how the body wall developed in the gad -deficient mouse fetus. ps a-c gaba mediated glutamate release from developing cerebellar cortex and ca sensitivity sachiko yoshida, miyuki ohshita, masakazu uematsu, shoichiro hirano, shinya tanaka, naohiro hozumi toyohashi university of technology, toyohashi, japan gaba (␥-amino butyric acid) and glutamate are known to play important roles as modulators in the survival and development of cerebellar neurons. during cerebellar development, gaba-mediated responses, gaba excitations, become depolarized inducing an increase in intracellular calcium concentrations, and are thought to have important trophic effects. many observations of gaba excitations using cultured cells have been reported, whereas few using acute slices. we recently reported the spatial nature of glutamate and gaba releases from acute slice with an enzyme-linked assay system and ccd imaging technology. in the present study, we evolved this measurement system to allow observations of spontaneous or gaba-mediated glutamate release from developing postnatal acute cerebellar slices. glutamate was released spontaneously, but gaba-mediated glutamate release appeared from postnatal to day in egl. its release, especially from premigratory zone, was inhibited by ni + , but cd + couldn't. we suggest that gaba excitation induces granule cell migration. ps a-c gabaergic fiber in the rat trigeminal motor nucleus reorganized following masseter nerve transection hiroyuki hayashi , hiroaki wake , junichi nabekura , osamu takahashi department of histology, kanagawa dental college, yokosuka, japan; national institute of physiological science, okazaki, japan it has been reported that gabaergic nerve terminals are seen in the trigeminal motor nucleus (vm) of the rat, and that there are primary afferent inputs from the muscle spindle of masticatory muscles to the vm cell bodies. we recently found that the number of these gabaergic fibers projecting to vm is markedly reduced in postnatal development. in this study, to elucidate the possibility that the re-arrangement of gabaergic circuits could be reproduced after neuronal injury, we examined the effect of axonal injury of the masseter axon on the gabaergic circuits in the vm. two to eight weeks after unilateral surgical transection of the masseter nerve of rats, gabalike immunoreactive (gaba-ir) varicosities were examined using immunofruorescence technique. the significant increase in number of gaba-ir varicosities were seen after eight weeks of the operation. this result suggest that gabaergic inputs may play one of important role for reorganization of afferent inputs in the vm. akiko arata , kunihiko obata , jonathan davies , mark bellingham , peter g. noakes lab. for memory & learning, riken-bsi, wako, japan; obata res. unit, riken-bsi, wako, japan; sch. biomed. sci., univ. queensland, queensland, , australia during embryonic development, approximately half of the motoneurons (mns) undergo programmed cell death. this process depends also on glycinergic and/or gabaergic synaptic activity, as suggested by increased mn number in gephyrin-deficient mice (banks et al., ) . we investigated the involvement of gaba alone in the mn death using gad -deficient mice, in which cerebral gaba is reduced to less than % of the wild-type. mn numbers at embryonic day (e) were counted by the method of banks et al. brainstemupper spinal cord blocks were prepared from e embryos and subjected to electrical recording from the c and c ventral roots and also gaba measurement. in gad -deficient embryos, increase in number of brachial mns ( %) and decrease in both spontaneous discharges in the c , c roots and gaba content (less than %) were observed, compared with those of the wild-type littermates. gaba might control cell death in developing network. abolghasem esmaeili, joe lynch, pankaj sah queensland brain institute, the university of queensland, australia the amygdala has key role in processing emotional information. distribution of gaba a receptor subunits is crucial for understanding physiology and pharmacology properties of these receptors in the amygdala. we examined the pharmacology of gaba a receptors by expressing different subunit combinations in hek cells and comparing the pharmacology with specific gabaergic inputs in the amygdala. dmcm blocked the actions of gaba at expressed ␣  ␥ and ␣  ␥ combinations ( % reduction) but had no effect at ␣  ␥ or ␣  ␥ . in slice recordings dmcm blocked ipscs by % in the lateral amygdala and had variable effects in the central amygdala. diazepam and zolpidem enhanced ipscs in the lateral whereas the response in the central amygdala was either reduction or enhancement. real time pcr and western blotting revealed differences in the distribution of gaba a receptor subunits between the lateral and central amygdala. we conclude that in the lateral amygdala all inputs have ␥ subunits whereas in the central amygdala some inputs contain ␥ while others contain ␥ subunits. masayuki kobayashi department of pharmacology, nihon university school of dentistry, tokyo, japan noradrenergic agonists have different effects on the excitatory neural transmission according to their subtypes in rat cerebral cortex. the present study aimed to explore what kind of second messengers and the precise site of synaptic membrane, pre-or postsynaptic, is involved in these noradrenergic modulation. the suppressive effect by activation of ␣ -adrenoceptors was mediated by protein kinase c, and excitatory effect by activation of -adrenoceptors was mediated by camp/protein kinase a cascade. phenylephrine suppressed inward currents evoked by puff application of glutamate, and it decreased mepsc amplitude and increased mipsc frequency. isoproterenol increased mepsc frequency and decreased mipsc amplitude. gaba-induced postsynaptic currents were suppressed by isoproterenol. these results suggest that phenylephrine may decrease postsynaptic currents through glutamate receptors and increase the release probability of gaba from presynaptic terminals. on the other hand, isoproterenol may facilitate glutamate release and suppress gaba a receptor-mediated postsynaptic currents. ps a-d hydrogen sulfide modulates synaptic transmission in rat hippocampal neurons mamiko tsugane , takashi iwai , yasuo nagai , junichiro oka , hideo kimura dept. mol. genetics, nat'l. inst. neurosci., ncnp, tokyo, japan; lab. pharmacol., fac. pharm. sci., tokyo univ. sci., chiba, japan hydrogen sulfide (h s), which is a well-known toxic gas and facilitates the induction of hippocampal long-term potentiation, has been proposed as a neuromodulator in the brain. the aim of this study is to understand the mechanism of regulation on synaptic transmission by h s. we examined the effect of h s on spontaneous excitatory postsynaptic currents (sepsc) as well as paired-pulse facilitations using both whole-cell and field potential recordings from rat hippocampal slices. sodium sulfide (na s), a donor of h s, reduced the amplitude of field excitatory postsynaptic potentials and increased the ratio of paired-pulse facilitation. the frequency and the amplitude of sepsc were initially reduced by na s then gradually increased, while the inward currents elicited by glutamate were not significantly suppressed by na s. these observations suggest that h s may modulate glutamatergic synaptic transmission by suppressing the release of a transmitter. several studies show that activation of locus coeruleus (lc) play an important role in the symptoms of opiate withdrawal. in this study the effects of lc inactivation on self-administration of morphine and on morphine withdrawal syndrome in rats has been investigated. male rats were anaesthetized and implanted with silastic catheters inserted in to the right jugular vein. after days animals were fitted and the external end of the catheter was connected with a syringedriven pump, then were placed in the self-administration apparatus. lc was inactivated by ( l) lidocaein ( %) min before training. animals were allowed to self administer morphine ( mg/kg per inf.) ten consecutive daily -h session. during all morphine self administration session lever pressing was measured. our results show that: ( ) lc inactivation produced a significant decrease in the initiation of morphine self administration during all session. after the last test session morphine withdrawal symptom signs (mws) precipitated by naloxone were measured. ( ) most of mws were decreased by lc inactivation in comparison with morphine group. these results suggest that extracellular atp plays a dual role in astrocytic ca + wave propagation with activation of distinct purinergic receptors in the hippocampus of the rats. the electrophysiological analysis of the rescue effect of  estradiol from glucocorticoid activity yuki oishi , suguru kawato department of physics, graduate school of science, university of tokyo, tokyo, japan; graduate school of arts and sciences, university of tokyo, tokyo, japan it is well known that stress reduces several activity of brain. especially, hippocampus is the largest target of stress. these phenomena are caused by glucocorticoids which are synthesized at adrenal when suffering stress. on the other hand,  estradiol is one of the neuro protective factors and rescues neural death caused by several neurotoxins, such as -amyloid, glutamate, glucocorticoids. in this study, we focused attention on the acute effects of steroid hormones and researched the effects of glucocorticoids and estradiol on rat hippocampal long term potentiation (ltp), which is the index of learning and memory. the results was that corticosterone (glucocorticoid of rat) acutely reduced ltp via glucocorticoid receptor.  estradiol rescued this reduction via estrogen receptor ␣ and . so we found that  estradiol affected not only neuro protection but synaptic protection from stress-induced suppression of synaptic transmission acutely. ps a-d the hypothalamic neuropeptide y neuron system of rats after long-term, high-dose dexamethasone treatment jinko konno, ayuka ina, sachine yoshida, hideki ohmomo, fumihiro shutoh, setsuji hisano lab. neuroendocrinol., graduate sch. comprehensive human sci., univ. tsukuba, ibaraki, japan effects of dexamethasone (dex) on hypothalamic neuropeptide y (npy) expression were evaluated with semi-quantitative in situ hybridization and immunohistochemistry. adult male wistar rats received an injection of dex ( . mg/ g b.w., sc) or sesame oil (vehicle control) everyday for - days. the two and intact rats (intact control) were decapitated, and the hypothalamus was dissected out, fixed and cut into paraffin sections. npy-immunoreactive axonal varicosities in the external zone of the median eminence were apparently more frequent in the dex-treated rat than in controls. npy hybridization signals in the arcuate nucleus were significantly higher in the treated-rat than in controls. no difference was found between both control animals. these results indicate stimulatory effects of dex on hypothalamic npy production and suggest enhanced npy influences on pituitary function. akiko shingo, idumi yamashita, shozo kito lab. of neuroscience, hyogo university, hyogo, japan we examined estrogen-like actions of isoflavones in the cerebral cortex and hippocampus on the basis of our previous data that estradiol induces igf- mrna expression, upregulates estrogen receptors and facilitates ere binding in these brain areas. materials are ovxed and non-ovxed rats. each group of rats were divided into the following groups. a: rats fed with phytoestrogen-free control diet, b: rats fed with diet with soy bean-derived estrogen and c: rats fed with control diet combined with chronic intraperitoneal injections of minimum dose of -estradiol. after feeding, rats were sacrificed to remove the cerebral cortex and hippocampus. expressions of mrnas of igf- , estrogen receptors ␣ and , and ere binding were analysed. as the results, it was revealed that isoflavones induced increased expression of mrnas of igf- and estrogen receptors in both ovxed and non-ovxed rats. difference between estrogen receptor ␣ and  in responses to isoflavones were analysed. isoflavones feeding increased ere binding as much as chronic injections of estrogen did in the ovxed rats. research funds: kampo science foundation, japan ps a-d mechanism of central metabolic control by tgf-beta in the rat brain: using the rat with depletion of hypothalamic noradrenaline teppei fujikawa, kazuo inoue, tohru fushiki division of food science and biotechnology, graduate school of agriculture, university of kyoto, kyoto, japan we have previously reported that activated transforming growth factor-beta (tgf-beta) increase in the rat brain during exercise. intracranial administration of tgf-beta induced an increase in fat oxidation, free fatty acid and keton body in the blood. these results suggest that activated tgf-beta in the rat brain participates in metabolic control of peripheral tissue by cns. it is, however, not known how tgf-beta increases in specifically fat oxidation. many investigations suggest that hypothalamus is essential for central metabolic control. in addition, some reports suggest that noradrenergic system in the hypothalamus may play important role for fat oxidation. in this study we measured concentration of extracellular noradrenaline (na) in the hypothalamus by using microdialysis after injection of tgf-beta. then, we measured respiratory exchange ratio and serum samples, after administration of tgf-beta in the rat with depletion of hypothalamic na by injection of -hydroxydopamine. ps a-d the effect of brain-derived neurotrophic factor (bdnf) on neuropeptide y (npy) neurons in the mouse corpus callosum: an examination using organotypic brain slice culture ryoichi yoshimura, kazuto ito, yasuhisa endo department of applied biology, faculty of textile science, kyoto institute of technology, japan the morphology of neuropeptide y (npy) neurons existing in the corpus callosum (cc) and the effects of brain-derived neurotrophic factor (bdnf) on the npy neurons were examined by using organotypic slice culture system. bdnf treatment significantly increased the number of the npy-immunopositive cell bodies and fibers in cc assessed with immunocytochemistry. electron microscopy demonstrated that the npy immunoreactivities were mainly localized in the regions associated with accumulating synaptic or cored vesicles in cc nerve fibers. the sectional area of npy-positive fibers was larger in the bdnf-treated culture than in the control culture. the number of nerve fibers adjacent to the npy-positive fibers was also larger in the bdnf-treated culture than the control. these results suggest that npy may play a key role in the neuronal regeneration, and bdnf takes part in the development of npy neuron fibers as well as the increase of the number of npy neurons in cc. reiji semba , kimi watanabe , munekazu komada institute for developmental research, aichi human service center, aichi, japan; graduate school of medicine, kyoto university, kyoto, japan d-serine is hypothesized to be a glia-derived neurotransmitter activating the nmda receptor because d-serine was reported to be formed and localized exclusively in astrocytes. however, we reported strong immunoreactivity of d-serine in some axons. to reveal which cells are producing d-serine in the brain, an in situ hybridization study of serine racemase, the enzyme producing d-serine from l-serine, was performed. using antibodies against neun, a neuronal marker, gfap, an astrocyte marker, and cnpase, an oligodendrocyte marker, type of the cells containing the mrna was examined. coincidentally with our immunohistochemical study of d-serine, strong signals for serine racemase mrna were found in some neurons while weak signals were found in astrocytes. present results suggest that d-serine will be a neurotransmitter activating the nmda receptors produced in a specific type of neurons. takatoshi hikida , , asif k mustafa , kenji hashimoto , kumiko fujii , , kazuhisa maeda , , hiroshi ujike , richard l. huganir , solomon h. snyder , akira sawa dept. of systems biology, obi, suita, japan; depts of neurosci. & psychiat, johns hopkins univ. med., baltimore, maryland, usa; chiba univ. forensic mental health, chiba, japan; dept. of psychiat, shiga univ. med. sci., shiga, japan; div. of neuropsychiat, tottori univ., yonago, japan; dept. of neuropsychiat, okayama univ., okayama, japan accumulating evidence from both genetic and clinical studies suggests a critical role of d-serine in schizophrenia (sz). we identified and characterized pick as a protein interactor of the d-serine synthesizing enzyme, serine racemase (sr). d-serine levels in the hippocampus and frontal cortex of pick knockout mice were significantly lower than those of their wildtype littermates at age of p , but not in adults, suggesting regulation of pick on sr at developing stage. in case-control association study, we observed an association of the pick gene with sz, which is more prominent in disorganized sz. our findings suggest that pick contributes to sr activity, d-serine production, and nmda neurotransmission in the pathophysiology of sz. ps a-d epileptiform activity is inhibited by taurine which can activate glycine and gaba a receptors in immature rat hippocampus akihito okabe , , werner kilb , ileana l. hanganu , taizhe qian , daiichiro nakahara , atsuo fukuda , heiko j. luhmann dept. of physiol., hamamatsu, japan; inst. of physiol., mainz, germany; dept. of psychol., hamamatsu, japan many studies indicate that the underlying mechanism of epileptic seizures differ between children and adults. the depolarizing gabaergic responses in immature neurons may contribute to higher epilepsy susceptibility. to investigate whether taurine, a neurotransmitter found in high concentrations in the immature cns, modulates epileptiform activity in immature hippocampus, we performed field-potential recordings in neonatal rat hippocampal ca region of an intact preparation. mm taurine blocked epileptiform activity induced by mg + free acsf and m -ap. this taurine effect was prevented by the glycinergic antagonist strychnine and the gaba a antagonist gabazine. inhibition of taurine uptake by ges also suppressed epileptiform activity in strychnine and gabazine sensitive manner. these results suggest that taurine mediates an inhibition in immature hippocampus via glycine and gaba a receptors that suppresses epileptiform activity. ps a-d responses of pge in undifferentiated and differentiated ng - cells kayoko matsushima , takashi imanishi , akinori kawaguchi , tetsuyuki wada , shigeru yoshida , seiji ichida school of pharm. sci., kinki univ., osaka, japan; school of pharm. sci., kinki univ., osaka, japan; school of pharm. sci., kinki univ., osaka, japan; school of pharm. sci., kinki univ., osaka, japan; school of sci. & eng., kinki univ., osaka, japan; school of pharm. sci., kinki univ., osaka, japan our previous findings showed that -ht-and bk-induced [ca + ] i increases were enlarged in differentiated ng - cells. for the next stage, we investigated the effect of pge , an inflammatory mediator for -ht and bk, on the cells. ng - cells were loaded with fura- /am, and the change in [ca + ] i was monitored by an image processor. the results showed: ( ) pge -induced response was decreased when ng - cells were differentiated by bt camp, ( ) − m ah and sc irreversibly inhibited pge -induced response by about % and %, respectively, while − m ah and sulprostone had no effect, and ( ) pge -induced response was abolished under ca +free conditions in about % of both ng - cells. these results indicate that the response to pge , via ep and ep receptors, significantly decreased during differentiation. mitsumasa murano, fumihito saitow, hidenori suzuki department of pharmacology, nippon medical school, tokyo, japan the most of cerebellar outputs are generated as a result of synaptic interaction in the deep cerebellar nuclei (dcn) and by the electrical membrane properties of dcn neurons themselves. this study aimed at examining mechanisms underlying the serotonergic modulations of both the gabaergic transmission at the purkinje-to-nuclear cell synapses and the membrane properties of dcn neurons using cerebellar slices prepared from -to -day-old rats. bath application of serotonin ( -ht) decreased the amplitude of stimulation-evoked ipscs in dcn neurons in a dose-dependent manner. furthermore, slow inward currents ware observed in dcn neurons during -ht application. under the current-clamp recording, -ht markedly depolarized and increased action potential discharges of dcn neurons. taken together, these results suggest that -ht facilitates the voluntary activity in dcn neurons by both pre-and post-synaptic mechanisms. ps a-d searching for endogenous ligands of trace amine receptors in mammals ( ) akira komatsu , airi yamaguchi , noriko makikusa , osamu koizumi dept. physiol., tokyo women's med. univ., sch. med., tokyo, japan; neurosci. lab., fukuoka women's univ. fukuoka, japan trace amine receptors were discovered in mammals, but their endogenous ligands have not yet been found. to search for them, we developed a new method to make antibodies against monoamines for immunohistochemistry (ihc). monoamines, phenylethylamine (pea), tyramine (ta) and histamine (ha), were conjugated to a hemocyanine, klh, using an imidoester cross-linker, dimethyl suberimidate (dms). rabbits were immunized by the conjugated macromolecule. the obtained antibodies were assayed by elisa and competitive elisa technique to check their antibody titer and specificity respectively. the antibodies recognized specifically the monoamine-dms part within the complex. for ihc, the rat brain was perfused by % dms, post-fixed by % formaldehyde and then frozen-sectioned. the antibody against ha revealed the immunoreactive neurons in the hypothalamus, showing that this method is effective to demonstrate the presence and localization of monoamines. the antibodies against pea and ta failed to reveal immunoreactive neurons in the rat brain. ps a-d effects of mg + on neural activity of cultured cortical neurons of the rat and mouse yuriko furukawa , , nahoko kasai , akiyoshi shimada , keiichi torimitsu , , kunihiko obata , yuchio yanagawa , , tadaharu tsumoto , ntt basic research laboratories, kanagawa, japan; sorst/jst, saitama, japan; neuronal circuit mechanisms research group, brain science institute, riken, saitama, japan; dept. of genetic and behavioral neurosci., grad. sch. of med., gunma university, gunma, japan it is well known that mg + plays an important role not only in energy metabolism, but also in neural information processing. however, the mechanism of such a role in cns is not well understood. previously we reported that neural activity and the intracellular ca + concentration are largely affected by mg + removal in cultured cortical neurons of the rat. transient glutamate release was also detected. in the present study, we investigated effects of the mg + removal on neural activity in cultured cortical and hippocampal neurons. in particular, we measured the intra-and extracellular mg + concentration and their actions on neural activity using a mg + indicator, kmg- -am together with fluo -am. we observed different effects of the mg + removal on gabaergic and non-gabaergic neurons by using gad -gfp knock-in mice. research funds: jst/sorst ps a-e transient zinc-positive terminations in the developing rat somatosensory cortical system noritaka ichinohe, daniel potapov, kathleen s. rockland lab. for cortical organization and systematics, bsi, riken, usa synaptic zinc (zn) is a neuromodulator used by a subset of nonthalamic glutamatergic connections, and associated with both experiencedependent and developmental plasticity. during development, transiently high levels of synaptic zn occur in both sensory and nonsensory cortical areas. by injecting the retrograde tracer sodium selenite into barrel cortex, we demonstrated a transient subset of zn + thalamocortical neurons from p -p . zn + cortical neurons were also labeled, intrinsic and extrinsic, from p . unlike in the adult, these were in layer , instead of layers , , and . at p , neurons occurred in layers , , and and, in some areas, layer . at p , zn + neurons first appeared in layer ; and at p , there is the adult lamination. as whisking and exploratory behavior commences in the second postnatal week, these transient zn + terminations may play a role in experience-dependent adjustments in cortical circuitry. research funds: bsi, riken and kakenhi no. ps a-e systematic comparison of the structure of the serotonin immunoreactive neurons between insect species masaaki iwano , , ryohei kanzaki , kei ito , center for bioinform., imcb, univ. of tokyo, tokyo, japan; dept. of mechano-inform., grad. sch. of inform, sci. and tech., univ. of tokyo, tokyo, japan; bird, jst, saitama, japan in the vertebrate central nervous system, the distribution of the serotonin immunoreactive neurons (sirns) is known to be preserved remarkably during evolution. systematic comparison of the invertebrate sirns has not been performed, on the other hand. in the current study we analyzed the morphology of the sirns in the brains of holoand hemi-metabolous insects including flies, bees, moths, beetles, crickets, dragonflies and cicadas. in spite of the large variation in the size and cell numbers of the brain, the number and distribution of the sirns were highly consistent between species. for example, we observed either one or two pairs of bilateral sirns with similar morphology that connect specific subregions of the lateral accessory lobe, a candidate pattern generator of the zigzag locomotion of the insect. variation was greater in the antennal lobe, the insect primary olfactory center, where sirns project either ipsil-or contra-laterally depending on the species. maki kagohashi , , taizo nakazato , shigeru kitazawa neurol, juntendo univ., tokyo, japan; physiol, juntendo univ., tokyo, japan in vivo voltammetry has been used for measuring neurotransmitter releases in the brain of behaving rats (e.g. nakazato, ) . however, task freedom was restricted by cables connecting the head and the measurement system. to overcome the difficulty we developed a wireless voltammetry system and examined its sensitivity in vitro (kagohashi et al., jns ). the system consisted of a wireless transmitter with a potentiostat and a signal receiver. in the present study, we reduced the size and weight and measured dopamine (da) currents in vivo with the wireless system mounted on the back of the rat. a single-step voltage pulse ( to mv for da; to mv for ht) was applied at hz through a carbon electrode that was chronically implanted in the striatum. after administration of l-dopa, da currents showed a gradual increase in good agreement with the data measured with conventional systems. the present wireless system would be applicable to measurement of neurotransmitters in various situations (e.g. social interaction). research funds: scientific research on priority areas (mobiligence) hiroyuki yamazaki, tomoaki shirao department of neurobiology and behavior, gunma university graduate school of medicine, maebashi, japan dendritic spines are multiple functional units that receive most of excitatory inputs in central nervous system. in the purpose of finding a novel molecule that is involved in regulation of dendritic spines, we have done a screening of a novel drebrin binding protein. yeast twohybrid system was conducted with drebrin as bait, and a novel drebrin binding protein was isolated. in neurons, this protein was localized primarily in nucleus and dendritic spines. hence, we named it spikar for its unique intracellular localization in spine and karyoplasm. we studied the role of spikar in spine formation. hippocampal neurons were transfected with shrna expression vector for spikar at several developmental stages. in early stage, spikar knock down (kd) did not affect the density of dendritic protrusions that were mostly filopodia. in contrast, spikar kd reduced spine density at the stage of synapse formation. these results suggest that spikar plays a role in the formation of dendritic spines, without affecting the filopodia formation. ps a-e time-lapse analysis of the translocation of drebrin-actin complex from dendritic spines to dendritic shafts by glutamate stimulation toshiyuki mizui , , yuko sekino , , tomoaki sirao dept. of neurobiol. & behav., gunma univ. grad. sch. of med., maebashi, japan; div. of neural network, inst. med. sci. univ. of tokyo, tokyo; crest, jst, kawaguchi, japan; jsps, japan we have shown that nmda receptor activation induced translocation of drebrin, with retaining its binding to f-actin, from dendritic spines to their parent dendrites. in the present study, we analyzed the time course of gfp-tagged drebrin a (gfp-da) dynamics after glutamate receptor activation. we prepared primary hippocampal cultured neurons, transfected them with gfp-drebrin a expression vector using microinjection methods at days in vitro (div), and analyzed the dynamic localization of gfp-da at div. glutamate stimulation started gfp-da translocating within s and completed in min. after washout of glutamate, gfp-da gradually re-accumulated in the spine, and the fluorescence intensity of gfp-da is fully recovered in min. these data suggest that translocation mechanism of drebrin from spines to shafts is different from that from shafts to spines. research funds: grant-in-aid for jsps fellows ps a-e distribution of the srf co-activator mal in developing mouse brain mitsuru ishikawa , jun shiota , hiroyuki tsutsumishita , hiroyuki sakagami , masaaki tsuda , akiko tabuchi dept. biol. chem., fac. pharm. sci., univ. toyama, toyama, japan; dept. cell biol., tohoku univ., grad. sch. medicine, sendai, japan the srf co-activator mal (megakaryocytic acute leukemia) plays an important role in controlling srf-dependent gene, whose expression is regulated by rearrangement of actin cytoskeleton. recent studies with conditional deletion of srf gene demonstrated that srf was required for inducing genes such as egr- , c-fos,-actin but also for neuronal migration and plasticity. in this study, we investigated the expression of mal in developing mouse brain and the role of mal for dendritic morphology. the in situ hybridization analysis revealed that mal mrna was highly and developmentally expressed in hippocampus and broadly expressed in cortex, olfactory bulb. staining of mal displayed cytoplasmic localization at cell bodies and apical dendrites. furthermore, dominant negative mal mutants and rnai led to a reduction of dendritic number, as well as a decrease of srf transcription. these findings indicate that mal is involved in the formation or the stability of dendrites. research funds: kakenhi ( ) to a.t. shoko shimizu , shinsuke matsuzaki , tsuyoshi hattori , ko miyoshi , masaya tohyama department of anatomy and neuroscience, graduate school of medicine, osaka university, japan; department of brain science, graduate school of medicine and dentistry, okayama university, japan disrupted-in-schizophrenia (disc ) was identified as a novel gene disrupted by a ( ; ) (q . ;q . ) translocation segregating with schizophrenia and affective disorders in a scottish family. kendrin was identified as a protein which interacts with disc at centrosome and residues - of disc (kendrin-binding region: kbr) were essential for the interaction with kendrin. in this study, we show that c-terminal of disc downstream of kbr is indispensable structure for kbr to interact with kendrin and also essential for disc to target to the centrosome. furthermore, we have shown that inhibition of the disc -kendrin interaction perturbs the tubulin network formation. these results suggest that the c-terminal region of the disc is important to the disc -kendrin interaction and that a truncated form of disc lacking the c-terminal downstream of the translocation breakpoint might affect the microtubule organization. tatsuro kumada, yasuhiko nakanishi, atsushi fukuda department of physiology, hamamatsu university school of medicine migratory cells exhibit dynamic morphological changes in the cell soma and process in both normal developmental program and tumor growth. the morphological changes in the cells are correlated with the rate of cell migration and ion transfer such as ca + or cl − . although the highly invasive migration of glioblastoma in the brain is known to be influenced by a variety of ion channels, there were a little evidence about the relationships among the morphological changes and ion homeostasis. to clarify it, we have developed a glioma cell culture system for the simultaneous observation of the cell movement and ca + and cl − imaging. we found that the relatively low density a glioma cells actively moved on the substrate. the movement has the correlation with intracellular ca + oscillation in the cells. the relationship between cell movement and intracellular ion levels is further studied. ps a-e involvement of ca + influx in the unpolarized non-vesicular release of fgf- hayato matsunaga, hiroshi ueda division of molecular pharmacology and neuroscience, nagasaki university graduate school of biomedical sciences, nagasaki, japan little is known of molecular basis mechanisms for the er-golgiindependent or non-vesicular release of fgf- lacking a conventional signal peptide sequence. we found that fgf- is co-released with s a , a ca + binding protein from cultured rat astrocytes upon the serum-deprivation stress. here, we report that fgf- is co-released with s a from the axon and dendrites in cultured rat hippocampal neurons upon depolarization stimulation, but serum-deprivation stress leads to release, which is seen in neurites as well as in soma. the interaction between fgf- and s a required ca + . the overexpression of s a - mutant lacking an ability of interaction with fgf- inhibited the their release, suggesting that s a is a cargo molecule. the release of fgf- upon either stimulation was abolished by voltage-dependent n-type ca + channel blocker. these findings suggest that ca + influx may be involved in the unpolarized non-vesicular release of fgf- . in neuron, intracellular calcium involves a large number of physiological phenomena, including cell migration, differentiation, and neurite outgrowth. pc cell is a useful model of neural differentiation and neurite outgrowth, and recently we demonstrated that -ht has an effect on neurite outgrowth via the increase in intracellular calcium concentration ([ca + ] i ) in pc cells. however, it is unclear how [ca + ] i regulates neurite outgrowth via actin cytoskeleton. in this study, we investigated effects of [ca + ] i on actin dynamics in pc cells transfected with yfp-actin. filopodial growth speed and actin retrograde flow were increased by treatment with calcium ionophore, a . treatment with calcineurin inhibitors decreased the filopodial growth speed, while treatment with camk inhibitor did not. these effects could contribute to -ht induced enhancement of neurite elongation. the actin cytoskeleton is a complex protein network that not only provides cellular structure but is fundamental for cellular dynamics. on stimulation of pc cells by ngf, proteins that directly interact with f-actin such as actinin rapidly translocate to the f-actin-rich cytoskeleton. clp is a pdz-lim protein which was originally identified as an actinin-interacting protein in skeletal muscles. here, we show that clp is endogenously expressed in pc cells and plays an important role in actin dynamics during ngf-induced neurite outgrowth. immunofluorescent studies showed that clp is accumulated in irregular cell surface and membrane extrusion soon after ngf-stimulation, where colocalized with actin filaments. we next performed rnai experiments to explore the role of clp in actin dynamics in growth cones and found that knockdown of clp expression lead to the suppression of ngf-mediated neurite outgrowth. in addition, we revealed using clp deletion mutants that both of pdz and lim domains are necessary for the proper function of clp . ps a-e screening of genes expressed preferentially in migrating gabaergic neurons of developing cerebral cortex toshiya kimura , tsuyoshi kobayashi , yuchio yanagawa , kunihiko obata , fujio murakami , grad. sch. of frontier biosci., osaka univ., osaka, japan; grad. sch. of medicine, gunma univ., maebashi, japan; bsi, riken, wako, japan; sorst, jst, japan neuronal migration plays a critical role in constructing brain architecture organization. however, molecular mechanisms underlying this process still remain elusive. in an attempt to identify molecules that regulate the motility of migrating neurons, we focused on migrating cortical interneurons, and performed subtractive hybridization, differential screening and in situ hybridization. subtraction was done between the embryonic and postnatal interneurons, because they robustly migrate prenatally but not postnatally. among the clones tested, two genes, neuronatin and seizure related gene (sez- ) attracted our attention. they were expressed in the subventricular zone of the embryonic cortex, implicating that these molecules are expressed in interneuron subpopulations. postnatally, mrna signals were hardly detectable. these results raise the possibility that they are expressed preferentially in subpopulations migrating cortical interneurons. yan zhu , , tomoko matsumoto , , sakae mikami , takashi nagasawa , fujio murakami , grad. sch. of frontier biosci., osaka univ., japan; sorst, jst, japan; inst for frontier med. sci., kyoto univ., japan long distance neuronal migration takes place typically along the tangential plane of the developing neural tube. the migratory behaviour and the underlying molecular mechanisms of tangential migration are poorly understood. we address these issues using the hindbrain precerebellar system as model system. precerebellar neurons, born dorsally in the lower rhombic lip, migrate in close association with the pial membrane (except inferior olive neurons) ventrally or rostroventrally. we therefore studied the role of pia-secreted chemokine sdf- and its receptor cxcr in the precerebellar migration. we show that cxcr is expressed in the migrating precerebellar neurons, and its expression is down-regulated towards the end of migration. in cxcr and sdf- knock out mice, migrating precerebellar neurons are less confined to the pial surface. more strikingly, the rostrally-directed migration of pontine precerebellar neurons is severely disrupted, leading to a caudalized ectopic pontine-like cluster. ps a-e involvement of an immunoglobulin superfamily molecule, neph /mkirre in the migration of precerebellar neurons kazuhiko nishida , , kazuhide nakayama , saori yoshimura , , fujio murakami , grad. sch. of frontier biosci., osaka univ., osaka, japan; sorst, jst, saitama, japan neural cell migration plays a crucial role in central nervous system development. in this study, we analyze the involvement of neph family transmembrane proteins of the immunoglobulin superfamily in the migration of precerebellar neurons (pcns). postmitotic pcns derived from the rhombic lip in the hindbrain first migrate tangentially along the pial surface, followed by radial migration to settle at their final positions (kawauchi, d., taniguchi, h., watanabe, h., saito, t., and murakami, f., development, in press ). in situ hybridization analysis showed that among neph family members including neph , neph /mkirre, and neph , only neph /mkirre was strongly expressed in pcns. expression of neph /mkirre was detected from e . when pcns migrate tangentially. the expression level became weaker at p , when pcns stop the radial migration, raising the possibility that neph /mkirre might be involved in the migration of pcns. we are currently analyzing the function of neph /mkirre in the migration of pcns. hiroki umeshima , , toshio ohshima , tomoo hirano , mineko kengaku lab. for neural cell polarity, riken bsi, wako, japan; department of biophysics, kyoto university, kyoto, japan; lab. for developmental neurobiology, riken, bsi, wako, japan during lamination of the cerebellar cortex, granule cells exit their final mitiosis at the external granular layer and migrate to the internal granular layer. we analyzed the molecular mechanisms regulating migration of granule cells. using an in vivo electroporation system followed by time-lapse confocal microscopy of a slice culture, we found a dominant negative form of cdk (cdk -dn) disrupted the morphology of granule cells during radial migration. recently, centrosome positioning is thought to be one of the important factors for neuronal migration. double-labeling of the centrosome and the whole-cell images by transfecting centrin -gfp and rfp enabled us to record dynamic movement of the centrosome during radial migration. we found that the motion kinetics of the centrosome was disrupted by cdk -dn. based on these results, we will discuss the role of centrosome during neuronal migration. keisuke ito , , takahiko kawasaki , , tatsumi hirata , division of brain function, national institute of genetics, mishima, shizuoka, japan; department of genetics, school of bioscience, sokendai newly generated neurons migrate through proper pathways toward their own targets, where they are integrated into specific neuronal circuits. we have analyzed a unique tangential migratory stream of early-generated cortical neurons designated as lot cells, and performed pharmacological perturbations to characterize the intracellular mechanism of the migration. among various drugs, we found that a protein kinase inhibitor, k a has the most interesting effect on the lot cell migration. during the normal migration, leading processes and cell bodies of lot cells move forward in a coordinated manner, but k a blocks the migratory movement of cell bodies without inhibiting the extension of leading processes. we also found that k a has a similar effect on cerebellar granule cells. these phenomena are quite intriguing because the drug seemed to switch the neurons from "whole cell migration" to "neurite extension" mode. we are now analyzing possible targets of k a, aiming for dissection of these phenomena. the conserved ser/thr kinase unc functions with unc- to regulate axonal transport in drosophila hiroaki mochizuki , hirofumi toda , , emiko suzuki , joseph gindhart , toshifumi tomoda , katsuo furukubo-tokunaga grad. school life and envir. sci., univ. tsukuba, tsukuba, japan; gene net. lab., natl. inst. genet. mishima; beckman res. inst., city of hope, ca, usa; dep. biol., univ. richmond, va, usa neural network develops through regulated guidance of axons and interconnection among them. despite intensive researches in the past years, genetic mechanisms of axonal development still remain unclear. we have identified the drosophila homolog of unc , which encodes a ser/thr kinase and is required for axonal formation in c. elegans and mouse. we found that unc is essential for neural development in drosophila. loss of function of drosophila unc results in reduced locomotion and axonal transport defects reminiscent of the phenotypes observed in kinesin mutants. we also found that unc genetically interacts with unc- , an evolutionarily conserved cytoplasmic protein that binds to kinesin heavy chain. in unc mutants, unc- was separated from synaptotagmin vesicles. these results suggest that unc coordinates kinesin-cargo interaction via unc- to regulate dynamic axonal transport. ps a-e change in microtubule polarity during the conversion of dendrites into axons kensuke hayashi , daisuke takahashi life science inst. sophia university, tokyo, japan; waseda university, tokyo, japan axons and dendrites of neurons differ in the polarity of their microtubules. the mechanism for the difference, however, is not well understood. we found previously that dendrites convert into axons in cultured neurons isolated from rat cerebral cortex. in this study, we examined whether microtubule polarity changes during the conversion. in dendrites of neurons before culture, microtubule polarity was nonuniform. after h of culture, we found that most of microtubules in the original dendrites had their plus ends oriented distal. this indicates that microtubules with their minus-ends distal disappeared during the culture. microtubule movement along actin filaments is a candidate for this mechanism among several types of microtubule movement reported in neuronal processes so far. however, the change of microtubule polarity within dendrites was observed even in the presence of actin polymerization inhibitors. our results suggest a rearrangement of microtubules by a yet-unreported movement in neuronal processes. research funds: kakenhi ( ) and kakenhi on priority areas ( ) ps a-e generation and analysis of region-specific rac -deficient mice hidetoshi kassai , masahiro fukaya , eriko miura , mizuho sakahara , masahiko watanabe , , atsu aiba div. cell biol., kobe univ. grad. sch. med., kobe, japan; div. physiol. sci., hokkaido univ. grad. sch. med., japan rac is a member of the rho family of small gtpases, and assumed to be involved in regulation of neuronal development through actin cytoskeletal reorganization. nevertheless, physiological role of rac in the cns is poorly understood because of the embryonic lethality of rac knockout mice. in this study, we generated and analyzed region-specific rac -deficient mice (emx -rac ko mice) by the cre-loxp system, in which a promoter for emx homeobox gene induces expression of cre recombinase exclusively in the dorsal telencephalon, including cerebral cortex, hippocampus and olfactory bulb. emx -rac ko mice showed partially abnormal layering of cerebral cortex, indicating impaired migration of neuronal cells during cortical development. furthermore, emx -rac ko mice lacked corpus callosum and anterior commissure, both of which connect the left and right cerebral hemispheres. these results suggest that rac regulates neuronal cell migration and axonal growth in cerebral cortex. previously we reported overexpression of map b containing nterminal amino acids promoted neuronal death. to reveal the mechanism of map b n-terminal induced neuronal death, we searched for the proteins that interact with n-terminal of map b by two-hybrid system. alpha-tubulin was found to interact with map b n-terminal and their in vitro interaction was proved with pull-down assay. the interaction of tubulin and map b n-terminal has not yet been reported. beta-tubulin was also found to interact with map b n-terminal. when  tubulin was divided in fragment at between amino acid and , there was no interaction between  tubulin fragments and map b n-terminal. interaction needs the continuous region over aa and . there were much proportion of round formed cos cells in n-terminal containing map b transfected cells than in n-terminal lacking map b transfected cells. there might be some interference in interaction between map b and tubulin in cells express map b containing n-terminal. otone endo , , masaaki mizuno , yasukazu kajita , jun yoshida department of neurosurgery, ja kainan hospital, aichi, japan; department of neurosurgery, nagoya university, nagoya, japan; department of molecular neurosurgery, nagoya university, nagoya, japan primate es cells have rather different character from rodent ones, but it is inevitable to elucidate mechanism for stable culture, purification and induction into object-oriented differentiation, because human es cells might show wide similarity to cynomolgus ones. we refined the way of large scale culture maintaining totipotency without contacting feeder cells indispensable for primate es cells. our super selective induction method for dopaminergic neurons is also refined, and induced neurons transplanted in vivo which survive without forming tumor such as teratoma for long period, are evaluated not only immunohistologically but eletrophysiologically and ethologically suggesting its enough stability, activity, ability to make neural network system and potentiality to improve clinical symptom of parkinsonism. differentiation of other types of neurons and development of fully functional neural network must be established. shigeki ohta , masae yaguchi , yumi matsuzaki , yoshiaki toyama , yutaka kawakami , hideyuki okano , masahiro toda , neuroimmunology research group, keio univ., tokyo, japan; physiology, keio univ., tokyo, japan; orthopaedic surgery, keio univ., tokyo, japan; institute for advanced medical research, keio univ., tokyo, japan; neurosurgery, keio univ., tokyo, japan we have shown that mouse dendritic cells (dcs) have the ability to induce the proliferation and survival of neural stem cells/progenitor cells (nspcs) in vitro. implantation of dcs into injured mouse spinal cord could improve the motor function through activation of endogenous nspcs in vivo. in this study, to identify an effective dc subtype for the treatment of spinal cord injury (sci), we analyzed the effects of different mouse dc subtypes on the proliferation of nspcs in vitro. among mouse splenic cd c + dcs, cd ␣ + dcs increased the number of neurospheres most effectively in vitro. furthermore, a significant functional recovery after mouse sci was induced by implantation of cd ␣ + dcs compared to cd c + dcs. these results suggest that cd ␣ + dcs can be an effective subtype of mouse dcs for the treatment of sci. ps a-e von hippel-lindau protein regulate the neurogenesis in skin-derived precursor cells atsuhiko kubo , hiroshi kanno , takaakira yokoyama , shuichi nakano , naoki sugimoto , nahoko kobayashi , tetsuhiko yoshida , isao yamamoto dept. of neurosurgery, yokohama city university graduate school of medicine, yokohama, japan; fiber and dept. of chemistry, konan university, kobe, japan; toagosei co., ltd. corporate research laboratory, nagoya, japan skin-derived precursors (skps), multipotent somatic stem cells, are preferred cell source for autologus cns cell replacement therapy. they are proliferated by the mitogens of egf and bfgf. to investigate the effects of von hippel-lidau (vhl) protein in the neural cell fate commitment, skps were inoculated with hsv vector expressing vhl protein. skps showed promotion of neurogenesis and inhibition of gliogenesis. to detect the intrinsic factors that control lineage commitment, vhl peptides fused with the protein transduction domain (ptd) were synthesized. the ptd-vhl peptides showed rapid cell internalization in nearly %, and peptide with the elongin c binding site (residues - ) showed a high ability of inducing neuronal differentiation by interacting with jak/stat pathway. these findings are important in its application to the cns cell grafting. ps a-e the effect of pueraria mirifica on erk / and s- following sciatic nerve injury in rats pornpen chaiworakul, supin chompoopong department of anatomy, mahidol university, bangkok, thailand to investigate the effects of pueraria mirifica (pm) compared with genistein (g) and estrogen (e ) on the expression of erk / and s- following sciatic nerve crush and transection in rats. protein levels of perk / and s- in distal segments of nerve at day were determined by western blot analysis. it was demonstrated that pm and g treatments, similar to e , caused a significant decrease in the expression of perk / levels in both nerve crush and transection injuries. however, transected nerves showed high and sustained levels of erk / phosphorylation. following treatments, levels of s- were significantly decreased both in crushed and transected nerves with respect to control group at p < . . this estrogenic effect was blocked by ici , . because of their structural similarity to e , pm may have therapeutic potential in nerve injuries which as previously reported to enhance sfi following sciatic nerve crush in rats after day . this study suggested that pm as well as g could enhance nerve regeneration like e by interfering with the injury-induced erk signaling pathway. yasuhiro kato , takafumi suzuki , kunihiko mabuchi department of advanced interdisciplinary studies, graduate school of engineering, the university of tokyo, japan; department of information physics and computing, graduate school of information science and technology, the university of tokyo, japan mems technologies have been established to fabricate a multichannel neural probe for interfacing with the nervous system. there is, however, no suitable probe for long-term neural recording and stimulation. one main reason is the death of brain tissues damaged by the probe insertion and implantation. thus, a new skeleton-like multichannel flexible neural probe coated with hybrid biodegradable polymer was fabricated. the skeleton-like probe was designed to minimize the volume of the flexible probe and buffer injurious micromotion between the probe and the tissues in a post-implantation. the probe was coated with mixed polyethylene glycol and microspheres with nerve growth factor (ngf) to improve the stiffness for the probe insertion, and deliver ngf for an optimal period to promote regrowth of damaged neural tissues around the probe. damage-induced neuronal endopeptidase (dine) is a newly identified nerve regeneration-associated molecule. it encodes neuronspecific membrane-spanning metalloprotease and belongs to nep/ece family which degrades/processes neuropeptides. although the precise mechanism of dine including substrate is still unclear, dine seems to play a protective role in damaged neurons. the most marked property of dine is a striking response to various kinds of nerve injury in both central nervous system and peripheral nervous system. to clarify the transcriptional regulation of dine after nerve injury, we analyzed untranslated region of dine gene. previously, we found that lif treatment and ngf deprivation additively increased dine mrna. in this study, promoter analysis showed that dine promoter activity was cooperatively up-regulated by atf- and stat , which were induced after nerve injury and activated at the downstream of lif treatment and ngf deprivation. this combination of transcription factors may be pivotal to promote gene expression, which is responsible for nerve regeneration. tomohiro miyashita, takekazu kubo, masashi fujitani, katsuhiko hata, toshihide yamashita department of neurobiology, graduate school of medicine, chiba university, chiba, japan wnt proteins are known as those concerning with formation of central nervous system. we tested whether they play a role in inhibition of axon regeneration after spinal cord injury. cerebral granule neurons from p - wistar rats were cultured. wnt proteins were added into the culture medium. twenty-four hours after culture, neurite length of each neuron was measured. immunohistochemistry was done employing anti-wnts antibody and anti-ryk (wnt receptor) antibody. anti-ryk antibody was injected continuously for two weeks into the subarachnoid space of contused rat spinal cord. locomotor behaviour was evaluated up to six weeks after injury. immunohistochemistry showed that several wnt proteins and ryk were upregulated after spinal cord injury. wnt proteins inhibited neurite outgrowth of cultured cerebral granule neurons. and this effect was abolished by y , a rho-kinase inhibitor, and anti-ryk antibody. suppression of wnt proteins may promote axon regeneration and improve locomotor behaviour after spinal cord injury. akihito takeda, richard goris, kengo funakoshi department of neuroanatomy, yokohama city university graduate school of medicine, yokohama, japan in contrast to mammals, spontaneous nerve regeneration after lesion of the spinal cord occurs in fishes. we examined tissue remodeling and axon regeneration after spinal hemisection in the goldfish. in the lesioned spinal cord, neurogenesis reached the maximum level days after the hemisection. glial cells positive for glial fibrillary acid protein (gfap) temporarily increased at the lesion site one day after. many gfap positive cells expressed somatostatin. serotonin ( ht) positive cells increased in number progressively from day to weeks after. six weeks after, the regenerated axons with glial fibers invaded fibrotic scar centered about the lesion site, and ht cells surrounded the axons and glia. thus, ht may promote these neural elements to invade the fibrotic scar. six weeks after the hemisection, projections from locomotion center in midbrain to spinal motoneurons were restored, and swimming ability was also recovered. these results suggest that the goldfish have ability to reestablish correct projections after the spinal injury. masao koda , yukio someya , ryo kadota , chikato mannoji , tomohiro miyashita , atsushi murata , masashi yamazaki department of orthopaedic surgery, togane hospital, department of ortopaedic surgery, graduate school of medicine, chiba university, japan; division of rehabilitation medicine, chiba university hospital, japan objective: anoikis is a type of apoptosis due to the detatchment from the extracellular matrix. preparation of graft cells for cell therapy includes dissociation of cultured cells, which may cause anoikis. here we tested the effect of bdnf for anoikis of schwann cell. methods: (in vitro) schwann cells were cultured from sciatic nerves of neonatal rats. schwann cells were transferred to suspension culture. bdnf was added into the culture medium. cell death was detected h after suspension culture. (in vivo) schwann cells were transplanted with or without bdnf treatment into contused rat spinal cord. immunohistochemistry was performed to detect survival of grafted cells. the olfactory bulb and its caudal extension are unique forebrain regions with the residence of neural stem cells and the ability of persistent neurogenesis. however, evidence for active functional involvement of neural stem cells is still very limited. this study was undertaken to know whether or not newly generated neurons are integrated in olfactory neuronal circuits in the neonatally bulbectomized rats that had been proved to show olfactory discriminative abilities. for this purpose, retroviral vector, a very useful tool to trace neural stem cells, was applied to the anterior part of the subventricular zone of the rats of which olfactory bulbs had been unilaterally ablated at the neonatal stage. we will show cell dynamics of newly generated neurons in the neonatally bulbectomized olfactory nervous system, with special reference to their neuronal circuits. koichi kawada, masanori yonayama, kiyokazu ogita dept. pharmacol., setsunan univ., osaka the subventricular zone (svz) contains undifferentiated cells, which proliferate and generate the olfactory bulb (ob) interneurons. throughout life, these cells leave the svz and migrate to the ob via the rostal migratory stream, where they differentiate. we have shown that trimethyltin (tmt) causes neuronal damage in the hippocampal dentate gyrus. in this study, we examined neuronal degeneration and regeneration in the ob after tmt treatment in mice. ddy mice were given tmt ( . mg/kg) to prepare slices for an immunohistochemical analysis using antibodies against single-stranded dna (ssdna), -bromo- -deoxyuridine- -monophosphate (brdu), neuronal nuclei (neun) and nestin. positive cells immunoreactive to ssdna markedly increased in the ob on days after tmt treatment. positive cells immunoreactive to brdu markedly increased in the ob on days after tmt treatment. double staining of brdu and neun in the ob revealed that almost brdu was not incorporated into mature neurons on day after the treatment. these results suggest possible enhancement of neurogenesis in the ob following tmt treatment. ps a-f early migration of human umbilical cord blood neural stem cells transplanted into rat brain miroslaw janowski , hanna kozlowska , marcin jurga , aleksandra habich , elzbieta wanacka , barbara lukomska, krystyna domanska-janik department of neurorepair, medical research center, warsaw, poland many neurological disorders result from progressive cell loss or rapid cell damage. as stem cell technology appeared there is an arising hope for cell replacement therapy and definitive cure. recently, in our laboratory human umbilical cord blood neural stem cell line (hucb nsc) was established. the aim of the study was to analyze the migratory potential of hucb nsc transplanted into intact rat brain. hucb nsc transfected with gfp gene was stereotactically transplanted (tx) into intact brain of csa immunosuppressed adult wistar rats. cell detection was performed h, h, h and days after transplantation using abs anti gfp, hla class i and numa. analysis of rat brains revealed viable gfp positive hucb nsc cells migrating from tx site and dispersed through the host brain tissue , , and days after grafting. immunohistochemical studies confirmed that these cells were of human origin: hla class i or numa. in future we plan to study their lesion directed migratory potential. heparan sulfate proteoglycans (hspgs) are considered to play roles in cns development, such as axonal guidance. however, little is known about the function of hspgs during nerve regeneration. in this study, we examined the expression of ext , one of the enzymes for heparan sulfate biosynthesis, after hypoglossal nerve injury. the upregulation of ext mrna was detected using in situ hybridization in injured hypoglossal motoneurons, and heparan sulfate glycosaminoglycan was also upregulated in the injured hypoglossal nucleus. we also examined the expression of mrna for hspg core protein. the mrnas for glypican- and syndecan- were upregulated in injured motoneurons. these results indicate that the synthesis of hspg is upregulated in injured motoneuron and hspg might be involved in nerve regeneration. masami watanabe , hiroe sagawa , masahiro ichikawa , yoshihito tokita dept. perinatol., int. dev. res., kasugai, japan; dept. ophthalmol., nagoya univ. sch. med., nagoya, japan; dept. neurosurg., nagoya univ. sch. med., nagoya, japan we examined whether rho/rock inhibitor, y , can make injured rgc axons regenerate into the crushed optic nerve (opn) of cats. methods: culture; retinal pieces were cultured in dmem for d. after fixation, the neurites were stained with anti-tuj antibody to obtain number and length of tuj neurites. crush; after an intravitreal injection of drug, the left opn was crushed with thread. on day , wga-hrp was injected into the vitreous. sections of opn were reacted for hrp with tmb reaction. masanori yoneyama, kiyokazu ogita dept. pharmacol, setsunan univ., osaka, japan in this study, we evaluated the effects of glutathione depletion on proliferative activity in neural progenitor cells of -days-old embryonic mice. neural progenitor cells were prepared from the hippocampus of -days-old embryonic mice by culturing in dmem/f medium for days in vitro (div). marked round spheres were formed from cells adhered to each other under the culture conditions in the presence of bfgf and egf, and then subsequently proliferated to form large neurospheres in proportion to the duration of cultivation. to evaluate the effects of glutathione depletion on proliferation in the neural progenitor cells, buthionine sulfoximine (bso) were exposed into cultured neural progenitor cells for a period of - div. treatment with bso resulted in a marked reduction in endogenous glutathione in the cells. mtt assay revealed that the deletion of glutathione led to a marked decrease in surviving neurospheres cultured for - div. these results suggest that glutathione would positively regulate proliferative activity and/or survival in neural progenitor cells of murine hippocampus. michio hashimoto, eisuke kawakita, masanori katakura, osamu shido dept. of environ. physiol., sch. of med., shimane univ., japan docosahexaenoic acid (dha), one of the main lipids in brain, plays crucial roles in the development and function of brain neurons. we examined the effect of dha on neuronal differentiation of neural stem cells (nscs) in vitro and in vivo. nscs obtained from rat embryos were propagated as neurospheres and cultured with or without dha for days. dha increased the number of tuj (+) neurons compared with the control, and the newborn neurons in the dha group were morphologically more mature than in the control. dha decreased the incorporation ratio of brdu, the mitotic division marker, during the first h period. thus, dha promotes the differentiation of nscs into neurons by promoting cell cycle exit. furthermore, dietary administration of dha significantly increased the number of brdu(+)/neun(+) newborn neurons in the granule cell layer of the dentate gyrus in adult rats. these results demonstrate that dha effectively promotes neurogenesis both in vitro and in vivo, suggesting that it has the new property of modulating hippocampal function regulated by neurogenesis. research funds: kakenhi ( ) ps a-f interaction among cues for visual depth motion perception tomokazu shimizu, akitoshi hanazawa kyushu institute of technology, fukuoka, japan when an object surface approaches or leaves us, we perceive visual depth motion. cues for this motion are change in binocular disparity, change in spatial frequency and optical flow. we investigated interactions among these cues by using visual stimuli in which the cues provides opposite depth motion direction. for the stimulus without optical flow component, spatial frequency was changed continuously by presenting uncorrelated random dot patterns filtered by different band-pass filters. when binocular disparity and spatial frequency was oppositely changed, subjects perceived depth motion corresponding to the change in binocular disparity or special frequency. for the stimulus with optical flow component, a random dot pattern filtered by a band-pass filter was expanded or contracted. when binocular disparity and the other two cues were oppositely changed, subjects perceived depth motion corresponding to the change in the other two cues. when depth motion was perceived from binocular disparity, stimulus image was perceived as changing its size. when from the other cues, depth perception from binocular disparity was suppressed. research funds: coe-j kazuyuki takahashi, akitoshi hanazawa kyushu institute of technology, japan in phenomena such as biological motion and structure from motion, a global structure is perceived by an integration of local motion signals. to clarify the fundamental mechanism of this motion integration process, we psychophysically examined the influence of directional motion coherency on motion grouping. moving dots were presented in three apertures that were aligned horizontally. before presenting these stimuli, subjects were instructed to detect a dot moving in a direction among noise dots presented in the central aperture. the dots moving in the same as or different from the instructed direction were presented in the side apertures. the performance of the subjects was the best when the dots in the side apertures moved in the same direction as the instructed one. the performance kept high when the directional difference was up to ± • and declined as the difference increased. the high performance would be due to the grouping of the dots presented in the central and side apertures that have the same or similar motion direction. the underlying motion grouping mechanism was suggested to integrate motion signals that have a certain directional variation. ps a-f effect of spatial context on structure-frommotion perception koshi makino, akitoshi hanazawa kyushu institute of technology, japan when viewing an orthographic projection of dots on the surface of a rotating cylinder, one perceives a transparent rotating d cylinder. this phenomenon is called structure-from-motion (sfm). the direction of the rotation is ambiguous. we investigated the influence of spatial context on the perceived direction of the rotation. three spatially separated stimuli were horizontally aligned. subjects reported in which direction the central random-dot sfm cylinder rotated. they perceived the same direction of rotation as the side stimuli when the side stimuli were corotating cylinders whose direction of rotation was disambiguated by binocular disparity. this effect was strong when the stimuli consisted of a small number of dots, and was attenuated as the number of dots increased. the perception was also influenced by translational motion stimuli that had front and back planes comprising oppositely moving random-dots whose depth was specified by binocular disparity. these results suggest that the neural mechanism determining the rotation direction of bistable sfm is strongly influenced by the d structure of surrounding stimuli defined by binocular disparity. ps a-f rotational motion aftereffect in positive direction for -dimensional random-dot pattern masako ono, akitoshi hanazawa kyushu institute of technology, kitakyushu, japan when viewing a unidirectionally moving pattern followed by a stationary pattern, we will see the stationary pattern moving in the direction opposite to the preceding movement. this phenomenon is well known as motion aftereffect (mae). this mae can be perceived for -dimensional motion such as rotating cylinders. we found that adaptation to the rotation of a stereoscopic random-dot cylinder generate mae like phenomenon in the same positive direction as the rotation of the adaptation stimulus (positive mae). this positive mae was strong when cylindrical random-dot was used as a stationary test stimulus. this aftereffect could not be perceived for uniformly distributed non-cylindrical random-dot. although ordinary mae declined in a few seconds, this positive mae remained for a few minutes. this is a new phenomenon that is different from known dimensional mae. this finding suggests that the visual system has a mechanism that detect -dimensional rotation direction specifically, and this mechanism has a property that gives a bias to the perception of stereoscopic rotation direction in an adapted direction. research funds: coe-j takanori uka, ryo sasaki department of physiology , juntendo university school of medicine, tokyo, japan crowding refers to a subjectǐs difficulty in identifying a target in the presence of distracters. as a first attempt towards identifying the neural mechanism of crowding, we investigated perceptual crowding using a random-dot kinematogram. human subjects were required to report the direction of moving dots within a center patch ( deg) of a center/surround display presented degrees to the left of fixation, and to ignore the dots in the surround. motion coherence of the dots in the center patch, as well as surround size varied randomly across trials. motion coherence of the surround was always percent. for each of subjects, we calculated direction discrimination thresholds (at % correct) at each surround size. consistent with crowding, thresholds increased when surround size was . and degrees, compared to those with no surround. surprisingly, however, thresholds decreased when surround size was and degrees, relative to degrees. our results show that the spatial resolution of motion direction discrimination improves when the area we have to ignore exceeds a defined size. ps a-f generation of receptive fields in higher visual areas based on v columnar structure: a model study yoshitaka toyoda, yoshiyuki shimizu, izumi ohzawa graduate school of frontier biosciences, osaka university, osaka, japan neurons in higher cortical areas of the visual pathway, such as v and v , respond to stimuli with complex shapes. how do these neurons integrate signals from v ? in particular, does the well-known columnar organization of v play a role in determining the shape selectivity of higher-order neurons? to explore these questions, we devised a feed-forward hierarchical model. in our model, higherorder neurons sum the activities of v neurons linearly according to a neural receptive field (nrf), a weighting function defined over the cortical surface. the manner a nrf sums over multiple columns determines its shape selectivity. since there is no physiological data regarding possible forms for nrf, we have tested simple functional prototypes, gaussians and gabor functions. reponses of these model neurons are examined using non-cartesian gratings and other stimuli, and compared to published physiological data. about % of model neurons exhibit responses similar to those of v and v neurons. odd-symmetric gabor nrfs tend to generate more of these neurons. taihei ninomiya, takahisa m. sanada, izumi ohzawa graduate school of frontier biosciences, osaka university, osaka, japan when images with different spatial frequencies (sfs) are projected onto the two retinae, a -d surface slant is perceived (blakemore, ) . the relationship between the binocular receptive fields (brfs) and sf tuning properties indicate that the early cortical neurons can signal slant-in-depth (sanada and ohzawa ) . however, their measurements of brf were conducted in the spatial domain, and the sf tunings were tested monocularly. in this study, interactions are examined directly in the sf domain between grating stimuli presented to the left and right eyes. frequency-domain brfs were measured by a reverse correlation technique. both binocular and monocular sf profiles were obtained by this method. we predicted binocular sf (bsf) maps from monocular sf profiles, and compared the prediction and the actual bsf maps to assess the binocular interactions. with this method, neural response properties which previous studies couldn't access were revealed. research funds: mext( ), jsps( ), coe ps a-f consistency of simple cell receptive fields: space and spatial frequency domain measurements yuka tabuchi , kota sasaki , izumi ohzawa , grad. school of frontier biosci., osaka univ., japan; grad. school of eng. sci., osaka univ., japan frequency-domain subspace reverse correlation and -d spacedomain dynamic dense noise have become increasingly popular for mapping receptive fields (rf) of early visual cortical neurons. however, it is not known whether results from these methods are mutually consistent. to examine this issue, we compared an rf in the space domain measured by -d noise stimuli and an rf reconstructed from the response in the spatial frequency (sf) domain measured by flash grating stimuli of various orientation (or), sf and spatial phase presented in rapid succession. we fitted these two rfs by gabor functions, and examined the consistency of their parameters. all parameters including sf, or, spatial phase, and size of the rf agreed well when an expansive nonlinearity is considered for each cell. the optimal sf obtained in the space domain increased over time to the same extent as that obtained in the sf domain. therefore, responses of a simple cell can be encapsulated in a concise framework of a linear filter followed by expansive nonlinearity. research funds: mext( ), jsps( ), coe ps a-f firing statistics and stimulus selectivity of inferior temporal cortical neurons in the monkey shunta tate , , hiroshi tamura , , ichiro fujita , graduate school of frontier biosciences, osaka university, osaka, japan; jsps, japan; crest, jst, japan inferior temporal (it) cortical cells are selective for visual shape, and vary in their spontaneous firing pattern among them. cluster analysis indicated that it cells were classified into five groups based on inter-spike interval (isi) histograms of their spontaneous firing. the first two groups showed a single peak at a long or a short isi in isi histograms. the other three had multiple peaks, whose positions and relative heights varied among the groups. principal component analysis and other analyses of visual responses showed that the five groups differed in their stimulus selectivity for a predetermined set of visual stimuli. stimulus selectivity was sharper in the single-peak groups than in the multiple-peak groups. one of the single-peak groups was modulated by natural images more strongly than the other groups. the results suggest that cells with different firing patterns carry different aspects of visual information, and may perform different functions in the coding of visual object images. supported by jsps and crest. research funds: kakenhi - ps a-f spatial-frequency dependency of receptive field size and surround suppression in lgn and v hironobu osaki , tomoyuki naito , osamu sadakane , masahiro okamoto , hiromichi sato , med. sch., osaka univ.; grad. sch. med., osaka univ.; grad. sch. front. biosci., osaka univ., osaka, japan in the primary visual cortex (v ), neurons change their responses depending on stimulus parameters such as orientation, size, spatial frequency (sf). we investigated how sf of stimulus affects on stimulus-size tuning property of responses of neurons in v (n = ) and lateral geniculate nucleus (lgn) (n = ) in anesthetized cats. first, we found that v neurons exhibited shifts of their sf tuning from high to low according to a change in stimulus size from small to large. second, we measured stimulus-area summation curve of responses and found that a higher sf stimulus caused a reduction of the receptive field (rf) size and an increase of the surround suppression. similar results were obtained for lgn neurons implying that the relationship between sf and area summation properties observed in v has its origin in lgn. these results suggest that the sf tuning of rf surround is broader than that of rf center and this center-surround mechanism reduces redundancy in visual information processing. hiroyuki nakamura , akichika mikami , kazuo itoh department of morphological neuroscience, gifu university graduate school of medicine, gifu, japan; department of behavioral and brain sciences, section of neurophysiology, primate research institute, kyoto university, inuyama, japan an extrastriate visual area v a is considered to be involved in the dorsal stream visual areas, however, its connections are not understood. to demonstrate the cortico-cortical connections of v a, we injected a bi-directional tracer biotinylated dextran amine into the v a. our results indicated that the v a has connections with the occipital, parietal and temporal cortices. the v a may thus be involved in the visual information processing of both the dorsal and the ventral stream visual areas. in addition to these connections, we found that v a has commissural connections with the v , the v a, the parieto-occipital area, the dorsal parietal area, and the ventral intraparietal area, and receives commissural projections from the dorsal and ventral aspect of secondary visual area v . these commissural connections may convey ipsilateral visual information near the vertical meridian representations. ps a-g activity of neurons in the isthmo-optic nucleus and its relationship with head movements hiroshi ohno, hiroyuki uchiyama department of information and computer science, faculty of engineering, kagoshima university, kagoshima, japan retinopetal neurons in the isthmo-optic nucleus (ion) send their axons to the contralateral retina in birds. the centrifugal visual projection is thought to be involved in attentional modulation of retinal output. we recorded activity of neurons in the ion in awake, headunrestrained japanese quails using an implanted electrode assembly. head movements were videotaped with a high-speed video camera ( fps), and were also monitored with a d or d accelerometer. we found two distinct types of activity pattern: phasic and tonic. the majority of neurons in the ion discharge in a phasic manner. phasic and tonic cells are also different one from another in relation to head movements. phasic cells show phasic elevation of activity - ms after end of head movements, while tonic cells show tonic suppression during head movements. we will discuss the activity profiles of neurons in the ion in terms of their possible role in visually guided behaviors. ps a-g timing of face specificity in fusiform gyrus responses to stimuli in different parts of the visual field yuka okazaki , , arman abrahamyan , catherine stevens , andreas a. ioannides , brain science institute, riken, saitama, japan; graduate school of life science and systems engineering, kyushu institute of technology, fukuoka, japan; school of psychology, university of western sydney, sydney, australia neuroimaging techniques have demonstrated the preferential responses to faces in the fusiform gyrus (fug). event related potential (erp) and magnetoencephalography (meg) studies have shown that such the responses specificity to faces occurs approximately ms (n ) after stimulus onset by comparing with the other objects. in the present study, we examined whether these and earlier fug activities, which have been already identified by our team (within ms), were selective for face. we achieved this by analyzing meg data elicited by static human faces, hands and shoes stimuli placed in fovea and four quadrants. we found robust statistically significant activities for faces in fug about ms after stimulus onset which depended on the stimulus location in the visual field. narihisa matsumoto , shoutaro akaho , kenji fujikumi , yasuko sugase-miyamoto , masato okada aist, ibaraki, japan; ism, tokyo, japan; university of tokyo, chiba, japan to understand the temporal aspects of information encoded at a population level in the inferior-temporal (it) cortex, we applied a cluster analysis method to the responses of neurons. each response was recorded while one of the visual stimuli that consisted of geometric shapes and faces of humans and monkeys was presented. population activity vectors of neurons for visual stimuli were clustered by a mixture of gaussian model. we estimated the number of clusters by using variational bayes algorithm. we assumed that the probability of the number of clusters depended on the one at one time step before. in the early period, the population vectors formed three clusters corresponding to global categories (human versus monkey versus shape). in the subsequent period, each cluster expanded to form sub-clusters corresponding to detailed categories. moreover, the number of clusters changed smoothly over time. these results suggest that the responses of it neurons represent different levels of categorical signals separated along the time axis. ps a-g relationship between color and shape selectivity in area teo of the monkey masaharu yasuda , , hidehiko komatsu , national institute for physiological science, okazaki, japan; sokendai, okazkaki, japan visual objects typically consist of multiple features such as color, shape, texture etc. it is reported that neurons selective for these object features exist in the inferior temporal (it) cortex of the monkey and some of them are selective for more than one of these features. however, little is known about the relationship between the selectivity for different features. last year, we have reported that there exist many neurons in the posterior part of it cortex (area teo) that are selective for both color and shape. to study the relationship between the color selectivity and shape selectivity, we tested the responses of each neuron using all combinations of the sets of colors and shapes, and conducted svd (singular value decomposition) analysis. we found that some teo neurons exhibited selectivities for color and shape that were independent (separable) each other, whereas in some other neurons they were not independent (nonseparable). these results suggest a possibility that color and shape informations interact at cellular level in this area. ps a-g neural correlates of stimulus shape detection in monkey inferior temporal cortex taijiro doi lab. cogn., neurosci., osaka univ., japan we searched for a neural "correlate" of conscious perception of shape by recording neuronal activities from inferior temporal (it) cortex while a monkey performed a -choice shape detection task. the monkey was required to judge whether or not a sample stimulus was presented immediately after a forward masking stimulus. when there was, the monkey was required to select the stimulus identical to the sample from three targets, two shapes and one small dot. trial-totrial variation of firing rates of many it neurons correlated with the monkey's seen versus not-seen choices. the mean choice probability (cp) of it neurons was . , a value significantly larger than the chance level. neurons with stronger visual responses exhibited larger cps. we also searched for temporal firing patterns within the spike train from a single neuron or across - simultaneously recorded neurons, but failed to find any temporal structure related to the monkey's behavioral choice. the results indicate a link between the firing rates of it neurons with conscious perception of stimulus shape. research funds: mext grant ( ) ps a-g behavioral visual performance of the zebrafish mutant, eclipse yuko nishiwaki , atsuko komori , tomonori manabe , toshihiko hosoya , hiroshi sagara , emiko suzuki , hitoshi okamoto , ichiro masai masai initiative research unit, riken, wako, japan; riken bsi, wako, japan; ims, university of tokyo, minato-ku, japan eclipse was identified as a visual zebrafish mutant that does not show both electroretinogram and optokinetic response. in the last meeting, we reported that the els gene encodes the ␣ subunit of cgmp phosphodiesterase (pde c), which functions in phototransduction in cone photoreceptors. since genetic mutations of pde c have not been reported in human patients of hereditary eye diseases, the els mutant is a good model for studying physiological roles of pde c. here we investigated whether the structural integrity of photoreceptors and visual sensitivity are affected in the els mutants. our electron-microscopic analyses revealed that photoreceptors do not undergo degeneration and are maintained in the els mutant until day-post-fertilization. however, we found that visual response to the contrast is slightly affected in larvae heterozygous for the els mutation. these data suggest that the level of pde c activity is important for the sensitivity of vision. ps a-g localisation of two markers of oxidative phosphorylation in the ageing human retina: an immunohistochemical study tapas nag, shashi wadhwa aiims, india the enzymes of oxidative phosphorylation are known to be affected by reactive oxygen species, which cause mutations in them, leading to reduced energy production. we examined the distribution of two markers of oxidative phosphorylation (nadh-ubiquinol oxidoreductase and cytochrome c oxidase) in the human retina at different ages. eyeballs of donors (age: - years) were fixed in paraformaldehyde, frozen retinal sections from macular to midperipheral regions cut and immunolabelled for nadh-ubiquinol oxidoreductase (complex i) and cytochrome c oxidase (complex iv; molecular probe, usa). complex i-immunoreactivity (ir) was moderately present in photoreceptors, outer plexiform layer and few ganglion cells from to years of age, and showed a decline and lack of ir in older retinas ( - years). complex iv-ir was intensely present in most ganglion cells, outer plexiform layer and photoreceptors from to years of age, and absent at years of age. thus, complex i and iv-ir decline with age, with the former showing an earlier reduction in its ir. the data signify a reduced mitochondrial activity in the retina with ageing. research funds: aiims ps a-g temporal characteristics of neural activity related to target detection during visual search tomoe hayakawa , norio fujimaki , toshihide imaruoka nict, kobe, japan; kit, kanazawa, japan meg and fmri experiments were conducted during the orientation singleton search task, and moment magnitudes of dipoles were estimated with an fmri-constrained meg-multi-dipole method to obtain differences between target-present and -absent conditions in each brain region for the whole time course. activity around the cas consisted of a prominent and a subsequent smaller but still obvious peak ( , ms); the first peak showed no difference between conditions while the second peak was significantly larger in the target-present. activity around the pfug had a prominent peak and subsequent small activity ( , ms), whereas the target's presence or not had no influence on either activity. the activity of the right intraparietal sulcus (ips) was significantly larger than that for the left ips at latencies around ms irrespective of the target's presence or not. the results demonstrate that neural activities of multiple regions had different temporal characteristics and the later activity around the cas was related to the target segregation from its surroundings. kaoru amano , , derek arnold , alan johnston , tsunehiro takeda univ. tokyo, chiba, japan; ntt cs lab., kanagawa, japan; univ. sydney, sydney, australia; ucl, london, uk when a moving border defined by small luminance changes (or by color changes) is shown in close proximity to moving borders defined by large changes in luminance, the low contrast border can appear to jitter at a characteristic frequency -a phenomenon we refer to as misc (arnold & johnston, ) . in order to reveal the neurophysiological substrates of this illusion, brain activities measured using magnetoenceohalography (meg) were compared with the perceived rate of illusory jitter measured psychophysically. the result showed that the perceived rate was around hz and matched with the alpha frequency of meg. as hz meg responses were enhanced in the presence of illusory jitter relative to the presence of isoluminant motion and physical hz jitter, we believe that the activity is related to illusory jitter generation rather than to jitter perception or to isoluminant motion per se. these results support our hypothesis that misc is generated within cortex by the dynamic characteristics of a cortical feedback circuit rather than by any physical stimulus properties. ps a-g the internal structure and the visual neuron projection patterns of the ventrolateral protocerebrum (vlpr) in the drosophila central brain kazunori shinomiya , , kei ito , , center for bioinform., imcb, univ. of tokyo, tokyo, japan; dept. comput. biol., grad. sch. frontier sci., univ. of tokyo, kashiwa, japan; bird, jst visual information processing in the insect brain has so far been analyzed mainly within the optic lobe. many visual pathways are known to project from the optic lobe to a central brain area called the ventrolateral protocerebrum (vlpr). the vlpr is therefore expected to be one of the major higher-order visual centers. the neural circuits in this area, however, remain essentially unknown. our study is to reveal the detailed internal structure of the vlpr, for the first time, using the drosophila brain as a model system. we have identified discrete glomerulus-like structures (gls) in the vlpr, among which at least five are innervated by the visual projection neurons from the optic lobe. we analyzed the detailed internal structure of these gls by visualizing single cells in each visual pathway using the combination of the gal enhancer-trap and the flp-out systems, and revealed the directionality of each pathway by specifically labeling the pre-and post-synaptic terminals. ps a-g dynamic reorganization of orientation maps in a late phase of the sensitive period kazunori o'hashi , , toshiki tani , shigeru tanaka , graduate school of life science & systems engineering, kyushu institute of technology, japan; laboratory for visual neurocomputing, brain science institute, riken, japan we have found that there are two phases in the sensitive period of orientation plasticity: an early irreversible phase and a late reversible phase. in this study, we attempted to elucidate how orientation maps are reorganized in the late reversible phase, performing intrinsic signal optical imaging several times from the same kittens. we observed the over-representation of the exposed orientation even one day after the onset of goggle rearing around the age of weeks. we also found that when the goggles were removed after or weeks of goggle rearing, drastically reorganized orientation maps returned to regular orientation maps that had been established before goggle rearing. these results suggest that once established orientation maps in an early phase serve as template maps to which later rapidly reorganized orientation maps are restored by the release of single orientation exposure. manavu tohmi, seij komagata, yamato kubota, masaharu kudoh, katsuei shibuki department of neurophysiology, brain research institute, niigata university, niigata, japan fourier analysis of intrinsic signals produced by periodic visual stimuli has been applied for constructing retinotopic maps (kalatsky and stryker, ) . in the present study, we used fourier analysis of flavoprotein fluorescence signals for constructing retinotopic maps in the mouse visual cortex. periodic bar stimuli that moved across the visual fields produced periodic fluorescence signals in the visual cortex of anesthetized mice. the fourier components of the signals locked with the periodic stimuli were calculated in each pixel regarding the magnitude and phase. retinotopic maps were constructed based on these components. vascular artifacts could be removed when the stimulus frequency was higher than . hz, since fluorescence signals but not vascular responses could follow up to these frequencies. combination of flavoprotein fluorescence imaging and fourier analysis is a powerful tool for investigating high-resolution retinotopic maps with short acquisition time in the mouse visual cortex. yoshitake kohei, manavu tohmi, masaharu kudoh, katsuei shibuki dept. neurophysiol., brain res. inst, niigata univ., nigata, japan we have reported that ocular dominance plasticity induced by monocular deprivation can be visualized in mice using transcranial flavoprotein fluorescence imaging. another condition for producing ocular dominance plasticity is strabismus, which causes an increase in the proportion of monocular cells in the visual cortex. however, this possibility has not been tested in mice, mainly because surgical operations for producing large and stable shifts in eye position are difficult in mice. in the present study, we designed a new prism goggle for mice. this goggle was attached on the skull of mice during the critical period. the neural responses in the visual cortex of these mice were investigated using transcranial flavoprotein fluorescence imaging. preliminary experiments suggested that the responses in the monocular zone of the visual cortex were not affected in the strabismic mice. however, binocular interaction, which was additive in the binocular zone of normal mice, turned to be more repulsive in the strabismic mice. ps a-g retinotopy-based morphing of brain activity hiroshi ban, hiroki yamamoto, jun saiki graduate school of human & environmental studies, kyoto university, kyoto, japan the topographic visual field map is a fundamental property of the primate early visual cortex. we propose a new method to represent and sample topographic activities in the space of visual field by extending our previous study (maeda et al., . neurosci. res.) . the procedure was as follows. first, eccentricity and visual angle representations were measured for each subject using standard phase-encoding stimuli. second, individual cortical surfaces were reconstructed. third, the transformation between the position in the visual field and that on the cortical surface was established. finally, by using this transformation, brain activities were sampled and then displayed as an image spanning visual field dimensions, each pixel of which represents the activity of neurons representing a given position in the visual field. this retinotopy-based morphing is useful to analyze brain activity related to spatial and form vision and is more reasonable to integrate individual data than normalizing methods based on stereotaxic coordinates and anatomical structures. masahiro yamada , yasuhiro enami , hiroshi jouhou , takehiko saito , kaj djupsund tokyo metropol. univ., hino, tokyo; astellas pharma. inc., osaka, japan; suny upstate med. univ., center for vision and ophthal., ny, usa; univ. kuopio, dept. neurobiol., kuopio, finland on-off type amacrine cells are intensely connected with each other by gap junctions (gjs), forming a syncytium with a wide receptive field. we studied effects of external ph (ph ) on the control of cell functions. photoresponses of the cells were recorded intracellularly. slits of light stimuli simplified the estimation of the current flow in the cellular network into a one-dimensional problem. by lowering ph only . units from the baseline of . , we found a remarkable reduction of the conduction velocity by - %, an increase of the length constant and a hyperpolarisation of the resting potential. based on our theoretical model, combined with measurements of conduction velocity and length constants of the receptive field, we could estimate both gj and plasmamembrane conductances of the cell. thus, we suggest that protons could contribute to the reduction of conductances, especially at the plasmamembrane but also at gjs. ps a-g analysis of the band-pass filtering of the retinal rod by the ionic current model it is known that the rod network behaves like a band-pass filter. it was found that the time to peak of the response was shorter in rods further away from a slit of light. the band-pass filtering behavior has been attributed to an inductance element, i h , or i k(ca) . however, biophysical mechanism underlying the band-pass filter is not fully understood. to analyze the functional roles of ionic currents in the band-pass properties of rods, a model of the rod network was developed. the model incorporates much of the known parameters in rods, i.e., the phototransduction cascade, ionic currents (i ca , i kv , i k(ca) , i h , i cl(ca) ), calcium system and gap junctions between rods. in simulation, the band-pass properties of the rod was analyzed. it was found that single rod itself behaves as a band-pass filter. the mechanism underlying the band-pass filter was examined by changing model parameters. the result suggests that i k(ca) , i cl(ca) and i h are responsible for the bandpass filtering. research funds: kakenhi ( ) ps a-g stimulus selectivity and correlated spontaneous activity of distant neurons in monkey inferior temporal cortex go uchida, mitsuhiro fukuda, manabu tanifuji bsi, riken, wako, japan in inferior temporal (it) cortices of anesthetized macaque monkeys, we have previously shown that spontaneous spike activities (sas) of % ( of ) of neuron pairs (inter-neuronal distance > m) are significantly correlated. in the present study, to investigate how the correlated sas relate to functional structure in it cortex, we measured stimulus selectivity for each neuron of the pairs and explored similarity of stimulus selectivity by calculating correlation coefficients of responses to visual stimuli. this analysis revealed that the pairs with correlated sas tended to show more similar selectivity than the pairs lacking correlated sas. in addition, model analysis showed that in % ( / ) of the pairs the correlation of sas reflect synchronous transition between two activity states: periods with high and low mean firing rates. these results suggest that a network underlying the synchronous state transition provides circuitry that functionally connects distant it neurons showing similar stimulus selectivity. toshiyuki ishii , , toshihiko hosoya bsi, riken, japan; dept. biomolecular science, toho univ., japan understanding the significance of single spikes can be of critical importance in the analysis of neuronal information coding. it is often assumed that the firing rate is the sole carrier of information. however, if fine temporal patterns of spikes would carry information, the system could have large encoding efficiency. the vertebrate retinal ganglion cells fire burst spikes, separated by hundreds of milliseconds of silent periods. here we show that temporal patterns of spikes within these bursts carry visual information. when three or more spikes are fired, the multiple interspike intervals encode the input in a cooperative, non-redundant manner. this suggests that the spike patterns are not sorely determined by slowly modulating instantaneous firing rates. we also found that millisecond-scale structures in the spike patterns encode light intensity waveforms over ms. we propose that the retina compresses hundreds of milliseconds of light sequences into spike patterns at the scale of milliseconds. kazuhiro shimonomura, takayuki kushima, tetsuya yagi osaka university, osaka, japan purpose of this study is to design a neuromorphic hardware model that emulates fundamental architecture and function in the primary visual cortex (v ). we have constructed a binocular vision system consisting of two silicon retinas and simple cell chips and fpga circuits. the silicon retina has a concentric center-surround laplacian-gaussian-like receptive field. the output image of the silicon retina is transferred to the simple cell chips. the simple cell chip aggregates analog pixel outputs of the silicon retina to generate an orientationselective response similar to the simple cell response in v . this architecture mimics the feed-forward model proposed by hubel and wiesel, and computes physically a two-dimensional gabor-like receptive field. the fpga circuits compute complex cell responses based on the disparity energy model. the system can emulate the neural image of the binocular complex cells responding to natural scene in real-time and is useful to verify computational models of v neurons. masayoshi tsuruoka , masako maeda , bunsho hayashi , ikuko nagasawa , tomio inoue dept. physiol. showa univ. sch. dent. tokyo, japan; dept. anestesiol. showa, univ. sch. dent. tokyo, japan the present study investigated the involvement of ventral root looping afferent fibers in visceromotor function. under halothane anesthesia, the t -l dorsal roots were cut bilaterally to eliminate thoracolumbar influences. an electromyogram (emg) of the external abdominal oblique muscle evoked by colorectal distention was measured. colorectal distention ( mmhg) was produced by inflating a balloon inside the descending colon and rectum. emg activity evoked by colorectal distention significantly increased when the colon was inflamed with mustard oil ( %, ml). the increased emg activity significantly reduced following bilateral l -s ventral rhizotomies. a baseline emg did not significantly alter when the l -s ventral roots were cut bilaterally prior to inflammation. following the development of inflammation, there was less of an increase in emg activities. these results suggest that looping afferent fibers in the ventral root are involved in visceromotor function during colon inflammation. ps a-g hypnotic modulation of the cerebral processing of human visceral sensation using positron emission tomography using positron emission tomography (pet), we examined cerebral processing to visceral perception during neutral, hyperalgesic or analgesic suggestion with standard hypnosis. activation within right dorsolateral prefrontal cortex (dlpfc) and right inferior parietal cortex (ba ) was significantly greater (p < . , uncorrected) during rectal distention with analgesic suggestion than with neutral suggestion. on the other hand, activation within right medial frontal cortex (mpfc) was significantly greater (p < . , uncorrected) during rectal distention with hyperalgesic suggestion than with neutral suggestion. this is the first evidence with pet for a modulation of cerebral processing during visceral stimulation by hypnotic suggestion. these results suggest a role of dlpfc and mpfc in the cognitive control of the interoception. the participation of bladder receptors sensitive to cold temperature has been proposed in overactive bladder for decades. bladder cooling reflex (bcr) which consists of immediate sense of urgency and detrusor contraction in response to ice water infusion may be a neuropathic cause of detrusor overactivity (do). recently, urothelial cells display a number of properties similar to sensory neurons and have many sensors including gene for transient receptor potential (trp). we detected cold sensitive receptor trpm in the urothelial cell by immunofluorescence in an animal model for boo. intravesical administration of trpm agonist (l-menthol: . - mm) in freely moving rats, increased the micturition pressure (mp) in either normal (n = ) or boo rat (n = ). the micturition interval (mi) did not change in normal rat, but decreased in boo that have do. the results suggest that bcr is enhanced in boo by increasing trpm on the urothelium cell of the urinary bladder. ps a-g caudate projection from the vagal responsive site in the thalamic parafascicular nucleus in monkeys shin-ichi ito , a.d. craig dept. physiol, shimane univ. sch. med., izumo, japan; atkinson res. lab., barrow neurol inst, phoenix, usa we investigated efferent projections to the forebrain, from the vagal afferent activation focus in the thalamic lateral parafascicular nucleus (pf) (ito & craig, j neurophysiol ) . evoked potentials were mapped in the right thalamus from stimulation of the left cervical vagus nerve, and fluorescent dextrans were iontophoretically injected at the response focus. the injection sites were all located in the ventrolateral part of caudal pf, lateral to the habenulointerpeduncular tract, medial to the basal ventromedial nucleus, and ventromedial to the centre median. labeled terminals were found in the caudate nucleus (cd) in all cases. terminal patches extended longitudinally in the head of cd, concentrated in its ventral aspect. dense terminal patches also occurred throughout the tail of cd. these results suggest that visceral information modulates the portion of the striatum that has been implicated in cognitive function, and they implicate the caudate nucleus in the control of heart rate and respiration. research funds: nih grant ns ps a-g ascending general visceral sensory pathways to the telencephalon via the medial inferior lobe in a percomorph teleost, tilapia masami yoshimoto, naoyuki yamamoto, chun-ying yang, hironobu ito, hitoshi ozawa department of anatomy and neurobiology, nippon medical school, tokyo, japan general visceral sense is relayed to the telencephalon via thalamic and hypothalamic centers in mammals and birds. in teleosts, an ascending connection that corresponds to the thalamo-telencephalic pathway is present. however, it remained unclear whether or not a hypothalamo-telencephalic pathway exists in teleosts. the medial inferior lobe (mil), which corresponds to part of the hypothalamus of other vertebrates, is known to receive general visceral sensory inputs from the rhombencephalon in a percomorph teleost tilapia. hence, telencephalic connections of the mil were studied in this study. tracer injection experiments into the mil revealed that this hypothalamic zone projects to the preoptic area, the ventral telencephalon (i.e., vs, vd, and vv), and the dorsal telencephalon (i.e., dm, rdc, and dl). these findings suggest that the mil corresponds to hypothalamic relay zones in mammals (e.g. ventromedial hypothalamic nucleus). tatsushi onaka, yuki takayanagi department of physiology, jichi medical university, tochigi, japan administration of prolactin releasing peptide (prrp) decreases food intake. we have previously shown that an icv injection of anti-prrp antibodies increases food intake. neurones producing prrp are activated after peripheral administration of cholecystokinin octapeptide, a satiety factor. it is thus possible that prrp may mediate satiety signals in the brain. here we examined effects of anti-prrp antibodies upon total amounts of food intake and meal patterns. an icv injection of anti-prrp antibodies increased the total amounts of food intake and amounts of food intake during a meal but did not significantly change meal frequency. these data suggest that prrp may play an important role in the short-term control of food intake and are consistent with a hypothesis that prrp is a satiety signal within the brain. research funds: grant-in-aid for scientific research (c) ps a-h fasting induced long-chain fatty acid receptor gpr expression in the anterior pituitary of mouse ryutaro moriyama, shingo imoto, shinya shano, nobuyuki fukushima department of life science, kinki university, higashiosaka, japan g-protein-coupled receptor (gpr ) is known as a receptor for unsaturated long-chain fatty acids. the present study investigated the effect of h fasting on gpr expression in several regions of male mouse by real-time quantitative pcr, in situ hybridization and immunohistochemical method. gpr mrna expression was highly observed in the anterior pituitary, lung, colon, rectum, skeletal muscle, adipose tissue and testis in normal fed animals. h fasting induced gpr mrna expression increase in the anterior pituitary, lung and rectum. in the anterior pituitary, gpr -like immunoreactive cells were only observed in fasting animals. these results suggest that long-chin fatty acid regulates endocrine function in the anterior pituitary via gpr at least fasting period. ps a-h ketone body sensing cells in the lower brain stem to regulate food intake and reproductive functions kinuyo iwata, mika kinoshita, hiroaki sato, hiroko tsukamura, keiichiro maeda laboratory of reproductive science, graduate school of bioagricultural sciences, nagoya university, nagoya, japan ketone bodies are used for energy in the brain under malnutrition, such as prolonged fasting. we have previously revealed that hydroxybutylate ( hb), one of ketone bodies, sensed by the ependymocytes lining the fourth ventricular walls ( v) in the rat brain to regulate reproductive functions and feeding behavior. the present study was aims to determine if the ependymocytes located on the wall of v respond to the change in hb. change in the intracellular calcium concentration ([ca + ] i ) in vitro was measured in dispersed ependymocytes taken from the v in rats. the present results showed that the [ca + ] i increased in response to hb, but the increase was blocked by ␣-cyano- -hydroxycinnamic acid, which is a monocarboxylate transporter (mct ) inhibitor. immunohistochemistry showed that mct -immunoreactivities were located on the v ependymocytes. these results indicate that the ependymocytes may sense hb through a mct -dependent mechanism. research funds: kakenhi ps a-h comparison of hypothalamic histamine release by leptin in normal mice and high fat diet-induced obese mice tomoko ishizuka, kouta hatano, atsushi yamatodani dept. med. sci. and technol, grad. sch. allied hlth sci., fac med., osaka univ., osaka, japan leptin is a satiety factor which is produced by the white adipose tissue. peripheral administration of leptin decreases body weight and food intake acting on the hypothalamus. circulating concentration of leptin is in proportion to body fat mass, however, in obese humans, elevated concentrations of endogenous leptin cannot prevent the accumulation of the adipose tissue. we previously reported that leptin decreases food intake via the activation of the histaminergic system. in the present study, the effect of leptin on hypothalamic histamine release was compared in normal and high fat diet-induced obese (dio) mice. leptin ( . mg/kg, ip) reduced food intake in normal mice but not in dio mice, suggesting that dio mice have resistance for exogenous leptin like obese humans. the same dose of leptin increased hypothalamic histamine release in normal mice, while it had no effect in dio mice. these results suggest that the lack of the activation of the histaminergic system partly contributes to obesity in leptin-resistant dio mice. tomoya kitayama, yuri onitsuka, katsuya morita, toshihiro dohi department of dental pharmacology, hiroshima university, hiroshima, japan parkinson disease (pd) is neurodegenerative disorder of the substantia nigra accompanied by depletion of dopamine levels. symptoms of pd include disorder of aspiration and mastication, and dysphagia. in this study, rats injected with -hydroxydopamine ( -ohda) resulted in an extension of feeding time and a marked increase in the amount of feed powder on cage floor after clump feeding at weeks after -ohda without affect on number of neuron in solitary tract. these rats were transplanted with neural progenitor cells at mm; anteroposterior, + mm; lateral and − and − mm; dorsoventral from bregma at weeks after -ohda injection. the treatment shortened feeding time and decreased the leavings on the cage floor, as well as achieving decrease of neuronal death in substantia nigra. however, neural progenitor cells were not detected in substantia nigra. these results suggest that transplantation of neural progenitor cells may better -ohda-induced eating disorders via protection of neurons. research funds: grant-in-aid for young scientists b most tools used by nonhuman animals are extension of their effectors (motor-tools), while humans can use a kind of tools as substitute for their sensory organs (sensory-tools). to understand biological bases of using such tools, we trained japanese monkeys to use a tool as an extension of the eyes, and analyzed its learning processes to proceed as follows: ( ) retrieving the food with a rake (a motortool), ( ) retrieving the hidden food with a mirror-attached rake, ( ) using the reflected image of the food on a mirror separated from the rake, placed stationally beyond hidden food, ( ) moving a mirror hung along the rail by hand to find the food, ( ) using a rake with a small camera mounted inside, with which the monkeys searched for the food using the live video image captured by the camera on the monitor. finally, they could use a hand-held camera (a sensory-tool) as a manipulable extension of their eyes. thus, acquisition of using the externalized eyes can be achieved by gradual transfer of their own vision to the distant visual cues via motor-tools to extend their body image. kaori sawada , , shigehiro miyachi , michiko imanishi , masato taira , masahiko takada div. applied system neurosci., nihon univ. sch. med., tokyo, japan; dept. system neurosci., tokyo metropol. inst. neurosci., tokyo, japan to investigate the outflow of information from the temporal lobe to the prefrontal cortex, we injected rabies virus into three prefrontal regions: medial area ( m), dorsal area ( d), and ventral area ( v). the retrograde transsynaptic labeling was examined in the temporal lobe cortex days after prefrontal injections when the second-order neurons were labeled. the labeled neurons were observed in the lateral and medial aspects of the temporal lobe. in the lateral temporal lobe, neuronal labeling from m, d, and v was arranged topographically in and around the superior temporal sulcus. the labeing in the medial temporal cortex was also topographically arranged, such that m, v, and d receive multisynaptic projections from the entorhinal cortex, area , and both, respectively. these results suggest that there are parallel streams of information flow from the temporal lobe to the prefrontal cortex. research funds: crest, japan science and technology agency ps a-h new neural activities of reward anticipation and task errors h. ogawa , h. ifuku , t. nakamura , s. hirata kumamoto kinoh hosp, kumamoto, japan; fac educ, kumamoto univ., kumamoto, japan; nat kikuchi hosp, kumamoto, japan; dept. psych, kumamoto univ. hosp, kumamoto, japan neural activities at reward phase were recorded from the primary (pgc: areas g, & - ) and higher-order (hgc: prco & ofc) gustatory cortices of a monkey engaged in a taste discrimination go/nogo task. a lever had to be pressed after led onset when nacl was delivered, but not to water delivery. reward was given ca s after led offset at correct trials. relations between cues and responses were reversed. of reward-related neurons found, . % showed on type responses and the rest usual expectation responses. three types of on responses were noticed; c-type (n = ) only at correct trial, i-type (n = ) at around possible reward onset only at incorrect trials, and c-i type (n = ) at both. two classes of the c-i type were found; class i increased discharges at correct trials but decreased them at incorrect, but class ii increased them at both. all types were found in both cortices, but most class i were found in pgc and most class ii in hgc. i-type and class ii c-i type may represent error signals and reward anticipation. hiroaki ishida, masahiko inase, akira murata department of physiology, school of medicine, kinki university, japan in the macaque monkey, the ventral intraparietal area (area vip) integrated visual-tactile information in the body centered reference frame. the receptive fields of these neurons mapped on the same body parts in each sensory modality, so this area contributes to own body representation often referred to as body image. recent psychological studies implied that shared body representation of self and other might be required in the brain for social interaction. this means other¸s body image is mapped on own body image in the same neuron. in our experiments, we studied visual-tactile receptive field of the bimodal neuron in vip, then recorded activity during observing the experimenter being touched. some of neurons that had receptive fields anchored on the monkey¸s body showed visual response while the experimenter was being touched on corresponding body parts. the results suggested that bimodal neurons in vip may be related to matching mechanism between own body image and others, then we discussed that this area may contribute to the human social ability such as imitation. daichi hirai , takayuki hosokawa , masato inoue , akichika mikami section of brain sciences, primate research institute, kyoto university, inuyama, japan; department of psychology, tokyo metropolitan institute for neuroscience, tokyo, japan amygdala is involved in stimulus-reinforcement association learning, and have neural responses related to prediction of rewarding and aversive outcomes. however, it remains unclear whether representation of reinforcement value in the amygdala depends on other available outcomes in a given trial block. to elucidate how rewarding and aversive infomation are coded in the amygdala, we recorded single neuronal activity in monkey amygdala during delayed color matching task. we compared the neural responses to cue that rewarding outcome in two different stimulus-outcome conditions; one included electrical stimulus as aversive outcome, and the other included only rewarding outcomes. we found amygdala neurons to code the relative preference of available outcomes in a given trial block. ps a-h neuronal correlates of expectation-evaluation based on previous and ongoing contextual memories in the monkey prefrontal cortex kyoko matsuda, toshiyuki sawaguchi lab. cogn neurobiol, hokkaido univ. grad. sch. med., sapporo, japan to expect future events based on the ongoing context and to evaluate it are important for flexible control of goal-directed behavior. to examine a possible involvement of the lateral prefrontal cortex (lpfc) in such functions, we recorded neuronal activity from the lpfc of monkeys that performed an oculomotor task. in this task, the target of a saccade was indicated by combinations of successively presented two cues; symmetrically allocated two objects (cue ), and centrally allocated one of the objects presented in cue (cue ). the frequency of which object was presented as cue , i.e., task context, was manipulated across blocks. we focused on cue period and found that a subset of neurons showed object preference depending on current task context (cc type) or previous task context (pc type). cc type and pc type activities may be neuronal correlates of expectation-evaluation based on current and previous contexts, respectively. thus, neuronal processes for expectation-evaluation based on previous and ongoing "contextual memories" may progress in the lpfc. ps a-h anterior insular cortex neurons in monkey are activated when reward might be delivered, such as occurs in gambling takashi mizuhiki , barry j. richmond , munetaka shidara , grad. sch. of tsukuba univ., ibaraki, japan; neurosci. ri., aist, tsukuba, japan; lab. neuropsychol., nimh, bethesda, usa the human insular cortex has attracted interest because it is activated during risk-taking or decision-making tasks in fmri studies. to identify related neuronal signals, we recorded single insular neurons while two monkeys worked in a reward schedule task in conditions: ( ) a cue is picked at random so it is uncertain whether a correctly performed trial will be rewarded [uncertain condition], ( ) a cue indicates whether the current trial will be rewarded or not [certain condition]. in the uncertain condition / neurons responded in all trials. in the certain condition / neurons responded in the rewarded trials only. of these showed significant differences in firing rate between in the first trials after reward and other trials. these insular neuron responses seem related to reward expectancy and recent reward delivery. these neuronal responses might underlie the activation identified in imaging studies during gambling and decision-making tasks. research funds: kakenhi (priority areas ), aist masamichi sakagami , , kosuke sawa , xiaochuan pan , bsrc, tamagawa university, tokyo, japan; senshu university, kanagawa, japan; presto, jst, japan reward prediction behavior based on integration of associative information was investigated. monkeys were trained to perform a sequential association task with symmetric reward by symbolic delayed matching-to-sample procedure. at first, they learned two sequences of stimuli: a -b -c and a -b -c . after monkeys could acquire the sequences, new pairs of stimuli (i.e., d and d , e and e , etc) were introduced to associated with b or b (d -b , d -b , etc). the asymmetric reward rule was instructed by pairing c (c or c ) with the reward. after this instruction, reward predictive behavior was tested by using trained sequences and new stimuli. monkeys could show reward predictive behavior for not only a and a , which were associated with c and c in trained sequences, but also new pairs of stimuli, which were not directly associated either with c or reward. these results suggested that monkeys could use reward predicting information by integration of association among trained sequences, c-reward association, and new stimuli. research funds: kakenhi ( ), hsfp, presto, jst ps a-h reward predicting activity of prefrontal neuron based on group of stimuli xiaochuan pan , , kosuke sawa , , masamichi sakagami , bsrc, research institute, tamagawa university, japan; presto, jst, japan; department of psychology, senshu university, japan ability to anticipate a reward based on grouped events is important for guiding appropriate behavior. the main purpose of this study is to examine the pfc neuronal mechanism involved in predicting reward using learned associations among groups of stimuli. monkeys performed a sequential association task with symmetric reward. at first, they learned two sequences of stimuli: a -b -c and a -b -c . the asymmetric reward rule was instructed by pairing c (c or c ) with the reward block by block. monkeys were also trained with two different orders of stimuli (b-c-a and c-a-b). out of neurons from the lateral pfc, % showed reward-related activity in the first cue period. and one third of them (sr type) predicted reward only when a preferred stimulus was presented as a first cue. interestingly, the preference was not based on visual properties of stimulus, but on stimulus-group. the results suggest that about % of lateral prefrontal neurons predict reward based on stimulus-groups that were formed through the associative learning. attention evoked by novel stimuli is important for behavioral adaptation to new environment. however, it remains unknown whether the novelty is processed in a specific region of the prefrontal cortex. we trained two monkeys on a pavlovian conditioning task interleaved with an instrumental conditioning task and recorded cell activity from the lateral and medial prefrontal cortex (lpfc and mpfc). in a block of the pavlovian task (pv block), a visual stimulus (cs) was paired with a liquid reward and the trial repeated times. in a following block of the instrumental task, the monkey searched a correct action to obtain the cs as positive feedback. the cs was alternated every pv blocks. in many lpfc cells, responses to the cs were enhanced immediately after the change of cs, while such enhancement was less popular in mpfc. this result suggests that lpfc more contributes to coding of stimulus-novelty than does mpfc. when an outcome of action is uncertain, a top-down attention is directed to the coming outcome. to clarify the neural mechanisms, we trained two monkeys on a task with secondary reinforcers and recorded single cell activity of the medial and lateral prefrontal cortex (mpfc and lpfc). in a pavlovian block (pv block), a visual stimulus was paired with a liquid reward. in a following instrumental block (inst block), the monkey searched a correct action based on the visual feedback. the same visual stimulus as the one presented in the preceding pv block followed a correct action, whereas another visual stimulus followed a wrong action. when the monkey made more than consecutive correct trials, a new pv block started. both mpfc and lpfc cells gradually increased their firing toward the visual feedback when the outcome was uncertain, while the onset of the activity was significantly earlier in mpfc than in lpfc. these results suggest that the top-down attention first occurs in mpfc and propagates to lpfc in individual trials. ps a-h neuronal activity in the presupplementary motor area during a bimanual sequential motor task toshi nakajima , hajime mushiake , jun tanji department of physiology, tohoku university school of medicine, sendai, japan; brain science research center, tamagawa university, machida, japan to investigate the involvement of the pre-supplementary motor area (pre-sma) in organizing bimanual sequential movements, we recorded neuronal activity while a monkey was performing a motor task consisting of pronation or supination of either arm, with an intervening delay. in this report, we focus on neuronal activity during a period when the monkey was preparing to start the -sequence movements in a memorized order. we made regression analysis of neuronal activity in this period. we found that neuronal activity in the pre-sma rarely reflected muscle activity. instead, we found neuronal activity representing forthcoming actions such as supination, regardless of the arm to be used. we also found neuronal activity that reflected the second movement in a preparatory period before the execution of the first movement. we would demonstrate typical examples of pre-sma neurons and discuss their functional implications. ps a-h neuronal activity in the putamen and cm thalamus during response bias and its complementary process yukiko hori, takafumi minamimoto, minoru kimura dept. of physiol., kyoto prefect univ. med., japan we showed previously that cm thalamus participates specifically in complementary process to response bias (minamimoto et al. ) . to study the roles of the putamen and cm in response bias and it complementary processes, we recorded activity of cm and putamen projection neurons from two macaque monkeys performing asymmetrically rewarded go-nogo button press task. instruction of go or nogo activated cm neurons (n = ) preferentially when the instruction was associated with small reward. the instructions activated groups of putamen neurons preferring small reward-(n = ), large reward-action (n = ) and both types of action (n = ). onset latencies of these putamen neurons and rts in large-reward-go trials were shorter than those in small-reward-go trials by - and - ms, respectively. putamen neuron activation lead that of cm neurons by - ms. these results suggested that the putamen plays a major roles in both response bias and its complementary process while cm participates in the complementary process in concert with the putamen. research funds: kakenhi ( ) ps a-h encoding expected total rewards and their errors through a series of action choices by dopamine neurons naoyuki matsumoto, kazuki enomoto, minoru kimura dept. physiol. kyoto pref univ. med., japan to examine how dopamine (da) neurons represent reward expectation and its error through a series of action choices, we recorded activity of da neurons in two japanese monkeys making trial-anderror and repetition choices to find a correct, rewarding target among three alternatives. there are trials of first (t ), second (t ) and third (t ) choices with reward probabilities of about , and %, respectively. monkeys got reward after they hit a correct target, and got one more time by choosing the same target in the next trial (r , %). most da neurons ( / ) responded to the start cue of each trial and reinforcer beep after the choices. magnitude of the start cue responses progressively increased from t to t and to t trials, then decreased in r trial. in another task with two repetition trials (r and r , %), magnitude of start-cue responses decreased gradually from t to r and to r trials. thus, the start cue responses may reflect expected total rewards through a series of action choices for a goal, while reinforcer beep responses may reflect their errors. research funds: kakenhi ( ) ps a-h striatal neuron activity during decisions and action selections for probabilistic, scheduled rewards hiroshi yamada , , hitoshi inokawa , minoru kimura dept. of physiol. kyoto prefect univ. med., kyoto, japan; jsps, japan to study roles of the striatum in decision and selection of actions for probabilistic, multiple rewards, we recorded striatal projection neurons from a monkey. after depressing a start button, the monkey chose of target buttons with correct rates at st, nd, rd and repetition trials of , , and %, respectively. correct choices were followed by reward water. neuronal firing rates at starting each trial were related either to expected reward probability or to schedule states to obtain reward twice ( / ) rather than to upcoming choice of target ( / ). during the target choice, another subset of neurons showed firings selective to choosing particular target ( / ) rather than to expected reward probability ( / ). after the target choices, another group of neurons fired related to expected reward ( / ) rather than to chosen action ( / ). our results suggested that striatal neurons encode expected reward probability, schedule states to obtain multiple rewards and choice of actions during decision and action choices for a goal. research funds: kakenhi ( ), jsps fellows ps a-h encoding of reinforcement after rewardbased action selection by tonically active neurons in the striatum hitoshi inokawa, hiroshi yamada, minoru kimura department of physiology, kyoto pref. univ. of med., japan to study the signals encoded by tonically active neurons (tans) in the striatum, presumed cholinergic interneurons, reward-based decision and action selection, activity of tans was recorded from the putamen and caudate nucleus of a japanese monkey. after depressing a start button, the monkey chose of target buttons at average correct rates of (first), (second), (third) and % (repetition choices). correct and incorrect choices were followed by high-tone beep, reward water and low-tone beep, respectively. about a half of tans ( / ) responded differentially to the high and low tone beep respectively. number of responsive tans and magnitudes of the responses to high-tone beep was highest at the first choices, then, decreased gradually at second, third and repetition choices. these results suggested that the tans may encode reinforcement after reward-based action choices which is modified by reward expectation errors and motivation. research funds: kakenhi ( ) ps a-i representation of value of action, action and its outcome in sub-populations of striate neurons y. ueda , k. samejima , k. doya , m. kimura dept. physiol., kyoto pref. univ. med.; brain sci. res. center, tamagawa univ.; irp, oist to know the mechanisms of reward-based action selection in the basal ganglia, we recorded activity of striatal projection neurons of two macaque monkeys performing a free choice task with probabilistic reward. after a s delay, monkeys chose between left-and right-handle turn, followed by water reward at probability of , or %. a linear regression of neuronal discharge rates showed: neurons encoded reward values of either action during delay period before go signal, with most ( %) of them not having the action value signal in other task epochs. another subset of neurons encoded action signal selectively during action selection after go signal (n = ), while other neurons encoded presence or absence of reward at reinforcer epoch after the action selection. neurons encoding action values were in more anterior part of putamen than the neurons encoding actions. these findings suggested that sub-populations of striate neurons process action values and selection of actions during rewardbased decision and action selection. research funds: kakenhi ( ) ps a-i delay period activity of the monkey striatum in duration discrimination task atsushi chiba, ken-ichi oshio, masahiko inase dept. physiol., kinki univ. sch. med., osaka sayama, japan neuronal activity was recorded from the striatum of a monkey during a duration discrimination task. two visual cues (a blue or red square) were presented consecutively followed by delay periods, and the subject then chose the cue presented for the longer duration. durations of both cues, order of cue duration (long-short or short-long), and order of cue color (blue-red or red-blue) were randomized on a trial-by-trial basis. striatal neurons phasically responded during the first cue (c ), first delay (d ), second cue (c ), second delay (d ), and response periods. activity during the d and d periods was analyzed in this study. firing rates during the d period linearly depended on c durations. on the other hand, d period activity depended on trial types (ls and sl), but not on the variety of c durations in each trial type. our results suggest that striatal neurons encode, in the delay periods, not only temporal information with monotonic dependence on cue durations to prepare a comparison to a forthcoming cue duration, but also encode discrimination results between two cue durations. research funds: kakenhi ( ) ps a-i neuronal activities in the anterior inferior temporal cortex of monkeys during an asymmetrical pair association task based on facial identity satoshi eifuku , ryoi tamura , teruko uwano , taketoshi ono dept. integrative neurosci., univ. toyama, toyama, japan; dept. molecular integrative emotional neuroscience, univ. toyama, toyama, japan to elucidate neuronal basis of face memory, neuronal activities in the area teav of monkeys were recorded during a pair association paradigm that involves recognition of facial identity (i-apa task). in the i-apa task, monkeys were required to memorize paired associates of patterns and facial identity. each association has a particular direction, either the 'face to pattern' direction in which a cue stimulus which is a face is associated with a test stimulus which is a pattern, or the 'pattern to face' direction in which a cue stimulus which is a pattern is associated with a test stimulus which is a face. during the i-apa task, neuronal responses to a particular paired associate were identified. many of these neurons showed asymmetrical activities during the delay periods which were dominant in the 'face to pattern' trials. this asymmetrical delay activity are indicative of the crucial role of the teav area in face memory. research funds: kakenhi ( ) ps a-i reflexive social attention elicited by biological motion in monkeys and humans yoshiya mori , mikio inagaki , wu lisa , taijiro doi , eishi hirasaki , hiroo kumakura , ichiro fujita osaka univ., japan; massachusetts institute of technology, usa determining where another individual is attending and preparing for his/her upcoming action is crucial for members of a social group. here we report that the walking direction of another individual elicits a reflexive shift of visuospatial attention in monkeys and humans. we examined how the reaction time to peripheral visual targets was affected by a prior, brief presentation of a walking biological motion (bm) stimulus. during the task, subjects responded to a target point after the disappearance of the bm stimulus and fixation point. the walking direction of the bm stimulus was not predictive of the target direction, and was irrelevant for performing the task. we found that the reaction times in congruent trials, where the walking direction of the bm stimulus and the direction of the target appearance were the same, were significantly shorter than those of incongruent trials. we believe the attention mechanisms driven by bm may be part of the intentionality inference system. research funds: grants from and takeda science foundation ps a-i response properties of posterior parietal neurons during a multidimensional visual search task tadashi ogawa, hidehiko komatsu natl. inst. physiol. sci., aichi, japan the posterior parietal cortex (ppc) is thought to be one of crucial areas to direct spatial attention toward the target in visual search. visual sensory information (e.g. stimulus features) might be integrated in ppc to form a saliency map that controls spatial attention. to examine this hypothesis, we recorded the neural activity from the lateral intraparietal (lip) and a areas of monkeys performing a multidimensional visual search task. the monkeys had to make a saccade to either shape or color singletons in a stimulus array depending on the instructed search dimension. ppc neurons increased their activity when the receptive field stimulus became the target. some neurons showed target enhancement depending on the stimulus condition (singleton type and stimulus features), whereas others exhibited it irrespective of the stimulus condition. the mixed existence of these two distinct types of activities suggests that ppc is one of critical stages that integrate feature-dependent signals to produce featureindependent signals identifying the target location toward which spatial attention should be directed. monkeys utilize visual information in social communication. to elucidate visual function to categorize sexes, ( ) performance of visually guided sex discrimination task and ( ) neuronal activity during the task in orbitofrontal cortex (obf), the region could be related to sex recognition and vision processes, were investigated. monkeys were trained to discriminate the sex of a monkey shown in a picture that was presented on the display. the monkeys pressed the right bar for pictures of males and the left for females to get water reward. as a result, the monkeys were able to discriminate the sexes of monkeys shown in pictures. extracellular recordings of neurons in obf during the task showed that some cells responded to the pictures in a sexspecific manner. the present results suggest that visual information alone sufficiently contribute to discriminate sex in monkeys. obf could be involved in visual categorization of sex. research funds: kakenhi (a) ( ) (sa) and coe program in kit from the mext ps a-i activities of bursting neurons during color discrimination task in the monkey prefrontal cortex naoki ishikawa , satoshi katai , masanori saruwatari , masato inoue , akichika mikami section of brain sciences, primate research institute, kyoto university, inuyama, japan; third department of internal medicine, shinshu university, school of medicine, matsumoto, japan the neurons in the prefrontal cortex of monkeys are involved in the behavioral control of saccadic eye movements. on the other hand, cerebral cortex consists of different types of neurons. in this study, we trained macaque monkeys to perform a delayed matching to sample task with saccadic eye movement. and we classified neurons whether they had burst episode or not, and then classified bursting neurons into fast spiking (fs), fast rhythmic bursting (frb), and intrinsic bursting (ib) neurons (katai et al. neuro ) . most of bursting neurons activated during the target presentation or during the saccade period were selective to the target location or saccade direction. these results suggest that the bursting neurons have the significant role in the target selection and decision-making of the eye movement toward the specific direction. atsushi matsumoto , tetsuya iidaka department of psychology, nagoya university, nagoya, japan; department of psychiatry, nagoya university, nagoya, japan several studies indicated that gamma band activity (gba: - hz) reflects the process to form mental representation of objects or information. we investigated whether the gba is observed during subliminal visual word processing as well as supraliminal word processing. gba were observed both in masked and unmasked condition. at the - ms time window, gba was significantly higher in the word condition compared to the nonword condition in the unmasked condition. similarly, in the masked condition, gba of the word condition was significantly higher than that of the nonword condition at that time window. these results indicate that the unconscious lexical processing was reflected in the gba at that time window. furthermore, at the - ms time window, gba induced by word was significantly higher than that induced by nonword. this effect was not observed in the masked condition. in addition we found the significant semantic priming effect, indicating that the information of briefly presented words was processed unconsciously. wakayo yamashita, junichi hayashi, tomoki murakami, gang wang department of bioengineering, kagoshima university, kagoshima, japan the purpose of this study was to investigate the dependency of view association learning on the separation of the views. each stimulus set included images ( objects × views). novel objects were generated by deforming a prototype in four directions. for deg-interval object sets, views were obtained by rotating each object with the interval of deg, deg-interval set and deg-interval set were with deg and deg interval respectively. task performances were evaluated while the subjects performed an object matching task, in which the subjects had to recognize one object from others regardless of the viewpoint. the performance across deg separated views was significantly higher in the trials with deg-interval sets than those with deg-interval sets. similarly, the difference was also found in the performances across deg separated views between those with deg-interval sets and deg-interval sets. the results suggest that the exposure of interpolated views significantly improved the association learning of the views. ps a-i brain regional activity during attention task ( the kana pick-out test, treated as inspecting higher brain function, has been proposed to be suitable for screening dementia, which is widely used among public health nurses in japan. however, few fmri studies while demonstrating the test have reported. we therefore assessed the effect of brain regional activity with computerized kana pick-out test projected on the screen with clicking a mouse button to pick kana out under fmri running. executing the test resulted in significant increases in bold signals in right prefrontal area, bilateral hippocampus and broca's area. the results indicate the existence of the attention pathway from and/or to prefrontal area as association mechanisms for execution of kana pick-out test, suggesting that this test is useful in screening dementia. ps a-i obsessive compulsive symptoms in middle school students and its association with tic disorder, body dysmorphic disorder and trichotilomnia in shiraz, iran, ashkan mowla, arash mowla shiraz university of medical sciences, iran aim: the aim of this study is to evaluate ocd symptoms, tic disorder, body dismorphic disorder (bdd) and trichotilomnia (ttm) among middle school students of shiraz, iran. methods: middle school students were selected in a cluster random sampling from the four educational regions of shiraz, iran.persion standardized moci was used to assess obsessional symptoms. for evaluating bdd, tic disorder and ttm symptoms, a semi-structured interview was done according to dsm-iv-tr criteria. results: students with more obsessional symptoms were more girls and demonstrated more positive family history.they were more likely to be from lower socioeconomic class and with lower school average. they also showed more association with body dysmorphic disorder and tic disorder. conclusion: girls especially those from lower socioeconomic class demonstrated more obsessional symptoms. this study, like pervious ones, confirmed bdd symptoms and tics to be more in individuals with ocd symptoms. it was seen that ocd symptoms would affect school performance. ps a-i sirna-induced nr knockdown causes hypofunction of nmda-r and cognitive deficit m. saji , , t. utida , a. ohnishi , k. noda , m. ogata , h. akita , n. suzuki , physiol, health sci. sch. kitasato univ., sagamihara, japan; brain sci., graduate sch. kitasato univ., sagamihara, japan blockade of nmda-r by antagonists causes psychomimetic effects, suggesting involvement of nmda-r dysfunction in mental disorders like schizophrenia. however, the relationship between mental disorders and molecular abnormality has not been cleared. to identify the role of nmda-r in brain function, we performed sirnainduced knockdown of nmda-nr using hvj-envelope vectors. we confirmed that marked down-regulation ( %) of nr expression occurred only in the hippocampus among various brain regions - days after intra-ventricular injection of sirna-vector complex. in the hippocampal slice from rats with the nr knockdown, the nr down-regulation prevented depressive effects of nmda on fepsps, while the treatment did not affect ltp or ltd. in rats with the nr knockdown, the nr down-regulation caused disruption of prepulse inhibition, while the same treatment did not affect locomotor activity. these results suggest that hypofunction of hippocampal nmda-r by sirna-treatment causes a deficit of cognition. ken hatanaka , , hiroshi ageta , ikuko yao , kaoru inokuchi , yutaka kirino , mitsutoshi setou , graduate school of pharmaceutical sciences, the university of tokyo, tokyo, japan; mitsubishi kagaku institute for life sciences, tokyo, japan; okazaki institute for integrative bioscience, national institute for physiological sciences, okazaki, japan schizophrenia is a severe psychiatric disorder that characterized by psychotic symptoms in particular delusions and hallucinations, reduced interest and drive, altered emotional reactivity and disorganized behavior. to know the molecular mechanisms of the disease, we screened altered gene expression on the brain of schizophrenic patients by using microarray analysis, and found that the expression of ubl mrna was significantly decreased in the enthorinal cortex, whose size is known to be reduced in some schizophrenic patients. ubl is a highly conserved protein, which has a ubiquitin-like domain (ubl domain) and caax motif which is a membrane localization signal. we found that ubl mrna was expressed in the hippocampus, and purkingie cells of the cerebellum. the putative molecular function of ubl wil be discussed. research funds: grant-in-aid for young scientists (b), presto ps a-i decreased interneurons in the pax mutant mouse limbic system hasumi haba , tadashi nomura , yoshinobu hara , , noriko osumi , div. dev. neurosci., ctaar, tohoku univ. sch. med., sendai, japan; crest, jst, japan core features of schizophrenia are impairments in certain cognitive functions such as working memory, in which a number of brain regions in the corticolimbic system are involved. recent studies have revealed abnormality in distribution of interneurons in these regions. we have previously found that pax heterozygous mutant rats show behavioral abnormalities including impairment in fearconditioned memory and sensorimotor gating. in the present study, we thus analyzed distribution of interneurons in several regions of pax heterozygous mutant mouse (sey/+) brain. we focused on three subpopulations of interneurons: parvalbumin (pv)-, calretinin-, and somatostatin-posive interneurons. immunohistochemical studies indicated marked decrease in pv-positive interneurons in two brain regions of sey/+ mice, i.e., the olfactory bulb and the amygdala. reduced number of pv-positive interneurons was observed in the sey/+ amygdala at weeks, but not at weeks. our results suggest that age-dependent decrease of pv-positive interneurons might underlie behavioral abnormalities in sey/+ mice. schizophrenia is a complex genetic disorder, characterized by multiple susceptibility genes. dysbindin (dtnbp ) is a susceptibility gene for schizophrenia. genetic evidence for the association between the disorder and the dysbindin gene has repeatedly been reported in various populations world wide. recently, decreased expression levels of dysbindin mrna and protein have been reported in postmortem brain in patients with schizophrenia. thus, we performed behavioral analysis in sandy mouse, which has a deletion in dysbindin gene and expresses no protein. sandy mouse showed decreased locomotor activity and time in the center in the open field test. and an acute treatment of atypical antipsychotic, olanzapine ( . mg/kg, i.p.), improved the decrease in time in the center. moreover, subtle behavioral abnormality was observed in elevated plus maze test and social interaction test in sandy mouse. our results suggest that dysbindin might be involved in anxiety-related behavior in novel environment. research funds: , ps a-i gene expression analysis of dysbindin mrna in peripheral blood in schizophrenia sachie chiba , , satoko hattori , hiroaki hori , tetsuo nakabayashi , hiroshi kunugi , ryota hashimoto department of mental disorder research, national institute of neuroscience; tokyo university of agriculture and technology department of biotechnology and life science, koganei, japan; musashi hospital, ncnp, kodaira, japan although many efforts have been spent to discover a biological marker of schizophrenia, no biological marker has been established. as genetic evidence suggested that dysbindin (dtnbp ) is a susceptibility gene for schizophrenia, we measured dysbindin mrna expression level in peripheral blood samples of patients with schizophrenia and age-sex matched healthy controls by a quantitative real time rt-pcr method. we quantified the expression levels of two major dysbindin transcripts among several known splicing variants. no significant difference in the expression levels of examined dysbindin transcripts was observed between control and schizophrenia. further examination measuring other dysbindin transcripts should be warranted to find a biological marker for schizophrenia. research funds: , ps a-i genetic variation in dysbindin influences memory and general cognitive ability ryota hashimoto , hiroko noguchi , hiroaki hori , tetsuo nakabayashi , satoko hattori , sachie chiba , seiichi harada , osamu saito , hiroshi kunugi department of mental disorder research, national institute of neuroscience, national center of neurology and psychiatry, kodaira, japan; musashi hospital, ncnp, kodaira, japan; tokyo university of agriculture and technology department of biotechnology and life science, koganei, japan dysbindin (dtnbp ) is a susceptibility gene for schizophrenia, a neuropsychiatric disorder characterized by cognitive dysfunction. we examined the possible association between genetic variants in the dysbindin gene and memory and iq in healthy volunteers and patients with schizophrenia. individuals who did not carry a protective haplotype had lower performance in several memory domains wms-r, although this haplotype did not affect iq measured by wais-r. a risk independent polymorphism for schizophrenia influences both memory and iq in the opposite direction. these data suggest that dysbindin gene may have impact on the cognitive function such as memory and iq and that memory might be an intermediate phenotype of dysbindin on risk for schizophrenia. research funds: , ps a-i detection of f-dopa signal in brainstem monoaminergic nuclei in schizophrenia yuri kitamura , nicola bright , toshio yanagida , masatoshi takeda , paul grasby department of physiology, osaka university, japan; department of psychiatry, osaka university, japan; cyclotron unit, imperial college, hammersmith hospital, uk we used f-dopa pet to investigate presynaptic dopamine dysfunction in schizophrenic patients. the object of this study was to test that a schizophrenic cohort would show elevated aadc activity in the substantia nigra, midbrain raphe and locus coeruleus compared to normal controls. all subjects and f-dopa scans were obtained from a database of scans published in mcgowan et al. , archives general psychiatry. the schizophrenic patients all met dsm-iv criteria on medication and healthy volunteers were compared. we attempted to improve the quality of the f-dopa signal by implementing a fbf-realignment movement correction method. significant increases in f-dopa uptake were found in the striatum, substantia nigra and raphe nuclei of schizophrenic patients (p > . ). our result suggests that an elevated presynaptic dopamine function is present in dopaminergic neurons that innervate striatal areas associated with enhanced dopamine activity in schizophrenia. in this study, we analyzed the p component of the visual eventrelated potential in patients with schizophrenia and healthy controls, and also performed loreta analysis. the ethics committee of kurume university approved this study. the p amplitude for the crying face was significantly smaller in patients than in controls. in controls, the p amplitude was significantly larger for the crying face than for the laughing face, while in patients, there was no significant difference in the p amplitude between the faces. loreta analysis demonstrated that there were significant differences in the activity in brodmann area between the faces in controls, while in patients, there was no significant activity difference between the faces. stimulation with crying face induced higher activities in the and right areas in controls than in the patients. these results indicated that the cognitive function was influenced by affective stimulus. ps a-j inappropriate input produces schizophrenialike working memory deficits in a simulated neural circuit kensuke nomura , shoji tanaka , koki yamashita , motoichiro kato , haruo kashima department of neuropsychiatry, school of medicine, keio university; department of electrical and electronics engineering, sophia university a number of studies indicate that the prefrontal cortex (pfc) is intrinsically linked to working memory (wm) and that dopamine critically modulates wm activity. according to the hypothesis proposed by goldman-rakic and her colleagues, we constructed an electrophysiological circuit model for wm which represents eight directions. the computer simulation with this model shows that the working memory activity is dampened by cue-irrelevant inputs and greater noise inputs lose the directional selectivity of the representation. a lot of studies suggested that increase of noise was related to schizophrenia, especially in wm disturbance. our study indicates that noise inputs cause wm impairment in patients with schizophrenia and that working memory performance is not always positively correlated with the neuronal activity of the pfc. ps a-j pericentrin is localized to the base of neuronal primary cilia in the developing cerebral cortex ko miyoshi, ikuko miyazaki, masato asanuma department of brain science, okayama university, okayama, japan we previously identified pericentrin, a mammalian centrosomal protein, as a binding partner of the product of disc , a candidate gene for schizophrenia. in this study, we analyzed in vivo expression of pericentrin in the mouse embryo. in the developing cerebral cortex, pericentrin mrna was highly expressed in migrating cells of the intermediate zone, though proliferating neuroepithelial cells and mature neurons revealed a low expression level of pericentrin. the pericentrin protein was shown to be localized to the base of primary cilia in the pre-plate of the developing cerebral cortex, in agreement with a recent study demonstrating the involvement of pericentrin in primary cilia formation. specific subtypes of receptors such as -ht are known to be localized to the plasma membrane of neuronal primary cilia in certain regions of the brain, and then our results raise the possibility that pericentrin dysfunction may result in perturbed chemosensory function of neuronal primary cilia and increased vulnerability to psychiatric disorders. dysregulation of gr has been thought to play an important role in the pathophysiology of mood disorders. two isoforms of human gr-alpha and -beta arise from alternative splicing of the pre-mrna primary transcripts. previously, we evaluated these two isoforms mrna level in the peripheral white blood cells of the patients with mood disorders. we found that the reduced gr-alpha mrna level in the patients with both bipolar and major depressive disorders, while gr-beta mrna level was not altered. these results suggest that dysregulation of alternative splicing play an important role in the pathophysiology of mood disorders. to test this, we evaluated mrna level of alternative splicing-related sr protein family, which regulate alternative splicing in several genes including gr, in the peripheral white blood cells of the patients with mood disorders. we did not find any differences in of the sr protein mrnas level in the patients compared to healthy controls and now, we are examining other sr family mrnas level. ps a-j alteration of neocortical long-term depression following electroconvulsive shock yoshifumi ueta , ryo yamamoto , shigeki sugiura , kaoru inokuchi , nobuo kato dept. integrat. brain sci., grad. sch. med., kyoto univ.; nara med. univ.; mitsubishi kagaku inst. life sci. electroconvulsive therapy is useful in treating drug-resistant depressive disorders, though its mechanism remains unclear. there have been a few reports that studied effects of electroconvulsive shock (ecs) on long-term potentiation. however, its effects on long-term depression (ltd) have not been investigated to date. the present experiments examined roles of ecs in inducing ltd at a variety of corticocortical synapses in rat cortex slices by using whole-cell patch clamp. following ecs, ltd magnitude at layer ii/iii-to-vi pyramidal cell synapses was significantly reduced in comparison to no-ecs subjects. as described in recent microarray studies, homer a/vesl- s was identified as one of the most up-regulated molecules after ecs. we therefore injected homer a protein by diffusion from patch pipettes. homer a injection, as well as with ecs treatments, reduced ltd magnitude only at layer ii/iii-to-vi pyramidal cell synapses, implicating that homer a may be a biological mediator of ecs effects. masanori kasai , nozomi miyagi , norio kawashiro , daisuke torizuka dept. of chem. & biosci., faculty of sci., kagoshima univ., kagoshima, japan; sanko shokuhin co., ltd., tokyo, japan it is well known that zinc is an essential mineral necessary for a multitude of body functions, including acuity of taste. to know a change of serum level in adjuvant-induced inflammation, we measured a zinc level in serum from male lewis rats received a suspension of complete freund's adjuvant ( . mg), injected intradermally into the tail. body weight, food intake and water intake were also measured. all rats showed signs of systemic inflammation (weight loss, hind paw swelling, nodules around eyes and penis) after the th day. the rats were sacrificed to measure the serum mineral contents (zn, na, cl, p, ca, k, mg) on the nd, th, th, st, th and th days. the serum zinc level was decreased on all of the measurement and the average of serum zinc ( . ± . g/dl, n = ) on the the day was significantly lower than that in intact rats ( . ± . g/dl, n = ). this decrease of zinc was correlated with weight loss but not hind paw swelling. other minerals did not show any significant changes throughout the measurement period. ps a-j molecular cloning of a novel candidate for ethanol-responsive genes, yy ap-related protein (yarp), in rat brain in order to elucidate the molecular mechanisms of etoh action on the cns, we investigated changes in gene expression in the adult rat brain after chronic etoh treatment. by means of cdna subtraction, we identified a candidate for etoh-responsive genes in the hippocampus. cdna cloning and sequence analysis revealed that this gene encodes a novel homolog of yy ap (yy -associated protein) and is well conserved in rats and humans. homology search for functional domains predicted that the yarp polypeptide contains nlss', a dnabinding motif, and a chromatin decondensation domain, as well as yy -binding and transactivation domains previously demonstrated in yy ap. in the brain, neurons such as hippocampal pyramidal cells were stained by in situ hybridization, and co-expression of yarp and yy genes was demonstrated in the same neurons. analogous to yy ap as a co-activator of transcription factor yy , it is postulated that yarp can regulate cerebral gene expression in response to etoh treatment. ps a-j excitotoxic degeneration of hypothalamic orexin neurons: involvement of nr b-containing nmda receptors and rescue by gaba a receptor stimulation hiroshi katsuki, shinsuke kurosu, toshiaki kume, akinori akaike department of pharmacology, graduate school of pharmaceutical sciences, kyoto university, kyoto, japan selective degeneration of orexin neurons, a pathological hallmark of narcolepy, is in part reproduced in hypothalamic slice cultures by application of quinolinic acid (qa), an endogenous nmda receptor agonist. we report here that nr b-selective nmda antagonists ifenprodil ( and m) and ro - ( . and m) markedly inhibited degeneration of orexin neurons induced by h application of nmda ( m) or qa ( . mm). we also show that stimulation of gaba a receptors by muscimol ( and m) or isoguvacine ( and m) potently inhibited qa cytotoxicity. in addition, the protective effect of gaba ( m) plus a gaba uptake blocker nipecotic acid ( mm) was abolished by a gaba a antagonist picrotoxin ( m). norepinephrine and serotonin did not provide a neuroprotective effect. thus, gabaergic inhibition may be decisive on survival of orexin neurons under excitotoxic stimuli mediated by nr b-containing nmda receptors. yoshika kurokawa, shinji tsukahara, hidekazu fujimaki national institute for environmental studies, tsukuba, japan to evaluate neurotoxicological influence of volatile organic chemicals (vocs), such as toluene, on hippocampal function, we attempted to develop an in vivo optical imaging technique for the hippocampus of mice with or without receiving voc inhalation. we dissected out the cerebral cortex in mice anesthetized with pentobarbital in order to prepare an optical window for monitoring the dorsal surface of the hippocampus, and stained the hippocampus with voltage-sensitive dye (rh ). we then monitored optical signals responding to electrical single-pulse stimulation to the parahippocampal region or hippocampal formation with a time resolution of ms. we also examined optical signals in the hippocampus during toluene inhalation. as a result, neural excitation of the superficial layer was observed in the hippocampal formation after electrical stimulation. on the other hand, acute perinasal exposure of toluene gas did not alter any signal pattern in the hippocampal formation. we will discuss the usefulness of this technique for examination of the neurotoxicological influence of vocs. ps a-j a simple method for fabricating electrodes array for multichannel neural recording -investigation of the alignment of the array and the measurement system-noriyuki taniguchi , osamu fukayama , takashi sato , takafumi suzuki , kunihiko mabuchi , dept. biomed. eng., univ. tokyo, tokyo, japan; dept. info. physi. comp., univ. tokyo, tokyo, japan various types of electrodes have been developed for use as brain-machine interface (bmi) to record signals from neurons. electrode arrays can be purchased from vendors. however, economic considerations and the adjustment of the array alignment for experimental design still make it worthwhile to develop fabrication methods inhouse. thus we developed a low-cost multichannel microwire array electrodes for recording from the cerebral cortex of conscious rats. the electrodes were able to align for the experimental paradigms. the effectiveness of the arrangement of the array as a bmi device was investigated. the electrodes were implanted in the primary motor cortex of wistar rats. we used a wheel-formed rat exercising kit to measure the walking speed of a rat. the neural signal of the rat and the rotating speed of the wheels were simultaneously recorded. and we evaluated the estimation of the walking speed by multiple electrodes with different alignments. ps a-j on-chip electrophysiological measurement of artificially constructed single-cell based neuronal networks ikurou suzuki , yasuhiko jimbo , kenji yasuda department of life sciences, graduate school of arts and sciences, university of tokyo, tokyo, japan; department of precision engineering, graduate school of engineering, university of tokyo, tokyo, japan we have developed a single-cell-based on-chip um-diameter multielectrode arrays with an agarose microchambers (amc) for topographical control of the network patterns of living neurons. this system enables flexible and precise control of the cell positions and the pattern of connections through photo-thermal etching. and sampling rates of measurement are khz in ch electrodes simultaneously. using this system, we formed a single-cell-based neural network pattern of rat hippocampal cells within the amc array and controlled the growth direction of axon/dendrite selectively using photo-thermal etching methods during cultivation, and recorded the spontaneous firings and evoked responses. moreover, we identified propagation along patterned neural network and found the effects of tetanic stimulation within this neural network. in the meeting we will present the results in detail and will discuss the potential of our method. yuichi yamashita , tetsu okumura , kazuo okanoya , jun tani lab. for behavior & dynamic cognition, riken-bsi, japan; lab. for biolinguistics, riken-bsi, japan how the brain generates and learns temporal sequences is a fundamental issue in neuroscience. the production of birdsongs, a process which involves complex learned sequences, provides researchers with a good biological model to study this phenomenon. bengalese finches (bf) learn highly complex songs that have grammatical structure. the underlying neural mechanisms that allow the birds to learn these songs are however not fully understood. to address this issue, we developed a neural network model of bf's songs that might explain how different regions of the brain work together. to test the model, we also conducted empirical experiments on the brains of bf. the model shows that complex grammatical songs can be replicated by simple interactions between deterministic dynamics of a recurrent neural network and random noise. moreover, comparison between the model and the empirical data on real birds shows similar trends. this work is a part of an integrated research project combining model simulations and empirical study. please see also the empirical component of this project as reported by okumura. ps a-k local administrations of muscimol into the nif alter song grammar of the bengalese finches (bf) tetsu okumura , yuichi yamashita , kazuo okanoya , jun tani behav & dynamic cognition, riken-bsi, saitama, japan; biolinguistics, riken-bsi, japan songs of passerines are learned behavior which used by males to attract females. their songs consist of several song notes, and these notes are produced in a fixed temporal order. among the passerines, however, bfs sing complex song which follows finite state syntax. the song control system of bf consists of a set of discrete nuclei including the hvc and nif. previous study showed that nif lesioned bfs sung simpler songs, with less phrases to phrases branching. therefore, nif-hvc connection may play important role in generating song grammar. in this study, we perfused nif with muscimol via microdialysis probes as a perturbation on nif-hvc system. following a local perfusion, song grammar was modified. some of chunks in their grammar were disappeared and introductorily notesǐ duration was elongated. nif is also known as one of auditory relay nucleus to hvc. part of the effects is possibly caused by disruption of auditory feedback. we also developed a neural network model of nif-hvc system. please refer yamashitaǐs poster for details of this model. the reason for the emergence of reward expectancy neurons suggested by a model using reinforcement learning and an artificial neural network katsunari shibata , shinya ishii , munetaka shidara dept. of e&e engineering, oita univ., oita, japan; grad. sch. of comprehensive human sci., univ. of tsukuba, tsukuba, japan in the experiment of multi-trial schedule task to obtain a reward, reward expectancy neurons, which respond only in the non-reward trials prior to the reward trial, have been observed in the anterior cingulate cortex of monkeys. it is difficult to explain directly by reinforcement learning why they do not respond in the reward trial. here, we interprets that such neurons emerge as an intermediate representation to generate appropriate value and actions in reinforcement learning by simulation analysis using a model that consists of an artificial recurrent neural network trained by reinforcement learning. the simulation result suggests that the reward expectancy neurons emerge to realize smooth temporal increase of the state value by complementing the neurons that respond only in the reward trial. [ ] s. ishii, et al., "a model to explain the emergence of reward expectancy neurons using reinforcement learning and neural network", neurocomputing, behavior is adjusted by outcomes of actions. to examine the neural mechanisms of the behavioral adjustment, we recorded single cell activity of the medial prefrontal cortex (mpfc) of two monkeys performing a behavioral adjustment task. the monkey searched a correct action (left or right lever press) on the basis of the two kinds of visual feedback, one (cs+) paired with a liquid reward and the other (cs−) that did not appear in a preceding pavlovian conditioning. cs+ followed a correct action and cs− followed a wrong action. when the monkey made more than consecutive correct trials, a new block of pavlovian conditioning started. we calculated the prediction errors provided by cs+ and cs− on the basis of a reinforcement learning model of action selection. we found that the neuronal activity corresponds to the prediction error of value of the selected action. this result suggests that mpfc contributes to behavioral adjustment by providing prediction errors of action values. makoto miyazaki , shinya yamamoto , sunao uchida , shigeru kitazawa , faculty of hum sci., waseda univ., tokorozawa, japan; neurosci. res. inst, aist, tsukuba, japan; faculty of sport sci., waseda univ., tokorozawa, japan; dept. of neurophysiol, juntendo univ. grad. sch. med., tokyo, japan; crest, jst, saitama, japan our judgment of temporal order of two sensory signals is not always fixed but subject to changes due to prior experiences, such as repeated exposure to a constant stimulus sequence. to date, such perceptual changes occurred so that signals in the order of the most frequent sequence are judged as simultaneous. in this study, we examined temporal order judgment of two tactile stimuli, delivered one to each hand, using stimulation intervals sampled from biased gaussian distributions (mean = ± ms, s.d. = ms). previous studies predict that the point of simultaneity would be shifted toward the peak of the gaussian, i.e. toward the most frequent interval. however, the point of simultaneity was shifted away from the peak by about ms. our results disagree with the previous studies, but conforms to a contrasting prediction from a bayesian integration theory. research funds: kakenhi ( ) ps a-k single measurement of oxy-and deoxyhemoglobin for a functional near infra-red spectroscopy ichiro shimoyama , fumiko sato , ken nakazawa , kenichi ono chiba university, japan; field of home economics, faculty of education, chiba univ., japan; department of integrative neurophysiology, graduate school of med. chiba univ., japan to study single dynamics for oxy-and deoxy-hemoglobin to a single task, we measured near infra-red spectroscopy (omm- , shi-madzu) over the frontal area ( channels) for volunteers ( - y). thirty tasks were presented visually every s, the subjects were asked to think about the question immediately following the sentences and asked not to think moreover if the question was difficult (e.g., how to cook curried rice? or how to fold paper into a turtle? etc). a comprehension-test was done just after the record. easy/difficult serial tasks were selected, and the oxy-and deoxy-hemoglobin differences between tasks were calculated to obtain correlation coefficients between the oxy-and deoxy-hemoglobin. grand averaged correlation coefficient was − . +/− . between the dynamics of the oxy-and deoxy-hemoglobin. the correlation should be considered in discussing neural activation for nirs. we thank shimadzu corp. for providing the nir station. kazuya ishibashi , , kosuke hamaguchi , masato okada , , department of complexity science and engineering, graduate school of frontier sciences, university of tokyo, kashiwa, japan; jst, japan; riken bsi, wako, japan a synfire chain is one of the networks which generate stable synchronous pulse packets. although the networks with a single stable synfire state is intensively analyzed by using several neuron models, the networks with several stable synfire states have not yet been investigated so thoroughly. by using leaky integrate-and-fire neuron model we construct a layered associative feedforward network embedded with several memory patterns. we analyse the network dynamics with the fokker-planck equation. first, we analyze the activity of the network when we activated one memory pattern of the first layer. we show that the layered associative network has stable synfire state. second, we investigate the activity when we activated different memory patterns. then we observe several characteristic phenomena, which are not observed in the conventional homogenous synfire chain. we will report the details of those phenomena. research funds: kakenhi ( ) and ( ) ps a-k auditory erps can be identified as corresponding stimuli by classifier with naive bayes method akitoshi ogawa , , sachiko koyama , , takashi omori , takashi morotomi research institue for electronic science, hokkaido university, sapporo, japan; japan science and technology agency, saitama, japan; graduate school of information science and technology, hokkaido university, sapporo, japan; sakushin gakuin university, utsunomiya, japan in an attempt to reversely estimate the input stimulus from measured erps, we developed computational classifier using naive bayes method. correct classification rates could be index values of the erp characteristic. in this study, we applied the classifier to identify auditory erps (n = ). the erps were elicited by tones ( hz) with different durations ( , , , , , ms) and gaps ( , , , , , ms) embedded in a continuous pure tone ( hz). to confirm the generality of the method, we used leave-one-out cross validation. erps of each subject were identified by the classifier which was constructed from the others' erps. as a result, the correct rates for and ms were high both for the tones ( ms, %; ms, %) and the gaps ( ms, %; ms, %). ps a-k determination of channel parameters for construction of a neural model of caenorhabditis elegans kazumi sakaa, akane andoh, taro ogurusu laboratory of bioscience, faculty of engineering, iwate university, iwate, morioka, japan caenorhabditis elegans (c. elegans) is one of the most suitable model animal for investigation of the relationship between the connection and the function of the neural network because its connection was revealed with the electronmicroscopy. on the other hand, it has been difficult to build a precise model neuron because the neuronal electrophysiological data of c. elegans has not been sufficient. we have been developing a precise neural model by extracting parameters required for model of voltage dependent channels from the electrophysiological data by the genetic algorithm with a neural simulator genesis and parallel genesis. using these simulation softwares, not only the optimum parameter set was determined for each channel but also the ratio of the conductances of several channles were determined. we report validity of obtained parameters and the possibility of the existence of unknown channel. supported by grant from jsps. ps a-k theoretical consideration about nmda current change and its effect on synaptic plasticity shigeru kubota, tatsuo kitajima department of bio-system engineering, yamagata university, yonezawa, japan it is well known that nmdar plays an important role in learning and memory. several experiments have shown that the property of nmdar epsc can change within a few weeks after birth, leading to the shortening of its decay time course. since the calcium current through nmdar is involved in ltp and ltd induction, it is possible that such change can work as the modulation of the plasticity rule or higher-order plasticity. here we show by the biophysical compartmental model that the alteration of nmdar property can modulate the calcium influx into the spine, which finally switches plasticity rule. we also show that this type of plasticity switch can promote synaptic competition and separate postnatal synapses rapidly into two groups of either strong or week ones. our results suggest that changing nmdar time course is very useful for the developing animals in order to promote fast and stable formation of the polysynaptic circuit. manish kumar jain department of psychiatry, r.d. gardi medical college, india introduction: i want to inform you regarding the some of challenges coming across my practice with the person with the psychiatric disorder in social rehabilitation like education and training, work and employment, family, groups, social, sexual, environmental and regional, coordination with the other health group and care giver, insurance problems, medical, physical, occipital vocational, languages problems mostly how to give oppurtinies with in the society and many more to be come in future. method: i keep the records with me since i join the medical college and my during practice but this is really challenging to calm down for question with their relatives and care givers. results: it is always to see the experience of the other people including self help groups in this regards and most challenging with near by perfect action and required more interaction with the rehabilitation groups because some are social problems in psychiatric disorder. conclusions: there is big challenge in the for social rehabilitation for the persons with psychiatric disorder as multifactor involvement s are there in this groups with early intervention and long term rehabilitation so that we can produced many working induals with in the society among the person with psychiatric disorder the more interaction among the society and care giver working in this field as well as neuroseiencents working in this field so that we will able to achieve almost complete social rehabilitation as till today we are not able to achieve social rehabilitation up to % till now. hepatic encephalopathy (he) refers to acute neuropsychiatric changes accompanying fulminant hepatic failure (fhf). in the present study we investigated changes in lipid composition of membranes isolated from cerebral cortex of rats treated with thioacetamide (taa), a hepatotoxin which induces fhf and thereon he. estimation of phospholipid fatty acid content in cerebral cortex membranes from taa treated rats revealed a decrease in monounsaturated fatty acid namely oleic acid and the poly unsaturated fatty acids ␥-linolenic acid, decosa hexanoic acid and arachidonic acid compared to controls. assesment of membrane fluidity with pyrene, , -diphenyl- , , -hexatriene, and -[ (trimethylammonio)phenyl]- -phenyl- , , -hexatriene revealed a decrease in annular membrane fluidity while the global fluidity was unaffected. the level of thiobarbituric acid reactive species-marker for lipid peroxidation also increased in membranes from taa treated rats indicating prevalence of oxidative stress. results from the present study demonstrate gross alterations in cerebral cortical membrane fatty acid composition and fluidity during taa induced he and their possible implications in the pathogenesis of this condition are also discussed. nagatoki kinoshita, shigenobu yonemura cellular morphogenesis, cdb, riken, kobe, japan rho-gtpases are well known as regulators of cytoskeletal reorganization and many cellular morphogenetic movements. however, little is known about their distributions and their physiological functions in vertebrates. immunohistology of chick embryos revealed apical accumulation of rho, rac and cdc in neural plate cells, especially in bending hinge points. after neural tube closure, the apical accumulation decreased. coordinately, activities of rho-gtpases and myosin ii in neural plate cells were higher during neurulation than after neural tube closure. inhibitions of actin filament formation, myosin ii-mediated contraction or rho-associated kinase activity affected neural tube formation. inhibition of rho activity induced the disruption of its apical accumulation and the defects of neural tube formation. these results suggest that rho-gtpases in an active form accumulate in the apical surface of neural plate cells and play important roles in neurulation. furthermore, we are screening regulators and effecters of rho-gtpases transiently expressed in neural plate cells during neurulation. setsuko sahara, dennis dm o'leary mnl-o, the salk institute, usa gradients of morphogens are postulated to establish the initial patterning of the mammalian forebrain, but little is known about their downstream targets and the mechanisms of patterning. here we report mouse buttonhead homogoues, the sp gene family, as candidates of downstream of those morphogens: sp expression correlates with wnts/bmps in the cortical hem, sp with fgfs in the cop, and sp with shh in the ventral midline and mge. by using in utero electroporation, we show that sp regulates anterior-posterior patterning of the cortex into areas by controlling distinct fgfs that having opposing effects. sp and fgf exhibit reciprocal induction, indicating that sp is a positive feedback regulator of fgf . surprisingly, though, ectopic expression of both sp and its dominant active form shift cortical areas in the opposite manner to fgf , suggesting that sp activates additional targets that overcome fgf function. our results indicate that fgf is an additional target of sp , showing effect on patterning similar to sp . these findings indicate that sp balance the proper cortical arealization through fgf and fgf . research funds: nihr ns ps p-c fyn-fak signal transduction is involved in the radial migration of late-generated neocortical neurons eiko nakahira , kotaro hattori , takeshi yagi , shigeki yuasa dept. ultrastructural res., nat. inst. neurosci., ncnp, tokyo, japan; kokoro biology group, fbs, osaka univ., suita, japan fyn tyrosine kinase posphorylates focal adhesion kinase (fak) that is involved in cell migration. taking into account the defective formation of neocortical layers ii-iii in fyn-deficient mice, fyn-fak signal transduction might be involved in the control of the migration of neocortical neurons. accordingly, we analyzed the neuronal migration in the mutant neocortex and compared the phenotypes to the changes induced by fak gene-knock down by foreign gene transfer by means of in utero electroporation. late-generated neocortical neurons exhibited defective radial migration in the mutant and this defect was rescued by the transfer of fyn-expression vector to the neocortical primordium. fyn and fak were colocalized in the migratory neurons, and fak sirna transfer into neocortical primordium induced migration defect similar to that in fyn deficiency. these findings strongly suggest that the coordination of fyn and fak is essential for the radial migration of late-generated neocortical neurons. noriyo ishibashi , kazuko keino-masu , tatsuyuki ohto , satoshi kunita , satoru takahashi , masayuki masu dept. of mol. neurobiol., grad. sch. of comprehensive human sci., univ. of tsukuba, tsukuba, japan; laboratory animal resource center, univ. of tsukuba, tsukuba, japan heparan sulfate (hs) proteoglycans regulate developmental patterning through the interactions with cell surface proteins and extracellular matrix molecules. these interactions are mediated by the specific hs structures generated by sulfation and epimerization. a recently identified extracellular sulfatase, sulffp , has been implicated in the regulation of growth factor/morphogen signaling through hs remodeling in vitro, but its physiological roles remain unknown. here we generated knockout mice lacking the sulffp gene, and examined the brain development. a previous study showed that the brain-specific disruption of the ext gene, which encode a hs synthesizing enzyme, led to severe brain defects including hypoplasia of the cerebral cortex and cerebellum. in this study, we thus examined the morphological changes of the cerebellum in the neonatal and adult sulffp -deficient mice. heparan sulfate (hs) proteoglycans play a crucial role in mediating important signaling by wnt, hedgehog and fgf. recently, novel sulfatases, sulffp /sulfatase- and sulffp /sulfatase- , which have hs -o-endosulfatase activity have been isolated. since these sulffps are detected in the extracellular space, sulffps are thought to regulate cell surface signaling through hs remodeling. in order to examine the function of sulffp genes in zebrafish, we isolated zebrafish sulffp and sulffp . here we report the isolation and the characterization of the third homologue, sulffp . sulffp has about % and % overall amino acid homology with sulffp and sulffp , respectively. at h postfertilization, sulffp is expressed in the ventral region of spinal cord, whereas sulffp is expressed only in the floor plate and sulffp is expressed in the lateral floor plate and ventral regions of spinal cord. detailed expression patterns of sulffp will be presented. masahiko ajiro, kenichi arai, mika maeda-sato, masuo obinata, wataru shoji dept. of cell biology, idac tohoku univ., japan collapsin response mediator proteins (crmps) are cytosolic proteins involved in neuronal differentiation and axonal guidance. a member of this family, crmp was shown to mediate the repulsive effect of sema a on axons. crmps appear to play more complex roles in axonal differentiation, elongation and branching during development. since less is known about their in vivo function, we studied their roles during development using transparent zebrafish embryos. at early axogenesis stage, zebrafish crmps are expressed in specific patterns. in trigeminal sensory ganglia, crmp , , , and are highly expressed. knocking down of these gene results in disorganization of the ganglia, separating into several clusters. however, their axonal patterns including direction, extension, and branching appears normal. same defects were observed in the knockdown of neuropilins, receptor component for class semaphorins. these results suggest that crmps may functionin keeping trigeminal neurons as a ganglia by mediating semaphorin-neuropilin signals. ps p-c developmental origin of diencephalic sensory relay nucley in teleosts y. ishikawa , n. yamamoto , m. yoshimoto , t. yasuda , k. maruyama , t. kage , h. takeda , h. ito nat. inst. rad. sci., chiba, japan; nippon med. sch., japan; tokyo univ., japan we propose a novel interpretation of the embryonic origin of cells of diencephalic sensory relay nuclei in teleosts, based on our studies in the medaka embryonic brain. it has been proposed that the relay system in teleosts is unique among vertebrates. teleost relay nuclei, the preglomerular complex (pg), have been assumed to originate from the basal plate (posterior tuberculum, pt) of the diencephalon, whereas relay nuclei in mammals are derived from the alar plate. our results show, however, that many pax -or dlx -positive cells migrate laterally and ventrocaudally from the diencephalic alar plate to the basal plate during development. massive clusters of the migrated alar cells become localize in the mantle layer lateral to the pt neuroepithelium, from which the pg appear to differentiate. we therefore consider most neurons in the pg are be of alar, not basal origin. thus, the teleost pg can be regarded as migrated alar nuclei. the organization of the diencephalic sensory relay system may have been conserved across vertebrates. hideyuki dekimoto, yoshihiro oomiya, satoshi kikkawa, toshio terashima, yu katsuyama department anatomy and developental neurobiology, kobe university graduate school of medicine laminaiton is one of features unique to the brain of vertebrates. to understand the evolution of layer formation in the vertebrate brains, we are studying genes which exhibit layer-specific expression. since one of ets family transcription factors, er is expressed specifically in the layer v of the mouse neocortex, we selected this gene for the purpose of our study. here we cloned zebrafish er homologue (zfer ), and found that the amino acid seuqence of the putative protein is highly conserved throughout the entire length. expression of zfer was observed in multiple sites of developing brain. the expression disappears sequentially in some sites, whereas it persisted in other sites until adult stage. er expressing sites in the brain was basically conserved between mouse and zebrafish, whereas expression pattern in each site (i.e. telencephalon, tectum) was different. based on these observations, evolution of the gene expression in the brain lamination will be discussed. hiroyuki koizumi, teruyuki tanaka, joseph g. gleeson university of california, san diego, usa doublecortin (dcx), encoding a microtubule-associated protein, is critical for neuronal migration, as mutations result in x-linked lissencephaly in hemizygous males and subcortical band heterotopia in heterozygous females, whereas in mouse, rnai-mediated knockdown but not germline knockout shows abnormal positioning of cortical neurons. dclk (doublecortin-like kinase) is one of the homologous genes of dcx, encodes for protein with an n-terminus that is % identical to dcx, but also additional c-terminal protein kinase domain. here, we report that the dclk functions in a partially redundant pathway with dcx in the formation of axonal projections across the midline and migration of cortical neurons in mouse. dosagedependent genetic effects were observed in both interhemispheric connectivity and migration of cortically and subcortically derived neurons. rnai-mediated knockdown of either gene results in similar migration defects. these results indicate the dcx microtubuleassociated protein family is required for proper neuronal migration and axonal wiring. hiraki sakuta , , hiroo takahashi , , takafumi shintani , , kazuma etani , masaharu noda , div. of mol. neurobiol., nibb, okazaki, japan; crest, jst, japan in the developing chick retina, the expression of bmp is relieved by that of bmp at around e with a change from a dorsal high to dorsotemporal high pattern, complementary to that of ventroptin, a bmp antagonist. we previously demonstrated that misexpression of ventroptin altered the retinotectal projection along both the dv and ap axes. here, we show that topographic molecules along the dv axis, together with ephrina , are expressed in a double-gradient fashion from e on like ventroptin and bmp . when bmp expression is manipulated by using the gene-specific knockdown and the reagent-inducible gene expression techniques, the expression patterns of these double-gradient molecules are all changed. moreover, in the bmp knockdown and ephrina -misexpressing embryos, the retinotectal projection is altered along the two axes. the expressional switching from bmp to bmp thus appears to play a key role in retinal patterning and consequently in topographic retinotectal projection, by changing the direction of the dv axis toward the posterior side during retinal development. noriyuki morita, teiichi furuichi lab. for molecular neurogenesis, riken-bsi, wako, japan the mammalian cerebellum is anteroposteriorly and mediolaterally compartmentalized at the level of neuroanatomy and also at the level of gene expression. to elucidate the molecular mechanisms underlying the establishment and the maintenance of functional cerebellar compartment, genes responsible for mouse cereballar development transcriptome were examined for patterned expression in cerebellum by whole-mount in situ hybridization. not a few known and novel genes were found to be expressed in parasagittal band pattern in the embryonic mouse cerebellum, which could be categorized as "early-onset-genes". parasagittally expressed genes were classified in comparison with the band pattern of en , wnt b and pcp /l gene expression in declival vermal lobule, to investigate the correlation between spatial expression profiles and transcriptional regulatory elements. our accumulating data suggest that not only patterning genes like engrailed and wnts, also genes related in later events in neural development such as synaptogenesis are expressed as earlyonset-genes. yasufumi tanaka, tomiyoshi setsu, hideyuki dekimoto, yu katsuyama, toshio terashima kobe university graduate school of medicine, japan the nissl staining of the brains of the adult reeler and normal mice showed that the size of the pontine nuclei (pn) was reduced in the reeler compared with the normal counterpart. the injections of dii and di- asp into the left and right hemicerebellum, respectively, resulted in that only a few pn neurons were doubly labeled in the control, but in the reeler most of pn neurons were doubly labeled. the placements of solutions of dii and di- asp into the left and right cerebellar peduncles of paraformaldehyde-fixed brains resulted in that dii-labeled or di- asp-labeled pontocerebellar fibers made a fascicular formation in the cerebellum of the normal mouse, but such a fascicular formation was not recognized in the reeler and labeled terminals of mossy fibers were randomly arranged along the course of the pontocerebellar projection. reelin mrna and reelin were both expressed in the pn of the normal mice. these data elucidate that the reelin may play a key role in fasciculuation and collateral formation of pontocerebellar projections in addition to cell positioning or migration of pn neurons. kudoh suguru , , takahisa taguchi aist, ikeda, japan; presto, jst the spatiotemporal patterns of spontaneous action potential were analyzed, using the multi-site recording system for extracellular potentials of neurons and the living neuronal network cultured on a -dimensional electrode array. the map of functional connections between neurons revealed that each culture contained some hublike neurons and the distribution of the number of functionalconnections approximated a power-law distribution. we confirmed that the spatiotemporal pattern of spontaneous action potentials became more complex pattern along with developmental stage, and the constant pattern of stimulation promote this developmental change. in addition, the spatiotemporal pattern and the functional connections between neurons were drastically re-organized by real-time feedback stimulation. these results strongly suggest that the network structure of the cultured hippocampal neurons is neither stable nor random, but is functionally dynamic and is suitable for certain types of information processing. research funds: presto, jst ps p-d laterality of the human cerebral hemisphere taiko kitamura, jinzo yamada department of anatomy, tokyo medical university, tokyo, japan it has been reported that some functional predominance is located in the right or left hemisphere of the human brain. especially, the speech center and the center related to thought and emotion are located in the left and in the right hemisphere, respectively. in this study, the laterality between the right and the left human hemisphere was investigated macro-anatomically. we measured the weight, the medial-lateral width (m-l), the anterior-posterior lenght (a-p), and the width of the medial surface in the right and the left human hemisphere using in anatomical practice for medical students. the weight of each hemisphere was roughly equal. the m-l was wider in the right side than the left side. the a-p was longer and the width of the medial surface was larger in the left side than in the right side. because of the longer a-p and the larger width of the medial surface in the left hemisphere, it appeared that the left hemisphere overspreads the medial-dorsal marginal surface of the right hemisphere by the naked eye. such overspreading suspects that the left hemisphere develops earlier and faster than the right hemisphere. ps p-d synchrony-induced transition behaviors organized under spike-timing dependent plasticity for retrieving the memorized patterns takaaki aoki , toshio aoyagi department of physics, kyoto university, kyoto, japan; graduate school of informatics, kyoto university, kyoto, japan temporally correlated spikes, such as spike synchrony, have been observed in relation to behaviors or cognitions. however, it is unclear how the neurons read out the incoming spike synchronization in the dynamical behavior of network. in this modeling study, considering a network of excitatory and inhibitory neurons organized under spiketiming dependent plasticity, we present a type of network model in which incoming spike synchrony causes a transition between learned activity patterns in the order they were experienced in the learning process. furthermore, using appropriate training patterns, this network exhibits a context-dependent transition, in which the network switches to multiple patterns from a single pattern depending on the temporal structure of neuronal activity at the onset of incoming spike synchrony. this ability of the network may provide one of mechanisms by which a neuronal system can be trained to carry out tasks in a context-dependent manner. shozo kito, maiko kitagawa, akiko shingo lab. of neurosci., hyogo univ., kakogawa japan in our previous studies, we showed that a part of nicotine's beneficial effects on hippocampal and cortical neurons were due to increased igf- mrna expressions. nevertheless, the situation may be somewhat different as far as nicotine's effects on the neuronal progenitor cell, which is still on the way of differentiation are concerned. to clarify this problem, nicotine was intraperitoneally injected into weekold wistar strain rats in several doses followed by successive injections of brdu for the next days. then rats were sacrificed and vertical sections of the hippocampus formation were offered for double immunohistochemical staining of brdu/psa-ncam, brdu/neun or brdu/gfap. as the results, numbers of both brdu(+)/psa-ncam(+) cells and brdu(+)/neun(+) cells were much decreased nicotine-dose dependently. on the other hand, as much as mg/kg was needed for nicotine to exert its effect on the number of brdu(+)/gfap(+) cells. these results reveal that nicotine inhibits neurogenesis and plasticity in the hippocampus of adult rats. ps p-d the establishment of the organotypic slice culture of postnatal rat forebrain involving egfp-labeled neural progenitors kaoru sato , james e. goldman division of pharmacology, national institute of health sciences, tokyo, japan; department of pathology, columbia university, new york, usa after injecting egfp-encoding retrovirus into p rat svz, sagital sections of forebrain were made at p and cultured for days. the migration pattern of the egfp-labeled neural progenitors in the cultured slices is almost same as that at the corresponding age. the expression patterns of the glial differentiation-markers were also in accordance with those at the corresponding age. when slices were cultured with anti-␣ integrin antibody, the migration of the neural progenitors inside svz was significantly enhanced along the rostrocaudal extent. these results suggest that the organotypic slice culture of postnatal rat forebrain is an efficient experimental system for pharmacological studies about migration and differentiation of neural progenitors. radial glia is involved in the contact guidance of neuronal migration and also the neuronal and astroglial precursors. to make clearer the role of radial glia, we developed a method for the selective ablation of a subset of radial glia. it has been reported that tenascin-c (tn-c) is one of the markers for radial glia. accordingly, diphtheria toxin (dt)gene and enhanced green fluorescence protein (egfp)-gene both driven by tn-c gene promoter were co-transferred into the ventricular zone cells of the mouse neocortical primordium by means of in utero electroporation. the numbers of egfp-labeled cells in that tn-c gene promoter and subsequently dt gene are activated selectively decreased by this approach. using this method, the examination of radial glial morphology and neuronal migration following selective ablation is in progress. takayuki manabe, kouko tatsumi, eri makinodan, manabu makinodan, takahira yamauchi department of nd anatomy, nara medical university, kashihara, nara, japan it has been well documented that neurogenesis persists at the subventricular zone and the subgranular layer of the dentate gyrus in the adult mammalian brain. in the adult mice, we demonstrated that cells around a cryo-injured cortical lesion had a proliferative activity (labeled with brdu in vivo) and formed neurosphere-like aggregates in the sphere-forming culture condition. significantly lager number of spheres was observed in the culture from the injured hemisphere, which excluded the neurogenic regions (i.e. the svz and hippocampus), than those cultured from the control (contralateral and intact) hemisphere. furthermore, the sphere-forming cells differentiated to neuronal-and glial-marker positive cells in vitro. these results suggest that the cells forming sphere-like aggregates in vitro may function as a kind of progenitor cells in the injured brain. if this is a case, it would be tempting to transplant these sphere-forming cells to cure brain injury or disease. further characterization of the cells is underway. ps p-d localization of neurotrophin receptors trka in pc cells: d reconstruction analysis of membrane proteins tomoki nishida , hiroshi jinnai , tatuo arii , akio takaoka , ryoichi yoshimura , yasuhisa endo department of applied biology, kyoto institute of technology, kyoto, japan; department of polymer science and engineering, kyoto institute of technology, kyoto, japan; national institute for physiological sciences, myodaiji, okazaki, japan; osaka university, mihogaoka, ibaraki, osaka, japan it was previously reported that trka (ngf receptor) was associated with caveolae, small invaginations on the cell membrane, but its subcellular localization is not clarified in detail. we performed immunocytochemistry of trka and caveolin- in pc cells, analyzed by high-voltage electron microscopy, and reconstructed d structure of their subcellular distribution by imod. our results indicated that localization of caveolin- , known as an integral membrane protein of caveolae, was never found in the invagination structure in pc cells, but trka and caveolin- immunoreactivities were mainly found as a mesh-like structure in the cytoplasmic matrix. kensuke shiomi, kazuko keino-masu, masayuki masu department of molecular neurobiology, graduate school of comprehensive human sciences, university of tsukuba, tsukuba, japan the wnt signaling plays important roles in cell growth, differentiation, polarity formation, and neural development. previously we identified ccd , a third-type of the dix domain-possessing protein, as a positive regulator of the wnt/-catenin pathway. ccd mrna was mainly detected in the neural crest derivatives and differentiated neurons in mouse embryos, suggesting the importance of ccd in the wnt-mediated neuronal development. there are three subtypes of mouse ccd gene products, ccd a, ccd b and ccd c, which are generated by different promotor usage. mouse ccd a as well as zebrafish ccd a has a calponin homology domain which can mediate the interaction with the actin cytoskelton. we found that in the ccdtransfected hela cells, only the type a ccd proteins co-localized with the actin filament. in order to examine the function of the type a ccd proteins, we are now doing overexpression and functional blocking experiments using zebrafish embryos and cell culture. research funds: kakenhi ( , ) ps p-d analysis of a role of r-spondin on proliferation of the cortical neuroepithelium yumiko hatanaka , masahiro yamaguchi , fujio murakami , masayuki masu grad. school of comprehensive human sci., univ. of tsukuba, japan; grad. school of med., univ. of tokyo; grad. school of frontier biosci., osaka univ r-spondin (rspo ) is a secreted activator of wnt/-catenin signaling (kazanskaya et al. ) . rspo is expressed in the developing medial cerebral wall and transgenic mice expressing rspo in the entire neuroepithelium show enlarged lateral ventricle with a slight increase of brain size (hatanaka et al. ) . since wnt a has a role for expansion of caudomedial cortical progenitor cells (lee et al. ) , these findings lead us to the idea that rspo may synergistically promote proliferation of cortical neuroepithlial cells together with wnt a. to clarify their role on proliferation of cortical neuroepithelial cells, we first introduced a -catenin/tcf reporter gene into these cells of embryonic day . mouse. an application of wnt a on these cells increased level of the reporter expression, and an addition of rspo further increased its level. we are now monitoring incorporation of brdu in neuroepithelial cells to know whether wnt a and rspo directly promote their proliferation. tae sun kim, hideki hida, tomoko narita, sachiyo misumi, hitoo nisino department of neurophysiology & brain science, nagoya city university graduate school medical sciences, nagoya, japan to investigate whether physiological low oxygen during development and cytokines expressed in the dopamine (da)-depleted striatum increase the number of da neurons from es-derived neural progenitor cells (npcs), npcs were treated with cytokine cocktail (il- , il- , lif, gdnf) or lowered o ( . %), followed by tyrosine hydroxylase (th) immunostaining. low oxygen increased total number of th (+) cells ( . -fold) as compared to normal o . cytokine cocktail significantly increased th (+) cells ( . -fold) compared to nontreated control. treatment of lif and il-  to npcs exhibited major contribution in the effect of cytokine cocktail. data suggest that physiologically relevant low oxygen in development and cytokines and trophic factors that were enhanced in da-depleted striatum cause in the increase of daergic neurons from es-derived npcs. ps p-d structural basis for reelin signaling: determination of receptor-binding site and its three-dimensional structure norihisa yasui , terukazu nogi , mitsuharu hattori , kenji iwasaki , , junichi takagi research center for structural and functional proteomics, inst for protein res., osaka univ., suita, japan; dept. of biomed. sci., grad. sch. of pharm. sci., nagoya city univ., nagoya, japan; core research for evolution and technology (crest) a large secreted glycoprotein reelin acts on target neurons through its receptors (apoer and vldlr), resulting in tyrosine phosphorylation of dab . in the present study, we have carried out structural and functional studies on the reelin signaling. first, we determined the structure of a single reelin repeat by x-ray crystallography. it had a horseshoe-like globular structure with some similarities to carbohydrate binding modules from many enzymes. moreover, electron micrographic d reconstruction of four-domain reelin fragment (i.e. r - ) revealed an elongated rod-like structure. next we determined minimum active unit within reelin. a fragment containing both the fifth and sixth reelin repeats (r - ) was capable of binding to the receptor (apoer ), and was also able to induce tyrosine phospholylation of dab in primary neuronal culture. ps p-d effects of astrocyte-derived factor and cell-cell communication on uni-directional differentiation from mouse embryonic stem cells into neural cells embryonic stem (es) cells uni-directly differentiate into neurons via neuroectoderm and neural stem cells by neural stem sphere (nss) method. cultured with astrocyte-derived factor, colonies of es cells give rise to nsss. we analyzed structure and gene expression of cell spheres formed under various culture conditions, in order to elucidate mechanisms of the uni-directional differentiation into neurons. quantitative real-time rt-pcr analysis demonstrated that the neuronal differentiation did not occur in the cell spheres. these results suggest that astrocyte-derived factor and cell-cell communication are necessary for the differentiation. we have previously established es cell differentiation system, by which we can derive neurospheres containing neural stem/progenitor cells (ns/pcs) with the identity of early caudal neural tube. taking advantage of this culture system, we have recently found conditioned medium of a stromal cell line (cmsc) has the activity to support the formation of neurospheres. this activity was more prominent when cultured at low cell density than when cultured at high cell density, suggesting that it supports the survival of ns/pcs. moreover, rt-pcr analysis of regional identities of the cmsc treated neurospheres revealed elevated expression of pax and pax compared with those of untreated neurospheres, indicating that cmsc promotes dorsalization of ns/pcs or selective proliferation of dorsal ns/pcs. elucidation of underlying mechanisms may provide important tools to derive early ns/pcs which can generate variety of projection neurons and be applicable to regenerative medicine. research funds: sorst jst ps p-d neudesin, a secreted factor, promotes neural cell proliferation and neuronal differentiation in mouse neural precursor cells neudesin expressed in adult mouse brain encodes a secreted signal with neurotrophic activity in neurons (j neurosci res : , ) . most neurotrophic factors are involved in neural cell proliferation and/or differentiation. however, the role of neudesin in neural development remains to be elucidated. neudesin mrna was expressed in the neural precursor cells before the appearance of neurons. therefore, roles of neudesin in neural development were examined using the neural precursor cells. neudesin significantly promoted neuronal differentiation. in addition, neudesin transiently promoted neural cell proliferation early in the developmental process. the differentiation was mediated though activation of the pka and pi- k pathways. in contrast, the proliferation was mediated through the mapk and pka pathways. the expression profile and activity indicate that neudesin plays unique roles in neural development. ps p-d fabp is required for maintenance of neural stem/progenitor cells in the postnatal hippocampus motoko maekawa , miho matsumata , , yuji owada , shigeki yuasa , noriko osumi , natl. inst. of neurosci., ncnp, tokyo, japan; tohoku univ. sch. of med., sendai, japan; crest, jst pax transcription factor is a key player for brain patterning and embryonic neurogenesis, and also expressed in the postnatal brain. we have previously shown that pax is necessary for keeping neural stem/progenitor cells in the hippocampus. in this study we have focused on a fatty-acid binding protein fabp , a downstream of pax , regulating maintenance of embryonic neural stem/progenitor cells (arai et al., ) . fabp was expressed in neural stem/progenitor cells in the hippocampal dentate gyrus (dg). % of fabp -expressing cells co-expressed gfap (a marker for early progenitors), and % of them co-expressed psa-ncam (a marker for late progenitors). fabp expression was also overlapped with pax , and expression of fabp was down-regulated in the dg of pax deficient rats and mice. finally, brdu-labeling analysis revealed decreased cell proliferation in the dg of fabp knockout mice. taking all together, it is concluded that fabp is required for maintenance of neural stem/progenitor cells in dg. ps p-d involvement of the psa-ncam expressing cells in early development of the vascular system of the forebrain momoko miyakawa, tatsunori seki department of anatomy, juntendo university school of medicine, tokyo, japan early development of the vascular system of the forebrain were studied in the chick embryo. staining of vascular endothelial cells by fitctomato lectin and immunohistochemical staining of the surrounding cells were performed on the same cryostat sections of embryos of embryonic day - . sections were examined under a confocal laser scanning microscope. capillaries were found in the lateral pallium and seemed to grow from psa-ncam-positive outer zone to negative inner zone of the pallium. psa-ncam is thought to be expressed in the immature neurons. the rims of capillaries were immunoreactive with psa-ncam in both zones. immunoreaction of doublecortin (neuronal marker) and punctate immunostaining of laminin also were observed on rims of capillaries. by immuno-electron-microscopy it appeared that the endothelium were covered with very thin processes of cells of which outer surface was immunoreactive with psa-ncam. psa-ncam expressing cells may be involved in the development of the vascular system of the forebrain by supporting or guiding the growing capillaries. masaharu kotani , , shiki okamoto , masato imada , kouichi itoh , atsushi irie , hitoshi sakuraba , hideo kubo department of molecular biologu, ohu univ., koriyama, japan; dept. deve. physiol., natl. inst. physiol. sci., okazaki, japan; dept. anatomy, nihon univ. shl. med., tokyo, japan; dept. mol. pharma., univ. tokushima bunri, sanuki, japan; dept. biochem. cell res., tokyo metro. inst. med. sci., tokyo, japan; department of clin. genet, tokyo metro. inst. med. sci., tokyo, japan; dept. med. biol, tokyo metro. inst. med. sci., tokyo, japan as randam- shows the highest expression level with the proliferating stage of neural stem cells (nscs), it is thought that the isolation of nscs based on the expression level of randam- is possible. in the present, we show that the isolated randam- high+ cells enrich nscs. the randam- high+ cells had the characteristics as the highly self-renewal capability and potential for multilineage differentiation into neural cells. in contrast, almost all of the randam- low+/− cells exhibited not only the extremely low self-renewability but the differentiation capability restricted to neurons. the results demonstrate that randam- is a usefule marker for the isolation of nscs by facs. yasuharu takamori , yasuhisa tamura , , yosky kataoka , , yilong cui , , hisao yamada department of anatomy and cell science, kansai medical university, osaka, japan; department of physiology, osaka city university graduate school of medicine, osaka, japan; morecular imaging reserch program, riken frs, saitama, japan lamins are major structural proteins of nuclear envelope. three lamin subtypes, a/c, b and b are mainly present in mammalian somatic cells. to investigate the pattern of lamin expression during neuronal differentiation, we immunohistochemically analyzed the existence of lamins in two neurogenic regions of rat brain; subgranular zone of dentate gyrus and subventricular zone, with confocal microscopy. gfap-positive primary progenitor cells possess lamin a/c (++), b (++), b (++), psa-ncam-positive subsequent progenitor cells possess lamin a/c (−), b (+++), b (+), and mature neurons possess lamin a/c (++), b (+), b (+++), in both neurogenic regions. these observation showed that the composition of lamin subtypes was distinct in particular differentiation stages during adult neurogenesis. yusuke tozuka , yuichi tanaka , tatsuhiro hisatsune department of integrated biosciences, university of tokyo, chiba, japan recent work has shown that nestin + neural progenitor cells exist in the adult brain, and suggested that neural activity itself could act directly on these progenitor cells. it has been unclear, however, how do adult progenitor cells sense activity signals from surrounding neural circuit. in the hippocampus where new neurons are continuously produced throughout life, nestin + adult progenitor cells received gabaergic inputs. the gabaergic activity depolarized these progenitor cells, and then promoted their neuronal differentiations. although neuronal production does not readily occur in the adult neocortex, nestin + neural progenitor cells exist in this area too. interestingly, these progenitor cells also received excitatory gabaergic inputs. this gabaergic inputs inhibited their cell proliferations. from these results, we here propose that adult progenitor cells are a direct target of gabaergic neuronal networks, and that this networkto-progenitor cell interaction influences progenitors development by regulating their cell proliferations and/or neuronal differentiations. ps p-e new migration pattern in the postnatal neurogenesis of the dentate gyrus takashi namba , , hideo namiki , tatsunori seki dept. of anat, juntendo univ. sch. of med., tokyo, japan; integrative biosci. and biomed. eng, sch. of sci. and eng, waseda univ., tokyo, japan in the hippocampus, granule cells continue to be generated from embryonic to adult stages. the early postnatal neurogenesis is a transitional state between the embryonic and adult neurogenesis. previously, we have suggested that the postnatal hilus contains astrocytic neural progenitors that divide and differentiate into neuroblasts, and that finally the neuroblasts settle in the granule cell layer (gcl). however, the questions remain how astrocytic progenitors divide and differentiate into neurons, and how the neuroblasts migrate to the gcl. to observe them, we developed a time-lapse imaging system. retrovirus-gfp was injected into the rat hippocampus at p . three days after the injection, the hippocampal slices were prepared for the time-lapse imaging. the present data show that neuroblasts migrate from the hilus to the gcl, changing the direction of their movement. this is inconsistent with the previous report suggesting simple radial migration (rickmann, et al., ) . the dividing pattern is currently under investigation. akiya watakabe , noritaka ichinohe , sonoko ohsawa , tsutomu hashikawa , kathleen s. rockland , tetsuo yamamori div. of brain biol, nibb, okazaki, japan; lab. for cortical organization and systematics, bsi, riken, wako, japan, lab. for neural architecture, bsi, riken, wako, japan by using gene expression profiles, we have tried to classify layer neurons in several areas of monkey neocortex. we previously reported that nurr , ctgf and sema e mrnas are specifically expressed in subsets of layer neurons. we further show here that cholecystokinin (cck) mrna is expressed in a subset of excitatory neurons in layer . by double ish, layer neurons in monkeys are roughly divisible into cck(+) and sema e(+) subgroups. each subgroup was further subdivided by other markers. tracer experiments showed that cck and sema e mrna expression correlate well with corticocortical and corticothalamic connectivity, respectively, but the correlation was only partial. from this, we infer that subtypes defined by gene expression may not directly correspond to classical neuronal types. the implication of our findings will be discussed in terms of constancy of laminar structure across areas and species. research funds: kakenhi ps p-e rbp-j regulates the cortical laminar formation kenji tanigaki , kazue muraki , norio yamamoto , tasuku honjo shiga medical center, research institute, shiga, japan; department of medical chemistry, kyoto university, kyoto, japan precise patterns of cell cycle exit and migration of neural progenitors are crucial for the formation of cortical layer structure. to examine involvement of notch-rbp-j signaling in the cortex laminar formation, we deleted rbp-j from neural progenitors in anatomically restricted areas by in vivo electroporation of cre-expressing plasmids. such studies revealed that rbp-j deficiency caused transformation of glutamatergic pyramidal neurons in layer ii/iii to layer iv neurons with concomitant loss of astrocytes. the loss of rbp-j accelerated neuronal differentiation and changed their laminar fates. in addition, time-lapse studies indicated the migration defect of rbp-j-deficient neurons. the results showed that notch-rbp-j signaling regulates migration of differentiated neurons as well as the timing of the cell cycle exit of neuronal progenitors to determine the laminar and cellular fates of neural progenitors. ps p-e search for the genes that define mammalian cortical progenitor cells using single-cell gene expression profiles ayano kawaguchi , tomoko ikawa , yuya kasukawa , hironori ueda , , kazuki kurimoto , michinori saitou , fumio matsuzaki , lab. for asymmetric cell division, cdb, riken, kobe, japan; functional genomics subunit, cdb, riken, kobe, japan; lab. for systems biology, cdb, riken, kobe, japan; lab. for mammalian germ cell biology, cdb, riken, kobe, japan; crest, jst, japan in the mammalian brain, cellular heterogeneity of the progenitor cells has largely hindered the molecular analysis of neuronal diversity. to overcome this problem, we randomly picked individual vz/svz cells of mouse embryos, and constructed cdnas from each of them by global pcr amplification method. we could classify these "single cell derived cdnas" into several groups retrospectively based on the expression of marker genes, including cell cycle related genes, transcription factors, and regional marker genes. samples that showed typical marker gene expression pattern of the groups were applied for genechip analysis. the obtained data were confirmed by quantitative pcr and in situ hybridization. by this strategy, we identified nine genes that were specifically expressed in the svz progenitor cells. research funds: kakenhi ( ) ryosuke tatsuno , tomoaki sai , , masahiro otsu , kuniko akama , takashi nakayama , tosifusa toda grad. sch. of sci. and tech., chiba univ., chiba, japan; lab. regener neurosci., tokyo metropol. univ. fac. health sci., tokyo, japan; dept. orthop. surg., jikei univ. sch. med., tokyo. japan; dept. biochem., yokohama city univ. sch. med., yokohama, japan; proteomics collab. res., tokyo metropol. inst. of gerontol., tokyo, japan embryonic stem (es) cells possess pluripotency and self-renewal. however, the proteomic analysis of neural stem cells and neurons differentiated in vitro from es cells has not so proceeded yet. we investigated the expression levels of proteins during in vitro differentiation of mouse es cells into neurons via neural stem cells by neural stem sphere (nss) method, using -d gel electrophoresis and maldi-tof ms. we identified vimentin, creatine kinase, atp synthase beta subunit, and some proteins with no annotation in murine brain the database, which were up-regulated in neural stem cells, and down-regulated in es cells and neurons. these results suggest that the neural stem cells have characteristic protein expression profile. ps p-e identification of se , a novel gene expressed in the nural progenitor cells shin-ichi sakakibara, kazuhiko nakadate, shiichi ueda department of histology and neurobiology, dokkyo university school of medicine, tochigi, japan identification of the genes regulating neural progenitor or neural stem cell functions is critical to understand the mechanisms of the adult neurogenesis and neurodegenerative disease. we compared the gene expression profile of proliferating neural stem cell cultures with those of differentiated cells. a subtractive library was constructed by using the suppression subtractive hybridization and the differential screening was performed. among two thousand of the differentially expressed subtracted clones, we identified genes that significantly upregulated in neural stem cell culture. these included several novel genes, in addition to the known genes involving in the cell cycle and signal transduction. in situ hybridization and the developmental northern analysis demonstrated that these mrnas were enriched in the germinal neuroepithelium, embryonic ventricular zone and the postnatal subventricular zone surrounding the lateral ventricles. we further analyzed the expression pattern of the novel gene se in developing and matured cns. teiichi furuichi , akira sto , , yukiko sekine , noriyuki morita , tetsushi sadakata , satoshi shoji , jin-hong huang , toshio kojima laboratory for molecular neurogenesis, riken brain science institute, japan; comparative systems biology team, riken genome sciences center, yokohama - , japan mouse cerebellum develops through a series of cytogenetic and morphogenetic events that are genetically coded within the first three weeks of life. we have extensively investigated the spatio-temporal gene expression profiles during the postnatal development of mouse cerebellum by differential display, rt-pcr, genechip, cdna microarray, and in situ hybridization. we have informatively systematized all the profiles in an online neuroinformatics database cdt-db (http://www.cdtdb.brain.riken.jp) with various search functions. we have demonstrated that the postnatal development of mouse cerebellum is genetically programmed by thousands of genes that exhibit differential expression patterns in time and space. further studies on a scale that includes the underlying expression of all genes and more detailed studies on their transcriptional regulation will shed light on the genetic basis for cerebellar development. miwako ozaki , makoto mizuno , kazuhisa sakai , yoshimoto kiyohara , kazuhiko yamaguchi , tsutomu hashikawa , hiroyuki nawa institute of biomedical engineering, waseda university, tokyo, japan; department of molecular neurobiology, brain research institute, niigata university, niigata, japan; laboratory for memory and learning, bsi, riken, saitama, japan; laboratory for neural architecture, bsi, riken, saitama, japan neuregulin (nrg), a neurotrophic factor, involved in the development, differentiation and repair of the nervous system, regulates the activation of ion channels and neurotransmitter receptors. in order to examine the molecular mechanism on the relationships between network, synapse formations and higher orders functions, we prepared ig-nrg knock out mice (nrg type i and iv were disrupted). the mutant mice showed motor disco-ordination and abnormality of synaptic structure in related areas in cerebellar nuclei and cortex. in addition, the number of vesicles in presynaptic neurons decreased in their synapses. the study on cerebellum that is very clear in the network input information would give some suggestions to the relationship between synaptic functions and behaviors. ps p-e psd- protein expression in rat oromaxillofacial motoneurons during postnatal development kohji ishihama , , satoshi wakisaka , shiho honma , akira ito , , kei azuma , , mikihiko kogo department of oral anatomy and developmental biology, osaka university graduate school of dentistry, osaka, japan; first department of oral and maxillofacial surgery, osaka university graduate school of dentistry, osaka, japan postsynaptic density (psd), which is composed of diverse proteins, involved in synaptic structure, neurotransmission and signal transduction. psd- implicates in formation and maturation of excitatory synapses. psd- regulates the localization of the nmda receptor by means of binding with nr . rhythmical oro-maxillofacial activities, such as suckling and chewing, are generated in the brainstem, and we showed that nmda receptors played critical role for the rhythm and pattern generation and signal transmission around the trigeminal motor nucleus during prenatal and early postnatal development. here we examined the temporal distributions of psd- protein using with immunohistochemical study, in developing rat brainstem from suckling to mature chewing stage. there was early emergence of psd- expression in the interneurons located at medial of the trigeminal motor nucleus. masami miura, masao masuda, toshihiko aosaki neural circuits dynamics research group, tokyo metropolitan institute of gerontology, japan the striatum, an input stage of the basal ganglia, contributes to habit formation as well as motor functions. recent studies suggest that striatal interneurons play an important role in processing of cortical input. we investigated the synaptic connections between interneurons using paired whole-cell recordings and immunohistochemical techniques. we found that fast-spiking (fs) interneurons sent gabaergic inhibitory input to cholinergic interneurons, which were gaba a receptor-mediated and suppressed by gaba b receptor agonist skf . in turn, cholinergic interneurons sent cholinergic excitatory input to fs interneurons. because the excitatory postsypnatic potentials (psps) were blocked by hexamethonium and dihydro--erythroidine, the psps were nicotinic acetylcholine receptor-mediated. these results suggest that gabaergic interneurons and cholinergic interneurons mutually influence their excitability and might modulate the activity of striatal local circuits. ps p-e ocular following responses (ofrs) to a brief background motin are modulated in relation to preparation for upcoming pursuit hiromitsu tabata, kenichiro miura, kenji kawano dept. integ brain sci., grad. schl of med., kyoto univ., kyoto, japan recently, our group reported that the ocular responses to a brief perturbation of a small target during fixation increased when subjects (humans, monkeys) were preparing for upcoming smooth pursuit eye movements (spems) rather than preparing for saccades or stationary fixation. here, we report that the increase in ocular responses based on the anticipation of spems was also observed in monkeys when a large-field visual stimulus (background) was moved briefly prior to pursuit. the result indicates that the visual region where the gain of the visuomotor transmission increased is not limited to a small region near the target but spreads to a larger field. in other words, the anticipation of upcoming spems could affect the generation of ofrs. furthermore, directionally biased ocular responses to the brief background motion were observed when the animals repeatedly performed spems toward one direction, implying that the prediction of the upcoming spem direction might cause the directional asymmetry of the visuomotor transmission gain. ps p-e comprehensive characterization of motor neurons related with locomotory central pattern generator in the earthworm by imaging toshinobu shimoi , kenji mizutani , hiroto ogawa , kohji hotta , kotaro oka ctr. for biosci. and info, keio univ., yokohama, japan; neuro, karolinska inst, stockholm, sweden; bio, saitama med. sch., saitama, japan in this study, we comprehensively identified and characterized motor neurons concerning with locomotory central pattern generator (cpg) in the earthworm by calcium imaging as multiple recording. the candidates of motor neurons were stained with dextran conjugated calcium indicators using retrograde labeling from projection nerves. we obtained the responses of up to cell bodies of motor neurons and sensory neurons on the ventral surface of the segmental ganglion ( % or less for all neurons on the ventral surface). we analyzed the activity patterns of the candidates of motor neurons using pattern matching method comparing between calcium responses or between calcium responses and locomotory motor pattern. as a result, we detected motor neurons as pairs of neurons having strong synchrony to each other neuron or to motor pattern. these results were great progress to identify motor neurons related with locomotory cpg in the earthworm. ps p-e three dimensional ( d) pursuit eye movement signals in cerebellar dorsal vermis takuya nitta, teppei akao, sergei kurkin, kikuro fukushima department of physiology, hokkaido university school of medicine, sapporo, japan for pursuit of a target moving in d space, signals for frontal and vergence-pursuit must be synthesized. studies in our laboratory have demonstrated that d pursuit signals are generated in the frontal eye fields, and also present in cerebellar floccular region. however, the majority of floccular purkinje (p-) cells discharged after onset of vergence-pursuit. cerebellar dorsal vermis is another cerebellar area for frontal pursuit. to examine whether d pursuit signals are present in this area, we examined simple-spike discharge of vermal pursuit p-cells in monkeys. of a total of p-cells that were examined during both frontal and vergence-pursuit, % discharged for both, % only for vergence, and % only for frontal pursuit. these results indicate that most of vermal pursuit p-cells discharged for vergence and that about half of them had d pursuit signals. majority ( %) of these p-cells discharged before onset of vergence eye movements with the typical lead time of ms, suggesting their involvement in the initiation of vergence-pursuit. research funds: kakenhi ( ) ps p-e information processing in fef-rnrtp pathway for smooth pursuit seiji ono, michael j. mustari division of sensory-motor systems, yerkes national primate research center, emory university, atlanta ga, usa the frontal eye field (fef) cortex is known to play a role in smooth pursuit (sp). this role is supported by fef projections to the rostral nucleus reticularis tegmenti pontis (rnrtp) which projects heavily to the vermis. using multiple linear-regression modeling, we have shown that sp neurons in rnrtp were biased towards eye acceleration. however, the functional characteristics of sp related fef neurons that project to rnrtp have never been described. therefore, we used micro-electrical stimulation to deliver single pulses in rnrtp to antidromically activate fef neurons. the majority of sp related fef neurons that we identified as projecting to rnrtp were most sensitive to eye acceleration and much less sensitive to eye velocity. the neurons in fef-rnrtp pathway carry signals that could play a primary role in sp initiation. our antidromic studies may help address a fundamental question regarding whether basilar pontine nuclei integrate signals from multiple cortical areas or mostly relay signals with little transformation to cerebellum. research funds: nih grants ey , rr aya takemura , yumi murata , , kenji kawano , neurosci. res. insti, aist, tsukuba, japan; dept. integ brain sci., grad. sch. med., kyoto univ., japan; grad. sch. compreh hum sci., univ. tsukuba, japan previous studies in monkeys suggest that the medial superior temporal (mst) area is involved in visual motion processing. to understand the role of the mst in optokinetic nystagmus (okn) and afternystagmus (okan), we examined the effects of bilateral chemical lesions in the mst in two monkeys. when each monkey was injected with ibotenic acid ( mg/ml, - l total), the initial rapid rise in okn was reduced. consequently, it took longer for the eye velocity to reach a steady state (i.e., an eye velocity close to the stimulus velocity). by contrast, the steady state okn was not affected and the okan persisted. the initial amplitude and falling time constant of the okan increased. the results suggest that the mst is part of the direct pathway for the initial rapid rise in the okn, but is not involved in the velocity storage mechanism for the steady state okn and okan. smooth pursuit is performed by coordination of eye and head movements. we have reported that the majority of fef pursuit neurons in monkeys with their head free to rotate about a vertical axis were modulated not only during eye-and gaze-pursuit but also head-pursuit to a moving reward feeder while the monkeys fixated an earth-stationary spot without gaze movement. to examine the origin of head-pursuit modulation, we moved the reward feeder in a ramp trajectory at • /s with random intervals. the majority of pursuit neurons discharged before the onset of head movements with the mean lead time of ms. discharge modulation during head-pursuit and passive whole body rotation was not correlated in most neurons. these results suggest that proprioceptive neck inputs or vestibular inputs are not the main origin of head-pursuit modulation. rather, our results suggest that the main origin reflects pursuit commands. ps p-e the local feedback loop of the saccadic system: an analysis of the eye movements induced by pdb stimulation rikako kato department of developmental physiology, national institute for physiological sciences, okazaki, japan saccadic amplitude are controlled by a comparator that calculates dynamic motor error. some models place the comparator in the superior colliculus while others assign this role to the reticular formation. to decide between the two hypotheses one would need to stimulate pathways in between their putative comparators. we stimulated collicular axons descending in the pdb. our data demonstrate that electrical stimulation of the pdb evokes saccades and they always terminate before the end of the stimulus train. the characteristics of evoked saccades are comparable to those spontaneously generated by the cat. our data clearly demonstrate that the feedback path of the local loop of the saccadic system closes downstream of the superior colliculus. katsuo fujiwara , kenji kunita , kaoru maeda , takeo kiyota department of human movement and health, graduate school of medical science, kanazawa university, kanazawa, japan; institute for health and sport sciences, osaka city university, osaka, japan we investigated changes in visual evoked potential (vep) during postural adaptation process while subjects maintaining standing posture on an oscillation floor with periodic vision shut. the subjects were undergraduate students. a shutter goggle was used as a vep stimulator which was opened periodically for ms with -ms intervals. the oscillation trial ( . -hz frequency and . -cm amplitude) ( - s) was repeated times. postural steadiness was evaluated by mean fluctuation speed of the center of foot pressure. the mean speed decreased as trial was repeated, and reached a plateau before the th trial. a significant correlation was shown between th- st trial differences in mean speed and vep amplitude (r = . ). this indicates that the role of visual information is different among subjects with various adaptation processes of postural control. ps p-e primary motor cortex contributes to generating manual following response toshitaka kimura , naoki saijo , hiroaki gomi , ntt cs labs, kanagawa, japan; erato shimojo implicit brain function proj, jst, saitama, japan a large-field visual motion during arm movements induces a shortlatency, involuntary arm response called as manual following response (mfr). the mfr exhibits similar features to the ocular following response (ofr) elicited by the similar visual stimulus, with respect to the stimulus-response directional characteristics and the spatiotemporal frequency tuning property. this suggests that computational mechanism is shared for both responses. however, the neural basis of the mfr motor command generation remains unclear, while ofr is known to be generated subcortically. here we show, by using transcranial magnetic (tms) and electrical (tes) stimulation over the primary motor cortex (m ), that ( ) an emg response evoked by tms was facilitated during mfr, while that by tes was not, and ( ) intracortical inhibition within m assessed by paired-pulses tms was reduced during mfr. these results suggest that mfr is generated through activity of interneuronal networks within m . such cortical mechanisms for mfr generation are distinct from the subcortical processes for ofr generation. naoki saijo , hiroaki gomi , ntt cs labs., kanagawa, japan; erato shimojo implicit brain function proj, jst, saitama, japan when a visual target is suddenly shifted during a reaching movement, we can quickly adjust the arm movement. however, the computational mechanism to generate quick adjustment is still unclear. here we investigated this mechanism from the viewpoint of visuomotor coordinate transformation. we observed the hand responses to the target shifts in radial directions applied during reaching. the data show that the direction of the initial phase ( - ms) of hand response acceleration was slightly biased from the corresponding target shift direction, whereas the direction of the late phase ( - ms) was little biased. additionally, when we use a target shift having less-motion energy, the response latency greatly increased and the directional bias significantly decreased. these results suggest that the on-line reaching adjustment would be generated by two different mechanisms: a reflexive controller which is induced by visual motion with short latency and generates spatially inaccurate response, and voluntary controller which generates spatially accurate response with long latency. ps p-e spatial relationship between gaze and reaching-target modulates manual following response naotoshi abekawa , hiroaki gomi , ntt cs labs., kanagawa, japan; erato shimojo implicit brain function proj, jst, saitama, japan to explore the functional mechanism of the manual following response (mfr) induced by a large-field visual motion during arm movement, we examine its modulation caused by the spatial relationships between gaze, target, and background. on a large vertical screen placed in front of the subject, full field checker pattern, two markers (upper and lower), and a gray mask around one of the markers, were displayed. in the first condition, subjects kept watching the upper marker, and pointed the upper (congruent) or lower (incongruent) marker instructed before every reaching. the checker pattern suddenly moved either rightward or leftward brief after reaching start. in the second condition, subjects did the same task with watching the lower marker. in both conditions, the mfr amplitude was significantly grater in the congruent condition than in the incongruent condition, whereas the mask location did not significantly affect the mfr amplitude. this suggests that the spatial relationship between gaze and target is important in modulating mfr. misako komatsu, eizo miyashita dept. compu. intelligence & systems sci., tokyo tech., yokohama, japan when a subject performed pointing to a remembered target under eyes fixated, we have reported that endpoints tended to sift closer to the fixation point. moreover, we have noted that the greater the distance between a target and the fixation point, the larger the errors. the result was consistent even when the position of the fixation point was changed. the above tendency was considered to occur in eye-or gaze-centered coordinates. it is open question, however, if the brain correctly compensates the difference of the relative position of eyes to the head? to answer this question, we investigated the dependency of the endpoint errors on the positions of a monitor and the fixation point. the subjects, sitting in front of the monitor, were asked to point a remembered target as accurately as possible using a computer mouse. all the results were consistent with the previous ones regardless of the position of monitor or the fixation point. these results suggest either the eye-position doesn't affect how we recognize the target position, or the brain correctly compensates the eye-position with a fixed head position. ps p-f influence of the coupling of muscle activity on rhythmic movements of ipsilateral hand and foot tetsuro muraoka , takashi obu , kazuyuki kanosue asmew, waseda university, saitama, japan; graduate school of human sciences, waseda university, saitama, japan; faculty of sports sciences, waseda university, saitama, japan the aim of this study was to investigate the influence of the coupling of muscle activity on rhythmic movements of ipsilateral hand and foot. the subjects (n = ) were supine, and their hand was prone. they performed cyclical flexion-extension coordinations of the hand and foot in the iso-(iso) or opposite-(oppo) directions, and those with an elastic load against wrist flexion (el-iso and el-oppo) at . , . , and . hz. over % success rate was observed in all tasks except oppo ( - %). the in-phase muscle activity of wrist and foot muscles was obserbed in all tasks except oppo. it was suggested that the in-phase muscle activity might be an important factor in a coordinated movement of ipsilateral hand and foot. research funds: the special coordination funds for promoting science and technology, mext, japan ps p-f simultaneous muscle activity stabilizes the coordinated movement of ipsilateral hand and foot takashi obu , tetsuro muraoka , kazuyuki kanosue , , faculty of human sciences, waseda university, saitama, japan; faculty of sport sciences, waseda university, saitama, japan; asmew, waseda university, saitama, japan in human, voluntary opposite-directional movement (antiphase) of ipsilateral hand and foot is more difficult than iso-directional movement (inphase). the purpose of the present study was to investigate the influence of the coupling of muscle activity on these movements. eight normal subjects lay in supine position with hand prone and their foot was forcedly moved by a dynamometer cyclically at , . , and . hz. they were asked to perform tasks, concentric/eccentric contraction of ankle dorsiflexors with in-phase/antiphase wrist extension/flexion. all tasks were performed successfully. muscle activity of hand flexors was observed in concentric-antiphase and eccentric-inphase tasks, indicating simultaneous muscle activity of hand and foot. it may be suggested that simultaneous muscle activity would make the movement easier regardless of the direction of movement. ps p-f activities of erector spinae muscles during jaw clenching in man kayoko yasunaga , , tadachika yabushita , kazuo toda , kunimichi soma orthodontic science, tokyo med. & dent. univ., tokyo, japan; div. integrative sensory physiology, nagasaki univ., nagasaki, japan recent studies focused the functional relationships between the masticatory and the posture system. the hypothesis of our present study is an existence of functional connections between the masticatory system and the spinal muscles which maintain the posture. therefore, we investigated the effect of the maximum jaw clenching on the spinal muscle activities. bipolar needle electrodes were inserted into erector spinae muscles to record the motor unit activities when the sitting subjects relaxed and performed maximal jaw clenching. as a result, the instantaneous frequencies of the spinal muscles decreased with clenching, compared with relaxed jaw position. our results suggested that there were some relationship between spinal muscle activities and jaw clenching. the effects of bipedal walking on the central nervous systems-influence of bipedal walking on the spinal reflex-naomi wada , sachiko motoyama , futoshi mori , shigemi mori department of veterinary physiology, yamaguchi university, yamaguchi, japan; national institute for physiological science, okazaki, japan the one of the biggest questions in the vertebrate evolution is how human got the highly developed brain. many investigators suggest that upright posture and bipedal walking caused remarkable development of brain and produced the human being. the purpose of our experiments is to show the influences of bipedal habits on central nervous systems. we have established the bipedal walking model using rats (rbm) by amputation of forelimbs and training of upright posture and bipedal walking. after training of upright posture and bipedal walking for - weeks, rats got abilities of the stable upright posture and bipedal walking with symmetrical hindlimb movements between left and right side. in the present experiments, we studied about the effects of bipedal habits on the lumbar spinal reflex. the results of out experiments showed that bipedal habits inhibit the spinal reflex pathways. ps p-f neuronal activity in primary motor cortex during quadrupedal locomotion of the japanese monkey katsumi nakajima , futoshi mori , akira murata , masahiko inase dept. of physiol., kinki univ. schl. of med., osakasayama, japan; dept. of vet. physiol., facult. of agr., yamaguchi univ., yamaguchi, japan to elucidate cortical mechanisms related to the control of primate locomotion, we recorded neuronal activity in m of the monkey walking quadrupedally on the treadmill. tungsten microelectrodes were inserted into m hindlimb region using a custom-made micromanipulator. we found that all neurons recorded in m modulated their discharge phasically time-locked to the step cycle or increased their discharge frequency tonically during simple locomotion. the neuron exhibiting phasic modulation peaked once or twice per step. the peak activity occurred at widely different times during the step cycle in different recorded neurons. as the treadmill speed increased, most of recorded neurons increased their discharge frequency. all these results suggest that m output in monkeys directly and/or indirectly acts on spinal circuitries generating a basic pattern of rhythmic activity during simple locomotion in a manner different from that in subprimates. research funds: kakenhi ( ) ps p-f activity of putaminal neurons receiving inputs from motor cortical areas in behaving monkeys sayuki takara , , nobuhiko hatanaka , , masahiko takada , atsushi nambu , school of life science, the graduate university for advanced studies, japan; division of system neurophysiology, national institute for physiological sciences, japan; tokyo metropolitan institute for neuroscience, japan the putaminal (put) neurons receive motor cortical inputs and change their activity in relation to movements. to investigate how these inputs contribute to put neuron activity in behaving monkeys, extracellular unit activity was recorded from identified put neurons during the performance of a memory-guided reaching task. based on orthodromic spikes evoked by cortical stimulation, individual put neurons were defined in terms of whether they receive input from the primary motor cortex (mi), the supplementary motor area (sma), or both. the results showed that mi-recipient neuron activity was responsive to the movement, while sma-recipient neuron activity was responsive to the cue stimuli and/or the delay period. the activity of neurons receiving convergent inputs was related to both the movement and the delay period. we previously reported that electrical stimulation of cerebrofugal fibers induced short latency facilitation and succeeding suppression on phrenic activities, while train pulse stimulation of caudal raphe nuclei (raphe magnus, rm, and raphe pallidus, rp) induced suppression or facilitation on respiratory neural activities in cats and rats. in this study, in order to analyze the cerebral and raphe projections to the respiratory neuron network, we examined the effects of stimulation of cerebrofugal fibers and caudal raphe nuclei on activities of ventral respiratory group neurons (vrgs) in the medulla and upper cervical inspiratory neurons (ucins). animals were anesthetized, immobilized and artificially ventilated. stimulation of cerebral peduncle (cp) induced short latency facilitation and succeeding suppression on activities of ucins. stimulation of rm or cp evoked inhibitory postsynaptic potentials in the caudal vrgs. these results suggest that rm and cerebral cortex directly inhibit main respiratory output neurons in vrg. ken muramatsu , sei-ichi sasaki , yuichiro cho , kenji sato anatomy and physiological science, tokyo medical and dental university, tokyo, japan; department of physiology, ibaraki prefectural university of health sciences, ibaraki, japan distribution of average diameters of external anal sphincter (eas) motoneurons and peripheral motor fibers were examined in cats. to identify eas motoneurons, horseradish peroxidase was applied to the central cut end of the anal branches of the pudendal nerve. eas motoneurons were found in the onuf's nucleus of s and s spinal levels. to examine size of peripheral motor fibers, ganglionectomy was performed onl -s spinal segments which contain afferent fibers of eas muscles. after weeks survival period, anal branches of the pudendal nerve was examined. histograms of the distribution of average diameters of cell body and motor fiber shows unimodal distri bution. also, distribution of muscle spindles of eas muscle were examined by serially sectioning the distal colon and staining with mayer's haemotoxylin and eosin. no muscle spindles were found. these results suggest that eas muscle is controlled without gamma loop. mariko miura, yoshiki iwamoto, kaoru yoshida neurophysiol., univ. tsukuba, tsukuba, japan saccade accuracy is ensured by an adaptation mechanism. the speed and magnitude of adaptation vary greatly across experiments even for the same subject. one factor that might cause this variability is adaptation history. the present study aims to clarify whether preceding adaptation influences subsequent adaptation over several days. gain decrease adaptation was induced in a monkey by stepping the target backward during saccades. adaptation experiments were repeated for consecutive days. we compared adaptation in day and that in day . the gain decrease for the first saccades in day ( . ± . ) was larger than that in day ( . ± . ) (p = . , n = , paired-t test). the rate of adaptation in day ( . ± . × − /sac) was higher than that in day ( . ± . × − /sac) (p = . ). the overall gain change ( saccades) in day ( . ± . ) was larger than that in day ( . ± . ) (p = . ). thus, both the speed and magnitude of adaptation were increased by preceding adaptation. the present study suggests that the memory of saccadic adaptation is retained for days and facilitates following adaptation. research funds: kakenhi ( ) ps p-f asymmetry of the anticipatory convergence eye movement haruo toda, takehiko bando div. integr. physiol., grad. sch. med. sci., niigata univ., niigata, japan typically, convergence eye movement is known as symmetric adduction of the both eyes. but asymmetrical convergence also found in the natural condition. these asymmetrical convergence may reflect asymmetries of central control of convergence eye movement. the lateral suprasylvian (ls) areas are extrastriate cortices which receive visual information from v . the ls has contralateral dominant receptive fields and convergence eye movements evoked from the long latency regions were asymmetrical. cats (n = ) were trained to start convergence by an alarm signal (buzzer sound or combination of buzz and blinking of led), preceding target movement by s. after training, ocular convergence was elicited by the alarm signal before target movement (predictive open-loop convergence) in % of trials. in three cats, we used training with obliquely approaching target. after training, asymmetrical anticipatory eye movements were observed. based on these findings, related ls neuronal activities and results from lesion study, we will discuss the role of ls in asymmetry of anticipatory and visually-evoked convergence eye movement. yusuke uchida , xiaofeng lu , , shogo ohmae , toshimitsu takahashi , , shigeru kitazawa , dept. of neurophysiol., juntendo univ. grad. sch. of med., tokyo, japan; crest, jst, tokyo, japan we examined reward related neural activity in the supplementary eye field (sef). for this purpose, two monkeys were rewarded after each visually guided saccade from a central fixation point to one of targets that were arranged in a radial pattern. a target appeared while the monkeys were fixating on the central point, and the monkeys made a saccade to the target when the fixation point disappeared and held on the target until the target turned off. reward was delivered during or after target-hold period. we found that many sef cells became active during the period of reward delivery (r-cell). more than half of r-cells showed enhancement of the neural discharge in the specific target directions but not other directions in which the same amount of reward was given (rd-cell). interestingly, most of rd-cells displayed activity with the clear directional tuning. these results demonstrate reward dependent activity specific to spatial direction in the sef, and further suggest that sef cells provide reinforcement mechanism. research funds: kakenhi ps p-f frontal pursuit area is involved in the retinalslip dependent adaptation of monkey post-saccadic pursuit eye velocity hiromasa kitazawa , soichi nagao , lab. for motor learning control, riken bsi, saitama, japan; sorst, jst, saitama, japan smooth pursuit is under learning control by several brain areas including cerebrum and cerebellum. smooth pursuit velocity is modifiable by repetition of target velocity for a brief period at its onsets. role of cerebellar vermis and hemisphere in the adaptive control of smooth pursuit is suggested by lesion experiments, but the role of frontal pursuit area (fpa) is not known. to reveal possible involvement of fpa in the adaptation of smooth pursuit, we identifying fpa by unit recording and microstimulation, and reversibly inactivated it by local injection of muscimol. we found that inactivation of fpa not only reduced of the velocities of pursuit in the ipsi-and contra-versive directions to the inactivated fpa, but also appreciably depressed its adaptation, suggesting that fpa is involved in the adaptation of smooth pursuit. shinji matsutani department of functional morphology, kitasato university school of nursing, kanagawa, japan distribution of terminals on individual centrifugal axons in the main olfactory bulb was studied using an anterograde tracer to elucidate function of the centrifugal system. the tracer was injected into olfactory cortical areas, and individual labeled axons were traced from serial sections. as already reported in the last meeting, the centrifugal axons had multiple terminals with discrete locations. distribution of these terminals was examined in reconstructed maps in which localization of the terminals was projected onto a sagittal plain. in most axons, the terminals were clustered to form a patch that was stretched in a rostrocaudal direction. it was also common that patches belonging to the same axon were found in distant locations and in both sides of the single bulb. while most of the terminals were seen in the granule cell layer, those located in the glomerular layer and in the external plexiform layer were found following injections into the anterior olfactory nucleus. the centrifugal fibers may couple the activity of discrete and distant subsets of bulbar neurons. ps p-f projection targets of the drosophila taste receptor neurons in the primary gustatory center of the brain takaaki miyazaki , , kei ito , , dept. of comput. biol., grad. sch. of frontier sci., univ. of tokyo, japan; center for bioinform., imcb, univ. of tokyo, japan; bird, jst, japan in order to figure out the way of information processing linking gustatory stimulus and taste-associated behavior, systematic knowledge about the underlying neural networks is required. drosophila melanogaster is an attractive model organism for this task, thanks to its relatively simple brain structure and a wide variety of molecular and genetic tools available. gustatory sensory neurons in the labellum of the mouth project their axons via the labial nerve to the suboesophageal ganglion (sog) of the brain. to understand the entire neural circuits of these first-order neurons in the primary gustatory center, we searched for the gal enhancer-trap strains that visualize specific neural fibers in the sog and the labial nerve. screening , strains, we identified about candidate lines. the projection targets of the labeled neurons were classified into seven areas. the terminals of the already identified sensory neurons appear to fall into specific subsets of these areas. research funds: bird, jst ps p-f immunoreactivity and voltage-gated channels of mouse taste bud cells kennji kimura , yoshitaka ohtubo , takashi kumazawa , kiyonori yoshii graduate school of life science and systems engineering, kyushu institute of technology, kitakyushu, japan; department of applied chemistry, saitama institute of technology, fukaya, japan mammalian taste buds comprise four heterogeneous cell types, type i to iv, and their collaboration seems to generate taste sensation. we investigated the electrophysiological properties of these cell types except type iv with taste buds preserved in mouse lingual epithelia. type i cells elicited smaller ttx-sensitive, tea-sensitive, and teainsensitive currents in magnitude than other cell types. type ii cells elicited a smaller tea-sensitive current and a larger tea-insensitive current than type iii cells. these results suggest that type ii and iii cells elicit action potentials with different ionic mechanisms, and that the difference results from the functional differences of these cell types. research funds: kakenhi ( ) and the st coe program (center # ) granted by mext of japan ps p-f inositol monophosphatase maintains synapse localization and regulates behavior in the mature nervous system of c. elegans yoshinori tanizawa , atsushi kuhara , hitoshi inada , eiji kodama , takafumi mizuno , ikue mori , lab. of mol. neurobiol., nagoya univ., japan; institute for advanced research, nagoya univ., japan inositol monophosphatase (impase) is suggested to be relevant to bipolar disorder. although lithium is believed to exert therapeutic effect by inhibiting impase in patients, the mechanism underlying lithium therapy is largely unknown. here we show that the loss of impase causes defects in behavior and localization of synapses in c. elegans. mutations in ttx- gene encoding impase exhibit defective thermotaxis behavior, which is attributable to the loss of impase activity in the most essential integrative interneuron ria in the nervous system. the ttx- mutations also cause mislocalization of synaptic proteins in ria. both behavioral and synaptic defects in ttx- mutants were rescued by expression of impase at adult stage and inositol application, and were mimicked by lithium application in wild type animals. these results suggest that impase is required in the mature nervous system for maintaining synapses of the central interneurons in order for animals to behave properly. research funds: kakenhi ps p-f postnatal alterations in expression of vesicular glutamate transporters in the main olfactory bulb (ob) of rats h ohmomo, f shutoh, a. ina, s. yoshida, h. nogami, s. hisano lab. neuroendocr., graduate sch., univ. tsukuba, tsukuba, japan olfactory information is conveyed to the brain by transmission from primary olfactory neurons to mitral or tufted cells. however, little is known about development of these ob glutamatergic neurons in early postnatal life. vesicular glutamate transporters (vglut) have been used as the best histological markers to identify glutamatergic neurons. we here studied expressions of two vglut isoforms (vglut and - ) during rat ob development from postnatal day (p ) to p by in situ hybridization and immunohistochemistry. at p vglut immunoreactivity (ir) was detected in all layers except the olfactory nerve layer, and thereafter its localization expanded and intensity increased. vglut mrna signals were detectable in the mitral cell layer from p to p . in contrast, vglut ir was prominent in the glomerulus at all days examined, and only at p and p in mitral cells. despite mitral vglut ir disappeared at p , the mrna signals were still detectable. these results suggest that glutametergic neurons in the rat ob continue to develop even after birth. ps p-f v r genes multiplied in amphibian and expressed in the main olfactory system atsuko date-ito , , masumi ichikawa , yuji mori , kimiko hagino-yamagishi tokyo metrop. inst. med. sci., tokyo, japan; the univ. of tokyo, tokyo, japan, tokyo metrop. inst. neurosci., tokyo, japan in rodent, v r gene family is expressed specifically in the vomeronasal organ (vno) and is thought to be responsible for pheromone reception. however, teleost fishes lacking for the vno have a single v r gene, which is expressed in the olfactory epithelium (oe). to examine when the v rs function as pheromone receptors in the course of evolution, we analyzed the amphibian xenopus tropicalis genome, and identified v r sequences. these v rs were not expressed in the vno, but most of them were expressed in the oe of the middle cavity, which is considered for reception of water-soluble odorants. from these results, we speculate that the amphibian v rs get a chance to receive diverse odorants such as pheromones by gene multiplication and sequence diversification. our results raise the possibility that pheromonal information is transmitted via the main olfactory system. ps p-f analyses of ligand binding sites and snps on sweet taste receptor system in human noriatsu shigemura, a.a. islam, yuki nakamura, shinya shirosaki, yuzo ninomiya sect. oral neurosci., grad. sch. dent science, kyushu univ., japan recent studies have shown that t r /t r heterodimer plays a role as a sweet taste receptor. but, mice lacking t r showed diminished but not abolished behavioral and nerve responses to sugars, suggesting t r -independent sweetener binding site also exist in mice. in this study, to predict binding sites on t r /t r and/or other sweet receptor in human, we measured sensitivity thresholds to various sweet compounds and examined the qualitative similarities. we also used gymnemic acid and ␥-cyclodextrin, which selectively inhibits sweet responses and reduces the inhibitory action of it. the ten sweet compounds were classified into five groups [( ) sucrose, glcose, fructose, ( ) saccharin, aspartame, acesulfame-k, glycine, ( ) d-phenylalanine, ( ) d-tryptophan, ( ) l-proline]. in sequencing analysis, four and two snps with amino acid substitution were revealed in t r and t r , respectively. these results suggest that there may be at least five binding sites in human sweet receptor system. the individual differences in sweet sensitivities may be due to these snps. keiko yasumatsu , sachiko saito , yuko murata , ding ming , tatsu kobayakawa , robert f. margolskee , yuzo ninomiya sect. oral neurosci., grad. sch. dent. sci., kyushu univ., fukuoka, japan; saito sachiko taste and smell research institution, ibaraki, japan; national res. institute of fisheries sci., kanagawa, japan; dept. of physiol. & biophys., mount sinai sch. med., new york, usa; national institute of advanced industrial science and technology, ibaraka, japan the effect of unsaturated fatty acids on taste responses was examined by measuring perceived taste intensity in human, behavioral short-term lick responses and electrophysiological taste responses recorded from the chorda tympani and glossopharyngeal nerves in mice. the results showed that dha and other polyunsaturated fatty acids inhibit responses to bitter taste compounds without affecting other taste stimuli. we also found fatty-acid inhibition on bitter responses in an in vitro g-protein activation assay using bovine taste membrane, but lack of the bitter taste inhibition in ggustducin ko mice. these results suggest that fatty acids specifically inhibit responses to bitter stimuli by suppression of activation of t r receptors which coupled with ggustducin. ps p-f newborn infant body odor attenuates their mother's postpartum moods shota nishitani , mayumi kokuryo , tsunetake miyamura , kazuyuki shinohara div. neurobiol. & behav., nagasaki university, japan; obstet. & gynecol. of miyamura hospital, japan mothers are attracted to the body odor of newborn infants, but little is known about its reason. in the present study, we examined whether the body odor of newborn infants exert effects on moods in postpartum mothers. the body odors of newborn infants were collected from their undershirts. postpartum mothers were exposed to odors of a part of the undershirt with control odors, their own infant body odors or other infant body odors. we used the poms to assess the effects of infant body odors on postpartum moods. this study was approved by the ethics committee of nagasaki university. the infant body odors significantly increased hedonics and friendliness scores, and significantly decreased anxiety, depression and fatigue scores, whether infant odors may be originated from their own infants or other infants. these results suggest that body odors of newborn infants attract their mothers because they have calming effects on postpartum mothers. research funds: japan science and technology agency (jst), research institute of science and technology for society (ristex) ps p-f human prefrontal activity in taste encoding: an fnirs study masako okamoto , mari matsunami , haruka dan , tomoko kohata , kaoru kohyama , ippeita dan national food research institute, tsukuba, japan; nippon suisan kaisha, ltd., japan taste remains one of the least-explored human senses. using multichannel functional near-infrared spectroscopy (fnirs), we examined the lateral prefrontal cortex (lpfc) of healthy volunteers (n = ) while they tasted and encoded the quaternary taste mixtures. the contrast between the cortical activation under encoding conditions and that under control conditions without memory requirement revealed activation in the bilateral ventro-lpfc and the right posterior portion of the lpfc. the activation pattern, which was in line with those that have been associated with intentional encoding of non-verbal materials of other senses, supported an amodal role of lpfc in intentional encoding, at least at a macro structural level. this study also demonstrates that, by using fnirs, lpfc functions on taste can be examined with experimental paradigms comparable to those used for other senses. recently, we performed simultaneous respiration and electroencephalographic recordings during odor stimulation. we sought to identify changes in respiratory pattern, inspiratory phase-locked alpha oscillation (i-␣) and location of dipoles estimated from the potentials. electroencephalographic dipole tracing identified the location of dipoles from the i-␣ in the limbic area and the cortex; the entorhinal cortex, hippocampus, amygdala, premotor area and orbitofrontal cortex. in this study, we compared the respiratory pattern during odor stimulations, i-␣, dipole localizations without habituation with those with habituation of odors. onset of inspiration was used as a trigger for averaging, and potentials were averaged before and after the habituation period. habituation of odor caused to return to the normal respiratory pattern, decrease of amplitudes of ␣, and entorhinal cortex, hippocampus, amygdala were less active. akio tsuboi, takaaki miyazaki, takeshi imai dept. of biophys. & biochem., univ. of tokyo, tokyo, japan vertebrate odorant receptor (or) genes are divided phylogenetically into two distinct classes, the fish-like class i and the terrestrialspecific class ii. in the present study, we systematically analyzed mouse class i or genes ( subfamilies) to elucidate the expression profiles in the olfactory epithelium (oe) and the projection sites of their olfactory sensory neurons (osns) in the olfactory bulb (ob). in situ hybridization (ish) revealed that most class i or genes ( subfamilies) were expressed in the dorso-medial zone (zone ) of the oe. furthermore, there appeared to be no significant differences in the distributions of osns expressing class i genes within zone . these results indicate that there is a clear boundary between zone and non-zone areas in the oe. some class i ors are known to possess ligand specificity for aliphatic acids, aldehydes and alcohols. our ish analysis has revealed that osns expressing the class i ors in zone tend to converge their axons on a cluster of glomeruli in an antero-dorsal domain that is assumed to be involved in responses to the aliphatic compounds on the ob. research funds: kakenhi ( ) ps p-g taste response characteristics of putative interneurons in the rat gustatory cortex tatsuko yokota, kunihiro eguchi, katsunari hiraba department of physiology, school of dentistry, aichi-gakuin university, nagoya, japan previous studies have indicated that the extracellular spike waveforms and discharge rate properties of cortical neurons differed between pyramidal cells and interneurons, the latter tending to have narrower spike-widths and higher discharge rates. taste-sensitive neurons in the rat gustatory cortex were classified according to ( ) best-taste profiles and ( ) spike-widths which were found to form a bimodal distribution (narrow and broad). narrow-spike neurons had a significantly larger response to nacl than broad-spike neurons, but no differences were found to other tastants. the proportion of narrow-spike neurons in the n-best neurons was higher than that in the h or nh-best neurons. these results indicate that putative interneurons may play an important role in the coding of salt taste information. research funds: kakenhi ( ) of japan to t.y. yuki sato, nobuhiko miyasaka, yoshihiro yoshihara laboratory for neurobiology of synapse, riken bsi, wako, japan in the fish olfactory system, individual olfactory sensory neurons (osns) are thought to express only one or at most a few different odorant receptors (ors) from the large or family consisting of ∼ members. here, we investigated the mechanisms underlying or gene choice by using transgenic zebrafish that carried a modified bac containing a zebrafish or gene cluster. replacement of the or coding regions in the bac transgene with reporter genes allowed the reporters to be expressed in a small population of osns in the transgenic fish. in situ hybridization analysis using or-specific probes revealed that or genes expressed in reporter-positive cells were mostly restricted within the same or subfamilies to which the replaced ors belonged. additionally, the reporter-expressing osns projected their axons to a topographically fixed cluster of glomeruli in the olfactory bulb. these findings suggest the hierarchical regulation of or gene choice, whereby an individual osn may express one or gene from a limited subpopulation that is chosen from the entire repertoire in advance. research funds: kakenhi ( ) ps p-g identification of perisomatic-targeting granule cells in the mouse olfactory bulb hiromi naritsuka , kazuhisa sakai , tsutomu hashikawa , kensaku mori , masahiro yamaguchi dep. physiol. grad. sch. med., univ. of tokyo, tokyo, japan; laboratory for neural architecture, bsi, riken, saitama, japan in the olfactory bulb (ob), odor information is processed by the local circuit that includes inhibitory interneurons. granule cells (gcs) are major interneurons in the ob, but their diversity is not well understood. in the ob of adult transgenic mice expressing gfp under the control of nestin gene regulatory regions, we observed gcs with strong gfp expression (referred to as type s cells). their dendrites branched and formed spines within the granule cell layer, internal plexiform layer and mitral cell layer but did not reach the external plexiform layer, where typical gcs make synapses with dendrites of mitral and tufted cells. type s cells had huge protrusions at their dendritic ends, which formed contact with mitral cell somata. electron microscopic analysis revealed the existence of reciprocal synapses between type s cell protrusions and mitral cell somata. characteristic morphology of perisomatic-targeting gcs indicates that they have functions distinct from typical gcs in the ob. keiko moriya-ito, kentaroh endoh, yuuki ishimatsu, masumi ichikawa department of neuroscience basic technology, tokyo metropolitan institute for neuroscience, fuchu, tokyo, japan a coculture system of accessory olfactory bulb (aob) neurons and vomeronasal neurons was established for studying the functional roles of aob neurons in pheromonal signal processing. in this study, the effect of vomeronasal neurons on the development of aob neurons was examined in a coculture system. the densities of dendritic spines were lower in the coculture than in single culture. the ratio of the density of synaptophysin-immunopositive spine/total spine density was larger in the coculture than in the single culture. the volume of spine head was larger in the coculture than in single culture. by electron microscopic observation, the synapses on dendritic shafts were decreased and the synapses on dendritic spines were increased in the coculture. the synapses between aob neurons and vomeronasal neurons were recognized in the coculture. these observations suggest that synapse formation of aob neurons is modified by synaptic contact with vomeronasal neurons. ps p-g nacl induced responses of mouse fungiform taste cells: existence of amiloride sensitive and insensitive taste cells ryusuke yoshida, tadahiro ohkuri, keiko yasumatsu, noriatsu shigemura, yuzo ninomiya sect. of oral neurosci., grad. sch. of dental sci., kyushu univ., fukuoka, japan previous electrophysiological studies showed that the chorda tympani nerve contains two types of nacl-responsive fibers, amiloride sensitive (n-type) and insensitive (e-or h-type) fibers, suggesting the existence of amiloride sensitive and insensitive taste receptor cells in fungiform papillae. in this study, we examined nacl responses of mouse fungiform taste cells in isolated taste bud and amiloride sensitivity of them. some taste cells respond to apical restricted nacl stimulation with increase in firing frequency and their responses were concentration dependent. amiloride mixed with apical nacl solution inhibited nacl responses in some taste cells [amiloride sensitive (as) cells] but not in others [amiloride insensitive (ai) cells]. ai cells responded to other electrolytes such as kcl and hcl. these results suggest the existence of at least two types of nacl sensitive cells, as and ai cells. n-or e-type fiber may selectively innervate as or ai cells respectively. research funds: kakenhi ( ), kakenhi ( ) ps p-g integration of olfactory and oral sensory input in the rat insular cortex hideki kashiwadani, kensaku mori department of physiology, university of tokyo, tokyo, japan axonal connections between olfactory cortex and insular cortex suggest that insular cortex integrates olfactory information and information originated from the oral cavity (taste, tactile, temperature). however cellular mechanisms underlying the integration of multimodality are poorly understood yet. in this study, we examined single-unit spike responses of insular cortical neurons to odor stimulation and intraoral water stimulation in urethane-anesthetized rat. we found that more than % of recorded neurons in the insular cortex responded to odors. about half of the odor-responsive neurons were activated by intraoral water stimulation, indicating the convergence of olfactory and oral sensory information onto individual neurons in the insular cortex. when odor stimulation and intraoral water stimulation were simultaneously applied, some neurons showed spike responses larger than the responses evoked by each stimulus. the integration of olfactory and oral sensory information in the insular cortex might contribute to form the flavor sensation. research funds: kakenhi ( ) ps p-g odor combination selectivity of the rat piriform cortex neurons ikue yoshida, kensaku mori dept. physiol. grad. sch. med., univ. of tokyo, tokyo, japan olfactory cortex is thought to integrate signals from different odorant receptors to form the olfactory image of objects. however, the manner of integration at the level of individual cortical neurons is not well understood yet. using single-unit recording method, we examined the response selectivity of individual neurons in a dorsocaudal part of the anterior piriform cortex (apc) to classes of odorous compounds, each class being present in odors from many different vegetables and fruits. individual neurons typically responded to more than classes of odorants. each neuron was uniquely tuned to a specific combination of odorant classes, and different neurons typically showed different odor combination selectivity. single-unit responses to odor mixtures showed mixture facilitation and mixture suppression. these results suggest that individual neurons in the apc can be characterized by the odor combination selectivity and that the apc neurons may integrate signals from different odorant classes. research funds: kakenhi ( gs ) ps p-g odor-driven activity in the anterior piriform cortex of an in vitro isolated whole brain with the olfactory epithelium takahiro ishikawa , takaaki sato , akira shimizu , ken-ichiro tsutsui , toshio iijima div. of systems neuroscience, grad. sch. of life sciences, univ. of tohoku, sendai, japan; res. inst. for cell engineering, aist, amagasaki, japan to examine the neural mechanisms underlying odor-induced response in the anterior piriform cortex (apc), we analyzed odorinduced local field potential (lfp) and multiunit activity in an in vitro preparation, isolated guinea-pig whole brain with the olfactory epithelium. in apc, odor-induced lfps consisted of a phasic initial component followed by a fast oscillatory activity in the beta range ( hz). by comparison a result of current source-density analysis with unit activity data, we confirmed that the initial component of odor-induced response has a characteristic temporal pattern, generated by a relatively weak direct afferent input, followed by an intracortical associative response, which was associated with a phasic inhibition. the beta oscillation might be generated by the repetition of these network activities. these electrophysiological data were consistent with the results of previous studies that used slice or anesthetized in vivo preparations. ps p-g chemotaxis of c. elegans to concentration gradient of an attractant superimposed on a uniformly distributed attractant lin lin, hiroyuki oikawa, miyako sasaki, tokumitsu wakabayashi, ryuzo shingai department of welfare engineering, iwate university, morioka, japan to investigate the informational interaction between pathways from different sensory inputs to the behavior in the nervous system of c. elegans, chemotaxis toward the concentration gradient of an attractant spotted on a uniformly distributed another attractant was investigated. lysine and chloride ions are water soluble chemoattractants. when m lysine was spotted on ammonium chloride background, . - . m and . m background did not influence lysine chemotaxis, while . m background augmented and . - . m background suppressed the chemotaxis. in contrast, when . m ammonium chloride was spotted on the lysine background, the background did not alter or suppressed the chemotaxis. interaction between informational pathways from different sensory inputs could be seen also in the presentation of an odorant spotted on chemoattractant background, and vice versa. ps p-g glutamate receptors are regulated by the ras-mapk pathway in neural circuit-dependent odor adaptation in c. elegans takaaki hirotsu , , , takeshi ishihara , eisuke nishida , yuichi iino dept. biol., fac. sci., kyushu univ., japan; mol. genet. res. lab., univ. of tokyo, japan; grad. sch. biostudies., kyoto univ., japan c. elegans shows a decrease in chemotaxis to odorants after exposure to the odorant for min. this plasticity, called early adaptation, requires aiy interneurons, which receive synaptic inputs from olfactory neurons, indicating that early adaptation depends on neural circuit. the ras-mapk pathway is activated by odorant exposure in aiy and plays essential roles for early adaptation. the function of glr- , a non-nmda type glutamate receptor, in aiy is also important for early adaptation. glr- appears to localize at postsynaptic sites in aiy. this localization was changed by odorant exposure in early adaptation. mutation of the ras-mapk pathway impaired localization of glr- . in vitro kinase analyses revealed the possibility that mapk directly phosphorylates glr- . these results suggest that the ras-mapk pathway controls odor adaptation by directly regulating glr- localization in aiy neurons. kohei ueno , yoshiaki kidokoro dept. behav. sci., grad. sch. med., gunma univ., maebashi, japan; inst. mol. cel. reg., gunma univ., maebashi, japan sodium chloride (nacl) is the major substance that induces nacl taste. in rodents, some strains prefer nacl solutions (∼ %), but others do not or even avoid them. although it is reported that the difference is based on the genetic background, the molecular information involved in the difference is not known. in the th ns annual meeting, we have shown that nacl preference in several wild-type strains of drosophila melanogaster is variable and p-element insertion in a single gene suppressed nacl preference. here, we carried out the sequencing analysis and found eight single-nucleotide polymorphisms (snps) in the gene. moreover, we found that one of the snps was correlated with nacl preference among wild-type strains. we generated transgenic flies and rescued the low preference phenotype of p-element insertion strain using the gal /uas system. finally, we examined the expression pattern of the gene and found the gene is expressed in taste organs. taken together, we suggest that the gene is a novel nacl receptor gene. ps p-g spatial and temporal organization of odor representation by moth antennal lobe output neurons shigehiro namiki , graduate school of life and environmental sciences, university of tsukuba, ibaraki, japan; department of mechano-informatics, graduate school of information science and technology, university of tokyo, tokyo, japan the antennal lobe (al) is the first relay station for olfactory information in the insect brain and is the anatomical equivalent of the mammalian olfactory bulb. both systems have common structures called glomeruli, functional units of olfactory processing. odor-evoked spatial and temporal patterns by an array of glomeruli are both important in olfactory coding. but the details of olfactory coding mechanisms are still unclear. we confirmed that projection neurons (pns, al output cells) innervating the same glomerulus had similar olfactory responses in the silkmoth. by pooling data from many pns that innervate identified glomeruli i reconstructed odor representations. i found that olfactory information is encoded by distributed spatiotemporal activity of a pn population and that there are no clear correlation between the similarity of slow temporal patterns of pns and spatial distances of innervating glomeruli. research funds: brain ps p-g medial nucleus amygdala neurons have morphologically and electrophysiologically heterogeneous properties makoto yokosuka , yoshinori sahara , shinichiro horie , masumi ichikawa , shun nakamura st. marianna univ. schl. med., kawasaki, japan; ntl. inst. neurosci., ncnp, tokyo, japan; tokyo metropol. inst. neurosci., tokyo, japan we characterize the electrophysiological and morphological properties of the medial nucleus amygdala (mea) neurons using whole-cell recordings in mice slice preparations. most mea neurons showed either tonic-bursting or adapting burst of action potentials to deporalizing currents. biocytin labeling showed that mea neurons possessed bipolar to multipolar cell bodies and dendritic fields covering projection areas from the accessory olfactory bulb. norepinephrine increased the frequency of spontaneous ipscs in some neurons, while serotonin increased spontaneous epscs in others. morphologically and physiologically heterogeneous mea neurons seem likely to produce multiplex outputs of many instinct behaviors. hideyuki matsumoto, kensaku mori department of physiology, graduate school of medicine, university of tokyo, tokyo, japan olfactory sensation sometimes lasts even after odorant stimulation has ceased. neuronal mechanisms for the olfactory afterimage are not well understood yet. single unit recordings from mitral/tufted cells in the mouse olfactory bulb (ob) showed that some neurons continued to discharge for more than s even after the cessation of odorant stimulation. the induction of the sustained spike discharge depended on the intensity of odorant stimulation, and showed an allor-none behavior. spike discharges during the sustained discharge mode phase-locked to the respiration cycle and the phase-locking pattern during the sustained discharge mode differed from that during odor stimulation. these results suggest that neuronal mechanism in the ob may be responsible for the induction of the post-stimulus sustained discharges. the respiratory-phase-locked sustained discharges were recorded from juxta-glomerular cells. this implies that neuronal interactions within the glomeruli are involved in the induction of the sustained spike activity of mitral/tufted cells. ps p-g synaptic transmission shows state-dependent change in the urethane-anesthetized rat olfactory bulb yusuke tsuno, hideki kashiwadani, kensaku mori department of physiology, graduate school of medicine, the university of tokyo, tokyo, japan olfactory cortex (oc) shows a state-dependent sensory gating that is controlled under the modulatory inputs from the basal forebrain and brainstem. since the olfactory bulb (ob) receives the modulatory inputs heavily, neuronal activity in the ob might change in a state-dependent manner. in the present study, we demonstrate a clear state-dependent change in the magnitude of the transmission of granule-to-mitral dendrodendritic inhibitory synapses and olfactory cortex-to-granule excitatory synapses. transmission of granule-tomitral synapses and olfactory cortex-to-granule synapses was facilitated during slow-wave state and suppressed during fast-wave state. in addition, we observed synchronous slow oscillations (about hz) in the granule cell layer of the ob, layer iii of the oc, and the occipital cortex. thus the ob shows state-dependent synaptic modulation and presumably receives top-down periodic signals from the cortex. research funds: kakenhi ( ) ps p-g rem sleep deprivation decreases na-k atpase phosphorylation gitanjali das, birendra n. mallick school of life sciences, jawaharlal nehru university, new delhi, india it has been hypothesized that "one of the functions of rem sleep is to maintain brain excitability" rem sleep deprivation increases noradrenaline in the brain that increases the na-k atpase activity causing increased brain excitability. however, the molecular mechanism of such increased na-k atpase activity was unknown; although it was known that dephosphorylated state is the active form of na-k atpase. rats were rem sleep deprived by flower-pot method; large platform and recovery from lost rem sleep were carried out as controls. at the end of experiment, brains were quickly removed by cervical dislocation and synaptosomes prepared, which were used for western blotting against phosphoserine and phosphothreonine antibodies as well as for na-k atpase activity. after rem sleep deprivation the activity increased, while the level of phosphorylated form of na-k atpase decreased in the same sample. this confirms our hypothesis that rem sleep deprivation induced increased activity is due to dephosphorylation of na-k atpase. research funds: icmr (govt. of india) and upoe (govt of india) takeshi fujii , , ken yoshikawa , yuki takatori , koichiro kawashima dept. of pharmacol., fac. of pharmaceut sci., doshisha women's coll., japan; dept. of pharmacol., kyoritsu univ. of pharmacy, japan stimulation of muscarinic (machr) and nicotinic (nachr) receptors with respective agonists induces ca + signals in t cells. in the present study, using rna interference approach, we investigated roles of machr and nachr subtypes in ca + signals in ccrf-cem (cem) cells, a human t cell line, as a model of t cells. cem cells express m , m , m and m machr subtypes, and ␣ , ␣ , ␣ , ␣ , ␣ , ␣ and  nachr subunits. transfection of anti-m , anti-m and anti-␣ small interfering rna (sirna) significantly down-regulated respective mrna expression, while no changes were observed in gene expression of other machr subtypes or nachr subunits. ca + signals evoked by oxotremorine-m, a non-selective machr agonist, were reduced by anti-m or anti-m sirna. ca + signals evoked by nicotine were reduced by anti-␣ sirna. these findings indicate that m , m machr and ␣ nachr subtypes play major roles in ca + signals to acetylcholine in t cells, and suggest that these receptors are involved in regulation of immune function. research funds: kakenhi ( ) ps p-g is "seronegative" mg explained by autoantibodies to musk? kazuhiro shigemoto , sachiho kubo , seiji matsuda , naoki maruyama dept. of preventive medicine, ehime univ. schl. of med., ehime, japan; dept. of mol. path., tokyo metro inst. for gerontology, tokyo, japan; dept. of integrated basic medical science, ehime univ. schl. of med., ehime, japan muscle-specific kinase (musk) is critical for the synaptic clustering of nicotinic acetylcholine receptors (achr). musk is activated by agrin, which is released from motoneurons, and induces achr clustering at the postsynaptic membrane. although autoantibodies against the ectodomain of musk have been found in a proportion of patients with generalized myasthenia gravis (mg), it is unclear whether musk autoantibodies are the causative agent of generalized mg. in the present study, rabbits immunized with musk ectodomain protein manifested mg-like muscle weakness with a reduction of achr clustering at the nmj. the autoantibodies activated musk and blocked achr clustering induced by agrin or by mediators that do not activate musk. thus, musk autoantibodies rigorously inhibit achr clustering mediated by multiple pathways, an outcome that broadens our general comprehension of the pathogenesis of mg. (shigemoto et al., j. clinical investigation, ) research funds: kakenhi ( ) ps p-g dynamic changes in the thalamo-cortical system associated with thalamic neurodegeneration shin-ichi kyuhou, hisae gemba department of physiology, kansai medical university, japan in purkinje cell degeneration (pcd) mice, degenerating thalamic neurons were found morphologically in the particular thalamic nuclei including the ventral medial geniculate nucleus around postnatal day . electrophysiologically, auditory evoked potentials in the primary auditory cortex began to decrease gradually in amplitude from postnatal day . analysis of spontaneous cortical field potentials by fast fourier transform, revealed that high frequency oscillation (hfo) of around hz appeared prominently in the auditory cortex. local injection of kynurenic acid, a glutamate receptor blocker, into the thalamus suppressed the hfo in the auditory cortex, indicating that the thalamus is involved in the generation of the hfo. the real time polymerase chain reaction analysis demonstrated the upregulation of the mrna of nmda receptors in the auditory cortex. these results suggested dynamic changes occurred in the thalamo-cortical system after thalamic neurodegeneration in pcd mice. research funds: grant c from kansai medical university ps p-h unusually folded sod species sequester specific motor molecules and inhibit the axonal transport of their cargos minako tateno , yumiko simazaki , fuminori saitoh , ryosuke takahashi , toshiyuki araki national institute of neuroscience (ncnp), tokyo, japan; dept. of neurology, kyoto university, kyoto, japan misfolding of mutant sod protein is thought to be responsible for the selective loss of motoneurons in sod -related familial amyotrophic lateral sclerosis (als), although the molecular mechanisms underlying the toxicity of such unusually folded sod species are not yet clarified. since we have detected accumulation of unusual sod species in motoneuronal axons from g a sod -tg mice, we fractionated the ventral white matter of spinal cords to isolate the unusual sod species. immunoprecipitation analyses revealed specific interaction of unusual sod species with certain kinds of motor molecules. moreover, the axonal transport of cargos mediated by those molecules was found to be significantly reduced in symptomatic mutant sod -tg compared with wt sod -tg mice. these data strongly suggest that the toxic property of unusual sod proteins is partially ascribable to the transport inhibition of specific cargos. research funds: grant-in-aid for scientific research c ( ) ps p-h relationship between the amount of the cathepsin d expression and the symptomatic manifestation of neuronal ceroid-lipofuscinosis in a mouse model masahiro shibata, masato koike, yasuo uchiyama department of cell biology and neuroscience, osaka university graduate school of medicine, japan mice deficient in cathepsin d (cd), a representative lysosomal aspartic proteinase, have been shown to be an excellent model of neuronal ceroid-lipofuscinosis (ncl). here we report that the phenotype of mice in which cd is partially expressed is decided depending on the amount of the protein expression of cd. the proteolytic activity and protein expression of cd in the mutant mice were approximately % of those in the wild-type mice, while the growth of the mice appeared intact until postnatal day . the mice started to show ncl symptoms on p , and their life span was prolonged for one to three days, compared to that of the cd-null mice. the protein expression of cd in the heterozygous mice was approximately half of that in the wildtype mice and the mice showed no pathological finding. these results indicate that a threshold of the cd expression required for the manifestation of ncl symptoms in the mice may be present in the range from % to % of that in the wild-type mice. research funds: kakenhi ( ) ps p-h neuronal toxicity of expanded polyglutamine depends on intracellular distribution among cells with similar expression levels mamoru satoh, atsuyoshi shimada, noriko kawamura, yoichi chiba, yuko saitoh, hiromi keino, masanori hosokawa dept. pathol., inst. develop. res., aichi human service center, aichi, japan we previously reported that expanded polyglutamine (polyq) tracts induced cellular toxicity of neuro a cells in the form of massive cytoplasmic aggregates but not of intranuclear inclusion. however, we did not rule out the possibility that such toxicity depends on the level of intracellular expression of polyq. in this report, we compared the toxicity of polyq among cells expressing polyq tracts with a variety of intracellular distribution but at similar expression levels. damages were most remarkable in cells with cytoplasmic massive aggregate in terms of shrunken cellular and nuclear sizes. cells with cytoplasmic homogeneous distribution, cytoplasmic punctate distribution and intranuclear inclusion of polyq tracts were relatively spared. these data suggest that the severity of cell damages depends on the type of intracellular distribution of polyq tracts in cells expressing polyq tracts at similar level. ayumi takamura , katsumi higaki , junichiro matsuda , yoshiyuki suzuki , eiji nanba division of functional genomics, research center for bioscience and technology, tottori university, tottori, japan; national institute of biomedical innovation, osaka, japan; clinical research center, international university of health and welfare, tochigi, japan g m -gangliodisosis is an autosomal recessive lipid storage neurodegenerative disorder. due to a deficiency of lysosomal -galactosidase, excessive lysosomal accumulation of gm is observed in patients and animal model brains. however pathogenesisi of this disease is still unclear. since gm is known to be a major sialoglycolipid constituent of plasma membrane (pm) in neuron, we examined the analysis of brain of mouse model. cerebellar granule cells from this mouse showed gm accumulation of lysosome and pm and the membrane fluidity was also reduced. gm -bound phosphorylated trka was markedly decreased in cultured neuron and brain tissues. subsequent plc␥, known as a downstream signal of trka, was also impaired. these results suggest that dysfunction of neurotrophin signaling may cause the onset of neurodegeneration in g m -gangliosidosis. katsuya inoue , , katsuaki endo , takamitsu fujikawa , seijyun fukuda , tatsuo nakamura department of physical therapy, university of aino, osaka, japan; institute for frontier medical science, kyoto university, kyoto, japan regeneration of spinal cord injury is an important thema in rehabilitation science as well as basic one. the experiment was designed to reveal the process after spinal cord injury by asphyxia. to establish the animal model of spinal cord injury produced by asphyxia, we used adult cats with aorta occulusion under deep pentabarbital anesthesia. twenty minutes after occulusion electrical reflex activity of spinal cord disappeared. after min occulusion, irreversible functional changes were observed, long term depression of reflex activities and disorders of motorsensory function. we also traced time course of electrical and functional changes after min occulusion. ps p-h development of a rodent behavioral model to study the direct interactions of reward and learning adam weitemier, niall p. murphy riken brain science institute, japan cognitive and reward processes often occur simultaneously, and perhaps interdependently. learning is a necessary condition in many experimental models aimed at assessing the rewarding value of a given stimulus. conversely, reward is often used as an experimental tool to engage mnemonic processes in studies aimed at investigating learning and memory. recent studies have demonstrated shared neurobiology between memory and reward. a direct behavioral interaction between reward and memory has never been studied. cognitive impairments observed in psychiatric conditions of dysregulated reward, such as drug abuse and depression, make this issue important, particularly in light of ongoing efforts to investigate higher brain functions. we are developing a rodent behavioral model with which to directly assess the influence of reward processes on learning and memory. we will introduce our recent progress with this new model, including two variations of the procedure designed to study the influence of reward on memory acquisition and memory recall. tetsuya ando , yuya kawanaka , minoru saito , hiroaki mochizuki , ken honjo , hirofumi toda , , toshifumi tomoda , akira sawa , katsuo furukubo-tokunaga grad. school of life & envir. sci., univ. tsukuba, japan; molecular physiol., tokyo metropolitan inst. neurosci. tokyo, japan; beckman res. inst., city of hope. california, usa; dept. of psych. & neurosci. johns hopkins univ. school of medicine. baltimore, usa the disrupted-in-schizophrenia- (disc ) gene, originally identified at the breakpoint of a chromosome ( ; ) (q . ; q . ) translocation in a scottish schizophrenia pedigree, is a promising candidate gene for schizophrenia and affective disorder. however, cellular and molecular mechanisms underlying cognitive impairments are yet to be elucidated. to address disc functions in vivo, we expressed disc in drosophila and examined developmental and behavioral phenotypes. overexpression of disc resulted in marked suppression of olfactory associative learning in flies whereas it caused no symptoms of neural degeneration even in aged animals. we anticipate that the drosophila system will serve as a novel model system amenable to a variety of genetic manipulations for the study of schizophrenia. ps p-h effect of hypothermia on discrepancy between memory learning ability and anatomical brain damages in rats with neonatal hypoxic ischemic encephalopathy yuji miyatake , ayumi kamo , kenji minato , hitoshi haruna , hiritsugu fukuda , yuji murata , takayoshi hosono department of bomedical engineering, osaka electro-communication university, japan; graduate school of medicine, osaka university, japan we investigated the effect of brain hypothermia on neonatal hypoxic ischemic encephalopathy (hie) in hie-model rats using olton t-maze and anatomy. the common carotid artery of of -day-old rats was ligated and cut under anesthesia. after the operation the rats were put in a box containing % oxygen at • c for min. after the insult, of the rats were put in a box at • c for h (hypothermia, h-group). the other rats were returned to their mother without hypothermia (normothermia, n-group). sham operations were performed on three rats (s-group). eight weeks after the operation, their learning and memory ability was assessed by olton t-maze, and no statistical difference was observed in either the working or reference memory in the three groups although the anatomical brain size in the n-group was significantly smaller than in the h-group and s-group. withdrawn ps p-h tau hyperphosphorylation in ts cje, a partial trisomy mouse model for down syndrome ebrahim abdul , a. shimohata , w. yu , m. yamaguchi , m. murayama , d. chui , t. akagi , t. takeuchi , k. amano , h.s. karthik , t. hashikawa , h. sago , c.j. epstein , a. takashima , k. yamakawa research scientist; lab. for neural arch.; lab. for alzheimers disease; div. of fetal med. ncchd; ucsf, usa although down syndrome (ds) or trisomy is the most common genetic cause of mental retardation, its neuropathology remains unclear. ts cje, a ds mouse model partially trisomic for chromosome , shows learning and behavioral abnormalities mimicking ds mental retardation. the trisomic segment, corresponding to parts of human chromosome q , has about genes. importantly, sod and app, which may contribute to the ds phenotype, are excluded from the ts cje trisomic segment. here we report that ts cje brains show hyperphosphorylation of tau in the absence of nft formation, as well as increased gsk  and jnk/sapk activities without alterations in app metabolism. our results suggest that genes on the trisomic ts cje segment other than app and sod can cause hyperphosphorylation of tau, which in turn may be critical in the pathogenesis of ds mental retardation. research funds: kakenhi number: ps p-h increased oxidative stress and mitochondrial dysfunction in ts cje, a down syndrome mouse model atsushi shimohata , ebrahim a. s. , m. yamaguchi , w. yu , h. sago , c.j. epstein , k. yamakawa lab. for neurogenetics, riken-bsi, japan; div. of fetal med. ncchd, japan; dept. pediatrics, ucsf, usa down's syndrome (ds), caused by chromosome (hsa ) trisomy, is the most common genetic cause of mental retardation and affects every major organ in the body. ts cje is one of a number of segmentally trisomic ds mouse models, and is triplicated for a region of mouse chromosome extending from sod to znf , containing genes syntenic with hsa . since these mice show learning and behavioral abnormalities mimicking ds mental retardation, ts cjespecific trisomic segment genes may be involved in the ds phenotype. in the present study, we observed increased levels of reactive oxygen species (ros), mitochondrial function impairment in primary cultured astrocytes and hippocampal neurons, and increased cabonylated proteins in ts cje brains. collectively, our results implicate dosage imbalanced genes other than sod and app in both ros generation and mitochondrial dysfunction, which in turn possibly contribute to the ts cje ds mental retardation-like phenotype. ps p-h polyinosinic-polycytidylic acid injection in early pregnancy causes the hypomyelination in the hippocampus, but not in the cortex manabu makinodan , , kouko tatsumi , takayuki manabe , takahira yamauchi , , eri makinodan , juro shimoda , toshifumi kishimoto , akio wanaka department of psychiatry, nara medical university, kashihara, japan; department of nd anatomy, nara medical university, kashihara, japan polyinosinic-polycytidylic acid (poly i:c) elicits maternal immune response similar to anti-viral ones. recent studies demonstrated that poly i:c injection into pregnant mice resulted in behavioral changes including deficits in prepulse inhibition in the offspring, rendering this system an animal model of schizophrenia. in the present study, we observed such behavioral abnormalities reproducibly in the experimental group born from poly i:c-injected mice, but not in the control group born from pbs-injected mice. they showed decreased myelination in the hippocampus at juvenile period with unaltered number of oligodendrocytes. on the other hand, myelination in the cerebral cortex did not significantly differ between the experimental and control mice. the hypomyelinaton in the hippocampus at the juvenile period may be a possible cause for the behavioral changes in later periods. joanna doumanis, ritsuko kazama, adrian moore, nobuyuki nukina riken brain science institute, japan the fruitfly drosophila melanogaster is well established as a model system in the study of human neurodegenerative diseases. to model the polyglutamine expansion disease, huntington disease (hd), we have established stable, inducible cell lines expressing n-terminal truncated huntingtin fused to egfp with an expanded ( q) polyglutamine repeat in a drosophila larval central nervous system-derived cell line. induction of expression results in the formation of protein aggregates, characteristic of hd. utilising rnai, we have carried out a high-throughput screen for modifiers of aggregate formation in these cells. genes, encompassing around % of the drosophila genome, were screened, resulting in the identification of candidates that either suppress or enhance aggregation. most candidates identified have mammalian orthologues, validating the use of drosophila to screen for genes relevant to human disease. we established in vivo models of hd by expressing polyq-egfp in the drosophila nervous system and are further characterising selected candidates in our model. the rodent model of harmaline-induced tremor has been used as an animal model of essential tremor. the present study investigated effects of harmaline on olivocerebellar systems of mice and rats. systemic administration of harmaline produced generalized tremors in both types of rodents. immunohistochemical studies revealed significant degeneration of purkinje cells that was associated with activated microgliosis in the cerebellar cortex, following administration of harmaline in rats but not in mice. however, in mice but not rats, microgliosis was induced following administration of harmaline in the inferior olivary nucleus (ion). numbers of neurons in the mouse ion did not decrease, suggesting the possibility that microgliosis in ion might not be a simple neurotoxic effect. presumably, differences in sensitivity of purkinje cells between rats and mice may be related to differences in functional alterations in their respective olivocerebellar systems induced by harmaline. recognition of these species-specific differences is an important consideration for experimental analysis of the rodent model of tremors. ps p-h analysis of ␣-synuclein expression in young mouse model of multiple system atrophy kimiko nakayama, yasuyo suzuki, ikuru yazawa laboratory of research resources, national institute for longevity sciences, aichi, japan multiple system atrophy (msa) is a sporadic neurodegenerative disease that affects oligodendrocytes and neurons in human central nervous system. glial cytoplasmic inclusions (gcis) are diagnostics of msa. gcis are shown to be abnormal accumulation of filamentous ␣-synuclein. yazawa et al. ( ) generated a transgenic (tg) mice overexpressing human wild-type ␣-synuclein in oligodendrocytes under the control of the , ,-cyclic nucleotide -phosphodiesterase (cnp) promoter. tg mouse study demonstrated that formation of gci-like ␣-synuclein inclusions in the oligodendrocyte leads directly to neuronal degeneration, as shown by motor impairment and novel accumulation of mouse ␣-synuclein in neuron. to elucidate the mechanisms of neurodegeneration in tg mice, we prepared primary cultures of neurons and glial cells from tg mice. the cells are examined the effects of ␣-synuclein accumulation. ps p-h dysregulation of sodium channel  subunit by expanded polyglutamine in huntington disease transgenic mice fumitaka oyama, haruko miyazaki, kazumasa okamura, yoko machida, kurosawa masaru, takashi sakurai, nobuyuki nukina laboratory for structural neuropathology, riken bsi, wako-shi, japan sodium channel  ( ) is a very recently identified auxiliary subunit of the voltage gated-sodium channels. we have identified  as an est that was significantly downregulated in the striatum of hd model mice and found that reduction in  started at a presymptomatic stage of the hd model mice. in contrast, spinal cord neurons, which generate only negligible levels of expanded polyq aggregates, maintained normal levels of  expression even at the symptomatic stage. expanded polyq with nls expression suppressed the promoter activity of  gene in pc cells. forskolin, an activator of the camp/pka pathway, did not affect b promoter activity, indicating that  is not camp-responsive gene. these findings strongly suggest that sodium channel  subunit is a novel molecule, which is an upstream non-camp-responsive gene in hd pathogenesis. ps p-h repeat length-and age-dependent changes in behavioral phenotypes of drpla transgenic mice harboring a single copy of a full-length human drpla gene kazushi suzuki , yuji takahashi , jun goto , mutsuo oyake , toshiya sato , shoji tsuji department of neurology, the university of tokyo, tokyo, japan; department of neurology, brain research institute, niigata university, niigata, japan; center for bioresource-based research, brain research institute, niigata university, niigata, japan we carried out detailed analyses of the behavioral phenotypes of drpla transgenic mice carrying an expanded cag repeat of (q ), (q ), (q ), or (q ). in the accelerating rotarod ( w), the latencies of q , q , q and q were %, %, % and %, respectively. in the open field, moving distances of q , q , and q were decreased to %, %, and %, respectively, while that of q was increased to %. home cage activity was decreased depending on the repeat length. the q mice, however, showed increased ratios of the activity during the light time to that during the total day at weeks ( %) and weeks ( %), suggesting that drpla mice display not only impaired motor coordination, but also changes in emotional behavior, and disrupted night and day activity patterns. ps p-h the mice lacking schnurri- show multiple behavioral abnormalities related to psychiatric disorders keizo takao , nobuyuki yamasaki , keiko toyama , tsuyoshi takagi , shunsuke ishii , tsuyoshi miyakawa hmro, kyoto university graduate school of medicine, kyoto, japan; riken, tsukuba, japan schnurri- (shn- ) is a zinc finger transcription factor, a mouse homologue of human hiv-ep , that binds to nuclear factor kappa b-binding site in the hiv long terminal repeat. shn- is known to play important roles in the mammalian immune systems. however, the role of shn- in the central nervous system (cns) is still unknown. to investigate the functional significance of shn- in mammalian brain, we analyzed the shn- knockout (ko) mice using a comprehensive behavioral test battery. shn- ko mice were dramatically hyperactive under novel environment and in their home cage. they also showed increased acoustic startle response and impaired prepulse inhibition, indicating their impairment in sensorimotor gating. anxiety-like behavior and depression-like behavior were also significantly reduced in shn- mice. our results demonstrate a critical role of shn- in cns and suggest that shn- ko mice may serve as an animal model of psychiatric disorders. research funds: kakenhi ( , , , ) , jst bird ps p-h comprehensive brain-behavior phenotyping of camkii␣ heterozygous knockout mice nobuyuki yamasaki, koichi tanda, keiko toyama, yasuyuki fukui, keizo takao, tsuyoshi miyakawa hmro, kyoto university graduate school of medicine, kyoto, japan ca + /calmodulin-dependent protein kinase ii (camkii) is a ubiquitous serine/threonine protein kinase that is abundant in brain as a major constituent of the postsynaptic density and critically involved in synaptic plasticity, learning and memory. several behavioral abnormalities of camkii␣ mutant mice were reported, but systematic assessments of behaviors of camkii␣ mutant mice have not been conducted. to analyze the behavioral effects of camkii␣ deficiency, we subjected camkii␣ heterozygous knockout mice to a comprehensive behavioral test battery. the mutant mice showed hyperactivity, decreased anxiety, decreased depression-related behavior, increased offensiveness, selective spatial working memory deficit, and dramatic periodic change of locomotor activity in home cage. to identify the mechanism underlying these behavioral abnormalities, gene expression analysis was conducted. the potential involvement of camkii␣ in pathogenesis/pathophysiology of psychiatric disorders will be discussed. research funds: kakenhi ( , , , ) , jst bird ps p-h effects of various factors on the results of a comprehensive behavioral test battery for genetically engineered mice: a factor analytic study hiroshi ougino, nobuyuki yamasaki, koichi tanda, keiko toyama, keizo takao, tsuyoshi miyakawa hmro, kyoto university graduate school of medicine, kyoto, japan we have been using a behavioral test battery to reveal unknown phenotypes of genetically engineered mice. for the adequate experimental design and interpretation of data, it is essential to know experimental variables which may potentially influence results, and various kinds of factors which underlie many indices measured in the tests. in this study, we investigated the effects of background strains (c bl/ j, c bl/ n, c bl/ c, svev, balb/c), body weight, age at test, and start time of test on the results of each test, by analyzing data of more than mice (, including wild type and mutant mice from strains of genetically engineered mice), which had been tested in our laboratory. also, we conducted factor analyses of a large set of data to examine the relationship between behavioral indices. the potential implications of our findings for the improvement of the behavioral test battery will be discussed. calcium-and calmodulin-dependent protein kinase iv (camkiv) is a protein kinase that activates the transcription factor, camp responseelement binding protein (creb). camkiv has been hypothesized to play a significant role in synaptic plasticity and in learning and memory. however, functions of camkiv in a variety of behaviors, e.g., motor function, nociception, fear, anxiety, depression, learning and so on, have not yet been fully elucidated. to gain more insight into behavioral significance of camkiv, we subjected camkiv−/− mice to a battery of behavioral tests. camkiv−/− mice did not display any deficit in spatial reference memory and working memory tests, but had mild performance deficit in fear conditioning tests. these results indicated selective and specific involvement of camkiv in regulating emotional behavior. research funds: kakenhi ( , , , ) , jst bird ps p-h comprehensive behaivoral analysis of ryanodine receptor type knockout mouse suzuko ohsako , koichi tanda , , nobuyuki yamasaki , keiko toyama , hiroshi takeshima , tsuyoshi miyakawa kyoto university graduate school of medicine, kyoto, japan; dep. of pediatrics, kyoto prefectural univ. of medicine, kyoto, japan; dep. of biochem. and mol biol., tohoku univ. graduate school of medicine, miyagi, japan ca + signaling is essential for the regulation of neuronal processes including synaptic transmission and transmitter release. ryanodine receptors (ryrs) are family of intracellular calcium channels and mediate calcium-induced calcium release from the endoplasmic reticulum. ryr is highly expressed in the hippocampus, caudate putamen, and thalamus. to investigate the behavioral effects of ryr deficiency, we subjected ryr knockoout mice to a battery of behavioral tests. ryr knockout mice exhibited hyperactivity and abnormal behavior in social interaction test, while they did not show any deficit in motor function, depression, attention, and working memory tests. these results suggest a role of ryr in regulating general locomotor activity and social behavior. research funds: kakenhi ( , , , ) , jst bird ps p-h comprehensive behavioral analysis of neuronal nitric oxide synthase knockout mouse keiko toyama , koichi tanda , , nobuyuki yamasaki , tsuyoshi miyakawa hmro, kyoto university graduate school of medicine, kyoto, japan; dept. of pediatrics, kyoto prefectural univ. of medicine, kyoto, japan nitric oxide (no) plays several important roles in the brain, including in regulation of synaptic signaling and plasticity. no is synthesized from the amino acid l-arginine by the enzyme nitric oxide synthase (nos). in neurons, no is produced by neuronal nitric oxide synthase (nnos), representing one of three nos isoforms expressed in most tissues. to elucidate function of nnos/no in a variety of behaviors, e.g., activity, motor function, nociception, attention, anxiety, depression, social interaction, learning and so on, we subjected nnos knockout mice to a battery of behavioral tests. nnos knockout mice exhibited increased locomotor activity and decreased depressionrelated behavior. furthermore, they displayed increased social contacts in novel environment and homecage. these results indicate that nnos/no is involved in regulation of their behaviors. research funds: kakenhi ( , , , ) , jst bird ps p-h primate model of attention-deficit/hyperactivity-disorders (adhd) shintaro funahashi , keiko shimizu grad. sch. human and environmental std, kyoto univ., kyoto, japan; primate res. inst., kyoto univ., inuyama, japan adhd is one of the prevalent childhood psychiatric disorders. children with adhd show hyperactive behavior and attention problems, suggesting prefrontal (pfc) contribution to adhd. adhd is also known as dopamine (da) related dysfunctions, because methylphenidate is the most effective drug for the treatment of adhd. pfc is the cortical area where the strongest da innervation is observed. injection of da-related drugs to pfc produces behavioral deficits in cognitive tasks. these suggest that da-related dysfunction in pfc could be a candidate of biological causes of adhd. to prove this notion, we injected -ohda into bilateral pfc to destroy da innervation in infant monkeys and examined whether these monkeys exhibited hyperactivity. -ohda injected monkeys showed significant increase of spontaneous activity in test cages. oral administration of methylphenidate reduced spontaneous activity in -ohda injected monkeys. these results suggest that monkeys injected -ohda into pfc are good candidates of the primate model of adhd. research funds: kakenhi ( ) ps p-i training-induced recovery of precision grip after primary motor cortex damage in the adult monkey yumi murata , , , noriyuki higo , , takao oishi , , , akiko yamashita , keiji matsuda , motoharu hayashi neurosci. res. inst, aist, tsukuba, japan; grad. sch. compreh. hum. sci., univ. of tsukuba, tsukuba, japan; crest, jst, kawaguchi, japan; dept. cell mol. biol., primate res. inst., kyoto univ., inuyama, japan; div. applied sys. neurosci., nihon univ. sch. med., tokyo, japan in the present study, we compared the motor recovery between monkeys that received daily training and that did not receive any training after lesion of the primary motor cortex (mi), in order to investigate the effects of postlesion training on motor recovery. we derived a hand representation map in mi, and ibotenic acid was then injected to destroy the digit region, which resulted in hand paralysis. after one or two months of postlesion training, skilled use of the affected hand including a precision grip was recovered. untrained monkeys also became able to grasp objects with their affected hand, but they couldn't use a precision grip. this suggests that recovery of precision grip requires postlesion training. research funds: a grant-in-aid for scientific research on priority areas from mext ( ) mouse mutants with behavioral abnormality are indispensable tools to elucidate molecular pathways underlying behavior. in order to develop numbers of novel behavioral mutants, we have been carrying out dominant behavioral screening in potential mouse mutants that was randomly induced point mutations by a chemical mutagen enu (n-ethyl-n-nitrosourea). we screened about , g animals (dba/ j × enu-treated c bl/ j) for home-cage activity, open-field activity, and passive avoidance response, and obtained lines of dominant behavioral mutants. by linkage analysis, the causative genes were mapped in of mutant lines. hyperactivity was predominant phenotype, and of mutants showed hyperactivity in home-cage and/or open-field. we will report the recent results of initial characterization and the progress of fine mapping in these enuinduced mutants. ps p-i ubiquitin signal in neurons of cathepsin ddeficient mouse brains with special reference to the autophagic process masato koike, masahiro shibata, yasuo uchiyama dept. of cell biol. and neurosci., osaka univ. grad. sch. of med., suita, japan we have shown that autophagy contributes to the accumulation of vacuolar structures in neurons obtained from cd−/− and cb−/−cl−/− mice, murine models for neuronal ceroid lipofuscinoses (ncls) (koike et al., ) . until recently, it remains unknown what signaling is essential for autophagosome formation. interestingly, in the conditional atg -knock-out mice where autophagy is absent specifically in the liver, numerous ubiquitinated aggregates are detected in the cytosol of hepatocytes (komatsu et al., ) , suggesting that protein ubiquitination may serve as a signal to the autophagic process. we therefore examined the immunohisto/cytochemical localization of ubiquitin and lc , and found that in our ncl model mice, positive signals for ubiquitin and lc were co-localized on the membranes of granular structures in the neuronal perikarya. these results suggest that protein ubiquitination may be involved in signaling for autophagosome formation in ncls. research funds: grant-in-aid for young scientists (b)( ) and creative scientific research ( gs ) ps p-i activation of medial prefrontal cortex neurons by systemic phencyclidine is primarily mediated via ampa/kainate glutamate receptors tadahiro katayama , eiichi jodo , yoshiaki suzuki , ken-yo hoshino , yukihiko kayama dept. of physiology, fukushima medical university, fukushima, japan; dept. of neuropsychiatry, fukushima medical university, fukushima, japan it has been shown that tonic activation of the medial prefrontal cortex (mpfc) plays a pivotal role in development of behavioral abnormalities induced by systemic phencyclidine (pcp). however, receptors mediating such activation are not clearly specified, though several studies indicate the increase of extracellular acetylcholine, dopamine, and glutamate in the mpfc. here, we examined effects of local application of those antagonists on increased firing activity of mpfc neurons by systemic pcp in anesthetized rats. after tonic activation of mpfc neurons by pcp had been established, cnqx, sch , mecamylamine or scopolamine was locally applied with iontophoresis or gas pressure on the recorded neuron. cnqx reduced pcp-induced augmentation of firing activity to the baseline level, while others gave little changes. these results suggest that pcpinduced activation of mpfc neurons be mediated primarily via ampa/kainate receptors. ps p-i increased depressiveness and decreased sensitivity to antidepressants in calcium/calmodulin-dependent protein kinase iv (camkiv)-knockout mice jiro kasahara , hiroyuki sakagami , hisatake kondoh , kohji fukunaga department of pharmacology, graduate school of pharmaceutical sciences, tohoku university, sendai, japan; department of histology, graduate school of medicine, tohoku university, sendai, japan calcium/calmodulin-dependent protein kinase iv (camkiv) is expressed abundantly in the nuclei of neurons and thought to regulate ca-dependent gene expressions mediated by the transcriptional factors such as creb. recently, we found that chronic treatments of the rats with antidepressants increased camkiv activity and creb phosphorylation in the prefrontal cortex, suggesting the importance of camkiv in the effects of antidepressants. this result led us to perform the behavioral assessments of depressiveness and the sensitivity to antidepressants in camkiv-knockout mice by some experimental paradigms. from the experiments, the increased depressiveness and decreased sensitivity to antidepressants were observed in the mice, suggesting the importance of camkiv for the regulation of depressiveness and the effects of antidepressants. ps p-i severity of audiogenic seizures is influenced by multiple factors in vlgr -mutated mice hideshi yagi , , makoto sato , division of cell biology and neuroscience, department of morphological and functional sciences, faculty of medical sciences, university of fukui, fukui, japan; research and education program for life science, university of fukui, fukui, japan epilepsy is a highly prevailed disorder and reports are accumulating that demonstrate that single gene mutation causes such disorders. we made vlgr -mutated mice and found that they showed high susceptibility to audiogenic seizure, one of the reflex seizures provoked by loud noise. to evaluate whether the genetic backgrounds influence on phenotype of the audiogenic seizure in our mice, we made c bl/ backcrossed vlgr -mutated mice and /svs backcrossed vlgr -mutated mice. these two backcrossed lines showed different susceptible periods and severity of audiogenic seizure from the original line. furthermore, phenotype of audiogenic seizure was altered by restraining mice from free moving while being exposed to loud noise. these observations suggest that genetic factors and environmental factors may modify the phenotype of seizures and our vlgr -mutated mice are good model of reflex epilepsies that are evoked by multifactors. ps p-i reduction in the density of parvalbumin-positive cells in the medial frontal cortex of rats behaviorally sensitized to methamphetamine tomoko kadota , ken kadota , department of bioenvironmental medicine, university of chiba, chiba, japan; chiba institute of psychiatry, chiba, japan our previous study demonstrated that the development of behavioral sensitization of rats to methamphetamine (map) corresponded in time with the progress of neurotoxic changes induced in the medial prefrontal cortex (mfc). the present study further examined morpholological changes of rats that were administered a daily dose of mg/kg of map i.p. for days (d d ) and then withdrawn from the drug for days (wd wd ). the regimen reduced the densities of parvalbumin positive cells (pac); these were probably gabaergic cells and distributed in the strata covering layers ii, iii and v in the anterior cingulate cortex (cg ) and mfc. the decrease in the density of pac was first observed in cg and then in mfc. the reduction began on d and advanced to higher levels on d and subsequently wd . these findings suggest that the behavioral sensitization regimen leads to the deterioration of inhibitory processes in the neural circuits in cg and mfc, particularly in layers ii and iii. ps p-i up-regulation of  -adrenergic receptor immunoreactivity in astrocytes in the spinal cord after dorsal rhizotomy teruyoshi kondo, yoshihiro ishibashi, kei-ichiro nakamura department of anatomy, division of microscopic and developmental anatomy, kurume university school of medicine, kurume, japan stimulation of  -adrenergic receptor ( -ar) induces astroglial proliferation and activation after brain injury, but little is known concerning the potential role of adrenergic receptors in the spinal cord. present study demonstrated that rhizotomy induced a marked and prolonged up-regulation of  -ar-immunoreactivity (ir) in the regions of the dorsal root entry zone and dorsal funiculus containing the central processes of the injured primary sensory neurons.  -arimmunoreactive cells coexpressed gfap-ir and were positive for nestin which is characteristic of reactive astrocytes. a population of  -ar-immunoreactive cells were labeled with ki- , a marker of cell proliferation, indicating some of them went into cell mitotic state. interestingly, a major population of  -ar-immunoreactive cells also exhibited fgf- -ir. these findings suggest that  -ar may play important roles in astrocytic activation and neuroprotection associated with induction of synthesis of growth factor such as fgf- . ps p-i effects of lateral fluid percussion injury (fpi) on the optical signals in dentate gyrus of the rat brain slice preparations shin yamashita , norihiro muraoka , hiroshi hasuo , takashi akasu , minoru shigemori dept. of physiology, kurume univ. sch. of med., kurume, japan; dept. of neurosurgery, kurume univ. sch. of med., kurume, japan we investigated the effects of experimental traumatic brain injury on the neuronal function in dentate gyrus (dg) using optical recording techniques with voltage-sensitive dye (rh ). horizontal hippocampal slices were obtained from the control and the fpi rats (one week after the single moderate impact). electrical stimulation of perforant path (pp) produced the optical signal spread in the molecular layer of dg. temporal change in the optical signal, obtained from an area on the propagation pathway, had two peaks (fast and slow peaks). increment of stimulus intensity ( - v) increased the amplitude of both fast and slow peaks. the intensity for producing the maximal response was - v. the amplitude of slow peak in fpi group was about % larger than that in control group, while the amplitudes of fast peak were not different in the two groups. these data suggest that the excitatory pp synapse onto granule cells of dg is facilitated after fpi. ps p-i comparative study of neural activities in mouse hippocampal slices by flavoprotein autofluorescence and ca + imaging chikafusa bessho, yasuharu mitsushima, ryo matsumoto department of physics, kyoto sangyo university, kyoto, japan recently k. shibuki et al. have succeeded in flavoprotein autofluorescence imaging of neural activities in the rat brain. we examined neural activities in mouse brain (hippocampal) slices by the modified method and ca + imaging. the slices ( m) were prepared from the block in an ice cold acsf medium using microslicer and incubated for h in the oxygenated medium at room temperature. a slice was placed on a recording chamber perfused with the medium at a flow rate of ml/min. green autofluorescence (> nm) of the slices illuminated by blue light ( - nm) was observed by an inverted microscope. images of the autofluorescence were recorded using a calcium imaging system. ca + imaging was also performed in the slices. slices were incubated in acsf medium containing m of fluo / am for h at • c. the ca + image was recorded with an excitation wavelength of - nmand an emission wave length of > nm. the autofluorescence and ca + responses wereobserved in slices perfused with l-glutamate ( mm). takuya hayashi , hiroshi sato , shinichi abe , takashi hanakawa , hiroshi watabe , hidenao fukuyama , babak aldekani , hidehiro iida department of investigative radiology, national cardiovascular center research institute, osaka, japan; human brain research center, kyoto university, kyoto, japan; nathan kline institute for psychiatric research, ny, usa we show connectivity pattern between cortex and striatum in macaque and human by using the non-invasive method of diffusionweighted magnetic resonance imaging (dwi). in macaque, the dwibased striatal connectivity of brodmann's area corresponded to that revealed by the tracer (mncl ) tractography. the dwi-based connectivity pattern also isolated a part of the ventral striatum corresponding to the histochemically-specific 'shell' region in both human and macaque. in addition, we confirmed the species-homology in intra-striatal topography of cortical connection by quantitatively analyzing the connectivity; however, we found that human striatum was more intensively connected to prefrontal cortex and less connected to extra-frontal cortices. these results suggest that human striatum has a dominant and specific role in processing prefrontal information. research funds: h -kokoro- ps p-i optical analysis of synaptic transmission by a fluorescent glutamate probe shigeyuki namiki, hirokazu sakamoto, sho iinuma, kenzo hirose department of cell physiology, nagoya university graduate school of medicine, nagoya, japan glutamate is an essential excitatory neurotransmitter in the central nervous systems. for optical analysis of glutamatergic synaptic transmission, we have developed a fluorescent glutamate probe called eos. by imaging with eos, we successfully detected the synaptically released glutamate following axon firings in cultured hippocampal neurons; the spatial distribution of the glutamate release was non-uniforml along dendrites. we also succeeded in monitoring the phorbol ester-induced potentiation of the glutamate release. furthermore, we found spontaneous and stochastic glutamate release which was confined to small regions. neither application of tetrodotoxin nor removal of extracellular calcium blocked the release. high concentrations of sucrose increased the frequency of the release. these features are reminiscent of those of miniature epsc in electrophysiological recordings and thus suggest that the spontaneous release is quantal vesicular release. in conclusion, our probe directly visualizes the presynaptic release. shingo miyata , , yasutake mori , , tsuya taneda , , hiroaki okuda , , masaya tohyama , department of anatomy and neuroscience, graduate school of medicine, osaka university, osaka, japan; st coe program, tokyo, japan local protein synthesis in neuronal dendrites is one of the mechanisms that may mediate a rapid and synapse-specific mobilization of proteins from the resident mrnas. a great deal of effort has been made in analyzing the dynamic state of protein synthesis in the living cells, chiefly by quantifying protein level. however, the protein level cannot mirror the spatio-temporal alteration of translation, because it cannot be affected only by protein synthesis but also by other factors like degradation. therefore, it is problematic to visualize the dynamic state of translation by the present methods. to solve the problem, we applied fret (fluorescence resonance energy transfer) technique to in situ detection of the assembly and disassembly cycle among a pair of translation initiation factors (eifs), thereby showing that bdnf and ephrin could potentiate local protein synthesis in the dendrites of hippocampal neurons. ps p-i a model selection of glm applied to fmri data using aic jobu watanabe , fumikazu miwakeichi , andreas galka , , ryuta kawashima , tohru ozaki , sunao uchida , institute for biomedical engineering, consolidated research institute for advanced science and medical care, waseda university, japan; department of medical system engineering, faculty of engineering, chiba university, chiba, japan; institute for statistical mathematics, tokyo, japan; institute of experimental and applied physics, university of kiel, keil, germany; new industry creation hatchery center, tohoku university, sendai, japan; faculty of sport sciences, waseda university, tokorozawa, japan in the general linear model (glm) that is widely used in analyses of functional neuroimaging data, several combinations of explanatory variables are possible. the akaike information criterion was applied as a basis of comparison and selection among several glms that analyze block-designed functional magnetic resonance imaging (fmri) data. the glms with/without a resting condition, head motion covariates, time derivatives and dispersion of hemodynamic response function were compared. we demonstrate that a combination of these explanatory variables can effectively improve the model and that aic is a useful tool for model selection in fmri studies. ryuzo shingai, katsunori hoshi, tokumitsu wakabayashi department of welfare engineering, iwateuniversity, morioka, japan to investigate the relationship between the behavior and function of the nervous system of caenorhabditis elegans, quantitative analysis of behavior that indirectly represents the internal states of the worm is necessary. we devised an automated analysis system of c. elegans locomotion. the system is well suited for detecting four locomotion states: forward or backward movement, curl and rest. the system was applied to a phenotype that when a worm is transferred from a seeded plate to a bacteria-free plate, the worm shows frequent backing and short duration of forward movement for - min and then a gradual increase in the duration of forward movement. accuracy of the state identification for wild type and several mutants was sufficiently high, indicating the system is robust in studies of locomotion. ps p-j flavoprotein fluorescence responses elicited by thalamic stimulation in slices obtained from the mouse barrel cortex daiki kamatani, ryuichi hishida, masaharu kudoh, katsuei shibuki dept. neurophysiol., brain res. inst., niigata univ., niigata - , japan we have reported that whisker trimming induced activity-dependent changes in the barrel cortex of rat cortical slices using flavoprotein fluorescence imaging. however, contribution of thalamo-cortical afferents in this plasticity was not clear, since specific stimulation of thalamo-cortical afferents was not possible in the coronal cortical slices obtained from rats. in the present study, we used the mouse cortical slices that kept thalamocortical connections to the barrel cortex intact. the cortical activities in layer iv were observed as fluorescence responses after thalamic stimulation. the magnitude of the fluorescence responses was increased as the amplitude of cortical field potentials was increased. these cortical responses were suppressed by antagonists of glutamate receptors such as cnqx and apv, and almost completely abolished in the presence of cnqx plus apv. in preliminary experiments, we confirmed that whisker trimming induced activity dependent changes in the barrel cortex of mice. ps p-j effects of implicit emotional processes on encoding-related activations of episodic memory: an eventrelated fmri study yayoi shigemune , , takashi tsukiura , hiroko mochizuki-kawai , chisato suzuki , , toshio iijima neurosci. res. inst., aist, tsukuba, japan; div. systems neurosci., tohoku univ. grad. sch. life sci., sendai, japan in this study, we investigated the effects of implicit emotional processes on encoding-related activations of episodic memory using fmri. nineteen healthy right-handed male participated in this study. we prepared emotional pictures with three kinds of emotional valence (negative: nega, neutral: neu and positive: posi) and line drawings for encoding. in the fmri scanning, subjects memorized line drawings, which were presented after the emotional pictures. after the scanning, subjects were presented with the names of line drawings, and were required to judge whether or not line drawings with the names were learned. we found significant activations of the right anterior cingulate gyrus specifically in the nega condition, the right lingual gyrus in the neu condition and the right amygdala and pulvinar in the posi condition. these results suggest that encodingrelated activations of episodic memory may be modulated by the implicit primer with emotional valence. ps p-j different neural correlates of stimulus-actiondependent and stimulus-dependent reward predictions revealed by fmri masahiko haruno , kenji kansaku , yu aramaki , mitsuo kawato atr cns, kyoto, japan; institute of physiology, okozoki, japan efficient decision making requires multiple reward predictions in switching different contexts and learning. we conducted a human fmri experiment (n = ) to examine stimulus-action-dependent and stimulus-dependent reward predictions. in condition a, each of two fractal figures specifies a monetary reward associated with a button push (left or right). if the button is pressed correctly, or yen is provided with a probability of . , but only with . if pressed wrongly. the key difference in condition b is that a fractal determines the reward but not the action. subjects had learned the two conditions fully before scanning. at the fractal onset, the putamen, lateral ventral and medial dorsal prefrontal cortices showed stronger activity correlated with the predicted reward (p < . ) in a, while it was more prominent in the caudate, dorsolateral prefrontal cortex and cerebellum in b. the striatum also showed a similar difference correlated with the reward prediction error at reward feedback, suggesting the different neural substrates for different reward predictions. research funds: nict ps p-j brain networks for communicative speech production: feeling inference and speech content production yuko sassa , , motoaki sugiura , hyeonjeong jeong , , keisuke wakusawa , , kaoru horie , shigeru sato , ryuta kawashima niche, tohoku university, sendai, japan; ristex, jst, tokyo, japan; miyagi university of education, sendai, japan; gsics, tohoku university, sendai, japan; department of pediatrics, tohoku university, sendai, japan; the lbc research center, tohoku university, japan communicative speech production often accompany inference of the targetperson's feeling. in this fmri study, we segregated the brain networks forthe feeling inference and speech content production processes incommunicative speech production. during presentation of a picture showingan actor's utterance in a balloon, normal subjects covertly talked to theactor (speech), inferred feeling (feeling), or described the action (des). greater activation in the contrasts speech-feeling was observed in themedial prefrontal cortex, and that in the contrast feeling-des wasobserved in the right superior temporal sulcus extending to the temporalpole. the results suggest that these two regions play roles in the speechcontent production and feeling inference, respectively. research funds: the st coe program ps p-j the construction of a brain-computer interface using the brain activity measured by near infrared spectroscopy takafumi miyoshi , yasuhisa fujibayashi , yoshiharu yonekura , tatsuya asai department of human and intelligence systems, university of fukui, fukui, japan; biomedical imaging research center, university of fukui, fukui, japan people with severe motor disabilities can increase the quality of life if they can communicate with the external world. a brain-computer interface using brain activity is one of the ways to provide such communication without depending on muscular controls. brain activity was measured non-invasively by multi-channel near infrared spectroscopy (nirs) during various motor tasks from healthy subjects. these spatial brain activities were fed to neural networks, and pattern learning was carried out by matching the tasks and the brain activities. we propose that nirs signals may be used to construct a brain-computer interface. ps p-j imaging of brain activity by near infrared spectroscopy in response to various sounds tatsuya asai , kuniyoshi shinya , tetsuo araki , masahiro kusakabe , yasuhisa fujibayashi , yoshiharu yonekura department of nuclear power and energy safety engineering, university of fukui, fukui, japan; department of human and intelligence systems, university of fukui, fukui, japan; biomedical imaging research center, university of fukui, fukui, japan brain activity can be monitored non-invasively by near infrared spectroscopy (nirs). in the present study, we measured changes in cerebral hemoglobin concentrations during a listening task using multi-channel nirs from healthy right-handed subjects, and hemispheric dominance for various sounds including verbal sounds was assessed. we have found asymmetrical brain activity when subjects listened to sounds with their left or right ear. these results suggest that hemispheric sound dominance may exist in addition to language dominance in healthy humans. kazuo kitamura , , winfried denk , michael hausser department of cellular neuroscience, graduate school of medicine, osaka university, osaka, japan; university college london, london, uk; max-plank institute, heidelberg, germany we describe a new approach for making targeted patch-clamp recordings from single neurons in vivo visualized using two-photon microscopy. the method involves using a patch electrode to perfuse the extracellular space surrounding the neuron of interest with a fluorescent dye, thus allowing the neuron to be visualized as a negative image and identified on the basis of its somatodendritic structure. the same electrode can then be placed on the neuron under visual control to allow gigaseal formation. we demonstrate the reliability and versatility of the method using recordings from principal neurons and interneurons in mouse and rat barrel cortex and cerebellum. we also show that the method can be used for in vivo juxtacellular labelling in identified cell types. this approach thus offers the prospect of targeted recording and labelling of single neurons in the intact native mammalian brain without the need to pre-label neuronal populations. research funds: wellcome trust, gatsby foundation, jsps and uehara foundation ps p-k analysis on viability of gabaergic neurons in cerebral cortical slices of adult mice yasuyo tanaka , yasuhiro tanaka , takeshi kaneko , dept. of morphological brain science, kyoto univ., kyoto, japan; crest, jst, kawaguchi, japan whole cell clamp recording and intracellular staining in adult brain slices are technically difficult because of their low viability. we analyzed the effect of slice cutting and incubation conditions on viability of cortical gabaergic neurons, using gad -gfp knock-in mice. we considered gfp positive cells as having survived. we observed more gfp-positive cells in the slices when nacl in cutting solution was replaced with n-methyl-d-glucamine (nmdg) chloride, choline chloride or sucrose. however, the viability was lower after h incubation in nmdg-based solution than in nacl-based solution. cutting at • c did not reduce the number of gfp-positive cells, but decreased gfp fluorescence in single neurons as compared with cutting at • c. the viability after h incubation was better kept at • c than at or • c. we thus recommend that slices be cut at • c in na-free solution, and incubated at • c in nacl-based solution. we thank dr yanagawa for his generous gift of knock-in mice. research funds: kakenhi ( , , ) ps p-k contribution of reduced and oxidized glutathione to signals detected by magnetic resonance spectroscopy as indicators of local brain redox state takumi satoh , yoshichika yoshioka faculty of engineering, iwate university, morioka, japan; iwate medical university, takizawa, japan we evaluated gsh signals by the mega-press (a frequencyselective refocusing technique) signals assessed by magnetic resonance spectroscopy (mrs). gsh gave a single positive signal ( . ppm) by mega-press. in contrast, gssg gave a multiplet of reversed signals ( . , . , and . ppm). a phantom solution mimicking the normal condition (gsh:gssg = : ) gave a single positive peak. gsh was prominent and gssg signals were minimal. thus, the signals originated from gsh, not from gssg. in the phantom solution (creatine: gsh: aspartate: gaba = : : : ), the creatine signal overshadowed the other signals. through mega-press, a single peak of gsh stood out over other signals. in vivo, the brains of healthy volunteers gave similar signals as the in vitro phantom solution, indicating that the signal originated from gsh. the estimated concentration of gsh in the human brain was . mm. in conclusion, mega-press allowed us to assess gsh levels in vivo non-invasionally. hiroshi kadota, hirofumi sekiguchi, yasoichi nakajima, yutaka kohno, makoto miyazaki department of sensory and communicative disorders, research institute, national rehabilitation center for persons with disabilities, tokorozawa, japan we investigated the brain regions related to the inhibition of habitual responses by using functional mri. we used the rock-paper-scissors game as an example of a familiar habitual behavior. it is considered that making positive attempts to lose when presented with the gesture of a rock, paper, or scissors is associated with the inhibition of habitual responses. in this study, the subjects were randomly assigned to one of the following two groups: the "win group" and the "lose group." a comparison between these groups showed that the lose group displayed activation of multiple cortical areas in the brain. with regard to the prefrontal cortex, the comparison revealed a higher activation in the left middle frontal gyrus (brodmann area ) and the right superior and middle frontal gyri (brodmann area ) in the lose group. these findings suggest that these regions play a role in the inhibition of habitual responses. ps p-k cortical commissural connection in macaque and human callosum using diffusion mri rishu piao , takuya hayashi , hiroshi sato , shinichi abe , , takashi hanakawa , hidenao fukuyama , hidehiro iida national cardiovascular center, osaka, japan; human brain research center, kyoto university, kyoto, japan we investigated the cortical commissural connection in human and macaque using the non-invasive diffusion-weighted magnetic resonance imaging (dwi). we used the probabilistic algorithm to track connection paths between a pair of the left and right homologous in subcortical areas. in macaque, the classification of callosum based on the highest interhemispheric connections paralleled with the results of tracer studies. however, the territory corresponding to the interfrontal connectivity extended more posteriorly than suggested in the tracer studies. the human interhemispheric connectivity showed similar topography in callosum as in the current macaque study, except that the connectivity territory of the frontal areas extended more posteriorly than in macaque. this study revealed that the commissural connectivity of the two species has a common intra-callosal topography. ps p-k optimal resolution of eeg/meg source imaging by spatial filtering wan xiaohong , niche, department of qutantum science and energy engineering, tohoku university, sendai, japan, niche, tohoku university, sendai, japan nowadays, electro-and magnetoencephalography (eeg/meg) is the sole invasive technique which is able to directly measure the human brain neural cortical dynamics. although we are well aware that it is impossible to accurately estimate the -d neural cortical activity using the -d eeg/meg surface potential topography, the upper limit of these techniques is not well described. during the past decades, various inverse approaches based on different criteria have been proposed, from the single dipole or multiple dipoles to the distributed current dipoles. however, it is difficult to systematically evaluate their efficiencies due to the different criteria and regularizations adopted in these methods. in this paper, we ask the question whether there exists an optimal approach based on a systematical criterion. this motivation firstly seems to be conflicted with the primary knowledge that there is no unique solution for the bioelectromagnetic inverse problem. essentially, here we are trying to find an optimal inverse solution that is closest to the real current distribution. ps a-c sensitivity of serotonin synthesis to synthesis inhibitor gtp cyclohydrolase i in senescence-accelerated mouse-prone inbred strain (samp ) nobuyuki karasawa , kazuko watanabe , keiki yamada seijoh universitry, tokai, japan, dept. physio., sch. med., gifu univ., gifu, japan, dept. anat., sch. health sci., fujita health univ., toyoake, japan to study the relationship between aging and levels of monoaminergic neurons, , -diamino- -hydroxypyrimidine (dahp), an inhibitor of monoamine synthesis, was intraperitoneally administered to senescence-accelerated mouse-prone (samp ) mice. time course of immunoreactive intensity for serotonergic ( -ht) neurons in the dorsal raphe nucleus, which were stained using laboratory-raised serotonin-specific antibody, was quantitatively evaluated using an image analysis system. results showed that -ht neruons are not highly sensitive to a synthesis inhibitor common to both catecholaminergic and -ht neurons. katsuya yamada , , , yoshihiro matsumura , takashi miki , makoto wakui ␣-smooth muscle actin + arterioles were fewer in kir . (−/−) barrel cortex than in wild-type one. in addition, whisker stimulation-induced increase in local cerebral blood flow was much smaller in kir . (−/−) barrel cortex than in wild-type one for short ( s, hz) but not long ( s) stimulation, suggesting crtical involvement of thin arterioles in a short-time neuro ps a-h learning to use sensory-tools by japanese monkeys yumiko yamazaki , hiromi namba support by public health research foundation (japan). acknowledgement supported by hkrgc. we wish to thank professor miyashita for valuable advice. ps p-h mechanisms for processing of intellectual excitement kazuhiko yanai, hongmei dai dept. pharmacol tohoku grad. univ. sch. med., sendai, japanthe aim of this study was to investigate the role of histamine h receptor (h r) in cognition in physiological and pathological conditions by using h r mutant (h −/−) mice. in normal condition, several behavioral studies indicated h −/− mice show impaired object recognition and spatial memory, improved conditioned fear memory. moreover, hippocampal long-term potentiation was reduced in h −/− mice. these results indicate h receptor is involved in memory process for which the frontal cortex, amygdala and hippocampus interact. in pathological condition, both h −/− and control mice were subjected to social isolation, an animal model of schizophrenia. social isolation impaired locomotion, prepulse inhibition of startle response and water maze performance in control mice, but not in h −/− mice. mutation of h receptor decreases isolation-induced hyperactivity of cortical dopaminergic neurons. these findings indicate blockage of h r attenuates social isolation-induced behavioral changes. in conclusion, blockage of h r impairs cognition in normal conditions, whereas h r blocking inversely improves cognition in disease models of schizophrenia.research funds: kakenhi ( ; ) ps a-h -ht a receptor gene polymorphism modulates activation in the human ventrolateral frontal lobe during go/no-go task michio nomura , , hirohito-m. kondo , makio kashino , department of psychology, tokai women's university, kakamigahara, japan; ntt communication science laboratories, ntt corporation, atsugi, japan; shimojo implicit brain function project, erato, jst, kawaguchi, japan impulsive behavior has been suggested to be due to a dysfunction of -ht neurotransmission. we examined whether this -ht a receptor gene polymorphism is involved in impulsive aggression by evaluating a reward-punishment go/no-go task using fmri. participants were required to learn to respond to active stimuli and inhibit their response to passive stimuli both under the reward-only (r) condition and the punishment-only (p) condition. the r condition, compared with the p condition caused right prefrontal activation mainly seen in ventrolateral regions. it has been reported that the possible involvement of the -ht a receptor gene polymorphism in impulsive behavior (nomura et al., ) , together with the present findings, this observation indicates the involvement of -ht a receptor gene polymorphisms in ventrolateral frontal lobe.research funds: shimojo implicit brain function project, erato, jst ps a-h role of cortical thin arterioles in neurovascular coupling; analyses of kir . -containing atpsensitive potassium channel-deficient mice ps p-g rem sleep deprivation increases serum ceruloplasmin level manoj jaiswal , chinmay k. mukhopadhyay , birendra n. mallick school of life sciences, jawaharlal nehru university, new delhi, india; special centre of molecular medicine, jawaharlal nehru university, new delhi, india rapid eye movement sleep (rems) is present across higher species and is essential for life. its loss predisposes one to several pathophysiological conditions. continuous loss of rems leads to several diseases and extreme loss may be fatal. rems loss is reported to increase metabolism and food intake though associated with hypothermia. hence, we proposed that the rems deprivation would affect acute phase response protein. in this study rats were rems deprived by platform method. free moving normal, large platform and recovery from rems loss were used as controls. blood was collected from the same rat before and after experimental as well as control periods. level of serum cruloplasmin, a positive acute phase response protein, was detected using western-blot analysis. the results showed that rems deprivation increased the serum ceruloplasmin level suggesting that the rems deprivation triggers an acute phase response at least in rats.research funds: council of scientific and industrial research, india and dst, india the indirect cytopathic effect in hiv- and the direct infection of hsv- are critical in their pathogenesis. we established murine neurosphere and evaluated with cocultivation of hiv- jrfl-infected macrophages or with hsv- . the generation of primary neurospheres did not suppressed by hiv- -infection or by hsv- infection at no more that moi . in the secondary neurospheres, cd + neural stem cells were intact in these infections, although beta- -tubulin + cells were decreased in hiv- infection and intact in hsv- -infection. in the differentiation assay, neun + nfp + neurons in hiv- -infection and gfap + s + astrocytes in hsv- infection were significantly decreased. the migration capacity of the neurosphere cells was suppressed in hiv- infection and in hsv- infection. we conclude that neural stem cells in vitro are resistant to cytopathic effect by hiv- and hsv- infection and their differentiation capacities are different in these infections. our assay will be one of the significant methods in neurovirological research.research funds: kakenhi grant-in-aid for young scientists(b) ps p-g effects of attraction to favorite opposite gender on nervous, endocrine, and immune systems masahiro matsunaga , , taeko yamauchi , toshihiro konagaya , hideki ohira department of psychology, nagoya university, japan; department of internal medicine, aichi medical university school of medicine, japan everybody can "fall in love". thus everybody knows that attraction to favorite opposite gender invokes positive feelings and often makes us energetic. to investigate effects of this positive emotion on the biological systems, we recorded various parameters, namely mood states, heart rate, skin conductance level (scl), serum levels of several hormones, and proportions of t cells and natural killer (nk) cells in the lymphocytes simultaneously when subjects viewed the video films of their favorite opposite genders. when the subjects were evoked their attraction to favorite opposite gender, they became more vigorous and felt better. as for the biological systems, scl and the proportion of nk cells in the lymphocytes significantly increased. these results suggest the possibility that attraction to favorite opposite gender may have a role in activating nk cell-related innate immune system by means of the activation of scl-related sympathetic nervous system. hiroko ikeshima-kataoka , shen jin-song , saburo saito , shigeki yuasa dept. mol. immunol., inst. dna med., jikei. univ. sch. med., tokyo., japan; dept. gene ther., inst. dna med., jikei univ. sch. med., tokyo, japan; dept. ultrastruc. res., natl. inst. neurosci., ncnp, tokyo, japanto investigate the role of tenascin (tn)-expressing astrocytes played in the injured brain, we analyzed tn knockout (tn/ko) mouse. we have previously reported that tn is one of the essential molecules for proliferation of the primary culture of astrocytes. from injured mouse brain model with stab wound, gfap expression was down regulated sharply at earlier stages in tn/ko mouse than in the wt mouse. some of the inflammatory cytokines are known to be expressed in injured cns, and also those receptors are expressed in the primary culture of astrocytes. to evaluate immune responses in the cns, some of the inflammatory cytokine production was determined in the lesioned mouse brain compared with tn/ko and wt mouse. from rt-pcr method, tn seemed to have the possible roles for some of the cytokine prodution at the cns lesion sites. we are currently investigating the function of tenascin for the cytokine production around the lesion site. aiko hori , tomoko yamamoto , kiyoshi matsumura , hiroshi hosokawa , shigeo kobayashi dept. of intelligence science and technology, grad. sch. of informatics, kyoto university, kyoto, japan; dept. of information science and technology, osaka institute of technology, osaka, japan intracerebroventricular (i.c.v.) injection of arachidonic acid (aa) evokes fever. this response has been thought to occur simply because aa is converted to prostaglandin e (pge ), the final mediator of fever. however, our recent study suggested that aa might not only be the precursor of pge but also induce an enzyme cyclooxygenase- (cox- ) that catalyses aa to form prostaglandins. we here examined in rats whether i.c.v. injection of aa induces cox- , and whether cox- is involved in aa-induced fever. two hours after i.c.v. injection of aa, cox- was expressed in the perinuclear region of brain endothelial cells. aa-induced fever was partly suppressed with a cox- specific inhibitor, ns- . these results indicate that aa itself or its metabolites induces cox- that accelerates the formation of pge from aa, and, hence, enhances fever. mitsunari abe , tatsuya mima , shinichi urayama , toshihiko aso , nobukatsu sawamoto , hidenao fukuyama human brain research center, kyoto university graduate school of medicine, japan; nano-medicine merger education unit, kyoto university, japanrepetitive transcranial magnetic stimulation (rtms) can induce lasting changes in the cortical excitability. however, its cellular mechanism remains unknown. diffusion weighted imaging (dwi) is a useful tool for measuring microscopic states of the brain tissue by probing water diffusion.we examined changes of dwi following rtms to further understand its effects. four healthy volunteers received rtms at . hz ( min; % of the rest motor threshold) applied over the left primary motor area (m ). we scanned sets of dwi (before, and min, min and min after rtms) using -t mr scanner, and calculated apparent diffusion coefficient (adc). in out of subjects, the adc decreased (mean . × − /mm s − ) in the left m just after the rtms, which recovered at min. it is possible that the rtms-induced change of adc might occur as the cellular response. further examination is needed for confirming this point. vahe poghosyan, andreas a. ioannides laboratory for human brain dynamics, brain science institute riken, wako-shi, japanretinotopic areas v and v a in macaques occupy almost the entire extend of the anterior bank of parieto-occipital sulcus (pos). v a located more dorsal has a larger receptive field size then v . both areas lack a foveal magnification. we used meg to record brain activity while human subjects were viewing stimuli presented at two different eccentricities in each quadrant of visual fields. to verify the reliability of results, for each subject, the experiment was repeated on three different days. tomographic analysis of meg signal, in each subject, identified highly reproducible activations throughout visual cortex in accord with the known organization. two new areas along the pos with a similar retinotopy to that of macaques v and v a were identified. in the ventral one, activations in response to each stimulus were spatially separated. the foveal magnification was much reduced compared to v in both areas and in the more dorsal area activations elicited by stimuli in the same quadrant could not be separated. given the above finding we suggest these areas as possible homologues of macaques v and v a.ps p-i measurement of magnetic evoked field of ratmeasurement of magnetic evoked field of rat using micro squid naohiro tsuyuguchi department of neurosurgery, osaka city university graduate school of medicine, japanthe study of neural activity in rodents would be enhanced by the stimulation of neuronal function in vivo. magnetoencephalography (meg) is used to study brain function in humans, but the limited resolution and sensitivity of conventional instruments have precluded the use of meg to study neuronal function in rodents. we demonstrate that micro meg developed for use with small animals, can be used to detect assess neuronal activity in conscious rodent brain. we used a micro -channel magnetometer consisting of a × matrix of superconducting quantum interference device (squid) with its integrated base of . × . mm to measure the visual evoked magnetic field (vef) and auditory evoked field (aef) of rats. we obtained the vef wave with - ms peak by the white led flashing stimulation and the aef wave with - ms peak by the tone and burst stimulation. this study demonstrate that micro meg can be used for serial assessment of neuronal function of individual, live animals with a minimal degree of invasiveness, has the potential for use in the study of brain function and plasticity. kentaroh takagaki, michael t. lippert, jian-young wu department of physiology and biophysics, georgetown university, washington, dc, usa voltage-sensitive dye (vsd) imaging is used to study visually evoked responses in rat visual cortex, in single trials without averaging. the signal is small, and a diode array with an effective dynamic range of bits was used, along with a "blue dye" (rh ) with small heartbeat artifact. a subtraction algorithm was used to further remove heartbeat artifact in the data. with the combination of the array, the blue dye and the algorithm, we were able to visualize sensory evoked wave activity with a high signal-to-noise ratio in single trials. the signals were . - . % of the resting fluorescence intensity. spatiotemporally, the evoked response manifested as propagating waves in the visual areas. there were large trial-to-trial variations in the propagating velocity and directions of the waves. the evoked wave apparently interacted with spontaneous waves in the cortex, and varied greatly according to anesthetic regimen. visualizing evoked waves may contribute to the understanding of cortical dynamics underlying sensory processing. masahito nemoto , yoko hoshi , chie sato , susumu terakawa tokyo institute of psychiatry, tokyo, japan; photon medical research centre, hamamatsu university school of medicine, hamamatsu, japanwe investigated interhemispheric interactions and neurovascular coupling by simultaneous recordings of neuronal and hemodynamic signals in rats. bilateral somatosensory cortices were activated with a stimulus time lag between test stimuli (electrical pulses to contralateral hindpaw) and conditioning stimuli (to a homologous somatosensory region of the contralateral hemisphere). we measured electrophysiological signals (local field potentials and multiunit activity) and optical intrinsic signals ( and nm, indicators of cbv and oxygenation), and analyzed the dependence of the signals on the time lag. the results showed that both neuronal and hemodynamic signals were suppressed around -ms time lag. average and trial-by-trial correlation analyses suggested that the hemodynamic signals reflected a balance of neuronal excitation and inhibition via callosal connections. we can infer some parts of underlying neural interactions by imaging of the hemodynamic signals. ps p-j multiple-site optical detection of spontaneous activity in the rat sensorimotor cortex akihiko hirota, shin-ichi ito department of physiology, shimane university school of medicine, izumo, japan multiple-site optical recording provides a powerful tool for the cerebral cortical neurophysiology, but its application has largely been restricted to reproducible, stimulus-evoked activation. we have developed the recording system with longer continuous recording capacity and larger signal-to-noise ratio to detect spontaneous activity in a single sweep. we applied this system to the sensorimotor cortex of rats anesthetized with a mixture of urethane and ␣-chloralose. the hindlimb region was exposed and stained with rh , a voltage sensitive dye. optical records, after compensation for pulsation artifacts, contained deflections time-locked to the high amplitude transient waves, characteristic to ␣-chloralose anesthesia, recorded with a wire electrode placed in the optically sampling area. as the transient in the electrocorticogram fluctuated, the optical signal also varied. this signal was distributed over a broad region, whose latency, amplitude or shape varied systematically within the region, probably reflecting the regional differences in the transient activity. yuko tanaka, r. allen waggoner, kenichi ueno, keiji tanaka, kang cheng bsi, riken, saitama, japanobjective: in this fmri study, we attempted to identify the brain regions involved in the process completing objects with degraded image information.method: fourteen healthy subjects were studied using a t mri scanner while performing a matching-to-sample task with three task conditions. the main condition required the subject to judge in a -s trial if a trial-unique, degraded animal image matched a contour image. in the comparison condition, we reversed the order presenting intact and degraded images (id epoch).results: comparing images acquired in di epochs with those acquired in id epochs, significant activation was found in the left parietooccipital cortex spanning the cuneus (ba ), superior occipital gyrus around the parieto-occipital sulcus (ba ) and superior parietal lobule (ba ). other activated foci include the left anterior cingulated cortex, left dorsal frontal gyrus and right middle frontal gyrus.conclusion: these results indicate that the parieto-occipital cortex is critically involved in the object completion with degraded images.ps p-j influence of task difficulty during meter inspection: an fmri study naoki miura , , makoto takahashi , jobu watanabe , , shinya uchida , , shigeru sato , kaoru horie , masaharu kitamura , toshio wakabayashi , katsuki nakamura , , ryuta kawashima crest, jst, kawaguchi, japan; niche, tohoku univ. sendai, japan; graduate school of engineering, tohoku univ. sendai, japan; bme institute, waseda univ., tokyo, japan; idac, tohoku univ., sendai, japan; lbc research center, tohoku univ., sendai, japan; department of animal models for human disease, ncnp, tokyo, japanthe purpose of the study was to analyze the cognitive process of a subject facing a human-machine interface (hmi) using fmri. we compared brain activation during meter inspection tasks with different task difficulty. during the meter inspection tasks, the subjects were instructed to inspect the three meters, and to press the button, if the subject found abnormal state. the task difficulty was devised by controlling the rate of change for the value to be displayed. in the right occipitotemporal area and the left cerebellar posterior lobule, activation during analog meter inspection was greater when the task difficulty was higher case. the results suggest that these regions are related to attention and perception of visual appearance of hmi.ps p-j neural connectivity among brain areas related to language function shinya uchida , , , naoki miura , , jobu watanabe , shigeo kinomura , kazunori sato , yasuyuki taki , kentaro inoue , ryoi goto , ai fukushima , kaoru horie , shigeru sato , katsuki nakamura , hiroshi fukuda , ryuta kawashima department of nuclear medicine and radiology, idac, tohoku university, sendai, japan; niche, tohoku university, sendai, japan; national institute of neuroscience, ncnp, kodaira, japan; japan science and technology agency, kawaguchi, japan; bme institute, asmew, waseda university, tokyo, japan; graduate school of international cultural studies, tohoku university, sendai, japanthe present study examined the neural connectivity of languagerelated regions using functional mri (fmri) and diffusion tensor imaging tractography (dtt). twenty subjects were participated. functional region of interest (roi) in the left inferior frontal gyrus (lifg) defined by fmri during speech production task was used as a seed point for dtt. in more than % of subjects, tracts between the roi and the left thalamus (lth) were estimated. post hoc fmri analysis showed activation in the lth during speech production tasks. therefore, cortical connectivity between the lifg and lth may have certain functional roles in speech production. keisuke wakusawa , , motoaki sugiura , yuko sassa , , hyeonjeong jeong , , kaoru horie , shigeru sato , hiroyuki yokoyama , kazuie inuma , ryuta kawashima niche, tohoku univ., sendai, japan; department of pediatrics, tohoku univ. graduate school of medicine, sendai, japan; miyagi univ. of education, sendai, japan; ristex, jst, tohoku univ., sendai, japan; gsics, tohoku univ., sendai, japan; lbc rc, tohoku univ., sendai, japanthis study examines the cortical mechanisms of comprehension of implicit social meanings such as irony and metaphor. healthy subjects judged whether the utterance in a picture such as irony, metaphor, or control expressions was situationally appropriate (s), or literally correct (l). greater activation during s than l task was analyzed to identify the activation for implicit meanings and neural responses to irony or metaphor were analyzed. the left medial prefrontal cortex showed higher activity during the s than l task. the medial orbitofrontal cortex and the right temporal pole showed responses selective to the irony; the responses in the former were observed during s task only, while the latter in both tasks. no selective response to metaphor was observed. keiichi onoda , yasumasa okamoto , kazuhiro shishida , akiko kinoshita , shigeru toki , kazutaka ueda , hidehisa yamashita , shigeto yamawaki department of psychiatry and neuroscience, hiroshima university, hiroshima, japan; training and research center for clinical psychology, hiroshima university, higashi-hiroshima, japan anticipation of emotional events may affect perceptual and cognitive processes when the events actually happen. we studied the effects of anticipation of positive and negative affective images on neural processes estimated with meg and event-related fmri. participants were presented emotionally positive or negative images with cue stimuli. the cue stimulus indicated the emotional valence of the image which followed a few seconds later. in meg study, visual evoked field (vef) was smaller for the anticipatable negative image than the anticipatable positive image. this result suggests that when the presentation of a negative image is anticipated before the event, neural processing for the image is depressed compared to when a positive image is anticipated. furthermore, we report the difference of brain activation between anticipation of positive images and that of negative images in event-related fmri study. makoto wada , , , kenji yoshimi , , noriyuki higo , yong-ri ren , hideki mochizuki , yoshikuni mizuno , shigeru kitazawa , , dept. of physiol, juntendo univ. schl. of medicine, tokyo, japan; dept. of neurol., juntendo univ. schl. of medicine, tokyo, japan; crest, jst, tokyo, japan; neurosci. res. inst., aist, tsukuba, japanwe developed a new method for comparing immunopositive cell densities across groups of animals and creating statistical parametric maps on standardized sections. as an example, we compared iba- positive glial cell densities in rats with and without unilateral injection of mpp+. immunopositive cell density map was automatically created in each animal over a coronal section in the midbrain (bregma − . mm). after the map was normalized to a template section, positive cell densities of the two groups were compared in each pixel and a statistical parameter was mapped on each pixel. we were able to detect significant increases of microglias in the side of the injection not only in the substantia nigra pars compacta but also in the white matters. the new method was proven to be useful for detecting significant changes of cell densities over the entire area of immunostained sections. key: cord- -kt gt t authors: nan title: poster session abstracts date: - - journal: pediatr pulmonol doi: . /ppul. sha: doc_id: cord_uid: kt gt t nan causing mutation is ∆f , making studies of nbd essential in understanding cftr function. crystal structures have been solved of wild-type (wt) and mutant cftr nbd , including variants containing ∆f (lewis et al. embo j. ; lewis et al. j. biol. chem. ; thibodeau et al. nat. struct. mol. biol. ) . cftr nbd contains a regulatory insertion (ri) and a regulatory extension (re) that contain pka phosphorylation sites. although the nbd core is very similar in the various structures, the ri and re in some structures of human wt nbd differ by °rotations, indicating that they are mobile. we have used nmr spectroscopy to study wt and ∆f nbd in the phosphorylated (phospho) and non-phosphorylated (non-phospho) states, and bound to different regions of cftr. despite their similar structures, differences are observed in the nmr spectra of wt and ∆f nbd . these changes, possibly due to altered dynamics or interactions of the ri and re with the nbd core, will be discussed. the inherent stability and function of nbd could be affected by these changes, accounting for part of the ∆f defect. different interactions between wt and ∆f nbd with the icls could also affect channel function. to identify residues comprising the icls, we generated a homology model of cftr based on the crystal structure of sav in which nbd and nbd form a productive dimer (dawson and locher. nature. ) . our data on a peptide corresponding to the first intracellular loop (icl ) indicate that binding to nbd requires phosphorylation of the ri. our homology model shows that the icl binding site is buried when the ri is bound to the nbd core. nmr data comparing phospho and non-phospho nbd indicates that phosphorylation of the ri and re disrupts their interactions with the nbd core, likely exposing the site for icl binding. the structure of the molybdate transporter modb c , with the nbds in an open conformation, shows icl binding is cognate nbd at a different interface (hollenstein et al. nature. ) . icl in modb c interacts with the nbd near y , the analogous residue to f in cftr. we will show binding data of icl with wt and ∆f nbd in different states and also present resonance assignments of nbd to map the icl interaction sites. since the sav and modb c structures were solved in different states, the different icl interactions may represent structural changes during the reaction cycle. the ∆f mutation may thus affect intramolecular associations in certain conformations, which would account for another part of the ∆f defect. probing differences in interactions of wt and ∆f nbd is critical for understanding the molecular basis of normal cftr function and of the altered cftr function in cystic fibrosis. shsps target ∆f cftr for erad via the sumo pathway ahner, a. ; brodsky, j.l. ; frizzell, r.a. . cell biology and physiology, university of pittsburgh, pittsburgh, pa, usa; . biological sciences, university of pittsburgh, pittsburgh, pa, usa the most frequent disease-causing mutation in cystic fibrosis (cf) is a deletion of phenylalanine at position (df ) in the first nucleotide binding domain of cftr. essentially all of the mutant protein, as well as % of wild type (wt) cftr, is retained in the er and targeted for erassociated degradation (erad) by the s proteasome. key mediators of cftr folding and degradation are molecular chaperones, which help protein substrates fold, but can target them for degradation if folding efficiency is compromised. to identify factors modulating erad of cftr we performed a microarray analysis, screening for transcription profile differences between yeast expressing cftr and control strains, and identified enhanced transcript levels for the small heat shock protein hsp in yeast expressing cftr. we then demonstrated that cftr degradation was blocked in strains lacking the genes encoding the two yeast shsps, hsp and hsp ( ) . to examine whether shsps regulate cftr biogenesis in mammalian cells, we asked which of the human shsp homologues are endogenously co-expressed with cftr using rt-pcr and western blot analysis. we detected shsps, including alphaa-crystallin and hsp , in calu- and primary hbe cells. co-expression of alphaacrystallin or hsp together with wt or df -cftr in hek cells selectively decreased the steady state levels of df -cftr. pulse-chase experiments showed that the rate of df -cftr degradation was enhanced when either shsp was over-expressed, but wt cftr biogenesis was unchanged. co-immuno-precipitation experiments in hek cells revealed that alphaa-crystallin and hsp interacted preferentially with df -cftr. thus far, our results suggest that shsps selectively increase df -cftr's accessibility to proteasome-mediated degradation pathways. previously, hsp was reported to interact with ubc , the sumo (small ubiquitin-like modifier) conjugating enzyme ( ) . sumo is covalently linked to its substrates, often at sumoylation consensus sites; similarly to ubiquitin, this occurs through a series of thiol transfer reactions. we confirmed this interaction by co-immuno-precipitation and found that ubc as well as the sumo specific protease, senp , are expressed in airway epithelial cells. over-expression of ubc decreased, while over-expression of senp increased df -cftr protein levels. pulse-chase experiments indicated that these enzymes regulate selectively the degradation of df -cftr, similarly to the shsps. preliminary studies in vitro and in vivo indicate that cftr, and particularly purified nbd , is sumoylated and that hsp facilitates this process. mutational analysis of the sumoylation consensus sites within cftr suggests a cycle of sumo modification that facilitates wt cftr biosynthesis but targets df -cftr for degradation, probably because the balance between sumo addition and removal is impaired by an excessive interaction of shsps with the mutant protein. [supported by grants from the nih and the cystic young, a. physiology and biophysics, chicago medical school, north chicago, il, usa efficient retrieval of wtcftr from the cell surface is mediated by components of the clathrin mediated endocytic pathway; however many aspects of cftr endocytosis remain to be elucidated. we have previously shown that endocytosis of wtcftr is dependent upon recognition of a tyrosinebased motif in the carboxyl tail of cftr by the ap endocytic adaptor complex. in contrast to wtcftr, less is known about the mechanisms whereby mutant cftr undergoes internalization. according to one model, df cftr undergoes accelerated endocytosis compared to wtcftr, whereas others have argued that endocytic rates of df cftr are comparable to those of wild-type cftr. to further define the function of proteins involved in the initial steps of wt and mutant cftr endocytosis, we investigated the role of the gtpase dynamin. dynamin has been proposed to play a key role in endocytosis by facilitating the scission of nascent clathrin coated vesicles from the cell surface. until recently, very few tools were available to modulate clathrin mediated events. we took advantage of dynasore, a newly discovered small molecule, non-competitive inhibitor of the gtpase dynamin. dynasore acts as a potent, rapid and reversible inhibitor of endocytic pathways known to depend upon dynamin, by blocking coated vesicle formation within seconds of dynasore addition. endocytosis of wtcftr was inhibited rapidly and efficiently upon addition of dynasore. furthermore, within minutes of dynasore washout, endocytosis of wtcftr resumed completely. also upon dynamin washout, normal trafficking and recycling of wtcftr was also observed. temperature correction of df causes expression of cftr at the cell surface, however such 'rescued' df cftr is not stabilized upon returning cells to c. application of dynasore prevented such rapid loss of mutant cftr from the cell surface. since maintenance of mutant cftr at the cell surface is critical for effective therapeutic intervention in patients with cf, our results provide insight into additional potential targets for pharmacological manipulation for the treatment of cf. richardson, j.m.; thibodeau, p.h.; watson, j.; thomas, p.j. physiology, ut southwestern medical center, dallas, tx, usa protein misfolding is the basis for a multitude of human diseases; however, the mechanisms underlying misfolding are not well understood. most cases of cystic fibrosis are associated with mutations-including the most common, deletion of phenylalanine (∆f ) in nbd -that interfere with the folding of the cystic fibrosis transmembrane conductance regulator (cftr). the resulting loss of functional cftr causes the disease. thus, elucidating how ∆f , affects the folding of cftr is critical to understanding the pathology of the disease. cftr is composed of five domains: two integral membrane transmembrane domains (tmds), a regulatory domain (r), and two nucleotide binding domains (nbds). ∆f occurs in the n-terminal nbd . both the murine wildtype and ∆f nbd can be expressed in bacteria and purified to near homogeneity. while the soluble expression yield of ∆f is lower than the wild type under identical conditions, ∆f achieves a native conformation similar to wild type as monitored by a variety of hydrodynamic and spectroscopic characteristics such as analytical size exclusion chromatography, circular dichroism, and fluorescence. recently, a non-native, but folded, species has been detected under mildly denaturing conditions. far-uv cd reveals a time and temperature dependent conversion from a mixed alpha/beta native conformation to a less helical non-native conformation, while fluorescence measurements reveal a parallel blue shift in the peak emission intensity from to nm. this non-native species is in a more open conformation as determined by both limited proteolysis and the change in retention time on analytical size exclusion chromatography. the conversion to this state is inhibited by the native state ligand (atp) and by the presence of the second site suppressors (g e, r m, and r k). notably, the ∆f nbd protein populates the non-native state under milder conditions than the wild type nbd . these studies reveal the properties of the native state and its conversion to a partially folded state that is affected by the ∆f mutation. this data highlight the proximal affect of the disease causing mutation and provide a means of assessing strategies designed to correct the defect. this work is supported by the cystic fibrosis foundation through a postdoctoral training grant and by the nih we have previously demonstrated that the stability of cftr is negatively modulated by the cftr interacting protein, cal. cal competes with nherf in binding to the pdz motif at the c-terminus of cftr and directs cftr trafficking to the lysosome. recently, silencing of cal or overexpression of nherf has been shown to restore chloride transport activity in ∆f -cftr cell lines. to identify the molecular machineries involved in cal dependent degradation of cftr, we investigated the role of the cal interacting snare protein syntaxin (stx ). stx protein expression and function were manipulated by sirna silencing, over-expression, and a dominant-negative mutant. we also performed a functional study of chloride transport activity in cystic fibrosis bronchial epithelial cell line cfbe o-stably expressing either wt-cftr or ∆f -cftr. the endogenous stx and cal protein expressions were knocked down by sirna techniques in multiple human cell lines including hek , hela cell stably expressing ∆f -cftr and cfbe o-stably expressing either wt-cftr or ∆f -cftr. in hek cells, knockdown of either stx or cal augments the expression of transfected gfp-tagged cftr (gfp-cftr), which is consistent with the observation that overexpression of either stx or cal enhances the degradation of cftr. stx interaction with cal was confirmed by a co-immunoprecipitation assay. because of this interaction, we hypothesize that stx negatively regulates cftr protein stability by interacting with cal which in turn binds to the c-ter-minal pdz motif of cftr. consistent with his hypothesis, stx knockdown has no effect on protein levels of a cftr mutant lacking the pdz motif (gfp-cftr∆trl). more importantly, cal knockdown eliminates the inhibitory effect of stx overexpression on gfp-cftr. a cell surface biotinylation assay and confocal microscopy showed significant increases in plasma membrane expression of cftr in stx knockdown cells without gross changes in the expression pattern. furthermore, consistent with its trans-golgi localization, stx knockdown has no effect on gfp-∆f -cftr expression. however, ∆f -cftr increases significantly in stx knockdown, temperature-rescued cells. to assess the effect of stx in bronchial epithelial cells, stx was knocked down in cfbe o-stably expressing wt-cftr. consistent with the observations in hek and hela cells, the expression of untagged wt-cftr is also augmented. knockdown of stx increases cftr expression in a does-dependent manner, reciprocal to the expression level of stx , without changing the level of expression of cal or gapdh. furthermore, stx knockdown lead to a does-dependent increase in cftr mediated short-circuit current that is stimulated by forskolin and inhibited by cftr chloride channel inhibitor cftrinh- . more importantly, in temperaturerescued cfbe o-stably expressing ∆f -cftr, stx knockdown tripled the cftr mediated short-circuit chloride current. these results not only delineate the effect of stx on post-golgi trafficking of cftr and its dependence on cal-mediated pdz-based interaction, but also points to it as a potential therapeutic target in conjunction with the er-quality control based rescue of ∆f -cftr. supported by the cystic fibrosis foundation and the national institutes of health. seavilleklein, g. ; evagelidis, a. ; amer, n. ; chappe, f. ; hanrahan, j. ; chappe, v. . physiology & biophysics, dalhousie university, halifax, ns, canada; . physiology, mcgill university, montreal, qc, canada cftr channel activity is regulated by phosphorylation and de-phosphorylation of the r-domain. direct pkc phosphorylation at specific sites in the r domain and nbd is essential and modulates cftr activation by pka. however, the molecular mechanism by which phosphorylation of the r domain induces channel activity remains unknown. we have reported biochemical evidence for the role of phosphorylation in domain-domain association using a deltar-split construct encoding the front (tmd -nbd ) and back (nbd -tmd ) halves of cftr as separate polypeptides ( ) . in agreement with the inhibitory role of the r domain, iodide effluxes and patch-clamp experiments confirmed that deltar-split channels were functional and constitutively active without camp stimulation. co-transfecting a plasmid that directs expression of the r domain restored the pka-dependence of re-assembled channels. co-immunoprecipitations of the halves of the deltar-split with a gst-r domain fusion protein in vitro or with the cotransfected r domain in bhk cells revealed that pka phosphorylation enhances the r domain binding to the rest of the channel. we have now investigated the effect of pkc phosphorylation on the r domain binding. our data demonstrate that pkc phosphorylation also induces a strong binding of gst-r domain in vitro or co-transfected r domain in vivo when bhk cells are stimulated with pma. interestingly, the combination of pkc+pka phosphorylation induced a stronger binding than pka alone. mutation of all consensus pkc sites on the r domain to alanine ( ca) eliminated specific pkc phosphorylation but did not alter pka phosphorylation in gst-rd. western blotting and confocal microscopy experiments confirmed that expression levels of all domains of the split- ca/rd are unchanged compared to split-rd and co-localize at the plasma membrane of bhk cells. basal activity of the split- ca/rd was similar to that of the split-rd in iodide efflux experiments. however, activation by camp+ibmx was delayed and significantly reduced ( . % of split-rd and . % of wt-cftr activation). these results confirm the functional re-assembly of the ca-rd with the front and back halves of the channel and the inhibitory role of the r domain. in co-immunoprecipitation experiments, the same level of interaction was observed between the ca/rd and rd with the split halves suggesting that basal, inhibitory interaction of the r domain is independent of pkc phosphorylation. however, ca/rd binding with the split channel was not increased by pkc or pka stimulation. we conclude that pkc phosphorylation is essential for pka-dependent binding of the r domain to the rest of the channel, and that the combined effect of these kinases further strengthens the r domain interaction. these structural changes are consistent with the essential role of pkc sites for cftr activation by pka as previously reported ( ) . supported by cihr, nshrf, dmrf & ccff. g.seavilleklein was supported by nserc. ( ) the in vivo folding mechanism of multidomain membrane proteins, including the cystic fibrosis transmembrane conductance regulator (cftr), is poorly understood. cftr, a member of the abc transporter superfamily, contains two symmetrical halves, each consisting of a membrane spanning domain (msd and msd ) and a cytosolic nucleotide binding domains (nbd and nbd ), connected by the regulatory (r) domain. to address whether cftr follows the archetypical domain-wise folding of multidomain soluble polypeptides and/or the cooperative domain folding mechanism, first we determined domain combinations that are necessary and sufficient to escape the endoplasmic reticulum (er) quality control. one-, twoor three-domain assemblies failed to be processed efficiently in cells, measured by biochemical and morphological assays. the smallest folding unit that escaped the er quality control was composed of a four-domain assembly, containing msd , nbd , r or msd as linear or split polypeptide. as a second approach we showed that cf-causing point mutations in the msd , nbd or msd provoked the er retention of the full-length cftr with severe conformational defect in the nbd , measured by limited proteolysis and immunoblotting, using domain spacific antibodies. the posttranslational folding kinetics of the four-domain folding unit was comparable to that of cftr, suggesting that cooperative domain assembly is essential for the channel biogenesis. this mechanism provides an explanation for the processing defect caused by numerous cf mutations and outlines a possible paradigm for cftr folding in living cells. cftr in post-golgi compartments. our results, jointly, uncover the role of n-glycans as a critical structural determinant of cftr conformational maturation/stability in a chaperone-independent manner. karamyshev, a.l. ; patrick, a.e. ; johnson, a.e. ; thomas, p.j. . ut southwestern medical center at dallas, dallas, tx, usa; . texas a&m university, college station, tx, usa cystic fibrosis (cf) is caused by mutations in cftr (cystic fibrosis transmembrane conductance regulator). cftr, a member of the abc transporter family, is a multispanning membrane protein critical to chloride and water movement in secretory epithelia. over one thousand mutations, in all parts of the cftr sequence, have been identified, many of which disrupt the function of the protein to cause the disease. some of the disease-associated mutations in the n-terminal portion of cftr were predicted to alter cotranslational interactions with factors involved in the membrane integration of cftr required for its proper function. to test this hypothesis, a photoreactive probe was incorporated into different positions of the n-terminal portion of cftr by introducing amber stop codons and translating these messages in vitro in the presence of the amber suppressor trna n ε -( -azido- nitrobenzoyl)-lys-trna amb . ribosome-associated nascent chains of a specific length containing the modified amino acid at a defined position were produced using truncated mrnas. some of these cftr nascent chains photocrosslinked with proteins of approximately and kda molecular mass. crosslinking to the ~ kda protein was observed only after the tm span had emerged from the ribosomal tunnel. moreover, truncation analysis demonstrated that tm is necessary for this interaction. in evaluating a range of nascent chains, the extents of photocrosslinking to the ~ kda and kda proteins often varied inversely. immunochemical and knockdown/depletion studies indicate the ~ kda protein is srp , a subunit of the signal recognition particle involved in targeting ribosome nascent chain complexes to the translocon in the endoplasmic reticulum membrane. highlighting the functional relevance of this interaction, in cells with reduced srp levels, nonglycosylated forms of cftr accumulate and total cftr levels decrease. notably, some cf-causing mutations in tm also reduce crosslinking to srp. these data reveal that mutational inhibition of nascent cftr association with srp likely contributes to or mediates some types of cf. aleksandrov, l.; aleksandrov, a.a.; riordan, j.r. the initial reversible steps of atp binding to the first (nbd ) and second (nbd ) nucleotide binding sites of cftr are divalent cation independent and dependent, respectively (aleksandrov et al, j biol chem , - , ) . subsequent to the initial binding, either mg or mn drive rapid hydrolysis at the second site while promoting trapping of the nucleotide at the first site. this occlusion at the first site of functional wildtype cftr is somewhat similar to that which occurs when the catalytic glutamates in both of the hydrolytic sites of p-glycoprotein are mutated. this occluded state has been proposed to be the result of dimerization of the two nbds and represent a transient intermediate formed during atp hydrolysis (tombline and senior, j bioenerg biomembr , - , ) . to test the possible relevance of this interpretation to cftr, we have now characterized the process by which nbd occludes [ p]- n atp and [ p]- n adp. we have now found that only n atp and not n adp can be bound initially at nbd in the absence of mg. consistent with this mg requirement for the ndp binding, n adp or adp is able to compete for the n atp binding site only when the divalent ion is present. despite the lack of a requirement for mg for atp binding, retention of the nucleotide triphosphate at the binding site at °c was dependent on the cation. however, at reduced temperature ( °c), n atp remains locked in the binding pocket with virtually no reduction over a hour period even in the absence of mg. these apparently paradoxical temperature effects hint that different molecular events can contribute to the occlusion of nucleotide at nbd . that the occlusion process occurs exclusively at nbd and does not depend on its interaction with nbd , was shown by experiments in which identical behavior was exhibited by ∆nbd constructs of cftr. thus, nucleotide trapping by wild-type cftr, unlike that by mutant p-glycoprotein, is accomplished by events at a single domain (nbd ), rather than by dimerization of the two nbds. (supported by the nih and cff). cftr consists of five domains or regions, two regions that span the cell membrane (the tmds), two cytoplasmic nucleotide-binding domains (the nbds), and a regulatory (r) domain. the r domain is unique to cftr: without phosphorylation of this domain, the cftr chloride channel does not open. the tmds and the nbds of cftr are similar to those found in many atp-binding cassette proteins, the majority of which are transporters. the domains are fused in a single polypeptide chain, linked in the sequence: tmd , nbd , r domain, tmd , nbd . the first nbd is the site of the location of the mutation that is responsible for the development of cystic fibrosis in the majority of human patients (loss of phenylalanine ). although there is a structure for nbd , we are still lacking a structure for the entire cftr protein. we have employed electron microscopy to provide low-to medium-resolution structures for the complete cftr protein which was expressed in bhk cells and purified by affinity chromatography. using single particle analysis combined with site-specific labelling using . nm diameter ni-nta nanogold, we have identified the locations of nbd and the region around residue of the r-domain within the low resolution d structure. by a process of elimination, this has also allowed the identification of nbd and the region occupied by the two tmds. these low resolution studies have now enabled us to interpret medium resolution d structural data obtained for cftr. progress in obtaining two-dimensional arrays (crystals a single molecule thick) should allow higher resolution structural data to be obtained for the entire cftr protein -sufficient for the identification of the transmembrane alpha helices. these data should allow a much clearer picture of the transmembrane channel formed by cftr and will inform molecular modelling approaches aimed at the development of novel drugs for the treatment of cystic fibrosis. we explored the gating process by introducing cysteines along the entire lengths of various transmembrane domains and extra-cellular loops, and studying their ability to chemically modified. we study the changes in their reactivity in closed and open channel states to characterize the changes in transmembrane domain conformations during gating. using membrane impermeant mts-reagents, we have characterized the conformational changes in tm -ecl , ecl -tm , ecl -tm , and ecl -tm of cftr. our results suggest that atp binding induces modest changes in the transmembrane domains and extracellular loops. channel opening involves a rearrangement of the tm helices leading to decreased hydration of residues near the extracellular end. this conformational change is either a part of, or allosterically coupled to the gating mechanism. physiological role of nherf in the regulation of cftr raghuram, v.; yang, y.; yaemsiri, s. laboratory of kidney and electrolyte metabolism, nhlbi/nih, bethesda, md, usa in the apical membrane of epithelial cells, cftr seems to exist within a multiprotein complex, in which its activity is regulated by interactions with other proteins. the c-terminus of cftr contains a pdz-binding motif, which binds to several pdz domain-containing proteins, including members of na+/h+ exchanger regulatory factor (nherf) family, nherf /ebp , nherf /e karp, cap /nherf , nherf , and cal/pist/gopc. pdz domain proteins act as scaffolds to assemble signaling proteins to form signaling complexes. these signaling complexes are central to achieve specificity and efficiency of signal transduction. nherf and nherf have a c-terminal erm domain, which interacts with cytoskeleton-associated erm proteins. this interaction may anchor cftr to the actin-cytoskeleton and restrict its movement within the membrane. in addition, several functional roles have been proposed for cftr-pdz domain interactions, including as an adapter to promote efficient pka phosphorylation of cftr, apical targeting and recycling of cftr, regulation of single-channel activity, organization of β-adrenergic, and lysophosphatidic acid- receptor signaling complex, and trafficking of romk channels. our current understandings of the role of cftr-pdz interactions are mostly obtained from heterologous expression studies. very few studies have addressed the role of pdz domain proteins in native epithelial cells or in organ physiology. we have used rna interference techniques to produce a knockout or knockdown (kd) calu- cell lines using a lenti-viral vector for stable suppression. using four different shrnas, we have generated four stable nherf -kd cell lines with varying levels of nherf suppression. in these cells, nherf expression ranged from - % of calu- nt cells (control cells). in nherf -kd cells, steady-state cftr protein level was greatly reduced, whereas its apical localization was unaffected. we also found structural defects in the apical microvilli. in polarized nherf -kd epithelia, membrane-permeable camp analogs were unable to stimulate cftr mediated short-circuit currents. however, forskolin induced response was nearly normal despite reduced cftr expression. examinations of βadrenergic-, and adenosine-receptor signaling pathways suggest that nherf plays a unique role in organizing the adenosine receptor signaling pathway. abnormal retention of the df cftr mutated protein in lung epithelial cells underlies the pathology in a large proportion of individuals with cystic fibrosis. a drug discovery alliance between cfft and biofocus dpi was initiated with the aim to identify novel genes which upon shrna-mediated knockdown were able to efficiently restore df cftr activity. a high-throughput assay based on an yfp halide reporter was developed in a human cystic fibrosis bronchial epithelial cell line (cfbe o-), whereby the rate of halide influx directly correlated to df cftr activity. biofocus dpi's proprietary adenoviral shrna libraries totalling , viruses were screened in this functional assay and yielded hits. these hits were repropogated to generate new adenoviral stocks and rescreened at three concentrations on the yfp halide reporter assay. genes were eliminated due to potential cytotoxic effects, and hit calling was performed on the remaining hits. a total of duplicate hits were confirmed ( %). of the confirmed hits had more than one shrnas which targeted the respective gene. confirmed hits were stronger in restoring df cftr function than the most potent positive control: syntaxin- . in order to elucidate which of these hits induce halide influx via direct rescue and activation of df cftr, the yfp halide reporter assay was performed in the presence or absence of the cftrinh . these cftr dependent hits will move forward into subsequent validation experiments whereby hits will be categorised into potentiators and correctors. the final goal of this program is to prioritize targets for entry into drug discovery. restoring expression and/or function of mutated cftr provides a means to treat cystic fibrosis. biofocus dpi -a galapagos company -in collaboration with the cystic fibrosis foundation therapeutics (cfft) has established a drug target discovery project that will enable the selection of proteins that rescue mutated cftr. by screening biofocus dpi's proprietary adenoviral shrna libraries in a high-throughput primary functional assay for cftr activity in human cells, we have identified and confirmed hits out of , that entered the primary screen. to further validate these proteins as potential drug targets, we have set up a series of experiments and assays focused on the rescue of cftr expression and its maturation by knock-down of specific targets. ) in order to establish whether the knock-down of a specific target was able to promote df -cftr plasma membrane localization, we performed immunofluorescence stainings of df -cftr in a patient-derived human cell line carrying the df -cftr mutation (cfbe o-) grown in air-liquid interface culture. a transduction protocol to deliver shrna adenovirus to this polarized airway cell model system has been optimized and preliminary results showed increased levels of expression of df -cftr at the plasma membrane level upon knock-down of syntaxin- , our positive control. experiments performed at οc also demonstrated redistribution of df -cftr to the apical plasma membrane at low temperature. we will show the results of all the shrna hits in this assay. ) changes in the post-translational maturation levels of df -cftr upon knock-down of specific proteins were evaluated by determining the accumulation of band b and c by using a western blot approach. in this way, we can select targets able to promote post-translational maturation of df -cftr and/or its escape from er in the cfbe o-cell line. accumulation of band b and c indicates maturation of cftr and will be considered as a beneficial effect. preliminary results show increased accumulation of band b upon knock-down of specific targets and overexpression of wild-type cftr induces band c accumulation. experiments performed at οc also demonstrated increased band b expression. we will show the effect of all the shrna hits in this assay on band b and c expression. we believe that the results obtained by the combination of those two approaches will guide us in obtaining very strong drug targets for entry into a drug discovery phase. because gene therapy as a corrective measure for cf is possible in the foreseeable future, it is important to understand whether the global effects of gene introduction will have significant detrimental impact on cell function. to address this question, global gene mrna expression profiles were created with the affymetrix u a & b chip sets. wild type cftr (n= ), ∆f (n= ) and g d (n= ) were transfected in separate ib cell cultures using lipofectamine ™. control expression profiles were generated from untreated ib cells; one from this experimental set and five from previous control experiments. differential gene expression was determined by anova using a % false discovery rate threshold. genespring was used for data analysis and visualization. genego was used to determine which functional categories were over represented in the list and further assisted in interpretation of the results. rt-pcr was used to confirm differential expression of selected genes and examine the response in more detail. all transfected constructs produced very similar gene expression profiles quite distinct from the untransfected cells. probe sets representing uniquely named genes were significantly different between transfected and untransfected cells. cftr mrna expression in all forms was increased an average of -fold. transfection of ib - cells resulted in upregulation of a variety of genes involved in several basic processes: inflammation, protein folding and cytoskeleton framework regulation. genego canonical pathways with a significantly enriched number of genes from the data set involved glucocorticoid signaling and regulation, cytoskeleton remodeling and adhesion plus a variety of other signaling pathways including il , chemokines, g-protein and mapk cascades. cell processes overrepresented in the list were predominately associated with protein folding, inflammatory responses and cytoskeleton regulation. geneontology categories over represented in the list were regulation of signal transduction, receptor mediated endocytosis and protein folding. the proinflammatory interleukins il and il were upregulated . fold and fold respectively. the genes most upregulated, samd ( -fold) and oasl ( fold), are not well annotated. as controls, rt-pcr analysis of cells treated with either lipofectamine ™ alone or any form of cftr dna alone did not show upregulation of il , il , samd or oasl. in cells transfected with lipofectamine containing dna, upregulation of il (n= ), oasl (n= ) and samd (n= ) was confirmed by rt-pcr. oasl is known to interact with methyl cpg-binding protein , a dna transcription repressor acting at methylated promoter regions. thus it is possible that the global effects noted here leading to dramatic upregulation of two novel genes oasl and smad could be previously unrecognized consequences of gene therapies. cystic fibrosis (cf) is a disease which is caused by mutations of the cf gene, which codes for the cftr chloride channel. the ∆f -cftr mutation results in a mutant protein that undergoes transcription but fails to localize to the apical membrane, where it normally functions as a camp-regulated clchannel. however, clchannel activity can be detected at the plasma membrane when ∆f -cftr is overexpressed, synthesized at a reduced temperature, or in the presence of chemical chaperones. thus, strategies that facilitate the translocation of ∆f -cftr to the plasma membrane by increasing cftr transcription may be beneficial for the treat-ment of cf. previous studies performed using fluorescence halide efflux measurements and short-circuit current voltage clamp have shown that treatment with pparγ (peroxisome proliferator activated receptor gamma) agonists, such as pioglitazone and fll (fmoc-l-leucine), resulted in an increased biosynthesis and trafficking of ∆f -cftr to the cell surface. this effect was at least partially due to increased ∆f -cftr expression. further studies were designed to investigate the effect of pparγ agonists on cftr promoter activity. two firefly luciferase-conjugated cftr promoter plasmids, containing either . kb or . kb fragments, were transfected together with a control plasmid containing a renilla luciferase in human alveolar basal epithelial (a ) cells. the ratio of the firefly to renilla luminescence was used to measure cftr promoter activity. pparγ agonists increased cftr expression acting both on . kb and . kb promoter fragments. these results were confirmed in quantitative rt-pcr studies. transcription factor binding site analysis using current online programs and matrices showed three putative ppre (peroxisome proliferator response element) consensus sequences in the cftr promoter. the interaction of these sites with pparγ was shown using emsa (electrophoretic mobility shift assay). interestingly, none of these consensus sequences appear in the . kb construct, which still maintained a significant pparγ activation response. therefore, we hypothesized that a novel pparγ agonist effector site(s) is involved in transcription activation of cftr. to characterize this novel site(s), a series of deletion constructs to the promoter region were created, using mutation-introduced restriction sites in the original promoter construct. the new constructs ranged from . kb to base pairs upstream of the transcription start site. in preliminary studies we found that at least one previously uncharacterized pparγ effector site was present in the . kb promoter fragment that regulated cftr expression. additional experiments were performed using emsa to determine the proteins and cofactors which controlled the optimal conditions for cftr expression. the results of this study show that pparγ agonists, currently prescribed to treat hyperglycemia and improve peripheral insulin resistance, may also have additional benefits for the treatment of cf. our studies also show that pioglitazone and other pparγ agonists, by increasing cftr expression, are potent activators of clflux in epithelial cells expressing ∆f -cftr. this work was supported by grant from the canadian cystic fibrosis foundation decreased by µm gsno. the effect of gsno on hop expression was post-transcriptional. in cfbe o-cells transfected with sirna duplex specific for hop ( hr), hop expression was modestly inhibited; however, gsno dramatically augmented the inhibition. we next showed that knockdown of endogenous hop by sirna increased the steady-state levels of immature and mature forms of cftr. though overexpression of hop did not significantly affect cftr expression (immature and mature forms increased . ± . and . ± . fold respectively), in the presence of µm gsno, expression of both immature and mature cftr forms significantly increased in the hop overexpressing cells ( . ± . and . ± . fold respectively). next, we treated cfbe o-cells with a pulse of gsno ( µm; hr); isolated cytosol, er and golgi fractions; immunoprecipitated cftr from these fractions; and studied whether hop, co-immunoprecipitated with cftr, was s-nitrosylated (using a biotin substitution technique followed by streptavadin isolation). we found that sno-modified hop coimmunoprecipitated with ∆f cftr in the cytosol and er at baseline; and that gsno decreased the association between sno-modified hop and ∆f cftr. we speculate that the effect of gsno to decrease hop-cftr association may ) result from increased hop s-nitrosylation; and ) be permissive for ∆f cftr maturation. these data may be of relevance to the development of no donor-based therapies for cf. supported by cf foundation and the nih. mouse models for cystic fibrosis (cf) provide new possibilities to study disease pathogenesis, to correlate genotype and phenotype, and to test novel cf therapies. we use the cftr tm eu mice homozygous for the f del mutation in a congenic fvb background as a tool in pre-clinical testing of therapeutic strategies aimed at the correction of the f del folding and trafficking defect. these mice show characteristic cf ion transport abnormalities such as reduced camp-and cgmp activated intestinal chloride secretion and an increase of the amiloride-sensitive sodium absorption in nasal epithelium. we developed an ex vivo assay for the rescue studies of the f del trafficking defect in murine intestine by proteasome inhibitors based on periodical measurements of camp-activated transepithlial chloride currents in the ussing chamber. muscle-stripped ileum was incubated at c in william's e glutamax medium supplemented with insulin ( µg/ml) and dexamethasone ( µg/ml), in the presence of vehicle or proteasome inhibitor. a partial gain of chloride secretory function was achieved by incubating the tissue for - h at c in the presence of the proteasome inhibitors (pi) epoxomicin ( µm), mg ( µm), nip-(leu) -vinyl and adalys(bio)ahx l vs (both µm) and bortezomib ( nm). exposure of the tissue for h at c to alln ( µm) revealed a functional recovery up to wt levels. immunohistochemical and western blot analysis of all pitreated tissues showed the appearance of cftr positive crypt cells and mature band c cftr protein. rescue by alln remained detectable following inhibition of the er to golgi transport by brefeldin a. our ex vivo experiments demonstrate that proteasome inhibitors can enhance the amount of mature, functionally active murine f cftr mutant protein in intact tissue by preventing proteolytic degradation and increasing its expression at the cell surface. in vivo treatment of homozygous f del mice with bortezomib at a dose of . mg/kg/day for days resulted in a high morbidity. administration of a lower dose ( . mg/kg/day ) was well tolerated and no mortality or significant weight loss could be observed. as outcome parameters for the in vivo treatment with pis we used a salivary secretion assay and bioelectric measurements of intestinal and nasal epithelium in the ussing chamber. there was no difference in isoproterenol-induced salivary gland secretion or and in cftr-mediated intestinal chloride currents between control -and bortezomib-treated animals. however we found a reduction ( . fold) of the amiloride-sensitive short circuit current (isc) in the nasal epithelium of bortezomib-treated mice, indicating a moderate correction of the enacmediated sodium hyper absorption, that was not paralleled by a gain of the forskolin-mediated transepithelial chloride secretion. current in vivo experiments focus on the effects of pis upon short term ( h- h) rather than chronic treatment, to allow a direct comparison with the ex vivo treatment data. supported by grants from the cf foundation, bethesda, the sophia foundation and the dutch stomach-liver-intestine foundation. rotterdam, netherlands; . physiology and biophysics, university of alabama birmingham, birmingham, al, usa; . physiology, school of medical sciences, university of bristol, bristol, united kingdom recent progress in the development of small molecule correctors and potentiators capable of restoring cftr function have increased the need for pre-clinical test models including cultured airway epithelial cells from human cf patients as well as cf mouse models. the validity of cf mice as a surrogate for human cf patients depends on the assumption that human and mouse cftr, despite their limited sequence homology ( %) and differences in open probability (p o ) and channel subconductance state, behave similarly in their response to pharmacological correctors and potentiators. to verify this assumption, we used the iodide efflux technique to compare the cftr chloride channel activity in (i) cho cells stably transfected with mouse f del-cftr, (ii) nih- t cells stably expressing human f del-cftr, and (iii) their wt-counterparts. forskolin ( µm)-stimulated iodide efflux was very low in both mouse and human f del cftr cells ( . respectively % of wt values) and was stimulated to a similar extent ( . and . fold) by the cftr potentiator genistein ( µm). preincubation for h at c, or at c in the presence of the vertex corrector cfcor- ( µm) caused a - fold increase in forskolin-activated i-efflux in both cell types that was further enhanced by . - fold upon addition of genistein. functional correction was paralleled by the appearance of mature, complex-glycosylated mouse or human cftr on western blots. surprisingly, two other, structurally unrelated compounds known to correct the gating defect in hcftr, i.e. the vertex potentiator cfpot- ( µm) and the uncharged nppb analogue nppb-am ( µm; cf. wang w et al. ; j biol chem : ) mimicked the effect of genistein on hf del-cftr but failed to potentiate mf del-cftr in the t cells. both cfpot- and nppb-am likewise failed to potentiate forskolin-stimulated, cftr-mediated transepithelial chloride currents ex vivo in muscle-stripped ileal mucosa from homozygous f del-cftr tm eu mice (congenic fvb) mounted in ussing chambers under conditions in which genistein ( µm) enhanced the current response by - fold. in contrast, preincubation of the tissue in william's e glutamax medium for h at c, or for h at c in the presence of cfcor- ( µm) enhanced the forskolin+genisteininduced transepithelial clsecretion up to - % of the response in cftr+/+ littermates, and was associated with the appearance of band c on western blots. these results indicate that mouse and human f del-cftr ( ) respond to low temperature incubation or to a small molecule corrector with a similar gain in surface expression and function, supporting the use of cf mouse models for in vivo tests of cftr correctors; ( ) respond differently to various classes of cftr potentiators, in line with their pronounced differences in gating behaviour, and emphasizing the specificity of cftr-potentiator interaction. further studies focussing on human/mouse cftr chimera and aimed to identify the domain(s) accounting for the differential response to the cftr potentiators are in progress. cell apoptosis is a highly regulated process which is central to the maintenance of pulmonary vascular homeostasis. recent investigations implicated cftr in the regulation of intracellular sphingolipid levels. our laboratory demonstrated that an imbalance in endothelial sphingolipid levels triggers apoptotic cell death. we therefore hypothesized that a dysregulated cftr function in human lung endothelial cells inhibits programmed cell death in response to stress. methods: patch clamp analysis was used to verify the expression of cftr on human lung endothelial cells and the effect of specific inhibitors. we inhibited cftr with dpc, cftr inh - , and nppb or specific sirna followed by treatment with prototypical stressors that induce endothelial cell apoptosis, staurosporine, h o , or tnf-α. apoptosis was quantified by caspase- activity or annexin/ propidium iodide staining. lipid extraction was performed by a modified bligh and dyer method and intracellular sphingolipids were measured by tandem mass spectrometry. results: patch clamp analysis confirmed functional cftr expression on endothelial cells and a complete inhibitory effect of dpc and nppb but no effect of dids. as expected, treatment with staurosporine, a general protein kinase inhibitor and h o , as an oxidative stress stimulus resulted in significant endothelial cell apoptosis. similarly, tnf-α treatment ( ng/ml) in combination with cycloheximide caused activation of caspase- associated with a significant upregulation of the pro-apoptotic sphingolipid ceramide. knock-down with specific cftr sirna was validated with western blot and whole cell patch clamp analysis. dpc, nppb, and cftr inh - consistently and significantly inhibited apoptosis in human lung and primary mouse lung endothelial cells. caspase- activity was decreased by - % (% decrease in caspase units/µg/min) in hlmvec treated with h o after pretreatment with dpc ( %), cftr inh - ( %), and nppb ( %). however, we observed that dids, a non-specific chloride channel inhibitor also inhibited staurosporine induced caspase- activation ( %), suggesting the possible involvement of other chloride channels. in conclusion, our data suggest a role of cftr in modulating endothelial cell apoptosis in response to various stresses. the absence of functional cftr impaired stress-induced apoptosis, a process which may be mediated via alterations in sphingolipid trafficking. this abnormal cftr function in cf leading to aberrant cellular responses to stress may perpetuate an activated, pro-inflammatory phenotype of the lung vasculature. in turn, aberrant vascular activation may have a significant impact to the abnormal airway remodeling and persistence of a chronic inflammatory state characteristic of cf. supported by: cystic fibrosis foundation clinical fellowship training grant hegedus, t. , ; serohijos, a.w. , ; dokholyan, n.v. ; he, l. , ; riordan, j.r. , . department of biochemistry and biophysics, unc at chapel hill, chapel hill, nc, usa; . cystic fibrosis treatment and research center, unc at chapel hill, chapel hill, nc, usa; . department of physics and astronomy, unc at chapel hill, chapel hill, nc, usa cftr (cystic fibrosis transmembrane conductance regulator) is a camp dependent chloride channel. the phosphorylation of its amino acid r domain by protein kinase a (pka) is obligatory for channel gating under normal conditions. r domain contains multiple pka phosphorylation sites, which participate in activation, but no specific site is essential for channel function. in spite of numerous studies of the role of r domain in cftr regulation, the mechanism is largely unknown. one reason for this is the lack of information on r domain structure and its interactions with other parts of cftr. it has been shown that r domain lacks well-defined secondary structural elements and is intrinsically disordered. in this study, we used computational methods to explore the structure and function of the disordered r domain. our results show a specific disorder-order pattern in r domain, which is conserved among species even though the primary structure is not conserved. this result implies a role of the disorder-order pattern in r domain function and provides a basis of a novel working model. to gain some insight into the three dimensional structure of r domain, we per-formed ab initio folding of r-domain using discrete molecular dynamics with an all-atom force field. as disordered domains can not be characterized with a single structure, an ensemble was generated by selection of structures with the lowest energy from a large number of folding simulations. these structures were clustered to determine the dominant r domain conformations. they reveal features of secondary and tertiary structure, the positions of phosphorylation sites in d, and residue contacts within the r region that might be important for structure and function. to computationally confirm the mechanism proposed in our working model, an ensemble of phosphorylated r domain structures was also produced and characterized. supported by nih (r dk ), cff (dokhol i ), and novartis ( ). physiology system that utilises the planar patch clamp technique (finkel et al ) . this format utilises perforated patch clamp recording of wells in parallel, permitting the generation of up to data points per day. we have employed this system to develop assays to detect modulators of mutant cftr. human cftr-g d and df , over expressed in bhk and cho cells respectively, were cultured to approximately % confluence before harvesting. df cells were temperature corrected at °c for hours prior to experimentation to facilitate channel trafficking to the plasma membrane. cells were placed on the quattro system and whole cell chloride currents measured using asymmetric chloride selective solutions such that the chloride reversal potential was significantly different from that of leak currents ( mv). currents were assessed using a voltage ramp from - to + mv applied prior and min subsequent to compound addition. to validate the assays we used the prototypical cftr potentiator, genistein, which was added simultaneously to cells under study with µm forskolin. after assessing the effects of genistein, the specific cftr inhibitor glyh , was added at µm to confirm the currents were mediated via activation of cftr. the ec values determined for genistein were . ± . µm (mean ± s.e.m, n= ) for df and . ± . µm (mean ± s.e.m, n= ) for g d. these values agree with literature values (bulteau-pignoux et al ) . two alternative potentiators, phenylglycine- (pg- ) and vrt- were also tested in the presence of µm forskolin and were active against both df and g d. the ec values for pg- were . ± . µm (mean ± s.e.m, n= ) for df and . ± . µm (mean ± s.e.m, n= ) for g d. the ec values for vrt- were . ± . µm (mean ± s.d, n= ) for df and . µm (mean, n= ) for g d. these values agree with published data from ion transport studies (pedemonte et al ; van goor et al ) . in summary the ionworks quattro can be used to identify compounds which modulate the activity of cftr. this assay has a number of advantages over other formats in that it is direct, sensitive and employs voltage control of the cells under study. these assays can be used for primary screening of small to medium compound sets or as a secondary screen to triage the output from higher throughput indirect formats such as membrane potential. references it is very well agreed that ∆f cftr is recognized as a misfolded protein by the er quality control and targeted for degradation by the proteasome. during the course of experiments for aav gene therapy for cf we created an aav / vector expressing a truncated cftr insert (∆ ) driven by a chicken beta actin promoter (raav-cb-∆ -cftr). ∆ -cftr contains the normal coding sequence of wt cftr except that it is missing the first amino acids. we observed that cos cells transiently transfected with raav-cb-∆ cftr express cftr at significantly lower levels as compared to cells similarly transfected with a wt-cftr construct. to examine why, cells were transfected with raav-cb-∆ -cftr and treated for hours with µm of proteasome inhibitor, mg . in the cells treated with mg , ∆ -cftr protein expression was significantly increased. because lysosomal inhibitors did not have a significant effect, the data suggest that a large frac-tion of ∆ -cftr protein is degraded in the proteasome. to evaluate the rate of degradation, cells were treated with cycloheximide. time course experiments showed that ∆ is more rapidly degraded than ∆f -cftr. importantly, in cos cells cotransfected with ∆f and ∆ -cftr, there was a significant increase in the mature c-band of ∆f cftr compared to cells transfected with ∆f alone. this suggests that ∆ -cftr enhanced the maturation of ∆f from b to c bands. enhanced maturation of cftr from b band to c band also occurred when ∆ -cftr was transfected into hela cells stably expressing ∆ cftr (hela ∆ ) and cfbe o-cells stably expressing ∆ (cfbe o-∆ ). a number of proteins participate in the er to determine the fate of cftr. ∆f -cftr is known to associate with retrograde translocation protein complex on the er membrane containing vcp (valosin-containing protein) and derlin . vcp is also found in aggresomes containing ∆f -cftr in association with cytosolic histone deacetylase, hdac . we hypothesized that since ∆ is more efficiently degraded as compared to ∆f -cftr it may have higher affinity with vcp protein complex. we verified that ∆ -cftr interacts more readily with vcp and also has a higher degree of association with hdac as compared to ∆f -cftr. our data suggest that ∆ is more readily degraded by the proteasome as compared to ∆f -cftr and lacks biosynthetic arrest in the er. ∆ -cftr binds more readily to vcp and associates avidly with hdac , two outcomes that are expected to enhance proteasomal degradation. by engaging key quality control proteins, ∆ -cftr allows more ∆f -cftr to mature. our data suggest that truncated forms of cftr (∆ ) may be useful for cf gene therapy by affecting the maturation of endogenous cftr. ∆f , the most common disease causing cftr mutation, has impaired conformational maturation during its biosynthesis, and thus is unable to exit the endoplasmic reticulum (er). a well-defined di-acidic er exit code has been identified within the first nucleotide binding domain (nbd ) of wildtype cftr (jcb : - , ) , the disruption of which prevents cftr from efficiently exiting the er. this is consistent with the role of the diacidic code in the copii-mediated cargo concentration process. however, it is not known whether other types of er-to-golgi trafficking signals play a role in cftr trafficking in the early secretory pathway. even less is known about the trafficking signal(s) that are responsible for the rescue of ∆f cftr by a number of means. to achieve a better understanding of the trafficking signals dictating the export of cftr from the er, we constructed a series of cftr mutants to probe the potential functional targeting signals within both wild-type and ∆f cftr. it has been shown that nbd is not required for the export of wild-type cftr from the er. we further found that an internal deletion mutant lacking membrane spanning domain (msd ) and nbd -r but retaining the n-terminal cytoplasmic region, msd and nbd does not have sufficient signal to allow er export, suggesting that the signal(s) that are responsible for cftr export largely reside within the nbd -r. unlike ∆f cftr, the di-acidic code mutants are not temperature-sensitive. dissecting the di-acidic code revealed that both acidic residues contribute to er export, and that simultaneous substitution of both leads to a much decreased export of cftr. we found that the temperature-dependent export of ∆f relies on the presence of the di-acidic code within the nbd , suggesting that this code has a functional role in the temperature-dependent rescue of ∆f cftr. in our effort to further examine the role of the di-acidic code in the rescue of ∆f cftr by second site mutations, we found that ∆f cftr rescue by the simultaneous disruption of two of the arginine-framed tripeptides (afts) is also dependent upon the di-acidic code. surprisingly, we found that while nbd is not necessary for the export of wild-type cftr, it is required for the aft-mediated rescue of ∆f cftr, suggesting a role for cytoplasmic inter-domain interactions in the aft-mediated rescue of ∆f cftr. a detailed biochemical analysis of these mutants provides valuable insights into the roles of different cftr "trafficking" motifs in the er-to-golgi transport of cftr, the specific conformational defects of ∆f cftr and the potential molecular mechanism underlying the rescue of ∆f cftr. supported by cff. sonawane, n.d. ; zhao, d. ; galietta, l. ; zegarra-moran, o. ; verkman, a. . medicine, university of california san francisco, san francisco, ca, usa; . genetica molecolare, istituto gianina gaslini, genova, italy cftr inhibitors are predicted to prevent intestinal fluid secretion in enterotoxin-mediated secretory diarrheas such as cholera. cftr inhibitors that are not absorbed across the intestinal wall are attractive for diarrhea therapy because they may provide safe oral therapy. we previously discovered low affinity glycine hydrazide (glyh, ic ~ µm) cftr inhibitors that block cftr at its external pore (muanprasat et al., j. gen. physiol. ; : - ) . in order to increase inhibitor potency and prevent washout during severe secretory diarrhea, we synthesized a series of glycocalyx interactive cftr inhibitors containing a malonic acid hydrazide (malh) cftr pore blocking moiety linked to a lectin (sonawane et al., gastroenterology ; : - . lectin conjugation improved cftr inhibitory potency by ~ -fold (ic to nm). high-affinity cftr inhibition was abolished by malh-lectin heat denaturation, protease digestion, or competition by mannose or unconjugated lectin. fluorescently labeled malh-lectin remained membrane-bound for > hours after washout, whereas washout occurred in a few minutes without the lectin. malh-lectins blocked cholera toxin-induced intestinal fluid secretion in closed intestinal loops in mice with ec - pmol, and greatly reduced mortality in a suckling mouse model of cholera. we recently synthesized mono-and divalent cftr inhibitors consisting of malh coupled via a disulfonic stilbene linker to flexible mono and bifunctional polyethyleneglycols (pegs) of molecular size . , . , , , , , , and kda, with calculated solution lengths of - nm, with the larger size pegs potentially spanning cftr dimers or inducing their formation. ic for inhibition of cftr chloride current was - µm for monovalent malh-pegs, but substantially lower and size-dependent for divalent malh-pegs, decreasing from . µm to nm with increasing peg size. the mechanisms responsible for the improved and size-dependent potency of divalent malh-pegs were studied by whole cell, single-channel patch-clamp and by functional analysis of multivalent malh-conjugated dextrans and asymmetric divalent pegs. whole-cell experiments revealed reversible voltage-dependent block of cftr currents, with outward (positive) currents being more strongly blocked. in outside-out patch-clamp experiments, inhibitors caused a reduction of the mean open time. for malh-peg kda-malh ( µm) , the mean open time decreased from ± to ± ms. the effect on currentvoltage relationship and channel kinetics are consistent with a mechanism involving occlusion of the cftr pore from the extracellular side. luminally added divalent malh-pegs blocked by > % cholera toxin-induced fluid secretion in mouse intestinal loops with ic s of < pmol/loop, and greatly reduced mortality in a suckling mouse model of cholera. nonabsorbable, multivalent cftr inhibitor-macromolecule conjugates may be useful as anti-secretory agents in the treatment of enterotoxin-mediated diarrheas. supported by cff and nih. activation of the cystic fibrosis transmembrane conductance regulator (cftr) clchannel is primarily controlled by pka-dependent phosphory-lation of the r domain. once the r domain is phosphorylated, atp binding and enzymatic activity at two nucleotide-binding domains (nbds) open and close the cftr clchannel. we hypothesized that r domain phosphorylation regulates cftr activity by modulating atp interactions within two nbds. to test this hypothesis, we studied the activity of wild-type cftr and five variants with deletions of portions of the r domain between residues and . to alter atp-dependent channel gating, we tested three cftr stimulators, pyrophosphate (pp i , mm), 'deoxy-atp ( 'datp, mm) and caatp ( mm) . consistent with previous studies, pp i , 'datp and caatp all increased current of wild-type cftr about two-fold. each stimulator produced a similar increase in the r domain variants. the similar effects of stimulators on the channel activities of wild-type cftr and r domain variants suggest that the r domain does not have a major role in regulating atp-dependent channel gating. the r domain deletions did not alter the single-channel current amplitude of the r domain variants. however, r domain variants missing the sequence between residues and markedly reduced the open state probability, suggesting that this region is required for normal gating. these data suggest that the r domain does not control cftr activity by modulating atp interactions with nbd binding sites. instead, we speculate that the c-terminal part of r domain might participate in the channel-gating machinery downstream of atp regulation. supported by the cystic fibrosis foundation and howard hughes medical institute. haggie, p.m.; verkman, a. medicine and physiology, cvri, u.c.s.f., san francisco, ca, usa it was recently reported that phagolysosomes of alveolar macrophages from cf mice acidify in a cftr-dependent manner and that defective phagolysosomal acidification impairs bactericidal activity (di et al., nat. cell biol. , : - ) . these findings suggested a unifying hypothesis for cf disease progression: defective phagolysosomal acidification in cf macrophages permits the initiation and promotes progression of bacterial infection in the lungs. to reassess the central finding of that study we measured phagolysosomal ph using a fluorescent ph indicator containing oregon green® (pka ~ . ) and tetramethylrhodamine covalently bound to zymosan. phagolysosomal ph was insensitive to cftr inhibition ( µm cftrinh- ) in j macrophages (ph . ± . vs. . ± . ), alveolar macrophages from mouse lung (ph . ± . vs. . ± . ), and alveolar macrophages from human lung (ph . ± . vs. . ± . ). phagolysosomal ph in alveolar macrophages from ∆f cf mice was not significantly different from that in alveolar macrophages from wild-type mice (ph . ± . vs. . ± . ). to account for their finding of defective phagolysosomal acidification in alveolar macrophages, di et al. reported that lysosomal acidification was cftr-dependent and that fusion of lysosomes to phagosomes (at ~ min after phagocytic uptake) was responsible for phagosomal acidification. we measured lysosomal acidification in j macrophages using a dextran-conjugate containing oregon green® and tetramethylrhodamine and found that acidification was not impaired by cftr inhibition (ph . ± . vs. . ± . ) . we also measured the kinetics of phagosomal acidification using a zymosan-conjugate containing -(and- )-carboxyfluorescein (pka ~ . ) and tetramethylrhodamine. phagosomal acidification in j macrophages and murine alveolar macrophages began within min of phagocytosis and reached steady-state by - min, in agreement with prior data in murine peritoneal and bone marrow-derived macrophages. acidification kinetics in j macrophages was not altered by cftr inhibition, nor was acidification kinetics different in wild-type vs. cf alveolar macrophages. our findings do not support the conclusion that phagolysosomal acidification in alveolar macrophages is cftr-dependent, nor that it is impaired in cf. the mechanism of phagosomal acidification proposed by di et al. is not in accord with our data or precedents in the literature. because phagolysosomal acidification is central to the proposed mechanism linking defective cftr chloride channel function with cf lung disease, our results do not support the involvement of cftr in defective macrophage function in the pathogenesis of cf lung disease. supported by cff and nih. pared to that of wild-type cftr. the hsp family of molecular chaperones play important roles in the protein quality control process within the er. hsp atpase activity is regulated by multiple co-chaperones such as hsp , bag- and hspbp . hsp , a high-molecular-weight member of the hsp family was recently shown to display nucleotide exchange activity for hsp s in vitro. furthermore, hsp was identified as a component of a cftr-associated multiple protein complex using a global proteomic approach (cell : - , ) . in an attempt to explore the role of hsp in cftr conformational maturation in the er, we over-expressed the co-chaperone in hek cells and quantitatively analyzed its effect on the maturation and degradation of cftr. consistent with its role as a nucleotide exchange factor for hsp s, over-expression of hsp inhibits the er export of wild-type cftr and promotes its degradation. however, in striking contrast, over-expressing hsp stabilizes ∆f cftr and promotes its er export at reduced temperature. this effect is less pronounced at physiological temperature. the apparently opposite effects of hsp on wild-type and ∆f cftr maturation and quality control, suggests distinct conformational maturation pathways for the two cftr molecules, and reveals a specific role for hsp in regulating ∆f cftr refolding. such conclusion is reinforced by rnai experiments and further supported by quantitative co-immunoprecipitation. rnai-mediated down-regulation of hsp expression destabilizes ∆f cftr and reduces its export at reduced temperature, and hsp shows more extensive association with the er form of ∆f cftr than that of wild-type cftr. further analysis revealed that hsp not only modulates the chaperone activities of hsp s but also alters their steady state levels within the cell, creating secondary effects on cftr maturation and quality control. further studies are necessary to achieve a better understanding of the machinery, pathway and mechanism of ∆f cftr conformational maturation at reduced as well as physiological temperatures, and this in turn will provide critical insights and key factors that are of potential value to the rescue of the trafficking defect of ∆f cftr. farinha, c.m. , ; pissarra, l.s. ; amaral, m.d. , . department of chemistry and biochemistry, faculty of sciences, university of lisboa, lisboa, portugal; . ctr hum genet, nat inst health, lisboa, portugal the most frequent mutation in the cystic fibrosis (cf) gene, f del, causes retention of its protein product, f del-cf transmembrane conductance regulator (cftr) in the endoplasmic reticulum (er) as a core-glycosylated intermediate that is rapidly degraded. therefore, most f del-cftr fails to traffic to the plasma membrane, where wild-type (wt) cftr normally functions as a chloride (cl-) channel. retention, however, is not due to lack of function, since the mutant still retains some function if it reaches the membrane. instead, it results from misfolding which is recognized by the er quality control (erqc) in which many intervenients, including molecular chaperones, participate. identification of molecular partners involved in the disposal of f del-cftr to the proteasome is therefore crutial. it was recently shown that casein kinase ii (ck ), a pleiotropic constitutively active protein kinase involved in several processes, such as neoplasia, cell survival and viral infections, binds wt-cftr near the f residue, phosphorylating its first nucleotide binding domain (nbd ) at s [ ] . interestingly, deletion of f abrogates this interaction, which is the first described f del-dependent protein-protein interaction. our aim here was to identify whether ck interaction affects the early steps of cftr biogenesis, turnover and processing. to this end, novel bhk cells were produced which stably express wt-or f del-cftr in which the consensus residue s was substituted by either a neutral (alanine -s a) or an acidic residue (aspartate -s d). initial biochemical analyses of these cell lines revealed that: ) cftr proteins bearing d or a at position are processed; ) despite producing equivalent levels of cftr transcripts, s a expressing lines consistently show lower levels of cftr protein. metabolic labelling and pulse-chase experiments followed by cftr immunoprecipitation were also performed in these lines. after quantification of bands b (immature form) and c (mature form) of cftr, these preliminary results show that substitution of s (by a or d) does not affect the turnover or processing of either wt-or f del-cftr. the effect of ck inhibition on the turnover and processing of cftr was also studied, by incubating cells with µm of the ck inhibitor tetrabromobenzotriazole (tbb) for min. pulse-chase experiments under these conditions show that: ) the steady-state levels of both wt-and f del-cftr are reduced; ) the turnover of wt-cftr (but not of f del-cftr) is increased; and ) processing of wt-cftr is decreased. such data are consistent with a putative stabilizing role for ck upon wt-cftr [ ] . however, in our preliminary results this effect does not appear to be dependent on residue (nor on the putative charge added by the kinase on this residue), thus suggesting that this effect may be indirect. further studies are underway to identify the mechanism by which ck affects the turnover and processing of cftr. . treharne et al ( ) pissarra, l.s. ; xu, z. ; farinha, c.m. , ; sheppard, d.n. ; amaral, m.d. , . department of chemistry and biochemistry, faculty of sciences, university of lisboa, lisboa, portugal; . dep physiology, univ bristol, school med sciences, bristol, united kingdom; . ctr hum genet, nat inst health, lisboa, portugal g e and rk (the simultaneous mutation of four arginine-framed tripeptides (afts): r k, r k, r k and r k) are second site mutations that rescue the processing and function of f del-cftr [ , ] . these revertant mutations rescue f del-cftr from retention within the endoplasmic reticulum by distinct mechanisms: g e likely alters the conformation of the first nucleotide-binding domain (nbd ), whereas rk plausibly allows f del-cftr to escape er retention/retrieval mediated by afts [ ] . both g e and one of the afts (r k) lie close to the residue g , where a common cf-causing mutation g d occurs, generating a correctly localized clchannel with a severe gating defect. our aim here was to assess whether the revertants g e and rk influence the folding, processing and gating behaviour of the g d mutation, by employing biochemical and functional approaches. to test this idea, we introduced the g d mutation into cftr cdnas containing either g e or rk in the pnut vector and stably expressed these constructs in bhk cells. preliminary results from biochemical studies indicate that the mature fully-glycosylated form of cftr protein (band c) of both g e-g d-cftr and rk-g d-cftr were present at similar levels to those of g d-cftr. analysis of cftr-mediated iodide efflux from these cells revealed that g e is unable to rescue the functional defect of g d. however, rk generated an efflux of iodide larger than that elicited by cells expressing g d, albeit smaller than that of cells expressing wild-type (wt) cftr. consistent with these data neither rk nor g e rescued the defective channel gating of g d-cftr. however, both revertants caused a small increase in g d-cftr activity by attenuating the prolonged interburst interval of g d. we conclude that f del and g d disrupt cftr channel gating by distinct mechanisms. altogether, our data suggest that at least when in cis with the g d mutation, the afts (together or individually) might have a direct effect on cftr channel gating. this raises the possibility that rk, in addition to its well-described effect on trafficking, may act on cftr structure and/or folding, as previously suggested for r k [ ] . work supported by grant pocti/sau/mmo/ / and pluriannual funding of cigmh (fct, portugal) and the uk cf trust. l. pissarra was a recipient of bd/ / doctoral fellowship (fct, portugal) . [ ] carvalho ac, gansheroff lj, teem jl ( ) j biol chem , - . [ ] chang xb, cui l, hou yx, jensen tj, aleksandrov aa, mengos a, riordan jr ( ) mol cell , [ ] [ ] [ ] [ ] [ ] [ ] [ ] roxo-rosa m, xu z, schmidt a, neto m, cai z, soares cm, sheppard dn, amaral md ( ) proc natl acad sci usa , - . [ ] teem jl, carson mr, welsh mj ( ) improves ∆f -cftr intracellular trafficking in cf epithelial cells, although the mechanism by which this occurs is not clear. to identify gene products with atlered abundance in response to pba, we performed a differential display rt-pcr screen on rna isolated from ib - cf epithelial cells treated with pba for - hours. we isolated a cdna encoding stip- , a putative human stat- (signal transducer and activator of transcription- ) interacting protein and confirmed that pba causes transiently increased stip- mrna and protein abundance after hours of exposure. stip- was originally described as a scaffold protein that is required for ligand-dependent activation of stat- . stip- is also identical to elongator protein (elp- ), a subunit of the multicomponent elongator complex that stimulates rna polymerase ii activity. interestingly, recent data suggests that elongator may also regulate polarized secretion (rahl, et al. ( ) , mol. cell, : - ). we therefore hypothesized that stip- /elp- would regulate ∆f -cftr intracellular trafficking. in ib - cells, stip- /elp- associates with ∆f -cftr in coimmunoprecipitation experiments after pba treatment. in ib - cells overexpressing stip- /elp- , immunofluorescence experiments suggested that ∆f -cftr trafficked to the plasma membrane even in the absence of pba treatment. stip- /elp co-localized with markers of the golgi ( kda golgi protein) and exocytic vesicles (snap- ) when overexpressed in these cells. in contrast, overexpression of a deletion mutant of stip- /elp- lacking the wd domain, or stat- binding region, blocks the improvement of ∆f -cftr trafficking in response to pba in immunofluorescence and surface biotinylation experiments. this stip- /elp- deletion mutant did not co-localize with golgi or exocytic vesicle markers. furthermore, immunofluorescence experiments also suggested that pba causes colocalization of stip- /elp- and elp- , another member of the elongator complex that is often defective in familial dysautonomia. these data are consistent with stip- /elp- regulating ∆f -cftr intracellular trafficking in response to pba. this regulation may occur in the context of pba stimulating assembly of elongator, which in turn may promote trafficking of exocytic vesicles carrying ∆f -cftr to the plasma membrane. supported by grants from niddk. fold [ , ] . however, functional studies demonstrate significant differences in the gating behaviour of hcftr and mcftr [ ] . a powerful approach to investigate cftr structure and function is to examine interspecies differences and identify regions of conservation and divergence. to understand the structural basis for the functional differences between hcftr and mcftr, we generated hmcftr chimeras containing mcftr domains on an hcftr backbone. for this purpose, we replaced all or part of nbd , nbd or the rdomain of hcftr with the equivalent regions of mcftr and investigated their biochemical and functional properties. the in vivo folding of these hmcftr chimeric proteins was indirectly evaluated from their maturation status, after their stable expression in novel bhk cell lines. like wt-hcftr, most chimeric proteins were processed within the cell. however, two chimeras failed to mature: clone b (mnbd , amino acid (aa) residues - ) and clone c (mnbd , aa - ). we compared the murine sequence of these two chimeras with that of hcftr to determine the respective physico-chemical distances (pcds) of their aa changes. changes with higher pcd values were selected and in vitro mutagenesis performed to introduce these aa alterations into hcftr cdna. for clone b the selected changes were: e q, e q (pcd = ); s t (pcd = ); k q (pcd = ), i t (pcd = ) and k e (pcd = ). for clone c the changes were: p t (pcd = ), k q (pcd = ), y n (pcd = ), s k (pcd = ), c y (pcd = ), d g (pcd = ) and e d (pcd = ). biochemical analyses of these mutants stably expressed in bhk cells revealed that for clone b, e q, s t, k q and i t were processed, whereas k e was not. for clone c, p t, k q, y n, d g and e d were processed. iodide efflux showed that s t, k q, i t, e d and d g were functional, but not k e. we consider that with additional functional analyses, our approach will identify critical residues responsible for conformational changes and hence, functional differences between hcftr and mcftr. work supported by the bbsrc and grant pocti/mgi/ / (fct, portugal) and pluriannual funding of cigmh (fct, portugal) . ac dapaula is a recipient of phd fellowship sfrh/ bd/ / (fct, portugal we recently reported that the upr decreases endogenous wild-type (wt) cftr expression. as a follow up of these studies, we investigated the role of the folding deficient, ∆f cftr on upr induction and identified a mechanism by which the upr decreases cftr expression. for these studies, we developed a cell line expressing recombinant ∆f cftr on the endogenous wt background (calu- ∆f) and established individual clones with different ratios of endogenous (wt) to recombinant (∆f cftr) expression. two clones which express : (calu- ∆fc ) and : (calu- ∆fc ) ratios of wt to ∆f cftr and cfpac- cells expressing endogenous ∆f cftr were tested as models. the upr was constitutively active in calu- ∆fc cells only. in calu- ∆fc induction of ∆f cftr expression resulted in upr activation, indicating that high expression of ∆f cftr is required for upr induction. furthermore, following pharmacological induction of the upr, endogenous cftr mrna decreased to undetectable amounts both in calu- ∆f and cfpac- cells. the decrease in cftr mrna levels was not the result of shortened mrna half-life. in contrast, using a human cftr promoter driven reporter (pcftr-pluc), we demonstrate suppression of the cftr promoter when the upr is activated. considering that correction of ∆f cftr is the main therapeutic approach for cf, it is important that ∆f cftr expressed at low levels, such as in vivo, does not activate the upr. however, our results also reveal that ∆f cftr correctors have to be tested for upr activation, since transcriptional inhibition in this setting may contribute to inefficient rescue in native cells or in vivo. genistein, a naturally occurring tryrosine kinase inhibitor, at low concentrations ( µm) stimulates wt -cftr-mediated chloride secretion in a variety of cell and tissue types by an adenosine ', '-cyclic monophosphate (camp)-independent pathway. genistein has been shown to potentiate the channel function of ∆f -cftr and g d-cftr channels by prolonging channel open time (hwanget al., am j physiol cell physiol : c -c , ; illeck et al. am j physiol cell physiol : c -c , ) patch clamp studies have provided indirect evidence that genistein may bind directly to cftr and potentiate channel open probability by modifying the function of the nucleotide binding domains (weinreich et al., pflügers arch : - , ) . the goal of the present study was to determine whether genistein activated purified and reconstituted cftr protein as an initial step toward defining its molecular mechanism of action. the effect of genistein on purified and reconstituted wt-cftr was first studied following its reconstitution into planar lipid bilayers. in the presence of µm atp, phosphorylated cftr exhibited a low open probability (po= . ). the addition of µm genistein, caused a significant increase to the open probability of cftr (po= . ). using an electrogenic flux assay on a population of purified and phosphorylated cftr molecules, we determined that µm genistein caused a potentiation ( % increase) of chloride channel activity in the presence of µm atp. these results suggest that this macroscopic assay of purified cftr function is sufficiently sensitive to detect the effect of this potentiator. currently, the consequences of genistein on the channel activity and atpase activity of purified reconstituted ∆f -cftr and g d-cftr are being assessed. preliminary results suggest that in the presence of low atp concentrations ( - µm), µm genistein inhibits the atpase activity of ∆f -cftr, suggesting that it interacts directly with the nucleotide binding domains to alter their function. previous studies indicated an acute coordination between the activities of cystic fibrosis transmembrane conductance regulator (cftr) chloride channel and the amiloride sensitive epithelial sodium channel (enac) so that both these channels are either activated or deactivated in a synchronous fashion in the human sweat duct (reddy et.al, ) . however, the mechanisms responsible for such cooperativity between these ion channels are unknown. previous studies indicated that: cytoplasmic ph controls cftr activity through effects on phosphorylation (reddy et.al. ) . at ph . , cftr chloride conductance was reduced to ± ms/cm , but incresed to ± ms/cm at ph . (n=number of ducts= ). enac channel activity is also a function of cytosolic ph in heterologous expression systems (chalfant et.al, ) . the objective of this study was to test the hypothesis that the cytosolic ph may mediate the cooperative effects that occur between cftr and enac. we used basolaterally a-toxin permeabilized apical membrane preparations of native human sweat duct which expresses enac and cftr robustly as an experimental system. we showed that while luminal ph had no effect, changes in cytosolic ph acutely affected enac activity. that is, acidic ph inhibited, while basic ph activated enac activity. alkalinizing cytosolic ph from . to . increased enac conductance (genac) by ± . ms/cm (n= ). ph regulation of enac activity appears to be independent of cftr and endogenous kinase activities because basic ph stimulated enac: a.) after deactivating cftr by removing camp and atp in normal ducts, b.) in the absence of cftr in cf ducts, and c.) after blocking endogenous kinase activity with the non-specific kinase inhibitor, staurosporine. na + /h + exchanger (nhe) may mediate changes in cytosolic ph as a function of intracellular na + activity. nhe mrna is expressed in the sweat duct and cytosolic ph responds to changes in na + gradients across the basolateral membrane. conceivably, when transport conditions are favorable and intracellular na + is low, alkaline ph would allow enac and cftr to cooperatively admit na + and clthrough the apical membrane. when conditions are unfavorable and intracellular na + is excessive, acid ph would limit na + and clentry to protect the cell from disruptive changes in cell volume. thus, changes in cytosolic ph may play a crucial role in coordinating the activities of enac and cftr during transepithelial salt transport. acknowledgements: kirk taylor and sucharitha madireddi for expert technical assistance. funded by nih-ro de , nih-ro hl and the nancy olmsted trust. the cystic fibrosis transmembrane conductance regulator (cftr) is an anion channel that is normally expressed in the plasma membrane but is mislocalized in cf. to monitor trafficking we inserted super ecliptic phluorin, a ph-sensitive variant of green fluorescent protein (gfp) (miesenbock g, de angelis da, rothman je ( ) nature , - ) into the fourth extracellular loop of cftr. this construct, which we call cftr-phluor, has weak fluorescence intracellularly that increases ~ -fold when it is inserted into the plasma membrane and the phlour becomes exposed to more alkaline extracellular ph . thus cftr-phluor should be less fluorescent than cftr with the normal gfp fused to the n or c termini when situated in intracellular vesicles. despite the insertion of an entire gfp in the middle of the channel protein, cftr-phluor trafficked to the membrane and patch clamp studies revealed little change in unitary conductance or open probability compared to wild-type cftr. cftr-phluor enabled visualization of vesicle insertion events at the plasma membrane by total internal reflection fluorescence (tirf) microscopy. faintly fluorescent vesicles containing cftr were observed under the plasma membrane and eventually fusing with it to generate a burst of fluorescence. phluorin inserted at the same position in ∆f -cftr, the predominant mutant in cf, was retained in the er where the phluor remained partially quenched. when the folding defect was corrected by culturing cells at °c or treating them with known correctors, ∆f -phluor trafficking was partially restored and total cell fluorescence increased. this enabled a quantitative analysis of ∆f -cftr correction based on the increase in fluorescence in individual cells by flow cytometry. cftr-phluor may be most useful for microscope-based high-content screening studies of cftr and ∆f -cftr trafficking. support: the breathe program, canadian cf foundation, canadian institutes of health research and cf foundation therapeutics (usa). tukaye, d.n.; guggino, w.b. physiology, johns hopkins university, baltimore, md, usa type iii secretion system (t ss) toxins of p. aeruginosa are important virulence factors in p. aeruginosa infections. one of the t ss toxins, exos, has been shown to facilitate uptake and invasion of p. aeruginosa at airway surface epithelium. in mouse models of pseudomonas pneumonia, infection with p. aeruginosa exos+ strains caused increased levels of fluid in lungs as determined at autopsy in contrast to infection with p. aeruginosa exosstrains. the exact molecular mechanism underlying these observations is not known. exos is a bifunctional protein with gtpase activating protein (gap) activity at the n terminal domain and adp ribosyl transferase (adprt) activity at the c terminal domain. we found that exos-gap activity increases total cellular levels of mature (c band) wild type (wt) cftr in cfbe o-cells as measured by western blot analysis. this increase was mediated by decreased delivery of wt cftr for lysosomal degradation. in contrast, exos-gap failed to increase total levels of ∆ cftr (b band). interestingly, exos-gap increased total levels of ∆ cftr, bands c and b, following rescue with -phenylbutyrate. this indicates that targets of exos-gap exist in cftr trafficking beyond the er degradation pathway. exos-gap also brought about a corresponding increase in surface levels of mature wt cftr as measured by surface biotinylation. in conclusion, we have shown that p. aeruginosa t ss exos-gap, upregulates total and surface levels of mature wt cftr by modulating cftr trafficking beyond er, most likely by decreasing lysosomal degradation. the decrease in lysosomal degradation could be occurring in part by inactivation of small molecular weight g-proteins involved in delivery of wt cftr to lysosomes. increases in total and hence surface levels of cftr could in part explain increased amounts of fluids seen in p. aeruginosa pneumonia mouse model treated with exos+ strains. the post-maturational trafficking and localization of cftr is regulated by a wide variety of proteins. among the most prominent are several pdz domain proteins that bind to the c-terminal residues of cftr. in particular, two of these proteins, nherf and cal, have been shown to mediate opposing effects on the apical membrane levels of the disease-associated ∆f -cftr mutant, by controlling the balance between endocytic recycling and lysosomal degradation. in particular, whereas overexpression of nherf rescues ∆f -cftr at the cell surface, overexpression of cal has the opposite effect on cftr. cal-mediated downregulation of cftr requires a functional pdz binding site. we have now shown that suppression of endogenous cal cooperates with temperature rescue to stabilize functional ∆f -cftr channels at the apical membrane in polarized bronchial epithelial cells. this work supports the hypothesis that the cal pdz binding site is a therapeutic target for treatment of cystic fibrosis. a more direct test of this hypothesis will require competitive inhibitors that can efficiently block the cal:cftr interaction. in addition, given the large-number of protein-protein interactions formed by both the cal pdz domain and the cftr c-terminus, these inhibitors should ideally exhibit bidirectional selectivity, neither disrupting the favorable nherf :cftr interaction, nor strongly displacing other membrane proteins that interact with cal. using a fluorescence polarization assay and peptide-array technology, we have now identified a high-affinity, cal-selective peptide inhibitor. compared to cftr, this sequence binds cal more tightly, but nherf more weakly. furthermore, we have shown that the cal:cftr interaction is the weakest among the known cal-binding proteins, and that cftr should thus be susceptible to selective displacement from the cal binding site. finally, we have designed cell-permeable peptides that allow us to test the hypotheses (a) that such compounds will specifically disrupt the cal:cftr interaction and (b) that such targeted stereochemical inhibition will stabilize the functional cell-surface expression of ∆f -cftr in airway epithelial cells. the pathways for the endoplasmic reticulum associated protein degradation (erad) of misfolded proteins in the mammalian cells are incompletely understood. we investigated the role of molecular chaperones in erad for the cystic fibrosis transmembrane conductance regulator (cftr) and its common folding mutant, ∆f cftr. we found that hsp and its cochaperone hdj- interacted significantly with ∆f cftr and wt-cftr in airway epithelial cells. hsp interacted with the immature form of cftr while hdj- associated with both immature form and ubiquitylated cftr. structure-function studies showed that hdj- recognized ubiquitylated cftr via its zn-binding domain. immunoprecipitation and gst-fusion protein pulldown experiments revealed that hdj- interacted with ubiquitin. in steady-state, over-expression of hdj- elicited increasing both immature and mature forms of wt-cftr, but it resulted in decreasing immature form of ∆f cftr. pulse-chase studies showed that co-expression of hdj- promoted ∆f cftr degradation, reducing its half-life from to min. in contrast, hdj- expression did not significantly affect wt-cftr biogenesis. these data are consistent with our finding that hdj- shows a selective physical interaction with ∆f cftr, which translates into a functional discrimination between the mutant and its wt counterpart. the hpd motif within the j-domain of hdj- , which is necessary for the co-chaperone to bind hsp and stimulate its atpase activity, was required for hdj- mediated ∆f cftr degradation. mutation of the hpd motif retarded the degradation of ∆f cftr and increased the steady-state levels of ∆f cftr -fold. a role for hsp in hdj- mediated ∆f cftr degradation was tested in which hsp knockdown increased ∆f cftr expression - fold. hjd- regulates ∆f cftr maturation and knockdown of endogenous hdj- promoted ∆f cftr and its camp-dependent anion conductance in airway epithelial cells. in contrast, we were unable to detect maturation of ∆f cftr in hsp knockdown experiments, indicating that the maturation of ∆f cftr mediated by reduced hdj- expression may be independent of hsp function. taken together, our data demonstrate that hdj- is a molecular sensor that can detect differences in the folding related to the ∆f cftr maturation. these data also highlight a novel pathway for hdj- linked, ubiquitin-dependent degradation of ∆f cftr. reduction of hdj- expression or its association with ∆f cftr promotes maturation of this mutant and therefore represents a potential therapeutic approach for cystic fibrosis. [supported by nih and cf foundation] how the loss of cftr function results in cholesterol accumulation within the cell is currently unknown. cftr activation is driven by the camp and we propose that cf cells respond to the loss of cftr activity by increasing the camp pathway in order to increase cftr expression. to test this hypothesis, epithelial cells were treated with the phosphodiesterase- (pde ) and pde inhibitors milrinone and rolipram, respectively. inhibition of pde function with rolipram in wildtype cells leads to peri-nuclear free cholesterol accumulation identical to what is observed in cf epithelial cells as viewed by filipin staining, strongly implicating a camp-dependent pathway in the regulation of cholesterol trafficking. the pde -selective inhibitor milrinone had no effect on cholesterol trafficking suggesting specificity for the pde pathway. our preliminary data support this hypothesis in that both cf-model hteo-pcepr cells and mouse nasal epithelium (mne) from cftr -/-mice exhibit reduced protein expression of the campspecific phosphodiesterases pde compared to wt controls. the proposed consequence of a chronic amplification in camp signaling is altered cholesterol transport regulation. to address this potential role of pde in cholesterol trafficking, free cholesterol was visualized using filipin staining in wild type cells that were treated with rolipram, a pde -specific inhibitor. conversely, treatment of cf-model pcepr cells with the pka inhibitor rp-camps significantly improves cholesterol processing, further pointing to the involvement of the camp pathway. we propose that the camp pathway influences cholesterol processing through the regulation of β-arrestin- (βarr ) according to the premise that chronic increase in the camp pathway would initiate an elevation in βarr expression. βarr is an important regulator of adrenergic receptor recycling and organelle trafficking. because βarr is pivotal in regulating endocytotic recycling pathways, it could also impact cholesterol processing. this predicted increase in βarr protein expression is observed in both cf-model pcepr cells and cftr -/-mne compared to respective controls. over expression of βarr in wt epithelial cells leads to cf-like peri-nuclear cholesterol accumulation further implicating a role for βarr in the development of this phenotype. altered cholesterol trafficking in cftr would lead one to expect different βarr localization throughout the cell. further understanding of the implications of altered camp signaling and its relationship to aberrant cholesterol accumu-rationale: we recently reported that vcp (valosin containing protein) physically interacts with gp /amfr (autocrine motility factor receptor) to couple retrograde translocation of ∆f -cftr to proteasomal degradation (vij et al. jbc ) . recent studies have revealed an alternative system to the proteasome for degradation of polyubiquitinated misfolded proteins termed the aggresome. histone deacetylase- (hdac ) is a unique cytoplasmic deacetylase capable of interacting with ubiquitin and mediating the accumulation of ∆f -cftr in aggresome bodies. hypothesis: vcp competes with hdac to dissociate ∆f -cftr perinuclear aggregates. methods: ib - (∆f /w x) cf bronchial epithelial cells were transiently transfected with ∆f -cftr and vcp-gfp constructs and treated with a hdac inhibitor (tubacin), proteasome inhibitor (ps- or mg- ), lysosome inhibitor (baflomycin a ) or nil-tubacin (control) for , or hrs. the effect of these inhibitors on ∆f -cftr and vcp:∆f -cftr interactions were quantified by immunoprecipitating these protein complexes followed by immunoblotting with vcp, hdac or clathrin antibody. ∆f -cftr levels were measured by metabolic labeling using tran- s-label ( µci/ml) for a min pulse and hrs chase. the subsequent effects of these inhibitors on vcp localization was detected by fluorescence microscopy of gfp moiety. results: the co-immunoprecipitation experiments showed that hdac inhibition by µm tubacin ( hrs) promotes vcp:∆f -cftr and prevents hdac :∆f -cftr interaction. the proteasome inhibition (mg µm) resulted in maximal hdac :∆f -cftr at hrs with minimal at hrs while vcp:∆f -cftr increased overtime ( to hrs). adding tubacin ( µm; hrs) reversed the proteasomal inhibitor effects. in pulse-chase experiments, mg- increased the accumulation of cftr bform and poly-ubiquitinated-∆f -cftr as compared to the untreated control. tubacin suppressed the levels of mg- induced poly-ubiquinated-∆f -cftr as well as ∆f -cftr b-form. the vcp localization by microscopy showed the accumulation of vcp-gfp in perinuclear aggregates in the presence of proteasome inhibitor (ps- µm). the µm tubacin blocked these ps- induced perinuclear aggregates. we observed that vcp is associated with endocytic protein-complexes containing clathrin in the presence of ps- indicating cytosolic re-localization of vcp. we further confirmed the cytosolic re-localization of vcp-gfp in the presence of lysosome inhibitor (bafilomycin a µm) by fluorescent microscopy. conclusion: we found that a small molecule inhibitor of aggresome function prevents hdac :∆f -cftr interaction, promotes vcp:∆f -cftr interaction, and suppresses the accumulation of poly-ubiquinated-∆f -cftr. wang, y. ; jiang, y. ; zhu, n. ; feng, x. ; yang, h. , ; ma, t. , . membrane channel research laboratory, northeast normal university, changchun, china; . biopharmaceutical center, liaoning normal university, dalian, china deletion of the codon encoding the phenylalanine residue at position (∆f ) in the cystic fibrosis transmembrane conductance regulator (cftr) is the most common mutation causing cystic fibrosis (cf). the ∆f mutation results in a cftr protein with impaired folding, trafficking and gating in human and rodents. the consequences of ∆f -cftr mutation in other species have not been studied. the purpose of the present study was to characterize the ∆f mutant of porcine cftr in cell culture model. cdna sequence encoding full-length porcine cftr (pcftr) was cloned from the lung by rt-pcr using primers designed according to porcine genomic sequences in two bac clones (genbank accession no. ac and ac ) containing all exons of the porcine cftr. pcftr encodes a -amino-acid protein that shows . % identity to human cftr. the phenylalanine residue at position is conserved in pcftr. functional analysis of pcftr stably expressed in fisher rat thyroid (frt) epithelial cells by short-circuit current assays indicated a campactivated cl-channel similar to human cftr except that the sensitivity of pcftr to the specific cftr inhibitor cftrinh- is -fold lower than that of human cftr. a ∆f mutation of porcine cftr was generated by site-directed mutagenesis. surprisingly, ∆f -pcftr expressed as a func-tional chloride channel activated by forskolin in stably transfected frt cells. western blot analysis of cos and bhk cells transiently transfected with cmyc-tagged ∆f -pcftr indicated a protein pattern identical to that of wildtype pcftr. immunofluorescence analysis of the transiently transfected cells demonstrated similar plasma membrane expression pattern of ∆f -pcftr and wildtype pcftr. these results clearly demonstrated unimpaired cellular processing of ∆f -pcftr. however, the sensitivity of ∆f -pcftr to forskolin activation (ic ~ . µm) was dramatically lower than that of wildtype pcftr (ic ~ . µm), indicating a defect of channel gating by pka phosphorylation. the present study provided the first evidence that the ∆f mutation in mammalian cftr does not result in impaired cellular processing that is closely associated with cf phenotype. we have optimized airway epithelial cell spheroid cultures for functional measurements of cftr chloride conductance in order to screen smallmolecule activators and inhibitors. spheroids, which are sealed spheroidal monolayers of airway surface epithelial cells with diameter - micron, were generated within to days after nasal brushing and suspension culture. yields of up to spheroids were obtained from nasal brushing of a non-cf or cf subject. approximately , spheroids could be prepared from one million freshly isolated human bronchial epithelial cells. we compared several electrophysiological and fluorescence methods to assay cftr function in spheroids. whole-cell current was measured in single spheroids following pipette immobilization and micropipette puncture of the solutionexposed apical cell membrane. large, forskolin-stimulated whole-cell chloride currents were measured in non-cf spheroids. as an independent approach, transepithelial potential difference was measured by micropipette insertion into the spheroid lumen. for fluorescence detection, cell cytoplasm was stained with the halide indicators mqae or lzq. fluorescently stained spheroids were immobilized on a coated coverglass or in a custom microfluidic chamber. halide flux across the apical surface was measured in response to chloride-nitrate or iodide-chloride solution exchange, in the absence and presence of camp activators. the fluorescence assay is suitable for medium-throughput screening of ∆f correctors and potentiators, with definitive verification of cftr chloride channel function by whole-cell current analysis. an airway spheroid cultures provide a powerful approach to prioritize the efficacy of ∆f correctors because of their rapid generation without prolonged culture, and their suitability for quantitative analysis of cftr function. supported by cff and nih. background and aims: cftr has been shown to be expressed and functional in ventricular cardiomyocytes. however, to date, no physiological role for cftr in the heart has been established. the aim of this study was to determine if cftr plays a role in the regulation of cardiomyocyte contraction rate. methods: cardiomyocyte contraction rate was assessed using the neonatal mouse beating assay. ventricular myocytes were isolated from neonatal mice and cultured for - days until they formed a functional in sitium. contraction rates were captured by video imaging and analyzed by metamorph in the presence and absence of beta-adrenergic receptor stimulation by isoproteronol ( µm). assessment of cftr activity was determined by pharmacological inhibition by glibenclamide ( µm), dpc ( µm), and cftr inh- ( and µm). the role of k atp channels was assessed by -hd ( µm). results: baseline contraction rate of vehicletreated (dmso or h o) cardiomyocytes was unchanged. addition of isoproteronol caused a significant increase in contraction rate, which was sustained for at least minutes. in contrast, application of glibenclamide to beating ventricular myocytes significantly, but transiently, inhibited cardiomyocyte contraction rate, with recovery occurring within minutes. glibenclamide did not affect the initial increase in contraction rate caused by isoproteronol stimulation, but inhibited sustained increases in isoproteronol-stimulated contraction rate. to determine if this was an effect of k atp channel inhibition we examined the effect of -hd on contraction rate. in contrast to glibenclamide, -hd had no inhibitory affect on basal or isoproteronol-stimulated contraction rate, indicating glibenclamide's inhibitory effect was due to its inhibition of cftr. to further verify this, we examined the effect of two other cftr inhibitors, dpc and cftr inh- on ventricular cardiomyocyte contraction rate. both dpc and cftr inh- elicited similar responses as glibenclamide, with a quick, but transient inhibition of baseline contraction rate and inhibition of sustained isoproteronol-stimulated increases in cardiomyocyte beating. summary and conclusions: using neonatal murine ventricular myocyte cultures inhibition of cftr leads to a significant, but transient inhibition of baseline ventricular cardiomyocyte contraction rate. additionally, while inhibition of cftr did not affect initial increases in beta-adrenergic receptor-stimulated contraction rate, cftr inhibition did prevent sustained increases in contraction rate. the data suggest that cftr plays a role in modulating ventricular cardiomyocyte contraction rate during resting and adrenergic-stimulated conditions. drevillon, l. , ; tanguy, g. , ; hinzpeter, a. , ; arous, n. , ; goossens, m. , ; fanen, p. , . génétique, inserm u. , créteil, france; . faculté de médecine, université paris , créteil, france cystic fibrosis is mainly caused by mutations that interfere with the biosynthetic folding of the cftr protein. the aim of this study was to find cellular proteins capable of interacting with cftr and modifying its processing or its trafficking. we have used a genetic screen in yeast to identify such proteins and identified commd that preferentially interacted with the third cytoplasmic loop of cftr. commd is a regulator of copper transporter atp b in hepatocyte and sodium epithelial channel enac, but its exact biochemical function and physiological relevance remain elusive. here, we report that first, commd interacted with wild-type and f del-cftr in epithelial cells and second, commd subcellular distribution was not only both nuclear and cytoplasmic, but also in cytoplasmic vesicular compartments. we wondered if commd was involved in the processing and/or the trafficking of cftr protein in epithelial cells. we demonstrated that commd was not involved in cftr processing but that cell surface expression of wild-type cftr at the plasma membrane was cut by half when commd expression was % reduced by rna interference. we drew the conclusion that commd is a major component of cftr trafficking and/or recycling at the plasma membrane, precise characterization of commd in these vesicular compartments is under process to decipher its function in cftr trafficking. commd has been labelled as the prototype of a newly described protein family that plays a role in inhibiting nf-kappab signalling. moreover, subcellular distribution of commd was different in cf and non cf cells, nuclear form of commd being increased in f del cells compared to wild-type cells. thus, this opens up a new area of investigation about commd nuclear function. finally, these data indicate that commd is involved in multiple cellular processes of outstanding interest in cf pathophysiology. understanding how its modulation modifies transepithelial transport and inflammation in cf versus non cf cells should give new therapeutic clues to reduce exaggerated inflammation and improve fluid secretion in cf patients. supported ; fanen, p. ; galietta, l.j. . lab. of molecular genetics, istituto giannina gaslini, genova, italy; . inserm u. , creteil, france class iii cystic fibrosis mutations cause reduced cltransport by impairing the process of cftr channel opening. typical class iii mutations include g d, g d, and f del, all of them affecting nucleotide binding domains. it has been reported that mutations residing in other regions of the cftr protein may also reduce channel activity. we investigated the extent of loss of function caused by such mutations, in particular by missense mutations localized in cftr intracellular loops, and the sensitivity to pharmacological stimulation with cftr potentiators. to determine cftr activity, plasmids carrying the coding sequence for wild type or mutant cftr were co-transfected in cos- cells together with the halide-sensitive yellow fluorescent protein yfp-h q/i l. the rate of cftr-dependent anion transport was measured after short-term stimulation with forskolin alone or in combination with potentiators (felodipine, phenylglycine pg- , or sulfonamide sf- ). processing of cftr protein was assessed by examining glycosylation status of cftr by immunoprecipitation experiments. when stimulated with forskolin alone, cftr mutants showed the following anion transport compared to the wild type protein: i t ( %), i v ( %), q k ( %), e k ( %), g r ( %), d h ( %). interestingly, all mutants having a reduced anion transport responded to stimulation with potentiators pg- , sf- , and felodipine ( µm) with an increase in anion transport that approached the level of normal cftr protein. the correction of defective cftr activity by potentiators was confirmed in frt cells expressing e k and d h mutants using short-circuit current measurements. our results indicate that some missense cf mutations, like e k and g r, really cause a severe reduction in cftr activity. others, like d h, cause only a partial loss of function which may explain their association with a milder form of the disease. conversely, some other amino acid changes (with the extreme example of i v that seems to cause a gain of function) do not impair cftr activity and therefore may represent polymorphisms and not cf-causing mutations. interestingly, all mutants having a mild or more severe anion transport defect were sensitive to potentiators. this finding indicates that cftr potentiators have a wide efficacy on many class iii mutants and therefore may represent a promising therapeutic strategy for a significant number of cf patients. supported by cfft and telethon-italy. cftr, a cl-channel membrane protein, is a member of the atp-binding cassette (abc) super-family. mutations in cftr result in a misfolded protein which fails to mature, leading to impaired flow of cl-ions across the membrane of the epithelial cells lining the airway, and to cf. the most common mutation leading to cf is df . currently available structural information relevant to cftr modeling includes low resolution structures of cftr ( Å) and p-gp ( Å) and high resolution structures of other abc transporters as well as nucleotide binding domains (nbd) . of particular relevance is the newly published structure of the bacterial multidrug abc transporter, sav , whose topology can serve as a starting point for cftr modeling. as a first step towards the development of cf therapeutics, epix has undertaken the modeling of the d structure of cftr (excluding the r domain) in its conducting (i.e., open-channel, nbd-dimer) state, using our proprietary membrane-protein modeling technology (predicttm) in combination with other modeling tools. to date, we have modeled the cytosolic part of the receptor, namely the nbds:icls construct and work is ongoing on modeling the transmembrane (msd :msd ) region. the now available partial model has been scanned for putative binding sites for df -cftr correctors/potentiators. several potential sites have been located, and epix has initiated its in silico screening technology in order to identify potential drug candidates. this work is supported by the cfft. the deletion of a phenylalanine residue at position (f del) in the first nucleotide-binding domain (nbd ) of cftr is the principal cause of cystic fibrosis (cf). the altered interaction of f del cftr with endoplasmic reticulum (er) quality control proteins, primarily chaperones, promotes its proteasomal degradation. however, it is believed that crucial cftr-interacting proteins (cips) remain unknown [ ] . moreover, there is little information currently available on the strength of cip-cftr interactions. our goals here are two-fold: ) to quantify cftr-cip interactions; and ) to isolate and characterise unidentified cips. to address our first goal, we used surface plasmon resonance (spr; biacore tm ) to quantify real-time binding of hsc (a well-known cip) to bacterially expressed wt-and f del-nbd . hsc /hsp was covalently immobilised onto the surface of carboxymethyl dextran (cm ) sensor chips ( µm) and the real-time binding of purified nbd and control proteins quantified. in control experiments, anti-hsc antibody b ( nm; against residues - ) bound specifically to immobilised hsc with high affinity (k d , . ± . nm; n = ) whereas bovine serum albumin ( µm) did not interact (n = ). given the difficulties associated with expression of hnbd carrying the f del mutation [ ] it was not used in our spr analyses. instead, we used purified murine (m) nbd to quantify the impact of f del on the strength of the hsc -nbd binding. in the presence of low concentrations of atp (≤ um), the affinity of nbd binding to immobilised hsc was strengthened when f was deleted (wt, k d app , . ± . µm; f del, k d app , . ± . µm; n = ; p < . ). interestingly, raising the concentration of mgatp weakened the binding of nbd ( . µm) to hsc (wt, k i , µm atp; n = ). furthermore, deletion of f dramatically increased the concentration of mgatp required to destabilise the nbd -hsc interaction (f del, k i , µm atp; n = ). in summary, we used spr to demonstrate that: (i) nbd of cftr binds specifically to hsc , and (ii) the f del mutation enhances the association of nbd with hsc . we are presently investigating the effect of co-chaperones and small molecules on the interaction of hsc/hsp with wt-and f del-nbd . to identify novel cips, purified nbd was immobilised onto metalaffinity resin and used to capture cips from epithelial cell lysates. by this approach, cips were captured from human respiratory cell (calu- ) lysates and analysed by d-electrophoresis. relevant protein spots so far identified by mass spectrometry include: ) raichu x (thr/ser kinase); ) profilin isoform b; ) annexin a ; ) ifapsoriasin (intermediate filament-associated ca + regulatory protein); ) mgc (member of the er reticulon family). current analyses are underway to determine their functional roles in cftr trafficking and function. [ ] amaral ( billet, a. ; melin, p. ; norez, c. ; bilan, f. ; vandebrouck, c. ; mettey, y. ; becq, f. . physiologie, cnrs umr , poitiers, france; . gmas umr , poitiers, france; . faculté medecine et pharmacie, poitiers, france in order to gain a better insight into the structure-activity relationship (sar) of cftr protein, we have functionally characterized a cystic fibrosis mutation: the cftr-g d, identified in patients with oligospermia (vankeerberghen et al., ) . we examined cftr chloride channel activity by patch-clamp analysis, in whole cell configuration, using cos- cells transiently transfected with gfp-cftr-wt or with the mutant gfp-cftr-g d. cftr channels were activated by µm forskolin (fsk), and, after stable activation µm cftrinh- was added. as gfp-cftr-wt, g d chloride channels elicit a time and voltage independent current in presence of µm fsk. gfp-cftr-g d current density at + mv ( . ± . pa/pf) is . fold less than gfp-cftr-wt current density at + mv ( . ± . pa/pf). g d mediated currents are blocked by µm of the specific cftr-inhibitor, cftrinh- (ma et al., ) . as expected, gfp-cftr-wt channels are stimulated by µm of the potent activator -butyl- -hydroxy- -chlorobenzo[c]quinolizinium chloride (mpb- , derand et al., ) with a current density at + mv of . ± . pa/pf. interestingly, no activation of g d channels is recorded in presence of mpb- : difference between currents recorded in basal condition or in presence of mpb- is not significant. using iodide efflux experiments, we showed that cftr-g d mutant does not respond to several benzo[c]quinolizinium derivatives. however, the mutant channel can be activated by forskolin, ibmx and genistein. thus, the mechanisms of activation by xanthine and isoflavone are not affected by the mutation. these results show that: ( ) cftr-g d channels are functional: activation by camp pathway, inhibition by cftrinh- . ( ) camp chloride currents elicited by cftr-g d protein are weaker than cftr-wt. ( ) cftr-g d channels are refractory to benzo[c]quinolizinium activation. the replacement of the glycine by a negative charged amino acid in position affects cftr chloride channel activity and prevents activation by benzo [c] quinolizinium. this amino acid g , localized in the interface of nbd and r domain between two β sheets (callebaut et al., ) seems to have a crucial role in pharmacological activation of cftr by mpbs. further experiments will be performed to evaluate the role of the charge and of the nature of the amino acid at position in cftr protein. supported by vaincre la mucoviscidose and cnrs. cysteine string protein (csp) is a j-domain containing protein that regulates the extent of wild type cftr maturation. over-expression of csp blocks the formation of mature, glycosylated (band c) cftr and increases the level of immature (band b) cftr, whereas knockdown of csp elicits a marked increase in cftr maturation (zhang et al., j. biol. chem. : , ) . thus, csp appears to block the exit of cftr from the er. when the er exit of cftr is blocked by a different mechanism, i.e. the expression of mutant sar -gtp (sar h g interferes with the formation of copii vesicles), the steady-state level of immature cftr is two-fold greater than that resulting from csp over-expression. this finding suggests that csp, in addition to blocking er exit of cftr, may facilitate the degradation of the immature protein. in agreement with this concept, csp expression increased cftr ubiquitylation. a mutation within the conserved hpd motif of the j-domain (h q) disrupts the ability of csp to interact with and stimulate the atpase activity of hsp . csp h q does not block cftr maturation (above ref); in addition, the level of immature cftr was ~ % higher when h q csp was over-expressed than the level observed with mutant sar . this finding suggests that the pro-degradative effect of csp on immature cftr requires hsp binding/activation. chip (carboxy terminus of hsp -interacting protein) is an e ubiquitin protein ligase that can target wt and ∆f cftr for proteasome-mediated degradation (meacham et al., nature cell biol. : , ) . we sought to determine weather chip was involved in the csp-mediated degradation of cftr. indeed, co-immunoprecipitation (co-ip) experiments showed that chip associated with csp. also, csp h q, which has reduced interaction with hsp , showed a similar level of chip interaction by co-ip, suggesting that their association did not depend on hsp as a linker between the two proteins. a csp mutant that is truncated after the cysteine string domain, which is its site of membrane attachment, blocked cftr maturation as did wt csp; however, this mutant did not decrease cftr band b levels like wt csp did. in addition, the csp mutant missing its cterminus did not associate with chip. these findings further implicate chip in the csp-mediated degradation of immature cftr, and they suggest that the c-terminus of csp is a chip binding domain. when a dominant-negative mutant of chip, which lacks the u-box needed for chip-mediated ubiquitylation (chip∆ub), was co-expressed with cftr, csp was no longer able to facilitate the degradation of cftr in the er. these data show that it is possible to separate the effect of csp on cftr maturation from its ability to mediate the degradation of immature cftr. thus, csp blocks the exit of cftr from the er, and this requires a j-domain mediated activation of hsp but not the csp c-terminus. the action of csp on cftr degradation requires both a functional j-domain and the csp c-terminus, and this may require a trimeric complex involving csp, hsp and chip. [ the cystic fibrosis transmembrane conductance regulator (cftr) represents the main clchannel in the apical membrane of epithelial cells for camp-dependent clsecretion. mutations of this channel causes cystic fibrosis disease ; thus discovery of pharmacological activators of cftr is crucial to design future medicament for protein therapy. recently, we reported on the synthesis and screening of a small library of -phenylpyrrolo [ , b] pyrazines (named rp derivatives) evaluated as activators of wild-type cftr, g d-cftr and f del-cftr clchannels (noël et al, ) . this preliminary structure-activity relationship study identified -hydroxyphenyl and -n-butyl as determinants required for activation of cftr (rp- and rp- ). here we studied structure-function relationship of more than compounds prepared by chemical synthesis, and the subsequent activation of cftr channels. within the -phenylpyrrolo [ , -b] pyrazines family, we observed by iodide efflux technique that rp- bearing a hydroxyphenyl substituant is more potent (ec = nm) than rp- having -hydroxyphenyl substituant (ec = nm). by whole-cell patch clamp recording analysis, we confirmed that nanomolar concentrations of rp- activate linear chloride current in cho cells stably transfected with human wild-type cftr. this current was inhibited by µm of cftr inh - . we also found significant stimulation of short circuit current (i sc ) by rp- (ec = nm) on colon of cftr +/+ but not of cftr -/mice mounted in ussing chamber. stimulation of i sc by rp derivatives was inhibited by glibenclamide. the structural analogue , was less potent (ec = nm). as for rp- compound, we found that the -nbutyl chain is crucial for rp- and rp- activity, and that the -hydroxyphenyl compounds without -n-butyl chain are unactive on cftr. in this study, we showed that the presence of an hydroxyl group at position , or of the pyrrolopyrazine cycle determined the highest activity on cftr. the most potent compound is the -n-butyl- -( -hydroxyphenyl) hpyrrolo [ , b] pyrazine . the potency of these agents indicates that compounds in this class may be of therapeutic benefit in cftr-related diseases, including cystic fibrosis. the most common mutation causing cystic fibrosis (cf) is the deletion of f in the cftr gene, which results in inefficient trafficking of the cftr protein from the endoplasmic reticulum to the plasma membrane. the surface expression of the cftr is usually quantified biochemically with western blot or functionally with electrophysiological assays. however, these methods are labor intensive. we explore using fluorescence-based techniques for monitoring cftr surface expression. wild-type cftr was tagged at the amino terminus with green fluorescent polypeptides (gfp-cftr). the gfp-cftr was either transiently or stably expressed in chinese hamster ovary (cho) cells. the cells were then treated with a red fluorophore that specifically labels plasma membranes. the fluorescent images were acquired with an olympus laser scanning confocal microscope. two image channels were taken simultaneously. the green channel was excited with a nm laser, and signals within - nm were collected. the red channel was excited with a nm laser, and signals within - nm were collected. for a single focal plane, each image was composed of * pixels. threshold values were given to both green and red channels. pixels with an intensity value above the threshold were marked as green/red pixels. the green pixels in the first channel represent the signal of gfp-cftr, and the red pixels in the second channel represent the location of the plasma membrane. thus, pixels that are both green in the first channel and red in the second channel are the cftr surface expression, and denoted as yellow pixels. the percentage of successfully trafficked cftr is thus quantitatively estimated by the ratio of yellow to green pixels in the single plane. alternatively, a stack of images from the same cell at different depth was collected and analyzed. the total amount and surface portion of cftr expression are then determined by summing the correspondent pixels. the percentage of cftr surface expression was then calculated and will be verified both biochemically and functionally. the approach is modified to study deltaf cftr trafficking. we anticipate that the method can be used to screen cftr correctors from a small or intermediate sized chemical database. supported by cfft and nih. the most common mutation of the cystic fibrosis transmembrane conductance regulator (cftr) gene (deletion of phe- (∆f ) in the first nucleotide binding domain (nbd )) causes retention of the ∆f protein in the endoplasmic reticulum (er). ∆f mutation prevents conformational maturation of cftr protein without altering profoundly the local structure of nbd , but possibly by disrupting the interaction between nbd and nbd . however, the individual role of nbd and nbd in biogenesis, folding, maturation and membrane stability of a full length cftr is still under debate. using splicing by overlap extension method (soe by pcr), we generated six recombinant cftr proteins with inverted or suppressed nbds. the native boundary domains of nbd and nbd were respected for all mutants (riordan j.r. et al. ) and their c-terminal tails preserved. the constructs were transiently or stably expressed in cos- and bhk cells respectively. immmunodetection and immunolocalization assays confirmed that deletion of nbd domain (n -cftr) did not alter the trafficking or the plasma membrane expression of cftr. both processes were abolished in the presence of ∆f mutation. however, using whole cell patch-clamp configuration, we did not detect any camp-stimulated clcurrent. pulse-chase experiments established that the turnover of n -cftr is - fold faster (t / ~ h) than its wild type counterpart (t / ~ h). when we replaced nbd with nbd ( n -cftr), we could detect a faint plasma membrane expression associated with a weak cl-channel activation. more surprisingly, the t / of n -cftr coreglycosylated form was around h. when nbd was deleted (n -cftr) or substituted with nbd domain ( n -cftr), the expression and functional pattern were similar to cftr ∆f . furthermore, we showed that the n -cftr folding was not attenuated as demonstrated by protease susceptibility and sdsresistant thermoaggregation tendency, supporting the notion that n -cftr membrane stability requires nbd -nbd interaction. altogether, our results suggest that nbd domain is essential to form a foldable cftr that satisfies er quality control (erqc) but correct inter-domain assembly is mandatory for its stability at plasma membrane. the results are also in agreement with previous suggestion that ∆f mutation alters the interactions between nbds and transmembrane domains of cftr. supported . inserm u , inserm, paris, france; . inserm u , inserm, paris, france; . inserm u , inserm, paris, france; . inserm u , inserm, paris, france; . inserm u , inserm, paris, france; . inserm u , inserm, paris, france; . inserm , inserm, paris, france; . inserm u , inserm, paris, france the physiological role of cftr in renal epithelium is not known. in the proximal tubule, cftr co-localizes with the sodium-phosphate cotransporter npt a, a protein involved in phosphate (pi) reabsorption. the aim of our work is to determine if there is a functional interaction between cftr and npt a. to this end, the arnm coding for cftr and the arn coding for npt a (bearing a myc tag in c-ter) were injected in xenopus laevis oocytes. electrophysiological or biochemical experiments were performed days after arns injection. currents induced by mm pi (ipi) were measured in voltage-clamped (- mv) oocytes that expressed npt a alone, or in oocytes co-expressing cftr + npt a. for immunoprecipitation experiments, - cftrab and myc ab were used. results ipi was significantly reduced in oocytes expressing cftr + npt a compared to that measured in oocytes expressing npt a alone. using a two-bath electrodes configuration during voltage-clamping did not change this observation. this result suggests that when expressed in oocytes, cftr is in functional interaction with npt a. kinetic analysis (ipi as a function of pi concentration) of npt a showed a reduction in vmax but not in phosphate km. this suggests that npt a membrane expression and/or activity is altered when cftr is co-expressed. co-immunoprecipitation experiments performed on protein lysate derived from oocytes co-expressing cftr and npt a suggested an interaction between the transporters. using a pka activating cocktail as a pkc activator induced cftr-mediated currents in cftr+npt a expressing oocytes. in these oocytes, ipi is decreased by pkc activation (as expected from npt a regulation) but is increased shortly after exposure to the pka activating cocktail. the increase of ipi observed under this condition is at variance to that expected from classical npt a regulation. in oocytes expressing npt a alone, both pka activation and pkc activation reduced ipi. these observations suggest that in oocytes, cftr expression modifies npt a regulation. our study suggests that, after expression in xenopus laevis oocytes, cftr interacts functionally with npt a, and modifies its regulation. an interaction is also suggested by the co-immunoprecipitation of cftr and npt a. these results cannot discriminate between a direct or an indirect (via a third protein) interaction. to determine if an intracellular chloride concentration change may participate to the functional interaction, we are now measuring in cftr+npt a expressing oocytes intracellular chloride activity (using chloride-sensitive microelectrodes) during the use of the pka activating cocktail, and measuring ipi when pka is stimulated in the presence of a specific cftr inhibitor. cystic fibrosis (cf), the most commonly inherited lethal pulmonary disorder in caucasians, is caused by mutations in the cystic fibrosis transmembrane conductance regulator gene (cftr). to date, more than mutations were identified in cftr and were associated with clinical disease. a phenylalanine deletion at position accounts for % of cf genotypes in caucasian populations, and determines cftr misfolding and degradation by proteasome. consequently, limited cftr abundance leads to multiorgan disease, affecting the lung, pancreas, gut, liver, sweat glands and the reproductive organs. the past several years have witnessed an explosion of information regarding the identification and elucidation of molecules and pathways that are regulated by cftr or that regulate cftr activity. genomics and proteomics technologies now offer the opportunity to examine global alterations in the mrna and protein expression patterns of cf cells and tissues to elucidate the pathways linking defective cftr to clinical disease. here we describe systems biology methods that integrate heterogeneous datasets, including protein-protein interaction networks, gene expression and mass-spectrometry profiles, and mutation and genetic variation information, in order to identify the regulatory circuits and active subnetworks that are responsible for the progression and the severity of cf disease. the study will focus on correlations that define the key events in the cftr folding and trafficking pathways that lead to pathogenesis and dysfunctions, and could provide insights and targets for intervention and therapy. the number of cftr cl-channels in the plasma membrane, and thus the transepithelial cl-secretion is controlled, in part, by the endocytic trafficking of cftr. rab gtpases regulate the endocytic trafficking by acting as molecular switches that cycle between the gdp-bound (i.e. inactive) and the gtp-bound (i.e. active) state, associate with target membranes, and recruit downstream effectors. rab facilitates cftr endocytosis and rab a facilitates the recycling of internalized cftr to the plasma membrane. furthermore, rab and rab facilitate cftr trafficking to the lysosome. the mechanism of sorting internalized cftr for either recycling to the plasma membrane or degradation in the lysosome is not well understood. recent evidence demonstrates that protein sorting occurs in rab specific sorting endosomes. presence of cftr in rab compartment has been previously demonstrated in human airway epithelial cells by electron microscopy. yet, the role of rab in cftr trafficking has not been determined in these cells. rab did not control cftr trafficking in fibroblasts (bhk cells). however, it negatively regulated the plasma membrane expression of cftr in the colorectal cells . the goal of this study was to elucidate the role of rab in the endocytic trafficking of cftr in polarized human airway epithelial cells (calu- cells and cfbe o-cells stably expressing wt-cftr or deltaf -cftr). endogenous rab coimmunoprecipitated with cftr in calu- cell and cfbe o-cells. to determine if rab plays a role in cftr trafficking, cfbe o-cells were transfected with either the flag-tagged, dominant negative (dn) rab mutant deficient in gtp binding (gdp-locked; flag-rab -n i) or with a non-specific cdna control (gfp). if rab sorts internalized cftr to the recycling pathway, the dn rab should inhibit recycling, and, thus, should decrease the plasma membrane expression of cftr. if, on the other hand, rab sorts internalized cftr for degradation the dn rab should inhibit cftr degradation, increase cftr recycling and thus, should increase the expression of cftr in the plasma membrane. the dn rab increased cftr expression in the plasma membrane as determined by cell surface biotinylation. these data are consistent with the role of rab in sorting internalized cftr for degradation. similar to overexpression of the dn rab , silencing endogenous rab with double stranded small interfering rna specific for the human rab sequence increased the plasma membrane expression of cftr and increased the cftr mediated cl-secretion across polarized cfbe o-cells in ussing chamber experiments. the plasma membrane half-life of deltaf -cftr is decreased compared to wt-cftr. however, after silencing rab , the plasma membrane half-life of deltaf -cftr was similar to that of wt-cftr, as determined by cftr disappearance from the plasma membrane following incubation with cycloheximide. our data are consistent with the role of rab in sorting the internalized cftr for degradation and are consistent with the changing paradigm for the role of rab in protein trafficking (supported by the nih grant p rr and the cff swiate qo). the conformational changes underlying activation of phosphorylated cftr by atp remain unclear. in the present study we assessed the utility of labeling endogenous cysteine(s) in cftr using an environmentally-sensitive fluorescent probe alexa- in monitoring such changes in structure. these studies were performed using purified and functionally reconstituted wild-type cftr. we labeled pka-phosphorylated cftr with µm alexa- maleimide prior to reconstitution in phospholipids liposomes. changes in fluorescence intensity were monitored in a suspension of proteoliposomes following addition of and mm mg-atp or mm mg-amp-pnp in order to evaluate nucleotide dependent changes in conformation. in order to identify the labeled cysteines, the protein was subjected trypsin-mediated proteolysis and analysis by mass spectrometry. we have shown that cftr is labeled using alexa- maleimide. a significant increase in fluorescence emission occurred in alexa- labeled cftr of ± (au) relative to empty liposomes one minute after the addition of activating nucleotide, mm mg-atp (n = ). the non-hydrolyzable analogue, mm mg-amp-pnp, failed to cause an increase in fluorescence of alexa- -labeled cftr. however, the higher concentration of mm mg-amp-pnp evoked an increase in fluorescence similar in magnitude to that of mm mg-atp suggesting that the fluorescence response reflects structural changes relating to atp binding to cftr. analysis by mass spectrometry identified the labeled residue as c . this cysteine resides at the junction between nbd and the "r" domain in a region which has been suggested to be highly dynamic on the basis of multiple crystal structures. our studies suggest that this flexible region around c of cftr undergoes conformational change upon mg-atp binding such that it moves the alexa fluorophore into a relatively more polar environment. this finding presents a first case where conformational changes in the cftr nbds induced by atp binding could lead to changes in the conformation of the r domain. further, these studies suggest that fluorescence methods can be used to probe dynamic conformational changes in purified reconstituted cftr. acknowledgements cystic fibrosis is caused by mutations in the apical chloride channel cftr with % of patients carrying at least one allele of the ∆f mutation. this mutant form of cftr is characterized by a trafficking defect in which it fails to exit the endoplasmic reticulum (er). we have previously reported that ∆f cftr is found in complex with hsp and its co-chaperones in cell extracts suggesting that either this mutant form of the protein accumulates in on-pathway folding intermediates which result in its accumulation in the er or that these intermediates include inhibitory factors leading to retention of ∆f cftr in this compartment. we have examined the role of one of these hsp co-chaperones, fk binding protein (fkbp ), in the life cycle of cftr. fkbp is a unique member of the immunophilin family of peptidyl-prolyl isomerases (ppiases) which mediate the cis/trans interconversion of peptidyl-prolyl bonds, which is thought to represent a rate limiting step in protein folding. in order to establish a functional role for fkbp in cftr stability and trafficking, we modulated the expression level of this protein in human bronchial epithelial cells endogenously expressing either wild type or ∆f cftr. a knockdown of fkbp by sirna resulted in a destabilization of both wild type and ∆f cftr as well as a concomitant loss in channel activity of wild type and temperature corrected ∆f cftr. conversely, over expression of fkbp resulted in a stabilization of the er glycoform of ∆f cftr in both mammalian and yeast systems. these data suggest that fkbp is an essential component of the hsp -mediated folding machinery for both wild type and ∆f cftr, which supports the hypothesis that ∆f cftr accumulates in the er in an on-pathway folding intermediate. dmh cftr folding involves a series of sequential, although potentially overlapping steps that involve i) formation, orientation and integration of (tms), ii) helical packing, iii) folding of cytosolic and extracytosolic domains, and iv) establishing proper domain-domain contacts. this process begins cotranslationally and is facilitated by the sec er translocation machinery and a diverse group of lumenal and cytosolic chaperones. the most common inherited cftr mutation, deltaf , disrupts one or more early steps along this folding pathway. a major obstacle in understanding how deltaf causes cf is that the native folding environment, comprised of lipids, cytosolic and er proteins, is not amenable to traditional biochemical and biophysical approaches. to overcome this problem we are developing a flu-orescence based analytical approach that enables one to directly access nascent ribosome-attached folding intermediates at virtually any stage of synthesis. the strategy requires incorporation of fluorescent probes into the substrate protein, which provide a highly selective means to monitor structural features at sub-nanomolar concentrations in highly complex biological mixtures. polypeptides are synthesized in vitro from truncated mrna templates to generate uniform cohorts of ribosome-bound nascent chains. progressive stages of folding can then be evaluated using mrna templates of different length. as proof-of-principle we have shown that fully synthesized gfp variants can be readily trapped in an unfolded state and complete their folding only after the entire nascent peptide has exited the large ribosomal subunit. this approach enabled us to measure real-time folding kinetics of extended peptide domains following synchronous ribosome release and compare fluorophore maturation of four fluorescent protein variants with different spectral properties cfp, gfp, venus and mcherry. to extend this approach to cftr and other general protein substrates, we are employing fluorescence resonance energy transfer (fret) using "donor" and "acceptor" probes incorporated simultaneously into the substrate protein. an in frame n-terminal fusion to cfp provides the "donor" fluorophore, while the "acceptor" fluorophore is incorporated at an engineered stop codon using a synthetic modified aminoacyl-suppressor trna. we are currently developing a 'molecular ruler' to correlate the efficiency of energy transfer with changes in the relative distance between probes in ribosome-bound and ribosome-released cftr folding intermediates. fret-based experiments are underway to evaluate folding of both n-terminal ( - aa) and nbd ( - aa) domains. these studies will provide new insight into the mechanism of cotranslational cftr domain folding and provide a potential means to identify agents that correct the deltaf folding defect. relationships between hypoxia, ado release, and prostenoid production in ariway cells are not defined. in the present studies, we examined mechanisms governing hypoxia-induced production of prostenoids in cfbe o-and calu- monolayers. using elisa-based detection of prostenoid and leukotriene production, both calu- and cfbe omonolayers treated with ado ( µm) or arachidonic acid (aa, µm, the parent molecule of prostenoids and leukotrienes) for hrs led to increases in pge levels ( - ± - pg/mg of total proteins vs controls ± pg/mg in calu- cells, - ± - pg/mg vs controls ± pg/mg in cfbe o-cells, p< . in both airway cell types compared with control). in contrast, ltb and ltc release were not stimulated by both maneuvers. we next used sirna techniques to knockdown a b adenosine receptor expression, and demonstrated durable knockdown of a b ar expression to~ % of scrambled sirna-treated controls following lipid-based transfection with sirna directed against the a b ar in cfbe o-cells. following sirna knockdown of a b ar expression, cells were exposed to hypoxic conditions (mucosal volume = ml in -well plate), normoxic conditions (mucosal volume = . ml, fio = . ), and ado ( µm). hypoxic stress led to high levels of mucosal pge production ( ± . pg/mg vs controls ± . pg/mg, p< . ) that was sensitive to a b ar knockdown ( ± . pg/mg, p< . ). exogenous ado also stimulated pge production, but this effect was small relative to hypoxia. we next examined basolateral effects of ado and aa on transmucosal clsecretion in cfbe o-and calu- cells grown as monolayers on permeable supports and conducted ussing chamber analysis under voltage clamp conditions. addition of ado and aa to the basolateral membrane ( µm) activated transmucosal clsecretion [ . ± . µa/cm (ado) and . ± . µa/cm (aa) for calu- cells; and . ± . µa/cm (ado) and . ± . µa/cm (aa) for cfbe o-cells expressing wtcftr] that was sensitive to basolateral blockade with barium cl -( mm). isc by either agonist was stimulat-ed in the presence and absence of apical glybenclamide ( µm), adenosine deaminase (ada - u/ml) and hexokinase (hexo - u/ml), confirming that the effects were specific for the basolateral membrane and could be accomplished with or without cftr activity. these results demonstrate that hypoxia is sufficient to stimulate pge production through a b ars, and that both ado and aa activate basolateral k + channels to promote transepithelial clsecretion. the findings further support a model in which ado (and prostenoids) regulate cltransport through effects on both the apical and the basolateral membrane, and elucidate molecules that couple the two membranes as part of the normal clsecretory response. hypoxia would be predicted to increase local prostenoid production through ado release and stimulation of a b ars, and further highlight a unifying model by which local hypoxia promotes inflammation in airway epithelia. supported by the nih and cff. low levels of tissue oxygen -observed in lung diseases such as cystic fibrosis -initiate a signaling cascade resulting in altered transcription of genes possessing a hypoxia response element (hre). we recently used gene chip mrna array, quantitative rt-pcr, protein analysis, and functional (short circuit current) assays to show that cftr expression is strongly repressed by hypoxia in vitro and in vivo. among several thousand human mrnas suppressed by low ambient oxygen, cftr was among the most strongly inhibited (by - fold) in a cell model system. however, cftr and the vast majority of other repressed genes lack a traditional hre. because hypoxia inducible factor (hif) acts primarily as a transcriptional activator, the mechanisms underlying hypoxia mediated suppression of mammalian genes are not well understood. we therefore tested the hypothesis that micrornas play a central role during transcriptional regulation of genes such as cftr. micrornas are evolutionarily conserved, short noncoding rna sequences believed important for repression of gene expression. some reports have estimated that at least % of protein-coding genes are regulated by micrornas. thousands of micrornas have been predicted in the human genome by available algorithms (e.g. palgrade, mirscan, and promir) and several hundred experimentally validated. the cftr gene contains predicted target sites for micrornas in the 'utr, including hsa-mir- (nucleotides - of the cftr 'utr), hsa-mir- (nt - ) and hsa-mir- (nt - ). however, no study to date has examined the effect of micrornas on cftr message levels, or the influence of micrornas during the cftr response to environmental perturbations such as hypoxia. we therefore performed a global, genome-wide analyses of both mrnas and micrornas expressed during hypoxia (using air/liquid or liquid/liquid interface culture systems) in ht (colonic epithelial) cells that robustly express cftr. we found that approximately % of micrornas were significantly altered by hypoxia, as judged by mirna profiling (ambion mirvana™ bioarray v , asuragen microrna expression profiling service). comparison of these hypoxia-related micrornas with expression profiles of their predicted targets indicated a much lower level of correlation than has been previously hypothesized by others. we show that none of the micrornas predicted to regulate cftr are altered in ht (in response to hypoxia). these findings are therefore consistent with the notion that highly expressed genes in a particular tissue type are not co-expressed with their predicted regulatory micrornas. our data also indicate that micrornas do not mediate the hypoxic repression of cftr via the ' utr. supported by nih and cff. . physiology, mcgill university, montreal, qc, canada; . biochemistry, mcgill university, montreal, qc, canada the ∆f mutation impairs cftr maturation and trafficking to the plasma membrane and results in a partially functional chloride channel that is retained in the endoplasmic reticulum and degraded. using our highthroughput trafficking assay, we previously showed that the quinoline km , a structural analogue of sildenafil, corrects the ∆f -cftr processing defect and leads to a significant increase in cftr surface expression after h treatment with µm and after h treatment with nm according to detection of mature cftr in western-blots. additional studies have now been carried out with other preparations to assess the functionality of ∆f -cftr following rescue by km treatment. different time and concentration exposures were tested on the cftr function using iodide efflux assays with both bhk and human bronchial epithelial cells over-expressing ∆f -cftr. exposure to µm km for h partially restored iodide efflux responses to µm forskolin + µm genistein in both cell line. after h incubation, rescue of the mutant protein in bhk cells still required µm, whereas nm was sufficient in the human cell line cfbe. iodide efflux assays were performed at different times following km wash out to functionally assess the stability of the rescued mutant protein. camp-stimulated iodide efflux was still detectable h after removing km , but responsiveness was lost after h in both cell types. electrophysiological studies were performed to examine the channel activity of ∆f -cftr rescued by km treatment. incubation with µm km for h increased camp-responsive ∆f -cftr current density in hek whole-cell patches ~ -fold. the conductance was time-and voltage-independent, anion selective, and strongly inhibited by glibenclamide. incubating polarized cfbe monolayers with µm km increased the forskolin + genistein-stimulated short-circuit current after h, and this response was inhibited by cftrinh ( µm). permeabilization of the basolateral membrane with nystatin confirmed the apical location of the forskolin + genistein-stimulation. ussing chamber experiments also revealed trafficking correction in ileum that had been excised from cftrtm eu mice homozygous for ∆f . when the ileum was pre-incubated with µm km for - h, the forskolin + genistein-stimulation of short-circuit current was significantly increased when compared to untreated intestine. these results confirm partial correction of ∆f -cftr by km in unpolarized cells and in human polarized epithelial cell monolayers and ex-vivo intestine isolated from ∆f -cftr mice. this work was supported by the breathe program funded by the ccff and cihr, cihr, cfft, and génome québec. primary sequence and overall structure of human cftr overlaps significantly with murine, rabbit and other homologues, a feature that has limited generation of conformation specific reagents to address cftr folding. because regions of ∆f cftr exposed extracellularly may exhibit aberrant topology (jbc : - ), probes that identify cftr from the extracellular surface could be useful to ) verify plasma membrane localization of ∆f cftr, ) discriminate wild-type from mutant protein, and ) provide a means of determining whether small molecule "correctors" of the ∆f processing lead to a native (wildtype) cftr configuration. our laboratories are investigating phage-display techniques suitable for detecting properly folded extracellular domains of cftr. two large phage libraries encoding or amino acid sequences (approximately phage per library) were panned against extracellular regions of cftr in hela cells. following incubation in blocking solution containing pbs and % bsa at °c, unbound fraction was incubated with recombinant helas expressing wild-type or temperature corrected ∆f cftr (protein expression verified biochemically and functionally). bound bacteriophage was dislodged after multiple washings by exposure to an acidic elution buffer and amplified to increase copy number (five rounds of antigen selection for each library, eur j biochem : - ). supernatants from microtiter wells expressing phage were analyzed for binding to parental (no cftr), wt or ∆f cftr expressing hela cells. using this method we obtained enrichment of phage at the surface of cells expressing wt or ∆f cftr. an example using a -amino acid sequence ( phage) with specificity for ∆f cftr (representative of four repeat experiments) is shown in a binding assay with hela cells/condition. reagents such as these can be further optimized by constructing second generation libraries using core recognition sequences (derived from the initial screen) and surrounding the core epitopes with random amino acid diversity. these experiments are intended to identify peptides with high binding affinity (nm binding constants) that recognize native cftr epitopes exposed extracellularly. because there is evidence that low temperature corrected ∆f cftr in the plasma membrane is misfolded, these probes may ultimately distinguish wild-type cftr from the surface targeted ∆f mutant and be useful as drug discovery reagents. supported by cff and nih. bridges, r.; nagubadi, s.; thakerar, a.; jia, y.; bradbury, n.a. physiology and biophysics, rosalind franklin university of medicine and science, north chicago, il, usa the goal of this project was to compare several reported correctors of ∆f -cftr biosynthesis for their efficacy on chloride channel activity under a standard set of experimental conditions. fisher rat thyroid (frt) cells were transfected with ∆f -cftr and a stable cell line selected with g . cells were grown on transwell filters and studied under short circuit current (i sc ) conditions in ussing chambers. i sc measurements were performed at °c. the basolateral membrane was permeabilized with α toxin and chloride currents were measured with a serosal to mucosal chloride gradient. atp ( mm) and gtp ( µm) were added to the serosal bath. cftr channel activity was stimulated with camp ( µm), ibmx ( µm) and genistein ( µm). cells were treated with correctors or vehicle (dmso) at and hours before the i sc measurements. a subgroup of cells were temperature corrected at °c for hours for comparison. because only modest currents were observed even in the temperature corrected cells ( - µa/cm ) cells were treated for hours with mm sodium butyrate and nm dexamethasone to enhance the observed currents. under these conditions temperature corrected cells displayed stimulated chloride currents of ~ µa/cm that were - -fold greater than cells maintained at °c. correctors c (n- ' ]bithiazolyl- '-yl]-benzamide) and c ( -( h-benzomidazol- -ylsulfanylmethyl)- -( -methoxy- -methyl-quinazolin- -ylamino)pyrimidin- -ol) caused a modest rescue of ∆f -cftr chloride currents compared to cells maintained at °c (c , . -fold; c , . -fold) but far below the effect of temperature correction. rescue by c was concentration dependent with a significant observable effect at nm and a maximum effect at µm. rescue by c was observed at only µm, lower concentrations had no effect and higher concentrations caused an inhibition of the stimulated chloride currents. compounds c -piperazin- -yl]-ethyl}- -piperidin- -yl-quinazoline) ( to µm), c ( -cyclohexyloxy- -{ - [ -( - methoxy-benzensulfonyl)-piperazin- yl]-ethyl}-quinazoline) ( to µm) and phenylbutyrate ( mm) did not significantly increase the stimulated chloride currents. all four compounds c -c caused significant decreases in the transepithelial resistances at the higher concentrations ( - µm) suggesting some degree of cytotoxicity. our results indicate only c and c demonstrate efficacy in frt cells and that these effects are modest compared to temperature correction. supported by the cystic fibrosis foundation. cftr contains two membrane-spanning domains (msd), two nucleotide-binding domains (nbd), and a regulatory domain that require proper assembly for chloride channel activity. the most prevalent disease causing mutation, cftr∆f , arises from deletion of phenylalanine in the nbd domain. the cftr∆f mutant is translated and inserted into the endoplasmic reticulum (er) membrane, but is unable to attain its native state and accumulates in a kinetically trapped conformation that is degraded by the ubiquitin proteasome system ( ) . the nature of the misfolded cftr∆f intermediate that is selected for premature degradation is not clear and is a topic of great interest ( ) . recent work in the cyr lab indicate that defects in cftr and cftr∆f folding are sensed by two different e ubiquitin ligase complexes that are located in the er membrane and cytosol ( ) . the membrane-associated derlin- /rma e complex appears to recognize early folding defects of cftr that may involve assembly of msd into a complex with the amino-terminal regions of cftr. in contrast, the cytosolic hsc /chip e complex appears to sense folding defects that occur after synthesis of the nbd domain. misfolded cftrs recognized at either checkpoint are retained and degraded via the proteasomal pathway. the presence of dual protein quality control (qc) checkpoints suggests a mechanism by which the folding status of cftrs membrane and cytosolic domains are sequentially monitored prior to its escape from the er. currently little is known about how these erqc complexes sense misfolded substrates. small-molecule chemical correctors have recently been identified which rescue the folding defect of cftr∆f in model cell culture systems. in the presence of these chemical correctors a subset of cftr∆f is able to localize to the cell surface and retain channel function. however, how the chemical correctors rescue cftr∆f folding is not clear. we have examined the extent to which the molecules correct folding defects that are recognized by the derlin- /rma or hsc /chip qc checkpoints. in addition, because it is unknown at what point of cftr biogenesis that the chemical correctors act to facilitate folding, we have determined how the chemical correctors affect the folding of different biogenic intermediates. our results indicate that the correctors act in a post-translational manner to influence assembly of cftr sub-domains. interactions with members of the qc pathway have also been used to monitor the folding and assembly process of cftr in the presence of chemical correctors. ultimately the results from these studies will enhance our knowledge of how chemical correctors permit passage of the cftr mutants past different erqc checkpoints. supported by -cystic fibrosis foundation combination therapy has proven successful in treating a wide variety of human diseases, including cancer, infectious disease, and diabetes. historically, combination treatments have been discovered through trial and error using drugs with proven disease modifying effects. we have developed a combination high-throughput screening (chts™) technology to systematically and efficiently find synergistic combinations that target multiple pathways underlying disease biology, and that may be developed into new therapeutics. we are applying chts technology to discover and develop combination therapeutics for the treatment of cystic fibrosis. we have optimized two high throughput screening assays for identifying correctors and potentiators of the cftr (cystic fibrosis transmembrane protein regulator) ∆f mutation. one assay uses the flipr membrane potential dye (fmp) and the other uses genetically modified yellow fluorescent protein yfp h q/i l to measure changes in halide flux through ∆f cftr. both assays were optimized and validated on the flipr tetra using the panel of cf correctors, potentiators and inhibitors provided by the cf foundation. using our automated chts, we systematically evaluate pairwise combinations of a library of ~ , approved drugs and other bioactive molecules to identify novel disease-relevant biological interactions. candidate single agents and combinations are tested in secondary assays and prioritized for testing in animal efficacy models. combination therapies can act on multiple pathways in a coordinated way. chts may prove to be a useful tool for identifying combination therapeutic candidates with novel multi-target mechanisms and a way to enhance co-therapy regimens for the treatment of cystic fibrosis. supported the highly variable clinical phenotypes of cf airway disease suggest that a number of genetic factors, other than cftr, play a role in its pathophysiology. some of these modifier genes are expected to play a role in the endoplasmic reticulum quality control (erqc), since the major defect caused by f del is misfolding, retention and degradation of the mutant protein by this cellular surveillance system. the nematode caenorhabditis elegans is an excellent multicellular genetic model, which has been successfully used in studies of human diseases. its ~ , -genes genome has been fully sequenced, it has short life-span ( - weeks) and life cycle (~ . days) and it is easily cultured and amenable for gene disruption, both by knockout or rnai. our goal is to generate a c. elegans model for the cftr folding defect, so as to take advantage of this model for the identification of genes involved in cftr folding and/or degradation. to this end, and because no cftr orthologue has been described in c. elegans, we constructed two previously described human p-gp-/cftr chimeras (p-gp/wt-cftr and p-gp/f del-cftr) [ , ] to be used as an in vivo folding substrate in this organism. the two chimeric cdnas and intact human p-gp cdna were cloned into the c. elegans ubiquitous expression vector ps and injected into mutant nematode strains for multidrug resistance genes [ ] . the effect on the nematodes phenotype was evaluated by an assay of heavy metal sensitivity ( . mm arsenite), as described [ ] . our quantitative results show that the p-gp/wt-cftr chimera increases the resistance to arsenite when injected into the pgp- /pgp- c. elegans double mutant, whereas p-gp/f del-cftr causes no effect. these preliminary results indicate it is possible to generate two distinct nematode phenotypes caused by each of the transgenic chimeras (p-gp/wt-cftr and pgp/f del-cftr). they also suggest that the same folding defect impairing f del-cftr function in cf may be responsible for the loss of heavy metal resistance function by the p-gp/f del-cftr chimera. these chimeras are currently under test in pgp- /pgp- double and in pgp- /pgp- /mrp- triple c. elegans mutants. analysis of these strains by rt-pcr showed that the mrnas from the two p-gp/cftr chimeras have low expression levels. this led us to produce more robust constructs, including: ) to test chimera expression under the c. elegans endogenous pgp- or intestinal-specific (potent) promoters; and ) to develop p-gp chimeras with a full cftr-nbd . these c. elegans models will be used in genome-wide rnai screens to identify genes involved in the erqc of the p-gp/cftr chimeras. work channel gating of cftr is regulated by atp binding and hydrolysis at the nbds and by pka phosphorylation at multiple sites, primarily within the intrinsically disordered regulatory (r) region. phosphorylation at multiple sites is required for full activation of channel function, but no one specific phosphorylation site is required. the phospho-regulation of r region interactions with nbd likely contributes to the regulation of cftr channel function by modulating r region interactions with nbd at the nbd /nbd dimerization interface, consistent with nbd crystal structures that include the regulatory extension (re) comprising the first residues of the r region ( ). this regulation likely occurs via a dynamic complex where multiple segments of nonphosphorylated r region with αhelical propensity transiently engage nbd and are released. pka phosphorylation disrupts r region α-helical propensity and decreases binding of each interacting segment ( ) . regulation of the ∆f cftr by pka is altered, with the rate of channel activation by pka -fold slower than for wild-type ( ). we hypothesize that the dynamic interactions between the r region and nbd are affected by the ∆f mutation. to characterize the differences in r region binding between wild-type and ∆f nbd at residue-level resolution, we use nuclear magnetic resonance (nmr) techniques. experiments are performed using either r region or nbd samples labeled with nmr-active nuclei and monitoring their structural changes upon the addition of unlabeled binding partner, allowing us to confirm r region binding to nbd at the nbd /nbd dimerization interface and to identify other potential interaction surfaces on both the r region and nbd . together, these experiments will allow us to characterize the dynamic associations between the r region and nbd in order to better understand the molecular basis for the pathogenesis associated with ∆f cftr. funded by the canadian institutes for health research, the canadian cystic fibrosis foundation, and the us cystic fibrosis foundation . lewis, h.a., et al, embo j. , - ( ) . . baker, j.m.r., hudson, r.p., kanelis, v., choy, w-y, thibodeau, p.h., thomas, p.j., and forman-kay, j.d. submitted. the ∆f -cftr mutation, the most common gene mutation in cystic fibrosis (cf), results in diminished plasma membrane expression of cftr, leading to loss of functional cftr and altered mucociliary clearance. this impairment promotes chronic infection of cf patients by the opportunistic pathogen, pseudomonas aeruginosa. we previously reported that a secreted protein from p. aeruginosa (cftr inhibitory factor (cif)) reduces the wt-cftr and ∆f -cftr-mediated chloride secretion and the plasma membrane expression of cftr by decreasing endocytic recycling of cftr. the aim of the current study was to investigate the mechanism by which cif is secreted by p. aeruginosa and delivered to human airway epithelial cells (cfbe o-). in this study, we show that cif is packaged in outer membrane vesicles (omv) for secretion from p. aeruginosa. interestingly, cif secretion via omv was increased when the bacteria were exposed to airway epithelial cells. changes in cif protein level or bacterial growth did not account for the increase in cif secretion. purification and application of p. aeruginosa omv to airway epithelial cells caused a decrease in plasma membrane expression of cftr. the decreased plasma membrane cftr expression was followed by increased ubiquitination and lysosomal degradation of cftr. cif delivery to the airway epithelial cells via omv showed a more robust decrease in plasma membrane cftr expression, in comparison with cif delivery as purified protein. to investigate the mechanisms whereby cif enters epithelial cells, optiprep gradient fractionation experiments were conducted in polarized airway epithelial cells. in airway epithelial cells treated with p. aeruginosa omv, cif was associated with airway cell lipid rafts. pharmacological inhibition of lipid raft formation with filipin iii blocked the decrease in cftr plasma membrane expression upon treatment with omv. these studies demonstrate that cif is released by p. aeruginosa via omv and enters the airway epithelial cells through lipid rafts. upon entry into the epithelial cell, cif increases the ubiquitination and degradation of cftr and redirects cftr trafficking from the recycling pathway to the lysosomal-degradative pathway. these data suggest that chronic infection of p. aeruginosa in the cf lung may reduce the efficacy of therapeutics developed to increase plasma membrane expression of the mutant ∆f -cftr (supported by the nih ro hl - and t -dk - ). . physiology, dartmouth medical school, hanover, nh, usa; . microbiology and immunology, dartmouth medical school, hanover, nh, usa we have identified and cloned a secreted protein from pseudomonas aeruginosa (p. aeruginosa pa and clinical isolates), designated cftr inhibitory factor (cif). cif reduces the apical membrane expression of cftr and inhibits the cftr-mediated chloride ion secretion by human airway epithelial cells expressing wt-cftr and df -cftr. cif does not have general effects on protein trafficking, as the localization and expression of gp , na + -k + -atpase, and the transferrin receptor were not affected. cftr is a member of the atp-binding cassette (abc) transporter superfamily. abc proteins are atp-dependent transporters involved in exporting a wide variety of cytotoxic agents across the plasma membrane. p-glycoprotein (pgp; abcb , mdr gene product), also an abc transporter, is one of the major drug-efflux pumps expressed in normal tissues, as well as in many human cancers. over-expression of pgp results in reduced intracellular drug concentration and reduced cytotoxicity, conferring multi-drug resistance (mdr) to cancer cells. the aim of the current study was to examine whether cif also affects pgp expression in the plasma membrane, which could be exploited as a potential therapeutic strategy for restoring the sensitivity to pgp-transported drugs in cancer chemotherapy. cif significantly reduced the apical expression of pgp in mdck cells stably transfected with gfp-tagged pgp (mdck-gfp-mdr cells) and in caco- and calu- cells, which express endogenous pgp in the apical plasma membrane. the drug sensitivity of mdck-gfp-mdr cells to doxorubicin, a pgp substrate, was evaluated in the absence and presence of cif by measuring the ec . cif reduced the cytotoxicity of doxorubicin by -fold. neither cif nor vehicle alone had a cytotoxic effect. the drug sensitivity of the parental cell line, mdck-c , that expresses -fold less pgp than the transfected mdck cells, to doxorubicin was also increased by -fold. by contrast, cif had no effect on the ec values of cisplatin (a substrate of mrp ) and etoposide (a substrate of mrp ), suggesting that the alteration of the sensitivity to doxorubicin was due to decreased apical expression of pgp, but not other abc transporters. these results suggest that although cif may be an obstacle to therapeutic attempts to restore the apical expression of cftr in the cf lung, it could be useful for the development of a novel class of inhibitors of pgp aimed at increasing the sensitivity of tumors to chemotherapeutic drugs (supported by the nih ( ro hl )). hamai, h. ; keyserman, f. ; worgall, t.s. , . pathology, columbia university, new york, ny, usa; . pediatrics, columbia university, new york, ny, usa cystic fibrosis (cf) is characterized by an inflammatory state and susceptibility to chronic lung infections. a common clinical finding is dyslipidemia characterized by altered plasma free fatty acid patterns, low plasma hdl levels and increased phospholipase a (pla ) activity. it is not clear how cftr mutations relate to lipid abnormalities. it was shown that defective cftr is associated with decreased uptake of sphingolipids (sl). sl homeostasis is tightly regulated and metabolites are relevant to lipid metabolism and cf. sl synthesis (sls) correlates with activity of srebp, a key transcription factor of lipid metabolism; ceramide- -phosphate, a sl metabolite, is a regulator of pla that is highly activated in cf. we investigated the hypothesis that sls is increased in cf. experiments were carried out in airway epithelial cell lines that express defective cftr (c / ib ), no cftr ( hbe sense / antisense) and overexpress cftr (a cells infected with adcftr or adcontrol). sls was assessed using radioactive tracers h-serine (de-novo sls) and h-sphinganine (recycling pathways). ceramide and sphingomyelin mass were determined enzymatically. neutral and alkaline sphingomyelinase activity was assessed using h-sphingomyelin as substrate. srebp mediated gene transcription was assessed using sre-promoter assays. cholesterol synthesis was evaluated by incorporation of h-acetic and h-mevalonic acid. cholesterol mass was determined by gas-chromatography. abca- expression was determined by western blot analysis. pla activity was measured using fluorescent phospholipid substrates. sls was post-transcriptionally increased in cells expressing defective or no cftr through the de-novo pathway ( % ± % for ib ; %± % for hbe-antisense; p< . ) and the recycling pathways ( % ± % or ib ; % ± % for hbe-antisense; p< . ). ceramide mass was increased by % (± %) in hbe antisense, % ± % in ib cells. sphingomyelin mass was increased -fold in ib , up to -fold in hbe antisense cells. neutral and alkaline sphingomyelinase activity was increased up to -fold in both cell lines. sre-mediated gene expression was increased up to -fold in ib and by % in hbe-antisense cells. overexpression of cftr in a decreased sls and srebp activity up to %. free cholesterol synthesis was increased by % in ib and by % in hbe antisense. expression of the cholesterol efflux receptor abca- was decreased in ib and hbe antisense compared to controls. sl mass, sre-mediated gene transcription and pla activity were decreased following incubation with inhibitors of sls in ib and hbe antisense cells. conclusion: defective cftr or lack of cftr expression correlates with ) increased sls synthesis and sl mass; ) increased sremediated gene transcription and free cholesterol synthesis; ) decreased expression of the abca- cholesterol efflux receptor. inhibition of sls decreases sre-mediated gene transcription and pla activity. dysfunctional sls is a newly recognized pathway associated with defective cftr and a possible therapeutic target in cf. the ability to make accurate, reproducible measures of mucociliary and cough clearance (mcc/cc) in cf patients is critical to assessment of new therapies designed to improve mcc/cc function, and thus decrease pulmonary infections and decline in lung function. for example, using mcc/cc methods developed in our laboratory, we showed that -week treatment with aerosolized hypertonic ( %) saline (hs) led to a sustained increase in mcc and improved lung function in cf patients (donaldson et al, n eng j med ; : ) . the mcc/cc technique involves patient inhalation of radiolabeled particles followed by gamma camera scanning to determine the rate of particle movement from the lungs. while a few cf centers have shown the capability to measure mcc/cc, the techniques across these centers are not standardized, making comparison of results difficult. it is vital to develop standard, simplified techniques for these measures to make larger cf patient populations available for such studies. to standardize a radiolabeled particle inhalation technique that can be easily used by other cf centers, we have compared three protocols in healthy and cf subjects. protocol # was identical to that used in recent studies, and incorporates a devilbiss jet nebulizer to generate µm mmad (gsd = . ), aqueous droplets containing suspended tc m-sulfur colloid particles. tidal volume and inhaled/exhaled flow rates were guided by a volume signal displayed on an oscilloscope. protocol # was further standardized and simplified to allow use at other centers. this protocol utilized the same nebulizer, but triggered by a commercial dosimeter (spira electro , finland) during inhalation as the subject breathed in time to a metronome ( breaths/min) at flow rate of . l/sec. subjects controlled inspiratory flow rate by feedback from a digital flow readout on the dosimeter. repeat measurements of mcc/cc were made in healthy subjects and mild cf patients to compare mcc/cc variables following radioaerosol inhalation using protocols # and # . there were no differences in particle deposition (i.e. central to peripheral ratio, or "c/p"), or clearance of particles through minutes and hours in either subject group. these data suggest that the new methodology (protocol # ) will be useful and easily employed by other cf centers to test effectiveness of new therapies for cf. the third protocol (# ) was then tested in five of the healthy subjects. this protocol used a nebulizer that generated very large droplets ( . um mmad, gsd = . ) and subjects inhaled at very slow flow rates (~ ml/sec). it was designed to provide greater bronchial airway deposition and thus an "improved signal" for mcc/cc evaluation. indeed, this methodology produced a larger average c/p and significantly greater particle clearance from the lung compared to the other standardized methodology (protocol # ) (c/p = . vs. . ; min clearance = % vs. %; hr clearance = % vs. %, respectively). interestingly, the greater fraction of clearance between min and hr may also reflect heightened ability to assess clearance from smaller airways, which could be particularly important in cf. protocol # will be tested further in cf patients. supported by cf foundation. previous clinical trials in cystic fibrosis (cf) indicate that anti-inflammatory therapy probably will not result in improvement in lung function, but will slow the rate of decline. this imposes constraints on study design for new anti-inflammatory agents, requiring that they use many patients over long periods. it is highly desirable to design a strategy for evaluation of antiinflammatory agents that will allow for the selection of only the most promising agents for phase iii trials. sputum induction (si) samples lower respiratory tract secretions and permits measurement of inflammatory markers. the purpose of this study was to assess the measurement of inflammatory markers in induced sputum as one such strategy by using ibuprofen as the test agent because it has demonstrated clinical benefit in cf. if changes in inflammatory markers are detectable in ibuprofen treated patients, then si might serve as a quick, noninvasive method by which to select the most promising anti-inflammatory agents for further study. methods: in this twoarm (ibuprofen and no treatment), open-label, parallel group study, cf patients >/= years of age with mild to moderate lung disease were screened at sites. si was performed on days , , and . patients in the ibuprofen group received drug on days - . subjects met eligibility criteria. were randomized to ibuprofen and to no treatment. subjects ( ibuprofen and no treatment) completed the study through day and comprise the intent-to-treat efficacy population. a maximum of subjects ( ibuprofen and no treatment) and a minimum of subjects ( ibuprofen and no treatment) across all markers had acceptable inflammatory marker values at both days and . within group and between group comparisons were made. results: with respect to the within group comparisons (before and after ibuprofen), most inflammatory markers slightly decreased in the ibuprofen group and increased in the no treatment group. for ibuprofen patients, mean changes were most apparent for il- (- . log pg/ml, p= . ) and % neutrophils (pmns) (- %, p= . ). with respect to the between group comparisons (ibuprofen vs. no treatment), differences were strongest for il- ( . log pg/ml, p= . ), percent pmns ( %, p= . ), and absolute pmn count ( . cells/ml, p= . ). fourteen days after discontinuing ibuprofen, inflammatory mediators tended to increase. there was no difference in aes between the two groups, and si was generally safe. conclusions: . overall, si is well-tolerated in patients >/= years, . a -week treatment period may not be long enough to study anti-inflammatory drugs, . with respect to changes in inflammatory markers associated with anti-inflammatory therapy, the effect size is small and the variance of the effect is small in clinically stable patients. . measuring inflammatory markers in induced sputum has potential as a screening tool for studying anti-inflammatory drugs in cf, . further studies of si in cf are warranted. supported by cystic fibrosis foundation therapeutics, inc. in preparation for our gene therapy clinical trial programme we are currently assessing a number of sputum biomarkers including viscosity and elasticity, total solids and dna content and hr sputum weight. we tracked and correlated these biomarkers in cf patients ( years and over) during a course of iv antibiotics (ab) by collecting samples on several occasions [visit (v) : at the start of ab treatment, v : at the end of ab treatment (generally after weeks) and interim periods]. to ensure adequate reproducibility of the results viscosity/elasticity measurements were carried out in triplicate using a csl rheometer which required a comparatively large volume ( mls) of spontaneously expectorated sputum. because of this requirement paired samples could only be obtained from approximately % of the patients. there was no change in viscosity/elasticity (n= ), solid or dna (n= ) content when comparing samples at the beginning and end of iv ab. in contrast, hr sputum weight was significantly (p< . ) lower at the end of the ab treatment (visit : . ± . g, visit : . ± . g, n= ). there was a strong and significant correlation between solid content and viscosity/elasticity (r= . , p< . ) and a modest correlation between hr sputum weight and % predicted fev (r=- . , p< . ) and hr sputum weight and patient scored symptom severity (r= . , p< . ). surprisingly, sputum dna content did not correlate with viscosity/elasticity, despite being generally thought of as a contributor to viscosity, which may in part be related to the assay not being able to discriminate between free and cell-enclosed genomic dna.in summary, after a course of iv antibiotics which lead to significant subjective and objective (fev ) improvement the overall quantity of expectorated sputum was significantly reduced but, based on analysis available to date, none of the other parameters (viscosity, elasticity, solid and dna content) changed significantly. considering the difficulties we encountered in collecting sufficient sputum during this period of an exacerbation, sputum viscosity/elasticity measurements may not be feasible parameters to measure in a gene therapy trial to which stable patients re likely to be recruited. this study was funded by the cf trust. chronic neutrophilic inflammation is a feature of cystic fibrotic (cf)related inflammation and serves as a significant contributor to morbidity and mortality associated with the condition. recently, our group has described a novel collagen-derived neutrophil chemoattractant, proline-glycine-proline (pgp), in a murine model of lipopolysaccharide-induced inflammation. the purpose of this study was to explore both the presence of this peptide in sputum from cf patients and the determination of a proteolytic system involved in pgp generation. we show that lower airway secretions from cf subjects have -fold increase in pgp levels compared to normal controls and that cf sputum (p< . ) is able to generate pgp from intact type i and ii collagen -fold above that seen from normal control sputum ex vivo (p< . ). we further demonstrate that production of pgp is dependent on two specific matrix metalloproteases (mmps), mmp- and mmp- . finally, we show that the generation of pgp can be significantly inhibited ( - % inhibition) by the use of either mmp- or mmp- specific inhibitors (p< . versus no inhibitor); a nonspecific mmp inhibitor (doxycycline) also demonstrates significant attenuation of pgp production. the determination of the requirements of these proteases in pgp generation allows for the identification of logical targets for disease-modifying therapeutics in cf. . cfrc, uab, birmingham, al, usa; . cfrc, university of north carolina, chapel hill, nc, usa cystic fibrosis (cf) lung disease is characterized by chronic neutrophilic inflammation. the discrete collagen breakdown product prolineglycine-proline (pgp) is a potent neutrophil chemoattractant thought to be generated by a specific proteolytic cascade present during cf pulmonary exacerbation (nat med ( ): , ) . high-mobility group box (hmgb ) is a potent inflammatory mediator found in sepsis, rheumatoid arthritis, and other inflammatory diseases characterized by neutrophilic inflammation. the role of pgp and hmgb as pmn attractants in cf is unknown. in this study we utilized specimens derived both from humans and scnn b-transgenic mice (βenac mice) in which overexpression of the βenac subunit in the airways leads to sodium hyperabsorption and airway surface liquid depletion, mimicking the cf muco-obstructive phenotype. pgp was quantified using tandem ms. neutrophil chemotaxis was measured in vitro following preincuabtion of sputum or balf with anti-hmgb neutralizing antibody. murine balf was assayed for hmgb by western blot and elisa. human sputum was similarly evaluated after normalizing for total protein concentration. in vivo hmgb activity was measured after intratracheal instillation in wt mice. βenac mice had % higher cell counts and a greater percentage of pmns ( . % vs. . %) in bal samples than wild-type (wt) littermates (p< . , n= /genotype). pgp was elevated in balf of scnn b animals ( . vs. below limits of detection in wt controls, p< . , n= ), increased in serum from cf subjects ( vs. pg/ml normal controls, p< . , n= ), and detected at high levels in cf sputum ( , pg/ml). balf screened for hmgb by western blot revealed elevated levels in βenac mice (p< . by densitometry, n= /genotype); elisa confirmed % greater hmgb concentration ( . vs. . ng/ml, p< . ). hmgb was also detected by western blot in human cf sputum ( of ) and at higher levels than secretions from normal subjects (p< . by densitometry; n= cf, control). purified hmgb induced dose-dependent pmn chemotaxis in vitro (peak - ng/ml, p< . , n= ) and cf sputum caused potent chemotaxis ( -fold over control, p< . , n= ) that was inhibited by preincubation with hmgb antibody (p< . ). balf from βenac mice (but not wt controls) was also chemotactic ( -fold greater than media control, p< . , n= ) and inhibited by anti-hmgb antibody (p< . ). exogenous hmgb ( ng) administered intratracheally to lps resistant mice (c hej, toll- receptor mutant) and balb/c mice caused pmn influx in balf at hours (c hej: . x pmns after hmgb vs. . x after vehicle, p< . ; balb/c: . x vs. . x , p< . ), and generation of pgp ( . vs. . pg/ml in c hej, p< . , n= ) . in summary, pgp and hmgb are elevated in balf of βenac mice that present with cf-like lung pathology; in cf airway secretions, hmgb is present in vivo at concentrations that induce neutrophil chemotaxis in vitro and pgp production in vivo, and is inhibited by blocking antibody. the role of pgp and hmgb in cf deserve further evaluation as they may be potential therapeutic targets of dysregulated inflammation. esther, c.r. ; jasin, h.m. ; collins, l.p. ; boysen, g. ; boucher, r.c. . pediatric pulmonology, university of north carolina at chapel hill, chapel hill, nc, usa; . cf research center, univeristy of north carolina at chapel hill, chapel hill, nc, usa; . center for environmental health and susecptibility, univeristy of north carolina at chapel hill, chapel hill, nc, usa biomarkers of airway inflammation are needed in cystic fibrosis (cf) to aid clinical management and assess the efficacy of novel therapies. we describe a simple and non-invasive method to measure airway purines, signaling molecules previously shown to be biomarkers of neutrophilic airway inflammation. airway secretions were obtained using exhaled breath condensate (ebc) collection, a technique that was easily performed in the outpatient setting on children as young as three years. the purines adenosine and amp in ebc were measured using ultra-sensitive liquid chromatography/tandem mass spectrometry (lc/ms/ms). in addition, we used lc/ms/ms to simultaneously measure urea as a dilution marker to control for the known dilutional variability of airway secretions within ebc. detection of adenonosine, amp, and urea was optimized using positive mode ms with selected reaction monitoring, and stable isotope dilution was utilized to improve quantification. detection was linear with concentration over four orders of magnitude, with coefficients of variation < %. the method was sensitive, with limits of detection for adenosine and amp (~ . nm) and urea (~ . µm), below expected ebc concentrations. applying the methods to ebc demonstrated that detection of purines and urea was reliable, with intraclass correlation coefficients greater than . between duplicate measures (n= ). we also measured purines and urea in ebc samples collected prospectively from children with cf (n= ) and healthy controls (n= ) during regular clinic visits. all samples were lyophilized and reconstituted to increase concentration -fold. adenosine, amp, and urea were detected in all samples. neither adenosine nor amp levels differed between groups, but urea was significantly lower in cf ( . ± . µm) versus healthy control ( . ± . µm, p= . ), suggesting that airway secretions were more dilute in cf samples. when dilutional variability was controlled using purine to urea ratios, the amp to urea ratio was found to be elevated in subjects with cf ( . ± . ) compared to healthy controls ( . ± . , p= . ), consistent with previous findings. the amp to adenosine ratio was also elevated in cf ( . ± . ) compared to control ( . ± . ), although the difference did not reach statistical significance in this small sample set (p= . ). experiments are ongoing to increase sample size and assess ebc purine levels before and after treatment of a cf exacerbation. these results demonstrate that lc/ms/ms analysis of ebc provides a non-invasive method to measure purine biomarkers of cf airway inflammation. given the flexibility of ms, this methodology could also prove applicable for study of other small molecule biomarkers. importantly, we also show that dilution of airway secretions in ebc may be altered in cf, and lc/ms/ms can be used to control for this variability through measurement of urea as a dilution marker. introduction: mucociliary clearance and antimicrobial peptide activity may be affected by airway surface liquid (asl) ph, which is regulated by epithelial ion transport. asl ph is abnormal in disease states in which il- a is elevated, including cystic fibrosis (cf), and the il- a receptor is expressed at the basolateral surface of bronchial epithelium. therefore, we investigated the effects of il- a on vectorial ion transport in well-differentiated hbe cells. methods: hbe cells were grown with an apical air interface and incubated with il- a at or ng/ml in the basolateral medium for hours prior to being studied with standard short-circuit current (isc) techniques. results: il- a treated cells had a minimal increase in resting and amiloride-sensitive isc compared to control cells, and had a dosedependent, statistically significant increase in forskolin-stimulated isc. at a dose of ng/ml, il- a doubled forskolin-stimulated isc ( . ± . µa/cm for control vs. . µa/cm for il- a). the increased isc was not bumetanide-sensitive, but was sensitive to acetazolamide and dnds, and was not present in hco --free solutions, suggesting it was due to hco secretion. to investigate whether this hco secretion was cl --dependent, we studied hbe cells in cl --free solutions, and there was no difference between untreated and il- a treated hbe cells. to test the hypothesis that il- a promoted cl -/hco exchange, we performed experiments in hbe cells mounted in cl --free solutions, and after addition of amiloride and forskolin, mm nacl was added to the mucosal bath and mm nagluconate was added to the serosal bath. under these conditions, one would predict a decrease in isc due to mucosal to serosal clmovement down its electrochemical gradient. in untreated hbe cells, we saw a decrease in isc followed by a recovery. in il- a treated cells there was a sustained increase in isc, suggesting that movement of cldown its electrochemical gradient resulted in exchange of clfor an anion at the apical membrane. in the absence of hco -, both untreated and il- a treated cells responded to claddition to the mucosal bath with decreases in isc. to assess the cftr dependence of the il- a-induced isc, we stimulated primary cf hbe cells treated with il- a. in cf cells, there was a minimal increase in amiloride-sensitive isc ( . ± . µa/cm for control vs. ± . µa/cm for il- a ) and in forskolin-stimulated isc ( . ± . µa/cm for control vs. . ± . µa/cm for il- a ). conclusion: the proinflammatory cytokine il- a induces cl -/hco exchange in hbe cells. notably, the observed cl -/hco exchange appears to be cftr-independent. however, cftr activation is required for maximum cl -/hco exchange as demonstrated by the smaller isc generated in cf cells. these data suggest that the cytokine milieu of the airway epithelium can alter ion transport and potentially asl physiology. furthermore, they suggest that cftr may interact with or regulate novel proteins in the presence of inflammation. supported by cff and nih periciliary fluid balance is maintained by the coordination of sodium and chloride (cl-) channels in the apical membranes of the respiratory epithelia. in the absence of the cystic fibrosis transmembrane conductance regulator (cftr), cl-secretion is diminished and sodium reabsorption becomes exaggerated. activation of non-cftr-dependent cl-channels can provide an alternate pathway for cl-secretion in the airways and gastrointestinal tract. the ph and voltage-dependent type- cl-channel (clc- ) is expressed in airway epithelial cell luminal membranes. we hypothesize that topically applied clc- agonists may restore cl-secretion in cystic fibrosis (cf) murine airways. using in vivo nasal potential difference measurements, we quantified clc- -mediated cl-transport in both cf and wild-type mice during nasal perfusion with lubiprostone (a prostone compound and specific clc- agonist; sucampo, pharmaceuticals, inc., bethesda md) or vehicle control. wild-type (c bl , n = ) and cf knock-out (cfko; n= (jax stock# )(jackson laboratories, bar harbor, me) mice were sedated and intubated to protect the lower airways from aspiration of perfusate. nasal and subcutaneous bridges were connected to fluid-filled silver chloride electrodes. baseline (in ringer's), amiloride-inhibited, low cl-(cl-free, gluconate-substituted ringer's with amiloride), and low cl-plus lubiprostone (with increasing concentrations of lubiprostone or vehicle) mice were perfused and potential differences were measured with a high impedance voltmeter (world precision instruments, sarasota fl) and continuously recorded. a clear dose-response relationship was detected in both wild-type and cf mice. at µm lubiprostone, wild-type mice showed hyperpolarization of - . ± . mv and cf mice responded with - . ± . mv hyperpolarization. a paired t-test of low cl-perfusion and lubiprostone perfusions revealed significant (p< . ) differences in both genotypes. five clc- cfko mice were similarly tested and showed no response to lubiprostone (+ . ± / mv). cftr inhibitor- ( µmol) (calbiochem, san diego ca) added to the low cl-perfusion in additional wild-type mice eliminated the low cl-response but did not abolish the lubiprostone response, confirming that clc- is present and independent of cftr regulation. we conclude that direct application of a clc- agonist in the cf murine upper airways restores near normal levels of cl-secretion. cftr is an apical membrane chloride channel whose activity is required to maintain airway surface liquid (asl) volume and efficient mucociliary clearance. cftr is activated by camp dependent stimulation of protein kinase a. however, studies have suggested that the actin cytoskeleton may also be required for regulation of cftr, by acting as a structural element in second messenger compartmentalization ( ) . to determine whether an intact cytoskeleton is required for asl volume homeostasis, adenosine (ado), atp, or isoproterenol (iso) (all at µm) were added apically to human bronchial epithelial cultures (hbec's) before or after the cytoskeleton had been disrupted by cytochalasin d exposure ( µm for h). in control cultures ado, atp and iso all resulted in a significant increase in asl height (∆asl) within min (∆asl; ado, . ± . µm; atp, . ± . µm; iso, . ± . µm). however, in cytochalasin d exposed cultures ado was without effect while atp and iso were still able to evoke an increase in height (∆asl; ado, - . ± . µm; atp, . ± . µm; iso, . ± . µm). however, we showed that a b receptor function was not compromised by cytoskeleton disruption as camp levels were seen to increase both after addition of adenosine and adenosine in the presence of cytochalasin d. thus, the effect of cytochalasin d on asl height appeared specific for adenosine-mediated cl-secretion. to further test the effect of cytochalasin d on the relationship between adenosine and cftr, we designed a fluorescent resonance energy transfer (fret) pair of cfp-cftr (labeled at the n-terminus) and yfp-a b receptor (labeled at the c-terminus). addition of adenosine ( µm) increased fret by . times (% efficiency; control, . ± . %, ado, . ± . %) compared to the vehicle control (n= ). cytochalasin d exposure ( µm for h) completely inhibited the increase in fret that was observed after adenosine addition (p< . ). in contrast, cytochalasin d had no effect on fret between cftr and the β adrenoceptor. to determine whether cytochalasin d treatment ( µm for h) had any direct effect on cftr protein levels and localization we used a cell line stably expressed with an exotope-tagged cftr (hb cells) ( ) . cftr protein levels were found to be significantly reduced as compared to nontreated controls by both western blot analysis and chemiluminescence detection (n= ). cells were also stained with an antibody against the external cftr exotope and imaged by confocal microscopy and a significant decrease in staining at the plasma membrane was observed after cytochalasin d treatment (n= ). we conclude that cytochalasin d exposure decreases cftr protein levels and specifically inhibits adenosine-mediated regulation of cftr activity in the airways, leading to disruption of asl homeostasis and inhibition of mucociliary clearance. numerous studies have reported evidence that cftr is expressed in the apical membrane of serous cells in the airway submucosal glands. thus, it has been reasoned that ( ) cftr plays some role in normal anion and fluid secretion by airway glands and ( ) loss of this channel's function in cystic fibrosis (cf) airways plays some role in the etiology of cf lung disease. a recent study by joo et al. (jbc : - , ) indicates that cf airway glands lose the ability to secrete fluid when treated with forskolin or vip but maintain the ability to secrete when treated with muscarinic agonists. this result suggests that vip and muscarinic agonists mediate secretion by cftr-dependent and cftr-independent pathways, respectively. the present study was undertaken to explore the dynamics of fluid secretion by individual submucosal glands to determine if further differences in their responses to these two agonists could be distinguished. pig tracheas were obtained from a local slaughterhouse. after removal of the cartilage, tissues were mounted in a warm ( °c) observation chamber that permitted exposure of the submucosa to krebs solution. the mucosal surfaces of the airways were dried with a stream of warm dry air and then layered with mineral oil. liquid secreted from the gland duct openings formed spherical aqueous droplets within the oil layer, and their volumes were estimated from spatial dimensions taken from sequential digital images. liquid secretion rates were estimated for individual glands present within a . mm region of interest. rates were determined for two experimental protocols. for one group of tissues, secretion rates were determined for a control period, followed by a period of exposure to µm vip, and then a period of exposure to both vip and µm acetylcholine (ach). in the second group, the order of vip and ach treatment was reversed. these agonist concentrations were expected to produce near maximum rates of secretion. when the agonists were applied first, secretion rates were significantly (p< . ) increased from the control period by both ach ( . ± . nl/min to . ± . nl/min) and vip ( . ± . nl/min to . ± . nl/min). the secretion rates for individual glands were highly variable with both agonists (ach: . - . nl/min; vip: . - . nl/min). when ach application followed vip treatment, the mean secretion rate was significantly increased further to . ± . nl/min; however, when vip application followed ach treatment, the secretion rate was only marginally and insignificantly increased to . ± . nl/min. we conclude that both ach and vip are efficacious secretagogues for porcine airway glands and that the rate of secretion from individual glands to either agonist is very heterogenous. because vip has no effect following stimulation by ach, we speculate that ach induces secretion by activating all transporters relevant to vip-induced secretion including cftr. however, ach must also activate channels and/or transporters not induced by vip since its effects are additive to vip. this most likely includes activation of a non-cftr anion channel. ( serous cells are thought to serve as the principal anion and fluidsecreting cells of airway submucosal glands. mucous cells primarily secrete gel-forming mucins. these respective physiological roles for these two cell types imply that the mucous cells, which secrete the thick mucus gel, should lie downstream of the serous cells so that their watery secretions can flush the mucins out of the ducts and onto the airway surface. early studies indicated that such arrangements existed within glands but reports of cell distributions have been descriptive rather than quantitative. because of resurgent interest in serous cell function, we revisited this issue to formally document the distribution of these cell types within the secretory tubules of the submucosal glands as well as at the surfaces of the glands, where serous cells are most accessible for study. a piece of porcine lung, containing a mm diameter bronchus, was treated with formalin fixative, and consecutive µm sections were taken. the apices of virtually all gland epithelial cells, except the cells lining the collecting and ciliated ducts, stain positive for pas indicating that these cells are either serous or mucous cells. however, pas staining does not allow distinctions to be made between these two cell types. consequently, all slide sections were stained with hematoxylin and eosin. with these stains, mucous cells were identified as cells containing lucent granules in their apices whereas serous cells were considered to be cells where the apical cytoplasm stained a uniform dark reddish color. first, the distribution of serous and mucous cells in the secretory tubules were determined. the acini of individual tubules were located in the slide sections, and cells were identified and counted in consecutive sections that approached the collecting ducts until the tubules were no longer distinguishable as discrete tubes. secretory tubules from individual glands were studied. in the acini, serous cells accounted for . ± . % of total cells whereas mucous cells accounted for . ± . % of total cells (remaining cells were not distinguishable as either cell type). there was a significant (p<. ) negative correlation of serous cell numbers with distance from the acini so that at µm from the acinus serous cells accounted for only . ± . % of total cells. there was a coincident significant positive correlation with mucous cell numbers so that at µm from the acini mucous cells accounted for . ± . % of total cells. next, we examined the cell distribution at the adventitial and mucosal surfaces and at the lateral margins of the glands. serous cells accounted for . %, . %, and . % of the total cells at these respective locales. mucous cells represented . %, . %, and . % of total cells in these same respective regions. we conclude that serous cells are by far the dominant cell type in secretory tubules of porcine submucosal glands. mucous cells are rare in acini but increase in frequency with distance from the acini. the outer surfaces of the glands are dominated by serous cells as well. at the lateral margin of the glands, serous cells outnumber mucous cells by -to- . (supported by nih hl ) . many of the membrane transporters that participate in this process have been identified; however, the identity of the class of kchannels that maintains resting membrane potential and/or cell membrane polarization during the secretion process remains poorly characterized. liquid secretion by porcine submucosal glands is insensitive to numerous k + channel blockers including ba + , tetraethylammonium, apamin, charybdotoxin, iberiotoxin, clotrimazole, penitrem a, -aminopyridine, and quinidine. recently, a new class of k + channels, the "tandem-pore" or k p channels, have been described. potassium channels of this class are insensitive to many traditional k + channel inhibitors but are blocked by local anesthetics, such as lidocaine and bupivacaine. additionally, many k p channels are sensitive to changes in extracellular ph. in the present study, we considered whether k p channels might participate in the liquid secretion response to acetylcholine (ach). intrapulmonary bronchi, excised intact from the lungs of pigs, were cleared of accessible luminal liquid, cannulated, and treated with µm ach to induce secretion. paired airways were pretreated for h with mm bupivacaine. bupivacaine blocked . ± . % of the liquid secretion response to ach. the ic for the bupivacaine inhibition was approximately µm. the inhibitory effect of bupivacaine was not due to nonspecific toxicity since tissues exposed to bupivacaine for h and then washed with fresh inhibitor-free buffer recovered to % the control achinduced secretory rate. the bupivacaine effect was not due to inhibition of voltage-gated na + channels in intrinsic neurons since µm tetrodotoxin did not inhibit ach-induced secretion. no significant effect was seen in the secretion rate when the extracellular solution ph was lowered from . ± . (normal hco --buffered krebs gassed with % co ) to . ± . by gassing with % co gas (balance o ). while we recognize the relative nonspecificity of this agent, the inhibitory effects of bupivacaine on liquid secretion by porcine bronchi are consistent with k p channel participation in this secretory response. the failure to demonstrate inhibition with acidification of extracellular solution could signify a k p channel subtype that is insensitive to low extracellular ph. ( . novartis, horsham, united kingdom; . rosalind franklin university, chicago, il, usa; . mount sinai medical center, miami, fl, usa; . genomics institute of the novartis foundation, la jolla, ca, usa enac activity in the human airway epithelium has been reported to be partially-sensitive to the broad spectrum trypsin-like protease inhibitors aprotinin and placental bikunin (bridges et al., am j physiol :l - ; donaldson et al., jbc : - ) . a low molecular weight inhibitor of the airway channel activating protease (cap) would represent a potential therapeutic approach to attenuating enac function in cystic fibrosis (cf) lung disease. the aims of the current study were to: ( ) further characterise the in vitro efficacy of cap inhibitors in human bronchial epithelial cells (hbes), ( ) to evaluate the relevance of a cap mechanism to the regulation of enac function in vivo, and ( ) to establish whether the inhibition of the airway cap can modulate mucociliary clearance in vivo. primary cultures of hbes (normal and cf), cultured under air-liquid interface conditions, demonstrated an amiloride-sensitive short circuit current (isc), that was sensitive to aprotinin but that was insensitive to inhibition by sbti, α -anti-trypsin and α -pdx. the amiloride sensitive isc was also attenuated by the low molecular weight cap inhibitor nvp-qau (hbc ) with an ic value of approximately nm. nvp-qau caused a time dependent inhibition of the isc with a t ⁄ of approximately min and this effect could be reversed by the addition of excess trypsin. in vivo, aprotinin attenuated the guinea pig tracheal potential difference (tpd) from - . ± . mv to - . ± . mv (n= - ) with an ed value of pmoles/kg, measured at hours following intra-tracheal instillation. the tpd was unaffected by either sbti or α -anti-trypsin at doses up to pmoles/kg. the combination dosing of an enac blocker with aprotinin did not attenuate the tpd beyond the effect observed with either agent alone, consistent with aprotinin attenuating enac function in this model system. intra-tracheal instillation of nvp-qau attenuated the tpd from - . ± . mv to - . mv with an ed value of µg/kg (n= - ). in the sheep, the administration of nvp-qau into the airways by aerosolisation of a mg/ml solution resulted in a - fold enhancement of the rate of clearance of mtc sulfur colloid from the lungs compared with the vehicle control. these studies indicate that the cap mechanism of enac regulation can translate through in vitro human assays, into in vivo models. cap inhibitors can attenuate tpd in the guinea pig in vivo and enhance the rate of mucociliary clearance in the sheep, thereby representing an approach to the therapeutic regulation of enac function in cf lung disease. cotton, c. pediatrics, physiology and biophysics, case western reserve university, cleveland, oh, usa cystic fibrosis is caused by mutations in the gene that encodes cftr, a camp-activated apical membrane anion channel. loss of cftr-mediated anion conductance is a primary defect in cf but secondary defects such a sodium hyperabsorption are also implicated in cf pathophysiology. it is generally accepted that mucociliary clearance is compromised in cf airways due to reduced fluid secretion and/or increased fluid absorption. the recent generation of a transgenic mouse model with enac overexpression that exhibits cf-like lung disease highlights the importance of sodium hyperabsorption. therapies designed to reduce mucus production and viscosity, increase lumenal water content, stimulate fluid secretion, and inhibit fluid absorption are under development for treatment of cf airways. although controversial, several recent studies have demonstrated that serca pump inhibitors partially correct the trafficking defect associated with delta f mutant cftr. the goal of this work is to determine if serca pump inhibitors affect enac-mediated sodium absorption. a renal collecting duct epithelial cell line and primary cultures of well-differentiated human tracheal epithelial cells (air/liquid interface culture) were used for these studies. amiloride-sensitive short circuit current was determined as a measure of enac-mediated sodium absorption. quantitative rt-pcr was used to evaluate expression levels for each of the subunits of enac (alpha, beta, and gamma). epithelial monolayers were treated for - hours with serca pump inhibitors (thapsigargin, - nm; dbhq, - um; and curcumin, - um). treatment with serca pump inhibitors decreased amiloride-sensitive short circuit current by - %. in contrast, campstimulated short circuit current was not reduced by treatment with serca pump inhibitors. the steady-state levels of alpha, beta, and gamma enac were reduced by - % in renal epithelial monolayers treated with thapsigargin whereas beta and gamma enac were reduced by - % with no significant change in alpha enac in airway epithelial cells. the results of these studies demonstrate that sustained depletion of endoplasmic reticulum calcium stores and/or elevation of intracellular calcium by inhibition of serca pump activity reduces enac mrna expression and enac-mediated sodium absorption. thus, inhibition of serca pump activity in airway epithelial cells of cf patients that carry the delta f mutation may provide dual benefit by promoting delivery of mutant cftr to the membrane . pediatrics, national jewish medical and research center, denver, co, usa; . integrated department of immunology, national jewish medical and research center, denver, co, usa; . department of medicine, cystic fibrosis/pulmonary research and treatment center, the university of north carolina at chapel hill, chapel hill, nc, usa increased cytosolic calcium ([ca + ] i ) initiated by the release of stored ca + from the endoplasmic reticulum (er) is a key signal to elicit a wide range of essential cellular responses, including secretory functions of the respiratory epithelium that are modified in cf. serca pumps are responsible for (re)filling the er ca + stores, and serca blockers such as thapsigargin are very potent and commonly used pharmacological triggers of ca + -signals. modulation of the activity of sercas can profoundly affect ca + homeostasis. although defective calcium homeostasis is a characteristic of several pulmonary diseases including cf, the role of serca is unknown. lung tissue samples (bronchus and distal lungs) from normal (n= ) and cf subjects (n= ) were evaluated by immunohistochemistry. serca expression was decreased in the bronchial and bronchiolar epithelia of cf. non-cf and cf bronchial epithelial cell line pairs including calu- and jme/cf , c- and ib - , hbe o-( hbe), cfbe o-(cf o-) and cfbe o-(cf o-) were also probed. given certain limitations of such cells, several consistent findings still emerged. a % and % decrease in serca expression was observed in cf o-and cf ocells as compared to hbe cells. immunocytochemical studies in these cells confirmed that the serca was localized in the er and that the decreased serca expression was not associated with decreased er content. reduced serca expression and activity ( . ± . vs . ± . and . ± . pmol/min/mg protein in hbe vs. cf o-and cf ocells respectively) was observed in the purified er membranes from cf cell lines. northern blot analysis revealed a parallel reduced mrna expression as well. decreased serca was accompanied by increase in the low affinity isoform serca ( . ± . vs. . ± . and . ± . arbitrary serca intensity/b-actin in respectively) . we have also evaluated a limited number of primary airway epithelial cells isolated from lung samples of normal and cf subjects for serca expression. serca expression in polarized tracheobronchial epithelial cell lysates from cf subjects was decreased by about % as compared to those from normal subjects ( . ± . vs. . ± . arbitrary serca intensity/b-actin units in cf and normal respectively, n= ). expression of serca could also be suppressed by inhibiting cftr with cftr inh in normal human bronchial epithelial cells ( various studies indicate that the airway surface fluid possess an antibacterial system based on the combined action of lactoperoxidase, h o , and scn -(thiocyanate). the enzyme lactoperoxidase (secreted by submucosal glands) utilizes h o and scnto generate oscn -(hypothiocyanite) a molecule with antimicrobial activity. h o is produced by dual oxidases expressed on the apical membrane of airway epithelial cells whereas scnis transported across the epithelium through anion transporters and channels. in particular, scntransport seems to occur through cftr and other ca +dependent clchannels. therefore, reduced scntransport in the airways of cystic fibrosis patients may contribute to impaired antimicrobial activity. in a recent study, we have found that cytokines, in particular il- , cause a strong increase in the ability of cultured bronchial epithelia to transport scnfrom the basolateral to the apical membrane. this effect is mediated by upregulation of ca + -dependent clchannels and of the slc a (pendrin) anion transporter. these findings suggest that under proinflammatory conditions the activity of the scn -/h o /lactoperoxidase system is potentiated. we evaluated the antimicrobial activity of airway surface by seeding bacteria on the apical membrane of human bronchial epithelia grown with an air liquid interface on a porous membrane. an inoculum corresponding to , cfu of s. aureus was added to the cells in µl of saline solution with and without lactoperoxidase ( . µg/ml). experiments were performed in the presence or absence of scn -( µm) in the basolateral solution. bacteria were recovered after four hours and plated on agar plates for colony counting. our preliminary results show that simple addition of lactoperoxidase to the apical surface decreases bacterial survival. in addition, bacterial killing is strongly enhanced by prestimulation of cells for hours with il- ( ng/ml). the effect of lactoperoxidase is dependent on h o as it is prevented by addition of catalase and is absent in cell-free experiments when h o is omitted from the reaction mixture. surprisingly, the presence of scnin the basolateral compartment did not increase bacterial killing and, in some cases, appeared to generate a protective effect. our results suggest that lactoperoxidase, in the presence of h o , is an effective antimicrobial molecule. the contribution of scnand the mechanism of the potentiation caused by cytokines is less clear and requires further investigation. a possibility is that, in the absence of scn -, lactoperoxidase generates an oxidant molecule that is more toxic to bacteria than oscn -. elucidation of this mechanism and comparison between cf and non-cf epithelia is in progress to assess the role of cftr and of other anion channels. supported by cfft and telethon-italy. cf patients become infected with pseudomonas aeruginosa, which release flagellin into the airway surface liquid to activate toll-like receptor and proinflammatory signaling. flagellin has been shown to inhibit na absorption by airway epithelia. we tested flagellin on cl secretion and proinflammatory signaling (nf-κb activation) by calu cells, a cftrexpressing, serous-like airway gland cell line. calu cells were grown on filters and either mounted in ussing chambers (clamped to zero mv) in the presence of a serosa-to-mucosa gradient of [cl] for measurements of transepithelial cl secretion (i cl ) or treated with an adenovirus expressing nf-κb-controlled luciferase to assay nf-κb activation. flagellin ( - g/ml) on either the apical or basolateral surface of cells increased apparent anion secretion that began in - mins and increased over - mins by - µa/cm . this i cl was blocked by glibenclamide and glyh , indicating that it resulted at least in part from activation of cftr. flagellin also stimulated fluid secretion by intact human tracheal glands. flagellin activated nf-κb (luciferase assays) in both calu- cells and in the cf cell line cf , while i cl was stimulated in calu but not in cf cells. flagellin-stimulated i cl in calu- cells was blocked % by sb (a p mapk blocker) and by a similar amount by wortmannin (pi kinase blocker). interleukin β and the tlr -agonist pam cys also activated i cl . the effect of flagellin was not due to increases in cytosolic [ca + ] (ca i ) because flagellin did not alter ca i . in contrast to the slow effects of flagellin, pam cys and il β, atp rapidly increased i cl (within secs, up to µa/cm ) followed by slower decrease to steady i cl = - µa/cm that had a similar time signature as the increases in ca i . forskolin (to increase cytosolic [cyclic amp]) increased i cl within mins to a steady value = - µa/cm . flagellin had small or no effects on i cl following maximal stimulation with either atp or forskolin. atp and forskolin on their own had no effect on nf-κb. flagellin increased phosphorylation of p mapk and of akt (down-stream kinase phosphorylated by pi k) with a time course similar to the increase in i cl . flagellin-activated nf-κb was reduced by roughly % by either wortmannin or sb . these results indicated that tlr agonists and inflammatory cytokines stimulate both nf-κb and proinflammatory processes and also cftr-dependent cl and fluid secretion by airway gland cells. these responses are mediated in part by activation of both p mapk and pi kinase. flagellin-tlr -activated increases in cftr-mediated cl secretion and reduction in enac-mediated na absorption will increase fluid secretion into the airways, which may facilitate bacterial removal by the mucociliary escalator and thereby reduce the proinflammatory stimulus. in cf it is expected that bacteria will activate tlr signaling to trigger innate immune responses, but cl and water secretion and the resulting "bacterial flush," will be missing, leading to sustained inflammation. ( , ) . treatment with hs improved several measures of lung function including mucociliary clearance. in one study amiloride was seen to have a negative impact on the benefit of hs ( ) . the long duration of action of hs and the paradoxical effect of amiloride are surprising and require further investigation. aims: our goal was to study the effect of exposure to a hypertonic challenge (hc) on amiloride sensitive na+ transport (ina) in primary cultures of human bronchial epithelial (hbe) cells from non-cf and cf patients. methods: hbe cells were grown at an air liquid interface and studied under short circuit current conditions. cells were challenged with hs by exchange of the apical or basolateral baths. in some experiments the timedependent effect of a small ( µl) isosmotic or hs volume at the airway surface was tested on ina and apical osmolality. results: application of hypertonic nacl and mannitol solutions from either the mucosal or serosal sides of the epithelium inhibited ina. the degree of inhibition was a saturable function of the imposed hypertonicity with an ic of (mannitol) and (nacl) mosmol/kg h o and a maximal inhibition of %. the inclusion of amiloride ( - µm) did not affect the hc-induced reduction in ina. the inhibition in ina by hc was rapid in onset and accompanied by a fall in the transepithelial resistance and the total transepithelial capacitance as expected for cell shrinkage. the inhibition in ina was only partially reversible (~ %) after returning to isotonic solutions for min. exposure of the airway surface to hypertonicity ( mosmol/kg h o) inhibited ina by - % at min of exposure. as osmotically-driven water moved from the serosal to the mucosal side, the apical volume increased concomitant with a decrease in its osmolality and a recovery of ina. the osmolality declined exponentially and reached the isosmotic value after ~ hrs. the recovery of ina lagged behind the recovery of apical osmolality by , and % at . , and hrs of incubation, respectively. after hrs of airway hc exposure, the osmolality and ina recovered completely. our results demonstrate that hc causes a rapid, dramatic and prolonged decrease in na+ absorption and that continuous presence of amiloride had no effect on the inhibition of ina or the recovery of apical osmolality. hc also causes the epithelium to shrink. no appreciable differences were observed between cf and non-cf hbe cells. conclusions: we propose cell shrinkage together with the continued influx of na+ increases intracellular na+ ([na]i) and lead to an inhibition of na+ transport. elevated [na] i is a known inhibitor of epithelial na+ channels ( ). the sustained nature of the inhibition in na+ transport to a hc may help explain the longer than expected duration of action of hs in the clinical trials ( , ) . explanation of the paradoxical effects of amiloride observed in a clinical trial ( ) requires further investigation. references: . n engl j med : - , ; . n engl j med : - , ; . physiol rev : - , reduced airway surface liquid (asl) volume resulting from enac mediated na+ hyperabsorption and cftr-mediated hyposecretion plays a critical role in cf lung disease pathogenesis. however, the mechanisms involved in enac/cftr regulation are poorly understood. using human bronchial epithelial cultures (hbecs) under thick film conditions, i.e. in the ussing chamber with the asl washed away, adenosine (ado) directly stimulates cl-secretion. however, in a cl-free environment, ado has no effect on the amiloride-sensitive current, which is mediated by enac. mean change in i sc following µm ado to the apical chamber was . µa/cm from baseline compared to vehicle alone (- . µa/cm ;ns;n= ) in hbecs. mean delta amiloride ( µm) was - . and - . µa/cm respectively (ns; n= each). in contrast, under thin film conditions we have previously shown that ado stimulates sustained asl secretion via activation of cftr in the presence of the protease-inhibitor aprotinin, whereas trypsin pre-treatment abolished this secretion, likely by activating enac and abolishing the electrical driving force for cl-secretion . further, ado-mediated asl secretion is significantly increased if hbecs (thin film conditions) are left with intact asl for h, an action that is abolished by acutely washing the apical surface . thus, we hypothesised that a soluble enac inhibitor was secreted into the asl under thin film conditions, which could accumulate sufficiently to inhibit enac and provide the necessary driving force for cftr-mediated cl-secretion after ado-addition. to search for such an inhibitor, we incubated asl with trypsin-coated beads and identified bound proteins by mass spectrometry (albumin-coated beads were used as a control to exclude non-specific binding). the major identified protein was plunc, a protein that is secreted into the asl both in vivo and in vitro which has no current known function. secretion of plunc into hbec asl was confirmed by western blot. to test whether plunc could alter asl volume homeostasis, we made an anti-plunc shrna retroviral construct which we used to infect hbecs. unlike the control anti-luciferase shrna infected hbecs, anti-plunc shrna-infected hbecs exhibited > % knockdown of plunc and a failure to regulate asl volume (ctrl, . ± . µm; ± . µm; n= ) . when co-expressed with enac in oocytes, plunc inhibited enac currents by % (n= ). cftr function in contrast was unaffected by plunc expression (n= ). thus,it is likely that ado stimulates cftr mediated cl-secretion but is not involved in enac regulation. however, experiments were performed in a very simplified environment i.e. static cultures with little mucosal atp or mucus and these findings will need to be expanded and further evaluated in native tissues. we conclude that plunc, rather than ado may be the soluble mediator in the asl which regulates enac function by reducing its activity. . tarran, r. et al., j. gen. physiol., . ( ) airway surface liquid (asl) absorption is mediated by epithelial na + channels (enac), which establish an osmotic gradient that drives fluid absorption. we and others have recently reported that a protease / anti-protease balance regulates enac in normal human bronchial epithelial cells (hbe) and provides a mechanism for auto-regulation of asl volume. in cf, this balance is disturbed, leading to constitutive proteolytic activation of enac and the pathological na + hyper-absorption characteristic of the disease. to determine if channel activating protease expression is regulated by changes in asl volume and is altered in cf, we examined prostasin expression in control and cf hbe under basal and asl volume expansion conditions using western blotting. prostasin migrates as kda and kda bands in apically biotinylated proteins, apical secretions, and whole cell lysates of primary hbe. following apical aprotinin exposure, only the kda prostasin molecular species was present in the biotinylated proteins, suggesting that the proteolytic conversion of prostasin zymogen to active enzyme occurs on the cell surface. following asl volume expansion, cell surface prostasin expression increased by > % (p= . , n= tissue donors, > cultures from each). as our recent studies indicated that increased proteolytic activation of enac occurs in cf airway epithelia, we next compared prostasin expression in cf and control hbe cells. prostasin expression in the apical biotinylate of cf hbe was . fold greater than in control hbe (p= . , n= tissue donors). furthermore, the ratio of the kda (active enzyme) to kda (zymogen) prostasin molecular species was . fold greater in cf (p= . ), indicating that increased activation of prostasin occurs in cf airway epithelium. we next determined whether increased prostasin activation in cf may reflect the deficiency of a protease inhibitor. serpin e , also known as protease nexin- (pn- ), forms an ~ kda complex with prostasin and permanently inactivates its protease activity. while no significant difference in pn- expression was observed between control and cf hbe, pn- was found to contribute to prostasin regulation by (i) forming an inactive prostasin complex, (ii) inhibiting the amiloride-sensitive short-circuit current across hbe, and (iii) preventing conversion of prostasin zymogen to active enzyme. these findings demonstrate that cellular mechanisms coordinate prostasin expression and activity with asl volume. accordingly, at times when the asl volume is high, prostasin expression increases, and this presumably augments na + and asl absorption to absorb the excess luminal fluid. these regulatory mechanisms govern the apical membrane expression and processing of zymogen to active enzyme. because prostasin is incapable of autocatalysis, these findings support the existence of a proteolytic cascade that controls enac activity during asl volume homeostasis, perhaps reflecting the recently reported matriptase-prostasin cascade. furthermore, the increased expression of prostasin in cf suggests that abnormal regulation of prostasin contributes to na + hyper-absorption in cf airways. supported by the nih, cff, and ala. we report that annexin (anx )-s a forms a functional camp/pka/calcineurin (can)-dependent complex with cftr. cell stimulation with forskolin/ibmx significantly increases the amount of anx -s a that reciprocally co-immunoprecipitates with cell surface cftr and can a. pre-inhibition of the cells with pka or can inhibitors attenuates the interaction. furthermore, we find that the acetylated peptide (stvheilcklsleg, ac - ), but not the non-acetylated equivalent n - , corresponding to the s a binding site on anx , disrupts the anx -s a /cftr complex. analysis of dids and cftr inh -sensitive currents, taken as indication of the outwardly rectifying cl-channels (orcc) and cftr-mediated currents, respectively, showed that ac - , but not n - , inhibits both the camp/pka-dependent orcc and cftr activities. can inhibitors (cypermethrin, cyclosporin a) discriminated between orcc/cftr by inhibiting the cftr inh , but not the dids, sensitive currents, by more than %. furthermore, peptide ac - inhibited acetylcholine-induced short-circuit current measured across a sheet of intact intestinal biopsy. our data suggests that the anx -s a /cftr complex is important for cftr function across epithelia. the content and water (h o) mobility in airway epithelium is tighly coupled to airway function. in cystic fibrosis (cf), h o epithelial permeability is reduced in airways. the demand of noninvasively imaging techniques with high spatial resolution potential is rising because such imaging tools would expedite anatomical and functional phenotyping in the genetically altered mice. magnetic resonance microscopy (mrm) is a noninvasive, inherently three-dimensional ( d) imaging technique capable of visualizing anatomical structures in the mouse and allows for interpretation of complex spatial relationships between substructures and h o. in this study, we explore different mr contrast parameters and signal-to-noise ratios at a µm pixel size to characterize microstructure and h o mobility in ex vivo trachea of cf transmembrane conductance regulator (cftr)-deficient (cftr knockout, cftr tm unc ) mice and their aged-matched wt littermates. this study is performed using a bruker mrm system at . tesla. we demonstrate for the first time the ability of d-mrm to map the h o content and mobility in trachea epithelium. from the d-mrm videoimages, differential h o content was visualized in different levels of trachea in wt and cf mice. t mrm images depicting the h o rotational mobility which is related to environmental viscosity of trachea epithelium will be also shown. finally, this d-mrm imaging method is a valuable method for measuring h o content and permeability in airways and can serve for assessing the effects of drugs on h o mobility in cf airways. supported by grants from inserm, upmc-paris , cnrs, and the french cystic fibrosis association (vlm). cystic fibrosis (cf) is a lethal inherited disorder caused by mutations in a single gene encoding the cystic fibrosis transmembrane conductance regulator (cftr) protein resulting in progressive lung oxidative damage. in this study, we evaluated the role of cftr in the control of ubiquitin-proteasome activity (ups system) and the nf-κb / iκb-αsignaling after lung oxidative stress. we exposed cftr deficient (cftr-/-) and wild type mice for h to hyperoxia-mediated oxidative stress. cftr deficient mice exhibited significantly higher lung proteasomal activity than cftr+/+ animals after oxidative stress. this was accompagnied by a strong reduction of lung caspase- activity and an absence of degradation of nf-κb inhibitor iκb-α. in vitro, human cftr-deficient lung cells also exhibited higher proteasomal activity and a lack of increased nf-κbdependent transcriptional activity compared to cftr-sufficient lung cells after oxidative stress. furthermore, inhibition of the proteasomal activity by mg in cftr-deficient lung cells restored the nf-κb/iκb-α signaling to that of cftr-sufficient lung cells. inhibition of caspase- by z-dqmd in cftr-sufficient lung cells mimicked the response profile of increased proteasomal degradation and lowered nf-κb transcriptional activity of cftr-deficient lung cells when exposed to oxidative stress. all together, these results suggest that cftr is a crucial molecule in regulating proteasomal degradation and nf-κb activity in lung epithelium under oxidative stress. staphylococcus aureus, one of the major pathogen involved in airway infections, releases in the airway lumen virulence factors that may impair the airway epithelial functionality. to date, the effect of s. aureus virulence factors on the loss of electrolyte homeostasis of the airway epithelium has not been investigated. we have previously shown that the combination of a corticosteroid and a long-acting beta agonist attenuated the airway epithelial cell inflammatory response induced by s. aureus virulence factors. the aim of the present work was to investigate the effect of s. aureus virulence factors on the airway epithelial tightness and on the chloride efflux of airway epithelial cells incubated or not with salmeterol hydroxynaphthoate × - m and fluticasone propionate × - m (sm/fp). the airway epithelial tightness was assessed by immunocytochemistry, western blotting and transepithelial resistance measurement. the chloride efflux was evaluated by dynamic imaging using the -methoxy-n-( -sulfopropyl) quinolinium (spq) fluorescent probe. s. aureus (strain - ) virulence factors were obtained by growing bacteria at × cfu/ml for h at °c. the bacteria supernatant containing s. aureus soluble virulence factors was then centrifuged, filtered and diluted at % in cell culture medium (a concentration that did not alter cell viability). human airway epithelial cells (mm ) were incubated with % s. aureus virulence factors for h and then co-incubated or not with sm/fp for h. we observed that the incubation with % s. aureus virulence factors alone did not significantly alter the epithelial integrity assessed by the expression of tight junction proteins and transepithelial resistance measurement. however, when the cells were incubated with sm/fp alone or with % s. aureus virulence factors plus sm/fp, the expression of tight junction proteins was significantly increased (p< . ) as compared to cells incubated with s. aureus virulence factors. interestingly, the chloride efflux in airway epithelial cells was significantly decreased in a time-dependent way by s. aureus virulence factors ( fold decrease after a h incubation). the co-incubation of airway epithelial cells with s. aureus virulence factors and sm/fp prevented the s. aureus virulence factorsdependent chloride efflux decrease. our results demonstrate that bacterial virulence factors induce the loss of the electrolyte homeostasis and suggest that the treatment by the combination of a corticosteroid and a long-acting beta agonist may preserve the airway epithelial functionality. supported by association vaincre la mucoviscidose and glaxo-smithkline nilsson, h.e. ; dragomir, a. ; ahlander, a. ; johannesson, m. ; roomans, g.m. . medical cell biology, uppsala university, uppsala, sweden; . women's and children's health, uppsala university, uppsala, sweden inhalation of hyperosmotic solutions (salt, mannitol) has been used in the treatment of patients with cystic fibrosis or asthma, but the mechanism behind the effect of hyperosmotic solutions on mucociliary clearance (mcc) is unclear. one explanation has been suggested, namely that hypertonic solutions open tight junctions, which may lead to increased water transport followed by an increased airway surface liquid (asl) volume. furthermore, the role in cftr-mediated hco -conductance in regulating asl ph has led us to investigate if ph changes may have an effect on the tightness of tight junctions. the effect of osmolarity was investigated on the hbe o-cell line by the addition of nacl, nabr, licl, mannitol or xylitol ( - mosm). the effect of ph was investigated on the hbe o-, calu- and t cell lines as well as the cystic fibrosis cell line cfbe o-. transepithelial resistance was measured as indicator of the tightness of the cultures. cell-cell contacts and morphology were investigated by immuno-fluorescence and by transmission electron microscopy, with lanthanum nitrate added to the luminal side of the epithelium to investigate tight junction permeability. the electrolyte solutions caused a significant decrease in transepithelial resistance from mosm on, when the hyperosmolar exposure was gradually increased from to mosm, whereas the non-electrolyte solutions caused a decrease in transepithelial resistance from mosm on. immunofluorescence micrographs showed weaker staining for the proteins zo- , claudin- in treated samples compared to the control. the ultrastructure revealed an increased number of open tight junctions as well as a disturbed morphology with increasing osmolarity, and with electrolyte solutions opening a larger proportion of tight junctions than non-electrolyte solutions. it was noted that during the exposure of the cultured cells to the hyperosmolar solutions, the ph of the medium increased from . to . . hbe o-cells exposed to both a rise in ph and hyperosmotic stress showed an overall lower teer and a significantly reduced ability to recover from stress compared to cultures at a ph hold constant at . . without exposure to hyperosmolar solutions, a rise in ph caused a significant decrease in transepithelial resistance in hbe o-cells, calu- and t cell lines but not in the cfbe o-cell lines, where the reaction was significantly less and delayed. in conclusion, hyperosmolar solutions caused a reversible opening of the tj in hbe o-cell cultures with electrolytes having stronger effects than non-electrolytes, where one of the effects on mcc may be due to increased water transport across the leaky paracellular space. an increase in ph caused a significant decrease in teer in the healthy cell lines compared to the cfbe o-cell line. we speculated that an impaired alkalinisation of the apical fluid due to a defective cftr also will cause the tight junctions to react different to other external stimuli, such as osmolarity, compared to healthy cells, which is also indicated by preliminary experiments on the effect of nacl on teer in cfbe o-cultures. extracellular nucleotides regulate surfactant secretion in alveoli and mucociliary clearance in airway epithelia, but the mechanism(s) of their release and their regulatory pathways remain incompletely understood. previously, we showed that hypotonic swelling of a epithelial cells induces ca + -dependent secretion of several adenosine and uridine nucleotides, implicating regulated exocytosis. in this study, we examined sources of intracellular ca + ([ca + ] i ) elevation evoked by acute % hypotonic stress and the role of autocrine purinergic signaling in ca + -dependent atp release. we found that atp release does not directly involve ca + influx from extracellular spaces, but depends entirely on ca + mobilization from intracellular stores. the [ca + ] i response consisted of slowly-rising elevation representing mobilization from thapsigargin (tg)-insensitive stores and a superimposed rapid spike due to ca + release from tg-sensitive endoplasmic reticulum (er) stores. the latter could be abolished by hydrolysis of extracellular tri-and di-nucleotides with apyrase; blocking p y /p y receptors of a cells with suramin; blocking udp receptors (p y ) by ppads; emptying tg-sensitive stores downstream with µm tg or mm caffeine in ca + -free extracellular solution; or blocking the ca + -release inositol , , -triphosphate (ip ) receptor channel of the er with µm aminoethyl diphenylborinate. these results demonstrate that the rapid [ca + ] i spike results from the autocrine stimulation of ip )/ca + -coupled p y /p y receptors, which accounts for ~ % of total ca + -dependent atp release evoked by hypotonic shock. our study reveals a novel paradigm in which atp release is amplified by the synergistic autocrine/paracrine action of co-released uridine and adenosine nucleotides. we suggest that a similar mechanism of purinergic signal propagation operates in other cell types. (this study was supported in part by the canadian institutes of health research and the canadian cystic fibrosis foundation (ccff). s.t. was the recipient of a ccff studentship). the maintenance of a thin liquid layer on the airways and alveoli surfaces is essential for normal lung physiology.yet, the mechanism sensing the height of the layer remains obscure. in this study, we examined atp secretion from human lung a epithelial cell monolayers mounted in a closed, flowthrough chamber ( . mm height) and found that passage of an air bubble over the monolayer caused transient (< . min) but significant atp release ( to , pmoles/ cells). air bubble-induced atp release was reduced by~ % from cells loaded with the intracellular ca + chelator bapta-am, and was completely abolished in n-methylmalemide-treated ( mm) cells, suggesting the involvement of ca + -dependent exocytosis. fura- intracellular ca + ([ca + ] i ) imaging experiments revealed transient [ca + ] i elevation during the passage of an air bubble over the cell monolayer, but the [ca + ] i response did not involve non-specific ca + influx from the extracellular space, e.g. due to cell damage, since similar responses were observed in ca + -free extracellular solution. ethidium bromide staining did not disclose any cell damage in these experiments. confocal fluorescence microscopy study showed reversible cell deformation (flattening) of % to % in height in the monolayer part in contact with the air bubble and confirmed that cell integrity was entirely preserved. these experiments demonstrate that in close proximity to the air-liquid interface (i.e. between the air bubble and the wet cell surface), surface tension forces are transmitted directly on cells, causing their mechanical deformation, elevation of [ca + ] i and subsequent atp release. we propose that a similar mechanism may operate in vivo in the airways, where surface tension forces acting directly on exposed epithelial cell surfaces may provide a fail-proof mechanism to protect proper airway surface liquid volume via mechanosensitive, ca + -dependent atp release and purinergic modulation of fluid secretion. this mechanism may be defective in cystic fibrosis due to excessive mucus layer covering the airways. introduction : cystic fibrosis is a genetic disease that reflects the consequences of mutations in the cystic fibrosis transmembrane conductance reg-ulator (cftr) gene, affecting anionic transport in epithelia. slc family members are anionic transporters involved in cl-and hco -absorption or secretion in epithelia. in addition, the activation of some slc family members by cftr has been demonstrated (nature cell biology, , : - ) . slc a is a poorly characterized member of this family, being the solely expressed in lung. putative interaction domains with cftr are also present in the slc a protein. in this study, we have investigated the transport properties of slc a (human) to determine the functional and pharmacological characteristics of this transporter. methods : to this aim, electrophysiological studies (two-electrode voltage clamp or current clamp methods and intracellular ph measurements using ph sensitive microelectrode) were performed in xenopus laevis oocytes expressing slc a proteins. complementary ( cl) transport studies were also undertaken. results : the protein expression results in the appearance of an anionic current showing a linear current/voltage relationship. cl influxes experiments confirmed the induced cl-permeability following slc a protein expression. the sequences of conductivity, cl->i-> no -≥ gluconate > so -and selectivity (px/pcl), i-> cl-= no -> gluconate > so -are found. the hco -conductance mediated by the slc a protein expression is low. using co /hco -containing ringer solutions, no intracellular phi changes were detectable in conditions (low chloride in external medium) favoring the cl-/hco -exchange whereas phi changes (alkalinization) were observed with the expression of the ae exchanger. however, phi changes could be detected in conditions largely favoring the driving force for hco -entry. dids and ns inhibited slc a associated currents. the specific cftr inhibitor (cftrinh ) or glybenclamide had little effect. elevation of intracellular camp (a cftr activator) was also ineffective while maneuvers increasing intracellular calcium blocked the slc a associated currents. conclusions : our study demonstrates that slc a presents an electrogenic anion conductance (characteristics of an anionic channel) when expressed in xenopus oocytes (however a transporter functioning as a ncl-/hco -exchanger can not be excluded). this function (channel) has also been described for another member of this family, slc a (j biol. chem, , : - ) . the physiological role of slc a , present in the bronchial cells of airway epithelia and its potential interaction with cftr has to be precised in situ or in oocytes and in mammal expression systems. medicine, women's & children's hospital, nth adelaide, sa, australia; . cf research & treatment center, university of nth carolina at chapel hill, chapel hill, nc, usa; [ ] [ ] [ ] [ ] [ ] [ ] jasri, hyogo, japan; . physics and synchrotron science, monash university, clayton, vic, australia; . paediatrics, university of adelaide, adelaide, sa, australia non-invasive imaging of lung (e.g. hrct, mri, pet) is a valuable modality for detection and monitoring of the effects of cf in human airways, but resolution is limited; e.g. airway hrct detects airways no smaller than ~ . mm dia. detection of wall or lumen changes in smaller airways, or the detection and monitoring of induced airway disease in live rodent models, demands significantly greater resolution coupled with rapid image capture. we report substantially increased airway resolution is achievable using synchrotron phase-contrast x-ray and can produce three-dimensional reconstructions encompassing the smallest mouse lung airways. nembutal-anaesthetised c bl/ mice (~ gm) were imaged at the spring- synchrotron in hyogo, japan. -d phase-contrast airway images were obtained on ccd detectors ( . µm pixels) with a cm phase-contrast propagation distance at kev; nose and trachea were imaged at or sec intervals ( - ms exposures) over mins. mice killed with nembutal overdose were imaged in axially aligned segments to obtain dimensional ct data voume of approximately x x mm with x x um voxels of nose, trachea, and lung (hammamatsu ccd detector). volume renderings were produced using volview or osirix software. live, -d nasal or tracheal imaging revealed airway-surface activity in some live mice consistent with dynamic mucociliary clearance activity. individual ct slices revealed the fine detail in the mouse lung, with dynamic ("fly through") sequences of lung ct slices permitting visual identification and tracking of lung tree branching from trachea (~ mm dia) into small airways (approx um dia). adjustment of x-ray contrast, opacity, and range in these volume reconstructions permitted selective display of the mouse lung conducting-airway tree and airway surfaces. selected regions of the lung could be examined statically, or in rotation through different orientations in high-resolution -d. synchrotron phase contrast x-ray provides a new option for non-invasive imaging of intact mouse lung, at a resolution that permits examination of small conducting airways in mice. combined with volume-reconstruction software these ultract images can provide a powerful option for understanding the structure-function relationships produced by airway disease. ultra-ct -d studies are underway to compare lung airways at high resoltuion in normal and transgenic mice having altered airway pathophysiology. the epithelial sodium channel, enac, has a vital role in the function of the pulmonary epithelia and significantly contributes to the pathophysiology of the cf airway. thus, strategies to repair mutant cftr dysfunction must also consider the influence of such repair on enac functional expression. the ∆f trafficking repair agent -phenylbutyrate modulates the expression of the kda molecular chaperones hsc and hsp in cf epithelial cells. we therefore assessed the role of these chaperones in the regulation of enac trafficking. in xenopus oocytes, we previously observed that overexpression of hsc inhibits murine enac (menac) functional and surface expression. in contrast, hsp can either enhance or inhibit menac functional and surface expression depending on the extent of overexpression [goldfarb et al. ( ), proc. natl. acad. sci. : - ]. here, we tested the hypothesis that these differential effects of hsc and hsp on menac expression also occur in epithelial cells. mdck cell lines with stable expression of α-ha, β-v , γ-myc-menac and tetracyclineinducible expression of either hsc , atpase-deficient hsc , hsp or atpase-deficient hsp containing myc/his epitope-tags were selected. all epitopes were fused to the respective c-termini. these cells were grown as high-resistance (> Ω/cm ) monolayers on permeable supports. doxycycline-induced overexpression of hsc decreased amiloride-sensitive i sc and the whole cell content of α-haand β-v -menac. in contrast, lower amounts of tetracycline-induced hsp overexpression increased amiloridesensitive i sc and whole cell α-haand β-v -menac expression; these effects were absent at higher levels of hsp overexpression. these data are consistent with our previous data in xenopus oocytes. interestingly, the atpase-deficient chaperones had the opposite effect on menac functional expression. modest doxycycline-induced expression of atpase-deficient hsc increased amiloride-sensitive i sc in these cells, while modest overexpression of atpase-deficient hsp decreased amiloride-sensitive i sc . these effects may result from "dominant negative" interference with hsc and hsp function, respectively. these data are consistent with hsc and hsp having different effects on menac functional expression in epithelial cells, and that these effects are dependent upon the atpase activity of the respective chaperones. supported by grants from niddk. we have studied survival, airway epithelia bioelectrics and lung pathology of mice over-expressing various combinations of the epithelial na + channel (enac) subunits (α, β and γ, genes scnn a, scnn b and scnn g). we generated double-transgenic mice and crossed them with single-transgenic mice to obtain litters comprising all possible genotypes. survival analysis revealed that overexpression of βenac in combination with either αenac or γenac significantly reduced survival in comparison to wild-type (wt) littermates. strikingly, at days of age, all genotypes were represented according to the expected mendelian proportion, except for the triple transgenic αβγenac, which was significantly under represented. in utero studies are ongoing to understand if over-expression of αβγ affects the fetuses or the newborn pups during the first hours post-partum. due to the high mortality of mice over-expressing βenac subunit combinations, we studied tracheas from pups at days of age. as previously reported, mice overexpressing β, but not the α or γ enac subunits, exhibit airways hyperabsorption of na + and lung pathology (mall et all ) . the βenac tracheas exhibited na + absorption [as measured by the change in short circuit current (isc) in response to amiloride in the ussing chamber] that was ~ . fold greater (∆isc . ± . µa×cm - , n= , means±sem) than wt ( . ± . µa×cm - , n= ). over-expressing αβ or αγ enac subunits resulted in rates of na + absorption that did not differ significantly from those of βenac or γenac subunit alone, respectively. however over-expressing the β and γ enac subunits together resulted in an amiloride sensitive isc that was ~ . fold greater (∆isc . ± . µa×cm - , n= ) than wt. we also studied the tracheal bioelectrics of the few day-old αβγenac pups available. tracheas from the αbγ pups exhibited na + absorption that was ~ . fold greater (∆isc ± . µa×cm - , n= ) than wt. due to the small sample size, we cannot determine if this response is significantly different from the response of the βγenac pups. analysis of lung pathology in day-old pups revealed that all the combinations that exhibited increased airway na + absorption and decreased survival in comparison to wt littermates, e.g. αβ, βγ and αβγ, also presented with alveolar space enlargement, maybe due to postobstructive air trapping, and airway epithelia necrotic degeneration associated with ab-pas negative bronchial obstruction. from these data, it appears that the rate of airway na + absorption negatively correlates with the survival of the pups. thus, it is likely that the mucus and airway surface liquid is more dehydrated as a function of the rates of na + absorption (aβγ > βγ > αβ, β > wt) which produces gradual failure to survive due to asphyxia secondary to airway obstruction. supported by nih scor p hl choi, j. , ; joo, n. ; wu, j. ; krouse, m. ; wine, j.j. . cystic fibrosis research laboratory, , stanford, ca, usa; . otorhinolaryngology, yonsei university, seoul, south korea submucosal glands, which produce most airway mucus, are mainly controlled by parasympathetic pathways that stimulate glands via airway intrinsic neurons distributed along the airway walls. the intrinsic neurons can be stimulated via axon reflexes from receptors in the mucosa. in previous experiments, it was shown that capsaicin applied to the mucosa stimulated gland secretion in the upper tracheas of wt but not cftr -/-mice (ianowski et al., j physiol, , , ) . it seemed important to assess a possible role of capsaicin-sensitive pathways in pigs and humans. we obtained pig tracheas following acute experiments carried out for other reasons, and human airways following lung transplants from donor tracheas and from the excised lungs of the transplant recipients. secretion from individual submucosal glands was quantified optically by time-lapse digital imaging of the growth of spherical bubbles of mucus in an oil layer in humans, commercial chili oil ( µm dispersed in µm mineral oil) stimulated mucus secretion from submucosal glands from both donors (n = glands from subjects, . ± . nl/min/gland) and disease control subjects (n = glands from subjects, . ± . nl/min/gland). however, there were no responses to chili oil in submucosal glands from cf subjects (n = glands from subjects, . ± . nl/min/gland). in pigs, we determined concentrationresponse relations for gland secretion in response to purified capsaicin (sigma). the threshold was ~ µm and the ec was . ± . µm. the response to µm capsaicin was partially blocked by high dose ttx (> µm). as in humans, the capsaicin response required cftr, because it was blocked ~ % by cftrinh- , (n = glands, from pigs, p < . ). cftrinh- is presently the most specific inhibitor of cftr that can be used with glands, where we do not have access to the apical membrane. in ferrets, intrinsic airway neurons express acetylcholine, vip and sp, often colocalized within the same neurons (dey et al., am j respir cell mol biol, , , ) . in pigs, all three of these transmitter systems appear to be involved in gland secretion to capsaicin, because the response was partially blocked by the nk- receptor blocker l (oxalate salt, µm), the vip receptor inhibitor l k ( µm), and the muscarinic receptor blocker atropine ( µm); each of these blocked to % of the response (n = to glands from to pigs). indeed, most of the local pathways are probably damaged, which may account for the relatively small responses to strong stimulation. relatively few experiments have been carried out in intact airways with uninterrupted innervation and circulation, but in those experiments the glands were highly responsive to modest mucosal stimuli (reviewed in wine, auto neurosci, , , ) . hence, we hypothesize that ( ) reflex stimulation of glands plays an important role in lung innate defenses; ( ) defects in these responses is an important reason that cf airways are prone to infection, and ( ) local reflexes may assume greater importance for maintaining the mucosal defenses of transplanted lungs. supported by niddk ro - (jjw), cff and cfri. most airway mucus arises from submucosal glands, which express cftr in their ciliated ducts and in serous cells. the airway glands secrete to agonists that either increase intracellular ca + (e.g. acetylcholine) or camp (e.g. vip).substance p(sp) also stimulates airway gland liquid secretion in pig (trout l et al., am j physiol lung cell mol physiol. , ,:l ) , suggesting that sensory afferents or local neurons that express sp play a role in stimulating submucosal glands. we used pharmacological methods to dissect the secretory mechanism of sp-induced gland secretion in comparison with responses to carbachol. tracheas were obtained from pigs after acute surgeries carried out for other reasons. the ventral mucosa with underlying glands was dissected from the cartilage, pinned mucosal side up at the gas/bath interface of a physiological chamber, and covered with oil. secretions from individual glands could be visualized as spherical bubbles in the oil, and secretion rates determined by optical monitoring of bubble diameters. concentration-response relations for gland secretion were determined for sp ( - bubbles at each concentration from pigs). the threshold was ~ nm for sp and the ec was . ± . µm. the maximum secretion rate to sp was . ± . nl/min.gland, which is only ~ / of the maximal secretion rate to carbachol ( . ± . nl/min.gland). the inhibition profile for responses to sp ( µm) was quite different from those to carbachol ( nm, a value chose to mimic the secretion rate to µm sp). gland secretory responses to µm sp ( . ± . nl/min.gland, glands from pigs) were strongly inhibited by µm cftrinh- ( . ± . nl/min.gland, glands from pigs, p< . ), and by µm clotrimazole ( . ± . nl/min.gland, glands from pigs), whereas µm niflumic acid did not inhibit ( . ± . nl/min.gland, glands from pigs). in contrast, secretory response to nm carbachol ( . ± . nl/min.gland, glands from pigs) were only weakly inhibited by cftrinh- ( . ± . nl/min.gland, glands from pigs, p< . ), and by clotrimazole ( . ± . nl/min.gland, glands from pigs, p> . ), but niflumic acid, which was ineffective with sp, inhibited the carbachol response ( . ± . nl/min.gland, glands from pigs, p< . ). gland secretion to sp depends at least partially on intracellular ca + release, because the response was partially inhibited by µm bapta-am ( . ± . nl/min.gland, glands from pigs, p< . ). there was no additional effect of sp on top of µm carbachol, but a subthreshold level of sp ( nm) showed synergic response ( . ± . nl/min.gland, glands from pigs) with subthreshold level of vip ( nm). we are presently testing how sp activates cftr-dependent mucus secretion. in initial experiments to determine if pkc was being recruited, however the pkc inhibitor (gf , sigma) did not suppress sp-induced mucus secretion. in contrast, the nkcc inhibitor bumetanide ( µm) markedly suppressed secretion to sp ( . ± . nl/min.gland, glands from pigs, p< . ). supported by niddk ro - (jjw), cff and cfri. mucus obstruction of airways is considered the most vicious agent of morbidity and mortality in cystic fibrosis (cf). to provide optimal defense of the small airways the volume/thickness of airway surface fluid must be controlled according to physiological demand. to date we have no clear understandings of how these fluids are maintained in a steady state between two pathological extremes (too little or too much) in native small airways. earlier studies have attempted to measure the ion transport properties of small airways of sheep (al-bazzaz et. al. ) and pigs (ballard et. al. ), but the complicated branching structure of small airways may have compromised electrical signals. to avoid dissection trauma wang et. al. ( ) examined the electrophysiological properties of undissected intact small airways from pigs in vitro by microperfusing bronchioles (diam. - mm) embedded in the lung parenchyma; however, these studies were limited to luminal manipulations and transbronchiolar electrical potentials only. to more rigorously define small airway properties, we designed a special micro ussing chamber ( area ≈ mm ). we excised and opened small airways (diam. - mm) of pig lung that we mounted as a flat sheet over a pvc supporting filter ( - µm holes). both sides of the tissue could then be bathed with different solutions at °c. the luminal side was isolated by pressing a pipette (≈ mm diam., tip fire polished) on to the apical surface of the tissue until it sealed electrically. constant current pulses ( . - . µa) were passed across the tissue. transepithelial potential (tep) and resistance in bilateral mm nacl ringers were, - . ± . mv and . ± . Ω.cm (mean ± se) respectively. when mm naglu ringers replaced the luminal solution, tep increased significantly to - . ± . mv (n= , p< . ) and resistance increased to . ± . Ω.cm (n= , p< . . adding forkskolin (fsk, µm) plus ibmx ( . mm) hyperpolarized the tep to - . ± . mv (n= , p< . ), but decreased the resistance to . ± . Ω.cm (n= , p< . ). luminal amiloride ( µm) depolarized the tep to - . ± . mv (n= , p< . ) but significantly increased resistance to . ± . Ω.cm (n= , p< . ). these results show that this system supports measurements of ion transport properties of airways smaller than mm diameter where cf lung pathology is thought to originate. these airway epithelia express a very high constitutively active clconductance and are apparently capable of secretory as well as absorptive functions. amiloride sensitive na + conductance (≈ ms/cm ) was not significantly affected by fsk added to activate secretion. the observation that stimulation with fsk did not significantly suppress na + conductance (≈ ms/cm ) (p= . ) may suggest that separate groups of reabsorptive and secretory cells comprise small airway epithelia. mucins are class of proteins uniquely characterized by large glycosylated domains. these proteins likely play a key role in cystic fibrosis (cf) lung disease as major constituents of mucus. in airways of both humans and mice, the gel-forming secreted mucins are represented by muc ac and muc b, and the transmembrane mucins are represented primarily by muc , muc , and muc . we have been interested in the role of the transmembrane mucins in airways and have previously demonstrated that lack of muc in a knock-out mouse model (provided by s. gendler) shows increased susceptibility to adenoviral-mediated gene transfer (stonebraker, j. virology, ) . to expand upon these findings, mice deficient for muc were developed using embryonic stem cell technology by deleting exon of the mouse muc gene, which contains the atg start codon and signal peptide. rna analysis reveals loss of exon in homozygous mutant mice and immunohistochemistry using a vntr antibody indicated a loss of full length muc protein. muc homozygous mice are viable and do not develop any spontaneous, readily detectable disease phenotype. although the muc deficient mice do have reduced fertility consisting of both reduced litter size and number, all major tissue systems where the muc protein is easily detected, including the eye, respiratory tract, intestine, and reproductive tracts, appear normal in year old mice after histological analyses. in contrast to the results we reported in the muc mice, the muc deficient mice do not show an increase in adenoviral mediated gene transfer, suggesting that muc is not involved in the barrier aspect of the airway glycocalxy, at least for adenoviral infection. interestingly, quantitative real-time pcr analysis revealed up-regulation of muc protein in the trachea of the muc deficient mice, suggesting that muc may be able to compensate for loss of muc . the expression of muc in human ciliated cells as revealed by recent antibody studies prompted us to evaluate the potential role of muc in ciliary function. ciliary beat frequency (cbf) was monitored in a shear chamber using microscopy image analysis. cbf under baseline conditions for this study (low shear stress) from muc deficient trachea (c bl/ j congenic n ) was significantly lower than that for littermate controls ( . vs. . , sem ± . , p = . ). application of shear resulted in increased cbf in both groups, although the wt response to shear was smaller in magnitude. thus, muc may have a role in supporting basal cbf and may make cbf less vulnerable to stimulation by shear, perhaps by reducing the inherent friction of ciliary motion. this finding underscores the usefulness of this model for dissecting out the normal roles that the transmembrane mucins play in airway biology. supported by nih (hl ) and cff (r -cr ). enac (joo et al. jbc , and jbc , ) . in a search for a model to test the hypothesis we found that ussing chamber short-circuit current(i sc ) measurements with freshly obtained airway mucosa provided useful information about enac activity in response to various drug treatments. in these studies we observed an as yet unidentified pathway that regulates surface enac activity. airway mucosa from sheep tracheas displays a large, amiloride/benzamil-sensitive i sc prior to stimulation. when stimulated with basolateral carbachol, the enac i sc was almost abolished, as indicated by a sustained decrease ( . ± . %, n= ) in i sc after an initial increase and left a small effect to subsequent amiloride or benzamil. in contrast, when carbachol was given after benzamil, it again produced a peak response followed by a sustained increase (~ %, n= ) in the i sc . enac inhibition by carbachol was dose dependent with a minimal sensitivity between . ~ µm and was slowly reversible. apical µm atp also inhibited enac i sc by . ± . % (n= ) and carbachol on top of atp further reduced the enac i sc by . ± . % (n= ). however, u/ml of apyrase pretreatment, in attempt to breakdown atp, failed to inhibit carbachol-induced enac inhibition, indicating that atp release is not on obligatory component of carbachol inhibition of enac. carbachol inhibition of enac could result from a direct action of carbachol on the surface epithelia, by activation of airway intrinsic neurons, or by activation of the glands, which were intact in these preparations and are strongly stimulated by carbachol. rabbit tracheas, which lack airway submucosal glands, displayed large enac currents and these were not inhibited by cholinergic stimulation (n= ). carbachol-induced enac inhibition, like gland stimulation but unlike stimulation of neurons, was mediated by muscarinic receptors since it was eliminated by atropine but not by the nicotinic receptor antagonist, hexomethonium bromide (n= ). however, inhibition of gland secretion with hepes/bumetanide did not eliminate the ability of carbachol to inhibit enac. the effects of carbachol on surface na + transport in our study are consistent with previous observations in microperfused sheep bronchiole preparations (al-bazzaz, ajp-lung , ). although we do not yet know the mechanism, it would be physiologically efficient if parasympathetic, cholinergic activation of airway gland secretion was accompanied by enac inhibition, which would minimize the fluid absorption by surface epithelia and increase clearance. it will be important to determine if cholinergic inhibition of enac is present in human tissues, and if so, if it is defective in cf. supported by cff and nih. introduction: lung disease accounts for more than % of the current morbidity and mortality associated with cf. the majority of research aiming to elucidate the reason(s) of severe lung disease has focused on airway surface liquid (asl) layer homeostasis or composition. the cftr gene encodes a camp-regulated clchannel located on the apical side of the epithelium and is instrumental in maintaining a hydrated asl layer and promoting effective mucociliary clearance (mcc). in patients with class i or class ii cftr genetic mutations, normal cftr clchannel activity in the epithelium is impaired resulting in na + hyperabsorption and consequently in the generation of a thick and static overlaying asl layer creating an environment that favors progressively worsening bacterial infections. in this study we investigated the potential contributory role of altered respiratory cilia function to inefficient mcc in cf airways allowing for bacterial infestations. our ex vivo model system was the nasal airways of the cftr gene knockout (cftr tm unc ) mouse in which key aspects of the phenotype of human cf lung disease, such as dehydrated asl layer and goblet hyperplasia, are observed. methods and results: ciliary beat frequency (cbf) was measured using a dual temperature controlled perfusion chamber, differential interference contrast microscopy and high speed digital video analysis system. we evaluated the cbf of nasal septa explants from homozygote cf (affected) and non-affected littermate wild type (wt) and heterozygote mice congenic on the c bl/ background. the basal cbf of homozygote cf mice ( ± hz, n= ) was significantly lower than that of wt mice ( ± hz, n= ) and also heterozygote mice ( ± hz, n= ) (p< . , anova, snk test). to determine whether the dehydrated asl layer was still present on the surface of the cf mouse nasal airway epithelium and as such impeding normal cilial beating, we fixed nasal septa (n= ) of cf and wt mice immediately following isolation using the pfc/oso fixation method to preserve the fragile asl layer and compared the height of the asl layer to nasal septa (n= ) from cf and wt mice fixed with pfc/oso immediately following cbf analysis. electron microscopy analysis revealed that although the asl layer was present at the time of isolation, there was no asl layer following the perfusion of solutions necessary for the cbf measurements. this finding demonstrated that the activity of cilia was not impaired in the cf nasal airway epithelium due to the presence of a dehydrated asl layer. cbf frequency was also significantly reduced in three different codes of mouse ciliated nasal airway epithelial cultures derived from affected cf mice compared to those derived from non-affected wt mice. conclusion: we show that the absence of the cftr gene product in the cf knockout mouse nasal airway epithelium is associated with significantly decreased basal cbf compared to the non-affected wt and heterozygote mice. this observation potentially provides an additional mechanism as to why lung disease persists in human cf lungs. ml and mba contributed equally. clinical studies have linked increased sputum and peripheral blood neutrophil mpo activity with increased airflow obstruction in cystic fibrosis (cf) patients of the same age, gender, airway bacterial flora, and cftr genotype. variations in the tgf-β gene associated with increased tgf-β production have been linked to worse airflow obstruction in cf patients of similar age, gender and cftr genotype. we hypothesized that in the presence of mpo, tgf-β production would increase in airway epithelial cells. we obtained normal human airway epithelial cells (hae) and cultured them in trans-wells. tgf-β mrna was measured by pcr. tgf-β protein was detected by immunofluorescence staining and immunoprecipitation. we found increased tgf-β mrna after hae were exposed to mpo (at activities found in cf sputum) normalized to mrna of constitutive beta actin. under identical conditions, tgf-β protein expression was increased in hae. we conclude that neutrophil mpo present on the apical surface of cultured hae induces transcription and protein synthesis of tgf-β . these results suggest that mpo induction of airway epithelial tgf-β is one molecular system linking the chronic neutrophilic endobronchitis seen in cf and subsequent airway wall fibrosis. are more oxidatively stressed than normal. as oxidative stress (os) is a potent activator of nf-κb, these data supported our hypothesis that cftr dysfunction causes alterations in the expression and/or modification of redox related proteins thereby enhancing activation of nf-κb. delineation of this interaction will produce good therapeutic targets. in this report we examine the role of os in cf inflammation. methods: to address our hypothesis, we used both in vitro and in vivo models of cf. our airway epithelial cell models are the hteo-pcep and pcep-r cell pair; and the hbeo-sense (s) and antisense (as) cell pair. additionally, we examined whole lungs from - week old b . s -cftr tm mrc mice (r h mutants back-crossed into the c bl j background). proteins were prepared from homogenates of whole samples, run on -d gels, and their densities compared. the gel spots of differentially expressed proteins were excised, subjected to in-gel trypsin digestion, and identified by lc ms/ms. for biochemical analysis, we tested for the levels of h o , glutathione, and lipid peroxidation. we correlated these data with measurements of nf-κb activity (elisa and cytokine production), and the activity of nrf- , an antioxidant (ao) response pathway transcription factor. we studied cultured cells in the presence and absence of inflammatory stimulation (tnf-α/il- β), and compared non-stimulated cf mouse lungs to lungs from normal litter mates. results: our proteomic analysis revealed that in the absence of inflammatory stimulation, a paradoxical decrease in ao proteins exists in cf cells by or more fold compared to matched pairs. this is despite a significant increase in intracellular os, which we confirmed by different biochemical measures. activity of nrf- was decreased by ~ % in cf cells vs. normal. the data predict an increase in h o , which is a potent activator of nf-κb, and we confirmed both a significant elevation in h o (up - fold in cf) and a related significant increase in nf-κb activity, as assessed by elisa for nuclear p , and il- and il- production. when we analyzed whole lungs from cf mice for antioxidant protein levels, we similarly observed decreases vs. normal littermates. when cells were stimulated, differences in protein expression and oxidative stress between cf and normal were further enhanced, corresponding to an excessive activation of nf-κb and increased production of il- and il- . treatment with the antioxidants nac or selenium decreased the activation of nf-κb to normal levels. members of the clc family of proteins form either voltage-gated chloride channels or cl -/h + exchangers; nine subtypes are found in mammals. among these, clc- is one of the most ubiquitous variants, which is expressed in several of the types of epithelial cells that also express the cystic fibrosis transmembrane conductance regulator (cftr). it has been postulated that clc- may be a suitable alternative chloride pathway in these cells; however the contribution of clc- to chloride transport in these cells is poorly understood. this is partly due to the lack of appropriate pharmacological tools that are capable of specifically inhibiting clc- . we describe here the isolation, from venom of the scorpion leiurus quinquestriatus hebraeus, of the first peptide inhibitor of any clc channel. this toxin, which we have named gatx , specifically inhibits the clc- channel. gatx bears primary sequence identical to that of a toxin thought to serve as a k + channel inhibitor, although no molecular target for this toxin was previously known. a homology model of gatx reveals that it adopts a similar fold to other scorpion toxins. gatx was prepared via solid-phase chemical synthesis, and the synthetic toxin inhibits clc- with higher affinity than any other inhibitor of clc- or any other chloride channel. the k d for gatx mediated inhibition of clc- in multichannel patches is only pm at v m = - mv, and pm at - mv. single channel experiments show that gatx does not alter single channel amplitude, but may alter channel gating. these experiments provide the basis for developing gatx into a useful tool that can be used to define the role of clc- in airway and intestinal epithelial cells. furthermore, the high affinity and specificity of inhibition suggest that gatx interacts intimately with the channel, and thus may the exploitation of alternative chloride channels to bypass the defect in cftr-mediated chloride secretion is an established therapeutic strategy in cf. in search of such potential therapeutic targets, we assessed camp/pkadependent chloride secretion across excised nasal epithelium of cftr null mice and wildtype controls, by measuring short-circuit currents (isc) in an ussing chamber set-up. all experiments were performed in the presence of amiloride to block the contribution of epithelial sodium channels to the isc. in cftr null mice of mixed background (c bl/ ; ), the camp agonist forskolin activated a bumetanide-sensitive rise in isc that was comparable in magnitude to the cftr-mediated isc response of littermate controls ( ± vs. ± µa/cm , respectively). the forskolin-induced isc in these null mice was inhibited by the general chloride channel inhibitor dpc, but appeared insensitive to the cftr inhibitors cftrinh- and glibenclamide, the orcc and calcium-activated chloride channel inhibitor dids, and the clc- inhibitors cadmium and pre-activated omeprazol. moreover, the isc response to forskolin greatly exceeded the response to the calciumlinked agonists ionomycin and carbachol. intriguingly, in nasal epithelium of cftr null mice with a different genetic background (fvb), no such forskolin-inducible secretory pathway was evident (cftr-/-: ± vs. cftr+/+: ± µa/cm ). when, in nasal epithelium of cftr null mice (c bl/ ; ), the basolateral membrane electrical resistance was negated by nystatin treatment, and in the presence of a transepithelial chloride gradient, forskolin failed to elicit a rise in isc, suggesting that camp/pka signaling targets a basolaterally located ion transport system. indeed, in intact epithelium, addition of chromanol b, a selective inhibitor of camp/pka-activated potassium channels, to the serosal bath, blocked the forskolin-induced rise in isc. we tentatively conclude that the hyperpolarization of nasal epithelial cells, which ensues from camp/pka-induced potassium efflux across the basolateral membrane, drives chloride exit across the apical membrane via a constitutively active anion conductive pathway that is absent from fvb cftr null mice. rt-pcr and immunostaining demonstrated that, among the candidate anion channels that fit the above pharmacological profile, clc-ka, but not clc-kb, was expressed in nasal epithelium of both cftr null mice and controls (c bl/ ; ), together with the accessory protein barttin. importantly, immunostaining suggested a strong increase in clc-k protein abundance in the cftr null mice, as compared to wildtype animals. on the basis of these findings, we postulate that clc-ka channels may serve as cftr bypass channels in nasal epithelium of cftr null mice that, albeit indirectly, are responsive to camp/pka signaling. future studies aim to characterize clc-ka and barttin in the airways from cf patients and controls, and to identify the post-transcriptional mechanism resulting in the up-regulation of clc-ka protein in cftr null mice. in the disease cystic fibrosis (cf), the most common mutation f del results in endoplasmic reticulum retention of misfolded cf gene proteins (cftr). the endoplasmic reticulum (er) f del-cftr protein retention is dependent upon chaperone proteins, many of which require ca + for optimal activity. an increase in cytosolic free ca + concentration is dependent on the activity of ion channels in the membrane of er and on extracellular ca + influx. this influx, named capacitative ca + entry (cce), induced by calcium store depletion, represents the major ca + influx mechanism in cells (putney et al., ) . during the cce, ca + influx can be mediated by one or several of the isoforms of transient receptor potential canonical channels (trpc) (putney et al., ) . aims & methods: in the present work, we studied the cce in human cf tracheal gland serous cfkm cells compared to non cf human tracheal mm cells. for this, we characterized the molecular identity of trpc channels responsible of cce by rt pcr technique and we compared global ca + influx in cf and non cf cells by using fluo -am probes. moreover, we measured the single channel activity of plasma membrane trpc channels by cellattached patch-clamp configuration in cf and non cf cells. we also studied the cce in cf cells after correction of the abnormal f del-cftr trafficking by miglustat ( h at µm of n-butyldeoxynojirimycin, nb-dnj) (norez et al., ) and low temperature ( h at °c) (denning et al., ) . results: we detected by rt-pcr technique the presence of trpc and trpc in both cell lines and we observed that cce was increased -fold in cf-km cells compare to non-cf mm cells. following stimulation by histamine, we recorded elementary ca + currents with similar amplitude ( . pa at - mv) and conductance ( ps) whatever the cells tested (cf-km or mm cells). however, we observed a -fold increase of the npo (number of channels in each patch × open probability of single channel) in cf cells compare to non-cf cells. in f del-cftr corrected-cf cells, the ca + influx stimulated by the ca + stores depletion was reduced to a similar level to non cf mm cells. the permanent contact of the airway epithelium with the external milieu induces frequent injuries caused by inhaled pathogens and particles. regeneration of the wounded airway epithelium needs to respect its prior structure in order to restore its defence functions. the underlying molecular mechanisms associated with the regeneration of the human airway epithelium are poorly described. the aim of our study was to determine, using a global expression approach, the transcriptional profile of human airway cells during the epithelial regeneration process. human airway epithelial cells collected from nasal polyps were seeded on type iv collagen-coated porous membranes, cultured up to confluence in liquid-liquid(ll) conditions, then put at the air-liquid interface (ali) allowing epithelial mucociliary differentiation. rna were extracted from epithelial cells at % confluence in ll conditions (epithelial cell proliferation and migration stage, step i), after days in ali conditions (beginning of epithelial cell differentiation, step ii), when first ciliated cells appeared (step iii) and when cultures were completely differentiated (step iv). our results demonstrated that between step i and step ii, genes were significantly modulated (ratio r<- or r> , p< . ), among them were activated and were repressed. between step ii and step iii, transcripts were up-regulated (tubulin, dynein, stath, microtubule associated proteins…) and only one gene (fatty acid desaturase- ) was repressed. finally, between step iii and step iv, except stath which was~ -fold down-regulated, we observed only few genes differentially and slightly modulated. all along the regeneration process, between step i and step iv, genes were significantly modulated with % of activated and % of repressed genes, among them a family of genes encoding proteins involved in the ciliary differentiation (foxj , tektin, dynein, tubulin, …), extracellular matrix proteins (collagen, laminin, …) and inflammatory-related genes like cytokines, growth factors and their receptors (ccl , vegfc, csf , il , il ra …). by elucidating the specific transcriptome involved in each step of the human airway epithelium regeneration, our study could allow to better understand the different cellular and molecular mechanisms, as well as the chronological events involved in these processes, and will further be applied to the study of the regeneration process in cystic fibrosis airway epithelium. supported by french association vaincre la mucoviscidose. electrolytes may be transported either through cells, the transcellular pathway, or between cells, the paracellular pathway. most research has focused on the channels and transporters of the transcellular pathway, while much less is known about paracellular transport. paracellular transport is regulated by tight junctions. claudins, the main component of tight junctions, are necessary and sufficient for tight junction formation and are important in establishing ion selectivity. the goal of this study was to further investigate the role of tight junctions in human airway epithelia. real-time pcr identified claudins , , , , , and in primary human airway epithelia. immunocytochemistry localized claudins , , , and to tight junctions. to measure the relative ion selectivity of tight junctions, we studied well-differentiated human airway epithelia in ussing chambers. to minimize transcellular transport, we used cf epithelia, which lack cftr and blocked enac with amiloride. we found the pna/pcl is . ± . , suggesting tight juctions are more permeable to cations than anions. the relative cation selectivity was na+ > k+ > cs+ > tma+ > mg + and the relative anion selectivity was cl-> br-≥ i-> no -> gluconate-. we compared these in vitro results to in vivo studies in people with cf and measured nasal voltage in the presence of amiloride. we found the pna/pcl is . ± . . tight junctions were slightly more selective for cations than anions in vitro and in vivo. to determine the effect of specific claudins on the selectivity of the paracellular pathway, primary human airway epithelia were infected with adenovirus expressing claudin or gfp. epithelia overexpressing claudin had a higher transepithelial resistance (ter) and the pna/pcl was . . however, infection efficiency was only %. this lead us to investigate if transformed human airway epithelial cells lines may be a better model to study permeability. cufi cells also express claudins , , , , , and . furthermore, like the primary human airway, cufi cells are more selective for cations; pna/pcl = . ± . . future studies will investigate the effect of claudin expression on the paracellular permeability of these cells. the results will help identify the properties of a pathway critical to epithelial transport and barrier functions. altered adenosine (ado) signaling is associated with chronic lung disease and increased asl ado levels are thought to drive inflammation by stimulation of a b receptors (a b-r) in airway epithelia, which can activate nfκb. when measured by lavage, asl ado is ≤ nm. due to its relatively low affinity to ado, a b-r are thought to be inactive under normal conditions (ec is ~ . µm), and only stimulated during chronic lung disease. however, we have previously demonstrated that the ado-receptor (ado-r) antagonist -spt inhibits asl volume regulation, and that a b-r is in close proximity to cftr. thus, we hypothesized that the asl ado concentration close to the apical membrane is higher than measured by asl lavage in bulk solution. we tested this hypothesis by looking for ado-r expression and function in well-differentiated human bronchial epithelia cultures (hbecs) and in vivo. by pcr analysis, we found that a b-r was the only ado-r expressed in hbecs. we then measured asl height over h ± ado-r antagonists by xz confocal microcopy when asl height was preset to ~ µm at t= . at µm, caffeine is a non-specific ado-r antagonist, which inhibited asl volume regulation ( h asl height was . µm vs. . µm for controls, n= ). similar inhibition was observed with alloxazine, which is relatively specific for a b-r (n= ). in contrast, dpcpx, zm & mrs , which inhibit a , a a & a -r respectively had no effect on asl volume regulation. asl height regulation was also ablated by the addition of adenosine deaminase (ada) to the asl (n = ). we next attempted to recapitulate the changes in asl height seen under control conditions, i.e. increase from to µm over the initial h followed by a plateau (the remainder of the h) by adding the non-hydrolyzable ado analogue neca to the asl in the presence of ada. nm neca was insufficient to regulate asl volume and height remained at µm over h. in contrast, addition of µm neca to the asl resulted asl volume regulation that was indistinguishable from control conditions (n= ), suggesting that near-membrane asl is much higher than has been measured by lavage. to confirm that a b-r stimulates cftr chloride secretion, we measured transepithelial potential differences of hbecs mounted in ussing chambers. a dose-dependent increase in cl-secretion was observed by ado addition with an ec of . +/- . µm, similar to the concentration of neca required to regulate asl height. to demonstrate which ado-r are expressed in human airways in vivo, we used laser capture to obtain ciliated cells from frozen tissue sections of excised donor bronchi. relative expression of a b-r by qpcr was found to be . +/- . fold higher than the a , a a-r, or a receptors (n= ). to confirm that ado-r activate cftr in vivo, we measured nasal potential differences. we found that ado activated chloride secretion at similar levels to isoproterenol. the effect of ado was inhibited by pre-exposure to µm caffeine (ado, . +/- . mv; ado + caffeine, . +/- . mv). we conclude that a b-r has an important role in asl homeostasis through regulation of cftr and it is active in human airway epithelia under normal physiological conditions. through osmotic forces, hypertonic saline (hs) may increase the volume of cf airway surface liquid, restore mucus clearance, and improve lung function. the na + channel antagonist amiloride is predicted to increase the duration of action of hs. however, patients taking amiloride with hs showed less clinical benefits than patients taking placebo with hs. in vitro data suggested that amiloride inhibits transepithelial water flux (pf) in cystic fibrosis airway epithelia. we have extended this study by looking at the effects of amiloride on aquaporins in a heterologous expression system (mdck cells) and have used immunofluorescence to determine which aquaporins are expressed in human airways in vivo and in vitro. inhibition of enac by aprotinin pretreatment had no effect on hs-induced pf (n= ), suggesting that amiloride did not exert its actions on pf by inhibiting enac. however, since hs-induced pf was also inhibited with hgcl , we speculated that amiloride directly blocked an aquaporin. to test this hypothesis, we measured the rate of cell shrinkage of wild-type mdck cells, and those stably-expressing aqp or , or expressing aqp (after h exposure to a mildly hypertonic media; mosm). following mucosal exposure to a mosm hypertonic ringer solution, mdck cultures grown to confluency on permeable filters shrunk by % after s (n = ), as measured by xz confocal microscopy following staining with calcein-am. in contrast mdck/aqp or mdck/aqp cultures shrunk by % over the same period and mdck/aqp cultures shrunk by % (all n= ). a min pretreatment with µm amiloride abolished hypertonic ringer-induced cell shrinkage in mdck/aqp cultures ( % at s; n= ) but had no affect on cell shrinkage in aqp or aqp expressing cultures. amiloride is fluorescent and can be imaged in epithelia by xz confocal scanning under uv excitation. amiloride was not internalized immediately (< s) before hypertonic ringer addition and did not block aqp -mediated cell shrinkage ( % at s; n = ). however, a min pretreatment with amiloride caused it be internalized and at this time aqp -induced cell shrinkage was blocked, suggesting that amiloride blocks aqp intracellularly. to confirm that aqp is present in airway epithelia, we performed immunostaining with an aqp antibody and found that aqp is highly expressed at the apical membrane of ciliated cells in human bronchial tissue and well-differentiated bronchial cultures. we conclude that amiloride blocks aqp , which is abundantly expressed in the superficial airway epithelia. supported we have previously observed that cftr, annexin (a ) and cytosolic phospholipase a (cpla ) are partially recruited in detergent insoluble microdomains (dim) upon proinflammatory stimulation. this is prevented by specific inhibition of cftr. a participates in the regulation of inflammation by inhibiting cpla activity. we have also shown that a expression is decreased in cftr-/-mouse tissues. we postulate that cftr, a and cpla participate in the regulation of inflammation by their dynamic interaction within dim. in this study we aimed to (i) demonstrate and characterize this interaction, and (ii) identify other potential partners of this complex in dim. by coimmunoprecipitation, we examined the interaction between cftr, a and cpla in human pulmonary epithelial cells calu . to search for additional partners we developed a new proteomic approach based on double sds-page (dsds-page), which is compatible with membrane protein analysis. calu cells and human epithelial bronchial cfbe cells (expressing either wt or f delcftr) were subjected or not to proinflammatory conditions (tnfα). dim were isolated by density gradient and analyzed by dsds-page. cftr immunoprecipitated with a and cpla in calu cells. dsds-page analyses showed significant differential expression of cytokeratin , actin and protein disulfide isomerase (pdi, involved in protein folding) in dim between control and proinflammatory conditions in calu cells, and between wt and f del cfbe cells, as determined by mass spectrometry. in conclusion, cftr, a and cpla interact in basal conditions. proinflammatory treatment causes differential expression of cytoskeletal proteins and pdi depending on the expression of normal and mutated cftr. this work suggests that cftr could participate in the regulation of inflammation within a complex with a and cpla . cytoskeletal proteins and pdi could be involved in the dynamics of this complex. this could be related to the abnormal inflammatory response characteristic of cf. we acute and chronic inflammation, common features of cf airways disease, have been linked to higher atp release and changes in atp metabolic patterns in vivo, e.g., in induced sputum and exhaled breath condensate from cf patients and bronchoalveolar lavage fluid from inflamed lobes (esther, ped pulm sppl , ) . we, therefore, investigated the contribution of airway epithelia per se to the raised atp concentrations on cf airway surface during inflammation. when exposed to supernatant of mucopurulent material (smm) from cf airways for h, or infected with respiratory syncytial virus (rsv) h prior to the study, basal atp release from well-differentiated primary human bronchial epithelial (hbe) cultures did not differ from that of control cultures. however, basal atp release was increased weeks after rsv infection when inflammation had waned (as indexed by il- secretion). the increase in basal atp release correlated with increased basal udp-glucose and mucin release, and goblet cell metaplasia. these data indicate that the increased basal atp release following rsv infection reflected increased goblet cell secretion. a different pattern was observed for stimulated atp release. hbes exposed to smm for h, or infected with rsv h prior, released - times more atp following hypotonic challenge than control cultures (okada, ped pulm sppl, ) . the increase in peak atp concentrations correlated with the degree of inflammation as indexed by il- secretion and the increase in intracellular calcium (ca + i ) mobilization in response to hypotonic challenge, but not with the goblet cell number. ca + i mobilization in response to hypotonic challenge in control hbes was almost completely due to extracellular ca + i influx which was inhibited by antagonists of a transient receptor potential v (trpv ) ca + channel. in contrast, an additional component of atp-induced ca + i release from endoplasmic reticulum (er) was observed in smm-treated hbes. the increase in hypotonicity-induced atp release in smm-treated hbes compared to control hbes was almost completely sensitive to chelation of ca + i by bapta ( µm; h), or inhibition of exocytosis by brefeldin a ( µm; . h), monensin ( µm; h) or n-ethylmaleimide ( mm; min), whereas these reagents were ineffective against hypotonicity-induced atp release from control hbes. these data suggest a positive feedback loop between the increased atp release linked to ca + i -dependent exocytosis and the increase in atp-induced ca + i mobolization linked to increased er ca + i stores (ribeiro, jbc , ) in inflamed hbes. in conclusion, we propose that basal and hypotonicity-induced atp release from hbes are regulated in distinct mechanisms during inflammation. basal atp release reflects continuous exocytosis processes independent of ca + i mobilization and differs as a function of cell type (ciliated vs goblet cell), whereas acute inflammation upregulates hypotonicity-induced atp release via increased ca + i mobilization and ca + i -dependent exocytosis. we conclude that airway epithelia exhibit multiple mechanisms of increasing atp release into the lumen in response to inflammation. supported by cff grant okada i to sfo and grants from nih. the main biological role of vitamin d is in control of bone and mineral metabolism, but it has also been discovered to have many immune system functions. vitamin d, a fat soluble vitamin, is poorly absorbed in cf leading to low serum levels. we sought to investigate the involvement of vitamin d in the control of inflammation in a cf model. methods cf tracheal epithelial cells (cfte) were pretreated with vitamin d ( , [oh] d ) or control and exposed to p aeruginosa lipopolysaccharide (lps) for hrs before collecting the cell supernatants. cytokine levels in the supernatants were assessed by cytokine array and subsequent elisas specific for il- , il- and mcp- . vitamin d was measured in the plasma of children with cystic fibrosis aged months to years. neutrophil numbers, neutrophil elastase (ne) activity and il- concentrations were measured in the bronchoalveolar lavage (bal) of these children. children with cystic fibrosis were deemed to be non-colonised, intermittently colonised or chronically colonised with p aeruginosa on the basis of the leeds criteria. pretreatment with vitamin d significantly reduced secretion of il- , il- and mcp- from cftes after challenge with p aeruginosa lps in a dose dependant manner. plasma vitamin d levels were highest in the noncolonised group and lowest in the chronically colonised group with intermediate levels in the intermittently colonised group. bal neutrophil counts, ne activity and il- concentration showed a reciprocal pattern. conclusions vitamin d acts on cf lung epithelial cells to protect against excessive cytokine production in response to lps. levels of vitamin d are inversely related to both p aeruginosa colonisation status and bal markers of inflammation. low levels of vitamin d may contribute to advancing lung disease in children with cystic fibrosis. more aggressive correction of vitamin d deficiency may have a role in protecting against excessive inflammatory response to infection. sleeping in a mist tent has been used as a treatment for cystic fibrosis (cf) patients, in order to hydrate the viscous mucus and make it easier to be removed by coughing (matthews et al., ) . however, the efficiency of the method has been questioned, and its use was largely discontinued. with a new method to measure the ion content of human nasal fluid the effect of sleeping in a moisture tent on the ion content of nasal airway surface liquid (asl) in cf-patients and healthy controls was determined. the cf-patients and healthy controls spent a night ( h) in a mist tent, and samples of the nasal asl were taken before the experiment, after the period in the tent, and then at each hour during h after the persons had left the tent. samples of nasal fluid were collected on sephadex g- beads that had been mounted on strips of filter paper, which were then put into the nasal cavity of the cf patients or controls for - min. the strips were removed, and the beads were isolated, dried, and analyzed by x-ray microanalysis (vanthanouvong et al., ) . the concentration of na, cl, and k in the nasal fluid of cf patients was , , and mm, respectively, before the patient entered the tent, significantly higher than the levels in the nasal fluid of the controls (na mm, cl mm, and k mm). during the period in the tent, the ion content decreased, to levels of na mm, cl mm, and k mm (cfpatients) and na mm, cl mm, and k mm (controls) immediately after leaving the tent. after leaving the mist tent the ion levels in the nasal fluid increased, reaching after h values of na mm, cl mm, and k mm (cf-patients) and na mm, cl mm and k mm (controls), which was for both groups higher than before entering the tent. no major changes in the ion content of the asl occurred after h. the salt content of the asl may be relevant in cf, since the asl is known to contain anti-bacterial proteins, defensins, which are sensitive to the salt concentration (bals et al., ) . hence, the higher salt concentra-tions in the asl of cf-patients may have negative consequences for the anti-bacterial defense system in the lungs, and conversely, the decrease in ion concentrations, caused by spending time in a mist tent, may have positive effects. (we have previously shown that none of our patients was chronically colonized with pseudomonas aeruginosa while regularly sleeping in mist tents.) however, with currently used procedures, the effect of sleeping in a mist tent on the ion content of the nasal asl is short-lived. bals background: antibody microarrays for clinical applications have been anticipated for more than a decade but the technology has only recently become sufficiently mature for reproducible, robust detection of low abundance proteins. the advantage of this new technology is the high throughput, parallel detection of identified, known, low abundance proteins. antibody microarray results are usually given in terms of ratios between two samples (e.g., "experiment" and "control"). however, given the printing and calibration issues inherent with antibody microarrays, this ratio'ing approach precludes statistically valid inter-array comparisons of the individual protein concentrations. we have therefore developed and implemented a different calibration strategy using a benchmark mixture that is applied to every array as an internal standard, thus controlling for any differences in antibody activities as well as printing imperfections. the amount of protein bound to each spot is determined relative to the respective protein in the internal standard. since all samples in the study are compared to the same internal standard, this semi-quantitative approach permits the comparison of multiple samples. all data for the same antibody on multiple arrays can then be used to calculate the average and standard deviation for a given population (such as patients or control groups) in a parallel fashion. methods: cf lung epithelial cultured cells ib - and the cftrrepaired ib - /s were biosynthetically labeled with a hour pulse of [s]methionine, and then chased with cold methionine for , or hours. we then isolated total protein from each sample, and labeled the proteins with cy . labeled proteins were applied to clontech® antibody arrays ( antibodies printed in duplicates) according to the manufacturer's protocols. the internal standard, consisting of a mixture of cell cultures and tissue extracts known to contain the cognate protein for each antibody on the array, was labeled with cy . the data were then used to compare the cf and the corrected cells in terms of amount, biosynthesis rate and degradation half-life (t / ) for each one of the features on the array. data analysis: for each spot on the array the ratio of cy /cy (after background subtraction) was calculated, normalized and stored for further analysis. for "pulse-chase" experiments the ratio of [s] counts to cy at each time point gives a measure of the fraction of newly synthesized (radiolabeled) to total protein (dye labeled) bound to each given antibody on the array. the addition of the cy labeled internal standard enables the calibration of these totals, allowing the comparison all the spots for any given antibody on multiple arrays. furthermore, the internal standard calibration makes it possible to compare not only t / values but also to calculate the relative degradation rates in different cell lines even when the amounts of that protein are different in the "control" and "experiment" cells. the new approach quantitatively identifies and subjects to analysis the effect of the cftr mutation on protein biosynthesis and degradation of the cellular signaling proteome. supported by nih no -hv- (hbp) and ro - (hbp) in cf patients, defective apical chloride secretion, due to lack of functional cftr, leads to dehydration of airway surface liquid (asl) and mucus plugging. this defect is likely ameliorated, in part, by an alternative route of chloride conductance, the calcium activated chloride channel (cacc) . female gender appears to be associated with more rapid decline of lung function and earlier death in cf. this suggests that gender differences in hormonal environments might influence asl height regulation, thus modifying the pulmonary phenotype. we hypothesized that changes in estrogen concentrations might alter asl hydration by affecting cacc. consistent with this, we have shown that estrogen attenuates increases in intracellular calcium and asl height that follow purinergic receptor activation in primary cultures of human bronchial epithelial cells (hbecs, see accompanying abstract by hengrui sun et al). here, we report correlations between estrogen levels and nasal ion transport in vivo and further probe hormonal effects on ion transport and asl height regulation in vitro. in vivo studies: in healthy, spontaneously menstruating females, nasal potential difference (npd) was measured (both nostrils) on two occasions during a single monthly cycle. based on a history of the most recent menstrual cycles, we measured npd on a day when estrogen levels were predicted to be low (generally - days following onset of menses) and subsequently on the day when the pre-ovulatory estrogen surge was predicted. the recording electrode was positioned at the location where maximal stable basal pd was detected and changes in pd recorded continuously during sequential perfusion with ] ringer's solution, ] ringer's solution containing amiloride (to inhibit sodium absorption via enac), ] modified (low chloride) ringer's containing amiloride and finally ] a similar solution also containing utp (to activate cacc). as predicted, blood estrogen levels were higher on the "pre-ovulatory" day ( . ± . vs . ± . nm, p< . ). utp-stimulated change in pd was significantly lower on the study day when estrogen levels were elevated ( . ± . vs . ± . mvs, p< . ). further, the amiloride-sensitive component of basal pd was higher on this day ( . ± . vs . ± . mvs, p< . ). the change in pd in response to perfusion with a low chloride solution was not different. in vitro studies: expression of estrogen receptor (er) α (but not β) was confirmed in non-cf and cf hbecs by real time pcr. erα was detected in hbecs from males and female donors. in cf and non-cf hbecs mounted in modified ussing chambers, exposure to nm e was associated with a significant (~ %) reduction in utp-stimulated short circuit current. e inhibited the atp-stimulated increase in asl height in male and female hbecs and this asl regulatory phenotype was not reproduced by testosterone or progesterone. in conclusion, estrogen inhibits utp-activated chloride secretion in vivo and in vitro suggesting that elevated pre-ovulatory estrogen levels may further impair mucociliary clearance in females with cf. all data mean±sem. supported by cff leroy matthews award [ ] . data demonstrating an upregulation of caccs when cftr is absent or defective [ , ] have triggered further studies of these channels and prompted further elucidation of the functional (and possibly also physical) interactions between these two channels. although searched for long, the molecular identity of caccs is still under debate. the role of bestrophin family proteins as putative candidates for caccs is discussed controversially. bestrophin (best- ) has been proposed to form ca + activated clchannels in epithelial cells. moreover, caccs have been shown to support cell proliferation, namely in the development of cancer [ ] . here, our goal is two-fold: ) to investigate the correlation between expression levels of best- and epithelial cell proliferation; and ) to pursue a search for interacting protein partners of best- to better understand the role and function of bestrophin(s) in epithelial cells. to study the correlation between best- expression and cell proliferation, we analyzed two populations of the t colonic carcinoma cell line with very different proliferation rates: the original (t -slow) and the spontaneously transformed t cells (t -fast). we observed that t -slow cells have a small proliferation rate and express low amounts of best- , while t -fast cells express high levels of this protein. best- is spontaneously active in t -fast cells. best- -rnai inhibited ca + activated clconductance and cell proliferation, therefore establishing a novel role of bestrophins in cell proliferation, which may be of relevance for the regeneration of the epithelia in cf and also for alternative therapies for cf. for the second objective, two putative cytoplasmic domains of best , polyhistidine -tagged (phis) n-term (aa - , best -n) and c-term (aa - , best -c), were cloned into the pet-sumo bacterial expression vector. these domains were then immobilized onto metal-affinity resin to capture interacting proteins from human t whole cell and sub-cellular lysates. protein-containing fractions recovered from best -n/c-coated and blank resins were subjected to d-gel and protein identification. it is expected that functional characterization of the interaction between the captured proteins and best- will give new insights into the biological role, plausibly of relevance to cf. work supported by dfg-sfb -a grant (germany) and pluriannual funding of cigmh (feder-eu and fct, portugal extracellular nucleotides acting on epithelial cell surface purinergic receptors regulate the mucociliary clearance process that removes noxious materials from the lung. however, how epithelial cells release nucleotides into the airways remains largely unknown. cftr has been suggested to mediate atp release in airway epithelia, but conclusive demonstration remains elusive. calu- is a human airway derived cell line often described as a model of airway gland serous cells. at least % of cells in a monolayer expressed high levels of cftr protein and activity at the apical membrane. atp, adenosine, and udp-glucose, the naturally occurring agonists for the p y , a b, and p y receptors, respectively, were quantified in airway surface liquid with nanomolar sensitivity using radiolabeling and fluorescence hplc-based techniques, and real-time atp measurements. atp (and udp-glucose) was found to be released constitutively and selectively onto the mucosal surface of calu- cells. however, the specific cftr inhibitor ( µm, - min) failed to decrease atp concentration in the mucosal bath of resting calu- cells. forskolin ( µm, min) stimulated cftr channel activity, but did not affect basal levels of atp (or udpglucose) release. in contrast, elevation of intracellular calcium using ionomycin ( - µm, - min) significantly increased atp (and udp-glucose) release into the mucosal (but not serosal) bath of calu- cells. calciummediated nucleotide release enhancement was not abolished by cftr inhibitor . maneuvers like pre-incubation in nominally free-calcium solution, bapta ( µm, min), or cytochalasin d ( µm, min) to disrupt actin cytoskeleton, which inhibited regulated exocytosis in calu- cells, also reduced calcium-mediated increase in nucleotide release. calu- monolayers presented a fraction of cells (< %) exhibiting negligible cftr immunostaining signal, but displaying airway goblet cell mucin granules. remarkably, stimulation of atp and udp-sugar release with ionomycin resulted in concomitant enhanced exocytotic secretion of muc ac. these results suggest that cftr is not involved in regulation of atp release in airway epithelial cells. more likely, nucleotides and nucleotide-sugars residing in the biosynthetic pathway involved in modification of apically targeted glycoconjugates (e.g. mucins) are released to the extracellular environment via calcium-triggered exocytosis. supported although the regulation of na+ transport is relatively well described in normal and cf airway epithelial cells, the impact of inflammation and infection, two major components of cf lung disease, on na+ transport has not been studied extensively. significant amounts of evidence suggest that bacterial by-products may influence na+ transport in airway epithelium. furthermore, the secretion of exoenzyme s by pseudomonas aeruginosa is enhanced during an exacerbation of lung disease in cystic fibrosis. in the past few years, our laboratory has shown that exposition of lung epithelial cells to p. aeruginosa lps leads to a rapid decrease in activity and expression of enac channel. the aim of our study was to evaluate the mechanism involved in the modulation of enac expression and activity by lps. primary culture of lung epithelial cells grown on filters were treated with lps ( µg/ml) for different time ( min, , , h). at each time point, the total and amiloride-sensitive short circuit current (isc) was determined in ussing chamber. in parallel, the cell proteins were collected to investigate by western blot the signalling pathways involved in this response to lps. a hours lps treatment leads to a significant decrease in alpha and gamma enac mrna to % and % of control respectively. similarly, the amiloridesensitive current was decreased by % of the current in untreated cells after a min exposure to lps. by western blot, we demonstrated the strong implication of the pi k/akt pathway in response to lps in the regulation enac mrna and the potential implication of tyrosine/kinase receptors signalling pathway through mtor in the regulation of enac channel activity. in conclusion, we demonstrate that inflammatory molecules can modulate the expression and function of the na+ transport mechanism in lung epithelial cells and could contribute to the modulation of airway surface fluid in the chronic infectious process associated with cf. serous acinar cells are thought to be crucial for secretion of airway surface liquid by submucosal glands in large airways, but the molecular mechanisms of serous cell ion and fluid transport are not well understood. to address this, we isolated murine nasal serous acinar cells and studied them using simultaneous dic and quantitative fluorescence microscopy to track changes in cell volume and intracellular concentrations of ions involved in fluid secretion ([cl -] i ; spq) and its regulation ([ca + ] i ; fura- ). previous results indicated that serous acinar cell volume changes were indicative of solute efflux (shrinking) and solute influx (swelling), reflecting the secretory state of the cell. acinar cells stimulated with µm carbachol (cch) exhibited a rapid increase in [ca + ] i and subsequent ~ % cell shrinkage due to efflux of ~ % of cell clcontent and parallel loss of k + and osmotically obliged h o. because murine serous cells are sites of apical cftr expression in the airway, we tested the requirement for cftr in cch/ca +activated shrinkage/clefflux. acinar cells isolated from either cftr tm unc-/mice or wt cells pre-treated with cftr inh exhibited identical rates and magnitudes of ca + -induced cell shrinkage and clloss compared to wt control cells. resting [cl -] i was also identical in wt and cftr tm unc-/serous acinar cells ( ± and ± mm, respectively), suggesting similar levels of resting clconductance. thus, murine serous acinar cells can secrete kcl/h o by ca + -dependent mechanisms in the absence of functional cftr, in agreement with studies of intact glands suggesting cch-induced glandular fluid secretion is cftr-independent. wt serous acinar cells were stimulated with camp-agonists, including µm forskolin, µm isoproterenol, µm adenosine, and µm vip. however, no camp-induced shrinkage was observed, even in the presence of a cocktail of inhibitors to block solute uptake. to determine if cftr activation could enhance cch/ca + -induced cell shrinkage at a sub-optimal [cch], serous acinar cells were pre-treated with forskolin followed by stimulation with µm cch in the continued presence of forskolin. however, similar rates and magnitudes of cell shrinkage were observed in forskolin-treated and control cells. these results suggest that the magnitude of the cftr clconductance in murine serous acinar cells is much smaller than the ca + -activated clconductance, below the resolution of these optical assays. more sensitive electrophysiological approaches are being used to determine the molecular identity of the non-cftr clconductance(s) and the role of cftr in murine serous acinar cell ion transport. females with cf exhibit more rapid decline in fev than males, which reduces their life span. normal airway epithelia regulate airway surface liquid (asl) volume and mucus transport by secretion through both cftr and the ca +-activated cl-channel (cacc), and inhibition of either of these pathways in isolation does not abolish mucus transport. however, a loss of both pathways is predicted to result in mucus plugging which contributes to lung disease. despite a loss of cftr, cacc is functional in cf airways due to mechanical stimulation of atp release and activation of the p y pathway which serves to raise intracellular ca +. we hypothesized that e may inhibit cacc, leaving females with cf more prone to lung disease than males due to a greater depletion of asl volume. using confocal microscopy, we found that acute pre-exposure with e inhibited atp induced asl secretion in normal and cf human bronchial epithelial cultures (hbecs) from both male and female donors. the ic for e -inhibition of atp-induced asl secretion was . nm. to better understand how estrogen receptors (ers) interact with the p y pathway, we transiently transfected er-α or er-β linked to an orange fluorescent protein in an er null bhk cell line. with the co-transfection of ha tagged p y -r, we observed that e addition did not induce internalization of p y -r, neither did it alter the internalization induced by atp. we then measured total cell inositol phosphates (ip) in cf airway cells +/-e pretreatment. cell ip levels were not different between groups (n= ), suggesting that the effect of e is downstream of ip production. the investigation of the changes of the intracellular ca + (ca +i) by recording fura- emission ratio over time in er-α or er-β expressed cells provided clues. after nm e exposure, the fura- emission ratio in untransfected cells (n= ) or er-β transfected cells (n= ) increased by . following um atp addition. this increase was significantly inhibited by~ % in er-α transfected cells (n= ), providing good evidence that er-α is respon-sible for inhibiting the p y pathway. we next measured ca +i in normal hbecs. while acute nm e addition had no affect on basal ca +i levels, which were around nm, this maneuver reduced the ca +i response to nm following atp addition (both n= ), suggesting that e addition altered ca + signaling in response to atp. thapsigargin induces ca +i release from the endoplasmic reticulum, but the magnitude of ca +i release after thapsigargin addition was not altered by e pretreatment, indicating that e works further up stream to affect the p y signaling pathway. together, these results suggest that e directly interacts with the p y signaling pathway through er-α by altering ca +i. we propose that this interaction leaves females more vulnerable to infections due to a reduced asl volume during periods of high e , such as occur prior to ovulation. the nasal potential difference (or npd) is a sensitive, real time bioelectric assay of cftr-dependent and independent ion transport that is a commonly used study endpoint in early clinical stages of drug development. technical limitations of the assay must be controlled to facilitate its use in multicenter trials and limited intra-site variability. two forms of equipment are commonly employed to evaluate nasal potential difference in humans: silver-silver chloride electrodes (agcl) with saline bridges or calomel electrodes with agar bridges; however, these techniques have never been directly compared. we repeatedly evaluated (within days) the basal potential difference of five normal subjects using the two available techniques, then compared variance and repeatability of the measures. basal pd was measured during perfusion of ringer's solution, and measured at . , . , . , . , and . cm within the inferior meatus, as indicated in the cf-tdn standard operating procedures. mean basal pd were within . mv using the two techniques, with very similar coefficients of variation (cvar; . vs. . in agcl vs. calomel, respectively). maximum basal pds were also similar between the two methodologies (mean difference . mv; cvar . and . , respectively). the correlation between first and second measures of mean pd was highly reproducible, and similar between the two techniques (r= . vs. . in agcl vs. calomel, respectively); however, when each individual basal pd measure was compared, correlation between first and second pd using the agcl device appeared superior (r= . vs. . respectively; n= measures per device). comparison of values obtained in cf subjects are currently in progress and will also be reported. next, we examined diagnostic npd tracings in cf subjects for whether the effect of amiloride might influence values obtained in the contralateral nare. an effect of cross-contamination by amiloride or other agent (gene therapy vectors or other nasal administered compounds, for example) could influence accurate measure of npd values. when basal pds were performed in both nostrils prior to amiloride exposure in either nostril, mean basal pd between nares remained stable (< mv, p=ns for change). however, if the basal pd in one nostril was measured after amiloride exposure in the contralateral side, mean pd depolarized by mv (from - to - mv; p< . ), indicating the presence of a 'cross-nostril' amiloride effect that might potentially affect interpretation of this endpoint if not performed in a sequence designed to avoid amiloride exposure. in summary, we show that calomel and agcl electrodes perform similarly in repeated measures from the same normal subjects, and that improper npd sequence can adversely affect basal pd measures. other potential impediments to the pd measurement (e.g. solution temperature, tonicity, ionic content, and sources of electrical offset) will also be discussed. these findings are relevant to the optimizing the use of the npd assay in clinical trials. supported by nih, cff, and cfft. the gene defective in patients with cystic fibrosis (cf) is the cystic fibrosis transmembrane conductance regulator (cftr), a camp-activated chloride channel in the apical membrane of epithelia, but alteration of expression and function of many genes undoubtedly contributes to the pleiotropic manifestations of cf. a hallmark of cf in airway epithelia is the hyperabsorption of sodium through the epithelial sodium channel (enac) in the apical membrane followed by extrusion through the na,k-atpase in the basolateral membrane. this results in airway surface liquid (asl) dehydration, mucus buildup and bacterial infection characteristically observed in the lungs of cf patients. previous studies have shown that the na,k-atpase is increased in cf airway epithelia, consistent with increased sodium transport along the enac/na,k-atpase axis. we have previously demonstrated that fxyd , a member of a small family of proteins known to regulate the na,k-atpase, is increased in s x cf mouse lung and nasal epithelia and in primary human tracheal epithelial cells (hte) after treatment with an inhibitor of cftr ( ). recent evidence has indicated that enac activation occurs through modification of the serine protease-protease inhibitor balance in asl. thus, we hypothesized that enac-mediated increases in sodium absorption may be partially responsible for the increase in fxyd expression observed in cf airway epithelia. we now report that increasing asl volume on hte cells upregulated fxyd expression (p< . ) after hours, an effect that was further increased after trypsin addition and abrogated by the serine-protease inhibitor aprotinin. treatment of hte cells with nystatin also increased fxyd expression measured by quantitative rt-pcr (p< . ). based on these results and the observation that mice overexpressing enac beta-subunit exhibit cystic fibrosis-like lung pathophysiology, we examined fxyd expression in the lungs of mice overexpressing the scnn b-transgene. contrary to our expectations, fxyd lung expression was decreased as assessed by quantitative rt-pcr and immunoblot analysis (p< . ), indicating fxyd may be alternatively regulated in epithelia under conditions of acute versus chronic sodium hyperabsorption. therefore, we measured fxyd expression in an epithelial cell culture model using an inducible, tri-cistronic vector capable of expressing enac alpha, beta and gamma subunits. we determined that fxyd expression is decreased after chronic enac induction. we conclude that fxyd expression is coordinately regulated based on acute versus chronic enac-mediated sodium absorption and suggest that fxyd is increased in cystic fibrosis airway epithelia as a result of an imbalance between increased inflammatory signaling and altered ion transport. picher, m.; van heusden, c. cystic fibrosis center, university of north carolina, chapel hill, nc, usa airway infection by the respiratory syncytial virus (rsv) is considered an important cause of pulmonary exacerbation and hospitalization in cystic fibrosis (cf) children. this disease is characterized by functional mutations in the cf transmembrane regulator (cftr). the inability of cftr -/-mice to clear rsv is associated with exaggerated inflammatory responses. a relationship was recently established between airway inflammation and chronically-elevated airway adenosine. this signaling molecule regulates key aspects of lung defenses, including bacterial clearance and inflammatory cells. high adenosine levels measured in bronchoalveolar lavage fluid of asthmatic or cf patients cause airway inflammation, remodeling and fibrosis in animal models. the objective of this study was to investigate the longterm effects of rsv infection on adenosine regulation and inflammatory responses in human airway epithelia. methods. polarized primary cultures of bronchial epithelial cells from healthy subjects and cf patients were exposed to vehicle, uv-inactivated or active green fluorescent protein (gfp)-rsv. the cultures were monitored days for infection (fluorescence microscopy), adenosine levels (fluorescence chromatography), the release of cytokines regulating adenosine tnfα) and enzyme activities (ecto '-nucleotidase [ecto '-nt: amp Ç adenosine] and adenosine deaminase [ada : adenosine Ç inosine]). results. (a) both normal and cf cultures exhibited detectable rsv infection over days, with peak density on day - . (b) we demonstrate that rsv enhances the release of all five cytokines from normal cultures over days. in contrast, cf cultures responded to rsv by a transient decrease in il- β, il- and tnfα (peak on day ), but a sustained decrease in il- , and a sustained increase in il- , secretion. (c) on normal epithelia, rsv transiently stimulated ecto '-nt (peak on day ) but caused a sustained increase in ada activity, resulting in chronically-low adenosine levels. in cf, rsv raised adenosine levels by the combined effects of a sustained increase in ecto '-nt activity and the lack ada response. (d) chronically-elevated adenosine generated by ada inhibition [erythro -( -hydroxy- -nonyl)adenine] stimulated the secretion of all cytokines, except il- . conclusion. these data support a complex relationship between adenosine and cytokine regulation in human airway epithelia. the il- -adenosine amplification pathways identified in rsv-infected cf epithelia are in agreement with animal models and their key role in chronic airway inflammation/remodeling. on the other hand, the data also suggest that ada activity is influenced by il- , known to stimulate the expression of a major protein anchoring soluble ada to epithelial surfaces: cd . altogether, this study suggests that an rsv infection in cf children may accelerate the progression of the disease by setting the stage for airway adenosine-cytokine amplification pathways. supported by the cystic fibrosis foundation (picher g ). cystic fibrosis (cf) airway submucosal glands are defective in mucus secretion rate (jayaraman, pnas, , , ; joo, j biol chem, , , ; joo, pediatr. pulmonol., , suppl. , ; wine, proc ats, , , ; thiagarajah, faseb j, , , ; song, ajp cell, ; salinas, faseb j, , , ) . one hypothesis is that loss of cftr results in reduced secretion of electrolytes leading to underhydration of mucins, thereby altering the mucus properties. it has been generally believed that the cf mucus in response to cholinergic agonists is thicker and more viscous. however, comparisons of pure gland mucus are difficult because of variability in gland secretion rates and a decline in mucus solids with repeated stimulations (wu, pediatr. pulmonol. , suppl. , ) . in the present study, we directly compared the solid content of airway submucosal gland mucus between control and cf individuals. human donor tracheas (hn) or disease control bronchi (dc) were dissected and mounted as previously described but without mineral oil. tissue was kept warmed and humidified in a chamber with two layers of saturated degree c / % co /o water vapor. the mucosal surface was blotted dry and coated with a thin layer of dimethylpolysiloxane to prevent re-absorption by the surface epithelium. the tissue surface was covered with a cover slip, leaving minimal air space between it and the surface to further reduce evaporation. a µl droplet of % nacl was placed at the side of every tissue as a standard to assess evaporation and measurement error. any basal secretions were removed prior to stimulation with µm carbachol. mucus secretion from individual glands was pooled using forceps and then drawn into a constant bore microcapillary (drummond) that had been alcohol-cleaned, dried, and weighed. stimulation time was allowed to vary so that at least µl of mucus was collected. after mucus collection, both the standard and the bath solution were refreshed. mucus filled capillaries were weighed with a mettler microbalance before and after overnight degree c oven drying to obtain nonvolatile solid content. the average solids measured in six repeated measures of % salt solutions was ± . % with no trend over time. results for hn (n= ) and dc (n= ) did not differ and were pooled as 'control'. the initial control secretion yielded . ± . % solids (mean ± sem, n= subjects) and required ± min to produce adequate mucus. the initial cf secretion gave . ± . % solids (n= ) in ± min. the second control secretion yielded . ± . % solids (n= ) and required ± min, whereas the second cf secretion gave . ± . % solids (n= ) and required ± min. in summary, cf gland mucus solids content was % higher than control following the first stimulation (p = . ). after the second stimulation, cf solids content was % higher than the control (p = . ). solids contents of controls observed in our experiment are much higher than was found for mucus collected from airway lumens after hr stimulation (c. %, trout, am j physiol, , , l ) . supported by niddk ro - (jjw) and cff. in patients with cystic fibrosis (cf) as well as in some animal models of cf lung disease, higher levels of prostaglandin e (pge ) have been detected compared to control subjects. it is not clear whether this excess of pge contributes to the airway pathology of cf, or whether it may serve a protective function against airway constriction. inhaled pge has been tried in asthma and may be of benefit as a bronchodilator. interestingly, pge has shown benefit in aspirin-sensitive asthmatics, a population that shares some features with the cf airway phenotype including nasal polyposis. the mechanism of airway relaxation to pge is mediated primarily by ep receptors signaling through camp. in cf, beta-adrenergic bronchodilators are commonly used, but are often only partially effective at reversing bronchospasm. beta-agonists and pge both signal through g-proteins coupled to camp and pka signaling, thus pge may offer another treatment when beta-agonists are ineffective at reversing bronchospasm in cf. in mouse tracheas, the beta-agonist isoproterenol (iso) will partially relax a pre-contracted airway, but ± % of contractile force remains (n= ). in contrast to the limited relaxation with iso, constricted mouse airways demonstrate significantly greater relaxation to pge , with only ± % of force remaining (n= , p< . compared to iso). this difference in relaxation responses suggests different intracellular signals are activated by each agonist despite both signaling through pka. to investigate phosphorylation events in response to iso or pge , we developed a new method for perfusing isolated mouse lungs. using this technique, tissues were perfused with buffered solution containing p for radiolabeling. lungs were then stimulated with either iso or pge , and phosphoproteins were compared from each condition. radiolabeling was effective under all three conditions. targets were chosen from each group (unstimulated, iso, or pge treated) that showed phosphorylation or dephosphorylation, and proteins were identified by mass spectrometry. over differentially phosphorylated proteins were identified, and two of these (rho gdi and inositol-requiring protein ) were selected as candidates to be tested in the context of relaxation to pge compared to iso. by identifying signaling targets involved in airway relaxation to pge , it may be possible to activate beneficial airway relaxation responses without the limitations seen in iso relaxation and without activating pge responses other than relaxation that may link pge to the airway pathology seen in cf. the parental ∆f /∆f cf bronchial epithelial cell line (cfbe o-) was compared to clones of cfbe o-complemented with . kb wild type (wt) or . kb ∆f cftr cdna in terms of their ion transport characteristics. the transepithelial resistance (rt) (~ (cm ) observed in the parental cfbe o-was maintained in the complemented cells. two clones complemented with wt cftr cdna (c - . wt and c - . wt) demonstrated rt ~ Ωcm , while another clone (c - . ∆f) complemented with the ∆f cftr cdna showed an rt of ~ Ωcm . two stable clones expressing wt cftr were selected based on electrophysiological characteristics and stability of cftr expression. forskolin-stimulated cl secretion in clone c - . wt was on average . ± . µa/cm and . ± . µa/cm in clone c - . wt. furthermore small camp-stimulated transepithelial current of . ± . µa/cm was observed in clone c - . ∆f indicating that ∆f cftr can traffic to the plasma membrane if present at high enough levels at physiological temperatures. the role that cftr plays in ca-activated cl conductance was investigated following treatment of the cells with atp in parental cfbe o-and wtcftr . -cfbe o-monolayers. in symmetrical cl-containing solutions, mucosal application of atp ( µm) elicited a small, transient cl secretory response in the parental cfbe ocells ( . ± . µa/cm ), while the magnitude of the atp-stimulated cl current was markedly enhanced in cftr-corrected cfbe o-cells with peak currents at . ± . µa/cm . recordings done in presence of a serosal-tomucosal cl gradient to increase the driving force for cl secretion showed a transient atp-dependent activation of cl currents in parental cfbe ocells with peak cl currents of . ± . µa/cm (n= ). cftr corrected cfbe o-cells showed a sustained activation of cl currents of similar magnitude ( . ± . µa/cm ; n= ). atp-stimulated cl currents were effectively blocked by the cftr inhibitor glyh ( µm). histochemical analysis showed prominent epithelial characteristics in all clones. these observations indicate that i) the apical driving force is limiting for atp-stimulated cl secretion and that ii) cftr participates in the cl secretory response to atp. furthermore, the findings suggest that these novel clonal isolates of the cfbe o-bronchial epithelial cell line can be useful for the investigation of cf therapies. this work was supported by grants from the cff, cfri, nih, elizabeth nash foundation, and pacfi. the lactoperoxidase (lpo) system functions in normal airways to prevent infection and may be compromised in cystic fibrosis (cf) due to defective transport of its substrate thiocyanate. a computational model of normal airway surface liquid suggests that hydrogen peroxide levels regulate lpo system activity and that the lower thiocyanate in cf airways may result in elevated levels of hydrogen peroxide that is likely produced by duox the major nadph oxidase in airway epithelial cells. to investigate the regulation of duox by bacterial products, passage normal human airway epithelial cells were grown and redifferentiated at the air liquid interface and treated for , or h with either pseudomonas aeruginosa flagellin or lps, applied apically. treated and mock treated cultures were analyzed for levels of duox and lpo mrna and protein, and for hydrogen peroxide production. quantitative pcr was performed using taqman kits, protein was measured by western blots, and hydrogen peroxide was quantified using amplex red-hrp coupled fluorescence. the data showed the duox and lpo mrna but not duox mrna, were significantly upregulated at all measured time points following apical treatment of the cultures with purified flagellin compared to mock treated. at h duox was increased . +/- . fold and lpo was increased . +/- . fold (means +/-s.e.m., n = lungs, - cultures of each lung). muc ac mrna was also upregulated by flagellin treatment ( . +/- . fold, n = lungs, - cultures of each lung). apical treatment with lps had no effect on any tested mrna and induced minimal il- secretion in these cultures. western blots using anti-duox antibodies (courtesy of f. miot, brussels, belgium) showed corresponding increases in duox protein following flagellin treatments. hydrogen peroxide production by flagellin treated cultures was increased in proportion to increases in duox mrna. application of flagellin alone to the apical surface of previously untreated cultures did not increase hydrogen peroxide production. thus, pseudomonas flagellin up-regulates duox and results in higher activity in normal human airway epithelial cells perhaps serving to increase innate host defense activity of the lpo system. chloride ion in phagolysosomes is an essential substrate for neutrophils to produce hypochlorous acid (hocl), an important bactericidal agent. we have previously shown that cftr was present in the phagolysosomal membrane of neutrophils and the cftr defect led to deficient chlorination of ingested pseudomonas aeruginosa (pa) (painter et al, biochemistry, : - , ) . these results suggested that chloride accessibility to the phagolysosomal lumen might be impaired in cf neutrophils thereby affecting neutrophil-mediated bacterial killing. to understand the role of chloride ion in the process, we first assessed the effect of extracellular chloride concentration on pa killing by normal neutrophils under iso-osmotic conditions. surprisingly, bacterial killing was strongly dependent on extracellular chloride concentration. the initial rate of killing of pa was~ fold less in a chloride-free medium than a physiological chloride medium. killing efficiency increased as the chloride concentration was raised in a dosedependent fashion and plateaued at~ mm chloride concentration. to determine what percent of the chloride-dependent killing was oxidantdependent, we applied the cftr inhibitor glyh- , the myeloperoxidase (mpo) inhibitor aminobenzoic acid hydrazide (abah) and the nadph-oxidase inhibitor diphenylene iodium chloride (dpi), respectively to bacterial killing assays. the results showed that the killing of pa could be divided into three discernible components: ( ) a component accounting for about - % of the total killing rate that was inhibited by glyh- , abah or dpi; ( ) a second component amounting to~ - % that was chloride-dependent and partially sensitive to glyh- and; ( ) a third accounting for~ % of the total killing activity that was not sensitive to oxidant inhibitors, or extracellular chloride concentration. dpi and abah had no effect on killing of pa in the absence of extracellular chloride suggesting that most of the oxidant-mediated killing activity toward pa was due to hocl. none of the inhibitors had any significant effect on phagocytosis. finally, the rate of pa killing by cf neutrophils was found to be significantly lower ( - %) than that seen for normal neutrophils and was comparable to the effect of glyh- on killing of pa by normal neutrophils. the data suggest that cftr plays a significant role in killing of pa by normal neutrophils and, when defective as in cf, may compromise the ability of neutrophils to efficiently kill pa. this research was supported by a cff grant to gw. %. the cf group demonstrated a significantly higher frequency of pseudomonas respiratory infections than the non-cf group. interestingly, no significant differences were detected in any infections from other systems including blood, sinuses, skin, wounds, oral cavity, bowel, eyes, peritoneal cavity, and urinary tract. moreover, the cf lung-transplant patients had significantly less time free from pseudomonas infections in the transplanted lungs. conclusion normal lungs transplanted into cf patients had significantly higher susceptibility to pseudomonas infections than those transplanted into non-cf patients, which strongly suggests that defective host defense mechanism(s) beyond the respiratory system contribute to cf lung pathogenesis. pyocyanin (n-methyl- -hydroxyphenazine, pcn) is produced by pseudomonas aeruginosa and is found in pulmonary secretions of infected cf patients at concentrations up to µm. pcn is a redox-active compound that generates superoxide and hydrogen peroxide in the presence of cellular reductants. since cftr was reported to be modulated by h o , we hypothesized that pcn, through its ability to redox cycle and to produce h o , would affect cftr-mediated cl transport. we therefore tested pcn and h o for effects on cltransport (transepithelial short-circuit currents in ussing chambers measured with serosal-to-mucosal clgradient) and cytosolic redox potential (imaging microscopy on cells transfected with a redox sensitive gfp, rogfp ) using cftr-corrected cf bronchial epithelial cells (wtcftr . . under resting conditions, acute exposure of the apical epithelial surface to µm pcn elicited a small clsecretory response ( . ± . µa/cm , n= ) that was completely blocked by the cftr inhibitor glyh ( µm). in contrast, in presence of the camp-elevating agonist forskolin, pcn caused a time-dependent decline of forskolin-stimulated cftr clcurrents by %. exposure to h o ( µm) elicited a more pronounced clsecretory response than pcn in resting cells and h o -stimulated clcurrents peaked at . ± . µa/cm (n= ). h o was less effective than pcn at inhibiting forskolin-stimulated cftr clcurrents and currents declined by %. both pcn and h o oxidized the cytosolic redox potential from a resting value of - mv by similar amounts ( ± mv for pcn vs. ± mv for h o ), but rates of oxidation were faster for h o ( . ± . mv/min) than for pcn ( . ± . mv/min). inhibition of camp-stimulated cftr clcurrents by oxidation was a linear function of the cytosolic redox potential, but pcn ( µa×cm - ×mv - ) was more potent than h o ( . µa×cm - ×mv - ). cf bronchial epithelial cells homozygous for ∆f -cftr did not show a clsecretory response to pcn or h o . these data suggest that cftr-corrected cf bronchial epithelial cells respond acutely to oxidative stress by turning on cftr activity probably as part of the innate host defense response and this mechanism is absent in cf airways. furthermore, prolonged exposure to pcn and h o is detrimental for the proper function of the camp-regulated cftr clconductance. we conclude that oxidative stress in p. aeruginosa-infected airways is a key factor that needs to be considered for successfully correcting cftr function in cf airways. supported by nih (nccam p at ), cff (fischer g ) the lung is continually exposed to environmental agents and pathogens. the lung epithelial lining fluid (elf) is first to encounter these agents and gsh is a major antioxidant found in this apical fluid. the cystic fibrosis transmembrane conductance regulator protein (cftr) is the only known gsh transporter that maintains lung elf gsh levels. cystic fibrosis (cf) patient have low levels of gsh in their elf and have copious and viscous mucus. hypertonic saline is used in cf patient to help clear mucous and improve lung function however the mechanism(s) by which it does so are poorly understood. the purpose of these studies was to examine whether hypertonic ( . %) saline can modulate apical gsh levels and protect lung epithelial cells against the oxidant, t-butyl hydroperoxide (t-booh). we used human lung epithelial cell lines sufficient (c ) and deficient (ib ) in cftr. the cftr deficient ib cells have % lower basal levels of apical gsh as compare to cftr sufficient c cells. cells were exposed separately and in combination to % saline, and t-booh ( um) for hours. the cftr deficient ib cells were much more sensitive to t-booh-mediated oxidative injury as measured by lactate dehydrogenase (ldh) release. hypertonic saline exposure was associated with an increase in apical gsh levels in both cftr sufficient and deficient cells and decreased t-boohmediated oxidative injury in both cells lines. hypertonic saline increased gsh in the apical compartment, which appeared to be largely cftr mediated. we examined this issue in mice given a clinically used dosing regimen of % hypertonic saline nebulized twice daily and examined changes in elf gsh levels hours after last treatment. mice receiving the hypertonic saline treatment had significantly higher gsh levels in their elf than untreated controls. this data suggests that cftr and gsh adaptive responses play an important role in lung's reaction to oxidants. we propose that factors, which interfere with the lung's capability to mount and maintain an adequate adaptive apical gsh response, may compromise and sensitize the lung to oxidative injury. (supported in part from nih grant hl ). the absence of functional cftr in airway epithelia of cf patients leads to abnormal airway surface liquid, which favors chronic bacterial infection. this chronic infection in cf lungs is associated with an exaggerated inflammatory response characterized by abnormal cytokines secretion and massive lung neutrophils infiltration. we have previous recapitulated the abnormal inflammatory response in cftr-/-(cf) mice by repeated administration of pseudomonas aeruginosa lipopolysaccharide (lps) (ped. pulmonology, supp. , ) . we demonstrated that the bronchoalveolar lavage (bal) fluid of cf and heterozygous (het, cftr+/-) littermate control mice when compared to the bal from wt mice shows ) higher numbers of neutrophils and ) higher concentrations of cytokines (il- alpha, il- , kc, mcp- , gm-csf, m-csf) as assessed using multiplex analysis of selected cytokines. of note, airway epithelial cells from cf patients are known to have elevated secretion of some of these cytokines. however to date, the contribution of immune cells to these abnormal cytokine levels has not been well characterized. this study aims to investigate the contribution of cf macrophages in this altered cytokine profile/response. we examined cytokine secretion from two different macrophage populations: alveolar macrophages (am) obtained from the bal fluid and bone marrow (bm)-derived macrophages differentiated in presence of m-csf. both macrophage populations were cultured in the presence or absence of lps, and the supernatants were tested for the concentration of the cytokines that we had shown to be abnormal in vivo. these macrophage populations were examined from cf, het and wt mice. in absence of lps exposure, bm-derived macrophages from cf mice have lower levels of il- and kc compared to wt and het mice. in contrast, am of cf mice have higher levels of il- and kc than am of wt mice. this difference may be due to am's prior exposure to antigens in vivo whereas the bm-derived macrophages are a truly naïve population. interestingly, after lps stimulation, il- a, il- , mcp- , kc, g-csf and gm-csf are higher in cf macrophages compare to wt and het mice suggesting that cf affected macrophages display an altered inflammatory response when compared to wt and het. the abnormal production of these cytokines was also detected at the transcriptional level using quantitative rt-pcr analysis. in conclusion, our data support the hypothesis that cftr-null macrophages may have an impaired cytokine profile at baseline as well as after stimulation. furthermore, cftr deficient macrophages may contribute directly to the abnormal inflammatory response in cf. (supported by cff, nih, niddk) cystic fibrosis lung disease is associated with an excess of free neutrophil elastase (ne) in the airway arising from persistent neutrophil inflammation. dx- is a potent small protein inhibitor of neutrophil elastase that could be a useful therapy for diseases involving neutrophilic inflammation such as cystic fibrosis and copd. dx- effectively inhibits ne activity in cf sputum sol (ic = pm). however the physical barriers posed by cf whole sputum may prevent dx- accessing and inhibiting ne ensconced within the mucus. the mucolytic dnase acts by cleaving extracellular dna in mucus thereby reducing viscosity. however dna binds and inactivates ne and therefore dnase treatment of mucus results in an acute increase in active ne. the lipid portion of pulmonary surfactant acts as a lubricant and our group has previously shown that bovine lung extract surfactant (bles) increases the fluidity of whole sputum. ( aim we aimed to show that, by reducing the surface tension of sputum, bles would enhance the anti-elastase effect of dx- in whole sputum. methods whole sputum ( . g per well in a -well plate) was pre-incubated with µl saline as control, µl dnase ( µg/ml) or µl bovine lung extract surfactant (bles, µg/ml) for minutes at °c followed by addition of µl dx- ( µm or µm) for h at °c. the contents of each well were aspirated, added to ml nacl ( . %) and centrifuged at , g for min. the resulting sputum sol was assayed for ne activity using the colorimetric substrate n-meosuc-aapv-pna. results ne activity in whole sputum was significantly inhibited by µm dx- (p= . ) and µm dx- (p= . ). µm dx- inhibited significantly more ne when sputum was pre-treated with bles (p= . ) treatment of sputum with dnase caused a significant increase in ne activity compared to untreated sputum (p= . ) whereas treatment of sputum with bles did not have this effect. conclusion dx- effectively inhibits elastase in cf sol. pre-treatment of cf whole sputum with bles enhanced the anti-elastase effect of dx- and treatment of sputum with bles alone did not cause an increase in ne activity in contrast to treatment with dnase. dx- inhibitory capacity in whole sputum could be improved by co-treatment with surfactant which increases fluidity of sputum and could allow greater access of drugs to sputum components. bovine lung extract surfactant, bles™ was a kind gift from bles biochemicals inc., on, canada. dx- was a kind gift from dyax corp, ma who also funded part of this research. table : results are summarised as neutrophil elastase activity µg/ml and show the mean and sem of n= experiments. cystic fibrosis lung disease is associated with an excess of free neutrophil elastase (ne) in the airway arising from persistent neutrophil inflammation. dx- is a potent small protein inhibitor of neutrophil elastase that could be a useful therapy for diseases involving neutrophilic inflammation such as cystic fibrosis and copd. dx- effectively inhibits ne activity in cf sputum sol (ic = pm). however the physical barriers posed by cf whole sputum may prevent dx- accessing and inhibiting ne ensconced within the mucus. the mucolytic dnase acts by cleaving extracellular dna in mucus thereby reducing viscosity. however dna binds and inactivates ne and therefore dnase treatment of mucus results in an acute increase in active ne. pulmonary surfactant acts as a lubricant and our group has previously shown that bovine lung extract surfactant (bles™) increases the fluidity of whole sputum. ( we aimed to show that, by reducing the surface tension of sputum, bles™ would enhance the anti-elastase effect of dx- in whole sputum. methods whole sputum ( . g per well in a -well plate) was pre-incubated with µl saline as control, µl dnase ( µg/ml) or µl bovine lung extract surfactant (bles™, µg/ml) for minutes at °c followed by addition of µl dx- ( µm or µm) for h at °c. the contents of each well were aspirated, added to ml nacl ( . %) and centrifuged at , g for min. the resulting sputum sol was assayed for ne activity using the colorimetric substrate n-meosuc-aapv-pna. results ne activity in whole sputum was significantly inhibited by µm dx- (p= . ) and µm dx- (p= . ). µm dx- inhibited significantly more ne when sputum was pre-treated with bles™ (p= . ) treatment of sputum with dnase caused a significant increase in ne activity compared to untreated sputum (p= . ) whereas treatment of sputum with bles did not have this effect. conclusion dx- effectively inhibits elastase in cf sol. pre-treatment of cf whole sputum with bles™ enhanced the anti-elastase effect of dx- and treatment of sputum with bles alone did not cause an increase in ne activity in contrast to treatment with dnase. dx- inhibitory capacity in whole sputum could be improved by co-treatment with surfactant which increases fluidity of sputum and could allow greater access of drugs to sputum components. we would like to thank prof. f. possmayer, on, canada and bles biochemicals inc., on, canada for the bovine lung extract surfactant, bles™. dx- was a kind gift from dyax corp, ma who also funded part of this research. table results are summarised as neutrophil elastase activity µg/ml and show the mean and sem of n= experiments. cf patients infected with p. aeruginosa (pa) have increased levels of proinflammatory cytokines, such as tnf-α, il- β, il- , and il- ; but have low levels of the anti-inflammatory cytokine, il- in their airways. the proinflammatory mediators in the cf airway recruit a large number of polymorphonuclear neukocytes (pmns), which release high levels of neutrophil elastase and human neutrophil peptides and damage the lungs. the intense neutrophil-dominated inflammatory response associated with chronic pa infection results in progressive airway obstruction, the cause of death of over three-quarters of cf patients. recent studies highlight the significance of sphingolipid metabolism in cell growth, differentiation, membrane traffic, apoptosis, senescence, and immune regulation. acid sphingomyelinase (asm) plays a critical role in sphingolipid metabolism by hydrolyzing sphingomyelin to ceramide. ceramide self-associates to form ceramide-enriched membrane platforms(also called lipid rafts). these platforms have been shown to be involved in the internalization of pa; induction of apoptosis and immune regulation. our in vitro and in vivo studies of asm confirm that there is a loss of induction of asm after pa infection in both cf bronchial epithelial cells and cftr knock out mice as compared with controls. in vitro, we examined the asm activity in human bronchial epithelial cells with normal cftr expression (s cells) and with mutant cftr expression (ib cells ) after pa infection at , , , , , and min. asm activity was up-regulated in s cells following pa infection after min and kept increasing until min, while asm induction was lost in ib cells. asm protein and ceramide levels also increased in s cells following pa infection compared with ib cells. in our in vivo studies, cftr knock out mice and control c bl/ mice were both administered various doses of pa orally ( x cfu to x cfu) . six hours after pa infection, lung homogenates from the c bl/ mice indicated increasing asm protein and ceramide production, which corresponded to the dose of pa given. in contrast, asm protein and total ceramide production remained at baseline levels in the lung homogenates of cf mice regardless of the dose of pa given. rna interference (sirna) assays were performed by transfecting a bp pre-designed sirna specific for human asm in s cells. these cells were then infected with pa for hrs to see if they had similar il- level compared with infected ib cells. we demonstrated a significantly elevated il- expression in s cells tranfected with asm sirna compare with s cells transfected with a scramble sirna (p< . ). our results suggest that the loss of asm induction, caused by mutant cftr, is involved in pa clearance and contributes to the exaggerated proinflammatory cytokine response in cf airway. we are conducting research on defining the roles of asm and the sphingolipids in pa infection; cell signaling; as well as cytokine induction, we are also looking at the therapeutic potential of aav.asm vectors in pa-infected cf bronchial epithelial cells and cftr knock out mice. work supported by nhlbi introduction: new therapies are critically needed to treat cystic fibrosis (cf) lung disease, which is characterized by chronic infection and inflammation. novel therapies to target airway inflammation are likely to improve the clinical outcome in cf. as yet there is no consensus regarding the molecular pathway(s) mediating inflammation in the cf lung. objective: to establish whether toll-like receptor (tlr) signalling is the key molecular pathway responsible for the inflammation seen in cf lung disease and to determine whether inhibitors of tlr signalling can reduce this damaging inflammatory response. methods and results: to quantify the airway inflammatory response, we used established cf and cf-corrected lung epithelial cells (ib - and c ). since artefacts may alter the inflammatory phenotype of cell lines, we validated all findings with fresh peripheral blood mononuclear cells (pbmcs) from cf patients (n= ) and healthy controls. we measured the inflammatory response (il , il , tnfα) of these cells following stimulation with: (i) cf pathogens (p. aeurginosa, b. multivorans, b. cenocepacia); (ii) purified tlr ligands. both cf respiratory epithelial cells and cf pbmcs had a greatly increased inflammatory response compared to matched controls when stimulated with whole bacteria and purified tlr ligands (p< . ). interestingly, the cf airway cells responded most vigorously to the tlr ligand, flagellin (p< . ). to investigate tlr as a potential anti-inflammatory target, we found that tlr mrna (p< . ) and protein (p< . ) were expressed at a higher level in cf compared to cf-corrected lung epithelial cells. finally, to determine the contribution of tlr activation to cf lung inflammation we exposed the airway cells to wild-type (pak & pao ) and isogenic flagellin-deficient strains of p. aeruginosa (pak∆flic, pao ∆flic, pao ∆flge & pao ∆flim) . strikingly, the absence of the tlr -flagellin interaction significantly reduced the proinflammatory response of cf respiratory epithelial cells to p. aeruginosa (p< . ). we show that tlr-mediated innate immune responsiveness is increased in both cf respiratory epithelial cells and fresh blood cells from cf patients. moreover, inhibition of the tlr -flagellin interaction markedly reduced proinflammatory response of cf respiratory epithelial cells following exposure to predominant cf pathogen p. aeruginosa. these data suggest that tlr activation may represent a novel anti-inflammatory target for improving cf lung disease. supported by the canadian cf foundation. cf airway cells (ib - ) were stimulated with wild-type (pak) and flagellin-deficient p. aeruginosa (pak∆flic). introduction: genetic polymorphisms for tgf-β have recently been identified as a significant modifier of cf lung disease severity. however, at present little is known about protein measurements of tgf-β in bronchoalveolar lavage fluid (balf) or serum obtained from pediatric cf patients during an acute respiratory exacerbation. hypothesis: tgf-β in cf balf and serum is increased in association with markers of cf lung disease severity, and is decreased after completion of iv antibiotic (iv abx) therapy. study design and methods: in a descriptive, prospective study of children with cf presenting for hospital admission to treat a pulmonary exacerbation, balf specimens were obtained from patients undergoing clinically-indicated flexible bronchoscopy on admission. balf was immediately analyzed for total and differential cell counts with the cell-free balf supernatant stored at - °c. serum specimens were obtained at the initiation and conclusion of iv abx and similarly stored at - °c. measurement of total tgf-β utilized a commercial elisa kit. t-test statistical analysis assumed significance at p<. . results: balf was obtained from cf patients with a mean age of . ± . years (range - years). three balf cultures were pseudomonas aeruginosa positive (psa+). balf tgf-β levels ranged from - pg/ml (mean ± . pg/ml) with the highest tgf-β measured in a patient with abpa and culture positive for m. abscessus. in balf, higher neutrophil counts (greater than the median, . x pmn/ml) were associated with increased balf tgf-β ( ± . pg/ml vs. ± . pg/ml, p<. ). serum tgf-β was collected in cf patients with a mean age of . ± . years (range - years), four of whom were psa+. serum tgf-β ranged from . - . ng/ml (mean . ± . ng/ml). in both balf and serum, neither patient age nor psa culture status was associated with increased tgf-β at admission. however, previous hospitalization in the last months was significantly associated with an elevated admission tgf-β in both balf ( ± . pg/ml vs. ± . pg/ml, p<. ) and serum ( . ± . ng/ml vs. . ± . ng/ml, p<. ). after completing the clinically-indicated course of iv abx, / psa-cf patients had a reduction in serum tgf-β (mean . ± . ng/ml to . ± . ng/ml, p=ns). in contrast, / psa+ cf patients had an increase in serum tgf-β (mean . ± . ng/ml to . ± . ng/ml, p=ns) after completing iv abx therapy. conclusions: tgf-β is measurable in balf and serum from pediatric cf patients and varies in association with clinical parameters. these preliminary results support our hypothesis that increased tgf-β may be associated with more severe lung disease as evidenced by ) association of increased tgf-β in balf and serum obtained from cf patients with previous hospitalization in the last months, and ) association of increased tgf-β in balf with increased cellular inflammation. the direction of change in serum tgf-β after iv abx therapy may be influenced by the presence or absence of pseudomonas infection. acknowledgments: supported by cff (harris ao). paula murphy and cassidy henegar provided technical support. background: cystic fibrosis is one of the leading genetic causes of end stage lung disease for which the treatment is lung transplantation. however, mortality due to chronic rejection known as obliterative bronchiolitis (ob) remains high due to small airway obliteration. ob is associated with increased levels of matrix metalloproteinase- (mmp- ), an interstitial collagenase, expressed by polymorphonuclear cells (pmns). pmns are among the first inflammatory cells to be detected in ob but very little is known about the role of pmn mmp- or the mechanisms used by pmns to migrate through extracellular collagen matrices. objective: to identify the molecular mechanism for neutrophil recruitment into the airway. we hypothesize that mmp- promotes accumulation of pmns in ob lesions by promoting their migration through the extracellular matrix protein barriers by its pericellular collagenase activity. method: the heterotopic airway transplant model was used to study the role of neutrophil mmp- in promoting pmn migration through extracellular matrices. mhc-disparate balb/c tracheas were subcutaneously transplanted into c bl/ wild-type (wt) or mmp- deficient (mmp- -/-) mice. to further dissect the mechanism of pmn migration in vitro their migration through collagen gels was studied. collagen gels were made in transwells using rat-tail collagen providing a highly cross-linked thick collagen barrier. bone marrow derived pmns from wt or mmp -/-mice were fluorescently labeled and stimulated to induce the surface expression of mmp- . pmns were placed on thick collagen gels in traswells. the transwells had polycarbonate membranes at the bottom of the gels. the pore size of the membranes did not allow pmns to pass through into the lower chambers that had flmp a chemo attractant. at hours and hours the gels were fixed and the polycarbonate membrane at the bottom of the collagen gel was gently peeled off. pmns on the membrane were counted under a fluorescent microscope. results: significantly decreased migration of pmns into the airway lumen was noted in the mmp- deficient mice (p< . ) weeks post-transplantation compared to wt mice in the heterotopic airway transplant model. in addition there was more than a three-fold increase in the pmns outside the lumen in the extracellular collagen matix (p< . ). wt-pmns were able to penetrate collagen barriers in greater numbers both at and hours compared to mmp -/-pmns in vitro (p < . for hours and . for hours). in addition, mmp inhibitor gm -treated wt cells demonstrated decreased migration through collagen gels ( hours p< . and hours p< . ). conclusions: mmp- -/-mice has significantly less pmns in the airway lumen due to decreased migration through collagen matrices providing protection against ob. this has great significance for conditions such as cystic fibrosis, lung transplant rejection (obliterative bronchiolitis) and other inflammatory diseases mediated by pmns. mmp- blockade can potentially be a novel therapeutic target to decrease neutrophil efflux into the airways and thus reduce airway inflammation. background: our laboratory has demonstrated alterations in cf-related regulation of cholesterol processing. hydroxycholesterols (oxysterols) are oxygenated derivatives of cholesterol formed from autoxidation of cholesterol and from de novo synthesis. oxysterols are known to be key regulators of cholesterol metabolism and cellular signaling. the role of oxysterols in the cellular pathways involved in cholesterol processing in cystic fibrosis has not been elucidated. based on our previous findings of increased de novo cholesterol synthesis in cf tissues, we hypothesize that oxysterol production in cf would be positively impacted. increased oxysterol production may lead to abnormalities in cellular signaling associated with oxidative stress and may influence the inflammatory response in cf. the goals of this research are to investigate the effect of oxysterols on inflammation in cf and to determine if there is a difference in oxysterol production in cf compared to wild type controls. results: the goal of these studies is to determine if cf cells respond uniquely to oxysterols compared to control cells. initial studies demonstrate that cf-model /hteo-pcepr cells failed to respond to -oh cholesterol ( -oh) with regards to nfκb activation. control /hteo-pcep cells, however, exhibited a significant -fold increase in nfκb activity upon -oh stimulation ( . -fold +/- . increase from il- , tnf alpha alone). however, il- promoter activation in response to -oh was not significantly altered in either cf or wt cells ( . -fold +/- . (cf) increase from il- , tnf alpha alone, . -fold +/- . (wt) increase from il- , tnf alpha alone). in order to verify il- promoter results, il- protein production was measured directly in response to pseudomonas aeruginosa. il- protein level in response to -oh was increased in cf cells ( . -fold) and decreased in wt cells ( . -fold), although not significantly in either group. oxysterol content and de novo synthesis are currently being examined in vivo in cf and wild type mouse models. mice lacking cftr on a c bl/ background and wild type controls were obtained from our animal core and the amount of oxysterol present in these mouse models is currently being studied. conclusion: these findings suggest that endogenous oxysterol production or other components of oxidative stress are pre-stimulating nfκb activation via the oxysterol-sensitive pathway. interestingly, although oxysterols robustly activate nfκb in wt cells, oxysterol-dependent pathways are not involved in il- production. these data suggest that il- is not a primary cause for increased oxysterol mediated inflammation. elucidating the role of oxysterols in cf will be an important contribution to our understanding of the inflammatory pathways in cf and will contribute to the growing body of evidence for aberrant cholesterol regulation in cf. this work is supported by grants from the cff and the nhlbi. cystic fibrosis (cf) is a common genetic disorder caused by a mutation in the cftr, a chloride transporter of the abc transporter family. innate immunity, the first line in host defense against infectious agents, is abnormal in cf patients. cf patients are commonly colonized with bacterial pathogens, which is the major clinical problem of this disease. despite a marked difference in the clinical outcome amongst cf patients infected with pseudomonas aeruginosa and burkholderia cepacia, the mechanisms under-lying these differences remain unclear. understanding the innate immune response, specifically the role of cftr, in p. aeruginosa and b. cepacia infections will lead to new insights into mechanisms that limit these infections. our objective is to develop a cf model in the simple organism, the worm, c.elegans, and to test the role of the cftr orthologue, mrp- , in host defense. using this model, we have compared the survival of c. elegans between virulent p.aeruginosa (pa ) and b.cepacia (bc). our experiments have demonstrated that, when compared to growth on their natural food source, e.coli op , worms fed on pa and bc die prematurely. specifically, worms survive up to hours on op , whereas pa fed worms die with in hours. death on pa also requires active infection, as heat inactivation of these bacteria destroys their killing capacity. two bc strains have been tested, a virulent strain ( ) and an avirulent strain ( ). worms infected with bc show an intermediate phenotype, surviving approximately hours. our future work will define whether synergy occurs during coinfection with p.aeruginosa and b.cepacia, as would be suggested by clinical data. in addition, we will define the transcriptional changes induced in c.elegans fed on pa and bc, and use these data to identify common and pathogen specific responses to these infections. finally, mrp- knock-down worms will be generated using rna interference, and the role of the cftr orthologue in infection will be tested. in conclusion, c.elegans offers a genetically tractable model organism in which the innate immune response to pseudomonas aeruginosa and burkholderia cepacia can be studied. in addition, this simple model system will allow us to define the role of the cftr orthologue in host defense. supported serine proteases released from neutrophils are considered central to the pathogenesis of cf lung disease, and have been obvious therapeutic targets. although intracellular serine proteases are important in host defense and bacterial killing, extracellular enzymes may contribute to bacterial persistence and promote airway inflammation. neutrophil elastase (ne) digests key opsonins present in the lung and has been shown to disrupt phagocytosis, allowing the bacteria to persist in the face of established pulmonary inflammation. in addition we have found that other proteases, like cathepsin g (cg), an abundant serine protease found in human and murine neutrophils, may also have other, critical roles in development of cf lung disease. indeed, using murine models of endobronchial inflammation, cg inhibits airway defenses and interferes with the host's ability to clear pseudomonas aeruginosa from the lung with effects quite distinct from ne. to test the hypothesis that differences in bacterial killing are due to defects in innate defenses created by excess, unopposed cg at the apical surface of the airway epithelium, we have examined profiles of proteins secreted into the airway lumen by epithelial cells, which are necessary for p. aeruginosa clearance and susceptible to cg proteolysis. analysis of lavage fluid utilizing -d page indicated a total of spots which were increased in pooled lavage fluid from infected cg-deficient mice as compared to wild-type littermates. tandem mass spectrometry analysis identified several novel proteins that were cleaved by cg, including inter-alpha inhibitor protein, selenium-binding protein, sec -like protein, beta- -glycoprotein, and annexin a . another protein candidate, serum amyloid p component (sap), is a novel opsonin and may be involved in airway defenses. immunoblot analy-sis demonstrates that cg cleaves sap. these results suggest that cg, like ne, plays an important role in the pathogenesis of lung diseases. we have previously demonstrated in ∆f -cf mice that in vivo treatment with azithromycin (azm) attenuates cellular infiltration in baseline and lipopolysaccharide (lps)-induced inflammation, and inhibits proinflammatory cytokine release in induced inflammatory condition (legssyer et al, ) . this study aimed at investigating macrophages isolated from wild type (wt) or ∆f -cf mice (van doorninck et al, ) and the effect of azm on these cells. purified peritoneal and alveolar macrophages were stimulated with lps (p. aeruginosa, . µg/ml) plus ifnγ ( . µg/ml), with or without azm ( µg/ml). macrophage inflammatory status was investigated by assessing different pro-and anti-inflammatory mediators at mrna level h after stimulation. pro and anti-inflammatory markers seemed to be reduced in naive alveolar cf macrophages. by contrast, a trend to overexpression of these markers was seen in naive peritoneal cf cells. pro-inflammatory status, as assessed by il- β, was induced in stimulated alveolar and peritoneal cf macrophages and in naive peritoneal cf cells. moreover, nos- was found to be overexpressed in stimulated alveolar cf macrophages while il- was downregulated. azm significantly reduced il- β expression in stimulated alveolar macrophages. in conclusion, peritoneal and alveolar macrophages exhibit distinct phenotype. a pro-inflammatory status was more prominent in stimulated cf alveolar and peritoneal cells. interestingly, azm modulates il- β overexpression only in cf stimulated alveolar macrophages, supporting the antiinflammatory activity of this macrolide and identifying, at least partly, alveolar macrophages as possible target cells for its effects. supported it has been recently reported by perez and coll. that the inhibition of function of w/t cftr produces an inflammatory profile that resembles that observed in cf patients [am j physiol lung cell mol physiol, ] , whereas we demonstrated that correction of f del cftr function with mpb- down modulates the pseudomonas aeruginosa (p.aeruginosa) dependent expression of the pro-inflammatory mediators il- and icam- in cf bronchial cells [am. j. resp. cell. mol biology, ] . since both evidence support a direct link between cftr function and inflammatory response in respiratory epithelial cells, we extended our investigation to other f del cftr correctors, such as miglustat [norez et al, febs letters, ] , which is an approved drug for gaucher disease, in comparison with an isomer without any correcting effect, namely nb-dgj. human bronchial ib - (cftr f del/w x), cufi- (cftr f del/f del) and nuli- (cftr w/t) cells were exposed to the laboratory strain of p.aeruginosa (pao ) or tnf-alpha or il- beta and the transcription of icam- and il- were quantitated by real time rt pcr. cftr function was assayed by single-cell fluorescence imaging, using the potential-sensitive probe disbac [renier et al, hum. gene ther. ]. analysis of binding of nf-kb and ap- transcription factors to labelled dna target was performed with electrophoretic mobility shift assay (emsa) [borgatti et al, j. biol. chem. ]. miglustat significantly reduced the expression of il- by % in ib - and by % in cufi- cells and of icam- by % in ib - and by % in cufi- cells, upon infection by pao . in parallel, correction of f del cftr function was observed after miglustat treatment in both ib - and cufi- cells. miglustat had no major effects on overall binding activity of transcription factors nf-kb and ap- , activated by pao in ib - cells. the inflammatory response induced by tnf-alpha or il- beta was also significantly reduced in ib - and cufi- cells treated with miglustat. nb-dgj, which is not a corrector of function of f del cftr, down modulated the induction of il- and icam- in ib - and cufi- cells. in addition, both miglustat and nb-dgj reduced the inflammatory response to pao in non cf nuli- cells. in conclusion, miglustat and nb-dgj have an anti-inflammatory effect in bronchial cells independently of the correction of f del cftr, by interfering with the pro-inflammatory signaling downstream the receptors for pathogens and pro-inflammatory cytokines. since miglustat is already approved for the treatment of gaucher disease and other glycosphingolipidoses, it may be an interesting candidate to ameliorate lung inflammation in cf patients. background: the primary cause of mortality and morbidity in cystic fibrosis (cf) is lung disease, which is characterized as a chronic cycle of infection and inflammation dominated by a neutrophilic infiltrate and increased levels of circulating mediators such as il- . a wide variation in the rate of progression of lung disease is observed in cf. hypothesis: we postulate the presence of a brisk systemic inflammatory response in cf patients and that this systemic response reflects the magnitude and progression of lung disease. methods: lung function and clinical data were obtained from cf patients treated and followed in the adult cf clinic, st paul's hospital. forced expiratory volume at s (fev ) and forced vital capacity (fvc) values dating back to were used in regression equations to group patients into quartiles based on their decline in predicted fev %. rate of decline in lung function was compared with clinical characteristics and various systemic inflammatory biomarkers, including wbc, band cell count, crp, il- β, il- , il- , mcp- , and gm-csf results: the quartiles exemplified significantly different rates of decline in fev /fvc% values. subjects with a rapid decline in lung function were younger and were colonized/infected more commonly with methicillinresistant staphylococcus aureus (mrsa) (p< . ). in addition, altered calcium, magnesium, and albumin levels were found in subjects with increased rates of decline in lung function (p< . ). preliminary results show that patients with cf have significantly elevated levels of wbc ( . e /l), crp ( . e ng/ml), and il- ( . pg/ml) in serum when compared to control subjects (p< . ). there were trends in relationships between levels of il- , il- and mcp- and rates of decline in lung function, however none reached statistical significance. conclusion: there is a variable rate in the decline in lung function among cf patients and one factor may be a heterogeneous systemic inflammatory response. circulating pro-inflammatory mediators may not only impact progression of lung disease, but could also be novel biomarkers to monitor and assess disease severity in cystic fibrosis. nutritional status in addition effects the rate of decline in lung function perhaps by modifying the inflammatory response. this research is supported by the bc lung association. overexpression of the epithelial na + channel β subunit (protein = βenac, gene = scnn b) in transgenic mice results in cf-like lung pathology, characterized by neonatal mortality, mucus obstruction and airway inflammation (mall et al., nat. med. ) . breeding βenac mice into gene-deleted mice enables quick and efficient determination of the specific pathways relevant to the development of lung pathology. tnfα is a pleiotropic pro-inflammatory cytokine released by many different cell types, including t cells, macrophages, granulocytes, and epithelial cells. tnfα levels are significantly elevated in bronchoalveolar lavage (bal) from βenac mice in comparison to wild-type (wt) littermates. we crossbred βenac mice (heterozygous, inbred line c - ) with tnfα knockout mice, generating four types of mice: wt, tnfα +/-; wt, tnfα -/-; βenac, tnfα +/-; and βenac, tnfα -/-. all mice had comparable, high survival, ranging between - %. the lack of tnfα did not prevent neutrophil and eosinophil infiltration and did not significantly modify the lung pathology characteristic of βenac mice, namely mucus plugs, mucous secretory cell hyperplasia, airway inflammation and emphysema. the absence of tnfα in bal from βenac, tnfα -/-mice, in comparison to βenac, tnfα +/mice, was confirmed by luminex cytokine assay. the levels of kc, a potent neutrophil chemoattractant, were significantly elevated in bal fluid from βenac mice, regardless of the presence or absence of tnfα. in wt mice, no granulocytic infiltrate or morphologic changes were observed and tnfα and kc were undetectable. similarly, the pathologic changes in βenac mice were not mitigated by crossbreeding to knockout mice for tnfα receptor (tnfαr ), the major mediator of biologic responses classically attributed to tnfα. these results suggest that tnfα per se does not have a critical pro-inflammatory role in the development of inflammation in the βenac mouse model. the production of alternative cytokines may compensate for the loss of tnfα bioactivity in the tnfα and tnfαr -/-mice. since tnfα has been suggested to play a role in the regulation of enac, ussing chamber studies are underway to test whether the bioelectric features of tracheobronchial epithelia from βenac mice are altered by the absence of tnfα in vivo. these data suggest that a tnfα-independent signaling cascade links airway surface dehydration to airway inflammation in the βenac mouse model. innovative pharmacological approaches to control the excessive neutrophil infiltrates into the bronchial lumen of cf patients are thought to be beneficial to reduce the extensive airway tissue damage. the activation of expression of proinflammatory genes by p.aeruginosa with bronchial epithelial cells is a central mechanism to be targeted with novel therapies. medicinal plants from the socalled traditional asian medicine are attracting a growing interest because of their potential safety, already tested in large scale applications in human diseases. however, due to the presence of different active principles in each plant extract, whose multifunctional effects may even result contradictory, understanding the effect of each component is mandatory to pursue selective and reproducible applications. a panel of medicinal plant extracts have been firstly screened for their capacity to interfere in the binding of nuclear transcription factor proteins (tf) with dna consensus sequences identified in the promoters of the pro-inflammatory genes, thus for their potential inhibitory action on gene expression. extracts from several medicinal plants have been screened for their ability to interfere with the tf nf-kb, ap- and creb, which are induced by p.aeruginosa, and some of them, such as emblica officinalis (eo), aegle marmelos (am), polyalthia longifolia (pl), have been shown to inhibit tf/dna interactions, opening the possibility of potential applications to downregulate expression of pro-inflammatory genes. extracts from eo, am, pl were tested in ib - cf bronchial cells exposed to the p.aeruginosa laboratory strain pao . eo, am and pl strongly inhibited the pao -dependent transcription of il- in ib - cells. pyrogallol, one active principle of eo, was tested in ib - cells, where it inhibited the transcription of the neutrophil chemokines il- , gro-alpha and gamma, of the intercellular adhesion molecule icam- and the pro-inflammatory cytokine interleukin , similarly to the whole eo extract, whereas a second active principle from eo, namely hydroxy-isoquinoline, had no effect. similar results were obtained with eo and pyrogallol in the monocyte-derived macrophage cell line thp- exposed to pao . in conclusion, extracts from plants of the traditional medicine can inhibit expression of pro-inflammatory genes and screening active principles purified from medicinal plants could result useful to identify safe and innovative pharmaceutical molecules to control lung inflammation in the lung of cf patients. supported by italian cystic fibrosis research foundation and by fondazione cariverona -bando -malattie rare e della povertà. massive infiltrates of neutrophils in the mucosal wall and lumen of the conductive airways of cf patients contribute to the progressive lung func-tion decline by releasing different proteases responsible for the progressive airway tissue damage. bacterial products and pathogens themselves within the mucopurulent material of the airway surface fluid induce the activation of transcription factors such as nf-kappab, ap- , sp , nf-il , nf-at, elk- , creb resulting in expression of chemo/cytokine genes driving the recruitment of leukocytes inside the bronchial lumen. to find new treatment options focused on the reduction of neutrophil chemotaxis, without abolishing the immune response against pathogens, we are exploring the transcription factor (tf) "decoy" strategy, in which oligodeoxynucleotides (odn) mimicking the consensus sequences for the tfs proteins identified in the promoters of different chemo/cytokines are delivered inside the cell in order to interfere with gene transcription. cf bronchial epithelial cells ib - have been exposed to the (i)p.aeruginosa(/i) strain pao . a clear pao -dependent activation of tfs such as nf-κb, sp , ap- , nf-at, nf-il was confirmed by electrophoretic mobility shift assay (emsa). in parallel, transcription of genes involved in innate immune response has been quantified by real-time rt pcr. transcription factor "decoy" odns directed against the consensus sequences identified in the promoters of different genes have been designed and validated by testing their interference in tf protein/dna binding assays. transfection of ib - cells with hiv- ltr and igk chain nf-κb odn "decoys" complexed with lipofectamine, performed hrs before challenge with pao , was shown previously to inhibit strongly pao -dependent transcription of il- but not gro, il- β, il- and icam- . therefore other tf "decoy" odns have been also tested: a) odn for nf-κb from il- promoter inhibited il- , gro-gamma and il- by %, b) odn for sp from hiv- genome inhibited il- by %, c) odn for ap- from il- promoter inhibited both il- and gro-gamma by %. a tf "decoy" molecule designed as peptide-dna chimera mimicking the consensus sequence of hiv ltr nf-κb strongly and selectively inhibited il- transcription. in conclusion, transcription of chemo/cytokines induced by (i)p.aeruginosa(/i) in cf bronchial epithelial cells (i)in vitro(/i) can be inhibited with different efficiency and selectivity by tf "decoy" molecules. these results provide useful hints for a gene-targeted anti-inflammatory approach and add further information on the regulation of expression of pro-inflammatory genes induced by (i)p.aeruginosa(/i) in bronchial epithelial cells. supported by italian cystic fibrosis research foundation and by fondazione cariverona -bando -malattie rare e della povertà. background: cf is characterized by hypersecretion of the pro-inflammatory cytokine il- from airway epithelial cells. however the mechanism by which il- gene expression is dysregulated in cf is not known. the expression of cytokine and chemokine genes is known to be regulated at multiple mechanistic steps including transcription, mrna decay, translation, and various post-translational steps. sequence-specific mrna degradation is now recognized to be an important site controlling the expression of several chemokine mrnas. this selective behavior is conferred by cisacting elements in the mrna composed of au-rich sequences (ares). the regulatory function of ares is thought to be mediated via rna-binding proteins that specifically recognize the are motifs. the steps required for mrna decay is comprised of deadenylation, decapping and body decay. deadenylation or removal of the poly (a) tail by poly(a)-specific ribonuclease (parn) appears to be the first and perhaps the rate-limiting step. this is accompanied by decapping or enzymatic removal of the ' methylate guanosine cap. subsequently, exonuclease activities in the ' to ' or ' to ' direction predominate to degrade the remaining mrna. hypothesis: we have hypothesized that defects in transcription as well as modulation of the post-transcriptional stability of il- mrna might contribute to hyper-production of il- protein in the cystic fibrosis. methods: il- mrna stability was assessed in cystic fibrosis ib - lung epithelial cells and in aav-cftr-repaired ib - /s cells, by measuring residual il- mrna levels at various intervals of time after addition of actinomycin-d to the culture. protein s extracts, prepared from the cf cell line as well as the cftr-corrected cell line, were analyzed by western blot. the expression levels of the various factors known to participate in are-mediated mrna decay, including ttp (an are-binding protein), parn and the exosome were compared in the two sets of s extracts. results: we find that the levels of il- mrna in the cf cells are greater than the levels found in the cftr-repaired cells. in addition, we found that the rate of decay of il- mrna in cf cells was significantly less than that in the repaired cell line. the levels of ttp, parn and exosome proteins were significantly reduced in the cf cells compared to the cftrrepaired controls. however, expression of ttp in ib - cells causes significant destabilization of il- mrna. conclusion: we conclude that the high levels of il- protein expression in cf lung epithelial cells can be partly due to enhanced stability of the il- mrna. consistently, the actual levels of il- mrna are higher in the cf cells compared to controls. understanding the mechanism by which ttp promotes enhanced destabilization of il- mrna in cf cells may be important for developing novel therapeutic targets to alleviate the pulmonary pathophysiology of this disease manifested by hyper-secretion of il- protein. understanding the mechanism of airway remodeling could lead to the identification of novel therapeutic targets for the prevention of irreversible lung damage in cf. we have investigated transcriptional responses to epithelial injury in a mouse model of cf that was developed in our laboratory (f del, cftr tm eur fvb backcross f ). to induce transient epithelial lung injury, homozygous normal and mutant age matched littermates were treated in parallel with naphthalene ( mg/kg ip) or carrier (corn oil) as control (n= x ). naphthalene causes an almost complete selective ablation of clara cells overnight, followed by migration of resistant ciliated cells and rapid proliferation of undifferentiated progenitor cells. two and seven days after treatment, lungs were collected for histological and transcriptome analysis. based on a previous affymetrix microarray analysis (n= ), a quantitative pcr array was designed containing genes differentially expressed after naphthalene injury. as expected, naphthalene injury results in a transient increase of cell proliferation markers (mki , cdc , cdc a etc), and a strong ( %) reduction of clara cell markers (cc /scgb a ; claudin /cldn ; cyp f ). in addition, we have now identified twenty-five genes that are increased (p< . ) two-to fifty-fold after naphthalene injury in both normal and mutant mice. this includes known and novel markers of epithelial tissue injury such as timp , retlna, mmp , serpin n, and lipocalin. in particular, a group of egf receptor agonists is strongly induced: amphiregulin (areg, fold), epiregulin (ereg fold) and heparin binding egf ( fold). amphiregulin mrna can be detected by in situ hybridization only in airway epithelial cells after injury. these egfr agonists are involved in stimulating epithelial repair, but can also activate cells in the underlying mesenchyma. indeed, we observe a substantial increase of major extracellular matrix mrna's two days after injury (co a : -fold, col a : -fold, elastin: -fold). whereas expression levels were substantially reduced in normal animals seven days after injury, in cf mutant animals two to three fold higher levels were observed for these three genes at day seven (p< . ). conclusions. we have designed and validated a gene array that can be used to study distal lung injury and repair. egfr agonists produced by epithelial cells are the most prominent growth factors after injury, involved in both epithelial repair and extracellular matrix production by mesenchymal cells. ecm production in cf mouse lung is sustained compared to normal mice, suggesting an inherent tendency towards fibrosis in cf lungs. our data suggest that the mesenchymal egf receptor and regulation of its agonists are important future targets of novel therapeutic strategies. supported by eec th fw projects eurocarecf, improved precision the mucosa of the proximal airways defends itself and the lower airways from inhaled irritants, allergens, and microbial and viral infections by several mechanisms. sensory nerves monitor the luminal microenvironment and trigger reflexes in the central nervous system (cns) that alter breathing, induce cough and stimulate mucus secretion when challenged with noxious stimuli. sensory nerves also release the tachykinins substance p (sp), neurokinin a and calcitonin gene-related peptide through axon reflexes in neighboring tissues, and these locally released tachykinins stimulate mucus secretion by binding to neurokinin receptors on submucosal glands. recently we reported that local fluid secretory responses to noxious stimuli are dependent on the clchannel cftr in mouse airway submucosal glands and are defective in glands from cftr knockout mice (ianowski et al., j physiol, , , ) . we have now tested the effects of sp directly and examined the possible role of cftr in mediating these responses using tracheas from congenic wild-type and cftr knockout mice (cftr m unc /cftr m unc ) that had been bred onto c bl/ j or balb/c genetic backgrounds. we compared single gland secretion rates using optical methods as described previously. the cftr genotype of each mouse was assessed by using pcr to amplify genomic dna from tail clippings obtained at age days. after the cftr knockout mice were weaned, intestinal obstruction was minimized by supplementing their water with peglite. capsaicinoids (chili pepper oil) increased fluid secretion from glands of wild-type mice from ± pl/min (n= tracheas, glands) to ± pl/min (n= tracheas, glands, anova p< . , tukey-kramer multiple comparison test p< . ). this response was abolished by exposing the basolateral surface of the tracheas to l- , ( µmol/l), a known sp (nk- ) receptor antagonist ( ± pl/min, not different from control, tukey-kramer multiple comparison test p> . ). secretion was stimulated from ± pl/min to ± pl/min (n= tracheas, glands, student's t-test p< . ) by the direct application of sp, and this response was strongly inhibited by pre-incubation with the cftr inhibitor cftrinh (saturating concentration, nominal µmol/l; n= tracheas, glands). finally, submucosal glands from cftr knockout mice failed to secrete when exposed to sp ( µmol/l) whereas wild-type littermates were responsive. these results indicate that sp mediates local responses to capsaicinoids through a cftr-dependent mechanism. loss of this local regulation in cf may contribute to the susceptibility of cf airways. support: canadian cf foundation, canadian institutes of health research, nih (dk ), and the cff (usa). chronically infected/inflamed cf human bronchial epithelia (hbe), or normal hbe exposed to supernatant from mucopurulent material (smm) from human cf airways, exhibit expansion of the endoplasmic reticulum (er) ca + stores and amplification of ca + -mediated inflammatory responses. we have shown that infection/inflammation of hbe triggers an unfolded protein response (upr) coupled to mrna splicing of x-box binding protein- (xbp- ). spliced xbp- (xbp- s) is a transcription factor that promotes er expansion to augment the protein folding capacity during increased protein synthesis. because we have shown that hbe inflammation couples to increases in protein synthesis, we hypothesized that hbe infection/inflammation-induced er ca + store expansion is mediated by xbp- s. to test this hypothesis, we constructed four retrovirally-tranduced stable hbe o-cell lines containing empty vector (ev), xbp- unspliced (xbp- u), xbp- s, or dominant negative xbp- (dn-xbp- ). cells were grown to confluence under an air-liquid interface and er ca + store expansion and il- secretion studied in the absence or following hr mucosal pbs or smm exposure. consistent with our hypothesis, expression of xbp- s in the absence of stimulation induced er expansion and increased mucosal utp-sensitive er ca + stores ( to confirm that these effects were mediated by upr activation, cells were transfected with a upr response element luciferase reporter plasmid, and luciferase activity was measured in the absence or presence of the upr inducer tunicamycin (tm). cells expressing xbp- s exhibited higher baseline luciferase activity than cells expressing ev or xbp- u, whereas luciferase activity was decreased in the dn-xbp- expressing cells. furthermore, while tm increased luciferase activity in cultures expressing ev or xbp- u, its effect was blocked in dn-xbp- expressing cells. these findings suggest that xbp- s is the major trigger of er ca + store expansion, which mediates the amplified ca + -dependent inflammatory response in infected/inflamed airway epithelia. although it remains to be established whether upr-dependent xbp- s is a beneficial or a maladaptive response in infected/inflamed airways, therapies aimed at manipulating this upr pathway may be beneficial for patients with chronic inflammatory lung diseases. luminal exposure of well-differentiated normal human bronchial epithelia (hbe) to supernatant from mucopurulent material (smm) from human cf airways increases the secretion of inflammatory mediators and triggers an unfolded protein response (upr) mediated by the mrna splicing of the x-box binding protein- (xbp- ). spliced xbp- (xbp- s) is a transcription factor that expands the endoplasmic reticulum (er) and protein secretory pathway during er stress induced by accumulation of unfolded proteins due to increases in protein synthesis. our previous studies in primary cultures of hbe and hbe o-cells stably expressing empty vector, inactive unspliced xbp- (xbp- u), xbp- s or a dominant negative xbp- (dn-xbp- ) suggest that induction of xbp- s by smm exposure promotes er ca + store expansion, which mediates a ca + -dependent hyperinflammatory response. the present studies were designed to determine if pseudomonas aeruginosa pak strain (p.a.) would reproduce the effects of smm in both in vitro and, importantly, in vivo models. for in vitro studies, hbe were exposed to broth (tsb) or to a % p.a. extract for hr, and inflammation, xbp- s, and er ca + store expansion investigated by il- secretion, southern blots and calreticulin expression, respectively. similar to smm, p.a. induced a . fold increase in il- secretion coupled to a fold increase in xbp- s and larger er ca + stores as compared with tsbexposed hbe. these data demonstrate the link between p.a. infection, upr activation and xbp- s in vitro. we next tested the relevance of these findings in vivo. wild-type mice airways were challenged with pbs or cfu of p.a., and airway epithelial er density was assessed by calreticulin staining hr later. in comparison with pbs challenges, p.a.-challenged mice exhibited airway epithelial er expansion, which correlated with the degree of airway inflammation, based on the presence of inflammatory cells. to test whether this p.a.-induced er expansion was linked to xbp- s, we utilized "er stress activated indicator" (erai) mice expressing a fusion protein consisting of xbp- u and the fluorescent protein venus. in erai mice, upr activation-dependent xbp- splicing leads to venus expression; hence, venus fluorescence is an index of xbp- s. consistent with our hypothesis that p.a. infection-triggered inflammation would induce upr-dependent xbp- s, venus fluorescence, after hr challenge with cfu of p.a., was increased in inflamed as compared with non-inflamed airways. these findings suggest that ) airway epithelia respond to bacterial infection-induced inflammation by up-regulating the er ca + stores and ) activation of the xbp- s pathway by bacterial infection may be relevant to airway inflammatory responses in vivo. funded by the cff. we have shown that luminal exposure of well-differentiated primary cultures of normal human bronchial epithelia (hbe) to supernatant from mucopurulent material (smm) from human cf airways increases total cellular protein synthesis, which reflects the increased secretion of inflammatory factors induced by the infectious and inflammatory process. in the present studies, we first investigated whether these hbe responses to smm were linked with an increased metabolic rate by measuring lactate accumulation into the serosal media. smm increased lactate production, and this effect was maximal within hrs ( . + . vs. . + . , and . + . vs. . + . mmol/l in and hr pbs vs. smm, respectively; n= - ), suggesting that the increase in protein synthesis couples to a hyper-metabolic state in infected/inflamed hbe. in agreement with this notion, or hr smm, as compared with pbs exposure, induced the expression of genes associated with amino acid transport and metabolism (n= ). in addition, or hr smm exposure up-regulated genes involved in oxidative stress (n= ). we hypothesized that these hbe responses were linked to an unfolded protein response (upr) mediated by activation of the pkr-like er kinase/pancreatic eif α kinase (perk)-induced activating transcription factor (atf ), since this pathway has been shown to confer protection against amino acid loss and oxidative stress in other cells. we first tested whether smm induced activation of perk/atf in hbe by performing western blot analyzes of the components of this pathway. twenty-four hr smm exposure induced perk activation, as indexed by phosphorylation of perk, in comparison with pbs-exposed hbe. on the other hand, total perk protein levels were unchanged in smm-treated hbe. phosphorylation of eif α, the downstream effector of perk, and increased atf protein levels, which depend on the phosphorylated status of eif α, provided additional evidence that the perk/atf pathway was activated by smm. these data are consistent with the hypothesis that induction of atf is triggered by upr activation resulting from increased synthesis of inflammatory factors. we next utilized rna microarrays to test whether atf target genes were induced by smm. six or hr smm exposure induced atf target genes (e.g., ero , an oxido-reductase that provides protection against the accumulation of endogenous peroxides during er stress; stanniocalcin , whose expression is associated with anti-apoptotic functions; and heme oxygenase ). these findings suggest that ) activation of the upr-dependent atf pathway is a compensatory component of the airway epithelial adaptive response to luminal infection/inflammation, and ) activation of the atf pathway protects against inflammation-induced amino acid loss and oxidative stress by up-regulating genes involved in amino acid transport/metabolism and oxidative stress responses. unraveling the functions of atf should help determine if therapies targeted to manipulate pathway activity would be likely to improve lung function in patients with cf or other chronic inflammatory airway diseases. funded by the cff. miller, t.j.; perez, a.; qian, y.; davis, p. pediatrics, case western reserve university, cleveland, oh, usa fxyd is a cell surface protein originally identified in a screen for molecular markers of tumorigenesis. increased fxyd expression was found in tumors from stomach, thyroid, colon, pancreatic, breast and lung cancers and correlated with down-regulation of e-cadherin and poor patient prognosis. recent studies have shown that overexpression of fxyd promotes cell motility, decreases cell-cell attachment and increases tumor metastasis. fxyd , also known as dysadherin, is a member of small family of proteins known to regulate the na,k-atpase. we now report that fxyd is upregulated in cystic fibrosis (cf) airway epithelia and modulates wound healing. we show by immunohistochemistry and immunoblot analyses that fxyd is increased in the lungs of s x cf mice, and demonstrate an almost -fold increase in fxyd expression in the nasal epithelia of cf mice compared to wild-type littermates (p< . ). furthermore, we show that fxyd is upregulated in nasal scrapings from human cf patients compared to controls (p< . ). immunofluorescence data show that flag-tagged fxyd co-localizes with the na,k-atpase in epithelial cells, suggesting that fxyd , similar to other members of the fxyd family, may regulate na,k-atpase function. it has previously been shown that expression and localization of the na,k-atpase is required for efficient polarization and suppression of cell motility in epithelial cells. the recurrent remodeling of pulmonary epithelium as a result of bacterial infection in cf requires that airway epithelial cells polarize and migrate to wound sites in order to maintain lung integrity. thus we hypothesized that fxyd may be involved in wound healing after infection. laser-capture microdissection and microarray analysis of murine lung epithelia after hours treatment with p. aeruginosa indicated a significant, -fold increase in expression of fxyd that was confirmed by immunoblot analysis. others have shown that fxyd may mediate expression of mcp- , a critical determinant in monocyte recruitment, through activation of the nf-kb pathway. treatment of human tracheal epithelial (hte) cells with a cftr inhibitor ( ) confirmed that loss of cftr function correlated with increased fxyd expression by quantitative rt-pcr (p< . ), an effect that was abrogated with treatment of pdtc, an inhibitor of nf-kb (p< . ). we speculated that fxyd -induced increases in cell motility may be due in part to phosphorylation at serine . in a murine airway epithelial cell wound healing model, serine to alanine (s a) mutations at serine inhibited wound healing compared to wild type fxyd overexpression, whereas aspartic acid (s d) mutations increased wound healing (p< . ). immunoblot and immunofluorescence analyses of these mutants suggest phosphorylation at ser regulates membrane localization. we conclude that fxyd is increased in cystic fibrosis epithelia due to increased inflammatory mediators and suggest that fxyd may modulate airway epithelia wound healing after infection with p. aeruginosa through phosphorylation at ser . the inflammatory response to bacterial infection in the cf airway is exaggerated compared to normal, leading to the accumulation of millions of necrotic neutrophils. extracellular neutrophil elastase (ne) activity in the cf airway not only compromises innate defences by cleaving opsonins and reducing ciliary activity, but amplifies the inflammatory response by stimulating expression of the neutrophil chemoattractant il- and increases mucus production, in addition to degrading the tissue matrix leading to fatal bronchiectasis. ne therefore represents an important target for the development of new therapies. however, this strategy requires consideration of the normal physiological function of this enzyme, since previous studies in knock-out mice indicated an essential role for ne in cell migration, bacterial phagocytosis and killing. our approach was to use the intracellular neutrophil elastase inhibitor gw a to 'knock-out' ne activity in neutrophils in normal human blood and test the function of isolated cells in chemotaxis, bacterial phagocytosis and killing assays. whole normal human blood was incubated with gw a, or pbs control, for h at °c. neutrophils were isolated by sedimentation of red blood cells (rbc) on dextran , purification on lymphoprep and hypotonic lysis to remove contaminating rbc. chemotaxis towards il- was measured using a modified micro-boyden chamber. phagocytosis was assayed by the depletion of staphylococcus aureus (sa), pseudomonas aeruginosa (pa) and e. coli (ec) in supernatants following culture in a : ratio with pmn for h at °c. supernatants were diluted and remaining organisms were plated on agar and grown overnight to count viable colonies. bacterial killing was assayed by incubating bacteria and cells in a : ratio for minutes to allow phagocytosis, washing off remaining organisms, and incubating for h at °c. neutrophils were lysed with water and lysates plated on agar to test for bacterial growth overnight. results; gw a inhibited intracellular ne dose-dependently and at µm . ± . % ne activity remained (n= ). there was no significant effect of µm gw a on neutrophil chemotaxis, bacterial phagocytosis or killing of any organism compared to pbs-treated controls. phagocytosis data is shown in the table. thus, in the absence of ne activity, human neutrophils remain wellequipped with other defence molecules including myeloperoxidase and defensins to successfully maintain the role of the neutrophil in innate immunity. however, mouse neutrophils which lack defensins require ne activity for optimal intracellular bacterial killing, and mice are not a perfect model for studies of human infection. the development of novel inhibitors of ne to treat lung disease in cf therefore remains an important goal. supported by the cf trust of great britain. background: sphingolipid signalling may differ between individuals with cf and healthy controls. the response to bacterial inflammation is different, and uptake and inactivation of sphingosine- -phosphate, an intracel-lular pro-inflammatory mediator, is reduced in cf cells. it may therefore continue to act on g-protein coupled receptors in the plasma membrane. (boujaoude et al, j biol chem ) . furthermore, ceramide originating from basolateral sphingomyelin hinders augmentation of cftr-mediated anion conductance across the apical membrane, resulting in reduction of transepithelial airway anion secretion (ito et al, bbrc ) . aim of study: to determine if there is a difference in the levels of alkaline, neutral or acid sphingomyelinase (smase), or in the levels of neutral or acid ceramidase, in the intestinal or bronchial mucosa and some other tissues, between wildtype, homozygous (+/+) and heterozygous (+/-) delta-f cftr mice. methods: enzyme activities (duan and nilsson meth enzymol ) were determined in intestine (and content) divided into four regions, liver, lungs, kidney and spleen from deltaf -cftr mice (+/+) and controls (wildtype, +/-). results: there was an increased amount of neutral ceramidase in spleens from deltaf -cftr mice (+/+) in comparison to control mice (p= . ). no other significant differences were seen. conclusion: delta-f mutation did not influence the levels of alkaline smase and neutral ceramidase acting as ectoenzymes, or the levels of intracellular smases and ceramidases, which may all generate bioactive sphingolipid metabolites in intestine and lungs. the implications of the increased level of neutral ceramidase in spleen are not known. in children with cystic fibrosis (cf) there is a clear correlation between the development of chronic p aeruginosa infection and acceleration in the decline of lung function. when chronically present, p aeruginosa takes on a mucoid phenotype and is impossible to eradicate. prior to this, when colonisation is intermittent, it is possible to eradicate it with aggressive antibiotic regimes. we sought to examine the degree of inflammation and innate defence status in the lungs of children with cystic fibrosis in various stages of colonisation by looking at a range of proteases, innate defence proteins and markers of inflammation in broncho alveolar lavage (bal). children with cf were allocated to one of three groups in relation to p aeruginosa infection; chronically colonised, intermittently colonised and non-colonised, on the basis of the leeds criteria. bal was collected as per ers guidelines as part of each patient's routine clinical care. bal was collected from control patients undergoing elective non-pulmonary surgical procedures. differential cell counts in bal were performed manually. secretory leukocyte protease inhibitor (slpi), elafin, alpha- antitrypsin (a at) and lactoferrin concentrations were measured by elisa. neutrophil elastase activity and cathepsin activity were assayed by colorometric activity assays. fifty two patients were included in the study ranging in age from months to years ( chronic, intermittent, non colonised and controls). neutrophil counts, neutrophil elastase activity and cathepsin activity were markedly increased in children chronically colonised with p aeruginosa compared to those in the intermittent and non colonised groups. in contrast, levels of the antiproteases slpi and a at and the antimicrobial peptides elafin and lactoferrin were highest in the control group and decreased as colonisation progressed, with levels in the chronically colonised group markedly lower than those with intermittent colonisation. this study demonstrates that in children with chronic p aeruginosa colonisation, there is a marked decrease in antiproteases and antimicrobial factors and a marked increase in protease activity and neutrophil influx in comparison with those who are non-colonised or intermittently colonised. these findings underline the importance of careful microbiological surveillance and early aggressive treatment of p aeruginosa infection in children with cf in order to avoid chronic colonisation. background : abnormal bronchial angiogenesis is responsible for hemoptysis in cystic fibrosis (cf). expression of vegf-a in airway epithelium induces bronchial angiogenesis in animal models. we have recently found that vegf-a and egf receptors (egfr) are increased in the airway epithelium of subjects with advanced cf lung disease. aims: to examine the effects of pa bacterial products and egfr inhibition on vegf synthesis in airway epithelium. methods: culture of non cf (nci-h ) and cf (cfte o-) human airway epithelial cell lines. stimulation with pa lipopolysaccharide (lps). assessment of vegf mrna and protein by rt-pcr and elisa. use of chemical inhibitors, blocking antibodies and sirna. results: pa lps increased vegf gene expression and protein production time-and dose-dependently in both cells lines. using chemical inhibitors, we show that egfr and erk / activation are required for lpsinduced vegf production. using blocking antibodies to egfr and its ligands, we show that tgf-alpha-dependent egfr activation mediates pa lps-induced vegf gene and protein synthesis. using pharmacological inhibitors (an ros scavenger and an nadph oxidase inhibitor) and using small interfering rna of dual oxidase (duox) and tnf-alpha converting enzyme (tace),we show that lps-induced vegf upregulation is dependent on duox -mediated ros release and tace activation. thus, pa products induce vegf synthesis in airway epithelium via a duox -ros-tace-tgf-alpha-egfr-erk / cascade. conclusions: these results describe a novel pathway by which bacterial products induce angiogenic signaling in cf and non cf airway epithelium. background: unlike bronchoalveolar lavage (bal), the airway mucosa has been under-investigated in cystic fibrosis (cf), despite the fact that irreversible airway wall changes (bronchiectasis) are a feature of endstage disease. cf is characterized by a neutrophil-dominated inflammation in bal, but little is known about the pattern of inflammation in the airway mucosa, especially in children with relatively early stage disease. we aimed to assess whether the pattern of inflammation seen in cf bal was also found in the airway mucosa in cf children. methods: to date, endobronchial biopsies and bal from children ( - years) with cf and control children ( - years) without lower respiratory disease have been assessed. bal cell differential was assessed on may-grünwald-stained cytospins. endobronchial biopsies were stained for neutrophils (neutrophil elastase, ne), t-(cd ) and b-(cd ) lymphocytes, eosinophils (eg ), and macrophages (cd ). area profile counts of immunopositive cells in subepithelial tissue were performed by investigators blinded to disease group. results: all cell types were increased in cf bal compared to controls. cf bal was characterized by an abundance of neutrophils ( x /ml vs. x /ml in controls, p< . ) with moderate numbers of lymphocytes ( x /ml vs. x /ml in controls, p< . ). in contrast, cf subepithelial tissue was characterized by a lymphocytic infiltrate ( cells/mm vs. cells/mm , p< . ) with only very few neutrophils ( cells/mm vs. cell/mm , p< . ). the lymphocytic infiltrate in cf consisted mainly of t lymphocytes ( %). eosinophil counts in subepithelial tissue did not differ between cf and controls. for all cell types, there was no correlation between counts in bal and counts in subepithelial tissue. conclusions: in contrast to the neutrophil-dominated inflammation in the airway lumen, cf is characterized by a lymphocytic inflammation in the airway mucosa. the lymphocytic infiltrate consists mainly of t lymphocytes, the pathophysiological function of which may be important and is being investigated in future work. support: ers long-term fellowship and swiss national foundation grant to nr the epithelium serves as a barrier to the penetration of foreign antigens, particles, and infectious agents across the airway. the integrity of this barrier is dependent, in part, upon the apical junctional complex consisting of the tight junction (tj) and the adherens junction. alterations in tj permeability have been linked to the pathogenesis of inflammatory bowel disease and this increased intestinal permeability may actually precede the onset of chronic inflammation. in cystic fibrosis (cf), the airway lumen is filled with high concentrations of inflammatory cells, bacteria, and inflammatory mediators. since tj barrier function can be significantly reduced by inflammatory mediators, we hypothesized that measures that enhance airway tj barrier function will decrease airway responses to the continuous presence of inflammatory mediators in the lumen. to test this hypothesis, we examined the relationship between lung inflammation and epithelial permeability in vivo using a lipopolysaccharide (lps) model of lung inflammation. pseudomonas aeruginosa lps was instilled intratracheally into the lungs of c bl/ mice which were then euthanized at , and hrs. lung inflammation was assessed by total cell counts using a hemacytometer and differential counts by wrights staining of cytospin preparations of the bronchoalveolar lavage fluid (balf). lps increased total cell counts and neutrophil concentrations that peaked at hours after lps administration, compared to saline controls. measurements of the proinflammatory murine cytokine kc in balf, by a cytokine antibody bead technique, showed an increase in murine kc at hr following lps administration, which correlated with the substantial increase in neutrophil concentration. changes in lung permeability with inflammation were assessed by elisa measurements of the levels of serum protein murine albumin in balf. correlating with changes in cellular inflammation and murine kc levels, albumin concentration peaked at hr after lps administration. this increase was subsequently resolved, consistent with the restoration of barrier function. an examination of frozen sections of lung from lps-treated animals showed a redistribution of the tight junction protein zo- consistent with the disruption of barrier function. since the p map kinase signaling pathway has been implicated in lps-induced airway inflammation, and an inhibitor of this kinase, sb has been shown to reduce this inflammation, the effect of this inhibitor on barrier function is being investigated. in initial studies, sb appears to reduce total and neutrophil cell counts by % in vivo. the effect of sb on murine albumin concentrations in balf and on tj protein localization is currently being evaluated. a reduction in these parameters will be used as indices of improved barrier function with sb . these studies will determine whether a reduction in lung inflammation correlates with a restoration of barrier function. the degree of protection provided by the p map kinase inhibitor sb could have important implications for inflammatory lung diseases such as cf. background: chronic pulmonary inflammation in cf is characterized by a robust neutrophil response associated with airway damage and failure to eliminate the pathogen, p. aeruginosa (pa). pa is a highly adaptable opportunist which quickly develops resistance to antimicrobials. thus, the development of specific immunotherapy targeting the neutrophil recruitment without ablating the host's immune response to infection or promoting pathogen resistance would be ideal. our group has identified il- as a prime target for the development of immunotherapy due to the central role that the il- /il- proinflammatory axis plays in neutrophil recruitment. however il- does not mediate the early neutrophil recruitment seen in response to infection. hypothesis: il- , acting synergistically with il- , is critical to early neutrophil recruitment during pulmonary pa infection. the primary effector cells are the il- -producing antigen presenting cells: alveolar macrophages (ams) and myeloid dendritic cells (dcs). methods: wt and il- -deficient mice were infected with pa at x cfu/ ul by intratracheal (it) inoculation for hours. bal inflammatory cell counts, and cytokines and chemokines were measured. am and dc cultures were infected for hours in vitro and supernatant cytokines/chemokines were measured by luminex and elisa; il- levels were measured by taqman. these studies were designed to elucidate the role of il- in the early neutrophil peak and define am-and dc-mediated cytokine/chemokine production. recombinant murine il- , il- , and il- + il- were instilled via it into wt and il- -deficient mice. bal inflammatory cell counts and cytokines/chemokines were measured at hours. these studies were designed to elucidate the role of il- and il- in the early neutrophil peak and define am-and dc-mediated cytokine and chemokine production. results: at hours post-infection, il- deficient mice had significantly lower percent neutrophils (p< . ) and lower mip α, kc, and il- (p< . ) in the bal. il- was undetectable. there was no significant difference in bacterial load that could account for these cytokine/chemokine differences. infected wt ams elaborated significantly more mip a, gm-csf, mcp- , il- , g-csf, ip- , kc and il- than the il- -deficient ams (p< . ) and the response was inoculum-dependent (p< . ). dcs elaborated no il- and exhibited il- -dependent differences in mip- α and mcp- production (p< . ). in vivo studies of il- and il- effect demonstrated a synergistic increase in bal neutrophil recruitment (p< . ) and cytokine and chemokine induction (p< . ). conclusion: the first wave of neutrophil recruitment seen during pa infection is il- -dependent and il- -independent. ams and dcs are critical to il- and il- indcution of this neutrophil recruitment. these studies identify il- as a key mediator of neutrophil recruitment in the early stages of infection as well as the proximate mediator in the il- /il- pro-inflammatory axis and suggest il- as a potential target for anti-inflammatory therapy in the treatment of pa pulmonary infection. supported by the cystic fibrosis foundation, american lung association and the nih the airways are under constant assault from air-borne pathogenic material. despite the intake of up to , bacteria per hour, the airways are sterile below the larynx in healthy individuals. the task of maintaining this sterility falls to the airway surface liquid layer (asl), the protective twophased system consisting of the viscoelastic mucus layer and the periciliary layer (pcl) through which cilia beat, sweeping the mucus layer away from the lungs. the mucus layer, which is responsible for trapping pathogenic material, is comprised of mucins (high molecular weight glycoproteins), cellular debris, dna, neurtifils, and more than other proteins. this chemically heterogeneous mixture forms a viscoelastic gel that is thick enough to trap pathogens of various sizes and surface chemistries, while not sticking to the underling cellular / cilia layer, allowing the transport of trapped pathogenic materials away from the lungs. the performance of this trapping / transportation system is defined by the rheological properties of the mucus layer and the force imparted on the mucus. therefore, understanding how mucins and other chemical components of the mucus layer interact with each other to form a successful mucus gel (i.e. one that is cleared from the airways) is crucial to understanding airway defense. here we present the results of physical and chemical composition studies of sputum samples collected from patients with chronic obstructive pulmonary disease (copd) and cystic fibrosis (cf). the rheological properties of each sample was assayed using parallel plate rheolometry, probing the materials non-linear viscoelastic properties such as viscosity, elasticity, and yield stress. the physical properties of the sample are then correlated to the sample's chemical properties such as percents solids (divided between salts, proteins, and mucins), as well as the relative concentrations of the key airway mucins muc b and muc ac. our results indicate that the physical properties of sputum are not well predicted from the total amount of biosolids in a given sputum sample, but by the relative concentrations of muc b and muc ac and the interactions of these molecules with themselves and the other proteins present. further, we establish that the heterogeneous physical properties within a given sputum sample correlate to differences in the muc b and muc ac concentrations. the gene modifier study (gms) was established as an effort to identify potential genetic modifiers of cystic fibrosis pulmonary disease and survival. during the course of this study over delta f homozygous cf patients classified as having mild lung disease, severe lung disease, or increased survival, have submitted both clinical data and blood samples for single nucleotide polymorphism (snp) analysis. the original snp analyses have shown a significant association between variants in the endothelin receptor a (ednra) gene, and cf survival, most markedly in female cf patients. sixteen additional snps within and around the ednra gene have now been genotyped, and have implicated the ' and ' untranslated regions of the gene as having the most significant association with pulmonary disease in females (p< . ), suggesting quantitative differences as a possible mechanism for the association with pulmonary phenotype. we are in the process of saturating the ' and ' regions of ednra with an additional snps to further delineate the genetic association. ednra binds endothelin- (et- ) in airway smooth muscle cells, causing increased cell proliferation, smooth muscle contraction, and stimulation of inflammatory molecules. because each of these effects is known to be deleterious to the cf lung, we hypothesize that the ednra variants found more commonly in "severe" cf females ("severe" alleles) are marking increased ednra expression compared to alleles found more commonly in "mild" cf females ("mild" allelels). because the genetic association was strongest in cf females, we used the matinspector software to analyze kb of ednra promoter sequence and found several putative binding sites for both estrogen and progesterone. we then used a brdu assay to measure cell proliferation after stimulation with both estrogen and progesterone. these experiments showed that the asm cells with the "severe" ' ednra genotype proliferated at levels approximately twice that of the asm cells with the "mild" ' ednra genotype following stimulation with either estrogen or progesterone. using a single base extension protocol and quantitative pcr on human airway smooth muscle cells, we were also able to compare ednra expression from the "severe" allele, and the "mild" allele. these comparisons of ednra expression demonstrate that expression levels appear to be approximately % higher from alleles found more frequently in the "severe" cf females. in addition, preliminary data suggest that stimulating the asm cells with estrogen increases ednra expression by approximately fold, and like the cell proliferation experiments, these increases are most pronounced in cell lines with the "severe" genotypes. these data suggest that the "severe" genotypes are marking alleles with increased expression, perhaps due to estrogen binding, that leads to increased et- functional effects that over time are deleterious to the cf lung. cf patients do present with variable spectra of lung disease, of which infections are most life-threatening. β-defensins have an antimicrobial activity against a broad spectrum of microorganisms and are chemotactic agents for cells of the adaptive immune system, and therefore assist in combating these infections. β-defensins - are part of a repeat region. this repeat region is polymorphic between individuals and therefore the dosage of these defensin genes/proteins varies. we developed a real time pcr assay to quantify the number of βdefensin repeats in this region. appropriate controls are needed for an accurate quantitative assay. therefore we made concatemeric constructs with copy of defb and a particular number of defb copies, which ranged from to copies. using these controls as standards, the number of defensin repeats could be accurately determined in dna samples. we then tested f del homozygous cf patients from belgian ( patients), czech ( patients) and south-italian ( patients) origin. the diploid number of repeats varied between and . for each patient group, a higher number of repeats was found in the group of patients with milder disease (fev > %) compared to the group of patients with more severe disease (fev < %) (student t test, p-values of . , . and . respectively). moreover, in our cohort of belgian cf patients, cf patients of years or older have a significant higher number of repeats than the cf patient group below (p-value . ). to evaluate this at the functional level, we cultured nasal epithelial cells from individuals with a low number of repeats (i.e. or repeats) and individuals with a high number of repeats (i.e. repeats). the cells where grown in air liquid interface cultures. after differentiation, the cells were stimulated with ng tnfα. in cells with a high number of repeats, defb expression, as measured by the extent of transcription, was strongly upregulated by tnfα. in cells with a low number of repeats, defb was not upregulated (p-value = . ). we also tested the antimicrobial activity of epithelial cells. we challenged epithelial cells from individuals ( repeats) with a laboratory strain (pa ) of pseudomonas aeruginosa and a clinical isolate ( - cfu), either in combination with tnfα or without tnfα. after h, surviving bacteria were counted by a plating out method. cells that were stimulated with tnfα h prior to the bacterial challenges were more bactericidal. the clinical strain was more vulnerable to the surface liquid than the laboratory strain. in epithelial cells from individuals having a low number of repeats, these effects were very variable from individual to individual. in summary, the β-defensin region is a modulator of cystic fibrosis lung disease. the pro-inflammatory response in cultured epithelium cells strongly correlates with the number of β-defensin repeats. cells with a higher number of repeats respond to tnfα treatment, which in turn results in a better antimicrobial activity of the surface liquid. rationale: studies of affected twins and siblings demonstrate that modifier genes are major contributors to variation in cystic fibrosis (cf) lung disease severity. we performed genome wide linkage analysis to identify regions likely to contain modifier genes affecting severity of cf lung disease. methods: individuals with cf from families were analyzed. pulmonary function data were collected from patient chart review and were supplemented with data from the us cystic fibrosis foundation patient registry. to minimize environmental variation, only data obtained while subjects were living with an affected twin or sibling were analyzed. the pulmonary phenotypes were defined using the best cf-specific percentile for fev (kulich, et al) within the last year of available pft data as a crosssectional measure (maxfev cf%) and using two longitudinal measures: the lifetime average cf-specific % for fev (avgfev cf%) and the estimated percent-predicted fev at age (estfev %pred@ yrs, schluchter, et al). longitudinal measures were derived from a minimum of years of pft data. short tandem repeat markers (strs) were typed in all affected individuals and their parents (marshfield genotyping center: markers or decode genotyping center: markers). two-point and multipoint linkage analyses were performed using sequential oligogenic linkage analysis routines (solar). results: patients represented the spectrum of lung disease severity, with maxfev cf%'s ranging from to , mean . ± . and avgfev cf% ranging from . to . , mean . ± . . the maxfev cf% was predictive of avgfev cf% (r= . , p< . ) for the individuals for whom both measures were available. the two longitudinal measures were also highly correlated (r= . , p< . ). linkage was found at chromosome for all three phenotype definitions. peak multipoint lod scores on chromosome occurred at cm for maxfev cf% and avgfev cf% (lod . and lod . , respectively) and at cm for estfev %pred@ yrs (lod . ). single point lod scores on chromosome peaked at marker aaat ( . for maxfev cf%, . for avgfev cf%, and . for est-fev %pred@ yrs). the region of linkage encompasses approximately megabases near the telomere of chromosome q. conclusions: chromosome appears to contain one or more genetic modifiers of cf lung disease severity. supported by the nhlbi, cff and genome canada through the ogi. cystic fibrosis-related diabetes (cfrd) is the most common extrapulmonary complication of cf and is an increasingly important contributor to morbidity and mortality as cf patients live longer. while pancreatic fibrosis and loss of exocrine and endocrine tissue are common in cf, - % of cf adults develop defects in insulin secretion and accumulation of islet amyloid polypeptide, features typical of type diabetes (t dm) in the general population. to test whether modifier genes play a role in cfrd, we compared concordance rates for cfrd in pairs of monozygous (mz) twins, sets of dizygous (dz) twins, and sets of or more siblings ( individuals with cf). criteria for defining cfrd included physician diagnosis, treatment with insulin/oral agent, and episodes of glucose ≥ mg/dl. mz twins were highly concordant for cfrd ( of pairs, %). the young age of dz twin recruits precluded analysis of this group in isolation ( of pairs were concordant). twelve of ( %) sibling pairs were concordant for cfrd. with heritability defined as: h = *(mz concordance -dz concordance), and including siblings as a proxy for dz twins, heritability is estimated as ~ . . the same results were obtained considering only same-sex dz twins and siblings, correcting for differences in age and duration of clinical follow-up, or restricting analysis to ∆f homozygotes. these data support a significant role for one or more modifier genes in development of cfrd. we then tested whether cfrd correlated with a strong family history of adult-onset diabetes (at least first-degree or second-degree relatives on the same side of the family). of those reporting family history of diabetes, of had cfrd, compared to of with no family history (or= . [ . - . ]; p= . ). this correlation persisted after adjusting for age, sex, and pancreatic insufficiency (or= . ; p= . ). thus, family history of diabetes correlated with increased risk of cfrd. we then tested whether variants in tcf l , a transcription factor in the wnt signaling pathway, that have been reproducibly associated with t dm in the general population were associated with cfrd in our study subjects. genotyping of four single nucleotide polymorphisms associated with t dm (rs , rs , rs , rs , here termed snpa-d) and transmission disequilibrium testing (tdt) of parent-parent-child trios revealed significant overtransmission for snpb ( : , p= . ) and snpc ( : , p= . ), and possible overtransmission for snpa ( : , p= . ) and snpd ( : , p= . ). in every case, the tcf l allele overtransmitted to patients with cfrd is the same allele that confers increased risk for t dm. furthermore, individuals with cfrd who were homozygous for risk alleles were diagnosed at a significantly earlier age (average . vs. . ; p= . ). these data support a key role for modifier genes in development of cf-related diabetes, and demonstrate that cfrd and type diabetes may share disease mechanisms such as alterations in wnt signaling. supported by nih dk , hd , dk and hl , and cf foundation grant cuttin p . cystic fibrosis (cf) phenotypes and survival are highly variable among df homozygous patients, pointing to the existence of modifier genes and/or environmental factors that contribute to this disease. studies to identify genetic modifiers of cf are being carried out using dna from homozygous df cf patients. important clinical features, such as severity of lung disease, liver disease and meconium ileus (mi) status, are well defined. tgfβ has been previously identified as a modifier of cf lung disease (drumm et al., nejm, ( ) : [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] ) , but it does not explain all of the genetic heterogeneity in this population. current evidence suggests that mucus is involved in the progression of cf, making the muc genes prime candidates as modifiers of lung disease and/or other phenotypes. methods: our approach to evaluate muc genes utilizes both variable number tandem repeat (vntr) polymorphisms and single nucleotide polymorphisms (snps). vntr polymorphisms in the muc and muc ac genes (n= and n= patients, respectively) were detected by southern blotting under conditions fully optimized to maximize the allele size resolution. to minimize gel to gel variation, a genomic dna mixture with the most common muc and muc ac alleles was used as internal markers. accuracy and reproducibility were evaluated by duplicating the southern on the critical dna samples for muc and muc ac genes (n= and n= patients, respectively). fifty snps in muc ac, muc b, muc , muc and muc genes are being tested using illumina technology in cf patients. results: preliminary analysis suggests there are significant differences between cf patients with "severe" and "mild" lung disease for both muc and muc ac allele distribution, which is mainly driven by the male population; exhaustive statistical data analysis still is underway. the vntr data also suggest significant association between the larger muc allele size and cf patients with mi. the ongoing vntr analysis will be complemented by muc gene snps being genotyped. conclusions: initial results indicate that we can reproducibly characterize muc and muc ac vntr alleles. additional characterization of muc and muc ac vntr alleles, coupled to snp data, will allow us to better define the significance of muc gene variations as modifiers of different cf phenotypes. supported by cff perezv g (jpv), cff knowle a (mk), cff r -cr (wko), nih rr , r hl , and cff drumm a . reporting for the gene modifier study group (mrk). we have previously reported that βenac transgenic mice, which overexpress the beta subunit of the amiloride sensitive sodium channel (scnn b) specifically in the airways, share common features with cf, including increased enac activity, reduced airway surface liquid, mucus accumulation and obstruction, inflammation, and death. interestingly, analysis of this model also suggested the existence of potential genetic modifiers of phenotype severity, and we speculated that identification of these modifiers would provide novel insights into disease phenotype. to establish a set of reagents that could be used to uncover genetic modifiers, we have bred βenac transgenic mice from two independent founder lines ( and , b :c background) onto several strains of inbred mice, including c bl/ n, c h/hen, balb/cj, fvb/j, and /svj. these studies revealed dramatic phenotypic differences as measured by survival ( to %) among strains and between lines. all lines thus far tested show ~ - fold increases in amiloride sensitive short-circuit current as measured by ussing chambers in the trachea. complete phenotypic characterization of c bl/ n line at backcross generation reveals high survival ( ± % in comparison to ± % of the mixed b :c background), yet the mice maintain the pulmonary features of the originally reported mice, including increased mucus plugging, mucous cell hyperplasia, neutrophilic and eosinophilic inflammation peaking at early timepoints ( days - weeks). lymphocytic nodules, which are not commonly seen in - weeks-old animals with mixed strain background, are a common feature in the c bl/ n congenic line. emphysema and early airway epithelial cell necrosis, two phenotypes initially not strongly associated with transgene expression, are also observed. line on balb/cj and c h/hen backgrounds has reduced survival compared to the c bl/ n background, and generation backcross lines are now being evaluated in these two strains for other phenotypic characteristics. analysis of backcross data from line , which has low survival on all genetic backgrounds tested to date, including c bl/ n, suggested that the transgene may have integrated onto a c h locus with a dominant negative effect on survival. genetic analysis using genome-wide snp genotyping revealed a region of chromosome linked to the transgene in line (lod score > . ). further analysis of this region is underway. in summary, backcrossing onto different genetic backgrounds is revealing genetic modifiers for phenotypes in the βenac overexpressing model. characterization of these phenotypic and genetic differences should provide clues about the mechanisms relevant to disease development. furthermore, inbred lines with variable phenotype will likely be an important reagent for the cf community as the utility of this model is evaluated in future studies. supported by nih (scor p hl ) and cff (mall go, oneal go). introduction: new york state screens newborns with immunoreactive trypsinogen levels within the top % of all infants, for common cf gene mutations. infants found to be heterozygote carriers are referred to a cf center to determine sweat chloride concentration. the proposed abnormal sweat chloride value for this group of newborns is ≥ mmol/l, ≥ standard deviation (sd) above the mean (farrell, ) . this retrospective study reports on the mean sweat chloride value + sd in cf heterozygote newborns who have been referred for evaluation to suny upstate medical university cystic fibrosis center, and regarding the genotype of these infants with abnormal sweat chloride levels. method: from october, to december, , infants were referred for positive cf screening, and of these ( %) were identified as heterozygote carriers by the screening program. at our center, these patients underwent pilocarpine iontophoresis, followed by collection of sweat (≥ µl) in macroduct ® coils. sweat testing was performed successfully in of the ( %) patients. the patients' age (mean ± sd) at the time of sweat testing was . ± . days. for infants with an initial sweat chloride level ≥ mmol/l, the sweat test was repeated within a week and a complete gene sequencing was requested (quest laboratory, ca). results: the sweat chloride level (mean ± sd) in newborns who were heterozygous for a cf mutation (excluding those who were found to have an additional mutation or deletion) was . ± . mmol/l (n = ). the mean + sd was mmol/l, which defined our minimal value for an abnormal test. eleven infants had sweat chloride values of - mmol/l (table) ; of them ( %) were subsequently diagnosed with cf by complete gene sequencing. four of the remaining patients ( %) had ∆f mutation coupled to the t variant on the opposite chromosome (sweat chloride levels, - mmol/l). conclusions: the reference range for sweat chloride in cf heterozygote infants appears to be significantly lower for some centers than previously reported. thus, each cf center should consider evaluating the cutoff values for the test at their site. moreover, the t polymorphism may account for sweat chloride elevations in heterozygote infants. method: we reviewed the charts of all patients with cf who were referred as a result of the new york state newborn screening program to the suny upstate medical university cf center from october, through april, . we included the patients who were identified by the state as heterozygote carriers of one of common cf gene mutations, and whose second mutation was identified only after complete cf gene sequencing. results: seven of the patients ( %) met inclusion criteria. six of the patients ( %) were compound heterozygous for a novel or a rare cf gene mutation. one patient was compound heterozygous for a large deletion in the cf gene. the genotype and clinical status of the patients are shown in table. all patients have been pancreatic sufficient to date. conclusions:the clinical effect of compound heterozygosity as a result of novel or rare cf gene mutations appears to be mild in early childhood. prior to newborn screening (with the exception of the patient with cftr deletion), these patients may not have been diagnosed with cf in the first few years of life. these patients may have subclinical airway inflammation and thus benefit from early treatment. patients with cf manifest symptoms in the pancreas, respiratory tract, male reproductive tract and sweat gland due to mutations in cftr. patients with non-classic cf have disease in a subset of these organ systems. most non-classic cf patients have two disease-causing mutations in cftr and at least one mutation permits residual cftr function. a subset of non-classic cf patients have only one cf-causing mutation after screening for a panel of common cf-causing mutations or following mutation scanning of the coding region of cftr. these patients present a diagnostic dilemma and a challenge for genetic counseling. we evaluated cf patients with only one cf-causing mutation identified after a screen of cftr mutations ( patients) or scanning of the coding region of cftr ( patients). nine of these patients, including one set of siblings, have non-classic cf with borderline or elevated sweat [cl -] plus lung disease (table) . one patient is pancreatic insufficient and has classic cf. many of these patients have features which are consistent with cftr dysfunction including a cf-like nasal potential difference (npd),p. aeruginosa infection, or congenital bilateral absence of the vas deferens (cbavd), suggesting that they have a second cftr mutation. mutations that are not detected by screening methods include insertions or deletions, mutations outside of the cftr coding region that affect rna splicing or expression, or mutations in the coding region of cftr that were missed by screening methods. to exclude the third possiblity, dna sequencing of the exons and flanking introns of cftr was performed. a second mutation was identified in the coding region of cftr in of the patients; had screening for known cftr mutations (genzyme), while the remaining had comprehensive scanning of the coding region of cftr by modified tgge (ambry). each of the mutations identified by sequencing has been previously described in patients with cf and is predicted to cause cftr dysfunction. these results reaffirm that patients with one cftr mutation who have biochemical and clinical features of cf are likely to have a second mutation in the coding region of cftr. thus, we suggest sequencing cftr in patients with only one mutation after mutation screening before employing other more complex genotyping methods (insertion/deletion or rna analysis). diagnostic criteria for confirming cf in symptomatic individuals includes two positive sweat tests or two known disease-causing cftr mutations. accurate sweat testing is performed at accredited cf centers, while cftr testing is available through national and specialty genetic labs. most labs offer analysis of a basic panel of cftr mutations as recommended by the american college of medical genetics (acmg), while specialty laboratories may offer an expanded panel or full sequencing/scanning. we report on one center's use of these methodologies to confirm the diagnosis of cf presenting in adulthood. the charts of patients diagnosed with cf at 〉 years of age were reviewed. were sweat tested. all were genotyped. results: twelve ( %) pts had two positive sweat tests (> mmol/l). an additional five ( %) had at least one borderline result ( - mmol/l). two pts had negative results ( - mmol/l). one refused sweat testing because dna analysis through the acmg panel had confirmed the diagnosis prior to initial consultation. genotyping results for the pts are summarized in the table below. rare mutations identifiable only through gene sequencing accounted for / alleles ( %) in our population. importantly, among this group, only one pt had a negative sweat test, a suggestive mmol/l. two pts with positive sweat tests and clinical symptoms failed to reveal any cf mutations after sequencing. we continue to follow pts without genotypic confirmation, based on their clinical presentation and sweat chloride levels, and have recommended additional evaluation, including nasal potential difference studies in our series of adult-diagnosed patients, sweat test results were positive, borderline, or suggestive in all cases tested. sweat testing costs $ -$ and results are ready in a day. genotyping costs $ -$ and takes several weeks. we acknowledge that circumstances may arise where reliable sweat testing is not conveniently available; but in our series, genotyping with the acmg panel would have diagnosed % of pts; using an expanded panel would have diagnosed %. genotyping is an important tool for genetic counseling, determination of eligibility for research studies, furthering knowledge of cftr dysfunction and cf pathophysiology, and for confirming a cf diagnosis after borderline or suggestive sweat test results. based on our findings and the dramatic difference in cost, we conclude that sweat testing should remain the first approach in the diagnostic workup of adult patients with a clinical presentation suggestive of cf. in colorado, infants with cf (non-meconium ileus) have been diagnosed with cf by a two tiered immunoreactive trypsinogen (irt/irt) based newborn screening approach. the irt/irt algorithm has been recently adopted by other screening programs with two mandatory screening tests. while most infants in colorado have been successfully identified, the program has had a missed case rate of approximately %. the more common approach to cf newborn screening is the irt/dna method in which the blood spot of infants with an initial elevated irt is tested for the most common cf mutations. the initial irt cutoff is lower in the irt/dna programs than in the irt/irt programs, resulting in a lower missed case rate. the considerable number of carriers identified through the irt/dna approach puts a significant burden on the genetic counseling community, as carriers are identified at a rate of / - / of positive irts we propose an irt/irt/dna newborn screening algorithm that will maximize sensitivity and specificity while minimizing the number of identified carriers. using new database technologies in the newborn screening lab we will be able to identify those infants with an elevated first irt (> ng/ml, approximately th percentile). all infants with an irt > ng/ml will have a repeat irt on their second state mandated blood-spot. if the second screen is also elevated (> ng/ml), the blood spot will be tested using a panel of mutations, including mutations specific to the hispanic community. infants with one or two cftr mutations will have a sweat test to confirm the diagnosis, or rule out cf. we compared the projected statistics of our current method irt/irt to the new irt/irt/dna method, and to irt/dna is presented in the table, based on , births per year in colorado. four infants ( . %) identified under the current irt/irt protocol would not have been identified by the mutation panel proposed in the new algorithm, out of genotyped, non-meconium ileus infants. three of these missed cases are hispanic. two would be identified using an extreme irt cutoff of the . th percentile ( ng/ml). the projected missed case rate would be < . % ( . - . %, % ci), using the irt/irt/dna algorithm, with carriers identified, maximizing both sensitivity and specificity. this algorithm may provide a better alternative to the irt/irt screening methods in states with two mandatory screening tests, and has advantages over both the irt/irt and irt/dna methods. newborn screening for cystic fibrosis (cf) is rapidly expanding and has been implemented in at least states. although most newborn screening assays are done using biochemical testing, many laboratories screening for cf include both biochemical and molecular testing of multiple alleles in the cystic fibrosis transmembrane conductance regulator gene. in response to the growing need for proficiency testing (pt) materials for molecular testing, the centers for disease control's newborn screening quality assurance program (nsqap) in collaboration with the university of wisconsin school of medicine and public health, the johns hopkins hospital, and case western reserve university, created a repository of dried-blood spot specimens with known mutations in the cftr gene to be used in a pt program. twenty milliliters of blood was collected voluntarily from adult donors with cf and sent to the nsqap laboratory. each specimen was adjusted to a hematocrit of % before being spotted onto whatman paper ( µl per spot), dried, and stored at - °c with desiccant. proficiency testing (pt) panels consisted of to blind-coded specimens from adult donors. the panels were sent quarterly to laboratories worldwide that test specimens for cf using molecular methods. laboratories were asked to report the genotype, method used, and the presumptive clinical assessment of each specimen. twenty-two laboratories participated during both quarters and , . the laboratories used different methods ranging from in-house assays to commercially available kits. most reporting laboratories tested the following alleles -∆f , g x, and g d. another twelve alleles were detected by most participants. nine more alleles were common among commercially available kits. laboratories were evaluated based on the clinical assessments. mutations that were not detected by a particular method were not evaluated. overall, the laboratories performed well. data compiled from both quarters demonstrated that there was incorrect clinical assessment and amplification failures. developing a pt program for dna-based testing is complicated by the number of methods and different alleles each laboratory chooses to test. though molecular testing for cf may be complex, pt monitors the laboratory's ability to test multiple alleles, including uncommon alleles, the limitations of various assays, and the different algorithms used for screening. the repository will also allow storage and access to rare specimens that may be useful for future research but are not readily available. who did could not reproduce. genetic counseling focused on a patient's parents, who were counseled about their recurrence risk at the time of the child's diagnosis. today, cf is a disease of adulthood. in , > % of cf patients in the us were > ; by it will be > %. together with advances in assisted reproductive technology (art), reproduction and recurrence risk are now important issues for adolescent and young adult cf patients. methods: a item questionnaire was developed from the results of prior semi-structured interviews with cf patients age - years. knowledge based questions (medical issues, inheritance, and reproductive options/risks) as well as communication patterns (preferred resources for learning about cf and preferred people with whom to talk about reproductive issues) were addressed. recruited from the uab cf clinic population, patients age - (mean ), male ( %), female ( %), completed the questionnaire. results: regarding autosomal recessive inheritance of cf, only % knew that two carriers have a % chance of having a child with cf, and % knew that two carriers have a % chance of having a child who is a carrier. however, % knew that two carriers could have a child who did not have cf, and % knew that two carriers could have a child who did not carry cf. on their own reproductive risks, % knew that a cf patient had a % chance of having a child with cf if their partner was not a carrier, but only % knew that all their children would be carriers even if their partner was not a carrier. in the scenario of a cf patient with a cf carrier partner, % knew that a child had a % chance of having cf, and % knew that a child had a % chance of being a cf carrier. most patients knew about their reproductive potential, as % responded that cf patients are able to have children. however, when asked about whether the chance for having children was different for males and females with cf, % answered that it was more difficult for males, % that it was more difficult for females, and % answered "not sure." while % reported that they knew that there were options for male cf patients who wanted to have children, only % knew of art. conclusions: despite widespread availability, the lack of knowledge of adolescents and young adults with cf about the genetics of their disease continues. furthermore, these patients are unaware of both modern technologies that could enable them to have biological children and the risk of those children having cf. this study illustrates the changing needs of patient education as medical knowledge progresses. cf patients would benefit from further genetic knowledge and counseling to enable them to make informed decisions about reproduction as they mature into adulthood. center at the university of minnesota is one of three sites in the state providing confirmatory testing and follow up services for newborns identified by screening. while cf nbs identifies children with cf, most of the infants with positive screening results are carriers. our goal is to provide genetic counseling to every cf nbs patient seen at our center, and we believe that a protocol incorporating genetic counseling in the initial care plan for infants both with and without cf is imperative. the literature has shown that families who obtain genetic counseling through the cf nbs process recall genetic information more easily and accurately and are more likely to have testing to determine parental carrier status. for parents of a child that is determined to be a cf carrier, it is especially important to find the optimal method and timing of genetic counseling as many of these families are from several hours away and therefore less likely to return to clinic. to provide genetic information and emotional support for the families of infants screening positive on cf nbs, our center has a genetic counselor who serves as the cf nbs coordinator and clinical contact for the family. this allows many opportunities to speak with the genetic counselor and ask questions, as well as learn about their child's diagnosis or carrier status and the subsequent carrier testing recommendations for the infant's parents and families. to assess the success and impact of the minnesota cystic fibrosis center's nbs follow-up program and the incorporation of genetic counseling, an anonymous questionnaire was developed for parents of infants who were seen at our center due to a positive cf nbs result. questionnaires were mailed to parents of all infants seen at our center for a sweat test and genetic counseling due to a positive cystic fibrosis newborn screening result. as of the last mailing, this totals families. two questionnaires were returned due to incorrect address and questionnaires were returned answered, indicating a response rate of % ( / ). responses overwhelmingly indicated that parents were satisfied with our center's algorithm for the cf nbs follow-up program and found the information and support provided through genetic counseling to be a useful and recommended portion of the program. as cf nbs continues, it is critical that we learn about the patient's experience with genetic counseling and the nbs program, as well as identify areas needing improvement. genetic counseling is vital to the comprehensive success of our center's program, and we will report on the responses gathered from the families identified through cf nbs this first year, as well as discuss the lessons learned from setting up such projects on a state-wide basis. background & aims: aberrant splicing and nonsense mediated decay (nmd) lead to dysfunctional mrnas by skipping exons and to a reduced number of functional mrna respectively. both mechanisms have a strong quantitative aspect and may determine whether a cf patient develops a classic or atypical disease phenotype. in order to approach these highly important questions we wanted to establish a new quantitative real-time pcr based assay which allows allele specific quantification on cdna level. using this assay we like to determine the exact proportions of the f del and non-f del cftr mrna in cf patients compound heterozygous for the f del mutation (for example in cf patients carrying the f del and a nonsense mutation such as the r x). material & methods: materials: we used genomic dna (gdna) and total rna (extracted from white blood cells and nasal epithelial cells respectively) from cf patients with compound heterozygosity for the f del mutation, homozygosity for f del mutation and from healthy individuals (controls). methods: the lasq (ligation dependent allele specific quantification) assay comprises reactions: . reverse transcription of cftr mrna into cdna using gene specific primers (all rna specific). . overnight hybridization ( - h) of the cftr cdna with either the f del specific or the wt specific oligo probe pair provided by jan schouten (mrc holland). . ligation of the hybridized oligo pairs using the ligase enzyme from mrc holland. . quantitative real-time pcr of the allele specific ligation products on the lightcycler (roche). results: in order to establish the lasq assay we first validated it using gdna instead of cdna as template. mixing experiments were performed to verify the accuracy of the assay. in brief, gdna (c= mg/l) of a f del homozygous and a f del compound heterozygous cf patient were mixed in such a manner that . , . amplification products of the f del and the wt allele (both bp long) were analyzed by gel electrophoresis (page) and direct sequencing (abi ) to control specificity. our results using gdna and cdna showed that there occurs unspecific hybridization/ ligation for both probe pairs. the proportion of unspecific amplification products varies between . and . and increases the lower the initial number of templates is. however, the specificity of this assay can be significantly improved by increasing the hybridization temperature and/or decreasing the ligation time. conclusion: although some minor limitations concerning allele specificity the lasq assay has been proven to be an accurate, reliable and reproducible method for allele specific quantification and may be applied for several important questions in cystic fibrosis such as the exact determination of the amount of nmd of cftr mrna containing a premature termination codon (ptc) or the allele specific determination of aberrant splicing of cftr mrna . background & aims: as clinical presentation varies significantly among cf patients with the same genotype, e.g. in the f del homozygous, it is evident that factors in addition to the cftr genotype such as modifier genes, are involved in determining disease severity. however, only one of several previously postulated modifier genes, the tgfβ gene, could recently be confirmed in a large association study. hence the identification of new modifier genes is a very important task in order to find new explanations for the heterogeneity of pulmonary disease in cf patients. we decided to search for new potential modifier genes applying a quantitative proteomic approach comparing the proteomes of a wild type ( hbe o-) and a f del homozygous bronchial epithelial cell line (cfbe o-). the main goal of this study is the identification of up or down regulated proteins in the cfbe cell line which may act as modifiers of cf disease. material & methods: materials: we used two bronchial epithelial cell lines, e.g. a wild type ( hbe o-) and a f del homozygous (cfbe o-) cell line which we obtained from dr. gruenert (california, usa). additionally, we also used nasal cells from f del homozygous cf patients obtained either from nasal brushings or nasal polyps. methods: proteome analysis was performed by making d-gels using high sensitive staining protocols (ruthenium and deep purple). quantitative analysis was accomplished applying the powerful dige (difference in gel electrophoresis) method. for each dige experiment we made gels whereby each cell line was twice labelled with cy and cy (= technical replicates). finally, identification of the protein spots was done by the use of a maldi-tof mass spectrometer. results: in a first step we established the proteomes of the two bronchial epithelial cell lines. we were able to optimize protein extraction and d gel electrophoresis in such a manner that the proteomes of the two cell-lines looked very similarly and the assignment of spots could easily be done. protein spots from both cell lines were analyzed using our mass spectrometry (maldi-tof ms) and allowed the identification of more than different proteins so far. in a next step we quantitatively compared the proteome of the two cell lines using the d-dige method leading to the identification of proteins which are down regulated and proteins which are up regulated at least twofold in the cfbe cell line. out of the aforementioned differently expressed proteins could already be identified because they were among the previously determined proteins. while glutathione s-transferase p and protein s -a (s calcium binding protein) were down regulated ( . and . -fold respectively), superoxide dismutase was up regulated ( . -fold) in the cfbe cell line. conclusion: comparative quantitative proteomics using the dige method is a promising tool in search of potential new modifier genes which may unravel one of the key problems in cf: the large heterogeneity of pulmonary disease in f del homozygous patients. background: cystic fibrosis is one of the most common autosomal recessive disorders among caucasians, and manifests a wide range of disease severity. although this range of disease expression can be attributed, in part, to specific mutations within the cftr gene, much of this variability has not been adequately explained. from the gene modifier study (gms-a multicenter study of , cf patients), ten genes were tested as potential modifiers of cf, and the tgfβ codon cc genotype was associated with lung disease severity. objective: to test whether the adverse codon cc genotype is associated with higher circulating levels of tgfβ , compared to the tt genotype in cf patients and healthy controls. if true, then a link will be established between genotype, disease severity, and circulating levels of tgfβ , and have implications for novel treatment of cf patients. methods: the study includes clinically stable cf patients and healthy control subjects equally distributed between the cc and tt genotypes. the cf patients enrolled are age and older, of both genders, and all ethnicities that fulfilled the standard diagnostic criteria for cf, using the genetic information from the gms. healthy controls are age and older, caucasian males and females, obtained from the environmental polymorphism registry (epr), a dna registry of , self-reported normal volunteers. we genotyped blood samples from the epr to define healthy control subjects for each of the cc and tt cohorts. subjects have a blood draw of ml. blood is divided into tubes and each tube is used to measure a different parameter: cbc with differential; tgfβ levels in platelet poor plasma by quantikine human tgfβ elisa kit; tgfβ levels in the buffy coatwhich includes platelets; and tgfβ mrna levels in lymphocytes from the buffy coat, using real-time pcr roche light cycler. cbc with differential is performed to quantitate lymphocytes and platelets in order to reference tgfβ protein and rna levels to the number of circulating blood cells. analyses will include graphical comparisons between the two groups, chi square analysis, student's two-sample t test, and one way analysis of variance (anova). results: pilot studies show that elisa tgfβ measurements are reproducible and mrna levels can be quantified. we have currently enrolled cc genotype and tt genotype of the cf subjects and cc genotype and tt genotype of the healthy controls. blood samples have been collected and processed. conclusion: genetic variants that predispose to more severe cf disease are potential targets for new therapies. we are testing the hypothesis that the adverse (codon ) cc genotype is likely to reflect increased transcription and/ or tgfβ protein synthesis/ secretion. if true, "anti-tgfβ " therapies could provide a novel therapy in cf. *reporting for the gene modifier study group. supported by cff knowle a , cff drumm a , gcrc rr , nih r hl . aim: centralized periodic evaluations of data from the screening laboratories and cf centers by afdphe (french association for screening and prevention of infant handicaps), were analysed to optimize the efficiency of the program. methods: the strategy combined d irt assay/dna analysis (kit elucigen cf- arms) /d -fail-safe irt. revised irt-cut off levels were decided in order to maintain the percentage of positive screens around a . % target. a questionnaire yearly sent to the cf centers collected the cf false negative cases. results: from to december, , cf cases were detected through nbs ( screened infants). the i period (p) (n= ); d -irt: µg/l and d -irt: µg/l showed a) . % infant above the d cut-off, generating an excess of costly dna tests b) % had a d -irt above the cut-off leading to a very high number of st (n= ) with an extremely low rate of cf (n= ). by increasing slightly both d /d irt cutoff levels ( µg/l, µg/l) during the ii p (n= ) a) the number of positive screens decreased to . % b) . % of infants with elevated d -irt had to be referred for st (n= ) with cf diagnosis. since the risk to had true cf remained very low among infants with no detected mutations, during the iii p (n= ), d -irt concerned only the ones with d -irt> µg/l and the percentage of infants requiring a st was reduced to . % (n= , cf). the incidence of cf detected during these p did not vary significantly ( / - / - / ). another point of concern was the false negative cohort; with a follow-up period over months, cf were detected on clinical symptoms ( . %) at a mean age of months. only were directly related to the modifications of the strategy. conclusion: centralisation of data made possible changes in the flow charts of the screening strategy to limit the number of false positive cases without significant alteration of the global performance of the program. there were three infants of mixed aa/caucasian origin diagnosed with cf, all had ∆ /genotype on screening. mutation data on the additional aa cf patients followed at the cf centers in new york was collected. six patients have not been genotyped. there were patients who were homozygous for ∆ , patients had only one mutation identified and were not found to have any cf mutations. ∆ ( %) and + g>a( %) were the most common mutations. there are five cf patients (including infants diagnosed by nbs) with mixed racial origin who are not included in this analysis. mutations results: the median (range) mbl plasma level was . ( . - . ) µg/ml in cf patients, compared to . ( . - . ) µg/ml in controls. mbl genotype frequencies were similar in patients and controls. lung function level was not correlated with mbl genotype or plasma level. the frequency of colonization with pseudomonas aeruginosa was % in mbldeficient (xa/o and o/o genotype) children with cf and % in mbl-sufficient (a/a and ya/o genotypes) children (p= . ). we found a trend of a decreased age of first onset of colonization with staphylococcus aureus, haemophilus influenza and pseudomonas aeruginosa in mbl-deficient cf patients (p= . , p= . , and p= . , respectively). conclusions: mbl-deficiency was associated with an increased frequency of pseudomonas aeruginosa colonization in children. mbl deficiency was not associated with lung function deterioration. in a larger cohort we hope to confirm that mbl deficiency influences age at onset of bacterial colonization in cf patients. acknowledgments although there is some evidence that cftr gene mutations may be associated with respiratory diseases, little is known about the relationship between cftr gene mutations and idiopathic bronchiectasis (ngiam et al. ) . we have recently showed that a rare allele (- g>a) in the minimal cftr promoter, previously reported in patients with idiopathic bronchiectasis ( using supershift assays, we demonstrated that these transcription factors bind cftr promoter in vitro. a functional analysis, by using co-transfection assays with expression vectors of each transcription factor in pulmonary epithelial cells, showed that nrf , irf significantly decrease cftr expression, whereas irf and sp increase it. in an attempt to further elucidate the mechanisms involving these factors in the cftr transcriptional regulation, for instance, to determine whether these factors could interact together in order to regulate cftr transcription, we started several experiments such as co-immnoprecipitation, multiple co-transfection and rnai. taken together, these data evidence that the variant - a in cftr promoter should be considered as an important risk factor in bronchiectasis pathogenesis. furthermore, we have identified a novel regulatory complex on the minimal cftr promoter, which will enlighten the understanding of the transcriptional regulation of the cftr gene. a better knowledge of cftr cis-and trans-acting elements will allow to consider new approaches to modulate and/or control more specifically cftr expression. this work is supported in part by the association vaincre la mucoviscidose. (nat genet, ) . a recent study reports strong genetic influences for mi (blackman et al., gastroenterology, ) , but it did not replicate the modifier locus on chr. q . inconsistency of results may reflect the variability of data reporting and different classifications of mi (i.e. surgically or medically treated). we tested the accuracy of reporting of mi on case report forms (crfs), as compared to primary source documents. methods: the crf for the gms has a checklist for past medical history. for example, we requested "yes" or "no" for mi (an obstruction of the terminal ileum at birth), but did not require source documents or identification of the type of treatment. to evaluate crf reporting of mi, we requested source documents for patients with reported mi, including a surgical or medical treatment report. if a written report from the time of birth was unavailable, a clinic note, detailing mi at birth, treatment, and evidence of a surgical, abdominal scar (if applicable), was required. verbal confirmation by the patient and evidence of a surgical scar was also accepted, if no written documentation was available. results: on crfs, of patients ( %) were initially reported to have mi. to date, source documentation has been obtained for patients, and of those ( %) have been confirmed to have mi ( % by written report, % by verbal report). of the with confirmed mi, ( %) had surgery, and ( %) had medical treatment. there were false reports of mi ( . %). mi could not be confirmed or refuted for patients ( . %) because of insufficient information (n= ) or confounding circumstances (n= ). to date, documentation for patients who were reported to have no mi has been obtained, and no false negatives have been found. additional documentation for mi from other sites is expected. conclusion: at least %, and perhaps as many as %, of the reports of mi on crfs were inaccurate. gene modifier studies must include rigorous documentation of mi to ensure an accurate correlation between phenotype and genotype. studies should also characterize different classifications (i.e. surgical vs. medical treatment) to assist the detection of gene modifiers of mi. reporting for the gene modifier study group (mrk); supported by cff knowle a , cff drumm a , nih r dk , nih r hl , and gcrc rr . any mutation that disrupts or diminishes the efficiency of the splicing process will have an impact on disease manifestation. in the cystic fibrosis (cf) transmembrane conductance regulator (cftr) gene more than , mutations were identified, most of them being disease-causing [ ] . among these,~ % are classified as missense and about % are classified as splicing mutations, given that they disrupt the consensus splice sites [ ] . however, the splicing mutation concept is evolving, as it nowadays also includes mutations other than just those within or close to the consensus splice sites, rendering prediction of mutation consequences a hard task. nevertheless, it is still very important for the clinical settings to determine the functional effect of gene mutations. our aim here was to study the effect of i v, a rare cftr missense mutation in nbd (exon ), directly in native tissues of a cf patient bearing f del in the other allele, so as to gain insight on how it influence the disease outcome. to look for a possible effect at the rna level, total rna was extracted from native nasal cells and colonic tissue, and cdna producd using random primers. rt-pcr amplification was performed in the region spanning exons - , being one of the primers fluorescently labelled. the products were analysed as described before [ ] . the i v( a>g) mutation which creates both a novel acceptor and a novel donor, was found here to cause alternative spliced cftr transcripts lacking the last nucleotides of exon , thus showing that only the novel donor is used in vivo. moreover, our data show that no normal (only alternatively spliced) transcripts result from this allele. we have also analysed the i v mutation at the protein level by producing a stable bhk cell line expressing the i v-cftr, after generating the respective mutant cdna construct by site-direct mutagenesis. protein expression and function was determined by immunoblot and iodide efflux assay, respectively. results show that i v-cftr protein is processed and functional. however, given the above-described absence of normally spliced mrna coding for this cftr variant, we have to conclude that this protein is not produced in vivo. indeed, since only the mrna coding for cftr lacking the last six aminoacids of exon was detected, we are currently characterizing in vitro the proprerties of this truncated protein. altogether, our data clearly demonstrate that the functional effect of this mutation is not due to the amino acid change but to abnormal splicing. in conclusion, characterization of the consequences of mutations in native affected tissues is important, not just because this provides unexpected information about the mechanisms underlying the basic defect but also for disease diagnosis and prognosis. (darrah et al, nacfc ) . the purpose of this study was to examine the influence of common genetic variation in the endothelin pathway on the cf phenotype. methods: patients were recruited from the cf clinics in dublin, belfast and seattle. serial clinical data were abstracted from medical charts and local clinical databases. studentized residuals of maximum fev , after adjusting for age, gender and height, was the phenotype of interest. twentyone maximally informative tagsnps were identified in the edn , edn , ednra and ednrb genes using the niehs environmental genome project and were genotyped using the illumina beadarray system. genotype and haplotype analysis were carried out using helixtree genetic analysis software. results: clinical and genetic data were available on cf patients ( from seattle and from dublin/belfast). in the combined cohorts, tagsnps in the ednra gene were significantly associated with differences in cf lung disease severity (p= . ). this was observed in both the irish cohort (p= . ) and the seattle cohort (p= . ). the effects were independent of gender and cftr genotype. four common haplotypes (haplotype frequency> %) were identified in the ednra gene. there was significant association between ednra haplotypes and cf lung disease severity (table ). there was no association between genetic variants in ednrb, edn and edn and cf lung disease severity. conclusions: the ednra gene is a genetic modifier of the cf phenotype. our findings were seen in two independent cohorts and verify existing associations found in other populations. the endothelin pathway may be a novel therapeutic target for the treatment of cf lung disease. background: cystic fibrosis (cf) is a recessive "monogenetic" disorder, but there is heterogeneity of lung disease severity and survival reflecting environment and non-cftr genetic modifiers. the unc/cwru multisite gene modifier study (gms) identified patients who were "severe" or "mild" as teenagers or young adults and pertinent cross-sectional data was collected; however, we did not collect all pertinent information about early (age < years) clinical features. objective: we sought to retrospectively evaluate the early clinical features of "severe" and "young mild" patients enrolled in the gms. methods: we obtained all cff registry data available on patients. there were "severe" (worst th percentile of birth cohort, age range - ) and "mild" (best th percentile, age range - ). initial analyses focused on cross-sectional plots of patient age versus multiple clinical features, including age at diagnosis, hospitalizations, cdc height and weight percentiles, presence/absence of ps. aeruginosa from respiratory cultures, and fev (% pred). results: there were . and . years of cff registry data per patient for "severes" and "milds," respectively. preliminary results indicate "severe" patients were diagnosed earlier in life than "mild" patients (mean: . vs. . years, p= . ). this difference is greater after omitting patients who had meconium ileus (diagnosis: . vs . years, "severes" and "milds" respectively, p= . ). between and years of age, "severe" patients were hospitalized more frequently than "mild" patients and, from age onwards, the disparity in frequency of hospitalization increased. "severes" and "milds" had similar cdc height percentiles (~ th percentile) until age , at which point percentiles increased more for "mild" versus "severe" patients. by age , "severe" patients already had lower cdc weight percentiles than "milds" ( nd versus st percentile) and this disparity increased throughout adolescence. from age to , "severe" patients had a - fold higher prevalence of ps. aeruginosa than "mild" patients. as early as years of age, fev (% pred) was ~ points lower in "severes" as compared to "milds." however, some "severe" patients had normal lung function at ages - and overlapped with mild patients; thus phenotyping of lung severity for young patients is not optimal. all of these results were similar for males and females. summary: retrospective analysis of the cff registry data indicates that patients classified later in life as being "severe" experience a worse course of disease from early in life. understanding the early clinical course may prove helpful in defining surrogate phenotypes for modifier studies, and help define appropriate therapeutic targets. cystic fibrosis is mainly caused by small molecular defects of the cftr gene; despite the genotype is defined in the majority of patients, a number of cf cases still remain uncharacterised. the cf mutation database lists more than large rearrangements that may escape detection using pcrbase techniques. the innogenetics assay, the dhplc and sequencing screening showed a mutation detection rate of . % in our population. we report here the results of mlpa screening for ctfr gene rearrangements, performed on the unidentified alleles of our cf patients. our sample population consists of unrelated italian cf patients (for a total of alleles), followed at cf centres of lombardia region. mlpa analysis was performed in patients who still had one or two unidentified alleles after extensive analysis of cftr gene. all patients studied had classical clinical cf symptoms. subjects presented with persistent or recurrent respiratory symptoms, failure to thrive, salt loss syndrome and gastro-intestinal findings. we characterized different deletions and a new duplication (dup promoter-ex. ). thus, . % ( / ) tested alleles had a large gene rearrangement. the deletion of exons - ( / ) was the most frequent in our cohort. all patients had positive sweat chloride values (above meq/l), except the patient carrying duplication who has borderline sweat chloride value. out of patients, ( %) had fecal elastase levels consistent with a preserved pancreatic function: of these patients, had mild mutation, had severe, and had unknown mutation. six patients present liver involvement. the results of the present study could indicate that compound heterozygosity for large rearrangements in cftr gene, is strongly associated with severe pancreatic disease as mutations in classes i, ii, and/or iii. l f is a missense substitution which changes from leucine to phenylalanine at position , resulting from a g/c transition at position in exon a of the cftr gene. it has been described in patients with disseminated bronchiectasis, recurrent idiopathic pancreatitis, sarcoidosis, newborns with hypertripsinemia,. recently derichs et al. reported one healthy -year-old girl homozygous for l f; therefore the pathogenic role of the variant is still unclear. in this study we present subjects compound heterozygotes with l f. no other mutations have been identified after molecular analysis performed using sequencing analysis of the whole coding region of cftr gene and mlpa technique in order to search for gene rearrangements.none of them presented ivs t allele. three of them had a severe mutation in trans (r x, aa>g, and n k), while the other three had a mild mutation on the complementary allele (d h, r q, and r w). individuals with a severe mutation in trans presented a remarkably different clinical picture compared to those with a mild mutation in trans. the formers were diagnosed at a mean age of . years, had an average sweat chloride of . meq/l; besides, all of them have respiratory symptoms, staphilococcus aureus in sputum cultures (one had pseudomonas aeruginosa as well), and in two the chest x-rays was abnormal. the three l f/ps mutation individuals were diagnosed at a mean age of . years, had an average sweat chloride of . meq/l; besides none of them had respiratory symptoms, abnormal chest-xrays, or positive sputum cultures, but two had a history of pancreatitis. these data seem to suggest that l f cannot in any case be considered a neutral polymorphism. the variability of its clinical expression seems to be influenced by the mutation in trans. further studies are needed in order to support our results. expression of the cystic fibrosis transmembrane conductance regulator (cftr) is tightly regulated both spatially and temporally, yet the molecular mechanism of this regulation is not well understood. because no tissue-specific regulatory elements were recognized in the basal cftr promoter, the crucial cis-regulatory elements are likely to be located elsewhere within the cftr locus. a number of the non-coding regions of the cftr gene were found by our laboratory to contain dnase i hypersensitive sites (dhs), suggesting the presence of regulatory elements at these sites. studies presented here investigate the role of an intron dhs (dhs ) in the tissue-specific regulation of cftr gene transcription. the elucidation of molecular mechanisms underlying the temporal and spatial expression of cftr may aid in developing more specific targeted therapies for cystic fibrosis (cf). footprinting analysis of cftr intron revealed a protected region within the core of dhs at + kb. in silico analysis of this sequence uncovered a number of transcription factors binding motifs, including a consensus binding sequence for hepatocyte nuclear factor (hnf ). we have previously identified a role for this factor in the regulation of cftr expression. results of electrophoretic mobility shift assays (emsa) demonstrate that hnf α specifically binds to the motif in cftr intron in vitro. in addition, chromatin immunoprecipitation (chip) analysis of cells expressing both cftr and hnf α factor shows that this factor binds to the intron site in vivo. when cloned as an enhancer, the dhs element was found to augment minimal cftr promoter activity in a luciferase reporter based assay. this increase in luciferase activity was abolished when two nucleotides within the core hnf binding site were mutated, suggesting a functional role for hnf α in cftr gene transcription. further experiments are underway to determine whether additional transcription factors can be recruited to the core of the intron dhs regulatory element and can interact with hnf and the cftr basal promoter to modulate tissue-specific cftr gene expression. the promoter of the cftr (cystic fibrosis transmembrane conductance regulator) gene is not solely responsible for its complex pattern of expression. to identify potential regulatory elements for cftr we previously mapped dnase i hypersensitive sites (dhs) across kb spanning the locus. in addition to intronic dhs, a number of sites were observed that flank the cftr gene. we hypothesized that these may include insulator elements that establish the chromatin expression domain, within which the cftr gene is regulated. insulators are elements that shield against the effects of regulatory elements from adjacent genes, and they may also block silencing of integrated transgenes. using a well-established insulator assay, in which dna regions of interest are assayed for their ability to interfere with communication between a chicken β-globin enhancer and a neomycin resistance gene, we demonstrated that two dhs, at - . kb from the cftr translational start site, and at + . kb from the ' end of the gene, exhibit enhancer blocking activity comparable to known insulator elements. electrophoretic mobility shift assays (emsa), demonstrated in vitro binding of the well-characterized insulator protein ctcf (ccctc-binding factor) at the - . kb dhs. this was confirmed in vivo in both cftr-expressing and non-expressing cell types using chromatin immunoprecipitation (chip). in contrast, although the + . kb dhs did not bind ctcf, we obtained in vitro evidence for the interaction of other factors that may be involved in insulator activity. furthermore, chip analysis of histone modifications across the cftr locus revealed striking differences between the - . kb and + . kb dhs, again suggesting mechanistic differences between these elements. the characterization of insulator elements flanking the cftr locus may be of direct practical relevance in the design of vectors for effective cf gene therapy. one of the problems encountered in gene therapy protocols is the relatively rapid loss of expression from the cftr cdna once it is introduced into mammalian cells. incorporation of the - . kb and + . aminoglycosides, particularly tobramycin, are primary antibiotics used to treat the airway infections in cystic fibrosis patients. lifetime, systemic exposure to these antibiotics is significant and can be associated with significant nephro-and ototoxicity. these toxicities have the potential to significantly reduce the quality of life in the aging adult cf population. we previously reported an incidence of aminoglycoside ototoxicity of greater than % with sensitive audiometric studies (i.e. pure tone audiometry and distortion product otoacoustic emissions). these studies indicated considerable variability in ototoxicity in patients with similar systemic aminoglycoside exposure. the literature suggests that genetic variability in mitochondrial dna can partially explain these differences. single nucleotide polymorphisms (snps) in the mitochondrial srrna gene are associated with aminoglycoside ototoxicity. we genotyped unselected adult cf patients for four of the mitochondrial s-rrna polymorphisms associated with aminoglycoside ototoxicity by direct sequencing of dna harvested from peripheral blood. four patients exhibited polymorphisms in this gene. two patients possessed the a g transition, while another patient each revealed a polymorphism at a g and c t transitions. the patient with the a g polymorphism died prior to audiometric testing. both patients with the a g had audiometric studies consistent with moderate to severe ototoxicity. while the patient with the c t had mild aminoglycoside ototoxicity. all four patients had severe airway obstruction and at least one full course of parenteral tobramycin at mg/kg/d. there was no history of toxic serum levels aminoglycosides during this therapy. both patients with the a g polymorphism had a family history consistent with the expected maternal inheritance pattern associated with the mitochondrial s-rrna gene. we found no evidence of the delt polymorphism in this population. these studies indicate a higher than expected frequency ( %) of mitochondrial s rrna polymorphisms associated with aminoglycoside ototoxicity. these studies demonstrate that genetic screen-ing provides valuable susceptibility information and may change clinical decision-making. support: rising hope foundation/national cf foundation. cystic fibrosis is a genetic autosomal recessive disease that is caused by deleterious mutations in the cftr gene. in its most severe form, cf results in abnormal sweat electrolytes, sino-pulmonary disease, male infertility, and pancreatic exocrine insufficiency. in fact, cf is the most frequent cause of pancreatic insufficiency in humans. carriers of cf (i.e., heterozygotes) do not express any of these classic symptoms. recent studies indicate that significant numbers of non-cf patients diagnosed with congenital bilateral absence of the vas deferens (cbavd) or idiopathic chronic pancreatitis (icp) are compound heterozygotes and carry two cftr mutations of varying severity. in dogs, especially german shepherds and rough-coated collies, exocrine pancreatic insufficiency (epi) has been observed with symptoms similar to those seen in human patients. epi in dogs is most often caused by pancreatic acinar atrophy (paa), a degenerative disease of the exocrine pancreas that has been shown to be inherited as an autosomal recessive trait. the locus responsible for canine paa has not been identified to date. we considered the possibility that cftr mutations might be responsible for this disease. a dog that had been diagnosed with paa and one that was a known carrier of paa were screened for cftr mutations. our samples also included dogs diagnosed with idiopathic pancreatitis as well as healthy dogs. we have established protocols for detecting putative mutations in the canine cftr gene using temporal temperature gradient gel electrophoresis (ttge). in the dog with paa and in the carrier of paa, our screening methodology identified mutations in of the amplicons that span the exons of the cftr gene. dna sequencing revealed silent mutations in exons , and . in exons (l ) and (l ), single nucleotide polymorphisms converted ctt codons into ctc codons, both leucine codons. in exon (p ) another single nucleotide polymorphism converted a ccc proline codon into a cct proline codon. the other identified mutations (amplicons , and , , , , , and ) were found in intronic sequences and have no predicted effect on cftr expression or function. based on these findings, we conclude that cftr mutations are not responsible for paa and the epi that it causes in dogs. hypothesis: specific polymorphisms in genes of the innate immunity may modify the severity and progression of cf pulmonary disease and influence pseudomonas aeruginosa colonisation. methods: single nucleotide polymorphisms (snps) analysis of multiple genes contributing to the innate immunity ( (mbl ), masp (mbl associated serine protease) Ç , fcn (ficolin) Ç , lbp (lipopolysaccharide -binding protein), cd , tlr (toll-like receptor Ç ) ) was performed in cf patients (age - years). spirometric data and bacterial colonisation status with pseudomonas aeruginosa were collected retrospectively. a decline in fev of % over a years period and a percent predicted fev value of % were used to discriminate mild from more severe affected adult cf patients (≥ years). the frequency of single and combined snps in well-defined subgroups of the cf population was compared. results: in adult cf patients with a mean fev lower than %, the combinations of snps of cd (promoter) with tlr (promoter), fcn with tlr (exon ), and masp with tlr (promoter) were more common than in adult cf patients with mean fev > % (odds ratio (or) , ( % confidential interval (ci) : , - , ) , or , ( % ci: , [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] ) and or , ( % ci: , - , ) respectively). the frequency of combination snps of masp with tlr and masp with tlr was significantly higher in cf adults with more than % decline in fev compared to cf adults with less than % decline in fev (or , ( % ci: , - , ) and or , ( % ci: , - , ) respectively). however, the single snps of masp , tlr , tlr were not more frequently found. in pseudomonas aeruginosa colonised cf patients, an increased frequency of combined snps of tlr (exon ) with tlr (exon ), lbp (exon ) with tlr (exon ) and mbl with tlr (exon ) (or , ( % ci: , - , ), or , ( % ci: , - , ), or , ( % ci: , - , ) respectively) was found. in contrast, for single snp tlr (exon ) no statistically significant differences were found between the groups (or : , ( % ci: , - , )). in general, the odds ratios for the combined snps were higher than for the single snps and remained significant when the different groups were subdivided according to cftr genotype. conclusion: certain combinations of snps of genes of the innate immunity are more frequently found in cf patients with a lower fev , a stronger decline in pulmonary function and pseudomonas aeruginosa colonisation. however, for the single snps this trend was less pronounced or absent. congenital bilateral absence of the vas deferens (cbavd) is a rare condition associated with mutations in cftr. cbavd lacks extra-genital organ system abnormalities; it has been viewed as a mild (variant) form of cf. the disease serves as an excellent model for studying the clinical and experimental ramifications of marginal cftr activity, and the functional thresholds necessary to confer a cf-related phenotype. recently, drumm et al. (nejm, ) found that the codon cc genotype of tgfβ- is associated with more severe pulmonary disease, particularly in individuals homozygous for ∆f cftr. tgfβ is also known to play a critical role during normal development and differentiation of the human vas deferens (han-nema et al., horm res, ) . because % of cbavd subjects carry at least one defective cftr allele (but millions of males worldwide are heterozygous for cftr mutations without developing cbavd), the present study was designed to test whether tgfβ polymorphism might serve as a genetic modifier underlying isolated vas deferens loss in the setting of cbavd. we sequenced the ' end of the tgfβ gene in eighty cbavd individuals possessing at least one cftr mutation, and compared genotype frequency with results previously published from demographically similar controls. when cc frequency in cbavd subjects was compared to pooled estimates from earlier trials, no significant differences were seen using a non-dominant model. compared to frequencies reported by arkwright et al. and garrote et al., a greater number of homozygous cc subjects ( . % vs. . %, p< . ) was observed. our preliminary results therefore indicate that if tgfβ- codon polymorphism is associated with cbavd penetrance, it does not represent the major modifier of disease phenotype. in order to evaluate this possibility further, we plan analysis of a larger (matched) control set of cbavd and non-cbavd individuals, studies of tgfβ- activity in vas deferens epithelial cells with and without cftr, and experiments to investigate the effects of tgfβ on cftr biogenesis and function. supported by nih and cff. newborn screening (nbs) for cf has been successful in identifying cf infants, particularly those who have positive sweat tests immediately following the positive screening result. tracking by personnel at the central nbs program facilitates resolution of diagnostic status because cf nbs presents a challenge to cf center (cfc) systems and primary care providers when apparently asymptomatic infants have inadequate (quantity not sufficient (qns)), borderline or indeterminate sweat tests. short-term follow up is more problematic when the screening algorithm requests earlier sweat tests and defines a lower cutoff for indeterminate sweat test results than is used for older infants, children and adults. the massachusetts (ma) cf nbs workgroup chose a low cutoff for indeterminate results of meq/l up to months of age, reverting to the accepted meq/l cutoff thereafter. follow up by the central nbs program identified infants whose diagnostic status remained unresolved more than weeks after a qns or borderline sweat test and eliminated the need for individual cfcs to follow infants tested at more than one cfc. the program sent informational letters and a reminder recommendation to finalize the diagnosis to primary care providers of infants with unresolved diagnoses. of the cf infants identified by ma cf nbs, had an initial sweat result that was indeterminate and another had an initial result that was qns. the central screening program has the capacity to track infant and child outcomes across cfcs; for example, we have been tracking growth outcomes of all cf infants identified by ma cf nbs, despite of the cf infants having moved from one cfc to another during their first years of life. tracking and outcomes data will be presented. when more than one cfc operates within a screening program, centralized tracking ensures capture of quality data. despite several educational campaigns cf is often not diagnosed in early infancy in poland (the mean age of diagnosis months, median is months). after several years of discussions,in september a neonatal screening for cf supported by the ministery of health was started as the third obligatory neonatal screening in poland. more than , neonates were screened in the first seven months using the existing screening infrastructure and spare blood spots after pku and congenital hypothyreosis screening. the protocol was two stage irt/dna (covering the most common mutations in the polish population). the final diagnosis was based on sweat tests (conventional pilocarpine-iontophoresis and conductometric nanoduct) as well as clinical examination. values of chloride in sweat > mmol/l and nacl > mmol/l were considered diagnostic for cf. children were confirmed as cf. although delta f is the most common mutation in the polish population, only two infants were delta f homozygotes, six were delta f heterozygotes and the remaining had different mutations ( children had + kbc>). half of the diagnosed group was still pancreatic sufficient. the children had confirmed cf diagnosis at the mean age of weeks. only children were free from any radiological changes in lungs. the most common pathogen in this group was staphylococcus aureus. at the first visit information for parents about cf was given along with physiotherapy and dietary education. anthropometric measurements, airways bacteriology and elastase - in stool were performed. psychologist consultations were available. neonatal screening for cf enabled earlier diagnosis and the introduction of complex therapy in comparision with symptomatic screening. objectives: the clinical course of cystic fibrosis (cf) varies widely among patients carrying the same cftr gene mutations supporting that additional genetic modifiers could affect the cf phenotype. as inflammation is a central contributor to the pathogenesis of cf lung disease, genes involved in the inflammatory response are potential modifier candidate genes. methods: we examined the influence of polymorphisms in genes involved in the inflammatory response (tnf, il- β, il- receptor antagonist (rn), il- , il- , il- and tgf-β ), on disease progression in a group of caucasian children with cf. the genotypes were tested for an association with changes in lung function tests, pseudomonas aeruginosa colonization and nutritional status by multivariable analysis. results: we found a significant association between tgf-β + t/c variants and decline in lung function measured as the forced expiratory volume in one second (fev ) and the forced vital capacity (fvc) (p= . and . respectively). il- gene polymorphisms (- a/t, + g/t and + t/c) were associated with the occurrence of pseudomonas aeruginosa colonization (p< . ). conclusions: this study suggest that il- variants may influence pseudomonas aeruginosa colonization in cf, however this need further confirmation. moreover, we provide additional support to the results of other trials and strongly suggest an association between tgf-β variants and lung disease phenotype in cf. if such a role of tgf-β is confirmed by functional studies, it may have important clinical impact for the identification of patients at risk to develop more severe respiratory manifestations and for the development of new therapeutic strategies aimed at adequately balancing tgf-β production and action introduction: cystic fibrosis (cf) is the most common monogenic disease in caucasian population, its frequency is about one in live born. the estimated frequency in hispanic population is about one in , with differences in mutation frequency especially for low frequent alleles. heterogeneity in pulmonary manifestations cannot be explained by the cftr mutation genotype. recent studies are focused in the study of modifier genes. these are genes different from the primary cftr gene that could influence the fq phenotype, acting trough mechanisms like inflammation, repair and remodeling, protease-anti protease balance, innate immune response, etc. this work shows some advances in cf diagnosis and modifier genes analysis in mexican population. methods: we tested the effectiveness of innolipa cftr kit (innogenetics) in clinically diagnosed cf patients. thirty six frequent mutations in cftr gene were analyzed by pcr and reverse hybridization with allele specific oligonucleotides probes in dna samples extracted from blood or oral swabs. for frequency analysis of modifier genes we tested about seventy dna samples from a dna bank of patients previously tested positive for cftr mutation. the polymorphism analysis of modifier genes was made by pcr-rflp. for alpha antitrypsin (aat), z and s alleles were analyzed according stanford and cols. ( ) . for tumor necrosis factor alpha (tnfα) - polymorphism in the promoter region was analyzed according chen and cols. ( ) . results: mutation was detected in in of patients ( % of effectiveness). df allele frequency was % (caucasian - %) followed by + g-a, s n, g x, g e and delt ( %). % of the mutant alleles remain undetected. preliminary results in modifier genes show low frequency for aat mutant alleles ( . % for aat s and z alelle respectively) and . % for tnf- . conclusions: df alelle frequency was %, and we found the low fequency alleles s n and + g-a. frequency of modifier genes mutant alleles in cf patients are low. this work is in progress of selection and recruitment of patients and controls, as well as standardization of the other genes modifiers, along with some other biomarkers that will allow to understand the physiopathology of cf and to define a possible genetic risk profile of severe pulmonary disease. over putative cystic fibrosis (cf)-causing mutations have been reported to the cf mutation database and almost half (~ ) are rare missense mutations that are predicted to substitute a single amino acid. to determine if any of these rare missense mutations cause cf by altering localiza-tion of cftr, we examined mutations in the cytosolic loops of cftr since localization signals have been identified in cytosolic loops of other ion channels. amino acids alignments comparing human cftr sequence to that of species revealed that cytosolic loop (cl ) was the most conserved loop by identity. furthermore, cl had the most naturally observed mutations: of the amino acids were found to be mutated in cf patients and sites had multiple missense mutations at the same amino acid. in addition, different substitutions of the same cl amino acid have been associated with different disease consequences, such as the missense mutations in the arginine at codon . patients with the r p mutation have pancreatic insufficient (pi)-cf, those with r w mutation have pancreatic sufficient (ps)-cf or cbavd, while patients with r q primarily had pi-cf. to determine whether the r mutations cause disease by affecting cftr localization, we used the flp-in system to create stable polarized mdck-gfp-cftr cells to produce isogenic clones with cftr expressed from the same integration site. confocal microscopy studies of stable mdck-frt-gfp-cftr-r mutant cell lines revealed that cftr-r p was cytoplasmic, cftr-r w was apical and cytoplasmic, and cftr-r q was apical. to confirm these observations, quantitative biotinylation studies were performed. biotinylation assays of mdck cells stably expressing cftr-r p confirmed that this mutant was absent from the apical surface, cftr-r w had a low level of fully glycosylated protein at the apical membrane, while cftr-r q had fully glycosylated protein at the apical membrane comparable to wild-type cftr. thus, cftr-r p and cftr-r w displayed properties in polarized cells that were distinctly different from wild-type cftr consistent with their associated cf phenotypes. however, the profile of cftr-r q (apical localization and cl-channel function (seibert et al. , mickle et al. ) appears inconsistent with a pi-cf phenotype. indeed, re-analysis of cftr genes bearing r q revealed that of carried a second mutation in cis (s x). in nine of these patients in whom cftr genotype and pancreatic status is known, presence of the nonsense mutation s x is associated with the pi-cf phenotype. since nonsense mutations are known to cause severe gene dysfunction by promoting decay of their rna transcript, we concluded that the s x mutation rather than r q is responsible for the pi-cf phenotype. the finding of s x explained the otherwise enigmatic functional studies of r q and clinical observations in patients bearing this mutation. this study also demonstrates that stable expression of cftr mutants in polarized mdck cells using the flp-in system provides a useful screen for evaluating the disease potential of rare missense mutations. the clinical course of cf is characterized by recurrent pulmonary infections and chronic inflammation. in cf patients, up-regulation of toll-like receptor- (tlr ) gene in airway epithelial cells is believed to enhance proinflammatory responses towards bacterial tlr ligands. we have recently shown that decreased methylation (demethylation) of the tlr promoter is responsible for cf-related up-regulation of tlr in bronchial epithelial cells (shuto. t. et al., faseb j, ) . however, the molecular mechanisms responsible for dna demethylation-dependent tlr gene upregulation in cf cells remain unknown. here, we identified minimum region of the tlr promoter critical for its expression at - /+ , which contains one putative binding site for sp . sp inhibitor mithramycin a treatment reduced tlr promoter activity and its expression in cf bronchial epithelial cells (cfbe o-), suggesting the importance of this sp site for the tlr gene regulation. moreover, bisulfite sequence analysis revealed the hypomethylation of adjacent this sp binding motif within tlr promoter in cfbe o-cells, implying the cf-specific demethylation of adjacent sp binding motif. although mithramycin a rarely affected basal expression of tlr gene in hbe o-cells (non-cf bronchial epithelial cells), mithramycin a inhibited tlr expression in -azacytidine (dna-demethylating agent)-treated hbe o-cells. taken together, our results suggest that sp is crucial for dna demethylation-induced gene upregulation of tlr in cf bronchial epithelial cells. mucus hypersecretion due to goblet cell metaplasia is a critical feature of cf lung disease, affecting both mucociliary clearance and drug delivery. various studies in cf lungs and cf primary cultures have shown increased expression of muc ac, the main gel-forming mucin produced by goblet cells; however, little is known about the role of muc ac in the progression of lung disease. to determine whether overexpression of muc ac alone is sufficient to induce lung pathology, we generated a mouse model overexpressing muc ac. the entire muc ac cdna (minus % of the predicted vntr sequence, final size ~ kb) was cloned and tagged with an internal gfp epitope at a site close to the vntr to preserve the integrity of mucin domains. gfp-muc ac was linked to the rat ccsp promoter to drive specific airway expression, primarily in clara cells since these cells are capable of secreting mucins. blastocyst injections were used to generate transgenic founder lines in c bl/ background. although muc ac-gfp mrnas were expressed in transgenic mice, lung histology did not show significant increases in inflammation, ab/pas-positive cells, or luminal secretion compared to wild-type littermates. gfp fluorescence from freshly excised lung was weak but immunohistochemistry showed that the distribution of gfp correlated with the expected expression pattern, with the majority of positive cells localized to the airways and few positive cells in the alveolar space. at higher magnification, gfp-positive granules were observed in dome-shaped cells that were also positive with cc antibody, suggesting proper muc ac-gfp packaging in clara cells. bronchoalveolar lavage samples separated by agarose gel western blots and probed with gfp antibody confirmed that each line secreted muc ac-gfp, with a migration pattern comparable to a mucin. to further characterize the secreted transgenic mucin, a pool of lavage samples was separated into supernatant and pellet. the pellet was resolubilized in m guanidine. both elements were passed through a s size-exclusion column. analysis by light scattering and refractometry to determine molecular weight and concentration revealed that the solubilized pellet contained an enormous but compact complex ( x g/mol and nm radius of gyration). gfp-rich fractions were pas-positive, indicating proper glycosylation of the transgenic muc ac-gfp. cscl density gradient studies revealed that the gfp-rich fractions had a high molecular weight but did not appear to be a prominent part of the megacomplex retrieved in lavages, unlike other tested mucins. in conclusion, we showed that the overexpressed muc ac-gfp was a high-molecular-weight protein that was glycosylated, packaged in clara cells and formed multimers. lack of discernable phenotype in the transgenic lungs suggests that mucin overproduction alone is not sufficient to trigger goblet cell metaplasia and mucus accumulation, supporting the protective role of mucins. currently we are crossing the muc ac-gfp with the scnn b mouse model to study muc ac-gfp under dehydrated conditions and we are conducting allergen-challenges in the transgenic mice. results of such studies will provide novel insights into the role of secreted muc ac in development of lung disease in these models. supported by grants from the cf foundation. the major characteristic in cystic fibrosis (cf) is acquirement of chronic lung infections with p. aeruginosa. once established the chronic infection can only be suppressed, not eliminated. the cf patients harbour one single strain of p. aeruginosa which only rarely is replaced by other strains. during the chronic lung infection there is a year long mutual impact of the bactaria on the host, and of the host response and antibiotics on the bacteria, resulting in a continous adaptation of the bactaria in the lungs of the cf patients with selection for different clones. in contrast, mouse models of the lung infection in cf is limited to two weeks! therefore, we introduced a new strategy infecting different groups of balb/c mice with pulsed field gel electrophoresis-(pfge) identical clones isolated from one cf patient during -years of p. aeruginosa lung infection. ) infecting the mice with non-mucoid isolate showed that the early isolates were significantly better in establishing an infection with reduced clearance of the bacteria from the lungs (p< . ), increased dissimination of the macroscopic pathology (p< . ) and a more acute type and a higher degree of inflammation (p< . ) as compared to mice infected with a late isolate. in addition, the pulmonary cytokine response was comparable with observations in cf where gm-csf and il- correlated to a milder disease, whereas g-csf, mip- (il- analog), and il- b correlated to severe disease. ) infecting the mice with the mucoid pfge-identical isolates from the same cf patient revealed a signicantly higher mortality in mice infected with the late isolates (p< . ). in addition, the pulmonary g-csf and mip- response increased in the groups of mice that were infected with the late isolates as compared to the early isolate (p< . ). moreover, the g-csf and mip- increased during the first three days of infection with the late mucoid isolates (p< . ), indicating an impairment of the host response in controlling the lung infection. ) when tobramycin treatment was initiated h after infection of mice with a mucoid isolate, the number of bacteria were cleared or reduced to a significantly lower level (p< . ), and a significant decrease of both g-csf and mip- at day one, two and three was observed (p< . ). in contrast, when treatment was initiated after h the number of bacteria did not change significantly, and the reduction in inflammatory cytokine response became significant for both late isolates only at day three of treatment (p< . ). moreover, cytokine levels were significantly lower in the h as compared to the h group (p< . ) confirming the importance of early and aggressive antibiotic treatment in cf. non-mucoid isolates were reduced during the first three days of infection independent of treatment. in conclusion, we have implemented a clinical important cf-time perspective (years) in experimental p. aeruginosa lung infection, useful for patho-physiological and treatment studies. cystic fibrosis (cf) is a complex disease that affects multiple organs and results in a wide range of phenotypes in humans. mouse models of cf display many of the phenotypes observed in human patients and provide necessary tools to dissect this multifaceted disease. however, due to the involvement of cftr in various organ systems, the ability to discern the contribution of each organ system to each phenotype of cf is unknown. for example, we have found that mouse growth is dramatically reduced compared to wildtype, and mechanisms ranging from neuroendocrine disruption, intestinal malabsorption and cacchexia have been proposed. we have found that cf mice consume more calories per gram body weight than non-cf mice and intestinal absorption of dietary lipids is similar between the two. cf animals absorb % and non-cf % (p > . ). to identify the source of the growth deficit, it would be advantageous to restrict expression of cftr in specific tissues to determine each tissue's contribution to the phenotype. to accomplish this, we have created a conditional, null murine cftr allele. a conditional null allele is one in which the gene of interest is induced to be non-functional in either a specific tissue or cell type, or at a specific developmental time. examination of cftr absence on a tissue-by-tissue basis will greatly facilitate our understanding of the disease. the conditional cftr allele was created by "floxing" exon of cftr, known to be critical for function, with flanking loxp sites that allow for the deletion of exon when bacterial recombinase cre is present. to date, mice stably transmitting the floxed allele have been created. these founder mice have been crossed with mice expressing cre from the promoter of the villin gene, which restricts transcription of cre to intestinal epithelium. first generation animals show appropriate recombination and deletion of exon in the gut. however, these first generation animals are heterozygous for the floxed allele. we are currently backcrossing these animals with the founders to generate animals with homozygous deletion of cftr in the intestinal epithelium. similar crosses are planned to target other tissues. this new animal model of cf will be available to the cf research community to further our understanding of cf pathophysiology. this work was supported by a grant from the cf foundation increased enac-mediated na+ absorption is a hallmark of cystic fibrosis (cf) airways. we demonstrated that mimicking na+ hyperabsorption by overexpression of βenac in mouse airways results in airway surface liquid (asl) depletion and reduced mucus clearance causing a spontaneous cflike lung disease with high pulmonary mortality, and airway mucus obstruction, mucous cell metaplasia and chronic airway inflammation in adult survivors (mall et al., nature med. : , ) . the aim of this study was to identify the initiating lesions and investigate the natural history of lung disease caused by asl depletion in βenac overexpressing (βenac-tg) mice. to achieve this goal, we performed longitudinal studies on lung morphology, airway mucus obstruction, bronchoalveolar lavage inflammatory cell counts, expression levels of mucins (muc ac, muc b, muc ) and proinflammatory cytokines (tnfα, kc, ifnγ, il- , eotaxin- ), lung volume, lung mechanics, and airway na+ transport in neonatal to adult βenac-tg mice and wild-type littermate controls. we show that airway mucus obstruction in βenac-tg mice originated in the trachea in the absence of mucous cell metaplasia in the first days of life, and was associated with hypoxic degeneration and necrosis of airway epithelium, and death. in surviving βenac-tg mice, mucus obstruction extended into the lungs and was accompanied by secondary mucous cell metaplasia, increased expression of muc ac, muc b and muc , and airway inflammation with transient increases in macrophages and tnfα, in eosinophils and il- , and persistent increases in neutrophils and kc in the lung. βenac-tg mice also developed emphysema with increased lung volumes, distal airspace enlargement, and increased lung compliance. several disease surrogate markers waned in parallel with βenac transcript levels and airway na+ absorption in adult βenac-tg mice indicating that the level of enac overexpression was critical in determining disease severity. in summary, our results provide several novel insights into the in vivo pathogenesis of lung disease caused by asl depletion. we show that (i) asl depletion is sufficient to initiate severe airway mucus obstruction in the absence of mucous cell metaplasia and/or mucus hypersecretion; and (ii) airway na+ hyperabsorption and mucus plugging-induced hypoxia is associated with airway epithelial necrosis constituting a mechanism that initiates airway inflammation in the absence of infection. further, our results point to a novel link between airway na+ hyperabsorption, eosinophilic inflammation, and emphysema. the high frequency of allergic airway inflammation and emphysema in cf patients warrants further studies of the mechanisms that link asl depletion with these associated pathologies. the balance of airway surface liquid (asl) at a height compatible with efficient clearance of mucus is a process regulated by two main pathways: the secretion of clvia cftr, and absorption of na + via the epithelial na + channel (enac). to study this interplay, an immortal cell line capable of growing at an air liquid interface and having properties of human bronchiolar epithelium would be desirable for its simplicity in preparation and ease of molecular manipulation. an immortal human bronchial adenocarcinoma cell line (hbac) was infected with a lcfsn retrovirus carrying either cftr cdna (hbac cftr ) or the blank vector (hbac mock ). - day monolayers, grown on transwell clear membranes, were mounted in ussing chambers and studied under open-circuit conditions. to study regulation of clsecretion, the equivalent short circuit current was calculated in the presence of amiloride (apical, µm). in hbac mock cells, forskolin ( µm, basolateral) had no effect upon i sc . in contrast, hbac cftr cells demonstrated a robust increase in i sc ; a rapid increase to a peak (∆~ µa cm ), followed by relaxation to a stable and sustained plateau. genistein ( µm) also stimulated i sc in hbac cftr (∆i sc µa cm ) but not hbac mock cells. amiloride resistant basal current was consistently higher, by - µa cm , in hbac cftr versus hbac mock cells, presumably reflecting low basal cftr activity. we next examined the ability of endogenous receptors present in hbacs to regulate transfected cftr. stimulation of i sc in response to apical adenosine ( µm) in hbac cftr was qualitatively and quantatively similar to the forskolin response. while the above experiments provided evidence to suggest that hbac cftr cells possess the bioelectric capability of sustaining anion transport via nucleoside-stimulated cftr activity, it does not guarantee a resultant vectorial flow of fluid. in the final series of experiments, we examined whether adenosine was indeed able to stimulate secretion across the hbac-cftr monolayer by assaying asl height. monolayers were loaded apically with fitc-labeled dextran ( µl), and the majority of dye was then aspirated. monolayers were then allowed to equilibrate over a hour period, and asl measured by xz scanning on a leica confocal microscope. ~ µm adenosine and ~ µm benzamil was then added apically in pfc and asl height measured after and minutes. in both hbac mock and hbac-cftr cells, the basal asl height was ~ µm. over minutes asl height of hbac mock cells did not change in response to adenosine. in contrast, adenosine stimulated hbac cftr cells demonstrated a doubling of asl height (to µm) over the same period. this response is consistent with the prolonged increase in i sc observed upon stimulation with adenosine. in summary, the hbac model which displays only absorptive currents endogenously, can be transformed into a cell line in which cftr-mediated secretion can be stimulated and sustained by endogenous receptor systems present in the apical membrane. this new model will allow us to study cftr-mediated fluid transport in a defined manipulatable system. despite the identification of the cftr channel more than years ago, many questions remain about its regulation and in vivo function. in order to better understand how cftr functions in vivo, numerous studies have searched for and identified potential interacting partners but to date, there is little evidence linking any of the known cftr interactors to its activity. during a forward genetic screen in zebrafish, aiming to identify genes regulating endodermal organ development, we identified a mutant, which we named baobab (bao), after the african tree that accumulates water during the wet season, which exhibits a dramatically enlarged fluid-filled gut tube. most interestingly, we have shown that various cftr inhibitors reduced the appearance of enlarged guts in bao mutant embryos, suggesting that the fluid accumulation phenotype resulted from an increase in cftr activity. to test whether increased cftr activity resulted from changes in cftr protein levels or localization we raised antibodies against zebrafish cftr. western-blot and immunolocalization studies revealed that the channel was expressed and localized as in wild type in bao mutant embryos indicating that the increased cftr function most likely resulted from the activation of the channel. these data, together with the recessive character of the mutation, suggest that the bao protein is a negative regulator of cftr activity. we have positionally cloned the bao locus and isolated the affected gene. bao encodes a cytosolic protein that has not been previously shown to regulate cftr. in agreement with the pharmacological data pointing to a gut-specific function of bao, in situ hybridization studies showed that bao is higly expressed in gut. we are currently investigating how the bao protein regulates cftr function. in addition, we are also investigating the function of cftr during zebrafish development. these studies lay the groundwork to take advantage of the zebrafish system to further investigate cftr biology. we establish a genetic model system for studying the regulation of the cftr channel that will contribute to the understanding of the pathophysiology of cystic fibrosis and several intestinal secretory conditions. acknowledegements: this work was supported by an embo long-term postdoctoral fellowship to mb and by nih grants to d.y.r.s.. we thank a. verkman for providing cftr inhibitors and k. brand for technical assistance. the trachea of the adult cf mouse (cftr tm unc or cftr tm kth ) expresses little cftr and exhibits neither a defect in camp-mediated clsecretion nor hyperabsorption of na + , both signature abnormalities in human cf airways. we generated a mouse exhibiting hyperabsorption of na + in the airways by over-expressing the β subunit of the epithelial na + channel (βenac, gene scnn b). βenac mice exhibit airway pathology (decreased mucociliary transport, mucus plugging, and airway inflammation) similar to human cf airways. to determine whether inactivation of cftr in βenac mice results in a more severe phenotype, we generated a cf/βenac mouse by crossing the ∆f (c bl/ n) mouse (cf mouse) with the βenac mouse (c bl/ n). survival analysis showed a drastic reduction in survival for cf/βenac mice in comparison to cf mice or βenac mice. we studied the airway bioelectrics of day-old pups of each genotype. the basal short circuit current (i sc ) was ~ fold greater in wild-type (wt) ( ± µa . cm - ; mean ± sem, n= ) vs. cf ( . µa . cm - , n= ) neonatal tracheas. as previously reported, the basal i sc in βenac preps was significantly elevated ( . ± µa . cm - , n= ) vs. wt tracheas. however, the basal i sc in the cf/βenac tracheas ( . ± µa . cm - , n= ) was intermediate between the cf and the βenac tracheas. the amiloride-sensitive i sc was elevated similarly in the tracheas of the βenac and cf/βenac pups and was ~ x greater than that exhibited by the wt or cf tracheas. the post-amiloride i sc was strikingly reduced in both cf and cf/βenac tracheas compared to the wt and βenac tracheas. we suggest that the post-amiloride i sc reflects constitutive cftr-mediated clsecretion, which may help hydrate airways in the neonate. unlike the tracheas of adult cf mice, neonatal cf tracheas exhibited the classic defect in camp-mediated clsecretion, with the forskolin response in the cf and cf/βenac tracheas being reduced ~ fold compared to the response in the wt or βenac preps. the response to utp (ca ++ -mediated clsecretion) was similar in the four genotypes. absence of cftr-mediated clsecretion does not appear to cause airway pathology in neonatal cf mice, but when this defect is coupled with na + hyperabsorption, it results in a significant increase in neonatal mortality. due to lack of cftr-mediated clsecretion, cf/βenac mice appear to experience greater mucus adhesion, producing death due to asphyxiation. in addition, the tracheas of cf mice (especially congenic c bl/ n) often appear constricted or collapsed due to a cartilaginous defect resulting in increased compliance. this anatomical defect does not appear to be detrimental to the cf mouse. however, in cf/βenac pups, that accumulate thick, sticky mucus in their tracheas, this tracheal constriction may contribute to their asphyxiation. these results demonstrate the importance of both abnormal na + absorption and decreased clsecretion in the pathogenesis of cf lung disease. supported by nih scor p hl . recently, we reported that species-specific differences in the airway biology of raav transduction exist between mice and human (am j respir cell mol biol, , : - ) . these differences significantly interfere with the use of mice as surrogate models for human lung gene therapy with certain raav serotypes. to survey which species might be an optimal animal model for raav preclinical testing, we utilized differentiated airway epithelia grown at an air-liquid interface (ali) from five species (human, non-human primate [nhp] , pig, ferret, and mouse) to test the efficiency of transduction with three serotypes of raav (type , , and ). our results demonstrated significant species-specific differences in raav transduction biology in airway epithelia. the serotype preferences of raav transduction from the apical membrane in human, ferret, and pig epithelia was raav >raav ≈raav , different from that seen in mouse epithelia (raav >>raav >>raav ). surprisingly, the profile of raav transduction with these three serotypes in nhp rhesus monkey airway epithelia (raav ≈raav >>raav ) was less similar to human than pig and ferret. additionally, raav lacked a bias in polarity of transduction in human airway epithelia, however, in nhp epithelia, raav transduced the basolateral membrane -fold more effectively than the apical membrane. furthermore, differences in the properties of raav receptors between these species was also seen. using neuraminidase treatment of the apical surface of ali cultures, we tested whether n-linked sialic acids were potential receptors for raav binding and/or transduction. these studies demonstrated removal of n-linked sialic acids inhibited raav transduction of human, nhp, and mouse airway epithelia, but not ferret and pig. however, raav binding in this setting did not correlate with changes in transduction; neuraminidase treatment dramatically reduced raav binding to nhp epithelia, but enhanced binding to human epithelia (~ fold). these findings suggest viral binding to airway epithelial cells may not be a major barrier for raav transduction. evidence that proteasome-modulating agents enhancing raav transduction - logs for all serotypes and species tested, suggest that the ubiquitin/proteasome pathway represents a common intracellular block in raav transduction of airway epithelia. these findings support the notion that viral interactions at the surface of airway epithelial cells (with co-receptors and receptors) influence intracellular processing aav virions that can alter the efficiency of productive transduction. enac surface density in cortical collecting duct (ccd) epithelial cells is established by camp/pka regulated insertion and retrieval of the channel at the apical membrane, together with the recycling of internalized enac back to the apical surface (butterworth et al. ( ) journal of general physiology : - ). ubiquitination by the nedd - ligase elicits endocytosis of surface enac, which may lead to its degradation. however, enac recycling to the apical surface requires ubiquitin removal by a deubiquitinating enzyme (dub). using a chemical probe approach, we identified ubiquitin carboxy-terminal hydrolase (uch-l ) as the predominant dub in vesicular endocytic and recycling compartments in mouse ccd epithelia. specific inhibition of uch-l ( µm of , , , -tetrachloroindan- , -dione) in filter-cultured ccd cells resulted in a rapid decline in enac currents, which was accelerated by forskolin stimulation, suggesting that uch is responsible for recycling enac to the apical surface. knockdown of uch-l significantly reduced both basal and camp-stimulated enac currents (from . ± . µa/cm in control sirna treated to . ± . µa/cm in uch-l knockdown cells). to determine whether the same dub was responsible for the regulation of enac in human bronchial epithelial (hbe) airway cells, differentiated cultures derived from cf and non-cf lung tissue were treated with the uch inhibitor. to address the possibility that protease cleaved enac may be regulated differently from unprocessed channels, differentiated hbe cultures were either maintained at air/liquid interface or were submerged under liquid/liquid conditions to alter the cleavage state of enac, as we have previously described (myerburg et al. ( ) journal of biological chemistry : - ). in all cases, no significant difference in enac-mediated na+ transport was observed between control and uch-l inhibited conditions. these data are in sharp contrast with the mechanisms of enac regulation observed in the ccd model system, and they support our previous findings that enac turnover and mode of regulation in hbe are distinct from the recycling mechanisms defined in the ccd epithelia (butterworth et al. primary human tracheobronchial epithelial (htbe) cells cultured at an air-liquid interface (ali) regulate ion transport, mucin secretion, and cilia beating to recapitulate directional mucus clearance, a key innate defense mechanism that fails in cystic fibrosis (cf). thus, ali htbe cell cultures are crucial for studying cf pathogenesis and for developing/validating novel therapies. different research centers use alternative protocols that enable reproducible creation of ali htbe cell cultures. however, limited primary htbe cell availability has driven the creation of cognate cell lines, accomplished mainly by the introduction of viral oncogenes. viral oncogenes disrupt normal cell physiology causing cellular hyperplasia, abundant apoptosis, genomic instability and, sometimes, poor polarization/differentiation, rendering the cells less useful than primary cells for assessing cftr function. furthermore, genetically unstable cell lines will continuously acquire changes independent of cftr genotype. bmi- is a polycomb group protein that controls cell cycle and cell identity via epigenetic regulation of chromatin, maintaining stem cells by suppressing expression of p , an inhibitor of cyclin dependent kinases. bmi- expression has been used to create cell lines that recapitulate normal cell structure and function better than viral oncogene-immortalized cell lines. using hiv lentiviral vectors, we introduced bmi- and the catalytic subunit of telomerase to enhance the growth of different ∆f homozygous cf and non-cf primary human airway epithelial cell preparations. all new cell lines grew for at least population doublings, while their normal counterparts senesced prior to a maximum of doublings. at passage - , the new cell lines had a diploid karyotype compared to grossly abnormal chromosomes in cells immortalized by viral oncogenes. ussing chamber analysis of ali cultures at passage (p) - revealed variable transepithelial resistances among the cell lines ranging from -> Ω*cm , but short circuit current (isc) responses stimulated by forskolin and inhibited by cftr were true to the cell's genotype and comparable to early passage primary cells, except for one non-cf cell line with relatively low cftr isc's at p . amiloride sensitive and utp-stimulated isc's were present but were more variable in magnitude in comparison to the currents observed in low passage primary cells. ali cultures exhibited a pseudostratified morphology with prominent apical membrane polarization, few apoptotic bodies, numerous mucous secretory cells and occasional ciliated cells. all cf and non-cf cell lines in culture produced similar levels of il- at baseline and cf and non-cf cells equally increased il- secretion in response to il- β, tnfα and the tolllike receptor agonist, pam cys. these novel cell lines will help fill gaps currently hindering cf research and therapeutic development. supported by cystic fibrosis foundation grant randel g . we recently considered that domestic dogs could represent a large natural reservoir to search for cftr mutations and, ultimately, to identify and breed animals with cf traits. the canine genome has been completely sequenced, and the canine cftr gene, found on chromosome , consists of exons spread over kb. an animal model that faithfully expresses human cf lung pathology is critical for the development of effective treatments for this most lethal aspect of cf disease. while transgenic mouse models of cf replicate numerous aspects of cf disease, they unfortunately do not develop the characteristic lung pathology including mucus plugging of airways, chronic bacterial infections, and bronchiectasis. the reason for this is not completely understood but it is likely related to significant differences in lung morphology between these species. to date, no non-murine genetic model of cf has been developed, and naturally-occurring, cf-causing mutations have not been identified in any non-human animal species. temporal temperature gradient gel electrophoresis (ttge), developed as a screening tool to identify sequence variations and mutations in the human cftr gene, was used to screen dogs for cftr mutations. dna was purified from whole blood received from veterinary clinics treating dogs for a variety of ailments. we have performed ttge analysis on dna from dogs, which includes dogs with pancreatitis and dogs with bronchiec-tasis, and have found hundreds of potential mutations amongst the thousands of amplicons tested. ttge can detect single base pair changes within a fragment of dna. as the temperature increases, the migrating dna becomes partially melted, creating single stranded regions (internal bubbles and branched ends) that decrease its mobility through the gel. the dna fragments have distinct melting profiles that are sequence dependent; therefore, changes in sequence of even a point mutation can alter the profile and result in a change in the rate of migration through the gel that can distinguish wild type from even a homozygous mutant. thus far exonic mutations have been sequenced but none have caused changes to cftr at the amino acid level (i.e., the mutations have all been silent). we conclude that ttge is a sensitive and effective method for screening large canine populations for cftr mutations. ( introduction: the search for "correctors" of defective protein processing of ∆f cftr has led to the development of high throughput screening assays to find compounds that promote the surface activity of the mutant protein. by necessity, primary screening assays typically use cell lines expressing recombinant ∆f cftr. however, "hits" identified in primary screening require scrutiny for false positives whose activity is either dependent on the ∆f cftr expression system or insufficiently robust to correct processing in the complex physiological setting of a native epithelium. to this end, we have developed a secondary functional assay that measures the shortcircuit current (isc) responses in organ cultures of gallbladder mucosa from ∆f cftr mice (cftr tm kth ). previous studies have shown that murine ∆f cftr recapitulates the protein processing defect including the capability to increase processing when mucosa is incubated at reduced temperature ( °c) (j. clin. invest. : invest. : , . gallbladder mucosa was chosen for this assay because the murine gallbladder can be removed aseptically for organ culture, the epithelium is essentially a monolayer, and the mucosal wall is transparent which facilitates morphological assessment. methods: using either wild-type (wt), ∆f cftr (df) mutant or cftr knockout (cftr ko) mice, excised gallbladders were opened longitudinally, rinsed and positioned on loose nylon mesh. the preparation was mounted between two halves of a polycarbonate culture cup with . mm aperture and placed in submersion culture for - days at either °c or °c using supplemented ham's f medium mixed : with nih t fibroblast-conditioned dmem containing % calf serum. after incubation, the culture cups were placed in custom designed ussing chambers for measurement of isc responses to a cocktail of µm forskolin and µm ibmx (camp isc) at °c. contribution of calcium-activated clchannels to the camp isc was eliminated by addition of the distilbene dids ( µm) to the luminal bath. results: the mean ∆camp isc responses (in µa/cm ) after two days incubation were as follows: wt at °c = . ± . (n = ); df at °c = . ± . (n = ); df at °c = . ± . (n = ); and cftr ko at °c = . ± . (n = ). in a pairwise comparison, the wt ( °c) was significantly greater than the df ( °c) and cftr ko ( °c) but not significantly different from df ( °c). conclusions: incubation of ∆f cftr mutant murine gallbladder mucosa at °c for two days in organ culture restored approximately % of the cftr-dependent isc responses to camp stimulation. the temperature-dependent effect is consistent with correction of murine ∆f cftr processing, indicating a physiological screening assay that is useful for preclinical testing of candidate drugs targeted at correcting the protein processing defect of ∆f cftr. supported by cfft and nih. the lack of a large animal model that replicates the lung disease found in humans with cystic fibrosis is a major impediment to understanding disease pathogenesis and the development of effective therapies. although cystic fibrosis is a multi-system disease, airway infection and inflammation currently cause most of the morbidity and mortality in patients. several homozygote null and missense cftr mutations have been developed in mice, but they do not exhibit the lung phenotype observed in humans. in contrast to mice, the structure and physiology of porcine lung and airways closely resemble those of humans. therefore, the goal of this project is to develop a swine model for cystic fibrosis by combining gene targeting and nuclear transfer to produce a pig with a cftr null genotype. we employed homologous recombination to disrupt exon of pig cftr with a neomycin resistance cassette. we generated primary male fetal fibroblasts that were positive for cftr-targeting at one allele as detected by multiple pcr screens. genomic southern blot analysis confirmed proper gene targeting and revealed most clones were free of random integration events. these cells were used for somatic cell nuclear transfer that resulted in the birth of ten cftr null heterozygote piglets. subsequent breeding of these male pigs to wild-type females has yielded at least female cftr null heterozygotes. breeding male and female heterozygotes will provide cftrnull homozygotes. our recent published and preliminary studies demonstrate that mouse slc a , a newly discovered member of slc family of anion exchangers, is expressed on the apical membrane of tracheal epithelial cells and gastric surface epithelial cells and in the tubulovesicle membranes of gastric parietal cells. the functional properties of mouse slc a were examined in xenopus oocytes and in stably transfected cultured cells. in xenopus oocytes injected with the slc a crna and studied with the voltage and/or ph sensitive microelectrodes, the rank order for anion selectivity at + mv was cl->i-> no ->gluconate > sulfate, and that of the selectivity (px/pcl) was i-> cl-> no -= gluconate > sulfate. inhibitor profile studies demonstrated that nppb at µm inhibited the current at min by % (p< . compared to control period, n= ). the effect of nppb was partly reversible. dids at µm had no significant effect on the slc a -mediated current (p> . compared to control period, n= ). niflumic acid at µm inhibited the current at min by ~ % (p< . compared to control period, n= ) which was partly reversible. cftr inh at µm inhibited the current at min by ~ % (p< . compared to control period, n= ) which was reversible. clamping currents in a mm k-gluconate medium and using mm k-cl or k-bicarbonate revealed very low bicarbonate conductance for slc a compared to that of cl-. no significant phi changes were observed in oocytes when favoring cl-exit by substituting perfusate cl-with gluconate in the presence of co /hco -. conversely we observed significant alkalinization in ae -expressing oocytes in the same experimental solutions. interestingly, in hek cells stably transfected with the mouse slc a , a significant intracellular alkalinization was observed upon switching to a chloride-free perfusate. the alkalization in slc a -expressing cells was inhibited by ~ % in the presence of µm dids. baseline intracellular ph was significantly increased in slc a expressing hek cells vs. non transfected cells. however, baseline phi was comparable in control and slc a -expressing cells in the presence of µm eipa, indicating the activation of endogenous nhe by slc a . in conclusion, slc a displays characteristics of an anionic channel in xenopus oocytes, with low bicarbonate permeability. in mammalian expression systems, slc a can also function in cl-/hco -exchange mode with moderate sensitivity to inhibition by dids. we propose that slc a plays an important role in anion (chloride) secretion and/or ph regulation in tracheal and gastric epithelial cells. no cell lines exist at present that robustly express endogenous wt and ∆f cftr on the same genetic background. zinc finger nucleases (zfns) allow the introduction of specific genetic changes at loci within the human genome (nature : [ ] [ ] [ ] [ ] [ ] [ ] ) . we have been using this technology in an attempt to generate human cells carrying the major cf-causing genotype -∆f -at the endogenous cftr locus. such lines could be expected to provide better understanding of the ∆f maturational processing defect, as well as new model systems for cell based assays of ∆f repair. we performed a bioinformatic analysis of cftr exon , and designed multiple distinct zfns against the ∆f dna sequence. all of these nucleases were assembled, cloned into mammalian expression vectors, and assayed for dna binding affinity and specificity. the zfns were then assayed for endogenous cftr locus editing in calu- and ht- cells using a novel, massively parallel methodology applied for the detection of genome editing events. the effort demonstrated that specific zfn pairs successfully bind to, and cleave, within exon of the endogenous cftr locus in both cell types. we will describe experiments that yielded optimized zfns for the cftr locus, and a robust viral platform to transiently deliver genome editing reagents to the human pulmonary cell line, calu- . supported by cff. the localization of slc anion transporters, as well as their function vary among members. some (slc a , slc a , slc a , slc a ) are widespread, implying specific physiological function in diverse tissues. others are expressed only in a single epithelia, e.g., slc a (prestin) in the inner ear. we have previously found that slc a is a highly electrogenic cl --hco exchanger with a large anion conductance. to determine the possible physiologic roles of slc a , we developed a chicken antibody (designed against sulfate transporter and anti-sigma domain, stas, of slc a ) to localize the protein. to test the antibody specificity for functional protein, we inject xenopus oocytes with crna for slc a , slc a or slc a and documented appropriate ph and membrane potential changes. only oocytes functionally expressing slc a were stained using our primary slc a antibody. further, specific staining was eliminated when either pre-immune serum or blocking peptide was used. slc a was only found in epithelia. in the respiratory system, slc a was in the apical membrane of nasal epithelial cells and tracheal submucosal glands. membranes of calu- cells, derived from human tracheal submucosal glands, stained indicating that our antibody also recognizes the human slc a protein. slc a is found in the proximal parts of the gastrointestinal tract (esophagus, stomach), with lesser amounts in the small intestine and colon. however, slc a is not present in liver. slc a is present in ducts of secretory epithelia such as salivary glands and pancreas. in the urinary tract, slc a is localized to the renal proximal tubule apical membrane but not in urinary bladder. in the reproductive system, we found slc a in epithelia of the uterus and seminal vesicles but not the vas deference or the prostate. finally, slc a is found on the eye surface, i.e., corneal epithelial cells. together, these results and the varied slc a physiology suggest that several epithelial function models will need to be re-evaluated to incorporate an electrogenic cl --hco exchanger and/or another apical anion channel. (support: romero- g , sindic- f ). a tgfβ- polymorphism modifies severity of cystic fibrosis lung disease in the setting of ∆f cftr homozygosity (drumm et al., nejm, ) . the mechanism(s) underlying this observation, and whether elevated or repressed tgfβ are responsible, are not known. in addition, the pathogenic role of tgfβ in other cf organs and in murine models of the disease has not been well studied. because the tgfβ codon cc genotype is associated with worsened cf pulmonary prognosis, and since high levels of tgfβ are also known to suppress cftr mrna expression and apical cftr protein expression in colonic epithelia (howe et al., exp cell res, ) , we developed a mouse model to test tgfβ/cftr interactions that may directly influence cf phenotype. we bred ∆f heterozygous mice (cftr tm kth , ∆f cftr) against the tgfβ receptor-deficient murine strain (mtrii ). mtrii mice express a dominant-negative mutant form of the tgfβ type ii receptor which lacks the intracellular (kinase) receptor domain. the receptor binds tgfβ, but fails to transmit a tgfβ dependent signal. the tgfβ receptor transgene was inserted downstream of a metallothionein-derived promoter and expressed in numerous murine tissues following induction with mm znso in drinking water. an antibody raised against active tgfβ (lc- - - , gift of dr. kathy flanders, national cancer institute) was used to establish tgfβ in murine tissues including lung and intestinal tract of the mtrii strain. rt-pcr confirmed high level transgene expression similar to gapdh internal control. nasal potential difference measurements in cftr +/+ mice expressing the tgfβ transgene indicated intact camp dependent cltransport following induction of the dominant-negative receptor. homozygous cf animals deficient for tgfβ signaling have recently been obtained and exhibit the expected litter sizes and distribution of the ∆f allele. studies of cf homozygous mice following induction with znso are in progress, and will be used to determine whether a failure to signal through tgfβ in vivo ) influences the severity of cf intestinal disease, ) alters airway or intestinal bioelectric findings characteristic of cf, or ) changes survival, histopathology or other features of the disease. these experiments are intended to provide an in vivo means of investigating tgfβ signaling and resultant cf modifier effects in vivo. supported by nih and cff. objective: to determine if the abpa phenotype observed in cftr mice was preventable by corrective gene replacement, we delivered aav cftr to the lung. in addition to further assess whether cftr-/-lymphocytes alone where responsible for the phenotype, we performed adoptive transfers of cftr-/-lymphocytes into cftr+/+ mice and proceeded to expose the mice to the abpa model. design/methods: cftr-/-mice underwent it administration of raav cftr or raav gfp. all mice were then sensitized with ug of crude af antigen via intraperitoneal route. non-injected mice were sensitized without prior virus exposure. challenges were performed on all mice, using an af solution delivered by aerosol inhalation in a closed chamber on consecutive days. serum ige levels were then obtained, as well as bronchoalveolar lavage fluid for differential cell counts, histologic and cytokine analysis. adoptive transfer experiments were done using spleens cells isolated from af sensitized delaf -/-and f +/+ mice and transfer into c /bl _rag-/-mice. after weeks post transfer c /bl _rag-/-mice with f -/-and f +/+ lymphocytes were then challenged with af. results: following challenge with af, raav -cftr mice had significantly lower total serum ige levels ( , ) as compared to raav -gfp controls ( , ) (p= . ). analysis of af-specific ige also revealed a two-fold reduction in the mice receiving the corrective gene therapy. curiously, gene transfer to the lung lowered the secretion of inflammatory cytokines from activated splenocytes of raav -cftr when compared to raav -gfp controls. these results suggest that correction of immune cells in the lung that traffic to the spleen may be partially responsible for the attenuation of the phenotype. in addition the transfer experiments showed that the hyper-ige syndrome can be partially transferred into b rag-/-by f -/-splenocytes. conclusions: correction of cftr deficiency by aav lung transfer attenuates the inflammatory phenotype associated with abpa. epithelial cells lining the vas deferens express cyclooxygenase (cox) when exposed to testosterone in vivo, although no functional link to ion transport has been established. we hypothesized that testosterone modulates cftr-mediated anion secretion across vas deferens epithelia that is stimulated via cox-dependent pathways. vas deferens epithelial cells were isolated from human and porcine tissues and cultured as monolayers on permeable supports until assayed in modified ussing chambers. rna was isolated concurrently for semi-quantitative gene expression analysis. bradykinin (bk) caused an increase in anion secretion (measured as short circuit current) and this effect was inhibited by indomethacin, which indicates cox dependency. testosterone treated monolayers exhibited a bkinduced response that is % greater than paired vehicle treated monolayers. qrt-pcr revealed that cox- mrna is times more abundant than cox- mrna in cultured vas deferens epithelial cells. the effects of bk on ion transport are inhibited by the b receptor antagonist hoe while the b receptor agonist des-arg -bk produced no effect. prostaglandin e and selective agonists of the ep ( -oh-pge ) and ep (butaprost) receptors produced concentration dependent increases in short circuit current whereas sulprostone, an agonist of ep and ep receptors, had no effect. taken together, these results suggest that testosterone plays a key permissive role for bk-stimulated anion secretion by inducing cox- expression. bradykinin acts at the b receptor to increase cox- activity and the synthesis of prostaglandins that ultimately bind to ep receptors to promote cftr-dependent hco -and cl-secretion that would be expected to modify the environment for sperm. the results further suggest that developmentally associated increases in testosterone levels during gestation and puberty will produce physiological changes in vas deferens luminal environment that are likely compromised in cystic fibrosis patients. supported by cystic fibrosis foundation schult po and ksu cobre nih rr- . background: recent studies in cystic fibrosis (cf) suggest a prominent t h profile; a facet that may interfere with normal bacterial clearance. this phenotype appears to increase secondary to infection with aspergillus fumigatus or pseudomonas aeruginosa and also, may interfere with macrophage activation. these same characteristics are present in cftr-deficient mice (cftr s x -/-fabp-hcftr (+/+), hereafter designated cf mice). as a study of pathogenesis of cf related diabetes (cfrd), we previously observed increased il- and splenocyte proliferation in cf mice subject to streptozotocin-induced hyperglycemia. herein, we hypothesized that differences in glucose concentration would increase t h profiles of splenocytes in vitro. methods: cf mice and c bl/ j mice (b mice; n= /group) were sacrificed at wk of age. splenocytes were collected and cultured at the following glucose concentrations: , , and mm. each mouse and glucose concentration was stimulated with cona, lps and cd /cd . supernatants were collected at hr and measured by luminex analysis. statistical analysis was performed by a one-way anova. results: stimulation by cona produced increased levels of il- , il- , tnf-a, gm-csf and il- in splenocytes of cf mice (p< . for all). stimulation by lps produced decreased concentrations of il- in cf mice(p< . ). stimulation of cf mice by cd /cd produced decreased concentrations of il- (p< . ). interestingly, no difference was observed in cytokine elaboration as a function of glucose concentration. data are presented in table as fold increase of cytokine concentration for cf mice compared to b mice. discussion: profound differences in cytokine production were measured in stimulated splenocyte responses of cf in comparison to b mice. the profiles did support the notion for a prominent t h response, with the potential to interference of normal macrophage activation. in addition, differences in cytokine production profiles appear related to the nature of adaptive and innate stimuli provided (cona vs. lps). the absence of influence by glucose concentration in vitro suggests that local/immediate concentration of glucose is not relevant to prolonged expression of cytokine responses to stimuli. further studies on the impact of chronic hyperglycemia and its relation to immune response are necessary. an improved understanding of these mechanisms holds hope for a better overall outcome in patients with cf and cfrd. studies from different laboratories to characterize ∆f cftr clchannel activity have yielded disparate results, likely due to variations in technique, cell type, or method of analysis. in the present study, we compared ∆f cftr activation in two well characterized polarizing cell models. fisher rat thyroid (frt) cells have been used in high-throughput drug screening to identify ∆f cftr active agents, whereas cfbe ocells have been transduced to provide robust ∆f cftr expression in an airway epithelial background. ∆f cftr biogenesis following low temperature correction was compared, indicating qualitatively similar levels of cftr transcripts and band b and c cftr. in paired studies in ussing chambers, distinct differences in isc were seen following exposure to cftr activating stimuli. in frt cell monolayers grown at °c, forskolin ( µm) or genistein ( µm) were strong stimuli of isc, producing mean isc of . +/- . (forskolin) and . +/- . (genistein) µa/cm . regardless of exposure sequence, both agents induced equivalent changes in maximal current ( % of . µa/cm by forskolin when administered prior to genistein; % of . µa/cm by forskolin in converse order). growth at °c for hours increased currents approximately . fold, but relative contributions of the two stimuli were similar to cells grown at °c ( % of total current stimulated by forskolin). in cfbe o -∆f cells grown at °c, forskolin-dependant currents were minimal and genistein-stimulated currents small ( . +/- . µa/cm , compared to zero current in parental, nontransduced cfbe ocells, p< . ). in temperature-corrected ∆f cfbe ocells, forskolin was a poor stimulus of cl-transport relative to genistein, contributing % to the total current produced by both agents (p< . ). genistein-stimulated currents did not require pre-treatment with forskolin to produce maximal effects ( . µa/cm with genistein alone vs. . µa/cm after forskolin and genistein), and forskolin did not cause further isc when added after genistein ( µa/cm ). levels of camp did not account for these differences, since forskolin markedly increased camp in both models. to determine whether excessive phosphodiesterase (pde) or phosphatase (pp) activity limited ∆f cftr activation by forskolin in the cfbe o -∆f cells, monolayers were pretreated with the nonspecific pde inhibitor papaverine ( µm) or the pp inhibitor endothall ( µm), each sufficient to activate isc in cfbe oexpressing wtcftr (papverine: . vs. . µa/cm with vehicle, p< . ; endothal: . vs. . ua/cm , p= . ). neither agent restored isc in ∆f cfbe oby forskolin ( . mv difference, p=ns). these results indicate that activity of ∆f cftr in polarizing monolayers is exquisitely dependant on the model chosen for study. substantial differences in ∆f cftr behavior in two commonly used cell models, and suggest that the failure of camp to activate ∆f cftr in cfbe ocells is due to cell-specific factors that may ) influence ∆f cftr folding and tertiary structure, or ) favor interactions of ∆f cftr with inhibitory binding partners that limit activation by camp. adverse immune responses to viral vectors or vector-encoded proteins may hinder the practical application of gene therapy. to date, little effort has been devoted to determining the immunological consequences of topically delivered lentiviral vectors to respiratory epithelia. previously, we demonstrated that an integrating feline immunodeficiency virus-based lentiviral vector pseudotyped with a baculoviral envelope glycoprotein (gp -fiv) readily transduced differentiated airway epithelia in vitro when applied to the apical surface. furthermore, using a luciferase reporter gene and bioluminescence imaging, we observed in vivo gene transfer to murine nasal epithelia following a single application of gp -fiv. longitudinal bioluminescence analysis documented persistent expression in nasal epithelia for greater than months without significant decline. by histological analysis, surface epithelial cells were transduced following a single nasal application of gp -fiv expressing beta-galactosidase. importantly, we observed that consecutive doses of gp -fiv delivered over consecutive days greatly increased the number of beta-galactosidase positive epithelial cells. using quantitative bioluminescent imaging, we observed that repeated doses given over consecutive days resulted in a linear additive increase in gene expression. in addition, we performed studies investigating gene transfer efficacy and immune responses following lentiviral vector readministration with greater intervening time periods. in control mice, we observed that giving nasally instilled doses ( weeks apart) of an e /e deleted ad vector resulted in significantly attenuated expression after the second dose. the attenuated expression correlated to the production of neutralizing antibodies. in contrast, using the same delivery protocol, doses of gp -fiv resulted in gene transfer without loss of expression of the forth dose. no significant production of neutralizing antibodies was observed following the lentiviral delivery protocol. we further report the additive increase in reporter gene expression following doses of lentiviral vector delivered over consecutive weeks ( dose/week), as well as the lack of production of systemic and local neutralizing antibodies. gp -fiv efficiently transduces and persistently expresses a transgene in respiratory epithelia and has the potential for repeat administration without eliciting adverse immune responses. these data have important implications for the application of lentiviral gene therapy to pulmonary diseases. uniform delivery of gene therapy vectors to all lung lobes is important for cf gene therapy. however, this important objective has not been achieved in large animals. to address this obstacle, we have developed an approach to specifically deliver vector into all lung lobes. in this strategy, an intracorporeal nebulizing catheter (aero-probe) is inserted into a bronchoscope to permit visual targeted aerosolization of vector specifically into each lung lobe. using this approach, ml of . % lpc (to transiently open tight junctions) containing x vp of hdad-k lacz was sequentially aerosolized into each of the six major lung lobes of a baboon. aerosolization was triggered by the ventilator upon inspiration at . ml/pulse at a rate of to pulses/min. halfway through the procedure a very slight, transient and fluctuating decrease in oxygen saturation of no more than % was noted that did not warrant supplemental oxygen. the entire procedure was otherwise uneventful and well tolerated with no clinical manifestations of toxicity. no changes in chest x-rays taken at h, h and at necropsy h post-vector were observed compared to baseline. x-gal staining of the lungs at h revealed extensive transduction of the epithelium in the large and small airways in all six major targeted lung lobes. x-gal histochemistry revealed substantial transduction that was exclusively restricted to the airway epithelial cells and submucosal glands, the target cells for cf gene therapy. assessment of the proximal major bronchi from each lung lobe revealed that~ % of the airway epithelial cells were transduced in the left upper lobe (lul), ≥ % were transduced in the lml, lll, rul, rml and rll, and~ % were transduced in the accessory lobe. approximately % of the airway epithelial cells in the tracheal sections examined were transduced. we also investigated the duration of pulmonary transgene expression. in these studies, we aerosolized a hdad expressing the baboon α-fetoprotein from the k promoter into the lungs of baboons. by measuring serum bafp levels, we found that pulmonary transgene expression from transduced airway epithelial cells can be detected for at least days post-vector. these results demonstrate for the first time that exceedingly high levels of transduction of the airway epithelial cells and submucosal glands throughout all lung lobes in a large animal can be achieved with negligible toxicity resulting in long term trangene expression and should pave the way towards successful clinical cf gene therapy. inefficient gene transfer after repeat administration of most viral vectors has limited their suitability for cf gene therapy. however, lentiviral vectors are reported to be less immunogenic. this property combined with their capacity to integrate into the genome of transduced cells and, therefore, allowing gene expression for the life-time of the cell (approximately months for airway epithelial cells and theoretically unlimited for stem cells) justify further investigation of these vectors for cf gene therapy. to ensure high transduction efficiency in the lung without the need for any pre-conditioning strategies we pseudotyped a simian immunodeficiency virus (sivagm)-based vector with the sendai virus f and hn proteins (f/hn-siv-gfp) and transduced mouse nasal epithelium ( x e tu/mouse). at this titre transduction efficiency along the nasal septum was up to % when measured and days after transduction (n= /time-point). the vast majority (approx. %) of gfp positive cells across all time-points were respiratory epithelial cells followed by neuronal and sustenticular cells (approx. %) in the olfactory epithelium. interestingly, we observed two types of duration patterns of gfp expression in mouse nose. in some mice the number of gfp positive cells decreased over time (day : ± cells/section, n= , day : ± cells/section, n= ). however, in other mice strong gfp expression persisted for up to days (day : ± cells/section, n= ), significantly longer than the proposed life-span of airway epithelial cells. in order to support the idea of f/hn-siv integration into nasal respiratory stem/progenitor cells we artificially induced cell division after siv vector transduction by damaging the nasal tissue with the detergent (polidocanol), which strips of the epithelium within a few hours, while retaining the basal cell layer. the epithelium completely regenerates over the course of a few days. seven and days after the vector transduction ( x e tu/mouse, n= ) the nasal tissue was perfused with % polidocanol ( µl/mouse) and gene expression was analyzed weeks after the last detergent treatment. gfp expression was detectable in various cell types of the regenerating epithelium including ciliated and basal cells. importantly, gfp-expressing cells were more clustered after polidocanol treatment, possibly indicating origination from a common progenitor. these data suggest that, f/hn-siv integration may have occurred into differentiated airway epithelial cells as well as into stem/progenitor cells involved in the regeneration of the airway epithelium. introduction: hypersecretion of mucus is a major cause of airway obstruction in cystic fibrosis, asthma and chronic bronchitis. though mucus is a complex, non-homogeneous mixture of secretions, one group of its constituents, the mucous glycoproteins or mucins, contributes greatly to the obstruction associated with airway diseases. currently, there are a limited number of therapeutic agents available for controlling mucin overproduction and hypersecretion. the objective of this study is to develop a sensitive, cost-effective technique for reliably quantifying total mucin concentration in a robust mucin-secreting cell line for use in high throughput screening of small molecule compounds with potential to inhibit mucin secretion. methods: an enzyme-linked lectin assay (ella) was adapted using a lectin from helix pomatia (hpa), which binds specifically to n-acetylgalactosamine residues. bovine sub-maxillary mucin was used for test development and later, as a mucin standard. the test sample was "sandwiched" between hpa pre-coated onto -well microtiter plates and biotinylated hpa lectin. the plate was developed with a fluorescent hrp substrate, and measured at nm using a standard fluorescence plate reader. using the ella, a number of carcinoma (hm , a , ls , nci-h ) and immortalized lung epithelial ( hbe, cfsme) cell lines were tested to determine their responsiveness to various secretagogues. preliminary experiments indicated that the nci-h cells, derived from a human mucoepidermoid lung carcinoma, were most suitable for adaptation to high throughput studies. nci-h cells were seeded into -plates at a density sufficient to produce confluence in approximately hours. then, individual wells were incubated for hour in either control or secretagogue-containing media. supernatants were collected and mucin concentration of individual wells was determined using the ella. results: optimization of the ella provided a sensitive calibration curve from ng/ml to . mg/ml. mucin secretory ratios (sr) were calculated by comparing mucin release from test and control wells. significant (p ≤ . ) secretory ratios were obtained following incubation with utp at µm ( . ± . ), vip at nm ( . ± . ), carbachol at . mm ( . ± . ), interleukin- β at ng/ml ( . ± . ) and heparin-binding egf-like growth factor at ng/ml ( . ± . ); all data expressed as sr ± sem, n = . histamine at µm did not result in significant mucin secretion. conclusion: a relatively simple and economical assay has been developed for high throughput screening of small molecules for detecting their effects on mucin secretion. cystic fibrosis (cf) is the most common lethal genetic disease in north america, where it occurs with a frequency of one in ~ live births. it is caused by mutations in the cystic fibrosis transmembrane conductance regulator (cftr) gene, the most common being the deletion of phenylalanine (delf ). the delf mutation causes a folding defect that inhibits maturation and trafficking to the plasma membrane, however the mutant still functions as a chloride ion channel when delivered to the plasma membrane, and partial rescue ( - %) may be sufficient to alleviate disease symptoms. thus cftr is an attractive target for the development of pharmaceutical therapies. we recently described a novel, cell-based assay for protein trafficking which directly monitors expression of delf cftr on the surface of baby hamster kidney (bhk) cells and is suitable for high throughput screening of small molecule libraries. in this study we have extended the approach to natural products by studying extracts derived from marine sponges that were collected from the coasts of indonesia and papua new guinea. sponges are a particularly rich source of novel bioactives and secondary metabolites. iterative use of our high throughput assay and chemical deconvolution of active fractions yielded four alkaloids jmcg , jmcg , jmcg and jmcg . these compounds caused a detectable increase in delf cftr trafficking to the plasma membrane within h, and after exposure to pm for h all four increased surface delf cftr expression - % and enhanced camp-stimulated halide conductance. we tested a series of analogues of the alkaloids and found three others jmcg , jmcg and jmcg that also caused trafficking correction and functional ion channels in the plasma membrane. these results were confirmed in functional studies of the human bronchial epithelial cell-line cfbe stably overexpressing delf cftr. these compounds increased surface delf cftr by - % within h when treated with nm. these results identify a promising group of delf cftr trafficking correctors that are now being actively pursued as potential pharmacotherapeutics for cystic fibrosis along with other hits from compound library screening campaigns. work supported as part of the breathe program jointly funded by the ccff and cihr, and grants from cystic fibrosis foundation therapeutics, (cfft). kim chiaw, p. , ; bear, c.e. background/rationale: biosynthetic misprocessing of the major cf mutant ∆f -cftr has been proposed to result from the disruption in domain-domain interactions (du et al. nature struct and mol biol, ) that may lead to the exposure of di-arginine (rxr) retention/retrieval motifs. simultaneous site directed mutagenesis of all four rxr motifs to rxk or kxr motifs overcame retention and promoted cell surface expression of ∆f -cftr (chang et al. mol cell, ) . we hypothesize that it is possible to compete inhibitory interactions with rxr motifs exposed in ∆f -cftr by delivering synthetically derived cftr peptides bearing one or a combination of rxr motifs (cf-rxr), each conjugated to cell penetrating peptides (cpp) to permit intracellular delivery. results: confocal immunofluorescence microscopy confirms that alexa- -tagged cpp-cf-rxr peptides can be taken up rapidly (within min) by bhk cells stably expressing recombinant human ∆f -cftr. fluorophore-conjugated peptides appear to be concentrated in vesicular compartments, consistent with the current hypothesis that cpp-peptides enter cells into endosomal vesicles via macropinocytosis from which efflux into the cytosol occurs (wadia et al. nature med, ) . we determined that treatment of cell cultures with cpp-cf-rxr peptide at a final concentration [ µm] for minutes is effective in inducing cell surface functional expression of human ∆f -cftr in bhk cells as observed by a . -fold increase in cyclic amp-mediated iodide efflux over control ∆f -cftr (n= ; p < . ). this rescue is specific to the cf-rxr peptide as controls (cpp alone or peptides bearing a kxk motif in place of rxr) show no functional rescue of ∆f -cftr (n= ; p > . ). preliminary cyclic ampmediated iodide efflux results also indicate that ∆f -cftr functional rescue by cpp-cf-rxr peptide is as effective as temperature ( o c) rescue. although we hypothesize that the peptide acts to facilitate trafficking of the major mutant to the cell surface, immunoblotting studies fail to show a clear conversion of the er-form (band b) to the fully processed, band c form of the protein. currently, we are developing more sensitive assays of cell surface expression as well as evaluating the possible efficacy of this peptide in potentiating function of ∆f -cftr. discussion: future efforts include investigating the relative efficacy of peptides corresponding to the four different rxr motifs in rescuing ∆f -cftr trafficking. the promising candidate described above is currently being evaluated with respect to functional rescue of the major mutant in primary human bronchial epithelial cells obtained from patients homozygous for ∆f -cftr (joseph zabner and phil karp, university of iowa) and in cftr-∆f mice. these studies may provide the basis for future pre-clinical studies of peptide based therapies of cystic fibrosis (supported by ccff (breathe program grant) and cihr -(strategic training program in the structural biology of membrane proteins linked to disease)). several promising methods of promoting ∆f cftr escape from er degradation increase cftr chloride channel activity at the cell surface. however, increasing data from a number of laboratories indicate that in both epithelial and non-epithelial cell lines expressing ∆f cftr, the rescued chloride channel is unstable at the cell surface compared to wild-type cftr. small molecule correctors have therefore become a widely used approach to enhance ∆f cftr function at the cell surface. in most cases, the exact mechanism of these promising compounds has not been established. in the present study, we use stably transfected, polarized cfbe o-cells to elucidate the effect of two of these compounds, cfcor- and corr- a, on the surface stability of wild type and low temperature rescued ∆f cftr. specifically, we cultured cells expressing ∆f cftr at c for hours to establish a large pool of surface-expressed ∆f cftr; we then monitored cftr internalization rates and half-life with and without small molecule treatment using surface biotinylation-based assays, performed as described previously (jurkuvenaite et al. j. biol. chem. : , ) . as a control, internalization and half-life of transferrin receptor was also followed. our results indicate that both cfcor- and corr- a slow ∆f cftr endocytosis from ~ %/ . min to ~ %/ . min, and this is similar to wild type cftr internalization rates. neither wild type cftr nor transferrin receptor internalization rates were affected by corrector treatment, suggesting that the effects of the correctors were specific for ∆f cftr. in the surface half-life studies, both correctors doubled ∆f cftr halflife from to hours, but they did not correct the half-life to wild type levels (~ h). again, the correctors had no effect on either wild type cftr or transferrin receptor surface half-lives. our results suggest that small molecule correctors may not only rescue cftr from erad during biogenesis, but can contribute to the stabilization of ∆f cftr at the cell surface, and this effect seems to be ∆f cftr-specific. the ∆f mutation in cftr causes defects in chloride channel gating and cellular processing. cell cultures models suggest the need for both a potentiator and corrector to restore ∆f -cftr channel function and processing, respectively. previously, we identified several classes of potentiators, including 'phenylglycines', which probably bind to cftr at the cell surface and restore normal channel gating (pedemonte et al., mol. pharm. ; : - . we also identified several classes of correctors, including 'bisaminomethylbithiazoles', which may bind to ∆f -cftr in the er and partially restore its folding and plasma membrane targeting (pedemonte, et al., j. clin. invest. ; : - . these compounds were identified from screening a diverse collection of , small molecules; the most active potentiator was phenylglycine pg { -[( - h-indol- -ylacetyl)-methyl-amino]-n-( -isopropylphenyl)- -phenyl-acetamide} and the most potent corrector was the bisaminomethylbithiazole corr- a {n- ( -[ -chloro- -methoxyphenylamino]- '-methyl- , '-bithiazol- 'yl) benzamide}. here we synthesized and characterized a hybrid molecule (corr- a-linker-pg ) containing both corrector and potentiator fragments. based on sar data of potentiators and correctors, a hybrid molecule was designed that incorporated an enzymatically hydrolysable linker to generate and deliver the potentiator and corrector-linker fragments. using synthetic organic chemistry we synthesized a hybrid molecule containing a pg -oh moiety and a corr- a-linker-cooh moiety, linked with an ethylene glycol spacer through an ester bond. the potentiator and corrector fragments (after cleavage) had low micromolar potency or better. however, the intact hybrid molecule was inactive, likely because of its large size ( daltons) and hence poor cell penetration. as proof-of-principle evidence that the hybrid molecule can be hydrolyzed to the active fragments, treatment with carbonic anhydrase or rodent stool specimens gave the appropriate potentiator and corrector-linker fragments identified by lcms. smallmolecule corrector-potentiator hybrids have potential utility as single drug therapy for cf caused by the ∆f mutation. supported by cff and nih. cftr is a chloride channel gated by atp binding and hydrolysis at its two nucleotide binding domains (nbd and nbd ). the two nbds may dimerize in a head-to-tail configuration, as observed in other abc transporters, forming two atp binding pockets (abp and abp ), with the atp molecules buried at the dimer interface. abp , formed by the walker a and b motifs of nbd and the signature sequence of nbd , was proposed as the site critical for the atp-dependent opening of the cftr channel, while abp (consisting of the walker a and b motifs of nbd and the signature sequence of nbd ) may contribute to the stabilization of the open channel conformation. g d, the third most common cf-associated mutation, is characterized by a very low open probability, and patients carrying this mutation present a severe phenotype. this mutation is located in the signature sequence of nbd (and thus in abp ). we have recently characterized this mutant channel and found that its low activity is atp-independent. this behavior corroborates with the idea that the occupancy of abp by atp is crucial for catalyzing the opening of the channel. interestingly, we now found that a high affinity atp analog, n -( -phenylethyl)-atp (p-atp), increases g d currents primarily by increasing the open time of the channel. this effect of p-atp is reduced when p-atp was applied together with atp, suggesting a competition between atp and p-atp for a common binding site. introducing the mutation y g (located at abp ), which reduces by more than -fold the atp-binding affinity in wild-type channels, does not alter the effect of p-atp in the g d/y g mutant. in contrast, when we introduce the mutation w g (located at abp ) in the g d background, the effect of p-atp is reduced remarkably, suggesting that abp is the binding site where p-atp binds to increase the activity of g d channels. these new results further confirm the idea that nucleotide binding at abp could stabilize the open channel conformation. since the competition experiments show that atp and p-atp are readily exchanged in the binding site, we conclude that the atp molecule is not occluded in abp , at least under the g d background. our observation that p-atp enhances the g d activity by binding at abp implicates that abp can potentially be a target for drugs to bind and increase the channel activity. supported by cff grant to s. bompadre and nih grants to t.-c. hwang (hl r , dk r ) and s. bompadre (dk k ). van goor, f.; hadida, s.; grootenhuis, p. vertex pharmaceuticals, san diego, ca, usa several cf-causing mutations, including ∆f , g d, and r h, cause gating defects in cftr that decrease the open probability (po) of the channel, resulting in decreased cl-secretion across epithelia of multiple organs, including the lung. a therapeutic strategy for the treatment of cf is to develop pharmacological potentiators that increase po leading to increased fluid transport in affected tissues. vx- was identified through cell-based screening and chemistry optimization as a potent potentiator of cftr, including ∆f -, g d-, and r h-cftr. in single-channel recordings of cftr expressed in fisher rat thyroid cells, vx- increased the po of ∆f -cftr from . ± . to . ± . . the po of g d increased from . ± . - . ± . . these levels are similar to the po of wild-type cftr, indicating that vx- restores normal gating at the single channel level. to assess the potency and efficacy of vx- in native airway cells, cl-secretion was monitored in ussing chamber studies using human bronchial epithelia (hbe) cultured from the bronchi of cf and non-cf donors. in the absence of the potentiator vx- , cftr-mediated clsecretion in ∆f /∆f or g d/∆f -hbe reached a maximum amplitude of . ± . and . ± . ua/cm , respectively. this is ~ % of that observed in wild-type-hbe ( ± ua/cm ) and consistent with the low level of residual cftr activity reported in some individuals with severe cf disease. in ∆f /∆f -hbe, vx- caused a -fold increase (peak response; . ± . ua/cm ) in cftr mediated cl-secretion with an ec of ± nm, whereas in g d/∆f -hbe, cl-secretion increased fold (peak response; ± ua/cm ) with an ec of ± nm. the improved efficacy observed in g d/∆f -hbe is consistent with a higher cell surface density of g d-cftr compared to ∆f -cftr, which has defective trafficking to the cell membrane. vx- was found to be highly selective for cftr and to have good oral bioavailability in multiple species with a half-life of - hours. the biological and pharmacokinetic data support the clinical development of vx- for the treatment of cystic fibrosis. we would like to acknowledge the cfft for their support and dr. joseph pilewski for providing cf hbe. the pari eflow ® rapid nebulizer is a member of the "soft mist" inhaler class for the administration of aerosolized antibiotics that eliminates the need for a compressor. objectives: to compare the pharmacokinetics and safety of tobramycin inhalation solution (tobi ® ) delivered via the pari eflow ® rapid electronic nebulizer vs the pari lc ® plus jet nebulizer with compressor. methods: we compared tobramycin levels in sputum and serum, incidence of bronchospasm and frequency of adverse events (ae) of mg tobi, administered twice-daily for weeks from these two delivery systems in a randomized, crossover study in cystic fibrosis patients. blood and sputum samples were collected over a dosing interval after the first and last dose on each device (days and ). tobramycin serum and sputum area under the concentration-time curves (auc) were derived and the therapeutic ratio (mean sputum auc / mean serum auc) calculated. pulmonary function tests were performed before and min after nebulization on days and , and ae were recorded. results: the total nebulization time was reduced by half for the pari eflow rapid vs pari lc plus ( . ± . min vs . ± . min [p< . ] on day , and . ± . vs . ± . [p< . ] on day , respectively). tobramycin predose serum levels > µg/ml or cmax > µg/ml, predefined as a potential for increased risk of systemic toxicity, were not exceeded in any patient during use of either nebulizer. as tabulated below, mean systemic exposure to tobramycin from the pari eflow rapid on day was slightly lower, by %, whereas mean sputum exposure was higher, by %, compared with the pari lc plus. consequently, the therapeutic ratio was nearly -fold higher for the pari eflow rapid due mainly to higher sputum concentrations. adverse events occurred in patients using the pari lc plus nebulizer (primarily headache and abdominal pain) and in patients using the pari eflow rapid nebulizer (primarily headache, cough, dyspnea). on day , no clinically relevant bronchospasm (defined as ≥ % decrease in fev ) was observed for either treatment. the mean percentage change in fev at mins from predose on day was - . % for the lc plus and - . % for the eflow rapid. conclusions: tobi delivered via the pari eflow rapid electronic nebulizer required shorter delivery times with similar safety and a higher therapeutic ratio (sputum/serum exposure) compared with the pari lc plus jet nebulizer. sponsored by novartis pharma ag which acknowledges support from pari gmbh introduction alpha- antitrypsin (aat) is a protein protecting lung tissues by inhibition of neutrophil elastase. the function of neutrophil elastase is to digest bacteria and other foreign objects in the lungs. in a person deficient of aat, the neutrophil elastase acts uncontrolled, destroying healthy tissue. the result of the damage is emphysema, which progresses if not treated. patients show a specific impaired breathing pattern of short inhalation followed by prolonged exhalation. current therapy consists of weekly i.v. infusion of aat of about - mg/kg body weight. by this method only about % of the administered dose is estimated to reach the lungs. since aat can currently be derived only from human blood serum by an expensive purification process, the worldwide supplies are limited and do not allow to treat all patients. an improvement of the delivery efficiency is highly desirable. inhaled treatment would offer a targeted therapy by delivering aat directly to the lungs and allow for a better use of the limited drug. the eflow ® , a novel electronic nebulizer, is more efficient than conventional nebulizers. it operates by means of a perforated vibrating membrane, applying no or only little stress to the nebulized substances. hence it is well suited for the pulmonary delivery of proteins. it has been shown previously, that a % aat solution can be nebulized by eflow ® without a loss of biological activity. we investigated two eflow ® configurations comparing the in-vitro delivered dose for different breathing patterns. method a highly purified % aat preparation (kamada ltd, rehovot, israel) was aerosolized with the eflow ® electronic nebulizer. initial studies were conducted using the l configuration of eflow ® . delivery performance is compared to a specially designed xl configuration, with increased aerosol chamber volume. aerosol delivery efficiency was determined by breath simulation using an emphysema breathing pattern (tidal volume ml, breaths/min, inh:exh ratio = : . ) generated by a pari compas™ breath simulator. as reference, a standardized regular breathing pattern (tidal volume ml, breaths/min, inh:exh = : ) was also investigated. the respirable fraction (drug in droplets < and . µm) was determined using the andersen cascade impactor at . l/min. samples were analysed for aat activity by an elastase inhibition assay. when the eflow ® l configuration was investigated for the standardized breathing pattern, a delivered dose (dd) of ± % of the loaded dose ( mg) was found. this value was reduced to ± % when the emphysema pattern was applied. with the new eflow ® xl configuration an improved dd of ± % was obtained. furthermore, when the emphysema pattern was applied, the dd did not drop strongly, but remained at values above %. with % of aerosol droplets in the respirable size range (< µm) and % < . µm, a high % of aat can be expected to reach both, the central and peripheral lungs of patients. conclusion inhaled aat has the potential to significantly improve the efficiency of aat replacement therapy. a customized eflow ® electronic nebulizer produced delivered doses > % even when breathing patterns of patients with impaired breathing capabilities were mimicked. the formulation is optimized for rapid aerosol administration through use of higher drug concentrations and is taste-masked. this study was conducted to evaluate the aerosol performance of novel higher dosed levofloxacin inhalation solutions in a customized eflow ® electronic nebulizer. methods two concentrations of mp- ( mg/ml and mg/ml of levofloxacin) were nebulized at fill volumes of and ml using the eflow ® l nebulizer configuration. the in-vitro delivered dose (dd) was determined by breath simulation using a standardized breathing pattern (tidal volume ml, breaths/min, inh:exh ratio = : ) generated by a pari compas™ breath simulator. the geometric droplet size distribution was determined by laser diffraction (ld). the aerodynamic droplet size distribution was determined using the andersen cascade impactor (aci). the invitro respirable dose (rd) was calculated by multiplying the respirable fraction (rf, %droplets < µm) derived from the cascade impaction experiment and the dd derived from breath simulation. nebulization time was determined by the electronic shut-off of the eflow ® control unit. all tests were conducted for three devices in duplicate, each (n= ). the eflow ® electronic nebulizer delivered mp- at both concentrations and fill volumes with equal efficiency, obtaining in-vitro dds of around ± %. the average nebulization times were between and minutes for the ml fill volume and between and minutes for the ml fill volume and were also independent of the concentration in the investigated range. determination of the mass median diameter (mmd) by ld resulted in values between . and . µm. values of the mass median aerodynamic diameter (mmad) obtained by cascade impaction were similar (between . and . µm). the average respirable fraction was in the range between % and % for both experimental methods. conclusion the eflow ® electronic nebulizer can be customized to deliver high doses of mp- . the short nebulization times achieved are patient friendly and should support high patient compliance. mp- , with the potential for once daily administration, provides significant advantages over currently avail-able aerosol antibiotics. clinical evaluation of mp- delivered by the eflow ® nebulizer in cf patients is in progress. lavange, l. ; engels, j. ; accurso, f.j. . university of north carolina, chapel hill, nc, usa; . inspire pharmaceuticals, inc., durham, nc, usa; . university of colorado, denver, co, usa introduction: percent change from baseline in a continuous outcome variable, such as lung function, is a useful descriptive measure in therapeutic clinical trials. while often favored by clinicians as the primary efficacy measure, the use of percent change as the basis for statistical comparisons raises a number of issues. defined as *(post-pre)/pre, where pre and post represent baseline and follow-up values, respectively, percent change is a ratio of random variables and as such, does not follow a normal distribution. analyzing percent change with standard methods can result in inefficiences and may be inappropriate without a suitable transformation (e.g., logarithmic). furthermore, the treatment effect estimated on the transformed scale is difficult to interpret. the purpose of this abstract is to describe an alternative method for analyzing percent change and illustrate its utility in cf clinical trials. methods: probability plots are generated to compare the distributions of percent change in fev from baseline to study endpoint among treatment groups. the plots are similar to kaplan-meier curves, with percent change on the horizontal axis in lieu of survival time. the percentage of patients on the vertical axis depicts the cumulative percentage of patients who had a percent change at least as great as the corresponding value on the horizontal axis. a log-rank test statistic provides an overall test for any difference in distributions among treatment groups. the methods are illustrated using data from a -day, phase , multicenter, randomized, double-blind, placebo-controlled clinical trial of denufosol in cf patients with a screening fev ≥ % predicted. results: a probability plot of denufosol (active doses combined, n= ) vs. placebo (n= ) is shown below. more denufosol patients ( %) experienced improvement compared to placebo ( %). the log rank test, adjusted for study site, showed that the overall distributions of percent change in fev between denufosol and placebo were significantly different (p-value= . ). conclusions: the proposed methods provide a convenient means for assessing differences in percent change and may be useful in cf clinical trials. comparisons of both a descriptive and inferential nature can be made with minimal assumptions, thereby avoiding the pitfalls in analyzing percent change with standard techniques. the methods are easy to implement with existing software packages (e.g., sas). trial supported by inspire pharmaceuticals, inc. and cf foundation therapeutics, inc. chen, x. , ; davis, p. compacted dna nanoparticles formulated with pegylated polylysine and plasmid dna transfect airway cells in vivo at high efficiency, so they have great potential for gene therapy. the mechanism by which dna nanoparticles enter the cell and get expressed is not well understood. we previously showed that rhodamine labeled dna nanoparticles enter primary human tracheal epithelial (hte) cells within min, and accumulate in the nucleus in hr, where the transgene can be expressed. dna nanoparticles do not enter via clathrin-mediated endocytosis (cme), which leads to the degradation of internalized material in lysosomes. further characterization of the kinetics and trafficking pathway may allow us to improve the formulation of the nanoparticles and facilitate its gene delivery. as they do in hte cells, rhodamine labeled dna nanoparticles enter hela and hbeo-, an immortalized human bronchial epithelial cell line, within min, and by - hr accumulate in the nucleus, especially the nucleolus, where they colocalize with nucleolin. expression of the gfp reporter gene in the nanoparticles was observed at hr, which confirms the functionality of the labeled dna nanoparticles. cellular entry is energy dependent, as little or no intracellular rhodamine was observed at °c by hr. we also applied rhodamine labeled nanoparticles and biotin-conjugated transferrin, a marker for cme, simultaneously. little colocalization was observed during a min to hr time course, so the nanoparticles do not follow this pathway. we then found a direct and strong relationship of cell surface nucleolin and the ability of the cells to take up and express dna nanoparticles. nucleolin directly binds to dna nanoparticles with kd= . nm. manipulations of cell surface nucleolin in hela cells affect transfection of dna nanoparticles with a positive correlation. we therefore costained nucleolin in both hela and hbeo-cells treated with the rhodamine labeled dna nanoparticle. the rhodamine fluorescence colocalizes with nucleolin both in the cytoplasm at early time points and in the nucleus/nucleolus at hr and hr, which further supports that nucleolin might be associated with the nanoparticles during their trafficking. to confirm this association, we performed uptake experiments with ms- , a mouse monoclonal antibody against nucleolin, alone or with rhodamine labeled nanoparticles over a min to hr time course. the ms- antibody enters hela cells at similar kinetics to the nanoparticles. at min, we observed substantial amount of cytoplasmic staining, while nuclear staining tends to increase by time until hr. it has little or no colocalization with transferrin in the cytoplasm, which suggests that this antibody also enters the cell via a pathway other than cme. in contrast, it showed extensive colocalization with dna nanoparticles in both cytoplasm and nucleus at all time points, which suggests that cell surface nucleolin and dna nanoparticles are associated or at least in close vicinity during their trafficking inside the cell. therefore, we suggest that dna nanoparticles enter the cell by binding to cell surface nucleolin and enter the cell via the same pathway as nucleolin antibody. chen, x. , ; davis, p. dna nanoparticles are non-viral gene delivery vectors in clinical trial for treating genetic disorders including cystic fibrosis. we previously discovered that cell surface nucleolin serves as a receptor for the dna nanoparticles, and is important for their gene delivery efficiency. as nucleolin has no signal sequence or membrane-spanning domain, it is not clear how nucleolin are expressed on the outer surface of plasma membrane or what signals increase its surface delivery. we initially observed that the transfection efficiency of dna nanoparticles in hela cell decreases by . % following hr serum-free medium treatment, which reduces cell surface nucleolin by . % as determined by cell surface biotinylation and streptavidin bead pulldown. since serum depletion inhibits cell proliferation and affects cell cycle progression, we examined the level of cell surface nucleolin at different stages of cell cycle. hela cells were arrested at s phase by high concentration of thymidine, and allowed to progress synchronously through cell cycle after removing this block. surprisingly, we observed substantial increase of cell surface nucleolin at the onset of m phase, about hours after release of thymidine block. it has been reported that nucleolin is phosphorylated by a cell cycle dependent kinase (cdk) cdc , and has eight consecutive cdk phosphorylation sites. therefore it is appealing to speculate that phosphorylation by cdc might increase the targeting of nucleolin to the cell surface. we then determined the potential export signal in nucleolin by serial deletions. deletion of c-terminal aa of nucleolin, including the c-terminal glycine/arginine rich (gar) domain and the four rna recognition motifs (rrm) does not affect its arrival at the cell surface. the n-terminal aa of nucleolin is sufficient to target a gfp protein to the cell surface. in contrast, when we further delete the cdk phosphorylation sites from aa to , cell surface expression of nucleolin is significantly diminished. furthermore, nucleolin lacking the n-terminal aa is not present on the cell surface. therefore, phosphorylation on the cdk sites may serve as a signal to enhance the cell surface expression of nucleolin. there has been recent interest in dry powder inhaled mannitol as a therapeutic agent in patients with cystic fibrosis (cf). it is has been shown to increase mucociliary clearance (mcc) by airway rehydration. whilst there has been one short term clinical trial of mannitol in adult subjects [ ] , to date no studies have been conducted looking at its potential as a therapy in children with cf. it could be argued that children may have the potential to benefit most from a therapeutic agent that acts relatively proximally in the cf pathogenic pathway. the aim of this study was to determine acute tolerability of inhaled mannitol in children with cf. we recruited children (aged to years) with cf. inclusion criteria were either rhdnase treatment or an fev > % and < % of mean predicted normal value. bronchial provocation challenges with incrementally increasing doses ( , , , , , , mg) of dry powder mannitol were carried out. subjects were pre-treated with bronchodilator minutes prior to the challenge ( mcg of salbutamol or mg of terbutaline). fev was measured following each dose increase up to a maximum cumulative dose of mg. oxygen saturation monitoring was carried out throughout. a positive challenge was defined by a drop in fev of > % from baseline. these subjects received bronchodilator treatment and had spirometry repeated at minute intervals until fev returned to within % of baseline. those children with a negative challenge had spirometry repeated minutes post-completion of the challenge results mean baseline fev was % predicted ( - , sd . ). / subjects ( %) had a positive challenge. mean pd (dose of mannitol required to cause a % reduction in fev ) was mg ( - , sd ). the mean time to complete the challenge was minutes ( - , sd . ) for negative challenges and minutes ( - , sd . ) to pd for those subjects with a positive challenge. we found no association between a positive challenge and age, sex, weight, height, baseline fev , pseudomonas aeruginosa colonization, bronchodilator reversibility, previous aspergillus fumigatus sen-sitization, atopy or corticosteroid use. there was a non-significant trend for lower fef - (means . versus . ; p= . ) and higher prevalence of aspergillus fumigatus cultured in sputum at baseline ( / versus / children; p= . ) amongst those children who went on to have a positive challenge. there was no significant drop in oxygen saturation in either group. although cough was common during the challenge, other adverse events were infrequent. we find that % of children with cf could not tolerate inhaled mannitol as compared with % of adult subjects as reported in the previous study [ ] . we could not identify factors predictive of a positive mannitol challenge in these patients. the most common cause of cystic fibrosis (cf) is the deletion of phenylalanine (∆f ) in the cf transmembrane conductance regulator (cftr) chloride channel [ ] . a major problem with ∆f cftr is that the protein is defective in folding so that little mature protein is delivered to the cell surface [ ] . expression of ∆f cftr in the presence of small molecules known as correctors or pharmacological chaperones can increase the level of mature protein [ ] [ ] [ ] [ ] . unfortunately, the efficiency of correctorinduced maturation of ∆f cftr is low and other approaches are needed to increase the therapeutic value of correctors. we postulated that expression of ∆f cftr in the presence of multiple correctors that bound to different sites in the protein may have an additive effect on maturation. in support of this mechanism, we found that expression of p-glycoprotein processing mutants (cftr's sister protein) in the presence of two compounds that bind to different sites (rhodamine b and hoechst ) had an additive effect on maturation. therefore we tested whether expression of ∆f cftr in the presence of combinations of three different classes of corrector molecules would increase its maturation efficiency. it was found that the combination of the quinazoline vrt- together with the thiazole corr- b or bisaminomethylbithiazole corr- a doubled the steady-state maturation efficiency of ∆f cftr (about % of total cftr was mature protein) compared to expression in the presence of a single compound. the additive effect of the correctors on ∆f cftr maturation suggests that they may directly interact at different sites of the protein. the use of multiple correctors has the potential to increase the therapeutic value of pharmacological chaperones. pharmacol. , - . the cftr protein is expressed at the surface of the airway epithelium, where it plays a critical role in maintaining airway hydration by secretion of chloride. for gene therapy of cf lung disease to be successful, it is critical that the endogenous transgene be expressed in the correct cell type and at levels sufficient to restore normal function. the expression of high levels of cftr has resulted in cftr mediated chloride secretion in a wide range of experimental systems. however, the cell type or types that need to be corrected and the level of cftr expression required to ameliorate the pulmonary manifestations of cf are not yet clear. because cftr has been localized to the ciliated cells of human airway epithelial cells, we have been investigating the use of ciliated cell-specific promoters to improve the efficiency and safety of gene therapy for cf and other airway diseases. in previous studies a fragment of the human foxj promoter was shown to target transgene expression specifically to ciliated cells. however, expression of cftr from this promoter in transgenic cf mice did not significantly improve the cf phenotype, as measured by the nasal potential difference (pd) technique (ostrowski et al, ) . this may be due to the inability of this promoter to correct the olfactory epithelium, which comprises approximately % of the mouse nasal cavity. in cf mice, the olfactory epithelium exhibits sodium hyperabsorption and an absence of cftr mediated chloride secretion, similar to the respiratory epithelium (grubb et al, ) . in this work, we used a fragment of the ciliated cell-specific promoter foxj to drive cftr expression specifically in human ciliated cells. replication deficient adenovirus expressing either egfp or cftr from the foxj promoter were used to transduce well-differentiated cultures of human cf cells following treatment with c to disrupt tight-junctions. cultures were studied hours after treatment, and those treated with ad-foxj /egfp showed strong expression of egfp that was dependant on ciliated cell differentiation. rna analysis demonstrated strong expression of cftr from the foxj promoter, and western blotting demonstrated levels of protein that were much greater than the level in normal airway cells (> -fold). immuno-localization of cftr with specific antibodies resulted in a strong signal at the apical membrane of ciliated cells. cultures treated with ad-foxj /cftr demonstrated a significant increase in forskolin-stimulated short circuit current (isc; . +/- . µa/cm , mean +/-sem, n= ), that was approximately -fold greater than the response in ad-foxj /egfp treated cultures ( . +/- . µa/cm , n= ). the increase in isc was blocked by inh , an inhibitor of cftr mediated secretion. under these conditions, ad-foxj /cftr restored approximately % of the forskolin response of normal human airway cells ( . +/- . µa/cm , n= ). because patients expressing low levels of normal cftr mrna ( - %) have mild disease symptoms, these studies demonstrate that the incorporation of the ciliated cell-specific foxj promoter into gene therapy vectors may be useful for treatment of cf. supported by nhlbi ro hl and the cff. there is a compelling need for safe and effective ai therapy for cf lung disease. low-dose methotrexate (mtx) has been used to treat inflammatory diseases and its use has been reported in cf. encouraging results previously described regarding mtx in cf prompted this study. the study objective was to determine if mtx safely reduces inflammation in the airways of cf patients. methods: this was a single-center, open label study of mtx in stable cf patients with mild to moderate lung disease. baseline levels of neutrophils, free elastase, pro-inflammatory cytokines, and bacteria were determined from an induced sputum specimen. subjects received mg/m /week of oral mtx (single dose). after weeks of treatment, a second induced sputum specimen was obtained for the same inflammatory indices. within subject comparisons (end of treatment vs baseline) were performed for the following primary endpoints: total white cell and neutrophil counts, percent neutrophils, and concentrations of free elastase, il- , il- , tnf-α, and il- β. sputum quantitative microbiology was obtained at baseline and end of therapy. routine laboratories and spirometry were performed monthly. pharmacokinetic testing was performed on the final day of treatment. results: thirteen subjects were screened with started on mtx. six completed the protocol and withdrew early secondary to adverse events (gastrointestinal [gi] complaints and pulmonary exacerbation). five of subjects completing the protocol had declines in pulmonary function: mean change in fvc was - . l (range - . to - . ); mean change in fev was - . l (range - . to - . ); change in fef - was . l (range - . to . ). mean change in weight was - . pounds (range - to . ). mean change in free elastase was . µg/ml (range - . to . ; sd . ). mean change in il- was pg/ml (range - to ; sd ). similar results were obtained for the other cytokines. analysis of cytology specimens is pending. esr and crp did not show significant changes. two subjects completing the protocol had significant gi side effects, including requiring admission for severe abdominal discomfort. quantitative microbiology specimens revealed an increase in colony counts in subjects, decreases in , and mixed results in the th. other safety laboratories were not remarkable. pharmacokinetic studies are pending. complete statistical analysis and safety assessment are also underway. conclusions: the small sample size of this study precludes definitive conclusions regarding the safety or efficacy of mtx. however, the data suggest that ) induced sputum inflammatory indices can be used to assess an ai in clinically stable patients, and ) mtx therapy may not be beneficial, and may be difficult to tolerate as along-term ai therapy for cf lung disease. additional analyses of cell counts and microbiology from this study are ongoing, and may provide additional insight into the effect of mtx in the cf lung. sponsored by the cf foundation , a cellular component of many gram negative bacteria (e.g. pseudomonas aeruginosa), is a common airway stimulus. dampening the inflammatory response in cf reduces the progressive decline in lung function, so anti-inflammatory agents have become both a cornerstone of cf clinical care and a focus of therapeutics research development. reactive oxygen species [ros] can play a role in proinflammatory signaling, including the activation of nuclear factor κb [nfκb] . research has demonstrated increased oxidative stress in cf epithelial cells, potentially highlighting one of the mechanisms responsible for the exuberant inflammation observed. we have previously shown that cddo, a novel anti-inflammatory agent, significantly limits nfκb activation in cftr deficient cell culture models at nanomolar concentrations. using cf mice, we have also shown that intra-tracheal cddo limits neutrophil accumulation and the concentrations of proinflammatory cytokines and chemokines in bronchoalveolar lavage [bal] fluid in response to lps. the synthetic triterpenoids have been shown to activate the nrf transcription factor, thereby inducing several genes involved in redox balance. we now present data demonstrating that cddo may inhibit inflammation in models of cf pulmonary disease by reducing the oxidative stress within cells. methods: b . s -cftr tm mrc cf mice received µl of µm cddo in pbs with %dmso or vehicle (control) daily. drug was administered intratracheally with an atomizer (penncentury) for three days before stimulation. in separate experiments, all mice received µg lps or free pseudomonas aeruginosa intratracheally. mice were sacrificed by co asphyxiation and cardiac puncture hours after stimulation and bal, serum and lung tissue were obtained. the lungs were incubated in lysis buffer containing protease inhibitors and immediately frozen. upon thawing, the tissue was homogenized and whole lung protein extracted. one mg of lung protein from each experimental animal was separated by two dimensional gel electrophoresis. gels were then imaged and analyzed with pdquest to identify protein spots that were differentially expressed in drugtreated mice compared with controls. spots of interest were excised from the gels, trypsin digested and analyzed by liquid chromatography/tandem mass spectroscopy. results: over proteins with greater than -fold differences in expression were identified. we categorized proteins appearing in repeated analyses and identified several affecting intracellular redox regulation; including glutathione s-transferase mu and , peroxiredoxin and , enolase (alpha non-neuron), contrapsin, alpha- antiproteinase and cu/zn superoxide dismutase. these proteins were expressed at significantly greater concentration in the lungs of mice treated with cddo rather than vehicle. conclusion: cddo exhibits anti-inflammatory effects in mouse models of cf pulmonary disease and one potential mechanism for this effect may be the upregulation of reducing proteins to combat oxidative stress. methods: lpc ( ul of . %, . % or . % (w/v) in pbs) was administered (trans-orally via a cannula) to trachea of c l/b mice. in sheep ( of completed) we targeted delivery of . ml of . % lpc dissolved in pbs to the main bronchus of the right lung at branch in a month old sheep (bronchoscope, via tracheostomy). one hour later ul (mice) or . ml (sheep) of a lv-lacz vector ( . x tu/ml) was administered in the same manner. one week post-exposure lungs were inflation-fixed in situ, removed, stained using x-gal, sectioned, and counterstained with saf-o or h/e. results: no lacz gene expression (blue cells) was found in lungs of control (pbs-treated) mice or in the left (untreated side) of sheep lung. blue cells were found in scattered punctuate groups, or in lines of stained cells in mice and sheep. mice given the highest lpc dose ( . %) had extensive gene transfer in larynx, trachea, carina, and in large, middle and small airways of most lobes of the lung, reaching % airway perimeter cell transduction in one animal. with decreasing lpc dose the distribution and number of blues cells was reduced, but transduced epithelial cells including nonciliated columnar cells, ciliated cells, and basal cells were seen in all cases. in sheep, blue cells appeared in the right main bronchus between branch and branch , and in the branch airway, and with highest expression found near branch and . cross-sections revealed that primarily ciliated columnar cells and basal cells (and no goblet cells or macrophages) were transduced. conclusions: lv gene transfer into mouse or sheep lung can be enhanced by pretreatment with lpc. in mice this early data suggests an lpc dose-dependency. for sheep, we await further results to confirm the encouraging finding in this first study; however, it does confirm that gene transfer into the airways of large animals with a lung size similar to humans can also be achieved using lpc pre-treatment and a vsv-g pseudotyped lentivirus. the transduction of both ciliated and basal lung epithelial cells in-vivo in both models is an encouraging funding for the development and understanding of our continuing efforts to produce life-long airway gene transfer suitable for a cf airway gene therapy. supported by: nh&mrc, usa cff, sa channel crf, philanthropic donations. increased airway na+ absorption is a characteristic abnormality in cystic fibrosis (cf), and is thought to play a key role in the pathogenesis of cf lung disease. we have previously demonstrated that mimicking na+ hyperabsorption by overexpression of βenac in mouse airways results in airway surface liquid (asl) volume depletion and reduced mucus clearance causing a spontaneous cf-like lung disease with high pulmonary mortality, and airway mucus plugging, mucous cell metaplasia and chronic airway inflammation in surviving βenac-transgenic (βenac-tg) mice (mall et al., nature med. : , ). in the present study, we used βenac-tg mice to test if inhibition of increased airway na+ absorption by the classic enac blocker amiloride has therapeutic effects on cf-like lung disease in vivo. specifically, we determined the effects of 'early' and 'late' enac blocker intervention on mortality, airway mucus obstruction and inflammation by starting intrapulmonary amiloride therapy in βenac-tg mice either at birth, i.e. prior to the onset of lung disease, or after mucus obstruction and inflammation had established. to achieve this goal, newborn, day, or week old βenac-tg mice and wild-type littermate controls were treated by intranasal instillation of amiloride ( mmol/l; µl/g body weight, tid) or vehicle (h o) alone for a period of days. initial deposition studies showed that this treatment protocol resulted in pulmonary amiloride concentrations sufficient for inhibition of enac. during amiloride therapy, growth and survival were monitored, and any loss in body volume due to diuretic side effects was substituted by subcutaneous injection of isotonic saline (nacl . %). after the day treatment period, mice were euthanized, bronchoalveolar lavage (bal) was performed to determine inflammatory cell counts, and lungs were processed for histology and morphometry to quantitate airway mucus obstruction and mucous cell metaplasia. we show that amiloride significantly reduced pulmonary mortality by~ % (p < . ), when therapy was started in newborn βenac-tg mice. further, early amiloride treatment significantly reduced airway mucus content (p < . ), mucous cell numbers (p < . ), and bal eosinophils (p < . ) compared to vehicle treated βenac-tg mice. in contrast, amiloride therapy had no benefits on airway mucus obstruction, mucous cell metaplasia or inflammation, if treatment was started in day, or week old βenac-tg mice with established lung disease. taken together, our results are consistent with previous human studies where amiloride lacked therapeutic benefits in cf patients with established lung disease, and demonstrate for the first time that early inhibition of na+ hyperabsorption is an effective therapy for cf-like lung disease in vivo. these results warrant an evaluation of more potent and longer acting amiloride derivatives in chronic lung disease in mice, and a clinical evaluation of preventive enac blocker therapy in human trials. supported by cff (mall g ) and ec (mext- - objective: hypertonic saline aerosol delivered intranasally is currently being studied to enhance mucociliary clearance. there is evidence of patients' reluctance to use concentrations above % due to potential discomfort. our study was performed to determine the short term tolerance of . % and % hypertonic saline versus normal saline delivered intranasally via a nebulizer/compressor system (pari sinustartm, pari respiratory equipment, midlothian, va). methods: using the sinustartm nasal aerosol delivery system, we administered concentrations of saline solution, ( . %, . %, %) to healthy, adult volunteers for minutes each. a washout period of minutes between treatments allowed volunteers to wipe their nose and cleanse their mouth with water. a question self-administered questionnaire was completed following each treatment using a point scale ( =high tolerability; =low tolerability). burning sensation, cold, cough, throat irritation, runny nose, and overall comfort were measured. the order of treatments was randomized and volunteers were blinded to the concentration. data: for all measurements . % and % were equally well tolerated compared to normal saline with burning, cough, and throat irritation measuring . or below. no variable measured more than . . out of volunteers stated they would continue this treatment on a regular basis. conclusions: our study indicates that during the time of treatment nasal aerosol delivery of hypertonic saline up to % is well tolerated in healthy adults. there appears to be no issue of discomfort associated with hypertonic saline that would prevent nasal aerosol treatment compliance. our overall research goal is to discover new drugs for clinical treatment of cystic fibrosis (cf) that will correct the biochemical defect in the predominant cf mutation, the ∆f form of the cystic fibrosis transmembrane conductance regulator (cftr) protein, which accounts for over % of all cf cases. this mutation leads to a misfolded protein which is rapidly degraded as well as changes in its function and half-life at the cell surface. however, it has been proposed that only a fraction of the normal surface expression level is needed to provide a clinically significant impact on the disease. thus, from a pharmacological standpoint, strategies that can partially but effectively correct these defects would be expected to have a clear clinical benefit to these cf patients, and these strategies may also be applicable to other cftr mutations. we previously demonstrated that several members of a particular class of related drugs, the anthracyclines, anthraquinones, and anthracenediones, significantly increased cftr cell surface expression and function in cell culture. in particular, we demonstrated that a non-cytotoxic concentration of doxorubicin (dox), a model anthracycline drug, significantly increased total cellular and membrane-associated cftr protein levels and cftr-associated chloride currents in human colon cancer t cells, and also caused a two-fold increase in ∆f cftr-associated chloride current in a canine mdck-c -derived cell line that expresses a stably transfected copy of human ∆f cftr. our previous studies also demonstrated that dox is able to impart structural integrity to ∆f cftr, increasing its half-life and decreasing its proteolytic sensitivity, which is indicative of efficient folding. additionally, two other structurally related drugs, i.e., the anthracycline, daunorubicin, and the anthraquinone, mitoxantrone, were shown to have similar effects on ∆f cftr expression. in the current work, we have extended these effects to two other related aza-anthracenedione molecules, bbr (pixantrone) which is a cancer chemotherapy drug with lower non-target toxicity than dox and which is in phase iii clinical trials, and a structurally related analog, bbr , which is essentially non-cytotoxic with a -fold lower cytotoxic potency than bbr . both compounds increased cftr-associated chloride currents to a similar extent as dox in cfbe human airway epithelial cells expressing ∆f cftr, as measured by ussing chamber experiments. the ability of the non-cytotoxic analog bbr to do so is particularly important, since it indicates that correction of the ∆f cftr defect is not directly tied to the toxicity of this class of compounds per se but rather is a result of other structural features. thus, it is likely that this and/or other non-toxic analogs in this chemical class can be developed that have potential as clinically useful agents for treatment of cf in patients. supported by a cystic fibrosis foundation (cff) grant to jwh and rm, and by the cff-supported dartmouth cf program. cystic fibrosis (cf) is caused by mutations in cystic fibrosis transmembrane conductance regulator (cftr). cftr is not only an epithelial chloride channel, but it also regulates other transporter and ion channels, including epithelial sodium channels (enac) and aquaporin water channels. in patients with cf, absent or dysfunctional cftr results in abnormal electrolyte and fluid content on the epithelial surface. treatments intended to normalize ion transport in cf airways through non-cftr dependent pathways represent attractive approaches to alleviate the underlying pathologic defect. calcium (ca +)-activated cl-channels have been proposed as rescue channel for the cyclic amp-dependent cftr cl-channels, offering a target for cf pharmacotherapy. increases in cytosolic ca + concentration activate epithelial cl-channels, but inhibit epithelial na+ channels, which is also beneficial in correcting the hyperabsorption of na+ in cf. herein we developed a high throughput screening (hts) assay for screening compound libraries with the intention of finding compounds which can increase intracellular ca +. in the pilot primary screening, we screened a mssp library which contains known bioactive compounds and natural products. compounds were classified as the real hits after the secondary screening validation with the hit percentage . %. we have found that the ec of these compounds are less than um in dose-response studies and none of those compounds were toxic to the cells. we have further discovered that those compounds stimulate cl-secretion through activating ca + dependent cl-channels in cf and non-cf human airway epithelial cells in short-circuit current experiments. in summary, our data suggest that modulation of intracellular ca + is a target for cf therapy. the compounds identified by our study will provide possible therapeutic leads in the treatment of cf. non-viral gene delivery particles with various synthetic polymer coatings have been developed for cystic fibrosis (cf). intracellular trafficking and in vivo tissue distribution of these particles must be carefully monitored to guide rational design of efficient delivery system. fluorescent semiconductor quantum dots (qds) allow sensitive, long-term and multi-target imaging in cellular environment, and so are a promising tool in tracing gene delivery materials in vitro and in vivo. however, application has been limited by the lack of efficient and reproducible techniques of qd bioconjugation. here, we describe a method of labeling dna nanoparticles with tunable qds that may enable us to study gene delivery in vitro and in vivo. highly fluorescent zns-coated cdse qds were first synthesized and encapsulated in amine-containing phospholipid micelles. the non-viral vector system we used is based on a polymer backbone consisting of lysines with a cysteine on the n-terminus (ck ) which is conjugated to polyethylene glycol (peg). this polymer vector condenses dna plasmids into stable nanoparticles that efficiently transfect airway epithelial cells in vivo. to introduce the aminecontaining qds, we utilized a hetero-bifunctional peg with an nhs ester at one end, which was first reacted with the amine groups of the qds and a maleimide group at the other end which was subsequently reacted with the thiol group in ck . excess reagents were removed from the final conjugates qd-peg-ck by filtration. conjugation was first confirmed by . % agarose gel electrophoresis. free qds were positively charged and migrated toward the cathode. pegylated qds migrated significantly slower than free qds, and qd-peg-ck conjugates migrated at still a different rate. to further demonstrate conjugation, we biotin-labeled the ck and assessed the binding of qd-peg-ck -biotin to avidin coated agarose beads by monitoring the yield of fluorescent beads. qds, pegylated qds, and physical mixture of qds and ck -biotin were also incubated with the agarose beads as controls. fluorescent beads were detected only when qd-peg-ck -biotin was present. to directly evaluate the ability of qd-peg-ck to bind double stranded dna, we also immobilized double stranded dna fragments to agarose beads. qd-peg-ck , but not unconjugated qd bound to the immobilized dna fragments as determined by the yield of fluorescent beads. qd-peg-ck was then used to compact luciferase reporter plasmids. electron microscopy images of the compacted dna showed that qds were integrated into some dna nanoparticles. we transfected hela cells with qd-labeled and unlabeled dna nanoparticles. luciferase assay and toxicity assay indicated that qd-labeled dna nanoparticles transfected hela cells, though with less reporter gene expression than unlabeled nanoparticles and more toxicity than uncompacted qd. in conclusion, we have developed a method to conjugate qd to dna nanoparticles. improvement of the quality and biocompatibility of the qd may be required for further in vitro and in vivo studies. this system is also versatile. other less toxic optical makers can be readily tested. this study was supported by dk and the cff. in cystic fibrosis (cf) the epithelial sodium channel (enac) hyperactivity plays a role in the pathogenesis of chronic lung disease. the missing enac regulation by the cf transmembrane conductance regulator (cftr) causes increased absorption of sodium ions and fluid across airway epithelia leading to the depletion of the perciliary liquid layer and to the consequent inhibition of mucus clearance. we developed a hiv-based lentiviral (lv) vector containing a sirna cassette to efficiently knockdown the expression and activity of enac in human respiratory cells. background: sildenafil has been implicated in the relocation of cystic fibrosis transmembrane conductance regulator (cftr) protein. the effect was observed in vitro and in the presence of doses roughly times larger than those commonly used for treating erectile dysfunction. aim: to evaluate in vivo therapeutic efficiency of clinical doses of sildenafil and vardenafil, two approved type v phosphodiesterase inhibitors, for correcting chloride transport defect in ∆f mice. methods: we measured transepithelial potential difference in vivo across the nasal mucosa as a readout for sodium and chloride conductance. the effect of a single intraperitoneal injection of sildenafil ( . mg/kg) or vardenafil ( . mg/kg) was investigated in df /df and normal homozygous mice. results: in df /df mice, chloride conductance, evaluated by perfusing the nasal mucosa with a chloride-free solution in the presence of amiloride and with forskolin, was corrected h after sildenafil administration. a more prolonged effect, persisting at least for h, was observed with vardenafil. the forskolin response was increased after sildenafil and vardenafil in both normal and df mutant animals. no effect on sodium conductance was detected in any group of animals. conclusion: our results provide preclinical evidence of effectiveness of both drugs for correcting chloride transport defects in the cf. acknowledgments: this work was supported by grants from the french cf association, vaincre la mucoviscidose and by an educational grant from pfizer belgium. there is a strong interest in developing small molecules able to correct the phenotypic effects of cystic fibrosis (cf) mutations. many mutations (e.g. ∆f ) impair the function of cftr protein, by altering the protein targeting to the plasma membrane and/or by causing an abnormally low open channel probability. drug-like organic compounds may restore the correct membrane localization ("correctors") or increase channel activity ("potentiators") of mutant cftr. in the last years, various research teams have identified molecules with activity as correctors (corr- a, vrt- , miglustat, curcumin) or as potentiators (tetrahydrobenzothiophenes, phenylglycines, sulfonamides, vrt- , , -dihydropyridines). however, these compounds have been tested using different assays and the results are sometimes controversial, with marked differences in declared efficacy and potency of compounds. we have tested a panel of correctors and potentiators to directly compare their effects under the same experimental conditions. for this purpose, we have used the functional assay based on the halide-sensitive eyfp-h q/i l to measure ∆f -cftr activity in frt and a cells. the assay for potentiators consisted in stimulation with the test compound ( . - µm) plus forskolin (on cells previously incubated at °c for hours). the assay for correctors consisted in hours incubation of ∆f cells with test compounds and then determination of cftr activity in the presence of forskolin plus genistein ( µm). our results indicate that all potentiators are active in our assay with a comparable maximal effect but with values of potency that vary significantly among compounds. the potency order measured in frt cells was: dhp- = pg- (ka~ nm) > sf- (ka = nm) > dhp- = dhp- (ka~ nm) > act- b (ka = nm) > felodipine (ka = nm) > vrt- (ka = . µm) > genistein (ka = . µm). a similar order of potency was found also in a cells expressing ∆f . the activity of correctors showed a more marked dependence on cell line. while the potency was comparable between frt and a cells, the maximal effects showed clear differences. in frt cells, corr- a, corr- b and corr- c generated a maximal effect that was . - -fold higher than that obtained by incubating the cells at low temperature. conversely, vrt- and vrt- were less effective (maximal correction equivalent to - % of low temperature rescue). in a cells, all compounds were instead less effective than low temperature, with vrt- being the molecule eliciting the highest activity ( - % of low temperature). our results indicate the , -dihydropyridine dhp- and the phenylglycine pg- among the most potent activators of the mutant cftr channel. the similar behavior of potentiators in two different cell lines is consistent with the assumption that all potentiators act with a similar mechanism, by interacting with the cftr protein itself. in contrast, the cell line dependence of correctors suggests that they act with indirect mechanisms, possibly by interacting with proteins involved in cftr biosynthesis and trafficking. supported by cfft and telethon-italy. we also thank cfft and rfums for providing chemical compounds. lung damage in cystic fibrosis (cf) patients is determined by mucus accumulation, pseudomonas aeruginosa infection and chronic inflammation. extracellular gsh is a scavenger of free radicals produced by neutrophils in inflamed tissues. glutathione transferases (gst) are a superfamily of dimeric proteins which conjugate glutathione to a wide range of substrates including oxidants and are involved the synthesis of leukotriens. clinical beneficial effects have been reported in cf patients following treatment with the macrolide azythromicin (azm); anti-inflammatory properties have been proposed as possible mechanism. the aim of this study is to investigate the regulation of the gstt and gstm activity and expression by azm. reductions of about % and % on gst enzymatic activity were detected in ib - and cfsmeo-cells respectively. gsts mrna expression in cf airway epithelial cell lines was analysed by quantitative pcr (qpcr). the level of gstt and gstm basal expression in cf cells ib - was significantly higher than in isogenic non-cf cells c . we found statistically significant decreases of gstt and gstm mrna of about % and % respectively in ib - cells after treatment with azm for hours, restoring the levels observed in c cells. in cfsmeo-cells after exposure to azm we observed % and % reductions in gstt and gstm mrna respectively. the macrolide jm, known to lack clinical anti-inflammatory properties, had no significant effects on gstt and gstm mrna expression in all cell lines. furthermore, azm did not alter the mrna expression levels of gstp , a glutathione-s-transferase not differentially expressed in cf and isogenic non-cf cells. decreased expression of % and % of gstt protein has been detected by immunoblotting in ib - and cfsmeo-cells, respectively, following treatment with azm. in the same conditions we found a drastic reduction of protein level of gstm in both cf cell lines. finally, gsts activity and the expression of gstt and gstm proteins in cf cells, were reduced approximately to the same level detected after treatment with interleukin (il- ), an anti-inflammatory cytokine, shedding light on a possible correlation between gsts inhibition and antiinflammatory properties of azm. the effects of azm described in this study suggest that downregulation of gstt and gstm expression may result in increased availability of intracellular gsh making cf cells less susceptible to oxidative stress induced by chronic inflammation. inhibition of gstt and gstm might provide a therapeutic approach for limiting the effects of inflammation critical for lung damage in cf patients. this study is supported by italian cf research foundation; comitato di vicenza-associazione veneta per la lotta contro la fibrosi cistica; azienda ospedaliera verona, italy. tradtrantip, l.; padmawar, p.; yangthara, b.; verkman, a. activation of cftr chloride conductance by gpcrs involves camp elevation and pka-mediated cftr phosphorylation. we developed a 'pathway screen' in which cftr-mediated iodide influx is used as a read-out of gpcr activation. the cell-based fluorescence assay utilizes multiply transfected epithelial cells expressing wildtype cftr, yfp-h q/i l and a specified gpcr. we recently used this assay to identify a new class of nanomolar-affinity, -aryl- -benzoyl- hydroxy- -( -arylethyl)- h-pyrrol- -one vasopressin- receptor antagonists (yangthara et al., mol. pharm. , in press ). additional screening of , diverse small molecules yielded novel chemical classes of inhibitors of cftr activation. the potential molecular targets of pathway inhibitors include the gpcr, gs or gi proteins, adenylyl cyclase, phosphodiesterase, pka and cftr. a series of target identification studies was done to classify the new pathway inhibitors, which involved the use of agonists acting at different sites in the activation pathway, and specific site-of-action assays. the pathway screen yielded new small-molecule cftr inhibitors that are unrelated to thiazolidinone and glycine hydrazide inhibitors. one interesting class of pathway inhibitors, thiophenecarboxylates, represent the first small-molecule phosphodiesterase activators, which strongly reduce camp and cgmp concentration in many cell types. the best thiophenecarboxylate greatly reduced intestinal fluid secretion in closed loop mouse models of cholera and travelers' diarrhea, and slowed cyst growth in a model of polycystic kidney disease. other pathway inhibitors, which are potential effectors of g-proteins and pka, are under evaluation. the gpcr-linked cftr pathway screen developed here is useful for high-throughput parallel identification of small-molecule inhibitors of multiple targets in the cftr activation pathway. potential uses of the inhibitors identified here include therapy of secretory diarrheas, polycystic kidney disease, and cyclic nucleotide-dependent tumor growth, as well as pharmacological creation of cf-phenotype in ex vivo human tissues and animal models. supported by cff and nih. inhibitors of intestinal caccs are predicted to have anti-secretory effects in certain diarrheas, and activators of airway and intestinal caccs are of potential use in cystic fibrosis therapy (activation of 'alternative' chloride channels). the purpose of this study was to identify small-molecule cacc inhibitors and activators that target caccs directly, rather than ubiquitous upstream processes such as calcium signaling. we screened a collection of , chemically diverse small molecules using a novel high-throughput screening assay involving lentiviral introduction of a yfp-based halide sensor in cacc-natively expressing human epithelial cells. cacc inhibitors were identified from iodide influx following cacc simulation by carbachol/atp. we identified five classes of cacc inhibitors with micromolar potency, including tetrahydro-cyclopentaquinolines, and -aryl- -(trifluoromethyl)-pyrazoles each of which was unrelated to known transport modulators. two classes of compounds inhibited calcium-activated halide flux following stimulation by multiple types of agonists, including thapsigargin and calcium ionophores, and by patch-clamp analysis appear to target cacc directly. structure-activity analysis of analogs of 'hits' yielded compounds with improved potency, which have been resynthesized and characterized for use in assays of antidiarrheal effiacy in rodent models of viral and drug-induced secretory diarrheas. screening for caccselective activators that act in a sustained manner (non-transiently) was accomplished using a similar cell-based fluorescence assay, but instead testing for increased halide influx. several classes of putative cacc activators with micromolar-potency were identified in screening of , small molecules, whose mechanism-of-action and specificity are under investigation. small-molecule modulators of cacc function that target caccs directly have potential clinical applications, and may be useful in defining the physiological roles and molecular identity of caccs. supported by cff and nih. introduction: inhaled hypertonic saline 〈hs〉improves lung function and decreases pulmonary exacerbations in older children and adults with cf. initiating therapeutic interventions in the youngest patients, particularly those that target the underlying cf defect of airway surface liquid volume depletion, has potential to preserve lung function and improve prognosis. subbarao et al performed baseline, post-albuterol and post-hs lung function testing in infants using the raised volume rapid thoracoabdominal compression technique 〈rvrtc〉and demonstrated no significant drop in lung function 〈 pediatric pulmonology, 〉. however, performing three sets of rvrtc maneuvers under the same sedation could prove difficult. before conducting a therapeutic trial of hs in this population, a simplified protocol must be possible at multiple centers. we sought to evaluate a simplified approach as well as to analyze changes in lung function and clinical findings after acute administration of hs. methods: in this ongoing study, clinically stable children with cf between the ages of months and years inhale . mg of albuterol prior to sedation with chloral hydrate. rvrtc and plethysmography are then performed before and after inhalation of ml of ‰ hs. fvc, forced expiratory volume in . seconds 〈fev . 〉, fef - , frc, rv/tlc ratio, respiratory rate, oxygen saturation, and chest exam findings are recorded. predefined stopping criteria include a % drop in fev . or in oxygen saturation to below ‰. results: six subjects 〈mean age . ± . years〉 have completed the protocol with ‰ hs. comparison of post-albuterol lung function to that obtained minutes after ‰ hs revealed no changes in mean fvc 〈 vs ml; p= . 〉, mean fev . 〈 vs ml; p= . 〉, mean fef - 〈 vs ml/sec; p= . 〉, frc 〈measured after each inhaled therapy in of subjects; vs ml; p= . 〉, or rv/tlc 〈measured after each inhaled therapy in of subjects; . vs . ; p= . 〉. respiratory rate, oxygen saturation and chest exam were unchanged. conclusions: results from this study demonstrate that a two-step protocol may used to evaluate the safety of hs. based on these findings, acute administration of ‰ hs is safe in children ages months to years with cf. despite the known improvement in mucociliary clearance, preliminary findings demonstrate a lack of an immediate response in lung volume measures. given the demonstrated benefits in older children and adults, a multicenter therapeutic trial of hs is warranted. supported by the cystic fibrosis foundation. methods and results: we have characterized aav serotypes - in addition to twenty novel vectors isolated from human or macaque tissues to transduce the murine airway epithelium in vivo. vectors [ e+ genome copies (gc)/mouse] expressing α- -antitrypsin (aat) and βgalactosidase (β-gal) were co-instilled into the mouse lung or nose. transgene expression levels were monitored by assaying aat concentration in serum as well as the number and cell-types positive for (β-gal) expression in lung and nasal airways. of all vectors tested aav and aav were the two most efficient vectors in conducting airways. when these aav vectors ( e+ gc) were subsequently evaluated on human ciliated airway epithelial cultures (haec), in contrast to our findings in mouse airways, aav failed to transduce haec, whereas aav resulted in~ % of the haec expressing transgene. since aav was the most efficient vector in mouse and human airway epithelium we performed structure-function analyses of the aav vector capsid and found two atypical capsid residues that were unique in otherwise conserved positions (f , k ). to generate a potentially fitter vector, residue f was mutated to its conserved state. residue k was found to confer lung tropism and was left unchanged. the resulting vector, aav . , transduced mouse lung and nasal airway with greater efficiency than all aav vectors tested. the increased transduction efficiency of this vector was also observed (~ %) in haec derived from six different human subjects. to continue our preclinical studies in a more relevant model, aav / . expressing egfp was tested in ciliated cultures derived from macaque airways and showed - % of cells expressing egfp one week after inoculation with e+ gc. confocal microscopy revealed that aav . targeted a significant number of ciliated cells: the airway cell-types that likely require cftr expression in cf patients. aav . expressing rhesus α-fetoprotein (rhafp) was then inoculated in the nasal airways of a rhesus macaque and transduction evaluated by monitoring concentration of rhafp in the nasal lavage fluid. we have found that rhafp expression remained high (~ ng/mg) and stable for at least days. conclusion: the enhanced transduction efficiency of aav / . vector in human and macaque airway cultures and its ability to stably transduce the nasal airway of a rhesus macaque in vivo demonstrates that aav / . is a good candidate gene transfer agent for the efficient expression of cftr in human cf airway epithelium. submitted for presentation at the american society of gene therapy. supported by gsk, cff, cfpo , p , mtcc. pharmacological correction of ∆f cftr cellular processing is a potential therapeutic strategy for cystic fibrosis. recent high-throughput screening has identified synthetic small molecules, such as bisaminomethylbithiazoles (corr- ), which partially restore chloride permeability in ∆f mutant cells. the purpose of this study was to examine the utility of natural compounds (chinese medicinal herbs) as ∆f correctors. a herbal compound fraction library was constructed from herbs most frequently used in traditional chinese medicine (tcm) that are believed to contain therapeutic compounds for a broad spectrum of human diseases including lung disease. for construction of the tcm fraction library, crude herbal extracts were first prepared by % ethanol extraction on soxhlet reflux apparatus followed by automated fractionation by preparative hplc. eighty fractions were collected from each of the herbs. each fraction contained to (average . ) individual compounds as determined by analytical hplc. collected fractions were dried and milligram of the material was dissolved in µl dmso to generate mg/ml solutions in -well plates. each -well plate contained fractions from one herb. high-throughout screening was done using the frt cell-based fluorescence assay developed previously (j. clin. invest. : - , . of , fractions screened, active fractions from herbs were identified, with positive fractions verified in secondary screening. the positive fraction did not increase halide transport in control non-transfected cells, and halide transport in ∆f -corrected cells was fully abolished by cftrinh- . we have fractionated some of the most active fractions by preparative hplc to identify which compound(s) conferred activity. for example, in one fraction there were single compounds, of which conferred corrector activity with ic s < µm and efficacy comparable to that of low temperature rescue. these results demonstrate the feasibility of ∆f -cftr corrector discovery from natural compounds. further fractionation, characterization and structure determination are in progress. the unexpectedly high 'hit'rate for the natural compounds suggests their further exploration in cf therapy. cystic fibrosis (cf) is the most common genetic disease affecting the caucasian population, with an incidence of approximately one in three thousand births. cf transpires as a result from a mutation in the cystic fibrosis transmembrane conductance regulator protein (cftr), which regulates ion transport across epithelial membranes. subsequently, patients afflicted with cf have an abnormally excessive incidence of chronic lung infection, with organisms such as pseudomonas aeruginosa. because cystic fibrosis is characterized by chronic bacterial infections, excessive neutrophil recruitment to the lungs, and a coinciding increase in pro-inflammatory cytokine production and nuclear factor-kappa b (nf-κb) activation, we hypothesized that exogenous addition of the nf-κb inhibitor iκbα might ameliorate this phenotype. we cloned the human iκbα gene as well as a mutated iκbα gene into plasmids with chicken-beta actin hybrid promoters. we then tested the new plasmids, paav .cb-hiκbα and paav .cb-hiκbαm, in vitro in the presence and absence of pseudomonas aeruginosa infection in the ib - and s cell lines. both plasmids produce iκbα at high levels as shown by enzyme linked immunosorbant assays (elisas). we also show that paav .cb-hiκbα transfected ib - cells, after infection with pseudomonas aeruginosa, express significantly reduced levels of interleukin (il)- β ( fold, p< . ), il- ( fold, p< . ), and tnf ( fold, p= . ) as well as nf-κb activation ( fold, p< . ) compared to p. aeruginosa-infected ib - controls as determined by human cytokine and nf-κb phosphoprotein bio-plex assays; cytokine expression and nf-κb activation levels in infected paav .cb-hiκbα transfected ib - cells were between levels found in infected ib - and s- cells, excluding il- levels which were below s- levels of expression. flavonoids are among the most potent cftr modulators known. equol [ hydroxy- -( '-hydroxyphenyl)-chroman] is a product of intestinal metabolism of dietary isoflavones such as daidzein, and has been of recent interest in studies of cancer, cardiovascular risk, and neurologic disease. equol is metabolically stable, and % circulates in the free (non-protein bound) form, which is considerably greater than the proportion of free daidzein ( %). structural differences such as modification of ring c (e.g. saturation at c- / , and absence of carbonyl group at c- ) and an absent hydroxyl at position c- distinguish equol from compounds previously reported to modulate cftr activity. in prior work by our center, we showed that equol activates wt and ∆f cftr in membrane patches excised from bhk cells and in cfbe o-monolayers studied in ussing chambers. activation by equol occurred only after r-domain phosphorylation in wt and ∆f cftr constructs, but was independent of rdomain phosphorylation in ∆r cftr, indicating activity may be related to dimerization of the nbds or other domain-domain interaction. because a molecule that alters nbd interactions might have effects on the aberrant processing of ∆f cftr, we screened equol and other flavonoids by preincubating compounds with cfbe o-cells expressing ∆f cftr for hours, and then tested for rescue of cftr dependent cl-channel activity after exchange of media solution. we found that preincubation with equol ( - µm) induced rescue of short-circuit current compared to vehicle treated cells ( vs. µa/cm , p< . , n= ). we then evaluated hour preincubation with equol ( um) for biochemical evidence of cftr processing correction in ∆f cfbe o-cells grown in polarizing conditions. immunoprecipitation and in vitro phosphorylation demonstrated minimal formation of band c compared to vehicle treated cells, but adaptation of a more sensitive avidin label/biotinylation assay specific for surface localized cftr revealed clear evidence that equol preincubation led to cftr at the plasma membrane. next, we examined equol in hela cells stably transduced with ∆f cftr. preincubation of equol led to dose-dependent increases in halide transport measured by fluorescence-based halide efflux (spq) after stimulation by genistein ( . and . fluorescence slope (∆%/sec) with equol and µm respectively, compared to . ∆%/sec in cells pretreated with vehicle alone; p< . , n= - cells/condition). immunohistochemical staining of ∆f hela cells for cftr with - c-terminus antibody showed rescue of surface localized protein with equol ( µm) preincubation for hours, while vehicle treated cells showed only perinuclear staining. in summary, we show functional, biochemical, and immunohistochemical evidence that the naturally occurring flavonoid equol corrects the ∆f processing defect in two model systems. a naturally occurring agent that both activates and corrects ∆f cftr deserves further exploration as a potential cf therapeutic, and may lead to new insights regarding domain-domain interactions that influence the activation and biogenesis of the mutant channel. supported by nih and cff. mote ∆f cftr maturation has been identified. although several small molecule agents have been described that overcome ∆f cftr processing defects in specific cellular models, few studies have directly compared the activity of temperature corrected and chemically corrected ∆f cftr in polarized cell systems. in the present study, we examined chemical and temperature corrected activity of ∆f cftr. correctors included all members of the cfft modulator library (c , c , c , and c ; rosalind franklin university, chicago, il). in a screen using ∆f cfbe omonolayers, maximal corrector activity across the two model systems exhibited a rank order of c >> c > c = c , using forskolin ( µm) and genistein ( µm) stimulated isc as a sensitive test for ∆f cftr activity at the plasma membrane. no change in isc was observed in matched control (parental) cells lacking ∆f cftr expression. based on these results, we further defined the activity of c in ∆f cfbe oand frt model systems, including dose/response and time dependence for peak isc rescue. in cfbe o-cells, exposure to µm c for hrs produced maximal reproducible correction of ∆f cftr processing, with loss of activity following prolonged or high concentration exposure; in frt cells, peak effects were seen at hours. ∆f cftr activity following small molecule treatment qualitatively mirrored temperature correction ( °c growth for - hrs) in both cell types. maximal currents produced by stimulation with forskolin ( um) and genistein ( µm) in ∆f frt monolayers following c pretreatment were % of that produced by temperature correction (p< . ). forskolin was responsible for % of maximal current in frt ∆f cells following chemical correction and % of maximal currents following temperature correction. in ∆f cfbe o-cells, maximal currents following chemical correction were % of that produced by temperature correction (p< . ). forskolin was responsible for . % of maximal currents in cfbe o -∆f cells following chemical correction and . % of maximal currents following temperature correction. these results illustrate dose and time response with a small molecule corrector in two polarizing epithelial model systems, and provide reassurance that observations based upon ∆f cftr following low temperature incubation are relevant to functional analysis after chemical correction. similarity in activation properties between chemical and low temperature correction suggest it is unlikely that the two maneuvers result in ∆f cftr with significantly different structural properties. the studies also indicate fundamental differences in ∆f cftr behavior in frt compared to cfbe ocells, and emphasize the importance of identifying an agent that can restore camp dependent regulation to bronchial epithelial cell types. supported by the nih, cff and cfft. participants: n= patients ( randomised to azm and to placebo) who had successfully completed a course of intravenous antipseudomonal antibiotics immediately before the trial (mean age: . years; mean fev : % of predicted). measurements and results: after treatment (mean dose of . mg/kg body weight once a week) pulmonary function declined in both groups compared to baseline (i.e. after cessation of iv antibiotics). the azithromycin group had signifcantly better results regarding the mean changes in serum crp (azm: + . mg/l, placebo: + . mg/l, p= . ), lipopolysaccharide binding protein in serum, lbp (azm: + . µg/ml, placebo: + . µg/ml, p= . ), serum interleukin- (azm: - . pg/ml, placebo: + . pg/ml, p= . ) and alginate in sputum (azm: + µg/ml, placebo: + µg/ml, p= . ). quality of life (german version of the cfq) showed significantly better results after azm in adolescents and adults. azithromycin was well tolerated with no increase in treatment-related adverse events. conclusion: once-weekly azithromycin ameliorated inflammatory reactions and improved quality of life. a decline of pulmonary function after cessation of intravenous antibiotics could not be prevented, however. this study has been sponsored pfizer gmbh, germany this open-label, multicenter study was conducted in the usa and australia to evaluate the clinical responsiveness of a patient-reported outcome measure, the cfq-r respiratory scale, by determining the minimal clinically important difference (mcid) following a -day course of tobramycin inhalation solution (tis). cf patients (n= [≥ to < yrs, n= ; ≥ yrs, n= ]) with pseudomonas aeruginosa and clinical symptoms predictive of a pulmonary exacerbation (increased cough, increased sputum /chest congestion, decreased exercise tolerance or decreased appetite) were enrolled. three efficacy measures were included: )change in forced expiratory volume in second (fev ) from baseline (day ) to end of treatment (day ) or end of study (day ); )one question about change in respiratory function (days and ; global rating of change questionnaire, respiratory domain, grcq rd; =no change; =maximal improvement or worsening);and )change in cfq r-respiratory scale (day , ). average change from baseline fev (mean [standard deviation, sd]) was . % ( . ) at day and . % ( . ) at day . based on grcq-rd at day , patients ( %) reported no change in respiratory symptoms (score < . ), ( %) a minimal change (≥ . to < . ), ( %) a moderate change (≥ . to < . ), and ( %) a large change (≥ . ). mean (sd) change from baseline cfq-r respiratory was . ( . ) at day and . ( . ) at day . at day , change in cfq-r was moderately correlated with change in fev and with grcq-rd; the correlation was stronger for patients with baseline fev < % of predicted fev values (see table) . mean change from baseline on the cfq-r respiratory scale was . at day for patients with grcq-rd scores indicating minimal change in symptoms (≥ . to < . ; n= ); this provided an estimate of the mcid for the cfq-r respiratory scale for those in exacerbation. this mcid value was consistent with estimates from distribution-based methods( ⁄ sd and standard error of measurement). the cfq-r was responsive to changes in pulmonary symptoms in patients in mild exacerbation following tis treatment; the mcid in this population was . points. responses on the cfq-r-respiratory scale were moderately correlated with changes in fev . funded by gilead sciences, inc. this was an open-label, multicenter study conducted in the usa. we determined the minimal clinically important difference (mcid) for the cfq-r, respiratory scale following a -day course of tobramycin inhalation solution (tis) in patients with cf and chronic pa infection (n= , children [< yrs]). patients had received ≥ courses of tis (mean = . ) within the previous year, however their respiratory symptoms were stable at study entry, with forced expiratory volume in second (fev ) between % to % of predicted values. efficacy measures included: ) percent change in fev (l) from baseline (day ) to treatment end (day ); ) a single question about change in respiratory function (day ; global rating of change questionnaire; respiratory domain, grcq-rd; =no change, =maximal improvement or worsening); ) change in cfq-r-respiratory scale (day to ); and ) change in log pa colony-forming unit (cfu) density in sputum (day to ). the mcid for cfq-r was estimated using three methods: ) change in cfq-r (day to ) for the patient subset with minimal change in respiratory function, as determined by the grcq-rd at day ; ) cfq-r standard error of measurement (sem) from a validation sample), and ) ⁄ standard deviation (sd) of the cfq-r respiratory scale scores. at day , change from baseline fev (mean [sd]) was . % ( . ), change from baseline cfq-r was - . ( . ), and change in log pa cfus was - . ( . ) . based on the grcq-rd at day , patients ( %) reported no change in respiratory symptoms (score < . ), ( %) a minimal change (≥ . to < . ), ( %) a moderate change (≥ . to < . ), and ( %) a large change (≥ . ). pearson r-values for the correlation of change in the cfq-r-respiratory scale and change in fev , log cfus and grcq-rd were . , . , and . , respectively. estimates of the mcid for cfq-r-rd ranged from . to . for adults/adolescent patients (table) . in patients with cf who had no immediate need for antipseudomonal therapy at study entry, the cfq-r-respiratory scale appeared responsive to changes in patient disease perception following days of tis treatment; the mcid for cfq-r was approximately points for the adolescent/adult patient population. funded by gilead sciences, inc. ex vivo chloride secretion measurements (intestinal current measurement, icm) in cf patients have been established over the past years to study the cftr-basic defect in more functional detail. modified micro-ussingchambers are used to registrate the transepithelial short-circuit current (isc) in freshly obtained human rectal suction biopsies as a measure of ion transport after stimulation with secretagogues. hereby, the cftr clchannel, its amount of residual function in cf and alternative cl-channels can be investigated by a standardised protocol.in the course of the development of cftr pharmacotherapeutics as well as agents activating alternative cl-channels, icm may function as an useful outcome parameter in preclinical and clinical trials. it is easy to perform repeatedly at all patients ages and comprises the safety advantages of an ex vivo method which is relevant especially for early study phases. aim of this study was to describe reference values and quantify the intraindividual variability of different icm parameters. methods: a total of n= rectal biopsies from n= individuals; with pancreatic insufficient (pi)-cf (n= ; mean age . years), pancreatic sufficient (ps)-cf (n= ; . years), excluded cf by icm diagnostics (n= ; . years) and healthy control (n= ; . years) were included into analysis. for calculation of intraindividual variability, - biopsies/patient were compared with respect to basal tissue resistance (rt basal), basal open circuit potential difference (pd basal), basal short circuit current (isc basal) and the isc responses to stimulation with carbachol ( - mol/l, serosal), -bromocyclic monophosphate (camp) ( - mol/l, mucosal+serosal) + forskoline ( - mol/l, serosal) and histamine ( x - mol/l, serosal).results:we determined icm reference values for the groups of pi-cf (isc basal . ± . µa/cm , ∆isc carbachol . ± . µa/cm ,∆isc camp/forskoline . ± . µa/cm ,∆isc histamine - . ± . µa/cm );ps-cf (isc basal . ± . µa/cm ,∆isc carbachol . ± . µa/cm ,∆isc camp/forskoline . ± . µa/cm ,∆isc histamine . ± . µa/cm ),and healthy control (isc basal . ± . µa/cm , ∆isc carbachol . ± . µa/cm , ∆isc camp/forskoline . ± . µa/cm , ∆isc histamine . ± . µa/cm ). for the total cohort, mean coefficients of variation were: rt basal %, pd basal %, isc basal %, ∆isc carbachol %, ∆isc camp/forskoline %, ∆isc histamine %. conclusion:this first comprehensive analysis of the intraindividual variability of icm basal tissue and cl-secretion parameters provides the basis for the method as an useful outcome measure for future clinical trials aiming to rescue the cftr basic defect. possible effects of pharmacological therapeutics in cf relevant human epithelia have to be adequately interpreted with respect to subject variability and laboratories reference data. ex vivo cl-secretion measurements have the potential of being an essential step in the evaluation process of cftr-correcting/potentiating agents on their way from laboratory screening to the application in human cf tissue without any risk of toxicity. center, placebo-controlled, double-blinded pilot study we assessed safety and tolerability of . mg/d moli versus placebo (normal saline) administered by inhalation (pari lc plus) once daily for days. patients included were ≥ years of age in phase i and ≥ years of age in phase ii, with a fev > % predicted and stable lung disease. overall, subjects received moli and placebo. exclusion criteria included abpa, b. cepacia infection and severe liver disease. the study involved clinic visits over a period of weeks to assess adverse events, spirometry, pulse oximetry and quality of life. a total of adverse events (aes) were observed in subjects ( ae in subjects receiving moli ), with only (productive cough) in the moli group being of severe intensity. the most frequent aes related to the study medication were (productive) cough ( x) and dry throat/throat irritation ( x), and most of these resolved within hour after inhalation. in the moli group no significant ae, defined as a decline of fev ≥ % from baseline accompanied by symptoms, a decrease in oxygen saturation to < % or a fall of % from baseline requiring therapeutic intervention, or a change in safety parameters judged to be clinically significant was observed. this trial was not primarily designed to show efficacy; however, the median change in fev from day to day was - % in the placebo group, and + % in the moli group, and this difference was significant (wilcoxon test, p= . ). similarly, there was a significant difference between the median change in fef - % from day to day in the placebo group as compared to the moli group (- % vs. + %, p= . ). no significant changes were observed for the other study days or for fvc and pulse oximetry. moli was well tolerated in this trial, with the observed aes generally being mild and of short duration. these encouraging explorative results are currently being further evaluated in explorative and confirmatory trials. introduction: results from published data elucidate that microbes found in the upper respiratory tract are similar or the same as those found in the lower airways of cf patients. inhaled, aerosolized drug delivery to the lower respiratory tract is an established treatment route. however, drug delivery systems capable of depositing drug to the paranasal cavities are not yet established and require evidence of deposition and efficacy. pari developed the vibrent™ paranasal drug delivery system to enable the aerosol and drug to penetrate into the nose and sinuses. objectives: this study was conducted to demonstrate that the pari vibrent™ pulsating drug delivery system is capable of ventilating the human paranasal sinuses of healthy volunteers. methods: mkr-gas was continuously ventilated through the nasal tract of three healthy non-smokers in front of a single-head gamma camera (diacam, siemens, germany), using the pari vibent™ pulsating drug delivery system. the nebulizer was coupled to the right nostril and a flow resistor to an output tube was inserted into the left nostril. during ventilation with the krgas (about sec) the subject closed their soft palate to transmit the pulsation and to prevent penetration into the lower respiratory tract. the gas supply of the vibrent™ was directly taken from the mkr-gas generator output channel. kr-gas ventilation imaging was performed with and without pulsation. serial images were recorded with anterior and lateral views. additionally, mri (magnetic resonance imaging) lateral slices of the subjects' head were recorded. the gamma camera images were superimposed to the mri images by adjusting the spatial resolution. with no pulsation from the vibrent™ no ventilation of sinus cavity was visualized by gamma camera images and radioactivity was detected in the nose only. when pulsation was added the maxillary sinuses can be visualized in the gamma camera images of all volunteers. conclusions: without pulsation no ventilation was observed. gas penetration to the paranasal sinuses can be demonstrated using the pulsating action of the pari vibrent™, potentially enabling drug delivery via aerosols. this confirms results of in vitro studies using a cast model. mkr-gas ventilation of the nasal cavities during sec breath holding in front of a planar gamma camera head (anterior) using the pari vibrent without (w/o, left image) and with (w, right image) the pulsation system. the delivery and the exhaust tubing of the kr-gas are shown together with the outline of the head, obtained from mri pictures. introduction: pediatric patients with cf were previously studied in clinical trial studies of denufosol, a novel selective p y agonist that enhances ciliary beat frequency and activates chloride secretion to hydrate the airways in the lung. pediatric patients are often discouraged from participation in clinical trials until later stages of drug development. aims: in order to evaluate the safety experience with denufosol in this population, we have retrospectively examined integrated data for pediatric cf patients with mild to moderate pulmonary disease that participated in three phase studies. demographic and baseline characteristics in addition to safety and tolerability results for cf patients aged - years old are reported. methods: three phase , multicenter, randomized, double-blind, placebo-controlled, parallel group studies were conducted. patients were randomized to receive either denufosol ( , or mg) or placebo (normal saline) tid for days by inhalation. only study included denufosol mg, while all studies included denufosol mg and mg. the fev predicted normal required to be eligible was > % (study ); %- %, inclusive (study ); > % (study ). all three studies included a one-week pre-randomization period during which reproducibility of fev (l) ± % was required in order to be randomized to double-blind study medication. patients were allowed to use bronchodilators, dornase alfa and corticosteroids in studies , and . patients were allowed to use oral antibiotics including macrolides and inhaled tobramycin solution in studies and . results: a total of cf patients - years old were randomized and dosed in three -day studies. eighty-one received denufosol (active doses combined) and received placebo. demographics were similar for all treatment groups -denufosol pediatric patients had a mean (sd) age of . (± . ) years old compared to placebo pediatric patients who had a mean (sd) age of . (± . ) years. denufosoltreated patients were % male and placebo-treated patients were % male. the mean (sd) percent predicted fev at baseline was similar between treatment groups [ . % (± . ) and . % (± . ) for denufosol and placebo, respectively]. the overall incidence of treatment emergent adverse events (ae) was similar between treatment groups ( % denufosol, % placebo). the most common ae was cough, reported by % and % of patients that received denufosol and placebo, respectively. seven percent of denufosol and % of placebo patients prematurely discontinued from the study due to aes. there were no differences in compliance with administration of study drug ( % in patients given denufosol and % in patients given placebo). conclusion: doses of denufosol up to mg given tid for days were well tolerated in pediatric cf patients - years old. these data demonstrate that inclusion of patients - years old is feasible regardless of administration of denufosol or placebo. a longer term phase study of denufosol in cf patients > years old in north america is currently ongoing. acknowledgements: this research was funded by inspire and the cystic fibrosis foundation. objective: pulmonary delivery of anti-infectives provides the potential to attain pk-pd indices exceeding those which can be achieved with systemic dosing. mp- is a novel formulation of levofloxacin that enables delivery of high concentrations over a short period, and provides taste masking. the objective of this study was to: i) determine the safety of aerosol doses of mp- and, ii) determine pharmacokinetics of levofloxacin in serum, urine, and sputum following aerosol doses of mp- using the pari eflow nebulizer in normal healthy volunteers (nhv) and patients with cystic fibrosis (cf). methods: nhv and patients with stable cf were enrolled in a single within-subject ascending dose study of dose levels (loaded doses of , , and mg) of inhalational mp- (levofloxacin solution for inhalation) or placebo. study participants were monitored for safety and changes in pulmonary function. serum, urine, and sputum (cf patients only) samples were collected at various times following the dose and assayed for levofloxacin concentration using hplc. in addition, each participant received a single iv dose of levofloxacin to determine the systemic bioavailability following aerosol mp- . noncompartmental and compartmental methods were used to determine serum, sputum and urinary pharmacokinetic parameters. pharmacokinetic deconvolution methods were used to determine the amount of the dose remaining in the lung over time. results: this study is ongoing. dosing in nhvs ( active mp- , placebo) has been completed. there were no serious adverse effects, and no significant changes in pulmonary function were noted. preliminary pharmacokinetic data from nhvs show a proportional increase in serum levofloxacin concentrations with increasing aerosol mp- dose; serum aucs were . , . , . , and . mg-h/l for the iv dose, low, mid, and high aerosol doses, respectively. deconvolution of the serum levofloxacin concentrations from aerosol mp- and iv dosing shows absorption of drug from lung over time. conclusion: preliminary results show that mp- is well tolerated following aerosol administration in normal volunteers. serum concentration data following aerosol dosing suggests that absorption of levofloxacin into the systemic circulation is a major route of elimination from the lung. studies in cf patients are in progress and will be presented. methods: a mg/kg aerosol dose of mp- was given using a microspray aerosol generation device. when placed just above the tracheal bifurcation, and activated, it delivers a bolus aerosol dose to the lung. a mg/kg intravenous (iv) dose of lvx was given as a slow bolus into the lateral tail vein. plasma lvx was determined in all rats up to sacrifice at , , , or hours, at which time rats were humanely euthanized and bronchialalveolar lavage (bal) performed. fluids were analyzed for lvx concentration using an hplc/ms method. results: the plasma lvx versus time profiles of both iv and aerosol doses were best described by a two compartment model. the plasma auc after an iv dose of levofloxacin and an aerosol dose mp- were similar ( . mg᭹hr/l vs. . mg᭹hr/l, respectively) suggesting near % bioavailability from the lung. after an aerosol dose, the mean residence time (mrt) was prolonged when compared to the intravenous dose ( . vs. . hours). this delay in absorption was associated with an increase in bal lvx auc - h in bal ( . mg᭹hr/l vs. . mg᭹hr/l for iv vs. aerosol dosing, respectively). conclusion: these data show that levofloxacin is highly available to plasma following a single microspray aerosol dose of mp- . the aerosol dose does produce a slightly longer mean residence time in plasma, suggesting delayed, but complete absorption from the lung; this delay in absorption was associated with increase in bal aucs. these data suggest that high concentrations of lvx can be attained in lung fluids following an aerosol dose of mp- . auc: area under the time-concentration curve; cl: clearance; cmax: maximum concentration; f: bioavailability; mrt: mean residence time. methods: animals were assigned to one of five exposure groups for all studies. for the -day toxicology studies, animals were exposed for up to four hours daily for twenty-eight consecutive days, followed by a -day recovery period. respiratory function and cardiovascular safety were conducted on the first and last day of the -day study in dogs. rats were exposed to , , or mg/kg/day, and dogs were exposed to , , or mg/kg/day. at the conclusion of the study, necropsy was performed and all respiratory tissues were harvested, weighed, and underwent gross and microscopic examination. a separate dog respiratory safety study was conducted in which dogs were exposed to , , or mg/kg. results: four week repeat dose inhalational exposure of mp- at target doses up to mg/kg/day in dogs and mg/kg/day in rats was not associated with any test article-related changes in any respiratory tissues. aerosol administration of mp- at doses of up to mg/kg in dogs was found to have no acute effects on minute volume, tidal volume, respiratory rate, or ecg. conclusion: local effects due to the inhalational administration of levofloxacin formulated as mp- were not observed in either rats or dogs. these data suggest that the risk of respiratory toxicity from nebulized doses of mp- is low. methods: bacteria were grown overnight in mueller-hinton broth (mhb) at °c and then sub-cultured into fresh mhb and allowed to reach log phase ( hours). female swiss mice were infected under anesthesia by intratracheal instillation of . ml of a x cfu/ml bacterial suspension. ip doses were selected to provide lvx exposures (as auc) comparable to that obtained with systemic dosing regimens. for the p. aeruginosa infection, treatment was initiated by either the ip or aerosol route hours post-infection. mice were euthanized and hours after the start of treatment, lungs harvested, and bacterial counts in lung determined. results: lvx plasma pharmacokinetic profiles were nearly identical following intraperitoneal or aerosol dosing. the geometric mean log cfu/lung pair (sd) for the p. aeruginosa are shown in the table below: in both the k. pneumoniae and p. aeruginosa lung infection studies, aerosol administration was more effective than systemic administration; for k. pneumoniae, the extent of bacterial killing at hrs was ca. . log cfu greater with aerosol mp- than with systemic lvx. conclusion: aerosol administration of mp- produces a greater extent of bacterial killing than systemic dosing of lvx in mouse models of pneumonia due to k. pneumoniae and p. aeruginosa. the majority of morbidity and mortality in cystic fibrosis patients is caused by chronic and persistent lung infections especially with pseudomonas aeruginosa. since galactosyl ceramide had been previously shown to be involved in pseudomonas internalization, ceramide levels in the plasma of cf patients were assessed and compared to healthy volunteers using hplc followed by mass spectrometry. the results demonstrated that cf patients displayed significantly lower levels of several ceramide species. also, cftr-knockout mice displayed diminished ceramide levels in cf related organs (lung, pancreas, and ileum) and plasma compared to wildtype controls. treatment with a semi-synthetic retinoid (fenretinide), which was previously reported to induce ceramide in neuroblastoma cell lines, was able to increase ceramide concentrations in cf related organs in cftr-knockout mice to the levels of wildtype mice. treatment also dramatically improved the ability of cftr-knockout mice to control pseudomonas infection. following infection with pseudomonas-impregnated agar beads, fenretinide treated cftr-knockout mice were able to clear bacterial infection as efficiently as wildtype mice. overall, these findings not only documented a novel deficiency of ceramide in cf patients but also demonstrated a pharmacological means to correct this defect in cftr-knockout mice. our data provides a strong rationale for clinical intervention that may benefit cystic fibrosis patients suffering from cf lung disease. recent reports show that adult bone marrow-derived stem cells can localize to and acquire phenotypic and functional markers of lung epithelium. these findings raise the novel possibility of stem cell therapy for multitude of lung diseases. however, only small numbers of adult marrowderived stem cells localize to lung and it is not clear whether these cells will be clinically useful. we investigated whether mesenchymal stem cells (mscs) obtained from human cord blood might have increased potential to participate in structural lung remodeling. cord blood was obtained from normal deliveries at the university of vermont. mononuclear cells were isolated and plastic adherent cells were expanded and characterized as mscs according to international society for stem cells research (isscr) criteria. following systemic ( x cells/mouse by tail vein) administration to sublethally irradiated ( . gy) immunodeficient (nod/scid) mice, lungs harvested day, weeks, or months later demonstrated small numbers of human b -microglobulin positive cells in the airway epithelium at all time points. small number of cells was found also stain positive for pancytokeratin (pan-ck) and rarely, we identified cells of b -microglobulin+/pan-ck+ in the airway epithelium of these mice after weeks. we are currently characterizing the phenotype of these cells with ccsp and cftr but these data suggest that cb-mscs may be a potential alternative source of stem cells for use in lung remodeling. high-throughput screening (hts) and other drug development programs have identified cftr activators and potentiators that require secondary evaluation and mechanistic confirmation. we recently evaluated the cfft modulator library (rosalind franklin university, chicago, il) and found some, but not all, cftr potentiators induced potent phosphorylation of the regulatory-domain (r-d), conventionally viewed as the first step in cftr activation. to assess whether this observation has functional significance regarding ion channel activation by these agents, we evaluated cftr potentiators in cfbe o-and fisher rat thyroid (frt) cells stabily transduced with ∆f cftr. cells were studied in modified ussing chambers under control conditions and after correction of ∆f cftr misprocessing using either low temperature ( οc x hrs) or pre-incubation with the chemical corrector c . total currents were determined following potentiator (at reported ec ), forskolin ( µm), and genistein ( µm) stimulation. in temperature corrected ∆f cfbe ocells, potentiator (p , cfpot- ), an agent that does not induce r-d phosphyorylation, caused modest activation when acutely administered ( µm) ( . vs . µa/cm in vehicle treated cells, p < . ). importantly, this agent potentiated forskolin mediated short-circuit current (isc: . vs . µa/cm , p < . ). forskolin accounted for % (vs. % with vehicle) of total current, demonstrating potent rescue of camp dependent cftr activity, an effect not previously reported in this cell type (p< . ). in contrast, two cftr potentiators that induce r-d phosphorylation, p (uccf- , a benzoflavone intermediate, µm) and p (uccf- , an isoxazole, µm) induced modest direct activation of cftr ( . and . µa/cm respectively, p= . and p< . ), but no evidence of forskolin potentiation (p : . µa/cm , % of total stimulated current p=ns; p : . µa/cm , %, p=ns). in ∆f frt cells grown at low temperate, p elicited strong potentiation of forskolin mediated currents ( . vs. . µa/cm with vehicle, p < . ), and modest direct cftr activation ( . vs. . µa/cm , p < . ). p and p were limited to minimal (not significant) activation and no evidence of forskolin potentiation. in cfbe oand frt cells pretreated with c (to chemically correct ∆f cftr processing), p again potentiated forskolin mediated current ( . vs. . µa/cm with vehicle, p< . in the frt model) but did not directly activate i sc . findings with p and p were otherwise as seen with low temperature corrected cells. in summary, in ∆f cftr polarized epithelia, p both activates and potentiates cftr activity (with potentiation being the predominant effect, a unique observation in cfbe ocells), while p and p confer activation of cftr without forskolin potentiation. given that p and p induce r-d phosphorylation while p does not, our findings suggest that agents that do not confer phosphorylation of the r-d may be better suited to rescue the endogenous camp mediated component of i sc . screening of potential therapeutic agents for effects on r-d phosphorylation may help predict utility at restoring ∆f chloride channel activity. supported by nih and cff. activation of cftr is conventionally viewed as a two step process: pka-regulated phosphorylation of multiple sites within the regulatory domain (r-domain), followed by atp dependent gating mediated by binding sites at the interface of the two nucleotide binding domains (nbds). cftr 'potentiators', small organic molecules that overcome mutant cftr gating defects at the cell surface, have been proposed as therapies for cf. although a number of these agents are advancing to the clinical testing phase, their mechanism(s) of action are not well understood. as a step towards better characterizing cftr potentiators available through the cfft modulator library or other resources, we are developing standardized biochemical and functional assays to evaluate the r-domain during cftr activation in living cells. we have previously described a gel-shift method by which phosphorylation of isolated r-domain (residues - ) can be monitored. using this method, we have confirmed that one potentiator, p (cfpot- ), does not induce phosphorylation of the r-domain ( % of forskolin response, n= , p = ns). unexpectedly, two potentiators, p (uccf- , a benzoflavone intermediate) and p (uccf- , an isoxazole), robustly confer phosphorylation of the r-domain (p : % of forskolin response, n= , p = . , p : % of forskolin response, n= , p = . ). we found the phosphorylation conferred by either p or p could be inhibited with the pka inhibitor h ( µm). maximal stimulation of phosphorylation occurs within minutes, indicating time dependence similar to forskolin. importantly, neither p nor p increased total cellular camp, a finding confirmed by a number of other laboratories. the results may therefore implicate compartmental inhibition of cftr-associated phosphotases (eg. pp a) or phosphodiestereases (eg. pde ) as an underlying mechanism by which isoxazole or certain flavone-derivative compounds stimulate cftr. to further test this hypothesis, p , p and p are being examined for effects on ∆r-cftr chloride channel activity. these studies provide a means by which novel cftr potentiators can be biochemically categorized based on r-domain phosphorylation, a measure of the first step of cftr activation. compounds working through distinct mechanisms may have particular relevance to certain cftr mutations, and could provide synergy in the clinical setting. supported by nih and cff. mucociliary clearance (mcc) is an innate defense mechanism that protects the lungs from bacteria and viruses. mcc requires maintenance of a thin layer of airway surface liquid (asl) to eliminate inhaled particles. the asl volume is tightly regulated by a balance of ion and water transport across the airway epithelia. in cf, the loss of cftr clsecretion coupled with unregulated na + absorption via enac results in asl volume depletion and impaired mcc. although significant progress has been made in the identification of the basic disease mechanism in cf, therapeutic approaches that address abnormal cftr biogenesis are not currently available. the extracellular nucleotides atp and utp are important mediators of asl volume and mcc. in the airways, secreted atp acts on the g-protein coupled p y receptors to fine-tune mcc via the regulation of apical ion transport, ciliary beating, and mucin secretion. we have previously demonstrated that atp controls asl volume by inhibiting absorption through enac and increasing secretion through apical membrane chloride channels. p y receptor agonists are good candidates to treat cf. however, the rapid hydrolysis of atp and utp on the airway surface of cf patients limits the effectiveness of this approach. consequently, inspire pharmaceuticals has developed di-nucleotide molecules which retain the ability to activate p y receptors, but are more resistant to hydrolysis by ectonucleotidases. denufosol, inspire's lead compound for cf treatment, potently activates p y receptors to stimulate cl -/water secretion, ciliary beating, and mucin release in epithelial tissues. however, the stability and potency of denufosol has not been determined using human bronchial epithelial (hbe) cells. in primary cultures of hbes, denufosol was significantly more stable than utp. under thin-film conditions, the initial hydrolysis rate of a therapeutic concentration of denufosol ( mm) was . nmol × min - × cm - with a half-life of approximately hour. by comparison, denufosol is more than times more stable than utp on the mucosal surface of hbes. importantly, the increased stability of denufosol translated into an increase in efficacy for this compound in vitro. equipotent concentrations of denufosol and utp significantly increased asl height. however, denufosol produced maximum asl height increases that were both greater ( . % versus . % maximum increase over basal asl for denufosol and utp, respectively) and longer-lasting ( . % versus . % increase over basal asl following a minute application of denufosol and utp, respectively) compared to utp. furthermore, the asl height increases are specific for denufosol as the addition of apyrase blunts the response to utp, but not denufosol. our data demonstrate that denufosol is more stable than utp on the mucosal surface of human airway epithelia, which results in larger and more sustained increases in asl volume. recent phase ii clinical trials show that administration of denufosol over days was well tolerated and associated with improved lung function in mild cf patients. taken together, these data suggest that denufosol is a promising candidate molecule for cf therapeutics. mucociliary clearance (mcc) is the primary airway host defense against chronic exposure to infectious and noxious agents. mcc is dependent upon ciliary beating and the volume and composition of the airway surface liquid (asl). asl volume is regulated via isotonic fluid transport which is dominated by na + absorption in the superficial epithelium. in cf, unregulated na + absorption through enac drives asl volume depletion and a subsequent decline in mcc. the accumulation of mucus in the airways of cf patients supports persistent and life-threatening bacterial infection. increasing airway surface hydration represents a promising therapeutic approach for treating cf. in vivo, aerosolized osmotic agents such as hypertonic saline (hs) improve mcc and lung function in cf patients. however, the benefits derived from hs treatments are transient, as nacl is relatively rapidly absorbed by airway epithelia. previously, donaldson et al. (nejm ) tested the hypothesis that blocking na + absorption with the enac inhibitor amiloride would increase the efficacy and extend the benefit of hs. surprisingly, amiloride blunted the response to hs, which resulted from a previously unrecognized property of amiloride to inhibit aquaporin-mediated water transport. while potent inhibitors of enac would likely be of great benefit to the cf treatment milieu, the compounds then available were not adequate for this purpose. parion sciences has developed novel -substituted pyrazinoylguanidine compounds that selectively inhibit enac and are > -fold more potent than amiloride. in the present study, we evaluated the ability of two compounds, - and , to increase asl volume under thin film conditions. in primary cultures of human bronchial epithelial cells (hbes), - alone produced a small, but significant increase in asl height. in cf cultures exposed to phasic motion that simulates the shear stress generated by normal tidal breathing, - alone increased asl heights by . %. additionally, we tested the effects of - pre-addition in combination with % hs. as expected, hs alone produced a rapid and substantial increase in asl height ( . % by minutes) which declined to % bỹ hour post-treatment. pre-treatment of cultures with - prior to hs was both more potent ( . % by minutes) and longer-lasting ( % initial response at ~ hours) than hs alone. on normal hbes cultures, the compound alone increased the basal asl height by . % following a hour exposure. similar to - , in combination with hs likewise extended the longevity of the hs effect (t / ~ h versus ~ h for + hs and hs, respectively). by blocking na + absorption, the parion compounds alone increased asl volume in both normal and cf hbes. strikingly, when used in combination with hs, the parion compounds enhance and sustain the increase in asl volume associated with hs alone. our data demonstrate that the parion compounds alone are sufficient to increase basal asl volume. furthermore, these data provide a proof-of-concept that combinational therapies utilizing osmotic agents and compounds that regulate ion transport will provide therapeutic benefits to cf patients. methods: a total of patients (with fev ≥ % of predicted) received mg of arikace by inhalation for days. drug was administered using the pari lc star nebulizer. laboratory parameters, adverse events and pulmonary function tests were collected for all study subjects in order to determine safety and tolerability of arikace™. sputum samples were collected to determine changes in bacterial density, and amikacin pharmacokinetic parameters were assessed at selected time points in urine, serum and sputum specimens. change in fev , fev % predicted, fef - % and fvc relative to baseline and change in log cfu on days and were assessed. results: on day , and , the observed change for fef - % was . (p < . ), . (p = . ) and . l/sec (p = . ), respectively. on day and , the observed change for fev was . (p = . ) and . l (p = . ), respectively, and was . (p < . ) and . l/sec (p = . ) for fev % predicted. significant relationships (p ≤ . ) between log cfu and serum auc:mic ratio, and between changes in log cfu and fev , fev % predicted and fvc were identified. treatment was safe and well tolerated with the most frequent adverse events being dyspnea and headache of mild to moderate severity. conclusion: inhaled arikace™ mg/ml was well tolerated and in select patients improved pulmonary function. together these clinically relevant changes from baseline likely represent drug effect and warrant further development of liposomal amikacin for inhalation in patients with cf. allergic bronchopulmonary aspergillosis (abpa) is a disease caused by hypersensitivity to aspergillus fumigatus (af). the prevalence of abpa is approximately %- % of patients with cf and can contribute to worsening of their pulmonary disease. the treatment of abpa consists of high-dose oral corticosteroids for many months and may include also antifungal antibiotic such as itraconazole. the clinical effectiveness of corticosteroids is usually shown by an improvement in clinical symptoms and radiological parameters, as well as a reduction in total ige. the prolonged period of systemic steroids may cause significant side effects such as cushingoid appearance, hypertension, glucose intolerance and more. over the past years, we have used pulse high-dose methylprednisolone in patients with cf. method:all patients were diagnosed as suffering from abpa by the standard criteria as the may have mucoid impaction or central bronchiectasis on chest radiography, an elevated ige (> iu/ml), the presence of specific ige anti af and an elevated eosinophil count. the patient may have a positive skin prick test to af allergen. they were treated once a month with iv pulse high-dose methylprednisolone ( mg/kg/a day) for days once a month for several months until the total ige decreased to normal values. three patients were treated also with itraconazole. in patients itraconazole was discontinues due to side effects. results: we treated patients age - years old ( m/ f). four patient are pancreatic insufficient and pancreatic sufficient. ige was +/- ( - ) at the time of diagnosis and decrease to +/- ( - ). fev increased from +/- to +/- . the patients also gained weight and improved their chest x-ray finding. side effect were minor and mainly during the treatment days and resolved - days after each treatment and included malaise during the infusions, glucose intolerance on infusion days in one case, and hypertension in another case. conclusion: iv pulse steroid treatment should be considered in abpa as it was found effective with fewer side effects that should be expected from prolonged oral corticosteroid treatment. lung pathology in individuals with cystic fibrosis (cf) is linked to sodium hyper-absorption. the defective regulation of the epithelial sodium channel enac is thought to be a major contributor to reduced airway surface liquid (asl) volume and impaired mucociliary clearance of the airways. thus, strategies designed to inhibit enac function may result in clinical benefit. rna interference (rnai), mediated by short, double-stranded rna molecules, can be used to target complementary rna sequences for degradation via the rna induced silencing complex (risc). we investigated the possibility of using rnai to reduce expression of enac in the mouse lung, as proof of principle for a strategy to reduce sodium hyperabsorption in the cf lung. potent sirna molecules capable of efficient knockdown of enac alpha, beta and gamma subunits were identified in an optimised cell culture system utilising mouse kidney m cells ( , cells per -well; pmol sirna complexed with lipofectamine ; hour harvest) in which enac expression was quantified using real-time rt-pcr. approximately % knockdown of each enac subunit was observed with the most potent sirna molecules in this system. subsequently, sirna molecules were delivered to the lungs of female balb/c mice via hydrodynamic tail vein injection ( µg naked sirna, n= - ). after hours, lungs were harvested, rna extracted and enac mrna measured using real-time taqman pcr. whereas, enac subunit mrna levels in mice treated with a negative control sirna were not different from untreated mice (p < . ), delivery of enac alpha and beta-specific sirna molecules resulted in a reduction to . ± . % or . ± . % of the expression levels observed in untreated mice, respectively (p < . , mann-whitney u). interestingly, despite efficient knockdown in cell culture, in vivo treatment with the most potent enac gamma-specific sirna molecule led to no reduction in enac gamma expression compared with untreated controls (p > . ). these data show proof of principle that enac expression can be reduced in the lung using sirna. further work is needed to assess the functional consequences of inhibiting enac in the lung. gene therapy for cystic fibrosis lung disease will likely require longterm transgene expression; however, in the lung, many gene transfer agents have resulted in only transient gene expression. previously we have shown that the choice of enhancer/promoter elements has a strong influence on the duration of reporter gene expression following delivery of non-viral vectors to the mouse lung (gill et al., , gene ther, , . however, it is also possible that other elements of plasmid vector design play an important role. using a clinically relevant mouse lung aerosol model, we have studied the effect of varying the plasmid cpg-dinucleotide content on the duration of expression from plasmids that had an identical enhancer/promoter sequence. firstly we constructed a cpg-free plasmid vector containing a synthetic cpg-free luciferase gene under the transcriptional control of the human cmv enhancer/ef α promoter. secondly, we constructed a similar vector containing an identical enhancer and promoter but with a small number of cpg motifs, in the luciferase gene ( cpgs) and in the backbone sequence ( cpgs). we then investigated the level and duration of transgene expression following aerosol delivery of these plasmids complexed with the genzyme lipid gl to the lungs of balb/c mice ( . mg/ml pdna, mm gl , ml, pari lc+ nebuliser). total lung extracts were assayed for luciferase activity at days , , , & (n= per time-point). the cpgcontaining plasmid initially directed approximately -fold higher levels of reporter gene expression than the cpg-free plasmid (p< . both days and post-delivery). however, while expression from the cpg-containing plasmid subsequently fell slowly to background levels, expression from the cpg-free plasmid increased -fold to day , and remained at the peak levels observed with the cpg-containing plasmid until the end of the experiment at day (cpg-free plasmid -fold, -fold and -fold higher expression at days , and post-delivery respectively; p< . for each). these data suggest that the ability of a promoter/enhancer sequence to direct long-term expression in the mouse lung is dependent by the sequence of the plasmid backbone. one explanation for this observation is that a specific, but as yet unidentified, sequence exists in the cpg containing backbone that triggers transcriptional silencing. alternatively, the well-described host inflammatory response to cpg-containing dna, may be involved and the cpg content of the vector could be responsible for these differences. we are currently evaluating the potency and duration of effect of cftr expressing, cpgfree, plasmids in late-stage pre-clinical studies prior to the initiation of a further round of clinical studies of non-viral gene transfer in cf patients. background: novel approaches to cf therapy by improving/restoring cftr chloride channel function using gene therapeutic tools or small molecules are currently undergoing phase i and phase ii clinical trials. there remains a great need for accurate and practical methods of measuring cftr function in vivo to act as primary outcome measures of efficacy. we propose that an in vivo assay measuring cftr function in sweat glands offers several potential advantages over the nasal potential difference test. objective: to develop a reliable test of sweat gland function that is capable of measuring the range of cftr function in vivo. methods: we are performing repeated measures of sweat gland function in healthy controls (n= ), obligate heterozygotes (n= ), pancreatic sufficient (cfps, n= ) and insufficient cf patients (cfpi, n= ). sweat secretion is stimulated by iontophoresis ( µa/cm ) with % pilocarpine. to elucidate the most sensitive and discriminatory parameter of sweat gland function within this study group we are performing simultaneous measurements of: a) the transglandular potential difference of a stimulated skin area using an ecg electrode (espd) and b) single sweat gland potential differences in single sweat glands (spd). using a wescor® collector cup we also measure: c) sweat secretion rate and (d) sweat chloride concentration (sweat cl-). results: we report interim results of our ongoing study (table) . sweat rate was not different between the groups, but a gender difference was observed (mean±sd; male: . ± . µl/min/cm ; female: . ± . µl/min/cm , p< . ) as previously described. our preliminary data show that espd as well as sweat cl-allow good discrimination among healthy controls, cfps and cfpi (p< . and p< . respectively). spd show similar trends to espd but with greater overlap (differences not significant). conclusion: these encouraging preliminary results, particularly the espd method and sweat cl-following stimulation with a lower iontophoretic current, justify further efforts to complete enrolment of subjects and to further refine technical challenges such as minimizing the effect liquid junction potentials across the ecg electrode. results will also be compared to the classical sweat test using higher iontophoresis current and npd measurements in these patients. this study is supported by the ccff and genome canada. ino- , a prodrug, has been demonstrated to inhibit nasal potential difference (pd)in human cf nasal airway epithelia and cf mice and this effect is more potent with repeated dosing. as such it is being developed as potential therapeutic for cystic fibrosis. ino- cell entry is facilitated by its hydrophobic propionoxy(methyl)ester protecting groups which can be hydrolyzed by intracellular carboxyesterases after the prodrug enters the cell. once the protecting groups are removed, the drug (ino- ) with the ether-linked octyl group is expected to be more slowly metabolized. however, the precise kinetics of the uptake of ino- , its conversion to its active metabolite, and methods. confluent cultures of hela or t cells were incubated for varying periods of time ranging from min to hrs with medium containing [ h] ino- ( x cpm/ml) and µm ino- cold carrier to ascertain the rate of uptake. in pulse chase experiments after cultures were incubated for hours, they were washed and incubated with media without radiolabel for varying periods of time. after the indicated times, the media was removed and the cells were washed, harvested, extracted and the aqueous and organic extracts subjected to sax and reversed phase hplc respectively. results ino- , a prodrug, has been demonstrated to inhibit nasal pd in human cf nasal airway epithelia and cf mice and this effect is more potent with repeated dosing. ino- cell entry is facilitated by its hydrophobic propionoxy(methyl)ester protecting groups which can be hydrolyzed by intracellular carboxyesterases after the prodrug enters the cell. once the protecting groups are removed, the drug (ino- ) with the ether-linked octyl group is expected to be more slowly metabolized. however, it is not known whether the mucus present in cf airways could impede its access to airway epithelia or whether ino- would cross the epithelial layer to enter the bloodstream. here, in order to address these issues we measured its uptake into human tracheal epithelial sheets in the presence and absence of mucus and compared its transepithelial permeation to mannitol. we studied the uptake and transepithelial fluxes of [ h]ino- in primary cultures of human tracheal epithelium with transepithelial resistance of from to ohms.cm . the mucosal surface of some tissues was rinsed three times with pbs to remove mucus for comparison with control cells which were not rinsed. elisa for human airway mucins performed on the rinsings confirmed that three rinses sufficed to remove the mucosal mucous blanket. the development of long-term, safe and efficacious gene therapies for lung disease will require efficient gene delivery to stem cells which produce the differentiated progeny of affected epithelial tissues. recombinant lentiviral vectors are being considered for gene therapy applications for the lung because they can efficiently transduce a wide variety of stem and progenitor cell types. however, it is of concern that strong constitutive viral promoters have produced oncogenesis in x-scid patient recipients of retroviral vectors in part due to the disregulation of proto-oncogenes by the strong viral promoter/enhancer located near the integration site. we propose that regulation of transgene expression to the tissue or cell type of interest will enhance the safety of gene therapy for respiratory disease. in this study we have investigated the potential for respiratory specific transgene expression from integrated lentiproviruses in cell lines in vitro and in mice in vivo. the lentiviral vectors constructed for this study contained regulatory elements predicted to produce lung specific transgene expression: the surfactant protein c promoter (spc) for alveolar epithelial type ii cell (aecii) expression, the clara cell kd protein (cc ) for clara cell expression in the airway, and the jaagskiete sheep retrovirus (jsrv) promoter for expression in both cell types. transgene expression from the spc and cc vectors was restricted to aecii and clara cell lines respectively, while expression from the jsrv vector was observed in multiple respiratory and non-respiratory cell types. following intra-tracheal delivery of lentivector supernatant to mice, transgene expression was observed in aecii from the spc lentivector (n= mice), and in clara cells from the cc promoted lentivector (n= mice). transgene expression was not detected in non-respiratory tissues following intravenous delivery of cc and spc lentiviral vectors to neonatal murine recipients (n= and n= respectively). in summary, incorporation of genomic regulatory elements from the spc and cc genes resulted in respiratory specific transgene expression in vitro and in vivo. these vectors will provide a useful tool for the study of lung biology and the development of gene therapies for lung disorders such as cf. cystic fibrosis (cf) is associated with chronic pulmonary inflammation and progressive lung dysfunction, associated with the formation of oxidants derived from neutrophil myeloperoxidase (mpo). indeed, respiratory tract levels of mpo are extensive, and have been found to correlate with decreases in respiratory parameters or disease severity in cf. mpo-derived oxidants thus may play a role in perpetuating the progressive lung dysfunction associated with cf. based upon these premises, we reasoned that mpo could represent a novel therapeutic target for the treatment of inflammatory diseases such as cf. herein we report that substituted urea and thiourea compounds are potent inhibitors of mpo. utilizing tetramethylbenzidine (tmb) as a classical heme peroxidase substrate, we screened a large number of structurally varied urea and thiourea derivatives. alkyl, cycloalkyl, and aromatic ureas and thioureas all proved to be effective mpo inhibitors, however, with a high degree of variability in potency. of those compounds examined, substituted phenylthioureas proved to be the most potent mpo inhibitors. various ortho-, meta-, and para-substituted phenylthioureas (ptus) were then assessed for inhibitor potency. the most potent inhibitor of mpo was -chlorophenylthiourea (ic = nm, ki = nm). the potency of ortho-ptus as mpo inhibitors showed a linear relationship with ortho sigma substituent constants (reflecting resonance, steric and inductive effects). moreover, cyclic voltametry experiments with ortho-substituted ptu compounds revealed a linear relationship between inhibitor potency and thiourea oxidation potential. this correlation is indicative of a substituent effect on the relative ease of sulfur oxidation within the thiourea moiety. this finding suggests that the redox-active sulfur plays a crucial role in mpo inhibition, perhaps via a direct interaction with the heme of mpo. thioureas proved to also inhibit the chlorinating activity of mpo, whereas sustituted ureas were not effective in this regard. both ureas and thioureas were also effective inhibitors of dityrosine formation by mpo. these findings will help facilitate the rational design and optimization of mpo inhibitors with even greater potency, which could serve as novel therapeutic agents for the treatment of cf. gene therapy is the transfer of a normal copy of the cftr gene and should, in theory, provide a long-term cure for cf. the development of prenatal screening programs for cystic fibrosis has provided an opportunity to identify cf patients in utero, and may provide an opportunity to treat the disease before birth, prior to the onset of disease symptoms. the long-term goal of our study is to investigate the therapeutic potential of delivering the normal cftr gene to epithelial stem cells in fetal cf mice by in utero administration of recombinant lentiviral vectors carrying the normal cftr gene. this would provide us with the unique opportunity to "correct" the chloride channel defect in the epithelial stem cells, which give rise to the epithelial cells in the tissues affected by cf at a very early stage. the aim of the current study is to optimize the in utero gene delivery of recombinant lentiviral vectors, carrying an egfp reporter gene to epithelial stem cells in fetal mice. mice have been used for these studies, as mouse strains with a variety of cf mutations are available. in all studies, the uterus was exteriorized following a mid-line laparotomy, and the injections were made directly into the amniotic fluid of individual fetuses. in the first studies, the survival of pregnant females and pups following saline injection at e . (n= ) and e . (n= ) was evaluated. all five females survived the procedure and an average of % ( / ) of the pups present at the time of injection were born alive. this experiment demonstrated that the surgical procedures were reasonably safe. in the next experiments, we injected . ul lentiviral supernatant (~ . x iu/pup) into the amniotic fluid of each pup and evaluated the effect of the gestational age pups on transduction efficiency. the pregnant mice were allowed to carry the fetuses to term. at p , p and p , - pups of each litter were sacrificed and skin, trachea, lung, stomach and intestines were harvested for analysis of gene transfer and egfp transgene expression. all but one pup ( / ) from the e . time point aborted prior to parturition. from the e . and e . injections, / and / pups were born naturally, respectively. at e . and e . , proviral sequences were observed in the skin, trachea and stomach, although the level of transduction varied among individual fetuses and tissues. immunofluorescence analysis on the skin of one mouse with a high proviral copy number demonstrated that some of the egfp positive cells were also positive for vimentin or f / , markers of mesenchymal cells and macrophages, respectively but negative for pancytokeratin. our preliminary studies suggest that in utero gene therapy with lentiviral gene delivery may be safe, however ongoing studies will provide more in depth analysis on safety and feasibility of delivering the normal cftr cdna to epithelial stem cells in the tissues affected by cf in utero. future studies will investigate the therapeutic potential of delivering a lentiviral vector containing the cftr gene to fetal pups in a murine model of cf. supported background & objectives: cystic fibrosis is characterized by progressive loss of lung function resulting from chronic bacterial infection and concomitant airway inflammation. while a number of bacteria are known pathogens in cf, mounting evidence suggests that the lungs of cf patients harbor numerous bacterial species, and that interactions within this ecosystem may affect outcomes. however, the composition of this community is not well characterized. we therefore initiated work to profile the bacterial diversity of the lungs of cf patients with end-stage disease by s rrna gene sequencing. methods: genomic dna was purified from snap-frozen explanted lung tissue of randomly selected cf patients undergoing transplantation in toronto between and . bacterial s rrna genes were amplified from each sample using broad-spectrum pcr primers, and pcr products used to generate s rdna libraries for each patient. s rdna profiles for each patient were generated by automated dna sequencing of up to randomly selected clones per library, and tentative species assignments made by comparison to known s rrna gene sequences using phylogenetically-based analyses. results: sequence analysis of s rdna sequences from this preliminary set of patients (avg. = sequences/patient; range = - ) demonstrates a range of complexity of the microflora in cf lungs, with some patients showing a single dominant organism and others showing multiple co-existing species. in total, over species have been identified to date in this set of patients. these include species identified previously by clinical microbiology, including pseudomonas aeruginosa (detected in / patients), and burkholderia species (detected in / patients known clinically to be infected). numerous organisms not previously reported in the lung were also identified, including several from the taxonomic order burkholderiales. interestingly, while organisms commonly associated with cf were detected in patients transplanted throughout the year, a cluster of four plant-associated species demonstrated striking seasonal variation in rates of detection. conclusions: the lungs of cf lung transplant patients harbor a diverse bacterial community. sequencing of s rrna genes is a powerful method to profile this diversity. correlation of these profiles with clinical information may help identify new patterns of infection and important interactions between species in this community. introduction and aims: pulmonary infection, primarily with pseudomonas aeruginosa, is the leading cause of morbidity and mortality in cystic fibrosis (cf) patients. we have demonstrated that anaerobic bacteria, belonging primarily to the genera prevotella, veillonella, propionibacterium and actinomyces, can be cultured from the sputum of cf patients. the aim of this study was to determine the antibiotic susceptibility of these anaerobic isolates grown planktonically and as biofilms. methods: the planktonic susceptibility of anaerobic isolates to antibiotics used in the treatment of cf (meropenem, piperacillin/tazobactam) as well as to antibiotics traditionally used to treat anaerobic infection (clindamycin, metronidazole and ampicillin) was examined using e-test® strips. selected anaerobic isolates were grown as biofilms for or hours in well trays prior to challenge with antibiotic. bacterial biofilm formation was determined using crystal violet. results: the planktonic susceptibility of anaerobic isolates ( prevotella, veillonella, propionibacterium, actinomyces and additional isolates) comprising different genera was determined. although all of the anaerobic isolates examined were sensitive to meropenem when grown planktonically, high levels of resistance to the anti-anaerobic drugs, metronidazole and clindamycin were observed. examining susceptibility by genera, the veillonella were the most resistant to piperacillin/tazobactam and the propionibacterium the most resistant to metronidazole. furthermore, several isolates, including of the prevotella isolates examined, were found to be resistant to multiple antibiotics. the susceptibility of anaerobic biofilms to antibiotics was found to be isolate and antibiotic dependent. generally antibiotic treatment of hour old biofilms was often able to eradicate biomass and prevent biofilm formation; however, antibiotic treatment after hours of biofilm formation with up to x the mic of an antibiotic often failed to eradicate the biofilm. conclusion: these results suggest that alternative antibiotic treatment regimes may be necessary to treat cf pulmonary infection if anaerobes are present. these results also suggest, based on in vitro susceptibility, that meropenem and not metronidazole or clindamycin would be the most effective antibiotic in treating any anaerobes present in cf pulmonary infection. early eradication protocols for the first appearance of pseudomonas aeruginosa (psa) have become standard practice in many cystic fibrosis (cf) clinics. the details of such protocols often vary significantly between clinics, making it important to compare efficacy from one approach to another. however, comparison of reported results is currently very difficult. this is not only because of variations in completeness of reporting, but especially because calculation of average time to regrowth cannot by definition include patients who remain free of psa regrowth at the time the "average" is calculated (nor those in whom a re-appearance of psa is found to be due to a different strain). such results would be better expressed using "time to event" (tte) statistics (analogous to "life tables") and different protocols can be better compared using this methodology. we report for the first time, results from our psa early aggressive eradication protocol for the period - , expressed using tte statistical analysis. children are seen in clinic on average times yearly for full assessment, and at each clinic visit sputum or pharyngeal cultures obtained by the physiotherapist. standard treatment for "first growth" pseudomonas aeruginosa consists of weeks of intravenous antibiotics (piperacillin mg/kg/day plus tobramycin mg/kg/day) followed by weeks of oral ciprofloxacin ( - mg/kg/day) and inhaled colymycin ( mg bid) for months. a total of treatment courses have been completed for the period - with a % clearance rate, defined as or more consecutive negative cultures for pseudomonas aeruginosa over months. since the initiation of this protocol, the percentage of patients colonized with psa in our cf pediatric clinic has declined from % in , down to % in . subsequent regrowth of psa occurred in / of patients. treatment courses may then be repeated a second, third, or fourth time. to determine whether these repeat psa isolates are newly acquired or identical to the previous isolate, rapd typing of these isolates is routinely assessed. a total of isolates have been rapd tested and have identified the same strain as the previous isolate in ( %) and a different strain in ( %)treatment courses. the average time to regrowth for those who had a recurrence of psa following this treatment protocol was months, but this does not include those patients who have had no further growth of psa for periods up to years. to reflect the results for all patients, including those who have not as yet had further regrowth of psa, we have constructed tte curves. these measures can be used to compare results using our protocol with results from other approaches to clearing pseudomonas aeruginosa in cf. retsch-bogart, g.z. inhaled antibiotics have been part of the therapeutic armamentarium for patients with cf for decades. we studied the effect of inhaled aztreonam on respiratory symptoms in cf in a phase , double blind, placebo (pl) controlled trial of azli, a novel formulation of aztreonam, which enrolled patients with cf from centers in the us, canada, australia, and new zealand. all patients have completed study participation. inclusion criteria included age ≥ years, pa in sputum or throat swab, fev ≥ % to ≤ % predicted, and no use of anti-pseudomonal antibiotics in the previous days. following a day screening period, patients were treated for days with either azli mg or pl. study drug was administered tid using the pari eflow® electronic nebulizer after pre-treatment with bronchodilator. concomitant standard cf therapies were allowed, with the exception of anti-pseudomonal antibiotics, azithromycin and hypertonic saline. the primary endpoint was change from baseline to day in scores from the cystic fibrosis questionnaire-revised (cfq-r) respiratory domain, a validated patient-reported measure of respiratory symptoms. other efficacy measures included change in pulmonary function (fev , fvc, and fef - ), number of hospital days and number of courses of iv, oral or inhaled anti-pseudomonal antibiotics. microbiological endpoints included change in pa bacterial density in sputum, change in susceptibility of pa to aztreonam, and emergence or disappearance of other pathogens. safety evaluations included adverse events, airway reactivity (defined as acute decrease in fev ≥ % at minutes after dosing) and clinical chemistry and hematology. complete efficacy and safety results from this trial will be presented. salam, a.p.; orchard, c.; wee, a.; hodson, m. introduction: oral ciprofloxacin is often used for pseudomonas aeruginosa (psa) infections in patients with cystic fibrosis (cf) not requiring intravenous antibiotics. oral chloramphenicol is commonly used at the royal brompton hospital (rbh) as an alternative to ciprofloxacin, but less so in other cf centres due to concerns about aplastic anaemia and uncertainty regarding effectiveness. this study aimed to address three questions: ) what proportion of psa is sensitive to ciprofloxacin and/or chloramphenicol? ) were there any adverse haematological effects with the use of oral chloramphenicol at rbh? ) how effective is chloramphenicol in comparison to ciprofloxacin? study design: we carried out a retrospective review from the rbh cf database ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) together with analysis of patients' notes. methods: ) psa sensitivities to ciprofloxacin and chloramphenicol were recorded from sputum samples taken in late . ) all blood dyscrasias in patients who had received chloramphenicol from to were reviewed. ) the database was searched for patients who had received a course of either oral chloramphenicol or ciprofloxacin from to . patients from each group were randomly selected. fev , fvc and oxygen saturations pre and post treatment completion were analysed, as were markers of symptom improvement. results: ) sputum samples from patients grew psa. some were sensitive to only one antibiotic, and there were some cross-sensitivities, but in total . % were sensitive to chloramphenicol and . % to ciprofloxacin. ) patients received oral chloramphenicol and oral ciprofloxacin from - . multiple courses were often given, but usually no more frequent than monthly with chloramphenicol. over years, cases of blood dyscrasia were recorded, none due to chloramphenicol, and all recovered. ) in the ciprofloxacin group: the mean oxygen saturations improved by . % (p . ), the mean fev improved by . % (p . ), and the mean fvc by . % (p . ). in the chloramphenicol group: the mean oxygen saturations improved by . % (p . ). ), the mean fev improved by % (p . ) and the mean fvc improved by . % (p . ). % of patients in the chloramphenicol group and % of patients in the ciprofloxacin group reported symptom improvement. a significant proportion of psa is sensitive to chloramphenicol and in over years there have been no documented cases of related aplastic anaemia. clinical improvements with choramphenicol comparable to ciprofloxacin were also recorded. our data shows chloramphenicol is safe and may be the oral antibiotic of choice in ciprofloxacin allergy or psa resistance to ciprofloxacin. a randomised prospective study is needed to further evaluate the efficacy of chloramphenicol in comparison to ciprofloxacin. pseudomonas aeruginosa lung infections are one of the main factors causing disease in cf patients. at present the generally recommended treatment is repeated courses of antibiotics. this has resulted in fewer infections and a markedly improved prognosis for the patients. but it has also severe drawbacks such as antibiotic resistance, disturbed normal flora and allergenicity. antibiotic resistance is an emerging problem world wide and alternatives are urgently needed. specific chicken antibodies, anti-pseudomonas igy, have potential both as an alternative and a complement to antibiotics (pediatr pulmonol ; : ) . anti-pseudomonas igy prevents p. aeruginosa from adhesion to epithelial cells and has affinity for flagellin in vitro. humans do not produce anti-igy antibodies and oral administration of igy is generally regarded as safe. the risk that bacteria should develop resistance to igy is extremely low. in total, cf patients have received antipseudomonas igy for up to years. since november, the drug is given to a group of cf patients (at present patients) on individual permissions granted by the swedish mpa and reimbursed by the swedish government. in our first study about the effect of igy there were . positive cultures/ months, compared to . / months in a control group. up to december , , nearly five years later, the effect is maintained with . / months for the whole study period. we are at present compiling the data from the control group during the same period. only two siblings in the igy-treated group have been chronically colonized (with an identical p. aeruginosa strain), which still is non-mucoid after . and . years. during the whole treatment period there have been no cultures positive for mucoid p. aeruginosa or b. cepacia and other pathogens (s. maltophilia, a. xyloxidans, atypical mycobacteria and a. fumigatus) have only appeared sporadically -possibly due to the relatively low use of antibiotics. all patients have preserved pulmonary functions and nutritional status. there have been no adverse events. thus treatment with anti-pseudomonas igy has diminished the number of positive cultures and delayed the onset of chronic infection. the need for antibiotics is reduced as well as their adverse side effects. in conclusion, igy is an important complement to antibiotics for prevention of p. aeruginosa infections in cf. introduction and aims: we have previously shown by culture that the lungs of clinically stable cystic fibrosis (cf) patients are not only colonised by commonly recognised aerobic bacteria, such as pseudomonas aeruginosa, but also by a range of potentially pathogenic anaerobic species. the aim of this study was to determine whether anaerobes are also present in the sputum of cf patients with an acute exacerbation of pulmonary infection. methods: sputum samples were collected and processed using strict anaerobic bacteriological techniques, prior to commencing and at the end of antibiotic therapy, from adult cf patients admitted for treatment of an acute exacerbation of pulmonary infection. bacteria within the samples were detected by plating on selective agars, quantified by total viable count and identified by pcr and sequencing of s ribosomal rna genes. as a control induced sputum samples were collected, using hypertonic saline, from healthy volunteers who did not have cf and processed using similar methods. results: anaerobes from a range of species including prevotella, veillonella, propionibacterium, actinomyces and gemella were detected in high numbers (up to x cfu/g of sputum) from all patients prior to commencing antibiotic therapy with the predominant primary pathogens (p. aeruginosa or b. cepacia complex) detected in similar numbers. anaerobic bacteria were also detected in sputum samples at the end of antibiotic treatment and for / ( %) patients they were present in lower numbers than detected before antibiotic treatment. moreover, in / ( %) patients there was greater than a one log reduction in the total viable count of anaerobes. similar data for detection of the predominant aerobic pathogens was also collected for of these patients. p. aeruginosa or b. cepacia complex was present in lower numbers following antibiotic treatment in / ( %) patients and in / ( %) patients there was more than a one log reduction in the total viable count. anaerobes were not detected in the induced sputum samples from volunteers and in the remaining samples they were present in much smaller numbers, ranging from to cfu/g of induced sputum, than detected in cf sputum. the most commonly isolated bacteria from the induced sputum samples were the actinomyces and streptococcus, with prevotella cultured from only samples. no veillonella and propionibacterium species were isolated from the induced sputum samples and combined with the low frequency of isolation of prevotella species, these results strongly suggests that the prevotella, veillonella and propionibacterium species recovered from cf sputum samples are not oral contaminants. conclusion: these results indicate that anaerobes are present in large numbers within the cf lung during an acute exacerbation of pulmonary infection. their presence could be of important clinical relevance to cf patients as they may contribute significantly to the inflammatory process. airway infection and inflammation cause the majority of morbidity and mortality in cystic fibrosis (cf). the microbiology of cf is complex. not only are the routinely identified pathogenic bacteria present, increasingly a number of other bacteria are associated with pulmonary disease. in addition, the normal microbiota likely contribute to airway disease in cf via intracellular signaling, but remains largely unstudied. the application of culture independent methods for bacterial identification in cf airway secretions provides for the first time the ability to characterize the bacterial communities in the cf airway comprehensively and efficiently. here we present the results of a two-year study of cf airway samples to describe the bacterial communities present by ribosomal rna sequences. we have examined airway specimens from subjects ( cf, controls). these results provide a unique perspective on cf airway microbiology. bacterial communities identified in sputum were more complex than communities identified in bronchoalveolar lavage fluid (balf). however, pulmonary status determined the level of complexity, with higher complexity observed during stable pulmonary function. we identified a collection of anaerobic bacteria that were present at high levels during pulmonary exacerbation in a subset of cf subjects. detection rates ranged from - % of cf subjects examined depending on the specific organism (table ). these organisms of interest are suspected to be involved in pulmonary exacerbation especially when cultures for standard cf pathogens are negative (~ % in our center). preliminary data will be presented from quantitative real-time pcr experiments to track these organisms. we also will present the application of new meth-ods for community comparisons. this analysis provides the ability to look at how clinical information from individuals with cf corresponds to the microbial communities that occur in the airway. overall, the results point to a much richer bacterial community associated with the cf airway than previously thought. the results also point to new opportunities to understand the differences within the cf population, and thus potentially improve individual patient outcomes. cystic fibrosis (cf) patients are characterized by persistent microbial colonization of the airways and recurrent pulmonary infection. however, due to the domination of bacterial communities present in cf sputum by a small number of species (typically pseudomonas aeruginosa), culture-independent approaches to characterize the depth of bacterial diversity in cf sputum have yielded limited information. here we describe application of a novel microarray, the s rrna phylochip to comprehensively describe the bacterial diversity present in temporal sputum samples of cf patients who experienced periods of exacerbation (defined as hospitalization) and remission. in addition, these samples permitted assessment of bacterial community dynamism during periods of antimicrobial administration in these patients. phylochip analysis identified bacterial taxa present across these patient samples, including a large number of known human pathogens. antimicrobial administration caused a profound decrease in bacterial diversity in all patients examined. despite this, a sizeable fraction of each community remained stable or proliferated during the period of antimicrobial administration, suggesting many of the bacterial taxa present were resistant to the antimicrobials administered. taxa exhibiting this behavior included p. aeruginosa, arcobacter cryaerophilus, streptococcus constellatus and burkholderia mallei amongst other pathogens. these results suggest that complex polymicrobial communities exist in cf sputum that exhibit significant antimicrobial driven dynamism and consist of multiple antimicrobial resistant pathogens. future studies will focus on whole community gene expression to determine which virulence systems are activated in response to bacterial community perturbations and contribute to pathogenic processes in cf airways. the lungs of chronically infected cystic fibrosis (cf) patients have been intensively studied. it is well documented that pseudomonas aeruginosa is the predominant pathogen in cf (høiby , baurnfeind et al. , koch , and that p. aeruginosa accumulates in heaps (aggregates), detected in cf sputum (høiby ) and intraluminal in the lung (baltimore et al. ) . the bacterial density is highest in the bronchi (potts et al ) and these p. aeruginosa microcolonies are embedded in a matrix (lam et al. ) . the morbidity of the infection is due to an extensive and ongoing pmn response which apparently does not eradicate the bacteria, instead leading to slow degradation of the lung. in addition, even highest deliverable doses of antibiotics fail to clear the bacteria completely, though survival has significantly increased the last years due to aggressive anti p. aeruginosa therapy (koch and høiby ) the present study was performed to investigate the significance of the aggressive therapy for the distribution of p. aeruginosa and how p. aeruginosa is organized and persists in the cf lung. the material used was: autopsies from dead short term colonized cf patients (n= ) obtained before today's aggressive antibiotic treatment , and explanted lungs from long term p. aeruginosa infected cf patients (n= ) (i.v. antibiotics every rd month since + inhalation of colistin daily since . histological sections of the lungs were investigated using he stain, gram stain, alcian blue, antibodies against alginate and p. aeruginosa specific pna-fish combined with bacuni pna-fish and with dapi as counter stains. due to the high specificity of the pna-fish probes employed in this study, our results provide strong evidence for that before the aggressive antibiotic therapy, p. aeruginosa infected and destroyed the cf lung due to fast spreading into the respiratory zone. today antibiotics suppress but can not eradicate the bacteria from the conductive zone, whereas the remaining respiratory zone is protected from massive biofilm infection for prolonged time. the conductive zone serves as a reservoir; here the bacteria are organized in microcolonies embedded in puss, a trait which is independent on the time course of the infection and amounts of antibiotics. these microcolonies consisted solely of p. aeruginosa. in addition, we found no bacteria adhering to the epithelial tissue. the pus consisted mainly of leukocytes, surrounding the microcolonies. a smear of dna and dead leukocytes were detected just around the microcolonies, possible due to the quorum sensing dependent rhamnolipid killing recently described by us (jensen et al. ) . we conclude that p. aeruginosa persists in the cf lung due to its ability to create microcolonies i.e. biofilms. within these biofilms the bacteria are protected against antibiotics and the host defence. respiratory viral infections, including influenza (flu) and respiratory syncytial virus (rsv) contribute to the morbidity of children with cf. additional human respiratory viruses have been identified in children such as human metapneumovirus (hmpv) and coronavirus (cov), but these have not been examined in cf patients. in our laboratory, pcr assays have been developed to identify both traditional and newer respiratory viruses in healthy and immunocompromised children. these assays are usually performed on nasal swabs or nasal wash samples. however, oropharyngeal (op) and sputum samples are routinely obtained in cf for bacterial culture. the goals of this trial are to determine whether these available samples can be used for viral pcr studies and to examine the epidemiology of respiratory viruses in cf. for this single center observational trial, cf patients, ages . to . yrs (mean . yrs), have been recruited. subjects provide op and nasopharyngeal (np) samples at quarterly clinic visits and with acute pulmonary exacerbations requiring hospitalization; sputum is also collected if patients can expectorate. np and op samples are pooled when both are available. viruses assayed are flu a and b, rsv, parainfluenza (piv) , , and , hmpv, cov, adenovirus (adv), and rhinovirus (rhv). subjects are being followed for two respiratory viral seasons. we report here the methodology of viral pcr detection in cf sputum, as well as the results of viral detection during the first respiratory viral season in the seattle community. the mean length of time subjects were followed was . yrs (median: . yrs, range: . - . yrs). overall, samples ( % of the samples analyzed) were positive. twenty-four subjects ( . %) had at least one viral infection detected and ( . %) had two viruses at different visits. both sputum and pooled op/np samples were available at visits and were concordant in % of cases. among the discordant cases, it was more common for sputum to be positive and op/np to be negative ( . %) than the converse ( . %). the most frequently identified viruses were rhv ( isolations); piv and cov were the next most prevalent with and isolations, respectively. in conclusion, sputum viral pcr shows promise for the diagnosis of both traditional and novel viral infections in cf. as part of ongoing clinical trials in cf, non-fermenting gram negative bacilli (nfgnr) identified at site laboratories are often sent to the tdn core microbiology laboratory for confirmation of identification. among nfgnr from cf patients, p. aeruginosa are the most frequently isolated and also are acknowledged to be the easiest to identify. however, in a survey of "p. aeruginosa" isolates sent to the laboratory in the past year, pcr identified ( %) as alternate species. these included: non-aeruginosa pseudomonads, stenotrophomonas maltophilia, achromobacter xylosoxidans, ralstonia spp., alcaligenes spp., brevidimonas spp., and bordetella bronchiseptica. identification of these nfgnr can be difficult; however, p. aeruginosa is an important isolate for the clinical microbiology laboratories in all cf centers to be able to identify. many methods are available including conventional biochemical testing and rapid identification kits. in the tdn core laboratory we previously demonstrated the importance of combining phenotypic identification (morphology plus a short set of biochemical tests) and genotypic identification ( s rdna sequencing of v and/or pcr using three primer sets) and developed an algorithm for testing. in this study, we examined all of the cf cultures performed in the clinical microbiology laboratory at chrmc over the past two years to determine the number of isolates for which it was necessary to perform genotypic identification in nfgnr. among this collection of nfgnr from clinical cultures sent to the microbiology laboratory from the seattle children's hospital cf center, pcr identified isolates as burkholderia cepacia complex. next most commonly identified were a. xylosoxidans ( ), p. aeruginosa ( ) and non-aeruginosa pseudomonas spp. ( ). three isolates remained unidentified, even with pcr sequencing of the v determinant. the clinical microbiology laboratory at chrmc has served as the core microbiology laboratory for tdn clinical trials for years and thousands of cf samples are processed annually. the result is that there is increased experience with cf isolates among the laboratory personnel. however, even with experienced technologists, a significant number of nfgnr required genotypic identification in the past two years. thus, it is important for clinical microbiology laboratories with less experience in the identification of cf nfgnr to utilize additional molecular techniques or send problematic isolates to a reference laboratory. based on these results we propose an algorithm for the most efficient means of identifying nfgnr from cf samples using a combination of tests including phenotypic (colony morphology, biochemical testing) and genotypic identification (pcr, sequencing). background: the epidemiology of s. aureus has been changing in both cf and non-cf patients. in non-cf patients, methicillin-resistant s. aureus (mrsa) has traditionally caused infections in patients with risk factors such as hospitalization, surgery, and anterior nares colonization. recently, community-acquired (ca-) mrsa infections have increased among patients without known risks. in cf, colonization of the anterior nares with methicillin-susceptible s. aureus (mssa) is associated with transmission between patients and household members. the prevalence of respiratory tract colonization/ infection with mrsa has increased in cf, but little is known about risk factors for mrsa, including possible transmission among families, and if strains are ca-or healthcare-acquired. methods: this is a case-control, multicenter study to investigate potential risk factors for mrsa among children with cf - years of age. subjects are enrolled from cf centers in the new york metropolitan area that have a mrsa prevalence ranging from % to %. cases are children with a positive respiratory tract culture for mrsa within the past years. controls ( age and center matched per case) are negative for mrsa. the aims of the study are: [ ] to determine the point prevalence of anterior nares colonization with s. aureus in children with cf and their household members; [ ] to assess potential risk factors for mrsa by administering a survey to the parents of subjects inquiring about factors such as crowding, pets, and participation in contact sports and by reviewing the medical record for antibiotic use, hospitalization, and surgery; [ ] to determine potential transmission of s. aureus within families and centers by assessing the molecular epidemiology of strains using pulsed field gel electrophoresis and meca type of mrsa isolates. results: to date, cf subjects ( cases, controls), mean age . years, and household members ( to per subject) have been enrolled. preliminary data demonstrate positive anterior nares cultures among cases, controls, and household members to be / ( %), / ( %), and / ( %), respectively. of the s. aureus isolates, were mssa and were mrsa ( cases, household members of cases, and household members of controls). during the past months, household members have been hospitalized and one has stayed overnight in a nursing home; had staphylococcal infections and had skin infections. data collection and molecular analysis are ongoing. conclusions: we expect this study to provide insight into risk factors for respiratory tract colonization/ infection with mrsa, the role of ca-mrsa in cf, and potential strategies to prevent mrsa. aims: . revise infection control guidelines for the institution . establish outpatient infection control policies consistent with those used for hospital inpatients . cohort young and recently diagnosed cf patients without pseudomonas or mrsa to clinic days separate from colonized patients . establish a protocol for eradication of mrsa in newly colonized patients. methods: working with representatives from hospital epidemiology, the cf center, inpatient units, outpatient clinics, respiratory therapy, and cf families, a uniform infection control policy for cf patients in all settings (inpatient, outpatient, home care) was drafted and revised. arrangements were made to cohort newly diagnosed patients and children < years free of pseudomonas and mrsa to separate clinic days from colonized patients. review of the mrsa positive patients in our pediatric population, for whom data was available, showed median age of acquisition of years (range mo to yrs). for patients the first culture obtained at diagnosis or transfer from another center was mrsa positive ( mo, yr, yr). an eradication protocol for newly acquired mrsa has been developed involving treatment with oral and topical antibiotics and phisohex or clorox washes for days, plus changing disposable respiratory care equipment at the beginning and end of the treatment period. surveillance cultures of nose, groin, and airway or sputum are done after completion of the treatment protocol and re-treatment initiated if still positive for mrsa. conclusion: using a three-tiered approach of infection control guidelines, cohorting, and mrsa eradication protocol we expect to decrease our overall rate of mrsa and protect our newly diagnosed and young cf patients from acquisition. objective: studies have shown that respiratory pathogens can be transmitted within cf centers. in a recent cross-sectional study at cf centers, we assessed the rate of bacterial shedding by cf patients during office visits and noted that % of patients carried respiratory flora on their hands. the current study was performed to assess the effectiveness of alcohol-based rubs on hand carriage of respiratory pathogens by patients during the course of office visits. methods: four bacterial organisms were chosen for study: pseudomonas aeruginosa (pa), staphylococcus aureus (sa) [including methicillin-resistant (mrsa) strains], stenotrophomonas maltophilia (sm) and burkholderia cepacia complex (bcc). at the beginning of clinic visits, hands of study patients were cultured using the "glove-juice" technique (gjt) [ ] . hand hygiene was then performed using an alcohol-based rub. at the end of the visit, hands were again sampled using the gjt. samples were sent to the microbiology laboratory at dartmouth-hitchcock medical center for culture and identification of study organisms. hand and respiratory tract isolates were compared using pulsed field gel electrophoresis (pfge). recovery experiments were also performed to assess the sensitivity of the gjt. results: samples were collected from encounters ( adult; pediatric). median encounter time was min (range . - . min). recovery experiments determined that the level of detection for each study organism using the gjt was ~ colony forming units. confirmation of matches between hand and respiratory isolates was determined by pfge. the number of patients with study organisms recovered from the respiratory tract was: pa, sa, mrsa, sm, and bcc. the overall hand contamination rate with a matched respiratory tract isolate at either culturing time was % ( % ci, . - . %). before use of the alcohol rub, hand contamination was . % ( % ci, . - . %). in those patients with hand contamination by respiratory pathogens prior to alcohol rub, . % ( % ci, - %) had negative cultures at the end of the clinic visit. however, there was strong evidence of hand contamination during the encounter, despite hand hygiene, as the overall rate at the end of visits was . % ( % ci, . - . %). there was a trend toward increased hand contamination in those suffering from pulmonary exacerbations versus clinically stable patients ( . % [ % ci, . - %] vs. . % [ % ci, . - %], p= . ). conclusions: contamination of patient hands by respiratory tract pathogens is observed in the outpatient setting in approximately % of patients. a single application of an alcohol-based hand rub, while safe and inexpensive, has limited antiseptic effectiveness during the full course of a typical cf clinic visit. repeated use of alcohol-based hand rubs during outpatient encounters would likely be more effective. these data support recent recommendations in the cf foundation consensus guidelines for infection control [ ] (nthi) is the most common initial bacterium infecting the airways in cystic fibrosis (cf). nthi infection usually precedes pseudomonas aeruginosa infection in many chronic lung diseases, including cf, emphysema, diffuse panbronchiolitis, and immotile cilia syndrome. it is hypothesized that nthi acts as a gateway organism paving the way for subsequent infection with p. aeruginosa, however, the mechanism by which this occurs remains poorly understood. we used a novel co-culture model of persistent nthi biofilm infection on airway epithelial cultures to study epithelial immune responses following nthi biofilm infections. objective: we hypothesized that prolonged exposure to nthi biofilms causes airway epithelia to become tolerant to inflammatory stimuli and show subsequent decreased innate immune responses. methods: nthi were inoculated onto the apical surface of calu air-liquid interface epithelia and grown in co-culture. rna from sets of paired airway epithelial cultures at , , and hours both with and without nthi inoculation was isolated and hybridized on custom affymetrix chips. data were normalized with rma and data biases were removed using the mixed model anova method. differentially expressed gene analyses were performed using anova, and pathways analyses were performed using gene set enrichment analysis (gsea). to test the hypothesis that prolonged nthi infection induces tolerance, epithelial cultures were pre-treated with either days of nthi or days of pbs before stimulation with pbs, il β ( ng/ml) or th cfus of live p. aeruginosa before subsequent measurement of epithelial responses. results: both rma and gsea analyses of microarray data showed many innate immune and pro-inflammatory responses at early timepoints that largely returned to baseline after days of co-culture, despite an increasing apical nthi biofilm infection. stimulation with il β or p. aeruginosa after days of nthi infection resulted in significantly decreased il production compared to uninfected controls. nthi treated cultures subsequently infected with p. aeruginosa also displayed increased tight junction integrity and resistance to cell culture death. conclusions: persistent nthi infections on airway epithelia resulted in increased tolerance to bacteria and inflammatory stimuli and increased resistance to bacterial toxicity. airway epithelial tolerance may contribute to a failure to clear subsequent bacterial infections. these phenomena would help to explain how early infections with nthi may promote chronic bacterial infections and p. aeruginosa acquisition in cf and other nthi related airway diseases. the colonization of cf airways by p. aeruginosa leads to intractable and persistent lung infections that resist permanent eradication by antibiotics. the lack of efficacy of current therapies is believed to be due to the formation of p. aeruginosa antibiotic resistant biofilms in the cf airways. however, little is known about biofilm formation on human airway epithelial cells. thus, we designed a continuous flow, live cell imaging system to grow p. aeruginosa biofilms on glass or on the apical surface of polarized human cells. cfbe ohuman airway epithelial cells homozygous for the ∆f mutation (cfbe), and cfbe o-cells complemented with wt-cftr (cfbe+wt-cftr) were grown as polarized monolayers at an air-liquid interface and gfp-labeled p. aeruginosa were applied to the apical surface of the cells. after hours biofilms did not form on glass but large bacterial macrocolonies developed on cfbe cells. surprisingly, biofilms also developed on cfbe+wt-cftr cells: however, p. aeruginosa biomass on cfbe+wt-cftr cells was significantly reduced compared to cfbe cells ( . ± . µm /µm vs . ± . µm /µm ). the minimal bactericidal concentration for tobramycin was > , µg/ml for p. aeruginosa grown on cfbe and cfbe+wt-cftr cells. these results explain why tobramycin given clinically, which is~ , µg/ml in bronchoalveolar fluid collected from cf patients, fails to eradicate the persistent infection by p. aeruginosa. p. aeruginosa also formed biofilms on human bronchial epithelial cells, which produce mucus. indeed, biomass was similar on cfbe+wt-cftr cells, which do not produce mucus, and human bronchial epithelial cells. to determine if human airway epithelial cells secrete factors that facilitate biofilm formation, conditioned medium was collected from cfbe and cfbe+wt-cftr cells and applied to p. aeruginosa grown on glass. under these conditions, biofilm formation was dramatically facilitated, with a biomass of . ± . µm /µm when p. aeruginosa was grown in conditioned medium collected from cfbe+wt-cftr cells and . ± . µm /µm when grown in conditioned medium collected from cfbe cells. the secreted factor(s) were greater than kda, as assessed by size exclusion. our results suggest that human airway epithelial cells secrete factor(s) that facilitate biofilm formation by p. aeruginosa, in the presence and absence of mucus, and that secretions from cf cells are more effective in promoting biofilms than secretions from non-cf cells. the ability of non-cf cells to facilitate biofilm formation appears to contradict the observation that non-cf lungs do not harbor p. aeruginosa. we propose that the innate immune response in the non-cf lung effectively eliminates p. aeruginosa from the lung, thus, biofilms do not form even thought the airway epithelial cells secrete factors that have the potential to facilitate biofilm formation. however, in cf when mucociliary clearance is compromised, p. aeruginosa accumulates in the lung and secretions by cf airway epithelial cells greatly enhances the formation of drug resistant biofilms (supported by the nih (ro ai , ro -hl- , p -rr , t -dk- ), and the cff (stanto ro, anders fo). ma, l. ; parsek, m.r. ; wozniak, d.j. . microbiology and immunology, wake forest university health science, winstonsalem, nc, usa; . university of washington, seattle, nc, usa bacteria in natural, industrial and clinical settings live in surface-associated communities termed biofilms, which are the source of persistent infection and resistance to antimicrobial treatment. biofilm development is initiated by the attachment of planktonic cells to a surface, followed by formation of microcolnies, and finally disperses swimming cells from microcolnies to occupy a new surface. to maintain the community structure, bacteria in a biofilm are usually enmeshed in an extracellular polymeric matrix, which consists of nucleic acids, proteins, and polysaccharides. exopolysaccharides (eps) have been known as a component of the biofilm matrix for years. however, little is know about how the eps matrix forms and develops and whether an eps can form a matrix independently. in the present report, we use lectins that specifically detect the mannose or galactose structure in pseudomonas aeruginosa psl eps. this technique allows us to visualize psl eps on the bacteria cell surface and in the biofilm matrix. our results indicate that psl eps is likely anchored on the cell surface in a helical pattern and clearly forms a matrix, which holds bacteria in the biofilm and on the surface. in a flat multiple-layer biofilm, psl eps is equally distributed in the entire biofilm. however, microcolonies reveal peripheral staining of psl eps with minimal staining of matrix in the center of the microcolonies. instead, this region has swimming cells indicating a biofilm development stage prior to dispersion. more strikingly, this area also has concentrated dead cells and/or extracellular dna, which fills up the viod spaces in the lower center of microcolonies at the stage prior to dispersion. these data provides a plausible mechanism for how p. aeruginosa sacrifices a portion of cells to make void spaces and free another portion of cells for future dispersion. in addition, our data also show that psl eps matrix and dna matrix are not overlapping, which suggests that these two components of the matrix work coordinately to encase bacteria in the biofilm. finally we show that the psl eps matrix is present in biofilms formed by mucoid p. aeruginosa, and formation of the psl eps matrix is independent of the production of alginate and pel, two eps that also contribute to p. aeruginosa biofilm formation. overall, our data indicates that psl eps functions as a primary scaffold, holding biofilm cells together in the matrix. moreover, our data along with published literature suggest a possible model for how p. aeruginosa biofilms persist in cystic fibrosis patients. . clinical microbiology, rigshospitalet, copenhagen, denmark; . biocentrum, technical university of denmark, kgs. lyngby, denmark; . department of paediatrics, copenhagen cystic fibrosis centre, rigshospitalet, copenhagen, denmark pseudomonas aeruginosa predominates chronic lung infections in patients with cystic fibrosis (cf). a hallmark of chronic p. aeruginosa lung infections in cf patients is the presence of mucoid p. aeruginosa biofilms surrounded by numerous polymorphonuclear leukocytes (pmns) in the lower airways. how p. aeruginosa escapes the bactericidal pmns is not fully understood. several virulence factors of p. aeruginosa is controlled by quorum sensing (qs); a density dependent mode of inter-bacterial communication based on signal transmitter molecules. active qs is present during chronic p. aeruginosa lung infections in cf patients and we have previously demonstrated a qs-regulated tolerance of biofilm bacteria to the antimicrobial properties of the pmns. the precise qs-regulated effect on the pmns is, however, unknown. in the present study, further elaboration using flow cytometry and microscopy revealed that qs-competent p. aeruginosa (pao and ∆rhli lasi complemented with c -hsl and -oxo-c -hsl) induces rapid necrosis of the pmns. this mechanism was also observed in in vitro biofilms and in mouse lungs infected with p. aeruginosa embedded in alginate. by using hplc fractionation the toxicity could be ascribed to a single substance. using lc-ms and d nmr this substance was identified as rhamnolipid b. in accordance, concentrations of rhamnolipid exceeding µg/ml were found in the toxic supernatants from wild-type p. aeruginosa (pao ) whereas non-toxic supernatants from qs-deficient knock-out mutants (∆rhlr lasr and ∆rhli lasi) and from ∆rhla mutants contained low amounts of rhamnolipids approaching µg/ml. our results demonstrate the potential of the qs system to facilitate infections with p. aeruginosa by utilizing rhamnolipid to disable a major first line of defense of the host -the pmns. furthermore, our study emphasizes the inhibition of qs as a target for the treatment of infections with p. aeruginosa. carlsson, m. , ; pettersson, a. ; andersson, c. ; wieslander, j. ; eriksson, l. ; segelmark, m. ; hellmark, t. . dept of microbiology, immunology, and glycobiology, university of lund, lund, sweden; . department of nephrology, lund university, lund, sweden; . heart lung division, the cystic fibrosis center, lund university hospital, lund, sweden; . wieslab analys ab, lund, sweden the clinical consequence of chronic pseudomonas (p) aeruginosa colonization in cystic fibrosis (cf) varies between individuals for unknown reasons. auto-antibodies against bactericidal/ permeability increasing protein (bpi-anca) are associated with poor prognosis in cf. we hypothesize that there is a correlation between the presence of bpi-anca, the biological properties of the colonizing bacteria and the clinical conditions of the hosts. we have compared isolates of p aeruginosa from two groups of cf patients: one with positive serum levels of bpi-anca and deteriorating lung disease, and one with negative bpi-anca levels and stable clinical conditions. epithelial cells (a ) and isolated polymorphonuclear granulocytes (pmns) were stimulated with the clinical isolates and cell death was analyzed with flow cytometry. interleukin- (il- ) released into the supernatant was measured by elisa. we found that the anca associated strains in most cases showed a pyocyanin negative phenotype. these strains also induced less inflammatory response than the non-anca associated strains as shown by the number of necrotic cells and il- release, yet elevated compared to control. we conclude that colonization with strains of p aeruginosa that induce a weak inflammatory response is associated with unfavourable outcome in cf. we speculate that inadequate control of pathogen proliferation through an insufficient inflammatory response results in a slowly increasing number of bacteria and accumulation of dying pmns in the airways, contributing to the progressive lung disease seen in many cf patients. . pulmonary critical care, northwestern university, chicago, il, usa; . microbiology/immunology, northwestern university, chicago, il, usa; . pulmonary medicine, children's memorial hospital, chicago, il, usa; . pediatrics, children's memorial hospital, chicago, il, usa purpose: most cystic fibrosis (cf) patients are infected with pseudomonas aeruginosa (pa) and have progressive loss of lung function. among individual patients, however, there are marked differences in rates of lung function deterioration. although the reasons for this variability are not completely understood, it is likely that microbiological factors play a role. type iii secretion in pseudomonas aeruginosa (pa) isolates from non-cf patients have been associated with poorer outcome. the aim of this study was to determine whether there is an association between type iii secretion properties and deterioration in lung function in cf. methods: we prospectively enrolled cf children and adults over years. demographics, clinical characteristics, spirometry and a respiratory culture were obtained at the first visit. the age at time of first postive pa culture was also determined. subsequently, spirometry and respiratory cultures were obtained and clinical characteristics recorded every months until december . from each sputum culture individual isolates were selected for evaluation and type iii secretion was evaluated for each isolate by western blot analysis. the children were subcohorted into those newly infected ( st positive pa culture) or those chronically infected (minimum sputum pa (+) > year duration). results: there were patients evaluated, of which were adults and were children. ninety percent of our population cohort was caucasian and % were female. overall % of the patients were on tobi and % on macrolides chronically. the mean age at time of a first pa positive culture was years for the chronically infected subjects(adults and children) and years for the st time infected subcohort. a total of sputum samples were collected for the current study. the prevalence of type iii secretion properties for the overall population was . %. foe adults, of ( . %) pa isolates were type iii secretion positive compared to of isolates ( . %) in chronically infected children and of isolates( . %) from newly infected children. at study entry the mean fev % predicted was +/- for the whole cohort. for the adult subcohort the fev % predicted was % +/- %, for chronically infected children it was % +/- % and newly infected children is was %+/- %. the average annual rate of fev % change was - . % for adults, - . % for chronically infected children and + . for newly infected children. analysis on the relationship between type iii secretion pa isolates and fev % decline is ongoing and will be presented at nacf. conclusion: prevalence of type iii secretion in pa isolates from cf patients decreases with age. there is a slower decline in fev % in chronically pa infected adults compared to chronically pa infected children. the relationship of type iii secretion to fev % change and pulmonary exacerbations will be presented at nacf. a limited number of bacterial species -in example pseudomonas aeruginosa, staphylococcus aureus, burkholderia cepacia complex, stenotrophomonas maltophilia -is typically found in the cf lung. these facultative anaerobic bacteria exhibit the capability to switch from aerobic to anaerobic metabolism, which enables them to survive in the hypoxic environment in the cf lung [ ] . absence of oxygen would also favour the growth of fastidious anaerobes and, indeed, sequencing of s rrna resulted in the detection of strict anaerobic species in cf sputum [ ] . thus, we identified and quantified strict anaerobes in cf sputum using conventional anaerobic microbiological methods, examined the recovery rate of identical species in sputum samples from the same patients, and determined the antibiotic susceptibilities. sputum samples were collected from cf children and cf adults (mean age . ± . yrs). samples were incubated aerobically on columbia agar, supplemented with % sheep blood, and anaerobically on brain heart infusion agar and schaedler agar supplemented with % mutton blood, for up to days. bacteria were identified (rapid ana ii identification system, remel, lenexa, ks) and cfu's were determined by dilution plate counting. fastidious anaerobes ( strains) were submitted to e-test ® susceptibility testing (ab biodisk, solna, sweden) using the anaerobically active antibiotics ceftazidime, clindamycin, meropenem, metronidazole, and piperacillin/tazobactam. in . % of the patients, the following strict anaerobes were detected with a mean of . x cfu/ml (range . x to . x cfu/ml): peptostreptococcus spp., clostridium spp., actinomyces spp., prevotella spp., wolinella spp., propionibacterium spp., streptococcus spp., lactobacillus spp., gemella spp., bacteroides spp. and eubacterium spp., whereas p. aeruginosa, s. aureus and b. cepacia revealed . x cfu/ml (range . x to . x cfu/ml). in sputum samples contaminated with p. aeruginosa together with strict anaerobes . x ± . x cfu/ml p. aeruginosa were counted, whereas in samples with p. aeruginosa without strict anaerobes only . x ± . x cfu/ml were found (p= . ). for s. aureus, no significant difference was observed. identical strict anaerobic species were detected in out of patients with two or more repeated sputum samples ( %). e-test® sensitivity testing for strict anaerobes yielded high sensitivity for meropenem (only . % resistant strains), piperacillin/tazobactam ( . %), and clindamycin ( . %), but not ceftazidime ( . %) or metronidazole ( . %). high numbers of strict anaerobes are present in the majority of cf patients. possibly, strict anaerobes promote growth of p. aeruginosa but not s. aureus. the high persistence of identical anaerobic strains reflects chronic lung infection and may be caused by their increased resistence against standard antibiotics such as ceftazidime. [ in sputum in the cf lungs bacteria such as pseudomonas aeruginosa have to metabolize anaerobically [ ] . for anaerobic energy generation, p. aeruginosa can use nitrate and arginine, but also pyruvate which is produced from glucose via anaerobic glycolysis [ ] and can be metabolized to lactate and vice versa. in order to investigate if p. aeruginosa may benefit from externally produced lactate, we measured the concentration of lactate in p. aeruginosa, staphylococcus aureus, burkholderia cenocepacia and polymorphonuclear neutrophils in vitro and in cf sputum. in sputum samples of cf patients and in neutrophils ( x /ml) from healthy donors lactate concentrations were determined. in addition, p. aeruginosa (starting with x cfu/ml in tryptone soy broth), s. aureus ( x cfu/ml), and b. cenocepacia ( x cfu/ml)were grown aerobically ( through hrs) and anaerobically ( through days). l-lactate was measured spectrophotometrically (detection limit: . mmol/l), and total lactate gaschromatographically. aerobic and anaerobic gene expression of p. aeruginosa strain pao was determined using affymetrix® microarrays. lactate concentrations in cf sputum amounted to . ± . mmol/l (range . to . mmol/l). concentrations were similar in sputum samples colonized with p. aeruginosa, s. aureus ( . ± . vs. . ± . mmol/l, p= . ) and b. cenocepacia ( . mmol/l). neutrophils produced . mmol/l. in all samples exclusively l-lactate was found. during in vitro experiments, p. aeruginosa did not generate any lactate at all, neither aerobically nor anaerobically. in contrast, anaerobically grown s. aureus produced up to . mmol/l lactate, and b. cenocepacia up to . mmol/l. a p. aeruginosa suspension [ x cfu/ml] spiked with mmol/l l-lactate did not change its concentration, indicating that p. aeruginosa does not metabolize lactate. similar results were obtained in our gene chip experiments: after three days of anaerobic growth, the genes encoding for the lactate dehydrogenases were downregulated (pa ldha - . fold, pa llda - . fold) or unchanged (pa lldd . fold). in contrast, the genes encoding for pyruvate decomposition to acetyl coa (pa and pa , both encoding for pyruvate dehygrogenase e components) were upregulated by and fold, respectively. we could demonstrate that p. aeruginosa does not benefit from externally produced lactate. we confirmed the important role of pyruvate metabolism for anaerobic p. aeruginosa energy generation. whether lactate production of neutrophils, s. aureus or b. cenocepacia contributes to cf lung pathophysiology still remains to be investigated. references: [ ] macleod, d. ; barker, l. ; gurgel, j. ; kenney, t. ; burns, j. ; baker, w. . gilead sciences, inc., seattle, wa, usa; . university of washington, seattle, wa, usa antibiotic resistance may severely limit therapeutic options in individuals with cystic fibrosis (cf) or bronchiectasis. because of frequent antibiotic treatment courses, resistance continues to emerge, even to newer agents. treatment with multiple antibiotics in a single aerosol formulation may be a promising approach to slow development of resistance. fosfomycin is a phosphonic acid antibiotic that is bactericidal against both gram positive and gram negative organisms. fosfomycin inhibits the first committed step in the synthesis of peptidoglycan, suggesting cross resistance to other cell wall acting antibiotics will not occur. the aminoglycoside tobramycin is one of the most commonly used antimicrobials in cf, with potent activity against gram negative bacteria and the majority of staphylococcus aureus isolates. a : (wt/wt) fixed combination of fosfomycin:tobramycin (gs- / ) was used to determine the in vitro susceptibilities of a panel of respiratory pathogens: cf pseudomonas aeruginosa ( ), s. aureus ( ), haemophilus influenzae ( ), stenotrophomonas maltophilia ( ) and burkholderia cepacia complex ( ), including minimal inhibitory concentration (mic) and time-kill experiments in the absence and presence of % porcine mucin. synergy was evaluated using the checkerboard method, and spontaneous resistance mutation frequencies were determined in antibiotic-containing agar ( x, x and x mic). in vivo drug efficacy was examined using a rat agar bead pneumonia model of either p. aeruginosa or s. aureus. all experiments compared gs- / to fosfomycin and tobramycin as single agents. gs- / had a lower mic than tobramycin for the s. aureus strains, % of which were methicillin resistant (mrsa). for p. aeruginosa, gs- / had a lower mic and mic than fosfomycin alone, but tobramycin was more active than either. for h. influenzae and s. maltophilia, gs- / , fosfomycin and tobramycin had similar mic and mic . b. cepacia complex were resistant to all three drugs. results in the presence of mucin were similar. time-kill studies showed a more rapid and prolonged killing of s. aureus and p. aeruginosa by gs- / compared with either agent alone at the same drug concentrations. gs- / was bactericidal and exhibited concentration-dependent killing. synergy studies showed no antagonism between fosfomycin and tobramycin, and the majority of p. aeruginosa and all of the s. aureus tested demonstrated indifference for the combination. at x mic concentrations the mutation frequency of gs- / was at least - logs lower than tobramycin and - logs lower than fosfomycin alone for s. aureus. for p. aeruginosa the mutation frequency of gs- / was - logs lower than fosfomycin and - logs lower than tobramycin. in the rat pneumonia model, gs- / and tobramycin alone demonstrated bactericidal killing of p. aeruginosa; both were more active than fosfomycin alone. in vivo killing of s. aureus by gs- / was also demonstrated. gs- / appears to have advantages over single agents for the treatment of both gram positive and gram negative bacterial lung infections in cf and bronchiectasis. britton, l.j. antibiotic resistance is becoming a major problem in the treatment of pulmonary exacerbations in cystic fibrosis (cf). organ-isms that are resistant to multiple antibiotics infect the airways of an estimated - % of adults with cf. as a result of the growing resistance in cf patients, many centers have been performing synergy testing of sputum cultures in addition to the conventional culture and sensitivity testing. the purpose of our investigation was to determine if antibiotic synergy studies and the use of synergistic combinations of antibiotics improve therapeutic outcomes in cystic fibrosis patients with acute pulmonary exacerbations. methods: this study is a retrospective chart review of cf patients who had antibiotic synergy testing performed while hospitalized for respiratory tract infections. eligibility criteria included cf patients hospitalized with respiratory tract infections from to . laboratory data from each hospital admission was reviewed for synergy among those antibiotics commonly used against pseudomonas aeruginosa. a review of medical charts ascertained each patient's pulmonary function (determined by fev before and after antibiotic therapy), weight z-score, organism cultured from sputum sample, time to next hospital admission, and the antibiotic(s) actually prescribed. primary endpoints were determined to be the change in fev and the time to next admission. results: four hundred seventy-five hospital admissions were analyzed. a total of cystic fibrosis patients, age birth to years, were included in the study. patients receiving antibiotic synergy experienced a significant decrease in mean time to next admission ( days with synergy vs. days without synergy, p= . ). no significant difference was found in the change in fev before or after antibiotic therapy, with an increase of . % with synergy vs. . % without synergy (p= . ). patients infected with non-mucoid p. aeruginosa experienced days to the next hospital admission, while patients infected with mucoid p. aeruginosa experienced days to the next hospital admission (p = . ). no statistically significant difference was observed between the synergy and non-synergy groups in regards to nutritional status and lung function prior to antibiotic therapy. speculations: antibiotic resistance in cystic fibrosis patients increases the morbidity and mortality caused by this disorder with each exacerbation the patient experiences. synergistic antibiotic therapies should improve patient outcomes through more efficient bacterial eradication and increased time to next hospital admission; however, we were unable to substantiate this assumption based on the results from this study. synergistic antibiotics did not show an improvement in the therapeutic outcomes of days to next admission or change in fev . background: expectorated sputum (es) technique is currently the most frequently used method for routine assessment of lower airway infection in patients with cystic fibrosis (cf). induced sputum (is) using hypertonic saline (hs) has been successfully used in cf patients unable to produce sputum spontaneously, but only limited data are available comparing the diagnostic yield of expectorated versus induced sputum in cf patients. while ultrasonic nebuliser have been used in the majority of studies, new high output jet nebulisers may offer a suitable alternative technique to induce sputum expectoration. aim: to assess the feasibility of sputum induction using a pari e-flow nebulizer and to compare the diagnostic yield of is and es in children with cf . methods: this is a preliminary report of an ongoing study in routine clinical care in sputum producing children with cf. es is being obtained before sputum induction. subsequently, sputum induction is performed using stepwise inhalation of nebulized ml of %, % and % hypertonic saline with an e-flow nebulizer (pari, starnberg, germany). lung function is assessed by portable spirometry before the procedure and after inhalation of each saline concentration. results: so far cf patients ( females) with a mean age of . years (range to years) and fev between - % predicted (mean %) have been included in the study. all subjects provided es samples and all produced sputum after induction. discrepancies in cultures between es and is samples were seen in cases ( . %). in cases additional cf pathogens were found in is samples, whereas in cases es yielded additional organisms compared to is. the number of distinct pathogens was similar in the remaining patient, but different bacteria were found with the techniques. in cases p. aeruginosa was detected only with one of the techniques ( es versus is). the spectrum of side effects was similar to previous reports using other nebulizer systems, with throat irritation being the most common adverse event of the is technique. all patients who had symptoms during the procedure became asymptomatic at the time of discharge from the clinic - minutes after the procedure. patients did not finish all cycles of sputum induction procedure due to symptoms of shortness of breath and/or drop in fev > % from baseline (in patients); all reversed after salbutamol inhalation. vomiting occurred in one patient. patients refused to complete the procedure due to unpleasant taste and/or throat irritation. conclusion: these preliminary results show discrepancies between expectorated sputum and induced sputum cultures in a significant proportion of cf patients. this may be explained by the previously described regional differences in lower airway infection in cf airways rather than by a higher diagnostic yield of one of the techniques. the results also demonstrate that the e-flow system is an efficient and safe device for sputum induction in sputum producing children with cf. seidler, m.j.; salvenmoser, s.; müller, f.c. dept. pediatrics iii, university heidelberg, pediatric pulmonology, cystic fibrosis centre & infectious diseases, heidelberg, germany background: the preferred growth form of bacteria is a biofilm. s. aureus, h. influenzae, and p. aeruginosa can produce an extracellular matrix (ecm) with implications in cystic fibrosis (cf) lung disease. the biofilm can protect against host defenses and antimicrobials. a. fumigatus is a frequent colonizer of the cf respiratory tract and can cause allergic bronchopulmonary aspergillosis (abpa). while antifungals in vitro are active against a. fumigatus, in vivo antifungal therapy is often complicated or resistance is observable. the aim of this study was to investigate the ability of a. fumigatus to form a biofilm-like matrix in vitro on polystyrene (ps), human bronchial epithelia cells ( hbe) and human bronchial epithelia cells with f del/f del (cfbe o-). methods: a. fumigatus atcc # was incubated in rpmi at different ph and concentrations of fbs. temperature, production time and different flow conditions were varied on ps, hbe and cfbe o-. dry weight measurement and antifungal drug susceptibility testing was performed. scanning electron microscopy (sem) and confocal scanning laser microscopy (cslm) images were analyzed. results: the thickest biofilm was produced on ps with rpmi (+ % fbs, ph= . ) at °c for h slightly rocking. biofilm dry weight on ps was . mg after h and . mg after h. the dry weight of produced biofilm exceeded . mg on hbe and . mg on cfbe o-cells after h of biofilm production. there was no significant difference in dry weight increase between the cell lines and ps. planktonic a. fumigatus was susceptible to itraconazole ( . µg/ml), voriconazole ( . µg/ml) and amphotericin b ( µg/ml). aspergillus in biofilm was resistant against all drugs (> µg/ml). the sem pictures displayed a network of hyphal structures and matrix at h. characteristic flow channels were observed at h. cslm images displayed conidia and hyphal structures embedded in matrix formations. a-alexafluor dyed polysaccharides of the cell wall and of the ecm in the biofilm. three dimensional constructs of the cslm pictures displayed biofilm on hbe and proofed viability of the cells after h co-incubation. differences in biofilm production between hbe and cfbe o-were not significant. conclusions: a. fumigatus is able to form a biofilm structure in vitro on ps, hbe and cfbe o-. a biofilm-like matrix produced by a. fumigatus was evidenced by dry weight measurement, sem, cslm and antifungal drug resist-ance in comparison to planktonic cells. potential clinical implications of a. fumigatus biofilm formation in vivo require further attention and investigations. etherington, c. ; peckham, d. ; conway, s. ; hall, m. ; denton, m. . seacroft hospital, regional adult cf unit, leeds, united kingdom; . microbiology department, leeds teaching hospitals, leeds, united kingdom susceptibility testing results are not predictive of clinical response to antibiotic therapy in chronic pseudomonas aeruginosa infections in cystic fibrosis. we assessed the impact of reducing the number of routine susceptibility tests performed on clinical outcome in these cases. in june we introduced a protocol of limiting susceptibility tests to p. aeruginosa isolates obtained from respiratory samples taken at the commencement of antibiotic therapy, when there was evidence of clinical failure, or routinely if not tested in the previous three months. at all other times, isolates were identified and reported as normal but p. aeruginosa isolates were not subjected to susceptibility tests. between st june and th november p. aeruginosa, was isolated on at least one occasion from patients attending our adult cf unit. in this six month period we reduced the number of susceptibility tests by % (from a projected , tests on samples to an actual tests on samples). this resulted in projected savings of $ , in consumables and hours (costed at $ , ) of laboratory staff time per annum, a total saving of $ , (£ , ) per annum. we assessed the response to intravenous antibiotic treatment between the study period in and the same period in . no significant differences in median change of fev , fvc, crp, white cell count, weight, or duration of intravenous antibiotics were observed. for cf units sending regular, routine sputum samples, a reduction in the number of susceptibility tests performed in cases of chronic p. aeruginosa can be carried out without impacting on clinical outcomes. we report our preliminary results for a total of strains obtained from patients ( patients harbored more than strains). these patients were classified in groups of respiratory insufficiency according to their fev : severe in patients (fev < %), moderate in patients (fev : - %) and mild in patients (fev > %). the clonal distribution was analyzed for the different strains of sa ( to ) isolated from patients sputum. these strains were analyzed for their antibiotic susceptibility and typed by pulsed-field electrophoresis gel (pfge) after smai digestion of chromosomal dna. thirty seven patients ( %) were colonized with mrsa. nine patients were both colonized with mrsa and mssa. among mrsa strains, / ( %) were also resistant to more than three other antibiotic family. strains harboring minor differences in the banding pattern (> % similarity as assessed by the dice coefficient) were considered clonal. our results show that % of the patients were colonized with a single persistent strain during the year of follow-up. consecutive isolates with different pfge profiles were obtained from only / patients ( %). pfge analysis revealed that mrsa isolated from patients were grouped in clusters. these results revealed a possible clonal relationship between mrsa isolated from different patients with cf. we did not find any difference in the distribution of sa strains in our cf patients among the groups of respiratory insufficiency. the study is ongoing in our adult cf population and in necker pediatric cf centre in paris. giusti, r. ; furfaro, s. . pediatrics, long island college hospital, brooklyn, ny, usa; . research, lumina fund, new york, ny, usa palivizumab(synagis) is a humanized monoclonal antibody to rsv. the redbook acknowledges that some patients with cystic fibrosis may be at increased risk of rsv infection but that there is insufficient data to determine the effectiveness of this therapy. the objective of this study was to assess: )practice patterns of cf physicians in the us and canada )the severity of rsv disease in cf infants during the past rsv season. )if there is a standard of care concerning the use of synagis for cf infants. methods a questionnaire was developed using a web-based commercial vendor. an embedded web link was distributed via an e-mail sent to all us and canadian pediatric cf center directors. respondents clicked on a link to respond to the survey and results were automatically tabulated in real-time. completed responses were received from center directors ( us and canadian) for a response rate of % in us and % in canada. most responders ( %) have prescribed synagis for infants with cf in the first rsv season, however only % routinely prescribed synagis for all infants with cf. only % expressed having had difficulty in obtaining insurance approval for this medication. many physicians indicated that synagis was frequently prescribed by the general pediatrician and that infants living at long distances from a cf center may be hospitalized at local hospitals. these issues may affect the accuracy of the data and result in an underestimate of actual synagis prescription and hospitalization rates. there were infants diagnosed with cf in the past year and of these infants were reported as symptomatic. there were cf infants ( %) reported as having a documented rsv infection and ( %) of these infants had received synagis. there were infants ( %) with documented rsv infection who responded to outpatient management.there were no deaths but patients were hospitalized and of these had received synagis. of the infants that required admission to an icu had received synagis. of the patients who were noted as having persistent chest x-ray changes had received treatment with synagis. there were infants with persistent wheezing and of these infants had been treated with synagis. conclusions despite the limitations of a retrospective survey, this data demonstrates that rsv can cause significant and prolonged pulmonary disease and is a significant precipitating factor resulting in hospitalization of cf infants. the data also notes that many cf infants infected during the past rsv season have a mild illness and respond to outpatient management. a surprising finding of this survey is that infants at cf care centers where synagis was prescribed for all cf infants continued to have significant rsv related hospitalizations, persistent wheezing or prolonged chest x-ray abnormalities. there are different opinions among cf physicians concerning the routine use of synagis and currently the data suggests that there is not a standard of care concerning the prophylaxis of cf infants with synagis. this results of this survey should encourage physicians to prospectively study the efficacy of synagis prophylaxis in preventing hospitalization, persistent wheezing and chest x-ray abnormalities in cf infants. milani, a. ; cisbani, g. ; macchi, r. ; vidal-aroca, f. ; bertoni, g. . biomolecular sciences and biotechnology, university of milan, milano, italy; . basilea pharmaceutica ltd., basel, switzerland with ever increasing frequency, we now observe several examples of bacteria being resistant to every clinically available drug. therefore this urgently calls for the development of novel and improved antibiotics that may escape the extant mechanisms of bacterial resistance. one recent and promising development of the genome-wide search for target functions for antibiotics led to the identification of essential genes of pathogens by interfering antisense rnas. the first step is the construction of shotgun antisense libraries (sals) as follows. genomic dna is extracted from the bacterial strain of interest, fragmented by shearing into short pieces of dna, blunt-ended and cloned in an expression vector under the control of a regulatable promoter. the library is then reintroduced into the cognate bacterial strain and screened by replica plating colonies both in the presence and the absence of an inducer of the vector promoter. by this method, insert sequencing of clones showing conditional growth phenotypes is expected to lead to the identification of essential genes that can be silenced via antisense rna activity. we adopted this technology in order to generate a panel of essential functions of the cystic fibrosis-related opportunistic pathogen pseudomonas aeruginosa. so far, we tuned the protocol for sal generation in p. aeruginosa and identified a number of sequences conferring different levels of growth inhibition we are now characterizing these putative antisense rnas in order to define the minimal sequence able to cause the toxic effect and, on the other hand, to understand the cellular role of their targets. pseudomonas aeruginosa (pa) is an extremely versatile microbe with a vast array of pathogenic and metabolic mechanisms that allow it to form persistent infections in select patient populations, especially patients with cystic fibrosis. an ineffective immune response is considered partially to blame for failure of pa eradication. we have discovered that some strains of pa express peptidylarginine deiminase (padi) activity. padi is an enzyme that post-translationally modifies peptidylarginine to peptidylcitrulline with ammonia as a byproduct. our lab has shown that human padi can modulate the immune system through downregulation of tlr and ikk-gamma signaling. characterization of the pa padi will offer insights into a completely novel method of immune modulation by pseudomonas aeruginosa. using a widely published colorimetric assay for padi activity we have found the specific activity of crude pa cell lysates is very low. however this is similar to the only other known prokaryotic padi described in porphyromonas gingivalis. a protein homology search with the porphyromonas padi has revealed the likely genetic locus of pseudomonas padi in a . kb operon that appears in the genome of the pathogenic pseudomonas isolate pa . this operon appears to contain two candidate genes for padi activity based on conserved motif searches (padi and padi ). both have been cloned into expression vectors, partially purified by affinity tag technology and tested in the colorimetric padi assay. curiously padi autocitrullinates itself while padi has not shown activity. reaction conditions and substrate specificities for pa-padi are not like either the human or porphyromonas padi. furthermore analysis of up to clinically diverse strains has demonstrated % carry the gene for padi . this work is only the second description of padi activity in any prokaryote. pa padi could clearly have a dramatic impact on the local inflammatory milieu if it can access the same targets as human padi. the description of this activity in pseudomonas will advance our knowledge of the human-pathogen relationship and give insight into new therapies. which functions by translocating toxins into the cytoplasm of host cells. these toxins cause disease by damaging the surrounding host tissue, promoting dissemination of the organism and paralyzing the phagocytic mechanism of macrophages. pcrv is a factor required for the translocation of the toxins. the bases of these studies were to evaluate pcrv as a protective antigen in "p. aeruginosa" pneumonia and cldc as a vaccine adjuvant. methods: mice were vaccinated , , or times with the pcrv antigen combined with either aluminum hydroxide (alum) or cldc by various routes of administration. efficacy of vaccination was evaluated by challenge with "p. aeruginosa" and evaluation of survival and/or measurement of various parameters associated with lung injury. results: increase in median survival time was highly significant when cldc/pcrv was compared with cldc or pcrv alone. following subcutaneous administrations cldc/pcrv showed an increase in median survival time ( hours versus hours) and overall survival benefit following intraperitoneal administrations ( % versus %). mice with anti-pcrv antibody levels above µg/ml were significantly protected. conclusion: the investigators establish the efficacy of cldc/pcrv vaccines via several parenteral routes of administration compared to no treatment as well as cldc and pcrv-only controls. differences were demonstrated between performance of cldc/pcrv and alum/pcrv in measures of lung injury, median survival, and overall survival. the results correlated with antibody levels and histological examination of the lung tissues. importantly, these studies indicate that protection can be achieved against "p. aeruginosa" infection by targeting an antigen associated with the type iii secretion system. background: there has been a recent increase in the number of reported cases of acute renal failure (arf) in cystic fibrosis (cf). our group have undertaken a national survey, which measured the incidence risk of arf in cf patients at between . and . cases / , cf patients / year. we have now conducted a case control study to determine which factors which are associated with an increased risk of arf. methods: in our initial survey we confirmed cases of arf, in cf patients from uk cf centres, presenting between & . using the uk cf database, we identified sex and age (within months) matched controls. informed consent was sought from the control patients, or their parents, for access to the case notes and clinical data were extracted. analysis of risk factors was by conditional logistic regression, using stata (version ) and by fisher's exact test. results: there were cases of arf ( male, median age y, range m- y) and controls ( male, y, m- y). in the group of patients with arf, / had received an aminoglycoside at the time of their episode of arf or in the preceding week, compared with only of the controls for the same time period (p< . ). the median number of days of aminoglycoside in the year prior to the index case developing acute renal failure was ( - ) for cases and ( - ) for controls. conditional logistic regression showed that the odds ratio for arf per each day of aminoglycoside was . ( % ci . to . , p= . it is well-known that pa senses the environment and changes its phenotype. for instance, it produces greater amounts of the extracellular polysaccharide alginate in the cf lung, characterized by a microaerobic environment. little is known about the changes in protein secretion induced by oxygen limitation in pao , the proto-typical pa laboratory strain. no data are available on this regard about pa clinical strains. our work was aimed to study the differential regulation of proteins secreted by pa strains grown in microaerobic or aerobic conditions. a pa clinical isolate and pao were grown overnight in aerobiosis and in microaerophilic conditions. the supernatants were collected and proteomic analysis was carried over by two-dimensional capillary chromatography -tandem mass spectrometry (mauri et al., faseb j ) to evaluate the differential protein expression. in the pa clinical isolate, we identified proteins down-regulated and proteins upregulated in aerobic conditions in comparison with microaerophilic culture while in pao proteins were down-regulated and were up-regulated. proteins were down-regulated and up-regulated both in the clinical and in the laboratory strain. these proteins can mediate different biological functions since they are enzymes, heat shock proteins, chaperones, proteins involved in adaptation, motility and in the transport of small molecules. among all these proteins, as the alkaline metalloproteinase is associated with tissue invasion not only by causing rupture of epithelial tight-junctions but also by degrading several chemokines, we decided to validate its up-regulation observed in aerobic conditions by zymography. we found that the proteolytic activity of the supernatants of pa grown in aerobic conditions was higher than in microaerophilic culture both for the laboratory and clinical strain, indicating the functional relevance of data obtained by proteomic analysis. the identification of proteins differentially regulated in aerobiosis and oxygen limitated conditions in pa laboratory and clinical strains might be helpful for the knowledge of the mechanisms of colonization and lung damage due to pa in cf patients. for instance, the validation of the upregulation of the alkaline metalloproteinase in aerobic condition may shed light on these mechanisms of cf lung disease. further studies are in progress to evaluate the function of other bacterial exoproducts regulated in this model and to extend the analysis to other clinical strains. supported by the italian cystic fibrosis research foundation (ffc-grant# / ), comitato di vicenza dell'associazione veneta per la lotta contro la fibrosi cistica and azienda ospedaliera di verona, italy. cystic fibrosis (cf) sufferers are subject to repeated lung infections most commonly with the bacterium pseudomonas aeruginosa. in spite of antibiotic treatment p. aeruginosa tends to become established giving rise to persistent chronic infection. in the lung environment, the bacterium grows as a highly structured biofilm consisting of a complex community of cells embedded within a self-secreted polysaccharide matrix. investigations of p. aeruginosa biofilm growth using model strains have elucidated mechanisms which appear to govern the biofilm life-cycle. our research aims to test whether observation of these mechanisms in planktonic culture can be related to the efficiency of biofilm formation. biofilm initiation has been linked to cell motility and in particular to the presence of flagella and pili, which are thought to be important for cell attachment and the formation of microcolonies. on testing a large, genetically diverse group of clinical p. aeruginosa isolates retrieved from the lungs of cf patients we found no definitive correlation between the degree of motility of an isolate in planktonic culture and its ability to form a biofilm in vitro. the development of biofilm architecture has been demonstrated in model systems to be coordinated by the production and secretion of n-acylhomoserine lactone (ahl) quorum sensing molecules. however among the clinical isolates tested we observed no obvious correlation between the amount of ahls produced in planktonic culture and the extent of biofilm formation. overall we have found observation of phenotypic characteristics in planktonic culture to be poor predictors of efficient biofilm formation. a proteomics approach was adopted to provide further insight into the physiology of biofilm growth of p. aeruginosa isolates by comparison with planktonic growth of the same isolates. two genetically unrelated clinical isolates, demonstrated as being capable of efficient in vitro biofilm formation, were selected from our culture collection on the basis of their diverse phenotypic characteristics when cultured planktonically. one displays both twitching and swimming motilities, is mucoid and expresses ahls, while the other is non-motile, non-mucoid and no ahls have been detected in planktonic culture. we have developed a simple flow-through bioreactor to provide sufficient quantities of biofilm for proteomic analysis. gel-based and gel-free techniques were employed to study protein expression patterns for both biofilm and planktonic cultures of each isolate by mass spectrometry. proteins specific for each growth phase could be detected and may prove suitable biomarkers for monitoring the physiological status of biofilm forming strains when a larger bank of clinical isolates are examined. liposomal amikacin (arikace tm ) is a liposome-encapsulated form of amikacin that is formulated to treat chronic p. aeruginosa infections in cystic fibrosis patients. these liposomes carry a zwitterionic surface charge and are composed of lipids found naturally within the lung. a key aspect of the activity of the formulation is the ability to penetrate to the sites of pseudomonas biofilm-like growth in the lung. experiments were designed to investigate the penetration of liposomes into p. aeruginosa biofilms and in vitro activity. methods and results: model liposomes of the same size and lipid composition as liposomal amikacin (arikace tm ) were prepared with membrane-associated or encapsulated fluorescent labels, a hydrophobic carbocyanine dye and calcein, respectively. a mucoid strain of pseudomonas aeruginosa (pa ) was used to establish biofilms in rectangular optical grade glass flow cells. biofilms were observed after four days of growth by confocal laser scanning microscopy using a focal plane set to view within the biofilm cluster or outside as a control. time dependent accumulation of fluorescent liposomes within the biofilms was measured by the spatial distribution of fluorescence intensity in regions within or outside of the biofilm. images indicated significant penetration of liposomes into the interior of biofilms under these conditions. the rate of penetration was considerably slower than typical rates for small molecules, consistent with the size of the liposomes. liposome concentrations were higher near the periphery than the interior. however, even the interior concentration was at least as high as the concentration of liposomes in the fluid outside of the biofilm, suggesting some binding or trapping of the liposomes within the biofilm. penetration of liposomes was observed under flow or static conditions. in a "washout" experiment, where medium is passed through the biofilms previously treated with liposomes, a significant portion of the liposomes remained associated with the biofilms for an extended period of time. the penetration of liposomes was reflected in the observation of killing of bacteria in colonies in the interior of agar beads. exposure of these cultures to liposomal amikacin resulted in a large reduction of viable bacteria throughout the beads as monitored by a fluorescent dna content assay. similar colony forming unit reductions in animal models (to be shown in other poster presentations) suggest that these principles also operate in vivo. liposomes similar to liposomal amikacin (arikace tm ) readily penetrate into biofilms of pseudomonas aeruginosa and may even have enhanced binding to biofilms. this binding along with localized release can explain the substantial efficacy observed in animal models. coates, a.l. adherence to recommended therapy in cf has always been a challenge, in part, due to the time demands of the daily therapy. while twice daily inhaled tobramycin for those infected with pseudomonas aeruginosa (pa) has become an accepted standard of care, as much as minutes a day may be consumed inhaling mg in ml of tobramycin (tobi ® ) from the pari lc plus ® breath enhanced jet nebulizer. the purpose of this study was to determine if equivalent levels of pulmonary deposition could be achieved in a much shorter time period using . ml of a more concentrated ( mg/ml) tobramycin solution delivered by a perforated vibrating membrane nebulizer (eflow ® membrane configuration l) both, developed by pari pharma; germany. methods with a goal to study children and adults, to date, the subjects are children years and older and adult males, all with an fev > % predicted, with stable cf. all were receiving inhaled tobramycin for positive sputum cultures of pa. following pretreatment with albuterol, they inhaled both preparations on two occasions with m tc-dtpa added to the tobramycin in the nuclear medicine facility. in vitro preliminary work demonstrated that the radiolabel tracked with both formulations of tobramycin. deposition was measured by a gamma camera taking both tissue attenuation and mucociliary clearance during nebulization into account (pediatric pulmonol suppl : a ; ) . in order to have a continuous rate of deposition, the pari lc plus ® was run for a timed minutes and then both the total deposition and time of nebulization "scaled up" from in vitro testing when the nebulizer was run to dryness. this was done by multiplying the deposition by the total output when run to dryness divided by the total output in minutes. (blood samples were taken for quantification of tobramycin in the serum but not yet analysed). the rate of output per minute was calculated from the minute run and the total time was total output from in vitro testing divided by the rate of output. the eflow ® pro-vides a continuous output and stops automatically at dryness. quality assurance was the agreement between total radioactivity pre nebulization (in the nebulizer) and post which included the subject, the nebulizer, the connectors and the expiratory filter. the pari lc plus ® delivered . ± . mg in . ± . minutes compared to . ± . mg in . ± . minutes for the eflow ® . only the time of delivery was significantly different with p< . (paired t-test). tolerability of the treatment was comparable for both inhalation regimes, but the shorter treatment was preferred by all patients. these results demonstrate the possibility of delivering equivalent levels of tobramycin in much shorter periods of time into the lungs of cf patients when using eflow ® , a very efficient electronic nebulizer. this time saving may improve adherence to recommended therapy. (pediatrics ; : ) . in order to properly interpret op cultures from nbs infants, especially those with non-classic cf mutations, we need to know the op flora of non-cf infants. we obtained op cultures from healthy infants under yr of age. op specimens were plated on standard cf culture media. exclusion criteria included a first degree relative with cf, respiratory illness at the time of culture, or positive newborn screen for cf. data on cigarette smoke exposure, animal exposure, and exposure to hot tubs/swimming pools was collected. samples have been collected to date. in healthy, non-cf infants, the most common finding is non-specific mixed gram negative and positive growth. however, infants have grown pa (ages months, months, and months). many infants have grown multiple organisms. the following bacteria have been found in (n) number of infants: s. aureus ( ), e. coli ( ), e. cloacae ( ) , h. flu ( ), klebsiella ( ), pseudomonas aeruginosa ( ), h. parainflu ( ), unidentified non-lactose fermenting ( ), other ( ). data on infants including correlation to environmental factors will be presented. conclusion: non-cf infants commonly have s. aureus and many gram negative organisms including pa in their oropharynx. these results may have some bearing on interpetting colonization and clearance of pa in infants identified through nbs and in epic study participants. # ) ). identification of smg by sputum cultivation represents a significant challenge because the organisms are phenotypically diverse, grow poorly on routine culture media, and are very difficult to discriminate from other members of the oropharyngeal flora. we have developed a solid media for the selective isolation of smg from sputum. the value of the media is highlighted by the identification of smg as the quantitatively dominant organism in sputum samples of three cf patients admitted to hospital for an acute pulmonary exacerbation. in all three, the smg species failed to be identified on routine or selective media currently described for the culture of cf-specific sputum pathogens. antibiotic treatment directed against the smg correlated with clinical resolution of acute symptoms as well the reduction of smg on daily serial sputum cultures during hospitalization. this novel selective media makes use of antibiotics (colistin, sulfadiazine and oxolinic acid) inhibitory to the growth of principal cf pathogens and much of the usual oropharyngeal flora. smg agar utilizes a colorimetric indicator to uniquely identify smg colonies. the sensitivity and specificity of the selective media has been evaluated by molecular methods using terminal restriction fragment length polymorphism analysis. smg organisms do not respond well to anti-pseudomonal therapy, therefore proper detection and culture-directed antibiotic therapy is paramount. we believe that smg represent significant respiratory pathogens in cf, and because of the inability to effectively culture and identify smg they have largely gone unrecognized. pseudomonas aeruginosa releases substantial amounts of the blue antibiotic pigment pyocyanin. in presence of a reductant (such as nadph), pyocyanin redox-cycles and generates superoxide and h o . in infected cf airways, pyocyanin concentrations can reach high micromolar levels and contribute to oxidative stress of the airways. the structural basis of the pyocyanin molecule that underlies the redox-cycling with reductants of the airways is not clear. we therefore investigated i) the ability of physiologically or pharmacologically relevant reductants of the airways to support the redox-cycle activity of pyocyanin, and ii) the molecular features of pyocyanin that support redox cycling. dose-and time-dependent h o production by pyocyanin was measured by amplex red oxidation in presence of horseradish peroxidase. rates of h o production by µm pyocyanin in presence of µm reductant were: nadph ( pmole/min) > l-ascorbate ( pmole/min) > reduced glutathione ( pmole/min) > α-tocopherol ( pmole/min). in contrast, lipoic acid, genistein, or resveratrol did not significantly support pyocyanin-mediated h o production. in absence of a reductant, pyocyanin showed no measurable formation of h o . to identify the structural characteristics of the pyocyanin molecule that allow for its redox-cycling activity we synthesized a number of new pyocyanins containing electron-donating or electron-withdrawing substituents. functional assays were performed in presence of l-ascorbate as reductant. molecular substituents that donated electrons to the positively charged core of pyocyanin, either by hyperconjugative or resonance effects, reduced the h o output of the corresponding pyocyanin. in contrast, a closely related analog ( -hydroxyphenazine- , dioxide) showed significantly increased activity ( . x compared to pyocyanin) suggesting that the electron-withdrawing effect of the n-oxide functionality led to an increase in the redox-cycle activity. these data indicate that the functional characteristics of pyocyanin as a redox-cycling compound are governed by its positively charged core. in the airways, pyocyanin is predicted to utilize several reductants that are present in the airway surface liquid or intracellularly, thus contributing to cf airway disease. because pyocyanin utilizes a variety of reductants, it appears prudent to test whether inhaled small-molecular cf therapeutics support pyocyanin function. supported by nih (hl- , p at ), cfri, philip morris usa inc and philip morris international, and cff (fischer g ). taccetti, g.; braggion, c.; ravenni, n.; zavataro, l.; neri, a.; festini, f.; campana, s. meyer hospital, university of florence, cf center of tuscany, florence, italy for practical purposes, after early eradication treatment, at least three consecutive negative respiratory cultures over a -month period would indicate that the organism has been eradicated (cf trust guidelines). this recommendation is based on opinion/clinical experience of respected authorities in the absence of directly applicable studies of good quality. aims: using molecular biological techniques, we evaluated whether this -month interval is really trustworthy for distinguishing between regrowth of the same strain, suppressed but not eradicated by treatment, and new pseudomonas aeruginosa (pa) colonization. patients and methods: cystic fibrosis (cf) patients were treated with oral ciprofloxacin and nebulized colistin at detection of pa. all pa colonization episodes were recorded in an appropriate database. molecular study of each bacterial isolate from each colonization episode was performed with the rapd-pcr. results: between between - of patients in follow-up in our center had repeated pa colonization. the patients' mean age at first pa colonization was ± . months. a total of episodes was observed (mean of . episodes per patient, median of , range - ). molecular typing on strains indicated that ( . %) were a different genotype from later colonization episodes while ( . %) isolates had the same genotype as those of the preceding episode. the same genotype as preceding colonization was observed in ( . %) of isolates from patients in which a successive colonization was verified in less than months, and was verified in ( . %) of strains in patients having a successive colonization in over months (or= . ). colonization was due to a genotypically diverse strain in % of cases where colonization occurred within months of eradication. during the observation period ( %) of patients acquired chronic pa infection. conclusion: re-colonization by pa following eradication therapy is mostly ( %) caused by strains with a different genotype, suggesting acquisition from an external source. a short pa-free period is mainly due to transient suppression of pa growth, and true eradication followed by acquisition of a new pa genotype occurred in most cases only after a pa-free period longer than months. those patients with a pa-free interval of less than months had a six-times higher chance that the pa was not eradicated compared to those with a germ-free interval of over months. this evidence demonstrates that the definition of successful eradication should be reconsidered, taking into account additional parameters such as molecular analysis. cystic fibrosis (cf) patients appear to have an increased risk of urolithiasis. while a number of possible explanations for this have been proposed and investigated, no definitive mechanism has yet been demonstrated. as cf patients frequently get respiratory tract infections they regularly receive ciprofloxacin treatment, often given in doses well in excess of conventional prescription regimes. there are occasional reports of ciprofloxacin crystals urine and stones in the urinary tract. here we investigate the hypothesis that ciprofloxacin excreted in urine might act as a promoter of crystallisation of calcium or magnesium salts and thereby increase the risk of kidney stone disease. the effect of ciprofloxacin was tested in artificial urine (au). in vitro crystallisation was tested using a well plate turbidity method, to identify a metastable limit of oxalate concentration (ml) and a growth and nucleation parameter, the turbidity rate index (tri). the nucleation ph of urine was examined by tritrating oxalate free au through a ph range of . to . and monitoring the solution/suspension turbidity. in au at ph . , ciprofloxacin, at , , or mg/l, had no detectable effect on initiation of calcium oxalate crystallisation (ml) or its progress (tri) (n= for each concentration). when au with mm ca, mm mg and mm po was titrated there were two distinct nucleation points; a slow event starting at about ph . and a much faster event at about ph . . omitting ca or mg confirmed that the first event was due to calcium phosphate precipitation and the second to struvite. including ciprofloxacin at , or mg/l did not alter these nucleation ph values, but the magnitude of the turbidity rise showed that the ciprofloxacin co-precipitated with the struvite. ciprofloxacin at mg/l and without ca or mg began to precipitate at ph . and could be held in solution until ph . when ca and mg were included. even at high concentrations, ciprofloxacin does not influence calcium oxalate crystallisation. nor does it promote calcium phosphate or struvite precipitation; on the contrary, while the calcium and magnesium remain in solution, they help to prevent precipitation of the ciprofloxacin itself. urinary ciprofloxacin does not appear to act as a stone or crystal promoter. pseudomonas aeruginosa is a significant cause of mortality in cystic fibrosis (cf) sufferers. cf patients were thought to acquire p. aeruginosa from the environment; however genotyping over recent years has revealed clonal strains in sputa from cf patients in the uk, australia, and canada that are transmitted person to person or from a common source. one clonal strain, australian epidemic strain- (aes- ), (formerly melbourne epidemic strain, m or pi) currently infects up to % of patients in five cf clinics on the eastern seaboard of australia. most cf clonal strains have been associated with increased virulence not fully explained by greater antibiotic resistance. both genotypic and phenotypic differences have been postulated as important in enabling transmission of clonal strains. in order to compare the expression profile of aes- to the type strain p. aeruginosa pao , the cf research group at the university of sydney compared the genome expression data of four clonal aes- isolates and pao , when grown as planktonic and as -hr biofilm cultures. in aes- , a set of significantly differentially expressed genes (all downregulated) were identified, including the quorum sensing genes lasa, lasb and rhll. in contrast, both upregulated and downregulated genes were differentially expressed in pao biofilm compared to pao planktonic culture. expression data was validated using quantitative real-time pcr. to compare biofilm growth at the phenotypic level, the four clonal strains and pao were grown as -hr biofilms in a double-blind study, and the size of ten randomly selected biofilms per isolate, stained with syto® green fluorescent stain was measured. at hr, the biofilms formed by aes- isolates were significantly larger (ca. -fold) than pao (average size: ± µm vs ± µm )(p< . ). the average thickness of three biofilms per isolate, measured by confocal microscopy, showed aes- biofilms to be approximately . -fold thicker than those formed by pao ( . ± . µm vs . ± . µm). the general gene downregulation observed in aes- biofilms suggests an adaptation to the cf host, while a larger biofilm would provide for more effective bacterial dispersal. thus the transmissibility of aes- may be linked to enhanced biofilm formation upon colonisation. background -objective: whilst influence induced by bacterial colonization in cystic fibrosis (cf) is established, risk induced by fungal colonization is less defined. prevalence of other species than aspergillus sp. or candida sp., and factors associated with fungal presence are also poorly documented. our preliminary study aimed to determine which fungal species were present in sputum collected from adult cf patients, and which factors were associated with fungal presence. methods: in a monocenter, transversal prospective study, cf adult patients were included to determine fungal presence in sputum using semi selective growing media. clinical parameters (shwachman score, respiratory function, nutritional status, gastro-oesophageal reflux, pancreatic insufficiency and diabetes); therapeutics used (including oral or intravenous antibiotics, systemic or inhaled corticosteroids or bronchodilatators, antifungal treatments); microbiological data of bacterial colonization and environmental parameters (potted plants or domestic animals presence) were determined for each patient. correlation between fungal, clinical, environmental, therapeutic or microbiological data was evaluated by mann-whitney non parametric u test. results: patients ( %) presented with fungal presence in sputum. % presented with yeasts species, % with moulds. aspergillus fumigatus and candida albicans were the predominant species in moulds and yeasts respectively, but less common mould species such as exophiala dermatidis or paecilomyces variotii were also recovered. factors associated with fungal presence were pancreatic insufficiency (p= , ); malnutrition (p= , ), bacterial colonization and inhaled corticosteroids. candida albicans was correlated with more severe shwachman score (p= , ), bacterial colonization (p= , ), notably with pseudomonas aeruginosa (p= , ) , and intravenous antibiotics use (p= , ). moulds species were significantly associated with inhaled corticosteroids (p= , ). antifungal use was associated with frequent resistance to azoles treatments ( resistant isolates out of patients treated). conclusion: fungal presence in cf appears frequent. some species could have been previously overlooked due to diagnosis difficulties. the effect of corticosteroids on moulds species, already found in other pathologies, appears important in cf. influence of fungal presence on cf course needs prospective studies, in order to establish if patients could benefit from antifungal treatments or preventive measures. dren on a ventilator without a previous diagnosis of cystic fibrosis is unknown. the aim of our study was to investigate the prevalence of these microorganisms in routine sputum cultures in young children on a ventilator in a pediatric intensive care unit (picu). methods: from all ventilated children aged - years admitted from - , sputum culture results obtained from tracheal aspirates within the first week of admission were retrospectively analysed. three patient subgroups were identified: respiratory failure due to pulmonary disease (group ), ventilation after elective surgical procedures (group ) and other ventilated children (group ). children with a previous intensive care admission or cf were excluded. the cf database was checked ( ) to identify any children with a new diagnosis of cf that were included in the study. results: . % of all ventilated children had a positive sputum culture with one of the "cf-bacteria" s. aureus, h. influenzae and p. aeruginosa these were found mainly in group (see table) . / of these children were admitted for treatment of respiratory syncytial virus (rsv) bronchiolitis. a sweat test was performed in children, all admitted with pulmonary disease and because of co-existing clinical signs or symptoms. one sweat test was positive and subsequently cf was diagnosed in this child. no other children out of the study group have since then been diagnosed with cf. as the population in the northern part of the netherlands is very stable, it is unlikely a diagnosis of cf has been missed. conclusion: s. aureus and h. influenzae are cultured frequently in ventilated children without cf, especially when ventilated because of pulmonary disease. the opportunistic pathogen p. aeruginosa causes both acute and chronic human infections. the balance between systemic infection and mortality or chronic persistence and morbidity depends on complex relationships in which the immunological status and genetic potential of the host but also the bacterial biodiversity are determinant factors. p. aeruginosa extensive genetic adaptation and microevolution have been repeatedly observed in chronic infections of cf patients in contrast to what it is documented in acute infections. whether these p. aeruginosa clonal variants differ in their pathogenic potential is not yet known. a total of clinical p. aeruginosa isolates from six cf patients which carried unique clonal lineage from the onset of colonization over - years were selected (bragonzi et al, ; montanari et al, ) . five p. aeruginosa environmental strains, which represent the source of acquisition for cf patients, and two laboratory strains pao and pa were also used as references. multiple genotypic analysis of sequential p. aeruginosa isolates which included pfge, atchip and multilocus snps showed intraclonal diversity with genome rearrangements, variations in pathogenic islands and acquisition of prothoadaptive mutations in the muc genes. the largest divergence was observed between the completely sequenced reference strains pao and pa , the latter represented in our panel by three cf isolates. p. aeruginosa virulence has been assessed by monitoring its capacity to induce bacteremia and to establish chronic infection in a murine model (bragonzi et al, ) . overall, p. aeruginosa environmental strains increased five times the risk of bacteremia when compared to clinical strains (test of proportion: p< . ). the high risk of mortality was also evidenced for p. aeruginosa cf strains isolated at the onset of the infection when compared with those isolated after years of colonization (p= . ) indicating that environmental strains or newly acquired strains were similar in their virulence. p. aeruginosa clinical strains isolated after years of colonization increased chronic persistence and reduced the risk of bacteremia (p= . ) when compared to strains isolated at the onset of the infection. the strain pa was found to be lethal in contrast to p. aeruginosa clonal strains of clinical origin that established chronic persistent infection in the murine lung. furthermore, our data showed that adaptive traits commonly associated with the chronic p. aeruginosa infections in cf patients, such as transition to the mucoid phenotype, did not confer a selective advantage to bacterial cells in colonizing the murine lung (p= . ). these results suggest that p. aeruginosa pathogenicity is independent of the strain's genotype but rather genetic adaptation to the cf airways plays an important role in the development of persistence and in the resistance to host defences. supported by telethon and italian cf research foundation. beringer, p. the combination of antipseudomonal beta-lactam and aminoglycoside antibiotics is frequently prescribed during acute pulmonary exacerbations. since the introduction of the beta-lactam compounds there has been numerous reports citing altered pharmacokinetics of several compounds (but not all) within the beta-lactam class. recently it has been suggested that substrate specificity for the renal transporter pgp may explain the variability in renal drug clearance observed in patients with cf. pgp is structurally related to cftr and evidence suggests it serves a complementary role in modulating alternative chloride channel function. in a cftr knockout model mdr expression was reported to be increased nearly four-fold. in an effort to elucidate the potential contribution of pgp to the renal clearance of drugs in patients with cf, we conducted a controlled clinical trial comparing the pharmacokinetics of fexofenadine (fx) in patients with cf and age matched healthy volunteers (hv). fexofenadine is not significantly metabolized and is a relatively specific substrate for the membrane transporters pgp and oatp. probenecid (pb) is a selective inhibitor of oatp and was used pharmacologically to block the activity of this transporter in-vivo. our hypothesis is that if pgp is upregulated within the renal tubules of patients with cf we would expect to see an enhanced renal clearance of fexofenadine in these patients when compared with control subjects. methods: (n= cf, hv) subjects underwent this prospective controlled study which fx was received alone or in combination with probenecid (pb). iothalamate was given each day to measure glomerular filtration rate (gfr). blood and urine samples were obtained at specified times over hours each study day for determination of fexofenadine and iothalamate concentrations. fexofenadine and iothalamate concentrations were assayed using liquid chromatography-tandem mass spectrometry and hplc-uv respectively. pharmacokinetic analysis was performed using a -compartment cumulative urinary excretion model with the adapt ii software. differences between groups were determined using a paired t-test or mann whitney-u. results: patients were included, cf patients were slightly older and had lower body mass index when compared to hv, (mean±sd, ± . vs. . ± years p= . , . ± . vs. . ± . p= . respectively) but did not differ in gfr ( ± . vs. ± ml/min- . m p= . respectively). no significant differences were found between cf and hv in clr of fx alone and fx in combination with pb clr ( . ± vs. . ± . ml/min- . m p= . and . ± vs . ± . ml/min- . m p= . respectively). conclusion: the results of this study indicate the enhanced clearance of certain antibiotics previously reported in patients with cf do not appear to be due to upregulation of renal tubular pgp or increased gfr. maceachran, d.p. department of microbiology and immunology, dartmouth medical school, hanover, nh, usa p. aeruginosa is the leading cause of morbidity and mortality in cystic fibrosis (cf) patients. shortly after birth cf patients are colonized by p. aeruginosa, which leads to a lifelong chronic infection. the mechanisms behind p. aeruginosa colonization of the cf lung are still poorly understood, however several secreted toxins have been associated with this phenomenon. cf is characterized by a loss of mucocilliary clearance, a key component of innate immunity in the lung, and an increase in sputum viscosity as a result of mutations in the gene encoding the cystic fibrosis transmembrane conductance regulator (cftr). we have previously shown that the novel p. aeruginosa secreted protein cif is capable of reducing apical membrane expression of cftr. we have begun to characterize cif expression and have identified a key regulator of cif transcription. mutations affecting the divergently transcribed putative tetr family repressor encoded by the pa gene result in a significant increase in cif expression. furthermore, we have demonstrated that the pa gene product is capable of binding the promoter region located immediately upstream of the cif-containing operon. this work has been supported by the nih t -dk - . ryall, b. ; davies, j.c. , ; wilson, r. ; shoemark, a. ; williams, h.d. . division of biology, imperial college london, london, united kingdom; . department of gene therapy, imperial college london, london, united kingdom; . host defence unit, royal brompton hospital, london, united kingdom; . paediatric respiratory medicine, royal brompton hospital, london, united kingdom pseudomonas aeruginosa is the most important respiratory pathogen in patients with cystic fibrosis (cf) and non-cf bronchiectasis. the factors which enable p. aeruginosa to establish chronic, debilitating respiratory infections are unclear. however p. aeruginosa is one of a limited number of bacteria that is able to synthesise hydrogen cyanide, a potent inhibitor of cellular respiration. this study examined whether hydrogen cyanide is produced by p. aeruginosa in cf and non-cf bronchiectasis airway infection. cyanide concentration was measured in fresh sputum from cf and non-cf bronchiectasis patients with and without p. aeruginosa lung infection using a cyanide ion sensing electrode. cyanide was detected in sputum from / cf and non-cf bronchiectasis patients with current p. aeruginosa infection, whereas it was not detected in any of the patients without this organism (p< . ). maximum lev-els were µm (mean ±se: ± . µm), which compares with levels of above µm which would be considered toxic in blood. concurrent lung function data were available on all p. aeruginosainfected cf patients; the group with measurable sputum cyanide (n= ) was not different from those without (n= ) on the basis of age, gender or p. aeruginosa phenotype (mucoid/ non-mucoid). however, those with detectable cyanide had significantly poorer lung function than those without (fev % predicted: . ± . % versus . ± . %, p< . ; fvc% predicted: . ± . % versus . ± . , p< . ). we have shown that cyanide is detectable at clinically significant levels in sputum from cf and non-cf bronchiectasis patients infected with p. aeruginosa and is associated with significantly impaired lung function. we have recently reported a dose regimen for once-daily tobramycin dosing in patients with cystic fibrosis which was based on a retrospective study (lam w et al., j antimicrob chemother ) . we present here an interim analysis of an ongoing prospective study to evaluate the proposed dosing in paediatric patients with cystic fibrosis. as part of this protocol pharmacokinetic parameters were determined in cystic fibrosis (cf) patients receiving intravenous antibiotic therapy for a pulmonary exacerbation. serum creatinine, audiometric testing, respiratory cultures and fev were assessed at baseline and after a week treatment period. serum tobramycin concentrations were interpreted by clinical pharmacists performing therapeutic drug monitoring (tdm) to optimize dosing. serum tobramycin levels were drawn after the first dose, repeated with each dose adjustment and weekly thereafter. the proposed sample size of this study is patients; an interim analysis was performed to identify trends for pharmacokinetic and safety outcomes from patients admitted from november through april . seven ( . %) patients achieved the target c max of - mg/l, ( . %) patients remained under the target range and patient was above the target range with the st dose. with nd, rd, and th tdm performed for either routine weekly levels or dose adjustment levels, , , and patients met the target c max respectively. the percentage of patients who achieved target c max increased to . % with the rd set of tdm levels and . % with the th set of tdm levels (p-value< . ). there were no significant changes in k e , t / and v d /kg for each patient from st to nd set of tdm levels over time. compared to the previous retrospective study the mean v d /kg was significantly higher ( . l/kg versus . l/kg, p= . ) and the mean t / was significantly shorter ( . hrs versus . hrs; p< . ). none of the patients' serum creatinine increased ≥ % from baseline. baseline audiometric tests were within normal limits in ( . %) patients. p. aeruginosa was isolated from respiratory specimens of patients on initial culture. of the patients with follow-up cultures, patients grew p. aeruginosa sensitive to tobramycin, demonstrating no decrease in sensitivity. our preliminary results suggest the pharmacokinetic data differ from the expected profile suggested by the retrospective study. all study patients treated according to the guidelines were clinically well after their course of therapy and did not experience clinically significant toxicity from once-daily tobramycin dosing. the once-daily tobramycin dosing will continue to be evaluated until the proposed sample size is achieved for final analysis. anderson, g.g.; o'toole, g.a. dartmouth medical school, hanover, nh, usa pseudomonas aeruginosa chronically colonizes the lungs of individuals with cystic fibrosis (cf). evidence suggests that this infection progresses through distinct phases, wherein initial colonizing strains undergo a switch to mucoidy that corresponds to formation of bacterial microcolonies in the airways and life-long persistence. currently, the environmental signals and mechanisms regulating conversion from the acute, highly virulent form to a less virulent biofilm state remain obscure. to investigate these issues, we developed a tissue culture model system for growth of p. aeruginosa biofilms on cf-derived human airway epithelial cells in vitro. biofilms grown on these cells appear similar to those found in cf airways as well as to abiotic biofilms. eventually, epithelial cells were killed by the p. aeruginosa biofilms. however, we discovered that addition of the antibiotic tobramycin preserved the monolayer integrity for at least hours. bacteria were not completely eliminated by this treatment, suggesting that tobramycin influenced the virulence of the biofilm bacteria. microarray analysis of cf cell grown biofilms treated with tobramycin revealed marked alterations in transcriptional profiles compared to untreated controls. two tobramycin-induced genes were identified from this screen which, when deleted, exerted altered virulence phenotypes on airway epithelial cells in culture. quantification of cytotoxicity of these mutants revealed either increased or decreased ability to kill the epithelial cells. complementation with full-length copies of these genes restored wild-type function. these studies suggest a complex relationship between acute and chronic forms of p. aeruginosa, and that antibiotic treatment may influence transitions between these modes. it is this interplay that may determine the virulence status of the microorganism upon initial infection as well as during cf exacerbations. neville, m.; sardaryan, g.; ejaz, s.; scotto, a.w.; gupta, r. transave, inc., monmouth junction, nj, usa purpose: during the course of treatment, patients with cystic fibrosis and chronic pseudomonas aeruginosa infections in their lungs may be treated with nebulized tobramycin (tobi®). reduction in treatment times or dosing frequency may improve compliance and outcome. nebulized arikace™ may offer advantages to tobramycin by producing sustained lung levels of drug, reducing dosing frequency and increasing antimicrobial efficacy. herein are presented biodistribution data and efficacy data in a chronic rat model of p aeruginosa comparing arikace™, soluble amikacin and tobramycin. method: female rats (charles river) received (~ mg / kg) of arikace™, soluble amikacin and tobramycin by inhalation. test articles were aerosolized in a pari lc star nebulizer at psi using devilbiss compressors (sunrise medical) for min. - rats were euthanized at time and hr post inhalation by co asphyxiation. blood was collected by a cardiac puncture. serum was collected from coagulated blood by centrifugation and stored at oc until analysis for drug content. in addition lungs, kidneys, intestinal contents, urine and brains were harvested. tissues and biological samples were homogenized in saline and antibiotic concentrations determined by tdx analyzer (abbott). chronic lung infections were produced in rats by intratracheal instillation of agar beads containing p. aeruginosa. three days post instillation, rats (n= /group) were treated with aerosolized saline or once daily with arikace™, or twice daily with tobramycin (total daily dose = mg /kg) for days. rats were euthanized days post final dose and the pseudomonas cfu counts per lung were determined. results: the biodistribution and pharmacokinetics of aerosolized arikace™ was significantly different from that of amikacin and tobramycin. the primary difference was the amount of antibiotic retained in the lungs. rats that inhaled soluble amikacin cleared approximately % of the amikacin from the lungs after hr with concomitant % increase in drug levels in the urine. rats that inhaled arikace™ cleared only % of the deposited amikacin from the lungs after hrs. this initial clearance of drug most likely reflected the amikacin released during nebulization. extended clearance of arikace™ from the lungs was measured by recovery of amikacin in the urine at later time points. in a separate longer-term study comparing arikace™ and tobramycin, auc ( - hr)for arikace™ in the lung was ug*hr/g while only ug*hr/ g for tobramycin which demonstrates a -fold increase in retention of antibiotic in the lung with arikace™. eradication of p. aeruginosa from the lungs of rats with arikace™ was equal to tobramycin (given equivalent daily doses). however, arikace™ reduced the number of cfu in the lungs with a single daily exposure to the same degree as inhalation of tobramycin twice a day. conclusions: inhalation of arikace™ achieved higher concentrations and increased retention time of the antibiotic in the lungs of rats when compared to inhaled amikacin or tobramycin. it is expected that these properties of arikace™ will reduce dosing frequency and improve antimicrobial efficacy in patients. nielsen, x.c. ; johansen, u.r. ; nørregaard, l. ; vandamme, p. ; høiby, n. . clinical microbiology, rigshospitalet, copenhagen, denmark; . laboratory for microbiology, university of ghent, ghent, belgium background: the burhkolderia cepacia complex (bcc) is a diverse group of bacteria with at least genomovars (gv) or species. accurate species identification is necessary for better understanding of the epidemiology and pathogenesis of bcc. species identification based on phenotypic characters is difficult when it is based on few if any differences. crossed immunoelectrophoresis (cie) has been performed routinely to identify all burkholderia and other difficult isolates in our department (høiby et al.: "taxonomic application of crossed immunoelectrophoresis." int. j. syst. bact. ( ): - .) . in this study, we compared the results obtained by cie with those obtained by the standard molecular biological method using reca gene pcr and rflp to identify bcc at the species level (golden standard). methods: between and , burkholdreia isolates were recovered from patients in the two danish cystic fibrosis (cf) centres. . cie method: immunological cross-reactivity between antigens from the reference strains and the strains isolated from our cf patients were compared by employing cie and rabbit standard-antibodies (purified igg) raised against water-soluble antigens from reference strains of the bcc. species identification was made based on matching coefficient (mc). . reca gene-based identification: bcc specific primers were used to amplify reca gene. species-specific pcr is then performed using b. multivorans specific primers to identify the b. multivorans gv ii. rflp patterns were generated for those non-multivorans bcc by digesting the bcc-specific pcr products with haeiii. the patterns were then compared with the patterns generated from reference strains in the database. pulsed field gel electrophoresis (pfge) genotyping was performed for all strains to detect patient-topatient transmission. results: / isolates were bcc-specific pcr positive. species-specific pcr identified isolates as b. multivorans (gv ii). the other three isolates were identified as b. cenocepacia (gv iii) based on rflp patterns. two isolates with negative bcc-specific pcr reactions were identified as b. gladioli by cie (confirmed by reference laboratory in belgium). results from cie showed that strains from bcc were immunologically closely related with only a few non-cross-reactive antigens. compared with the standard reca gene-based method, cie method resulted in correct identifications in isolates ( . %), misidentifications in isolates ( . %) and uncertain identifications in isolates ( . %). genotyping by pfge showed unique patterns for all isolates except for those from two sisters. this suggested that patient-to-patient transmission of bcc among the danish cf patients has remained very rare. conclusions: reca gene-based pcr and rflp is a quick and reliable method for identification of bcc at the species level. cie proved useful in initial classification of bcc strains, but additional information is obtained by the reca gene-based species identification and pfge method. background: in childhood, the cystic fibrosis airway is characterized by persistent inflammation with high quantities of neutrophils. in this setting, recurrent exposure to low numbers of pseudomonas aeruginosa occurs routinely. chronic infection by p. aeruginosa is associated with a significant increase in morbidity and mortality in cf, and is believed to occur by formation of a biofilm in the cf airway. previously, we have shown a significant neutrophil dependent enhancement of p. aeruginosa biofilm density that is greatest for low initial concentrations of p. aeruginosa. biofilm enhancement is facilitated via neutrophil-derived dna and f-actin, which form a framework that p. aeruginosa can exploit for growth. dna and factin polymerize via positively charged molecules such as histones. p. aeruginosa then associates with these polymers, which results in increased early biofilm density, displaying an increase of over fold at hours compared to p. aeruginosa in the absence of neutrophils. hypothesis: compounds that disperse dna and/or f-actin can disrupt neutrophil-enhanced biofilms. methods: poly(aspartic acid), a negatively charged amino acid chain with the capacity to disrupt f-actin, was examined singly and in combination with dnase and/or antibiotics (ciprofloxacin and tobramycin). the nunc-tsp system was used to allow for high throughput assessment of the density of biofilm formation in the presence of neutrophils and the effect of various agents in disrupting the biofilm. the biofilm assay is independent of cellular settling, as the reactor is vertically suspended in the culture. p. aeruginosa strains tested were pao , and two isogenic cf isolates recovered from an initial infection (early), and following established infection ~ years later (late). biofilms were formed by incubation of p. aeruginosa with human neutrophils for to hours in rpmi with % heat inactivated platelet poor plasma. results: antibiotics were incapable of disrupting biofilms. poly(asp) significantly disrupted neutrophil-induced biofilms. in the presence of neutrophil products this action is sensitive to proteolytic degradation, but can be protected by the presence of protease inhibitors. dnase also disrupts a biofilm formed in the presence of human neutrophils. the combination of poly(asp) + dnase resulted in an increase in biofilm disruption for a hour biofilm. biofilms allowed to form for hours were more resistant to disruption by dnase and poly(asp) as single agents, but the combination of both demonstrated a synergistic effect. similar disruption of biofilms were observed when pao was compared to early and late cf strains. conclusions: pseudomonas aeruginosa biofilms formed in the presence of neutrophils in vitro can be disrupted by agents that disperse the f-actin and dna framework. support: cff, max and yetta karasik foundation cystic fibrosis (cf) patients may suffer increased morbidity and mortality through colonisation, allergy and invasive infection from fungi. the black yeast, exophiala dermatitidis (synonym wangiella dermatitidis) has been found with increasing frequency in sputum specimens of cf patients, with reported isolation rates ranging from between . and . %. at present, no species-specific diagnostic pcr exists to aid with the clinical laboratory detection and identification of this organism. a novel species-specific pcr-based assay was developed for the detection of e. dermatitidis, based on employment of rdna operons and interspacer (its) regions between these rdna operons. two novel primers, (designated exdf & exdr) were designed in silico with the aid of computer-aided alignment software and with the alignment of multiple species of exophiala, as well as with other commonly described yeasts and filamentous fungi within cf sputum, including candida, aspergillus and scedosporium. pcr employing exdf (forward primer [ -,mer], '-ccg cct att cag gtc c- ') and exdr (reverse primer [ -mer], '-tct ctc cca ctc ccg c- '), was employed and optimized on extracted genomic dna from a well characterized culture of e. dermatitidis, as well as with high quality genomic dna template from a further unrelated fungi, including candida albicans, c. dubliniensis, c. parapsilosis, c. glabrata, scedosporium apiospermum, penicillium sp., aspergillus fumigatus, aspergillus versicolor, pichia guilliermondii, rhodotorula sp., trichosporon sp., aureobasidium pullulans, fusarium sp., mucor hiemalis, bionectria ochroleuca, gibberella pulicaris. results demonstrated that only dna from e. dermatitidis gave an amplification product of the expected size, whilst none of the other fungi were amplifiable. subsequent direct employment of this primer pair detected this yeast in the sputum of / ( %) adult cf patients, employing a nested pcr approach with panfungal outer flanking primers of the its -its region. these two patients were the only patients who were previously shown to have a cultural history of e. dermititidis from their sputum. e. dermatitidis is a slow-growing fungus, which usually takes up to two weeks to culture in the microbiology laboratory and therefore is slow to detect conventionally, with the risk of bacterial overgrowth from common co-habiting pan-and multiresistant bacterial pathogens from sputum, namely pseudomonas aeruginosa and burkholderia cepacia complex organisms, hence this species-specific pcr assay may help detect this organism from cf sputum more specifically and rapidly. overall, employment of this novel assay may help in the understanding of the occurrence, aetiology and epidemiology of exophiala dermatitidis, as an emerging fungal agent in patients with cf. cystic fibrosis patients are particularly susceptible to infection by strains of pseudomonas aeruginosa and burkholderia cepacia complex. since , the pseudomonas genome database has been a resource for peer-reviewed, continually-updated annotations for the pseudomonas aeruginosa pao reference strain's genome and, more recently, comparative analyses to several closely-related pseudomonas species. in order to facilitate better cross-strain and cross-species genome comparisons, we have developed or are incorporating methods to improve the identification of orthologs (genes diverged due to speciation) and identify genes undergoing unusual selection. our analysis of several completely sequenced pseudomonas genomes has revealed that approximately % of ortholog predictions by the classic "reciprocal best blast hit method" are likely incorrect. we have also performed an analysis of unusually large intergenic regions in p. aeruginosa pao that appear to be essential (according to saturation transposon mutagenesis) or involved in virulence (according to signature tagged mutagenesis). we identified at least new putative protein-coding regions and non-coding rnas that were not previously annotated and may play critical roles in microbial pathogenesis or viability. we are also focusing our attention on facilitating better cross-strain comparisons between recently sequenced burkholderia cepacia complex genomes through development of a new online burkholderia genome database. in addition to improved ortholog prediction, this new database will provide access to a very flexible boolean search feature that allows researchers to search and compare annotations within or between the genomes of burkholderia strains, returning annotations from multiple genomes suitable for simultaneous viewing and downstream analyses. we have also included new, very accurate protein subcellular localization predictions for the deduced proteome from each of these genomes -predictions that can aid the identification of new cell surface or secreted therapeutic targets or vaccine candidates. further comparative analyses with other newly-sequenced, related strains should provide insight into strain-specific features that may be exploited to better understand virulence and antimicrobial resistance exhibited by cf-relevant pathogens. funding provided by cystic fibrosis foundation, therapeutics (usa). popova, a.p.; verma, r.; zanni, r.l.; sembrano, e. pediatrics, monmouth medical center, long brnach, nj, usa background: cystic fibrosis (cf) is a chronic, progressive, genetic disease leading to poor lung function, chronic bronchial infection with certain bacteria and recurrent pulmonary exacerbations. despite evidence of inflammation within the airway, it is unclear which circulating inflammatory biomarker best reflects the airway inflammation and lung function in cf. objective: to determine the utility of c-reactive protein (crp) value in evaluating patients with cystic fibrosis pulmonary exacerbation. design: a retrospective chart review was carried out on patients followed at the cystic fibrosis center at the children's hospital. information about serum crp values, pulmonary function test results(pft), presence of pulmonary exacerbation as documented in the chart, sputum culture results, current medications, as well as demographic data, genetic compositions and weight percentiles between january and march was collected from the charts and was analyzed. results: of patients whose charts were reviewed, were excluded because they underwent lung transplant, were excluded because they had other mild infection, not diagnosed as pulmonary exacerbation. for patients crp was not measured and they were also excluded from the analysis. total of patients were included in the study. for patients information about crp was obtained on more than one occasion. the patients were divided into groups based on the presence of pulmonary exacerbation and elevation of crp values -in the group with pulmonary exacerbation, patients had elevated crp and had normal crp and in the nonexacerbation group had elevated crp and had normal. fisher's exact test (two -tail) was applied (p= . ) and confirmed statistically significant elevation of crp in patients with pulmonary exacerbation of cystic fibrosis. the means and standard deviations of the crp values for the patients without pulmonary exacerbation and the patients with pulmonary exacerbation were calculated and analyzed using independent group t-test (two tail). mean crp value for the exacerbation group was . with standard deviation of . and mean crp value for the non-exacerbation group of . with a standard deviation of . . statistically significant difference between the two means was noted (p= . ). conclusion: serum crp values in patients with cf pulmonary exacerbation were significantly higher than in patients without pulmonary exacerbation. serum crp may be a useful marker of pulmonary exacerbation in children. in the absence of pulmonary exacerbation, cf is not associated with elevated crp values in children. the findings of this study could aid in identifying patients with pulmonary exacerbation earlier and initiate appropriate therapy sooner. further studies are needed to determine whether crp values are associated with the severity of the underlying disease, to what extent the pft are affected, the age of the patient, the type of bacterial pathogen, and genetic factors. background pulmonary infections with nontuberculous mycobacteria (ntm) are commonly associated with structural lung diseases or airways clearance disorders such as cystic fibrosis and primary ciliary dyskinesia. we follow approximately individuals with ntm pulmonary disease who share common clinical features but have no identifiable systemic immune defect or clearly defined predisposing conditions. the pulmonary disease and clinical presentation suggest these individuals may have disorders of ion transport or ciliary function. while some cftr mutations and ciliary abnormalities are difficult to detect through standard genetic and ultrastructural analyses, in vivo physiologic tests of ion transport and measures of ciliary function may allow us to identify ion transport abnormalities in subjects with no identified cftr abnormalities, determine the functional significance of novel cftr mutations, and distinguish disorders of ciliary function from other airways clearance disorders. methods we evaluated the medical and family histories, chest and sinus ct's, sputum cultures, cftr genotypes, and sweat chloride levels of individuals with nontuberculous mycobacterial pulmonary disease and individuals with chronic airways disease and bronchiectasis. we systematically tested the pulmonary function (pft), nasal nitric oxide production (nno), and nasal potential difference (npd) of each subject. nasal mucosal scrape biopsies were performed and sent to unc-chapel hill for assessment of ciliary ultrastructure as part of the genetic diseases of mucociliary clearance consortium. pulmonary function, nno, and npd were measured on healthy controls. results all subjects had sino-pulmonary symptoms consistent with those associated with cf and pcd. subjects had low nno ( . ± . nl/min) consistent with levels reported in subjects with pcd (< nl/min, noone ). one subject with variant cf (∆f /r h) displayed low basal pd (- . mv), elevated na+ absorption ( . mv), and no cftrmediated cl-conductance (- . mv). subjects with cftr mutations, subject with isolated elevated sweat chloride ( mmol/l), and subject with isolated borderline sweat chloride ( mmol/l) displayed normal basal potential difference (- . ± . mv) and na+ absorption ( . ± . mv) but decreased cftr-mediated cl-conductance (- . ± . mv) relative to controls (- . ± . mv). conclusions ntm patients heterozygous for cftr-mutations or with elevated sweat chloride (and no identified mutations with coding region sequencing of cftr) may have detectable subtle differences in cftr clconductance, while measurement of nno may identify disease associated with ciliary abnormalities. these findings suggest subtle abnormalities in airways clearance may predispose to airway infection with environmental organisms such as ntm. support: intramural funds, national institute of allergy and infectious diseases muhlebach, m. ; goodrich, j. ; sutton-shields, t.n. ; wedd, j.p. ; henegar, c. ; miller, m.b. ; gilligan, p.h. . dept. of pediatrics, unc chapel hill, chapel hill, nc, usa; . university of north carolina hospitals, chapel hill, nc, usa; . school of public health, university of north carolina hospitals, chapel hill, nc, usa; . dept. pathology and laboratory medicine, university of north carolina hospitals, chapel hill, nc, usa introduction: proportion of methicillin resistant (mrsa) vs. susceptible sa infections increased from % in to % in in us hospitals. similarly, the prevalence of mrsa in the community has risen in recent years. these community acquired (ca) mrsa isolates are genotypically different, have different antibiotic susceptibility, and often carry the pvl virulence gene. this virulence gene is associated with more invasive, mostly skin infections in healthy individuals. reports on impact of mrsa in cf show conflicting data. , . the aim of this study was to determine the prevalence of ca-mrsa strains at our pediatric and adult cf center and to assess the impact of these infections on clinical outcome. methods: all mrsa isolates recovered from routine clinical cultures, were prospectively collected and analyzed for months ( / - / ). using molecular assays to determine the pvl status and to identify the sccmec (staphylococcal cassette chromosome) type, the organisms were classified as ca or ha. classification as ca mrsa was based on presence of pvl and sccmec iv type. clinical outcomes included lung function measurements in patients reliably performing spirometry and nutritional parameters (bmi%). patients who had undergone lung transplant were excluded as their lung functions vary depending on transplant outcome. results: of the patients identified in this month period, % had ha and % ca mrsa. neither current age (mean . yrs. in ha vs. . years in ca) nor age at acquisition of mrsa ( . vs. years) was significantly different. sixty-eight % of all mrsa infected patients had chronic p. aeruginosa infection, ( % in the ha and % in ca, relative risk . of having ha in presence of chronic p. aeruginosa infection). crosssectional comparison of patients infected with ca vs. ha acquired strains showed no difference in nutritional status (bmi vs. %). lung function (fev and fef - ) was not different in ca vs. ha patients, neither when including all mrsa infected patients or those who had no concomitant infection with other organisms (n= ). we will present further comparisons to age and gender matched children with ossa infection in the final report. conclusion: comparison to ossa in matched patients will assess clinical outcomes of infections with mrsa. initial data collected at our center did not show a significant difference in outcomes between patients infected with ca vs. ha mrsa. a multicenter study would most efficiently assess the impact on clinical outcomes and determine regional differences of mrsa patterns (ca vs. ha) in different cf centers. ref : the stringent response is a global regulatory mechanism used by bacteria to adapt to nutrient limitation and other environmental stresses, and is mediated by the signaling molecules (p)ppgpp (phosphorylated guanosine nucleotides). increases in (p)ppgpp repress metabolic processes and growth, and regulate genes involved in long-term stress and starvation survival in several species ((i)e. coli, m. tuberculosis(i)), bacterial multicellular behavior ((i)myxococcus xanthus(i)), and pathogenesis ((i)p. aeruginosa(i)). while many studies have investigated bacterial functions (such as attachment and motility) involved in biofilm development, less is known about the physiological adaptation occurring in biofilm growth. when cells grow to high densities entrapped in a polymer matrix, they must adapt to heterogeneous micro-environments where gradients in nutrients, oxygen and metabolic waste exist. nutrient may thus become depleted as they are consumed by overlying cells, and some biofilm subpopulations are likely starved. we hypothesized that the stringent response mediates adaptation to nutrient starvation in (i)p. aeruginosa(i) and is important for biofilm formation. we tested a (i)rela(i) deletion mutant unable to synthesize (p)ppgpp in response to amino acid starvation, and its isogenic wildtype parent pao . we measured the starvation survival of planktonic bacteria in phosphate buffered saline and observed that the (i)rela(i) has a to fold greater loss in viability compared to wild type after h. we also tested biofilm formation in a static biofilm assay using polystyrene peg lids and estimated biomass accumulation by crystal violet staining. in this system, the (i)rela(i) accumulates times less biomass than wild type. in a flow-cell reactor system, the wildtype strain forms complex mushroom-like biofilm structures, but the (i)rela(i) mutant forms flat, thin biofilms. we determined cell attachment to polystyrene by crystal violet staining, and to glass surfaces by direct visualization under microscopy. the attachment to both surfaces is similar in the (i)rela(i) and wildtype strains, and the strains have identical planktonic growth rates. our findings suggest that the stringent response is involved in starvation survival, a physiological condition relevant to growth in biofilm. furthermore, (i)rela(i) inactivation impairs biofilm formation, suggesting that the stringent response may be an important stress adaptation in biofilm. belfast, united kingdom; . northern ireland public health laboratory, belfast city hospital, belfast, united kingdom; . northern ireland regional adult cf unit, belfast city hospital, belfast, united kingdom; . department of respiratory medicine, queen's university, belfast, united kingdom pseudomonas aeruginosa (pa) is a clinically significant bacterial pathogen responsible for increased morbidity and mortality in patients with cystic fibrosis. small non-coding micro(mi)rna species (generally ~ nts or less) in prokaryotes are involved in numerous cellular processes as is the case with eukaryotes. as in eukaryotes, these mirnas act by base pairing with target mrnas imposing translational and stability changes of mrnas and thus culminating in the control and regulation of target mrnas, crucial for bacterial stress responses and virulence to changing environments surrounding the infected zone or host cell (e.g. human airway epithelial cells). we recruited a commercial kit (biodynamics dynaexpress mirna cloning kit av, tokyo, japan) and isolated mirnas from a variety of pseudomonas pathogens from plant, soil / agricultural environment (pseudomonas syringae -ps), type strain (pseudomonas aeruginosa -pa) and a collection of clinical isolates of pseudomonas aeruginosa (cfpa) from adult cf patients, who were chronically infected with p. aeruginosa. the results on the mirna clones obtained exhibited a wide variation in the occurrence of mirnas in this bacterium. the putative mirnas obtained, cfpa in particular, yielded small non-coding rnas of sizes from as little as nts to well over nts. an initial search using the bioinformatic tool srnapredict (www.waldorlab/tufts.edu) has not as yet, fully revealed the genomic annotations for these novel srna sequences in the inter-generic regions of the pathogenic pseudomonad group of bacteria. we present our data on these unknown cellular mirnas cloned from cfpa isolates from cf patients and discuss their significance as novel regulators to bacterial stress responses and in the context of the bacterium's involvement as the predominant pulmonary pathogen, associated with cystic fibrosis. airway function is diminished in infants with cf diagnosed clinically but whether this is true for those identified by newborn screening remains unclear. we investigated whether lung function is diminished in infants with cf diagnosed by newborn screening and if this occurs in association with pulmonary infection. methods:lung function was measured in asymptomatic infants with cf following sedation with chloral hydrate ( - mg/kg) using the raised volume rapid thoracic compression technique at an inflation pressure of cmh . we measured forced vital capacity (fvc), forced expired volume after . seconds (fev . ) and forced expired flow at % expired fvc (fef ) which were then expressed as z scores. broncho-alveolar lavage (bal) was performed under general anesthesia within forty-eight hours following infant lung function testing. three separate aliquots of ml/kg sterile saline were instilled into the right middle lobe and a further aliquot in the left lingula and the bal fluid analyzed for bacterial, fungal and viral pathogens using routine culture techniques. results: bal was successful in all infants studied aged . to . yrs (median . yrs) and lung function in infants. median (range) z scores for fvc, fev . and fef were - . (- . to . ), - . (- . to . ) and - . (- . to . ) respectively. these decrements in lung function were all significant (p< . ). bal cultures were positive in ( %) infants of whom ( %) had evidence of infection with s. aureus and ( %) with p. aeruginosa. in infants there was no growth in bal or growth only of mixed oral flora. viruses were not identified in any infants. there was no association between any of the lung function parameters and pulmonary infection detected by bal. lung function was successful in a subgroup of of infants who were less than . yrs (median age . yrs) when tested. diminished airway function was identified in this younger subgroup even though only one infant had evidence of pulmonary infection with respiratory organisms in bal. in this younger subgroup median fev . z score was - . which was significantly below that predicted ( % ci: - . ,- . and p= . ). conclusions: airway function is diminished in infants with cf diagnosed by newborn screening and occurs irrespective of infection identified by bal. lung function is abnormal in very young infants before pulmonary symptoms develop and when no evidence of pulmonary infection can be detected in bal. inhaled high-dose tobramycin appears to transiently clear p. aeruginosa (pa) from airway secretions in young children with cf, though inflammation may not be markedly reduced (am j resp crit care med ; : ) . because inhaled antibiotics may not reach some areas of infection, we hypothesized that intravenous (i.v.) antibiotics may be more effective than inhaled high-dose tobramycin for reducing lower airways inflammation in cf children with early pa infection. study design: we initiated a single-center, randomized, prospective study comparing the effects of two antibiotic treatment regimens on bronchoalveolar lavage fluid (balf) inflammatory markers. clinically stable cf children with a recent isolate of pa from surveillance cultures are randomized to receive weeks of tobi® (group ) or weeks of i.v. ceftazidime and tobramycin (group ). balf is obtained just before treatment, and repeated - weeks after completion of treatment. the primary study outcome is change in balf % neutrophils (pmn). multiple secondary outcomes are assessed including quantitative bacterial cultures and cytokines in balf. this report summarizes interim analysis at the halfway point of the study. results: to date, subjects have enrolled. five subjects ( in group , in group ) dropped out after the initial bronchoscopy due to a decision by the primary cf physician to use other treatments based on balf culture results ( ) , or because of the development of a new respiratory illness before the second bronchoscopy ( ). a total of adverse events ( significant, unrelated to protocol) were reported in subjects, none resulting in a change of protocol request by the irb or data safety monitor. of the subjects completing the protocol to date, were in group , and were in group . the groups were well matched in terms of age, pa in initial balf, and initial balf %pmn. a majority of subjects in each group were children with their first isolation of pa on surveillance cultures. there was a tendency for group , but not group , to have mild reductions in balf %pmn (- ± %) after treatment. interim efficacy analysis did not suggest a significant difference between the treatment groups at this stage. similar changes were seen in group for pmn/ml (- ± x /ml) and several cytokines including gm-csf (- ± pg/ml) and mcp- (- ± pg/ml). two group subjects were unable to have stable i.v. access established and were treated with oral ciprofloxacin and inhaled tobi® for weeks as alternative systemic antibiotics, per protocol. dropping these subjects from group data did not change data trends. conclusions: the protocol appears to have adequate safety. parents of children with first-time pa infection may be more likely to consent to treatment randomization, than those with repeated pa infection. interim data analysis suggests that continuation of the study toward the original sample estimate is appropriate. acknowledgements: supported by cff(noah a ) and nih (gcrc r ). we thank tracy callahan rn, lupe haynes rrt, justin hubbard, paula murphy, cassidy henegar, nancy lee rn, benjamin butler rn, and sheree berckmans rn for logistical and technical support. methods: our local cf database was searched for patients born - . patients were included in the analysis if they were followed in our cf center before the age of . patients were separated into two birth cohorts, born - (cohort ) and - (cohort ). yearly peak fev % predicted from ages - were obtained using knudson reference equations. yearly peak bmi% and weight-for-age% at each age were also obtained. type tests of fixed effects and repeated measures analyses were used to assess mean peak bmi%, mean peak weight-for-age%, mean peak fev % predicted at first pft, mean peak fev % predicted from ages - , and mean rate of change (slope analysis) in peak fev % predicted from ages - . chi-square test for association was used to analyze differences in demographic parameters. wilcoxon rank-sum test was used to assess differences in age at diagnosis. wilcoxon non-parametric test was used to analyze differences in mean clinic visits per year. chi-square testing with a two sided alpha of p < . was used to determine significance. results: cf patients were included in the analysis. there were no significant differences between birth cohorts and for age of diagnosis (p= . ), sex (p= . ), race (p= . ), genotype (p= . ), pancreatic insufficiency (p= . ), or the mean number of clinic visits per year from ages of - (p= . ). there was a significant difference in the mean number of clinic visits per year from ages of - (p=. ; see table ) . birth cohort had a significant effect on the rate of decline in lung function from ages - . birth cohort and age had significant effects on the mean weight-for-age%, mean fev % predicted at first pft, and mean fev % predicted from ages - . sex had a trend for significant effects on lung function parameters. adjusted for age and sex, there were significant differences between birth cohorts and in mean bmi% and mean weight-for-age% at ages and , mean fev % predicted at first pft, mean fev % predicted from ages - , and in the mean rate of decline in fev % predicted (see table ). conclusions: improvements in lung function and nutritional outcomes in our cf center are associated with an increase in the mean number of clinical visits per year for cf patients ages - . the frequency of clinical follow-up is a marker of cf care that may have positive impacts on cf outcomes. background: current management of cf airway disease includes the use of key pharmacological therapies designed to combat the chronic cycle of obstruction, infection, and inflammation. randomized controlled trials have demonstrated that maintenance therapy with azithromycin (azm), dornase alfa (dnase), and tobramycin for inhalation (tob) each improves pulmonary function and reduces the frequency of exacerbations in patients with p. aeruginosa. the purpose of this study was to determine if cf patients in an uncontrolled environment (clinical setting), experience a reduced rate of pulmonary function decline when receiving azm + dnase + tob compared to those not on all three therapies. methods: adult cf patients were selected based on positive sputum cultures for p. aeruginosa, pulmonary function test results (fev % predicted) for the past year, and receiving one of the key pharmacological therapies. in addition data was collected on cfrd, depression, medication adherence, airway clearance, and nutritional status. patients were stratified according to baseline pulmonary function: mild (fev ), moderate (fev of - %), and severe (fev < %). patients were then sub-grouped as to whether they were receiving all or ≤ of the key therapies. the annual rate of decline in % predicted fev was determined using linear regression on non-exacerbation data and was categorized as stable (< . % decline), intermediate ( . - . %) , or rapidly declining (> . %). time to intermediate or rapid decline in pulmonary function was determined using cox proportional hazard modeling. results: patients ( males/ females, median age years) were included in this study; with mild, with moderate, and with severe pulmonary disease. overall, the mild and moderate groups experienced median declines of . % and . %, while the severe group experienced a . % improvement in fev % predicted respectively. the use of regimens containing azm therapy significantly delayed time to intermediate or rapid decline in lung function in the mild group (median days vs. . days, hr= . , p= . ). similarly, regimens containing dnase + azm + tob significantly delayed time to intermediate or rapid decline in lung function in the mild group (median days vs. days, hr= . , p= . ). no significant differences were seen in the moderate or severe group for patients receiving all or individual therapies. con-clusion: administration of pharmacological regimens containing dnase + azm + tob, or azm + tob or azm + dnase significantly delay progression of pulmonary disease in cf patients with mild lung disease. sawhney, v. ; arthur, b. ; seltzer, r. ; kraynack, n.c. . internal medicine, akron general medical center, akron, oh, usa; . pediatrics, akron children's hospital, akron, oh, usa; . biostatistics, neoucom, rootstown, oh, usa; . pulmonology, akron children's hospital, akron, oh, usa early and efficient diagnosis of pulmonary exacerbations (pex) in patients with cystic fibrosis (cf) is crucial in the management of cf. in oct , we implemented a pulmonary exacerbation scoring (pes) system to uniformly and consistently identify pex in cf patients as part of a quality improvement project at the cf center at akron children's hospital. we have previously reported improved pulmonary function outcomes in pediatric patients at our center after implementation of the pes. we now describe the impact of the pes on clinical practice in the management of pex and pulmonary function in our adult cf patients. the pes was developed by our multidisciplinary cf team after an extensive literature review and was implemented in oct . the pes has been previously described and consists of clinical questions divided into three domains: systemic and pulmonary symptoms and signs, and objective measurements. these elements are scored individually and a cumulative pes of ≥ (range - ) is considered a pex and treatment with antibiotics is recommended. the course and choice of antibiotic regimen is left to the physician's discretion. we examined median percent predicted fev decline from quarterly percent predicted fev measurements obtained from port cf. we calculated this for all the patients seen in our adult cf clinic (age> ) from oct till sep during which the pes was in use. we compared this rate of decline to median percent predicted fev decline for the same cohort of patients over the preceding two years (oct till sep , during which the pes was not in use. the percent utilization of the pes was calculated individually for all adult cf care providers. the influence of individual components of pes in decision to treat was also studied. we found no significant difference between the rates of decline in percent predicted fev during the pre-pes and post-pes periods (mean = -. , sd = . vs mean = -. , sd = . , p= . ) as measured by paired samples t-test. logistic regression analysis was done to evaluate the relative importance of each pes component in predicting decision to treat a pex. we found that changes in fev (or= . ), cough (or= . ), sputum (or= . ), and chest examination (or= . ) are most likely to affect the decision to treat where as dyspnea (or= . ) has the little effect on decision to treat. we found a high degree of correlation between adult provider use of the pes and an independent chart reviewer (correlation coefficient . - . ; p< . ). in contrast to cf patients ages - years, we found no significant effect of use of the pes on pulmonary function in our adult population, despite the high frequency of use by our adult providers. it is unclear why this is the case given the robust effect of the pes on our pediatric population. this study suggests that a simplified score that includes fev , cough, sputum, and chest exam may be useful in identifying a pex in adult cf patients. special thanks to tasha capozzi for her help with chart review. this project was supported by the akron children's hospital foundation. objective chronic pulmonary infection with pseudomonas aeruginosa is the main cause for morbidity and mortality in cystic fibrosis (cf) patients. tobramycin and colistin, widely used inhaled antibiotics in cf patients, were found to affect elastase activity in vitro. we showed recently that cxcr on neutrophils mediates bacterial killing, but is cleaved in cf airways by elastase. therefore, we examined the effects of inhalation with tobramycin and colistin on cxcr expression and antibacterial capacities by airway neutrophils in cf patients in vivo. methods cxcr expression was quantified by flow cytometry on neutrophils in peripheral blood and induced sputum of cf patients without inhalation therapy, cf patients with tobramycin and cf patients with colistin inhalation therapy. the longitudinal effect of a weeks tobramycin or colistin inhalation period on cxcr expression, free elastase levels and bacterial killing by airway neutrophils was assessed. results inhalation with tobramycin increased, while inhalation with colistin decreased cxcr expression on neutrophils in induced sputum of cf patients. neutrophils isolated from sputum of cf patients after tobramycin therapy had higher, whereas patients after colistin therapy had lower bacterial killing capacity and α-defensin release as compared to neutrophils before antibiotic inhalation therapy. conclusions tobramycin and colistin differentially modulate the pulmonary host defense in cf patients in vivo via cxcr on neutrophils. these findings may have clinical implications when considering antibiotic treatment in cf patients. sharp, j. ; sheehan, d. ; ren, c.l. . pediatrics, women & children's hospital, buffalo, ny, usa; . pediatrics, university of rochester, rochester, ny, usa background: airway infection and inflammation occur early in infants with cf. the raised volume/rapid thoracoabdominal (rv/rtc) technique is more sensitive than older infant pulmonary function test (ipft) methods in detecting airflow obstruction in these patients. however, there are limited data regarding lung function measured by rv/rtc in infants with cf nbs. we hypothesized that abnormalities of lung function measured by rv/rtc are present in cf nbs infants. to test this hypothesis, we reviewed the ipft data from cf nbs infants followed at our cf centers. methods: this study was a retrospective chart review. we identified infants with cf nbs followed at our cf centers who had had at least ipft performed in the first year of life. ipfts were performed using the collins ipl. forced expiratory flows were measured using rv/rtc as described by feher et al (j appl physiol : , and fractional lung volumes were measured by whole body plethysmography (bp) as described by castile, et al (ped pulm : , ) . data from the cf infants were compared to normal data reported by jones, et al (ajrccm : , ) for rv/rtc and castile, et al for bp. results: we reviewed data from infants. their mean age was . m (range= - ). pancreatic insufficiency was present in ( %) infants, and ( . %) had been treated for pseudomonas infection. their pft findings are summarized in the table. fvc and fev . were not significantly different from the normal predicted values. however, the mean fef and fef - z scores were significantly below the mean of normal infants (p= . and . respectively). gas trapping was present, as evidenced by significantly increased frc, rv, and rv/tlc (p< . for all measures). six infants ( %) had an fef z score of < - . , and ( %) had an frc > % predicted. conclusions: infants with cf nbs have abnormal lung function early in life. our results suggest that pfts in cf nbs infants can be useful in detecting early changes in lung function. we speculate that early therapeutic interventions may improve lung function in this group of patients. table. pft findings in infants with cf nbs. forced expiratory flows are reported as z scores, while fractional lung volumes are reported as % predicted. objective: clinicians use pfts to assess effectiveness of therapy in hospitalized cf patients. however, the variability of multiple pft efforts has not been described in this group of patients. previous study of within day variation of pfts has been reported in healthy adults and children (enright, , and nickerson, ) and clinically stable outpatients with cf (nickerson, , and cooper, ) . nickerson and cooper found coefficient of variations (cv) for fvc of . - %, fev of . - . %, and fef - of . - . % for adults and children with cf. no one has studied the within day variation of pfts in cf patients during pulmonary exacerbations. our objective was to determine the within day variation of pfts during hospitalizations for cf pulmonary exacerbations. we hypothesized that within day variation would improve during treatment. methods: we retrospectively reviewed pft data for all patients admitted to childrens' hospital and regional medical center for a cf pulmonary exacerbation in . patients performed pfts under the direction of experienced pulmonary function technicians. the test was complete when the patient performed efforts that met ats/ers criteria for acceptability. if the patient was unable to produce efforts because of coughing, efforts were accepted if the fvc and fev were within % or ml of each other. for analysis, we included only those pfts: ( ) with at least - flow-volume curves that fulfilled ats/ers criteria, and ( ) were performed within days of hospital admission or discharge. for patients with multiple admissions, we used pft data for their first admission only. for patients with admission and discharge pfts available, we compared the coefficient of variation (cv) for pfts (fvc, fev , ). there were at least days between admission and discharge pfts. the wilcoxon rank-order test was used to compare the cv for admission and discharge pfts. data: sixty-one patients were admitted times in . excluding repeat hospital admissions, patients had individual pft trials available at the time of admission and patients at the time of discharge. seven admission and discharge pfts included only trials. three patients at admission, and patients at discharge, were unable to produce pft curves that met ats/ers criteria, despite previously having done so when well. nine patients had paired admission and discharge pfts from the same hospitalization. the cv for fvc at discharge was significantly better than at admission, p = . (see table) . conclusions: within day variation of pfts during hospitalizations for cf pulmonary exacerbations is comparable to published outpatient data. cv for fvc improves significantly during exacerbation. changes in fvc > %, fev > %, and fef - > % can be considered a significant spirometric response to therapy in hospitalized cf patients. background: lung clearance index (lci), a measure of ventilation heterogeneity, can be calculated from multiple breath washouts of inert gas. in cross sectional studies it is a more sensitive measure of early airway dysfunction in cf than spirometry alone. in normal subjects, the technique is reproducible, with a narrow normal range of . - . . we hypothesized that it may be helpful in detecting change longitudinally, and may have value in assessing novel therapeutic interventions such as the upcoming gene therapy by the uk cfgt consortium. methods: patients aged years and over, presenting with an exacerbation requiring intravenous antibiotics, were recruited. lci was assessed in triplicate by multiple breath washout of . % sulphur hexafluoride (sf ), using a novel gas analyser (innocor, denmark) and custom-built software within the first hours of starting treatment. the test was repeated at the end of parenteral antibiotic therapy and again - weeks later when clinically stable. patients were recruited at three sites and assessed using standardised equipment and a data analysis protocol. results: paired lci measurements were available in patients, mean age (range - ) years, male. in a further patients, technically acceptable washouts could not be obtained at one or other time point. mean (sd) lci improved from . ( . ) to . ( . ), p< . . in / patients there was an improvement in lci of > % after antibiotics. patients showed a > % deterioration in lci. mean fev was significantly greater after treatment ( . v . l/s, p< . , n= ). there was a weak correlation between percent change in fev and lci (r = . , p< . ). patients also had lci measured at the follow up. mean (sd) lci at this visit was . ( . ), p< . vs visit , no significant difference vs visit . conclusions: lci can be used to demonstrate changes in the lung after antibiotic therapy. this is the first study in cf to demonstrate a lon-gitudinal improvement after intervention in a marker of ventilation heterogeneity, and offers different but complimentary information to spirometry. the higher sensitivity of lci may make possible measurement of improvement in mild patients after novel therapies such as gene therapy. this work was funded by the cystic fibrosis trust. aim: inhaled hypertonic saline positively affects sputum production in patients with cystic fibrosis (cf) by improving mucociliary clearance. this study was conducted to compare the aerosol delivery performance of various nebulizers upon aerosolization of hypertonic saline (hyper-sal™) . % and %, available as preservative-free, sterile ml solution in single dose, patient friendly blow-fill seal vials. method: ml hypertonic saline (hyper-sal™) % was nebulized by three jet nebulizers the pari lc plus®, lc star® and hudson rci micro mist as well as the eflow® for hypertonic saline (hs) generating aerosols via a perforated vibrating membrane. hs . % was characterized in the eflow®hs. droplet size distribution patterns were assessed by laser diffraction at a flow rate of l/min and aerosol delivery performance by breath simulation tests mimicking a sinusoidal breathing pattern. the delivered dose (dd) was determined by gravimetric analysis of the inhalation filters. inhalation and exhalation filters were changed after min and the drug delivery rate (ddr) was calculated as nacl found on inhalation filters per min. respirable drug delivery rate (rddr; ddr x respirable fraction < µm) indicates how much nacl can reach the lungs per minute. results: aerosol characteristics of the different nebulizers after aerosolization of ml hs % are summarized in the table. data for hs . % nebulized by the eflow®hs were as follows: dd= . %, ddr= . %, nebulization time = . min and comparable to data for hs %. the lower recoveries for the jet nebulizers are probably associated with increased evaporation and longer nebulization times. conclusion: about - % of the charged sodium chloride from ml hs was delivered and % - % of these droplets were in a respirable size range < µm resulting in vitro respirable doses of about % - % of the label claim. ddr and rddr are about -to -fold higher for eflow®hs indicating a much higher delivery efficiency compared to conventional jet nebulizers. further studies will be needed to demonstrate that about ml hs may be sufficient to deliver a comparable dose to the lungs using an eflow®hs compared with jet nebulizers. this bears the potential to further reduce nebulization time and increase acceptance by patients. objectives: individuals with cf frequently have complicated and time consuming treatment regimens consisting of inhaled antibiotics, mucolytics, and airway clearance. patients and clinicians may have difficulty determining which specific therapies are effective for any individual patient or whether a particular treatment is causing adverse effects. this study assessed the utility of home spirometry for providing an objective measure of pulmonary status in between scheduled clinic visits. methods: consecutive adults with cf were recruited at a single academic medical center and were asked to measure fev twice daily for six months using the piko- meter (nspire health™, llc). subjects agreed to participate and completed the study. subjects were given diaries to record changes in respiratory symptoms and changes in medications during the study period. subjects were evaluated in clinic at least every three months and fev values obtained in the pulmonary function lab were compared to home fev from the same day using linear regression and bland-altman analyses. graphical displays of home fev measures were visually inspected for changing trends and these were compared with medication changes. results: the mean fev measured at home was . ± . l. the mean fev in the pulmonary lab was . ± . l. the home fev was significantly correlated with the clinic value from the same day, r = . , p< . . fev measures fell within the limits of agreement (± sd) by bland-altman plot. in of subjects ( . %) there was at least one sustained change in fev that correlated with either stopping or starting a particular treatment. the figure shows two significant changes in fev in one patient related to medication changes. there is an increase in fev at the initiation of aerosolized % saline and a fall in fev that corresponded to inhaled tobramycin use. the improvement on hypertonic saline increased this patient's enthusiasm for this treatment. conclusions: home fev monitoring is feasible and appears to provide accurate measurement of pulmonary function. it can provide clinicians with objective data to tailor treatments without necessitating a clinic visit. feedback from home monitoring may improve medication adherence and may allow treatments to be individually tailored based on patient responses to the medications. . respiratory unit, royal hospital for sick children, glasgow, united kingdom; . diagnostic imaging, nhs greater glasgow & clyde, glasgow, united kingdom; . medical statistics, royal hospital for sick children, glasgow, united kingdom; . medical physics department, gartnavel hospital, glasgow, united kingdom background: there have been calls for caution regarding the suggested use of regular chest computed tomography (ct) scans for monitoring disease progression in cf . consideration of the associations of higher ionizing radiation exposure must be made before regular chest cts are performed on the wider cf population. aim: we tested the hypothesis that higher ionizing radiation exposure from chest radiographs (cxr) and chest cts would be associated with markers of severity including gender, genotype (class i/ii or iii-v), height sds, weight sds, bmi, fev sds, length of hospital admission (days), scottish index of multiple deprivation (simd) scores and pa/bc microbiological infection (chronic or intermittent colonisation with pseudomonas aeruginosa and/or burkholderia complex). methods: effective doses of ionizing radiation (msv) from cxrs and chest cts (including hr scans) were determined for all cf children at our centre between st january and th july ( weeks). the most recent anthropometric and lung function data to july were used. simd scores report a data zone that encompasses ~ individuals and comprises indicators in six domains (current income, employment, health, education, housing and access). results: cf patients ( male: female) underwent radiation exposure procedures (mean of . procedures per patient per year). the average effective doses were calculated for of the procedures ( . %) including cxrs and chest cts. lung function data were available on patients ( %). univariate analysis revealed that higher ionizing radiation exposure was significantly associated with fev sds (p< . ), length of stay (p< . ) and pa/bc infection (p< . ). the regression line indicates that for each drop of two fev sds, each child receives an extra msv in radiation exposure from cxrs and chest cts alone. similarly, each child receives an extra msv in radiation exposure from cxrs and chest cts for every days of hospital admission. multivariate analysis with backward selection demonstrated that length of hospital admission (p< . ) and pa/bc infection (p= . ) were associated with higher ionizing radiation exposure. discussion: in cf, higher ionizing radiation exposure from cxrs and chest cts in childhood are associated with poor lung function, pa/bc infection and length of hospital admission but not other indicators of severity such as gender, severe genotype, anthropometric measures or socio-economic status. using effective doses of ionizing radiation as an outcome marker in cf is an alternative means of assessing the impact of the disease and its management on the child. background: exhaled volatile compounds have attracted attention as potential non-invasive biomarkers of chronic lung disease. early sustained airway inflammation and bacterial infection are characteristic features of pulmonary involvement in cystic fibrosis (cf). the cellular response is dominated by neutrophils, which have been shown to generate oxygen-and halogen-derived radicals as part of their host defense armamentarium. aims: to gather pilot data on the pulmonary output or uptake of selected organic trace gases in cf patients and healthy controls; and to explore the impact of pulmonary exacerbations and antibiotic treatment. methods: samples of ambient air and exhaled breath were collected from children, adolescents and young adults with cf and from healthy controls. the patients' age ranged from - years and their fev between - % predicted, were pseudomonas positive. twelve patients were studied at the start of i.v. antibiotic treatment for a pulmonary exacerbation; repeated sampling after the course was performed in subjects. all samples were analyzed on a customized gas chromatography system with flame ionization and electron capture detectors. after determining the mixing ratios of a variety of volatile organic compounds, alveolar gradients were calculated from the corresponding exhaled and ambient values. results: compared to healthy control subjects, cf patients showed a significantly lower respiratory output of methanol, ethanol, acetaldehyde and dimethylsulfide but a higher net release of benzene, methylchloride, trichloromethane and trichloroethene. these differences were most pronounced in patients with acute exacerbations. a relevant scatter and overlap between groups was noted for ethane, pentane, acetone, isoprene, toluene, and tetrachloromethane. after antibiotic treatment, the output of benzene, methylchloride and trichloromethane decreased by - %. conclusion: although important pathways remain incompletely understood, the local biochemical milieu in cf lungs may be informative and accessible through non-invasive breath testing. in this context, the diagnostic potential of exhaled organic traces gases (especially oxygenated, halogenated and aromatic compounds) should be further explored. supported by: german research foundation (dfg ba / - ). toy, e. ; weiner, j. ; sacco, p. ; duh, m.s. . health economics, novartis pharmaceuticals, east hanover, nj, usa; . analysis group, inc., boston, ma, usa objective: to review up-to-date economic outcomes data in patients with cystic fibrosis (cf), and in particular, costs related to respiratory infection by pseudomonas aeruginosa (pa), which is the leading cause of morbidity and mortality in cf patients. methods: a systematic search of the medline database from - was conducted to identify major and review articles that contained the terms "cystic fibrosis" and "cost" and which were published in english language journals. selected conference abstracts were searched, and additional articles were identified through bibliographies of retrieved articles. recent articles that contained economic data on antibiotic and mucolytic therapies were selected for in-depth review. cost estimates were converted to us dollars for comparability. results: approximately articles fulfilled the initial search criteria, and were selected for in-depth review. articles were divided into four categories: economic impact of inhaled tobramycin ( articles), the effect of home-vs. hos-pital-based antibiotic therapies for pulmonary exacerbations ( articles), economic impact of recombinant human deoxyribonuclease (rhdnase) ( articles), and cost-of-illness for cf ( articles). inhaled tobramycin (actual or recommended dose of mg/ ml) has been associated with reductions in health care costs; these cost savings offset between % and % of the cost of this therapy. antibiotic therapy for pulmonary exacerbations generally resulted in lower health care costs when administered in a home setting rather than a hospital setting. use of rhdnase led to reduction in health care costs, with higher reductions observed in patients with higher levels of use; the cost savings offset between % and % of the drug cost. cost-of-illness studies have been conducted in different countries; the economic estimates varied widely across studies due to differences in treatment patterns, health systems, methodologies, and patient populations. most cost-of-illness studies were retrospective observational studies from the perspective of a hospital or third-party payer, and the cost of cf ranged from $ , to $ , per patient per year. the largest cost categories included hospitalizations, out-patient visits, rhdnase and antibiotics, while disease severity and presence of pa infection were common determinants of cost. cost-of-illness studies have underestimated societal costs because they have rarely considered patient time costs and none have considered indirect costs (e.g., burden on lost productivity or informal caregivers). conclusions: studies show that inhaled tobramycin and rhdnase can partially offset medical costs; home-based iv antibiotic therapy is likely to reduce costs; and direct costs associated with cf can be high but vary widely across countries and analytical methodologies. areas for future research include direct comparisons of inhaled antibiotic therapies, examination of the relationship between treatment adherence and economic outcomes, and estimation of societal cost-of illness. this study was sponsored by novartis pharmaceuticals corporation, east hanover, nj. background: sputum interleukin (il ) and myeloperoxidase (mpo) are advocated as markers for infective exacerbations in cf. we have previously shown that the neutrophil protein, calprotectin, is a sputum biomarker of disease activity in cross sectional and longitudinal studies. in the present study we evaluate longitudinal measurement of sputum and serum calprotectin during infective exacerbation. methods: sputum and venous blood samples were taken at the beginning (visit ) and end of a course of iv antibiotics (visit ), and at time of clinical stability following the infective episode (visit ). processed sputum supernatant was analysed by elisa for calprotectin, il and mpo. serum calprotectin was assayed by elisa. results: paired sputum samples samples were available for visits and and paired samples for visits and . all data are presented as mean(sem). sputum calprotectin decreased from ( ) to ( ) µg/ml, p< . , from visit to . there was a non-significant increase to ( ) µg/ml at visit . sputum il did not change from visit to [ . ( . ) ( . ) ]. serum calprotectin decreased from ( . ) to . ( . ) ng/ml, p< . (n= paired samples) from visit to . at visit there was an increase to ( ) ng/ml, p< . (n= ). discussion: calprotectin appears a valuable marker both in sputum and serum for tracking changes in lung inflammation during an exacerbation of cf and may be more sensitive to change than il or mpo. on stopping antibiotic therapy sputum and serum calprotectin increase implying that this marker may be sensitive to small sub-clinical changes in lung inflammation. these data suggest that sputum and serum calprotectin might be employed to assess responses to drug therapies in cf such as gene therapy. this research was funded by the cf trust uk. chronic lung disorders are usually associated with a hypoxia driven increase in red cell mass. however, patients with cystic fibrosis often have normal or decreased haemoglobin levels. the present prospective observational study in cystic fibrosis (cf) patients was performed to determine which factors were involved in alterations in the hematopoetic response to corresponding arterial oxygen pressure. sixty adult patients (age - ) with stable cf were included. they all had vitamin a, d, e and k but no vitamin b supplementation. patients were on oral fe + ( mg/day). resting arterial blood gases, lung function, complete blood counts, parameters of iron status, crp, sputum microbiology and serum erythropoietin were measured at recruitment and after and months. patients had varying degrees of pulmonary functional impairment and % were hypoxemic (arterial oxygen pressure < mm hg). low-grade systemic inflammation (crp > . mg/dl) was present in % of the patients, who all had bacterial colonization. none of the patient had erythrocytosis and patients had anemia . there was no significant difference in iron status between patients with or without chronic iron supplementation and erythropoietin levels were normal. during the months observation period no significant changes occurred. the patients exhibited an impaired erythropoietic response to hypoxemia with normal or low hematocrit in spite of chronic lung disease. linear multivariate regression analysis (table) revealed crp and iron levels but neither iron substitution, nor erythropoietin levels nor lung function parameters as independent determinant of haemoglobin levels. cf may be associated with anemia of variable severity as expression of the chronic inflammation present in these patients. the therapeutic consequences are to treat the underlying inflammation rather than to supplement iron. variables included in initial model: background improvement in the treatment and survival of females with cf has led to many leading a normal adult lifestyle, and some can now expect to raise a family. however, pregnancy can have a profound impact on wellbeing, and caring for a young child can affect compliance with cf treatment. to look at this further, we surveyed the experience of pregnancy and motherhood in cf women attending our large adult cf centre ( patients). methods we reviewed the records of cf women who chose to remain pregnant ( babies [ male] from pregnancies), looking at changes in lung function, bmi, respiratory exacerbations during pregnancy, clinic attendance, and oral and iv antibiotic therapy (home and inpatient care) during pregnancy and also in the first year post delivery. we devised a questionnaire to examine how patients coped with pregnancy and the demands of motherhood, focussing on changes in compliance with therapy, worries regarding the effect of cf treatments on the baby, and support given by the cf team. results at conception mean fev was % predicted (range to ) and mean bmi (range to ).there were no miscarriages or neonatal mortalities and all children remain well, but the sickest mother died at years. mean fev following pregnancy was % [range to ] (p= . ), with mother losing % by one year post delivery. at one year, bmi was unchanged (mean , range to ). rates of respiratory exacerbations and iv antibiotics were similar pre and during pregnancy, although during the first year post delivery mothers avoided hospital based treatment preferring home care. six mothers' clinic attendances increased during and post pregnancy ( attended outpatients times in the first year post delivery). nine mothers worried about the effects of cf medication on their unborn child: the remaining mother was in her nd pregnancy. all managed their health differently during pregnancy: whilst increased compliance, worked harder to stay healthy, and increased clinic attendances; avoided clinic, postponed treatment, avoided any medication in the first trimester, and reduced oral antibiotic use to prevent harm to their unborn child. following delivery, mothers spent less time on their own health due to motherhood demands, and missed treatments during the first year: missed all except iv antibiotics. seven mothers ceased chest clearance and nebuliser therapy, and whilst missed oral therapies only avoided iv therapy. one mother who returned to work early felt this had contributed to her post natal depression. nine mothers indicated that cf team support was adequate, but the remaining mother felt that she should have been offered more frequent home visits post delivery due to lack of family support. conclusions the experience of pregnancy in our centre has positive outcomes for most mothers. however, compliance with treatment was affected and a number of mothers lost significant pulmonary function due to pregnancy. our study has shown that the demands of motherhood especially in the first year challenge the management of their own health. we offer a proactive service to women considering pregnancy, with early participation of the multidisciplinary team, and have increased home visit frequency to during the first year post-delivery. geller, d.e.; kesser, k.c. research, nemours children's clinic, orlando, fl, usa background: alpha- antiprotease (α- ap) is being developed as an inhaled drug in cf to inactivate excess elastase in the lungs. it is estimated that tens of milligrams of α- ap need to be deposited in the lungs to be effective. delivering a high dose with conventional jet nebulizers may be too time-consuming. we speculated that slowing and prolonging inspiration could optimize α- ap delivery. the i-neb® adaptive aerosol delivery (aad® system, respironics) is a portable, electronic, battery-powered, mesh nebulizer that uses the aad algorithms to deliver drug in the first portion of inspiration. interchangeable mouthpieces allow the i-neb to be used in a conventional tidal breathing mode (tbm) or in the target inhalation mode (tim) that guides the patient to inhale very slowly. the low residual volume after nebulization reduces drug waste and optimizes efficiency. we compared delivery efficiency of α- ap in vitro with the i-neb in the tbm vs. tim modes. methods: we studied new i-neb devices using tbm (tidal volume [vt] . l, respiratory rate [rr]= , i-time= seconds), tim- (vt . l, rr= , i-time= sec), and tim- (vt . l, rr= , i-time= sec) using a breath simulator. nebulizers were filled with . ml α- ap ( mg/ml). nebulized drug was captured on a filter and measured via spectrophotometer (inhaled dose), and particle size was measured with an insitec laser system. results: median particle sizes for the modes were similar ( . - . µm) . the dose emitted from the i-neb was very high, and similar between modes ( - % of nominal dose). predicted lung doses for these experiments were calculated from a previous scintigraphy study (nikander ) that showed % of the emitted dose deposited with tbm and % with tim. predicted lung dose of α- ap (as % of nominal) and delivery times were tbm: . % in . min, tim- : . % in . min, and tim- : . % in . min. discussion: the i-neb has a very high predicted lung dose with both tbm and tim. however, tim reduces the time of administration to as little as / that of tidal breathing. if larger predicted lung dose is necessary to see a clinical effect, either the initial drug volume or drug concentration may be increased. we conclude that slow, deep, controlled inspirations using i-neb is a very efficient method to deliver α- ap, and is faster than tidal breathing. we also speculate that tim has the additional potential advantage of better distribution of drug in the lungs because of slower flow rate and particle velocity. objective: early inflammation was observed in infants with cf regardless the occurrence of bacterial infection. lung function (lf) is a tool to assess bronchial inflammation consequences. few data are available on the pronostic value of impaired lf in infancy. methods: in infants, airflow obstruction (t ptef/te , curve shapes, tidal volume, functional residual capacity (frc, nitrogen washout technique) were assessed with the sensor medics and interpreted with stocks reference values. at y, fev and pulmonary flows (spiro , nova medical sc, zapletal reference values) and frc he , were measured. symptomatic children were defined when cough, wheeze or rattle were recurrent. all symptomatic infants have been treated with inhaled steroids as any infantile wheezer. results: screened infants ( ∆f /∆f , ∆f /other, other/other) were investigated at mean age mo and years. ( %) had respiratory symptoms (rs); the mean tidal volume was normal ( . ml/kg), ( %) had a marked obstructive curve shape, ( %) a decreased t ptef/te , and ( %) hyperinflation. then, ( %) had normal lf, ( %) ao, ( %) hyperinflation and ( %) both. at age , % had rs, and lf with mild alteration as mean flows ≥ %, and frc he = . %. accurate analyses showed that ( %) had normal lf, ( %) ao and ( %) small airway diseases (only distal ao and/or hyperinflation). frc in infancy and at age were correlated (r= . ; p= . ). conversely, no correlation between ao parameters in infancy and at age was found. the lf alteration was independent of the genotype, bacterial colonization and recurrent symptoms at both ages. conclusion: ao in infancy is frequent as found in half of the screened cf infants. the lack of correlation with the ao observed at age suggested that ao in infants might be due to a non specific inflammation (i.e viral), transient and then relevant from a treatment. hyperinflation, although mild, is conversely an early marker of the cf disease as persistent between infancy and age . we describe the efficacy of a novel system using an electronic real-time distant home monitoring of symptoms and spirometry in cf patients with the aim of early detection of p exs patients and methods: this is a month prospective observational study. all patients were provided with a set consisted of a mobile phone-palm pc (the x daii, o -uk) and with a spirometer (vitalograph) with an attachment to the pc. the pc contained a purpose-built software on which cf symptoms and spirometric values were recorded once daily. these were then sent on a real time to a computer server where the data were plotted on a time-magnitude graph. the site was accessed by a password-permitted cf physicians. patients were asked to score four symptoms (cough, sputum, breathlessness and fatigue) once a day and to record at least three attempts of spirometry. these were accepted by the software only if they met the ats criteria. preset criteria for p exs were stipulated. when the recordings met the criteria for p exs patients were invited to come to the cf clinic for review and were managed by either watchful waiting, oral or iv antibiotics. a prodrome phase is deemed to be present when a patient had an increase in one or more symptom for one week prior to the p ex. twenty one evaluable patients were included in the study. in ( %) there were an increase in one or more than one recorded symptoms throughout the study. a total of p exs median . per patient were detected within the months study period. in ( %) p exs, symptom increase and/or decline in fev met the preset criteria for p ex and in cases patients presented despite that symptom changes were not sufficient to meet the pre-set definition of p ex. prodromal phase prior to p exs were seen in ( %) patients. adherence and technical issues were the main two problems throughout the study. distant daily monitoring of patients symptom is easy and sensitive. it can be used to monitor symptoms and early detect p exs. the vast majority of our patients have an increase in their symptoms even when they perceive their disease to be stable. a prodormal phase preceded p ex is seen in the majority of patients. the pre-set criteria for defining p ex may have been too stringent. the research was funded by o telecommunication, uk the monitoring system used in the study. a combination of a mobile phone-palm computer (x daii, o uk) and a spirometer (vitalograph uk) .. the aim of this research was to examine whether systemic inflammation is present in adult patients with cf during their stable status. patients and methods: blood samples were obtained from a total of cf patients female, mean age , range( - ) males mean . range - and from control subjects, female, mean age . , range( - ) males,mean age . yrs, range( - ) all cf patients were included when they were at least weeks away from any pulmonary exacerbations. all control subjects were not known to have any pulmonary or systemic disease. venous blood samples were taken and spun at rpm.separated sera was aliquted and frozen at - degrees c. they were analysed in batches for cytokines il- b,il- ,il- , il- , ilp ,and tnf-alpa using a cba human inflammatory kit(bd bioscience). whitney-u test was used to compare values of serum cytokines between stable cf patients and control subjects.. serum il- and il- were raised in cf patients compared to control subjects. the mean value for il- was . pg/ml (sd . ) for patients and . pg/ml (sd . ) for controls, p< . . similarly, the mean value of serum il was . pg/ml (sd . ) in cf patients compared to . pg/ml (sd . ) in the control group, p< . (figure below). there was no difference between cf patients and control with regards to serum il- b, il- and il- p . conclusion: serum il- and il- were raised in cf patients compared to an agematched control subjects. inflammatory markers were raised in the sera of cf patients even during disease stability. even during disease stability, the lungs remain an inflamed organ which could explain the decline in lung function tests and the countinued daily systemic symptoms experienced by patients with cf. serum il and il in cf patients with stable disease compared to the control group (please see abstract body) we have shown that even during disease stability, an increase in inflammatory markers are present in the sera of cf patients compared to an agematched control group. this longitudinal study examines changes in serum inflammatory markers during p exs. we investigated cf patients female, mean age , range - years. their mean baseline fev was . % of predicted (range . - %). a baseline venous blood sample was withdrawn and sera were stored at - degree c from patients during disease stability. out of those there experienced p exs. blood samples were obtained at the beginning and at the end of the p exs.the following inflammatory markers were analysed: white blood counts, c -reactive protein (crp), il- , il- , il- , il- b, il- p and tnf-α. patients recorded their symptoms and their fev on a daily basis on an electronic diary and the data were beamed daily to a research centre where data were analysed. p ex was defined as ). an increase in two symptoms for successive days, or ). an increase in symptoms and % reduced fev from baseline over successive days or ). % decline in fev over successive days or ).when a patient felt that he/she was going through a p ex. paired t tests were used to analyse changes in inflammatory markers. there was no change in the number of circulating neutrophils, lymphocytes or eosinophiles at the start compared with the end of p ex. there was an increase in mean crp values at the start of p ex and reduction at the end of p ex, but this did not attain statistical significance. mean (sd) of serum il increased from . pg/ml ( . ) at baseline to . pg/ml ( . ) at the start of p ex (p= . ) and returned to base line . pg/ml ( . ) at the end of p exs (p= . compared to values at the start of p ex). (figure ) there were no changes in the values of serum il- , il- , il- b, il p or tnf-α at the start and at the end of p exs. serum il increased at the start of p ex and returned at the baseline at the end of p ex. crp was the closest inflammatory marker to follow that pattern. none of the other inflammatory markers changed with the diagnosis of p ex's. this and other studies call for the price of analysis of il- to be reduced to become available in routine clinical practice. background. abpa can be a therapeutic challenge in children with cf. aim was to describe the tolerability of voriconazole and nebulised liposomal amphotericin in the treatment of abpa in children with cf. methods. we performed a retrospective case note review of voriconazole and nebulised liposomal amphotericin use, in the treatment of abpa in children with cf, at our centre. results. children diagnosed with abpa according to the current criteria, failed to improve despite previous treatment with itraconazole and steroids, and received voriconazole as a second line agent. the dose of voriconazole was as recommended by the manufacturer's summary of product characteristics and the duration of treatment was two weeks. significant side effects noted in children. observed side effects included visual hallucinations (n= ), photopsia (n= ), sleep disturbances (n= ), severe headache (n= ), influenza like symptoms (n= ), gastrointestinal side effects (n= ) and elevation of alkaline phosphatase (n= ). of these children, one child tolerated voriconazole when concomitant treatment with omeprazole was withdrawn. a second course of voriconazole, given at a lower dose and without the loading dose was tolerated by two children. voriconazole blood levels were not measured in any patient. we used nebulised liposomal amphotericin in of these children. one child received amphotericin as a second line agent when voriconazole had to be stopped because of significant side effects. children had active abpa despite treatment with first line (itraconazole and steroids) and second line (voriconazole) agents and received amphotericin as a third line agent. amphotericin was used at a starting dose of mg nebulised twice daily, with weekly increments as tolerated, up to a maximum dose of mg twice daily for four weeks. the starting dose and all subsequent increments in dose were supervised. all children received salbutamol immediately before nebulised amphotericin. the standard formulation available for intravenous infusion was made up into single doses for nebulisation by the pharmacy. this was stored at c and diluted with water for injection for use with the nebuliser. the only side effect, experienced by all children was a dry irritant cough at the start of nebulisation that settled soon without need for discontinuation of the medication. improvement in clinical symptoms, lung function and total serum ige was noted in all children. the improvement in fev and fvc, expressed as mean (range) of percentage predicted were and respectively. the mean (range) fall in serum total ige and aspergillus specific ige were ku/l ( - ) and mg/l ( - ) respectively. conclusion. voriconazole use was associated with significant side effects. avoidance of drugs that may potentially result in interaction is important. omitting a loading dose and using a lower maintenance dose may help selected patients tolerate voriconazole. nebulised liposomal amphotericin was tolerated by our children with cf and abpa who did not tolerate or responded poorly to voriconazole. recent studies have shown that high resolution computed tomography (hrct) is a more accurate method of evaluating early lung disease in cystic fibrosis (cf) than pulmonary function tests (pfts) or chest x-rays. since young children can not perform the breathing maneuvers required for high quality hrct, lung volume control is necessary. positive pressure facemask ventilation using chloral hydrate allows full inspiration and end exhalation images, but requires special expertise. controlled ventilation can also be produced with general anesthesia (ga). ga has not been reported in children undergoing hrct for cf research. the advantages of ga over sedation include rapid onset, faster recovery and reliability of scan completion. the undesirable effects of sedation are unpredictability and the higher failure rates. additionally, failed sedation contributed significantly to parental dissatisfaction. (malviya, et al., ) . the primary outcome of our study was to examine the safety of using ga for hrct scans and the quality of images obtained. a secondary outcome was parental satisfaction. methods: participants were recruited from a larger nih/cffti funded multi-site randomized clinical trial focusing on behavior and nutrition treatment to improve growth in preschoolers with cf. hrct in subjects to years old (mean age ± months) was performed with ga. parents accompanied the child during induction and left after the child was asleep prior to the intravenous (iv) being placed. after induction of anesthesia, a proseal®, specialized laryngeal mask airway (lma), was placed which allowed ventilation with higher pressures and the ability to suction the stomach. volumetric thin section ct scans were obtained. ga using inhalation (sevoflurane) and/or iv (propofol) agents was used and out of families who were eligible ( % recruitment rate) participated in the sub-study. results: the quality of the hrct scans in all subjects were optimal. there were no anesthetic complications upon emergence and none of the patients experienced any side effects after follow-up phone calls at hours. parents also expressed that the clinical interpretation of the hrct scan provided beneficial information about their child's current lung disease status. the mean time from completion of scan to discharge was . ± minutes. conclusions: the use of ga in radiology departments is becoming more common in pediatric centers. our initial findings of this study demonstrate that ga can be safely administered for children with cf. in addition to very good quality scans being obtained, parental satisfaction was also noted. the use of the proseal® lma for hrct may improve the image quality and aid in the diagnosis of early lung disease in preschoolers with cf. supported by nih grant r dk and a cf foundation clinical research grant. whey protein is rich in sulfhydryl (sh) groups and is recognized for its ability to increase glutathione and reduce oxidative stress. hyperbaric pressure treatment of whey protein increases its digestibility, promotes the release of novel peptides for absorption, increases intracellular glutathione in healthy subjects (zavorsky et al, int j vit nutr res, ) , and reduces in vitro production of il- (vilela et al, inter immnuopharmacol, ) . we present here the initial results of a pilot study of supplementation with pressurized whey in cf patients. children and adults with cf were supplemented with hyperbaric whey protein for month ( gm/day in patients less than years of age, gm/day in older patients). anthropometric measures, pulmonary function, serum c-reactive protein (crp) and plasma lipid peroxides were measured before and after supplementation. the results of the first patients to have completed the study are reported, with further data to be presented. in / subjects weight increased and in / subjects lean body mass increased. there were no significant changes in fev or fev/fvc ratio. in / patients the crp fell ( / by more than %) and in / the crp increased. in the remaining two patients, the crp was undetectable at baseline and did not change at follow-up. plasma lipid peroxides decreased in the three patients in whom it could be measured ( / to undetectable levels). these preliminary results suggest that supplementation with pressurized whey protein can reduce systemic inflammation in cf patients and has the potential to beneficially modulate the inflammatory response in cf patients. supported by the breathe initiative of the canadian cf foundation with pseudomonas aeruginosa. soon after its introduction, the mesh technology based pari eflow® nebulizer gained preference over jet nebulization in the cf community because of its short nebulization time, portability and noiseless operation. concerns were raised about the durability of the device due to expected vulnerability of the mesh. the aim of this study was to compare the particle size distributions in the aerosol, delivered doses and output rates from the eflow and the pari lc plus®(lc+)with paritur-boboy compressor before and after use in daily practice. lc+ nebulizers in use by the adult patients taking part in the study were taken in for testing and new eflows, cleaning instructions and a disinfection device were provided. after months use ( cycles of x doses tsi: mg/ ml), the eflows were collected by the local cf center. particle size distributions were measured at a constant flow rate of l/min with a laser diffraction apparatus. because of uncertainty regarding the cleaning procedure after last use, additional e-flows were collected from the same patients after another period of months use. conclusions: new eflows produce slightly larger droplets in a narrower size range than new lc+ nebulizers. after use in daily practice, droplet size distribution changes for both devices. output rate (delivered dose per minute) decreases for both devices. for used eflows delivered dose is lower than for used lc+ nebulizers, because the eflow is programmed to switch off after min, even when nebulization is still incomplete ( out of tested eflows). this may result in a considerable reduction of delivered dose. however, when a mesh is replaced, performance is the same as that of a new device, suggesting that the changes in performance originate in the vibrating mesh. for daily practice these results indicate: ( ) the performance of both nebulizers changes during use but the changes are smaller for eflow than for lc+ ( ) changes in droplet size distribution are relatively small for both devices;in contrast with output rate and delivered dose ( ) proper functioning of eflow can be improved by timely replacement of the mesh, ultimately when eflow switches off after minutes. without replacement, the delivered dose is unpredictable. mean values for size distribution, delivered dose, nebulization time and output rate (x is volume median diameter) backgrounds: lung surfactant protein a (sp-a) belongs to the family of collagen containing, c-type lectins, called collectins. sp-a plays a critical role in the innate immune defense of the lung. sp-a binds calcium dependent to carbohydrate structures on pathogens via its carbohydrate-binding domain (crd). cystic fibrosis (cf) lung disease is characterized by chronic inflammatory and proteolytic processes in the airways, leading to destruction of the lungs and early death. aims: we hypothesized that sp-a might have a reduced function in cf. this might result from sp-a degradation by neutrophil proteases, an altered macromolecular organization of sp-a, or inhibition of crd-binding by airway components. methods: we studied bronchoalveolar lavage fluid (balf) from clinically stable patients with cf and from children suffering from bronchitis. balf samples were purified by carbohydrate-binding assay on fucose attached to sepharose resin with and without calcium. in the absence of calcium sp-a cannot bind via its crd. each fraction was analysed for sp-a and igg by immunoblotting. macromolecular organization of sp-a was determined by gel chromatography. results: only % of total sp-a of cf balf bound to the fucose column, whereas % bound from the bronchitis balf. the structural organization of the sp-a oligomers did not differ between the non-binding and the binding fractions of both groups. of the cf balf had sp-a at kd that did not bind to fucose. these balfs had a high percentage of neutrophils (> %). in vitro human leucocyte elastase decreased sp-a to less than % of its initial levels and sp-a fragments at kd were detected. conclusions: neutrophil proteases degrade sp-a in cf lungs. the carbohydrate dependent binding of sp-a is diminished due to a reduced function of the crd. these alterations contribute to the reduced ability in cf to remove specific pathogens from the lungs. chiron, r. , ; murris-espin, m. , ; crampette, l. , ; varrin, m. ; didier, a. , ; wallaert, b. , ; chanez, p. , ; leroy, s. , . crcm, chu, montpellier, france; . crcm, chu, toulouse, france; . orl, chu, montpellier, france; . iurc, chu, montpellier, france; . crcm, chu, lille, france; montpellier, france in cystic fibrosis (cf) patients, upper airways involvement is frequent but its impact and relationships on the course and severity of the disease has been rarely investigated. objective: to relate upper airways involvement with lower airways parameters and quality of life in cf patients. methods: clinical and radiological prospective study of subjects with cf at french cf centres. clinical rhino-sinusitis (rs) was defined by the presence of more than episodes of nasal symptoms during the last year, current nasal obstruction or rhinorrhea. nasal investigations included: anterior rhinoscopy, sinus ct scan analysis and rs quality of life questionnaire. lower airways were assessed using a thoracic ct scan (bhalla score), spirometry and sputum culture and a cf quality of life questionnaire (cfq +). results: ( f/ m) patients aged . ( - ) years old were enrolled. endoscopy demonstrated polyposis in % and nasal hyper secretions in . % of the patients. spirometry, bhalla score, sputum colonization did not discriminate patients with (n= ) or without rs (n= ), and regardless the presence of polyps. cfq + was significantly lower in patients with of rs (p= . ). conclusions: rs involvement altered quality of life in cf patients, although, they do not have a more severe pulmonary disease. a specific attention and treatment to rs should be paid in cf patients. the impact on different outcomes deserves to be examined in longitudinal studies. as part of the uk cystic fibrosis gene therapy consortium "tracking study", ebc was measured (ecoscreen, jaeger) in adult and paediatric patients presenting with an infective exacerbation requiring iv antibiotics. exacerbation was defined by pre-determined criteria. ebc was repeated at the end of parenteral antibiotic therapy and again - weeks later. patients were recruited at three sites and assessed using standardised equipment. samples were obtained according to ers guidelines. exhaled breath ph was assayed (shindengen ph meter, camlab, uk) immediately after collection without a de-aeration step. results: paired ebc measurements at the start and end of antibiotic therapy were available in patients. mean age was yrs (range - ). data from patients were excluded because at least one ph measurement was > , suggesting salivary contamination. for the remaining patients mean ebc (sd) ph increased from . ( . ) to . ( . ), p< . . of these patients, also had measurements available at a third visit, - weeks after completion of antibiotics. were excluded because of salivary contamination. no difference in mean(sd) ebc ph was found ( . ( . ) vs. . ( . )) conclusions: ebc ph increases after treatment with antibiotics, and may offer a non-invasive means of assessing airways inflammation in chest exacerbations. further work is required to follow the longitudinal change in ebc ph and other inflammatory markers in clinically stable patients, as ebc may be a useful tool in assessing response to novel treatments, such as gene therapy. this work was funded by the cystic fibrosis trust. background: children with cystic fibrosis (cf) undergo multiple procedures exposing them to ionizing radiation in hospital. some specialist centres have advocated regular chest computed tomography (ct) as a means of better quantifying lung pathology. the clinical benefit of this approach is unproven and previous calculations of risk have failed to account for other ionizing radiation exposure procedures . aim: to quantify the exposure of ionizing radiation in the pediatric cf population of a tertiary level children's hospital. to determine the potential difference in exposure of introducing biannual chest ct scans. methods: effective doses of ionizing radiation from chest (cxr) and abdominal (axr) radiographs, fluoroscopy and cts (sinuses and chest including hr scans) were determined for cf children at our centre between st january and th july ( weeks). the maximal effect was tested i.e. introduction of biannual chest ct scans from age years abolishing the requirement for cxrs and the assumption was made that axrs, sinus cts and fluoroscopy rates were unchanged (median exposure values calculated for age groups -< year, -< year, -< year, -< year, -< year, > year) . results: cf patients (n= , gender ratio : ) underwent a mean of . ionizing radiation exposure procedures per patient over the study period (total of procedures). the average effective ionizing radiation doses (msv) were calculated for ( . %) procedures. each cf child received a mean (median) effective radiation dose of . ( . ) msv/year with a range of . - . msv/year. the reason for the skewed distribution was identified by analysing the proportions of the procedures to the total effective dose in this population: fluoroscopy . %, chest ct . %, sinus ct . %, cxrs . %, axrs . % and hrct . %. it was calculated that these patients undergo . cxrs per patient per year. we calculated that an average patient diagnosed by newborn screening could expect to receive . msv from ionizing radiation procedures between birth and age . biannual ct chest scans in addition to median exposures for each age group from axrs, sinus cts and fluoroscopy would potentially result in . msv by age , an fold increase. discussion: there is a in lifetime risk of developing a solid or haematological cancer with each exposure to msv of ionizing radiation . although fluoroscopy forms the most significant contribution to a pediatric cf patient's ionizing radiation exposure in our centre, introducing biannual chest cts would markedly increase exposure. as cf survival improves, cf physicians must be cognisant of the potential health cost of survival. references : previous studies have used generic measures of hrqol, which are not sensitive to important changes that may occur as a result of treatment. this study assesses the impact of iv antibiotic treatment for a pulmonary exacerbation on both generic and disease-specific hrqol of children and adolescents with cf. method: participants included patients (m age = . years) from the cincinnati children's hospital and university of florida cf centers. fiftyfour percent of the sample were male. patients completed two hrqol measures, the pedsql™ (generic) and cfq-r child or teen/adult version (cf-specific), which have excellent reliability and validity. these measures were completed within hours of hospital admission (pre) and hours of discontinuation of iv antibiotics (post). note that approximately % of patients went home on iv antibiotics and % remained in the hospital for approximately weeks. results: significant improvements were found in pulmonary functioning (fev % predicted) pre to post iv antibiotic treatment (paired t ( ) = - . , p < . ), with an average of % increase in fev % predicted. paired t-tests were conducted to examine changes in hrqol scores, using holm's procedure to correct for multiple comparisons. significant improvements were found on the cfq-r respiratory (paired t ( ) = - . , p < . ) and weight scales (paired t ( ) = - . , p < . ). trends were also noted for improved emotional functioning (paired t ( ) = - . , p = . ) and vitality (paired t ( ) = - . , p = . ), and worse treatment burden (paired t ( ) = . , p = . ). on the pedsql, only emotional functioning improved significantly (paired t ( ) = - . , p < . ). conclusions: results of this study confirm the effectiveness of iv antibiotics for the treatment of pulmonary exacerbations, with significant improvements found for both pulmonary functioning and hrqol. these results also highlighted the importance of using a disease-specific hrqol instrument, which proved to be more sensitive than the generic measure. on the cfq-r, significant improvements were observed in respiratory symptoms and weight, aspects of functioning that are not assessed on generic measures. fukushima, l.k.; hsu, e.; woo, m.s. division of pediatric pulmonology, childrens hospital los angeles, los angeles, ca, usa do hispanic and caucasian cf patients share the same frequency of pulmonary exacerbations? increased frequency of pulmonary exacerbations is linked to increased risk for cf morbidity and mortality. we have previously reported that pediatric hispanic cf patients have greater mortality than our caucasian cf patients who attend the same accredited cf center. we performed a retrospective review of cf pulmonary exacerbations (defined as physician prescription of intravenous or oral antibiotic therapy) in our cf patients who routinely attended the cf clinic during the period of january , through december , . data collected included demographics, number of intravenous antibiotic courses and number of oral antibiotic courses. unpaired student's t-test was used to compare age of the groups and chi-square was used to compare use of intravenous and oral antibiotics between hispanic and caucasian cf patients as well as between males and females. during the study period, there was a total of cf patients ( males: females; mean age . ± . years). of the patients, ( %) were classified as hispanic ( males: females; mean age . ± . years) and ( %)were classified as caucasian ( males: females; mean age . ± . years; p= . , ns). hispanic cf patients ( patients had episodes; males: females) had significantly greater number of pulmonary exacerbations (received treatment with intravenous and/or oral antibiotics) than caucasian cf patients ( patients had episodes; p = . ). hispanic cf patients who required intravenous antibiotic therapy were significantly younger ( . ± . years vs . ± . years; p< . ) than the caucasian cf patients who were treated with intravenous antibiotics. there was no significant difference in age between hispanic and caucasian cf patients who received oral antibiotic courses ( . ± . years vs . ± . years; p= . , ns). gender did not have a significant impact on pulmonary exacerbation risk in our population (hispanic iv use by gender p= . ; hispanic oral use by gender p= . ; caucasian iv use by gender p= . ; caucasian oral use by gender p= . ). we conclude that hispanic cf patients had increased incidence of pulmonary exacerbations than caucasian cf patients who attend the same cf clinic. hispanic patients who were treated with intravenous antibiotics were younger than the caucasian cf patients. we speculate that hispanic ethnicity has a major impact on the incidence of pulmonary exacerbations. other factors, such as modifier genes, environmental exposures, or inflammatory responses, may play a role in the increased risk of pulmonary exacerbations in the hispanic cf population. introduction: one of the main goals of the multidisciplinary team of the cf association of minas gerais, brazil is to find strategies to preserve lung tissue from the bacterial exacerbation in cystic fibrosis patients. objective: to investigate clues on onset of bacterial exacerbation. to our knowledge, no studies were found regarding the identification of the early warning signs of bacterial exacerbation with cf patients. we wanted to know whether cf patients present early warning signs before onset of fever and/or breathing difficulties. methods: telephone interviews were carried out with the cf patients registered in the association, by a physiotherapist academic who did not know the patients and just ask two questions. the first question was about the use of antibiotics during the year of and the second was related to early signs before the onset of fever. results: from the patients, only patients replied. patients who were using antibiotics therapy during and had fever: / patients who were using antibiotics and did not have fever: / patients who had fever and did not use antibiotics: / patients who did not have fever and did not use antibiotics: / the results revealed the following warning signs and also that some patients had more than one sign. tiredness, quietness, slowness ( / ); increased cough ( / ); lack of appetite ( / ); sticky sputum ( / ); sleepiness ( / ); headache ( / ); increased excitement, nervousness ( / ); crybaby ( / ); dry mouth ( / ); clammy ( / ); difficult to walk ( / ); gastric upset ( / ); itchy throat ( / ); abdominal pain ( / ) ; itchy eyes ( / ); breathing difficulties ( / ); tremulous ( / ); paleness ( / ); burning ear ( / ); chest pain ( / ); hissing ( / ) ; and syncope( / ). conclusions: it is helpful to know these early warning sings. we assume that the earlier the diagnosis of the exacerbation is made, the easier it is to resolve the problem and the less destruction of the lung tissues. reference: lung line, national jewish center for immunology and respiratory medicine . background: cystic fibrosis patients are experiencing increasing survival and are being treated with more chronic therapies. how these changes are reflected in day to day clinical symptoms has not been evaluated. objective: we examined data from the epidemiologic study of cystic fibrosis, a large longitudinal observational study, to characterize the change in respiratory symptoms over time. for each year between and data from patients with at least one visit recorded as occurring while clinically stable were included. data from all visits, including sick as well as stable, were used for each year in which at least one clinically stable visit was recorded. patients were separated into age groups less than , to , to , and or older. note that in data collection changed, potentially confounding the results. results: the average number of patients per year was . the percent of patients with no reported cough progressively increased over time (table) . results were similar for pateints with no reported sputum production at clinically stable visits. when sick visits were added there were similar results. the frequency of cough reported at greater than percent of visits progressively decreased over time (table) . again the results for sputum production were similar. these findings were seen in all age groups although older patients are more likely to experience these symptoms. conclusions: these data suggest that cf patients are experiencing fewer respiratory symptoms of cough and sputum production over the last decade. this improvement has occurred in patients with either intermittent or chronic symptoms. how clinical care has impacted on these symptoms has yet to be determined. also, more people are being diagnosed with cf at an earlier age or with milder disease, potentially resulting in the entire cf population having fewer symptoms. lung disease in children with cf begins in early childhood, with intermittent and then chronic infection, associated with an exaggerated inflammatory response. the extent to which inflammation contributes to bronchiectasis has not yet been identified. the aim of this study was examine relationships between inflammation and early lung damage methods: twenty eight children with cf were assessed at diagnosis and annually thereafter with a bronchoalveolar lavage (bal). bal fluid was used to assess micro-organisms and inflammation, including total and differential cell counts. a three slice hrct technique (inspiration and expiration) was performed under general anesthesia at age - y, immediately preceding the annual bal. hrct scans were scored by an independent and experienced radiologist using a modification of the brody score. results. twelve children ( %) had evidence of bronchiectasis on hrct. inflammation present in bal at months of age predicted bronchiectasis at - y. total cell count at mo, predominantly neutrophils, was higher in those who developed bronchiectasis compared to those who did not ( . vs . x cells/ml bal; p= . ). the total number of infections detected in bal was not related to bronchectasis. however, children with bronchiectasis had a higher incidence of pseudomonas aeruginosa within the first years of life ( % vs. %). conclusions: bronchiectasis is evident within the first five years of life and appears to be associated with an exaggerated inflammatory response and the early acquisition of pseudomonas aeruginosa. supported by: cystic fibrosis foundation (usa), national health and medical research council (australia). fortunately the survival of patients with cystic fibrosis has improved remarkably over the last few decades. many patients can life quite "normal". therefore family planning becomes more important. several reports docu-mented the good maternal and fetal outcome in pregnant women with cystic fibrosis. besides the data from the french cf registry no longterm data about the impact of paternity of male patients with cystic fibrosis are available until now. in a retrospective analysis we gathered data about all our male patients who became father while being followed-up in our cystic fibrosis center for adults. from male patients, patients became father between and . patient no. became father twice (second time father of twins); patients no. became also father of twins .we looked at fev % year prior to childbirth, perinatal and and years after the childbirth. for the fev % one year prior to birth as well as for fev % and years after birth, we calculated the difference to the fev % at time of birth. between and , male adult patients with cystic fibrosis became fathers of a total of children, all by the assistance of reproductive techniques. their fev % are shown in table . although the male patients are not directly influenced by a pregenancy (as the pregnant women are by growth of the uterus), there seems to be more than ususal differences in this lung function parameter. there were also more fluctuations in fev per year. the biggest differences in fev % were seen year after birth of their children. further investigations in a larger group (in cooperation with other centers) are planned; especially with a focus on the frequency of infections and quality of life compared to male patients without children. intravenous (i.v) aminoglycosides are widely used in cystic fibrosis (cf) patients as treatment for pulmonary exacerbations. in an effort to reduce undesiderable effects of these antibiotics it is recommended to measure drug serum levels. recent experience suggests that tobramycin (to) may be administered as a single daily dose with equal efficacy as multiple doses but less risk of nephrotoxicity. aim: we evaluated the efficacy of once-daily i.v to treatment to improve pulmonary function expressed as forced expiratory volume in one second (fev ) in cf patients with pulmonary exacerbations compared to the same group of patients who were previously treated with to in multiple daily i.v doses, as historic control. we evaluated baseline and peak drug serum levels of to administered once daily compared to multiple dose levels. methods: all patients were treated with i.v to in combination with a beta-lactam antibiotic for two weeks. respiratory exacerbation was defined according to cff criteria. all cf patients were colonized by non-fermentative gram-negative bacteria. serum concentrations of to were measured (fluorescent polarisation immunoassays) before the fifth infusion (basal) and minutes after the fifth infusion (peak). reference to blood levels were considered to be ≤ mg/l (basal) and < mg/l (peak) for multiple daily doses, and ≤ mg/l (basal) and - mg/l (peak) for single daily dose. treatment efficacy was evaluated on the basis of improved clinical condition and increased fev values compared to the beginning of therapy. results: patients with respiratory exacerbation were evaluated, with a mean (± ds) age of . years (± . ; median . , range . - . ) . out of patients were chronically colonized by pseudomonas aeruginosa. the total mean (± sd) daily dose of to administered in multiple doses was (± . ) mg whereas the mean single dose was . (± . ) mg. of those patients given multiple doses of to, only one patient ( . %) had basal to values outside the range and patients ( . %) had peak values outside the range. of those patients given a single daily dose, no patients had basal to values outside the range whereas out of ( %) had peak values of less than mg/l and patients out of ( %) had peaks between and mg/l. the mean (± sd) increase in fev was . % (± . ) in patients treated with a single dose and % (± . ) in those treated with multiple doses. the mean (± sd) period between fev measurements before and after exacerbation was . days (± . , median , range - ) in case of monodose therapy, and . (± . , median , range - ) in case of therapy with multiple doses. conclusions: both single and multiple-dose i.v to therapy caused a comparable fev increase and were effective in resolving the respiratory exacerbation. although clinical improvement was observed, the mean i.v to dose of mg/day in patients treated with once-daily i.v to determined peak values between and mg/l in % of subjects. intravenously administered aminoglycosides are efficacious for the treatment of pulmonary infections of cystic fibrosis (cf) patients, with the principal adverse effects of these molecules being acoustic nerve damage and nephropathy. damage to the eighth cranial nerve varies from . to % in cf patients treated with multiple daily doses of aminoglycosides. recent experience suggests that tobramycin can be administered as a single daily dose, resulting in less nephrotoxicity but equal efficacy. patients and methods: we evaluated the prevalence of oto-and nephrotoxicity due to aminoglycosides administered to cf patients followed at the tuscan regional center where we are currently following patients. cochlear damage was evaluated using tonal audiometry (amplifon amplaid s). renal damage was evaluated by measuring patients' creatinine clearance. results: ( %) patients ( males and females) between the ages of and (mean age . years, median , sd ) were tested with audiometry. all these patients had been repeatedly treated with multiple daily doses of intravenous aminoglycosides. ( . %) ( males and females) out of patients between and years (mean age . , median . , sd . ) had their creatinine clearance measured. we found that patients ( . %) had an abnormal audiometry, typically attributable to aminoglycoside ototoxicity (high frequency deficit). two patients required a hearing aid. one patient with normal cochlear function had labyrinth deficit. one out of patients tested had pathological creatinine clearance values. conclusions: our data indicate that ( . %) of our patients had damage to the eighth cranial nerve due to repeated multiple daily doses of aminoglycosides. we recommend that cf patients under aminoglycoside therapy be routinely given audiometric and creatinine clearance exams in an attempt to reduce the incidence of undesirable side effects of these drugs. davies, j. , ; voase, n. , ; dewar, m. , ; mullard, k. , ; gammie, f. , ; saunders, c. , ; horsley, a. , ; gray, r. , ; macleod, k. , ; somerton, l. , ; higgins, t. , ; donovan, j. , ; cornish, n. , ; hansell, d. , ; aziz, z. , ; ashby, d. ; geddes, d. , ; greening, a. , ; cunningham, s. , ; innes, a. , ; alton, e. , . gene therapy, imperial college, london, united kingdom; . western general hospital, edinburgh, united kingdom; . royal hospital for sick children, edinburgh, united kingdom; . royal brompton hospital, london, united kingdom; . queen mary, university of london, london, united kingdom; . uk cf gene therapy consortium, edinburgh, london, oxford, united kingdom the improving clinical status of patients with cf and the slow rate of decline of conventional outcome measures such as fev necessitate the development of clinically-relevant surrogate outcome assays for use in clinical trials. in our forthcoming clinical trial of cftr gene therapy, the uk cf gene therapy consortium will use both established, clinically-available assays and more novel measures designed specifically for this purpose. both groups of assays are being subjected to rigorous testing prior to use to establish their utility as disease biomarkers. in this study, we examined their performance in the context of an infective exacerbation treated with iv antibiotics. this abstract will report the response of established, clinically-available assays; available data from novel assays will be reported separately. children ( years and above) and adults with cf were recruited from three centres at the time of a clinician-defined infective exacerbation requiring iv antibiotic treatment. exclusion criteria included fev < % predicted and requirement for supplemental oxygen. a panel of assays was performed at the start and end of treatment, which was most commonly days. data are presented as mean (sd) or median (range) and differences compared with either parametric or non-parametric paired analysis as appropriate. clinical assays included spirometry, sputum microbiology, sputum cell count and differential, serum inflammatory markers (crp, white blood cell (wbc) count,) and chest ct. patients also completed a symptom score sheet. to date, patients (mean age . [ . ] years, male) have paired data available from the start and end of the course of ivs. at baseline, were infected with p. aeruginosa, with b. cepacia complex organisms and with s. aureus ( mrsa). significant changes from baseline were observed in fev ( . [ . ] the clinical response to any novel intervention, for example, cftr gene therapy is difficult to predict. prior testing of experimental assays in a study such as this provides data on the variability of the measurements within the disease population and the degree of change observed with an intervention known to lead to clinical benefit. this should aid the design of rational, powered clinical trials. funded by the cf trust influenza can worsen cf and lead to serious complications and mortality. influenza vaccine is safe and effective in cf patients. in france, annual influenza vaccination is recommended for all cf patients aged over months and for healthcare workers in regular and prolonged contact with high-risk patients. objective: to estimate the influenza vaccination coverage rate for - season in cf patients and healthcare workers in cf care centres of the great south region of france. methods: a multicentric observational study between february and september was conducted. healthcare workers were contacted by telephone and were questioned about their - influenza vaccination status, and the reason for not getting vaccinated. for patients aged over months whose vaccination booklet was available, the physician filled in a written questionnaire. results: a total of professionals were interviewed. the survey included . % of doctors, . % of nurses, . % of dieticians, . % of physiotherapists, . % of social workers and . % of psychologists. the overall influenza vc rate was . % and varied considerably according to profession: . % of doctors, . % of physiotherapists, . % of dieticians, % of psychologists, . % of nurses and . % of social workers. the overall influenza vc rate in cf patients was . % ( . % in children (age < years old) versus . % in adults; p< . ). receiving a voucher for free vaccination from the national insurance increased the influenza vc rate. for healthcare workers, the main reason for non vaccination was that flu was considered as a benign disease (useless vaccine); for patients, it was a lack of time. conclusion methods: adult patients with cf were studied. patients completed a standardised questionnaire. the questionnaire identified which changes and duration of symptoms would cause them to contact the cf team and/or change treatment. results: all patients would contact the cf team for haemoptysis but % would wait for more than hours and % would wait for more than hours before doing so. up to % of patients would wait a week or more before contacting the cf team because of sputum purulence. % of patients would not contact the cf team if they developed new chest pain. conclusion: there appears to be a disconnect between recognizing a change in symptoms and the length of time before acting on the change. this has major implications for the delivery of cf care. supported by cystic fibrosis association of ireland. aim: to determine the efficacy of standardized desensitization protocols in treating antibiotic allergies in adults with cystic fibrosis (cf). background: dependence on high dose and multiple combination intravenous (iv) antibiotics to treat pulmonary exacerbations in adult cf patients has resulted in an increased frequency of allergic reactions when compared with the general population. ( , ) strategies to address these allergies are essential to maintain effective antibiotic treatment in this population. antibiotic desensitization is the process by which one induces immune tolerance by incremental exposure to the antibiotic, which may induce stabilization of mast cells. the mechanism remains undetermined but may be mediated via ige or memory t-cells. prior to the study period, standardized antibiotic desensitization protocols were developed by an allergist/immunologist based on published reports and were approved through the hospital's pharmacy and therapeutics committee. methods: the toronto cf database was accessed to generate a list of hospital admissions for iv antibiotics during the -year study period ( ) ( ) ( ) ( ) ( ) ( ) . patients who underwent desensitization were identified and each of their case notes underwent retrospective analysis. data were collected with respect to age, organism requiring antibiotic therapy, antibiotic allergy requiring desensitization, reactions during desensitization, reactions post desensitization, treatments required to treat these reactions, and the completeness of antibiotic therapy post desensitization. statistical analyses were performed using graph pad prism®. results: during the study period the cf unit dispensed and initiated approximately courses of iv antibiotics. a total of desensitizations were performed in patients ( female). although some patients were desensitized to only one drug or to the same drug more than once, there were patient-drug combinations with a median of (+/= . ) unique combinations per patient. of the desensitizations protocols initiated, patients developed documented reaction to the antibiotic during desensitization however only desensitization protocol was not completed due to an adverse event. there were no episodes of anaphylaxis either during or after desensitization. a total of patients developed documented reactions during the subsequent antibiotic course - required termination of the antibiotic and completed the course of iv antibiotics with management of allergic type reactions with antihistamines. conclusions: standardized desensitization protocols were initiated in patients during the study period. the success rate for desensitizations in these patients was % however when the data is analyzed for true allergic phenomenon, the success rate rises to %. this demonstrates the efficacy of these standardized protocols in the treatment of ige-mediated allergic reactions to antibiotics in the adult cf patient. references: . burrows, j., toon, m., bell, s., ( ) . antibiotic desensitization in adults with cystic fibrosis. respirology, : - . . ramesh, s. ( ) . antibiotic hypersensitivity in patients with cf. clin rev allerg immun, : - . surgery for symptomatic disease. there is a long-held suspicion that poorly-controlled chronic sinusitis negatively impacts the respiratory status of cf patients, but whether sinus surgery improves the clinical course of cf lung disease is unclear. aim: assess the impact of functional endoscopic sinus surgery (fess) on respiratory exacerbation rate and lung function in adult cf patients methods: this is a single-center, retrospective study based on chart review. during the study period, - , adult patients underwent separate fess procedures. this abstract contains results so far from the analysis of patients that underwent a total of surgeries. primary outcomes were -year change pre to post fess in pulmonary function parameters and pulmonary exacerbation rate. results: at the time of surgery, the mean fev was % of predicted (ppd), the mean fvc . ppd, and the median age was . years (range . - . ). . % of patients were female. the median number of iv antibiotic courses for respiratory exacerbations in the months prior to fess was (range - ) compared to (range - ) in the year after surgery (p = . ). further,there were fewer days on intravenous (iv) antibiotics for respiratory exacerbations in the months prior to fess than in the month post-operative period (median vs. days), but this difference was not statistically significant (p = . ). the use of oral antibiotics for respiratory flares was comparable in the months before and after fess (median vs. days, respectively, p = . ). the median number of hospital admissions and median number of days hospitalized for respiratory exacerbations did not differ in the months pre vs. post-fess (p = . and p = . , respectively). the mean fev and fvc in the months pre and post surgery did not differ significantly ( . vs. . ppd, p = . , and . vs. . ppd, p = . ). the linear rate of change in fev and fvc did not differ significantly in the months prior to and following fess (-. l/month vs. +. l/month, p = . and -. l/month vs. +. l/month, p = . , respectively). there were no significant differences (p > . ) for any of the above comparisons when the analysis was limited to months before and after fess. there were no significant differences (p > . ) in antibiotic use and hospitalizations when the analysis was limited to the surgeries in which patients were experiencing both respiratory and sinus symptoms. this latter group had lower fev and fvc ppd in the month post-fess period ( . vs. . ppd, p = . , and . vs. . ppd, p = . , respectively). however, the rate of decline in fev and fvc in the months before and after surgery did not differ significantly ( p = . and p = . , respectively). conclusion: fess did not significantly affect the rate of respiratory exacerbations or rate of decline in pulmonary function in adults with cystic fibrosis. cumulative decline in lung function as measured by fev in cystic fibrosis (cf) pulmonary exacerbation is a major cause of cf-related morbidity and mortality. high-dose extended interval aminoglycoside (hdei ag) use may be more effective in improving lung function than traditional multiple daily dosing in cf-related pulmonary exacerbation. intermittent dosing of beta-lactam antibiotics are often used with hdei ag in patients with cf pulmonary exacerbation, which does not optimize betalactam pharmacodynamics. the primary purpose of this study is to determine the effect of hdei ag plus continuous infusion beta-lactam (cibl) on patients' return to best baseline fev from the previous months. this study was conducted at university of kentucky healthcare. this was a concurrent observational trial with patients serving as their own historic controls via review of the medical record. all pediatric and adult patients with cf respiratory disease hospitalized with an acute pulmonary exacerbation between november , and may , , with pseudomonas aeruginosa in their respiratory culture and were followed for at least one year were included. excluded were patients with known hearing disability or renal insufficiency or an inability to reliably perform spirometry (ie.less than six years old). patients served as their own historic controls via conventional treatment (control) versus hdei ag plus cibl (treatment). the primary endpoint was to determine the percent of patient courses that return to best baseline fev in the last twelve months after treatment with hdei ag plus cibl. secondary endpoints included determination of patient courses that returned to mean baseline fev in the last twelve months after study treatments, and description of beta-lactam dosing characteristics. thirteen patients were included in the pilot analysis which totaled patient courses (mean . courses per patient). average patient age was ± . years with a mean fev of %; . % were female. after hdei ag plus cibl no patients showed a return to their best baseline fev in the previous months with the exception of one patient whose value was unchanged. average overall percent change in best baseline fev group was - . ± . at follow up. % of patient courses exhibited a return to their mean baseline fev . average overall percent change in mean fev baseline was . ± . at follow up. five of the ( . %) study patients who had serum beta-lactam concentrations obtained were able to achieve at least four times the mic of the target organism. more data will be collected on subsequent courses and follow up lung function. our preliminary data suggest that our patients lost % of their best fev with an acute exacerbation and returned to % above mean baseline after treatment with hdei ag and cibl therapy. this evidence suggests that this might be an alternative to intermittent beta-lactam therapy. follow up data on remaining patients may provide sufficient evidence to validate this hypothesis. respiratory exacerbation in cystic fibrosis patients is characterized by increased sputum that may become more purulent. the detection of the exacerbation is based mainly on clinical subjective parameters. fev measurements are accepted as gold standard to be used for the life-time of the patient but do not adapt fast enough after resolution of an exacerbation. we compared parameters that are affected by the uneven distribution of ventilation in the lung due to increase sputum. methods: a zen body plethysmograph was used to measure tlc, fev and dlco (co/ch mixture). tlc was also calculated by the volume of ch , the angle of the slope of phase iii was calculated in cf patients at the beginning of the respiratory exacerbation and after weeks of antibiotic treatment. patients were - years old ( m/ f). results: fev changed by . + . % at the end of the antibiotic treatment, the tlc(pleth) however was stable and changed by only . + . %. the rv/tlc decreased by . + . % and the tlc(gas) was increased by + . %. the difference between tlc (pleth) and tlc (gas) before and after the antibiotic treatment decreased from . + . % to . + . %. conclusion: the changes in the parameters affected by the unevenness of distribution of ventilation (m/p reflecting the increased sputum in the airway) are more pronounced than fev and may be used as a more sensitive parameter for assessing the course of respiratory exacerbation. fibreoptic bronchoscopy was performed in children aged - years: with cf, with non-cf bronchiectasis (bx), with asthma, and control children without lower respiratory disease. endobronchial biopsies were taken and stained with haematoxylin and eosin. asm content, myocyte number and size were quantified using stereology. results: the volume fraction of asm in subepithelial tissue (median; iqr) was . ( . - . ) in controls. by comparison, it was . ( . - . , p< . ) in cf, . ( . - . , p< . ) in bx, and . ( . - . , p< . ) in asthma. myocyte number (cells per mm of reticular basement membrane) was ( - ) in controls, ( - , ns) in cf, ( - , ns) in bx, and ( - , p< . ) in asthma. myocyte size (um , mean; sd) was ( ) in controls, ( , p< . ) in cf, ( , p< . ) in bx, and ( , p< . ) in asthma. in cf, the volume fraction of asm in subepithelial tissue was related to myocyte number (r= . , p= . ), but not to myocyte size. conclusions: asm remodeling occurs in cf children. this, however, is not disease-specific and is also found in non-cf bronchiectasis and in asthma. both myocyte hypertrophy and hyperplasia contribute to increase in asm in cf. support: ers long-term fellowship and swiss national foundation grant to nr purpose: ct scans are increasingly being ordered on a routine basis in patients with cystic fibrosis. while prior studies have investigated their clinical utility in children, there is limited and conflicting information on their utility in adults. the purpose of this study was to determine the relationship between clinical disease severity as assessed by spirometry and ct findings as well as initial ct score ability to predict change in lung function in a nonselected group of adult cf patients. methods: a retrospective review of cf patients (mean age . +/- . , male %) who had undergone a routine chest ct was performed. the ct scans were scored using the brody method by a blinded reader with the degree of bronchiectasis, mucous plugging, peribronchial thickening, parenchymal disease, and air trapping assessed individually and a composite score generated. the ct metrics were first compared with spirometric test that were temporally related to the date of the ct scan, and then with future spirometric tests. results: for the cohort the patients had a wide range of severity of disease with the average fev % . +/- . , and the average fvc% was . +/- . . no significant correlation was found between initial fev % and total ct score ( . p value . ), or initial fvc% and total ct score (- . p value . ). for ct metrics and future spirometry, the average time from ct to pft's was days +/- with median of days. predictors for a significant decline in pft's were a decreasing bmi and male sex (p-values . and . respectively). further, for patients with a low fev % (< % at baseline), the fvc% change over time was associated with total ct score, male gender, and bmi (p-values . , . , and . respectively) conclusions: in adult cf patients, ct scan findings did not correlate with lung function. however in longitudinal analysis, bmi and gender had an effect on fvc change. for patients with low lung function, total ct score did have an association with change in fvc along with bmi and gender. pulmonary exacerbations (pes) are episodes of worsening of respiratory symptoms that commonly occur in patients with cystic fibrosis (cf). in clinical trials of new therapies in cf, pulmonary exacerbation is widely used as a primary or secondary endpoint. despite its importance, no single clearly agreed upon definition exists. some are based on patient reported symptoms while others require hospitalization or antibiotic prescription. use of different definitions makes it difficult to compare the results across studies and also to plan future studies. moreover, the rate of pes (proportion of patients with at least one exacerbation over a given time-period) is affected by baseline patient characteristics such as age, fev and medication use at the start of the clinical trial; factors which also contribute to the difficulty in comparing trials. using data from the cf therapeutics development network (tdn) data bank, we examined several pe definitions and baseline characteristics for their effect on pe rates. we find that pulmonary exacerbation rates vary substantially depending on both the pe definition and the baseline characteristics. for example, in one study, the proportion of subjects hospitalized and/or prescribed antibiotics during the month treatment period was %, while the proportion who met a symptoms based criteria for pe during the same period was %. we also examined the implications of varying control group exacerbation rates on sample size requirements for clinical trials and find that the control group pe rate has a large impact. for illustration, we assume a two group study with two-sided significance level . and % power. if the control group rate is %, then approximately subjects per group are required to detect a relative rate reduction of % (corresponding to a treatment group rate of %). however, if the control group rate is %, then only approximately subjects per group are required to detect the same relative rate reduction of % (corresponding to a treatment group rate of %). thus, a pe definition that selects for a greater pe rate in the control group will require fewer study subjects to detect the same relative rate reduction given that all other variables remain equal. sample size requirements can be further reduced by analyzing the number of events or the time to first event instead of the proportion of subjects who have at least one pe during a given time period. supported by cystic fibrosis foundation therapeutics, inc. (ram-sey y ), the national center for research resources (ncrr mo -rr ) and csl behring. briglia, h.; hilliard, j.; chmiel, j.f.; krenicky, j.; konstan, m.w. pediatrics, case western reserve university, cleveland, oh, usa cystic fibrosis (cf) is characterized by a vicious cycle of obstruction, infection and inflammation. the inflammatory response, which is profound and excessive relative to the burden of bacteria, is characterized by a massive neutrophil (pmn) influx. cf patients often experience intermittent declines in lung function associated with increases in both bacterial burden and pmn influx. these pulmonary exacerbations require treatment with antibiotics. there are substantive issues in the identification of pulmonary exacerbations and the assessment of therapies. a marker which indicates the inflammatory state of the lung would be useful to identify infective/inflammatory exacerbations, as opposed to worsening due to pulmonary vascular disease, or simply upper airway infection (cold), and might guide therapy for intensity and duration. g-csf and gro-α are cytokines produced in the lung by a variety of cells, including macrophages and epithelial cells. g-csf is involved in the proliferation, differentiation and activation of pmn precursors, while gro-α has known chemotactic and activating effects on pmns. transfer of these cytokines through the basolateral membrane into the bloodstream results in increased pmn production and activation in the bone marrow. the plasma concentrations of g-csf and gro-α might serve as potential biomarkers for a pulmonary exacerbation. in this pilot study we measured g-csf and gro-α in the plasma of healthy volunteers (n= ), clinically stable cf patients (n= ) and cf patients experiencing a pulmonary exacerbation, both before (n= ) and following (n= ) - days of antibiotic (abx) therapy. cytokines were measured using commercially available elisa kits (r&d systems, minneapolis, mn) . t-tests were performed on natural log transformed cytokine data (sigmastat v . , systat software inc., san jose, ca). ᭹ the lack of a significant difference between g-csf and gro-α plasma levels in healthy and clinically stable cf patients suggests that these cytokines are not indicators of chronic inflammation. ᭹ both cytokines are increased during pulmonary exacerbations in patients with cf and decrease to baseline values following antibiotic therapy, suggesting that these cytokines might serve as potential biomarkers for cf pulmonary exacerbations. ᭹ although not statistically significant, there was a trend towards an inverse correlation between both cytokines and fev during pulmonary exacerbations in this small study (data not shown). ᭹ further studies are warranted to increase our understanding of g-csf and gro-α as potential biomarkers of pulmonary exacerbation in cystic fibrosis. this work was supported in part by the cystic fibrosis foundation through an institutional research development grant. pletcher, s.d. ; koff, j.l. ; kleinhenz, m. . otolaryngology, university of california, san francisco, san francisco, ca, usa; . medicine, university of california, san francisco, san francisco, ca, usa introduction: while sinus disease is common in adult patients with cystic fibrosis (cf), the severity of sinus symptoms and relationship of sinus disease to other manifestations of cf are relatively unknown. objectives: to evaluate the severity of sinus symptoms in adult patients with cf and correlate these findings with other cf manifestations. methods: twenty-six consecutive adult patients with cystic fibrosis were surveyed during routine clinic visits for sinus specific and overall quality of life outcomes using both the sino-nasal outcome test (snot- ) and the cystic fibrosis questionaire (cfqr). analysis of snot- and cfqr scores were compared to %fev and sputum culture growth of pseudomonas for all patients. results: snot- scores ranged from . to . with a mean of . on a - point scale. for the purpose of data analysis, the patients were divided into two patient cohorts: group with minimal sinus symptoms (snot- < . ) and group with mild to moderate sinus symptoms (snot- > . ). the mean %fev for group was significantly higher than the mean %fev for group ( . % vs. . % respectively, p< . ). patients in group were more likely to have sputum culture growth of pseudomonas although this trend did not reach statistical significance ( % vs %, p= . ). cfqr scores differed significantly among the two groups with group patients reporting more disability in the physical, role, vitality, body, eating, health, respiratory, weight, and digestion domains but not in the emotion, social, and treatment domains. conclusions: this cohort of adult cf patients show minimal to moderate symptoms of sinonasal disability. those patients with mild to moderate sinonasal symptoms have decreased pulmonary function and decreased overall quality of life compared to cf patients with minimal sinus symptoms. , . internal medicine, university of iowa hospital and clinics, iowa city, ia, usa; . translational lung imaging program, university of iowa hospitals and clinics, iowa city, ia, usa; . pulmonary and critical care medicine, mount sinai medical center, miami beach, fl, usa mucociliary transport is an important component of airway host defense against inhaled particles and microbial pathogens. mucociliary clearance is hypothesized to be defective in cystic fibrosis allowing for airway colonization and infection. current methods for measuring mucociliary transport and clearance lack the ability to measure mucociliary transport in specific proximal and distal airway segments. using a -slice high resolution ct scanner (somatom , siemens), we are developing a method to track radiopaque particle movement in the swine airway. swine were induced with ketamine/acepromazine, intubated, and then anesthesia maintained with propofol infusions. animals spontaneously breathed humidified air with the endotracheal tube cuff deflated. radiopaque teflon/bismuth trioxide disks ( mm diameter, . mm thick) were instilled via a catheter into the airways. serial ct images ( . mm slice thickness and slice increment of . mm) were obtained every minutes for a total of minutes. airway tree segmentation was performed using pulmonary workstation . (vida diagnostics, iowa city, ia). this program allows for fully automated airway tree segmentation. baseline mucociliary transport rates ( -dimensional) were . ± . mm/min. following aerosol delivery of human sputum leukocyte elastase ( mu), mucociliary transport was markedly diminished ( . ± . mm/min) at minutes. thus far, we are able to measure mucociliary transport down to the rd generation airways. future studies include more distal deposition and measurement of particle movement. in summary, this novel method measures mucociliary transport in defined airways and will allow us to examine the effect of altered airway epithelial function on mucociliary transport. lung transplant. purpose: to design a ct scoring system to quantify structural abnormalities on ct scans from cf patients with severe advanced lung disease (sald) and to investigate the correlation between ct scores and survival. materials and methods: ct scans of cf patients screened for lung transplant between and were collected from transplant centres. all scans were scored with the brody ii scoring system. to design a new scoring system sensitive for the specific changes in sald, a panel of experts reviewed a random set of ct scans on eligible items to be used in a pilot analysis. the resulting sald scoring system consisted of four items: bulla/cysts; areas with consolidation/mucous; areas with hypo perfusion/air trapping and hyper/normal perfusion. in each ct slice ( mm spacing) and for each of the items the surface area of corresponding lung tissue involved was estimated on a - % scale. total surface area for the items and each slice added up to %. the final sald score was a mean volume estimate of abnormal and normal lung tissue. results: pilot analysis of a set of ct scans showed a a spectrum of abnormalities, ranging from predominantly bronchiectasis to predominantly mosaic perfusion. a moderate correlation was found between the sald components inflammation and hypo perfusion/ air trapping and brody score (p= . , r= . and p= . , r= . ). the inter-observer variability of both scoring systems was comparable. (ri ranging from . to . ) analysis of the complete cohort with the brody ii score showed a significant correlation with survival with a hazard ratio of . ( % ci of . to . ) for dying on the waiting list for every ten point increase in brody ii score. (p= . ). the brody component scores for bronchiectasis, airway wall thickening and parenchyma showed a similar correlation with hazard ratios ( % ci's) of respectively . ( . - . ), . ( . - . ) and . ( . - . ) for every ten point increase in score. (p= . , . and . ). the preliminary results with the brody score suggest that it is useful to include ct information in prediction models. it is likely that the predictive value of the ct can for survival can be further improved using the dedicated sald-ct score. scoring of the remaining scans according to the sald system is ongoing and will be completed shortly. snell, g. . physiotherapy, the alfred, melbourne, vic, australia; . allergy, immunology and respiratory medicine, the alfred, melbourne, vic, australia; . epidemiology and preventative medicine, monash university, melbourne, vic, australia; . radiology, the alfred, melbourne, vic, australia background despite the widespread use of airway clearance (ac) techniques to clear excessive secretions and improve lung function, little is known about their efficacy following lung transplant (lt). this study aimed to compare the effects of two ac strategies (proactive vs reactive) on a range of clinical outcomes following lt. methods a prospective randomized trial design with stratification for suppurative pre-lt disease was used. consecutive patients at month post lt were eligible for inclusion. subjects were excluded if medically unstable, ventilator dependent or had a contraindication to performing positive expiratory pressure (pep) therapy. patients performed ac using pep either twice daily (proactive strategy) or only in the presence chest infection (reactive strategy). lung function (fev and fvc), chest radiography (brasfield score), exercise capacity ( minute walk) and bronchoscopic airway characteristics (anastomotic healing, patency and secretions) were assessed at , and months post lt. patient adherence and satisfaction were measured. results of consecutive patients, ( in each group) were recruited and completed the study. both groups improved lung function (fev ± % to ± % p< . ; fvc ± % to ± % p< . ), brasfield scores ( . ± . to . ± . p< . ) and minute walk ( ± m to ± m p< . ) over the study period. no significant differences between groups for any outcome were found. the vast majority had fully healed, % patent anastomoses without secretions at months. there were no significant differences between airway characteristics and incidence of chest infection. adherence to both strategies was high ( % proactive, % reactive). conclusion in the absence of significant differences in outcomes, it is recommended that ac only be performed in the presence of chest infection based on greater treatment cost and treatment time required by a proactive strategy. further studies in those with reduced anastomotic patency and in those with recurrent chest infections later post-transplant are warranted. supported by an alfred trusts grant. munro, p.e. ; button, b. , ; bailey, m. ; holland, a. , ; snell, g. . physiotherapy, the alfred, prahran, vic, australia; . allergy, immunology and respiratory medicine, the alfred, melbourne, vic, australia; . epidemiology and preventative medicine, monash university, melbourne, vic, australia; . school of physiotherapy, la trobe university, melbourne, vic, australia background the optimal duration and structure of pulmonary rehabilitation (pr) for lung transplant (lt) recipients is not known. this study aimed to describe changes in functional outcomes in lt recipients who participated in a post lt pr program at the alfred, melbourne, australia. methods prospective, repeated measures design. functional exercise capacity (six minute walk test- mwt), lung function (fev , fvc) and quality of life (sf ) were assessed at , and months following lt. following discharge, all subjects attended a hour supervised outpatient exercise training class days per week until weeks post lt and education sessions. patients with post-operative complications were excluded. data were analysed using descriptive statistics and anova with repeated measures. results consecutive lt recipients from sept to mar were assessed for inclusion. ( % male) subjects, mean age ± yrs were recruited and completed the study. % had undergone bilateral lt. % had cystic fibrosis, % chronic obstructive pulmonary disease. significant improvements were demonstrated in mwt ( ± m to ± m, p< . ), fev ( ± % to ± %, p< . ), fvc ( ± % to ± %, p< . ) and all quality of life domains, p< . . the greatest changes in mwt and lung function were seen between and months. conclusion functional exercise capacity, lung function and quality of life improved significantly over the first months in lt recipients who participated in pr at our institution. these data will allow benchmarking with other centres and program structures. supported by an alfred trusts grant. does gender play a role in perception of cf quality of life domains before and after lung transplantation? the cf quality of life (cfq-r) for patients м years of age is a disease-specific instrument. previous studies using this instrument have reported that cf females have higher scores in weight and body perception than cf males. to determine the impact of lung transplantation on gender-based perception of quality of life, we reviewed the cfq-r on all cf patients м years of age and who underwent successful lung transplantation at our center. cfq-r was administered - months prior to transplant surgery and then months after lung transplantation while the patients were in the outpatient clinic setting. domains measured: physical, role, vitality, emotion, social, body image, eating, treatment burden, health status, weight, respiratory symptoms, and digestion. the cfq-r responses were computer-scored and domain scores were then generated (score - , =better). during the ⁄ year study period, cf patients underwent successful lung transplantation ( males: females; mean age . ± . years). prior to lung transplantation, female cf patients had lower physical domain scores but higher scores in health status perceptions than the male cf lung transplant candidates (see graph). all patients had marked improvement in all domains months after lung transplantation (see graph). after transplant surgery, cf males had higher scores than cf females in emotion and in eating domains. scores for body image domain were similar for both genders before and -months after lung transplantation. we conclude that although there were differences in physical and health domain perceptions prior to lung transplantation, both male and female pediatric cf patients had marked improvement in all domains after lung transplantation. however, female cf lung transplant recipients had lower emotion and eating domain scores after transplantation. we speculate that lower scores in these domains may reflect increased post-traumatic stress and depression in female lung transplant recipients. further study on cf quality of life in pediatric lung transplant recipients is on-going. background cf liver disease (cfld) with severe cirrhosis develops in - % of patients, usually within the first decades of life. most patients with cfld suffer from complications of portal hypertension but hepatocellular function usually remains intact for many years, even decades. patients with cfld referred for lung transplantation (tx) may be offered lung tx, lung-liver tx or be excluded from lung tx. however, variceal hemorrhage due to portal hypertension can be managed by banding, sclerotherapy or shunt procedures without liver tx. the aim of this study was to examine outcome of cfld patients who underwent lung tx and the subsequent progression of cfld, particularly with the use of potentially hepatotoxic immunosuppressive drugs. we conducted a retrospective cohort study to compare cf patients undergoing lung tx with and without cfld. b. cepacia-negative cf patients undergoing lung tx at toronto general hospital from - were eligible for inclusion. liver cirrhosis was defined by histology, esophageal varices on endoscopy or imaging evidence of splenomegaly. each patient with cfld was matched for age and year of tx with cf patients without cfld. demographic data, survival and liver function tests (lfts)(ast, alt, alp, inr, albumin, protein at week , month , , , , , , , , and post tx) were obtained by chart review. results of the groups were compared using unpaired t-test (parametric data) or mann-whitney u test (non-parametric data). results b. cepacia-negative cf patients underwent lung tx over this period, ( . %) having cfld with cirrhosis. data from the cfld patients vs. matched cf patients without cfld were analyzed. no significant difference was found between the groups in pre-tx age, gender, bmi, pi status, cfrd status, fev % predicted, -minute walk distance or lfts. in the cfld group albumin was lower week post-tx ( . + . g/l vs. . + . g/l, p< . ), alp was higher month post lung tx ( . vs. . , p< . ) and alt was higher months post-tx ( . + . u/l vs. . + . u/l, p< . ). there was no significant difference in the other blood tests. azathioprine was changed to mycophenolate mofetil in cfld patients due to liver test abnormalities. cfld patient developed hepatic encephalopathy and ascites years post lung tx and is being assessed for liver tx. no difference was seen in the number of episodes of acute rejection or post-tx survival ( . % vs. . % alive at years). discussion cfld patients undergoing lung tx did not have a worse prognosis than patients without cfld. transient elevation of lfts were seen in the immediate post-tx period but settled either spontaneously or after discontinuation of azathioprine. we conclude that selected patients with cirrhosis due to cfld may be safely offered lung tx without concomitant liver tx. infectious mononucleosis, commonly called mononucleosis, or "mono," is an illness caused by the epstein-barr virus. mononucleosis has been nicknamed the "kissing disease" because the epstein-barr virus commonly is transmitted in saliva during kissing. however, sneezes and coughs also can transmit the virus occasionally. the epstein-barr virus (ebv) permanently infects more than % of the people on earth, but it causes symptomatic mononucleosis only in a small number. in developed nations, such as the united states, mononucleosis most often occurs between the ages of and . unfortunately ebv can not only infect, but can also transform, b cells after transplant and may lead to lymphoma with a resulting ~ % mortality rate. the source of ebv is presumed to be passenger lymphocytes in the donor tissue and those at greatest risk are patients who are ebv naive before transplant. since most cf patients with endstage lung disease in the developed world are now being referred for transplant given the success of this intervention, we reviewed our entire lung transplant database to determine the nature of ebv infection and the likelihood of developing lymphoma following lung transplantation. of first-time lung transplants, ( %) had cf. ebv serology was not available in ( cf, others) before transplant. of ( %) cf patients were ebv seronegative before transplant as compared to of ( %) patients with other end stage lung diseases (composed mostly of copd and ipf) in the control population (chi square p < . ). the cf ebv seronegative cohort was (sd ) years old on average, an age when most seroconversion has already occurred in our society. of the cf ebv seronegatives, survived beyond months and were thus at risk for post transplant lymphoma. of these , developed lymphoma (incidence = %) in comparison to of the ebv seronegative controls (incidence = %, p = ns). all lymphomas were stage iv at presentation and the majority ( of ) arose in the first post transplant year. the cf ebv seronegatives who developed lymphoma did not differ from those who did not with regard to levels of immunosuppression (cyclosporine, methylprednisolone or prednisone) or doses of antiviral therapy (i.e., ganciclovir which is active against both cmv and ebv). in addition, the cessation of anti-lymphocyte antibody induction therapy in the summer of , somewhat surprisingly, had no impact on the development of lymphoma. two cases of lymphoma developed in cf ebv seropositives (incidence = . %) and no cases of lymphoma developed in the control ebv seropositives (p = ns). thus ebv seronegativity raises the lymphoma risk fold in cf patients (p < . ). two of the ( %) cases of lymphoma resulted in death (one cf and one other) and one cf patient probably died of complications related to chemotherapy even though he was tumor free at that time. lymphoma outcome was not stage or clonality dependent. in conclusion, cf lung transplant recipients are at an increased risk for lymphoma largely due to their lack of exposure to ebv before transplant. kissing more before transplant may lower the risk of lymphoma afterward. university hospital uzb, brussels, belgium; . chest medicine, erasme university hospital, brussels, belgium; . dermatology, erasme university hospital, brussels, belgium; . gynaecology, erasme university hospital, brussels, belgium; . gastroenterology, erasme university hospital, brussels, belgium; . pathology, erasme university hospital, brussels, belgium hpv infection is an underestimated phenomenon in organ transplantation (tx) recipients, being unable during immunosuppression (is) to mount an adequate anti-viral immune response and risking genital/anal warts (condylomata acuminata) as well as pre-cancerous/cancerous lesions. we retrospectively assessed the incidence/treatment of genital/anal hpv-associated lesions in cystic fibrosis (cf) lung tx recipients. the files of all tx patients transplanted in the ulb center between and / ( men/ women, median age ± . , range - yrs) were reviewed. maintenance is consisted in chronic triple therapy combining a calcineurin inhibitor, a cell cycle inhibitor and corticosteroids. median survival was . ± . months (range - ). ten of sexually active patients ( %) who survived ≥ months post-tx developed hpv-associated genital/anal lesions: men ( - yrs) and women ( - yrs). genital/anal hpv-pcr proven condylomata were diagnosed between - months in the men and between - months post-tx in the women. all underwent local treatment (cryotherapy ± laser ± topical imiquimod), / patients underwent multiple treatments under general anesthesia. recurrence of the condylomata was high. one male patient presented high-grade anal dysplasia and women moderate to high-grade cervical dysplasia; underwent conization and one complete hysterectomy after conizations. one of the females presented both condylomata and a high grade cervical dysplasia. these retrospective data in a cf population indicate that ) hpv infection may cause significant morbidity in young subjects with chronic disease needing organ tx. it may even compromise life expectancy after tx. ) data on the effectiveness of treatment strategies -including topical immunomodulators -have to be collected ) the potentially protective effects of the now available hpv vaccination in females and males? with chronic disease at risk for future organ tx, when administrated before sexual activity and before tx, should be rapidly evaluated in a multi-center effort. ballmann, m. ; pfister, e. ; schlueter, k. ; becker, t. ; melter, m. ; junge, s. . pediatric pulmonology and neonatology, medical school, hannover, germany; . pediatric nephrology and hepatology, medical school, hannover, germany; . visceral and transplant surgury, medical school, hannover, germany liver disease is an important comortality and comorbidity in cf patients even in the pediatric age group. liver transplantation (ltx) is an accepted option in end stage liver disease. nevertheless the clinical outcome in cf patients is still under discussion. here we reported the clinical outcome months after isolated liver tx in pediatric cf-patients.since n= ltx were performed in children (male n= , female n= ; age (mean±sd) , ± years) . immunosuppressive therapy was done with ciclosporin ± basilixumab and steroid. we followed pulmonary function, cn-score, nutritional status (bmi z-score) and body com-position, airway cultures and igg.results:all were still alive months after ltx. before ltx all patients had a mild to moderate pulmonary disease: fvc(%pred) , ± ( , %- ), fev (%pred) , ± , ( - ), mef (%pred) , ± , ( - ) , cn-score ± , points. nutritional status: bmi(mean) , ± , % (range: , %- , %, z-score: , ± , ), upper arm circumference(cm)(mean±sd) , ± , , triceps skin fat fold(cm) , ± , . body composition: , ± , % of bodyweight were fat. one year after ltx all patients were still in a stable pulmonary situation: fvc(%pred) , ± (range: , %- %), fev (%pred) , ± , (range: %- %), mef (%pred) , ± , (range: , %- %), cn-score , ± , points. there weren't any significant changes in the airway microbiology under immunosuppressive therapy (steroid, ciclosporin±basiliximab), the serum igg levels declined significant from , ± , g/l to , ± , /l(p< . ). the growth over the year was , ± cm and the increase of weight come to , ± , kg, while the bmi didn't increase in this first year after transplantation. certainly we found an increase of the body fat mass (to , ± , % of body weight), of the upper arm circumference (to ± , cm) und of the triceps skin fat fold (to , ± , cm) .conclusion:liver transplantation is an effective therapy even in children with cf related liver disease and stabilise pulmonary function and improves nutritional status in patients with cf and mild or moderate pulmonary involvement before ltx. (neglia jp et al., n engl j med ; : - ) , and colon cancer has been described in young adults with cf (chaun h et al., can j gastroenterol ; : - ) . three of lung transplant (ltx) recipients from our center developed colon cancer after successful bilateral lung transplant for end-stage lung disease. data from these individuals are given in table below . an additional recipients with cystic fibrosis were screened via colonoscopy. colonic polyps were detected in and included lesions up to cm in diameter. five years after a colonoscopy that had shown a mm polyp, one patient underwent a nd colonoscopy that revealed multiple polyps, including a cm diameter sessile polyp in the sigmoid colon. colonic malignancies appear to arise from mucosal polyps, and screening via colonoscopy can detect and remove premalignant lesions. lung transplant recipients have an increased risk of gi malignancies and tumor surveillance is impaired by post-transplant immunosuppression, colonoscopic screening should be considered for transplant recipients with cystic fibrosis. additionally, colonic malignancy should be considered as a potential cause of unexplained gastrointestinal symptoms such as constipation. nossent, g. ; kastelijn, e. ; teding van berkhout, f. ; zanen, p. ; van den bosch, j. ; van de graaf, e. . respiratory dis, university medical centre, utrecht, netherlands; . respiratory diseases, st. antonius hospital, nieuwegein, netherlands objective: in the netherlands lung allocation is based on waiting time. to prioritize patients with a poor prognosis and to reduce waiting list mortality the high urgency (hu) status was introduced in . to be considered for hu status there has to be a limited life expectancy on clinical judgement and all three transplantation centers have to audit this decision. the aim of this study is to analyze the potential effect of las on patients with cystic fibrosis (cf) who became hu or died on the waiting list. it would be expected that patients who became hu had a higher las. methods: from till in all cf patients placed on the hu waiting list, the median las ( th, th) was calculated at the moment of listing and at the moment the patients became hu. also for the cf patients who died on the waiting list the mean las (sd) was calculated at the moment of listing. results: from the patients on the waiting list patients had cf. of these patient were placed on hu list and died on the waiting list. in the cf patients that became hu the median las on the moment of listing was . ( . ; . ) . at the moment they were placed on the hu waiting list the median las was . ( . ; . )(p= . ) . the median las of the patients who died on the waitinglist was . ( . ) . this las did not differ significantly from the las of listing of the patients that became hu (p= . ). conclusion: the las does not detect the deterioration in patients with cf as diagnosed by clinical judgement. this may be due to the exclusion of specific prognostic factors of cf in the calculation of the las. besides that, the las does not differentiate cf patients on the waiting list with a high chance of mortality. introduction: lutx recipients have one of the highest rates of ia in solid organ transplantation causing high morbidity and mortality rates. nowadays antifungal prophylaxis is extensively used but clinical trials are rare. methods: all patients who received lutx in our hospital were included in this retrospective study. aspergillus airway colonization was defined as aspergillus cultured twice from airway specimens in the absence of ia or tracheobronchitis (tb). definition of ia and tb was according to literature. after the death of cf-patients due to ia, a prophylaxis protocol was introduced containing avoidance of use of the cell saver during the operation in pretx colonized patients to avoid hematological fungal spreading and targeted prophylaxis with itraconazole or voriconazole in all pre-and posttx colonized patients during months and inhaled amphotericin during hospital admission. results: a total of lutx were performed (in patients) ( % cf patients) from - - until - - . patients ( . %) were colonized with aspergillus pretx and patients posttx. only patients were colonized before and after tx. before introduction of the protocol patients ( / = . %) ( % cf patients) developed an aspergillus infection: ( . %) patients developed a. fumigatus tb and ( . %) patient suffered from ia: cerebral a. fumigatus infection (proven ia). two patients, both cf patients, ( / = %) died due toaspergillus infection (cerebral and tb).after introduction of the protocol only one a. fumigatus tb occurred ( / = . %) but no invasive fungal infection. conclusions: the rate of fungal infections in our lutx patients was comparable with data from literature. since introduction of azole prophylaxis protocol and avoidance of use of cellsaver no ia occurred. fischer, a. ; müllinger, b. ; arendt, t. ; bernhardt, t. ; hannah, k. ; scheuch, g. . activaero gmbh, gemuenden, germany; . biological products, bayer healthcare, leverkusen, germany; . activaero gmbh, gauting, germany; . talecris biotherapeutics, research triangle park, nc, usa background patient compliance during a study is an important factor in view of assessing the clinical effect of a treatment. this is especially true when patients administer the drug at home. usually, patient records, counting of returned doses or mechanical counters are used to track compliance, which may be biased by the study subjects. this report is about an aerosol study in cf which used a device for controlled breathing (akita® inhalation system, activaero, germany). the device works with a patient-individual smart card that records every single breath during a treatment including a date/time stamp in an encrypted manner. each patient's inhalation protocol can be displayed by loading the smart card into the proprietary "compliance manager" software. methods for this report, the data set of a recent controlled study (griese et al., ) was analyzed. cf-patients were instructed to inhale with the akita inhalation system at home for days. after the study, the smart cards were returned to activaero, and the inhalation records on the chip were analysed. we analysed the compliance of patients who participated at least days ( out of patients, others deemed to be drop-outs . most of the patients showed a dsc which is lower than the tdc, indicating that they had missing treatment days, which were compensated by additional inhalations on other days. we found patients with a dsc more than % lower than their tdc (max difference: . %). conclusion this aerosol study with home treatment demonstrated a high compliance rate for tdc and dsc. this information is more reliable for compliance than study participation in days alone. for future studies it is recommended to define in the protocol not only a minimum of study participation in days but also a minimum in tdc and dsc. in addition the compliance thresholds may be defined with regard to the drug's pharmacokinetic profile. in general, the inhalation record of the akita smart card provides an unbiased view of the inhalations treatments during a study especially when the subjects perform the inhalations at home. compliance calculations as shown above may be performed and linked to other outcomes of a study for validation. compliance data like these may also be used routinely by the treating physician in order to guide and supervise his patients. introduction: adaptive aerosol delivery (the i-neb ® aad ® system) has been developed to deliver precise, reproducible doses of aerosol into inhalation. i-neb utilizes a medication chamber, which incorporates a metering chamber to deliver a preset (metered) dose of aerosol. inhalation of hypertonic ( %) saline has been shown to aid mucociliary clearance in patients with cystic fibrosis. [ ] we performed an in vitro test to determine the emitted, delivered and residual doses of hypertonic saline from the i-neb aad system with . ml metering chamber, and a conventional jet nebulizer (lc plus ® ) with a ml fill. the results were then entered into a model to predict the likely lung dose from the in vitro tests, in order to determine the equivalent i-neb dose to the ml conventional jet nebulizer fill. methods:an i-neb device configured to operate at power level (of ) was fitted with a metering chamber designed to deliver a preset dose of . ml. the i-neb was weighed, loaded with ml of % saline and run on the cen simulated breathing pattern. this process was repeated using an lc plus nebulizer loaded with ml of % saline. aerosol was collected on a filter placed between a harvard pump and the device. emitted dose and residual dose were determined by gravimetric output, and delivered dose was determined by ion analysis. all tests were conducted in triplicate. results: as shown in table , the emitted dose approximated the delivered dose for i-neb, whereas for lc plus the emitted dose was approximately twice the delivered dose, due to the wastage of aerosol upon simulated exhalation. as i-neb has a delivered dose of mg, and i-neb has been shown to deposit . % of the dose in the lung, [ ] this would result in a lung dose of mg. the pari lc plus has a higher delivered dose, but the overall lung deposition is only % of the fill volume, i.e. mg. [ ] conclusion: the results of this test suggest that two treatments would be required in order to deliver an equivalent lung dose of hypertonic saline from an i-neb device fitted with a . ml metering chamber, compared with a ml fill in an lc plus jet nebulizer. references introduction: inhaled colistimethate sodium can be used for the eradication of pseudomonas aeruginosa in patients with cystic fibrosis. the tonicity of colistimethate sodium inhalation solution has been linked to the occurrence of bronchospasm in some patients. it has been suggested that this bronchospasm can be avoided by using an isotonic solution of colistimethate sodium. [ ] this can be complicated when using conventional jet nebulizers, since evaporation during nebulization tends to change the tonicity of the solution remaining in the nebulizer. however, because very little evaporation occurs within the i-neb medication chamber during nebulization, the tonicity of the loaded solution at the beginning and end of nebulization remains almost constant. aim: we investigated the effects of various saline concentrations upon the tonicity of two concentrations of colistimethate sodium solution ( . miu/ml and miu/ml). methods: diluent containing sodium chloride concentrations of: % (water for injection), . %, . %, . %, and . % (normal saline) were used to reconstitute vials of colistimethate sodium (promixin ® , profile pharma, uk). each vial was reconstituted with either or ml of diluent to make up miu/ml or . miu/ml colistimethate sodium solution, respectively. the tonicity of each diluent concentration/colistimethate sodium concentration combination was made up and tested in triplicate using an advanced micro osmometer (advanced instruments, norwood, usa). the results were plotted on a graph along with a line of best fit, to determine the diluent concentration that gave a mean tonicity closest to that of an isotonic solution. results: tonicity increased linearly with increasing concentrations of saline solution for both miu/ml and . miu/ml concentrations. the tonicity of the miu/ml solution was approximately mosm higher than the . miu/ml solution across the range of saline concentrations tested. the best fit line passed through the isotonic solution point ( mosm) closest to the . % saline concentration point. conclusions: the diluent that produces the closest approximation of isotonicity for the mean of . miu/ml and miu/ml colistimethate sodium concentrations was . % saline. references . transave, inc., monmouth junction, nj, usa; . activaero, gmbh, gauting, germany arikace™ is an inhalation formulation of a liposomal amikacin suspension that is designed to treat chronic pseudomonas aeruginosa infections in cystic fibrosis patients. liposome encapsulation of amikacin reduces nonspecific binding of this cationic aminoglycoside drug to the negatively charged mucus and biofilm surfaces and allows penetration and delivery of packets of highly concentrated drug to the otherwise protected bacteria. as nebulized and delivered to the lungs, arikace™ comprises % liposomal amikacin and % 'free' amikacin that is not entrapped by liposomes; free drug is produced by liposome leakage during nebulization. this profile provides an initial high peak concentration of amikacin followed by a sustained level as drug leaks from the liposomes. nebulized arikace™ with this profile was evaluated previously in human clinical studies using the lc star® nebulizer. although the . µm liposomes of arikace™ are efficiently loaded with drug, the expected effective dose in humans will require relatively large volumes of drug solution to be administered. to minimize treatment time and improve patient convenience we sought to find a nebulizer that would efficiently deposit drug and produce aerosol at a high output rate, while still producing the same level of free drug during nebulization. methods: nebulization of liposomal amikacin was compared using several nebulizers, including jet nebulizers: lc star® and lc sprint® (both from pari), and electronic mesh nebulizers: microair® ne u v (omron), aeronebgo® (nektar), and eflow® (pari). additionally, meshes of different pore sizes were examined for the microair® and eflow® devices. output rates were measured by gravimetric differences. droplet size distribution was assessed by a laser light scattering method and by cascade impaction; mass median aerodynamic diameters (mmad), geometric standard deviation (gsd), and fine particle fraction (fpf) (i.e., % droplet mass < µm). amikacin association with the liposomes was measured using a centrifugation-filtration assay. results: in terms of output rate, the order of performance was eflow® (~ . g/min) > lc sprint® > lc star® > aeronebgo® > microair®. while the aeronebgo® and microair® mesh nebulizers performed well with saline, their performance declined dramatically when nebulizing the liposomal amikacin solution, and, in fact, the microair device clogged. in additional studies with the eflow® where different meshes were compared, the l mesh was selected as it provided ideal aerosol properties (mmad = . µm, gsd = . , fpf > %) with a ratio of entrapped to free amikacin of approximately %/ %. importantly, from in vitro breath simulation studies it is estimated that about twice of the nominal drug dose is expected to deposit in the lungs compared to the lc star®. conclusions: for nebulization of liposomal amikacin, the eflow® configured with a l mesh provided optimum aerosol characteristics with a high output rate and ideal size for distribution throughout the lung. the eflow® reduces dose administration time not only because of its faster output rate but also because its higher deposition factor greatly reduces the amount of drug needed. zeman, k.l.; bennett, w.d. cemalb, university of north carolina, chapel hill, nc, usa many inhaled, therapeutic drugs have their site of action in the conducting airways, but may deposit elsewhere in the respiratory tract, resulting in unwanted side effects and waste. the quantity and location of particle deposition in the respiratory tract depends on both the particles' aerodynamic size and breathing pattern. in subjects with normal lung function, we evaluated three methodologies for their ability to deliver radiolabeled particles ( mtc-labeled sulfur colloid) to the conducting airways: . inhalation of µm mmad particles (devilbiss jet nebulizer) using a resting tidal volume and flow (mean . l and . lps); . inhalation from the same nebulizer using a very large inspiratory capacity volume and higher flow rate (mean . l and . lps); and . inhalation of larger . µm mmad (heart jet nebulizer) particles with a large tidal volume and very low flow rate (mean . l and . lps). gamma scintigraphy gave an estimate of % conducting airway deposition (%cad) by multiplying the percent of activity in the lungs immediately post-deposition relative to the total deposition (i.e. lung+mouth +esophagus+stomach) times the percent of activity cleared from the lungs over hours. %cad was %, %, and % for the three methods, respectively, significantly greater for the large particle, very slow inhalation technique when compared to the other two methods. the amount deposited in the mouth and larynx was %, and %. in addition, we evaluated protocols and above in a preliminary group of cf patients. for protocol , mean inhaled volume and rate was . l and . lps. mean inhaled volume and flow were . l and . lps for protocol . the %cad for the two protocols and were % and %, respectively. the amount deposited in the mouth and larynx was % and %. higher therapeutic value of a medication delivered to the airways where the primary defect is associated with cf disease, and with fewer losses to the extrathoracic airways, can be obtained by using a "very large particle/slow inhaled flow" routine when compared to normal or higher flow breathing associated with typical nebulizers. supported by cf foundation. positive effects of physical conditioning in cystic fibrosis (cf) have been reported for quality of life (qol) in addition to effectis on fitness and lung functions. the objective of this study was to identify the effects of different modes of training on qol in cf. cf-patients (age - yr., fev > %pred.) were randomized into groups: germany: unsupervised homebased training, hours per week, free choice of training mode and means (ut, n= ) and controls (con , n= ); switzerland: aerobic training (at, n= ) or weight training (wt, n= ) in a fitness center, x min per week, or controls (con , n= ). subjects in the training groups were asked to train for months. at study entry and after and months, qol was assessed by questionnaire and maximal physical working capacity (pwc, %pred) was determined during cycle ergometry (godfrey protocol). changes from baseline values were calculated and anova for repeated measures was used to test for differences among groups. weight training resulted in a significant decrease in qol (generic dimension) at and months compared with all other training modes including controls. there was no significant difference between at or ut and controls in qol during the program. however, there was a positive association between the change in pwc and the change in qol (r= . , p< . ) in the entire sample. in conclusion, physical training may not always result in an increase in ql in cf. possibly, the decrease in qol in the wt group may have resulted from tiredness induced by the training. thus, a different outcome might have been found with less intense training. positive effects may be achieved by improving aerobic fitness. supported introduction: exercise and exercise training programs are felt to be important to improve aerobic exercise capacity, muscle strength, lung function, and quality of life in cf-patients. recent studies in children and adults with cf have demonstrated an increase of motor performance after exercise training. less is known about trainability of motor performance in younger children with cf. the aim of this study was to assess the effects of a training program on motor performance in preschool children with cf. methods: preschool children with cf ( female and male patients, mean age . yrs; range - yrs, fev %pred. , ± . rage from . - . ) participated. at baseline (t ) and at the end of the training program (t ) the "motorik test (mot)" was used to assess motor performance and to evaluate training effects. components of motor performance tested with the mot are balance, agility and flexibility, strength, coordination, fine motor skills, reactivity, accuracy of movement). lung function in patients with cf was measured by spirometric techniques. the duration of training was - weeks ( times/week, minutes per session) with different kinds of sport activities. results: at t , strength and balance had increased significantly (p< . ). no other components of the mot increased, but the increases in strength and balance were enough to cause a significant (p< . ) increase overall motor performance, as indicated by an increase of . % in mq. the mq is the sum of all test-items of the mot. no correlation was found between lung function and motor performance. discussion: to our knowledge this was the first study that examined the effects of an exercise training program on motor performance in preschool children with cf. main findings of this study were that a training program of - week of duration improved some aspects of motor performance. the improvements may be explained by different kinds of sports activities during training when compared with activities before training. classification of motor performance of the tested children was normal (mq = . ± . ). this could be the reason that only some aspects of motor performance increased. parents of the children were asked about habitual physical activity at home. all children were physically active and some of the children participated in organized sports. a recent study showed that with an increase in age lower motor performance in children and adolescent with cf was seen compared to healthy children. we speculate that time spent in physical activity in preschool children with cf is comparable to healthy children. conclusion: an exercise program offered to preschool-children with cf may lead to an improvement in motor performance. pre-school children with cf have normal motor performance. to prevent a decline of motor performance, exercise programs should be available for preschool children. gruber, w. ; braumann, k.m. ; orenstein, d.m. ; huels, g. . dept. for sports and exercise, clinic sattelduene for children and adolescent, nebel/amrum, germany; . institute for sport and exercise medicine, university of hamburg, hamburg, germany; . school of medicine, university of pittsburgh, pittsburgh, pa, usa; . dept. of medicine, clinic sattelduene for children and adolescent, nebel/amrum, germany introduction: a decline in physical activity with age is one of the main problems in patients with cf. as a consequence, lower exercise capacity and a more rapid decline in lung function may be seen in those patients who are physical inactive. furthermore, motor performance is lower in children and adolescents with cf compared to healthy children of the same age. little attention has been paid to younger children with cf and motor performance. the aim of the study was to compare motor performance in preschool-children with cf with healthy children of the same age. methods: children with cf (mean age . yrs; range - yrs) and healthy children (mean age . yrs; range - yrs) participated. the "motorik test" (mot) was used to assess motor performance. the mot consisted of test-items which evaluated seven different components of motor performance (balance, agility and flexibility, strength, coordination, fine motor skills, reactivity, accuracy of movement). lung function in patients with cf was measured by spirometric techniques results: results of the mot showed no difference either for single testitems or for parameter "motorische quotient (mq)" (p> . ). this parameter classified motor performance of all aspects tested with the mot. classification of children with cf and of healthy children was normal. mean value for parameter mq (cf: . ± . vs. healthy . ± . ) was slight higher in patients with cf than in healthy children (p> . ). differences between healthy and cf for different components of motor performance were not significant (p< . ) discussion: the mot is a reliable and valid test method in germany to determine motor performance in younger children. to our knowledge this was the first study to examine motor performance in preschool-children with cf. furthermore, we compared patients with cf and healthy children. we observed no differences between groups for motor performance. in older children with cf, lower motor performance has previously been found compared to healthy children of the same age. we do not have any information about the level of habitual activity in tested healthy children. we therefore speculate that time spent in physical activity in cf is similar to that of healthy children at this age. a recent study showed a decline in physical activity in cf when age and problems during physical activity increased. we think that motor performance is normal in children with cf up to an age of yrs. when age increased, time spent in physical activity decreases (treatment, school) and therefore motor performance decreases. conclusion: motor performance is normal in preschool-children with cf compared to healthy children. to prevent a decline or to maintain motor performance it is recommended to motivate cf-children from early infancy to participate in sports activities or other physical activities to maintain or to increase their physical fitness. . respiratory medicine, the hospital for sick children, toronto, on, canada; . respirology, st. michael's hospital, toronto, on, canada; . respiratory medicine, montreal children's hospital, montreal, qc, canada purpose: the habitual activity estimation scale (haes) questionnaire has been shown to be a feasible tool to measure physical activity however the reliability has yet to be determined in cf populations. we therefore assessed the reliability and validity of the haes questionnaire in paediatric and adult cystic fibrosis (cf) populations. methods: fourteen ( male, female) patients aged . + . yrs with cystic fibrosis from the cf clinics at the hospital for sick children and st. michael's hospital participated in this study. participants were clinically stable at the time of the study (fev > % predicted) and participating in their 'habitual' physical activity. to assess test-retest reliability, patients completed each of the haes (schneiderman-walker et al., j pediatr. ; ( ): - .), and a validated -day activity diary (bratteby et al., eur j clin nutr. ; ( ) : - ), and wore an actigraphtm tri-axial accelerometer for two consecutive weeks. validity was assessed by comparing the activity results of each of the three instruments over a single week time period. results: intra-class correlation coefficient estimates of reliability for the haes, diary and accelerometer were . (p< . ), . (p< . ), . (p< . ), respectively. validity was estimated by comparing the results from each instrument using a one-way anova with block design. there were no overall differences among the participants' activity results as estimated by the haes, diary and accelerometer. furthermore, there were no significant differences between activity measures among the instruments when broken into morning, afternoon, or evening periods, or between measures from weekday or weekend days. while there were no significant differences among the instruments when recording 'very active' intensity levels, they did detect differences during the inactive / somewhat inactive (p = . ) and somewhat active (p = . ) intensity levels. conclusion: the findings of this study suggest that the haes questionnaire is a reliable and valid instrument that can be used to assess activity levels in patients with cystic fibrosis. in agreement with others, our patients found it easiest to recall and record accurately those periods in which they are participating in vigorous activity. acknowledgement: this research was supported by the canadian cystic fibrosis foundation. . respiratory medicine, hospital for sick children, toronto, on, canada; . respiratory medicine, montreal children's hospital, montreal, qc, canada; . respirology, st. michael's hospital, toronto, on, canada objective: previous work showed a relationship between habitual physical activity (hpa) and decline of lung function (fev ) in cf females (sneiderman-walker, j peds : , ) . this study only included a small number of patients and the follow-up period was limited to months. the purpose of this study was to evaluate daily activity levels and decline in fev in a larger cohort over a longer period of time. methods: a total of (n= female) patients with cf (age - yrs) were studied over a six year period at the hospital for sick children, st. michael's hospital and montreal children's hospital. at each quarterly cf clinic visit, patients performed pulmonary function tests to determine fev and completed the habitual activity estimation scale (haes). weekday total activity (wdta), expressed as hours/day (h/day), was calculated from the sum of 'very active' (wdva) and 'somewhat active' (wdsa) categories as previously described. a repeated measures linear regression was performed to evaluate the relationship of fev and hpa over time. results: at baseline, mean fev was % predicted (range - % pred). mean baseline wdta was . h/d (wdva = . h/d and wdsa = . h/d) similar to that previously reported. subjects were divided into high and low activity groups based on the group median wdta. those in the low activity group had a significantly (p= . ) steeper rate of decline (- . % predicted per year) compared to those in the high activity group (- . % predicted per year) . unlike in the in the previous study this difference was observed in males as well as females. conclusions: six year follow-up of this patient cohort has shown that higher activity levels are clearly associated with a slower rate of decline of fev . we are currently evaluating whether strategies aimed at increasing physical activity will have an impact on lung function decline in cf patients. background: knowledge of exercise principles, benefits, and techniques has not been assessed in children with cystic fibrosis (cf) or their parents. regular exercise is a recommended cf treatment regimen that is often not followed, but one which might improve cardiovascular fitness, lung function, sputum clearance, and health-related quality of life. further, aerobic fitness, measured as peak oxygen uptake (vo ), is positively related to survival in cf. construction of a reliable and valid measure of exercise knowledge is necessary to assess level of knowledge and study its relationship to exercise behavior. the aim of this study was to develop and test a measure of exercise knowledge for pediatric patients with cf and their parents. methods: we constructed a -item exercise knowledge test (ekt) and then examined its psychometric properties. a comprehensive review of the exercise literature guided the formulation of objectives and development of items. to establish content validity, a panel of experts (cf physician, two exercise physiologists, and a clinical cf nurse) evaluated the objectives and test items for relevance, clarity, and accuracy. after feedback from the panel, we revised the ekt through an iterative process. in consultation with a reading level expert, we wrote the ekt items so that the test could be administered to children and adults. a measurement and evaluation expert also reviewed the test, and additional revisions were made. during routine clinic visits, the ekt was administered to subjects with cf and parents. the cf subjects included boys and girls, age to years, fev range: - % predicted. the parents had an average age of years and most (n= ) were female. results: the ekt includes items, true-false and multiple choice. it is self-administered, yields a single summed score, and has a flesch-kincaid rd grade reading level. the means were . + . ( % correct) and . + . ( % correct) for the cf subjects and parents, respectively; % of the parents scored between and . internal consistency reliability estimates were . for the children and . for the parents, reflecting both the multidimensionality of the material and the lack of variability in the parent scores. item difficulty ranged from . to . for the cf subjects and from . to . for the parents. in both groups, the discrimination values were positive for all items with difficulty values less than . . conclusions: we were able to develop a measure of exercise knowledge with an acceptable reading level for children with cf and their parents. this sample of cf subjects and parents showed substantial exercise knowledge. the ekt can be a useful tool to identify gaps in exercise knowledge in cf patients and their parents, develop educational programs to increase knowledge, and examine how knowledge relates to exercise behavior in children with cf. supported by cystic fibrosis foundation therapeutics, inc. purpose: although physical activity is routinely recommended for adolescents with cystic fibrosis, past research suggests that many patients do not regularly participate in physical activity. there may be a variety of reasons, both related to disease and otherwise, for this inconsistent participation. in this study, qualitative methods were used to describe factors that facilitated or served as barriers to physical activity in adolescents with cf and compared these factors to those in a healthy peer group. methods: ten subjects with cf and ten subjects without cf aged - volunteered for this study. each subject participated in two, open-ended semi-structured interviews with - weeks between each interview. subjects were questioned about their current and past physical activity as well as the benefits and barriers to participation. the adolescents with cf were also asked about their perceptions of physical activity on their own health and other adolescents with cf. interviews were transcribed and coded by three investigator groups. analysis was conducted using the grounded theory approach at two points in the study, following each interview and again at the completion of all interviews. at the first point, individual interviews were coded using line-by-line coding, and similar information was grouped into categories. in the second interview, categories were confirmed and subjects were also asked to elaborate or clarify points from the first interview. interviews were continued until themes reached a saturation point. upon completion of all interviews, data for each group were organized by question. data categories appearing throughout the interviews became major themes describing facilitators and barriers to exercise. the major themes were identified by the three researcher groups and then through collaborative group analysis. results: the central theme for both groups was perceived importance of physical activity for health benefits. for the cf group, facilitators and barriers were both psychological and physical. physical benefits were categorized as either relating to their general health (such as feeling energized) or as benefiting their disease (such as improving breathing). in the non-cf group, facilitators included social factors in addition to general health-related and psychological benefits. in the non-cf group, barriers were categorized as either internal or external, with the internal barriers being similar to the cf group (physical and psychological), however, the non-cf participants also indicated external barriers (such as time) not articulated by the cf group. conclusions: in general, we found very similar facilitators and barriers to physical activity in adolescents with cf and those without. however, the adolescents with cf strongly indicated that physical activity would have positive effects on their physical health. further work is needed to determine ways to accentuate facilitators and remove barriers to improve physical activity levels in adolescents with cf. this project was supported by a research grant from the cardiovascular & pulmonary section, apta. lloyd, e. ; dodd, m. . paediatric cystic fibrosis service, central manchester and manchester children's university hospital, manchester, united kingdom; . adult cystic fibrosis centre, wythenshawe hospital, university hospital of south manchester nhs foundation trust, manchester, united kingdom background: training in the skills of communication and self care are considered important components of transitional care . physiotherapists work to involve families and young people to encourage this independence. the cf manager training programme (cfmtp) is a recognised tool for measuring age appropriate tasks in cf care. aim: the aim of the study was to survey the current levels of autonomy in all patients with cystic fibrosis in the north west of england from birth to years to provide a baseline measure for encouraging autonomy in preparation for transition. method: three areas of the cfmtp were selected for the survey: physiotherapy (p), inhalation therapy ( it) and communication (c) for the age groups: - . , . - , - , - , - , - , - , - , - ,and + years. physiotherapists from all the north west cf clinics were invited to assess all the patients under their care. data collected were age, gender and whether the patients skills fell "below", "same" or "above" the age appropriate self management tasks as described in the cfmtp. 'not applicable' was indicated where the skills did not apply. results: / clinics responded. out of a total of patients in the nw were assessed, three patients were excluded. population by clinic ranged from - . population by age category: ( - . years) ( . - years) ( - years) ( - years) ( - years) ( - years) ( - years) ( - years) ( - years and ( + years). / ( %) were male. for p (n= ), it (n= ), c (n= ), %, %, % = below, %, %, % = same, %, %, % = above respectively. there were no gender differences. conclusions: overall scores revealed that / rd of patients were gaining age appropriate independence in all areas. there was the least autonomy with it. it was rare for a child to be setting up and cleaning equipment regularly at - years, but this continued throughout the adolescent years. overall % were 'below' for p and it; this was highest after the age of years. autonomy with physiotherapy appeared slow to develop. some categories in cfmtp were different from our practice for e.g. patients are not invited to be seen alone in our clinics until years v - years and this may account for the lower scores in communication below this age. this study has highlighted that some of our patients are not fully independent in the time up to transfer. we need to encourage independence from an early age with all aspects of care and integrate the process into our routine clinical practice. refs mcdonagh and viner bmj ; : - , . parents' guide to the cf manager training programme. children's hospital of eastern ontario planner, s. ; morrison, l. . physiotherapy, gartnavel general hospital, glasgow, united kingdom; cystic fibrosis (cf) causes a deterioration in lung function and exercise tolerance. assessing exercise capacity is therefore essential in order to monitor changes in disease severity. the -minute walk test ( mwt) and minute step test ( mst) are validated and widely used outcome measures for identifying exercise capacity in patients with cf. exercise testing is part of annual review of all patients in the west of scotland. previous studies identified that the mst was a sensitive measure of exercise tolerance, but did not successfully challenge patients with mild or moderate lung disease as defined by fev(sub) (/sub)> % (morrison ) . further analysis of the mst, where step height was increased to inches did not show any difference in sao(sub) (/sub). decline or rate of perceived exertion (rpe) in patients with mild disease. the chester step test (cst) was developed to assess aerobic fitness which is used in the uk cardiac population for exercise prescription (sykes ) . it is a minute progressive, sub-maximal test with a variable step height ( - inches) , making it suitable for those with a wide range of exercise capacities. in addition, cst results extrapolate to a mets value (oxygen utilisation: met = rate of oxygen utilisation at rest) as an outcome measure which can facilitate exercise prescription based on established values for physical activities. we aim to determine whether the cst is a more sensitive measure of exercise capacity than the mwt or mst when considering decrease in sao(sub) (/sub) and rpe. patients ( aged - mean fev(sub) (/sub) . l) with mild-moderate cf performed the cst and mwt and results were correlated with fev(sub) (/sub). decrease in sao(sub) (/sub), increase in hr and rpe scores were also compared between the cst, mwt and recent mst data. patients were exercised to a symptom limited maximum which differed from the cardiac cst where patients are exercised to % of maximal hr. the cst appears to be more challenging as only patients completed it, all with an fev(sub) (/sub)of > % predicted, whereas in the mst and the mwt all patients completed the test. mets levels for the cst were weakly correlated with fev(sub) (/sub)(r= . ) and the mwt (r= . ) . this could be explained by the narrow range of mets data available or the size of the sample population. there were significantly greater rises in hr (p= . , p= . ), decrease in sao(sub) (/sub) (p= . , p= . ) and rpe (p= . , p= . ) , between the cst, mst and mwt respectively, suggesting that the cst is a more sensitive measure of cardiorespiratory effort. conclusions the cst appears to be a more challenging exercise test for those with mild to moderate disease and therefore may be a better reflection of exercise capabilities. cst results are useful for prescribing exercise and monitoring programmes appropriately for this patient group. further study could determine whether the cst is also suitable for those with severe disease. lee, a.l. ; button, b. ; holdsworth, m. ; holland, a. . physiotherapy, the alfred, melbourne, vic, australia; . latrobe university, melbourne, vic, australia musculoskeletal pain is prevalent in cystic fibrosis (cf). while the chest and back are frequently reported regions of acute or chronic pain, it is unclear if pain in these areas is related to disease severity or respiratory symptoms. manual therapy and postural advice has been shown to alleviate pain, but the effects of a combined approach of musculoskeletal physiotherapy and massage has not been studied. the aim of this study was to identify the primary regions of musculoskeletal pain, the relationship between pain severity and respiratory symptoms and to examine the effect of musculoskeletal physiotherapy techniques and soft tissue therapy, including remedial massage on pain and ease of breathing (eob) in adults with cf. one hundred and twenty-nine adults with cf ( with acute exacerbation, post lung transplant) aged ± years (mean ±sd) with fev(sub) (/sub) of ± % participated in this study. following assessment of primary pain regions, each subject underwent a single treatment session including spinal joint / intercostal mobilisation and soft tissue therapy. pain and eob on a visual analogue scale were measured before and after treatment. changes were compared using paired-samples t-tests. pain was frequently reported in the thoracic spine region ( % of subjects), followed by the shoulder region ( %), cervical spine region ( %) and chest wall region ( %). equivalent ratings of pain and eob prior to treatment were reported for all regions of pain. eob rating prior to treatment were worse in those with low bmi (r=- . , p= . ) and low fev(sub) (/sub) (r=- . , p= . ). a single treatment session was associated with reduction in pain ( %ci . to . , p< . ) and improvement in eob ( %ci . to . , p< . ), irrespective of clinical status. improvement in pain was equivalent for all primary pain regions. however, greater improvement in eob was evident in subjects with shoulder pain compared to other regions ( %ci - . to - . , p= . ). a combination of musculoskeletal physiotherapy techniques and soft tissue therapy reduces musculoskeletal pain and improves eob in adults with stable cf, during an acute exacerbation and post lung transplantation. service cystic fibrosis is characterized by an excessive production of airway secretions responsible for bronchial obstruction and recurrent infections of the respiratory tract. the transport by ciliary activity and cough may be dependent on the unsteady rheological properties of the respiratory mucus such as thixotropy and shear-thinning properties which correspond to a decrease of viscosity with time or flow rate, respectively. we have previously shown that respiratory mucus with high shear-thinning and thixotropic properties is better transported by the cough mechanism (zahm et al, eur. respir. j., ) . to promote mucus clearance and to finally expectorate the mucus by a cough manoeuvre, an innovative device, the airhelp, has been developed and aimed to improve chest physiotherapy effectiveness. during a prolonged expiratory manoeuvre, the airhelp delivers negative pressure variations (maximal amplitude: - kpa) at an oscillatory frequency of hz. the aim of the present work was to test the effect of the airhelp device on the in vitro clearance of mucus by airflow. a plexiglass tube of mm in diameter and mm in length has been connected by one part on the airhelp and by the other part on a l thank mimicking the lung compliance. a µl drop of mucus simulant at different concentrations (viscogum at . % or . % with actigum at . % to . %) or of respiratory mucus, was deposited within the plastic tube and the distance travelled by the sample under a sec-airflow was measured. twelve respiratory mucus samples collected from cf patients were analyzed and the viscosity of the samples was measured before and after the airhelp experiment. we observed a significant (p < . ) and negative relationship (r = - . ) between the viscosity of mucus simulants and oscillatory airflow transport: the lower the viscosity, the higher the mucus transport by oscillatory airflow. using cf respiratory mucus samples, we observed that without the oscillatory airflow, no transport occurred, whereas when the samples were submitted to the oscillatory airflow, a significant increase in mucus transportability was measured. in addition, the viscosity of the cf respiratory mucus samples was significantly decreased after oscillatory airflow treatment ( ± pa.s to ± pa.s, p < . ). these results demonstrate that the mucus transport efficiency by an oscillatory airflow is dependent on mucus viscosity and that the improvement in mucus transport is related to the thixotropic and shear thinning properties of the airway mucus. we conclude that the airhelp device may improve chest physiotherapy effectiveness. potter, e. ; nufer, j. ; cullina, j. , ; quinton, h. ; jain, m. , ; mccolley, s.a. , . northwestern university feinberg school of medicine, chicago, il, usa; . children's memorial hospital, chicago, il, usa; lebanon, nh, usa; . northwestern memorial hospital, chicago, il, usa socioeconomic status (ses) significantly impacts health outcomes in cystic fibrosis (cf). the cystic fibrosis foundation (cff) is currently using median income estimated by zip code from the us census of the population for ses risk adjustment of data reported publicly from the cff patient registry. median income estimated by zip code is an "ecologic" variable that may misclassify patient and family income. in order to more fully assess the impact of ses on cf health outcomes, the cff began to ask centers to collect data on household income directly from patients and families. these data were captured for . % of patients and families in and . % in in the national registry. we report a method of collecting ses data from children and adults implemented at the children's memorial hospital and northwestern university cystic fibrosis center, and compare directly collected income to income based on zip code. methods: a short survey with the questions on household composition, income, and education level from the cff patient registry was developed. a letter from the center director and adult program director was mailed to patients and families informing them of the survey's purpose and confidentiality measures, i.e. responses would be seen only by the individual entering the data and identified by cff id number. the survey was distributed to cf patients and families in clinic by clerical staff. completed surveys were placed in an envelope and sealed by the patient or family. both directly collected income and income from zip code was classified into groups (<$ k, - k, - k, - k, - k, - k, - k, - k, and > k) . percent concordance and spearman's rank correlation were calculated. data were used for patients who provided data in ; otherwise, data were used. each patient's data was used only once in the analysis. results: unique patients were identified, pediatric and adult patients. eighteen percent of pediatric and % of adult patients preferred not to answer income questions or did not complete the survey. the mean income by zip code was $ k for patients who did not report income data and $ k for the entire group. for the responses, reported income data was moderately correlated with income by zip code (rho= . , p< . ). concordance between the categories was %. the correlation was stronger for pediatric patients (rho= . , p< . ) than for adults (rho= . , p= . ). re-classifying income as "low", "medium" and "high" (<$ k, $ - k, >$ ) led to a concordance of %. conclusion: survey methods that protect patient and family confidentiality result in good return rates of full ses data from patients and families. zip code derived median income shows moderate concordance with reported income. comparing the associations of the forms of income data with important cf outcomes needs to be done in a larger data set. we acknowledge the cystic fibrosis foundation, gerald o'connor, kathryn sabadosa, and participating patients and families. the patient registry provided a unique opportunity to assess changes in the epidemiology of respiratory pathogens in cf. while the incidence of p. aeruginosa remained stable, the prevalence decreased, suggestive of the impact of antibiotic eradication strategies. in contrast, the increasing incidence and prevalence of s. aureus and mrsa may reflect improved microbiological surveillance and laboratory techniques. the trends noted for b. cepacia complex may reflect successful implementation of infection control strategies. future studies are needed to better define the association between these observed trends and improving care for cf patients. vanderwyden, a. , ; drumm, m.l. , ; schluchter, m. . pediatrics, case western reserve university, cleveland, oh, usa; . epidemiology and biostatistics, case western reserve university, cleveland, oh, usa; . genetics, case western reserve university, cleveland, oh, usa in the genetic modifier study (gms) of lung disease of cystic fibrosis (cf), patients, homozygous for the ∆f mutation, were classified as having either severe or mild lung disease, as defined by the lowest or highest quartile of forced expiratory volume in one second (fev ) and a linear model was fit from these patients' longitudinal data (drumm , schluchter . while these models successfully dichotomized patients for the association study, we assessed whether a single slope model could be improved. here we have begun to compare models with one, two and three slopes, each representing different age ranges, for their use in this type of association study. as an example, interleukin- (il- ), a neutrophil chemoattractant, associates with the severe lung inflammation seen in cf patients. we have observed the tt genotype of the il- snp rs , correlated with severe pulmonary function in the gms sample of patients, relative to the aa and at genotypes of this snp. in a replicate study of patients, with cftr genotypes associated with exocrine insuffiency, longitudinal linear mixed models were used to further characterize the decline in lung function over time as associated with the il- snp, rs . three linear regression models of fev % predicted of patients were fit over age and compared with likelihood ratio tests. the three models differed with respect to the number of nodes. the first model had a single slope while the second and the third were piecewise linear models, with nodes at age , and ages and , respectively. age was chosen as the first node, as by this age most cf patients have reached puberty, an event believed to coincide with changes in pulmonary function. the second node, age , was arbitrarily chosen as a point in the third decade, as survivor effect becomes a significant issue during this period. when compared to the single slope and two slope models, the three slope model with nodes at age and age better explained the pulmonary function of this study (p< . , - loglikelihood chi-squared tests). when the explanatory variable, il- rs genotype, was added, the single slope model and the three slope model were again compared. the three slope with nodes at age and age again resulted in a better fit of fev % predicted over age as correlated with rs genotype (p< . , - loglikelihood chi-squared test). estimates of fev % predicted at ages , , , and for each rs genotype (aa, at/ta, or tt) were determined using the three slope model (p< . for all estimates). other explanatory variables, like gender and survival (p< . ), were added to further elucidate the relation between il- genotype and lung disease phenotype. we propose to use this piecewise three slope model with nodes at age and age to look at the pulmonary function of patients in the cf foundation registry data to better explain the decline in fev % predicted over time seen in cf patients. (supported by hl- and grants from the cf foundation) stephenson, a.l. purpose: the purpose of this study is to determine the annual incidence of hospitalizations for individuals with cf living in ontario and to examine predictors of hospitalization, specifically gender. methods: this is a retrospective cohort study from to , using newly linked clinical and administrative databases. the canadian cf foundation patient data registry (cpdr), contains detailed clinical information on all cf patients receiving care at one of accredited cf centres across canada. the institute for clinical evaluative sciences (ices), holds linked administrative databases containing information on all publicly reimbursed health care services delivered in ontario. canadian institute for health information (cihi) discharge abstracts database (dad), ontario health insurance plan (ohip), and homecare databases were used for this study. the cpdr was probabilistically linked (automatch software) to the ices administrative database using name, gender and date of birth. all individuals were then linked to the cihi dad, ohip, and homecare databases using unique numeric identifiers. with these databases, longitudinal records for each individual were created. cihi-dad, ohip, and homecare claims data were used to calculate the number of hospitalizations per year for pulmonary infections. males and females were analyzed separately to elucidate any gender disparities. other predictor variables include age, geographical location, number of clinic visits, socioeconomic status (income quintile) as well as clinical variables such as pulmonary function tests, nutritional status, diabetes mellitus, sputum bacteriology and pancreatic status. results: the cpdr contained data on individuals with cf followed within canada between - . of those individuals, could be found within the ices administrative databases using probabilistic linkage. of those individuals matched to the administrative databases, ( . % female) had hospitalizations. a total of , ohip claims were made during this -year period which represents any encounter between an individual with cf and a physician in ontario. multivariable analysis of hospitalization predictors is in progress. zhang, z.; lai, h. uw-madison, madison, wi, usa the cff practice guidelines recommend adjusting for genetic potential when evaluating height status in children with cf. however, calculation of adjusted height percentile is not recommended due to the complexity of method involved in such calculation. instead, a simple method to estimate a target height based on mid parental height was recommended. however, this target height includes a -cm confidence interval, which spans most of the channels on the growth chart. therefore, a child's unadjusted height may be considerably below the target height, yet remains above the lower bound of the target height range. in addition, there is a paucity of data on the associations between adjusted height to lung disease outcomes to justify whether adjustment for genetic potential is necessary. the objective of this study is to compare two methods of incorporating genetic potential in identifying short stature, namely, the cff's target method and the method developed by himes et al (pediatr ; : - ) , which applies positive adjustments to children with short parents and negative adjustments to those with tall parents. data from - cff registry were utilized. a total of children born between - who had complete height data from age - years and their parental height data were analyzed. short stature was defined by unadjusted height < th percentile, unadjusted height below the lower bound of cff's target height, or himes' adjusted height < th percentile. our results showed that adjusted height percentiles are consistently lower than unadjusted height percentiles, with an average difference of . z-score unit between ages - years and . z-score unit between ages - . proportionately more children were classified as short stature based on himes method compared to unadjusted height ( % vs. %, p = . ). in contrast, proportionately fewer children were below the lower bound of cff's target height ( %) compared to unadjusted height < th percentile ( %), p < . . among children with average parents, unadjusted and adjusted height percentiles were similar. however, among children with short parents (< th percentile), the percentage children with of short stature decreased from % with unadjusted height to % with himes' adjusted height and % with cff's target height method. among children with tall parents (> th percentile), the percentage of short stature increased from % with unadjusted height to % with himes method and % with cff's target height method. taken together, these results demonstrate that, without incorporating genetic height potential, - times more cf children who have short parents would be classified as short stature, while times fewer cf children who have tall parents would be classified as short stature. further analyses show that, compared to unadjusted height percentile, himes adjusted height percentile is more sensitive to, and has stronger association with, percent predicted fev- . in conclusion, our findings provide evidence that genetic potential should be incorporated in evaluating short statue in cf children, particularly for children with short or tall parents. the cff's target height method and himes method differ significantly in identifying short stature in cf children with short parents. cohort study of individuals in the cff patient registry from - . all individuals years of age or older and diagnosed before age were included. the new, persistent mrsa infection cohort was defined by having at least two cultures negative for mrsa over a two-year lead-in period followed by at least two mrsa positive cultures. individuals with only one mrsa culture (transient infection) were excluded. we developed multiple linear regression models using generalized estimating equations to assess the effect of mrsa on fev %predicted (%fev ) and rate of change of %fev . results: during the study period , individuals cultured mrsa. of these, , ( %) cultured mrsa only once (transient infection) and were excluded from subsequent analysis. , individuals met the criteria for new and persistent mrsa infection. , met the criteria for never having mrsa resulting in a total study cohort of , . participants were followed for a median of years. the median time to first isolation of mrsa in the persistent mrsa cohort was . years and the mean number of positive mrsa cultures per patient was . . a comparison of baseline characteristics between the persistent mrsa and never mrsa cohorts revealed the mrsa cohort to be younger ( . vs . years p< . ), equivalent in lung function (%fev . % vs . % p=ns), and with higher likelihood of methicillin sensitive staph aureus colonization ( % vs. . % p< . ). . years into the study period (the median time of acquisition for new mrsa), the mrsa cohort had a lower %fev ( . % vs . % p< . ), and averaged twice as many hospital admissions per year ( . vs . p< . ) and home iv courses per year ( . vs . p< . ). in an unadjusted analysis, the presence of mrsa was associated with a mean %fev that was . % lower than the mrsa cohort ( %ci . %- . %). however, after adjusting for sex, age, pancreatic status, p. aeruginosa, and b. cenocepacia, the presence of mrsa was associated with a mean %fev . % lower ( %ci . %- . %). most importantly, an analysis investigating the interaction between mrsa and time, after adjusting for confounders, indicated no statistically nor clinically meaningful difference in the mean rate of lung function decline between the two groups. conclusions: approximately one-third of patients who culture mrsa do so only once and do not subsequently develop persistent mrsa infection. mrsa infection occurs in a group with more severe structural lung disease as measured by fev , and frequent hospitalizations and iv antibiotics are strongly associated with mrsa acquisition. after adjusting for confounders, mrsa infection is not statistically significantly associated with a more rapid decline in %fev over time. simmonds, n.j. , ; macneill, s. ; cullinan, p. , ; hodson, m.e. , . department of cystic fibrosis, royal brompton hospital, london, united kingdom; . national heart and lung institute, imperial college, london, united kingdom introduction: disentangling the influence different factors have on long-term survival in cf is complex. there are a myriad of influences (including environmental, healthcare-related, psychological and socioeconomic) which may be important, as there is generally a poor genotype-phenotype correlation. the aim of this case-control study was to identify these factors. the cf database at the royal brompton hospital was used to identify long-term survivors. they were classified as cases and were patients who had reached years of age (without transplantation). each case was agematched with at least one control who died of cf (or a cf-related condition) or was transplanted before reaching years of age. late diagnosis patients were excluded. conditional logistic regression models were used to identify potential influences on survival. results: cases and controls were analysed, producing matched pairs. % of the patients were pancreatic insufficient. of the many factors investigated, those resulting in increased probabilities of survival included: increased body mass index (or= . , % ci . - . ), fev (or per % increase= . , ) at transfer to the adult clinic (after adjusting for age and sex) and the use of oral antibiotics before attending the adult clinic (or= . , . - . ) after adjusting for age at first attendance. factors associated with a reduced probability of survival included: cf diagnosis < years old (or= . , . - . ); initial presentation of chest symptoms or malabsorption (or= . , . - . and or= . , . - . , respectively); referral from a paediatric clinic in a deprived area (or= . , . - . ); pneumothorax before transfer to the adult clinic (or= . , . - . ) after adjusting for age at first attendance; and s. aureus or p. aeruginosa colonisation before years of age (or= . , . - . and . , . - . , respectively). factors not influencing survival included: sex, h. influenzae colonisation before years of age, development of diabetes before years of age, major haemoptysis before transfer to the adult clinic, parents' occupation, number of siblings (cf and non-cf) and school achievements. in a very carefully matched study we failed to identify major non-clinical determinants of long-term survival in early diagnosis cf. findings suggest that the majority of significant factors were directly related to optimal physical parameters, the use of oral antibiotics, avoiding bacterial colonisation and a low pneumothorax rate. adler, a. , ; bilton, d. ; haworth, c. ; gunn, e. ; shine, b. . addenbrooke's cambridge university hospitals, cambridge, united kingdom; . papworth hospital, papworth everard, united kingdom; . oxford centre for diabetes and endocrinology, oxford, united kingdom background and aims: although the prevalence of diabetes in patients with cf (cfrd) is high, few prospective studies exist, and even for genet-ic factors, cross-sectional studies may bias the magnitude of associations. this study sought to identify risk factors for cfrd from a predominantly white cohort of individuals with cf in the united kingdom. methods: , individuals aged - were identified from the uk cf database, a registry of patients coordinated by the uk cystic fibrosis trust with data from over specialists centres from england, scotland and wales, countries which provide medical care free of charge. of the patients, , had no diabetes at baseline ( - ) , and also had at least one follow-up annual visit. diabetes was defined as either a physician diagnosis of diabetes, a blood glucose of > . mmol/l two hours following an oral glucose tolerance test, or treatment with insulin or oral hypoglycemic drugs. follow-up was calculated as time from baseline to the first detection of new diabetes or censoring. risk factors included clinical, genetic (functional classes i,ii,iii vs iv,v), and anthropometric characteristics measured at baseline. survival analyses were limited to patients with complete data. cox proportional hazards modeling was used to estimate the magnitude of the association between potential risk factors and incident diabetes. one-way interactions with sex were tested. results: the median age of entry into the cohort was years and bmi was . kg/m . % were male, and % were white. patients developed diabetes during , person-years of follow-up (mean . years); the incidence was . % per year. patients who developed diabetes were more likely in univariate analyses to be older, have a high body mass index (bmi), bacterial pulmonary infections, liver and pulmonary function abnormalities, poor oxygen saturation, a history of organ transplantation, supplemental feeds, and take pancreatic enzymes or bile acids. in addition, patients with class iv,v genotypes were less likely to develop diabetes relative to patients with other genotypes, as were patients screened for cf at birth. not associated with incident diabetes was age at diagnosis of cf and ethnicity. in multivariate analyses (n= , ) age (hazard ratio . per year, % ci . - . ), female sex ( . ( % ci . - . ), % predicted forced expiratory volume in second ( . , . - . per % decrement), dose of pancreatic enzyme replacement ( . , . - . ), above vs below median dosage, use of bile acid supplements ( . , . - . ) , and genotype class i,ii, iii ( . , . - . ) relative to class iv/v. these associations were not confounded by bmi or history of transplantation. there were no significant interactions between sex and other risk factors. conclusions: the study confirms the high incidence of cfrd, and shows that patients with class iv/v genotypes are less likely to develop diabetes independent of other cf-related complications. females are disproportionately at risk for diabetes which is not explained by a higher prevalence of known risk factors nor by effect modification. cystic fibrosis children are at increased risk for hearing disorders due aminoglycoside exposure, mucopurulent upper respiratory tract infections, and increased inflammatory responses. pure tone audiometry is the standard instrument used to determine hearing loss. however, it can be expensive and time-consuming to use audiometry for routine hearing screening. hearing questionnaires have been used as a screening tool to assess possible hearing loss in otherwise healthy populations. what are the results of hearing questionnaires administered to pediatric cf patients? does cf patient age or ethnicity increase the likelihood of hearing loss as determined by a hearing questionnaire? a hearing questionnaire (english and spanish versions) was administered to pediatric cf patients over a week period at our accredited cf center. data collected included demographic information. inclusion criteria: age м years and in baseline health status. possible hearing loss was defined as at least "yes" responses (> ) to the quesions administered to the patients. fisher's exact test was used to perform the analyses. a total of cf patients were seen in the cf clinic during the study period. cf patients completed the hearing questionnaire. mean age was . ± . years (median . years). there were males and females; were classified as hispanic and were classified as caucasian. patients were older than . years (the median age) and patients were less than . years. patients who were older than age . years had a score of > and patients < age . years had scores > (p= . , ns). thus, of cf patients ( %) were classified as having possible hearing loss as per their questionnaire scores. of hispanic cf patients had hearing scores > and of caucasian patients had hearing score > (p= . , ns) . we found that a significant proportion ( %) of our cf children may require formal hearing evaluation. we speculate that hearing questionnaires may be useful to screen for hearing loss in the cf patient population. hispanic cf patients have increased morbidity and mortality risks compared to caucasian cf patients who attend our accredited cystic fibrosis center in southern california. is the increased morbidity/mortality risk due to decreased access to outpatient cf care? we performed a retrospective review of all patient visits to our cf clinic between january , through december , . data collected included demographic information (age, ethnicity, gender) , and the number of clinic visits during the study period. data was analyzed by unpaired student's t-test as well as by chi-square. during the study period, a total of cf patients ( males: females; mean age . ± . years; range months to years) were routinely followed in our cf clinic. these patients had a total of outpatient visits to the cf clinic ( . clinic visits/patient over the year study period). there were hispanic cf patients ( males: females; mean age . ± . years) and caucasian cf patients ( males: females; mean age . ± . years; p= . , ns). there was no difference in gender distribution between the group (p= . , ns). hispanic patients had a greater number of cf clinic visits ( . visits/patient) as compared to caucasian patients ( . vists/patient; p= . ) during the same period. we conclude that the increased morbidity and mortality of hispanic cf patients in california cannot be attributed to barriers to outpatient care. in fact, hispanic cf patients were seen more frequently in cf clinic compared to caucasian cf patients who attend the same clinic. we speculate that hispanic cf patients are seen more frequently in cf clinic due to increased severity of cf disease manifestations. duguépéroux, i. ; scotet, v. ; audrézet, m. , ; blayau, m. ; parent, p. ; journel, h. ; boisseau, p. ; férec, c. , . inserm u , brest, france; . dep. of genetics, brest, france; . dep. of genetics, rennes, france; . unit of medical genetics, brest, france; . unit of medical genetics, vannes, france; . dep. of genetics, nantes, france aim: the aim of this study was to describe -year experience of prenatal diagnosis (pd) for cf in brittany (western france) and to assess its impact on incidence. method: we registered, by the genetic laboratories of our region, all the pds performed in women living in brittany over the period - . we described the number of pds made for each reason (way by which the one-in-four risk was identified: previous affected child, family testing, echogenic bowel, etc). we reported the proportion of cf-affected fetuses and of consecutive terminations, and assessed the incidence modification due to pd. results: over the -year period, a total of pds were performed in couples living in brittany. most of them were done in couples already having cf child(ren) (n= , . %). extended testing in families -a practice largely proposed in brittany -led to the identification of new onein-four risk couples among the relatives of cf patients who opted for pd times over the study period ( . %). the other pds were made in couples without previous history of cf. the one-in-four risk was mainly identified following the detection of an echogenic bowel during pregnancy ultrasound examination ( pds were done directly following the positive ultrasound ( . %), whereas others were done for subsequent pregnancies ( . %)). the other pds were consecutive to the detection of an heterozygote through newborn screening (n= , . %) or for an other reasons (n= , . %). over the study period, the number of pds per couple varied between one and five, the mean being . . overall, a total of cf fetuses were identified, among whom were terminated ( . %). the inclusion in the incidence calculation of these pregnancy terminations led that to an incidence modification of . % over the study period. conclusion: this study shows that pd for cf is commonly used in brittany and highlights the impact of family testing and of routine ultrasound examination of pregnancies in that region. supported context: hispanics with cystic fibrosis (cf) in the united states experience an % increased annual risk of death from cf compared to non-hispanic patients. when adjusted for socioeconomic status this disparity persists. studies characterizing this at-risk population do not exist. objective: to characterize the center-reported us hispanic population with cystic fibrosis through a cystic fibrosis foundation (cff) patient registry analysis and to elucidate factors present within this hispanic population that may affect outcomes. design: retrospective cross sectional study of the cff patient registry. patients: all , patients in the cff patient registry were included in the analysis. there were no exclusion criteria. the hispanic population is defined as those who were entered by their respective care center as hispanic ethnicity regardless of race. main study measures: prior to analysis, study measures were selected to demographically and genetically characterize the hispanic cf population. genotype, state of residence, mean age and mean age of diagnosis were obtained in the entire population. maternal education (for patients < years of age) and insurance status data were captured as indicators of socioeconomic status. complication rates, fev percent predicted (under years of age) and absolute fev (over years old of age) were obtained as measures of clinical status. t-tests and logistic regression analyses were used to compare measures between hispanic and non-hispanic groups. odds ratios (or) and % confidence intervals are reported. results: center-reported hispanic patients with cf were identified. over % of hispanic patients resided in california, florida, texas or new york. the most common genotype was delta f homozygous, accounting for % of the population. mean age was . years +/- . years for hispanic patients and . years +/- . years for non-hispanic patients. hispanic patients were diagnosed at an earlier age ( . years vs. . years, p= . ). they were more likely to have mothers with education at less than a high school level (or . ( . , . ) ) and have government insurance (or . ( . , . ) ). complication patterns differed between the two groups; non-hispanics were more likely to have complications reported that were related to depression (or . ( . , . ) ), bone health (or . ( . , . ) ) and cf related diabetes (or . ( . , . )) compared to hispanics. hispanics however, were more likely to have complications reported from cf liver disease (or . ( . , . ) ). pulmonary function, as measured by fev percent predicted in children (p< . ) and absolute fev for adults (p= . ) were lower for hispanic patients. conclusions: differences exist between hispanics and non-hispanics with cf with respect to maternal education, insurance status, complications rates and pulmonary function. the lower reported rates of depression, bone complications and cf related diabetes may represent ascertainment bias. further study is needed into the etiology of health outcome disparities in this population, and to design interventions for this high-risk population. mastella, g.; baldo, e.; forneris, m.; furnari, m.; lucidi, v.; manunza, d.; marinelli, i.; messore, b.; neri, a.; raia, v.; salvatore, d.; buzzetti, r.; cairo group, italian cf research foundation, verona, italy with our work we reviewed the international scientific literature coming from cf registries worldwide. our aims were: to verify what has been produced in scientific literature thanks to the material derived from fc registries and to see which clinical problems have been tackled and which clinical questions were answered correctly and exhaustively. a search in medline and embase produced articles (our strategy was "cystic fibrosis"[mesh] and ("registry"[mesh]) or registr$) updated to the / / ). out of these articles have been selected by two independent assessors on the basis of their pertinence (primary studies that used data taken from a formally established registry, at least national, to test some hypothesis of research). each article has been examined with the help of a pre-defined form that in particular evaluated skills of different registry data, selected clinical characteristics of the study populations and statistical methods. more than one half of cases data came from usa-cff foundation patient registry, the remaining coming from canadian, uk, french, italian, german, and other european registries. the main focuses of the articles were included in the following topics: ) incidence/prevalence of the disease and survival ( studies); ) neonatal screening, growth and nutrition ( ); ) genotype/phenotype correlation/ different ethnical groups and twin/brothers ( ); ) complications and outcomes (abpa, diabetes, nasal polyposis, pregnancy and paternity, etc) ( ); ) microbiology ( ). our assessment scores were good/excellent for / studies for the relevance of the problem addressed; / for the usefulness of the outcomes and / for the usefulness of the implications for research from the italian registry. thirty-two articles gave a significant contribution to the analysis of the studied problem, but of them left a partial uncertainty, while left complete uncertainty. cf registries are a very important source of data to provide a powerful tool for clinical and research questions. however, further comparable studies have to be planned to assess registry data as a platform for improvement in clinical practice. vandenbranden, s.l. ; mcmullen, a. ; konstan, m. ; morgan, w. ; wagener, j. ; schechter, m. ; watts, k. , ; mccolley, s. , . children's memorial medical center, chicago, il, usa; . emory university, atlanta, ga, usa; . university of arizona, tuscon, az, usa; . case western reserve university, cleveland, oh, usa; . northwestern university feinberg school of medicine, chicago, il, usa; . university of colorado, denver, co, usa; . university of rochester, rochester, ny, usa background: although life expectancy in cf has dramatically improved over the last years, most of the improvement is due to improved survival during early childhood. adolescence and young adulthood continue to be a time of worsening pulmonary disease and mortality. study objective: data from a large longitudinal observational study, the epidemiologic study of cystic fibrosis (escf), was examined to characterize lung function decline in subjects with cf during the periods preceding and after the age of . design: escf data from individuals with cf collected during the period of - were analyzed. inclusion criteria required individuals to have a minimum of encounters in each of the . year time periods before and after the age of . the cohort was strati-fied by disease severity based on the best fev at the th year (+/- months). one hundred thirty seven subjects had severe lung disease (fev < % predicted), had moderate-severe lung disease (fev -< % predicted), had mild lung disease (fev -< % predicted), and had very mild disease (fev > % predicted) the study compared the annualized rate of fev decline during the adolescent period, defined as ages - . years, and in the young adult period defined as . - years. results: in the entire cohort, the annualized rate of decline was less in the young adult period than in the adolescent period (p< . ). the most dramatic improvement in slope of decline was seen in the severe group, and significant improvements were also noted in the moderate group (p< . ). the mild group demonstrated insignificant change; the very mild group was the only group that demonstrated a more negative annualized rate of the decline in young adulthood than in adolescence (p= . ). conclusions: these data suggest that, overall, young adults have a slower rate of decline in fev in the period after age . this "stabilizing" of lung function is most prominent in those individuals with moderate-to-severe lung disease. only those with very mild lung disease have a slightly more rapid rate of decline between the ages of and . overall there is less variation in the rate of fev decline across disease strata after the th birthday. further analysis is underway to better understand the factors leading to these findings. . department of paediatrics, university of florence, cystic fibrosis centre, florence, italy; . italian cystic fibrosis microbiology group, florence, italy; . institute of medical microbiology and hygiene, tübingen, germany mrsa is now a worldwide public health problem, due to the increasing rate of infection in several settings as well as in cf patients. mrsa was first recognized as being acquired from hospitalized patients (ha-mrsa), but the onset of mrsa infection outside the hospital setting, in communityassociated strains (ca-mrsa), has recently been described with increasing frequency. ha-mrsa are known to be responsible for infections in hospitalized patients, although highly virulent ca-mrsa are increasingly reported worldwide as the cause of severe outbreaks, replacing ha-mrsa strains in nosocomial infections. little evidence is available about the characterization (sccmec type) and epidemiology of community-acquired mrsa (ca-mrsa) or hospital-acquired mrsa (ha-mrsa), transmissibility, antibiotic susceptibility and virulence in the cf population. the present multicenter study investigated the susceptibility pattern, epidemiology, and sccmec type of mrsa strains isolated from nine italian cf centers. the susceptibility pattern was determined by the disk diffusion method and pulsed field gel electrophoresis (pfge) was performed for epidemiological purposes. the sccmec type in order to identify hospital-and community-acquired mrsa strains (ha-mrsa and ca-mrsa respectively) was determined with molecular methods, and the presence of gene encoding panton-valentine leukocidin (pvl) was tested. during the study period, mrsa strains were isolated from out of ( . %) patients attending these cf centers. of the sccmec type representing ha-mrsa, our data showed an high prevalence of sccmec i ( . %) while the prevalence of sccmec ii, (reported as the most represented among ha-mrsa) was only . %. furthermore a high prevalence ( . %) was found of sccmec iv (suggestive of ca-mrsa strains). epidemiological analysis showed that ( . %) out of analyzed mrsa strains belong to the same pfge clone shared among six centers, belonging to the sccmec type iv, suggestive of ca-mrsa isolates. twenty-four ( . %) out of these strains were sccmec type iv, indicative of ca-mrsa, as suggested by the good susceptibility rate to trimethoprim-sulfamethoxazole and rifampin. surprisingly all the isolates belonging to this epidemic clone were negative for the pvl genes. these results show that ca-mrsa is now spreading in the cf population, documenting the first epidemic of ca-mrsa in cf patients who are considered at risk for ha-mrsa acquisition due to frequent hospitalizations. these data show a peculiar picture of mrsa epidemiology indicating that further studies are required to clarify the role of ca-mrsa in global epidemiology, their pathogenicity and clinical impact on cf patients. we thank the fondazione per la ricerca sulla fibrosi cistica-onlus for its grant (ffc# ) . there are concerns regarding an increased risk of cancer in patients with cystic fibrosis (cf). this study aimed to review the occurrence of malignant disease in a large population of adult patients with cf. the case notes of patients attending the leeds adult cf unit were reviewed. demographics and data regarding a diagnosis of malignant disease were recorded. a total of eight patients ( male, female) were diagnosed with a malignant condition over the year period. the median (range) age of diagnosis of a malignant condition was . years ( - years). five patients were diagnosed with malignancy post-transplantation. within this group of patients there were two cases of lymphoproliferative disease, one case of basal cell carcinoma, one case of liver cancer and one case of small cell lung cancer related to the donor lung. the median (range) time to diagnosis post-transplantation was two years ( - years) with a median (range) age of years ( - years). three patients were diagnosed with a malignant condition without solid organ transplantation. this group consisted of one case of pancreatic carcinoma, one case of oesophageal carcinoma and one case of bowel carcinoma. the median age of diagnosis in this group of patients was years ( - years) . prior to transplantation we found all malignant diagnoses were related to the gastrointestinal tract. post-transplantation we found a wider variety of malignant diagnoses, with lymphoproliferative disease being most common. awareness of the increased risk of malignant disease is important and appropriate investigations should be undertaken particularly in patients with atypical symptoms. background: patient outcomes can be improved by implementing evidence-based guidelines for initiating therapies. in our cf center, practitioners have traditionally prescribed medications without a centralized database to screen patients for evidence-based criteria. goals: the primary goal of this project was to increase cf practitioner, family and patient awareness of evidence-based medication guidelines to facilitate discussions regarding therapies. the secondary goal of this quality improvement (qi) project was to determine if cf practitioner prescribing patterns would be altered by increasing awareness. methods: the cf steering committee at cincinnati children's hospital medical center (cchmc) identified oral azithromycin, inhaled tobramycin, and dornase alpha as target medications for this qi project. an evidence-based medicine committee was formed, including a parent advocate, to direct the project. a consensus was reached among cf practitioners for when to initiate therapy discussions. dornase alpha was recommended for cf patients ages six and older. inhaled tobramycin was recommended for cf patients ages six and older with chronic pseudomonas (defined as positive cultures in past months). oral azithromycin was recommended for cf patients ages six and older, with chronic pseudomonas, and a weight of kilograms or more. the cf database was screened to determine each patient's medication eligibilities. eligibilities were reviewed weekly at our cf chart conference. medication specific family and patient handouts were developed, reviewed by the parent advocate, and given to eligible families and patients on arrival to clinic. data was collected to document the number of discussions facilitated at clinic visits, the decisions regarding medications, and the number of new treatment prescriptions that were facilitated. after seven weeks of the initiative, statistical analysis was conducted using mcnemar's test for differences. a chi-square test with a two sided alpha of p < . was used to determine significance. results: of the cf patients in our center were eligible for pulmonary medications using our evidence-based criteria. there were individual prescribing opportunities in these patients (taking each medication for each patient as a unique prescribing opportunity). at the start of the project, . % ( / ) of the prescribing opportunities were fulfilled. % ( / ) of patients were prescribed all of their recommended medications. during the first seven weeks of the project, cf practitioners discussed initiating new pulmonary medications with cf families and patients. the proportion of prescribing opportunities fulfilled increased from . % ( / ) to . % ( / ) (p value < . ; % ci . %- . %). the proportion of patients prescribed all of their recommended medications increased from % ( / ) to . % ( / ) (p value < . ; % ci . %- . %). conclusions: increasing awareness of evidence-based pulmonary medication recommendations with cf practitioners, families and patients facilitates therapy discussions, increases the proportion of therapies prescribed and increases the proportion of patients on all recommended therapies. purpose: our purpose was to optimize lung function (fev ) by consistently and aggressively diagnosing and treating pulmonary exacerbations (pex) in all cf patients at our center. it was important to work on this because our families wanted standardized care and we felt our patients' fev 's were suboptimal. methods: our core llc team included two parents of cf patients and one adult patient. we met weekly, received coaching every other week, and learned and applied quality improvement techniques. physicians began monthly meetings, and monthly meetings of the entire cf team continued. the fab met every other month. we identified our specific aims, and approached them systematically. the providers, starting with criteria used by several authors, agreed upon the definition of pex and created a pulmonary exacerbation score sheet (pexss). we selected a score of as the threshold for defining a pex. after using the pexss for three months, all providers reviewed the individual pexsss and validated our original choice of . run charts were produced and reviewed periodically to display each provider's percent of pexsss used. when the use of the pexss was not standardized after a year, it was reprinted on bright yellow paper to make it stand out. to educate the families and patients about the early symptoms of pex, a refrigerator magnet was designed and distributed that included all pex criteria. results: we standardized the use of our pexss by all providers on all patients seen in cf clinic with ongoing measurement of its use and regular feedback. many patients and families gained greater knowledge about what a pex was, and the importance of early recognition and treatment. the cf nurses note that ) many patients call with milder pex symptoms than they did before, ) providers treat pex earlier, ) many patients call with their pex score as well as their symptoms. we hospitalized more patients for pex after this project began, with an average of patients/year in the years prior, and in . the median fev % predicted of our - conclusions: participating in the llc changed the culture and functioning of our cf team. limitations to our study include: ) fev change may not have been due solely to this project, ) the groups who's fev 's were averaged over the various years are different. we will continue to optimize and standardize ) prevention of pex, ) inpatient care, ) care of pex in cf providers' private offices, and ) follow up of patients with pex. patient adherence to airway clearance techniques (act) in the treatment of cystic fibrosis is known to be sub-optimal resulting in deterioration of lung function. the global aim of our project was to improve median fev . the specific aim was to improve adherence to act. we believed that these goals could be acheived by implementing a program which included re-education of airway clearance techniques (react) for our cystic fibrosis patients. methods: the initial phase of this project began with the administration of an anonymous survey to patients/families. this survey was designed to assess act practices, knowledge of the rationale for performing act and barriers that prevent patients/families from adhereing to act. the next step included an in-clinic questionnaire and patient/family demonstration of current aerosol therapy and act. based on individual results patients were identified as: adherent with correct technique, adherent with incorrect technique or non-adherent. we defined adherent as correct performance of act one to two times daily as prescribed. then all patients entered the react program which included a standardized tutorial on proper act and correction of techniques if necessary. in the non-adherent group barriers to performing act were discussed and problem solving techniques were used to overcome barriers. the patients in the non-adherent group were re-evaluated in clinic monthly while the adherent patients returned on an every two month schedule. results: analysis of our initial survey revealed that while the majority of patients/families reported performing act daily, the duration of treatments was less then medically prescribed. many patients/families reported barriers which decreased adherence to therapy. to date % ( / ) of our patients have completed the in-clinic questionnaire and entered the react program. the results of these evaluations and patient demonstrations revealed: % ( / ) were adherent with correct technique, % ( / ) were adherent with incorrect technique and % ( / ) were non-adherent. in the six months since the implementation of the react program, patients/families are self-reporting an increased adherence to act and the median fev for our center has increased form . % ( cff registry data, ages - years) to . % (cff registry, first quarter, , ages - years). conclusion: while adherence to act in our center was poor, we were able to improve adherence and ultimately improve the median fev by implementing a program of re-education for act (react). we believe re-assessment of adherence to therapy and reviewing correct technique is necessary at every clinic visit. continuous quality improvement (cqi) techniques have recently been employed in medicine to improve quality of care. although there is evidence that cqi improves efficiency in hospitals, little is known about effects of cqi on improving patient outcomes. we have previously reported creation of a unique nutritional risk pathway (nrp) to care for cystic fibrosis (cf) patients who are at nutritional risk. the goal of this study is to assess perceived impact and objective clinical efficiency of our nrp. patient data was recorded from all children attending the cf clinic over an -month observation period (between sep and mar ). bmi percentiles were assessed and nutritional risk zones -green: acceptable (bmi > %ile), yellow: at risk (bmi - %ile) or red: at urgent risk (bmi < %ile) -assigned at each visit. the visit intervals were every to weeks based on severity of nutritional risk. patients that remained in the red zone for an average of to months with no improvement were considered for gastrostomy tube (gt) placement. the cf dietitian updated the data weekly and e-mailed quarterly to cf physicians for review of their patients' status. patient/family perceptions and understanding of the nrp was assessed by questionnaire. of the children in the study, ( %) were identified to be at nutritional risk and were enrolled into the nrp: in red and in yellow zones; ( %) male, ( %) female; ( %) age < yrs, ( %) age ≥ yrs. over the -month observation period, ( %) showed improvement ( moved from red to yellow, from red to green and from yellow to green zones). thirteen ( %) females improved, whereas ( %) males improved. in the under yrs age group, ( %) improved compared to ( %) in the ≥ yrs age group. as a result of participation in the nrp, ( %) children had gt placed: six of the showed improvement with four moving from the red to green zone. from the survey responders, % stated they understood the meaning of the cf nrp. all responders were extremely to somewhat motivated to focus on their child's nutrition when asked to return for growth monitoring or follow-up visits. having specific goals for weight gain, calorie requirements and bmi were considered extremely helpful by % of families. when gt placement for their child was recommended, % felt they lacked all the necessary information to make a decision. our new cf nutrition education booklet was not received by % of families. some of the physicians who received the quarterly nutritional patient data provided positive feedback and found this information beneficial for patient care. nutritional cqi in our cf center utilizing a specific nrp and monitoring of nutritional risk status resulted in improved patient outcomes. overall perceptions of our nrp were positive from both patients and cf physicians. these results suggest that this cqi strategy improved clinical outcomes in our patient population. patients with cf require frequent healthcare visits to optimize growth, initiate early interventions and delay progression of lung disease. the cff recommends evaluation at a cf center at least quarterly. as part of a llciv quality improvement (qi) initiative, our cf team set a goal to increase the percent of cf patients attending clinic or more times a year to % or greater by developing strategies to monitor and improve adherence rates. methods: we reviewed cf patients who attended clinic times or less in to determine patterns of non-adherence in regards to age, gender, distance from center, season, physician or insurer. a patient survey was used to assess clinic attendance barriers from a patient/family perspective. a brainstorming session identified other barriers. a fishbone diagram was drafted into barrier categories: external, internal, communication, and patient/family perceptions. after reviewing all barriers, we concluded the most effective improvements could be made to internal clinic barriers. clinic processes were then assessed including making of appointments, reminder letters/calls, a time cycle analysis, and number of cf appointments available per patient per physician. our family advisory board participated by focusing on the importance of or more clinic visits in their newsletter and by assignment of a member to the llciv team. we discovered a lack of a standardized process to follow-up (fu) patients who "did not keep appointments" (dnka). a spreadsheet was created to monitor physician dnka rates. a primary fu strategy was devised to identify patients who dnka through daily auto-emails to the cf office assistant and utilization of a rescheduling decision tree for reappointment of these patients by our appointment center. two secondary fu strategies were developed. the first involves the clinical coordinator printing a monthly report off port cf entitled "patients due a visit" then emailing this list to the cf team to contact and reappoint. the second involves the cf social worker tracking patients seen per quarter on a spreadsheet and emailing the spreadsheet quarterly to the cf team as well as posting on our datawall. results: in , and , %, %, and % of our patients attended clinic or more times, respectively. in in , and , %, %, and % of our patients were at nutritional risk, respectively. in and , mean fev % predicted was . % and . %, respectively. conclusion: the number of cf patients attending clinic quarterly at our center increased by implementing processes to track dnka patients and reappoint them to clinic promptly. however, some of the early improvement in clinic attendance was partially due to an increased awareness of our qi efforts by our clinic staff, cf team and patients/families. we speculate that improvements in nutritional status and lung function are at least partially related to more frequent clinic attendance by our patients. continued monitoring of the number of dnka patients and patients who attend clinic quarterly is required to sustain these gains in improved patient outcomes. data when we first measured patient/family satisfaction in , parents and children were given questionnaires to measure their satisfaction with services provided at the clinic. at that time, responses in our survey were very positive. with regards to our new model we received very positive feedback, encouraging us to continue with the model. parents and children liked the elimination of duplication in questions and they also like having the whole team involved in the meeting and problem-solving. final results for our second satisfaction questionnaire will not be available until june of this year as the questionnaires are being given at clinics over a month period this spring. the children complete a questionnaire with the help of their parents if needed and the parents also complete a survey. results to date are very positive for both the parents and the children. of the children who have completed the survey to date, all stated they would like to continue to meet with the group as a whole as opposed to meeting individually with team members. of the parents surveyed to date, all are either very satisfied or satisfied with the group format. conclusion: we have not yet completed the survey but results to date clearly show that both the patients and their families are happy with the round table model. results will be compiled early this summer and would be ready for the poster and/or presentation in the fall. the cff clinical care guidelines recommend routine clinic visits for assessments, interventions, monitoring, education and counseling every months or more often as indicated. adherence to these guidelines is important in ensuring better clinical outcomes. in reviewing our cf center data from the cff registry report, . % of pediatric patients ( - yrs.) and . % of adults ( yrs. and older) adhered to the guidelines. national goals were not provided then; instead data was given for the top ten cf centers ( . % and . % for pediatric and adult patients respectively) as well as the national rates ( . % and . % for pediatric and adult patients respectively). our cf care center acknowledged the need to improve adherence to these guidelines and at the beginning of the year , embarked on a quality improvement project to address this issue. following the pdsa (plan-do-study-act) steps, we agreed on the objective of increasing the percentage of pediatric and adult patients adhering to the guidelines by at least % and at the same time, to exceed the national average in year or by the end of the year . the physicians and social worker obtained feedback from patients and/or families during a clinic visit regarding barriers in complying with scheduled clinic visits. suggestions were elicited to overcome these barriers. the team members then identified barriers in meeting the guidelines and implemented measures to improve compliance with clinical care guidelines. among these measures were sharing the information about clinical care guidelines and importance of adherence through our cf newsletter and at every clinic visit, reminder calls to patients or caregivers at least days prior to the visit, appointments for the next visit given prior to discharge from the clinic, and same day calls to patients who failed to keep their appointment for rescheduling. our social worker identified patients with difficulties or problems in consistently keeping appointments and helped address these problems. progress made in this endeavor was shared with parents and caregivers during the "parents' night" held in the fall of . indeed, with consistent implementation of these measures, we have seen improvement in the percentage of patients adhering to guidelines. in our cf care center data, . % of pediatric patients and . % of adult patients adhered to recommended clinical care guidelines, approximating and in fact for the adults, exceeding, our target of % increase in the percentage ( % and % increase respectively for pediatric and adult patients). these data were better than the national average for ( . % and . % for pediatric and adult patients respectively) but for pediatric patients, still % below the national goal of %. adults approximated the national goal of %. we are committed to continually engage in the process of quality improvement in further improving adherence to clinical care guidelines and to hold on to the gains. weight z-scores and height z-scores were monitored prospectively for all infants, children, and adolescents at a pediatric outpatient cf clinic during an ongoing, multidisciplinary quality improvement (qi) initiative. all cf team members participated in this qi project which emphasized the importance of good nutrition and adequate growth for optimal lung health. specific strategies for improvement included education to families at each visit with a written nutrition action plan, increased emphasis on liquid supplements and enteral feeds as needed, and a consist message from all cf clinic staff in encouraging attention to nutrition. the study objective was to determine if multidisciplinary qi interventions to improve nutritional status had any effect on weight z-scores and/or height z-scores over a -year period. the null hypothesis was that z-scores for weight and height would either not improve or worsen. inclusion criterion for this study was attendance at cf oupatient clinic during the first months of the qi initiative (time ) with a subsequent clinic visit years later (time ). patients not attending clinic at both time and time were excluded. a cohort of patients ( male, female) met the inclusion criterion. the mean time to time interval was . ± . years (range . to . years) with mean patient age at time of . ± . years (range . to . years). pancreatic enzyme replacement therapy was used for ( . %) of the cohort. paired sample t-tests indicated significant improvements for weight z-score (p <. , t ( ) = . ) and height z-score (p <. , t ( ) = . ) after the -year intervention. at time , children ( . %) were underweight or less than th percentile while children ( . %) were normal-weight, at or more than th percentile for age. mean differences in both weight and height z-scores in the initially underweight patients were greater than those for the normal-weight patients over the year interval. no significant differences were found for the initially normal weight-for-age children between time and time for weight z-score ( p = . ) or height z-score (p =. ). children who were not underweight at time grew normally, without acceleration over the yr intervention. therefore, we conclude that a longterm multidisciplinary qi project to enhance nutritional status resulted in significantly improved weight z-scores and height z-scores for those individuals who were initially underweight for age. the most common cause of morbidity and mortality for patients with cystic fibrosis (cf) is pulmonary exacerbations. these exacerbations lead to hospital admissions and treatment with intravenous (iv) antibiotics. all cf patients at our facility are admitted to a teaching service which includes house officers and students. standardized order forms are used to improve accuracy and timeliness of medications and therapies. the efficiency of this process was brought into question in / , when several parents complained about a delay in the receipt of first dose iv antibiotics after hospital admission. the objectives of this study were to determine what were the actual delivery times of the iv antibiotics to the patients (both retrospectively and prospectively), and areas where improvements could be made. our predetermined target time was < hours. two analyses were performed in order to answer the objectives. the first analysis was done in a retrospective fashion. this analysis included cf patients admitted from / / - / / . a total of patients were included. total median time from admission to receipt of first dose of iv antibiotics was analyzed. a second collection of blinded prospective data was performed between / / - / / . only the pulmonologist, pharmacist collecting data, and pharmacy clinical manager were aware of the data collection. a total of patients were included in the prospective project. each part of the process model were recorded and analyzed so that areas of improvement could be realized. the median time from hospital admission to first dose of antibiotics was hours (range - hours) (retrospective cohort). the prospective cohort median time from hospital admission to first dose of antibiotics was hours (range min- hrs). the median time from admission to order entry by pharmacy: hrs, min (range min- hrs, min). the median time from the medication order being written by medical practitioner to order entry by pharmacy: minutes (range min- hrs, min). the median turn-around time by the hospital pharmacy of order entry until reaching the nursing unit: min (range min- min). the median time required by the nursing staff to administer the antibiotic: hrs, minutes (range min- hrs). this project was initiated in response to parental concerns. from the results of the retrospective and prospective analyses, it was determined that a delay is occurring in the receipt of iv antibiotics, which translates to hours in the hospital without therapy. this is both inconvenient and uncomfortable to the family, and wasteful of medical resources. the findings of this project were presented to the pharmacy and therapeutic committee of the hospital, to the nursing directors, and to the parents of cystic fibrosis patients. the delay is greater than expected before this project was initiated. as a result of these findings a "fast track" medication order set was developed to expedite initiation of inpatient therapy. four key areas included in this order set are sputum, iv access, diet, and iv antibiotics. we speculate that time to first drug delivery will decrease and patient satisfaction with improve. the pediatric intermountain cystic fibrosis center (imcfc) provides multidisciplinary cystic fibrosis (cf) care to pediatric patients from utah and parts of idaho, wyoming, and nevada. historically the imcfc has embraced early intervention. as new therapies have been added to the cf armamentarium it has become necessary to have a method of keeping track of which patients are candidates for various therapies and also which medications have been prescribed, utilized or even should be discontinued. in order to address this quality issue, the imcfc decided to develop and implement the use of a medication tracker (mt). the imcfc decided to focus on key therapies which were felt to be at risk for variability in prescriptive practice. the five therapies included were: pulmozyme<®>, tobi<™>, hypertonic saline(hs), azithromycin and asthma medications (inhaled corticosteroids and/or leukotriene receptor antagonists). ibuprofen was intially included and later removed from the mt. prior to implementing the mt, the imcfc met to review literature, medication trackers from other centers, and develop consensus among team members. six versions of the mt have been developed between / / - / / . each mt has been tested in a pdsa cycle and revised. since initating the mt, patients have been seen at least once. there have been mt encounters. the mt is used only after the first visit to the imcfc and reflects changes to established cf care. there have been a total of changes in prescribed therapies in different patients( % of patients seen). a minority of patients were receiving therapies that did not meet our pre-determined consensus for prescription. the most common therapies in this category were tobi™ and azithromycin. primary reasons cited for prescribing were: chronic suppressive antibiotic use, pseudomonas positive and < years old, and significant changes on chest imaging. reasons patients were not prescribed clinically indicated therapies were: patient refusal, participation in clinical trials, cost, and lack of compliance with other therapies. changes in asthma therapy included the discontinuation of medication in encounters and the initiation of therapy in . the use of a specific cf patient mt allows consistent practice in selection of therapies. it can be used by all team members and helps to identify discrepancies in therapy. assuring all patients receive information and are considered for new therapies is increasingly important as our treatment options expand. the mt also highlights the need to consider discontinuing therapies. we speculate the mt will become an increasingly valuable tool over time and improve patient care. rationale: recent reports document increasing prevalence of antimicrobial resistance across a range of pathogens in patients with cf. the increase in multidrug resistance of these organisms complicates the therapeutic management of these patients. objective: to describe patterns of antibiotic use among physicians treating cf patients. methods: for the original purpose of evaluating site characteristics that act as effect modifiers on the relative efficacy or cost of novel therapies, we administered a survey to acquire information on practice patterns and characteristics of physicians participating in a clinical study of an investigational drug for patients with cystic fibrosis. chi-square and spearman rank test were used to evaluate associations between physician characteristics and patterns of reported antibiotic use. results: fifty-nine physicians, representing study sites, completed the survey. ninety percent reported a medical specialty of pulmonology and % reported practicing at a teaching hospital. on average, respondents reported that about half of their practice consisted of patients with mild cf (defined by fev ≥ % of predicted normal.). forty-two percent of respondents reported prescribing oral antibiotics and % of respondents reported prescribing inhaled antibiotics for maintenance therapy for at least % of their mild cf patients. eight-five percent and % of respondents reported prescribing iv antibiotics for pulmonary exacerbation and pulmonary 'clean out', respectively, with about a third of these cases being prescribed for outpatient iv use. physicians that prescribed iv antibiotics for pulmonary clean out were more likely to pre-scribe oral (p= . ) and inhaled antibiotics (p=. ) for maintenance therapy than physicians that did not prescribe iv antibiotics for pulmonary clean out. conclusions: antibiotic use for maintenance therapy and iv antibiotic use for pulmonary exacerbation and pulmonary clean out acted as 'complements' rather than 'substitutes' as there was a group of physicians who reported less use of antibiotics. our findings warrant further research as well as additional exploratory analyses with a larger and broader sample to examine associations between physician characteristics and patterns of antibiotic use. understanding the relationship between physician characteristics and antibiotic use has the potential to influence the approach to management of cf patients in an environment of increasing antimicrobial resistance. introduction: hypertonic saline (hs) has been shown to increase mucus clearance and improve lung function in cf, and is now increasingly being used as part of routine therapy. one of the goals of our cf center quality improvement (qi) initiative is to increase use of hs among all eligible patients. objectives: to assess adherence to treatment with hs, parental opinions on treatment, perception of side effects, and the effect of initiating hs on the use of other nebulized medications in pediatric cf patients started on hs therapy during . methods: patients under years of age were identified as initiating hs therapy during . a telephone survey of their parents using a -item multiple-option questionnaire was administered in april as part of our cf center qi initiative. results: parents ( %) completed the survey: ( %) patients were still using hs. of those still reporting use of hs, ( %) were using it twice a day; ( %) once a day; and less often than daily. among the patients who discontinued hs, ( %) stopped following recommendations from their pulmonologist; ( %) stopped due to side effects; ( %) felt hs took too long to administer; and ( %) felt hs solution was too difficult to mix. the most common side effect reported was cough in patients ( %); ( %) reported no side effects. the majority of patients ( %) spent - minutes per day on hs therapy. thirteen ( %) patients discontinued another nebulized medication when they initiated hs; ( %) of those stopped pulmozyme. reasons for stopping pulmozyme included: time to nebulize both medications was too long ( %), perception that hs was working better for their symptoms ( %); and doctor's instructions ( %). overall, ( %) parents felt hs improved their child's symptoms; ( %) saw no difference; and ( %) believed hs made symptoms worse. there was a significant difference in the proportion of patients that reported improvement of their symptoms in the group using hs twice a day ( / , %) when compared to those using it once a day ( / , %) (p= . ). discussion: the majority of patients continued to report the use of hs therapy several months after our initial intervention, with the most parents reporting improvement in their child's symptoms. physician recommendation was the most common reason cited for discontinuation of hs therapy. further study of physician opinions on hs therapy, particularly the timing of initiation and discontinuation, is warranted. one-half of patients reported using hs only once a day; however those reporting twice daily use were more likely to report subjective improvement of symptoms. since treatment complexity in cf increasing, determining the effectiveness of once daily hs therapy, either alone, in place of, or in combination with other therapies such as pulmozyme is essential. long-term follow up of these hs-initiated patients will focus on effects on exacerbations, pulmonary function, and hospitalizations. the uab/children's hospital (chs) cf center participated in learning and leadership collaborative ii. as part of the qi journey, the chs cf qi team thoroughly evaluated the current system of care in an effort to identify areas of needed improvement. one area assessed was clinic attendance. it was discovered that % of patients failed to attend clinic appointments every week. according to cff care guidelines, patients with cf should be seen a minimum of times each year at the cf center. the cff has also found that those centers that see their patients more frequently have improved outcomes for fev and nutritional status. aim to decrease the number of missed appointments at the chs cf clinic. the chs cf center administered a qi survey to identify the reasons patients and families were missing appointments. this survey was a item questionnaire that evaluated: reasons for missed appointments, if the families rescheduled missed appointments, difficulties with rescheduling, the need for reminder calls, distance traveled to the cf center, and insurance status. results completed surveys were collected. % of the families reported medicaid as their insurance provider. % of families live more than miles away from the cf center. % of the families confirmed that they had missed an appointment in cf clinic over the last months. the reasons for the missed appointments were widely variable; however, . % of those who missed appointments reported that they did not have access to transportation to get to the appointment. overall, families did not report difficulties in rescheduling appointments. % of those that missed appointments identified medicaid as their insurance which can be correlated with lower income families and . % of those that missed lived greater than miles away from the cf center. % of families reported that a reminder call would be helpful for them. interventions after evaluation of the survey data, the center quickly initiated reminder calls for cf clinics. a student employed by the cf center made these telephone calls to the patients scheduled for clinic every week. the majority of the time the student caller left a message for the family. with the implementation of this simple intervention, missed appointments have decreased from % to % which is a % improvement rate. reminder calls are now a routine part of the cf center's system of care. other ongoing interventions include increased involvement of primary care doctors for patients lost to follow up for more than months and a system for quickly rescheduling patients. next steps in addition to reminder calls, the cf team will now determine where patients who routinely miss appointments live. if there are pockets of patients in common areas of the state, the team will attempt to maximize services for those patients such as linking them with community transportation resources, community health advocates, or providing satellite clinics. this will be accomplished through geomapping of the alabama cf population based on zip code. we designed a scoring system to uniformly identify patients experiencing a pulmonary exacerbation. the elements of this pul-monary exacerbation score (pes) have been previously described. at the time this project began the median fev % predicted of the cf patients at our center - years old was below the national average ( cf registry data). our hypothesis was that greater uniformity of identification of pulmonary exacerbations, would improve pulmonary function in the pediatric age group at our center. this abstract describes continued improvement in the pulmonary function of this age group at our center now that the pes has become standardized in our clinical practice. methods: beginning in october , a pes was calculated for all patients presenting to our center age - years of age. any patient with a pes of > was defined as having a pulmonary exacerbation, and treatment with antibiotics was recommended. the patient's cf physician dictated the ultimate decision for treatment and specific course of treatment. median fev of all patients age - years was calculated quarterly to determine the effect of the pes use on pulmonary function. in october the pes was incorporated into our cf clinic medical record and its use was standardized. the use of the pes, the adherence to the recommendation for treatment, and quarterly fev of the population has continued to be monitored. results: from / to / (the duration of the original qi project), patient visits occurred, with a pes calculated on (utilization of . %). patients were treated for a pulmonary exacerbation of the patients with a pes of > (adherence of . %). . % of exacerbations were treated with oral and/or inhaled antibiotics, and . % were treated with intravenous antibiotics. since standardization of the pes in / , visits have occured. use of the score has decreased slightly ( . % utilization), however adherence to the score has increased ( . % of pes > have been treated). use of oral vs. iv antibiotics has been similar ( . % oral vs. . % iv). the median fev of our patient population has improved from an average of . % predicted during the months before implementing the pes to an average of . % predicted during the ensuing months of the original qi project (a . % improvement). since standardization of the pes in / , the average median fev of the population has continued to increase to . % predicted (an . % increase from the baseline period). this improvement in median fev is reflected in our cf registry data. summary: implementation of a pulmonary exacerbation score, designed to encourage uniform identification of pulmonary exacerbations in patients with cf, is associated with an improvement in median fev in children - years of age. standardization of the pes has resulted in continued improvement in pulmonary function of this population at our cf center. this work has been supported in part by the cff and akron children's hospital. our cf family council parent and patient advisory group has been involved in the development and implementation of this project. utilizing data from the cff patient registry, we determined that our center has had nutritional outcomes consistently below the national average for pediatric and near the average for our adult patients. as a result, we developed the global aim to improve and maintain optimal growth and nutrition in all patients at our cf center. our specific aims included: )to record growth parameters at all outpatient visits; )to identify patients at nutrition risk, categorizing them based on anthropometric data; ) to develop and implement a specific nutrition plan based on nutritional category; and ) to educate staff, patients,and families on the benefits of normal growth and nutrition. we surveyed our patients to determine their overall attitudes toward nutrition and nutritional care received at our center. we discovered that many patients and families did not feel that nutrition was addressed at every cf visit nor that they had a nutritional plan at each encounter. this led to the development of an intake form/home-going sheet for our patients to complete that included data elements necessary for assessment of nutrition and that clearly outlined a nutrition action plan. a standardized nutritional assessment score (nas) was devised that incorporated weight, height/length, bmi and weight for length percentiles as well as weight loss, failure to gain or maintain, and failure to remain in the expected growth channel. the nas was developed after examining the medical literature, reviewing work done by other qi teams, and acquiring input from our pediatric gastroenterologist and endocrinologist. we then developed nutritional algorithms to address nutritional intake, malabsorption, evaluation for cfrd, and short stature. these four algorithms were used to develop a nutrition action plan for each individual. beginning in march , a nutritional category (optimal/acceptable, concerning, at risk or failure) was assigned to each patient at each visit based on their nas. an individualized nutrition action plan was developed with each patient. at the time of project implementation, patients age - at our center had a median bmi %ile of . % with . % of our patients with a bmi < th%ile. our patients > years had a median bmi of . with . % having a bmi< th%ile. after one month of implementation of the nas, % of our patients had been categorized with % of our patients having individual nutrition action plan developed. to track our progress, we will monitor median bmi, % of patients with bmi < %ile, and % of our patients in each nutritional category quarterly using portcf. our goal is to have all our patients assessed and a nutrition action plan implemented by the end of . we have developed a nutritional assessment score and nutrition treatment algorithms for pediatric and adult patients with cf as part of a qi initiative to improve nutritional status of patients at our center. the project has been supported, in part, by akron children's hospital. we are indebted to lori lundquist, a parent of two of our cf patients, who has been an integral part of our nutriton qi team. in germany there are about , cf patients who are usually treated in special centres by a team of trained and experienced health care professionals. the result of this structured approach to patient care, including frequent clinical assessments, monitoring, and aggressive interventions is an improved mean survival and a higher quality of life of pediatric and adult cf patients. however, a recent survey showed that german reimbursement schemes cover only about % of resources used within these centres. objective to assess and evaluate direct costs of outpatient treatment of cf patients in germany. a micro-costing approach was used to record resource use data directly within representative cf centres. results of the evaluation may be used to initiate and support discussions between health care providers and insurances about adequate reimbursement schemes in germany. outpatient care was evaluated in seven different centres for pediatric and adult cf patients. patient reported outcomes, clinical patient data, resource utilisation, and physician related time consumption for treatment was recorded for every patient during one representative month in . cost data for staff, materials, medical equipment, rooms, etc. were assessed within the respective centres. data for cf patients were collected. about half of them were children and adolescents, and half of them were adults. a comparison of the resource uses to the actual remuneration of these services indicates that only about % of the costs are covered by the reimbursement system. correlation analysis identified significant cost drivers like the age of the patient, comorbidities like pancreatic insufficiency and hepatobiliary complications, lung function (% fev ), and the presence of certain pathogens in the lungs. as the actual reimbursement covers only about % of the resource usage costs in germany, the existence of the cf centres and sufficient treatment for cf patients is uncertain in the future. in order to ensure the existence of the centres it would be necessary to agree to a cost-covering reimbursement at minimum. this may be based on a lump-sum payment that could be differentiated for pediatric and adult centres or according to comorbidities or clinical parameters. the data calculated in this study could be used to trigger and support discussions between health care providers and insurances about a new cost-covering reimbursement system for the out-patient treatment of cf patients in germany. the impact of the new us cystic fibrosis foundation (cff) nutrition practice guidelines, i.e., discontinuing the use of percentage of ideal body weight (%ibw) to define "nutritional failure" and setting body mass index (bmi) below the th percentile to define "nutritional risk", on evaluating nutritional care practices is unclear. methods: data from children reported to the cff patient registry were analyzed to compare the rates of malnutrition among cf centers. results: nationally, eliminating %ibw < % as a criterion for underweight resulted in a . % reduction (from . % to . %) in "nutritional failure" rate. misclassification of "nutritional failure" by %ibw < % ranged . - . % among centers and was greater for centers having a larger proportion of tall patients. one-third of centers switched to a different tertile ranking, after correcting for misclassification by %ibw. use of bmi < th percentile led to the classification of . % of patients as "nutritional risk". more than half of the centers had different tertile rankings on "nutritional risk" compared to "nutritional failure". a total of . % ( - . % among centers) of patients who had height < th percentile but bmi ≥ percentile were not considered at nutritional risk. the cystic fibrosis questionnaire-revised (cfqr) is a disease specific quality of life measure that is currently undergoing clinical validation and may prove to be a useful adjunct to provider assessments. we proposed the routine use of the cfqr in a busy adult cystic fibrosis (cf) clinic would assist in identifying patients in need of limited clinic resources. this performance improvement project focused on new and infrequently-seen patients (i.e. fewer than three outpatient clinic visits/year) to determine the frequency of patients with domain scores that fall more than one standard deviation below our clinic mean. methods: the cfqr is routinely self-administered to all adult cf patients in clinic at least once every year. these assessments occur after vital signs are obtained and prior to any other clinical assessments. patients were deemed to be at baseline if there were no changes made to their pharmacologic or chest physiotherapy regimens. the mean and standard deviation of each domain of baseline cfqr assessments were determined. the frequency of rarely seen and new patients with domain scores less than one standard deviation below the mean was determined. results ( ). of these baseline assessments, forty five were obtained from patients who were rarely seen or new to the clinic. . % (n= ) of these patients had one or more domain scores falling more than one standard deviation below the mean. . % (n= ) of these patients had two or more domain scores below these cutoffs. % (n= ) of the rarely evaluated patients had domain composite scores (i.e. the sum of the individual domains) that fell below the standard deviation cutoff. the most common outlying domains in these infrequently seen or new patients were body ( ), weight ( ), physical ( ), health perceptions ( ) and the composite domain ( ). conclusions: the cfqr is easy to administer and score during routine adult cf clinical visits. as many as % of our infrequently evaluated patients had multiple scores that were more than one standard deviation below our clinic mean. we conclude that the cfqr is a useful adjunctive measure to provider assessments and can help focus limited personnel resources. aminoglycoside antibiotics are known to be toxic to the inner ear. however, they continue to have a leading role in the treatment of certain infections and in the treatment of pulmonary exacerbations in patients having cystic fibrosis (cf). while there is ample evidence in the literature of ototoxicity, and there are established protocols for monitoring the effects of toxic agents on hearing, less is known about the prevalence of vestibular toxicity among patients who are exposed, and there are no commonly accepted protocols for monitoring vestibular system function. oscillopsia and unsteadiness when standing and walking are the two most commonly reported and disturbing symptoms associated with bilateral loss of vestibular system function. oscillopsia is the perception that viewed stationary objects or surroundings move coincident with head movement. when it is severe, oscillopsia can prevent an individual from having clear vision with even the slightest head movement. the unsteadiness that accompanies vestibular loss can range from mild to disabling. because of the referral of a number of patients to the vestibular testing center (vtc) at the university of michigan in whom severe bilateral vestibular loss was evident secondary to apparent aminoglycoside toxicity and our concern about quality of life issues, we initiated a quality improvement clinical protocol with our cf patients. vestibular system function and hearing are evaluated at the time of admission for pulmonary exacerbations and coincident with the three month follow-up visit with the pulmonologist. our goal is to understand the incidence of ototoxicity in this patient population, and to determine how best to measure incremental changes over time. the ultimate desired outcome is to investigate the efficacy of protective agents for limiting the toxic impact of these drugs on hearing and vestibular function. to date, we have completed vestibular testing on patients with cf ranging in age from - years, some of whom were referred prior to the initiation of this monitoring program. of the sample, individuals have completely normal vestibular test results, and of the had no prior aminoglycoside exposure. of the remaining patients, have bilateral vestibular loss ranging from mild to severe, two have evidence of a unilateral vestibular loss, and each of the others has non-lateralizing evidence of vestibular involvement. we have also documented change in the three individuals we have seen for repeat testing. specifically, repeat testing has shown incremental decline in vestibular function with repeated exposure to aminoglycosides. interestingly, only individuals in the group have documented hearing loss, and although most report that they do not and have never had any problems with dizziness or balance, many patients have evidence of oscillopsia or abnormal postural control test results. it is clear from our limited data that it is important to monitor vestibular system function whenever potentially toxic agents are used. while monitoring hearing is also warranted, our data suggest that a monitoring program that includes only hearing is insufficient. background: the correlation between bmi, lung function, and longterm outcomes in cystic fibrosis patients has been well documented. based on this data the cf foundation (cff) has established adult patient care guidelines for bmi of or greater for women and or greater for men. in our cf center implementation of nutrition therapies in cf adult patients has been difficult due to lack of acceptance of proposed interventions. due to the importance of optimal nutrition to attain the cff recommendation for bmi, university of cincinnati adult cf center implemented a weight management quality improvement protocol to improve the nutritional status of patients below the cff recommended bmi. our aim is to improve to the nutritional outcomes in our center through patients' acceptance of our nutrition interventions. strategies: as part of the weight management protocol we developed a nutrition action plan, nutrition algorithm, and weight management education record and learning log. the action plan took a systematic approach to weight management, classifying patients into different risk categories based on bmi. patients were categorized as mild nutritional insufficiency (bmi - ), moderate nutritional insufficiency , and severe nutritional insufficiency . each category required a specific action to address weight loss or inability to gain weight. areas of focus for the action plan include; intake assessment through computerized dietary analysis, assessment of pancreatic function, pancreatic enzyme replacement therapy (pert) regimen assessment, hour stool collection for malabsorption, assessment of glycemic control, assessment for gerd, and enteral/parental feeding. the dietitian completed a chart review and nutrition assessment with the patients and classified them into one of the three nutritional risk categories. eleven patients with a bmi below were initially identified. nine ( %) of the eleven patients with a bmi of or less have been approached with the protocol and algorithm. patients completed a three day food journal and oral intake was assessed via computerized dietary analysis. oral enteral supplements were recommended - times per day. g-tube placement was discussed and an educational material on tube feeding was distributed. the protocol and algorithm guided identification and assessment of contributing factors to weight loss. results: of eighty patients seen in the adult center since the beginning of october, thirty-seven of the patients ( %) have a bmi below and eleven patients ( %) have a bmi below . out of the nine patients approached with the protocol % (n= ) completed and returned the three day food journal. of the patients that completed their food journal % (n= ) agreed to a peg placement, one of the patients is still contemplating peg placement while sorting through some psychosocial issues, and the other patient has had a differential diagnosis that might negate the necessity for a peg placement. conclusion: we developed a systematic approach to weight management within our adult cf program. this systematic approach to weight management, which includes patient involvement and specific education, has improved the overall acceptance of nutrition interventions in our adult population. the inpatient care of adults with cystic fibrosis can be a challenging given complicated medical regimens, specialized respiratory care orders, and unique dietary requirements. a large proportion of these patients are admitted to academic centers with rotating house staff that may have never treated an adult with cystic fibrosis. to improve the quality of care of our adult patients we have implemented a standardized electronic order set, a recurring educational program, and a "pocket card" to help the house staff. the adult cystic fibrosis program at vanderbilt cares for over patients, with - inpatient admissions per month. vanderbilt's inpatient order system is completely electronic. we have devised an order set based on our agreed standard of care for inpatients with pneumonia. the order set guides the house staff through most aspects of inpatient care including culturing, antibiotic choice and dosing, dietary consultation, nutritional recommendations, and respiratory care orders. this order set not only educates the house staff on our standard of care but also provides direct access to each order they may wish to enter. we have reiterated this educational process with a pocket sized handout that guides them through the typical issues in adults with cf as well as provide phone numbers to members of the cf team. these resources are presented in short talk given at the beginning of each month to incoming house staff. a preliminary data analysis suggests that over % of all adults with cf are admitted using some component of this order set. an informal survey of the house staff also concludes that the pocket card and lectures have been helpful. all three aspects of this quality improvement have been implemented since december of . we plan to formally test the impact of this intervention when a meaningful number of house staff have been through the service. * background: malnutrition is a common complication of cystic fibrosis (cf). the median bmi percentile in cf patients between the ages of and years is . % but ranges between various centers from . to . percent. previous research has shown a trend toward improvement of pulmonary disease and a significant improvement in weight, height, and bmi z-scores after nutritional supplementation via a gastrostomy tube ; furthermore, fev is positively correlated with bmi percentile. higher weight at three years of age predicts better pulmonary function at six years of age. the effect of gastrostomy tube feedings on pulmonary exacerbation frequency has not been well defined. hypothesis: the incidence of pulmonary exacerbations in children will decrease after the initiation of supplemental gastrostomy feedings. methods: a retrospective chart review of all pediatric patients seen at the cmh cf center, and who received gastrostomy tubes between and was performed. data was evaluated in six month time intervals for two years prior and two years after gastrostomy tube placement. we assessed weight, height and bmi percentiles, fev percent predicted, number of courses of exacerbation therapy (intravenous and oral) and microbiologic data. results: we identified patients, females and males, with ages ranging from months to years of age ( . years mean) at time of gastrostomy tube insertion. all patients had increases in their weight-forage percentile (mean of all patients of . percentile pre to . percentile post, p< . ), height-for-age percentile ( . percentile pre to . percentile post, p< . ), and bmi percentiles ( . percentile pre to . percentile post, p< . ). the mean fev percent predicted declined overall from . % to . % ( . percent predicted per year) over four years in patients. there was a trend of exacerbation reduction after gastrostomy tube placement from to total courses (p= . ). this decline in exacerbations after gastrostomy tube placement was most dramatic in those patients growing pseudomonas aeruginosa without other organisms, with a decline from total courses to total courses in patients (p= . ). discussion: weight, height and bmi percentile improved after initiation of gastrostomy tube feedings. lung function (fev % predicted) decreased at the rate predicted in spite of gastrostomy feedings. there is a trend toward decrease in exacerbation frequency after improved nutrition with gastrostomy tube feedings, especially in patients infected with pseudomonas aeruginosa. limitations of this study include the small sample size, relatively short follow-up time, and the retrospective nature of the study which did not allow assessment of adherence. conclusion: improved nutrition through gastrostomy tube placement may decrease the frequency of pulmonary exacerbations. evidence of an association of improved pulmonary function with improved nutritional status has motivated aggressive nutritional intervention. percutaneous endoscopic gastrostomies (pegs) are employed increasingly in cystic fibrosis to optimize nutritional status. we reviewed our center data over the past years to assess changes in our peg practice and effects on nutritional and pulmonary outcomes. we hypothesized that our change in practice to earlier peg intervention improved patient's nutritional and pulmonary outcomes. we conducted a case controlled retrospective study of pediatric cf patients spanning the past years divided into two periods: p (peg placed before ) and p (peg placed or later) compared with their respective age/sex matched controls. we compared peg vs. control cf patients with respect to age, nutritional and pulmonary status. we compared weight, height and bmi directly preceding peg placement and at approximately one-year post peg. we compared available data for best yearly fev and fvc before peg and at , and -years post-peg. we then compared data between the two time periods. results: peg patients in p (n= ; mean age . + . yrs.) had lower bmi ( z = - . v. - . , p< . ) pre-peg placement compared to controls. by year post peg, the bmi normalized and equaled controls (z=- . v. - . , p= . ). peg patients' fev %ile prior to placement was lower than controls ( v. , p= . ) as was their fvc %ile ( v. , p= . ) and remained lower at year post-peg. following peg placement, absolute bmi increased ( . kg/m , p= . ), as did bmi z-score ( . , p= . ). we did not see significant changes in pulmonary function tests (pft). peg patients in p , (n= ; mean age . + . yrs.) also had lower bmi (z= - . v. - . , p= . ) pre-peg than controls. at year post-peg, the peg patients still had a significantly lower bmi (z = - . v. - . , p= . ). fev was lower in peg patients compared to controls in pre-, year, and -year post-peg data (p< . ). variability in pfts and small sample size limited our analysis. pre-peg weight, height, bmi, fev and fvc were lower in p patients compared to the younger p patients. at -year post-peg, similar bmi zscores (- . , p= . ) was obtained between the two periods. variability in pfts among the patients within each period prevented valid comparisons, although p tended to have better pft values. conclusions: peg placement was effective at improving nutritional status in patients with cystic fibrosis. placement in younger patients with better pre-peg nutritional and pulmonary status allowed more complete nutritional recovery. we speculate that improved height z-scores with apparently stable fev is consistent with increased pulmonary growth. demonstration of an attenuated rate of pft decline to correlate with this earlier intervention will require a larger population and longer observation period. a multi-center longitudinal outcome study would help determine optimal timing and criteria for peg intervention. . pediatric gastroenterology and hepatology, university medical center groningen, groningen, netherlands; . biochemistery, erasmus medical center, rotterdam, netherlands background: ursodeoxycholic acid (udca) treatment is frequently applied for cystic fibrosis-related liver disease (cfld). it has been hypothesized that udca is beneficial through its choleretic activity, i.e. its capacity to induce bile flow. the hepatic expression of the cftr protein is restricted to the cholangiocytes lining the bile ducts. cholangiocytic cftr is involved in the generation of ductular bile flow. it is not known to what extent the choleretic activity of bile salt treatment depends on expression of competent cftr protein, and whether or not udca differs in this respect from other bile salts. we evaluated the role of cftr in the acute and chronic choleretic effect of bile salt treatment, through comparative studies in cftr-null, homozygous f del, and corresponding wild type control (wt) mice. methods: bile flow was determined after gallbladder cannulation. bile flow during the first minutes after acute interruption of the enterohepatic circulation was regarded as basal bile flow. after min, taurocholic acid (tca) or tauroursodeoxycholic acid (tudca) were iv administered in stepwise increasing dosages, up to nmol/min/ g bw, to cftr-null mice and wt littermates fed a standard chow diet. other cftr-null, homozygous f del mice and their respective wt littermates were fed either the standard chow, or the same diet supplemented with cholic acid (ca, . wt%) for weeks. finally, cftr-null mice and wt littermates were fed the standard chow or the same diet supplemented with ursodeoxycholic acid (udca, . wt%) for weeks. : upon a regular chow diet, the basal bile flow was similar in cftr-null and homozygous f del mice, compared to their respective wt littermates (cftr-null, . ± . vs. . ± . ; and, f del(∆/∆), . ± . vs. . ± . µl/min. g bw, resp.; ns). iv administration of tca or tudca to cftr-null mice and wt littermates increased bile flow to similar extents. dietary ca treatment increased basal bile flow significantly less in cftr-null and homozygous f del mice than in their respective wt littermates (cftr-null, . ± . vs. . ± . , p< . ; and, f del(∆/∆), . ± . vs. . ± . µl/min. g bw, p= . ; resp.). interestingly, dietary udca treatment increased basal bile flow more profoundly than ca treatment, and to similar levels in cftr-null mice and wt littermates (+~ %, . ± . vs. . ± . µl/min/ g bw, resp.; ns). conclusion: upon chronic treatment, udca is highly choleretic in mice, independently of the presence of functional cftr. the independence of functional cftr is udca specific, since the choleretic activity of chronic ca treatment is diminished in cftr-null and homozygous f del mice. we speculate that this specific, cftr-independent choleretic effect of chronic udca treatment could be therapeutically important for cystic fibrosis. most cystic fibrosis (cf) patients have mild to moderate focal portal tract changes (ductal obstruction and cholangitis) and hepatosteatosis. etiology of severe liver disease (ld)with cirrhosis and portal hypertension which occurs in - % of cf patients is unknown. the c bl/ j congenic cf mice develop progressive cf-like ld. as a useful therapeutic model, we have shown that dietary addition of docosahexaenoic acid (dha) significantly ameliorates inflammation but has little effect on ductal obstruction (am j physiol gastrointest liver physiol : g -g , ) . we hypothesized that treatment with udca,a hydrophilic bile acid will ameliorate portal tract ductal obstruction and have an additive effect with dha in preventing ld. methods: thirty-day old wild-type and cf littermates are fed peptamen and either olive oil or dha, with and without . % udca ( groups, mice/group). mice are killed following days of treatment and bile salts, serum liver enzymes and serum and tissue lipids analyzed. h&e stained liver tissues are coded and assessed blindly by an expert hepatopathologist (jp) for evidence of hepatosteatosis, inflammation, bile duct obstruction, fibrosis and other signs of liver pathology. an arbitrary scale of to is used, with representing no pathology and being the most severe. we present interim statistical analysis performed for pathology results only for untreated mice (wt, n= ; cf, n= ); treated mice with dha, (wt, n= ; cf, n= ), treated mice with udca (wt, n= ; cf, n= ) and treated mice with udca and dha (wt, n= ; cf, n= ). results: mean pathology scores from groups of mice are pooled to examine the effects of genotype, treatment and diet (table) . at present,the bile,lipid and liver biochemical test data are insufficient to perform statistical analysis. udca-treated mice have significantly increased bile duct obstructed scores compared to the untreated mice which subanalyzed (not shown) reveals increased bile duct obstruction in the wild-type littermates and no effect in cf mice. hepatosteatosis and inflammatory scores in the untreated mice (fed olive oil) are increased compared to the mice treated with dha. summary: we anticipate completing and performing final analysis within six months. however, these preliminary data confirm that dha reduces inflammation in c bl/ j mice livers and suggest that udca therapy has an adverse effect in wt mice and is not effective in ameliorating liver disease in cf mice. udca with dha therapy does not appear to have an additive effect in ameliorating cfld in this mouse model. supported by axcan pharma inc. background the cf mouse intestine has an innate response associated with small intestinal bacterial overgrowth (sibo). a previous affymetrix genechip analysis of the cf mouse intestine showed altered expression of several eicosanoid metabolic genes. eicosanoids are biologically active arachidonic acid metabolites with a variety of pro-and anti-inflammatory actions as well as effects on mucus production, electrolyte transport, and gastrointestinal motility, all of which are of potential importance to the cf phenotype of the intestine. methods wild type (wt) and cf mouse (cftr tm unc ) littermates congenic on the c bl/ j background were fed an elemental liquid diet (peptamen). intestinal rna was isolated for quantitative rt-pcr of expression levels of genes involved in eicosanoid metabolism to confirm and extend the genechip analysis. various eicosanoids were measured in small intestinal lavage fluid using specific enzyme immunoassays. results we confirmed microarray results by qrt-pcr that the following genes were differentially expressed in the cf small intestine relative to wt: cyp a (cytochrome p a ; -hete synthesis; % of wt); ltb dh (leukotriene b dehydrogenase; metabolizes prostaglandins and leukotrienes; % of wt); cyp c (cytochrome p c ; -hete synthesis; % of wt); ltc s (leukotriene c synthase; ltc synthesis; % of wt); hpgd (hydroxyprostaglandin dehydrogenase ; metabolizes prostaglandins; % of wt); and pla g (group phospholipase a ; potentiates arachidonic acid generation; % of wt). non-significant or less than -fold changes in gene expression were measured for: cyclooxygenase (cox) genes, pge synthase, pgi synthase, and -and -lipoxygenases. as measured by their stable metabolic products, pge and pgf α were significantly increased in the cf mouse intestine ( % and % of wt, respectively). the following eicosanoids were significantly decreased in the cf mice: -hete ( % of wt), -hete ( % of wt) and -hete ( % of wt). there were not significant changes in the levels of pgi , pgd , ltb , cys-lts, or lxa . the changed levels of eicosanoids are generally consistent with the differential gene expression. conclusions there are significant changes in expression of eicosanoid metabolic genes and certain eicosanoids in the cf mouse small intestine. the increase in pla g and decrease in cyp expression levels are expected to make more arachidonic acid available for eicosanoids except hetes. decreases in degradative genes (hpgd, ltb dh) will prolong availability of prostaglandins. altered levels of eicosanoids are expected to play important roles in the pathophysiology of the cf intestine. elevated pge may contribute to increased mucus secretion which is characteristic of cf. pgf α and pge regulate intestinal motility and small intestinal transit is slower in cf patients and in cf mice. the hetes are mostly known for their effects on vascular tone and electrolyte transport in the kidney but are less-well characterized in the intestine. their dramatic decrease in the cf intestine may affect blood flow, electrolyte transport, intestinal motility, or other gi functions. the specific effects of the altered eicosanoid metabolism in cf remain to be explored. supported by cff grant delisl g . for the scandinavian study consortium. background: progressive pulmonary disease, correlating with igg levels, is a major cause of morbidity and mortality in cf patients. recently, immunosuppressive effects of vitamin d and a potential role of vitamin d deficiency in the pathophysiology of autoimmune diseases have been described. we investigated whether vitamin d deficiency could contribute to the chronic proinflammatory state characterizing cf and the higher occurrence of immune-hyperreactivity-related conditions in cf patients. methods: multiple linear regression analysis, taking the confounding factor of age into account, was used to evaluate the relationship between cross-sectionally determined vitamin d variables and: lung function parameters, inflammatory parameters and immune hyperreactivity markers in patients followed at the cf centers in sweden, norway and denmark. vitamin d intake was based on a -day precoded dietary food record, calculated in the national food databases. findings: in the population of all patients included in the study (n= ), blood vitamin d positively correlated with fev (p< , ) as well as with fvc (p< , ). negative correlation was found between blood vitamin d and igg (p< , ), supplemented vitamin d per kg bw and igg (p< , ), total vitamin d intake per kg bw and igg (p< , ). additionally, lower blood vitamin d levels were related to both presence of pathological ogtt (p< , ) and occurrence of cfrdm (p< , ). correspondingly, food vitamin d (p< , ), food vitamin d per kg bw (p< , ), supplemented vitamin d (p< , ), supplemented vitamin d per kg bw (p< , ), total vitamin d intake (p< , ) and total vitamin d intake per kg bw (p< , ) all correlated negatively with hba c values. most of the studied vitamin d variables were lower in patients with endomysial antibodies positivity (n= ) and higher in patients with abpa (n= ) than in the rest of cf population studied (ns). interestingly, in children (n= ) blood vitamin d correlated negatively with transglutaminase levels (p< , ) and there was also a significant negative correlation between hba c and vitamin d intake. surprisingly, in adult cf patients (n= ) iga positively correlated with total vitamin d intake (p< . ) and total vitamin d intake per kg bw (p< . ). in the population of stockholm cf-center patients (n= ), where it was possible to allow for the influence of both age and compliance, negative correlation between blood vitamin d and igg (p< . ) was found. after allowing for age, compliance showed significant correlation only with igg (p< . ) and esr (p< . ), and with none of the rest of the studied variables. conclusions: vitamin d deficiency might contribute to the continuous shift towards inflammation as well as to the higher prevalence of dm and celiac disease in cf patients. new recommendations for vitamin d supplementation and monitoring of blood vitamin d levels are needed, in order to improve immunological balance and decrease the need for anti-inflammatory therapy in cf patients. supported by swedish heart lung foundation, stiftelsen frimurare-barnhuset i stockholm, norwegian and swedish cystic fibrosis associations, and by an unrestricted grant from solvay pharma. von drygalski, a.; biller, j.a. medical college of wisconsin, milwaukee, wi, usa anemia is associated with increased morbidity and mortality in other chronic diseases, but little is known about anemia in cf. recognition and correction of anemia in cf might lead to better outcomes. chronic inflammation, iron deficiency and malabsorption may be important mechanisms contributing to the anemia in cf patients. we hypothesized that anemia might be correlated with poor lung function. methods: clinical charts and portcf.org visit logs of patients of all ages ( m to yrs) with cf were reviewed for as many years as charts permitted (range - yrs). the following data were extracted: cbc, iron studies, pulmonary function tests (pfts), vitamins a, d, and e levels, creatinine, pertinent medical history, and current medications. anemia was defined by age-and gender-specific world health organization (who) criteria. patients were considered anemic if low hemoglobin(hb) was present on two separate occasions at least two months apart, or average annual hb met who criteria. pfts in patients (all > yrs) included forced expiratory volume (fev ) and forced vital capacity (fvc) as percent predicted of normal and were considered a reflection of patient performance. the most representative annual pft from at least annual tests was chosen for analysis since acute infection adversely influences daily performance. results: of patients were anemic (prevalence %). prevalence of anemia increased with age from . % in patients < age to % in patients > age and was significantly higher in patients with vitamin deficiencies. % vitamin-deficient patients vs. . % non-vitamin deficient patients were anemic (p= . ). mean hb was . mg/dl (range . - . ). roughly two thirds of patients had moderate and severe anemia (hb < mg/dl) with no gender difference in regard to prevalence or severity identified. complete iron studies were available in / patients. were identified as iron deficiency anemia (ida), as anemia of chronic illness(aci), and as combined; others had renal failure. in patients, iron studies were incomplete, but renal failure, hemoptysis, hematochezia and solid organ transplants were contributing factors in half. pfts obtained in patients ≥ yrs were compared in anemic and non-anemic patients. mean fev and fvc at all ages were statistically significantly poorer in the anemic patients (p < . ). complete iron studies were also available in non-anemic patients with impaired lung function. / patients had ferritins < ng/ml suggesting relative iron deficiency. conclusions: the prevalence of anemia in cf patients is high and increases with age. in patients for whom iron studies were available, ida was the prominent underlying mechanism, followed by aci or a combination of both. in addition, a group of non-anemic patients with poor lung function had relative iron deficiency. the strikingly significant association between anemia and poor pulmonary function as well as the higher prevalence of anemia in vitamin deficient patients uncover anemia as significant co-morbidity in cf. identification of iron deficiency in anemic as well as in non-anemic patients with poor lung function could be important since iron supplementation might allow hb levels to increase as an appropriate response to hypoxia. oral administration of dha to a cf mouse model corrected the lipid imbalance and reversed certain pathological manifestations in tissues affected by cf. the aim of this study was to investigate the metabolism of la through the n- pathway, and to determine the effect of dha supplementation on la metabolism in cultured cells with a cf phenotype. methods: sense (wt) and antisense (cf) cftr hbe cells were cultured in mem containing % horse serum. cells were supplemented with various concentrations of la (ranging from to µm) and dha ( , , and µm) for one week. cellular lipids were extracted from confluent monolayers, and fatty acids were methylated and analyzed by gc-ms. metabolism through the n- pathway was studied by incubating the cells with . µm [ c] la for hours, and quantitating its downstream metabolites by hplc. results: the levels of la and dha were significantly decreased in cf cells compared to wt cells (la in wt: . ± . , cf: . ± . %, p< . ; dha in wt: . ± . , cf: . ± . %, p< . , mean ± sem for each). la supplementation resulted in an increase in la and aa levels in both wt and cf cells. at most la concentrations tested, la levels were significantly lower and aa levels were significantly higher in cf cells, indicating an increased production of aa from la in cf cells. the study of [ c] la metabolites showed an increased production of multiple downstream fatty acids including : n- ( . fold), aa ( . fold), and : n- ( . fold) in cf cells compared to wt cells, further substantiating an increased metabolism through the n- pathway. supplementing hbe cells with dha for week resulted in a significant increase in la levels and a corresponding decrease in aa levels in wt and cf cells. when supplemental dha was added in combination with la, dha inhibited the la-derived increase in aa levels in cf cells and normalized them to the wt values. the formation of [ c] la metabolites was inhibited after -hour supplementation with dha. this inhibitory effect of dha was greatest on aa production, and it was more prominent in cf than in wt cells (wt: . fold, cf: . fold decrease in aa production). conclusions: decreased la levels in cf are at least in part related to increased la metabolism through the n- pathway. the fact that dha supplementation decreased the flux from la to fatty acid metabolites in the n- pathway, especially to aa, may partially explain dha's mechanism of therapeutic benefit. these data would also suggest that dha combined with la in the diet may normalize fatty acid metabolism in cf patients. background: decreased levels of linoleic acid (la) and docosahexaenoic acid (dha) have been found in the blood and tissues of cf patients. studies on cultured cf cells and cftr-/-mice have indicated that decreased la levels in cf might be related to its increased metabolism through the n- pathway. other potential mechanisms of the observed decreased la and dha levels in cf could be related to altered cellular uptake of these two essential fatty acids. the goal of this study was to investigate the uptake of la and dha into cultured cf cells, and their distribution among the major lipid classes. methods: sense (wt) and antisense (cf) cftr hbe cells were cultured in mem containing % horse serum. the uptake of la and dha into the cells was studied by incubating the cells with . µm [ c] la or [ c] dha for hour and hours, followed by measurement of radioactivity in the supernatant and cell lysate. lipid fractions from the cells (total phospholipids, triglycerides, cholesteryl esters, and free fatty acids) were separated by thin layer chromatography (tlc), and the fatty acid composition was determined by gc-ms after methylation. incorporation of la and dha into cellular lipid fractions was determined by incubating the cells with . µm [ c] la or . µm [ c] dha for hour and hours, followed by tlc separation of lipids and measurement of radioactivity associated with the various fractions. (all data are mean±sem). results: the levels of la and dha were significantly decreased in cf cells compared to wt cells (la in wt: . ± . , cf: . ± . %, p< . ; dha in wt: . ± . , cf: . ± . %, p< . ). fatty acid composition analysis of lipid fractions indicated that la levels were significantly decreased in the phospholipid and triglyceride fractions of cf cells ( . % and . % of the wt values, respectively), and dha levels were significantly decreased in the phospholipid fraction of cf cells ( . % of the wt values). the uptake of la was significantly higher into cf cells compared to wt cells at hour (wt: . ± . , cf: . ± . dpm/µg protein, p< . ) and hours (wt: . ± . , cf: . ± . dpm/µg protein, p< . ). the uptake of dha was also significantly higher in cf cells compared to wt cells at hour (wt: . ± . , cf: . ± . dpm/µg protein, p< . ) and hours (wt: . ± . , cf: . ± . dpm/µg protein, p< . ). incorporation of la and dha was increased in the phospholipid, cholesteryl ester, and free fatty acid fractions of cf cells at hours. dha, but not la, incorporation was significantly increased in the triglyceride fraction of cf cells. conclusions: low la and dha levels in cf cells are associated with a decrease in cellular lipid fractions, the largest of which is in total phospholipids. there is an increased uptake of la and dha into cf cells, and into most lipid fractions of these cells. the decreased levels of la and dha in cf cells, despite their increased cellular uptake and esterification in lipid classes, indicate that there is increased mobilization of these fatty acids in cultured cf cells. perez, a.; issler, a.; davis, p.b. pediatrics, cwru, cleveland, oh, usa even though lung disease continues to be the primary cause of death in cystic fibrosis (cf), the considerable increase in life expectancy of the cf patient over the last decades, has made management of the diverse gastrointestinal and nutritional complications in the adult cf increasingly important. malnutrition and fatty acid deficiency has been a hallmark of cf and its impact in long-term lung health has been established. data obtained by us using affymetrix mg-u av arrays from ileum and liver of pristine -week-old male mice bearing the y x cf mutation and their normal littermates, indicates that there is a profound disturbance in genes associated with lipid metabolism, many of which are regulated by the peroxisome proliferator-activated receptor-gamma (pparγ), a ligand-regulated transcription factor that regulates lipid metabolism and possess anti-inflammatory properties. we hypothesize that lack of cftr function results in altered expression of pparγ regulated genes in the gut and liver, leading to the fatty acid metabolism abnormalities observed in cf. pparγ mrna and protein levels were measured in ileum, colon, liver, and fat of week old female mice bearing the cf mutation s x and its wt littermates (c bl/ j background), and fabpcftr mice (gut corrected, mixed background) after the administration of ethanol or pioglitazone (a pparγ agonist, mg/kg) by gavage every h for days, sacrificed h after last dose. highest pparγ mrna levels were found in fat, followed by colon, liver, and lowest in ileum. at basal state, mrna levels were higher only in fabp ileum vs. wt and fabp colon vs. cf. after treatment, higher expression was seen in cf and fabp ileums and cf liver vs. controls and wt treated mice. however, there were striking differences at the protein level, especially in the colon. at basal state, pparγ was localized, mainly at the surface of the epithelia in wt, in crypts in cf, and mainly in crypts but extending towards the surface in fabp mice. upon treatment, pparγ location in wt colon moved almost exclusively to the crypts, and in the cf and fabp mice, although expression remained mainly in crypts, pparγ could now be seen at the surface. changes in pparγ protein were also seen in ileum and especially liver, between wt and cf, at basal and upon treatment, but changes were subtle and will require more sensitive methods to further examine them. we also examined mrna and protein levels for several genes involved in fat processing that could be related directly or indirectly to pparγ. mrna and protein levels for aqp and cubilin, and protein levels for megalin, apoa- , and apob- were reduced in the ileum of cf mice compared to wt. our data indicates a pparγ dysfunction in the gastrointestinal system of cf mice, which could be responsible for the gastrointestinal manifestations observed in cf. understanding the association between pparγ, cftr, and fat processing may help us to develop new therapies directed towards improving the nutritional status of cf patients that, in the long run, will influence their lung health. cf is characterized by low linoleic acid (la) and docosahexaenoic acid (dha) levels. although this could be due to a primary abnormality in fatty acid biosynthesis, this could also be explained by alterations in phosphatidylcholine (pc) formation. pc are formed by two pathways. the most utilized is the de novo conversion of choline to pc via the cdp-choline pathway, which preferentially forms pc containing saturated and monounsaturated fatty acids. in the other pathway phosphatidylethanolamine (pe) is converted to pc through three methylation steps utilizing the enzyme phosphatidylethanolamine n-methyltransferase (pemt). this pathway preferentially forms pc containing polyunsaturated fatty acids. thus decreased pemt activity would be predicted to result in decreased dha and la levels. this is supported by results seen in pemt knockout mice (watkins et al, j nutrition, ) showing decreased dha and la levels, and variable to increased levels of arachidonic acid (aa). furthermore, innis et al have shown that cf patients have decreased choline levels making them more dependent on the pemt pathway. we hypothesized that alterations in fatty acid levels in cf may be due at least in part to decreased pemt activity. methods: cftr -/mice (cf) and wt littermates were fed with either a diet containing choline (control) or, in order to mimic choline status in humans, an otherwise matched choline deficient (cd) liquid diet for days. choline levels were measured using a kit. livers were perfused and microsomes prepared. to measure pemt activity, microsomes were incubated with radioactive s-adenosylmethionine in the presence of dimethyl pe. the pc product was extracted and radioactivity measured. fatty acids from liver and pancreas were analyzed by gc/ms. results: there was no difference in choline levels in serum or liver comparing wt and cf mice. there was no difference in pemt activity in livers from cf mice compared to wt on control diet. cf mice on cd diet had decreased pemt activity in the liver compared to wt mice on cd diet (wt: ± ; cf: ± cpm/mg protein/min; p< . conclusion: induction of choline deficiency in cf mice to mimic the situation in cf patients, leads to decreased pemt activity. this resulted in low la levels compared to wt mice in both the liver and pancreas and indicates that the fatty acid abnormality in cf is at least in part due to defective pc metabolism through the pemt pathway. ( ). urolithiasis appears to be more common in adult cystic fibrosis (cf) patients than in the general population. a variety of urinary findings have been observed which could account for this, including low volumes, raised oxalate and low citrate, but not all the reports are consistent. most cf patients have pancreatic insufficiency and a plausible explanation for an elevated oxalate excretion in these patients is malabsorbed fat in the intestinal tract sequestering calcium and permitting more oxalate to be absorbed from their food. if this were the mechanism for the elevated stone risk in cf patients then we would expect to observe differences in stone incidence and oxalate excretion between cf patients with exocrine pancreatic sufficiency (ps) and pancreatic insufficiency (pi). methods diagnosis of cf is by clinical and genetic criteria and ps or pi status is based on faecal elastase measurement. patients and healthy volunteers (hv) provided h urines for analysis of volume, ph, ca, mg, na, k, po , oxalate, citrate, creatinine, urea. supersaturations were calculated using equil . there is a history of renal stone disease in out of cf patients attending our unit. patients have pi and include of the stone formers. the remaining stone former is amongst the ps patients. no significant differences in urine chemistry or derived supersaturations were found between the cf patients without a history of stones that were ps (n= ) or pi (n= ). nor were any such differences found when stone forming cf patients were included (ps, n= ; pi, n= ) or when the healthy volunteers were included in the ps group (ps, n= ; pi, n= ). amongst the cf patients, only citrate and creatinine excretions were significantly different between stone formers (n= ) and non stone formers (n= ) (medians (mmol/ h), citrate, . vs. . , p= . ; creatinine, . vs. . , p= . ) . the difference in creatinine excretion can be accounted for by the difference in body mass of these two groups (median (kg) . vs. . , p= . ) compared to the hv, the cf patients with a history of stones had significantly lower citrate excretion and higher calcium oxalate supersaturation (medians, citrate (mmol/ h), . vs. . , p= . ; . vs. . , p= . ) conclusions there is no evidence to suggest that the prevalence of stones is different in the ps and pi groups nor could we detect any difference in excretion of oxalate or any other urine variable related to pancreatic sufficiency. comparing those with and without stones, the only consistent finding was a lower citrate excretion by stone formers. between the hv and stone formers this was also associated with a significantly reduced calcium oxalate supersaturation. our evidence does not support the hypothesis that fat malabsorption by the majority of cf patients contributes to an elevated risk of renal stone disease. we undertook assessment of the use of a hypopharyngeal sensor for detection of laryngopharyngeal reflux in infants and children. gastric reflux in the airway, or supraesophageal reflux, commonly takes a gaseous form that cannot easily be measured using conventional technology. the miniaturized ph sensor at the tip of the dx-ph probe is the only sensor able to measure ph in the airway. fifteen infants and children referred to the pediatric pulmonary center for assessment of chronic cough, hoarse voice, or uncontrolled asthma unresponsive to usual medical intervention underwent - hour to placement of the dx-ph probe in supplementation of radiologic assessment of gastresophageal reflux. the probe was placed transnasally and visualzed in the hypopharynx with the red light at the tip. drops in ph < were correlated with food and symptom diaries. positive results were found in / patients, leading to surgical intervention in three patients. one patient with cystic fibrosis age months with failure to thrive underwent study for symptoms of cough. upper gi demonstrated no gross gastroesophageal reflux or anatomic obstruction.gastric emptying scan was mildly prolonged. outpatient supraesophageal ph probe demonstrated drops to ph< associated with respiratory symptoms. patient underwent nissen and g tube placement with resolution at six week follow-up.the patient returned at age six months with onset cough and underwent repeat study. there were no drops in ph and the cough responded to oral steroid burst. the device was tolerated in / patients with early removal in one young adult with endstage neuromuscular disease and in an infant with "colic" due to a hysterical parental reaction.as a result of our experience we have incorporated the use of supraesophageal ph monitoring into our practice, with particular attention to infants with persistent asthma and children with histories suggestive of gastric asthma. we have now incorporated it into our vivometrics lifeshirt, an ambulatory physiologic monitoring device which will allow us to examine supraesophageal ph and its relationship to sleep apnea, cough, tachycardia, tachypnea, and hypoxemia. aim: monitoring and adjusting dose requirements of pancreatic enzyme replacement therapy (pert) are an integral part of the dietetic assessment of patients with cf. we investigated characteristics of enzyme use in our adult clinic population and determine the extent to which inappropriate enzyme use contributed to poor nutritional and clinical state. method: information was collected using an annonymous self-administered questionnaire developed to measure patient practice, knowledge and beliefs on pert. exclusion criteria included pancreatic sufficiency, < units lipase/kg/d, and fev < %. results: out of potential clinic population of patients completed the questionnaire ( - y, % male, fev - %). % of participants reported to never miss enzymes with meals; this was considerably lower for snacks ( %). those patients who omit enzymes with meals also missed enzymes with snacks (r = %, p< . ). a more appropriate use of pert was observed in patients with lower as opposed to higher bmi. despite intensive dietetic input % of patients missed pert with foods containing fat and % took pert inappropriately with food and drink that did not contain fat. the results identified potentially better practices for measuring pert behaviour and knowledge: ) taking pert with all meals, ) taking pert with majority of snacks ) swallowing capsules intact rather than splitting them open, ) carrying enzymes around with them and ) the ability to titrate pert in accordance to the fat content of food. patients were scored on the basis of the above criteria to differentiate between prudent enzyme users and those who are compromising therapy. in conjunction with their nutritional status score (bmi and gastrointestinal symptoms) risk for intervention can be assessed. discussion:underweight patients have more optimal enzyme use, suggesting greater dietetic involvement in these patients. schall et al also found this to be the case in children. the findings emphasised the need for targeted and effective input in patients where problems are less obvious. the questionnaire has been a useful research tool, and has been adapted as a screening tool for dietitians to gain a subjective perspective of patient's enzyme management and identify patients who need support. the combination of patient's pert usage and their nutritional status could help capture and identify risk objectively and quickly and allows resources to be allocated most effectively. schall . ), nutritional failure, measured as bmi< , (p> . ), or lung transplant status (p> . ) between patients with and without colonic wall redundancy. there was no significant difference in cftr function, per sweat chloride values, between groups (p> . ). conclusion: we report a previously undescribed colonic wall abnormality seen on ct in patients with cystic fibrosis: proximal colonic wall redundancy was found in % of adults with cystic fibrosis, but not in children, appeared to be chronic, and was associated with non-∆f cftr gene mutations, particularly the g x mutation. recognition of the ct appearances will prevent erroneous diagnosis of acute colonic disease in this patient population, and stimulate investigation of a biologic basis for this finding as a recognizable feature of adult cystic fibrosis. intro: infants and children with cystic fibrosis, both pancreatic sufficient and insufficient, are at risk for developing hyponatremia due to an excess loss of salt though skin during sweating. these children should receive daily supplemental sodium. children without cf require - meq/kg of sodium daily; and one could argue children with cf need more. a historical recommendation is / tsp table salt daily for infants; this contains to meq of sodium based on the density of the table salt. parents may measure the salt inaccurately, and it may be difficult logistically to distribute the salt throughout the day. if supplementation is given as sodium chloride solutions distributed through pharmacies, as suggested in a recent consensus conference, co-pays or full payments may be required. our aim is to provide a simple, inexpensive, and more accessible method for accurately giving sodium supplementation. do pre-packaged salt packets contain a precise amount of sodium and thus could be recommended to fulfill the daily requirement of sodium in patients with cf? method: pre-packaged table salt packets were collected with permission from four national fast food restaurants and one commercial salt producer. contents (sodium chloride) of ten packets from each source were weighed individually. measurements were made using a scientific scale with the ability to measure up to / , of a gram. weights of sodium chloride from individual packets were converted to meq sodium. means, ranges and standard deviations were calculated for the data using microsoft office excel ™ program. see table conclusions: there is some variability in the content of pre-packaged salt packets, both between and within brands, though some brands have less variance than others. measurements showed that packages contained mean values of approximately to meq of sodium. there is a range in the sodium dose recommendations, and spot urine sodium can be used to determine if patients are sodium depleted. pre-packaged salt packets lack the precision of pharmacological dosing but are inexpensive, practical, and supply a reasonably predictable dose of sodium. background: ten to forty percent of people with cf have vitamin d deficiency secondary to pancreatic insufficiency. obtaining optimal vitamin d levels ( -ohd) has been a challenge. for vitamin d deficient individuals, current supplementation strategies ( , iu weekly for months) have been mostly unsuccessful. objectives: to identify individuals at risk for vitamin d deficiency and to evaluate the efficacy of a week repletion course of high dose ergocalciferol in children and young adults with cf. methods: as part of a quality improvement initiative, a prospective cohort study was performed from january to april . phase i included querying our cf practice database to identify patients who were due for annual routine blood tests or who had recently documented levels of -ohd less than ng/ml. in phase ii, , iu of daily ergocalciferol was prescribed for a day period as part of routine inpatient or outpatient care. during phase iii, a post treatment -ohd level was obtained. baseline subject characteristics were obtained at entry and included age, gender, pubertal status, pancreatic status, and fev %. post -ohd levels were classified as sub therapeutic (< ng/ml), therapeutic ( - ), high therapeutic ( - ), or potentially toxic (> ). a paired t test was performed to evaluate pre and post intervention differences. the impact of age, gender, and lung function on the response to vitamin d supplementation were also analyzed. all values are expressed as mean ± standard deviation. results: eighteen individuals with cf participated in the study. the mean age was years with a range of to years. . % were male, and % were pancreatic insufficient requiring pancreatic enzyme replacement. fev % was . ± . %. all participants had -ohd levels less than ng/dl pre-treatment. -ohd levels increased from . ± . to . ± . (p< . ). of the participants ( . %) became therapeutic over the two week interval. an increase of . ± . (ng/ml) or % was seen in the two week period. no correlation was seen on the extent of increase in -ohd and baseline lung function. pre and peri pubertal individuals had a greater increase in -ohd levels than post pubertal individuals ( . ± . vs . ± . , p< . ) although the magnitude of change did not reach significance. the degree of response also appeared related to age (r=- . ). no impact of gender was seen. seven individuals achieved normal therapeutic values while were in the high therapeutic range. no participants had toxic levels. conclusions: the results of this study demonstrate that high dosing of ergocalciferol over a day period is an effective strategy in achieving therapeutic levels of vitamin d. it is unclear whether a pubertal effect on the degree of response exists or if this response is merely age related. further research is needed to evaluate whether this strategy is able to maintain therapeutic levels after completion of the intervention. additional studies that monitor compliance and evaluate responders and non-responders are needed. foundation consensus committee established guidelines for optimizing bone health in individuals with cf, including screening for vitamin d insufficiency and a treatment protocol for achieving and maintaining normal serum vitamin d concentrations. however, since the guidelines were published, there has been additional research assessing various vitamin d treatment regimens and their effect on serum vitamin d concentrations. in addition, vitamin d assay variability has recently been well documented. the objective of this study was to evaluate variability of vitamin d surveillance methods as well as treatment protocols used to treat low vitamin d concentrations at cf centers. methods: a survey was created via survey monkey, and was sent to the cf nutrition listserv, which includes over cf healthcare providers. spss version . was used for statistical analysis. results: fifty-nine listserv members responded to the survey. the majority ( %) of respondents measured oh vitamin d concentrations, and % considered patients to be vitamin d deficient when serum concentrations were below ng/ml. however, % were monitoring only , oh vitamin d or a combination of both concentrations. there was considerable variability in vitamin d supplementation protocols for treating low oh vitamin d concentrations. only % were following the consensus guidelines of , units of ergocalciferol once/week for ages through adults. one third indicated that they followed consensus guidelines for patients less than age ; however nearly half of the remaining two thirds were not sure what is prescribed or have not observed low vitamin d concentrations in this age group. fifty-two percent of respondents were aware of vitamin d assay variability. despite this knowledge, the majority were unaware of the type of vitamin d assay used to measure serum vitamin d concentrations at their institution. conclusions: there is considerable variability in the measurement and treatment of vitamin d concentrations across cf centers despite guidelines provided via a cff consensus report. the consensus guidelines for measuring vitamin d concentrations and treatment of vitamin d insufficiency should be revisited based on recent studies and expert opinion. children with cystic fibrosis (cf) who suffer from sub-optimal growth are offered overnight enteral feeding via gastrostomy tube. this study aimed to assess the nutritional impact of overnight feeding over a year period using a retrospective study design. data, including height, weight, fev , pancreatic function were collated for all patients with cf who had a gastrostomy tube placed in the past years. data were collected at baseline and then every months for years post-gastrostomy insertion. data for height, weight and body mass index (bmi) were converted into z scores using centre for disease control reference ranges and the lms method ( , ) . a total of patients ( . % of the clinic population) were identified as having a gastrostomy tube. two were no longer receiving feeds and there were incomplete data for subjects. on placement of the tubes, there were substantial deficits in nutritional status with mean z scores for height - . (sd . ), weight - . (sd . ) and bmi - . (sd . ), with compromised lung function (mean fev %, range - ). using a repeated measures anova with fev as a co-variate, there were no significant differences in weight, height or bmi from baseline to years (at years post placement: mean z scores for height - . (sd . ), weight - . (sd . ) and bmi - . (sd . ). fev remained stable over time. these results indicate that gastrostomy feeding can potentially halt the decline in nutritional status that is a feature of cf. however, patient expectations that over night enteral feeding will lead to an increase in nutritional status need to be sensitively managed. background : cystic fibrosis (cf) is the most common life threatening autosomal recessive disorder in caucasians.following a landmark paper by corrie et al a high fat,high calorie diet has promoted a normal growth pattern.improved nutritional status together with prevention and early treatment of respiratory infections has contributed to improved survival. objective: the aim of this study was to assess the relationship between lean body mass and disease severity in children with cf. methods: the nutritional status and body composition of children with cf was measured using a harpenden stadiometer, calibrated electronic scales and a ge lunar prodigy densitometer. the following indices were calculated; body mass index (bmi), fat mass (expressed as logarithm % total body fat (ln%tbf) and fat free mass (ffm). body composition data were expressed as z-scores using dutch reference values. correlations were performed between indices of nutrition (bmi, height, ffm, tbf) z scores and markers of disease severity (percent predicted fev and shwachman kulczycki (sk) scores).the nutritional status of these children was also compared with healthy controls. statistical analyses were performed using microsoft excel . results: there was a weak but significant correlation between sk scores and height z score and ffm (r = . , p< . & r = . , p= < . respectively). there was also a weak but significant correlation between % predicted fev and bmi and ffm (r= . , p < . , r= . , p< . ). height and weight z scores were significantly lower in children with cf (- . and - . respectively) than in control subjects ( . and . respectively) with p < . in both groups. conclusion: this study demonstrates that there is an association between indices of nutrition and disease severity in children with cystic fibrosis. muscle mass (ffm), assessed by dual energy x-ray absorptiometry (dxa) correlates with lung function and with sk scores. the strongest correlation between markers of disease severity (sk scores and % predicted fev ) was with ffm. ffm may be expected to be associated with respiratory function tests.however,sk scores are a more general assessment of well being and less obviously directly related to muscle mass. ffm and growth may reflect long term nutrition and health in contrast to bmi and ln%tbf which may reflect acute deterioration or short term nutritional intervention. therefore ffm may prove to be a useful tool to assess quality of nutrition and may be predictive of respiratory and general decline. body composition assessment has an important role in chronic conditions such as cystic fibrosis, in order to identify nutritional depletion and evaluate nutritional interventions. it is important that reliable and accurate methods are used. the usefulness of non-invasive methods, such as skinfold thickness measurements (sft) to measure change in body composition has not been evaluated in cf. this study aimed to compare changes in fat-free mass (ffm) and fat mass measured using sft and dual-energy x-ray absorptiometry (dxa) in adults with cf. methods: adults with cf ( % male, % pancreatic insufficient, mean age at baseline . (sd . ) years, mean fev at baseline . (sd . ) % predicted) were studied. they underwent body composition assessment using dxa and sft, at baseline and a mean of . (sd . ) years later. durnin and womersley equations were used to estimate ffm and fat mass from sft. estimates of change in ffm and fat mass obtained using sft were compared with dxa using paired t-tests, univariate analysis and bland and altman analysis. results: at baseline, mean ffm was . (sd . ) kg; mean fat mass was . (sd . ) kg and mean bmi was . (sd . ) kg/m . mean ffm, fat mass and bmi were not significantly different at follow-up, indicating no change in nutritional status for the population overall. individual change in ffm ranged from - . to + . kg, while change in fat mass ranged from - . to + . kg. the table shows mean change (∆) in ffm and fat mass by each method, correlations (r ) between sft and dxa, and the % limits of agreements (loa) between sft and dxa for change in ffm and fat mass. mean change in ffm and fat mass estimated using sft did not differ significantly from dxa. mean bias between the methods was small, and overall correlations between sft and dxa were strong for changes in both ffm, suggesting good agreement for the overall population. however the % loa between the two methods were wide for change in both ffm and fat mass. this suggests that sft will not accurately predict changes in body composition in all cf patients. conclusion: sft measurements do not accurately estimate change in ffm or fat mass over time in individual adult cf patients compared with dxa. body composition changes measured using sft tended to more closely reflect changes detected by dxa in male patients compared with females. caution should be exercised when interpreting the results of serial measurements of body composition using sft in individuals with cf. analysed were significantly correlated with age-cml or srage levels (age, gender, bmi, presence of cf-related diabetes mellitus (cfrd) or hba c level). conclusions: serum levels of ages are elevated in adults with cf. elevation is not restricted to those with cfrd. the levels of srage reflect ability to respond to this pathway and are associated with poorer lung function. modification of the diet in cf may reduce this mediator and may have the potential to modify lung and renal injury. mouse models of cf generated by expression of improperly processed cftr (∆f ) or lacking cftr expression (ko) demonstrate selective induction of sult e in the liver (falany et al., biochem. j. : , ) . sult e is responsible for the sulfation and inactivation of β-estradiol (e ) at physiological concentrations. the increase in sult e expression is specific to the hepatocyte, whereas cftr is selectively expressed in cholangiocytes. the induction of sult e activity is associated with changes in levels of e regulated proteins in cftr (-/-) liver (li and falany, j cystic fibrosis : , ) . increased sult e activity is correlated with low body weight and decreased igf- message levels in livers of cftr(-/-). the mechanism for the induction of sult e in hepatocytes by cholanigiocytes lacking cftr expression, and the mechanism for the inhibition of igf- expression by increasing sult e expression was investigated in human cells. human mmnk- cholangiocytes in which cftr expression was inhibited by short interfering rna (sirna) induced sult e expression in human hepg hepatocytes when cocultured in a permeable membrane separated system. sult a expression was not altered in this model. the data suggests that loss of cftr function in cholangiocytes is capable of selectively modulating sult e expression in hepatocytes. to investigate the ability of increased sult e activity to modulate expression of igf- , hepg cells were stably transformed with sult e /pcdna to activity levels intermediate to the levels observed in cftr(-/-) mice. the increased sult e activity was associated with decreased igf- message expression in the hepg cells. since a major pathway for igf- regulation involves growth hormone (gh) stimulation of stat b phosphorylation, the effects of e and increased sult e activity on gh stimulated stat b phosphorylation were examined. stat b activation was identified using immunoblot analysis with a rabbit anti-tyrosine phosphorylated-stat b antibody. treatment of control hepg with nm e prior to the addition of gh increases stat b phosphorylation. in hepg cells expressing increased sult e activity, the stimulation of stat b phosphorylation by nm e was significantly decreased. e had an apparent rapid action on stat b phosphorylation that is not attenuated by the estrogen receptor antagonist, ici , . e was effective at increasing stat b phosphorylation when applied to hepg cells - min before gh. increased stat b phosphorylation was observed in control hepg cells at nm e although -fold higher e concentrations were required in the sult e -hepg cells consistent with the increased e sulfation. no differences were observed in total stat b in any of the studies. this study demonstrates that human cholangiocytes with low levels of functional cftr are capable of inducing sult e expression in human hepatocytes and the increase in e sulfation inhibits the gh stimulation of igf- expression via a decrease in stat b phosphorylation. supported by grants from the cystic fibrosis research, inc. and nih (gm ). williams, j.e. ; benden, c. ; jaffe, a. ; suri, r. ; wells, j.c. ; fewtrell, m.s. . mrc childhood nutrition unit, institute of child health, london, united kingdom; . portex respiratory medicine unit, great ormond street hospital, london, united kingdom background: patients with cystic fibrosis (cf) are at high risk of malnutrition. to our knowledge, no study so far has employed a reference fourcomponent model(sup) (/sup)( cm) to assess body composition (bc) in cf children, which allows accurate evaluation of both fat mass (fm) and the components of fat-free mass (ffm; mineral, protein, water) and most studies have been cross sectional. methods: cf subjects, aged - yrs, were compared with age-and sex-matched healthy controls for assessment of bc using a reference cm. comparison between groups was performed using paired t-tests and general linear models to adjust for age, height and pubertal status. in addition fm index (fmi; fm/height ) and ffmi (ffm/height ) standard deviation scores (sds) were calculated using measurements performed in a reference population of healthy children aged to yrs. repeat measurements were made in children with cf ( boys) after yrs and the change in fmi sds and ffmi sds investigated. results: for the initial measurement at baseline, boys with cf (n= ) were significantly shorter (mean (se) height sds: - . ( . , p< . ) compared to uk reference data; girls with cf were lighter, bmi sds (- . ( . ), p< . ). at baseline, comparison of cf children with pair-matched controls indicated that there was no difference in the boys but cf girls had less fm (cf minus control) (- . ( . )kg, p< . ) and mineral mass (- . ( . )kg, p< . ) after adjustment for age, height and puberty. fmi sds adjusted for age and puberty was also significantly lower in girls with cf (- . ( . ), p< . ) whereas ffmi sds was not. for comparison with the large reference population there was no difference in the boys at either time point for fmi sds and ffmi sds (paired t-test). for girls however, there was a significant difference at both baseline fmi sds (- . ( . ), p< . ), ffmi sds (- . ( . ), p< . ) and yrs fmi sds (- . ( . ), p< . ) and ffmi sds (- . ( . ), p< . ). paired t-test analysis between baseline and yr followup measurements indicated that fmi sds and ffmi sds were not significantly different at each point in time in either boys and girls. conclusion: at baseline, the bc of cf boys appears to be within normal range, but the cf girls already have lower fm and mineral than their healthy pair matched controls, even when adjustment for size and puberty is made. follow-up measures in cf children indicated that these descriptions of nutritional status of cf boys and girls remained consistent over the following years, with no evidence of any further deterioration in girls. however, nutritional surveillance is important in both sexes in cf at around puberty to address potential deterioration and this study suggests that the cm is a useful tool for this purpose. fuller background: although lung function (lf) in children with cystic fibrosis (cf) has been reported to be related to their nutritional status, particularly body fat, the latter has usually been assumed from anthropometric measurements and not measured using an appropriate reference method. habal, h. ; al-turkmani, m. ; freedman, s. ; laposata, m. . pathology, massachusetts general hospital and harvard medical school, boston, ma, usa; . medicine, bidmc and harvard medical school, boston, ma, usa background: increased dietary intake of fatty acids is recommended for cf patients. it is unknown if this results in increased incorporation of all fatty acids-saturated, monounsaturated, and polyunsaturated-into cells with a cf phenotype to a greater extent than into non-cf cells. docosahexaenoic acid (dha) supplementation of cftr-/-mice has been shown to reverse the pathologic cf phenotype. aim: to investigate whether the uptake of multiple predominant fatty acids and the release of membrane-bound arachidonic acid (aa) are altered in cultured epithelial cells with a cf phenotype; in addition, to determine whether fatty acid uptake is selectively inhibited by dha supplementation. methods: sense (wt) and antisense (cf) cftr hbe cells were cultured in mem containing % horse serum. aa release from membrane phospholipids (pls) was studied by incubating the cells with µm [ c] aa for min, and then with serum free medium containing . % fatty acid free human serum albumin in the presence or absence of µm adenosine for min, followed by measurement of radioactivity in the supernatant. the uptake of fatty acids into the cells was studied by incubating the cells for different time points with . µm [ c] linoleic acid (la), [ c] aa, [ c] dha, [ c] palmitic acid or [ c] oleic acid, in the presence and absence of µm or µm dha, followed by measurement of radioactivity in the cell lysate. results: basal and adenosine-stimulated aa release from membrane pls was significantly higher in cf cells compared to wt cells (basal wt: . ± . , cf: . ± . dpm/ cells, p< . ; stimulated wt: . ± . , cf: . ± . dpm/ cells, p< . ; mean±sem for each). the uptake of la, aa, dha, palmitic acid and oleic acid were significantly higher into cf cells compared to wt cells. dha significantly decreased the elevated uptake of these fatty acids into cf cells, but not into wt cells. data are shown in the table below. conclusion: in this cell culture model, these data demonstrate that aa uptake and release are increased in cf cells, which may suggest elevated mobilization of this fatty acid by cf cells. our data also show that cftr dysfunction leads to increased uptake of all tested fatty acids into the cells. downregulation of this increased uptake may be one mechanism by which dha exerts its therapeutic benefits. low bmi in cf correlates directly with worsening lung function and is an independent risk factor for mortality. the causes for this decreased bmi are incompletely understood but could be related to levels of the orexigenic peptide ghrelin, and to levels of leptin, an anorexigen. studies assessing plasma levels of leptin in cf have shown contradictory results, while the data on ghrelin in cf are scarce. we assessed both leptin and ghrelin plasma levels in mild (fev > % predicted), moderate (fev % to % predicted), and severe (fev < % predicted) adult cf patients. we compared these levels in cf subjects to age matched controls with normal bmi (range . to . kg/m ). we conclude that leptin levels are decreased, while ghrelin levels are increased only in cf patients with severe disease and low bmi. plasma levels of ghrelin and leptin did not differ from normals in those with mild and moderate disease. gastroesophageal reflux (ger) is common in cf before and after lung transplant (ltx). laparascopic fundoplication (lapf) is used when patients fail on conservative medical therapy. aims: to review longer term clinical outcomes of lapf in patients with cf in terms of lung function, body mass index (bmi), reflux symptoms, patient satisfaction with surgery and complications. method: retrospective review of patient records and patient questionnaire to gather relevant information. results:thirty-two patients with cf underwent lap.f recently; transplanted (ltxgroup), female (mean age [ - ]) years, and not transplanted (cf group), female ( ). the median time between ltx & lapf was ( ; ) days, range - . endoscopy was undertaken in patients prior to surgery and all underwent hr ph monitoring. complete (nissen) lapf was undertaken in % patients and a partial (toupet) in the other %. total median time in the operating theatre was ( - ) minutes. the average length of stay in hospital (in days) in the ltxgroup: pre-op. was . ; post-op. was . days; and in the cf group was: . and . respectively. lung function (fev and fvc in percent predicted) and bmi were measured at minus(-) , plus (+) , + and + days from lapf. patient satisfaction with lapf was a mean of % in the ltx group and % in the cf group and improvement in reflux symptoms of % in both groups. nocturnal cough reduced markedly in the cf group and there was an overall improvement in quality of life in both groups. complications consisted of port site hernias, failed lapf with re-operation months later; esophageal dilatations and patient with lower esophageal sphincter hypotonia. there were deaths on day , and post fundoplication. one was in a non-transplanted patient with recurrent episodes of hemoptysis. she succumbed to a catastrophic hemoptysis episode not related to lapf. the other were in transplanted patients. conclusions: in this relatively small series of patients there was a small but significant drop in fev in the ltx group days after lapf, but not in the cf group who remained stable throughout the review period. there was a significant drop in bmi in both groups after lapf, but the cf group returned to pre lapf values at + days. patient satisfaction with the procedure was high. fundoplication is an expensibe complicated procedure not without risk. a long term prospective study is warranted. historically this phenomenon has been attributed to intestinal malabsorption and cachexia as a consequence of chronic lung infection. however, cf mice exhibit similar growth and weight deficits but are not afflicted with malabsorption or lung infection, suggesting other mechanisms are participating as well. we hypothesize that small size is in part a result of increased β-oxidation in adipose stores. to test this we utilized a mouse model to study lipid metabolism. our study includes female cf mice with a null mutation in the cftr gene. these mice are congenic on the c bl/ j background as well as age matched with their controls (wt) to - days. liver, adipose and skeletal muscle (calf) were dissected from wt and cf mice in a fed state. tissues were immediately flash frozen in liquid nitrogen and used for rna and protein lysate harvest. quantitative pcr (qpcr) was performed using primers for the lipogenic isoform of acetyl-coa carboxylase α (acc α) in the liver and adipocytes, whereas the non-lipogenic isoform acc β was assessed in the skeletal muscle. primers for acyl coa oxidase (aox), carnitine palmitoyltransferase (cpt ) and long acyl-coa dehydrogenase (lcad) were used as gene markers for β-oxidation steps in the peroxisome, carnitine shuttle and mitochondrion, respectively. western blots were performed to detect acc (active) and phospho-acc (inactive) forms. the results show that there is a decrease in the total amount of acc protein in the fat as well as the skeletal muscle of the cf mice and the ratio of acc:phospho-acc is substantially lower in the cf tissues. phosphorylation and/or decreased expression of acc results in decreased expression of malonyl coa, leading to a cascade of events that increase β-oxidation. in addition, increased expression of aox and lcad, two downstream β-oxidation genes, is also observed. hyperactivity of the β-oxidation pathway could account for the decreased adipose stores seen in cf mice compared to the wt. such patterns of expression are reminiscent of starvation. caloric intake and intestinal absorption of dietary lipid were measured and cf animals consumed approximately % the calories of wt animals (per gram body weight) and lipid absorption was not significantly different ( % for cf, % for wt), indicating the cf animals have sufficient access to nutrition. we will continue to investigate fatty acid synthesis and degradation pathways during both the fed and fasted states to delineate the mechanism. this research was supported by grant hl . effect of gender on dha levels in p and rbc with lower values for male cf patients. acknowledgments: this work was supported by the belgian cf association (ablm), the french cf association (vlm) and by a grant from frs (ucl). data are expressed as means ± sd. * and indicate a significant difference with controls and cf ps patients respectively, p< . . background: deficit of antioxidant systems and increased oxidative stress have been demonstrated in cf patients with respect to healthy controls. the origin of this imbalance is known to be multifactorial. objective: the aim of our study was to assess changes in antioxidant systems and oxidative stress parameters in cf pediatric patients in presence or absence of pancreatic insufficiency material and methods: we recruited patients ( with pancreatic insufficiency pi and with pancreatic sufficiency ps) attending the cystic fibrosis centre of turin ( ) ( ) . during the annual routine control visit were performed antropometric measurements, pulmonary function tests (fev ), and were collected fasting blood samples and last days food record. vitamin e, a, c, selenium, glutathione (gsh, gshpx, gssg), dehydroascorbic acid, pcr were assessed. data were analysed using the two sample t-test with equal variances. results: there are not statistically significant differences between two groups for sex, age and fev . / of pi patients and / of ps patients took oral vitamin supplementation. serum levels of alpha-tocopherol and beta-carotene resulted within the local reference range either in pi or ps patients showing no significative differences. the mean serum levels were respectively suboptimal and optimal for vitamin e and a (biesalsky ' ). pi and ps patients did not show significant intergroup differences of water soluble and enzymatic antioxidants levels as well. serum levels of vitamin c, selenium, erythrocyte reduced glutathione (gsh) and glutathione peroxidase activity (gshpx) resulted within the local reference range. plasma vitamin c concentrations resulted slightly inferior to the optimal values (biesalsky ' ) in both groups. oxidative stress parameters (erythrocyte glutathione disulfide-gssg, dehydroascorbic acid) and inflammatory parameters (pcr) did not differ significantly, presenting values within the local reference range, in both groups. nutritional data revealed significantly reduced zscore for weight (p< . ) and height (p< . ) in pi patients vs ps patients. our data did not evidence significant differences in total energy (kcal/kg) and macronutrients (% of total kcal) intake in both pi and ps patients. dietary intake of antioxidant vitamins resulted slightly superior the rda (larn- ) presenting no significant intergroup differences. conclusions: this study shows that pancreatic insufficiency do not influence oxidant/antioxidant balance in cf patients at least in paediatric age. until innovative supplementation guidelines will be proposed for application, nutritional education and monitoring and correct dietary habits are useful to optimize antioxidant status in cf patients. there are more than cf mutations that are responsible for the spectrum of disease severity in cystic fibrosis (cf). the most common, f del mutation, is associated with pancreatic insufficiency (pi), and a classic phenotype. . yet even within a group of f del homozygotes, there is a small minority of patients ( . %) who are not taking pancreatic supplements, and are therefore presumably pancreatic sufficient (ps) (data from * cff registry). this observation prompted us to look more closely at enzyme use in cf patients with non-f del homozygous genotype. we reviewed enzyme use in cf patients who were fully genotyped and whose enzyme use was documented in the * cf registry. heterozygotes with one or two non-f del mutations were included in this analysis. using the assumption that the milder mutation is dominant, if the patient was ps and heterozygous with one f del mutation, the second mutation was classified as ps; if the person was pi, the second mutation was classified as pi. we also reviewed the pancreatic status and genotype of patients participating in the wisconsin newborn screening study. in this randomized control trial (rct), pancreatic status was determined by hour fecal fat analysis. enzyme use was extracted from the database for the year . there were patients in the cff registry with a total of different mutations. twenty-one of these mutations were associated with pi. this finding correlated well with the per cent of patients who were on enzyme supplements (range of per cent patients on enzymes by mutation - %). however when we looked at the six mutations associated with ps, the distribution of patients on enzymes vs. not, was much closer. see table below. possible reasons for enzyme supplement use by patients with ps mutations include a) treatment for recurrent pancreatitis, b)the fact that this is a cross-sectional analysis and patient may be on enzymes temporarily, c) patient may truly be pi, d) overuse of enzymes due to unreliable test to measure pancreatic status in patient, e) patients who are on enzymes are older, and have changed from ps to pi phenotype. this analysis suggests a need for better assessment of pancreatic status in patients with cf. * this analysis was originally done by request for the ecfs conference at garda on cftr genetics, and focused on the suggested " main mutations", and will be updated with the cff registry data. (supported by nih grants dk , dk , mo rr , mo rr ) number of patients in rct with each mutation: , , , , , respectively. dutta, a. ; woo, k. ; fitz, j.g. ; feranchak, a.p. . department of pediatrics, ut southwestern medical center/children's medical center, dallas, tx, usa; . medicine, ut southwestern, dallas, tx, usa extracellular atp is an important signaling molecule contributing to bile formation by the liver through binding biliary epithelial cell (cholangiocyte) membrane p receptors and stimulating cl-efflux. importantly, atp appears to work through cl-channels unrelated to cftr, suggesting it may be a potential way to modulate bile flow in cf liver disease. however, the signaling pathways linking receptor binding to channel activation in cholangiocytes are unknown. consequently, the aim of the present study was to identify the pathways responsible for atp-stimulated increases in [ca +]i and membrane clpermeability in a human biliary epithelial cell model. studies were performed in mz-cha- human biliary cells; [ca +]i was measured by fura- and membrane cl-currents by patch-clamp techniques. results: exposure of cells to atp ( µm) resulted in a rapid increase in [ca +]i to . ± . nm (n= ) which was abolished by prior depletion of intracellular ca + by thapsigargin ( . ± . nm; n= ), but unaffected by removal of extracellular ca + (egta, . ± . nm; n= ). in parallel studies, atp ( µm) increased current density from - . ± pa/pf to - . ± . pa/pf (n= ) . currents reversed at mv (ecl-), were outwardly rectifying, and were inhibited by the cl-channel inhibitor nppb (- . ± . pa/pf, n= ), but not the cftr inhibitor cftrinh . atp failed to activate currents after depletion of intracellular ca + stores by bapta-am (- . ± . pa/pf, n= ). the p y receptor antagonist suramin inhibited atp-stimulated increases in [ca +]i ( . ± . nm; n= ) and cl-channel activity (- . ± . pa/pf; n= ). however, the p x receptor antagonist brilliant blue g did not affect the magnitude of atp-stimulated cl-currents (- . ± . pa/pf, n= ). together these findings suggest that atp activates cl-currents primarily through p y, but not p x, receptors in human biliary cells. since p y are g-proteincoupled receptors and modulate [ca +]i by phospholipase c (plc) generation of inositol , , -trisphosphate (ip ), a series of experiments were designed to directly assess the effects of ip receptor inhibition or stimulation on atpstimulated cl-currents. first, the plc inhibitor, u blocked atp-stimulated cl-currents ( . ± . pa/pf; n= ). second, the specific ip receptor blocker -apb, inhibited both the atp-stimulated increase in [ca +]i ( . ± . nm, n= ) as well as membrane cl-currents (- . ± . pa/pf, n= ). lastly, intracellular dialysis with purified ip during whole-cell patch clamp activated cl-currents with identical properties to those activated by atp (- . ± . pa/pf, n= ). together these studies demonstrate that extracellular atp stimulates ca +-activated cl-channels in cholangiocytes primarily through p y receptor binding and plc/ip -dependent release of intracellular ca + stores. thus, the p y-ip receptor signaling complex represents a potential target to increase cholangiocyte secretion, and hence augment bile flow, in the treatment of the cholestatic liver disease associated with cf. supported by grants from the nih niddk and the cff to a. feranchak. background: supplemental pep therapy is used to treat cf patients with epi to assist with digestion, food absorption. this open-label, multipledose, single-treatment, multicenter study evaluated efficacy and safety of eur- for malabsorption treatment in young patients with cf and epi. eur- is a novel, zero-overfill, highly-stable formulation of porcinederived pancreatic enzymes, and was specifically developed for use in very young children allowing the product to be sprinkled on food. methods: eligible patients were < years with cf and epi, in acceptable nutritional status, needing peps and clinically stable. patients consumed a standard cf recommended diet, could not take drugs affecting gastric ph or motility. the trial involved a -day dose stabilization period and a -day treatment period. patients switched from their baseline enzyme treatment without washout. the optimal dose of eur- , determined during dose stabilization, was used during treatment. primary endpoint compared malabsorption of fat after eur- vs. previous treatment. clinical symptoms were also evaluated, as was physicians' and parents' judgment on the control of malabsorption signs and symptoms. safety measures included aes, physical exams, vital signs and changes in clinical laboratory findings, including cholesterol levels and fat-soluble vitamins. results: nineteen patients ( male) enrolled and completed all study phases. mean age was . years (range - ). percentage of responders (patients without steatorrhea (< % fecal fat content) and without signs and symptoms of malabsorption after and weeks of treatment) was . at baseline, . (p= . vs. base) after stabilization and . (p = . vs. base) at study end. frequency and oily stools showed a significant decrease vs. baseline at study end. the mean number of stools per day was . stools at screening and . stools during treatment (p< . ). the mean proportion of stool samples with visible oil or grease at screening was . % and . % during treatment (p= . ). a statistically significant improvement was seen in the incidence of moderate bloating. physicians characterized % of patients as "improved" in the control of epi signs and symptoms vs. previous therapies and % as "unchanged" at the end of the treatment phase, while parent assessments were % "improved" and % "unchanged." vitamin k absorption (measured through pivka) improved with eur- treatment over prior pep treatment measured at baseline. eur- was safe and well tolerated, with minimal aes. no aes led to discontinuation of study drug interruption. no incidences of uric acid toxicity or fibrosing colonopathy following eur- treatment. no trends were seen in lab parameters, physical examination, or vital signs. conclusion: eur- is effective, safe, well-tolerated in the treatment of epi in young cf patients. treatment controlled malabsorption and clinical symptoms of epi in young patients with cf. safety and efficacy of eur- in this trial is consistent with the pivotal phase iii trial. there is an association between the presence of cf and alterations in fatty acid metabolism, consisting of a decrease in linoleate ( : n- ). more recently, a deficiency of docosahexaenoate ( : n- , dha) has arisen as a prominent fatty acid abnormality in cf. an established breeding colony of exon cftr knockout (cftr-/-, cf, n= ) and wild type (wt, n= ) mice was used for this study. all mice were weaned at days of age and then raised on peptamen and water until days of age, and then continued for days with ml/day of peptamen to homogenize the nutritional status. after this period, some mice (n= per group) were subjected to dha treatment ( mg/day) for one week. after sacrifice, pancreatic tissue was collected, homogenized and extracted. lipid classes were separated by sequential elution in aminopropyl columns. the eluted fractions were transmethylated and injected into a hewlett-packard a gas chromatograph with a flame ionization detector for quantitative analysis. the vast majority of fatty acids were mainly distributed in phospholipids, except those with -carbons. wt and cf mice were studied a gender gap in the survival of cystic fibrosis (cf) is well-documented. however, little is known whether there is a gender différence in pubertal growth pattern in cf children. delayed and attenuated pubertal growth are commonly observed in adolescents with cf. however, characterizing height velocity (hv) pattern is very challenging because of errors associated with calculating hv, e.g. interpolation and extrapolation between heights measured in irregular intervals, as well as difficulties in determining the location and the magnitude of peak height velocity (phv). in this study, we applied a new statistical method, namely, the semi-parametric shape-invariant model, to characterize the hv pattern of cf children. this method is based on the assumption that all individuals' growth curves have a common shape. therefore, this method attempts to shift and stretch (squeeze) individual's growth data until they can be modeled by the common shape curve. the semi-parametric shape-invariant model has the advantage of fitting measured heights directly, thereby eliminating the errors associated with calculating hv. the study population included cf children who had height measurements between ages to years documented in the - cff registry. because fitting each individual height curve is computing-intensive, boys and girls were randomly chosen for this preliminary analysis. our results showed that girls exhibited a typical growth deceleration during late preadolescence, followed by accelerated hv and a single phv during adolescence (figure) . unexpectedly, cf boys exhibited a notable hv peak during late preadolescence in addition to the typical adolescent phv (figure) . overall, the mean magnitude and age of phv was . cm at . years for boys and . cm at . years for girls. when compared to phv of non-cf children based on tanner's reference (pediatr. , : ) , cf boys showed a greater delay in the age of phv while cf girls showed a greater attenuation in the magnitude of phv. further analyses are being performed to characterize the hv patterns of all children to confirm these results. (supported by nih-dk ). anelli, m.; foresti, r.; peloso, l.; ortenzi, g. medical affairs, eurand, pessano con bo, italy background: given their inherent instability, the dose units of all pancreatic enzyme preparations (peps) have always been "overfilled" to compensate for enzyme degradation over time and assure at least % of the label lipase content at the end of their shelf life. this overfill issue was first examined by whitehead who reported overfills ranging from to % after analyzing several commercially available peps (whitehead am. pharm weekbl sci. ) . the usp presently allows for pep overfills up to %. thus, the actual active lipase content of a pep capsule labelled at , iu might vary from , to , units according to its "freshness." as a result, patients taking peps receive a product with variable potency, which can lead to efficacy issues, increased pill burden, unnecessary drug interactions, and product switching. the fda has also noted the potential safety risk posed by overfilling and, in a recently published guidance, it imposed a zero overfill requirement for all peps to be marketed in the usa after april . methods: over a period of two years, we ran a series of tests similar to those conducted by whitehead to evaluate overfill on commercial peps currently available in the u.s. and europe. results: no finished product was formulated to % of the labelclaimed lipase enzyme activity. overfill on commercially available peps ranged from % to %, with a median value of %. the peps analyzed were to months old on average; therefore, the actual amount of overfill is likely underestimated. conclusion: our findings confirm those reported earlier by whitehead and demonstrate that none of the currently available peps complies with the zero overfill requirement of the fda guidance. these findings highlight a potential cause of suboptimal therapy with currently available peps. references: -whitehead am. study to compare the enzyme activity, acid resistance and dissolution characteristics of currently available pancreatic enzyme preparations. pharm weekbl sci. feb ; ( ): - . -food and drug administration. guidance for industry: exocrine pancreatic insufficiency drug products -submitting ndas. april . heubi, j. ; boas, s.r. ; blake, k. ; nasr, s.z. ; woo, m.s. ; graff, g.r. ; hardy, k.a. ; amaro-galvez, r. ; latino, m. ; lee, c. . children 's hospital medical center, cincinnati, oh, usa; . chicago cf care, glenview, il, usa; . nemours, jacksonville, fl, usa; . michigan u, ann arbor, mi, usa; . children's h, los angeles, ca, usa; . penn state h, hershey, pa, usa; . children's h, oakland, ca, usa; . texas u, tyler, tx, usa; . eurand, milan, italy; . eurand, vandalia, oh, usa background: supplemental pep therapy helps prevent maldigestion and malabsorption in cf patients. eur- is a novel, zero-overfill, highly-stable formulation of porcine-derived pancreatic enzymes developed to treat epi in cf patients. this phase iii, randomized, doubleblind, placebo-controlled, two-treatment, crossover, multicenter trial evaluated the efficacy and safety of eur- to treat epi in cf patients. methods: patients with confirmed cf and epi, age ≥ years, good nutritional status and weight ≤ kg were enrolled. no drugs affecting gastric ph or motility were allowed. after open-label dose titration, patients were randomized to receive eur- or placebo over a week-long period. following another open-label normalization, all patients were crossed over to the alternative treatment arm. primary endpoint was change in coefficient of fat absorption (cfa) following oral administration of eur- vs. placebo. secondary endpoints were change in coefficient of nitrogen absorption (cna), cholesterol, fat soluble vitamins, body weight, bmi and epi symptoms following oral administration of eur- vs. placebo. safety endpoints included aes, clinical laboratory parameters, physical exams and vital signs. results: thirty-four patients were enrolled ( female); were evaluated. mean age was . years (range - ). eur- treatment was associated with statistically significant (p< . ) increases in both cfa and cna vs. placebo. eur- treatment was associated with decreases in pivka ii (an indicator of vitamin k status). epi symptoms consistently improved during eur- treatment, including a statistically significant reduction in stool frequency and fewer soft and watery stools. treatment with eur- improved the signs and symptoms of malabsorption even in the small subset of patients with cfa values greater than % under placebo treatment. there were no notable changes in serum lipids, vitamin a and e values, mean weight or bmi in patients who received eur- . eur- was safe and well-tolerated. there were no unexpected or significant differences in the frequency or type of aes between eur- and placebo, and no trends were seen in lab parameters or vital signs. no patient discontinued from the study due to an ae. two serious aes were considered unrelated to the study drug and both resolved. conclusion: in this randomized, double blind, placebo-controlled study, eur- was safe, well tolerated and effective in the treatment of cf patients with epi, with clinically and statistically significant improvements in cfa, cna, and epi signs and symptoms in the absence of any concomitant treatment affecting gastrointestinal motility and ph. the therapeutic benefit of eur- was seen even in patients with very high cfa values (> %). the relative infrequency of cystic fibrosis related diabetes (cfrd), until recently, means little is known about hypoglycaemia ('hypos') associated with its treatment. insulin is frequently taken by those who have cfrd, for whom the risk of hypoglycaemia may be high due to dramatic changes in insulin sensitivity that can occur with pulmonary exacerbations and a decreased ability to secrete glucagon. a number of studies involving people with type diabetes mellitus (t dm) have reported on their experiences of hypoglycaemia, but whether results from such investigations apply to cfrd is unclear. an exploratory investigation was conducted to compare the type, frequency and severity of hypoglycaemic symptoms experienced by patients who had cfrd with patients who had t dm. method a cross sectional study was conducted, involving adults ( - years) with t dm or cfrd, recruited from two hospitals in england. a questionnaire, sent to patients with t dm and with cfrd, included investigator-developed items about knowledge and frequency of hypoglycaemia and the edinburgh hypoglycaemia scale (ehs), a standardised measure that assesses autonomic (e.g. sweating, hunger) and neuroglycopenic (e.g. confusion, poor co-ordination) symptoms associated with hypoglycaemia. questionnaire data were entered into spss for analysis. comparisons were conducted using t-tests, mann whitney tests or chi square tests as appropriate. results questionnaires were returned by patients with t dm and with cfrd. five of those with t dm were over years of age and, therefore, excluded from analysis. almost all participants had experienced a 'hypo' ( %). comparisons between the two groups are reported in table . conclusion patients with cfrd reported fewer and tended to have less severe episodes of hypoglycaemia compared to those with t dm. fewer neuroglycopenic symptoms in the former could be related to their shorter duration of diabetes or to how cfrd was managed; patients with cfrd were given shorter acting insulin, making them less prone to 'hypos' during the night, and had a high sugar diet to ensure weight maintenance. differences between the groups could also be related to specific characteristics of these two forms of diabetes. table : data from questionnaire insulin resistance. the purpose of this study was to investigate the relationship between pem and the risk of developing glucose intolerance and/or cfrd and the effect on lung function in children ≤ years of age whose history suggests pem. patients whose diagnosis of cf was suggested by failure to thrive(ftt) and whose growth parameters remain < % for weight and height for age were identified as having incurred pem. a retrospective analysis of our cf registry database was performed. cf patients ≤ years old were identified. of patients( . %) had cfrd. ( . %) of these plotted < % for weight and height (pem)and ( . %) were diagnosed with cfrd. further review of the patients showed whose diagnosis of cf was suggested by ftt and ( . %) of those had cfrd. statistical analysis did not show a clinical significance, but a strong relationship is implied. pulmonary function, fev %, was significantly lower in the pem/cfrd patients (mean fev . % ± . %) than the pem only group, (mean fev . % ± . %)(p= . ). this review provides further support that patients who have pem are at increased risk of developing cfrd. a significant risk was not established; however a strong association is suggested. assessment of a larger cohort of patients may show a stronger relationship. findings from this study would suggest that further research into the physiological relationship between pem and cfrd should be initiated. cfrd screening should be considered at a younger age than the current cff recommended age of > years. diagnosing cfrd/glucose intolerance early may contribute to improve growth parameters, preserve lung function, and improve survival. based on this review, our center is initiating cfrd screening for all patients ≥ years, and considers screening for all pem patients ≥ years. background: patients with cf are thought to have a rapid postprandial rise in plasma glucose which may be followed by a delayed and prolonged insulin response and hypoglycemia. we sought to estimate the prevalance, cumulative one year incidence, and factors associated with hypoglycemia during oral glucose tolerance testing in non-diabetic adults with cf. we also compared the prevalence of hypoglycemia to a geographically matched cohort of young adults without cf. methods: we performed a prospective study of individuals aged over years with cf followed in clinic providing population-based, specialized care in cambridgeshire, england. we excluded patients with previously-diagnosed diabetes or taking anti-diabetic medications. we evaluated individuals who underwent g hour oral glucose tolerance testing (ogtt) in . of these, individuals had neither new diabetes nor hypoglycemia in and were retested using ogtt in . we testing the association between biochemical and anthropometric factors measured in with the prevalence of hypoglycemia in , and incident hypoglycemia in . biochemical hypoglycemia was defined as a blood glucose between < . mmol/l ( mg/dl), but greater or equal to . mmol/l ( mg/dl) and severe hypoglycemia as a value lower than . mmol/l on either the fasting or post glucose challenge value. for comparison, we used data from the cambridgeshire-based young ely study of individuals age - years who underwent g ogtt testing in - . results: in , in patients with cf, the prevalence of fasting hypoglycemia was . %. no patients had fasting severe hypoglycemia. following glucose challenge, . % and . % of patients had glucose values consistent with hypoglycemia and severe hypoglycemia respectively. there were no diferrences in age, hepatic, pulmonary or renal function, but patients with hypoglycemia at two hours were five times more like to be male (odds ratio . , % ci . - . ) and more likely to have a higher bmi. the prevalence of a hours value of < . mmol/l in patients with cf at . % ( / ) was not different than in controls . % ( / ). of patients who did not have hypoglycemic readings in , at one year, patient developed fasting hypoglycemia and individuals developed post-challenge hypoglycemic for a cumulative incidence . %. of these . % ( ) had values in the range of severe hypoglycemia. there were no differences between age, sex, bmi, liver, renal, or pulmonary function in those who did and did not develop hypoglycemia. conclusions: hypoglycemia appears common in patients with cf but is also common in a healthy population. male sex and bmi were associated with post load hypoglycemia in patients with cf in cross-sectional analyses only. further research needs to assess whether these low blood glucose values are associated with symptoms. it is well recognised that cystic fibrosis related diabetes (cfrd) is a poor prognostic indicator in cf, and therefore early recognition is imperative to allow effective treatment in an attempt to prevent the associated decline in pulmonary function and nutritional status. although the oral glucose tolerance test (ogtt) is the accepted method of detecting diabetes mellitus in non cf individuals, glucose intolerance and lack of insulin can be variable in cf patients such that the reliability of ogtt in the diagnosis of cfrd has been questioned. under these circumstances, other methods of monitoring glucose intolerance, such as serial post prandial glucose monitoring (sgm), may be more appropriate. to look at this further, we prospectively compared ogtt with sgm in a group of adult cf patients. fourteen patients with no previous history of cfrd ( ], df homozygous, male) admitted for acute pulmonary exacerbations were evaluated. all were exocrine pancreatic insufficient, and was on long term oral steroids. eleven patients received mg/day oral prednisolone in addition to iv antibiotic therapy for the duration of their admission. ogtt was performed in the standard fashion (ingestion of . g glucose/kg body weight [maximum g] within minutes, after an overnight fast). sgm was performed hours post prandially and before bedtime over the period of the admission. the local ethics committee approved the study and informed consent was obtained from all patients. results sgm revealed elevated postprandial blood glucose values in all patients. there were episodes (mean per patient [ to ]) with glucose > . mmol/l (frank diabetes, who criteria) and episodes (mean per patient [ to ] ) with glucose . to . mmol/l (impaired glucose tolerance, who criteria). however, ogtt revealed impaired glucose tolerance in only one patient ( hour glucose value . mmol/l). in the remaining patients, ogtt revealed normal hour glucose values (mean . mmol/l [range . to . ]). fasting plasma glucose values were also normal (mean . mmol/l [ . to . ]) in all patients. we have shown a high prevalence of hyperglycaemia in our adult patients admitted for pulmonary exacerbations that was only detected using serial glucose monitoring. this study suggests that sgm may be more sensitive at detecting early cfrd than the ogtt. further work needs to be carried out to look at the best methods of diagnosing cfrd, to facilitate early treatment and thereby improve the prognosis. the diagnosis of cystic fibrosis related diabetes (cfrd) is associated with a decline in pulmonary function and nutritional state and is a poor prognostic indicator: the risk of its development increases with age. furthermore, the development of cfrd is due to a gradual loss of insulin production, and the disease may therefore be preceded by a period of glucose intolerance. despite this, the incidence of hyperglycaemia in cf patients is unknown. to study this further, we monitored the blood glucose profiles of adult cf patients admitted for the treatment of a pulmonary exacerbation. we looked at consecutive cf patient admissions for iv antibiotic therapy to our large adult unit. of these, had established cfrd and were excluded. the remaining patients (mean age years [range to ], male) formed the study population. none were on long term oral steroids, but received mg/day prednisolone as part of their routine exacerbation treatment. all patients underwent blood glucose measurement hours after each meal and before bedtime as part of a standard assessment protocol in our clinic. thirty patients ( %, / on steroids) demonstrated post prandial hyperglycaemia: exhibited values between . and . mmol/l (impaired glucose tolerance, who criteria) and > . mmol/l (frank diabetes, who criteria). there were no significant differences in age, genotype, fev , fvc, bmi, or duration of hospital stay between the two groups. of the hyperglycaemic patients, ( %) had abnormal glucose levels detected before bed time, significantly more than hours post breakfast ( %, p< . ), post lunch ( %, p< . ), but not post dinner ( %, p= . ). this study has shown a high prevalence of hyperglycaemia in adult cf patients treated for a pulmonary exacerbation who were not otherwise known to suffer from cfrd. nearly all our patients demonstrated glucose intolerance later in the day, when the recommended screening test for cfrd (the fasting oral glucose tolerance challenge) cannot be used. post prandial glucose monitoring of such patients during times of stress may therefore be advisable, allowing the introduction of insulin treatment at an earlier stage in the disease process. whittaker, l.a.; tilluckdharry, l.; christian, r. medicine, university of vermont, burlington, vt, usa objective: bone disease is a recognized complication of cystic fibrosis (cf). there are currently no guidelines for bone mineral density (bmd) testing or tools for fracture risk assessment in cf adults. we piloted a survey designed to identify cf-specific risk factors for low bone density and fracture. methods: a question survey was completed by patients prior to measurement of bmd at the hip, spine and radius by densitometry (dxa). the survey was comprised of questions on calcium and vitamin d intake, exercise, family history, medications, reproductive health, ultraviolet exposure, cf related diabetes (cfrd), weight loss, severity of cf lung disease and quality of life. z scores (bmd adjusted for age and gender) were used so that male and female data could be analyzed collectively. patient perceived health (pph) was rated by four questions and totaled for the pph score, which ranged from - with the highest score indicating the best health. functional status (fs) was rated by three questions that reflect the degree to which cf interferes with school, work or play. the total fs score ranged from - , with the highest score representing the least interference with life. questions on pph and fs were derived from a validated clinical assessment tool, the cf questionnaire (cfq) ( ) . results: all cf adult patients (n= , age and older) were offered participation in the study. dxa of total hip and lumbar spine was performed on patients ( %); ( %) of these patients also completed the survey. % of patients had low bone density on dxa (z score <- ). % of male cf adults had low bone density vs % of females. fracture was reported by % of patients who completed the survey ( % female and % male). lower pph score was associated with lower z score at the hip and spine. lower fs score was associated with lower z score at the hip and spine. low body mass index was associated with low z scores at both hip and spine. patients with bmi below kg/m were most likely to have osteopenia. conclusions: osteopenia and history of fracture were common in our adult cf population regardless of gender. bmi, pph and fs predicted bone density. risk stratification by a cf-specific bone health survey may guide bone density screening strategies for cf adults. prospective studies in cf adults are needed to determine whether a cf-specific survey can predict fracture risk as well as bone density. references : as life expectancy in cystic fibrosis (cf) increase, osteoporosis has become more prevalent. vitamin d status is one of many factors that contribute to optimal bone health. previous data has shown, the majority of cf patients are deficient in vitamin d despite being prescribed daily cf specific vitamin therapy. previous research has also shown that the current recommendations for vitamin d supplementation and repletion provided by the cystic fibrosis foundation bone health consensus ( ) are not sufficient to achieve optimal vitamin d levels in the majority of patients. moreover, the recommendations fail to sustain levels in the optimal range for bone health. purpose: we demonstrate a successful supplementation algorithm for individualized high dose vitamin d with continued high dose maintenance therapy which achieves and sustains appropriate levels. methods: all patients followed at the pediatric center are prescribed routine cf vitamin supplementation. vitamin d levels are obtained as part of routine clinical care via mass spectroscopy. if serum vitamin d level is < ng/dl, vitamin d therapy with ergocalciferol is initiated one time per week. patients < years of age receive iu/ dose and > years of age receive iu/dose. at the next routine quarterly visit, vitamin d levels are re-assessed via mass spectroscopy to determine efficacy of once weekly supplementation. if levels are > ng/dl, patients continue high dose supplementation time a week as maintenance therapy. however, if levels remain < ng/dl, the ergocalciferol is increased by time a week in month increments until adequate levels are obtained. once adequate serum vitamin d levels are obtained, patients remain on the level of supplementation needed to achieve levels > ng/dl as maintenance therapy. all patients are instructed to take vitamin d and their cf vitamins with enzymes and food to optimize absorption. results: after implementation of the algorithm, . % of patients, with varying levels of supplementation, had vitamin d levels > ng/dl (mean= . ng/dl). of the patients that achieved goal vitamin d levels, % were able to achieve and sustain adequate vitamin d levels without additional vitamin d supplementation while on standard cf vitamins (mean vitamin d level = . ng/dl). the remaining patients required chronic maintenance vitamin d: % required vitamin d supplementation once per week with a resulting mean vitamin d level of . ng/dl, . % required maintenance vitamin d supplementation twice per week with a resulting mean vitamin d level of . ng/dl, and, . % required vitamin d supplementation or more times per week with a resulting mean vitamin d level of . ng/dl. another % of patients take vitamin d supplements other than ergocalciferol and were not included in this study. conclusions: individualized vitamin d repletion and maintenance therapy as outlined in our algorithm is a successful mechanism to obtain optimal vitamin d levels in patients with cf. inhaled tobramycin ( %). sixty-two percent reported using at least different types of oral medications on that day including pancreatic enzymes ( %), oral antibiotics ( %), anti-reflux medication ( %), azithromycin ( %), anti-histamine ( %), and a decongestant ( %). twenty percent reported taking at least one medication for pain. forty-nine percent reported performing airway clearance at least twice during the day with % using a vest, % performing standard chest physiotherapy, and % using a flutter or acapella device. there was no difference in the reported number of medications or the reported time needed to complete therapies based on respondent age. although respondents with severe lung disease (fev < % predicted) reported a higher median number of therapies ( vs. , p= . ), the reported time for completing therapies was not associated with fev (p= . ). conclusion: daily treatment burden and complexity in all adults with cf is high, and is only marginally increased in those with worse pulmonary function. given this already high load of daily therapies, efforts to assess the treatment burden of new cf therapies are warranted. the impact of this high treatment burden on overall health related quality of life for adults with cf needs to be assessed. objective: the cystic fibrosis foundation (cff) has a network of over accredited care centers throughout the united states. data is collected from these care centers to help better understand the disease and describe changes in survival, standards of care, and health outcomes. when the cff was founded in few children survived to school age. now the predicted survival extends beyond the mid 's (cff, ) . with this change has come the need for age appropriate care in many areas including reproduction. the adult cystic fibrosis center at morristown memorial hospital presently follows adults. of those, women( %) with cf have had children affected with cystic fibrosis. each woman was diagnosed after her child. the cf data registry does not have any means of identifying if a patient has an affected child. as the average age of survival of cf patients has increased, so has the likelihood of cf patients having cf offspring. what is the estimated incidence of cf patients having cf affected children attending accredited cf centers in the united states? method: questionnaires were sent to care centers and satellite centers via standard mail or facsimile based on information from the cff directory. one month after the last questionnaire was mailed, the cf foundation sent out a reminder email to the nurse coordinators. questions asked were as follows: ) name of center ) do you presently care for any patients with cf who have given birth to, or fathered a cf affected child? ) how many patients at your center have had cf affected children? ) was the parent diagnosed before or after pregnancy was confirmed? ) if parent's diagnosis came after conception, was this after child was diagnosed? ) was the parent's diagnosis a result of prenatal testing? results: a total of questionnaires were mailed or faxed. this netted a result of responses. after email reminder, an additional questionnaires were returned for a total of responses, an % response rate. name of center was checked to remove duplicate information. the number of parents with cystic fibrosis who have had children with cf numbered . of those, ( %) were diagnosed before pregnancy; ( %) were diagnosed after pregnancy. of those diagnosed after pregnancy, the majority ( %) were diagnosed as a result of their child's diagnosis. only ( %) were diagnosed as a result of prenatal testing. conclusion: based on the responses from accredited cf care centers, patients with cf have children also affected with this disorder. prior to conceiving their child, % knew that they had cf. having cf did not appear to be a deterrent to delivering cf offspring. this study did not look at the age of the parent or child; they may have conceived their child before genetic screening was common for spouses. as the median survival of this population increases, the incidence of cf adults with cf children may also increase. the cff may choose to add a question to the cf data registry, identifying cf patients with cf offspring. the potential physical and psychosocial impact can then be tracked. anderson, a.; popli, k.; stewart, j.; heslop, k.; gascoigne, a.; bourke, s. adult cystic fibrosis centre and fertility centre, royal victoria infirmary, newcastle upon tyne, united kingdom as the outcome of patients with cf improves fertility issues become increasingly important. almost all men with cf are infertile because of azoospermia due to congenital absence of the vas deferens. these men can attain genetic paternity by sperm aspiration from the epididymis or testis with intracytoplasmic sperm injection (icsi), but few men with cf seek fertility treatment to assess their knowledge and attitude towards fertility issues a questionnaire was sent to men with cf: ( %) responded; ( %) declined to give information and ( %) replied in full. overall % thought that nearly all men with cf were infertile, % did not know how many were affected, and % could describe the exact nature of infertility. only % knew that treatment for infertility was available: % of these thought it was rarely successful and % were unsure of success rates. although % had thought of having children at some stage, only % had sought investigation of infertility. two men had children, one by icsi and one by donor insemination. overall % feared that the child could have cf and % worried that their health would affect their ability to function as a parent. in terms of receiving advice % thought that this should be given by a fertility specialist, % by a cf physician and % by a primary care physician. with regard to fertility treatment % thought that decisions should be made by the patients and their partners, % by the patient alone, and % by a doctor. the reluctance of men with cf to seek fertility treatment appears to be due to many factors including lack of knowledge of available treatments, fears about the child inheriting cf, and concerns that their health would adversely affect their role as a parent. in addiition to information provided by the cf team advice from a specialist in fertility treatment may help to improve the provision of reproductive counselling to these patients. nash, e.f. , ; coonar, a.s. ; stephenson, a.l. , ; delgado, d.h. ; singer, l.g. , ; tullis, e. , ; chaparro-mutis, c. studies have shown varying degrees of right ventricular (rv) dysfunction in adult cf patients with severe lung disease. left ventricular (lv) dysfunction is much less common with conflicting reports as to its prevalence. pulmonary hypertension (ph) is commonly seen, the severity correlating with fev . one group reported that ph was more prevalent in b. cepaciacolonized patients, albeit in a small sample. the aim of this study was to determine the true prevalence of ph and cardiac dysfunction in cf patients with severe lung disease. we carried out a retrospective cohort study of all adult cf patients referred to the lung transplant program at toronto general hospital from - . adult patients referred with non-cf bronchiectasis were included for comparison. demographic data, lung function, -minute walk, micro-biology, doppler echocardiography and resting and stress radionuclide ventriculography (muga) results were analyzed by students t-test (parametric) or mann whitney u test (non-parametric). results adult cf patients ( % non-b. cepacia, % b. cepacia) and non-cf bronchiectasis patients were included. data from these groups are summarized in the table. we found higher minute walk and bmi values and a higher percentage of males in the b. cepacia compared to non-b. cepacia group. ph was present in % of the cf patients in whom pasp could be obtained. b. cepacia patients had lower first pass rvef compared to non-b. cepacia patients. % of the b. cepacia patients had an abnormal lvef response to exercise (defined as < % increase on exercise) compared to % of the non-b. cepacia patients. we found a high prevalence of ph as well as rv and lv dysfunction in cf patients with severe lung disease. b. cepacia-colonized patients had worse rv function compared to non-b. cepacia-colonized patients on first pass radionuclide ventriculography. *non-cf bronchiectasis group older than cf groups (p< . ) b. cepacia cf group higher than non-b. cepacia cf group (p< . ) b. cepacia cf group lower than non-b. cepacia cf group (p<. ) perobelli, s. ; dorazio, c. ; assael, b. ; tamanini, a. ; castellani, c. . cystic fibrosis center, verona, italy; . molecular biology laboratory, verona, italy cf neonatal screeening (nbs) can identify neonates with elevated irt, normal or borderline sweat chloride concentrations, and one cf mutation plus an additional cftr sequence variant, or, alternatively, two cftr sequence variants. the combination of hypertrypsinogenemia and two cftr gene defects is consistent with the presence of a cftr-related disorder (cftr-rd). although these neonates are usually healthy at diagnosis, the long-term phenotypical consequences may be highly variable. such variability makes impossible to predict the clinical outcome, and provide satisfactory genetic counselling for the family. the diagnosis of such a vague condition could potentially cause considerable family stress. this study aimed at assessing how parents of infants diagnosed with a cftr-rd through nbs perceive their children's health. a questionnaire for parents was designed, consisting of three sections. the first section asked about information given to the family in the neonatal period, and about emotional reactions to the cftr-rd communication of diagnosis. the second section investigated the present perception of the child's health, and the parents level of anxiety/concern. the third section focused on the impact of the diagnosis on family planning, on relationships inside the couple and with the child, and on emotional distress; this section was concerned also with the use of carrier testing in relatives. parents were also asked to fill in a standardized form on child behaviour (achenbach child behaviour check-list). the questionnaire was distributed to two populations, the parents of children diagnosed with cftr-rd through nbs (group a), and the parents of children diagnosed with cf through nbs as the life expectancy of children with cf increases new issues arise that have not been dealt with in the past. in the past,issues related to the support and care of a young adult with cf were rarely raised because only small numbers of children with cf lived into adulthood. however, with an increasing young adult population, young adults without family support have surfaced in significant numbers and their needs should be identified and understood by the cf center care team. these young adults face the same range of issues others with cf face related to health insurance, government benefits, housing, education and employment. however, they do not have a family to help them wiht support, health insurance benefits and care needs. method in october , the cf legal information hotline ("hotline") began to provide free and confidential information to people with cf, family members and healthcare providers in the u.s. the hotline is staffed by attorneys, beth sufian and james passamano(bs and jp) and is accessed by dialing a toll free number or by e-mail. information about the hotline is available at cf care centers, in cf publications and on the web. information regarding the content of each call is recorded on an intake form and includes caller name, address, phone number, age of person with cf, cf center attended, and information related to the caller's question, attempts made to access legal information prior to calling the hotline, relation of access to information to access to care. calls and e-mails are tracked by seven major categories which are divided into subcategories. if a caller has more than one question, the call is tracked using the caller's primary issue. results from november to april the cf legal information hotline has received calls from young adults with cf who do not have family support and have issues related to obtaining health insur-ance, government benefits, educational oppornties or employment opportunities. as well as issues related to lack of support for care. the hotline has also received calls from cf care centers team members with questions related to the needs of young adults without family support. over an eight year period the cf hotline received _______calls. the categories for the calls were: social security benefits ( , ) , health insurance ( , ) , educational ( ), employment ( ), long term disability benefits ( ), family law ( ) and miscellaneous legal issues ( ). discussion people with cf face obstacles to care if they are unaware of their legal rights especially in the areas of health insurance and government benefits. healthcare providers are busy addressing the medical needs of patients and are often not well equipped to provide detailed information about the legal rights of their patients. the hotline provides important information to both patients and healthcare providers that can optimize both patient care and outcomes. access to legal information can result in individuals obtaining health insurance, medicaid, medicare or other state coverage that in turn results in better access to treatment. legal information can also lead to important modifications in the educational or employment environment for the individual with cf. these modifications can be critical in allowing those individuals access to important care and treatments. providing information that can help individuals obtain social security or long term disability benefits can result in a monthly benefit and health insurance that then allows the individual to devote more time to their healthcare. many people with cf and healthcare providers lack information about the legal rights of individuals with cf. understanding the legal rights of people with cf can reduce obstacles to obtaining care, optimize education and employment, and increase access to retirement benefits. further work needs to be done to identify ways to make individuals with cf and healthcare providers aware of the hotline. the cf hotline provides a unique service to cf patients and healthcare providers by providing information regarding the legal rights of people with cf. identification of ways to assist young adults without family support should be identified and cf center xcare teams shoudl be given the tools to allow them to help their patients access government assistance and other programs that can help make sure that the young adults have acces to care and support they need to live with cf. the dvd gives healthcare staff, patients and families a rare glimpse of ten couples with cystic fibrosis (cf) interacting at a cf care center-sponsored 'couples support group'. they discuss the impact this forum has in their daily lives for gainingo information about how cf affects their lives as individuals, and as couples, and how the 'non-cf partner' learns from others. o skills at networking and personal coping strategies. o reflections on 'family life and cf', and o observations on ways cf care centers can join them in greater health partnership. lastly, this dvd provides a 'starter kit' for ways to establish such a group. one of the realities of a life-threatening illness can be that of social isolation, and of separation from others who share the same disease. having the opportunity of participate in a support group can significantly reduce that sense of isolation and give the participants an ability to share experiences and give support to others with this disease. cystic fibrosis as an illness has growing ranks of 'survivors' who live longer, fuller lives, thanks to the advances made on multiple-research fronts. historically, adults with cystic fibrosis have not had much occasion to share person-to-person experiences within the structure of their cf care centers. justifiably, with the potential for cross-infection, the cystic fibrosis foundation has urged caution for the cf population in having face-to-face interactions. in , our cf care center recognized this need and sponsoredwith guidance from staff facilitators-an on-going adult support group. prior to the formation of the group pre-and on-site group ic protocols were developed. at the pre-group stage, the physician initially certifies that the patient is free from aggressive pathogens. each time the patient rsvp's permission to attend a group session, the physician must 'recertify' this status. on-site protocols emphasize a number of ic precautions to minimize crossinfection. the center recognized that by developing such a support group, 'patient and family centered care' could be enhanced. a goal in the group's formation was to create 'mutually beneficial partnerships among patients, families, and healthcare providers'. group structure/successes to date: the group is limited to participants. two cf care center staff act as facilitators. this is a support, not a therapy group. compliance with therapies is reported to have increased as one outcome of this forum. formal topics are purposefully not predetermined; the group's success is predicated on the personal responsibility of each participant to bring topics/issues from their daily life experiences. the group has gained identity and trust and has evolved over three years into two separate groups: an individual cf group and a couples cf group. ownership of these groups by those who attend is apparent. both the care center and the couples support group are forums for learning 'how to live with cf'. future goals will call for using identified quality of life tools to measure psychosocial and emotional growth in individuals through their participation over time in the group. sawicki, g. ; asher, d. ; dill, e. ; sellers, d. ; robinson, w. . children's hospital, boston, ma, usa; . education development center, newton, ma, usa background: advance care planning (acp) is an important tool to promote alignment of the patient's care with his or her needs, goals and values, and is particularly useful if the patient's ability to make decision becomes compromised. the purpose of acp is to develop a treatment plan promoting a high quality of life for those near the end of life. such planning is especially appropriate in persons with life limiting disease. minimal research exists on the advance care planning process of adults with cf.
methods: in the fifth survey wave of the project on adult care in cystic fibrosis (pac-cf), an ongoing longitudinal panel study of cf adults from cf centers in the us, participants were asked to report on their opinions and experiences with advance care planning. the survey asked about opinions on advance care planning documents, communication about advance care planning with family and clinicians and whether a directive had been completed. clinical data were obtained from the participating centers. preliminary bivariate analyses were conducted to examine factors associated with completing an advance care directive.
results: / ( %) surveys were completed. the mean age was ± years, % were female, and the mean fev (% predicted) was ± . % reported that they had spoken to someone about the care they would want if they became too ill to make their own decisions in the future, % had thought about whom they would want as their healthcare proxy, and % reported having specific wishes about the types of medical treatment they want or would not want if they became too ill to make decisions for themselves in the future. however, only % reported completing a healthcare proxy form, living will or any other type of written instructions concerning advance care directives. % of respondents reported feeling comfortable talking to their clinician about acp. % of subjects said that their cf clinicians have asked them about acp, and only % reported that they have discussed acp with their cf clinician. the following characteristics were significantly related (p<. ) to completion of an advance care directive: female gender ( % vs. %), age (mean years vs. years), having a college degree ( % vs. %), being married currently or in the past ( % vs. %), having children ( % vs. %), having diabetes ( % vs. %), having specific wishes about treatment decisions ( % vs. %), and reporting that a clinician had discussed acp with them ( % vs. %).
discussion: though the majority of cf adults report thinking about, communicating with family and deciding on their advance care wishes, only a minority report completing any legal documentation supporting their decision. additionally, very few cf adults report being asked about acp by their clinicians and even fewer report discussing acp with them. formulating specific wishes and discussing acp with a clinician are strongly associated with completing an advance care directive, suggesting that if clinicians were more active in talking to their patients about acp, patients are much more likely to complete advance care directives. efforts to improve clinician communication with cf adults around the issues of advance care planning are needed. objectives: caregiver involvement in children's cf treatments fosters better adherence and improved health outcomes. several studies have suggested that parents of children with cf report elevated symptoms of depression (quittner et al., ) , however, there is no data on the effects of caregiver depression on adherence. adherence to treatment is difficult to measure in patients with cf, and prior research has relied primarily on self-report, which is likely to be inflated. the purpose of this study was to examine the effects of caregiver depression on adherence to enzymes using both parent-reported and electronically monitored enzyme adherence over a three-month period. in addition, we examined the relationship between depression, rates of enzyme adherence and short-term changes in weight. methods: as part of a larger intervention study at cf centers, children with cf ages to and their parents were recruited. parents completed a standardized measure of depression (cesd) at enrollment and were provided with mems caps that recorded the date and time of each bottle opening. three months later, the electronic data from the pill caps was downloaded and parents completed a structured interview reporting their child's adherence to all components of the cf treatment regimen. only the section addressing enzyme adherence was included in this analysis. standard health outcomes, such as weight and height, were also assessed at enrollment and months later. results: preliminary analyses indicated that caregivers reported elevated levels of depression, with % scoring in the clinical range. rates of adherence to enzymes were poor ( % at home and % at school). caregiver depression was negatively associated with adherence, with depressed caregivers demonstrating lower rates of adherence ( percentage points). adherence to enzymes was associated with changes in weight, with a % adherence translating into percentile points of weight gain. final analyses will also include parent-reported adherence to enzymes. conclusions: a substantial number of parents scored in the clinical range on a depression screening measure. in addition, depression was associated with poorer adherence. rates of adherence to enzymes were surprisingly low, both at home and at school. furthermore, poor adherence was associated with a decrease in weight three months later. caregiver depression appears to be under-diagnosed and these results suggest that screening and intervention may be warranted. adherence to enzymes should also be targeted in clinical interventions. funding was provided by nih grant #hl of non-compliance with medical treatment and greater behavioral and emotional distress. adherence is particularly problematic during adolescence, however, few family-based interventions have been developed to target adherence behaviors in this population. the goal of this study was to compare the effects of behavioral-family systems therapy (bfst), an empirically-supported treatment, to both family education (fe) and standard care (sc). methods: as part of a larger intervention study, adolescents with cf ages to and their parents completed the conflict behavior questionnaire (cbq) and the parent-adolescent relationship questionnaire (parq) developed by robin and foster ( ) . adolescents completed these measures for each parent and both parents, if available, completed them with respect to their teen. the problem-solving, communication, and beliefs subscales from the parq were administered. participants were then randomly assigned to one of three treatment arms: bfst, fe or sc. families in the bfst group received ten -minute sessions over a month period, including family problem-solving, communication skills training, and cognitive restructuring. families assigned to the fe group received ten minute psychoeducational sessions over months aimed at increasing knowledge about cf and its management. those in the sc group received their usual care at the cf center. the cbq and parq were completed pre, post and , and months following the intervention. results: adolescents reported that the bfst intervention, relative to the other two, improved their communication with their primary caregivers (p < . ) and caregivers improved their communication with their adolescents (p < . ). in addition, caregivers reported improved problem-solving with their adolescents (p < . ). on the cbq, bfst reduced both adolescent and primary caregiver conflict and fe reduced caregiver conflict. no significant improvements were found for those in sc. conclusions: preliminary findings suggest that bfst is effective in improving communication skills and reducing conflict in adolescents with cf and their caregivers. future analyses will evaluate the effectiveness of bfst and fe on adherence and other family functioning measures over the course of the study. funding was provided by nih (hl # ) background: cases of diabetes among people with cystic fibrosis (cf) have increased as life expectancy for these patients improves, yet the impact of this additional illness on daily functioning is under-researched. to explore this issue, a study was conducted comparing the views of patients with either cystic fibrosis related diabetes (cfrd) or type diabetes mellitus (t dm) about being diagnosed and living with diabetes. methods: qualitative research was used because the study was concerned with understanding diabetes from patients' perspectives. purposive sampling was employed to achieve maximum variation in terms of duration and type of diabetes experienced by participants. data were collected via semi-structured interviews, all of which were taped and transcribed verbatim. recruitment continued until no new ideas or insights emerged from additional participants. a framework approach to analysis was adopted. this involved coding and summarising interview data into charts to explore and develop main themes. initial analysis was undertaken by two researchers (st and cd) and amended after comments from the remaining authors. results: interviews, carried out with cfrd and similarly aged t dm patients, lasted for an average of minutes (range - minutes). the following themes were derived from the data collected: evolving vs fracturing identity; diabetes in context; self-management motivators. for patients with cfrd, diagnosis represented a progression in their health status, which called on them to adapt existing treatment regimens to accommodate this additional condition. in contrast, interviewees with t dm had to re-evaluate their previous sense of self as 'healthy' and adjust to manag-ing a long-term complaint. these individuals were more likely to talk about diabetes in relation to a range of competing commitments (e.g. work or family related) and to describe feeling psychologically low due to diabetes compared to patients with cfrd, the latter depicting demands from their primary illness (cf) as a major obstacle to caring for diabetes. participants with cfrd recalled feeling lucky when told they would not face strict dietary restraints, which they associated with other forms of diabetes, and seemed less concerned about diabetic complications than those with t dm. for interviewees with t dm, a desire to reduce future health risks motivated their self-management efforts, whilst those with cfrd were driven by the negative effect poor control of diabetes had on their chest and weight. conclusions: both sets of interviewees found diabetes time consuming and, on occasions, frustrating to accommodate into daily life. findings from the study act as a reminder that patients manage their condition in the real world, against a plethora of other demands on their time and energy. in the narratives of participants with cfrd, these demands were often related to the existence of their primary illness. ing the course of development. the purpose of this study is to examine rates of medication adherence across a wide age span using an objective measure of adherence and to compare adherence to health outcomes. methods: patients with cf age years or older who are prescribed azithromycin, colistin, hypertonic saline, pulmozyme, and/or tobi for at least one year are eligible. recruitment is ongoing. the previous year's medication refill records were requested from participant-identified pharmacies. a medication possession ratio (mpr) was calculated for each drug and was defined as the sum of all days of medication supply received during the months divided by the number of days the medication was prescribed. values were truncated to % and averaged across all medications to obtain a composite score. medical records were abstracted to identify the prescribed drug regimen and health outcomes (e.g., lung function, exacerbations, bmi) over the same time period. results: thus far, patients have joined the study ( % participation rate), resulting in pharmacy records requested ( unique pharmacies). of the first participants with complete pharmacy data, the mean age was . years (sd= . ; range= - ) and % were female. the table presents mpr data for each drug and composite score overall and stratified by child, adolescent, and adult participants. the complexity of the drug regimen and age were significantly correlated with the composite score (rho= -. and rho= -. respectively; p<. ). future analyses will compare the composite score and each drug's mpr with health outcomes. con-clusions: participants had suboptimal medication adherence similar to that reported in the literature. as mpr provides the maximal possible level of adherence, actual adherence may be even lower. poor adherence spanned the age groups and was associated with regimen complexity. children had the highest adherence, but also the least complicated drug regimen. these results suggest that obtaining pharmacy records is a viable means to objectively assessing medication adherence. moreover, results suggest that interventions targeting medication adherence may be a strategy for improving health outcomes, particularly for adolescents and adults. * fewer than participants within this cell (cf) whose exchange capacities for oxygen and carbon dioxide are already diminished. lack of rem and delta sleep have significant implications for the daytime functioning of pediatric patients with cf, potentially leading to impaired memory and attention which would reduce their ability to complete typical activities, such as school work and disease-specific tasks related to self care and adherence. based on these clinic findings, the authors are beginning a prospective study on the effects of sleep on neuropsychological functioning, well-being and compliance in pediatric patients with cf. future research should examine the relationship between decreased slow wave, rem sleep and psychosocial outcomes, including attention, memory, and health-related quality of life. there are diagnostic challenges related to non-classic cf (knowles and durie, ) particularly patients presenting in adolescence or adulthood with obstructive azoospermia, chronic sinopulmonary disease and chronic or acute, recurrent pancreatitis. practitioners are often unable to provide clarity to patients about diagnosis, disease course, and prognosis. potential harms and benefits of relaying a clear or unclear diagnosis may be psychological rather than physical and have long-term implications for mental and social well-being. the aim of the current study is to assess the psychological impact of diagnostic information for adults presenting with a cf phenotype, be it confirmatory (cf or most likely not cf) or unclear. method: we administered self report measures pertaining to psychological state, cognitive appraisal and uncertainty in a welldefined cohort of adult patients. presenting symptoms of patients include chronic sinopulmonary disease, obstructive azoospermia, pancreatitis, and those presenting with more than more cf phenotype. patients completed the self-report measures on two occasions: at the time of diagnostic testing and months after being counseled and notified of the diagnostic results. at the time of the initial assessment, the subjects were unaware of the test results and had not been seen by a cf physician. results: we provide interim observations on patients (sinopulmonary (n= ), obstructive azoospermia (n= ), pancreatitis (n= ), and multiple phenotypes (n= ) who completed the initial assessment. at the time of diagnostic testing, the level of depression, hostility, anxiety and interpersonal sensitivity were elevated in each group (t score > ); male patients reported significantly greater depression, anxiety and interpersonal sensitivity than female patients. compared to published normative data of patients with mixed chronic illnesses, patients presenting with sinopulmonary or pancreatic disorders reported significantly more uncertainty(p < . ). to date, of these patients have completed the measures months after receiving the diagnostic information. preliminary review of results shows that the level of depression, hostility, anxiety and interpersonal sensitivity remained elevated while the degree of uncertainty decreased. among the patients who completed assessment at months, patients were told that it was unlikely that they had cf, were told that they had mild cf, while were given an unclear diagnosis. those who were told that they did not have cf reported more uncertainty than those who were told they had cf (p < . ). detailed analyses related to outcome diagnosis will be conducted at the time of study completion (october ) . when this study is complete, the information will assist in our understanding of the psychological impact of a genetic diagnosis at an older age, identify issues confronting those individuals and help to establish appropriate paradigms for delivering complex information about genetic diseases. supported by grants from niddk [scor] and the canadian cf foundation. ( ). in accordance with these guidelines, we report our experience in using our nutrition action plan (nap) in patients with suboptimal nutrition. objective: to determine if implementing our nap would improve the bmi of these patients. methods: our nap is a written contract between the patient, caregiver and cf team. for clarity of nutritional goals, the nap is based on the colors of a traffic light. the red zone indicates a bmi less than the th percentile and poor nutritional status. the yellow zone indicates a bmi between the th and th percentile and fair nutritional status. the green zone indicates a bmi greater or equal to the th percentile and desired nutritional status. within each color zone, specific recommendations to improve nutritional status are listed. the ultimate goal for each patient is reaching the green zone. the caregiver, patient and nutritionist review the current nutritional status and a mutually accepted weight gain goal is established. the goal is recorded on the nap which is then signed by the nutritionist and patient. this contract establishes a commitment to achieve the goal by the next clinic visit. a reward system is implemented in conjunction with the nap. for patients reaching their goal, a previously agreed upon prize is given. patients achieving some weight gain but falling short of their goal receive a gift such as candy or small toys that serve as an incentive for continued weight gain. patients chosen for inclusion into this study demonstrated a bmi less than the th percentile; those on growth hormone, appetite stimulants or chronic systemic steroids were excluded. results: twenty-one patients, male and female, with a mean age of . years (range to years) were included in this study. fifteen patients demonstrated an improvement in their bmi with use of the nap: improved by - %; improved by - %; improved by - %; improved by - %; improved by - %. six participants had a negative change in bmi which we attribute to family upheaval, missed appointments with the cf team, exacerbations of disease and non-adherence. conclusion: we intervened in patients with a bmi less than the th percentile using a nap and a reward system. the results of this pilot study suggest that our nap and reward system may be of benefit in achieving positive gains in bmi. reference: ( ) previously it was rare for women with severe cf to undertake pregnancy and doctors tended to advise against pregnancy if the forced expiratory volume in one second (fev ) was less than %. the attitudes of doctors and patients are changing and many women living their lives with cf nowadays chose to undertake pregnancy despite the severity of their health problems. over the last years females (age range - years) have attended the newcastle adult cf centre. there have been a total of pregnancies involving women with live births ( ectopic). a further women had termination of unwanted pregnancy. mean age at the time of pregnancy was years. we compared an age-matched cohort of never pregnant women to see if there were differences in key variables making successful pregnancy more likely. the mean fev was % (range - %) and % (range - %) of predicted in the pregnant and non-pregnant group respectively (p= . ); rates of chronic pseudomonas aeuriginosa infection were ( %) and ( %) respectively (p= . ). cases of burkholderia cenocepacia were identified in the non pregnant group. there were no cases in the pregnant group (p= . ); ( %) had diabetes with patients having cf related diabetes prior to pregnancy and patients developing gestational diabetes; this compared with ( %) of non pregnant women (p= . ); there was impaired nutritional status within both groups with a mean bmi of . pre pregnancy (range - ) and . in the non pregnant group (range - ) (p= . ); ( %) and ( %) patients had had previous gastrostomy feeding (p= . ). pancreatic insufficiency was present in ( %) and ( %) patients in respective groups (p= . ). no child had cf but one child had complex congenital heart disease. there were no maternal deaths but patients subsequently underwent lung transplantation and patients died leaving young children (ages and years). patients in the non-pregnant group have also died. conclusion the profile of women with cf undertaking pregnancy is now similar to the overall cf population. the woman's decision to undertake pregnancy is often not directly related to the severity of her disease. amongst those who successfully completed pregnancy maternal and fetal outcomes are generally favorable. objectives: health-related quality of life (hrqol) instruments provide important information on disease progression and are increasingly used as patient-reported outcomes (pros) for behavioral and pharmacological trials (goss & quittner, in press) . recent advances in medical care have improved the long-term stability of pulmonary functioning, which has made it more difficult to detect small improvements in fev % predicted. new outcome measures are needed; the cystic fibrosis questionnaire-revised (cfq-r; quittner et al., ) , a disease-specific hrqol measure, has shown promise in this regard. the purpose of this study was to examine the longitudinal relationships between fev % predicted, weight percentiles, and cfq-r scores for adolescents with cf. methods: as part of a larger study evaluating adherence interventions, adolescents with cf, ages to , and their parents were enrolled at six centers. parents reported on their adolescents using the cfq-parent version, and adolescents completed the cfq-teen/adult version as a selfreport. measures were completed pre and post treatment and at , , and month follow-up visits. spirometry and weight data were collected at these same time points. results: empirical bayes estimates were used to examine change over time. preliminary results indicated that adolescents' scores on the cfq-r respiratory scale were significantly and positively correlated with changes in pulmonary functioning. parents' scores on the cfq-r vitality scale were also correlated with changes in pulmonary functioning, with higher cfq-r vitality scores associated with better lung function. in addition, parents' scores on the cfq-r weight scale were significantly and positively correlated with changes in adolescents' weight. conclusions: these results support the validity and sensitivity of the cfq-r to changes in both pulmonary functioning and weight for adolescents with cf. additional analyses will be conducted to determine the relative stability of hrqol scores over time and to examine other cfq-r scales that may reflect positive changes in adolescent functioning (e.g., reduced treatment burden background: development of cf specific patient symptom tools as outcome measures is a critical step toward the development of potential new therapies for cf. we have developed a novel instrument to assess respiratory symptoms in cf patients. methods: we conducted in-depth qualitative interviews using the day reconstruction method and cognitive debriefing interviews ( adults, youth, and parents) at two cf programs, the university of washington and children's hospital and regional medical center. interviews were conducted until no new symptoms were mentioned by interviewees (i.e., data saturation). the interviews were audio-recorded, transcribed, coded and analyzed for themes. results: six pulmonary symptoms were identified in the interviews: cough, sputum production, wheeze, chest tightness, difficulty breathing/shortness of breath and fever. in addition, the most commonly cited activity and emotional impacts identified in the interviews were also included on the questionnaire. these emotions included: frustration, sadness/depression, irritability, worry, difficulty sleeping, and activities included time spent sitting or lying down, reduction of usual activities, and missing school or work. in all, eight symptom items were selected for inclusion on the self-administered questionnaire (see table) . as a result of the cognitive debriefing interviews, we changed initial language and adjusted the response options on some of the items in order to create greater distinctions between them. no important issues were felt to be omitted by patients who were interviewed. conclusions: using qualitative inductive methodology, we have created a novel patient reported outcome measure for cf. further study will be required to assess validation of the instrument. or more signs and symptoms occurred for more than two days, antibiotics were ordered with physician input and phone follow-up in days. if symptoms occurred less than two days, chest clearance was increased with phone follow-up in hours. the initial algorithm (ia) was piloted by nurses on patient calls on the emr and reviewed for adherence. subsequently, the cf phone note was revised, expanding the symptoms to include gi issues, last visit date and last hospitalization. the revised algorithm (ra) was used for a one month trial period with the entire nursing staff. a total of patient calls relating to pulmonary exacerbation were again reviewed for adherence to the algorithm. results: the review of phone notes following the ia found ( %) of the phone notes to be adherent to the assessment and treatment plan on the algorithm. several exceptions included calls received for gi issues, medications, complaints of general pediatric symptoms, and lab results. the phone follow-up within - hrs was also difficult to accomplish secondary to nursing responsibilities and difficulty locating the patient. the review of calls following the ra found ( %) notes were adherent to the algorithm. discussion: the revised algorithm and cf phone note for pulmonary exacerbation improved consistency in nursing assessment of signs and symptoms, evaluation of the current plan of care, and triggered appropriate interventions based on symptoms and length of illness. this improvement was particularly pronounced among the part-time rns who found it easier to obtain a more accurate history of patient's illnesses without asking for assistance from other cf team members. since the algorithm and cf phone note are newly implemented, tracking for consistency with the cf phone note will continue, revisions will be made as needed, and phone notes for conditions other than pulmonary exacerbation may be developed. methods: in , helen devos children's hospital integrated research coordinators (rc) from the spectrum health research department into the cf care team. this model incorporates two key aspects: . a shared resource of experienced, centrally educated and trained research coordinators (rc) with three rc's having a primary cf focus . incorporation of rc's into helen devos cf care centers' multidisciplinary team for weekly contact with patients. using the cf registry consent as a starting point of research discussion with the patients and families, the rc's present new and upcoming opportunities and answer questions about research. with attendance at local and national cf meetings, rc's have expanded their cf knowledge, networked with other rc's and explored new research trials. our site also integrated education about various aspects of research into both the clinical staff and cf family education meetings. a review of the research database was completed to assess our cf center's research program. results: over the years reviewed, participation in clinical research trials has seen significant growth and patient participation has exceeded patients enrolled into clinical studies. conclusion: utilizing a team of dedicated rc's from the research department has proven to be effective in advancing our cf center's clinical research program. discussion: to meet the growing demand of patients needed to participate in clinical trials, cf centers will need to provide research education to both the patients and families and the clinical team, improve on skills needed to identify and enroll our patients into clinical trials and include the rc's into the multi-disciplinary cf patient care team. centralized research represents an excellent model for cf centers that want to excel in clinical research. integration of rc's into the cf care team, along with education initiatives for both clinical staff and patients and families has led to successful participation and enrollment in clinical trials. further studies need to be done to better identify barriers that patients and families have with participation in research. the goal of "cystic fibrosis transition care" is to ensure that youth with cystic fibrosis (cf) are adequately prepared to participate in the management of their health condition into adulthood and as they graduate to the adult health care system. in canada, it is unknown if, or to what extent, cf transition care is practiced in individual clinics. we therefore surveyed all canadian pediatric cf clinic coordinators (n= )using a brief standardized questionnaire; surveys ( %) were completed and returned for analysis. findings: all responding clinics transfer their pediatric patients to a distinct adult cf clinic, however almost half of these share one or more team members between the two clinics. transition care is recognized and practiced by % of responding clinics: % follow a formal transition program, and % follow an "informal program". "informal" practices vary widely from one clinic to another. formal programs were created by individual clinics, and share the common properties of being based on current research, having set goals, and utilizing a tool to document the process. the average age of transfer to the adult clinic is years. most clinics have "rare exceptions only" to delaying the age of transfer, the most commonly cited reason being "very ill/palliative patient". a small number of clinics also cited "intellectually challenged patient" and "reluctant patient" as reasons to delay transfer. only % of pediatric clinics hold a formal "transition" or "graduation" clinic, which allows adult team members to be introduced to youth before the actual transfer of care takes place. conclusions: although sharing team members between pediatric and adult clinics may ease the change from pediatric to adult care, it can also create barriers to the transition process. therefore, it is encouraging to learn that the majority of canadian pediatric cf clinics view adult care as distinct from pediatric care, and that there is a consistent age of transfer across the country. transition preparedness is also recognized as an important component of care, as evidenced by the number of clinics following a formal or informal transition process. it remains undetermined, however, whether in general there is adequate preparation for adult care. with only % of clinics following a formal transition process (and % not following any type of transition program), further assessment seems warranted and may reveal inadequacies requiring remediation. future direction: to strengthen cf transition care in canada, a "patient readiness to transition" questionnaire has been developed and will be administered to canadian cf patients transferring to adult cf clinics in . analysis of this questionnaire will provide insight into how well canadian cf clinics are preparing their youth for transfer to adult life, and may provide directions for further improvement. nearly all females with cf now survive into adulthood, and advice regarding pregnancy and contraception is becoming increasingly important as part of their sexual health education. we set out to identify the level of knowledge of these issues in patients attending our large adult cf unit. using a structured questionnaire, we surveyed consecutive cf females (age range to years) attending routine cf clinics. we asked about knowledge and usage of contraception, and issues relating to pregnancy and its possible effect in cf, ensuring the opportunity for a one to one discussion with the individual on completion of the questionnaire. only ( %) claimed to have had previous advice relating to contraception and ( %) pregnancy education. of these, ( %) stated that this was from adult cf nurse specialists, ( %) from adult cf physicians and the remainder from other sources (general medical practitioners, written literature, and the internet). only patients ( %) recalled that they had been given information in the paediatric sector before transition. the majority of all advice was given verbally. of the ( %) who were using some form of contraception ( [ %] condoms, [ %] combined pill, [ %] depot injection, [ %] a coil, and [ %] the mini pill); ( %) had experienced problems, and in some cases patients had been misinformed about the reliability of their contraceptive choice. as regards pregnancy, ( %) had undergone previous termination and ( %) already had children. thirty six ( %) had considered becoming pregnant but ( %) said they would discuss this with a member of the cf team beforehand: ( %) were aware this might impact on their health. of these, ( %) believed a reduction in lung capacity to be the biggest possible problem. other problems identified were alteration of medication, tiredness, and weight loss or gain. overall, ( %) felt they did not have enough information regarding pregnancy in cf and ( %) that insufficient contraceptive advice was given, expressing a wish for further knowledge. conclusion this survey has shown that a significant number of adult females with cf require further education about contraception and pregnancy. in some cases the advice they had already been given may not have been appropriate for patients with cf. furthermore, few patients appeared to have had effective counselling in the paediatric sector, despite the risk of pregnancy. we are working with patients and paediatric colleagues to improve the education in this important area for our women with cf. the state of minnesota added cystic fibrosis to the newborn screening panel on march , . one-year follow-up to date reveals infants have screened positive, of the screen positive, have been diagnosed with cystic fibrosis, infants are pending sweat chloride tests and evaluation. of this population infants are being followed by the minnesota cf center. the university has a comprehensive, interdisciplinary team available to provide care to infants, children and adults with cystic fibrosis. in response to newborn screening for cystic fibrosis, the center has implemented a prophylactic care program to include clinical care through early intervention and education within the first two weeks of life. benefits of collaborative care have been previously identified in the literature, showing improved quality of care with increased patient satisfaction, lower mortality, and improved outcomes. infants who are identified by newborn screening and receive earlier treatment have the potential for improved physical health and development. the inclusion of comprehensive genetic consultation as an integral component of cf education, allows for informed reproductive decisions. the newborn screening intervention plan is supervised by a nurse practitioner and consists of initial evaluation, initial therapy implementation, and a teaching/consultation schedule, which covers a minimum of four appointments scheduled over weeks. follow-up visits are scheduled within hours of referral and then at one week, days and weeks post referral. the schedule is adjusted based on infant acuity and family needs. initial evaluation consists of the confirmatory sweat chloride testing and/or genetic mutation analysis, serum laboratories, chest x-ray, stool for fecal elastase, and nasopharyngeal culture. general, supportive teaching with evidence from the literature is provided. preliminary results to date demonstrate that our infants have had no pulmonary exacerbations, no pulmonary hospitalizations with pulmonary functions obtained in over half of the infants. nasopharyngeal cultures obtained at the time of ascertainment demonstrate newborn screening patients have positive cultures for staphylococcus aureus, haemophilus influenzae, escherichia coli, streptococcus pneumoniae, klebsiella pneumoniae, acinetobacter junii and alcaligenes faecalis. one infant was culture positive for pseudomonas aeruginosa at the first out-patient clinic visit. this demonstrates colonization of organisms prior to clinic exposure. through minnesota's early intervention and education plan, the families will be more adherent to prescribed care, which will result in improved cf infant outcomes as monitored by number of exacerbations, pulmonary functions and potential complications. a detailed outline of medical intervention, education, and outcomes will be presented. cf-pediatric centre we developed a malabsorption blood test (mbt) using odd-chain length fatty acids, pentadecanoic acid (pa) and heptadecanoic acid (ha), to assess fat absorption in subjects with cf. pa is a free fatty acid, while ha requires hydrolysis of triheptadecanoic acid (tha) by pancreatic enzymes for absorption. objective: to determine the mbt reproducibility in healthy subjects and subjects with cf. methods: subjects with cf ingested a liquid test meal including fats (pa and tha) on occasions at least days apart. for each subject, a standard dose of pert ( , lipase units) or the subject's usual dose (if higher) was given with each test meal. serum was analyzed for pa and ha levels at baseline and then hourly for hours. a non-compartmental pharmacokinetic analysis was performed using pa and ha concentration-time data from subjects with cf. c max was directly observed from the individual subject profiles, and auc was calculated using the linear trapezoid rule. summary statistics (mean±s.d.) were calculated for all parameters. reproducibility of the mbt was also assessed in healthy adult controls tested on occasions. between subject (bsv, %) and within subject variability (wsv, %) was calculated for pa, ha and the ha/pa ratio. results: bsv (%) and wsv (%) of pa and ha absorption are presented in the table for subjects with cf (age . ± . y, females) and healthy adult subjects (age . ± . y, females). as expected, the mbt had greater reproducibility in healthy controls than in subjects with cf. in healthy controls, the reproducibility for pa and ha absorption was comparable; there is less wsv than bsv, and the ha/pa ratio has the best reproducibility. in subjects with cf, pa and the ha/pa ratio show less wsv than ha. conclusion: wider variability in fat absorption in subjects with cf reflects the complex interactions of the biliary, pancreatic and intestinal factors background: the international, multi-center gene modifier study of cystic fibrosis liver disease (cfld) initially enrolled patients with severe liver disease (i.e. cirrhosis and portal hypertension). for the initial population despite severe liver disease with portal hypertension, many of these patients had normal liver biochemical function tests and/or inr ( - %), depending on the test. the mean age of enrollment into the study was years, with the mean age of cfld diagnosis of years. candidate gene testing revealed an increased prevalence of the alpha- antitrypsin z allele in cfld patients, particularly in females, as compared to cf patients (> yrs.) without cfld, and an association with tgfβ variants (- and codon ) in males smoothing reference centile curves: the lms method and penalized likelihood hepb) and inactivated hepatitis a (hepa) vaccines in cystic fibrosis (cf) patients chronic liver disease (cld) remains the second mortality cause in cf. we evaluated the immunogenicity of hepa-and hepb-vaccines in cf-patients because it is described lower in patients with cld. patients and methods: blood samples of cf-patients aged . - . y (mean . y) were tested twice (time interval y) by chemiluminescent microparticle immunoassay to asses the presence of hbs-ab and ha-igg, and their vaccination status was recorded. seronegative patients for hbs-ab and/or ha-igg were vaccinated in between tests with engerix®, havrix® or twinrix®. results: / ( . %) patients tested positive for hbs-ab. / cld ( steatosis and cirrhosis). / tested positive at first but negative at present data are incomplete for hbs-ab and ha-igg for and patients. conclusions: . immunogenicity of hepa-and hepb-vaccines is comparable in cf-patients and healthy subjects. . cf-patients are at risk for cld and seroconversion must be checked after vaccination. . vaccination records are important as numbers of antibodies may decline beneath the detection limit in time methods: lf was assessed in children with cf, aged - yrs, using forced expiratory volume (fev(sub) (/sub)% predicted). body composition was measured using a reference four-component model(sup) (/sup) ( cm) allowing accurate evaluation of both fat mass (fm) and the components of fat-free mass dual-energy xray absorptiometry (dxa; lunar prodigy, g.e. medical systems) was used to measure fat, lean (non mineral) and bone mass and skin-fold thicknesses (bicep, tricep, sub-scapular) were measured. strength of relationship was assessed using pearson's correlation coefficient (r) and significant body composition components were fitted into a regression model. difference in fev(sub) (/sub) between the sexes was assessed with an independent t test. results: boys with cf did not differ from the reference population conclusion: our results confirm that body fat, but not ffm or bone mass, are related to the severity of impaired lf in children with cf. it is likely that the significant association between fev(sub) (/sub)and fat in girls but not boys reflects the poor body composition of the girls with cf. longitudinal follow-up of these children should indicate whether this sex difference persists after puberty. given the fact that prognosis is worse in girls the sex difference we've identified merits more attention. fuller nj et al. four-component model for the assessment of body composition in humans: comparison with alternative methods and evaluation of the density and hydration of fat-free mass conclusions: these results show that pancreatic status plays an important role in relation with pufa status in cf patients and particularly in (the initial fatty acids in the n- and n- pathways) in neutral lipids, nonesterified fatty acids, and phospholipids this may, in some way, account for the fact that : n- is more readily metabolized, and therefore depleted, in cf animals than in wt mice. conclusion: dha supplementation increased the localization does an integrated clinical and nutritional approach prevent this case control study examines the impact of clinical approach on pre and post-cfrd clinical course. patients with cfrd (mean age . + . yrs) matched to cf controls ( . + . yrs) for age, sex and pseudomonal status had parameters of clinical status and nutritional intervention recorded annually from six years pre diagnosis of cfrd to two years post. weight and body mass index (bmi) were lower at all time points to diagnosis of cfrd (ns) but were stable as a % of control values intravenous antibiotic treatment intensified, peaking at one year post diagnosis . days/yr (cfrd) v . (cf) ns but there was no difference in nebulised antibiotic use. an aggressive clinical approach prevents nutritional decline and delays respiratory decline until the year preceding diagnosis of cfrd data show mean (sd) or % , ; battezzati with advancing age insulin secretory defects and insulin resistance cause glucose intolerance and diabetes in an increasing proportion of cystic fibrosis (cf) patients. prediction of diabetes development in cf is made difficult by unique features: many patients are normoglycemic or even hypoglycemic after overnight fast, and there are repeated changes of glucose tolerance status from normal to diabetes and vice-versa, for many years and for unclear reasons. aim of the study was to detect predictive factors of definitive cf related diabetes (cfrd) development in patients undergoing oral glucose tolerance test (ogtt) evaluations routinarily. methods. starting from , all patients followed at the cf center in milan aged > years and without established cfrd undergo ogtt yearly. among those who received their first ogtt between and , had developed definitive diabetes by µu/ml, and . ± . vs . ± . ng/ml). glucose (p= . ) and insulin aucs (p= . ) were the most important predictive variables, respectively directly and inversely related to cfrd development (pseudo r for the model: . , p< . ). glycated hemoglobin and baseline glucose concentrations were directly related with outcome at univariate analysis whereas c-peptide concentrations were inversely related. in contrast, no relationship emerged between insulin-sensitivity indexes and outcome. anthropometric (weight, height z scores, bmi) and pulmonary function indexes were also unrelated. conclusions. insulin secretory defects are an important determinant of subsequent cfrd developement response rate was / in group a and / in group b. group a included males and females, mean age was months, sd . ; group b included males and females, mean age months, sd . . followgroup b parents do ("poor-very poor" health % vs %, p< . ), but over time health perception in parents of children with cf improves, and the gap vanishes ("poor-very poor" health in last year % in group b). post-diagnosis anxiety, depressive symptoms, sleep disturbances, mood changes, and nervousness are less frequently reported in group a ( % vs % p< . ), and the diagnosis was considered to affect the parent-child relationship more in group b than in a (p< . ). the two groups did not differ in the assessment scores of internalizing, externalizing and total behavioural/emotional problems. however, group b performed worse than the general control population average (mean . sd . vs mean sd . , p< . ). thirty-five% of parents in group a and % in group b changed their family planning projects following the diagnosis physical health and anxiety symptoms: does monitoring mediate the relation? results: physical health, monitoring, and anxiety symptoms were related. children who perceived themselves to be relatively healthy were less likely to have an information processing style characterized by high levels of monitoring (r = -. , p < . ), and they reported fewer trait anxiety symptoms (r = -. , p < . ). monitoring, as hypothesized, was associated with increased trait anxiety (r = . , p = . ). low bmi, a potential indicator of poor nutritional status and physical health among youth with cf, also was related to higher monitoring (r = -. , p = . ) and trait anxiety (r = -. , p = . ). tests of mediation indicate that monitoring partially mediated the relation between poor health and trait anxiety for both child report of physical health (goodman test: z = . , p = . ) and for bmi (goodman test: z = . , p = . ), but not when parental report or fev were used to assess youth's physical health, or when parental report of youth's internalizing symptoms was used as the outcome. conclusion: youth who tend to generally scan for and are attentive to changes in their environment, which may include their internal physical environment, were found to report more anxiety. moreover, this monitoring style appears to partially mediate the relation between physical health and anxiety among youth with cf. if monitoring is found to be a risk factor leading to an increase in future anxiety symptoms among youth with cf data was analysed using a thematic analysis: "framework" results: young people say that befriending is fun, offers opportunities for new experiences, a confidant outside the family, and gives them a boost psychologically. carers see befriending as confidence building for their children, providing time out for themselves, and helping with the big questions. befrienders see their role as mentoring, broadening young people's horizons and providing a safe place physically and emotionally. challenges include: forming and ending relationships, having multiple befrienders, ongoing support and training for befrienders, maintaining boundaries, sibling rivalry, and cost. conclusions: befriending is a new innovation in cf, and has the potential to make a difference to young people's lives. careful planning at the outset, ongoing support for befrienders and regular evaluation are essential factors in ensuring its success good practice in befriending services for people with learning disabilities qualitative research practice sage london ଙ in addition, there was high use of non-cf vitamins (n= ) and protein shakes (n= ) which were not included as a cam therapies. when prayer was excluded (which was used by % of respondents), . % of the patients are still using some form of cam. the most commonly used cam therapies in cf were relaxation therapies (n= ), massage therapy (n= ), chiropractor care (n= ), herb/plant product therapies (n= ), homeopathy (n= ), and yoga (n= ). an analysis of the relaxation therapies revealed that deep breathing exercises (n= ) were the most common and frequently combined with other relaxation techniques: meditation (n= ), progressive relaxation (n= ) and guided imagery (n= ). the most common herb/plant products used were: echinacea (n= ), garlic (n= ), selenium (n= ), ragweed or chamomile (n= ) and ginseng (n= ). the use of possible cf specific cam therapies were fish oils/omega fatty acids (n= ), glutathione (n= ), docosahexaenoic acid (n= ) and curcumin (n= ). in conclusion, cam is widely used by the cf population with "prayer for health" being the most common modality, however, many patients are utilizing multiple cam therapies (data not presented) within the framework of a weighted satisfaction model of quality of life, we investigated the importance ratings of adolescent and adult patients regarding disease-specific aspects of living with cf. method: outpatients (aged - years, m= . , sd= . ; fev - %, m= . , sd= . ) repeatedly filled in the cf-specific module of the questions on life satisfaction (flz-cf) to measure satisfaction with nine cf-specific aspects of life in relation to the subjective importance of each life domain. a ranking list of the most important life domains across the study group was determined, and intra-individual changes of the importance scores were analysed. associations of importance ratings with changes of pulmonary functioning (fev %) were examined. results: the most important aspects (% of "very important" or "extremely important" answers) are sleep ( . %), integration of therapy into daily routine ( . %), breathing ( . %), gastrointestinal functioning ( . %), and eating ( . %), less important is understanding by others ( %) , ; durieu, i. , . département d'information médicale, hospices civils de lyon dr. von hauner children`s hospital helios hospital, e.v. behring, berlin, germany supported by the cystic fibrosis foundation leroy matthews physician scientist award, the national heart, lung, and blood institute cystic fibrosis foundation therapeutics inc. symptoms patient-reported respiratory symptoms in cystic fibrosis: initial validation children's hospital and regional medical center the first author is supported by a second year clinical fellowship from razvi, s. ; quittell, l.m. ; sewall, a. ; marshall, b.c. ; saiman, l. . pediatric pulmonary medicine, columbia university, new york, ny, usa; . cystic fibrosis foundation, bethesda, md, usa significance: significant improvements have been made in diagnostic and therapeutic strategies for cf patients in recent decades. we hypothesized that these changes could potentially impact cf respiratory microbiology. thus, we examined longitudinal trends in the annual incidence and prevalence of cf respiratory pathogens from to .methods: cf foundation patient registry data, estimated to include % of the cf population in the u.s., was utilized in this analysis. patients were included if results from at least one respiratory culture were in the registry from january to december . patients were excluded after organ transplantation. to avoid misclassification of incident cases, a retrospective review of registry data from to was performed to establish the culture status of included patients prior to . thus, incident cases were subjects with first detection of a given pathogen in a given year. prevalent cases were defined as subjects with at least one positive respiratory culture for a given pathogen in a given year. all submitted culture results were included.results: the number of cf patients submitting registry data increased from , in to , in . the proportion of subjects meeting inclusion criteria remained relatively constant ( % to % per year). the median age of the study cohort increased from . years in to . years in . during the study period, the incidence of hemophilus influenzae remained stable ( . % to . %) as did the prevalence ( . % to . %). the incidence of pseudomonas aeruginosa ranged from . % in to a peak of . % in . the prevalence of p. aeruginosa declined from . % in to . % in . there is a trend for both an increasing incidence and prevalence of staphylococcus aureus; the incidence increased from . % in to . % in and the prevalence increased from . % in to . % in . the age specific prevalence of s. aureus remained highest in children aged - years. the incidence of methicillin-resistant s. aureus (mrsa) increased from . % in to . % in , with a parallel increase in prevalence from . % to . %. the highest prevalence of mrsa was noted in subjects years of age and older. the incidence of burkholderia cepacia complex decreased from . % in to . % in , while the prevalence remained relatively stable (range . % to . %). both the incidence and prevalence of stenotrophomonas maltophilia increased (incidence: . % to . %, prevalence: . % to . %).this retrospective study aims at assessing the respiratory function and the cystic fibrosis (cf) co-morbid conditions according to the patients' nutritional status.method: data were collected from the french observatory (onm) (n= ) (vlm, ined). the last year data for weight (w), height (h), fev , fvc were extracted from the database (exclusion criteria: transplan-tation); were also analysed the genotype, pancreatic enzymes consumption, pseudomonas aeruginosa (pa) colonization, cirrhosis, diabetes, pulmonary transplantation and mortality (sas . ). we used international age-adjusted standards for bmi z-score for children (c) and bmi for adults (over years) (a) (cole tj bmj ) to define underw (uw), normal w (nw), overw (ow) and obesity (ob) .results: data are available for patients in the c cohort and for patients in the a cohort ( %). nutritional status subgroups prevalence (%) is: . , . , . and . in c and . , . , . and . in a for respectively uw, nw, ow and ob. mean age in a is significantly increasing with bmi (p<. ). frequency of del/ del in c and a is lower in ow/ob (p<. ) as well as the use of pancreatic enzymes (p<. ). ow/ob patients have the best fev and fvc values whatever the gender and the age, with significantly less pa colonization in a (p<. ). we could identify a positive correlation between the pulmonary function and bmi. the cf co-morbid conditions demonstrated a lower prevalence of diabetes ( / ) and cirrhosis ( / ) in ow/ob. none of the ow/ob c or a are on a transplant waiting list versus . % in uw/nw (n= / ) and none died versus % in uw/nw (n= / ).conclusion: the observed prevalence of ow and ob in cf is respectively . and % whereas the french obepi study collected and %. our results suggest that increased bmi is associated with better fev , fvc, lower prevalence of pa, cirrhosis and diabetes. the potential risks of chronically high bmi have not been studied in this population yet, but justify further investigations in this longer life expectancy cohort. materials and methods: we retrospectively identified abdominal ct scans of consecutive patients ( females, males, mean age of years) with cystic fibrosis and a control group of consecutive patients ( females, males, mean age of years) scanned as potential renal donors. three readers reviewed all scans and recorded the presence and location of colonic wall redundancy, and the wall thickness of the ascending, transverse, and descending colon. clinical information on cystic fibrosis patients, including cftr gene mutations, was queried from our cystic fibrosis patient registry database. additionally, medical records of all cystic fibrosis patients were reviewed to determine the indication for abdominal ct. results: colonic wall redundancy was seen exclusively in patients with cystic fibrosis, and was noted in of patients each for reviewers , , and (p< . ). colonic wall redundancy was seen in of adult patients with cystic fibrosis ( %), but was not seen in any children age or younger (p< . ). excellent agreement was found for the ct identification of colonic wall redundancy among readers (kappa= . , p< . ). cystic fibrosis patients with colonic wall redundancy had significantly thicker ascending colonic walls (mean . mm) vs. those without wall redundancy (mean . mm) or controls (mean . mm), (p= . ). three patients with colonic wall redundancy had follow-up ct, and all showed temporal stability (mean of months). among adult cystic fibrosis patients, cftr gene mutations were available for of patients with and of without colonic wall redundancy. while the common ∆f mutation was the predominant mutant allele among patients with normal colons at ct (only of patients, or %, had an identified mutation other than ∆f on either allele), a higher prevalence of less common non-∆f mutations was seen patients with colonic wall redundancy ( of patients, or %, p< . ). the g x mutation was seen exclusively in patients with colonic wall redundancy ( of patients, or %, p= . ). there was no significant difference in the proportion of patients with abdominal pain (> . ), pancreatic insufficiency (p> . ), diabetes mellitus (p> . ), history of meconium ileus background: airway inflammation in cf is associated with marked remodelling and bronchiectasis. pro-inflammatory mediators such as advanced glycation end-products (ages) and the soluble receptor for age (srage) may perpetuate this response in the lung and in other organs such as the kidney . the total body burden of ages reflects exogenous sources from the diet, endogenous production by the body, tissue degradation and renal clearance, which may be reduced in renal impairment. the accumulation of endogenous age is accelerated in conditions of high oxidative stress and inflammation, and ages have been implicated in the pathogenesis of diabetic nephropathy. the burden and significance of ages in cf has not been determined, but if elevated, dietary modification of ages may represent a novel anti-inflammatory approach for cf. the aim of this study was to determine serum levels of age and srage, and identify clinical correlates of age and srage levels.methods: adults with cf (n= , males, % pancreatic insufficient, mean age . ± . years (range to years), mean fev %predicted . ± . %predicted (range - )) and healthy adults (n= , male, mean age . ± . years (range - years)) were studied. cf participants provided - serum samples each over a six month period, while healthy controls provided a single sample. serum was analysed for levels of advanced glycation end-product (age-cml) and soluble receptor for age (srage) (elisa). for each cf participant, levels in the multiple samples were averaged, and the median for the study sample reported. clinical data, including bmi, presence of cf-related diabetes mellitus (cfrd) and serum hba c level were collected from the medical record. mann-whitney tests were used to compare cf and control levels, while spearman rank correlations were used to identify clinical correlates of age and srage.results: prevalence of cfrd was . %. mean bmi was . ± . kg/m and mean hba c level was . ± . %. median (iqr) levels for age-cml were ( , ) background: osteopenia is diagnosed in cystic fibrosis (cf) using dual-energy x-ray absorptiometry (dxa). areal bmd from dxa is subject to error when bones are smaller in volume than reference standards (t-scores). normalization of bone size by use of zscores in cf is controversial and not widely utilized, thus comparing to larger bone areas. reports in cftr-deficient mice (cf mice) reveal osteopenia when measured by dxa. we hypothesized that use of pqct, which eliminates bias of size, would more accurately analyze bmd.methods: femurs were collected at necropsy from cf mice and c bl/ j (b ) mice at wks, and cf mice and b mice at wks (all female) for pqct. time points were chosen to coincide with pre-pubertal and adult ages for comparison to human disease. total mineral, trabecular and cortical densities were measured. student's t-test was used to detect significant differences (p< . ).results: femur measurements from both cf mice and b mice are listed as mean ± sd in the table (below). length was greater in b mice compared to cf mice at and wks. total area at the metaphysis and diaphysis were greater in b mice at and wks. data are consistent with larger bones in b mice. total mineral density , trabecular density and cortical density , however, were greater in cf mice compared to b ( & wks, wks, wks).discussion: our study demonstrates greater bmd in cf mice when volumetric data are analyzed and size differences are accounted for by use of pqct. these findings persisted at adult age, suggesting a normal deposition of bone. in clinical management, dxa imaging is readily available compared to pqct, and can be used as a predictor of bmd and fracture risk. correct reference ranges must be utilized to minimize erroneous values secondary to size. z-scores allow correction for differences in bone area and size, even in adults. more studies into understanding bone mineral deficit and tools of measurement are needed in cf. meanwhile, bone measurements by radiographic imaging must be taken in context of overall health, pubertal progression, and size of individuals. gainesville, fl, usa; . nemours children's clinic, orlando, fl, usa; . suny upstate medical university, syracuse, ny, usa; . case western reserve university, cleveland, oh, usa; . genentech, san fancisco, ca, usa background: greater growth rates, bmi and fat-free mass are associated with improved lung function in individuals with cf (pedreira, et al. pediatr pulmonol. ) . current treatments for weight gain have focused on nutritional supplements and appetite stimulation, and not underlying issues of catabolism or chronic disease. we hypothesized that anabolic effects of gh would not only improve weight and height, but lbm as well.methods: sixty-seven prepubertal children with cf and height less than or equal to the th percentile were randomized to daily gh (n= ) or observation (n= ) for a period of year, followed by an off-treatment observation period of months. children randomized to gh received somatropin injections daily at . mg/kg/wk. height and weight were measured every three months. height was evaluated as height standard deviation score (sds) to control for differences in age and sex. lbm was measured by dexa scan at time zero, at and months, and at the end of the -month observation period. in addition, for lbm, change from baseline was calculated for subjects for whom the same dexa equipment was used at both time points. the preliminary results of the first months of the study are presented here.results: data for subjects in the observation arm and subjects in the gh arm, who completed months, are available. as shown in the table below (listed as mean ± sd), gain in height, weight and lbm were significantly greater in the gh treated group than in the observation group over the month period.discussion: gh improves growth in prepubertal children with cf as measured by height sds. in addition, gh significantly improves weight gain and this gain is, in part a result of the significantly greater increase in lbm in the gh-treated group than in the observation group. the relationship between the improvement in lbm and other outcomes in cf deserves further exploration. the national cystic fibrosis(cf) registry database notes cystic fibrosis related diabetes mellitus(cfrd) as a complication in . % of cf patients at all ages. cfrd is associated with poorer nutrition and increased pulmonary morbidity. the cause of cfrd is not completely known but insulin resistance may be associated with fibrotic damage to pancreatic islet cells due to chronic inflammation. other studies suggest protein energy malnutrition(pem) in early life leads to impairment of insulin secretion and cystic fibrosis-related diabetes (cfrd) accounts for increased morbidity and mortality in patients with cf and occurs in approximately % of patients by the age of years. cfrd net (network for epidemiology and trials) is a consortium of four large uk cf centres caring for adult patients with cf. the aim of this group is to undertake research into the diagnosis, investigation and management of cfrd. in this abstract, we estimate the prevalence of diabetes in a screened population of adults with cf. methods annual review data were collected on attending patients with cf aged and above during . g ogtts were performed after fasting for at least hours on patients without known diabetes. each ogtt was categorised as either normal ( hour glucose . - . mmol/l), impaired ( hour glucose ≥ . mmol/l and < . mmol/l) or diabetic ( hour glucose ≥ . mmol/l). all patients with a diabetic ogtt were followed up with serial bm monitoring to determine whether or not they had cfrd. fasting plasma glucose (fpg) was considered to be elevated ≥ . mmol/l, isolated impaired fasting glucose (igf) was defined as a value between . mmol/l and . mmol/l and hypoglycaemia was defined as a blood glucose < . mmol/l. in addition, we obtained information on age, sex and prescription of anti-diabetic medications subsequent to ogtt. in three of four centres, hba c was routinely performed on all patients. of patients (median age years, % male), ( %) had established diabetes and were therefore excluded from further screening. of the remaining patients, ( %) underwent formal ogtt testing. in this latter group ( %) had a diabetic ogtt, ( %) an impaired ogtt and ( %) had evidence of reactive hypoglycaemia at hours. of the patients with a diabetic ogtt, ( %) only had an abnormal hour value, ( %) only had an elevated fpg and (< %) had both. all patients with diabetic ogtts underwent blood glucose monitoring and ( %) went on to treatment with hypoglycaemic agents in the calendar year. none of the patients with an isolated elevated fpg had diabetes.hba cs were available in of newly diagnosed patients with diabetes (in the centres which performed hba c). in this group median hba c was . %. this study confirms the high prevalence of diabetes among screened patients and the growing burden of diabetes management in adult cystic fibrosis clinics. it further highlights the importance of performing screening for diabetes in this population; the majority of patients were identified on the basis of abnormal hour values. despite this only half of all suitable patients underwent ogtts in the four centres committed to screening. these patients merit further study, as previous work suggests that the decline in clinical status occurs several years before diabetes becomes apparent. cystic fibrosis related diabetes (cfrd) occurs in up to % of patients with cystic fibrosis (cf), the incidence rising with increasing age. the likelihood of developing long-term complications secondary to diabetes increases with poor glycaemic control and duration of diabetes. as survival has improved for people with cf those who develop cfrd may live with diabetes for several years. aim: to establish the frequency of diabetic complications in patients with cystic fibrosis related diabetes.method : patients with cfrd attending the adult cf service at the royal brompton hospital between april -march were screened for diabetic complications and cardiovascular risk factors. a total of patients (male/female: / ) including post transplant patients were screened. mean age was . years ( - ), mean hba c . % ( . - . % ) and average duration of diabetes was . years (< - years).as immunosuppressive therapy can also cause many of the complications associated with diabetes the results are presented separately for nontransplant and post transplant patients (table ) . patients in total had retinopathy: background retinopathy, proliferative retinopathy undergoing laser therapy and maculopathy. the average duration of diabetes in those with background retinopathy was . years with only patient having diabetes for > years. the patient with proliferative retinopathy (non-transplant) had been diabetic for years. a raised creatinine level was identified in all transplanted patients with microalbuminuria but none of the non-transplanted patients. patient (nontransplant) had macroalbuminuria. post transplant patients were on treatment for hypertension and a further patients ( post transplant and nontransplant) had elevated blood pressure (> / ) at screening requiring follow-up. none of the patients had evidence of cardiovascular disease or stroke.conclusion: macrovascular complications were not seen. microvascular complications occurred but were less common than the reported incidence in type and type diabetes. this may reflect the relatively short duration of diabetes (mean . years) of patients in this study. a further study comparing cfrd patients with a non-cf diabetic control group of similar duration of diabetes is warranted. cystic fibrosis (cf) is a disease that leads to serious disturbances in nutritional status and bone calcification. comparison of two methods for assessment of bone mineralization: dexa and hand radiograms in diagnosing osteopenia or osteoporosis. study was performed in a group of cf patients ( f, m), aged - yrs. nutritional status was assessed using bmi, cole's index and bmc. radiograms of non-dominant hand were assessed according to normalized optical density comparing to aluminum standard. bone density was also assessed using dexa. for statistical analysis, backward stepwise binary logistic regression (wald's test) was used. analysis of data revealed that using bmc, cole's index and hand radiograms markers we can diagnose bone mass disturbances (z score < sd) with precision up to . % comparing to dexa. sensitivity and specificity of this method was respectively . % and . %. false negative results were obtained in patients and false positive were also in patients. hand radiograms method could be an alternative for dexa in screening of bone density disturbances in cf patients. the study was partly supported by grant of ministry of science and higher education no p e .in a very recent publication( ) a high rate of fasting ( %) and reactive hypoglycaemia ( %) was described in a group of n= cf patients older than years who received an oral glucose tolerance test. reactive hypoglycaemia was related to the minute glucose concentration in the ogt test. we were surprised by the high percentage of asymptomatic hypoglycaemic situations in cf patients. as a part of a prospective intervention study in cf patients with cfrd, we screened more than patients years or older with cf for cfrd. in this multi-centres population ogt was performed in the morning after overnight fasting according to who standards. using the same definition for fasting hypoglycaemia (glucose < mg/dl; . mmol/l) and reactive hypoglycaemia (glucose < mg/dl; . mmol/l at minute during ogt test)as in the other study we calculated the percentage of cf patients with fasting or reactive hypoglycaemia in our cohort. results: ogt were done in patients with cf. age(mean±sd) , ± , years; bmi z-score - . ± , ;height z-score - , ± , and weight z-score - , ± , . ogt was categorised according to ada criteria. normal ogt (n= all ; fasting hypoglycaemia (fh) n= ( , %), reactive hypoglycaemia (rh)n= ( , %),ifg (n= all, fh n= , rh n= ( , %), igt(n= ,fh n= ( , %), rh n= ),fgt (n= all, fh n= ,rh n= ) and diabetes mellitus (n= all, fh n= ( , %)). fh was observed in ( , %) and rh in ( , %) out of ogt tests. there were no difference related to age, bmi z-score, height z-score and weight z-score comparing those patients with fh or rh to those without fh or rh in all categories of ogt tests.in this large multi-centres cohort of cf patients we were neither able to confirm the high percentage of fasting nor of reactive asymptomatic hypoglycaemia which was reported recently on a small group of cf patients. nutritional status measured by bmi z-scores, height and weight z-scores were unaffected by hypoglycaemia in our cohort as noticed also in the small group. as the ogt tests in the other study were done in a single centre it might by that a centre specific situation influenced the frequency of hypoglycaemia in that small group. we conclude that large numbers of investigations might be needed to come up with firm conclusions related to frequency of specific aspects of glucose disturbance in cf patients.fh does not exclude diabetes mellitus or igt. as survival from cf improves, surveillance to identify and treat complications associated with longevity is an important component of management. renal disease has been reported in adults with cf. risk factors may include cf-related diabetes mellitus (cfrd) and use of nephrotoxic medications. diabetic nephropathy has been observed in the absence of cfrd , highlighting the need for screening for renal impairment. this study aimed to measure renal function in a sample of adults with cf, and to compare estimated glomerular filtration rate (egfr) and urinary creatinine clearance (urcrcl) as markers of renal function.methods: adults with cf aged years or over (n= , % male, % pancreatic insufficient, mean age . ± . years) underwent screening for renal impairment. of these patients, ( males) had cfrd, and ( males) had no history or clinical features of cfrd. hour urine collections were analysed for urcrcl, with renal impairment being defined as < ml/min. serum creatinine level was used to calculate egfr (mdrd formula), which was classified renal function as normal or mildly impaired (> ml/min); moderately impaired ( - ml/min) or severely impaired (< ml/min). sensitivity of egfr for identification of renal function was calculated using urinary creatinine clearance as the reference method. unpaired t-tests were used to compared cfrd with non-cfrd patients.results: mean urcrcl was ± ml/min, and eight patients ( . %) had renal impairment (< ml/min). of these, only had egfr suggestive of renal impairment. the sensitivity of egfr for identifying renal impairment in this sample was %. a further patients had moderately impaired egfr, but normal urcrcl. five of the patients with impaired urcrcl had cfrd. age, gender, cfrd, fev %predicted and hba c level did not correlate with urcrcl. the table compares cfrd patients with non-cfrd patients. hba c level was higher in cfrd patients. there were no other significant differences in clinical or renal function parameters.conclusion: renal impairment, is common in the adult cf population and is not confined to patients with cfrd. screening using egfr is poorly predictive of impaired urcrcl in this population. these results suggest that surveillance to monitor renal function is indicated in the adult cf population, including in patients without cfrd, and that determination of urcrcl should be included in the screening process. cystic fibrosis (cf) patients suffer from pancreatic insufficiency resulting in malabsorption of fat soluble vitamins including vitamin d. many fac-tors contribute to low bone mass and fractures in patients with cf; however, chronic vitamin d deficiency plays a major role. the prevalence of vitamin d deficiency has been reported as high as % in some specialized care centers. in addition, reports of occult vertebral fractures have been reported as high as %. we sought to determine the prevalence of vitamin d deficiency (defined as -hydroxyvitamin d ( (oh)d) < ng/ml) and of vertebral fractures at our cf center. we obtained irb approval to review the records of all patients seen at our cf center during [ ] [ ] . we collected information related to bone health including (oh)d, bone mineral density and lateral spine or chest x-rays to examine for the presence of a vertebral fracture. we reviewed the records of subjects who were seen at our center during the study period. the mean age of the subjects was ± years. subjects had a mean bmi of . ± and an fev % predicted of . ± %. the percentage of subjects having an annual (oh)d level checked was only % and % in and , respectively. the mean (oh)d was . ± and . ± ng/ml in and . the prevalence of vitamin deficiency was % and % in and . about one quarter of the subjects had bone mineral density testing with half of the tests showing osteopenia or osteoporosis. twenty-seven percent of subjects had vertebral abnormalites detected on lateral chest x-ray. we sought to determine whether any factors were associated with vitamin d deficiency. we found that taking a multivitamin did not significantly protect against vitamin d deficiency. however, not taking a supplement containing vitamin d other than a multivitamin was associated with a % risk of vitamin d deficiency (p= . ). subjects having (oh)d levels determined in the winter or spring was associated with a % risk of vitamin d deficiency (p= . ). in summary, we found annual testing for vitamin d status was inadequate in our cf center and that when (oh)d level was determined, over % of subjects were vitamin d deficient. nearly one quarter of our adult patients already had evidence of vertebral compression fracture seen in lateral chest x-ray. we urge greater screening for vitamin d deficiency in the cf population. effective protocols to prevent and/or treat vitamin d deficiency are urgently needed in the cf population. improved vitamin d status in cf patients is one factor that may reduce the high prevalence of vetebral fractures. support for this study was provided by proctor and gamble pharmaceuticals background: more cf patients are surviving into adulthood, in part due to the increasing use of new therapies and more aggressive management of chronic respiratory and gi disease. as a result, the recommended daily treatment regimens for most cf adults are both complex and time consuming. objective: to assess the self-reported daily treatment burden of cf therapies in a cohort of adults with cf. methods: in the sixth survey wave of the project on adult care in cf (pac-cf), an ongoing longitudinal panel study of adults with cf from us cf centers, respondents were asked to report the type of medications, inhaled therapies, and airway clearance therapies they used during the day prior to completing the survey, as well as estimate the time generally required to complete each type of therapy.results: of the respondents (response rate %), the median number of therapies reported was (iqr - ) and the median reported amount of time usually spent on treatments was minutes per day (iqr - minutes). forty-eight percent reported using or more different inhaled therapies or using inhaled therapies or more times during that one day. the most commonly reported inhaled therapies were a bronchodilator ( %), pulmozyme ( %), an inhaled steroid ( %), hypertonic saline ( %), and objective: caring for a child with cf is stressful and often leads to adverse effects for both children and caregivers. support from family and friends can help reduce the adverse effects of chronic stress, however, there is little research documenting types of support provision as well as sources of support. moreover, social support may be related to important health outcomes. this study described the types of support provided to caregivers of children with cf and examined relationships between support and health outcomes.method: as part of a larger intervention study to improve adherence to medical regimens, children with cf ages to and their caregivers were enrolled across cf centers in florida: university of florida, nemours children's clinic, and all children's hospital. caregivers completed the norbeck social support questionnaire (nssq; norbeck, ) in addition to other measures at baseline and follow up. this questionnaire provides information about the number and type of people providing support, as well as its type and quality.results: caregivers received the majority of support from their families, spouses and friends and characterized these relationships as longer in duration, with more frequent provision of support. other individuals who provided support included the cf team, religious leaders, counselors or psychologists, and work associates. overall, caregivers rated family members and spouses as providing the greatest amount (i.e., "quite a bit") of emotional support. spouses received the highest ratings for tangible support; they were perceived as providing "quite a bit" of support in comparison to families (i.e., moderate) and friends (i.e., a little). in terms of size of the support network, caregivers in this study had similar networks to those of a normative sample of healthy adults. in addition, there was no difference in amount of emotional or tangible support received by these caregivers. preliminary baseline results indicated that social support was positively related to both adherence, children's growth (i.e., height and weight percentile), and family income.discussion: caregivers of young children with cf reported similar support networks as other healthy adults. however, there was variability in the amount of support provided by source, with spouses providing the greatest amount. social support appeared to be a protective factor, and lack of support was related to negative health consequences for their children. more attention should be focused on the potentially beneficial effects of social support.funding was provided by nih grant # befriending is reported to be valued by those who have been befriended, offering opportunities for social activities and new experiences and can impact positively on self confidence and self esteem . whilst government policy supports befriending, few schemes collect evidence to demonstrate the effectiveness of these services . young people with cf may suffer social isolation due to chronic illness and treatment demands and a befriend-ing service was offered to those considered socially vulnerable in the edinburgh area.this project aims to evaluate the effectiveness of this befriending service on young people with cf and their carers. methods liverpool, united kingdom; . child mental health, university of liverpool, liverpool, united kingdom; . child health, university of liverpool, liverpool, united kingdom; . psychology, university of miami, miami, fl, usa; . psychology, birkbeck college, university of london, london, united kingdom; . mathematics and statistics, university of lancaster, lancaster, united kingdom; . psychiatry, university of manchester, manchester, united kingdom introduction: existing treatments for cystic fibrosis (cf) are time-consuming and labour intensive and biomedical advances are likely to lead to further novel interventions. there is concern amongst clinicians that a high care burden associated with these interventions may compromise the wellbeing of caregivers, reduce adherence to the treatment protocol and increase the likelihood of inadvertent errors in the delivery of interventions. at present, there is no measure of 'treatment burden' and therefore no way to assess the impact of new and increasingly more complex interventions conducted by lay caregivers at home. furthermore, trials are hampered by the lack of measurable patient-reported outcomes beyond a broad quality of life assessment. to address this gap we have developed a measure of treatment burden for cf using qualitative methods (focus groups, action research, in-depth interviews) with participating parents and a working group of cf team professional staff. this measure addresses the time, effort, meaning and ease of management for caregivers of children with cf up to years and post the first year following a diagnosis. methods: we report here the pilot phase of the validation of this instrument, which involved (i) cognitive interviewing with n= caregivers; and, (ii) n= caregivers completing the clcf instrument together with a quality of life measure at time points. this yielded data for stability, reliability and coherence of the hypothesized constructs. results: the clcf takes minutes to complete. face validity and acceptability are established. for the first pilot sample the age of the child ranged from . - . years. treatments took, on average minutes per day to complete and an average of minutes of this was devoted to administering enzymes particularly for carers of infants. a prominent concern amongst these parents was their child's height and weight and their efforts were directed at optimising growth. for the child however, management of nebulised medications and physiotherapy were more frequently flagged as a concern. these data suggest this is a reliable, coherent and useful, although complex tool. conclusion: the caregiver challenge for cf cannot be understood simply in terms of time and effort involved in delivery of treatments. it also involves contextualising interventions for their meaning at multiple levels of explanation for the individual concerned. structural equation modelling would be an appropriate way to proceed with the main validation when such complex relationships occur between latent variables and would serve as a confirmatory factor analysis for the hypothesised relationships. sponsored by: royal liverpool children's nhs trust and university of liverpool. funded by: national institute for health research (nihr) research for patient benefit programme.introduction: the french cf practice recommendations, published at the end of in parallel with the creation of cf reference centres, advise that each patient should be seen at least every months at a cystic fibrosis reference centre.objective: to investigate the impact of these recommendations on the effectiveness of the follow-up of patients at the four reference centres in the rhône-alpes region.methods: all patients with cystic fibrosis attending one of the four cf centres between and were retrospectively included. the total number of visits was recorded for each patient and each year of the study period. to determine the evolution slope for each patient followed for at least years, a negative binomial regression of number of visits versus time was carried out (confirmed with a repeated model). to estimate the impact of the recommendations, the analysis was restricted to patients with at least two visits before and after and a second model was adjusted with a new intercept in to estimate the change in slope as of this point.results: a total of patients were included in the cohort. the average number of visits per patient rose from . in to . in (p< . ). the proportion of patients with at least visits per year increased from % to %. the negative binomial regression for the patients having had at least years follow-up confirmed this trend with an average slope of + . (sd= . ). a total of patients were evaluable for the change in slope. no significant change in trend was observed in : only % of patients had a higher rate of growth (this change was significant for only subjects). at the last follow-up visit, patients with increasing rates of number of visits were older ( yrs vs. yrs, p< . ), had lower %fev ( vs. , p= . ), had a similar average number of visits before ( . vs. . , p= . ), and a similar weight-for-age z-score (- . vs. - . , p= . ) .conclusion: the number of visits per patient is regularly increasing. since the publication of the practice recommendations in , the growth has tended to slow down: clinicians were already convinced by the need for closer follow-up and had begun to increase the rate of visits. methods: patients > years who presented for routine healthcare voluntarily completed four surveys: ) the revised eating attitudes test (eat), validated for cf patients by abbott and colleagues, where higher scores reflect worse attitudes towards eating; ) the rosenberg self-esteem scale (rse), where higher scores reflect better self-esteem; ) the body image scale (bis), developed for cf patients by wenninger and colleagues, where higher scores reflect better body image; ) the cystic fibrosis questionnaire (cfq), developed by quittner and colleagues, where higher scores reflect better hrqol. also, fev %, body mass index (bmi), and pancreatic sufficiency or insufficiency (based on the need for pancreatic enzymes) was recorded. regression analyses controlling for age, gender and bmi were used to examine the associations between the surveys. results: this study included patients with % males and a mean age of years. the eat was negatively associated with the rse (p= . , adjusted r = . ), bis (p= . , adjusted r = . ) and cfq (p= . , adjusted r = . ). the rse was positively associated with the cfq (p= . , adjusted r = . ). the bis was positively associated with the rse (p= . , adjusted r = . ) and cfq (p= . , adjusted r = . ). neither bmi nor pancreatic function was associated with the surveys (p=ns). fev % was positively associated with the cfq (p= . , adjusted r = . ) but was not associated with the other surveys.conclusions: more negative attitudes towards eating predict worse selfesteem and body image, while more positive body image predicts better self-esteem. also, attitudes towards eating, self-esteem and body image are significant predictors of hrqol with body image being the most important predictor.clinical importance: hrqol is an important clinical outcome measure in cf. clinicians need to be sensitive to attitudes towards eating, self-esteem and body image in cf patients, because they are important predictors of hrqol. riekert, k.a. ; mogayzel, p.j. ; bilderback, a. ; hale, w. ; boyle, m.p. . medicine, johns hopkins university, baltimore, md, usa; . pediatrics, johns hopkins university, baltimore, md, usa background: existing research suggests that self-reported adherence to all aspects of the regimen is likely suboptimal and objective measures suggest even poorer adherence. there is little empirical data on how much adherence is necessary to achieve desired health outcomes. moreover, there is limited data on how adherence changes dur-objective: cf may be associated with pain attributed to several etiologies. this study evaluates the prevalence of pain symptoms in adult cf patients and the influence on patients` lives.methods: patients of adult cf centers in germany completed a validated, self report questionnaire during a routine clinic visit assessing characteristics of chronic pain (prevalence, duration, location, quality and intensity of pain symptoms). furthermore, the impact of pain on different aspects of life was explored using a numeric scale from to with being the worst. every-day-life was divided into categories: duties at home, recreation, social activities, occupation, sexual life, autonomy, vital activities and cf-therapy. the average intensity of pain within the last weeks was correlated to fev , bmi and age.results: patients ( male) completed the questionnaire. the age was - years (mean . ± . ). patients ( %) aged - years (mean . ± . )reported pain within the last months. bmi was . - . kg/m (mean . ± . ), fev - % (mean . ± . ). if asked for pain within the last weeks ( %) reported painful episodes lasting from - days with a mean of . days (± . ). most patients described pain occurring at more than one site with the head being the most localized site, followed by chest and abdomen. concerning the quality of pain . % characterized their pain episodes as attacks whereas . % reported them as continious, . % had continious pain combined with pain attacks. patients desribed the intensity of pain as . (± . ) on a scale from to with being the most severe pain. the category of life negatively influenced most of all by pain episodes was recreation, followed by occupation and duties at home. female patients were more limited in their acitivites by pain symptoms than male patients with the highest difference being reported in sexual life. there was no correlation of the average intensity of pain within the last weeks to fev , bmi or age.conclusion: the prelevance of pain in cf is often underestimated. painful episodes can be the cause of worsening the quality of life for adult cf patients. assessment of pain should be routinely performed as part of care in cf centers. objective. sleep has been examined in a number of pediatric conditions, with impaired sleep resulting in worse neurobehavioral functioning (beebe, ) . recent research has shown that slow wave sleep is critical for memory (marshall, helgadottir, molle, & born, ) . to the authors' knowledge, no studies have examined slow wave and rapid-eye-movement (rem) sleep in pediatric patients with cystic fibrosis. methods. retrospective medical record review revealed two patients who underwent clinically indicated polysomnography (psg) who reported feeling tired and lacking energy. patients were females with cystic fibrosis, ages and years. results. psg revealed an increase in stage sleep for both patients, resulting in increased risk and observed hypopneic episodes. patient -a experienced . % in stage , . % in stage , and % in stages and combined. this patient also had no rem sleep. patient -b experienced % in stage , . % in stage , and % in delta wave sleep, with no rem sleep. respiratory disturbance index (rdi) for patients a and b were calculated at . and . , respectively. respiratory effort related arousal (rera) contributed to rdi by . and . points in patients a and b, respectively. in addition, endtidal co for patient a and b were maxed at mm hg and mmhg, respectively. their epworth sleepiness scales were scored at and for patients a and b, respectively. conclusions. structure of sleep was abnormal in both patients with decreased slow wave (delta) sleep in patient b and lack of delta sleep in patient a. neither patient experienced rem sleep. compensatory increased stage (nrem) sleep could cause respiratory related abnormalities, such as hypopnea and apnea in patients with cystic fibrosis background: understanding and advancing the application of tools to measure patients' symptoms is critical to advancing our evaluation of potential new therapies used to treat cf. we performed initial evaluation of the measurement properties of a cf-specific respiratory symptom daily questionnaire. methods: we planned to enroll cf subjects, stratified by age, at three cf programs, the university of washington medical center and children's hospital and regional medical center in seattle and mary bridge children's hospital in tacoma in a prospective assessment of a novel cf specific respiratory questionnaire. cf subjects years and older were eligible for enrollment. patients (parents for children under the age of ) completed a daily symptom questionnaire during two -day periods of clinical stability and one -day period when patients were ill for a total of days. the ill state began when patients/parents sought medical attention for respiratory symptoms. the questionnaires were completed using a secure web-based application or via paper for those patients without access to the internet. two health related quality of life measures (generic and cf specific) were completed by the subjects at the end of each -day period to assess the relationship between the novel questionnaire and health related quality of life. patients also used pedometers during the well and ill states to assess relationship between symptoms and activity level.results: cf subjects have been enrolled to date. at the time of this interim analysis, patients had completed questionnaires. of this total, only individual questionnaire entries have been missed in patients (< % of possible questionnaires). interim review of cross-sectional data suggests clear differences in symptom reporting between the ill and well periods. examples include % ( / ) noted difficulty breathing and % ( / ) noted tightness in the chest in the preceding hours during the initial well period compared to % ( / ) noting difficulty breathing and % ( / ) noting tightness in the chest during the ill period.conclusions: interim evaluation of this novel instrument demonstrates feasibility of deploying this instrument via the internet with an extremely high completion rate. using a cross-sectional analysis, the instrument can discriminate between the ill and well state. further data will be presented regarding within patient variability and discrimination of the instrument.supported by the cystic fibrosis foundation leroy matthews physician scientist award, the national heart, lung, and blood institute (hl - ), national institute of health (rr- - ), cystic fibrosis foundation therapeutics inc. background: improved communication in today's health care delivery system is a critically important aspect of patient care. nurses are in an effective position to improve communication due to their close interaction with patients. as a result, they are able to identify potential problems, communicate them to the healthcare team and improve patient care and satisfaction.hypothesis: improving communication between the patient/family and the health care team will improve patient/family satisfaction.method: as a member hospital partnering with the institute for healthcare improvement's (ihi) initiative, transforming care at the bedside (tcab), the children's hospital of philadelphia (chop) introduced the use of daily patient goal sheets (dpgs) as a vehicle to improve communication between members of the healthcare team, patients and families and the dpgs was refined to meet the needs of an inpatient medical unit to which cf patients are admitted. using rapid plan-do-study-act (pdsa) cycles, a multidisciplinary team engaged in small tests of change in different stages. initially, patient goals and discharge criteria generated in daily rounds were posted in each room on easel paper. then large "whiteboards" were placed in each patient room for documenting patient goals, discharge criteria, names of the care team members and questions from patients and families. as a final step in the process, we shifted the health care team rounds to the patient's bedside and implemented bedside shift to shift nurse report and safety checks. patient satisfaction was continuously monitored over this process using the press ganey survey.results: over an eight month period, the press ganey survey results for the unit showed an average increase of %. we attribute this increase to better information sharing among patient, family and hospital staff regarding the plan of care for the patient's hospitalization. the health care team also had an improved dialog with the patients/families. conclusion: patient/family and healthcare team communication and satisfaction were improved using an organized and stepwise pdsa process to develop daily patient goal sheets on a medical unit. background: the uab/chs pediatric cf center provides care for approximately patients. the pulmonary division has full-time rns and part-time rns who rotate phone triage for all calls (including cf) of patients seen by the faculty physicians. historically, patient calls received during office hours are taken by the receptionist and put into the emr, then returned by the rn on call. the rn contacts one of the staff md's, gives report of the patient's condition,treatment orders are received, and the patient notified. there are several issues with this phone triage system, which include inconsistencies in rn and md response as well as differences in documentation. global aim: to improve the consistency among rns in phone triage of sick cf patients.method: an algorithm for pulmonary exacerbation phone triage was developed by the cf center director and lead rns to improve consistency in symptom assessment and treatment plan. a specific list of systemic and pulmonary signs and symptoms was formed including length of illness. if three objective: patients with cf often require intravenous antibiotics for treatment of pulmonary exacerbations. patients often receive peripherally inserted central catheter (picc) lines or totally implantable venous access devices (tivads) for venous access. few studies have examined complications of tivad implantation and little published data exists concerning picc line complications in cf patients. this study sought to assess the complication rates of both tivad and picc lines as well as to identify possible risk factors for developing complications.methods: this retrospective study included patients from cf centers in northern new england. data was obtained from each patient's local medical record, port cf, and patient interviews. demographic data was recorded for all cf patients between / / - / / . for each tivad or picc line, the following information was recorded: type of line placed, history of prior line placement during the study period, patient age, history of use for blood draws, method of line flushing, and status of the line at the end of the study (if still in use). complications were defined as catheter occlusion, vascular thrombosis/stenosis, infection, or other local inflammatory reactions.results: data was collected for pediatric and adult cf patients during the defined study period. seventy-three tivads and picc lines were placed during the study period in patients. the tivad and picc line complication rates were % ( / ) and . % ( / ), respectively. in pediatric patients, % ( of ) tivads and . % ( / ) picc lines had a complication. of the tivad complications, three were systemic infections and three were catheter occlusions. of the picc line complications, there were two venous thromboses, one line occlusion, two systemic infections, and nine minor incidents of localized phlebitis. in adults, complications were recorded in % ( / ) of tivads and . % ( / ) of picc lines. of the tivad complications, there were five systemic infections, seven catheter occlusions, and four venous thromboses. of the picc line complications, there were three venous thromboses, three line occlusions, and four minor incidents of localized phlebitis. all adult patients who developed a deep venous thrombosis(dvt) associated with tivad implantation were homozygous for the deltaf mutation. the presence of diabetes or burkholderia cepacia complex(bcc) lung infection was associated with dvt with odds ratios of . ( % ci . - . ) and . ( %ci . - . ), respectively. conclusions: complications of picc lines were uncommon and usually minor. the rate of tivad complication observed was more common over the lifetime of the catheter and was similar to previously published reports. we identified potential risk factors for the development of dvt associated with tivads and picc lines, specifically cf related diabetes, bcc infection, and homozygous deltaf genotype. the mechanism by which these factors are associated with catheter complications is unclear and warrants further investigation.