key: cord-0000979-afgvzxfx
authors: Rogers, Scott W; Myers, Elizabeth J; Gahring, Lorise C
title: The expression of nicotinic receptor alpha7 during cochlear development
date: 2012-08-23
journal: Brain Behav
DOI: 10.1002/brb3.84
sha: 2aad3ed695f9313c14066aa2c385092c7cf814b2
doc_id: 979
cord_uid: afgvzxfx

Nicotinic acetylcholine receptor alpha7 expression was examined in the developing and adult auditory system using mice that were modified through homologous recombination to coexpress either GFP (alpha7GFP) or Cre (alpha7Cre), respectively. The expression of alpha7GFP is first detected at embryonic (E) day E13.5 in cells of the spiral prominence. By E14.5, sensory regions including the putative outer hair cells and Deiters' cells express alpha7GFP as do solitary efferent fibers. This pattern diminishes after E16.5 in a basal to apex progression, as Hensen's cells and cells of the spiral ligament acquire alpha7GFP expression. At birth and thereafter alpha7GFP also identifies a subset of spiral ganglion cells whose processes terminate on inner hair cells. Efferent fibers identified by peripherin or calcitonin gene-related protein do not coexpress alpha7GFP. In addition to cochlear structures, there is strong expression of alpha7GFP by cells of the central auditory pathways including the ventral posterior cochlear nucleus, lateral lemniscus, central inferior colliculus, and the medial geniculate nucleus. Our findings suggest that alpha7 expression by both neuronal and non-neuronal cells has the potential to impact multiple auditory functions through mechanisms that are not traditionally attributed to this receptor.

Numerous neurotransmitter systems contribute to the normal development and function of the auditory sensory (cochlear) apparatus and the circuitry of the central nervous system. This includes members of the excitatory ligand-activated nicotinic acetylcholine receptor family (nAChR; Albuquerque et al. 2009 ). The nAChR subunit family consists of 16 distinct subunits that in various pentameric combinations form ligand-activated ion channels that each exhibit uniquely specialized pharmacological and functional properties (Albuquerque et al. 2009 ). One of these is the homomeric alpha7 nAChR (a7) whose functional uniqueness is in part due to its expression by both neuronal and non-neuronal cells in many tissues throughout the body and because it is responsive to multiple agonists (including acetylcholine and choline as well as nicotine). This results in its ability to modulate a diverse range of cellular functions including cell growth, cell survival, neurotransmission, and inflammation (Gahring and Rogers 2005; Levin et al. 2006; Albuquerque et al. 2009 ).

Members of the nAChR family contribute to essentially all aspects of the auditory sensory system function and development (Morley and Happe 2000; Morley 2005) . This includes widespread changes in expression during embryogenesis that optimizes their contribution to signal transduction, fine-tuning of sensory hair cells, and modulating central auditory circuit neurotransmission (Elgoyhen et al. 1994 (Elgoyhen et al. , 2001a Happe and Morley 1998; Vetter et al. 1999 Vetter et al. , 2007 Morley and Happe 2000; Katz et al. 2004; Morley 2005) . This functional diversity is in part accomplished through strict spatiotemporal control of different nAChR subunit expression, as has been extensively described for the nAChRs composed of either homomeric (a9) or heteromeric (a9 + a10) subunits (Elgoyhen et al. 1994; Vetter et al. 1999 Vetter et al. , 2007 Elgoyhen et al., 2001b; Murthy et al. 2009) . Less is known about the role of other nAChRs including a7, although this receptor is implicated in modifying longer lived stimulation by high-frequency sound and supporting survival of spiral ganglion cells during development (Morley and Happe 2000; Morley 2005) . Because the measurement of a7 expression and function can be compromised by low receptor expression levels or the absence of conditions that best reveal its modulatory role (Gahring and Rogers 2005; Albuquerque et al. 2009 ), the participation by this receptor as an important contributor to the development and normal auditory sensory function remains to be fully explored.

In this study, we examine a7 expression during development of the auditory sensory system. This was done using mice that were modified though methods of homologous recombination to introduce, at the a7 gene 3′ end, a hemagglutinin (HA) protein tag to the a7 receptor protein and a bicistronic IRES-driven tau + enhanced-GFP fusion protein reporter (a7 GFP ). An advantage of the tauGFP reporter construct is that the tau component directs GFP into the axon of cells expressing a7 GFP . Also generated was a mouse in which Cre-recombinase replaces the tauGFP. The expression of a7 GFP in these mice reveals extensive spatial and temporal remodeling of receptor expression during embryonic and postnatal development of the cochlear sensory structures. Furthermore, a7 GFP expression continues in both neuronal and non-neuronal cells of the adult cochlear structure and the central ascending auditory pathway. This suggests that a7 has the potential to impact functionally on auditory processes through multiple pathways and mechanisms that could impact upon the adult function in ways not traditionally attributed to this receptor.

All animals were used and housed in accordance with protocols approved in advance by the Institutional Animal Care and Use Committee at the University of Utah . This includes adherence to the Guide for the Care and use of Laboratory Animals of the National Institutes of Health.

Generation of alpha7-HA-IRES-tauGFP and alpha7-HA-IRES-Cre mice

The construction of the a7 protein and gene (Chrna7) reporter mouse lines; Chrna7-HA-IRES-tauGFP (a7 GFP ) and Chrna7-HA-IRES-Cre (a7 Cre ) have been described in detail . Briefly, as diagramed in Fig. 1A , the methods of homologous recombination were used to introduce an epitope hemagglutinin (HA) and stop codon extension to the a7 C-terminus and a bicistronic IRES-tauGFP reporter cassette ). This generated the a7 GFP mouse (Fig. 1A) , which expresses the tauGFP protein as a marker of Chrna7 transcription. The Speed Congenic Program of the Jackson Laboratory was used to achieve 98% C57BL/6 background congenicity . For conditional cell ablation of the cells expressing Cre as in the a7 Cre mouse, we crossed this mouse with the LoxP conditional diphtheria toxin (DTA) mouse lines as described previously .

Embryo (E) timing was based upon identification of coital plugs (equal to E0.5). Immunohistochemical methods were as described . Embryos were fixed in PBS/2% paraformaldehyde/5% sucrose, cryoprotected with sucrose in PBS to a final of 30%, embedded and sectioned using a Microm EM550 microtome. The 12-lm sections were mounted on glass slides, blocked, and permeabilized with 1% deoxycholate and 0.2% Triton X-100 in PBS, and then incubated overnight at 4°C with the appropriate primary antibodies. After washing, sections were incubated with secondary antibodies conjugated to fluorescent markers (Jackson Immuno-Research, West Grove, Pennsylvania) for 1 h at room temperature. The sections were again washed, and mounted in prolog gold antifade reagent (Invitrogen, Grand Island, New York; P36930) and cover-slipped before being photographed using fluorescence microscopy . Images were collected using a Microfire 24-bit CCD camera (Optronics, Goleta, California) and imported into Photoshop C2 for preparation of figures.

The antibodies used were commercially obtained. These were anti-calcitonin gene-related protein (CGRP; rabbit; 1:30; Chemicon/Millipore, Temecula, Californa AB5920), anti-GFP (chicken; 1:800, Aves Labs, Tigard, Oregon GFP-1020), anti-HA (rabbit; 1:200; HA.11 Covance, Princeton, New Jersey PRB-101P), anti-peripherin (rabbit; 1:100; Abcam, Cambridge, Massachusetts #1530), anti-S100beta (rabbit; 1:100; Abcam ab868), rabbit anti-beta-III tubulin (TUJ1; 1:3000; Covance MMS-435P). Detection of GFP offers superior sensitivity that is well over background fluorescence ( Fig. 1B and C) . For this study, some inconsistent signal detection or autoflourescence was occasionally observed and these sites identified in the individual figures. We find the expression of GFP and HA are similar, although anti-HA expression is detected predominantly on the surface of cells identified by anti-GFP expression (Fig. 1D ).

The expression of a7 exhibits distinct spatiotemporal patterning in developing cochlear structures. Previously, we demonstrated the earliest expression of a7 in the developing embryo to be in rhombomeres 3 and 5 of the E9.0 embryo ). Thus, we initiated studies of a7 GFP staining at this time. From E9.5 through approximately E12.5, the otic and cochlear structures did not express detectable a7 GFP ( Fig. 2A and not shown, see . The earliest detected expression of a7 GFP in the cochlear structures was at E13.5 in cells of the spiral prominence (SP; Fig. 2B ). The SP retains a7 GFP expression throughout embryonic and post-natal development (see below). By E14.5 ( Fig. 2C and D) , a7 GFP expression extends to cells in the sensory domain of the lesser epithelial ridge near the site of the presumptive outer hair cells (OHC) and Deiters' cells (Morsli et al. 1998; Lanford et al. 1999; Kiernan et al. 2005a,b) . Light staining of the greater epithelium ridge was also present from E14.5 and thereafter, although this staining is inconsistently observed (Fig. 1B and C and not shown).

Coincident with this expression was strong staining of pioneering efferents that become separated into individually distinguished processes as they progress through the spiral ganglion (SG) to reach the external face of this sensory domain ( Fig. 2C ; see below). The staining of the epithelial cells of the lesser epithelial ridge intensifies thereafter (e.g., E15.5 in Fig. 2E ). At this stage, expression of a7 GFP by cells of the SG was in general only weakly observed in scattered cells (Fig. 2E ). By E16.5, a7 GFP expression continues to increase in cells of the lesser epithelial ridge of the prosensory domain where OHC and Deiters' cells can now be distinguished (Fig 2F and G and insert) . Cells throughout the SG were also revealed by expression of a7 GFP by this developmental stage. Pillar cells do not express a7 GFP and there were no identifiable efferent processes labeled by the expression of this receptor at this stage or thereafter (see the following sections). the a7 gene (Chrna7) was modified using homologous recombination to add a C-terminus epitope tag (hemagglutinin [HA] ) and inserted into the 3′ terminus of Chrna7 a reporter bicistronic internal ribosome entry sequence (IRES)-tau fusion to enhanced green fluorescent protein (eGFP) fusion protein cassette (a7 GFP ; see Methods and ). This construct was subsequently altered by replacing the tau:GFP cassette with the Cre-recombinase gene (a7 Cre ). (B, C) The visualization of the Chrna7 transcription using immunological detection of GFP compared with background. Shown are sagittal sections of the cochlear sensory structures of an E16.5 a7 GFP embryo in (B) and at greater magnification in (C). The panels on the left are stained for GFP expression (see Methods), whereas the image on the right shows an adjacent serial section that received the same staining treatment, only primary antibody was omitted. Photographs were collected at the same gain and exposure. The asterisk identifies cochlear ducts and the arrow points to the spiral prominence and the arrow head points to cell giving rise to the outer hair cells and Deiters' cells. Abbreviations are SG, spiral ganglion; and tg, trigeminal ganglion. In (B), the bar = 100 lm and in (C), the bar = 400 lm. (D) Examples of colabeling for a7 GFP (green) and anti-HA (HA) in cells associated with the spiral ganglion at E16.5. Examples of double-labeled cells are identified by with arrows. Some processes are also colabeled (arrow head). Bar = 50 lm.

The pattern of a7 GFP expression in the E18.5 cochlear structure undergoes significant remodeling as both sensory hair cells and the associated supporting cells complete their differentiation ( Fig. 2H and I). This includes a decrease of a7 GFP expression by OHCs and underlying Deiters' cells that progresses away from the inner hair cells and proceeds in a basal-to-apical direction (next section). This is coincident with the appearance of signal in Hensen's cells that are most proximal to the outer line of OHCs (returned to below). Ganglionic afferent fibers ending at the base of the inner hair cells are also detected (see subsequent sections). In the postnatal mouse, as shown in the P6 cochlear sensory structure ( Fig. 2J and K), the expression of a7 GFP becomes limited to Hensen's cells immediately adjacent to the most distal OHC. Cells of the spiral ligament also acquire a7 GFP expression, while the spiral prominence remains unchanged. In the SG, the expression of a7 GFP is well established and the projections from these labeled cells can be followed to the vicinity of the inner hair cells (IHC) where their terminals appear to surround the base of the inner hair cell (IHC; Fig. 2J and K). A summary diagram illustrating the expression of the a7 GFP during these major developmental stages is shown in Fig. 2L . Remodeling of a7 GFP in the cochlear structure after E16.5 is in a basal-to-apical direction

The remodeling of the sensory cell region of the cochlear structure between E16.5 and E18.5 as suggested by the progression in changing a7 GFP expression was examined further. Through E16.5, all otic structures exhibit a similar a7 GFP expression pattern (Fig. 3A) . This was not the case in the E18.5 cochlear structure where the loss of a7 GFP expression by OHC and Deiters' cells and acquisition of staining by Hensen's cells was first observed in the most basal structures and it then appears in the more apical structures successive developmental stages ( Fig. 3B and C and not shown). This generates a striking contrast in a7 GFP expression between cochlear structures at the apex relative to the base with intermediary turns, exhibiting the progressive stages of this change in a7 GFP expression (Fig. 3B ). By P4, this gradient was not evident (not shown) and the mature a7 GFP expression pattern first observed in the E18.5 basal cochlear structures was present across the entire structure. In Fig. 3D , a diagram depicts the remodeling of a7 GFP expression seen in the E18.5 developing cochlear structure.

Nonsensory cells of the cochlear structure express a7 GFP As suggested by the preceding discussion, there was expression of a7 GFP by both neuronal and non-neuronal cells (Fig. 4) . This is particularly clear in the postnatal mouse (e.g., P6-P12), where the predominant expression of a7 GFP in neuronal cells was by cells of the SG (Fig. 4A) . The strongest labeling of cochlear structures was restricted to Hensen's cells and the spiral prominence ( Fig. 4A-E) . Evident at the P6 stage was a7 GFP signal in individual cells of the spiral ligament ( Fig. 4C and D) . Also evident were the extended branching that is characteristic of the morphology of type II fibrocytes located in this region (Fig. 4D ; Spicer and Schulte 1991; Sun et al. 2012 ). In the P12 cochlear structure, the branches were more abundant and form a 'feathered' structure that emanates from cell bodies defined by a7 GFP expression (Fig. 4E) . Cells of the stria vascularis or other members of the cell family composing the structures of the lateral wall and surrounding cochlear duct were not observed to express a7 GFP in these later stages of development (Fig. 4) .

The expression of a7 GFP during innervation of the developing cochlear structure

Innervation of cochlear sensory cells follows a series of distinct steps that were in part revealed by a7 GFP visualization (Fig. 5 ). As noted, the first detection of a7 expression was in the prominently labeled efferent processes that appear to form bundles upon entering the SG and then disperse into small solitary fibers (E14.5; Figs. 5A and 2C,D). These solitary processes exhibit a beaded structure as they proceed to the base of the developing sensory cells (Fig. 5B) . The origin of these efferent fibers was examined in serial sections of the E14.5 hind brain. These fibers appear to originate from a cell grouping in the basal brain stem caudal to trigeminal nucleus V that could be distinguished by their transient a7 GFP expression (Fig. 5C ). These cells occur in clusters (Fig. 5C insert) and their prominently labeled processes can be followed using serial section sets to the cochlear structure where they give rise to the fiber bundles and the point of dissemination associated with the SG (Fig. 5C and insert) . The anatomical location of these cells suggest that these cells are within the forming olive complex, which is consistent with the reports of pioneering fibers that originate from the developing olive complex and extend to the developing cochlea (Zuo et al. 1999 ). These fibers were not detected after E15.5. During the E15.5-16.5 period, there was essentially no labeling of neuronal processes by a7 GFP (Fig. 5D-F) .

However, ongoing innervation of cochlear sensory cells was identified using peripherin labeling ( Fig. 5E ; see Simmons et al. 1996; Hafidi 1998; Huang et al. 2007) or for olivocochlear efferents that were identified by labeling for calcitonin gene-related protein (CGRP; Fig. 5F , Fritzsch 2003). By E18.5, the SG a7 GFP signal was present in afferent processes that extend to the base or near vicinity of the IHCs (Fig. 5G) .

At birth and thereafter (P0-P12 analyzed), the expression of a7 GFP was strongly detected in SG afferent fibers where they terminate near or at the base of IHC sensory cells ( Fig. 5H and I) . This basic pattern of a7 GFP expression was reinforced during the remaining postnatal period as fibers continue to form a dense plexus that appears to surround the base of the IHCs. The other efferent fibers not detected by a7 GFP continue to be trimmed and also associate with their final targets (Merchan-Perez and Liberman 1996; Simmons et al. 1996; Hafidi 1998; Huang et al. 2007 ). The outcome of this remodeling was evident by P12 when the SG1 afferent terminals surrounding the IHC were distinguished by strong a7 GFP staining of the terminal clusters ( Fig. 5I and inset) . This was approximately the same time hearing onset occurs in mice (~P10; Kros et al. 1998 ). Processes originating from SG cells identified by peripherin expression that were not colabeled with a7 GFP form distinct efferent terminals on or very near OHCs cells and on the terminals that end on the IHC afferent terminals identified by a7 GFP labeling ( Fig. 5I ; Huang et al. 2007 ). While not entirely evident from the images shown, not all SG cells at P12 expressed a7 GFP , suggesting this could identify a functionally distinct subpopulation ( Fig. 5I ; Happe and Morley 1998) . Again, no a7 GFP labeling of olivocochlear efferents was detected. A diagram summarizing these findings is shown in Fig. 5J .

Ablation of the a7 Cre -expressing cell lineage confirms a7 GFP expression during cochlear development Although a7 GFP expression was not detected in the developing cochlear structures until E13.5 (Fig. 2B) , as reported previously the earliest a7 expression we have defined is at P9.0 in rhombomeres 3 and 5 . Because cochlear morphogenesis includes signaling from rhombomere 5 (Liang et al. 2010) , the possibility of a7 GFP contributing to the development of this structure was examined. This was done using embryos from a7 Cre mice crossed with mice harboring the conditional ROSA26-loxp (diphtheria-A toxin (DTA; termed a7 Cre: DTA ; . In these embryos, a7 Cre: DTA -expressing cells and their direct lineages were ablated, thus revealing expression that could have been be missed by a7 GFP measurements ). An example of the cochlear structure at E16.5 taken from a7 Cre:DTA crosses is shown in Fig. 6 . Because there is only occasional overlap with a7 GFP (see Fig. 5E ), we used peripherin expression to aid in examining the fate of non-a7-expressing cells (Fig. 6A and B) . The overall patterning of the cochlear structure and the formation of major boney structures of the cochlea inclusive of the otic capsule and modiolus were intact, albeit somewhat distorted. The cochlear ducts were collapsed (Fig. 6B) , probably due to the absence or severe thinning of the distal lateral wall. Also absent was the sensory cell domain Figure 5 . The a7 GFP expression during cochlear innervation. Innervation of the developing cochlear structure is revealed by a7 GFP labeling. (A) An E13.5 sagittal section shows a group of efferent processes (arrow) that distribute to solitary fibers that are strongly labeled for a7 GFP expression (arrow heads). Cells of the putative sensory region (sr) and the spiral prominence (SP) are identified. (B) At greater magnification, these fibers (arrow heads) have a beaded appearance and project towards the base of sr. (C) The possible origin of the pioneering efferent fibers is suggested by the intense expression of a7 GFP in the E13.5 cell groups (arrow) located caudal to the trigeminal sensory nucleus (V) consistent with the early olive in this horizontal section through the posterior brain stem. At increased magnification (Insert), the cell clustering (arrow) and their projections (arrowhead) are identified. Serial sections (not shown) reveal continuity between these cells and those entering the cochlear structures (arrow heads). (D) E15.5 a7 GFP expression and colabeling with other neuronal process markers (red). The processes that express peripherin (arrow) end mostly in the vicinity of the inner hair cells (IHC). Occasional solitary fibers (arrow) extend towards the base of the outer hair cells (OHC) at the dorsal boarder of the Deiters' cells (D). (E) The E16.5 cochlear innervation pattern looks much the same as E15.5, although the peripherinlabeled fibers (arrow) are more distinct. These processes lack detectable a7 GFP expression. (F) Olivocochlear efferents identified by calcitonin generelated proteins (CGRP; arrows). (G) The E18.5 embryo exhibits afferents detected by a7 GFP expression (arrows). These extend from SG cells that are not colabeled with peripherin (not shown). (H) At birth (P0), there are distinctly labeled a7 GFP afferents (arrowhead) and peripherin-labeled efferents that extend to the Deiters' cells (D) and then turn (arrows) to contact the base of the OHCs. Hensen's cells are noted (H). (I) The P12 innervation pattern is similar to the P0. In this merged image of a7 GFP expression (green) and peripherin (red), many spiral ganglion (SG) cells and processes are labeled, but the labels only rarely overlap in the same processes (see insert). The a7 GFP identify mostly processes reaching the IHCs (arrow). Peripherin-labeled processes mostly terminate at the base of the outer hair cells (OHC) or onto the a7 GFP -labeled afferent fiber near the base of the IHC. Hensen's cells expressing a7 GFP is identified (H). The inset shows the sensory cell region at increased magnification. The arrows identify the a7 GFP -expressing afferent ending at the base of nonlabeled IHC, whereas the double arrow heads point to the peripherin-labeled terminal. Other peripherin processes extend to the base of the OHCs (individual arrow heads). (J) Diagrams as in Fig. 2 depicting the basic innervation patterns observed in this study. Green is a7 GFP and red is peripherin. Afferent (af) and efferents (ef). Bars = 50 lm containing presumptive OHCs and Deiters' cells ( Fig. 6C and D), as expected from results of a7 GFP expression (Figs. 2, 5) .

The SG of a7 Cre:DTA embryos is reduced in size and the majority of cells remaining give rise to mostly peripherinlabeled efferents (see Fig. 5E ). These fibers also appear to be more densely aggregated relative to the a7 GFP control mouse ( Fig. 6A and B) . While peripherin-identified processes still project to the presumptive sensory cells (both IHC and OHC), they were less branched and those that did project to the former OHC target fields often turn and proceed backwards towards the vicinity of IHCs ( Fig. 6C and D) . These results are consistent with the earliest expression of a7 being after major cochlear structures are determined, and there was the expected selective ablation of OHCs and Deiters's cells. The necessity of the presence of the target sensory cell to coordinate the innervation process is also suggested by these findings.

The results of studies examining a7 expression using in situ hybridization and functional measurements using electrophysiology have shown that this receptor is an important contributor to various nuclei of the central auditory system (Happe and Morley 1998; Vetter et al. 1999 Vetter et al. , 2007 Morley and Happe 2000; Morley 2005 ). The a7 GFP mouse system offers an excellent opportunity to view these central systems and their connections as shown in Fig. 7 . The connections between the SG and the cochlear nuclei were strongly identified at E18.5, presumably due to the dense projections from SG cells expressing a7 GFP that extend processes both to the IHC (Fig. 2 ) and the developing cochlear nuclei of the brainstem (Fig. 7A) .

The expression of a7 GFP appears to intensify after P10, and by P12 signal is consolidated almost exclusively in the ventral-posterior cochlear nucleus (Fig. 7B ). This is in agreement with reports from in situ hybridization studies reporting the strong expression of a7 in this nucleus, whereas other major cochlear nuclear divisions exhibited only weak or sporadic labeling (Yao and Godfrey 1999; Morley and Happe 2000) . Also consistent with those studies was that the cells identified by a7 GFP expression resemble octopus cells (Fig. 7B, insert) . Essentially, no expression of a7 GFP was detected in the dorsal cochlear nucleus, although some dispersed and weakly stained cells were present in the granular aspect. Also evident was the strong staining of neuropil, presumably in part due to terminals of SG cells associated with the eighth cranial nerve (Fig. 7B, inset) . This strong labeling of the P12 SG and OHC afferents is consistent with other reports (Morley and Happe 2000) .

The expression of a7 GFP also persists into the adult animal. This is apparent in the ascending central auditory system nuclei and their fibers (Fig. 7C) . After the cochlear nucleus, a7 GFP is present in the ventral lateral lemniscus, on through the dorsal lateral lemniscus, and to the inferior colliculus where dense staining of a7 GFP is present ( Fig. 7C ; Morley and Happe 2000; Yao and Godfrey 1999) . The commissural fibers of the inferior colliculus are also identified by a7 GFP expression (Fig. 7D) . Thereafter, efferents follow the brachium of the inferior colliculus to the medial geniculate nucleus where scattered cells expressing a7 GFP were seen. Not shown is that the expression of a7 GFP in the adult auditory cortex appears restricted to cells of layer 1. Labeling of olivocochlear fibers was not detected.

This study extends the reports of spatiotemporal regulation of a7 expression during mouse embryonic development to include the cochlear sensory structure, as well as confirms the extensive expression of this nAChR in the ascending central auditory system. The novel finding that in addition to expression of a7 GFP in developing sensory cells of the cochlear structure and neuronal cells of the spiral ganglion, there is also considerable expression by nonsensory cells. Cells of the spiral prominence and ligament, Deiters' cells, and some Hensen's cells. Despite overall agreement between our studies and those using in situ hybridization (e.g., Happe and Morley 1998; Morley and Happe 2000) , these nonsensory cells were not reported previously to express a7. However, these comparisons are incomplete because the earlier studies did not necessarily show the comparable structures or the developmental stages at times where we observed peak a7 GFP expression. Also, our method of detecting GFP as a marker of a7 expression offers improved sensitivity and resolution that has previously not been available for this nAChR.

The nicotinic receptors a9 and a10 are particularly well characterized in the auditory system (Elgoyhen et al. 1994 (Elgoyhen et al. , 2001a Vetter et al. 1999 Vetter et al. , 2007 Katz et al. 2004; Morley 2005) . Comparing the expression of a7 GFP to the results from these studies of the sensory hair cells and the nonsensory cells of the cochlea indicate that there are significant spatiotemporal differences during development between the expression of a7 versus a9 and/or a10. The a9 KO mouse also exhibits auditory deficiencies that are not observed in the a7 KO mouse, which is largely devoid of a phenotype in this sensory system under normal physiological conditions (Liberman and Brown 1986; Simmons and Morley 1998; Morley 2005; Lustig 2006 ). The a7 GFP is not detected in IHCs, which is consistent with a9 nAChR being the principle target of alpha-bungarotoxin in this cell type (Uziel et al. 1981; Glowatzki and Fuchs 2000) . Collectively, this suggests that functional redundancy between these receptor subtypes is unlikely (see also . This is also supported by the extensive studies by the Morley group Morley 1998, 2004; Morley and Happe 2000; Simmons and Morley 2011) who showed that multiple receptor subtypes are expressed in the cochlear and central auditory systems, but each exhibits distinct spatiotemporal patterns that likely preclude substantial or sustained functional overlap.

Noteworthy is that the functional contribution of a7 towards modulating physiological systems may not be revealed unless the system is imbalanced as by genetic . Central auditory systems express a7 GFP . Central auditory nuclei identified by a7 GFP expression. (A) At E18.5 in this sagittal image of the entire otic complex and the adjacent basal brainstem is included. The cochlear nucleus (C) and the eighth cranial nerve (8n) are visible as is the fifth cranial nerve (5n), the trigeminal nucleus (TGN), and trigeminal ganglion (TGG). Also noted are cochlear ducts (asterisk) and a spiral ganglion (SG). (B) At P12, a7 GFP expression of cochlear complex reveals the strongly labeled cells of the ventralposterior cochlear nucleus (VCP). The dorsal cochlear nucleus (DC) and ventral-anterior cochlear nucleus (VCA) are identified and is the eighth nerve (8n) and a cochlear duct (asterisk). The inset shows the VCP at increased magnification. Cells clusters expressing a7 GFP (arrowhead) and individual cells that resemble the morphology of octopus cells described previously (Morley and Happe 2000; Morley 2005) to express a7 (arrow) are noted. (C) Another P12 sagittal section reveals a7 GFP expression in the ascending central auditory pathways. (D) The expression of a7 GFP in the inferior colliculus (CIC) of this horizontal section reveals staining of the commissural fibers (arrow). Structures identified are the brachium of the inferior colliculus (BIC); dentate gyrus (DG), inferior colliculus, central nucleus (CIC); lateral lemniscus, dorsal nucleus (DLL); lateral lemniscus, ventral nucleus (VLL); medial geniculate nucleus (MGN), and the substantia nigra (SN). Bars = 100 lm (A, B0; 20 lm (B-insert), and 1 mm (C, D). deficiencies, sustained exposure to pharmacological compounds, or other events such as inflammation (e.g., Faustman et al. 1992; Gahring and Rogers 2005; Venables et al. 2007; Albuquerque et al. 2009; Brown 2011; Severance et al. 2011) . For example, the dysfunction of a7 is implicated in several psychiatric syndromes associated with certain forms of autism and schizophrenia (particularly in patients who hallucinate) whose spectrum of disorders include abnormal sensitivity to sensory stimuli including an abnormal auditory gating phenotype (Khalfa et al. 2001; Veuillet et al. 2001; Araki et al. 2002; McEvoy and Allen 2002; Freedman et al. 2003; Lippiello 2006; Martin and Freedman 2007; Wallace and Porter 2011 and references therein) . Also, the association of certain auditory deficits and nicotine abuse, mostly associated with cigarette smoking, has further focused speculation on the role of a7 in these pathologies and the possible advantages of therapeutically targeting this receptor for symptomatic relief in these cases (Araki et al. 2002; McEvoy and Allen 2002; Simosky et al. 2002; Freedman et al. 2003; Levin et al. 2006; Lippiello 2006; Martin and Freedman 2007; Wallace and Porter 2011) . In this context, our results suggest additional lines of investigation. For example, in a 7Cre:DTA cell lineage ablation there are collapsed cochlear ducts and abnormal innervation indicating that the cells express a7 and the cells that do so contribute an obligatory role in the successful development and long-term function of these structures. The a7 receptor could also participate in auditory performance after birth, including functions related to the central auditory pathways. This study also adds the possibility of an effect by a7 on the performance of the spiral ligament. These cells exhibit a cholinergic response that is most often described in terms of muscarinic acetylcholine receptors (Khan et al. 2002; Maison et al. 2010) , and their dysfunction is related to several pathogenic auditory deficiencies (Spicer and Schulte 1991; Slepecky et al. 1995; Kikuchi et al. 2000; Sun et al. 2012) . The role of a7 has, to our knowledge, not been examined in these cells. Collectively, the potential for a7 functional pleiotropy in the auditory system is similar to other tissues we have recently examined . Thus, multiple defects that impact upon adult function could be expected depending upon the timing, duration, and nature of the receptor dysfunction.

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Distinct contributions from the hindbrain and mesenchyme to inner ear morphogenesis

Physiology and anatomy of single olivocochlear neurons in the cat

Nicotinic cholinergic antagonists: a novel approach for the treatment of autism

Nicotinic acetylcholine receptor structure and function in the efferent auditory system

Muscarinic signaling in the cochlea: presynaptic and postsynaptic effects on efferent feedback and afferent excitability

Schizophrenia and the alpha7 nicotinic acetylcholine receptor

The importance of nicotinic acetylcholine receptors in schizophrenia, bipolar disorder and Tourette's syndrome

Ultrastructural differences among afferent synapses on cochlear hair cells: correlations with spontaneous discharge rate

Nicotinic cholinergic intercellular communication: implications for the developing auditory system

Cholinergic receptors: dual roles in transduction and plasticity

Development of the mouse inner ear and origin of its sensory organs

Activity of nAChRs containing alpha9 subunits modulates synapse stabilization via bidirectional signaling programs

Nicotinic receptor alpha7 expression during tooth morphogeneiss reveals functional pleitropy

Ablation of the nicotinic receptor alpha7 cell lineage produces lumbosacral spina bifida that is modified by maternal diet

Coronavirus immunoreactivity in individuals with a recent onset of psychotic symptoms

Differential expression of the alpha 9 nicotinic acetylcholine receptor subunit in neonatal and adult cochlear hair cells

Spatial and temporal expression patterns of nicotinic acetylcholine alpha 9 and alpha 10 subunits in the embryonic and early postnatal inner ear

Olivocochlear innervation of inner and outer hair cells during postnatal maturation: an immunocytochemical study

Nicotinic agonists and psychosis

Posttranslational modifications of tubulin suggest that dynamic microtubules are present in sensory cells and stable microtubules are present in supporting cells of the mammalian cochlea

Differentiation of inner ear fibrocytes according to their ion transport related activity

Functional interaction between mesenchymal stem cells and spiral ligament fibrocytes

Development of cochlear potentials in rats

Prenatal influenza exposure and delivery complications: implications for the development of schizophrenia

Role of alpha9 nicotinic ACh receptor subunits in the development and function of cochlear efferent innervation

The alpha10 nicotinic acetylcholine receptor subunit is required for normal synaptic function and integrity of the olivocochlear system

Abnormal peripheral auditory asymmetry in schizophrenia

Targeting the nicotinic alpha7 acetylcholine receptor to enhance cognition in disease

Immunolocalization of alpha4 and alpha7 subunits of nicotinic receptor in rat cochlear nucleus

Visualization of alpha9 acetylcholine receptor expression in hair cells of transgenic mice containing a modified bacterial artificial chromosome

This work was supported by National Institutes of Health grants AG017517, DA025057, and AG029838.

None declared.