Translational research in bipolar disorder: emerging insights from genetically based models FEATURE REVIEW Translational research in bipolar disorder: emerging insights from genetically based models G Chen1, ID Henter1 and HK Manji1,2 1Mood and Anxiety Disorders Program, National Institute of Mental Health, National Institutes of Health, Bethesda, MD, USA and 2Johnson and Johnson Pharmaceutical Research and Development, Titusville, NJ, USA Bipolar disorder (BPD) is characterized by vulnerability to episodic depression and mania and spontaneous cycling. Because of marked advances in candidate-gene and genome-wide association studies, the list of risk genes for BPD is growing rapidly, creating an unprecedented opportunity to understand the pathophysiology of BPD and to develop novel therapeutics for its treatment. However, genetic findings are associated with major unresolved issues, including whether and how risk variance leads to behavioral abnormalities. Although animal studies are key to resolving these issues, consensus is needed regarding how to define and monitor phenotypes related to mania, depression and mood swing vulnerability in genetically manipulated rodents. In this study we discuss multiple facets of this challenging area, including theoretical considerations, available tests, limitations associated with rodent behavioral modeling and promising molecular–behavioral findings. These include CLOCK, glycogen synthase kinase 3b (GSK-3b), glutamate receptor 6 (GluR6), extracellular signal- regulated kinase-1 (ERK1), p11 (or S100A10), vesicular monoamine transporter 2 (VMAT2 or SLC18A2), glucocorticoid receptors (GRs), Bcl-2-associated athanogene-1 (BAG1) and mitochondrial DNA polymerase-c (POLG). Some mutant rodent strains show behavioral clusters or activity patterns that cross-species phenocopy objective/observable facets of mood syndromes, and changes in these clustered behaviors can be used as outcome measures in genetic–behavioral research in BPD. Molecular Psychiatry (2010) 15, 883–895; doi:10.1038/mp.2010.3; published online 9 February 2010 Keywords: mania; depression; bipolar disorder; animal model; lithium Introduction Bipolar disorder (BPD) is a severe, disabling and often life-threatening psychiatric disorder that affects ap- proximately 1–2% of the general population.1,2 Phenotypically, BPD is a very complex disease in which patients alternate between episodes of mania and depression1,2 (Figure 1). Accumulating data support the theory that BPD arises from the complex inheritance of multiple genetic variants3–5 (Figure 1). In susceptible subjects, physical and psychosocial stressors can facilitate the progress of BPD into a stage of vulnerability and trigger mood episodes.6 Such episodes can also be induced by psychostimulants, monoamine depletion and corticosteroids; an intrin- sic biological trigger is also suspected for ‘sponta- neous’ mood episodes.1,2 Recently, large-scale candidate and genome-wide association studies have generated a rapidly growing list of risk genes for BPD, including ANK3 and CACNA1A.5,7 More key findings are expected from copy-number variance and in-depth sequencing stu- dies of thousands of individuals. Advances in genetic studies create an unprecedented opportunity to finally understand the pathophysiology of this devas- tating illness at the molecular level, and to develop novel therapeutics capable of relieving mood symp- toms as well as preventing recurrences. However, both existing and future association studies have at least three key issues that remain unresolved: (1) whether, and to what extent, risk variance causes gene dysfunction; (2) whether risk genes are causally linked to behavioral abnormalities in BPD and (3) if so, what are the precise mechanisms that lead to behavioral abnormalities. Animal studies have long had a pivotal role in attempting to resolve these issues. Currently, two types of behavioral approaches are used to analyze the pathogenesis of BPD; broadly, these can be thought of as the animal model and model animal approaches.8 The animal model approach begins by identifying a behavioral stress or chemical treatment that induces specific behavior(s) thought to cross- species phenocopy one or a few mood symptoms (for example, immobility in the forced swim or tail suspension tests, escaping deficit in the learned helplessness paradigm9 or social avoidance or re- duced sweet solution preference in the social defeat Received 8 November 2009; revised 26 December 2009; accepted 31 December 2009; published online 9 February 2010 Correspondence: Dr G Chen, MAP, NIMH-IRP, NIH, Building 35, Room1C-912 (main office) and 1BC-108, 35 Convent Drive, MSC 3711, Bethesda, MD 20892, USA. E-mail: guangchen@mail.nih.gov Molecular Psychiatry (2010) 15, 883–895 & 2010 Macmillan Publishers Limited All rights reserved 1359-4184/10 www.nature.com/mp http://dx.doi.org/10.1038/mp.2010.3 mailto:guangchen@mail.nih.gov http://www.nature.com/mp paradigm10). The biological basis of induced beha- viors can then be studied. In contrast, the initial step of the model animal approach is to introduce specific gene or pathway alterations implicated in BPD into animals. These animals are then evaluated using batteries of behavioral tests to examine whether the abnormality is causally linked to behaviors or to behavior clusters that cross-species phenocopy mood symptoms, mood syndromes or mood swing vulner- ability observed in euthymic BPD patients. Once these causal relationships are established, the im- plicated biological alterations to behavior can be further analyzed. Although both animal models and model animals can be used to evaluate novel therapeutics, the increased use of model animals is expected, given the unique ability of such animals to evaluate pathophysiology driven target treatments. The term ‘cross-species phenocopy’, which we use throughout this review, is used to emphasize apparent phenomenological similarities that can result from the same or different underlying mechanism(s) in humans and rodents.11 To be cautious with regard to animal research, investigators have used the term ‘cross-species phenocopy of depression or mania’ rather than ‘model of depression or mania’ to reflect the fact that true behavioral models for a given illness stem from established pathology, and that the pathol- ogy of BPD is still largely unknown. Below, we discuss the strengths and limitations of each animal approach and describe recent promising findings from several mutant strains. Specifically, we discuss (1) the advantages and drawbacks associated with current batteries of tests that characterize behavioral clusters or activity patterns related to the Bipolar I Bipolar II Genetic factors Environmental factors Early life maltreatment Psychostimulant Monoamine depletion Circadian shift/sleep deprivation Social-psychological stress S100A10 (p11) SLC18A2 (Vmat2) CLOCK GluR6 ERK1 GSK-3 betaGR BAG1 Predisposed abnormalities (Intrinsic & inducible switch) pathophysiology pathophysiology Mania Depression Susceptible stage Mood stabilizers Antidepressants Figure 1 (a) BPD has unique episodic and dual-directional features in which mood states can be either elevated (red, mania), suppressed (blue, depression) or rapidly alternate between the two extremes (mixed episodes). Euthymic/remitted phases occur between the episodes in which patients, especially those with residual mood symptoms, remain vulnerable to the recurrence of full-blown mood episodes. Mood stabilizers, such as lithium, valproate, carbamazepine, lamotrigine, as well as atypical antipsychotics, are effective therapeutic agents that gradually relieve the symptoms of mania and/or depression. They also prevent the recurrence of mood episodes. Recent data from the Systematic Treatment Enhancement Program for Bipolar Disorder (STEP-BD) study found that approximately 60% of patients recover after appropriate treatments, and approximately 50% of recovered patients experience a recurrence within 2 years; recurrence is highly associated with residual mood symptoms at initial recovery. BPD-I: patient has one or more manic episodes or mixed episodes. Often individuals have also had one or more major depressive episodes. BPD-II: patient has at least one hypomanic episode and at least one major depressive episode. (b) Conceptually, the course of BPD can be divided into two major parts: vulnerability and episode stages; the illness is further characterized by a transition period from vulnerability to episode stage. Genetic variants and environmental stressors, and the interaction between the two (G � E), contribute to the predisposed abnormalities underlying the vulnerability stage. These two factors and their interaction may also contribute to the intrinsic (or spontaneous) switch mechanism. Psychosocial and biological stressors can trigger mood episodes in susceptible individuals. The use of psychostimulants and experimental monoamine depletion can cause mania or depressive relapses in subjects with a personal or family history of mood disorders, or with certain genetic variants. Unique pathophysiological mechanisms are believed to underlie depression, mania and mixed episodes. The color reproduction of this figure is available on the html full text version of the manuscript. Translational research in bipolar disorder G Chen et al 884 Molecular Psychiatry three major components of BPD: mania, depression and vulnerability to mood swings; (2) the relevance of behavioral clusters or patterns to depression and mania, and how these can be further validated through treatment with antidepressants or mood stabilizers; and (3) how these behavioral clusters or patterns can be used as major behavioral outcome measures in studies that use the model animal approach to address issues related to genetic–beha- vioral relationships in BPD. It is worth noting that although rodents are nocturnal animals, the behavioral tests or measures discussed in this review are currently conducted during the light phase (with the exception of home cage wheel running12 and home cage activity11 scans). Furthermore, animals are bred in the laboratory for experimental purposes, and these conditions are far from natural. This adds a further dimension to our ability to extrapolate the results of one or a few tests to the overall state of rodent activity, and from rodents to humans. Future studies should address alternative behavioral tests and measures, as well as the logistical problems of animal holding and behavioral facilities for experiments that are more in line with the nocturnal nature of rodents. The animal model approach A comprehensive list of current animal models for depression or mania can be found in several recent, well-written reviews.13–16 These animal models are valid for evaluating mood stabilizers as well as ‘me- too’ and novel types of antidepressants.13,14 For instance, the forced swim test is sensitive to most direct monoamine manipulating antidepressants13,14 as well as other clinically effective antidepressant agents such as ketamine,9 lamotrigine17,18 and lithium.19 Because the outcome measures of these models are sensitive to treatment with mood stabi- lizers or antidepressants,13–16 they are often collec- tively referred to as antidepressant- or mood stabilizer-sensitive behavioral paradigms. Two terms describe experimental outcomes from these para- digms: (1) mood-stabilizer-like or antidepressant-like behavioral actions and (2) the behavioral effects of mood stabilizers or antidepressants. It should be noted that these terms are not directly equivocal to mood stabilizing or antidepressant actions. Presently, it remains unknown whether or not the administra- tion of mood stabilizers or antidepressants elicits different behavioral actions through the same me- chanisms. These animal models are associated with other advantages and weaknesses, and these have been well addressed in previous reviews.13–16 The model animal approach The ‘reverse translation’ model animal approach is being used in the study of BPD with increasing frequency. Loosely defined, reverse translation shifts the focus from creating animal behaviors that pheno- typically resemble aspects of mental disorders (that is, animal models) to instead using what we have learned about the potential pathological changes found in BPD from human genetic, post-mortem and other studies (that is, creating model animals).8 Potential pathological changes include genetic, epi- genetic and other biological alterations. This subtle distinction shifts the focus from creating animal models to understanding the role of those specific changes already implicated in BPD. Thus, instead of approaching this issue from the perspective of creating an ‘animal model of BPD’, researchers can focus on understanding whether and how specific changes contribute to a ‘mania-like phenotype’, ‘depression-like phenotype’ or ‘vulnerability-like phenotype’. Studies using this approach can ulti- mately provide fundamental information for design- ing novel therapeutics that are able to target causally linked changes, or the biological consequences of the changes. The animals created from this type of research can, in turn, serve as tools for the compre- hensive evaluation of the effectiveness of novel therapeutic methods for BPD at both the neurochem- ical and behavioral levels. In the model animal approach, biologically predis- posed animals are generated using genetic (for example, gene transgenic, knockout or mutation– knock-in manipulation) or other biological means (such as viral vector-based gene overexpression or knockdown). These predisposed animals are then studied using batteries of behavioral tests for pheno- copies related to different components of BPD. Some of these tests are highlighted in Tables 1–3. It should be emphasized that the translational study of BPD using the model animal approach rests upon making the same assumptions that are made in studies using animal models. First, the basic structure and biochemical function of the gene being studied is thought to be, to a certain degree, similar in both humans and rodents. It is important to recognize, however, that although the proteins encoded by the genes of human and rodents are highly homologous, the regulation of gene transcription by transcription factors or epigenetics, or the regulation of gene translation by microRNAs, can vary extensively between humans and rodents. Second, the gene– behavior relationships of some behaviors may be conserved only to a certain extent between the human and animal genomes. Clearly, human brains are far more complex than those of rodents, and rodents should not be viewed as miniature humans.20 We now turn our focus to research approaches that use model animals, as well as findings from these studies. Mania Mania can affect mood, thinking (processing and content) and behavior. Although many genes impli- cated in BPD are certainly being studied (see Craddock and Sklar21 and Kato22 for a review), some Translational research in bipolar disorder G Chen et al 885 Molecular Psychiatry Table 1 Clinical features of mania and rodent phenocopies of components of excessive behavioral excitement Domains Clinical diagnostic features Suitable animal tests Expected outcomes mCLOCK GluR6 �/� ERK1 �/� Mood Elevated, expansive or irritable mood Unknown NA NA NA NA Thought Inflated self-esteem or grandiosity Unknown NA NA NA NA Flight of ideas or racing thoughts Unknown NA NA NA NA Behavior More talkative than usual or pressure to keep talking Unknown NA NA NA NA Increase in goal-directed activity (either socially, at work or school or sexually) or psychomotor agitation Open field test Novel object exploration Increased investigation Yes Yes Yes Social interaction test Resident–intruder test Increased social interaction, constructive or destructive No data Yes No data Mating activity Increased sexual activity No data No data No data Sweetened solution preference test Wheel running activity FUST CPP ICSS Increased hedonic/ pleasurable activity Yes Yes Yes Forced swim or tail suspension tests Learned helplessness paradigm Increased escaping activity Yes Yes Yes Excessive involvement in pleasurable activities that have a high potential for painful consequences (e.g., engaging in unrestrained buying sprees, sexual indiscretions or foolish business investments) Center activity in open field Elevated plus maze or zero maze Light/dark Reward/aversion test Novelty-induced hypophagia More risk-taking (or less anxiety- like) activity Yes Yes Yes Decreased need for sleep (e.g., feels rested after only 3 h of sleep) Home cage activity monitoring Home cage wheel running More activity and less rest in home cage No data Yes Yes Attention Distractibility (i.e., attention too easily drawn to unimportant or irrelevant external stimuli) There are potential tests, but these need to be further validated. More distractible No data No data No data Exclusions Manic symptoms are not due to neurological, endocrine or metabolic diseases Brain morphology, biochemical tests and battery of motor-sensory tests No obvious changes Circadian dysfunction Yes Yes Psycho- stimulant Increased use Psychostimulant- induced locomotion No change or increased Yes Yes Yes Abbreviations: CPP, conditioned place preference test; ERK1, extracellular signal-regulated kinase-1; FUST, female urine sniffing test; GluR6, glutamate receptor 6; ICSS, intracranial self-stimulation; NA, not applicable. Translational research in bipolar disorder G Chen et al 886 Molecular Psychiatry T a b le 2 C li n ic a l fe a tu re s o f d e p re s s io n a n d ro d e n t p h e n o c o p ie s o f d e p re s s iv e s y m p to m s D o m a in s C li n ic a l d ia g n o st ic fe a tu re s S u it a b le a n im a l te st s E x p e c te d o u tc o m e s p 1 1 � /� V m a t2 � / þ D e p re s s e d m o o d D e p re s s e d m o o d m o s t o f th e d a y , n e a rl y e v e ry d a y , a s in d ic a te d b y e it h e r s u b je c ti v e re p o rt (e .g ., fe e ls s a d o r e m p ty ) o r o b s e rv a ti o n s m a d e b y o th e rs (e .g ., a p p e a rs te a rf u l) U n k n o w n N A N A N A In c h il d re n a n d a d o le s c e n ts , c a n b e ir ri ta b le m o o d U n k n o w n N A N A N A A n h e d o n ia M a rk e d ly d im in is h e d in te re s t o r p le a s u re in a ll , o r a lm o s t a ll , a c ti v it ie s m o s t o f th e d a y , n e a rl y e v e ry d a y (a s in d ic a te d b y e it h e r s u b je c ti v e a c c o u n t o r o b s e rv a ti o n s m a d e b y o th e rs ) H o m e c a g e a c ti v it y s c a n S w e e te n e d s o lu ti o n p re fe re n c e F U S T W h e e l ru n n in g L e s s L e s s D e c re a s e d D e c re a s e d N o d a ta Y e s N o d a ta N o d a ta N o d a ta Y e s N o d a ta N o d a ta W e ig h t o r a p p e ti te c h a n g e S ig n if ic a n t w e ig h t lo s s w h e n n o t d ie ti n g o r w e ig h t g a in (e .g ., a c h a n g e o f > 5 % o f b o d y w e ig h t in a m o n th ), o r d e c re a s e o r in c re a s e in a p p e ti te n e a rl y e v e ry d a y . N o te : In c h il d re n , c o n s id e r fa il u re to m a k e e x p e c te d w e ig h t g a in s W e ig h t m o n it o ri n g E a ti n g a n d d ri n k in g m o n it o ri n g V a ri e d V a ri e d N o d a ta N o d a ta N o d a ta N o d a ta S le e p d is tu rb a n c e s In s o m n ia o r h y p e rs o m n ia n e a rl y e v e ry d a y H o m e c a g e a c ti v it y s c a n P a tt e rn c h a n g e N o d a ta N o d a ta P s y c h o m o to r a c ti v it y c h a n g e P s y c h o m o to r a g it a ti o n o r re ta rd a ti o n n e a rl y e v e ry d a y (o b s e rv a b le b y o th e rs , n o t m e re ly s u b je c ti v e fe e li n g s o f re s tl e s s n e s s o r b e in g s lo w e d d o w n ) H o m e c a g e a c ti v it y s c a n O p e n fi e ld te s t S lo w /p a tt e rn c h a n g e S lo w /p a tt e rn c h a n g e N o d a ta Y e s N o d a ta Y e s L o s t p h y s ic a l s tr e n g th F a ti g u e o r lo s s o f e n e rg y n e a rl y e v e ry d a y H o m e c a g e a c ti v it y s c a n T re a d m il l O p e n fi e ld te s t F o rc e d s w im o r ta il s u s p e n s io n te s ts L e s s L e s s L e s s M o re im m o b il it y N o d a ta N o d a ta Y e s Y e s N o d a ta N o d a ta Y e s Y e s L o w s e lf e s te e m F e e li n g s o f w o rt h le s s n e s s o r e x c e s s iv e o r in a p p ro p ri a te g u il t (w h ic h m a y b e d e lu s io n a l) n e a rl y e v e ry d a y (n o t m e re ly s e lf -r e p ro a c h o r g u il t a b o u t b e in g s ic k ) U n k n o w n N A N A N A C o g n it iv e d is tu rb a n c e s D im in is h e d a b il it y to th in k o r c o n c e n tr a te , o r in d e c is iv e n e s s , n e a rl y e v e ry d a y (e it h e r b y s u b je c ti v e a c c o u n t o r a s o b s e rv e d b y o th e rs ) U n k n o w n N A N A N A H o p e le s s n e s s R e c u rr e n t th o u g h ts o f d e a th (n o t ju s t fe a r o f d y in g ), re c u rr e n t s u ic id a l id e a ti o n w it h o u t a s p e c if ic p la n , o r a s u ic id e a tt e m p t o r a s p e c if ic p la n fo r c o m m it ti n g s u ic id e U n k n o w n N A N A N A E x c lu s io n s T h e s y m p to m s a re n o t d u e to th e d ir e c t p h y s io lo g ic a l e ff e c ts o f a s u b s ta n c e (e .g ., a d ru g o f a b u s e , a m e d ic a ti o n ) o r a g e n e ra l m e d ic a l c o n d it io n (e .g ., h y p o th y ro id is m ) B io lo g ic a l te s ts G e n e ra l h e a lt h c h e c k li s t N o m a rk e d c h a n g e Y e s Y e s T re a tm e n t L it h iu m , la m o tr ig in e T re a tm e n t E ff e c ti v e N o d a ta N o d a ta A n ti d e p re s s a n ts T re a tm e n t E ff e c ti v e Y e s Y e s K e ta m in e T re a tm e n t E ff e c ti v e N o d a ta N o d a ta A b b re v ia ti o n s : F U S T , fe m a le u ri n e s n if fi n g te s t; N A , n o t a p p li c a b le ; V m a t2 , v e s ic u la r m o n o a m in e tr a n s p o rt e r 2 . Translational research in bipolar disorder G Chen et al 887 Molecular Psychiatry T a b le 3 A ff e c ti v e v u ln e ra b il it y in h u m a n s a n d c ro s s -s p e c ie s p h e n o c o p ie s o f a ff e c ti v e v u ln e ra b il it y in ro d e n ts T y p e A ff e c ti v e v u ln e ra b il it y in h u m a n s S u it a b le te st /o b se rv a ti o n O u tc o m e s G R tr a n sg e n ic B A G 1 tr a n sg e n ic B A G 1 þ /� G e n e ra l A b s e n c e o f n e u ro lo g ic a l d is e a s e s G ro w th a n d a p p e a ra n c e G e n e ra l b e h a v io r N o c h a n g e N o c h a n g e N o c h a n g e N o c h a n g e E u th y m ic m o o d B a tt e ri e s fo r d e p re s s io n a n d m a n ia p h e n o c o p ie s N o c h a n g e N o c h a n g e N o c h a n g e N o c h a n g e V u ln e ra b il it y to d e p re s s io n S tr e s s c o p in g d e fi c it s Im m o b il it y in fo rc e d s w im a n d ta il s u s p e n s io n te s ts R e s p o n s e to h e lp le s s n e s s in d u c ti o n R e c o v e ry fr o m h e lp le s s n e s s R e s p o n s e to c h ro n ic s tr e s s R e c o v e ry fr o m c h ro n ic s tr e s s N o c h a n g e /m o re In c re a s e d L e s s /l o s t In c re a s e d L e s s /l o s t In c re a s e d N o d a ta N o d a ta N o d a ta N o d a ta N o c h a n g e N o c h a n g e E n h a n c e d N o d a ta N o d a ta N o c h a n g e N o c h a n g e R e d u c e d N o d a ta N o d a ta M o n o a m in e d e p le ti o n c a u s e s d e p re s s io n re la p s e R e s p o n s e to m o n o a m in e d e p le ti o n in te s ts fo r a n h e d o n ia -l ik e b e h a v io rs In c re a s e d N o d a ta N o d a ta N o d a ta V u ln e ra b il it y to m a n ia P s y c h o s ti m u la n ts c a u s e m a n ia re la p s e P s y c h o s ti m u la n t c h a ll e n g e te s t In c re a s e d N A E n h a n c e d re c o v e ry N o c h a n g e B e h a v io ra l s e n s it iz a ti o n In c re a s e d In c re a s e d R e d u c e d In c re a s e d C ir c a d ia n s h if t/ je tl a g tr ig g e rs m a n ia re la p s e C ir c a d ia n s h if t/ s le e p d e p ri v a ti o n -i n d u c e d b e h a v io ra l c h a n g e s In c re a s e d N o d a ta N o d a ta N o d a ta T re a tm e n ts fo r d e p re s s io n a n d m a n ia A n ti d e p re s s a n ts B e h a v io ra l re s p o n s e to a n ti d e p re s s a n ts E it h e r (1 ) n o t e ff e c ti v e ; (2 ) p re v e n t th e in d u c ti o n o f d e p re s s io n -l ik e b e h a v io rs ; o r (3 ) w o rs e n m a n ia - li k e b e h a v io rs In c re a s e d N A N o d a ta M o o d s ta b il iz e rs B e h a v io ra l re s p o n s e to m o o d s ta b il iz e rs N o t e ff e c ti v e o r p re v e n ti v e N o d a ta N A N o d a ta A b b re v ia ti o n s : B A G 1 , B c l- 2 -a s s o c ia te d a th a n o g e n e -1 ; G R , g lu c o c o rt ic o id re c e p to r; N A , n o t a p p li c a b le ; V m a t2 , v e s ic u la r m o n o a m in e tr a n s p o rt e r 2 . Translational research in bipolar disorder G Chen et al 888 Molecular Psychiatry genes have been more comprehensively evaluated in behavioral experiments and seem to be most convin- cing in terms of their ability to encompass the multiple behavioral aspects of mania; for research purposes, these behavioral aspects are collectively referred to as excessive behavioral excitement.11 Any working definition for phenocopies of excessive behavioral excitement should include the following: the animal shows hyperactivity in multiple tests, increased goal-directed behavior, increased risk-tak- ing activity, distractibility and reduced sleep or rest; furthermore, these deficits as a cluster can be experimentally distinguished from locomotor unrest, which is another behavioral abnormality, and cannot be alleviated by treatment with psychostimu- lants. Table 1 lists some of the tests proposed for monitoring these behaviors. The most promising genes believed to be associated with excessive behavioral excitement include: glycogen synthase kinase 3 (GSK-3), CLOCK, glutamate receptor 6 (GluR6 or GRIK2, one of the kainate receptors) and extracellular signal-regulated kinase-1 (ERK1, also known as MAP kinase-3). Ample evidence implicates all of these genes in the pathophysiology of mood disorders. For instance, polymorphisms of GSK-3b have been associated with age of onset in BPD,23 as well as with therapeutic response to lithium treatment or sleep depriva- tion.24,25 Some studies suggest that polymorphisms of CLOCK and other components (ARNTL (BMAL1), BHLHB2, CSNK1E and PER3) of the molecular clock are associated with BPD risk,26,27 recurrence28 and age of onset.29 GluR6 (or GRIK2) resides on chromosome 6q21, which has been linked to BPD in several studies;30–34 however, whether GRIK2 polymorphisms per se contribute to the genetic risk of BPD in general or in some BPD pedigrees requires further investiga- tion. It is interesting to note that mRNA levels of GluR6 were found to be significantly lower in the brain tissue of individuals with BPD.35,36 Finally, ERK1 and the ERK pathway are activated by treatment with mood stabilizers,37–40 and single gene and genome-wide association studies show significant and strong associations between BPD risk and poly- morphisms of ERK pathway-modulating genes, such as BDNF (brain-derived neurotrophic factor),41 DISC1 (disrupted-in-schizophrenia 1),42,43 RASGRP1 (RAS guanyl releasing protein 1),7 EGFR (epidermal growth factor receptor)44 and GluR6.30–34 It is worth noting that genetic findings are not consistent across all studies, or consistent with findings from genome- wide association studies. Although reasons for such inconsistencies remain a matter of considerable debate, the interplay between risk genes and environ- mental factors are key issues of consideration. As Table 1 highlights, behavioral studies used to assess cross-species phenocopies of different facets of excessive behavioral excitement have revealed that in mice, GSK-3b overexpression,45 mutant CLOCK (mCLOCK),46 GluR6 knockout (KO)11 and ERK1 KO12 all resulted in hyperactivity in the open field or in the locomotor activity test, and reduced immobility in the forced swim or tail suspension tests. mCLOCK, GluR6 KO and ERK1 KO mice also showed increased hedonic and risk-taking activity.11,46,12 In addition, GluR6 KO mice showed increased aggression during home cage observation, the social interaction test and the resident–intruder test, as well as changes in rest/ activity pattern in their home cages.11 However, the interpretation of such behavioral data is complicated. For instance, although it is a featured abnormality of manic behavioral disturbances, hyper- activity per se is not specific to mania, and can also be observed in models of several other disorders, most notably attention deficit/hyperactivity disorder; it should also be noted that psychostimulants induce manic episodes in euthymic BPD patients47 and relieve symptoms in attention deficit/hyperactivity disorder patients.48 Consistent with the effects of psychostimulants in patients with BPD, psychosti- mulants aggravate, but do not relieve, hyperactivity in GluR6 KO, ERK1 KO and mCLOCK mice. Table 4 Genes and behavioral patterns Genes Mutant stains Open field test Immobility tests Anxiety-like/ risk-taking tests CLOCK mCLOCK Hyperactive Less immobile More risk-taking-like GRIK2 (also known as GluR6) GluR6�/� Hyperactive Less immobile More risk-taking-like MAP3 (also known as ERK1) ERK1�/� Hyperactive Less immobile More risk-taking-like GRIN2D (also known as NMDAR2D or GluRe4) GluRe4�/� Hypoactive Less immobile More risk-taking-like OPRM1 mu-opioid receptor KO No change Less immobile More risk-taking-like OPRD1 Oprd�/� Hyperactive More immobile More anxious-like GABBR1 GABAB(1)�/� Bell-shaped activity display Less immobile More anxious-like GABBR2 GABAB(2)�/� No data Less immobile More anxious-like Abbreviations: GABBR1, g-aminobutyric acid (GABA) B receptor, 1; GABBR2, GABA B receptor, 2; GRIK2, glutamate receptor, ionotropic, kainate 2; GRIN2D glutamate receptor, ionotropic, N-methyl D-aspartate 2D; MAP3, mitogen-activated protein kinase 3; OPRD1, opioid receptor, d-1; OPRM1, opioid receptor, m-1. Translational research in bipolar disorder G Chen et al 889 Molecular Psychiatry In addition, discussion and interpretation of these results have been aided by studies that clarify the ability of mood stabilizers to relieve these symptoms. For instance, in humans, lithium can reverse many of these manic symptoms, but there are no sufficient or convincing clinical data to support the notion that lithium therapy is effective in treating attention deficit/hyperactivity disorder. Furthermore, most be- havioral abnormalities observed in mCLOCK and GluR6 KO mice respond to chronic treatment with lithium.11,46,12 The amphetamine supersensitivity noted in ERK1 KO mice also responded to chronic treatment with either lithium or valproate, as well as acute treatment with olanzapine; in addition, treat- ment with either chronic valproate or acute olanza- pine reduced the baseline activity of ERK1 KO mice. Notably, lithium treatment did not significantly reduce the baseline activity of ERK1 KO mice, suggesting that ablation of ERK1—which is a target of mood stabilizers—compromised the behavioral effects of lithium. These treatment data also support the link between the phenocopies showed by these mice and a manic behavioral syndrome. The beha- vioral effects of mood stabilizers on behavioral disturbances in GSK-3b overexpressing mice have not yet been reported. It is important to note that even if behavioral phenocopies of model animals do not respond to treatment with existing mood stabilizers, the data can still support (though less conclusively) the role of implicated or suspected changes in affective regulation in BPD; this is related to the fact that existing mood stabilizers are not effective for all BPD patients. Indeed, such an outcome may yield clues as to the particular mechanisms of action of these physiological changes. Another related issue is the extent to which hyperactivity showed in the open field test confounds results from tests for other behaviors, including risk- taking behaviors, aggression and escaping activity (in the forced swim or tail suspension tests). It is notable that behavioral clusters or activity patterns assessed through multiple tests seem relatively gene specific (Table 4). For instance, mCLOCK, GluR6 KO and ERK1 KO mice show similar patterns of behaviors— for example, hyperactivity in the open field test, less immobility in the forced swim test and more risk- taking or less anxious-like behaviors in anxiety- related tests. In contrast, deletion of the NMDAR2D (N-methyl-D-aspartate (NMDA) receptor type 2D) in rodents reduced total activity in the open field test, reduced immobility in the forced swim test and increased risk-taking behavior in the elevated plus maze.49 Deletion of d-opioid receptors did not affect open field activity, but did increase activity in the open arms during the elevated plus test and reduced immobility in the forced swim test.50,51 Deletion of d- opioid receptors increased open field activity in the open field test and increased anxious-like beha- viors.51 Deletion of the g-aminobutyric acid (GABA) B(1) and B(2) receptors did not change overall activity in the open field test, but did induce anxiety-like behaviors and reduced immobility.52,53 As Table 4 illustrates, the behavioral patterns showed in the open field, anxiety-related and immobility tests differ between strains of mice harboring different genetic alterations. Therefore, a cluster of behaviors high- lighting a pattern of increased activity in the open field arena, increased activity in the open arms of the elevated plus maze and reduced immobility is likely to be specific for manipulating certain genes or pathways. Bipolar depression Depression affects mood, thinking and physiological and social–psychological functioning. The diagnosis of depression requires one of two core symptoms, that is, either depressed mood or anhedonia. Because mood cannot be directly assessed in rodents, anhe- donia-like behaviors have become critical in evaluat- ing behavioral clusters of cross-species phenocopies of depressive symptoms (see Table 2) to link the behavioral clusters being studied with clinical de- pression. Behavioral clusters without such a link to the core symptoms of depression provide evidence that is much more circumstantial. In humans, anhedonia is loosely defined as an impaired capacity to experience or anticipate plea- sure, and in rodents tests that assess anhedonia-like behaviors traditionally focus on a particular reward- seeking activity assessed in a given context; for example, sweet solution consumption or preference, wheel running, female urine sniffing or lever pressing or wheel running in the intracranial self-stimulation paradigm. Home cage and other activities (for exam- ple, novel object exploration or social interaction) can also provide useful information. Theoretically and empirically speaking, the outcomes of these tests or observations (Table 2) can be confounded by factors that by themselves do not constitute depression, such as locomotive dysfunction, lack of explorative curi- osity or neophobia, specific sensory alterations, low sexual drive and loss of appetite. For all of these, one operational definition of anhedonia-like behaviors in rodents is that experimental animals show concurrent reductions in multiple activities known to be pre- ferred by suitable control animals, and these reduc- tions cannot be explained by any single cause alone. This definition means that anhedonia-like behaviors should be evaluated as a behavioral cluster or pattern assessed through multiple tests or tasks, but not as one type of activity in any given context. Anhedonia and other symptoms of depression can be observed in other neurological and psychiatric disorders, includ- ing schizophrenia, parkinsonism and dementia. Therefore, additional tests should be considered to examine whether genes associated with anhedonia- like behaviors/depression-like alterations are depres- sion specific. Furthermore, the effects on such anhedonia-like behaviors of therapeutic agents used to treat various illnesses are also useful in evaluating this issue. Translational research in bipolar disorder G Chen et al 890 Molecular Psychiatry The diagnostic criteria for a depressive episode are the same for both BPD and major depressive disorder. Therefore, diagnosing bipolar depression requires both the occurrence of a current depressive episode as well as knowledge of an individual’s previous course of illness, for example, whether mania or hypomania had previously occurred. Bipolar depres- sion can be treated with mood stabilizers such as lithium and valproate and, as suggested by a recent independent meta-analysis and meta-regression of individual patient data from five randomized trials, lamotrigine, although the overall effect of lamotrigine is modest.54 Treating bipolar depression with anti- depressants alone can cause dysphoric symptoms, and even induce switch to a manic episode,55 and there is no convincing evidence suggesting that a combination of mood stabilizers and antidepressants is beneficial in treating bipolar depression. Very recent data suggest that the N-methyl-D-aspartate antagonist ketamine, which is added on to mood stabilizers, may rapidly relieve depressive symptoms in patients with bipolar depression.56 Although suitable tests exist for monitoring animal behaviors that cross-species phenocopy behavioral facets of depressive symptoms (Table 2), further refinement and validation of these tests are needed. In addition to the behavioral tests listed in Table 2, physiological and biological methods, such as electroencephalo- gram, can be used to monitor sleep disturbances. It is important to note that some rodent behavioral displays, such as motivational deficits, ‘behavioral despair’, ‘helplessness’, submissiveness and anxiety- like abnormalities, are thought to be linked to clinical alterations frequently observed in depression, despite the fact that not all are diagnostic symptoms. Several animal models target these behaviors and are sensi- tive to chronic or repeated treatment with anti- depressants, including the learned helplessness paradigm, the forced swim and tail suspension tests, the social defeat paradigm and the novelty-induced hypophagia test.13 In addition, some investigators have suggested that the behavioral effects of chronic treatment with antidepressants in the novelty-in- duced hypophagia test reflect the anxiolytic effects of chronic treatment with these agents observed in humans.57 Notably, much of the data used to assess these paradigms were generated from models de- signed to shed light on major depressive disorder or depressive disorders in general rather than bipolar depression in particular. However, conclusive data are lacking regarding whether or not the learned helplessness paradigm, the social defeat paradigm and the novelty-induced hypophagia test are sensitive to mood stabilizers and to lamotrigine administered in clinically relevant regimens. Many candidate genes are being studied in anti- depressant-sensitive behavioral paradigms or animal models. The influence of some genes (such as brain- derived neurotrophic factor and cAMP response element binding (CREB)) on behavior is brain region specific.58,59 For instance, blocking brain-derived neurotrophic factor and CREB in the hippocampus attenuated the effects of antidepressants, whereas in the ventral tegmental area, amygdala or nucleus accumbens, the blockage itself produced antidepres- sant-like effects or potentiated the effects of anti- depressants. It remains unknown whether or not functional changes of these genes cause anhedonia- like behaviors. The model animal approach is also used in transla- tional studies analyzing the relationship between suspected genes and phenocopies of depressive symp- toms. Functional manipulation of p11 was recently found to result in a phenotype conferring anhedonic- like behaviors and behavioral despair. Also known as S100A10, p11 is a member of the S100 protein family that mediates cell surface localization of the 5-HT1B receptor and several ion channels.60 p11 levels were lower in the brain tissues of depressed patients and of helpless animals, and higher in the brain tissues of animals treated with antidepressants or electroconvul- sive therapy. p11 KO mice showed increased anxiety- like measures (thigmotaxis) in the open field test, and immobility in the tail suspension test. The mice also consumed less palatable 2% sucrose solution than their wild-type littermates. Thus, the mice showed a behavioral phenotype of depressive symptoms with two critical features: anhedonia-like behaviors and behavioral despair. It is presently unknown whether these features of p11 KO mice respond to treatment with various mood stabilizers, and it remains to be elucidated whether p11 alterations are involved in bipolar depression or, indeed, whether bipolar and unipolar depressive episodes share a common patho- physiological pathway. Another promising finding was obtained from the study of vesicular monoamine transporter 2 (VMAT2) heterozygous (HET) KO mice. In brief, treatment with reserpine, which is an irreversible inhibitor of VMAT that transports cytoplasmic monoamine into secretory vesicles,61 depletes vesicular monoamine stores and precipitates depression in susceptible individuals. A specific haplotype of VMAT2 (also known as SLC18A2) was significantly associated with depressive symptoms in elderly men.62 VMAT2 HET KO mice were hypoactive in the open field test, showed less preference to sweetened but not quinine solutions and showed increased immobility in the forced swim and tail suspension tests.61 Treatment with antidepressants reduced the immobility of VMAT2 HET KO mice. However, the effects of antidepressants on motor retardation/lack of explorative behavior, anhedonia and increased immobility remain unknown, as do the behavioral effects of mood stabilizers and lamotrigine in VMAT2 HET KO mice. Direct evidence for the involvement of VMAT2 in the pathophysiology of bipolar depression is still lacking. Vulnerability to BPD Patients with BPD remitted from a manic or depres- sive episode are sometimes considered to be in a Translational research in bipolar disorder G Chen et al 891 Molecular Psychiatry euthymic state in which they are vulnerable to relapse2 (Figure 1). Relapses can be triggered by physical stressors (such as lack of sleep or jetlag), social–psychological stressors,2 experimental use of psychostimulants47 and experimentally induced monoamine depletion63 (Figure 1). Interestingly, experimentally induced monoamine depletion causes some mood changes in healthy controls, but a full- blown depression in only individuals with a personal or family history of mood disorders.63 These unique clinical responses can be collectively considered as an affective vulnerability. Several animal paradigms can be used to study the roles of candidate genes in affective vulnerability in animals13–16 (Table 3). These paradigms contain components of behavior induction and outcome observation. It should be noted that these paradigms encompass previously proposed animal models of depression or mania;64 however, their focus under the model animal approach is to analyze the interaction between implicated or suspected biological changes in BPD in tandem with behavioral inducers (such as foot shocks or psychostimulants) on outcome mea- sures. Under such a design, the effects of these factors alone on outcome measures are not the focus of the investigation. Some of the most promising candidates include glucocorticoid receptors (GRs), and B-cell lymphoma 2 (Bcl-2)-associated athanogene (BAG1). Considerable clinical data have shown that gluco- corticoids are one of the few agents that are capable of triggering both depressive and manic episodes in individuals with BPD.2 Furthermore, alternative GR expression variants have been implicated in major depressive disorder by two independent groups of studies.65,66 Upbinding to glucocorticoids, GRs trans- locate from cytoplasm to nuclei and then regulate gene expression.67 This trafficking process is regu- lated by chaperones and co-chaperones including FKBP5 (FK506 binding protein 5, also known as FKBP51).67 Some, but not all, genetic studies show that variants of FKBP5 are associated with increased recurrence of depression,68 response to antidepres- sants,68–70 increased risk for BPD71 and symptoms of posttraumatic stress disorder in adults with a history of childhood abuse.72 These clinical data support the role of GRs in mood regulation. GRov mice, a strain in which overexpression of GRs in neurons was achieved through use of a neuron- specific promoter,73 showed anxious-like behaviors in the elevated plus maze and light–dark box tests, and increased immobility in the forced swim test, suggest- ing that the mice were sensitive to stressful environ- mental conditions. Interestingly, the mice also showed enhanced cocaine-induced behavioral sensi- tization. Taken together, these results suggest that GRov mice seem to show an altered affective-like lability. BAG1 is another co-chaperone involved in GR signaling.74 Through interaction with Hsp70/Hsc70, this protein regulates both glucocorticoid binding and gene transactivation of GRs.74 BAG1 is one of the common brain targets of chronic treatment with lithium and valproate,75 both of which upregulate BAG1 mRNA and protein levels and alter BAG1 function, including GR trafficking to nuclei. Mice with selective brain overexpression of BAG1 show less anxious-like behaviors in the elevated plus maze test, accelerated recovery from amphetamine-induced hyperlocomotion, blunted response to cocaine in the psychostimulant-induced behavioral sensitization paradigm and rapid spontaneous recovery from help- lessness in the learned helplessness paradigm.76 BAG1 HET KO mice showed enhanced response to cocaine in the psychostimulant-induced behavioral sensitization paradigm and increased tendency to become helpless in the learned helplessness para- digm.76 These data suggest that BAG1 regulates affective lability and resilience. Additional considerations Cognitive deficits Clinically, to diagnose BPD, symptoms that can be explained by other medical conditions, such as organic brain diseases, must be excluded.2,77 Mild cognitive impairments in working memory and attention are known to be present in at least some patients with BPD, and these phenomena are often observed in schizophrenia. To address whether gene manipulation affects mood regulation as well as broadly influences multiple domains of brain func- tion, predisposed animals can be examined using batteries of tests to assess general health, motor, sensory, learning and memory functioning and other neurological functions. These have been well- described elsewhere.20 Mood swings Because the process of switching from depression to a state of mania or hypomania is a unique and core feature of BPD, another key issue is that of sponta- neous mood swings. Although often triggered by particular stressors or medications, spontaneous mood relapses can occur in BPD without a clear external triggering event.2 If these mechanisms do exist, they must closely interact with, or even be integrated into, the mechanisms that underlie vulner- ability. To study candidate genes for internal sponta- neous swing mechanisms, long-term home cage activity monitoring of mice predisposed to known BPD risk factors can be helpful. Accumulating data from both human genetic and post-mortem studies imply that mitochondrial dys- function contributes to the etiology of BPD (for a review, see Kato22). Interestingly, a mutation (D181A) of mitochondrial DNA polymerase-g(POLG) does not affect this enzyme’s polymerase activity; instead, it abolishes its 30–50 exonuclease activity and causes mitochondrial DNA deletion.78 Transgenic mice with brain-specific expression of mutant POLG accumulate brain mitochondrial DNA abnormalities similar to those found in the post-mortem brain of individuals Translational research in bipolar disorder G Chen et al 892 Molecular Psychiatry with BPD.78 Although the mutant POLG mice seem normal on a variety of short-term tests, long-term home cage monitoring revealed that these mice have a unique activity pattern. Namely, they show altered activity immediately before and after the dark phase (which is the active phase for rodents) of the circadian cycle.78 This alteration can be further deteriorated by treatment with a tricyclic antidepressant known to induce mood switches in individuals with BPD. Female mutant POLG mice also show robust activity swings concurrent with the estrous cycle that can be stabilized by treatment with lithium. Taken together, the findings support the notion that mitochondrial dysfunction influences diurnal activity rhythms and the magnitude of rhythmic activity. Whether and how mitochondrial dysfunction drives these abnormal mood swings requires further investigation. Closing remarks This is an unprecedented time in BPD research. Owing to significant advances in human genetics, the identity of true susceptibility genes is starting to emerge. The influence of genetic risk variants on gene and gene network function continues to be exten- sively analyzed at the molecular, neural circuitry and behavioral levels. Although all animal approaches have well-known limitations in their ability to mirror human conditions, there is no doubt that animal behavioral research in general—and the model animal approach in particular—will have an irreplaceable role in analyzing the causal relationship between biological abnormalities resulting from genetic BPD risk variants, early-life environmental factors and behavioral manifestations of BPD. At the same time, a significant need exists to form a consensus regard- ing how to phenotype animals in mood disorders research, and how to improve the ability of behavioral research to monitor these alterations and the manner in which they relate to mood disorders. The reverse translation model animal approach outlined in this review can and should be viewed as one of many research avenues in overall BPD research. Future research advances in both biological information and behavioral methodology will be essential for the rapid development of true novel therapies for rapidly relieving mood symptoms, preventing mood relapses and halting the progression of this devastating disorder. Conflict of interest The authors declare no conflict of interest. Acknowledgments We gratefully acknowledge the support of the Intramural Research Program of the National Institute of Mental Health. References 1 Belmaker RH. Bipolar disorder. N Engl J Med 2004; 351: 476–486. 2 Goodwin FK, Jamison KR. Manic-Depressive Illness: Bipolar and Recurrent Unipolar Disorders, 2nd edn. Oxford University Press: New York, 2007. 3 Baum AE, Akula N, Cabanero M, Cardona I, Corona W, Klemens B et al. A genome-wide association study implicates diacylglycerol kinase eta (DGKH) and several other genes in the etiology of bipolar disorder. Mol Psychiatry 2008; 13: 197–207. 4 Kato T. Molecular genetics of bipolar disorder and depression. 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Translational research in bipolar disorder G Chen et al 895 Molecular Psychiatry Translational research in bipolar disorder: emerging insights from genetically based models Introduction The animal model approach The model animal approach Mania Bipolar depression Vulnerability to BPD Additional considerations Cognitive deficits Mood swings Closing remarks Acknowledgements References