Neurobiology of Disease 40 (2010) 66–72

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Neurobiology of Disease

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Review

Use of zebrafish as a model to understand mechanisms of addiction and complexneurobehavioral phenotypes

Priya Mathur, Su Guo ⁎Department of Bioengineering and Therapeutic Sciences, Programs in Human Genetics and Biological Sciences, University of California San Francisco, CA 94143-2811, USA

⁎ Corresponding author. Fax: +1 415 502 8177.E-mail address: su.guo@ucsf.edu (S. Guo).Available online on ScienceDirect (www.scienced

0969-9961/$ – see front matter © 2010 Elsevier Inc. Adoi:10.1016/j.nbd.2010.05.016

a b s t r a c t

a r t i c l e i n f o

Article history:Received 19 April 2010Revised 7 May 2010Accepted 11 May 2010Available online 20 May 2010

Keywords:ZebrafishAddictionAnxietySchizophreniaAutism

Despite massive research efforts the exact pathogenesis and pathophysiology of addiction andneuropsychiatric disorders such as anxiety, schizophrenia and autism remain largely unknown. Animalmodels can serve as tools to understand the etiology and pathogenesis of these disorders. In recent yearsresearchers are turning to zebrafish as it allows easy access to all developmental stages and imaging ofpathological processes as well as automated behavioral quantification coupled with large scale screening andmutagenesis strategies. This review summarizes studies conducted over the last few years whichdemonstrate the relevance of the zebrafish model to human diseases including addiction and neuropsychiatricdisorders.

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Contents

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 66Use of zebrafish to model addiction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 67Use of zebrafish to study autism. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 68Use of zebrafish to study fragile X syndrome . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 69Use of zebrafish to study schizophrenia . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 69Use of zebrafish to study stress, fear, and anxiety. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 70Concluding remarks . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 70Acknowledgment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 70References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 70

Introduction

Neuropsychiatric disorders have a relatively high prevalence inmodern society and can affect individuals at all life stages. They oftenhave severely disabling symptoms and represent a huge burden onindividuals, families and society as a whole (Kessler et al., 2005).Neuropsychiatric disorders are genetically complex and there are novalidated biological markers for distinguishing and characterizing thedifferent disorders (Hyman, 2008). Despite immense research theprecise risk factors and pathogenesis of neuropsychiatric disorders arestill unknown. Almost no new therapeutic mechanisms or new drugtargets have been identified in the last fewdecades (Nestler et al., 2009).

Human experimental neurobiology is mostly limited to non-invasive and indirect methods of investigation. An animal model givesus the opportunity to study how genetic and environmental factorscan lead to the neuropsychiatric disorder by allowing us tomanipulate molecules and confirm their role in the disease process.

The most commonly used animal models are small mammals(rats and mice). Although they have several advantages, thesemodels are expensive to maintain and use in large-scale genetic orchemical screens and they are not readily accessible for studies at theembryonic stages. The small body size, large brood size, externaldevelopment, and optical transparency of zebrafish allow us toovercome these limitations of mammalian models and provide apowerful tool kit, which includes large-scale genome mutagenesisand gene mapping, transgenesis, protein overexpression or knock-down, cell transplantation and chimeric embryo analysis, andchemical screens (Veldman and Lin, 2008), making this organism

67P. Mathur, S. Guo / Neurobiology of Disease 40 (2010) 66–72

particularly well-suited to the molecular genetic analysis ofvertebrate neurodevelopmental mechanisms relevant to neuropsy-chiatric disorders. It is possible to visualize complete neurotrans-mitter systems in the whole zebrafish brain at 5 days postfertilization when the fish already displays a sophisticated repertoireof behaviors (Panula et al., 2006). Magnetic Resonance Imaging(MRI) on living zebrafish embryos for molecular screening ormolecular tracking over time (Canaple et al., 2008) and MagneticResonance Histology (MRH) of the zebrafish brain have also beencarried out (Ullmann et al., 2009). Therefore, the relative simplicityof the nervous system in fish larvae facilitate circuit analysis (Burgessand Granato, 2007).

An additional round of genome duplication occurred early in theevolution of teleosts but not all duplicated genes were retained byzebrafish. Current estimates for zebrafish in Zv8 zebrafish genomedatabase is 24,000 protein coding genes, only about 20% more thanhuman. While this can lead to uncovering redundant, species-specificinformation, duplicate genes can also be advantageous in studies ofsub-functionalisation, since zebrafish co-orthologs represent selectedexpression patterns and developmental functions of mouse orthologs(Bergner et al., 2009; Key and Devine, 2003).

Zebrafish show similarity to humans in terms of the structure ofthe nervous system, which includes a fore-, mid- and hind-brain,including diencephalon, telencephalon (Mueller and Wullimann,2009) and cerebellum and a peripheral nervous system with motorand sensory components, enteric and autonomic nervous systems.

Table 1ZF disease models.

Neuropsychiatric disorder Example of ZF model/assay Pheno

AddictionAlcohol dependence Adult ZF brain neurotransmitter levels Increa

Microarray analysis of brain samples ChangAdult ZF brain AchE IncreaVideo recording of locomotor activity in adult ZF Increa

of novfan mutant ReducVideo recording of locomotor activity in larval ZF RetardVideo recording of locomotor activity in larval ZF Increa

disperAlcohol withdrawal Novel tank diving test FreezinCocaine dependence dum, gts, jpy mutants ReducCocaine withdrawal Video recording of locomotor activity in adult ZF HyperMorphine dependence Nociceptin receptor expression in embryos Decrea

tof mutant Reduc

Morphine withdrawal Novel tank diving test FreezinAmphetaminedependence

nad mutant Reducache/+ mutant Reduc

Nicotine dependence bdav/cct8 and hbog/gabbr1.2 AltereMicroarray analysis of brain samples Chang

Diazepam withdrawal Novel tank diving test FreezinSalvinorin A dependence Locomotor activity and CPP Prefere

Autism MO knockdown of SUSD4 DeveloMO knockdown of Met/HGF Altere

Fragile X syndrome fmr1 knockout using TILLING No obvMO knockdown of FMR-1 AbnormiRNA transgenes against fmr1 gene Abnor

Schizophrenia Knockdown of disc-1 AltereKnockdown of nrg-1 Loss oophelia mutant Reduc

Fear/anxiety Light/dark preference task AvoidExpose adult ZF to predators or images of predators ErraticExpose adult ZF to alarm substance JumpinExpose adult ZF to hypoxanthine 3-N oxide IncreaDirect and indirect contact with predators IncreaExpose adult ZF to novel tank Increa

Aggression Pair-house male ZF after separation Chasessubord

ZF = zebrafish, AchE = acetylcholinesterase, MO = morpholino, CPP = conditioned place

However, the telencephalon has only a rudimentary cortex. Althoughsimplified compared to human behaviors, zebrafish do exhibit ‘higher’behaviors and integrated neural functions including memory, condi-tioned responses and social behaviors (for example, schooling)(Lieschke and Currie, 2007).

In the following sections we highlight various neuropsychiatricdisorders including addiction, anxiety and depression, autism andschizophrenia, and discuss current efforts toward creating zebrafishmodels for these disorders (Tables 1 and 2). These disorders arecovered in this review because of our interests as well as the presenceof available data in zebrafish. As always, it is worth noting that not asingle model is able to recapitulate all aspects of the complex humanconditions.

Use of zebrafish to model addiction

The zebrafish model has been widely used to better understandthe complex processes involved in the development of addiction. We(Guo, 2009) and others have previously reviewed studies of thepsycho-stimulatory (Bretaud et al., 2007; Darland and Dowling, 2001;Gerlai et al., 2000; Lau et al., 2006; Lockwood et al., 2004; Ninkovic etal., 2006) and reinforcing (Bretaud et al., 2007; Darland and Dowling,2001; Lau et al., 2006; Ninkovic et al., 2006) effects of alcohol anddrugs of abuse in zebrafish.

Numerous studies in recent years have focused upon modelingalcohol addiction in zebrafish and the findings have striking parallels

type and/or studies of disease pathogenesis References

sed dopamine and serotonin levels Gerlai et al. (2009)es in gene expression following conditioning Kily et al. (2008)sed AchE levels Rico et al. (2007)sed swim speed, increased time in upper zoneel tank

Gerlai et al. (2000)

ed melanocyte response to ethanol Peng et al. (2009)ed transition in activity from dark to light MacPhail et al. (2009)sed swim speed, thigmotaxis, melanocytesion

Lockwood et al. (2004)

g bouts and erratic movements Cachat et al. (2009)ed cocaine-induced CPP Darland and Dowling (2001)activity Lopez Patino et al. (2008)sed nociceptin receptor expression Macho Sanchez-Simon and

Rodriguez (2009)ed morphine-induced CPP Lau et al., 2006; Bretaud et al.,

2007g bouts and erratic movements Cachat et al. (2009)

ed amphetamine-induced CPP Webb et al. (2009)ed amphetamine-induced CPP Ninkovic et al. (2006)d locomotor sensitization to nicotine Petzold et al. (2009)es in gene expression following conditioning Kily et al. (2008)g bouts and erratic movements Cachat et al. (2009)nce for Salvinorin compartment Braida et al. (2007)pmental abnormalities Tu et al. (2010)d cerebeller growth and cell type specification Elsen et al. (2009)ious phenotype den Broeder et al. (2009)mal axonal branching and craniofacial abnormalities Tucker et al. (2006)mal neuronal morphology and connectivity Lin et al. (2006)d cranial neural crest migration and differentiation Drerup et al. (2009)f olig2-positive cerebellar neurones Wood et al. (2009)ed prepulse inhibition Burgess and Granato (2007)light compartment Maximino et al. (2010a,b)movements and freezing Gerlai (2010)g, erratic movements, increased shoal cohesion Speedie and Gerlai (2008)sed jumping and erratic movements Parra et al. (2009)sed whole body cortisol Barcellos et al. (2007)sed bottom-dwelling Levin et al. (2007)and bites by dominant male and retreats byinate male

Larson et al. (2006)

preference, TILLING = targeted induced local lesions in genomes.

Table 2Examples of neuropsychiatric disease-related genes studied in ZF.

Disease Gene Technique used References

Drug dependency(ethanol)

fan ENU mutagenesis,cloning

Peng et al. (2009)

Drug dependency(amphetamine)

calcineurin B Microarray analysis Kily et al. (2008)

Autism SUSD4 MO knockdown Tu et al., 2010neuroligin Studied expression

patternsRissone et al. (2010)

neurexin Studied ZF homologs Rissone et al. (2007)Met/HGF MO knockdown Elsen et al. (2009)

Fragile X syndrome FMR-1 TILLING den Broeder et al.(2009)

MO knockdown Tucker et al. (2006)miRNA transgenes Lin et al. (2006)

Schizophrenia disc-1 MO knockdown Drerup et al. (2009)nrg1 MO knockdown Wood et al. (2009)ndel1a, ndel1b Studied expression

patternsDrerup et al. (2007)

ophelia Mutagenesis Burgess and Granato(2007)

ZF = zebrafish, MO = morpholino, TILLING = targeted induced local lesions ingenomes.

68 P. Mathur, S. Guo / Neurobiology of Disease 40 (2010) 66–72

with the effect of ethanol in mammals. Acute alcohol exposure hasbeen shown to result in significant changes in dopamine, serotoninand related metabolites in adult zebrafish brain (Chatterjee andGerlai, 2009). Different strains of zebrafish have been found to showdifferences in response to a zebrafish shoal and predator whileundergoing alcohol withdrawal and this is correlated with differencesin neurochemical (including dopamine) responses (Gerlai et al.,2009). Withdrawal after chronic exposure to ethanol in adultzebrafish has been profiled in terms of freezing bouts and erraticmovements in the novel tank diving test and correlated with changesin whole body cortisol (Cachat et al., 2009).

In addition to alcohol, the effects of drugs of abuse includingcocaine, amphetamine, and morphine have also been studied inzebrafish. Withdrawal symptoms following euphoria are part of thecycle of drug abuse for drugs like cocaine. In zebrafish, cocainewithdrawal produces an anxiety-like state which develops earlier inthe female but is more robust and persistent in males and isaccompanied by a decrease in dopamine transporter and an increasein dopamine levels (Lopez Patino et al., 2008b). Thus the zebrafishprovides new opportunities to study genetic and endocrine gender-related factors involved in cocaine withdrawal symptoms. Studies inzebrafish have also shown that exposure to drugs such as morphineduring development changes the expression level and localization ofnociceptin receptors indicating that nociceptin (NOP) plays a role indevelopment of dependence/addiction to drugs (Macho Sanchez-Simon and Rodriguez, 2009). Recently the mu-opioid receptor fromzebrafish was shown to have a pharmacological profile similar to thatof mammalian mu-opioid receptor (de Velasco et al., 2009).Additionally, the effects of a potent psychoactive drug Salvinorin-Ahave been documented in zebrafish. Salvinorin-A produces rewardingeffects independently of its effects on zebrafish locomotor activity andthese effects aremediated by kappa-opioid and cannabinoid receptors(Braida et al., 2007).

The above-mentioned studies have established a similarity betweenzebrafishandmammals in termsof their behavioral responses toalcoholand drugs of abuse and possibly also the underlying neural substrates.However, in order to exploit the strength of zebrafish to understand theunderlyingmolecular and cellularmechanisms, assays amenable tohighthroughput screening need to be developed to reveal novel molecularand cellular insights.

Recent studies (Petzold et al., 2009) using larval zebrafish addressthe question of how genetic variation accounts for differentialpredisposition to nicotine dependence. Four-day old larval zebrafish

show increased locomotor activity in response to a water stimulusand pretreatment with nicotine (2.5–50 μM) results in significantlygreater locomotor activation. Zebrafish display sensitization due toprior nicotine exposure and the nicotine response is blocked bynicotinic receptor antagonists. This nicotine behavioral assay has beencoupled with zebrafish forward genetic screens using gene-breakingtransposon mutagenesis to identify mutants with defects in Chaper-onin Containing Protein 8 (CCT8) and GABA-B receptor Subunit 1.These genes provide candidates for human association studies ofpredisposition to nicotine addiction.

The ethanol-modulated camouflage response of zebrafish, which issubjected to regulation by a variety of neurotransmitters andneuropeptides, has been used as a screening assay for understandingthe biological effect of ethanol. A mutant which displays reducedcamouflage response as well as behavioral sensitivity to ethanol hasbeen identified to disrupt the evolutionarily conserved adenylylcyclase 5 (ac5) gene (Peng et al., 2009). The reduced sensitivity ofthe mutant to the stimulatory effects of ethanol can be mimicked inwildtype zebrafish by partial inhibition of phosphorylation ofExtracellular Regulated Kinase (ERK). These studies demonstratecritical roles of AC5 and ERK signaling in behavioral sensitivity toethanol. It will be of importance to determine whether polymorph-isms in the human counterparts of these genes may predisposeindividuals to alcoholism.

Combining a zebrafish forward genetic screenwith CPP andmicro-array analysis leads to the discovery of the “no-addiction” or nadmutant which does not exhibit preference for the compartmentpaired with amphetamine (Cadet, 2009; Webb et al., 2009). Many ofthe genes involved in reward pathways and brain development andassociated with neurogenic zones of the adult brain respondedinappropriately to amphetamine in the nad mutant, indicating thatdrugs of abuse can hijack the brain's natural reward system. Inaddition, the transcription factors identified in this study can be usedto study the link between neurogenesis and addiction (Cadet, 2009;Webb et al., 2009).

Microarray analysis has been coupled with the Conditioned PlacePreference (CPP) assay in adult zebrafish to study whether prolongednicotine/ethanol exposure in zebrafish results in changes in behavioras well as changes in gene expression at the level of the brain (Kilyet al., 2008). Using zebrafish as a model system, candidate moleculesand pathways that underlie neuro-adaptation to both ethanol andnicotine were identified and these include glutamate receptors,benzodiazepine receptors and molecules associated with synapticplasticity (Kily et al., 2008). It will be of importance to determine,using high throughput zebrafish technologies, if any genes andpathways discovered in the profiling study are causally related tothe change of behavior.

Taken together, zebrafish show great promise as a model forunderstanding the molecular and cellular mechanisms underlyingbehavioral responses to addictive substances. Continued exploitationof this model organism shall provide valuable insights into addiction.

Use of zebrafish to study autism

Just like with any animal model for autism spectrum disorder(ASD), the repertoire of zebrafish behaviors is limited and cannotrecapitulate all aspects of human behaviors that are impaired inautism. However, zebrafish are a useful tool to study the in-vivofunction of genes implicated in autism, since the larvae developexternally and rapidly, large numbers of larvae can be generated andused for molecular and genetic screens and the larvae are transparentenabling observations of brain development in living embryos atsingle cell resolution. Indeed, reviews discussing the potential of thezebrafish system for modeling autism (Tropepe and Sive, 2003), thezebrafish homologs of genes implicated in autism and assaysdeveloped to measure social interaction in zebrafish (Guo, 2009)

69P. Mathur, S. Guo / Neurobiology of Disease 40 (2010) 66–72

have been published previously. Zebrafish behavioral assays thatcould be used to study social behaviors affected in autism include so-called “measures of personality” including social distance, activity inopen field under social isolation and distance from predator (Daddaet al., 2010); novelty induced responses (Blaser and Gerlai, 2006);inhibitory avoidance (Blank et al., 2009); social interaction (Delaneyet al., 2002); Courtship behavior (Colman et al., 2009) and dominant–subordinate relationships (Larson et al., 2006). Unvarying, repetitivebehavioral patterns with no obvious function (stereotypy) (Lopez-Patino et al., 2008) and comorbid conditions in autism includingaggression (Colman et al., 2009) and seizure disorders (Hortopanet al., 2010) have been modeled in zebrafish.

Roughly one percent of cases of autism are associated withdeletions within a single region of chromosome 16, which containsnearly 30 genes (Eichler and Zimmerman, 2008). Of these 30 genes, atleast 25 have clear homologs in zebrafish (sfari.org). Using morpho-lino antisense oligonucleotides (Nasevicius and Ekker, 2000), whichinhibit maturation of the mRNA corresponding to each gene, it may bepossible to identify genes required for the formation of normal brainstructure or neurons, and assess interactions between genes. Recently,zebrafish morpholino knockdown experiments provided the firstinsight into the important physiological roles of Sushi domain-containing protein 4 SUSD4, which is deleted in a subset of patientswith autism. SUSD4 is highly expressed in the central nervous system(CNS) in different species including human, mouse and zebrafish, andplays an essential role in zebrafish development (Tu et al., 2010).However, high-resolution analyses of the neuronal phenotypes werenot carried out in the study.

Mutations in Neuroligin 3 and Neuroligin 4 genes in humans havebeen linked to mental retardation and autism (Talebizadeh et al.,2006) and neuroligins along with neurexins are involved in synapticfunction and maturation (Boucard et al., 2005). Studies in zebrafish(Rissone et al., 2007; Rissone et al., 2010) have revealed that sevengenes in the zebrafish neuroligin family are very similar to theirhuman homologs suggesting that these genes are subjected to a verystrong evolutionary pressure to preserve their function. Neuroliginsare expressed throughout the nervous system of the zebrafish and thismodel system provides an excellent opportunity to further explorethe role of neurexins and neuroligins in the development of ASD.

Changes in cerebellar structure, and disrupted cerebellar geneexpression have also been associated with ASD (Fatemi et al., 2008).The role of the Met (proto-oncogene associated with cellularmetastatic cancer)/HGF (hepatocyte growth factor) signaling path-way in cerebellar development has been studied in zebrafish tounderstand the developmental basis of autism (Elsen et al., 2009).These studies in zebrafish revealed that Met signaling is critical forcerebellar morphogenesis, including normal growth and cell typespecification, and plays an important role in hindbrain cell migration.These recent studies demonstrate that zebrafish is a viable modelsystem for future exploration of the underlying molecular and cellularmechanisms of autism.

Use of zebrafish to study fragile X syndrome

Fragile X syndrome (FraX) is a leading heritable cause of mentalretardation that results from the loss of FMR1 (fragile X mentalretardation) gene function. The zebrafish embryo has been estab-lished as a model for fmr1 loss-of-function analysis using amorpholino antisense oligonucleotide approach (Tucker et al.,2006). The zebrafish has genes orthologous to the human FMR1gene family as well as fmr1 interacting proteins and can be used toexamine phenotypes that are difficult or inaccessible to observationin other model organisms. Treatment with mGluR antagonist MPEP[2-methyl-6-(phenylethynyl)-pyridine] rescues the neurophysiolog-ical abnormalities and alleviates craniofacial defects associated withthe loss of zebrafish Fmr1 and a model has been proposed for the

interaction of Fmr1 and mGluR in control of axonal branching,guidance and fasciculation that has implications for the synapticmorphology component of fragile X syndrome (Tucker et al., 2006).

In support of this model loss-of-function transgenic zebrafishexhibiting abnormal neuron morphology and connectivity similar tothose in human FraX have been generated using manmade miRNAtransgenes directed against the fish fmr1 gene (Lin et al., 2006).

With the advance of such amiRNA-mediated loss of-fmr1-functionzebrafish model, it will be possible to investigate the molecular,pathological and neurobehavioral changes that are common in humanpatients. Recently this approach has been extended with thegeneration of two independent fmr1 knockout alleles in the zebrafishusing TILLING (targeted induced local lesions in genomes) (denBroeder et al., 2009). Surprisingly, these knockout zebrafish (with nodetectable FMR1 protein) did not display any phenotype, which wasin contrast to that of the morphant reported in the previous study. Itremains to be resolved whether the difference is due to themorpholino's off-target effect (making the morphant phenotype apotential artifact), the morpholino's ability to target multiple fmr1-like genes (while the knockout only targeted a single gene), oralterations in the genetic background used in each study.

Use of zebrafish to study schizophrenia

Schizophrenia is a devastating disorder caused by both genetic andenvironmental factors that disrupt brain development and function. Itis distinguished as a neurodevelopmental disorder in part due to earlycognitive impairments, behavioral dysfunction in childhood andadolescence, and abnormalities in central nervous system develop-ment with no neurodegenerative component (Lewis and Levitt,2002). The zebrafish model system provides an opportunity tostudy both the genetic and developmental basis of schizophrenia aswell as various pathological processes affecting neurogenesis, cell-fatedetermination and neuronal migration (Morris, 2009).

Neuropsychiatric disorders including schizophrenia are associatedwith disturbances in pre-pulse inhibition (PPI), which affects theindividual's ability to filter out extraneous information from theenvironment (Braff et al., 2001). Larval zebrafish have been found toexhibit PPI which modulate the acoustic startle response in a mannersimilar to mammalian PPI. Larval PPI is disrupted by dopamineagonists and this disruption can be corrected by antipsychotic drugssimilar to the mammalian situation. A zebrafish screen has isolatedthe Ophelia mutant, which has reduced PPI (Burgess and Granato,2007) and further high-throughput zebrafish screens could helpunderstand the genetic basis for defects in PPI observed inschizophrenia.

A study of the in-vitro effects of anti-psychotic drugs on zebrafishbrainmembranes has revealed that these drugs alter ectonucleotidaseactivity which can in turn modulate adenosine levels and affect thepathophysiology of schizophrenia (Seibt et al., 2009). Another studyemploying both in-vitro and in-vivo studies in zebrafish found thatantipsychotic drugs produce alterations in acetylcholinesteraseactivity and this could reveal molecular mechanisms related tocholinergic signaling in schizophrenia (Seibt et al., 2009).

Disrupted in schizophrenia 1 (disc1), a well-documented schizo-phrenia-susceptibility gene has recently been characterized inzebrafish cranial neural crest (CNC). Disc1 is a potent regulator ofsox10 which is an oligodendrocyte related schizophrenia susceptibil-ity gene. Understanding the basic functions of Disc1 in transcriptionalregulation, cell migration and cell differentiation in the zebrafishneural crest might give insight into the cellular processes that, whendisrupted, predispose individuals to mental illness (Drerup et al.,2009).

Other recent findings in the zebrafish embryo reveal neurode-velopmental connections that may exist between key candidateschizophrenia susceptibility genes like DISC1 (Drerup et al., 2009),

70 P. Mathur, S. Guo / Neurobiology of Disease 40 (2010) 66–72

NudE-Like (NDEL1/NUDEL) (Drerup et al., 2007), NRG1 (Wood etal., 2009), OLIG2 and ERBB4 and suggest that disc1 and nrg1function in common or related pathways controlling developmentof oligodendrocytes and neurons from olig2-expressing precursorcells (Wood et al., 2009). Future studies will continue to exploit thepotential of zebrafish to elucidate further the roles of Disc1 andNeuregulin-ErbB signaling pathways in early brain development andto generate new experimental systems in which neurodevelop-mental mechanisms and genetic pathways of relevance to schizo-phrenia can be dissected.

R1117X, a mutation in the gene encoding the synaptic proteinSHANK3 identified in families of schizophrenia patients has beenvalidated in zebrafish. Knockdown of zSHANK3 orthologous genes inzebrafish produced behavioral and differentiation defects (Gauthieret al., 2010).

Use of zebrafish to study stress, fear, and anxiety

Anxiety disorders and phobias represent a large unmet medicalneed in the human population (Garner and Baldwin, 2008). Zebrafishhave a single glucocorticoid and mineralocorticoid receptor whichhave been cloned and sequenced and the zebrafish corticoid signalingpathway which is involved in stress is similar to that of mammals(Alsop and Vijayan, 2008; Denver, 2009).

At the behavioral level, fear reactions such as jumping, erraticmovements and increased shoal cohesion can be elicited in adultzebrafish by exposing them to alarm substance extracted from theskin of zebrafish (Waldman, 1982; Speedie and Gerlai, 2008) and alsoby synthetic substances such as hypoxanthine 3-N oxide (Parra et al.,2009). This zebrafish alarm response provides the opportunity toanalyze fear at multiple levels from genes to circuits (Jesuthasan andMathuru, 2008). Combining this assay with a genetic screen couldallow the discovery of mutations leading to enhanced or reduced fearand could also be used to screen chemicals which alter the fearresponse.

Zebrafish have been observed to exhibit anti-predatoryresponses including erratic movements and freezing when exposedto animated images or live Indian leaf fish on the basis of visualcues alone. These responses can be quantified by automatedmethods (Gerlai, 2010). Both direct and visual contacts withpredators have been shown to increase whole body cortisol inzebrafish (Barcellos et al., 2007).

When exposed to novelty (new tank) zebrafish dive to the bottomand do not initially explore the environment. In the wild this responsecould help zebrafish escape from predators. The Novel tank divingresponse (Bencan and Levin, 2008) has been developed in recentyears to characterize the fear/anxiety response in zebrafish (Bencanet al., 2009; Bergner et al., 2009). Fish spending more time at thebottom of the tank and freezing or swimming slowly or exhibitingincreased erratic movements are considered to be fearful or anxious.Pre-treatment with anxiolytic substances like nicotine decreasesbottom-dwelling (Levin et al., 2007). Acute exposure of adultzebrafish to substances like ethanol, nicotine, fluoxetine or diazepamresults in anxiolytic effects in this assay (Bencan and Levin, 2008;Bencan et al., 2009; Egan et al., 2009), while exposure to alarmsubstance or caffeine had anxiogenic effects. Chronic ethanol andfluoxetine treatment improve habituation to novelty in zebrafishwhile anxiogenic agents like pentylenetetrazole and alarm phero-mone attenuate habituation (Wong et al., 2010). Thus, zebrafish noveltank diving response can serve as an inexpensive, high-throughputmodel for screening anxiolytic drugs.

When given a choice between light and dark compartmentsadult zebrafish show a significant preference for the dark andavoidance of the light compartment. (Maximino et al., 2010b; Serraet al., 1999). This response is only seen if the tanks are open at thetop and only the walls and floor are light/dark. Those zebrafish

which show higher avoidance of the light compartment display acharacteristic freezing response if confined to the light side and thisresponse may be a reliable behavioral measure of anxiety (Blaser etal., 2010). Avoidance of the light compartment did not show intra-or intersession habituation (Maximino et al., 2010a). The light/darkpreference task in zebrafish can be used to investigate anxiety-related behavior in a simple, high throughput manner (Maximino etal., 2010b).

Concluding remarks

Similar to humans, zebrafish (both larvae and adult) gatherinformation about the environment by means of specialized sensoryorgans including eye, olfactory system and ear (zebrafish possess anadditional sensory organ the lateral line). This information isprocessed by the nervous system and can result in a large repertoireof behaviors. To date zebrafish, in particular larval zebrafish that areespecially amenable for screening and cellular level analysis, havebeen successfully used as a model system to elucidate genetic(Table 2), molecular and behavioral (Table 1) mechanisms underlyingseveral neuropsychiatric disorders including addiction (Cachat et al.,2009; Cadet, 2009), aggression (Larson et al., 2006), depression(Roger et al., 2010), anxiety (Bencan et al., 2009; Blaser et al., 2010),schizophrenia (Wood et al., 2009) and autism (Elsen et al., 2009).Zebrafish possess homologs of several genes implicated in thesedisorders (Renier et al., 2007). Zebrafish are ideal organisms fortranslational research as they can be used to study mechanisms at thegenetic, cellular and developmental level (Bergner et al., 2009).Improved behavioral phenotyping and development of mutant andtransgenic lines of zebrafish will allow researchers to model theseneuropsychiatric disorders with greater accuracy (Bergner et al.,2009). A high-throughput, behavior-based approach to neuroactivesmall molecule discovery is uniquely possible in zebrafish (Kokelet al., 2010) and behavioral profiling using zebrafish has revealedconserved functions of psychotropic molecules and predicted themechanisms of action of poorly characterized compounds (Rihel et al.,2010).

Human neuropsychiatric disorders can involve several genes andsymptom clusters and a particular mutant zebrafish strain mayrepresent only a sub-population of individuals diagnosed with thedisorder. Any potential treatment may need to be tested on severalstrains of zebrafish using several different behavioral assays.

All these findings suggest that our understanding of the genetic,cellular and developmental mechanisms underlying addiction andneuropsychiatric disorders can be vastly improved by using zebrafishas a genetically tractable model system.

Acknowledgment

The research in our laboratory is supported by grants from theNational Institutes of Health.

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