Developing zebrafish models of autism sp

Review article

Developing zebra

fi

sh models of autism spectrum disorder (ASD)

Adam Michael Stewart

a,b

, Michael Nguyen

c

, Keith Wong

d

, Manoj K. Poudel

a

, Allan V. Kalueff

a,

⁎

a_{ZENEREI Institute and Zebrafish Neuroscience Research Consortium (ZNRC), 309 Palmer Court, Slidell, LA 70458, USA}

b_{Department of Neuroscience, University of Pittsburgh, A210 Langley Hall, Pittsburgh, PA 15260, USA} c_{Department of Biomedical Engineering, University of Virginia, 415 Lane Road, Charlottesville, VA 22908, USA} d_{University of California San Diego (UCSD) School of Medicine, 9500 Gilman Dr, La Jolla, CA 92093, USA}

a b s t r a c t

a r t i c l e

i n f o

Article history:

Received 30 September 2013

Received in revised form 22 November 2013 Accepted 28 November 2013

Available online 6 December 2013

Keywords:

Autism spectrum disorder Behavioral tests Social deficits Translational research Zebrafish

Autism spectrum disorder (ASD) is a serious neurodevelopmental disorder with complex symptoms and unclear, multi-factorial pathogenesis. Animal (rodent) models of ASD-like behavior are extensively used to study genet-ics, circuitry and molecular mechanisms of ASD. The evolutionarily conserved nature of social behavior and its molecular pathways suggests that alternative experimental models can be developed to complement and enhance the existing rodent ASD paradigms. The zebrafish (Danio rerio) is rapidly becoming a popular model organism in neuroscience and biological psychiatry to study brain function, model human brain disorders and explore their genetic or pharmacological modulation. Representing highly social animals, zebrafish emerge as a strong potential model organism to study normal and pathological social phenotypes, as well as several other ASD-like symptoms. Here, we discuss the developing utility of zebrafish in modeling ASD as a new emerg-ingfield in translational neuroscience and drug discovery.

© 2013 Elsevier Inc. All rights reserved.

Contents

1. Introduction . . . 27

2. Traditional experimental models relevant to ASD . . . 28

2.1. Genetic rodent models relevant to ASD . . . 28

2.2. Pharmacological rodent models relevant to ASD . . . 29

3. Zebrafish models relevant to ASD . . . 30

3.1. Behavioral and pharmacological models . . . 30

3.2. Physiological correlates . . . 31

3.3. Genetic models relevant to ASD . . . 33

3.4. Environmental models potentially relevant to ASD . . . 33

4. Conclusion . . . 33

Acknowledgements . . . 34

References . . . 34

1. Introduction

Autism spectrum disorder (ASD) is a serious debilitating mental ill-ness affecting approximately 1–2% of the general population (Evans, 2013; Mayes et al., 2011; Wing et al., 1967). Recently revisited by the American Psychiatric Association (2013), ASD represents a neurode-velopmental disorder characterized by impaired social communication, repetitive behavior and cognitive de_ficits (seeTable 1andFig. 1for details of clinical phenotypes associated with ASD). In addition to these core symptoms, ASD shows high (~90%) heritability, representing one of the most heritable brain disorders (Crawley, 2012; Edvardson et al., 2013). Notably, ASD is a polygenic disorder with multiple genetic determinants

–

Abbreviations:ADHD, attention deficit hyperactivity disorder; ASD, autism spectrum disorder; AVP, arginine vasopressin; DSM-5, Dianostic and Statistical Manual of mental disorders, 5th edition; FXS, fragile X syndrome; GABA, gamma-aminobutyric acid; MDMA, 3,4-methylenedioxy-N-methylamphetamine; NMDA, N-methyl-D-aspartate; PCP, phencyclidine; SERT, serotonin transporter; SFARI, Simons Foundation Autism Research Initiative; SSRIs, selective serotonin re-uptake inhibitors; V1aR, V1a receptor.

⁎Corresponding author at: ZENEREI Institute, 309 Palmer Court, Slidell, LA 70458, USA. Tel./fax: +1 240 328 2275.

E-mail address:avkalueff@gmail.com(A.V. Kalueff).

0278-5846/$–see front matter © 2013 Elsevier Inc. All rights reserved.

http://dx.doi.org/10.1016/j.pnpbp.2013.11.014

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Progress in Neuro-Psychopharmacology & Biological

Psychiatry

and candidate genes. For example, the SFARI Gene database (https:// gene.sfari.org) currently lists 546 genes associated with ASD. Thus, ASD has a complex, poorly understood pathogenesis and associated genetic/ environmental risk factors, aberrant brain circuits and disordered molec-ular pathways (Edvardson et al., 2013; Geschwind, 2008; Kesli et al., 2014; Matson et al., 2012).

Despite recent progress in dissecting the neural underpinnings of ASD (Kéïta et al., 2011; Kujala et al., 2013), its pharmacological therapy is complicated by the lack of efficient, disorder-specific and safe medica-tion (Benvenuto et al., 2013; Farmer et al., 2013). For example, the Food and Drug Administration approves two drugs (atypical neuroleptics ris-peridone and aripiprazole) for treating ASD-associated irritability (Crawley, 2012). Other agents, such as methylphenidate, selective serotonin re-uptake inhibitors (SSRIs), valproate, atomoxetine,α2 ad-renergic agonists and olanzapine, can also treat some ASD symptoms, but are not effective in alleviating others (Benvenuto et al., 2013; Myers, 2007).

Animal (experimental) models of brain disorders are an indispens-able tool for drug discovery and dissecting the pathogenic mechanisms of brain disorders (Kalueff et al., 2007; Kas et al., 2013; Silverman et al., 2012, 2013). Complementing traditional rodent models of brain disor-ders, new experimental approaches using zebrafish (Danio rerio) are rap-idly gaining popularity in neuroscience research (Gerlai, 2010a, 2011, Kalueff et al., in press). Can zebrafish be used to model complex brain dis-orders? For decades, zebrafish have been viewed as‘simple’organisms with relatively primitive, instinctively-driven behaviors suitable mainly for screening drugs, genetic mutations or developmental defects (see

Gaikwad et al., 2011; Kalueff et al., 2013;Stewart and Kalueff, 2012for discussion). However, mounting recent experimental evidence shows that zebra_fish possess high genetic and physiological homology to mam-mals and display complex affective, social and cognitive responses which are similar to those observed in rodents and humans (Gerlai, 2010b, 2011; Kalueff et al., 2013 Pather and Gerlai, 2009;Stewart et al., 2013). Taken together, this suggests that zebrafish models can be used exten-sively in translational neuroscience research (Gerlai, 2010a; Kalueff et al., 2013, in press).

While various experimental (e.g., genetic or pharmacological) ma-nipulations model certain symptoms and/or disordered pathways of ASD, they do not reflect the entire disease state. However, several social, motor and cognitive phenotypes (Table 2) are commonly assessed in rodent models of ASD, providing important mechanistic insights into its neurobiology (Crawley, 2012; Kas et al., 2013); also see discussion of this further in the text. The evolutionarily conserved nature of social behavior and its molecular pathways suggests that novel experimental models can be developed to complement and enhance the existing rodent ASD paradigms. Can we use zebrafish to study ASD-related path-ogenesis? Addressing this important question, here we discuss recent advances and outline future promising directions of research in the

field of novel zebrafish models of ASD-like states.

2. Traditional experimental models relevant to ASD

2.1. Genetic rodent models relevant to ASD

Laboratory rodents are highly sociable animals, and therefore are useful to study normal and pathological social behaviors (Brodkin, 2007; Crawley, 2012; Fairless et al., 2013; Kas et al., 2013; McFarlane et al., 2008; Moy et al., 2008; Ryan et al., 2010; Silverman et al., 2012). With mouse and rat genomes now being fully characterized, various ge-netic models of ASD and related disorders have been developed. For ex-ample, the Fragile X Syndrome (FXS) is an inherited mental retardation disorder caused by a single mutation in the FMR1 gene in the X chromosome. FVB and C57BL/6 mice withFMR1genetic knockout dis-play some FXS social and behavioral symptoms (Bernardet and Crusio, 2006; Pietropaolo et al., 2011).

Loss-of-function mutations in theNlgn4 gene in mice encoding for Neuroligin-4 impair social behavior and vocal communication, establishingNlgn4mice as a genetic model of ASD-related pathogenesis (El-Kordi et al., 2012; Jamain et al., 2008) relevant to human genetic data linking this gene to ASD (Pampanos et al., 2009). Shank proteins are also involved in modulation of synaptic communication and

Table 1

Diagnostic criteria for autism spectrum disorder (ASD), according to the Diagnostic and Statistical Manual of Mental Disorders (DSM-5) of the American Psychiatric Association (2013).

A. Social deficits

Persistent deficits in social communication and interaction, including: i) deficits in social–emotional reciprocity and social approach, reduced sharing of interests, failure to initiate/respond to interactions; ii) deficits in nonverbal communication, poorly integrated verbal/nonverbal communication, poor eye contact and body language, deficits in understanding/use of gestures; a deficit in facial expressions and recognizing facial affect; iii) deficits in developing, maintaining and understanding relationships, problems with adjusting behavior to various social contexts, making friends and developing interest in peers.

B. Behavioral and cognitive perseverations

Restricted, repetitive patterns of behavior, interests or activities, manifested by: i) stereotyped/repetitive motor movements, use of objects or speech (e.g., simple motor stereotypies, idiosyncratic phrases); ii) insistence on sameness, routines, ritualized patterns in verbal/nonverbal behavior (e.g., distress at small changes, rigid thinking patterns, stable rituals); iii) highly restricted interests abnormal in intensity or focus (e.g., strong attachment to objects, excessively circumscribed or perseverative interest); iv) hyper/hyporeactivity to sensory input or sensory aspects of the environment (e.g., indifference to pain/temperature, adverse response to specific sounds or textures, excessive smelling/touching objects, visual fascination with lights or movement).

C. Symptoms' trajectory

Symptoms must be present in the early developmental period (but may not become fully manifest until social demands exceed limited capacities, or may be masked by learned strategies in later life); these symptoms cause clinically significant impairment in social, occupational, or other important areas of current functioning.

Mood

Sleep

Anxiety

ADHD

Seizures

Irritability

(aggression, self-aggression)

Social

deficits

Repetitive

behaviors

Language

impairment

Core clinical

symptoms

Cognitive deficits

*

*

*

*

*

*

*

*

*

complex with Neuroligin. Loss-of-function mutation in theShank3gene, encoding for Shank protein 3, inhibits synaptic transmission and im-pairs mouse social behaviors, modeling the 22q13.3 deletion syndrome associated clinically with delayed development and speech impairment (Bozdagi et al., 2010).SHANK3is also of particular interest here because of its potential role in ASD (Gauthier et al., 2010) given recent human genetic studies linkingSHANK3to this disorder (Boccuto et al., 2013).

Serotonin transporter (SERT) gene knockout in C57BL/6J mice re-sults in increased grooming (Kyzar et al., 2012b) and causes social deficits in both SERT−/−mice and rats (Kalueff et al., 2010). SERT

variant Ala56 mice display differential serotonin neuronfiring patterns with altered social behavior, communication and repetitive behavior (Veenstra-VanderWeele et al., 2012). Integrinβ3 receptor interacts with SERT (Carneiro et al., 2008), and theITGB3knockout mice also show excessive grooming in novel environments (Carter et al., 2011).

In addition to mutant mice, several non-mutant mouse strains are relevant to ASD. For instance, the BTBR T+_{tf/J (BTBR) inbred mice}

display core behavioral phenotypes of autism, including social deficits and stereotypic behavior (McFarlane et al., 2008; Meyza et al., 2012; Scattoni et al., 2012), which have been used in many studies relevant to ASD (McTighe et al., 2013; Schwartzer et al., 2013; Silverman et al., 2012). Systemic diazepam (Pobbe et al., 2011) or _fluoxetine (Chadman, 2011) administration normalizes social activity in BTBR, but not in control C57BL/6 mice. Environmental factors, such as enriched housing in BTBR mice, reduce grooming duration, suggesting that enrichment normalizes certain repetitive behaviors (Reynolds et al., 2013). Social environmental factors also affect behavioral outcomes in mouse ASD models. For example, adolescent BTBR mice reared in the same housing as_‘social_’C57BL/6 strain exhibit markedly increased sociability, compared to BTBR adolescents reared with other BTBR mice. In contrast, adult BTBR mice do not show improvement in repetitive (self-grooming) behavior, suggesting that behavioral recov-ery differentially affects the social but not the motor ASD-like pheno-type in this model (Yang et al., 2011).

Likewise, the C58/J strain exhibits a behavioral phenotype re-sembling core ASD symptoms, including excess locomotor activity, social deficits, abnormal repetitive behaviors (Ryan et al., 2010), poor T-maze performance and motor stereotypies (Moy et al., 2008). BALB/c mice also exhibit low sociability, exaggerated aggres-sion, enlarged brain mass, low serotonin levels (Brodkin, 2007) and decreased passive social behavior (Fairless et al., 2013), suggesting them as a genetic model of ASD-related states. Systemic treatment of BALB/c mice with MK-801, an allosteric inhibitor of the gluta-matergic (NMDA) receptors, elicits stereotypic circling behavior (Burket et al., 2010), whereasD-serine, a NMDA receptor agonist,

in-creases sociability (Jacome et al., 2011). Together, this suggests the involvement of glutamatergic pathways in the pathogenesis of locomo-tor stereotypies and social impairment in the BALB/c mouse model of ASD-like behavior. Interestingly, acute administration of oxytocin does not alter social behavior, whereas its sub-chronic administration in-creases sociability 24 h post-treatment in BALC/cByJ mice and for 1–2 weeks in C58/J mice (Teng et al., 2013). Oxytocin receptorOxtr

knockout mice exhibit decreased social ability but maintain normal locomotor activity (Pobbe et al., 2012). Mice lacking theCd38gene (which encodes for CD38, an oxytocin secretion regulator;Bartz and McInnes, 2007) exhibit oxytocin signaling defects, behavioral impair-ments and de_ficits in social recognition (Higashida et al., 2011), similar to those exhibited by other mouse models with oxytocin deficits (Higashida et al., 2012; Higashida et al., 2011). Moreover, the arginine vasopressin (AVP) system also plays an important role in social behavior, and the V1a receptor (V1aR) gene has been linked to social de_ficits (Egashira et al., 2007). Subchronic phencyclidine (PCP) in rodents im-pairs social interaction and reduces density of brain V1aR (Egashira et al., 2007), whereas V1aR−/−mice display social deficits, and may

there-fore be a useful model of ASD-like behavior and other disorders associat-ed with social deficits (Egashira et al., 2007).

2.2. Pharmacological rodent models relevant to ASD

In addition to genetic models, rodents are valuable in studying drug-evoked responses relevant to ASD. For example, as social withdrawal is one of the symptoms of schizophrenia and ASD, the social approach test has long been used to study the psychopharmacology of social pheno-types (Hanks et al., 2013).D-Amphetamine disrupts rodent sociability without stimulating locomotor activity (this effect was not reversed by antipsychotics), whereas the gamma-aminobutyric acid (GABA)-A inverse agonist FG-7142 reduces sociability, reversed by the GABA-A antagonist,flumazenil (Hanks, Dlugolenski, 2013).

Antipsychotic drugs antagonize both dopaminergic-induced and glutamatergic N-methyl-D-aspartate (NMDA) antagonist-induced be-haviors (Corbett et al., 1995). A typical glutamatergic antagonist, PCP evokes social withdrawal in rats housed socially without affecting their locomotion (Corbett et al., 1995). SCH 23390, raclopride, haloperidol, chlorpromazine and risperidone fail to reduce PCP-evoked social withdrawal, whereas clozapine and olanzapine signifi -cantly reverse this deficit, demonstrating pharmacological sensitivity of rodent social phenotypes to various drug classes relevant to treating clinical ASD (Corbett et al., 1995). PCP also reduces social behavior in ro-dents following chronic treatment_—an effect which lasted for several weeks after discontinuation, and is reversed by clozapine but not haloper-idol (Qiao et al., 2001). Ketamine, another glutamatergic antagonist, also causes similar social de_ficits as PCP (e.g., disrupting social recognition for several days), which were attenuated by chronic haloperidol or olanzapine (Gao et al., 2009).

While rodent models appear to be a valuable tool to study ASD genetics and pharmacology (Crawley, 2012, Kas et al., 2013), there is a growing need for alternative, time/cost-efficient and high-throughput models of this disorder. Furthermore, domain-oriented approaches to complex human brain disorders focus on various domains (e.g., cogni-tive, affeccogni-tive, motor or social) that represent clusters of various individ-ual behaviors, and overlap during brain pathogenesis (seeKalueff et al., 2008for details). Emphasizing the importance of cross-domain model-ing of CNS pathobiology, these‘integrative’approaches assess multiple disordered phenotypes as a system, and are particularly useful in study-ing diseases such as ASD (which is polygenic and displays a wide range of phenotypes with different severity) (Kalueff et al., 2008). In addition, this allows the researchers to analyze multiple co-expressed behaviors, simultaneously generating rich behavioral data in several distinct neurophenotypic domains (Warnick et al., 2011). Finally, inter-species trait genetics approaches (Kas et al., 2013) recognize the importance of cross-species modeling of complex human brain disorders. Given evolutionarily conserved mechanisms of social behavior, it is therefore

Table 2

Comparison of rodent phenotypes relevant to autism spectrum disorder (ASD; selected fromCrawley, 2012; Kas et al., 2013) and zebrafish behaviors (also seeTable 3–4and

Kalueff et al., 2013for a comprehensive zebrafish behavioral catalog).

Mouse phenotypes Examples of relevant zebrafish phenotypes and models

Lack of preference for social novelty

Social preference test (Fig. 2A)

Reduced social interactions Social interaction test, shoaling, social preference tests, mirror stimulation test (Fig. 2A–C)

Reduced pup ultrasonic vocalizations

Not available

Reduced adult vocalizations Not available Reduced vocalizations Not available

Impaired learning and memory Various learning/memory tasks Impaired social recognition Social learning/recognition tasks Increased repetitive

self-grooming

Not available

Repetitive stereotyped circling behavior

important to foster innovative modeling of ASD, expanding the spec-trum of model organisms for its translational research (Kalueff et al., 2007).

3. Zebrafish models relevant to ASD

3.1. Behavioral and pharmacological models

The zebrafish (Danio rerio) is a new popular model organism in bio-medical research. Thesefish are particularly useful for translational neu-roscience because of their high physiological and genetic homology to humans, ease of genetic manipulation, fully characterized genome, and rapid development (Gerlai, 2010a, 2010b, 2011; Kalueff et al., in press; Lopes et al., 2013; Suen et al., 2013). Zebra_fish are also highly so-cial animals, spending the majority of their time in schools (Fig. 2_–3) and staying very close to conspecifics. It has recently been suggested that the zebrafish system may contribute to understanding ASD patho-biology and genetics (Tropepe and Sive, 2003). Here, we discuss how zebrafish models can be applied practically for experimental studies of autism.

Tables 1_–3show that many of the clinical and rodent ASD-like phenotypes can be successfully modeled in zebrafish. For example, sim-ilar to a mouse social interaction protocol (Kas et al., 2013), in the

zebrafish social interaction test two unfamiliar zebrafish introduced together explore each other, demonstrating a wide range of quanti_fiable social behaviors (Table 4) which can easily be assessed in terms of dura-tion and frequency of various types of social contacts and approach.

In the zebrafish social preference test, also adapted from rodent studies (Kas et al., 2013), a target_fish given a choice between staying close to the empty vs. conspecific zone, spends significantly more time near the conspecific area (Fig. 2A). In other modifications of this model, zebra_fish typically spend more time near a group of zebra_fish (vs. a singlefish), also showing kin recognition/preference (Table 3) and spending more time during social investigation of novel (unfamil-iar) zebra_fish (Fig. 2A). Similarly, zebra_fish models can also take advan-tage of the availability of various color variants, assessing the duration and frequency of social contacts with such phenotypically distinct strains of_fish. For example, while the wild type zebra_fish typically avoid shoals of white-skin stripelessnacremutants, their social prefer-ence fornacrefish increases by systemic injections of oxytocin (and its

fish analog isotocin), paralleling its similar pro-social effects in rodents (Capurro, 2010); see discussion further.

In the shoaling test (Fig. 2B,Fig. 3), zebrafish spent most of their time swimming in dynamic groups (schools), characterized by short

inter-fish distance, smaller zebra_fish group area size/diameter, as well as rel-ative polarizations. In contrast, disorganized social structure in zebrafish is characterized by reduced polarization offish shoals, looser and larger schools, and higher percentage of_fish leaving the group and spending time outside the shoal (Fig. 2B and3;Kalueff et al., in press).

The mirror stimulation test, quite specific for aquatic models, is based on mirror presentation and the fact that_fish perceive their own reflection as another zebrafish (Fig. 2C andTable 3; seeCachat et al., 2013for review). This situation can evoke both social behavior and aggression, which will be robustly disrupted if the_fish display social de_ficits. For example, increased head-butting or mirror biting in this test may represent elevated aggression and/or sociability in zebrafish, whereas abnormally low responses can be used to quantify ASD-like symptoms (Cachat et al., 2013).

In addition to social deficits (Fig. 1), behavioral perseverations are also an important ASD-related phenotype (Table 1). Therefore, it may be critical to assess the availability of zebra_fish behavioral stereotypies that can be measured in various tests. While zebrafish do not display patterned and complex grooming behavior, they do show several common behavioral stereotypies, including repetitive, stereotypic

‘thigmotaxic_’swimming near the walls, or specific circling behavior, involving rotational swimming in tight circles (Fig. 2D,Table 4). Thus, zebra_fish models are fully capable of displaying both hallmark behavior-al symptoms of ASD_—social de_ficits and behavioral perseverations, indi-cating high translational potential of zebrafish models for ASD-related states.

Interestingly, zebra_fish circling behavior can be induced by selected psychotropic drugs, such as glutamatergic antagonists MK-801, PCP or ketamine (Kyzar et al., 2012a), paralleling rodent drug-evoked circling locomotion already discussed above in relation to ASD. In line with this, various pharmacological agents have also been shown to potently modulate zebrafish social behaviors. For example, zebrafish shoaling is markedly disrupted by various psychoactive drugs (Fig. 3;Maaswinkel et al., 2013), resembling the effects on ASD-related social behavior de-scribed for these agents in rodents (see above). Collectively, this sup-ports the evolutionarily conserved nature of ASD-related social and motor phenotypes in various species, again emphasizing the transla-tional value of zebrafish models to study human ASD.

As mentioned earlier, there is a general lack of clinically efficient ASD-speci_fic drugs, stressing the importance of developing novel com-pounds to treat various symptoms of ASD. For example, risperidone (clinically effective to treat irritability and motor/aggressive behaviors in ASD patients) does not reduce their social de_ficits and repetitive/ obsessive behaviors (Crawley, 2012). While risperidone was not tested infish social tests, it potently affects zebrafish motor and physiological

Target fish

Conspecific Empty zone Group Target fish Conspecific

Target fish Kin

Non-kin Unfamiliar Target fish Familiar

Normal school Disrupted, loose school

Mirror

C

B

A

D

activity (Prieto et al., 2012); also see similar results for aripiprazole (Lee et al., 2013). Fluoxetine, also commonly used in clinical and rodent ASD studies (Chadman, 2011), albeit not yet tested infish social tests, also strongly modulates zebrafish motor and emotional behavior (Egan et al., 2009). Collectively, this indicates a generally high sensitivity of zebrafish to compounds clinically relevant to correcting some ASD symp-toms, suggesting a potential predictive validity of zebrafish screens to anti-ASD drugs. Clearly, future studies examining in-depth this applica-tion of zebrafish models to ASD research merit further scrutiny.

Cognitive deficits are widely recognized as an important clinical aspect of ASD, and are often observed in rodent models of this disorder (Kas et al., 2013). Zebra_fish possess excellent cognitive abili-ties, which can be comprehensively evaluated in various cognitive tasks (Oliveira, 2013; Stewart and Kalueff, 2012). In addition to zebra_fish social cognitive models (e.g., social or kin recognition, Fig. 2a,Table 4), various other paradigms can be used to assess non-social cognitive deficits relevant to ASD in this organism. For example,

habituation to novelty (reflecting spatial working memory) can be reli-ably measured in zebra_fish, and is affected by various pharmacological agents (Wong et al., 2010a). Memory performance in various mazes can be assessed in zebrafish in a manner similar to rodent studies (Gaikwad et al., 2011; Stewart and Kalueff, 2012). Since attention de_ficit hyperactivity disorder (ADHD) is frequently comorbid with ASD (Roy et al., 2013; Sprenger et al., 2013), and is related to both behavioral and cognitive clusters of ASD symptoms (Fig. 1), the possibility of modeling ADHD-like phenotypes in zebrafish (Norton, 2013) becomes important and translationally relevant to ASD (also seeParker et al., 2012a,2012b, 2013for details on recent progress in experimental models of attention in zebra_fish).

3.2. Physiological correlates

In addition to behavioral biomarkers, ASD is often accompanied clini-cally by altered physiological (e.g., neuroendocrine) functions. For

MDMA 160 mg/L

Control

PCP 5 mg/L

Control

Reserpine 10 mg/L 7 days

Control

Ethanol 4% vol/vol

Control

Fig. 3.Examples of pharmacological modulation of zebrafish social (shoaling) phenotypes. Note that zebrafish shoaling responses are markedly impaired by acute 20-min exposure to phencyclidine (PCP, 5 mg/L), 3,4-methylenedioxymethamphetamine (MDMA, 160 mg/L), ethanol (4% vol/vol) and chronic reserpine (10 mg/L for 7 days) in representative 4-fish shoals (Kalueff et al., 2009–2013 unpublished studies; also seeGreen et al., 2012, vs. drug-free controls). PCP, a dissociative glutamatergic hallucinogen, is well-known to affect social behaviors in humans and rodents (see text for details) and evokes similar effects in zebrafish. A serotonergic psychedelic drug MDMA disrupts social behavior in rodents and zebrafish (Green et al., 2012). Alcohol at mild-to-high doses is commonly known to cause social withdrawal in human and rodents, also showing a similar profile in zebrafish. Reserpine, a plant alkaloid which depletes brain monoamines, is known to impair social behavior in rodents (Bisong et al., 2010, 2011), also causing social deficits in zebrafish in the shoaling test used here. Overall, these data support sensitivity and face validity of zebrafish autism spectrum disorder (ASD)-related social phenotypes to pharmacological modulation by drugs known to evoke ASD-like symp-toms clinically and in experimental (rodent) models. In addition to drugs evoking ASD-like behavior, it is logical to expect that zebrafish models can be used to study agents that modulate zebrafish behavior in the opposite direction, i.e., for screening for novel potential anti-ASD compounds (see discussion of zebrafish sensitivity to risperidone, aripiprazole andfluoxetine in the text).

example, a dysregulated oxytocinergic system has been strongly impli-cated in social deficits common for ASD (Carter, 2007; Insel et al., 1999; Jacob et al., 2007). Therefore, continued investigation into the develop-mental and functional role of oxytocin is critical. The zebra_fish homologue

of oxytocin, isotocin, facilitates numerous facets of social behavior in tel-eosts, including social approach, fear, reproduction-related vocalizations and courtship behavior (Braida et al., 2012; Goodson and Bass, 2000; Thompson and Walton, 2004). Recently, peripheral administration of either oxytocin or isotocin (equally potent relative to each other), or an oxytocin antagonist, has been shown to modulate social preference and anxiety-related behavior in zebrafish (Braida et al., 2012), similar to humans (Ebstein et al., 2012; Kumsta and Heinrichs, 2013; Neumann and Landgraf, 2012) and rodents (Carter et al., 2008; Lim et al., 2005). Since the oxytocinergic system is increasingly recognized as a potential therapeutic target in the treatment of ASD (Harony and Wagner, 2010; Modi and Young, 2012; Striepens et al., 2011), zebra_fish may provide use-ful tools to investigate the pharmacological profiles of oxytocin-related compounds, as well as to test the potential of novel anti-ASD drugs targeting the oxytocinergic neuroendocrine system.

Another potential physiological correlate of ASD may include altered cortisol levels, often reported in autistic patients. While some species differences may exist in mineralocorticoid vs. glucocorticoid action of this hormone (Pippal et al., 2011), zebra_fish offer an excellent model for cortisol screening, as they not only possess a robust neuroendocrine stress axis, but also utilize cortisol (like humans), which can be easily and reliably quanti_fied in_fish (Egan et al., 2009). Likewise, seizure be-havior is commonly observed in ASD in humans (Tuchman, 2013) and rodents (Gilby, 2008; Greco et al., 2013). Importantly, experimental sei-zures can be easily evoked in zebra_fish (Hortopan et al., 2010; Stewart et al., 2012; Wong et al., 2010b) and analyzed (like other ASD-related symptoms,Fig. 1) via high-throughput screening, further supporting the use of zebra_fish as an ef_ficient model for ASD and drug discovery.

Table 4

Selected zebrafish behaviors potentially relevant to autism spectrum disorder (ASD; based onKalueff et al., 2013, also seeTable 3for a general framework).

Behaviors Brief description

Aggression Complex behaviors (including approach,fin raise, biting, charging, chasing and circling) directed at conspecifics in adult zebrafish; may appear in the context of establishing dominance (social interaction)

Charge Movement towards a secondfish with increasing acceleration; establishes social dominance and marks the resolution of a zebrafish‘fight’ (Oliveira et al., 2011).

Circling Repetitive swimming in a circular direction; can be defined by their diameter (e.g., 2 body lengths/~5 cm) and quantified by the number of complete circles per trial, the number (%) of animals showing circles and the direction of circling (left- or right-rotations); automated methods may also quantify turn angle and angular velocity (Kyzar et al., 2012a; Riehl et al., 2011)

Display Social behavior used to establish dominance/hierarchy (Spence et al., 2008). Lateral display involves twofish lined up parallel to each other head to tail, raising dorsalfins (fin raise), extending caudalfins and circling. Frontal display involves twofish approaching each other from the front with the attempt of biting (Schneider, 2011)

Fighting Agonistic confrontation between two individuals to establish social dominance (Oliveira et al., 2011). During thefirst phase, thefish assess each other by exhibiting display, biting/nipping, which continues until thefirst chase/flee occurs. Next, the‘winner’(chaser) initiates all agonistic behaviors, while the‘loser’displaysfleeing, submission behavior or freezing (Oliveira et al., 2011)

Habituation Tendency to show a decreased response upon repeated exposure to a novel stimulus/environment (reflecting cognitive ability of zebrafish). Includes inter-trial (inter-session) and

intra-trial (intra-session) habituation, quantified by calculating the ratios of behavioral activity during the initial vs. latest trials, or by assessing the behavioral profile offish across the trial(s) (Stewart et al., 2010; Stewart et al., 2013)

Kin preference and recognition

The preference for kin vs. unrelated zebrafish, particularly robust in juvenile zebrafish. Zebrafish are able to seek/recognize kin from unrelated zebrafish; this behavior involves approach/attraction and leads to increased time spent near kin (kin preference) (Spence et al., 2008) Mirror stimulation response Complex behaviors evoked infish by mirror exposure; most likely linked to aggression and social investigation; typically includes approach,

head-butting, biting the mirror and chasing own reflection (Cachat et al., 2013; Oliveira et al., 2011)

Place preference The tendency to establish a preferred location in which thefish spends more time; can be induced by social reward (e.g., sight of the conspecific) (Al-Imari and Gerlai, 2008)

Polarization Behavioral characteristic of adult zebrafish reflecting the degree to which members of the group are moving in the same direction; is high in established zebrafish groups (Miller and Gerlai, 2012b)

Shoaling Formation of a group (shoal) of adult zebrafish, held together by social pressures (Miller and Gerlai, 2011; Miller and Gerlai, 2012a,2012b). Zebrafish shoaling can be quantified manually or using automated video-tracking systems, assessing several endpoints, including the average inter-fish distance; shoal area size; proximity (time each member of the shoal spent within a specified distance from each other); nearest and farthest neighbor distances; time spent in shoal; time spent away from shoal; number of animals leaving the shoal and polarization (heading) Social interaction Normal social behavior of zebrafish, a reciprocal change in zebrafish behavior influenced by the presence or actions of other conspecifics

(Al-Imari and Gerlai, 2008; Miller and Gerlai, 2011; Oliveira et al., 2011). Some examples includefighting/aggression, shoaling, courtship, approach/boldness (social investigation), social recognition and social preference

Social preference and recognition

A natural tendency to spend time close to conspecifics; can be observed as part of shoaling behavior, kin recognition, social recognition, or preference of the‘conspecific’vs.‘empty’zones (Al-Imari and Gerlai, 2008; Spence et al., 2008). Zebrafish also recognize familiar from unfamiliar zebrafish (social recognition) and distinguish between own vs. phenotypically different other strain (Spence et al., 2008) Stereotypic locomotion A pattern of rigid, repetitive behaviors (e.g., swimming from corner to corner, stereotypic jaw movements) evoked in zebrafish under some

conditions (e.g., treatment with psychostimulants and PCP;Cachat et al., 2013; Kyzar et al., 2012a)

Submissive behavior A social behavior following aggressive confrontations. Submissivefish stays immobile (withfins retracted), typically near the bottom or near the surface, with the caudal part of the body oriented downward (Oliveira et al., 2011)

Table 3

Overview of zebrafish phenotypes relevant to autism spectrum disorder (ASD).

Clinical symptomsa _{Relevant zebrafish phenotypes}b

Social deficits Reduced social interaction in the shoaling test (e.g., less time spent in shoals, increased shoal size, increased average inter-fish distance and farthest neighbor distance). Reduced social interaction and/or investigation time and frequency in the shoaling, mirror stimulation and the social preference tests (e.g., reduced time spent near conspecific, reduced time investigating new (vs. familiar) conspecific, reduced time and contacts with mirror). Repetitive behaviors and

cognitive inflexibility

Presence of specific behavioral stereotypies (e.g., cycling behavior,figure 8 swimming, stereotyped thigmotaxis-like swimming and other motor stereotypies, such as repetitive jaw movements) and/or cognitive deficits (e.g., detected in various motor and cognitive tasks, such as impaired habituation in the novelty tests or aberrant patterns of alternation in Y or/T-mazes)

Developmental nature of observed symptoms

Aberrant developmental trajectories of zebrafish social behaviors (e.g., in shoaling test across lifespan)

a_{Based on DSM-5; seeTable 1for details.}

3.3. Genetic models relevant to ASD

Considering the high heritability of ASD and the ease of genetic manipulations in zebrafish, this species is also emerging as a useful tool to study ASD genetics. For example, the ASD-linked human 16p11.2 chromosomal area has a homologous region in zebrafish, consisting of several genes important for brain development (Blaker-Lee et al., 2012), including KCTD13, which is responsible for head size and neurogenesis in zebra_fish, and was identi_fied as a major contributor in some cases of autism in humans (Golzio et al., 2012). Similarly, the ASD susceptibility genemetregulates zebrafish cerebellar development and motor neuron migration.Metencodes a tyrosine kinase receptor, im-plicated in autism and brain development in mice and zebrafish (Elsen et al., 2009). Zebrafishmetis expressed in the cerebellar primordium (later localizing in the ventricular zone), and its morpholino knockdown reduces the size of the cerebellum and affects its cellular morphology, thereby paralleling the correlation between altered MET regulation and ASD (Elsen et al., 2009). Reflecting a neurodevelopmental aspect of ASD pathogenesis (Table 1), this phenotype illustrates the potential of zebrafish in modeling ASD (Blaker-Lee et al., 2012; also see Norton, 2013for a review).

The AUTS2 locus has also been strongly implicated in ASD and other human brain disorders, such as ADHD, epilepsy, dyslexia, motor delay and language delay (Oksenberg et al., 2013). The knock-down ofauts2

in zebra_fish results in a smaller head size, neuronal reduction and de-creased mobility. Collectively, this suggests that AUTS2 plays an impor-tant role in neurodevelopment, consistent with both clinical and zebra_fish phenotypes relevant to ASD (Oksenberg et al., 2013). Like-wise, mounting evidence has recently implicated neurexins and neuroligins in neurodevelopmental disorders, including ASD and

psychoses (Wright and Washbourne, 2011). These genes have been extensively characterized in zebra_fish, demonstrating high sequence conservation with the human genes, therefore suggesting that zebrafish models (with their simpler circuitry and high-throughput capacity) may prove extremely useful in identifying therapeutic strategies to treat ASD (Wright and Washbourne, 2011).

The potential role ofSHANK3in human ASD (Boccuto et al., 2013) has recently been paralleled by zebrafish models, where the genetic knock-down of zebra_fish zshank3orthologous genes (zs3.1and zs3.2) by morpholino results in a reduction in the head size and markedly impaired swim responses to touch (Gauthier et al., 2010). The fact that this gene is strongly implicated in CNS function in zebra_fish, rodents and humans (Gauthier et al., 2010) strongly supports the value of zebrafish models to study CNS pathogenesis, including ASD.

In addition to genetically modi_fied_fish, recent studies on strain differences in shoaling have revealed important insights into the genetics of zebrafish social behavior. For example, based on strong differences between a wild-derived strain offish and the laboratory AB strain, quantitative trait loci (QTL) analysis identified a genomic region on chromosome 21 responsible for shoaling behavior (Wright et al., 2006;also seeWright et al., 2003). Overall, these results not only con_firm the potential of zebra_fish for genetic mapping of complex be-havioral traits, but also establish genetic determinants of social behavior in this species (Wright et al., 2003, 2006), generally consistent with the notion of high heritability of ASD.

3.4. Environmental models potentially relevant to ASD

In both humans and rodents, environmental factors play a signifi -cant role in the development of ASD (Kawicka and Regulska-Ilow, 2013; LaSalle, 2013; Thirtamara Rajamani et al., 2013). Recent studies suggest that ASD-like state may also be modulated by environmental stimuli in zebrafish. For example, it has been suggested that zebrafish also have gene-by-environment epigenetic regulation of complex be-haviors, such as ASD-like phenotypes (Norton, 2013). Consistent with this, exposure to a pesticide chlorpyrifos induces ASD-like behaviors in zebrafish (Richendrfer et al., 2012). Thus, while the environmental modulation of ASD-like behavior in zebra_fish has not yet been thoroughly investigated, future studies of environmental effects on zebrafish phenotypes relevant to ASD may be important. Given the well-recognized history of zebra_fish tests in developmental pharmacol-ogy and toxicolpharmacol-ogy, the possibility of developing _‘environmental_’ zebrafish models of ASD-like pathogenesis can represent an interesting direction for future research in this_field.

4. Conclusion

In summary, zebra_fish emerge as an important model species in translational neuroscience and the neurogenetics of ASD. The presence of key behavioral phenotypes which parallel human and rodent ASD, as well as their sensitivity to major groups of pharmacological agents used clinically to treat (or evoke) ASD, also supports their role in experimen-tal modeling of autism. The availability of recently developed video-tracking tools enables ef_ficient, objective and data-dense quanti_fication of zebrafish social phenotypes (Green et al., 2012; Maaswinkel et al., 2013). A highly significant correlation for manual vs. automated analy-ses in such studies demonstrates the capacity of video-tracking technol-ogy to assess zebra_fish social behaviors in a high-throughput and reliable manner, further fostering zebrafish models relevant to ASD (Green et al., 2012).

Notably, zebra_fish behavior is strongly reliant on visual sensory infor-mation about their social environment, which offers a possibility of using standardized still images and‘virtual reality’videos of individual or group zebra_fish rather than utilizing live_fish as social stimuli (e.g.,Gerlai et al., 2009; Luca and Gerlai, 2012; Pather and Gerlai, 2009). Unlike rodent ASD models (which typically require presentation of live animals), zebrafish

Robotic fish Zebrafish

A

B

Fig. 4.Additional models for zebrafish social behaviors. A: An experimental setup utilizing standardized automated (vs. live) stimulusfish image presentation in zebrafish sociability models relevant to ASD. These approaches enable a better, standardized control of exper-imental conditions across all groups and eliminate potential confounding effects on the testedfish (e.g., resulting from aberrant stimulusfish behavior). In addition, they also en-able testing multiple additional experimental parameters (which can be easily adjusted using computers), such as size, shape, number of subjects, color, skin pattern, body orien-tation/angle, position in the tank, as well as swimming speed and trajectory of the present-ed stimulusfish. B: The potential application of roboticfish (e.g.,Polverino et al., 2012) for modeling zebrafish social behaviors, such as shoaling, relevant to ASD-like behavior (note that the same approaches can also be applied to zebrafish social preference tests shown in

Fig. 2).

social models based on automated stimulus presentation (Fig. 4A) can therefore be better standardized and controlled experimentally, leading to improved reproducibility and reduced variance of data. Moreover, recent evidence suggests the utility of automated roboticfish (Fig. 4B) to study social and other behavioral responses in zebrafish (Abaid et al., 2012; Polverino et al., 2012). For example, the ability of zebrafish to display robust social behaviors (e.g., shoaling or preference) when exposed to a robotic zebrafish provides an excellent opportunity to study_fish social behavior while fully controlling the stimulus presenta-tion by manipulating the robotic zebrafish‘behavior’(Fig. 4B).

Clearly, each experimental animal model of complex brain disorders has various limitations (Kalueff et al., 2007). For a complex multi-faceted brain disorder like ASD, it is impossible to develop an animal model that recapitulates all aspects of its pathogenesis. However, animal models are a valuable tool to dissect various aspects of brain pathobiology, explore the role of specific neural pathways or discover genetic determinants. Thus, the question of which models or model organisms are‘better’is misleading, since the answer will only be limited to specific research questions, phenotypes in question or putative mechanisms to study. Therefore, complementing the existing rodent models relevant to ASD (Crawley, 2012), zebrafish can be used complementarily, to advance our understanding of ASD and related behavioral de_ficits.

Moreover, ASD is a neurodevelopmental disorder (Kas et al., 2013), and this aspect is critical for experimental animal models of ASD-like states (Tropepe and Sive, 2003). It was already mentioned that zebra_fish have a rapid development, which enables time- and cost-efficient modeling of this important pathogenetic aspect of ASD (note, however, that social behavior is not seen in larval_fish, and therefore larval models and screens may not be useful to study social phenotypes in zebrafish). Finally, the ease of genetic and pharmacological manipulations, as well as the low cost, high-throughput and potential for targeting develop-mental trajectories of ASD, make zebra_fish a promising new model (Green et al., 2012; Kalueff et al., in press; Tropepe and Sive, 2003) to complement traditional rodent tests (El-Kordi et al., 2012; Kas et al., 2013). As noted recently, the_field of experimental modeling of ASD has taken thefirst step in a long and challenging journey (Crawley, 2012). So far, the mounting experimental evidence presented here indicates that zebra_fish models relevant to ASD will provide an exciting new chap-ter for this endeavor.

Acknowledgements

The authors' research has been supported by the International Zebra_fish Neuroscience Research Consortium (ZNRC) and the ZENEREI Institute.

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