Developmental Psychobiology

Robert Gerlai Department of Psychology University of Toronto Mississsauga 3359 Mississauga Road North, Mississauga, Ontario, L5L 1C6 Canada E-mail: [email protected]

Embryonic Alcohol Exposure: Towards the Development of a Zebrafish Model of Fetal Alcohol Spectrum Disorders ABSTRACT: Fetal alcohol spectrum disorder (FASD) is a devastating disease of the brain caused by exposure to alcohol during prenatal development. Its prevalence exceeds 1%. The majority of FASD cases represent the milder forms of the disease which often remain undiagnosed, and even when diagnosed treatment options for the patient are limited due to lack of information about the mechanisms that underlie the disease. The zebrafish has been proposed as a model organism for exploring the mechanisms of FASD. Our laboratory has been studying the effects of low doses of alcohol during embryonic development in the zebrafish. This review discusses the methods of alcohol exposure, its effects on behavioral performance including social behavior and learning, and the potential underlying biological mechanisms in zebrafish. It is based upon a recent keynote address delivered by the author, and it focuses on findings obtained mainly in his own laboratory. It paints a promising future of this small vertebrate in FASD research. ß 2015 Wiley Periodicals, Inc. Dev Psychobiol 57:787–798, 2015. Keywords: zebrafish; FASD; ARND; fetal alcohol exposure; social behavior; shoaling; learning and memory; dopamine; tyrosine hydroxylase

FASD: A DEVASTATING DISEASE WITH MAJOR SOCIETAL IMPACT According to the US Centers for Disease Control and Prevention, excessive alcohol (ethanol, ethyl alcohol) consumption kills about 88,000 people and costs over $220 billion (US) each year in the United States alone (http://www.cdc.gov/features/alcoholconsumption/). Alcoholism and alcohol abuse present an enormous societal burden because of health care and loss of productivity related costs but also because of the amount of human suffering these diseases cause. Among all of

Manuscript Received: 15 February 2015 Manuscript Accepted: 8 April 2015 Correspondence to: Robert Gerlai Contract grant sponsor: NIH/NIAAA Contract grant number: R01 AA14357-01A2 Contract grant sponsor: NSERC Contract grant number: 311637 Article first published online in Wiley Online Library (wileyonlinelibrary.com): 16 June 2015 DOI 10.1002/dev.21318  ß 2015 Wiley Periodicals, Inc.

the alcohol related diseases perhaps the most troubling are the fetal alcohol spectrum disorders (FASDs) (Abel & Sokol, 1991; Lupton, Burd, & Harwood, 2004; Popova et al., 2013; Popova, Stade, Bekmuradov, Lange, & Rehm, 2011). FASD results from alcohol exposure during fetal development (Jones, Smith, Ulleland, & Streissguth, 1973; Lemoine, Harousseau, Borteyru, & Menuet, 2003; Wattendorf & Muenke, 2005), which leads to life-long suffering. Depending on how much, for how long, when, and how frequently alcohol is consumed during pregnancy, the disease may manifest with differing severity (Cartwright & Smith, 1995; Maier, Chen, Miller, & West, 1997). FASD ranges from full blown fetal alcohol syndrome (FAS), in which gross physical, that is anatomical, abnormalities accompany the significant behavioral and cognitive impairments, to milder forms of the disease sometimes labeled “Alcohol Related Neurological Disorder” or ARND (Stratton, Howe, & Battaglia, 1996 and references therein). In the latter, obvious physical signs may be absent, although the significant abnormalities, for example, in social behavior and in cognition, continue to be observable

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(Carmichael Olson et al., 1997; Mattson & Riley, 2000; Roebuck, Mattson, & Riley, 1999; Streissguth et al., 1991). The prevalence of FASD was estimated to be 1 in 100 live births (Sampson et al., 1997), but more recent studies suggest that this is likely a significant underestimate, and the actual number of children suffering from the disease may be as many as 5% of the population (May et al., 2009). One reason why recording the exact number of FASD cases is difficult is that the majority of FASD patients fall in the category of milder forms of the disease (Sampson et al., 1997), and thus are likely misdiagnosed or not diagnosed at all. Given the long history of acceptance of alcohol drinking in human history and given the numerous alcohol related customs engrained in our culture, it is likely that alcohol consumption will not subside, and with it the devastating effect of alcohol on developing human fetuses will continue to be a major problem. Thus, in addition to concerted efforts to educate people to reduce the prevalence of the disease, we also have to study the biological consequences and the mechanisms of fetal alcohol exposure. Better mechanistic understanding of the disease may be beneficial for two main reasons. One, utilizing such knowledge one may be able to develop effective biologybased (e.g., pharmaceutical) treatment strategies, which are currently lacking. Two, one may be able to identify biomarkers that could aid proper diagnosis. Currently, several forms of FASD require information on, or evidence of, drinking during pregnancy (Stratton et al., 1996). This piece of information, however, is not easy to obtain because it represents a sensitive subject that is often avoided by the young mother, or an event about which the mother has no recollection. Briefly, alcohol use in pregnancy is notoriously underreported (MorrowTlucak, Ernhart, Sokol, Martier, & Ager, 1989). Clearly detectable biomarkers would thus be highly useful, even in the absence of pharmacological intervention, because the diagnosed children may be malleable to environmental enrichment strategies or to educational and training programs custom tailored for them. How can one discover biomarkers and the way alcohol affects the developing human embryo? This is not an easy question to answer for two main reasons. One, alcohol is a “dirty” drug from a pharmacology standpoint. It directly or indirectly interacts with a large number of biological targets, and it does so in a dose and exposure regimen dependent manner, a topic on which 10 of 1,000 of research papers have been published. It is safe to say that the effect of alcohol on the brain is rather complex in the adult, but one may expect even more complexity when this drug interacts with the developing fetus. The second reason is that mechanistic studies are difficult to perform in

Developmental Psychobiology

humans. Due to the above, animal models have been proposed. In this review, I focus on the zebrafish and will ignore other animal models not because I claim these other models have not successfully advanced our knowledge. On the contrary, mammalian as well as non-mammalian models have allowed making important discoveries about how alcohol works in the brain and how it affects embryonic development too. However, exactly for these reasons, excellent reviews have been published on these topics (e.g., Lovinger & Crabbe, 2005; Patten, Fontaine, & Christie, 2014), and thus I do not need to reiterate previously published findings here. Instead, I will focus on zebrafish, a relatively novel translational tool, which I argue will also significantly contribute to the understanding of the mechanisms of alcohol’s actions in the brain and in the developing vertebrate fetus.

ZEBRAFISH: AN UPCOMING TRANSLATIONAL TOOL Bony fish (Osteichthyes) represent the evolutionarily oldest and most species rich vertebrate taxon (Leveque, Oberdorff, Paugy, Stiassny, & Tedesco, 2008; Nelson, 2006), yet these species rarely make it to the laboratories of biomedical research scientists with one exception, the zebrafish. Why has the zebrafish become so popular? Early studies about four decades ago already started using zebrafish and, for example, analyzed numerous behavioral responses of this species (e.g., Bloom & Perlmutter, 1977; McCanna, Koehna, & Klinea, 1971). Nevertheless, zebrafish was only one of the few obscure exotic organisms, at least in science, until developmental biology and genetics discovered some of its advantages (Kimmel, 1989; Streisinger, Walker, Dower, Knauber, & Singer, 1981). Zebrafish are fertilized and develop externally, embryonic development is rapid (completes within 5 days), and the developing embryo is practically transparent. Due to these features scientists started to utilize zebrafish, and developed numerous genetic tools with which the biological mechanisms of organogenesis may be studied. By now, zebrafish has become one of the most preferred model organisms of geneticists (Eisen, 1996). Perhaps due to the developed genetic tools and the accumulated genetic information on the zebrafish, disciplines other than embryology and genetics have also taken notice of this species. For example, the number of publications identified in PubMed using the keywords “zebrafish” and “behavior” shows an exponential increase over the past decade, a rate of change that dramatically outpaced that of papers on “mouse

Developmental Psychobiology

and behavior” or “rat and behavior”, mammalian model organisms that represent the bedrock of biomedical research (Kalueff, Stewart, & Gerlai, 2014). The reason why the zebrafish enjoys such newly gained attention is due to evolution. Throughout the process of evolution changes are built on previously existing features, “designs”. Thus, core features or mechanisms found in evolutionarily more ancient organisms, such as fish, tend to remain conserved, at least to a certain degree in more “modern” evolutionarily younger species, such as us, humans. The degree to which this conservation occurs is difficult to measure in complex traits such as behavior and brain function, but it is very simple in the case of genes. The nucleotide sequence homology between zebrafish and corresponding human genes is usually found to reach, and sometimes to exceed, 70%. This level of similarity is enough for one to identify human homologs of newly identified zebrafish genes. And this is exactly where translational relevance of the zebrafish lies: this species allows the efficient discovery of genes and the functions of these genes with a promise that the discovery can be quickly translated to human. Why is discovery of such genes potentially more efficient using the zebrafish? It is because of the several practical features of this species, including its small size, prolific nature, and ease of maintenance in the laboratory. But more importantly, it is because fish are evolutionarily older than mammals and thus are also simpler in “design”. This simplicity represents a reductionist approach, one which may allow the investigator to focus on the most important, evolutionarily ancient, core of mechanisms (Gerlai, 2014a). Another general point I would like to make, before going into specifics, is the argument that even if a particular study species has only few advantages over another model organism, it is worth adding this species (zebrafish in this case) to the mix of discoveries. This is because it allows one to discover the overlaps across multiple species (Gerlai, 2014a). These overlaps, similarities among features and mechanisms across multiple study species, are expected to represent the functionally most relevant aspects of the studied phenomena, ones which are likely to cut across a variety of species, including our own, thus leading to the increase of translational relevance.

ZEBRAFISH IN FASD RESEARCH: A PARTICULARLY GOOD FIT I argue that the zebrafish is particularly suited for FASD research. There are many reasons for this. Most importantly, alcohol can be delivered in this species in

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a simple and controlled manner. Fish, or the developing embryo inside the egg, may be exposed to a precisely predetermined concentration of alcohol by immersing the organism into the alcohol solution for a specific period of time and at the desired developmental stage (for dechorionated embryo exposure see Bradfield, West, & Maier, 2006; and for intact embryo/egg exposure see Fernandes & Gerlai, 2009 and Mahabir, Chatterjee, & Gerlai, 2013a). Also importantly, the immersion may be performed using a large number of subjects (eggs, embryos, or adult) in a uniform manner at the same time. For example, we use a plastic cup device, with or without sub-compartments depending on the application, whose bottom is made of a fine plastic mesh (Fig. 1). This device allows us to immerse even the small eggs or hatched larvae of zebrafish, hundreds of them at a time, by lowering the cup into

FIGURE 1 Zebrafish eggs and embryos may be exposed to alcohol (ethanol, EtOH, or ethyl alcohol) in a simple and efficient manner by lowering them into the appropriate alcohol solution, larger cylinder, using the smaller cylinder with a thin mesh on the bottom. Using this method, we exposed zebrafish eggs to alcohol for an hour at their 24th hr post-fertilization (hpf) stage, and subsequently measured how much alcohol diffused into the egg. The bar graph shows the alcohol concentration inside the egg as a function of the external concentration, and demonstrates that approximately 1/20th to 1/30th of the external alcohol concentration reached the egg. Modified from Fernandes & Gerlai (2009).

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the alcohol solution for a set period of time (onset) and then removing the subjects still inside the cup (offset) and immersing them into a wash solution (e.g., system water). The fact that the embryos develop externally may be viewed as a problem with the model. Human fetuses depend upon and very much influenced by the mother’s physiology. In fish, there is no placenta to filter the blood, no maternal intra-uterine environment to influence the development of the embryo, and no maternal liver to detoxify teratogenic compounds like alcohol. However, this is exactly the reason why zebrafish is effective. It represents a reductionist approach that allows us to investigate the effect of alcohol on embryonic development and later adult neural function without the complicating aspects of mammalian maternal physiology.

MODELING MILD ALCOHOL EXPOSURE DURING EMBRYONIC DEVELOPMENT IN ZEBRAFISH: THE FIRST STEPS I emphasize that we are at the beginning stages of building the FASD zebrafish model, but I also argue that we have a good rationale for it. ARND, the milder form of FASD, is three times more prevalent than the severe forms of the disease (Sampson et al., 1997). Also, children with ARND or with even less severe forms of FASD are most likely to be undiagnosed or misdiagnosed, and thus can remain untreated. It is thus crucial to model the milder forms of the disease. The zebrafish was successfully utilized in FASD research before we started to develop our model (Arenzana et al., 2006; Bilotta, Barnett, Hancock, & Saszik, 2004; Bilotta, Saszik, Givin, & Hardesty, 2002; Carvan, Loucks, Weber, & Williams, 2004; Loucks & Carvan, 2004; Reimers, Flockton, Tanguay, 2004). However, these earlier studies utilized higher alcohol concentrations and/or administered alcohol for prolonged periods of time. The effect of these treatments often included robust anatomical deformities, cyclopia, heart defects, and/or increased mortality, alcohol induced developmental abnormalities that recapitulated human FAS, the most severe form of FASD. Instead of this route, we decided to focus on the more prevalent and less severe form of the disease, and exposed the developing eggs of zebrafish only to small doses of alcohol and only for a short duration of time (Fernandes & Gerlai, 2009). We employed concentrations .25, .50, .75, and 1.00% (vol/vol % of EtOH) and bathed the zebrafish eggs in the given alcohol solution for 2 hr, a relatively short, but not unreasonably short, period of time for the zebrafish that develops into a fully functional little fish within 5 days after

Developmental Psychobiology

fertilization (Fernandes & Gerlai, 2009). It is also notable, that when we measured the alcohol concentrations inside the egg, we found it to be about 1/20th to 1/30th of the external bath concentration, an alcohol level that is around the legal limit of driving in North America (Fernandes & Gerlai, 2009; Mahabir, Chatterjee, Buske, & Gerlai, 2013b) (Fig. 1). Thus, we argued that our alcohol exposure regimen was in line with what one may expect in the milder forms of FASD, mimicking what happens when pregnant women drink moderately and/or infrequently during their pregnancy. We first employed this alcohol exposure regimen at the 24th hr post-fertilization (hpf) in the zebrafish embryo. Our rationale for targeting this embryonic age was that the 24th hpf represents the end of the segmentation and the beginning of the pharyngula stage of zebrafish development, which corresponds approximately to the late 1st trimester or early second trimester of human fetal development (Kimmel, Ballard, Kimmel, Ullmann, & Schilling, 1995 also see http://zfin.org/zf_info/ zfbook/stages/ and http://www.ehd.org/virtual-humanembryo/). At this stage, the brain has started to develop, for example, neuronal progenitor cells have started to specialize and develop into neurons, and have begun to migrate and establish connections, but the development of the brain is far from complete. It was largely a guess on our part, as systematic analysis of the potential differential effects of the timing of alcohol exposure during embryonic development in zebrafish had not been performed before. Nevertheless, upon examination of the consequences of alcohol exposure at this stage of zebrafish development we realized that the choice we made represented a good start. Alcohol exposure exerted significant effects that we could quantify using behavioral test paradigms, and the behavioral abnormality we observed appeared rather specific (Fernandes & Gerlai, 2009).

EMBRYONIC ALCOHOL EXPOSURE DIMINISHES RESPONSES TO SOCIAL STIMULI IN ADULT ZEBRAFISH In response to the embryonic alcohol exposure, we found no obvious signs of defects in the embryo or in the adult fish (Fernandes & Gerlai, 2009). Fish from even the highest alcohol concentration group (1% EtOH bath application) appeared healthy, developed apparently normally without any gross observable anatomical abnormalities, and with growth rate that did not differ from that of control. According to casual observation, all experimental zebrafish seemed unaffected by having been exposed to low levels of alcohol for 2 hr at their 24th hpf developmental stage. The lack

Developmental Psychobiology

of alcohol effects could not have been due to inability of alcohol to reach the embryo (for e.g., one could theorize that the eggshell did not allow alcohol to diffuse into the egg), because we did detect measurable amounts of alcohol inside the egg (although only less than 1/20th of the external dose, Fernandes & Gerlai, 2009; Mahabir et al., 2013a). We theorized that the low dose and short duration of exposure perhaps led to only mild changes in the developing brain that one could detect only with the use of sensitive behavioral test paradigms. But what behavior should we test? Children with ARND have been found to have cognitive deficits, including learning disability (Hamilton, Kodituwakku, Sutherland, & Savage, 2003; Mattson, Riley, Delis, Stern, & Jones, 1996; Mattson & Roebuck, 2002 also see Kodituwakku, 2007), and they often suffer from abnormal social behaviors as well (O’Connor et al., 2006; Rasmussen, Becker, McLennan, Urichuk, & Andrew, 2011). We decided to focus on the latter first, because we have appreciated a species-specific feature of the zebrafish. Zebrafish are highly social. They form groups called shoals in nature as well as in the laboratory (Gerlai, 2014b). Their shoaling behavior is thus easy to elicit and also easy to measure (Miller & Gerlai, 2007; Saverino & Gerlai, 2008). We have developed numerous methods for its induction and quantification (Miller & Gerlai, 2008, 2011a,b, 2012; Qin, Wong, Seguin, & Gerlai, 2014), and decided to utilize one of these paradigms for the analysis of potential functional changes of the brain induced by embryonic alcohol exposure (Gerlai, Chatterjee, Pereira, Sawashima, & Krishnannair, 2009). The paradigm is rather simple, but efficient (Fig. 2). We noticed that upon presentation of conspecifics, a zebrafish swimming alone in the test tank will robustly decrease its distance to the conspecifics. We also discovered that it does not really matter whether the stimulus conspecifics are live fish in the test tank (providing cues of all modalities), live fish outside of the test tank (providing only visual cues but reacting interactively to the test fish), video-recordings (providing visual cues alone and are not interactive but move in 3D), or computer animated zebrafish images (providing visual cues only and move only in 2D) (Qin et al., 2014). The test fish responded to all of the above stimuli with a robust decrease of distance from the stimulus: they immediately approached the stimulus and stayed about 5–10 cm away from it as long as it was shown, a distance that is similar to the average distance zebrafish keep from each other in freely swimming shoals (Buske & Gerlai, 2011a). Notably, this robust shoaling response was not seen towards animated images that did not resemble zebrafish (e.g.,

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FIGURE 2 An automated computerized method of inducing and quantifying social behavioral responses in zebrafish. The single experimental subject is placed into a 40 L test tank flanked by two computer monitors. One of the monitors projects animated (moving) images of conspecifics for a predetermined period of time in an experimenter controlled manner (speed, location, size, and number of images). Among other behavioral parameters, the distance between the test subject and the stimulus monitor showing the images is quantified using video-tracking. Modified from Scerbina et al. (2012).

moving rectangles containing scrambled pixels from a previous zebrafish photograph, or elongated images of zebrafish) (Saif, Chatterjee, Buske, & Gerlai, 2013; Saverino & Gerlai, 2008). It is also notable that image presentation was accomplished with the use of a custom software application developed in house (Saverino & Gerlai, 2008), and the response of experimental zebrafish was also recorded and analyzed using a software application, e.g., Ethovision (Noldus Info Tech., Wageningen, The Netherlands). Thus the paradigm was fully automated and required minimal experimenter intervention, an important point for two reasons. One, experimenter induced fear was minimized, and two, the task was scaleable and thus appropriate for large scale mutagenesis and/or drug screening (Gerlai, 2010a, 2002, 2012; Stewart, Gerlai, & Kalueff, 2015; Gerlai, 2014c). Using this shoaling paradigm, we tested sexually mature adult zebrafish that were exposed to one of five concentrations of alcohol (.00, .25, .50, .75, or 1.00%

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emphasize that we measured the behavior of fully grown adult (about 6–8 months old) zebrafish that were exposed to alcohol only once for 2 hr during their 24th hr post-fertilization stage. The observed reduction of shoaling response is notable because this was the first embryonic alcohol

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alcohol) at their 24th hpf stage for 2 hr (Fernandes & Gerlai, 2009). The results (Fig. 3) revealed a near perfect linear dose dependent decrease of shoaling response. The higher the concentration of alcohol was, the less experimental zebrafish reduced their distance to the stimulus upon stimulus presentation (Fg. 3). I

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FIGURE 3 Two hour long exposure of zebrafish embryos to alcohol at their 24th hr post fertilization stage dose dependently impairs their ability to respond to social stimuli when they grow up. The line graphs show the distance between the adult (6–8 month old) test zebrafish and the stimulus screen as a function of time. The period of stimulus presentation is indicated by the black filled horizontal bar just above the X-axis of the line graphs. The white, unfilled, horizontal bar above the X-axis represents the periods when the stimulus screens were blank (no conspecifics shown). The thin horizontal dashed line shows random chance location (25 cm, i.e. the middle of the tank). Note the robust reduction of distance seen in response to the presentation of the conspecific images in the control fish that were exposed to no alcohol (0%) during their embryonic development. Also note the concentration dependent reduction of this shoaling response (diminishing reduction of distance) among fish that received alcohol during their embryonic development. The lower graph shows data derived from the upper graph. It shows the reduction of distance in response to the presentation of the conspecific images for the five treatment groups. The reduction of distance is calculated as the average distance of the test fish from the stimulus screen during the period of the stimulus presentation minus the average distance during the period that preceded the stimulus presentation (habituation period). Note the almost perfect linear dose dependent decrease of the reduction of distance. Also note that fish that received 1% alcohol during their embryonic development (last bar) did not reduce their distance to the stimulus below random chance, i.e. in these fish the shoaling response was abolished. Modified from Fernandes, & Gerlai (2009).

Developmental Psychobiology

exposure induced abnormality seen in zebrafish that was not accompanied by any detectable gross anatomical aberration. Nevertheless, this impairment may be due to several factors, only one of which may be abnormal social behavior. For example, if the affected fish suffered from motor dysfunction, they may not have been able to swim towards the stimulus. Alternatively, if the alcohol treated fish suffered from impaired vision, they may not have been able to see, and thus respond to, the animated conspecific images. Analysis of our results and subsequent studies, however, suggested that these alternative hypotheses were not correct (Buske & Gerlai, 2011b; Fernandes & Gerlai, 2009; Fernandes, Rampersad, & Gerlai, 2015). We found no motor impairment in the alcohol treated fish. Their swim speed and swimming pattern (e.g., angular velocity, location of swimming, vertical exploration, etc.) were statistically indistinguishable from control. Our results also suggested that visual impairment was unlikely a contributing factor. For example, we observed that in response to the shoal images all fish, alcohol treated fish included, responded with a reduction of swimming speed, and the magnitude of reduction was statistically indistinguishable among all our treatment groups including control (Fernandes & Gerlai, 2009). We thus concluded that all fish were able to see and respond to the presented images, but the response did not include swimming closer to the images in case of the alcohol treated fish. It is also notable that in a subsequent study, we observed impaired shoaling in freely moving shoals of embryonic alcohol treated zebrafish as compared to control. The alcohol treated fish exhibited increased interindividual distances, that is formed looser shoals compared to control fish (Buske & Gerlai, 2011b). Notably, shoaling fish perform normal shoaling behavior even in the complete absence of visual cues, because they can utilize their lateral line similarly to echolocation and keep the appropriate distance from each other (Gerlai, 2014b and references therein). Thus any putative impairment in vision could not have contributed to the impaired shoaling behavior we observed in these freely moving shoals of zebrafish (Buske & Gerlai, 2011b; Fernandes & Gerlai, 2009; Fernandes et al., 2015). If performance factors, motor function, and vision did not lead to the observed shoaling response impairment, what else could explain the alcohol effect? There may be many possible explanations as to how embryonic alcohol induced changes of the central nervous system may affect social behavioral responses of our experimental zebrafish. For example, the alcohol treated zebrafish may not have been able to recognize their conspecifics as such due to abnormalities in the

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functioning of the social behavioral network, distributed anatomical sites argued to play roles in vertebrate social behavior (Goodson & Kingsbury, 2013). It is also possible that neurobiological mechanisms subserving motivational processes may have been disrupted by the embryonic alcohol treatment, and thus although the affected zebrafish recognized their shoal mates, they remained unmotivated to swim close to them. It is also possible that neural mechanisms associated with fear responses may have been altered. An important adaptive role of shoaling is predator avoidance (Gerlai, 2010b, 2013, 2014b), and under mildly aversive conditions shoal cohesion increases (Miller & Gerlai, 2007; Speedie & Gerlai, 2008). At this point we cannot conclusively distinguish among these possibilities, but our preliminary data suggest that fear responses of the alcohol treated zebrafish are comparable to those of control fish. Could conspecific recognition or motivation be the culprit? While we do not yet have any empirical information on the former possibility, we do have evidence for the latter.

MECHANISMS OF EMBRYONIC ALCOHOL INDUCED SOCIAL BEHAVIOR IMPAIRMENT IN ZEBRAFISH Understanding of the mechanisms of the impaired shoaling response induced by embryonic alcohol in zebrafish will require systematic analysis of processes at many levels of biological organization. For example, one could conduct a thorough analysis of the transcriptome and compare expression of all known zebrafish genes across the alcohol treated and control fish using DNA micorarrays (Pan, Mo, Razak, Westwood, & Gerlai, 2011). Similar in principle to microarrays, one could also utilize the latest deep sequencing methods that may provide even better resolution for discovering differentially expressed genes. One could also map neuroanatomical sites showing differential activation using immediate early gene expression and/ or calcium sensitive dyes. These and other recombinant DNA-based or neuroanatomy-based approaches are now all feasible with zebrafish (Gerlai, 2012; Guo, Wagle, & Mathur, 2012; Rinkwitz, Mourrain, & Becker, 2011 also see Mushtaq, Verpoorte, & Kim, 2013). For now, however, we have taken a less systematic and more hypothesis driven approach. We decided to first focus our attention on a particular and well defined mechanism, the dopaminergic system. We explored this neurotransmitter system as a proof of principle to show how one may be able to utilize zebrafish in the investigation of the mechanisms underlying milder forms of FASD.

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We discovered that presentation of shoals is rewarding for zebrafish (Al-Imari & Gerlai, 2008). For example, the sight of conspecifics is a sufficient reinforcer in appetitive associative learning tasks (Karnik & Gerlai, 2012; Pather & Gerlai, 2009). We have also shown that the appearance of conspecifics, but not the appearance of other moving images, induces a rapid increase of the level of dopamine and DOPAC (dopamine’s metabolite) in the brain of zebrafish (Fernandes et al., 2015; Saif et al., 2013). Importantly, the response appears to be specific to the dopaminergic system as, for example, the amount of serotonin or of 5HIAA (serotonin’s metabolite) did not change in response to the shoal image (Saif et al., 2013). We have also shown that disruption of the functioning of the dopaminergic system with the use of a D1-R selective antagonist leads to disruption of shoaling (Screbina, Chatterjee, & Gerlai, 2012). Thus, we concluded that the dopaminergic system plays an important role in mediating the shoaling response in zebrafish (Gerlai, 2014b). Subsequently, we started to investigate whether the dopaminergic system is affected by embryonic alcohol exposure in zebrafish. First, we analyzed the adult fish and investigated the amount of neurotransmitter dopamine and its metabolite DOPAC using high precision liquid chromatography (HPLC) with neurochemical detection (Buske & Gerlai, 2011b). We found significant reduction of the amount of these neurochemicals in response to embryonic alcohol treatment in the brain of adult zebrafish. Subsequently, we examined the developmental trajectory of changes in the levels of these neurochemicals in the brain of control zebrafish, and discovered a significant age-dependent increase (Buske & Gerlai, 2012; Mahabir et al., 2013b). Importantly, we also discovered that this age dependent increase was significantly blunted by embryonic alcohol

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exposure in a dose dependent manner (Fig. 4): the higher the concentration of alcohol was during the embryonic exposure, the more blunted the age-dependent increase of neurochemical levels was (Mahabir et al., 2013a). It is notable that the changes induced by the embryonic alcohol exposure in the amount of dopamine and DOPAC were not accompanied by gross anatomical abnormalities or overall alteration of brain weight. Subsequently, we also examined whether the reduction of dopamine and DOPAC levels found in the embryonic alcohol exposed adult fish was due to altered baseline levels of these neurochemicals or whether it was the result of the inability of this neurotransmitter system to respond to stimulation. Interestingly, we found evidence for the latter (Fernandes et al., 2015). In order to investigate this question we isolated our experimental zebrafish for 24 hr. The rationale behind this was that we knew that the appearance of conspecifics acts as a reward (Al-Imari & Gerlai, 2008) and activates the dopaminergic system (Saif et al., 2014), and we expected the absence of conspecifics around the test fish to return the activity of this neurotransmitter system to baseline. Analysis of dopamine and DOPAC levels in the brain of socially isolated zebrafish showed no effects of embryonic alcohol treatment as compared to control fish. However, upon presentation of conspecific images, we found a robust dopamine and DOPAC increase in the control fish, a response that was absent in the fish exposed to the highest alcohol dose (1% alcohol bath for 2 hr at 24 hpf) and blunted in the fish exposed to the intermediate dose (.5% alcohol bath for 2 hr at 24 hpf) (Fernandes et al., 2015). This is a notable set of findings as it shows a correlation between the impaired ability of the dopaminergic system to respond to social stimuli and the impaired reduction of distance from the social stimuli both seen in the adult embryonic alcohol treated

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FIGURE 4 Age and embryonic alcohol concentration dependent changes in the amount of dopamine as measured from whole brain extracts using HPLC in zebrafish. Note the agedependent increase of dopamine amount (relative to total brain protein weight) in the control fish (0% alcohol exposure during embryonic development). Also note the alcohol concentration dependent reduction of this age-related dopamine increase. Modified from Mahabir et al. (2013a).

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zebrafish. Taken together, these findings strongly suggest that one of the mechanisms mediating the embryonic alcohol exposure induced impairment of shoaling is the dopaminergic system. Although suggestive, our findings are only correlative and preliminary, and may reflect only a small fraction of potential mechanisms that underlie the embryonic alcohol induced behavioral changes. For example, alcohol in the brain is known to interact with and affect a large number of molecular targets directly and indirectly, among which are several neurotransmitter systems other than the dopaminergic system. In the developing brain the effects may be even more complex as alcohol may interfere with numerous fundamental processes including cell division, migration, and the establishment of neuronal connections. In line with these arguments is our finding showing that embryonic alcohol exposure reduced the amount of serotonin as well as its metabolite 5HIAA as examined across multiple age-groups of zebarfish (Mahabir et al., 2013a). It is thus too early to say how important a role the dopaminergic system may play in the impaired shoaling response seen in the embryonic alcohol exposed fish. Although the effects of alcohol on the developing vertebrate brain may be complex, as discussed at the beginning of this section, we have numerous tools with which we can start unraveling this complexity. For example, as a proof of principle, we have already conducted a comprehensive DNA microarray analysis, not of developmental effects of alcohol, but of the effects of chronic exposure to this substance (Pan et al., 2011). Although not directly relevant for FASD research, our results demonstrated that over 50% of the entire genome was expressed in the brain of adult zebrafish at the time of our sampling. This DNA microarray analysis allowed us to detect numerous (close to 2,000) genes whose expression significantly changed as a result of our experimental manipulation, and 60% of the differentially expressed genes were novel with no annotation, that is no functional information on them. Thus, microarray or deep-sequencing based analysis of embryonic alcohol exposure induced changes in the transcripome of the zebrafish will likely be a useful approach. But one can proceed in several other ways too. For example, one can compare neuronal activation of different brain regions of embryonic alcohol exposed fish using c-fos immunohistochemistry or in situ hybridization, or can also investigate apoptotic cell death (alcohol induces apoptotic cell death) and whether this cell fate is differentially affected across specific neurotransmitter systems in the embryonic alcohol exposed zebrafish brain. Another entirely new line of research one can follow is based upon some interesting differences we (Mahabir et al., 2013a)

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and others (Loucks & Carvan, 2004) have discovered among zebrafish strains. For example, Mahabir et al. (2013a) found that zebrafish of the TU strain were not vulnerable to exposure to alcohol during embryonic development, that is these fish did not show the alcohol dose-dependent blunting of the age-related increase of neurotransmitter levels as seen in AB fish. Discovery of such strain differences may be important for two principally different reasons. One, such differences give us a tool for discovering genetic mechanisms that may protect against or may modify the deleterious effects of alcohol during embryonic development. A discovered strain difference allows one to conduct a quantitative trait locus (QTL) analysis, and subsequently to identify the locus/loci and eventually the genes underlying the observed phenotypical differences between or among zebrafish strains. Given the availability of high resolution genetic markers in zebrafish (Currie, Schilling, & Ingham, 2008; Beier, 1998) as well as of increasingly powerful DNA sequencing methods (Henke, Bowen, Harris, 2013), efficient identification of such genes is now a reality. The second reason why discovery of strain differences is useful is that it allows us to pick and choose the strain best suited for pharmacological studies or for mutagenesis studies. In summary, the number of ways one can investigate the mechanisms of the effects of embryonic alcohol exposure in zebrafish is already large, and with the renewed interest in this species it is rapidly increasing. We are at the very first steps on the road leading to mechanistic discoveries, but the results of recent studies, our own included, suggest that the future is bright for the zebrafish (Gerlai, 2011): this species may become one of the important laboratory organisms with which human FASD is modeled and its mechanisms revealed.

NOTES I would like to thank all my students and research assistants without whom none of the work conducted in my laboratory would have been possible. I would also like to thank the International Society for Developmental Psychobiology for the opportunity to deliver the John Wiley Distinguished Lecture at the Society’s 2014 Conference in Washington DC. Last, I would also like to thank Dr. George F. Michel, Editor-in-Chief, for inviting me to write this review. The author’s research is supported by NIH/NIAAA (R01 AA14357-01A2) and NSERC (311637).

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