Developmental stage and genotype dependent behavioral effects of embryonic alcohol exposure in zebrafish larvae

Journal Pre-proof Developmental stage and genotype dependent behavioral effects of embryonic alcohol exposure in zebrafish larvae

Amira Abozaid, Lidia Trzuskot, Zelaikha Najmi, Ishti Paul, Benjamin Tsang, Robert Gerlai PII:

S0278-5846(19)30541-X

DOI:

https://doi.org/10.1016/j.pnpbp.2019.109774

Reference:

PNP 109774

To appear in:

Progress in Neuropsychopharmacology & Biological Psychiatry

Received date:

3 July 2019

Revised date:

29 August 2019

Accepted date:

2 October 2019

Please cite this article as: A. Abozaid, L. Trzuskot, Z. Najmi, et al., Developmental stage and genotype dependent behavioral effects of embryonic alcohol exposure in zebrafish larvae, Progress in Neuropsychopharmacology & Biological Psychiatry(2018), https://doi.org/10.1016/j.pnpbp.2019.109774

This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

© 2018 Published by Elsevier.

Journal Pre-proof

Developmental stage and genotype dependent behavioral effects of embryonic alcohol exposure in zebrafish larvae

Amira Abozaid1, Lidia Trzuskot 1*, Zelaikha Najmi2*, Ishti Paul2, Benjamin Tsang1, &

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Robert Gerlai1,3

1 Department of Psychology, University of Toronto Mississauga

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2 Department of Biology, University of Toronto Mississauga

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3 Department of Cell & System Biology, University of Toronto

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* These authors contributed equally to this study

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Correspondence may be addressed to:

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Robert Gerlai Department of Psychology, Rm CCT4004 University of Toronto Mississauga 3359 Mississauga Road, Mississauga Ontario, CANADA, L5L 1C6 office: (905)569-4255 lab: (905)569-4257 fax: (905)569-4326 e-mail: [email protected] e-mail: [email protected]

Abstract Fetal Alcohol Spectrum Disorders (FASD) represent a worldwide problem. The severity and types of symptoms of FASD vary, which may be due to the genotype of the fetus and the

Journal Pre-proof developmental stage at which the fetus is exposed to alcohol. The most prevalent forms of FASD present less severe symptoms, including behavioral and cognitive abnormalities, and arise from exposure to low amounts of alcohol consumed infrequently. Treating or diagnosing FASD patients has been difficult because we do not understand the mechanisms underlying FASD. Animal models, including the zebrafish, have been suggested to answer this question. Here, we present a proof of concept analysis studying the behavioral effects of embryonic alcohol

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exposure in one-week old juvenile zebrafish. We exposed zebrafish embryos at one of five

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developmental stages (8, 16, 24, 32, or 40 hours post-fertilization) to 0% (control) or 1%

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(vol/vol) ethanol for 2h, and tested the behavior of these fish at their age of 7-9 days post-

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fertilization. We employed two genetically distinct zebrafish populations, a quasi-inbred AB derivative strain, and a genetically variable WT population. We report significant developmental

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time and genotype dependent effects of alcohol on certain measures of motor function and/or

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anxiety-like responses. For example, we found embryonic alcohol exposed AB fish to swim faster, vary their speed more, stop moving more often and turn less compared to control fish,

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alcohol induced changes that were absent or less robust in WT fish. We conclude that our results

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open new avenues to the identification of genetic mechanisms that mediate or influence alcohol induced developmental alteration of brain function and behavior, which, on the long run, may allow us to identify diagnostic biomarkers and treatment options for human FASD.

Key words: Alcohol, anxiety, ethanol, FASD, genotype x development interaction, motor function, zebrafish

Journal Pre-proof Introduction Fetal Alcohol Spectrum Disorder (FASD) is a devastating life-long disease that affects children and adults whose mother has consumed alcohol during pregnancy. FASD presents a range of behavioral, cognitive, and physical deficits (Wilhoit et al., 2017). Although FASD would be completely preventable by abstinence from alcohol, globally about 8 out of 1000 children suffer from FASD (Lange et al., 2017). In addition to human suffering, FASD also

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represents a significant financial burden. For example, just in Canada, this disease costs 1.8

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billion dollars per year (Popova et al., 2018). There may be many reasons for the unreduced high

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prevalence of FASD. Mothers may not be aware of their pregnancy until the end of their 1st

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trimester (Santelli et.al., 2003). Societal attitudes towards drinking in developed countries tolerate, if not promote, alcohol consumption. Last, lack of appreciation of the teratogenic

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effects of alcohol on the developing fetus also hinders prevention. In summary, FASD remains a

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major societal and medical problem, a major unmet medical need. One way to alleviate this need would be to understand the mechanisms of the disease and

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develop evidence-based therapeutic applications. However, understanding these mechanisms is

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hindered by two main problems. One, analyzing alcohol induced changes at the mechanistic (e.g. molecular or neurobiological) levels is difficult with humans. Two, alcohol is known to interact with a large number of molecular targets directly and influences an even larger number of biochemical processes indirectly (e.g. Abrahao et al., 2017). To tackle this complexity, animal models have been proposed for the analysis of human FASD (Kelly et al., 2009; Gerlai, 2015; Patten et al., 2014). The most frequently utilized model organisms in this endeavor have been the house mouse and the rat. For example, prenatal ethanol exposure has been found to induce impulsivity,

Journal Pre-proof disrupt attention, and lead to hyperactivity in rats and mice. (Kim et al 2013), as well as abnormal motor function including reduced grip strength (Brys et al., 2014). Although less closely related to human than these latter two species, the zebrafish has also been employed in the analysis of the effects of alcohol administered during embryonic development (e.g., Facciol et al., 2019; Seguin & Gerlai, 2018; Fernandes & Gerlai, 2009; Loucks & Carvan, 2004). There are several reasons for this, perhaps the most important among

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them is the fact that the externally fertilized and externally developing embryo of the zebrafish

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allows unhindered control over the timing, length and dose of alcohol exposure (Facciol et al.,

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2019). In rats and mice, alcohol dosing is complicated by the lack of direct access to the embryo

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as well as by maternal physiology that affects absorption, distribution, metabolism and excretion of alcohol. Furthermore, in rodents only about half of the embryonic development that would

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correspond to human intrauterine development is actually inside the uterus, the other half is

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outside.

In recent years, the zebrafish has been proposed and successfully utilized in a number of

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psychopharmacology studies as well as neurotoxicological assays (Linney et al., 2004; Peterson

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& MacRae, 2012; ) among them in the analysis of the effects of embryonic alcohol exposure. For example, prolonged exposure to and/or administration of high doses of alcohol during embryonic development in zebrafish were shown to recapitulate several gross morphological deficits found in the most severe forms of human FASD, fetal alcohol syndrome, or FAS (Zhang et al., 2014; Bilotta et al., 2004; Arenzana et al., 2006). More recently, the milder, and more prevalent, forms of FASD, often called Alcohol Related Neurological Disorder or ARND, have also been modeled in zebrafish. For example, immersion of zebrafish eggs into up to 1% (vol/vol) alcohol bath for 2 hours at 24 hour post-fertilization (hpf) was found to induce a robust

Journal Pre-proof and dose dependent deficit in the response to social stimuli (conspecifics) when tested in sexually mature adult zebrafish (Fernandes & Gerlai, 2009; Buske & Gerlai, 2013). This behavioral deficit remained observable even at the old age of zebrafish (Fernandes et al., 2015). Importantly, this deficit was not accompanied by motor or perceptual abnormalities (Fernandes & Gerlai, 2009), and fear and anxiety responses were also unaltered in the alcohol exposed fish compared to control (Seguin et al., 2016). Thus, the abnormality was argued to be specific to

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shoaling (group forming) (Seguin et al., 2016). It is also notable that abnormal social behaviors

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and impaired social cognition have recently been considered central to the symptoms of mild

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human FASD cases (Kully‐Martens, et.al., 2012; Stevens et al., 2015). Last, the actual concentration of alcohol that reached the embryo inside the egg in the above zebrafish studies

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was found to be approximately 1/15 – 1/25 of that of the external bath concentration, which, in

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case of 1% external alcohol bath is just below the blood alcohol concentration limit for driving in

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North America.

The developmental trajectory of embryonic alcohol exposure induced behavioral changes

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has not been explored, and thus we do not know how soon after the exposure the behavior

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altering effects of alcohol may manifest. Furthermore, it is likely that alcohol exerts its teratogenic effects dependent upon the stage of development at which the substance reaches the embryo. For example, during periods of rapid developmental changes involving neuronal and/or glial cell proliferation, migration and differentiation and/or during major connectivity organizational processes of the brain including axonal outgrowth and establishment of topographic connections in the brain, alcohol may exert more robust deleterious effects that may manifest as significant behavioral or cognitive deficits later on. To map such potential critical periods, and to investigate whether induced behavioral changes are detectable early in life of the

Journal Pre-proof zebrafish, we administered alcohol to the embryo of zebrafish at five different developmental time points (each embryo receiving only one administration). We tested the potential behavioral consequences of this treatment in what we call juvenile zebrafish, i.e. at about one week of age. Furthermore, to investigate potential genotype dependence, we performed the above analysis using two genetically distinct populations of zebrafish: a wild-type, genetically variable and highly heterozygous population (WT), and a quasi-inbred strain, ABsk that is known to be

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homozygous at the majority of its loci (Coe et al., 2009). The effect of genotype in the severity

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of symptoms of FASD has been suspected (Eberhart & Parnell, 2016), and strain dependent

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alcohol effects have been demonstrated in the zebrafish (Loucks & Carvan, 2004) (Mahabiret al.,

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2014). Thus, discovering the potential effects of genes (population origin) on embryonic alcohol induced changes in zebrafish would represent a proof of principle. In addition, finding strain or

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population differences in zebrafish would allow subsequent mechanistic analysis of the

transcriptome studies.

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underlying mechanisms, for example, with the use of quantitative trait locus, or detailed

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Last, because shoaling behavior was reported not to emerge until two weeks of age in

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zebrafish (Buske and Gerlai, 2011), we focused our behavioral analyses to the characterization of swim path parameters in an open field, and thus measured motor function-related and anxietylike responses. Here, we report significant alcohol induced behavioral impairments that were dependent upon the developmental time point at which alcohol was administered as well as upon the genotype of our experimental fish. We regard our study as a proof of concept analysis, one that is aimed at revealing the phenomena of developmental stage dependency and or genotype x

Journal Pre-proof alcohol interaction without the ability to pinpoint specific mechanisms, a question that will require future follow up studies.

Methods Animals, housing and alcohol treatment Zebrafish obtained from a local pet store (Big Al’s Aquarium Warehouse, Mississauga)

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we designate as WT (wild type) and ABsk zebrafish obtained from the Hospital for Sick Children

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(Zebrafish Facility, Toronto, ON Canada) were bred in our facility to obtain fertilized eggs. The

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rationale for using pet store fish for breeding our wild type (WT) population is to maximize

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genetic variability and minimize homozygosity. Because Big Al’s Aquarium obtains their fish from commercial breeding facilities where the effective population size (the number of breeding

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individuals) is large, one may expect minimal level of inbreeding. Thus, these wild type fish are

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expected to approximate the prototypical zebrafish found in nature. The AB zebrafish strain, on the other hand, was established more than four decades ago and is known to be homozygous at

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over 80% of its loci (Johnson & Zon, 1999; Trevarrow & Robison, 2004 and references therein).

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This strain is one of the most frequently employed strains in a variety of zebrafish studies and its genome is well characterized. Unfortunately, due to severe import restrictions in Canada, we could not obtain AB zebrafish from the Zebrafish International Research Center or ZIRC (Eugene, OR, USA), where these fish are maintained, e.g. cryopreserved with genotype constancy. We call our AB population ABsk because these fish although originated from the AB strain of ZIRC, have been bred at the Hospital for Sick Children Research Institute’s zebrafish facility for over ten generations. Thus, these AB fish may differ, due to genetic drift, for example, from the standard AB strain.

Journal Pre-proof Adults of both strains (ABsk and pet-store WT) were housed in 9.5 litre tanks on system racks designed by Aquaneering, Inc (San Diego, CA, USA). Two days prior to breeding, the fish were separated by sex and placed into 2.8 litre tanks on system racks. Two females and two males were placed in each breeding tank. The breeding tank had a perforated bottom that allowed collection of eggs. Both WT and ABsk zebrafish were placed in breeding tanks at the same time, i.e., the evening (at 17:00h) before the scheduled spawning the next day. The automated light-

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cycle system at the zebrafish facility mimics dawn, and slowly turned on the lights next morning

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starting at 6:30h reaching full lighting at 7:30h, the start of spawning. Subsequent to the

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spawning event, the fish were removed from their breeding tanks at 9:00 am. In our study, the

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post-fertilization hours (the age of our experimental subjects) were counted from this time point. The eggs were kept in system water, deionized and sterilized water supplemented with

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Instant Ocean Sea Salt (Big Al’s Aquarium Warehouse, Mississauga, ON, Canada). In order to

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investigate potential developmental stage-dependent effects of embryonic alcohol exposure, we immersed the eggs, selected randomly, at one of five developmental time points (8, 16, 24, 32,

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40hpf) for 2 hours in either 1% or 0%v/v ethanol. Each egg was immersed in alcohol or system

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water only once, i.e., we employed a between subject experimental design and had 5 alcohol treated and 5 corresponding control groups. Immediately after the 2-hour bath, both the control and alcohol treated eggs underwent a 1-minute system water rinse and were subsequently transferred to a new system water bath for a final 20-minute immersion. This procedure ensured removal of alcohol in the alcohol treated groups and exposed the control groups to the same immersion/washing procedure as the alcohol groups. Subsequently, eggs were transferred to petri dishes and allowed to develop. Water chemistry was checked daily, and parameters, including conductivity (250S), pH (6.5-7.5), temperature (26-28oC), nitrate (0ppm), nitrite (0ppm), and

Journal Pre-proof ammonia (0ppm), were maintained at optimal levels for zebrafish. The developing eggs and fry were kept on a 12-hour photoperiod via the Wattstopper lighting program (Legrand North America) that mimicked natural sunrise and sunset, with sunrise occurring between 06:30 to 07:00, and sunset between 20:30 to 21:00. Zebrafish fry reached free swimming stage at their age of 5 days post-fertilization and were started to be fed API 100 (Zeigler®, Gardners, PA) fry

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food at that age. At age 7-9 days post-fertilization (dpf) all fish underwent behavioral testing.

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Quantification of behavior

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The 7-9 dpf old juvenile experimental fish were singly pipetted into individual 3.5cm

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diameter Petri dishes. We employed 4 video-cameras (JVC GZ-MG330HV) placed 30 cm above the petri dishes. Each camera viewed 5 petri-dishes from above. That is, at any given point of

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time, we recorded the behavior of 4 x 5 subjects. All developmental time point (5 levels) x

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alcohol (2 levels) x strain (2 levels) groups, i.e. 20 groups, were tested simultaneously and in a randomized manner with regard to the specific petri dish location of the test fish. Randomization

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was achieved by following placement of the fish according to a number list generated by a

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random number generation algorithm. Each fish was tested only once, i.e., we employed a between subject design. We note that no behavioral change resulting from ontogenesis has been reported between 7 and 9 dpf for zebrafish, and similarly, we do not expect significant alterations of neurobiological mechanisms during this short developmental period. The petri dishes were lit from below using a Picker Light-table to reduce overhead lighting glare and enhance video tracking. Illumination level was found to be 1800 Lux as quantified by the Lux Light Meter Pro v2 (developed by Elena Polyanskaya for the Apple Iphone). Video-recordings were obtained in an automated manner and were uploaded onto a Dell computer running Windows 7 operating

Journal Pre-proof system, and they were later quantified blindly using a video-tracking system, Ethovision XT 13 (Noldus Info Tech., Wageningen, The Netherlands). The following behaviors were quantified, they were chosen as non-redundant measures of the swim path of zebrafish that characterize numerous behavioral functions including locomotor activity, exploratory activity, and anxiety (Blaser & Gerlai, 2006; Ahmed et al., 2011): swim speed (cm/sec), intra-individual temporal variance of swim speed (cm2/sec2), angular velocity ( o/sec), duration of immobility (sec),

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frequency of immobility, duration of time spent within the thigmotaxis zone (sec, the zone that is

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within 5 mm from the wall of the petri dish), frequency of entering the thigmotaxis zone. The

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time-course of potential changes in the above specified behavioral measures is plotted and

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analyzed with 1-min time bins for the 30 min long recording session, as employed before, for

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example, for the analysis of the effects of acute alcohol administration (Tran & Gerlai, 2013).

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Statistical Analysis

SPSS (version 24) written for the PC was used for statistical analysis. Repeated

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measures variance analyses (ANOVAs) were performed to test the effects of Interval (the within

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subject repeated measure factor with 30 levels, the number of 1 min intervals), Alcohol with 2 levels (1% vs 0%), Developmental time point of exposure with 5 levels (8, 16, 24, 32, 40hpf), strain with 2 levels (WT and AB) and the interactions among these factors. The overall ANOVAs were followed by post hoc repeated measures ANOVAs whose aim was to investigate the effect of alcohol in each specific strain and developmental time point of exposure group separately. Significance was accepted when the probability of the null hypothesis was less than 5% (p < 0.05).

Journal Pre-proof Results Figure 1 shows the swim speed (cm/sec) of AB zebrafish exposed to either 1% alcohol (alcohol group) or 0% alcohol (control group) at their age of 8, 16, 24, 32, or 40 hpf (panels A-E) and of WT zebrafish treated the same manner (panels F-K). The results suggest that AB zebrafish treated with alcohol showed pronounced hyperactivity, especially when the fish were exposed to alcohol at their 24th, 32nd or 40th hpf age. On the other hand, WT zebrafish did not

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seem to show such an alcohol effect. Furthermore, WT fish appeared to swim faster, in general,

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compared to AB fish. ANOVA confirmed these observations and found a significant Interval

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effect (F(29, 14268) = 136.20, p < 0.001), a significant Alcohol effect (F(4, 492) = 4.85, p <

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0.01), and significant effect of Strain (F(1, 492) = 82.57, p < 0.001). The interaction terms Alcohol x Strain (F1, 492) = 13.01, p < 0.001), Interval x Developmental time point of exposure

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(F(116, 14268) = 1.30, p < 0.05), Interval x Strain (F(29, 14268) = 2.38, p < 0.001), Interval x

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Alcohol x Developmental time point of exposure (F(29, 14268) = 1.37, p < 0.01), Interval x Alcohol x Developmental time point of exposure x Alcohol (F(116, 14268) = 1.31, p < 0.05)

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were also found significant, while the other interaction terms were non-significant. These results

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demonstrate that embryonic alcohol exposure affected swim speed depending upon at what stage of development alcohol was administered and whether the fish had the AB or the WT genotype. To confirm this conclusion and to further investigate the question at which developmental time point of exposure alcohol administered led to significant alteration of behavior of our fish, we conducted separate ANOVAs for each strain and each developmental time point of exposure group. This analysis confirmed what Figure 1 suggested. If the alcohol administered at age 8 or 16 hpf, AB fish developed no hyperactivity, i.e., neither the effect of Alcohol nor the Alcohol x Interval interaction was found significant. However, if the alcohol was administered at 24hpf

Journal Pre-proof (F(1, 54) = 10.68, p < 0.01), 32hpf (F(1, 56) = 8.12, p < 0.01) or 40hpf (F(1, 55) = 5.36, p < 0.05), significant hyperactivity resulted as measured by swim speed at the age of one week of these fish. It is also notable that the Alcohol x Interval interaction term was found nonsignificant for all age groups of the AB fish suggesting that the significant embryonic alcohol effect found for the latter three developmental time point of exposure groups was consistent throughout the behavioral recording session. ANOVA detected no significant alcohol effect in the wild type

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fish of any developmental time point of exposure group, but it did find the Alcohol x Interval

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interaction significant for the 8hpf (F(29, 1334) = 2.25, p < 0.001) and the 32 hpf (F(29, 1334) =

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2.62, p < 0.001) groups: alcohol treated fish of the former group exhibited decreased swim speed

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and alcohol treated fish of the latter group increased swim speed compared to their control counterparts during the second half of the recording session.

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Next, we analyzed Variance of Swimming speed. We emphasize that this variance

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represents within individual temporal variance, i.e., it is a measure of the inconsistency of speed with which the fish swam. The results shown in figure 2 depict a fairly similar picture to what

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we found for swim speed. It appears that alcohol administration during embryonic development

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increased the variability of swim speed in AB fish but not in WT fish. Furthermore, although the alcohol effect on AB appears strongest for developmental time point of exposure groups 24 and 32 hpf, all AB groups appear to respond robustly to this substance. ANOVA confirmed these observations and found a significant Interval effect (F(29, 14268) = 20.77, p < 0.001) and significant effect of Strain (F(1, 492) = 5.96, p < 0.05), but the main effect of Alcohol was nonsignificant. The interaction terms Alcohol x Strain (F1, 492) = 21.78, p < 0.001), Interval x Alcohol (F(29, 14268) = 1.47, p < 0.05) and Interval x Strain (F(29, 14268) = 2.78, p < 0.001) were also found significant, while the other interaction terms were non-significant. These results

Journal Pre-proof suggest that embryonic alcohol exposure affected variance of swim speed in a strain dependent manner, but at what stage it was administered did not make a difference. However, it is notable that ANOVA has been found insensitive (underpowered) to detect significance of interaction between main effects (Wahlsten, 1990), and thus to confirm these conclusions we conducted separate ANOVAs for each strain and each developmental time point of exposure group. This analysis confirmed what Figure 2 suggested. If the alcohol administered at age 8, 16 or 32 hpf,

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AB fish developed no increase in variability of speed, i.e., neither the effect of Alcohol nor the

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Alcohol x Interval interaction was found significant. However, if the alcohol was administered

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at 24hpf (F(1, 53) = 4.29, p < 0.05) or 40hpf (F(1, 55) = 6.15, p < 0.05), significantly elevated

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variance of swim speed was detected. It is also notable that the Alcohol x Interval interaction term was found nonsignificant for all age groups of the AB fish suggesting that the significant

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embryonic alcohol effect found for the latter two developmental time point of exposure groups

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was consistent throughout the behavioral recording session. ANOVA detected no significant alcohol effect in the wild type fish of any developmental time point of exposure group, but it did

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find the Alcohol x Interval interaction significant for the 8hpf group (F(29, 1334) = 1.79, p <

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0.01): alcohol treated fish of this group exhibited decreased variability of swim speed compared to control after the first third of the recording session. Angular velocity is shown in Figure 3. Similarly to what we found in the analysis of the previously discussed behavioral measures, administration of alcohol during embryonic development did not affect angular velocity in WT fish. Angular velocity also did not seem to change in AB fish, except perhaps in the group that received alcohol at 24th hpf. ANOVA revealed a significant Interval effect (F(29, 14268) = 49.12, p < 0.001), but the main effect of Alcohol and Strain was non-significant, whereas the effect of Developmental time point of

Journal Pre-proof exposure was (F(4, 492) = 2.61, p < 0.05). The interaction terms Alcohol x Strain bordered but did not reach the level of significance (F1, 492) = 3.29, p = 0.07). The interaction terms Interval x Strain (F(29, 14268) = 2.18, p < 0.001), Interval x Alcohol x Strain (F(29, 14268) = 1.87, p < 0.01) and Interval x Developmental time point of exposure x Strain (F(116, 14268) = 1.398, p < 0.01) were also found significant, while the other interaction terms were non-significant. These results suggest that embryonic alcohol exposure affected angular velocity in a strain dependent

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manner, but at what stage it was administered did not make a difference. Because ANOVA has

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been found insensitive to detect significance of interaction between main effects (Wahlsten,

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1990), and because we found significant triple interaction terms and a borderline significant

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Alcohol x Strain interaction, we conducted separate ANOVAs for each strain and each developmental time point of exposure group. These analyses confirmed what Figure 3

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suggested. For AB fish we found the effect of Alcohol to be significant only when it was

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administered at 24 hpf (F(1, 53) = 4.41, p < 0.05). The Interval x Alcohol interaction was nonsignificant for all developmental time point of exposure groups. For WT, the alcohol effect was

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non-significant for all developmental time point of exposure groups but the Interval x Alcohol

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interaction was significant for the 32 hpf group (F(29, 1334) = 1.94, p < 0.01). The results we obtained for intra-individual variance of angular velocity were highly similar to those of angular velocity and thus they are not shown here. Figure 4 shows the duration of time zebrafish stayed immobile. The figure suggests that AB zebrafish that received alcohol during their embryonic development remained immobile for shorter duration of time compared to their control (non-alcohol treated) counterparts, but WT zebrafish responded the opposite manner: alcohol treated WT zebrafish appear to show elevated immobility compared to control WT zebrafish. It also appears that the developmental time point

Journal Pre-proof at which alcohol was administered influenced the alcohol induced behavioral changes. ANOVA confirmed these observations and revealed a significant Interval effect (F(29, 14268) = 34.18, p < 0.001), as well as significant main effect of Strain (F(1, 492) = 25.52, p < 0.001) and of Developmental time point of exposure (F(4, 492) = 4.65, p < 0.001). Although the main effect of Alcohol was non-significant, interaction terms Alcohol x Strain was found highly significance (F1, 492) = 78.72, p < 0.001). The interaction terms Interval x Strain (F(29, 14268) = 6.13, p <

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0.001) and Interval x Alcohol x Strain (F(29, 14268) = 1.56, p < 0.05) were also found

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significant, while the other interaction terms were non-significant. Subsequent ANOVAs

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conducted separately for each strain and developmental time point of exposure showed that,

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although the strength of the effect of alcohol appeared to differ across the developmental time point of exposure groups of AB zebrafish, alcohol significantly decreased the duration of

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immobility irrespective of at what developmental time point it was administered (Alcohol main

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effect F(1, 51-56) > 4.42, p < 0.05), and the effect was consistent across the behavioral session (no significant Interval x Alcohol interaction). A similar set of findings was obtained for WT

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fish but in the opposite direction. All developmental time point of exposure groups exhibited a

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significant and interval independent increase of immobility (F(1, 45-46) > 4.06, p < 0.05), except fish that received alcohol at 32 hpf (for these WT fish the main effect of Alcohol was nonsignificant, but the Alcohol x Interval interaction was (F(29, 1334) = 2.88, p < 0.001). Figure 5 summarizes the results obtained for the number of times (frequency) zebrafish stayed immobile for every minute of the 30 min recording session. The figure suggests that AB zebrafish that received alcohol during their embryonic development stopped moving more frequently compared to their control (non-alcohol treated) counterparts, but WT zebrafish responded the opposite manner: alcohol treated WT zebrafish appear to show reduced frequency

Journal Pre-proof immobility compared to control WT zebrafish. It also appears that the developmental time point at which alcohol was administered influenced the alcohol induced behavioral changes. ANOVA confirmed these observations and revealed a significant Interval effect (F(29, 14268) = 59.26, p < 0.001) as well as significant main effect of Strain (F(1, 492) = 12.37, p < 0.001) and of Developmental time point of exposure (F(4, 492) = 7.90, p < 0.001). Although the main effect of Alcohol was non-significant, the interaction terms Interval x Alcohol (F(29, 14268) = 1.54, p

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< 0.05), Interval x Developmental time point of exposure (F(116, 14268) = 1.27, p < 0.05),

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Interval x Strain (F29, 14268) = 10.43, p < 0.001), Alcohol x Strain were found significant (F1,

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492) = 88.60, p < 0.001), while the other interaction terms were non-significant. Subsequent

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ANOVAs conducted separately for each strain and developmental time point of exposure showed that alcohol increased the frequency of immobility in AB zebrafish exposed to this

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substance at all developmental time points of exposure (F(1, 51-56) > 5.01, p < 0.05) except at 8

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hpf in which case its effect only bordered but did not reach significance (F(1, 51) = 3.84, p = 0.056). The interaction between Interval and Alcohol was non-significant for all Developmental

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time point of exposure groups in AB fish. For the WT zebrafish ANOVA found all

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developmental time point of exposure groups to exhibit a significant decrease of frequency of immobility (F(1, 45-46) > 4.69, p < 0.05), an effect that was independent of Interval except for WT fish treated at 16 hpf, for which the Interval x Alcohol interaction term was found significant (F(29, 1305) = 1.50, p < 0.05). Figure 6 shows the results obtained for the duration of time for which zebrafish stayed near the wall, a response termed thigmotaxis. The figure suggests that both AB and WT zebrafish responded to embryonic alcohol treatment in a manner that was dependent upon the stage of development at which the alcohol was administered. ANOVA confirmed this

Journal Pre-proof observation and found a significant Interval effect (F(29, 14268) = 19.47, p < 0.001), a significant main effect of Strain (F(1, 492) = 31.06, p < 0.001) and of Developmental time point of exposure (F(4, 492) = 5.15, p < 0.001). Although the main effect of Alcohol was nonsignificant, the Interval x Alcohol x Strain (F(29, 14268) = 3.31, p < 0.001), the Interval x Alcohol x Strain x Developmental time point of exposure, the Alcohol x Developmental time point of exposure (F(4, 492) = 3.22, p < 0.05) and Developmental time point of exposure x Strain

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(F(4, 492) = 2.48, p < 0.05) interactions were all significant. Subsequent ANOVAs conducted

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separately for each strain and developmental time point of exposure showed that AB fish

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receiving alcohol at their 8th hpf (Alcohol F(1, 53) = 4.27, p < 0.05) or 24th hpf developmental

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time point of exposure significantly reduced their thigmotaxis duration (Alcohol F(1, 53) = 5.91, p < 0.05), and in the latter the reduction was more pronounced at the first two thirds of the

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session and subsided by the end (Alcohol x Interval interaction F(29, 1537) = 3.27, p < 0.001),

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while AB fish receiving alcohol at other developmental time points of exposure did not significantly change their thigmotaxis duration. On the other hand, unlike AB, WT zebrafish

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treated with alcohol increased their thigmotaxis but only when the alcohol was administered at

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the 16th hpf stage (Alcohol F(1, 45) = 6.85, p < 0.05), a change that was more prevalent for the first half of the session and subsided by the end (Interval x Alcohol F(29, 1305) = 2.43, p < 0.001). Alcohol delivered at other stages of development had no significant effect on thigmotaxis. The pattern of results for thigmotaxis frequency (figure 7), i.e. the number of times the fish entered the thigmotaxis annulus area, was highly different from that obtained for duration of thigmotaxis. AB zebrafish exposed to alcohol during embryonic development showed an apparent increase of the number of visits to the thigmotaxis area, while WT zebrafish seemed to

Journal Pre-proof remain unaffected by embryonic alcohol exposure. These observations were corroborated by ANOVA, which found a significant Interval effect (F(29, 14268) = 7.97, p < 0.001), a significant main effect of Strain (F(1, 492) = 74.04, p < 0.001) and of Developmental time point of exposure (F(4, 492) = 9.98, p < 0.001). Although the main effect of Alcohol only bordered significance (F(1, 492) = 3.74, p = 0.054), the Interval x Alcohol x Strain (F(29, 14268) = 1.51, p < 0.05), Developmental time point of exposure x Strain (F(4, 492) = 3.21, p < 0.05) and Interval x

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Developmental time point of exposure x Strain interaction (F(116, 14268) = 1.36, p < 0.01)

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terms were all significant. Subsequent ANOVAs conducted separately for each strain and

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developmental time point of exposure showed that all AB zebrafish except those receiving

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alcohol at their 8th hpf significantly increased their thigmotaxis frequency (Alcohol F(1, 53) > 5.40, p < 0.05) and this increase was consistent across the recording session (Interval x Alcohol

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interaction was non-significant). On the other hand, unlike AB, WT zebrafish treated with

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alcohol did not alter the frequency of entries to the thigmotaxis area The Alcohol effect and

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Discussion

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Interval x Alcohol interaction were both found non-significant.

Previously, embryonic alcohol exposure employed at 24 hpf in zebrafish was found to lead to lasting behavioral changes affecting how sexually mature young adult zebrafish respond to social stimuli (Fernandes & Gerlai, 2009; Buske & Gerlai, 2011; Fernades et al., 2015). The impaired social response demonstrated in these fish was found not to be accompanied by any other defects. Motor function, perception, (Fernandes & Gerlai, 2009) and anxiety-like or fear responses (Seguin et al., 2016) were found to remain unaltered in zebrafish exposed to embryonic alcohol compared to control, alcohol un-exposed, fish. Furthermore, the alcohol

Journal Pre-proof exposed fish were found to develop normally, exhibited no signs of anatomical alterations and showed no increase of mortality (Fernandes & Gerlai, 2009). Importantly, the exposure method, and the highest dose of alcohol employed in these previous studies, corresponded to our current method, except that in the current study we investigated the effect of embryonic alcohol exposure not only when this exposure was conducted at 24 hpf, but also before or after this developmental time point of exposure.

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Our current study demonstrates that embryonic alcohol does alter motor function, and

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perhaps anxiety-like behaviors too, in zebrafish, and that these alterations are detectable as early

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as by the 7th day post-fertilization stage of the developing juveniles. Alcohol was found to affect

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a number of swim path parameters of the juvenile zebrafish in a complex and behavior measure dependent manner. Furthermore, the alcohol effects were also developmental time point of

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exposure and strain dependent. For example, ABsk zebrafish exposed to alcohol during their

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embryonic development showed increased swim speed, increased temporal variance of swim speed, reduced angular velocity, reduced duration of immobility and increased frequency of

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immobility. In other words, although the alcohol exposed ABsk fish swam generally faster, they

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also varied their speed more, stopped more often and swam more in a straight line compared to control, alcohol unexposed, fish. The effect of alcohol on this strain of zebrafish was found to be generally strongest when alcohol was administered at or 8 hours before or after the 24th hpf stage. Thigmotaxis, a behavior often interpreted as a sign of fear or anxiety in rodents (Simon et al., 1994), was also affected by embryonic alcohol exposure. Frequency of visits to the thigmotaxis zone was increased fairly consistently across the AB sk zebrafish exposed to alcohol at different stages of their development, while the duration of time they spent in the thigmotaxis

Journal Pre-proof zone was affected by alcohol in manner that was dependent upon the developmental time point at which this substance was administered. Importantly, WT zebrafish treated and measured in a manner identical to AB sk, either showed less robust alterations or occasionally responded to embryonic alcohol treatment opposite to how alcohol exposed AB sk zebrafish behaved. For example, swim speed and the variability of swim speed either did not increase or somewhat decreased depending on when WT

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fish received the embryonic alcohol treatment. Duration of immobility increased while

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frequency of immobility decreased in WT groups exposed to alcohol, an effect opposite to what

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was found for ABsk. Last, thigmotaxis frequency was unaffected by embryonic alcohol in WT

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fish, and thigmotaxis duration responded in a way opposite to what we found for AB sk. In

exposure dependent alcohol effect.

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summary, this pattern of results demonstrates a genotype and often developmental time point of

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The genotype x alcohol x developmental time point of exposure interactions revealed in this study represent important possibilities for future research. First, genotype dependent alcohol

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effects imply that one may be able to identify, using quantitative trait locus analysis for example,

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genes and subsequently the biochemical mechanisms that mediate or influence alcohol effects during embryonic development. Identification of such mechanisms may lead to development of therapeutic applications and/or discovery of biomarkers that would aid diagnosis. Second, the developmental time point of exposure dependency of alcohol effects is also an important discovery as it may allow one to identify sensitive periods of embryonic development during which the developing human fetus is particularly vulnerable to the teratogenic effects of alcohol. From this proof of concept analysis with zebrafish, it appears that for this species, the 24 th hpf developmental time point of exposure and perhaps also the 16th and 32nd hpf stage represent

Journal Pre-proof particularly sensitive periods. Why these periods are particularly sensitive, i.e. what mechanisms alcohol engages, and how they alter brain development and behavioral performance at later stages of the development of the zebrafish are questions to which the answers are only speculative at this point. At and around 24th hpf, the brain of zebrafish has already started to form but has not completed its structural development (Kimmel et al., 1995) At this stage precursor cells start to differentiate into neurons (Tropepe & Sive, 2003). Cell migration is

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robust, and neurons start growing axons and dendrites, and start establishing connections

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(Devine & Key, 2003; Kimmel et al., 1995). In other words, a myriad of developmental

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processes, along which a large number of molecular and biochemical changes, occur at this stage

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of development in the zebrafish brain. It is thus not surprising that a complex drug, like alcohol, exerts a particularly robust effect when administered at this time point of ontogenesis. Although

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pinpointing all the molecular, or other neurobiological, mechanisms altered by alcohol

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administration in the developing zebrafish brain is not possible at this point, we do know that embryonic alcohol exposure increases apoptotic cell death (Chatterjee et al., unpublished

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results), disrupts the development and later functioning of the dopaminergic neurotransmitter

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system (Buske & Gerlai, 2011; Fernandes et al., 2015), and reduces the expression of numerous neuronal markers, e.g. NCAM, BDNF and synaptophysin, throughout the brain, known to be involved in neuronal plasticity, synaptic function and drug addiction (Mahabir et al., 2018). Although the correspondence between zebrafish and human embryonic developmental stages is not in complete agreement with regard to all organs, tissues or even brain areas, the zebrafish developmental stages we found to be most sensitive to alcohol exposure are approximately equivalent to the beginning to the middle of the second trimester of pregnancy in humans. Nevertheless, even our limited behavioral results suggest that there may be no period of

Journal Pre-proof embryonic development during which alcohol may be consumed safely. We found alcohol to have the least severe effect perhaps when administered at 8 hpf, but even for these fish certain behaviors showed abnormality. Thus, the alcohol effects were dependent upon not only the strain and the developmental timing of its delivery but also on what behavioral measures we analyzed. In summary, although may not be maximally teratogenic outside the most sensitive periods, alcohol may affect numerous developmental processes throughout the ontogenesis of the

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embryo leading to distinct behavioral abnormalities that depend upon at what stage of

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development this substance reached the embryo.

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The next question we consider is why WT fish may have been less affected by embryonic

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alcohol compared to ABsk. Strain differences in responses to acute and chronic alcohol exposure (Pan et al., 2012; Pannia et al., 2014; Chatterjee et al., 2014; Gerlai et al., 2009), as well as in the

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effects of embryonic alcohol exposure (Mahabir et al., 2014; Loucks & Carvan, 2004) have been

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demonstrated in zebrafish, and are thought to mimic, in general, what is found in mammals including humans. Genetically heterogeneous populations in which individuals exhibit higher

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degree of heterozygosity have often been found to be “buffered” against environmental insults.

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The phenomenon is analogous to what is called hybrid vigor, or heterosis, in agriculture (Baranwal et al., 2012). The WT zebrafish we tested in the current study originate from commercial breeding facilities where a very large number of individuals are bred. Increasing the effective population size (the number of individuals that breed) decreases inbreeding, thus the WT fish we used in the current study are expected to show high degree of heterozygosity. In contrast, the majority of the loci of the genome of the AB strain is known to be in a homozygous form (Coe et al., 2009). Thus, we argue that the blunted alcohol effects we found in our WT fish are likely due to genetic buffering resulting from high degree of heterozygosity.

Journal Pre-proof Last, we consider why alterations in motor function and anxiety-like responses we found in juvenile zebrafish were found undetected in previous studies using adult zebrafish (e.g. Fernandes & Gerlai, 2009). The answer to this question is speculative at this point. At 7 dpf the brain of the developing zebrafish is similar to that of the adult in terms of gross neuroanatomy. Nevertheless, the connectome of this young brain is likely far from fully established. It appears that by the time the zebrafish reaches sexual maturity, motor function and anxiety-like responses

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in the alcohol treated fish become restored, perhaps due to compensatory mechanisms associated

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with altered connectivity and/or molecular mechanism that counteract the early deleterious

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effects of embryonic alcohol exposure.

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In summary, the current study demonstrated significant and behavior specific embryonic alcohol exposure induced changes manifesting in altered motor function and anxiety-like

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responses in zebrafish that depended upon the genotype of the fish and the developmental time

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point at which alcohol was administered. Although the mechanisms underlying the developmental time point of exposure and genotype dependency are unknown at this point, this

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proof of concept analysis demonstrates that zebrafish may be an appropriate tool with which one

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may investigate these questions.

BT & RG conceptualized, designed, and oversaw the study AA, LT, ZN, IP & BT performed the experiments and conducted behavioral testing AA & ZN performed behavioral quantification RG & AA performed statistical analysis BT & RG prepared and wrote the manuscript with input from all authors

Journal Pre-proof Acknowledgements Supported by NSERC Discovery Grant (311637) and University of Toronto Mississauga ROP funding to RG.

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Figure legends Figure 1. Swim speed (mean + S.E.M) as a function of 1-min time intervals is shown. The embryonic alcohol treatment (black squares 1.0 % vol/vol; or grey circles 0% vol/vol, control) is indicated by the legend. The upper row of graphs shows results for AB strain zebrafish, the lower row of graphs for the WT population. The developmental time point

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(DevelStage) at which alcohol was administered to the embryo is indicated by the number

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in hours post-fertilization along with the strain/population origin of the fish above each graph. Note that swim speed was found significantly affected by embryonic alcohol

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treatment in a developmental time point of exposure and strain dependent manner.

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Figure 2. Variance of swim speed (mean + S.E.M) as a function of 1-min time intervals is

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shown. Note that this variance represents the within individual temporal variability of behavioral performance, i.e. is a measure of how inconsistently the fish swam. The

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embryonic alcohol treatment (black squares 1.0 % vol/vol; or grey circles 0% vol/vol,

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control) is indicated by the legend. The upper row of graphs shows results for AB strain zebrafish, the lower row of graphs for the WT population. The developmental time point

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(DevelStage) at which alcohol was administered to the embryo is indicated by the number in hours post-fertilization along with the strain/population origin of the fish above each graph. Note that variance of swim speed was found significantly affected by embryonic alcohol treatment in a developmental time point of exposure and strain dependent manner. Figure 3. Angular velocity, i.e. the speed of turning, (mean + S.E.M) as a function of 1-min time intervals is shown. The embryonic alcohol treatment (black squares 1.0 % vol/vol; or grey circles 0% vol/vol, control) is indicated by the legend. The upper row of graphs shows results for AB strain zebrafish, the lower row of graphs for the WT population. The

Journal Pre-proof developmental time point (DevelStage) at which alcohol was administered to the embryo is indicated by the number in hours post-fertilization along with the strain/population origin of the fish above each graph. Note that angular velocity was found significantly affected by embryonic alcohol treatment in a developmental time point of exposure and strain dependent manner. Figure 4. Duration of Immobility (or freezing) (mean + S.E.M) as a function of 1-min time

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intervals is shown. The embryonic alcohol treatment (black squares 1.0 % vol/vol; or grey

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circles 0% vol/vol, control) is indicated by the legend. The upper row of graphs shows

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results for AB strain zebrafish, the lower row of graphs for the WT population. The

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developmental time point (DevelStage) at which alcohol was administered to the embryo is indicated by the number in hours post-fertilization along with the strain/population origin

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of the fish above each graph. Note that duration of immobility was found significantly

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affected by embryonic alcohol treatment in a strain dependent manner. Figure 5. Frequency of Immobility (or freezing) (mean + S.E.M) as a function of 1-min time

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intervals is shown. The embryonic alcohol treatment (black squares 1.0 % vol/vol; or grey

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circles 0% vol/vol, control) is indicated by the legend. The upper row of graphs shows results for AB strain zebrafish, the lower row of graphs for the WT population. The developmental time point (DevelStage) at which alcohol was administered to the embryo is indicated by the number in hours post-fertilization along with the strain/population origin of the fish above each graph. Note that frequency of immobility was found significantly affected by embryonic alcohol treatment in a developmental time point of exposure and strain dependent manner.

Journal Pre-proof Figure 6. The amount of time fish spent in the Thigmotaxis zone (mean + S.E.M) as a function of 1-min time intervals is shown. The embryonic alcohol treatment (black squares 1.0 % vol/vol; or grey circles 0% vol/vol, control) is indicated by the legend. The upper row of graphs shows results for AB strain zebrafish, the lower row of graphs for the WT population. The developmental time point (DevelStage) at which alcohol was administered to the embryo is indicated by the number in hours post-fertilization along

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with the strain/population origin of the fish above each graph. Note that Thigmotaxis

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duration was found significantly affected by embryonic alcohol treatment in a

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developmental time point of exposure and strain dependent manner.

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Figure 7. The number of times fish entered the Thigmotaxis zone (frequency) (mean + S.E.M) as a function of 1-min time intervals is shown. The embryonic alcohol treatment (black

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squares 1.0 % vol/vol; or grey circles 0% vol/vol, control) is indicated by the legend. The

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upper row of graphs shows results for AB strain zebrafish, the lower row of graphs for the WT population. The developmental time point (DevelStage) at which alcohol was

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administered to the embryo is indicated by the number in hours post-fertilization along

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with the strain/population origin of the fish above each graph. Note that Thigmotaxis frequency was found significantly affected by embryonic alcohol treatment in a developmental time point of exposure and strain dependent manner.

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Highlights The behavioral effects of low concentration embryonic alcohol exposure are studied Embryonic alcohol effects were detected as early as 7 days post-fertilization in zebrafish The effects dependent upon the strain and developmental time of alcohol exposure The effects were most robust in ABsk fish that received alcohol around 24 hours postfertilization WT, highly heterozygous, zebrafish were less affected by embryonic alcohol treatment

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Ethical statement All experiments described in this manuscript were performed in compliance with local (University of Toronto), Provincial (Ontario) and Federal (Canada) guidelines for the ethical use of animals in research. All experiments have been reviewed and approved by the Local Animal Care Committee (LACC) of the University of Toronto Mississauga and University Animal Care Committee (UACC) of the University of Toronto.

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