Alcohol exposure during embryonic development: An opportunity to conduct systematic developmental time course analyses in zebrafish

Neuroscience and Biobehavioral Reviews 98 (2019) 185–193

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Alcohol exposure during embryonic development: An opportunity to conduct systematic developmental time course analyses in zebrafish Amanda Facciola, Benjamin Tsangb, Robert Gerlaia,b, a b

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Univeristy of Toronto, Department of Cell and Systems Biology, 3359 Mississauga Road, Mississauga, Ontario, L5L 1C6, Canada University of Toronto Mississauga, Department of Psychology, 3359 Mississauga Road, Mississauga, Ontario, L5L 1C6, Canada

A R T I C LE I N FO

A B S T R A C T

Keywords: Anxiety Fetal alcohol spectrum disorder Learning and memory Social behaviour Zebrafish

Ethanol affects numerous neurobiological processes depending upon the developmental stage at which it reaches the vertebrate embryo. Exposure time dependency may explain the variable severity and manifestation of lifelong symptoms observed in fetal alcohol spectrum disorder (FASD) patients. Characterization of behavioural deficits will help us understand developmental stage-dependency and its underlying biological mechanisms. Here we highlight pioneering studies that model FASD using zebrafish, including those that demonstrated developmental stage-dependency of alcohol effects on some behaviours. We also succinctly review the more expansive mammalian literature, briefly discuss potential developmental stage dependent biological mechanisms alcohol alters, and review some of the disadvantages of mammalian systems versus the zebrafish. We stress that the temporal control of alcohol administration in the externally developing zebrafish gives unprecedented precision and is a major advantage of this species over other model organisms employed so far. We also emphasize that the zebrafish is well suited for high throughput screening and will allow systematic exploration of embryonic-stage dependent alcohol effects via mutagenesis and drug screens.

1. Introduction Exposure to alcohol (ethanol, ethyl alcohol, EtOH) during embryonic development resulting in Fetal Alcohol Spectrum Disorders (FASD) remains the leading cause of preventable form of mental disability in the western society (Clark and Gibbard, 2013). For example, as many as 30% of women in the United States admit to have drunken alcohol during their pregnancy, with approximately 5% of pregnant women being characterized as alcoholics (McHugh et al., 2014). The resulting global prevalence rate for FASD is staggering: It is estimated to be between 1–11% of the population depending on region or country examined (Lange et al., 2017). Not surprisingly, the most prevalent subset of FASD patients fall within the categories of the milder forms of the disease termed, e.g., Alcohol Related Neurodevelopmental Disorders (ARND) or partial alcohol (Roozen et al., 2016). Despite global popularity of alcohol, the question of what neurobiological mechanisms alcohol alters in the developing vertebrate brain remains largely unanswered. Although abnormalities in the most severe forms of FASD are clearly observable, including cranio-facial deformities (small eyes, thin lips, small head), symptoms in the less severe forms are also devastating and include a variety of less obvious problems, e.g. growth-related, and behavioural or cognitive deficits (Jones et al., 1973; Streissguth et al.,

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1978). For example, FASD patients are more likely to have low birth weight, suffer from problems in school, including social behaviour problems, learning and memory issues, and are more likely to abuse drugs, including alcohol, later in life (Streissguth, 1997). Although the solution for FASD should be simple, abstinence from alcohol at least during pregnancy, complex societal attitudes towards alcohol consumption make it impossible to protect the developing fetus from this substance. For example, some believe small amounts of alcohol in the early or late stages of pregnancy can be harmless (Kelly et al., 2013), or even beneficial, as it is believed to aid with the onset of labour. While consuming one alcoholic drink is not enough to cause severe facial dysmorphia, there is no confirmed “safe” amount or time to consume alcohol during pregnancy, as alcohol easily crosses the blood brain barrier, exposing the brain of the developing fetus to teratogenic effects of this substance (Banks, 1999; Lovley et al., 2016). Studying humans with FASD has proven useful in understanding various aspects of FASD. Nevertheless, human studies are mostly limited to questionnaires, correlative analyses and non-invasive measures due to ethical considerations and/or technical issues. Therefore, research in recent years has turned to animal models, including rodents, and more recently zebrafish, to further our understanding of the mechanisms that underlie FASD (Patten et al., 2014). Animal models allow

Corresponding author at: 3359 Mississauga Road, Mississauga, Ontario, L5L 1C6, Canada. E-mail address: [email protected] (R. Gerlai).

https://doi.org/10.1016/j.neubiorev.2019.01.012 Received 14 September 2018; Received in revised form 9 January 2019; Accepted 11 January 2019 Available online 11 January 2019 0149-7634/ © 2019 Elsevier Ltd. All rights reserved.

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produce marked hyperactivity (Schneider et al., 2011), whereas prenatal exposure during GD 7–18 of mice produced no significant increase in activity (Downing et al., 2009). In learning and memory, fear conditioning seems to be most significantly altered by exposure of the rodent embryo to alcohol in early postnatal periods (Hunt et al., 2009; Kelly et al., 2009), whereas passive avoidance deficits were produced with late gestational exposure (Schneider et al., 2011). However, significant impairments in the Morris Water Maze task were seen with both prenatal (GD 1–22) (Gianoulakis, 1990) and postnatal (PD 4–9) (Kelly et al., 1988, 2009) alcohol exposure in rodents. Differential effects of alcohol have also been demonstrated by varying the timing of exposure within the post-natal period, as alteration in fear conditioning in adult rodents was shown to be greater when pups were exposed during PD 4–6, and less robust with exposure between PD7-9 (Hunt et al., 2009). Similarly, social behaviour was also found to be altered with both prenatal (Hamilton et al., 2010; Lugo et al., 2003) and postnatal exposure (Patten et al., 2014). The fact that learning and social behaviour are altered by embryonic alcohol exposure in a developmental stage dependent manner, but at the same time alcohol having significant effects on these behaviours both when it was administered pre-as well as postnatally confirms these behaviours may not be dependent upon a single developmental mechanism, but instead rely on multiple developmental processes.

better control of numerous factors potentially influencing the outcome of alcohol exposure. For example, inbred strains and the well controlled laboratory environment allow researchers to parse out the effects of genetic and environmental factors, ensuring reliability and validity of results. An important conundrum of FASD research concerns why there is large variability in the expression and severity of the symptoms among FASD patients. For example, children of mothers who binge drink have been found to exhibit a variety of different impairments that also vary in terms of severity despite similar amount of alcohol consumed recorded for their mothers (Warren et al., 2011). Among several factors, a possibly important one is timing, i.e., the developmental stage at which the fetus is exposed to alcohol. May et al. (2013) surveyed mothers on the quantity, frequency, and timing of alcohol consumption, and found correlations between onset of drinking and the severity of FASD symptoms. For example, earlier drinking (within the first trimester) was found to be associated with more severe expression of FASD symptoms compared to the effects of drinking during the second or third trimesters. Nevertheless, detrimental effects of alcohol were found to be associated with drinking during all trimesters, supporting the notion there is no “safe time” to drink alcohol during pregnancy (May et al., 2013). In this review we focus on the question of developmental stage dependency of alcohol effects in a particular model organism, the zebrafish, and on a particular cluster of phenotypes, behaviour. Our rationale for this focus is as follows. One, although the zebrafish is a novice in FASD research, it possesses numerous features that we argue will significantly advance our understanding of FASD. Two, we also argue that this research perhaps is best conducted with comprehensive large-scale investigation of the behavioural consequences of embryonic alcohol exposure. Before we delve into these specific questions, however, we provide a succinct review of the more expansive mammalian literature.

1.2. Neurobiological mechanisms underlying embryonic alcohol exposure induced abnormalities in mammals Alcohol is a highly complex drug from a pharmacology standpoint, as it is known to directly interact with a large number of molecular targets (Abrahao et al., 2017) and is also known to indirectly affect a cascade of biochemical mechanisms (Kane et al., 2013). These complex effects may alter embryonic developmental processes in a manner that is potentially highly developmental stage-dependent. Nevertheless, systematic analysis to address this question has not been conducted. Here, we briefly mention some relevant examples of mechanisms known to be engaged by embryonic alcohol exposure. The vast number of processes alcohol may affect include molecular targets subserving apoptosis, neurogenesis, and epigenetic mechanisms (Isayama et al., 2009; Kane et al., 2013). For example, alcohol has been found to affect the levels of BAX and BLC-2, proteins that mediate cell death (Young et al., 2003). Hyperactivation of GABA-A receptors, in addition to the blockade of NMDA receptors by alcohol, is also believed to contribute to apoptotic cell death (Olney et al., 2002). Changes in levels of neurotrophic factors including brain-derived neurotrophic factor (BDNF), nerve growth factor (NGF) and Insulin-Like Growth factor (IGF) induced by embryonic alcohol resulting in abnormal developmental processes including neuronal cell migration and neurite growth have also been demonstrated. The ability of ethanol to bind to and activate GABA-A receptors is also hypothesized to result in robust structural alterations in the developing brain, including abnormal development of cortical layers and neuronal circuitry (Ledig et al., 1988). Although several studies have focussed on the devastating effect of embryonic alcohol exposure on the GABAergic system, numerous other neurotransmitter systems have also been found to be robustly affected in rodents (Isayama et al., 2009; Olney et al., 2002). Furthermore, using a histamine H3 receptor antagonist, Savage et al (2010) have been able to ameliorate prenatal alcohol exposure induced spatial learning deficits in rats, suggesting the involvement of the histaminergic system. Importantly, similar to behavioural deficits, molecular targets in animal models of FASD have also been found to be differentially influenced depending on timing of exposure to alcohol (Kane et al., 2013). The latter authors examined ethanol exposure in rodents during the second trimester equivalent (GD 11–21) and found loss of cortical neurons with no effect on thalamic neurons, whereas later exposure (PD 4; third trimester equivalent) led to loss of Purkinje neurons in the cerebellum. Ethanol exposure during PD 6–8 and PD 13–15 resulted in

1.1. Traditional animal models: Rodents in alcohol research Rodents, particularly rats and mice, the favourites of biomedical research, have been the most commonly used animal models in alcoholrelated research (Patten et al., 2014; Valenzuela et al., 2012). Developmental stage-dependent effects of alcohol exposure have also been extensively studied using rodents. Embryonic developmental stage-dependent effects of alcohol have been demonstrated in both brain morphology (Valenzuela et al., 2012) and on a variety of behaviours, including hyperactivity (Downing et al., 2009; Schneider et al., 2011), social behaviour (Hamilton et al., 2010; Lugo et al., 2003; Marquardt and Brigman, 2016; Patten et al., 2014) and learning (Gianoulakis, 1990; Hunt et al., 2009; Kelly et al., 1988; Marquardt and Brigman, 2016; Patten et al., 2014; Schneider et al., 2011; Brady et al., 2012), alterations that mirrored behavioural deficits found in humans with FASD. Exposure to alcohol during late gestational stages (equivalent to the human second trimester) was found to reduce both brain and body weight, however, exposure during postnatal days (PD) 2–10 (third trimester equivalent) selectively reduced brain weight without appreciable alteration in body size (Tran et al., 2000). Exposure to alcohol during PD 1–10 has been shown to have the most deleterious effects in both rats and mice, resulting in significant arrest of brain development (particularity affecting the cerebellum and forebrain) (Maier et al., 1997, 1999) and also leading to increased apoptotic cell death (Dunty et al., 2001), impairment of learning and memory (Hunt et al., 2009; Patten et al., 2014; Wagner et al., 2014) and abnormal social behaviour (Kelly and Tran, 1997; Kelly et al., 2009). Furthermore, rat pups exposed to ethanol during PD 4–6 suffered from robust memory impairment when tested at their adult stage, but pups exposed to the same concentration of ethanol between PD 7–9 had less impairment of memory when tested in adulthood, suggesting critical periods of development within the third trimester equivalent (Hunt et al., 2009). Neonatal alcohol exposure of rodents as well as primates was found to 186

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1996) on the mother leading to abnormal maternal care (e.g. lower levels of pup licking and grooming) can also influence or interact with the effects of alcohol on the behaviour of the offspring later in life (Brancato et al., 2016; Marquardt and Brigman, 2016; Wilson et al., 1996; but also see Allan et al., 2014), complications not present for zebrafish, a species that does not provide parental care for their offspring, and one which breeds via external fertilization and whose offspring develop outside of the mother. Could one thus regard the zebrafish as a realistic model of human FASD? After all, none of the above listed important factors associated with the intrauterine development of the human fetus and the subsequent maternal care influence the effects of alcohol administered during development in the zebrafish. From this perspective, the zebrafish is clearly not an appropriate model. However, as is the case in all preclinical studies, animal models are not employed to recapitulate all features of the human disease or to include all factors that influence it. We argue that the zebrafish FASD model allows us to focus on the most important among the disease-causing factors, the direct effects of alcohol on the fetus, and thus this model allows one to dissociate such effects from the effect of all other factors we mentioned above as being present in mammals but absent in the zebrafish. We also have to mention several features of rodents that represent clear advantages of these species over the zebrafish. For example, the behaviour of rodents is much better characterized than that of the zebrafish, and a larger number of behavioural paradigms has been developed for the former species too, although the zebrafish is gaining increasing momentum in this context (Stewart et al., 2015; Kalueff et al., 2014). Furthermore, decades of research have been conducted characterizing altered brain function using non-behavioural methods in rodents. Although the zebrafish is somewhat behind, it too is amenable to such analysis. In fact, the zebrafish brain can be imaged in vivo (Liao et al., 2018; Moeyaert et al., 2018), given that the fish remain practically transparent throughout their development. Adult fish may also be amenable to sophisticated in vivo imaging given that some zebrafish strains, e.g. the casper strain, are pigment deficient (Antinucci and Hindges, 2016; Fernandes et al., 2019). Although the zebrafish brain is much smaller than the rodent brain, and thus may be more difficult to image and manipulate, an entire field is emerging using optogenetic techniques to study and modify in vivo brain function based upon the relative optical transparency of the zebrafish (Simmich et al., 2012; Umeda and Shoji, 2017), a major advantage compared to rodents.

reduced expression of BDNF, whereas glial cell derived neurotrophic factor (GDNF) was only sensitive to exposure to ethanol during PD 6–8 (Kane et al., 2013). Developmental ethanol exposure related epigenetic changes have recently become the focus of a number of studies. Although not yet investigated in terms of developmental stage-dependency, epigenetic mechanisms such as alteration in histone acetylation, chromatin remodelling, DNA methylation and microRNAs have all been shown to be associated with behavioural deficits observed in FASD models (Kane et al., 2013). An in depth summary of these molecular targets and processes is given by Kane et al. (2013). In summary, there is substantial evidence from the mammalian (rodent, non-human primate and human) literature demonstrating the developmental stage dependency of embryonic alcohol effects both on behaviour and on some neurobiological processes. However, the connection between behavioural changes and neurobiological alterations remains unclear, and the sporadic analyses conducted for specific developmental time points make it difficult to draw conclusions about the reasons for developmental stage dependency of the alcohol induced changes. Studies in which the potential developmental stage-dependency of alcohol’s effects is systematically analyzed are needed. Although rodents clearly have numerous merits in such analyses, we argue that the zebrafish is particularly appropriate for this purpose for practical reasons. 2. Rodents: excellent models but not without practical complications Although rodents have proven to be excellent translational models for human FASD, and although they have become the primary laboratory organisms of biomedical research, their use is not without limitations. For example, the methods of administration of alcohol and of several other drugs are often not ideal for these species, requiring invasive procedures including injections or oral gavage. Less invasive methods, e.g. spontaneous (as opposed to forced) ingestion (via either food or water) or forced vapour inhalation have also been successfully employed (Kelly et al., 2009; Patten et al., 2014; Schneider et al., 2011). However, these latter methods do not allow precise control of the amount of alcohol administered, and also make the control of timing of alcohol delivery complicated. Another issue with rodent models of FASD, at least from the perspective of alcohol administration methods, is that rodent embryos develop intra-utero, at least for a substantial proportion of their development. The rodent (mouse or rat) embryo is born at a developmental stage that corresponds to the beginning of the 3rd trimester of the human fetus. Thus, the human third trimester is equivalent to post-natal days 1–10, during which the rodent embryo is developing outside of the mother (Patten et al., 2014). During this stage, alcohol may be administered directly to the embryo, but prior to this stage, alcohol delivery to the embryo has to be achieved via administration of this substance to the mother. This represents complications for two reasons. One, maternal physiology, e.g., the mother’s genetic makeup, health status, etc., may significantly affect how much alcohol and how soon may reach the rodent embryo developing in utero. Second, comparison of alcohol effects before and after post-natal day 1 is complex, due to the presence vs. absence of the effects of the above-mentioned maternal factors. Furthermore, during early embryonic development, rat and mouse pups rely primarily on their mothers for food and care. Notably, adult behaviour in rodents has been demonstrated to be dependent upon early life experience during maternal care (Fleming et al., 1999), which thus also represents a confound for investigating the effects of developmental alcohol exposure on behaviours later in life (Pueta et al., 2008). Last, it is also notable that when alcohol is administered to the pre-natal rodent pups through injecting the substance into the mother, indirect effects of stress (Weinstock, 2005) or of alcohol (Wilson et al.,

3. The zebrafish: A potentially good choice for systematic and large-scale analyses of embryonic alcohol exposure induced changes in vertebrates One can list several arguments for the use of the zebrafish in FASD research. One of these, the comparative perspective, is often underappreciated. According to this perspective, even if this particular species has no major advantages over rodents, or over other laboratory species, it should still be included, because comparing more than two species (usually humans and mice) is expected to enhance translational relevance (Gerlai, 2014a). This idea is not new. Entire scientific societies have been formed based upon this principle, and comparative studies have shown their power across a broad range of subdisciplines in biology. Discussing all the arguments and providing a comprehensive list of examples would be beyond the scope of this focussed review. Instead, the reader is referred to one of the most seminal papers in this topic (Beach, 1950), and is given a brief recount of some of the main arguments, we find particularly relevant, from the perspective of using zebrafish in FASD research. If one assumes at least some level of evolutionary relatedness among vertebrates, an assumption that is certainly correct according to the overwhelming evidence supporting Darwinian evolution, then one should also accept that some features of biology should be shared among the studied species. The question then becomes how one could 187

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rodents for another reason. The embryo can be directly manipulated without invasive procedures. For example, compounds and drugs may be administered by simply immersing the eggs or the hatched embryo into the drug solution, and thus the developmental timing, the length, and the dose of drug exposure can be precisely controlled (Magno et al., 2015; Tran and Gerlai, 2013). Although alcohol administration via oral gavage allows such precision with rodents, it is invasive (stressful) and it can only be employed with the mother or the newly born pup, and not intra-utero. Even such issues as ADME (absorption, distribution, metabolism and excretion) characteristics of the administered compound may be less complicated in zebrafish as compared to mammals, because the immersion-based drug administration procedure practically “clamps” the dose, i.e. it maintains a steady drug level inside the embryo as long as it is immersed in the drug solution. A further important benefit of immersion-based drug administration is the ability to avoid anxiety, fear or pain induced by human handling typical in mammalian models (Clark et al., 2011; Patten et al., 2014). Another advantage of external fertilization and external development of the zebrafish embryo is that this development, as we emphasized above, is not divided into internal and external phases, or pre- and post-natal periods as in rodents (Patten et al., 2014). Nevertheless, a complication with zebrafish, somewhat analogous to the pre- and post-natal phase issue in rodents, is that the zebrafish embryo remains inside the egg only until its age of 70 h post-fertilization (hpf), after which the fish hatch and continue their embryonic development outside of the eggshell (chorion) (Kimmel et al., 1995). The chorion is a protective barrier and, for example, alcohol has been found not to be able to penetrate it very easily. Recent analyses showed the concentration of alcohol inside the zebrafish egg to be about 1/10th to 1/25th of the external bath concentration after prolonged period of immersion (Fernandes and Gerlai, 2009; Mahabir et al., 2014). Thus, if one wants to use consistent alcohol concentrations throughout the development of zebrafish, i.e. up to their age of 120 hpf, one has to factor in the protective effects of the chorion before hatching and the lack of it for the subsequent stage of development. However, given that most major CNS developmental steps are complete before hatching (i.e. by 70th hour post-fertilization), we view this complication as somewhat minor. Furthermore, dechorionation, and allowing the dechorionated zebrafish embryo to develop to fully form fish, is a standard and regularly employed procedure (Henn and Braunbeck, 2011). Furthermore, injecting the chorionated embryo is also routinely done with zebrafish (Ali et al., 2011; Rosen et al., 2009; Wang et al., 2007). Thus, controlling alcohol delivery throughout any stages of ontogenesis of zebrafish is simple. For the above reviewed advantages of the zebrafish, it is not surprising that the effect of embryonic alcohol exposure has started to be explored using this species (Table 1). However, most of these studies utilized high doses of alcohol (up to 10%, vol/vol) and administered alcohol for prolonged periods of time (up to 7 days, i.e. throughout the entire embryonic development of the fish) (Lovley et al., 2016; Parker et al., 2014). This administration regimen and dosing produces highly severe and thus easily observable deficits in zebrafish mimicking what happens to the human fetus as a result of the most extreme forms of binge or chronic alcohol drinking in pregnant women. It is notable, however, that such extreme Fetal Alcohol Syndrome (FAS) cases are rather rare. Milder forms of the disease termed, e.g., Alcohol Related Neurodevelopmental Disorders (ARND), are more frequent. Administration of low concentration and short duration dose of alcohol may better recapitulate these milder and more prevalent forms of FASD. Such short and low dose ethanol exposure protocol has recently been developed and employed using zebrafish (Bailey et al., 2015; Fernandes et al., 2015b; Lovley et al., 2016; Parker et al., 2014; Fernandes and Gerlai, 2009). This alcohol exposure regimen has been found to produce little to no observable morphological deficits, but has been demonstrated to lead to significant and long-lasting behavioural and cognitive deficits, including impaired social behaviour (Buske and Gerlai, 2011;

identify these common features. The main point about comparing more than two species is that such comparison may allow one to identify the most evolutionarily conserved features. In fact, the larger number of species we use in such a comparative approach, the more likely we will be able to find the evolutionarily most conserved and thus most fundamental features. The zebrafish is an evolutionarily old “design”. That is, the last common ancestor between fish and mammals lived approximately 400 million years ago, and since then the basic anatomy and other biological features of fish remained less altered than those of mammals. In other words, having zebrafish in the mix of comparisons across species increases the chance for one to find evolutionarily old, fundamental aspects of biology. This is important from a translational relevance standpoint. A crucial, and valid issue often raised about the use of “model organisms” is that they do not model anything. They are unique species on their own right. How could we use a nocturnal rodent, adapted to an entirely different environment, having an incomparably smaller brain with species-specific structure and function to conclude about human CNS disorders? The criticism is even more valid for zebrafish research. Yet, countless studies show that due to evolutionary conservation, it is indeed possible to use laboratory organisms to model some aspects of human disorders. The question is how we can tell what aspects of the biology of the model organism is shared with our own features. This is where a comparative approach, utilizing more than just two species, enjoys the biggest advantage. A recent example of how such a comparative approach may yield powerful and translationally relevant results is a study conducted by Choi et al. (2018) in which the authors discovered a gene encoding a novel chemokine-like protein expressed in the zebrafish brain, and identified its cellular and synaptic function, found homologous processes subserved by the protein in zebrafish and mice, and identified human patients suffering from CNS disorders in which this gene carried a mutation. Another point of using the zebrafish is about employing a reductionist approach. The fish brain is incomparably simpler than a human brain, yet possesses the same basic vertebrate structural layout, neurotransmitter systems and several other homologies with human features at multiple levels of its organization, including the nucleotide sequence of genes expressed in this organ. Briefly, it is simpler to study. Furthermore, given that it is evolutionarily more ancient, it is expected to yield information about the core mechanisms and features of the studied phenomena (Gerlai, 2014a), an argument that brings us to another point: ethical considerations. An important guideline in the ethical use of animals in research is reduction. An example of reduction is when one uses evolutionarily less advanced, simpler model organisms. Fish are the simplest, evolutionarily most ancient vertebrates one can employ in biomedical research. Although relatively new to psychopharmacology in general and alcohol research in particular, the zebrafish is becoming increasingly popular in a variety of subfields of biology, including in neuroscience, and in pharmacological and behavioural studies. A number of reviews have been published that examine the use of zebrafish in FASD related research (i.e. Lovley et al., 2016; Patten et al., 2014). There are many aspects of this teleost that may be viewed as an advantage compared to traditional rodent laboratory research species, including their prolific nature, transparent eggs, external fertilization, fast ontogenesis, as well as non-invasive drug/alcohol administration techniques, to name but a few. For example, the prolific nature and rapid growth of zebrafish make this species a key candidate for high-throughput drug and mutation screens (Fernandes et al., 2015a).The transparency of their embryo throughout their development allows unprecedented analysis of organogenesis from the moment of conception. Due to the external fertilization of zebrafish eggs, the embryo develops outside the mother. Furthermore, zebrafish exhibit no parental care. Thus, as mentioned above, confounding effects associated maternal physiology or with parental care, as typical in most mammals including rodents, are absent. External fertilization represents a major advantage compared to 188

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Table 1 List of studies in which the effects of alcohol administered during embryonic development were analyzed using zebrafish. The table shows the developmental stage (time point) at which alcohol was administered in hours post-fertilization (hpf), the length of time during which alcohol was administered (measured in days or hours as indicated), the concentration as given in the original publications (vol/vol % or micromolar), the strain of zebrafish employed, and the behavioural characteristics and morphological features tested. Note that although most of these studies conducted dose response analyses, and although the alcohol was administered at a range of developmental stages, developmental stage-dependent effects were not analyzed systematically within any of these studies, and the inconsistencies in procedures also make comparing the results across studies difficult. Timepoint

Duration

Concentration

Strain

Behaviour

Morphology

Molecular (if applicable)

Reference

4hpf

6 days

0mM 3mM 10mM 30mM 100mM 300mM 0% 1% 3%

Unreported

Spatial Learning Anxiety (startle response)

Skeletal mophometric (Alcian Blue staining)

Apoptosis (AO staining)

Carvan et al., 2004

AB

Anxiety (novel tank; tapping) Spatial learning

Eye size Brain malformation

N/A

Bailey et al., 2015

0% 1% 3% 0% 0.25% 0.5% 0.75% 1% 0% 0.25% 0.5% 0.75% 1% 0% 0.5% 1% 0% 1% 0% 0.25% 0.5% 0% 0.25% 0.5% 0.75% 1% 0% 1% 0mM 20mM

AB

Novel tank dive task

Eye size Brain malformation

Shh and retinoic acid signalling (morpholinos)

Burton et al., 2017

AB

Associative learning

N/A

N/A

Fernandes et al., 2014

AB

Locomotor activity Shoaling Fear response

N/A

N/A

Fernandes and Gerlai, 2009

AB

Shoaling

N/A

Dopamine and DOPAC (HPLC)

Fernandes et al., 2015a

AB

Shoaling

N/A

N/A

AB

Shoaling

N/A

Dopamine and DOPAC Serotinin and 5-HIAA (HPLC)

Fernandes et al., 2015b Buske and Gerlai, 2011

AB TU

N/A

N/A

Dopamine and DOPAC Serotinin and 5-HIAA (HPLC)

Mahabir et al., 2014

AB

N/A

N/A

Mahabir et al., 2018

TU

Novel tank dive task Shoaling

N/A

BDNF NCAM Q-PCR

8-10hpf 24-27hpf

2 or 3 hours

8-10hpf 24-27hpf

2 or 3 hours

16hpf

2 hours

24hpf

2 hours

24hpf

2 hours

24hpf

2 hours

24hpf

2 hours

24hpf

2 hours

24hpf

2 hours

48hpf

7 days

Parker et al., 2014

development of the zebrafish brain.

Carvan et al., 2004; Fernandes et al., 2015b; Fernandes and Gerlai, 2009), anxiety-related behaviours (Burton et al., 2017; Parker et al., 2014) and learning and memory (Carvan et al., 2004; Fernandes et al., 2014), changes that we will review in detail below. Despite the increasing number of studies reporting morphological and/or behavioural deficits resulting from embryonic ethanol exposure in animal models, a systematic comparison of how the timing of alcohol exposure may differentially alter the development of the vertebrate brain leading to abnormalities of behavioural characteristics is lacking. This question may be best addressed by precisely controlling the onset and offset of alcohol exposure during embryonic development, and by limiting the exposure period to a short time window. We will limit our discussion on studies in which low concentrations of alcohol were employed, and the resulting changes were analyzed using behavioural methods, a specific focus that is justified given that the most prevalent forms of FASD result from low amount of alcohol consumption (Lange et al., 2017; Roozen et al., 2016, and references therein). We admit that only limited amount of information has been gathered with zebrafish to answer the above questions. Our goal with this review is to draw attention to the need to address this limitation and persuade others about the utility of the zebrafish in this research. Before we delve into the above questions, however, we briefly review what is known about the

3.1. Zebrafish brain development Numerous processes change throughout brain development in zebrafish, ranging from altered gene expression to structural modification of the brain. To fully understand the developmental-stage dependent effects of alcohol exposure, one should consider all these factors. Here, we only briefly consider this question, and given the complexity of the issue, we only discuss two aspects of brain development as examples: structural development and neurochemical changes associated with the ontogenesis of the zebrafish brain. 3.1.1. Structural development of the zebrafish brain Hour-by-hour ontogenetic changes in the developing healthy zebrafish embryo have been well characterized. For example, Kimmel et al. (1995) review such changes in detail from 0 h post-fertilization (hpf) to 72 hpf, identifying 7 major stages, or periods of development. These stages are organized approximately as follows: 1, Zygote period (0–1 hpf); 2, Cleavage period (1–2 hpf); 3, Blastula period (2–5 hpf); 4, Gastrula period (5–10 hpf); 5, Segmentation period (10–24 hpf); 6, Pharyngula period (24–48 hpf); and 7, Hatching period (48–72 hpf). 189

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The first 4 stages (zygote to gastrula period) involve rapid cell division. In the zygote period, the egg is in a one to two-cell stage, which lasts for approximately 45-min post fertilization, and during which the blastodisc forms and the first zygotic cell division cycle occurs (Kimmel et al., 1995). Subsequently, the embryo enters the cleavage phase where the development of the embryo progresses through the first six mitotic cell division cycles (2–64 cells) (Kimmel et al., 1995). In the blastula period, the embryo develops from blastodisc into the shape of a ball, and epiboly movement leads to the formation of the blastoderm (Kimmel et al., 1995). Following the blastula stage, the gastrula stage marks the development of two germ layers, which form a primitive gut as epiboly movement finishes with the emergence of the tail bud at 10 hpf (Kimmel et al., 1995). It is not until the segmentation period that the zebrafish embryo starts to differentiate both morphologically and neuroanatomically (Kimmel, 1993). There is little distinction between brain regions until approximately 18 hpf, a time point when there is rudimentary compartmentalization of the telencephalon, diencephalon, and mesencephalon (Kimmel, 1993; Kimmel et al., 1995). Hindbrain rhombomeres are also present, which eventually form the rhombencephalon (Kimmel, 1993). However, by the end of the segmentation period (24 hpf), neuroanatomical morphogenesis has progressed greatly, with clear differentiation of the hypothalamus, pineal gland, and cerebellum (Kimmel, 1993; Kimmel et al., 1995). Ventricles have formed and earlier rudimentary compartmentalization has now been replaced by clear distinction between the telencephalon, mesencephalon, and diencephalon (Kimmel, 1993). In terms of morphology, a rapid increase in body length is accompanied by the first evidence and formation of a tail (Kimmel et al., 1995). By the end of the pharyngula period (48thhpf), neural differentiation appears almost complete, but there is an increase in neurogenesis and synaptogenesis (Wilson et al., 1990). With regard to gross morphology, the bilateral body plan is well developed (Kimmel et al., 1995). Brain structures are now divided into the major lobes with a fully formed notochord (Kimmel et al., 1995). The heart begins to beat, visible pigmentation is beginning to occur, and fins begin to form (Kimmel et al., 1995).

temporal effects of developmental alcohol exposure, with focus on behavioural changes induced by alcohol exposure between 10 and 30hpf. Given the myriad anatomical and molecular changes that accompany zebrafish ontogenesis, and given the complexity of how alcohol may interact with several of these changes, it is difficult to forecast what mechanism, neuroanatomical structure, function, or which biochemical pathway or gene product one should focus on when analyzing the consequences of embryonic alcohol exposure. We argued that, for this reason, perhaps comprehensive behavioural phenotyping of the effects of alcohol exposure may be the best way to start this investigation (Gerlai, 2002, 2015). We have also argued that behavioural phenotyping may be the best way to start such systematic screens, as behavioural analysis would represent unbiased analysis with which functional alterations in the brain, whatever their underlying mechanisms may be, could be discovered (Gerlai, 2002, 2014b, 2012, 2010, 2002; Gerlai and Clayton, 1999). Such comprehensive behavioural studies have not yet been performed. However, the first pieces of evidence suggesting developmental stage dependent effects of alcohol exposure on the behaviour of zebrafish, have been obtained. Below we review these pioneering studies. 3.2. Zebrafish in FASD research FASD is a complex disorder induced by exposure of the developing human fetus to alcohol with many contributing factors influencing the symptoms and severity of the disease. Two of these factors are the dose and timing of alcohol exposure. Although the primary focus of this review is on how the timing of alcohol exposure relates to later behavioural deficits, the effect of alcohol dose is just as important for understanding the mechanisms of alcohol in the early developing brain. First, we briefly discuss literature pertaining to concentration dependent behavioural effects of embryonic alcohol exposure, and only subsequently review what is known about the influence of the timing of embryonic alcohol exposure in zebrafish. Our focus on behaviour is somewhat due to our personal bias as we are behavioural neuroscientists. Nevertheless, there are some objective reasons why this bias is justified. We will not review all points but instead refer the reader to some of our prior publications that delve deeper in these questions (Gerlai, 2002; Gerlai and Clayton, 1999). Here, we only briefly emphasize that behavioural analysis we regard as most useful as a start of characterizing the effects of alcohol as well as the last step in testing mechanistic questions. It is useful to start such characterization with behavioural analysis because this analysis is not restricted by techniques to particular functions or areas of the brain. Yet, it has the capacity to reveal changes in the way the brain works and thus may allow one to narrow his/her focus to mechanistically betterdefined alterations. Briefly, behavioural analysis may be an unbiased, relatively simple and efficient, yet sensitive method to reveal changes in brain function, induced, in this case, by embryonic alcohol exposure. It is also a useful analysis once more mechanistic details have become available and, for example, one wants to test a candidate mechanism underlying a particular developmental process engaged by alcohol, or a candidate drug developed for ameliorating the effects of thus substance. The endpoint of most CNS drug trials is behaviour, after all the patient does not care why he/she has improved memory or motor function, or reduced fear and anxiety. Nevertheless, behavioural analysis is far from being the only excellent method with which alcohol induced alterations in brain function or structure may be revealed, an area of research, however, we will not review here.

3.1.2. Neurochemistry Significant differentiation of the telencephalon and diencephalon during the segmentation and pharyngula periods relates to the development of neurotransmitter systems and associated neurochemicals in the zebrafish brain (Kimmel, 1993; Kimmel et al., 1995). The zebrafish telencephalon contains the pallium and subpallium, the former of which is believed to be the zebrafish equivalent of the human hippocampus and amygdala (anterodorsolateral pallium and dorsomedial pallium, respectively) (Cheng et al., 2014). The zebrafish subpallium contains glutamate decarboxylase (the enzyme responsible for converting glutamate to GABA) as well as choline acetyltransferase (the enzyme responsible for synthesis of acetylcholine) (Cheng et al., 2014). The zebrafish diencephalon includes the evolutionarily conserved habenula (Cheng et al., 2014) that connects to a number of brain areas via specific neurotransmitter systems, including the raphe nuclei (serotonin), locus coeruleus (norepinephrine), and the ventral tegmental area (dopamine) (Cheng et al., 2014). Choline acetyltransferase positive neurons have also been found in the habenula (Cheng et al., 2014). It is clear that much of the zebrafish neuroanatomical development occurs during the segmentation and pharyngula phases (10–48hpf), suggesting this may be a key exposure time period or developmental stage range upon which embryonic alcohol exposure studies should focus (Kimmel, 1993; Kimmel et al., 1995). Recent studies have started to examine these periods (Bailey et al., 2015; Burton et al., 2017; Buske and Gerlai, 2011; Carvan et al., 2004; Fernandes et al., 2015a, b; Fernandes and Gerlai, 2009), however, they did not go beyond 30 hpf, and also have not systematically mapped the temporal pattern of alcohol effects. Below, we will review studies that have investigated the

3.2.1. Dose-dependent behavioural effects of embryonic alcohol exposure in zebrafish Exposure to alcohol during embryonic development has been found to lead to lasting changes in behaviour that are detectable in the adult as well as in the old zebrafish, with higher doses of alcohol usually producing more severe behavioural deficits. This dose-dependency has 190

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behavioural differences in the novel tank task or a predator avoidance task in adult zebrafish that were exposed to alcohol at 24hpf. Whether exposure to alcohol at an earlier or later stage of development would have had a differential effect on anxiety-like responses has not been systematically analyzed.

been demonstrated in social and anxiety-related behaviours in zebrafish. For example, Bailey et al. (2015) showed a more robustly altered anxiety response in adult zebrafish that were exposed to 3% ethanol during early development compared to those exposed to 1% alcohol. Fernandes and Gerlai (2009) showed a quasi-linear dose-dependent deficit in responding to social stimuli in zebrafish exposed to 0%, 0.25%, 0.5%, 0.75%, or 1% (vol/vol) alcohol at 24hpf. However, when the same concentrations were employed, but at 16th hour post-fertilization, Fernandes et al. (2014) found a flat dose response curve, with doses from 0.25% (vol/vol) alcohol to1% alcohol inducing a similarly severe learning deficit across all concentrations. The different dose response curves found with the 24thhpf exposure for social behaviour and with the 16thhpf exposure for learning performance suggest distinct underlying mechanisms of embryonic alcohol exposure whose identity is unknown at this point. We stress again that although the above results are suggestive and potentially interesting, systematic analyses of how the developmental timing of alcohol exposure, the concentration of alcohol employed and what behaviour(s) is/are affected most, have not been conducted. Systematic manipulation of the developmental time of alcohol exposure coupled with comprehensive behavioural phenotyping of the potential effects of alcohol exposure will be needed to map sensitive periods of development. Such research may aid one in the future to uncover specific distinct mechanisms via which alcohol exerts its teratogenic effects during the ontogenesis of the vertebrate brain, a question we further discuss below.

3.3.2. Learning and memory The findings surrounding the influence of embryonic alcohol exposure on learning and memory in zebrafish have also been inconsistent. Bailey et al. (2015) examined how 1% ethanol administered at either 8–10hpf or 24–27hpf differentially affected learning and memory. These authors found that acquisition of spatial memory in young adult zebrafish (2 months) did not change (compared to control) as a result of alcohol administered during either period (Bailey et al., 2015). However, other studies contradicted these findings and have demonstrated impaired learning and memory induced by exposure to alcohol during embryonic development. For example, Fernandes et al. (2014) found that exposure to alcohol at 16hpf for 2 h impaired associative learning as demonstrated by reduced time spent near a conditioned stimulus that was previously paired with food reward. Carvan et al. (2004) exposed zebrafish to low doses of alcohol continuously for a period between 4–24hpf, and observed that the exposed fish required more time to learn the location of food reward. Detailed and systematic analyses of the potentially developmental stage-dependent effects of alcohol, however, have not been performed. Notably, because Fernandes et al. (2014) found significant effects of alcohol even when the exposure period was only 2 h long, mapping of potential developmental stage-specific effects of alcohol will likely succeed. It is possible that around the 16thhpf stage the brain is undergoing such developmental changes that alcohol is particularly damaging, but alcohol exposure before (e.g. 8–10 hpf) or after (e.g. 24–27 hpf) this point has less deleterious consequences (Bailey et al., 2015), a working hypothesis that requires the systematic developmental time course analyses we advocate. The fact that social behaviour has been found to be significantly altered by alcohol exposure at 24hpf (Fernandes et al., 2015a,b) when learning and memory is not, and that learning and memory is altered at 16hpf (Carvan et al., 2004; Fernandes et al., 2014) but social behaviour is only mildly affected (Fernandes & Gerlai unpublished results) also suggests possible short-term critical periods of development during which alcohol exposure may have well defined and specific behavioural consequences.

3.3. Are developmental stage dependent alcohol effects specific to particular behaviours? The fundamental assumption underlying all behavioural phenotyping studies is that the behavioural alterations quantified reflect modification of brain function and thus should aid the discovery of the neurobiological changes induced. From this line of reasoning it also follows that if embryonic alcohol exposure is expected to engage different mechanisms dependent upon the developmental stage at which the embryo was exposed to this substance, then one should be able to find behavioural effects idiosyncratic to the given developmental stage of alcohol exposure. However, thorough and systematic behavioural phenotyping of changes induced by embryonic alcohol exposure has not been performed with zebrafish. Below, we review the sporadic studies that focussed on distinct behavioural phenotypes as examples to illustrate the infancy of this research field and to emphasize the need for systematic studies.

3.3.3. Social behaviour There are several forms of social behaviour of zebrafish (e.g. courtship, aggression, schooling), almost none of which has been studied in the context of embryonic alcohol exposure research. One form, shoaling, however, has been extensively investigated. Shoaling, or group forming, is a robust and species typical feature of the zebrafish (Miller and Gerlai, 2007). This behaviour has been observed in nature as well as in the laboratory. It can be induced reliably and measured relatively easily. There are two distinct methods one can use: one, a single fish is presented with a social stimulus (either live conspecifics or video-recorded or computer animated images of conspecifics) and the distance between the test fish and the social stimulus is quantified (Saverino and Gerlai, 2008.); two, a group of freely swimming zebrafish is allowed to explore a large water tank and the distances among shoal members are quantified using an overhead camera and video-tracking system (Miller and Gerlai, 2008; Saverino and Gerlai, 2008). Both of these methods have been utilized to detect alterations in brain function induced by embryonic alcohol exposure. Fernandes and Gerlai (2009) exposed zebrafish to alcohol for 2 h at doses ranging between 0.25 and 1.00% (vol/vol %) ethanol at the 24thhpf stage of development and found an almost perfect liner dose response with adult fish that were exposed to the highest dose during their embryonic development exhibiting the strongest reduction in their response to moving images of conspecifics. This finding was replicated using live shoals and

3.3.1. Anxiety responses A number of tasks have been employed to quantify anxiety-related responses in zebrafish, one of the most popular being the novel tank task (Bailey et al., 2015; Norton and Bally-Cuif, 2010). When placed into a new environment, zebrafish exhibit a variety of anxiety-like responses, including elevated amount of freezing, increased bottom dwelling and wall hugging (thigmotaxis) (Norton and Bally-Cuif, 2010). Zebrafish that spend more time near the top or center of the tank are considered having diminished anxiety response (Bailey et al., 2015). Exposure of zebrafish to alcohol has been found to have a developmental-stage dependent effect on anxiety-like responses. Bailey et al. (2015) examined anxiety-related behaviours in zebrafish exposed to0%, 1%, or 3% alcohol at 8–10hpf or at 24–27hpf. Zebrafish that were exposed to alcohol between 24–27hpf exhibited significantly diminished anxiety responses compared to control: both 1% and 3% alcohol exposed fish swam more near the surface (reduced anxiety) during a novel tank task (Bailey et al., 2015). Sterling et al. (2016) exposed developing zebrafish embryos to low doses of ethanol (0.25 or 0.5%) for 2 h at 24hpf, and also found decreased anxiety-related responses and increased exploratory behaviour when they tested the exposed fish at their adult stage in a novel tank test (Sterling et al., 2016). However, a study by Seguin et al. (2016) found no anxiety- and fear-related 191

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Acknowledgements

measuring the inter-individual distance among shoal members by Buske and Gerlai (2011), a study that also confirmed the previous conclusion suggesting the impairment was not due to such performance factors as motor function or perception. Interestingly, preliminary results (Fernandes & Gerlai, unpublished) suggest that the strength of impairment is developmental stage specific: fish exposed to ethanol at 24thhpf show the strongest alcohol effect, and fish exposed earlier or at a later stage of development show a blunted effect, confirming the speculation that, at least for some behaviours, critical or sensitive periods of development for alcohol exposure do exist, likely within the 16–48hpf time window, i.e. when the zebrafish brain is undergoing rapid development. Although the mechanisms underlying the behavioural effects of embryonic alcohol exposure demonstrated in zebrafish remain largely unexplored, the first studies of this question already present some promising results. For example, impairment of the dopaminergic system has been demonstrated to accompany the observed shoaling behaviour abnormalities. While baseline dopamine and DOPAC levels appeared unaltered, embryonic alcohol exposed fish when tested at their adult age exhibited a lack of dopaminergic response (lack of increased dopamine and DOPAC levels) in response to the appearance of social stimuli (conspecific images) (Fernandes et al., 2015a). Lasting changes in a number of proteins known to be involved in brain development as well as neuronal plasticity (including BDNF, NCAM and synaptophysine) have also been demonstrated resulting from embryonic alcohol exposure in zebrafish (Mahabir et al., 2018). Last, increased apoptotic cell death has been demonstrated in the brain of zebrafish embryos induced by early developmental exposure to alcohol (Mahabir, Chatterjee and Gerlai, unpublished results). Developmental stage dependent alcohol exposure effects on these processes and molecular targets, however, have not been explored.

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4. Conclusion The effect of alcohol on the developing brain remains a highly complex problem requiring multidisciplinary analyses. A simple solution, abstinence from alcohol use, although available, appears practically impossible as societal attitudes towards alcohol use remain unaltered. Thus, investigation of the effects of embryonic alcohol exposure and the mechanisms that underlie the induced changes is warranted. The characterization of embryonic alcohol exposure induced changes in the brain could start with thorough behavioural phenotyping. Results of sporadic studies have already demonstrated developmental-stage specific behavioural effects of embryonic alcohol exposure. Some results imply the existence of critical periods during which alcohol may be particularly deleterious, and also suggest potentially distinct developmental stage-dependent mechanisms via which alcohol induced changes may occur. However, as of today, no zebrafish study has been conducted to systematically analyze the potentially differential developmental stage dependent effects of alcohol exposure. Nevertheless, given that lasting behavioural as well as neurochemical changes induced by very short (2 h long) exposure to alcohol during embryonic development have been demonstrated, mapping the time-of-exposure dependent effects of this substance in zebrafish should be possible. Such studies may be fairly complicated if conducted with mammals, because alcohol cannot be directly administered to the embryo, unlike in the zebrafish that develops externally. We suggest that administering alcohol for short periods of time and at a systematically varied developmental stage will significantly contribute to our understanding of how alcohol induces developmental and functional changes in the brain, and why these changes manifest in a varied way at the level of behavioural performance in humans. Declarations of conflict of interest None. 192

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