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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 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 exposure in one-week old juvenile zebrafish. We exposed zebrafish embryos at one of five developmental stages (8, 16, 24, 32, or 40 hours post-fertilization) to 0% (control) or 1% (vol/vol) ethanol for 2h, and tested the behavior of these fish at their age of 7-9 days post-fertilization. We employed two genetically distinct zebrafish populations, a quasi-inbred AB derivative strain, and a genetically variable WT population. We report significant developmental time and genotype dependent effects of alcohol on certain measures of motor function and/or 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, alcohol induced changes that were absent or less robust in WT fish. We conclude that our results 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.

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... Because Big Al's Aquarium obtains their fish from commercial breeding facilities where the number of breeding pairs is large, we assume that the decreased level of inbreeding meets the above criteria. We have also argued that the increased genetic variability and high heterozygosity ratio makes the WT population more representative of species-typical features of zebrafish found in nature [44,71]. Unlike this genetically heterogeneous population, standard zebrafish strains may have unique, strain-specific idiosyncratic features. ...
... Immobility here is defined as fewer than 20% of the number of pixels corresponding to the total visible surface area of the subject changing from one video-frame to the next (with 30 frame per s temporal resolution). Although intuitively total distance travelled and immobility duration may seem to be redundant, negatively correlating measures, empirical data have suggested that they represent independent behavioral responses [41,44,71,75]. Frequency of immobility (number of immobility episodes) was also quantified as it may represent a behavioral strategy independent of the duration of immobility. ...
... Here, however, we found a linear dose dependent reduction of distance moved in fish to whom buspirone was administered. The literature, however, does provide some controversies as swimming activity has also been found to increase, rather than decrease, in response to fear inducing stimuli in some zebrafish studies [71,77]. Immobility has also been used to quantify effects of fear and anxiety inducing stimuli or drugs. ...
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Anxiety continues to represent a major unmet medical need. Despite the availability of numerous anxiolytic drugs, a large proportion of patients do not respond well to current pharmacotherapy, or their response diminishes with chronic drug application. To discover novel compounds and to investigate the mode of action of anxiolytic drugs, animal models have been proposed. The zebrafish is a novel animal model in this research. It is particularly appropriate, as it has evolutionarily conserved features, and drug administration can be employed in a non-invasive manner by immersing the fish into the drug solution. The first step in the analysis of anxiolytic drugs with zebrafish is to test reference compounds. Here, we investigate the effects of buspirone hydrochloride, an anxiolytic drug often employed in the human clinic. We utilize two genetically distinct populations of zebrafish, ABSK, derived from the quasi-inbred AB strain, and WT, a genetically heterogeneous wild-type population. We placed juvenile (10–13-day, post-fertilization, old) zebrafish singly in petri dishes containing one of four buspirone concentrations (0 mg/L control, 5 mg/L, 20 mg/L or 80 mg/L) for 1 h, with each fish receiving a single exposure to one concentration, a between subject experimental design. Subsequently, we recorded the behavior of the zebrafish for 30 min using video-tracking. Buspirone decreased distance moved, number of immobility episodes and thigmotaxis, and it increased immobility duration and turn angle in a quasi-linear dose dependent but genotype independent manner. Although it is unclear whether these changes represent anxiolysis in zebrafish, the results demonstrate that behavioral analysis of juvenile zebrafish may be a sensitive and simple way to quantify the effects of human anxiolytic drugs.
... The larval stage is often used to evaluate the motor activity and anxious behaviour. The open field, which allows free swimming, was used in most studies [34][35][36][37], with an apparatus as simple as Petri dishes [34,36] and 96- [35] or 6well plates [37]. Motor activity was also assessed under light challenge, based on the fact that zebrafish larvae increase locomotor activity under light, allowing fast changes in motor activity in a light/dark challenge test [38], which can be performed in specific automatised chambers using 6 to 96well plates to allocate several larvae simultaneously [39][40][41][42]. ...
... The larval stage is often used to evaluate the motor activity and anxious behaviour. The open field, which allows free swimming, was used in most studies [34][35][36][37], with an apparatus as simple as Petri dishes [34,36] and 96- [35] or 6well plates [37]. Motor activity was also assessed under light challenge, based on the fact that zebrafish larvae increase locomotor activity under light, allowing fast changes in motor activity in a light/dark challenge test [38], which can be performed in specific automatised chambers using 6 to 96well plates to allocate several larvae simultaneously [39][40][41][42]. ...
... Motor activity was also assessed under light challenge, based on the fact that zebrafish larvae increase locomotor activity under light, allowing fast changes in motor activity in a light/dark challenge test [38], which can be performed in specific automatised chambers using 6 to 96well plates to allocate several larvae simultaneously [39][40][41][42]. Decreased locomotion [36,40,41,43] was the main outcome detected at the larval stage, but some studies did not find any effect on motor activity or even an increase in motor activity after early ethanol exposure [34,37,44]. This variability was attributed to the developmental time point and regimen of ethanol exposure, as well as genotype differences and visual impairment [34,36,42]. ...
Article
Background The consequences of mild to severe exposure to alcohol during brain development is still a matter of debate and scientific investigation. The long-term behavioural effects of ethanol exposure have been related to impaired social skills and cognition. Zebrafish have become a suitable animal model to investigate the effects of early ethanol exposure because it is very feasible to promote drug delivery during early development. Objective The goal of the current report is to review existing behavioural studies addressing the impact of early alcohol exposure using zebrafish to determine whether these models resemble the behavioural effects of early alcohol exposure in humans. Methods A comprehensive search of biomedical databases was performed using the operation order: “ZEBRAFISH AND BEHAV* AND (ETHANOL OR ALCOHOL)”. The eligibility of studies was determined using the PICOS strategy, contemplating the population as zebrafish, intervention as exposure to ethanol, comparison with a non-exposed control animal, and outcomes as behavioural parameters. Results The systematic search returned 29 scientific articles as eligible. The zebrafish is presented as a versatile animal model that is useful to study FASD short and long-term behaviour impairments, such as anxiety, impaired sociability, aggressiveness, learning problems, memory impairment, seizure susceptibility, sleep disorders, motivational problems, and addiction. Conclusion This systematic review serves to further promote the use of zebrafish as a model system to study the pathophysiological and behavioural consequences of early alcohol exposure.
... The zebrafish (Danio rerio) has been attracting attention in the scientific community due to the genetic variations between populations used in research and its 70-80% homology with humans (Barbazuk et al., 2000;Howe et al., 2013). Although its genetic background and specific mutations have yet to be identified, studies suggest the existence of genetic, behavioral, and neurochemical differences in response to alcohol in zebrafish populations commonly used in laboratories worldwide, such as inbred lines (AB), Tuebingen (TU), and locally purchased animals, considered outbred due to their highly heterogeneous genome (Dlugos and Rabin, 2003;Loucks and Carvan III, 2004;Gerlai et al., 2008;Gerlai et al., 2009;Pan et al., 2012;Pannia et al., 2014;Chatterjee et al., 2014;Abozaid et al., 2020). For instance, TU embryos are supposedly more sensitive to alcohol in terms of survival while displaying lower cell death compared to AB (Loucks and Carvan III, 2004). ...
... In regard to research on alcohol's effects, other authors sought to identify which zebrafish populations are more sensitive or resistant to the drug (Dlugos and Rabin, 2003;Loucks and Carvan III, 2004;Gerlai et al., 2008;Gerlai et al., 2009;Pan et al., 2012;Pannia et al., 2014;Mahabir et al., 2014;Chatterjee et al., 2014;Abozaid et al., 2020). However, natural variations in the genome of these populations (Brown et al., 2012;Coe et al., 2009) make it difficult to elucidate these questions. ...
Article
Alcohol abuse is one of the most dangerous and serious problems for patients and society. Interpopulation studies are important in understanding how genetic background contributes to the effects of alcohol. In this study, we applied a chronic alcohol exposure protocol in three zebrafish populations (Danio rerio; both sexes; AB, TU, and outbred fish - OB). We analyzed the behavioral responses and mRNA expression involved in neurotransmitter metabolism - th1, tph1, ache, ada1, gaba1, gad1b, and bdnf. Locomotion patterns were similar between populations (increased speed after acute alcohol and unaltered locomotion after chronic and withdrawal treatments). All populations exhibited increased expression of genes associated with locomotion (th1, gad1b, and gaba1) after acute alcohol exposure. Anxiety-like responses increased in AB and TU fish during withdrawal and decreased in AB fish after acute alcohol exposure. Genes related to anxiety-like behavior (tph1 and ada1) were overexpressed in AB and TU fish after acute and withdrawal treatments, while OB fish exhibited unaltered responses. Bdnf levels decreased during withdrawal in AB and OB fish, while TU showed upregulated levels in both chronic and withdrawal treatments. Our results suggest that zebrafish populations respond differently to alcohol exposure, which may contribute to understanding the mechanisms underlying alcohol use and dependence. Moreover, we found that a more diverse genetic background (OB) was related to higher variability in behavioral and mRNA expression, demonstrating that inbred populations (AB and TU) may be useful tools in identifying alcohol use and abuse mechanisms.
... Native from Asia, the zebrafish is a teleost that has high genetic and physiological homology with humans (Lieschke and Currie, 2007), by this reason, this species has been used as a research animal model for different lines such as embryology and development (Hao et al., 2013;Keller et al., 2008), oxidative stress (Choi et al., 2010;Marcon et al., 2018), behavior (Abozaid et al., 2020;Nabinger et al., 2021;Reis et al., 2020) and genetic (Nasevicius and Ekker, 2000;Falcão et al., 2021). Moreover, this aquatic animal is a very interesting environmental bioindicator used in toxicology and ecotoxicology research, due to its capacity to simulate the conditions of an animal in its natural ecosystem (Asharani et al., 2008;Park et al., 2020;Valadas et al., 2019). ...
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Ochratoxin A (OTA) is a mycotoxin produced by species of filamentous fungi widely found as a contaminant in food and with high toxic potential. Studies have shown that this toxin cause kidney and liver damage, however, data on the effects of exposure to OTA on the central nervous system are still scarce. Zebrafish (Danio rerio) is a teleost often used in translational research due to its physiological, genetic, and behavioral homology with mammals, in addition to being useful as an environmental bioindicator. Thus, this study aimed to investigate the effects of exposure to OTA on behavioral and neurochemical parameters in adult zebrafish. The animals were treated with different doses of OTA (1.38, 2.77, and 5.53 mg/kg) and submitted to behavioral evaluations in the open tank and social interaction tests. Subsequently, they were euthanized, and the brains were used to assess markers associated with oxidative status. In the open tank test OTA induced changes in distance, absolute turn angle, mean speed, and time-freezing. However, no significant effects were observed in the social interaction test. Moreover, OTA also induced alterations in neurochemical parameters with changes in non-protein thiols (NPSH), glutathione peroxidase (GPx), glutathione-S-transferase (GST), and glutathione reductase (GR). This study showed that OTA can affect neurobiological aspects in zebrafish even at low doses.
... The diminished exploratory activity induced by ethanol treatment replicates previous results described by Baggio et al. (2018) in adult FASD zebrafish. Other authors have also reported embryonic ethanol exposure led to higher anxiety levels both in larvae zebrafish (Ramlan et al., 2017;Abozaid et al., 2020) and juvenile rats (Brolese et al., 2014;Diaz et al., 2016;Ramlan et al., 2017;Rouzer et al., 2017;Balaszczuk et al., 2019). In the present study, we corroborate with cited embryonic ethanol effects showing that behavioral responses can be observed during early stages (10dpf), what contributes to the syndrome thorough understanding and suggests that the attentive look at the first phases of growth may help to diagnose and initiate early treatment for FASD cases. ...
Article
Aims: Fetal alcohol spectrum disorder (FASD) is an umbrella term to describe the effects of ethanol (Eth) exposure during embryonic development, including several conditions from malformation to cognitive deficits. Zebrafish (Danio rerio) are a translational model popularly applied in brain disorders and drug screening studies due to its genetic and physiology homology to humans added to its transparent eggs and fast development. In this study, we investigated how early ethanol exposure affects zebrafish behavior during the initial growth phase. Methods: Fish eggs were exposed to 0.0 (control), 0.25 and 0.5% ethanol at 24 h post-fertilization. Later, fry zebrafish (10 days old) were tested in a novel tank task and an inhibitory avoidance protocol to inquire about morphology and behavioral alterations. Results: Analysis of variance showed that ethanol doses of 0.25 and 0.5% do not cause morphological malformations and did not impair associative learning but increased anxiety-like behavior responses and lower exploratory behavior when compared to the control. Conclusion: Our results demonstrate that one can detect behavioral abnormalities in the zebrafish induced by embryonic ethanol as early as 10 days post-fertilization and that alcohol increases anxious behavior during young development in zebrafish.
... EAE had no detectable impact on food consumption in adult males, suggesting that it is an unlikely mechanism for increased adiposity ( Figure 4B). Multiple studies have demonstrated that EAE affects zebrafish swimming behaviors (35)(36)(37)(38). To determine whether reduced locomotion was present in adults after EAE, we assessed activity level (35,(39)(40)(41)(42)(43)(44). ...
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Prenatal alcohol exposure (PAE) affects at least 10% of newborns globally and leads to the development of fetal alcohol spectrum disorders (FASDs). Despite its high incidence, there is no consensus on the implications of PAE on metabolic disease risk in adults. Here, we describe a cohort of adults with FASDs that had an increased incidence of metabolic abnormalities, including type 2 diabetes, low HDL, high triglycerides, and female-specific overweight and obesity. Using a zebrafish model for PAE, we performed population studies to elucidate the metabolic disease seen in the clinical cohort. Embryonic alcohol exposure (EAE) in male zebrafish increased the propensity for diet-induced obesity and fasting hyperglycemia in adulthood. We identified several consequences of EAE that may contribute to these phenotypes, including a reduction in adult locomotor activity, alterations in visceral adipose tissue and hepatic development, and persistent diet-responsive transcriptional changes. Taken together, our findings define metabolic vulnerabilities due to EAE and provide evidence that behavioral changes and primary organ dysfunction contribute to resultant metabolic abnormalities.
Article
Fetal Alcohol Spectrum Disorder (FASD) is characterized by a variety of morphological, behavioural and cognitive deficits, ranging from mild to severe. Numerous animal models, including the zebrafish, have been employed to better understand the onset, expression and progression of this disorder. Embryonic ethanol-induced deficits in learning and memory, anxiety, social responses and elevated alcohol self-administration have been successfully demonstrated in zebrafish. Studies in zebrafish have also shown the expression of these behavioural deficits depends upon the developmental stage of ethanol exposure, the age of observation, as well as the genotype (strain or population origin) of the tested zebrafish. Here, we investigate how the genotype and age of observation may influence embryonic ethanol-induced alterations in anxiety-like responses in zebrafish. Zebrafish embryos exposed to either 0% or 1% (vol/vol) ethanol at 24hpf were tested in an open tank at one of three stages: larval (6–8 days post fertilization (dpf)), mid-larval (16-18dpf), or juvenile (26-28dpf). Two genotypes were tested in this manner, ABNS (a quasi-inbred strain) and ABSK (a mix of AB, TU and TL strains). We found embryonic ethanol induced behavioural changes to significantly differ depending on the genotype and age of observation. For example, significant differences between control and ethanol exposed zebrafish in both genotypes were observed in juvenile zebrafish, but few significant treatment effects were observed in larval zebrafish. Additionally, ethanol appeared to alter anxiety-like behaviours in the ABNS genotype but did not have as robust of an effect on the ABSK genotype. Lastly, there were significant behavioural differences between unexposed (control) zebrafish of the two genotypes, suggesting baseline behavioural differences despite a common AB genetic origin.
Article
Zebrafish are increasingly being utilized to model the behavioral and neurochemical effects of pharmaceuticals and, more recently, pharmaceutical interactions. Zebrafish models of stress establish that both caffeine and ethanol influence anxiety, though few studies have implemented co-administration to assess the interaction of anxiety and reward-seeking. Caffeine exposure in zebrafish is teratogenic, causing developmental abnormalities in the cardiovascular, neuromuscular, and nervous systems of embryos and larvae. Ethanol is also a teratogen and, as an anxiolytic substance, may be able to offset the anxiogenic effects of caffeine. Co-exposure to caffeine and alcohol impacts neuroanatomy and behavior in adolescent animal models, suggesting stimulant substances may moderate the impact of alcohol on neural circuit development. Here, we review the literature describing neuropharmacological and behavioral consequences of caffeine and/or alcohol exposure in the zebrafish model, focusing on neurochemistry, locomotor effects, and behavioral assessments of stress/anxiety as reported in adolescent/juvenile and adult animals. The purpose of this review is twofold: (1) describe the work in zebrafish documenting the effects of ethanol and/or caffeine exposure and (2) compare these zebrafish studies with comparable experiments in rodents. We focus on specific neurochemical pathways (dopamine, serotonin, adenosine, GABA, adenosine), anxiety-type behaviors (assessed with novel tank, thigmotaxis, shoaling), and locomotor changes resulting from both individual and co-exposure. We compare findings in zebrafish with those in rodent models, revealing similarities across species and identifying conservation of mechanisms that potentially reinforce co-addiction.
Article
Ethanol consumption is a worldwide problem. Sensitivity to acute effects of ethanol influences the development of chronic ethanol abuse and ethanol dependence. Environmental and genetic factors have been found to contribute to differential effects of acute ethanol. Animal models have been employed to investigate these factors. An increasingly frequently utilized animal model in ethanol research is the zebrafish. A large proportion of ethanol studies with zebrafish have been conducted with adult zebrafish. However, high throughput drug and mutation screens are particularly well adapted to larval zebrafish. These studies are often carried out using the 96-well-plate that allows monitoring large numbers of fish efficiently. Here, we investigate the effects of acute (30 min long) ethanol exposure in 8-day post-fertilization (dpf) old zebrafish. We compare four genetically distinct populations (strains) of zebrafish, measuring numerous parameters of their swim path in two well sizes, i.e., in the 96-well-plate (small volume wells) and in the 6-well-plate (large volume wells). In general, we found that the highest dose of ethanol (1% vol/vol) reduced swim speed, increased duration of immobility, increased turn angle, and increased intra-individual variance of turn angle, while the intermediate dose (0.5%) had a less strong effect, compared to control. However, we also found that these ethanol effects were strain dependent and, in general, were better detected in the larger volume well. We conclude that larval zebrafish are appropriate for quantification of acute ethanol effects and also for the analysis of environmental and genetic factors that influence these effects. We also speculate that using larger wells will likely increase sensitivity of detection and precision in screening applications.
Chapter
The zebrafish has been utilized for the analysis of fundamental developmental processes for over four decades. More recently, this species is also employed in a variety of subdisciplines of biology, because it possesses evolutionarily conserved features and thus allows modeling of a variety of human disorders. One of these disorders is fetal alcohol spectrum disorder (FASD). FASD represents a cluster of symptoms with differing features and severity all arising as a result of women drinking during their pregnancy. In this chapter we will review a particular zebrafish model whose aim is to mimic the milder, and more prevalent, forms of FASD. To achieve this, zebrafish eggs (embryos) are immersed in low alcohol concentration bath (up to 1% vol/vol) for only a short period of time (2 h). The advantage of this dosing regimen is that, just like in the milder forms of FASD, it does not lead to structural deformities or increased mortality. Instead, it affects brain development in a way that manifests in abnormal behavioral responses. This chapter, focusing mainly on the results obtained in the laboratory of the author, reviews these abnormalities and also the admittedly limited amount of information we know about potential underlying mechanisms, including alterations in neurotransmitter systems, apoptotic cell death, and neuronal and glial cell phenotypes.
Article
The objective of this work was to determine whether folic acid (FA) reduces the embryonic ethanol (EtOH) exposure induced behavioral and morphological defects in our zebrafish fetal alcohol spectrum disorder (FASD) model. Teratogenic effects, mortality, the excitatory light-dark locomotion (ELD), sleep (SL), thigmotaxis (TH), touch sensitivity (TS), and optomotor response (OMR) tests were evaluated in larvae (6-7 days post-fertilization) using four treatment conditions: Untreated, FA, EtOH and EtOH + FA. FA reduced morphological defects on heart, eyes and swim bladder inflation seen in EtOH exposed fish. The larvae were more active in the dark than in light conditions, and EtOH reduced the swimming activity in the ELD test. EtOH affected the sleep pattern, inducing several arousal periods and increasing inactivity in zebrafish. FA reduces these toxic effects and produced more consistent inactivity during the night, reducing the arousal periods. FA also prevented the EtOH-induced defects in thigmotaxis and optomotor response of the larvae. We conclude that in this FASD model, EtOH exposure produced several teratogenic and behavioral defects, FA reduced, but did not totally prevent, these defects. Understanding of EtOH-induced behavioral defects could help to identify new therapeutic or prevention strategies for FASD.
Article
FASD results from the developing fetus being exposed to alcohol, and is characterized by morphological, behavioural and cognitive deficits. However, the expression, severity and age of onset of these symptoms has been found to show variation. This variation may partly be due to the developmental stage at which alcohol reached the developing fetus. Previously, alcohol was shown to lead to significant concentration dependent behavioural as well as neurochemical changes detected in adult zebrafish when this substance was administered at 24 h post-fertilization (hpf) for 2 h. This alcohol exposure method arguably mimicked the milder, and more prevalent, forms of human FASD. However, whether the observed changes depended upon the developmental stage, i.e., the timing, of alcohol exposure has not been systematically analyzed. Here, we employ the same alcohol dosing regimen, where zebrafish eggs are immersed into 0% or 1% (vol/vol) alcohol for 2 h, but we perform the immersion at 5, 10, 16, 24, 36, or 48 hpf. We previously developed a sensitive HPLC method to quantify neurochemicals, and found levels of dopamine, serotonin and their metabolites DOPAC and 5-HIAA to be affected by embryonic alcohol treatment. Here, using the same method, we compare whole-brain levels of these neurochemicals in the embryonic alcohol exposed and control zebrafish at their age of 30 days post-fertilization (dpf). Consistent with previous reports, we found significant reduction of levels of dopamine, serotonin and their metabolites in the fish exposed to alcohol at 24 hpf. However, we also found significant dependency on the developmental stage at which alcohol was administered with particularly robust impairments when the exposure was at the early or middle of the developmental periods probed. Our results now demonstrate that one can detect functional abnormalities in the zebrafish brain induced by embryonic alcohol as early as 30 dpf and that the neurochemical deficits are dependent upon the developmental stage at which alcohol is administered.
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Ethanol is one of the most commonly abused drugs. Although environmental and genetic factors contribute to the etiology of alcohol use disorders, it is ethanol's actions in the brain that explain (1) acute ethanol-related behavioral changes, such as stimulant followed by depressant effects, and (2) chronic changes in behavior, including escalated use, tolerance, compulsive seeking, and dependence. Our knowledge of ethanol use and abuse thus relies on understanding its effects on the brain. Scientists have employed both bottom-up and top-down approaches, building from molecular targets to behavioral analyses and vice versa, respectively. This review highlights current progress in the field, focusing on recent and emerging molecular, cellular, and circuit effects of the drug that impact ethanol-related behaviors. The focus of the field is now on pinpointing which molecular effects in specific neurons within a brain region contribute to behavioral changes across the course of acute and chronic ethanol exposure.
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Fetal Alcohol Spectrum Disorders (FASD) includes a continuum of disorders that occur in children as a result of their mothers’ consumption of alcohol during pregnancy. The most severe of these disorders is Fetal Alcohol Syndrome (FAS). FASD presents differently in every child, but all children with FASD have intellectual and/or behavioral impairments. There is no cure for FASD, but research shows that early intervention and life-long support help those born with FASD to manage the difficulties that come with it. This paper examines the characteristics, complications, and treatment for FASD.
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Background: Fetal alcohol spectrum disorders (FASD) may vary in symptoms and severity. In the milder and more prevalent forms of the disease, behavioral abnormalities may include impaired social behavior, for example, difficulty interpreting social cues. Patients with FASD remain often undiagnosed due to lack of biomarkers, and treatment is unavailable because the mechanisms of the disease are not yet understood. Animal models have been proposed to facilitate addressing these problems. More recently, short exposure of the zebrafish embryo to low concentrations of alcohol was shown to lead to significant and lasting impairment of behavior in response to social stimuli. The impairment may be the result of abnormal social behavior or altered fear/anxiety. The goal of the current study was to investigate the latter. Methods: Here, we employed the alcohol exposure regimen used previously (exposure of 24th hour postfertilization embryos to 0.00, 0.25, 0.50, 0.75, or 1.00% vol/vol alcohol for 2 hours), allowed the fish to reach adulthood, and measured the behavioral responses of these adults to a novel tank (anxiety-related behaviors) as well as to an animated image of a sympatric predator of zebrafish (fear-related behaviors). Results: We found behavioral responses of embryonic alcohol-exposed adult fish to remain statistically indistinguishable from those of controls, suggesting unaltered anxiety and fear in the embryonic alcohol-treated fish. Conclusions: Given that motor and perceptual function was previously shown to be also unaltered in the adults after embryonic alcohol exposure, our current results suggest that the impaired response of these fish to social stimuli may be the result of abnormal social behavior.
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Children with fetal alcohol spectrum disorders (FASD) show sociobehavioral impairments; however, the social cognitive profile contributing to these impairments is poorly understood. This study compared social perspective taking and empathy in children with FASD versus typically developing controls (TDC). Thirty-seven children with FASD and 21 TDC participated. Measures included parent-rated CBCL and SSIS, and NEPSY-II Theory of Mind, Test of Social Cognition and Index of Empathy. Parents rated the FASD group higher than TDC on indices of behavior problems and lower on indices of social skills and empathy. Children with FASD scored significantly below TDC on tasks requiring complex social cognition. The majority of correlations between social cognition and parent-ratings were not significant in FASD and TDC, with the exception of a negative correlation between self-reported empathy and parent-rated behavior difficulties in TDC. FASD subgroup analyses revealed lower theory of mind and empathy scores among children with ARND than pFAS/FAS. With regard to sex, males with FASD were rated as having more behavior difficulties than females, whereas TDC females obtained higher empathy ratings than males. In both groups, females scored higher on theory of mind and empathy indices. On theory of mind tasks, older children with FASD performed below younger, whereas younger TDC children performed more poorly than older. Children with FASD show reduced functioning on indices of sociobehavioral and social cognition, and the effects are influenced by sex and age. These findings provide insight into the clinical and social profile of children with FASD. (JINS, 2015, 21, 74-84)
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Background: The zebrafish is a powerful neurobehavioral genetics tool with which complex human brain disorders including alcohol abuse and fetal alcohol spectrum disorders may be modeled and investigated. Zebrafish innately form social groups called shoals. Previously, it has been demonstrated that a single bath exposure (24 hours post-fertilization) to low doses of alcohol (0, 0.25, 0.50, 0.75 and 1% v/v) for a short duration (2 hours) leads to impaired group forming, or shoaling, in adult zebrafish. Methods: In the current study we immersed zebrafish eggs in low concentration of alcohol (0.5 % or 1% v/v) for two hours at 24 hours post fertilization, and let the fish grow and reach adulthood. In addition to quantifying the behavioral response of the adult fish to an animated shoal, we also measured the amount of dopamine and its metabolite DOPAC (3,4-dihydroxyphenylacetic acid) from whole brain extracts of these fish using high-pressure liquid chromatography (HPLC). Results: Here we confirm that embryonic alcohol exposure makes adult zebrafish increase their distance from the shoal stimulus in a dose dependent manner. We also show that the shoal stimulus increases the amount of dopamine and DOPAC in the brain of control zebrafish but not in fish previously exposed to alcohol during their embryonic development. Conclusions: We speculate that one of the mechanisms that may explain the embryonic alcohol induced impaired shoaling response in zebrafish is dysfunction of reward mechanisms subserved by the dopaminergic system. © The Author 2015. Published by Oxford University Press on behalf of CINP.
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Rationale Decline of attentional performance as a function of time engaged on a task and hyperactivity are features shared by children and adults with fetal alcohol syndrome or attentional deficit and hyperactivity disorders. Objective To investigate the effects of prenatal exposure to two doses of ethanol on developmental milestones, locomotor activity and attention. Methods Wistar rats born from dams exposed to one of four maternal treatments during pregnancy were used: A35 - liquid diet with 35% ethanol-derived calories; A10 - liquid diet with 10% ethanol-derived calories; control - ethanol-free liquid diet; chow - laboratory chow and water. Results A35 performed worse in grip strength than control and chow (postnatal day - 14, p < 0.05) but not in negative geotaxis (postnatal days 7-10); A35 also showed more locomotor activity than control and A10 (p < 0.05). Regarding attention, acquisition of the five choice reaction time task was similar between groups, but, the percentage of omission errors from A35 group was greater than other groups at baseline parameters, at shorter (2 s) and longer (7 s) inter-trial intervals and at a shorter stimulus duration (0.5 s) (p < 0.05). The percentage of omissions was larger in A35 as the blocks progressed in sessions with either longer or shorter inter-trial intervals (group × block p < 0.05). Animals from A10 group did not show any impairment in the tasks performed. Conclusions our study demonstrates that as well as developmental impairments, prenatal ethanol can produce deficits associated with an increase in attentional demand in rodents, analogous to those observed in fetal alcohol syndrome and attentional deficit and hyperactivity disorders.
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Prenatal ethanol exposure (PNEE) has been linked to widespread impairments in brain structure and function. There are a number of animal models that are used to study the structural and functional deficits caused by PNEE, including, but not limited to invertebrates, fish, rodents, and non-human primates. Animal models enable a researcher to control important variables such as the route of ethanol administration, as well as the timing, frequency and amount of ethanol exposure. Each animal model and system of exposure has its place, depending on the research question being undertaken. In this review, we will examine the different routes of ethanol administration and the various animal models of fetal alcohol spectrum disorders (FASD) that are commonly used in research, emphasizing their strengths and limitations. We will also present an up-to-date summary on the effects of prenatal/neonatal ethanol exposure on behavior across the lifespan, focusing on learning and memory, olfaction, social, executive, and motor functions. Special emphasis will be placed where the various animal models best represent deficits observed in the human condition and offer a viable test bed to examine potential therapeutics for human beings with FASD.
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Fetal Alcohol Spectrum Disorder (FASD) is a preventable disease of the child resulting from alcohol (ethanol) consumption by pregnant women. Despite being preventable, FASD represents a prevalent problem throughout the world. Embryonic alcohol induced abnormalities in behavioral responses to social stimuli have been shown in humans and zebrafish. The neurobiological mechanisms underlying the abnormalities remain obscured. Here we start a mechanistic analysis by investigating the effect of embryonic alcohol exposure on the neurochemistry of zebrafish. The differeing severity of symptoms seen in FASD may be partially due to genetic factors. To explore such genetic effects, here we analyzed two distinct zebrafish strains: AB and TU. Zebrafish were exposed to one of the following concentrations of alcohol, 0.00%, 0.25%, 0.50%, 0.75%, 1.00% (vol/vol %) at 24hours post-fertilization (hpf) for 2hours. From whole brain extracts we analyzed the amount of neurotransmitters dopamine and serotonin and their metabolites across 4 different developmental time points: 15, 40, 70 and 102days post- fertilization (dpf) using high performance liquid chromatography (HPLC). AB zebrafish exhibited a significant dose dependent embryonic alcohol exposure effect which increased in robustness with age. However, TU showed no such concentration effect: the levels of neurochemicals remained mainly unaltered by embryonic alcohol exposure in all age groups. We also analyzed the amount of alcohol reaching the embryo in the two strains and ruled out the possibility that TU has a more protective chorion. We conclude that the uncovered strain differences are due to genetic differences that protect TU from the deleterious effects of embryonic alcohol exposure.
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Prenatal exposure to alcohol has consistently been associated with adverse effects on neurodevelopment, which is collectively called fetal alcohol spectrum disorder (FASD). Increasing evidence suggest that prenatal exposure to alcohol increases the risk of developing attention deficit/hyperactivity disorder-like behavior in human. In this study, we investigated the behavioral effects of prenatal exposure to EtOH in offspring mice and rats focusing on hyperactivity and impulsivity. We also examined changes in dopamine transporter and MeCP2 expression, which may underlie as a key neurobiological and epigenetic determinant in FASD and hyperactive, inattentive and impulsive behaviors. Mouse or rat offspring born from dam exposed to alcohol during pregnancy (EtOH group) showed hyper locomotive activity, attention deficit and impulsivity. EtOH group also showed increased dopamine transporter and norepinephrine transporter level compared to control group in the prefrontal cortex and striatum. Prenatal exposure to EtOH also significantly decreased the expression of MeCP2 in both prefrontal cortex and striatum. These results suggest that prenatal exposure to EtOH induces hyperactive, inattentive and impulsive behaviors in rodent offspring that might be related to global epigenetic changes as well as aberration in catecholamine neurotransmitter transporter system.
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The biological mechanisms of human social behavior are complex. Animal models may facilitate the understanding of these mechanisms and may help one to develop treatment strategies for abnormal human social behavior, a core symptom in numerous clinical conditions. The zebrafish is perhaps the most social vertebrate among commonly used laboratory species. Given its practical features and the numerous genetic tools developed for it, it should be a promising tool. Zebrafish shoal, i.e. from a tight multimember groups, but the ontogenesis of this behavior has not been described. Analyzing the development of shoaling is a step towards discovering the mechanisms of this behavior. Here we study age-dependent changes of shoaling in zebrafish from day 7 post fertilization to over 5months of age by measuring the distance between all pairs of fish in freely swimming groups of ten subjects. Our longitudinal (repeated measure within subject) and cross sectional (non-repeated measure between subject) analyses both demonstrated a significant increase of shoaling with age (decreased distance between shoal members). Given the sophisticated genetic and developmental biology methods already available for zebrafish, we argue that our behavioral results open a new avenue towards the understanding of the development of vertebrate social behavior and of its mechanisms and abnormalities.
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There is substantial evidence that genetic variation, at both the level of the individual and population, has a significant effect on behaviour, fitness and response to toxicants. Using DNA microsatellites, we examined the genetic variation in samples of several commonly used laboratory strains of zebrafish, Danio rerio, a model species in toxicological studies. We compared the genetic variation to that found in a sample of wild fish from Bangladesh. Our findings show that the wild fish were significantly more variable than the laboratory strains for several measures of genetic variability, including allelic richness and expected heterozygosity. This lack of variation should be given due consideration for any study which attempts to extrapolate the results of ecotoxicological laboratory tests to wild populations.
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Zebra fish may be an ideal vertebrate model system for numerous human diseases with which the genetics and biological mechanisms of the disease may be studied. Zebra fish has been successfully used in developmental genetics, and recently, neurobiologists have also started to study this species. A potentially interesting target disease amenable for analysis with zebra fish is drug addiction, e.g. alcoholism. Although genetic tools to manipulate the genome of zebra fish are available, appropriate phenotypical testing methods are often lacking. In this paper, we describe basic behavioral tests to investigate the acute effects of alcohol on zebra fish. These behavioral paradigms will be useful for the genetic and biological analysis of acute and chronic drug effects as well as addiction. In addition to presenting findings for the acute effects of alcohol, we briefly describe our strategy for generating and screening mutants. We hope that our pilot work will facilitate the future development of behavioral tests and the use of zebra fish in the genetic analysis of the biological effects of drugs of abuse.
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This chapter outlines the current issues surrounding the construction, maintenance, and use of genetically defined lines of zebrafish. The increasing number of zebrafish strains used for research raises a number of significant issues. Careful identification of zebrafish lines is critical in establishing the generality of research results. Each line of zebrafish is usually derived from a separate founder stock, and therefore each might harbor a unique genetic background. Through epistatic gene action, the genetic background of a given line can have marked effects on experimental results. Differences among the backgrounds of different wild-type lines could conceivably result in a given mutation displaying different phenotypes, which could have a variety of effects. This is particularly important in developmental biology because of the extensive use of forward genetic mutagenesis screens. The goals of a breeding program can be organized into three broad categories: genetic uniformity, maintenance of genetic variation, and selection for desirable phenotypes. Genetic monitoring of standard lines has made use of morphological, behavioral, immunological, and general line performance traits. Distribution of a line should provide the line's entire genetic background.
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Fetal Alcohol Spectrum Disorder (FASD) is one of the leading causes of mental health issues worldwide. Analysis of zebrafish exposed to alcohol during embryonic development confirmed that even low concentrations of alcohol for a short period of time may have lasting behavioral consequences at the adult or old age. The mechanism of this alteration has not been studied. Here, we immersed zebrafish embryos into 1% alcohol solution (vol/vol%) at 24 hours post‐fertilization (hpf) for 2 hours, and analyze potential changes using immunohistochemistry. We measured the number of BDNF (brain derived neurotrophic factor) and NCAM (neuronal cell adhesion molecule) positive neurons and the intensity of synaptophysin staining in eight brain regions: lateral zone of the dorsal telencephalic area, medial zone of the dorsal telencephalic area, dorsal nucleus of the ventral telencephalic area, ventral nucleus of the ventral telencephalic area, parvocellular preoptic nucleus, ventral habenular nucleus, corpus cerebella and inferior reticular formation. We found embryonic alcohol exposure to significantly reduce the number of BDNF and NCAM positive cells in all brain areas studied as compared to control. We also found alcohol to significantly reduce the intensity of synaptophysin staining in all brain areas except the cerebellum and preoptic area. These neuroanatomical changes correlated with previously demonstrated reduction of social behavior in embryonic alcohol exposed zebrafish, raising the possibility of a causal link. Given the evolutionary conservation across fish and mammals, we emphasize the implication of our current study for human health: even small amount of alcohol consumption may be unsafe during pregnancy. This article is protected by copyright. All rights reserved.
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IMPORTANCE: Prevalence estimates are essential to effectively prioritize, plan, and deliver health care to high-needs populations such as children and youth with fetal alcohol spectrum disorder (FASD). However, most countries do not have population-level prevalence data for FASD. OBJECTIVE: To obtain prevalence estimates of FASD among children and youth in the general population by country, by World Health Organization (WHO) region, and globally. DATA SOURCES: MEDLINE, MEDLINE in process, EMBASE, Education Resource Information Center, Cumulative Index to Nursing and Allied Health Literature, Web of Science, PsychINFO, and Scopus were systematically searched for studies published from November 1, 1973, through June 30, 2015, without geographic or language restrictions. STUDY SELECTION: Original quantitative studies that reported the prevalence of FASD among children and youth in the general population, used active case ascertainment or clinic-based methods, and specified the diagnostic guideline or case definition used were included. DATA EXTRACTION AND SYNTHESIS: Individual study characteristics and prevalence of FASD were extracted. Country-specific random-effects meta-analyses were conducted. For countries with 1 or no empirical study on the prevalence of FASD, this indicator was estimated based on the proportion of women who consumed alcohol during pregnancy per 1 case of FASD. Finally, WHO regional and global mean prevalence of FASD weighted by the number of live births in each country was estimated. MAIN OUTCOMES AND MEASURES: Prevalence of FASD. RESULTS: A total of 24 unique studies including 1416 unique children and youth diagnosed with FASD (age range, 0-16.4 years) were retained for data extraction. The global prevalence of FASD among children and youth in the general population was estimated to be 7.7 per 1000 population (95% CI, 4.9-11.7 per 1000 population). The WHO European Region had the highest prevalence (19.8 per 1000 population; 95% CI, 14.1-28.0 per 1000 population), and the WHO Eastern Mediterranean Region had the lowest (0.1 per 1000 population; 95% CI, 0.1-0.5 per 1000 population). Of 187 countries, South Africa was estimated to have the highest prevalence of FASD at 111.1 per 1000 population (95% CI, 71.1-158.4 per 1000 population), followed by Croatia at 53.3 per 1000 population (95% CI, 30.9-81.2 per 1000 population) and Ireland at 47.5 per 1000 population (95% CI, 28.0-73.6 per 1000 population). CONCLUSIONS AND RELEVANCE: Globally, FASD is a prevalent alcohol-related developmental disability that is largely preventable. The findings highlight the need to establish a universal public health message about the potential harm of prenatal alcohol exposure and a routine screening protocol. Brief interventions should be provided, where appropriate.
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Fetal Alcohol Spectrum Disorders (FASD) represent a large unmet medical need. Exposure of the developing human embryo to alcohol can lead to life-long suffering. Despite the well documented deleterious effects of alcohol on the developing fetus, pregnant women continue to drink alcohol, and FASD remains the leading cause of preventable mental retardation and other behavioral abnormalities. Particularly prevalent are the milder forms of the disease cluster, representing children who do not show obvious physical signs and who may be undiagnosed or misdiagnosed. To develop treatment and diagnostic tools, researchers have turned to animal models. The zebrafish is becoming one of the leading biomedical research organisms that may facilitate discovery of the biological mechanisms underlying this disease and the identification of biomarkers that may be used for diagnosis. Here we review the latest advances of this field, mostly focussing on the discoveries made in our own laboratory and others with zebrafish employed to analyze the effects of moderate to low level of exposure to alcohol. We argue that the zebrafish represents unique advantages, and adding information obtained with this species to the mix of other animal models will significantly increase translational relevance of animal biomedical research for the analysis of human FASD.
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The term "fetal alcohol spectrum disorders" (FASD) defines the full range of ethanol (EtOH)-induced birth defects. Numerous variables influence the phenotypic outcomes of embryonic EtOH exposure. Among these variables, genetics appears to play an important role, yet our understanding of the genetic predisposition to FASD is still in its infancy. We review the current literature that relates to the genetics of FASD susceptibility and gene-EtOH interactions. Where possible, we comment on potential mechanisms of reported gene-EtOH interactions. Early indications of genetic sensitivity to FASD came from human and animal studies using twins or inbred strains, respectively. These analyses prompted searches for susceptibility loci involved in EtOH metabolism and analyses of candidate loci, based on phenotypes observed in FASD. More recently, genetic screens in animal models have provided an additional insight into the genetics of FASD. Understanding FASD requires that we understand the many factors influencing phenotypic outcome following embryonic EtOH exposure. We are gaining ground on understanding some of the genetics behind FASD, yet much work remains to be carried out. Coordinated analyses using human patients and animal models are likely to be highly fruitful in uncovering the genetics behind FASD.
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Fetal alcohol spectrum disorder (FASD) is a devastating disease of the brain caused by exposure to alcohol during prenatal development. Its prevalence exceeds 1%. The majority of FASD cases represent the milder forms of the disease which often remain undiagnosed, and even when diagnosed treatment options for the patient are limited due to lack of information about the mechanisms that underlie the disease. The zebrafish has been proposed as a model organism for exploring the mechanisms of FASD. Our laboratory has been studying the effects of low doses of alcohol during embryonic development in the zebrafish. This review discusses the methods of alcohol exposure, its effects on behavioral performance including social behavior and learning, and the potential underlying biological mechanisms in zebrafish. It is based upon a recent keynote address delivered by the author, and it focuses on findings obtained mainly in his own laboratory. It paints a promising future of this small vertebrate in FASD research. © 2015 Wiley Periodicals, Inc. Dev Psychobiol. © 2015 Wiley Periodicals, Inc.
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The zebrafish is increasingly utilized in the analysis of the effects of ethanol (alcohol) on brain function and behavior. We have shown significant population-dependent alcohol-induced changes in zebrafish behavior and have started to analyze alterations in dopaminergic and serotoninergic responses. Here, we analyze the effects of alcohol on levels of selected neurochemicals using a 2 × 3 (chronic × acute) between-subject alcohol exposure paradigm randomized for two zebrafish populations, AB and SF. Each fish first received the particular chronic treatment (0 or 0.5 vol/vol % alcohol) and subsequently the acute exposure (0, 0.5 or 1.0 % alcohol). We report changes in levels of dopamine, DOPAC, serotonin, 5HIAA, glutamate, GABA, aspartate, glycine and taurine as quantified from whole brain extracts using HPLC. We also analyze monoamine oxidase and tyrosine hydroxylase enzymatic activity. The results demonstrate that compared to SF, AB is more responsive to both acute alcohol exposure and acute alcohol withdrawal at the level of neurochemistry, a finding that correlates well with prior behavioral observations and one which suggests the involvement of genes in the observed alcohol effects. We discuss correlations between the current results and prior behavioral findings, and stress the importance of characterization of zebrafish strains for future behavior genetic and psychopharmacology studies.
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The zebrafish (Danio rerio) is rapidly becoming a popular model organism in pharmacogenetics and neuropharmacology. Both larval and adult zebrafish are currently used to increase our understanding of brain function, dysfunction, and their genetic and pharmacological modulation. Here we review the developing utility of zebrafish in the analysis of complex brain disorders (including, for example, depression, autism, psychoses, drug abuse and cognitive disorders), also covering zebrafish applications towards the goal of modeling major human neuropsychiatric and drug-induced syndromes. We argue that zebrafish models of complex brain disorders and drug-induced conditions have become a rapidly emerging critical field in translational neuropharmacology research.
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The zebrafish has been proposed as a model organism to study genetic effects influencing behaviour and also as a tool with which the mechanisms of the action of alcohol (ethanol or EtOH) in the vertebrate brain may be investigated. In the current study we exposed zebrafish from two genetically distinct strains (WIK and TU) to a computer animated image of a natural predator of this species, the Indian leaf fish. We measured the subjects' behavioural responses in the presence of different acute doses of alcohol (0.00, 0.25, 0.50, and 1.00% vol/vol) using an observation based event-recording method. We found fish of both strains to exhibit an atypical predator inspection response during the presentation of the animated predator image coupled with a classical fear response, increased jumping frequency. We found numerous alcohol induced behavioural changes and more importantly also revealed alcohol induced strain dependent changes as well, including different dose-response trajectories for WIK versus TU in predator inspection response, general swimming activity, location of swimming (top vs. bottom half of the tank) and freezing. The results suggest that zebrafish of the TU strain may be more tolerant at least to lower doses of alcohol as compared to WIK. The characterization of strain differences in zebrafish will aid the identification of possible molecular mechanisms involved in alcohol's actions in the vertebrate brain.
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It makes sense to attribute a definite percentage of variation in some measure of behavior to variation in heredity only if the effects of heredity and environment are truly additive. Additivity is often tested by examining the interaction effect in a two-way analysis of variance (ANOVA) or its equivalent multiple regression model. If this effect is not statistically significant at the α = 0.05 level, it is common practice in certain fields (e.g., human behavior genetics) to conclude that the two factors really are additive and then to use linear models, which assume additivity. Comparing several simple models of nonadditive, interactive relationships between heredity and environment, however, reveals that ANOVA often fails to detect nonadditivity because it has much less power in tests of interaction than in tests of main effects. Likewise, the sample sizes needed to detect real interactions are substantially greater than those needed to detect main effects. Data transformations that reduce interaction effects also change drastically the properties ofthe causal model and may conceal theoretically interesting and practically useful relationships. If the goal ofpartitioning variance among mutually exclusive causes and calculating “heritability” coefficients is abandoned, interactive relationships can be examined more seriously and can enhance our understanding of the ways living things develop.
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The zebrafish is becoming increasingly popular in behavior genetics because it may allow one to conduct large scale mutation and drug screens facilitating the discovery of mechanisms of complex traits. Strain differences in adult zebrafish behavior have already been reported, which may have important implications in neurobehavioral genetics. For example, we have found the AB and SF strains to differ in their behavioral responses to both acute and chronic alcohol exposure. In the current study, we further characterize these strains using semi-quantitative RT-PCR to measure the expression of ten selected genes and HPLC to measure the levels of nine neurochemicals. We chose the target genes and neurochemicals based upon their potential involvement in alcohol and other drugs of abuse related mechanisms. We quantified the expression of the genes encoding D1-R, D2a-R, D4a-R dopamine receptors, GABA(A)-R, GABA(B)-R1, GAD1, MAO, NMDA-R (NR2D subunit), 5HT-R1bd and SLC6 a4a. We found the gene encoding D1 dopamine receptor over-expressed and the genes encoding GABA(B1) receptor and solute family carrier protein 6 (SLC6) 4a under-expressed in SF compared to AB. We also found the level of all (dopamine, DOPAC, Serotonin, GABA, Glutamate, Glycine, Aspartate, Taurine) but one (5HIAA) neurochemicals tested decreased in SF as compared to AB. These results, combined with previously identified behavioral differences between the AB and SF strains, demonstrate the importance of strain characterization in zebrafish. They now also allow formulation of working hypotheses about possible mechanisms underlying the differential effects of acute and chronic alcohol treatment on these two zebrafish strains.
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As the current paradigms of drug discovery evolve, it has become clear that a more comprehensive understanding of the interactions between small molecules and organismal biology will be vital. The zebrafish is emerging as a complement to existing in vitro technologies and established preclinical in vivo models that can be scaled for high-throughput. In this review, we highlight the current status of zebrafish toxicology studies, identify potential future niches for the model in the drug development pipeline, and define the hurdles that must be overcome as zebrafish technologies are refined for systematic toxicology.
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Individuals gestationally exposed to alcohol experience a multitude of sociobehavioral impairments, including deficits in adaptive behaviors such as social skills. The goal of this report is to critically review research on social skills deficits in individuals with prenatal alcohol exposure, including individuals with and without fetal alcohol spectrum disorders (FASD). Social deficits are found in alcohol-exposed children, adults, and adolescents with and without a clinical presentation. These deficits tend to persist across the lifespan and may even worsen with age. Social deficits in this population appear to be independent of facial dysmorphology and IQ and are worse than can be predicted based on atypical behaviors alone. Abnormalities in neurobiology, executive function, sensory processing, and communication likely interact with contextual influences to produce the range of social deficits observed in FASD. Future investigations should strive to reconcile the relationship between social skills deficits in FASD and variables such as gender, age, cognitive profile, and structural and functional brain impairments to enable better characterization of the deficits observed in this population, which will enhance diagnosis and improve remediation.
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Fetal alcohol syndrome (FAS) is a devastating disorder accompanied by numerous morphological and behavioral abnormalities. Human FAS has been modeled in laboratory animals including the zebrafish. Recently, embryonic exposure to low doses of ethanol has been shown to impair behavior without any gross morphological alterations in zebrafish. The exposed zebrafish showed reduced responses to animated conspecific images. The effect of embryonic ethanol exposure, however, has not been investigated in a real shoal and the potential mechanisms underlying the behavioral impairment are also unknown. Here we show that a 2h long immersion in 0.25% and 0.50% (vol/vol) alcohol at 24h post fertilization significantly increases the distance among members of freely swimming groups of zebrafish when measured at 70 days post fertilization. We also show that this impaired behavior is accompanied by reduced levels of dopamine, DOPAC, serotonin and 5HIAA as quantified by HPLC from whole brain extracts. Our results demonstrate that even very low concentrations of alcohol applied for a short period of time during the development of zebrafish can impair behavior and brain function. We argue that the observed behavioral impairment is not likely to be due to altered performance capabilities, e.g. motor function or perception, but possibly to social behavior itself. We also argue that our neurochemical data represent the first step towards understanding the mechanisms of this abnormality in zebrafish, which may lead to better modeling of, and ultimately perhaps better therapies for human FAS.
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Zebrafish are becoming increasingly popular in behavioral neuroscience as investigators have started to realize the benefits of sophisticated genetic tools specifically developed for this species along with the pharmacological tools already available for other laboratory model organisms. The zebrafish has been proposed as an in vivo tool for the analysis of vertebrate fear responses as well as human psychopathological conditions such as anxiety. We have been developing behavioral tasks for zebrafish that could be utilized for screening mutation or drug induced changes in fear responses. In this paper we present a modified version of a previously developed predator avoidance paradigm that now allows the induction and quantification of avoidance reactions that we previously could not elicit. Most importantly, in the current paradigm zebrafish are now shown to respond to the appearance of a moving image of a sympatric predator, the Indian leaf fish, by increasing their distance from the image, a robust reaction that is easy to quantify in an automated manner. Unexpectedly, however, another fear response, the "diving" response, was seen robustly only at the beginning of the test but not in response to the predator stimulus. We discuss the implications of these results and conclude that although zebrafish fear responses are complex and context dependent, the current paradigm is a significant step towards high throughput screening for alterations in fear responses of zebrafish.
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Animal models of fetal alcohol spectrum disorder (FASD) have been used to demonstrate the specificity of alcohol's teratogenic effects and some of the underlying changes in the central nervous system (CNS) and, more recently, to explore ways to ameliorate the effects of alcohol. The main point of this review is to highlight research findings from the animal literature which point to the impact of the social context or social behavior on the effect(s) of alcohol exposure during development, and also to point to research questions about the social environment and effects of prenatal alcohol exposure that remain to be answered. Alcohol exposure during early development alters maternal responding to the exposed pup in a variety of ways and the alteration in maternal responding could alter later stress responsivity and adult maternal and social behavior of the exposed offspring. Environmental enrichment and voluntary exercise have been shown to ameliorate some of alcohol's impact during development, but the roles of enhanced social interactions in the case of enrichment and of social housing during voluntary exercise need to be more fully delineated. Similarly, the role of social context across the lifespan, such as social housing, social experiences, and contact with siblings, needs further study. Because of findings that alcohol during development alters DNA methylation patterns and that there are alterations in the maternal care of the alcohol-exposed offspring, epigenetic effects and their relationship to social behavior in animal models of FASD are likely to become a fruitful area of research. Because of the simpler social behavior and the short lifespan of rodents, animal models of FASD can be useful in determining how the social context impacts the effects of alcohol exposure during development.
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Zebrafish is becoming an important research tool for the analysis of brain function and behavior. It has been proposed to model human alcoholism as well as fetal alcohol syndrome. Previous studies investigating the consequences of exposure to ethanol during early development of zebrafish employed robust dosing regimens (high ethanol concentration and long exposure) that may model a rare situation in the human clinic. These studies found major structural abnormalities developing in the exposed fish. Here we hope to avoid such gross changes and administer only low doses of ethanol (0.00, 0.25, 0.50, 0.75, 1.00 vol/vol %) at 24-hour postfertilization and for only a short period of time (for 2 hours). We analyze the behavior of exposed fish at adult stage using computerized stimulus presentation and automated videotracking response quantification. Despite the short ethanol exposure period and the modest concentrations, significant behavioral alterations were found: fish exposed to higher doses of ethanol swam at an increased distance from a computer-animated zebrafish shoal while their activity levels did not change. Although the interpretation of and the mechanisms underlying this finding will require further investigation, the results suggest that zebrafish will be an appropriate model organism for the analysis of the effects of moderate to mild prenatal ethanol exposure.
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When mice are introduced into an open-field, they are inclined to explore mainly the peripheral zone of this open-field. This tendency to remain close the walls, called thigmotaxis, decreases gradually during the first minutes of exploration. We have considered the degree of thigmotaxis during this period of decrease as an index of anxiety in mice. This hypothesis has been validated with several reference anxiogenic drugs (dexamphetamine, pentylenetetrazole, yohimbine, idazoxan) which increased thigmotaxis; and with anxiolytic drugs (buspirone, phenobarbital), which reduced it. On this test the selective or non-selective indirect dopamine agonists GBR 12783, dexamphetamine and cocaine induced an increase of thigmotaxis. Finally, the simultaneous involvement of D1 and D2 dopamine receptors has been evidenced in the anxiogenic-like effect associated with an increase of dopaminergic transmissions.
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The concept of unintended pregnancy has been essential to demographers in seeking to understand fertility, to pub- lic health practitioners in preventing unwanted childbear- ing and to both groups in promoting a woman's ability to determine whether and when to have children. Accurate mea- surement of pregnancy intentions is important in under- standing fertility-related behaviors, forecasting fertility, es- timating unmet need for contraception, understanding the impact of pregnancy intentions on maternal and child health, designing family planning programs and evaluating their effectiveness, and creating and evaluating community-based programs that prevent unintended pregnancy. 1 Pregnancy unintendedness is a complex concept, and has been the subject of recent conceptual and method- ological critiques. 2 Pregnancy intentions are increasingly viewed as encompassing affective, cognitive, cultural and contextual dimensions. Developing a more complete un- derstanding of pregnancy intentions should advance ef- forts to increase contraceptive use, to prevent unintended pregnancies and to improve the health of women and their children. To provide a scientific foundation for public health efforts to prevent unintended pregnancy, we conducted a review of unintended pregnancy between the fall of 1999 and the spring of 2001 as part of strategic planning activities with- in the Division of Reproductive Health at the Centers for Disease Control and Prevention (CDC). We reviewed the published and unpublished literature, consulted with ex- perts in reproductive health and held several joint meetings with the Demographic and Behavioral Research Branch of the National Institute of Child Health and Human Devel- opment, and the Office of Population Affairs of the De- partment of Health and Human Services. We used standard scientific search engines, such as Medline, to find relevant articles published since 1975, and identified older references from bibliographies contained in recent articles; academic experts and federal officials helped to identify unpublished reports. This comment summarizes our findings and in- corporates insights gained from the joint meetings and the strategic planning process.
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The zebrafish has proven to be an excellent model for analyzing issues of vertebrate development. In this review we ask whether the zebrafish is a viable model for analyzing the neurodevelopmental causes of autism. In developing an answer to this question three topics are considered. First, the general attributes of zebrafish as a model are discussed, including low cost maintenance, rapid life cycle and the multitude of techniques available. These techniques include large-scale genetic screens, targeted loss and gain of function methods, and embryological assays. Second, we consider the conservation of zebrafish and mammalian brain development, structure and function. Third, we discuss the impressive use of zebrafish as a model for human disease, and suggest several strategies by which zebrafish could be used to dissect the genetic basis for autism. We conclude that the zebrafish system could be used to make important contributions to understanding autistic disorders.
Article
Prenatal exposure to alcohol has been shown to produce the overt physical and behavioral symptoms known as fetal alcohol syndrome (FAS) in humans. Also, it is believed that low concentrations and/or short durations of alcohol exposure can produce more subtle effects. The purpose of this study was to investigate the effects of embryonic ethanol exposure on the zebrafish (Danio rerio) in order to determine whether this species is a viable animal model for studying FAS. Fertilized embryos were reared in varying concentrations of ethanol (1.5% and 2.9%) and exposure times (e.g., 0-8, 6-24, 12-24, and 48-72 h postfertilization; hpf); anatomical measures including eye diameter and heart rate were compared across groups. Results found that at the highest concentration of ethanol (2.9%), there were more abnormal physical distortions and significantly higher mortality rates than any other group. Embryos exposed to ethanol for a shorter duration period (0-8 hpf) at a concentration of 1.5% exhibited more subtle effects such as significantly smaller eye diameter and lower heart rate than controls. These results indicate that embryonic alcohol exposure affects external and internal physical development and that the severity of these effects is a function of both the amount of ethanol and the timing of ethanol exposure. Thus, the zebrafish represents a useful model for examining basic questions about the effects of embryonic exposure to ethanol on development.
Article
At a time when common regulatory pathways are being identified in several different species and genomics is beginning to allow comparisons of genes, how they are arranged on chromosomes and how they are regulated, zebrafish has emerged as a valuable and complementary vertebrate model. Some of the characteristics that prove of value are described and illustrated. Fluorescent transgenic lines of zebrafish embryos are presented for time-line studies with neurotoxicants. While genetic knockout technology has yet to be developed for the model, the anti-sense, morpholino approach allows for knockdown of expression of genes for the 3 day, embryonic period. This can provide for phenocopies of mutant genes for those genes essential to embryonic development or it can provide for a limited inhibition of gene expression that allows subsequent development of the fish. With the zebrafish genomic sequencing effort, microarray technology is now developing for the model system. These resources and technologies allow one to challenge the system with toxicants, and to view the immediate effects of the toxicants with transgenic embryos that fluoresce in part or all of the nervous system. Behavioral and learning protocols have been developed for the organism so that early exposures can be assayed for effects upon adult fish. Microarray technology should allow for one to identify specific genes and pathways affected by a neurotoxicant. In the future, these approaches should provide a working protocol for exploring molecular mechanisms of neurotoxicants. This type of complementary approach should then allow for more efficient examination and testing of mechanisms in mammalian models.
Article
Ethanol intake during pregnancy can produce a wide range of adverse effects on nervous system development including fetal alcohol syndrome (FAS). The most severe congenital malformation observed in newborns with FAS is cyclopia. In this study, we have exposed zebrafish embryos to different ethanol concentrations (2.4%, 1.5% or 1.0%) during eye morphogenesis in four zebrafish strains (AB, EK, GL and TL). In addition, we have studied the survival rate of the cyclopic animals to the end of larval development. The zebrafish strains GL and AB generated the higher percentage of cyclopic animals after exposure to 2.4% ethanol, while EK showed the higher percent cyclopic animals using 1.5% and 1.0% ethanol. The EK strain showed the higher percent survival during the larval period at all ethanol concentrations (2.4%, 1.5% and 1.0%). Moreover, we have investigated cytoarchitectural alterations in the main components of the visual pathway-retina and optic tectum-and ethanol treatment affects both the retina and the optic tectum. The lamination of neural retina is clearly delayed in treated larvae 3 days postfertilization and the thickness of the pigmented epithelium is considerably reduced. With regard to the optic tectum, treatment with ethanol alters the normal pattern of tectal lamination. The use of zebrafish EK strain is a suitable in vivo vertebrate model system for analyzing the teratogenic effect of ethanol during vertebrate visual system morphogenesis as it relates to both cyclopia and FAS.
Article
The zebrafish has been popular in developmental biology and genetics, but its brain function has rarely been studied. High-throughput screening of mutation or drug-induced changes in brain function requires simple and automatable behavioral tests. This article compares three behavioral quantification methods in four simple behavioral paradigms that test a range of characteristics of adult zebrafish, including novelty-induced responses, social behavior, aggression, and predator-model-induced responses. Two quantification methods, manual recording and computerized videotracking of location and activity, yielded very similar results, suggesting that automated videotracking reliably measures activity parameters and will allow high-throughput screening. However, observation-based event recording of posture patterns was found generally not to correlate with videotracking measures, suggesting that further refinement of automated behavior quantification may be considered.
World Health Organization International Study on the Prevalence of Fetal Alcohol Spectrum Disorder (FASD): Canadian Component. Centre for Addiction and Mental Health
  • S Popova
  • S Lange
  • A E Chudley
  • J N Reynolds
  • J Rehm
  • P A May
  • E P Riley
Popova, S., Lange, S., Chudley, A.E., Reynolds, J.N., Rehm, J., May, P.A., Riley, E.P., 2018. World Health Organization International Study on the Prevalence of Fetal Alcohol Spectrum Disorder (FASD): Canadian Component. Centre for Addiction and Mental Health. CAMH Education, Toronto, Canada, pp. 83.
Stages of embryonic A. Abozaid, et al. Progress in Neuropsychopharmacology & Biological Psychiatry 97 (2020) 109774 development of the zebrafish
  • C Kimmel
  • W Ballard
  • S Kimmel
  • B Ullmann
  • T Schilling
Kimmel, C., Ballard, W., Kimmel, S., Ullmann, B., Schilling, T., 1995. Stages of embryonic A. Abozaid, et al. Progress in Neuropsychopharmacology & Biological Psychiatry 97 (2020) 109774 development of the zebrafish. Dev. Dyn. 203, 253-310.
  • A Abozaid
A. Abozaid, et al. Progress in Neuropsychopharmacology & Biological Psychiatry 97 (2020) 109774