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Behavioural effects of acute ethanol in larval zebrafish (Danio rerio) depend on genotype and volume of experimental well
Abstract
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.
... Increased thigmotaxis may also be effective in nature as proximity to physical objects (the wall of the tank in the lab or the side of the trunk of an immersed tree in nature) reduces the area of attack by, and makes the prey fish less detectable to, the approaching predator [42]. Indeed, these behavioral measures have been successfully employed to quantify anxiety-like responses and changes in such responses induced in psychopharmacology analyses [43][44][45][46]. We note, however, that anxiety-like responses have been found to represent a rich behavioral repertoire, and how exactly zebrafish respond depends upon the environmental context as well as the nature of the aversive stimulus [39]. ...
... 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. ...
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.
BXD recombinant inbred (RI) lines represent a genetic reference population derived from a cross between C57BL/6J mice (B6) and DBA/2J mice (D2), which through meiotic recombination events possesses recombinant chromosomes containing B6 or D2 haplotype segments. The quantitative trait loci (QTLs) are the locations of segregating genetic polymorphisms and are fundamental to understanding genetic diversity in human disease susceptibility and severity. QTL mapping represents the typical approach for identifying naturally occurring polymorphisms that influence complex phenotypes. In this process, genotypic values at markers of known genomic locations are associated with phenotypic values measured in a segregating population. Indeed, BXD RI strains provide a powerful tool to study neurotoxicity induced by different substances. In this review, we describe the use of BXD RI lines to understand the underlying mechanisms of neurotoxicity in response to ethanol and cocaine, as well as metals and pesticide exposures.
Alcohol use disorder (AUD) exacts an immense toll on individuals, families, and society. Genetic factors determine up to 60% of an individual's risk of developing problematic alcohol habits. Effective AUD prevention and treatment requires knowledge of the genes that predispose people to alcoholism, play a role in alcohol responses, and/or contribute to the development of addiction. As a highly tractable and translatable genetic and behavioral model organism, Drosophila melanogaster has proven valuable to uncover important genes and mechanistic pathways that have obvious orthologs in humans and that help explain the complexities of addiction. Vinegar flies exhibit remarkably strong face and mechanistic validity as a model for AUDs, permitting many advancements in the quest to understand human genetic involvement in this disease. These advancements occur via approaches that essentially fall into one of two categories: (1) discovering candidate genes via human genome-wide association studies (GWAS), transcriptomics on post-mortem tissue from AUD patients, or relevant physiological connections, then using reverse genetics in flies to validate candidate genes' roles and investigate their molecular function in the context of alcohol. (2) Utilizing flies to discover candidate genes through unbiased screens, GWAS, quantitative trait locus analyses, transcriptomics, or single-gene studies, then validating their translational role in human genetic surveys. In this review, we highlight the utility of Drosophila as a model for alcoholism by surveying recent advances in our understanding of human AUDs that resulted from these various approaches. We summarize the genes that are conserved in alcohol-related function between humans and flies. We also provide insight into some advantages and limitations of these approaches. Overall, this review demonstrates how Drosophila have and can be used to answer important genetic questions about alcohol addiction.
We know from human genetic studies that practically all aspects of biology are strongly influenced by the genetic background, as reflected in the advent of 'personalized medicine'. Yet, with few exceptions, this is not taken into account when using laboratory populations as animal model systems for research in these fields. Laboratory strains of zebrafish (Danio rerio) are widely used for research in vertebrate developmental biology, behaviour and physiology, for modelling diseases, and for testing pharmaceutic compounds in vivo. However, all of these strains are derived from artificial bottleneck events and therefore are likely to represent only a fraction of the genetic diversity present within the species. Here we use Restriction site-Associated DNA sequencing (RAD-seq) to genetically characterize wild populations of zebrafish from India, Nepal and Bangladesh, and to compare them to previously published data on four common laboratory strains. We measured nucleotide diversity, heterozygosity and allele frequency spectra, and find that wild zebrafish are much more diverse than laboratory strains. Further, in wild zebrafish there is a clear signal of GC-biased gene conversion that is missing in laboratory strains. We also find that zebrafish populations in Nepal and Bangladesh are most distinct from all other strains studied, making them an attractive subject for future studies of zebrafish population genetics and molecular ecology. Finally, isolates of the same strains kept in different laboratories show a pattern of ongoing differentiation into genetically distinct substrains. Together, our findings broaden the basis for future genetic, physiological, pharmaceutic and evolutionary studies in Danio rerio.
The zebrafish is becoming increasingly utilized in behavioral neuroscience as it appears to strike a good compromise between practical simplicity and system complexity. Particularly in alcohol (ethanol) research, the zebrafish has been employed as a translationally relevant model organism. However, the majority of studies investigating the effects of alcohol on brain function and behavior has used adult zebrafish. In the current study, we utilize 6-8 post-fertilization day old larval zebrafish (fry) to investigate the effects of a 40 min-long, acute, immersion into the alcohol bath. We measure the behavioral responses of the fry during the immersion session in relatively large arenas, the petri dish, instead of the often employed 96 well plate, and report on significant modification of behavior induced by alcohol. For example, we found the intermediate dose of alcohol (0.5%, vol/vol) to exert a stimulant effect manifesting as slight elevation of swim speed, robust increase of turning, temporal variability of swim speed and turning, and diminished frequency of staying immobile. We also found the high dose of 1% alcohol to elicit an opposite response, a sedative effect. This biphasic dose response of alcohol mimics what has been found in mammals, including humans, and thus we conclude that a few day-old zebrafish fry may be a cost effective and efficient tool with which one can screen for small molecules or mutations with alcohol-effect modifying properties.
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.
Social isolation can be used to study behavioral, neural, and hormonal mechanisms that regulate interactions in social animals. Although isolation effects have been reported in social mammals and various fish species, systematic studies with isolated zebrafish are rare. Here, the authors examined behavior (social and nonsocial), physiological stress (whole-body cortisol levels), and neurochemicals (serotonin, dopamine, and their metabolites), following acute and chronic social isolation in adult zebrafish. To observe how isolated fish respond behaviorally to social stimuli, they exposed zebrafish to live conspecifics or animated images after acute (24 hr) or chronic (6 months) social isolation. The authors observed that isolation did not affect locomotor activity, but acute isolation had weak nonsignificant anxiogenic effects in adult zebrafish. They also found that all isolated fish responded to both live and animated social stimuli, and the stress hormone, cortisol was lower in chronically isolated fish. Finally, neurochemical analyses showed that serotonin levels increased when fish were exposed to social stimulus after acute isolation, but its metabolite 5HIAA decreased in response to social stimulus following both acute and chronic isolation. Levels of both dopamine and its metabolite DOPAC were also reduced in fish exposed to social stimulus after acute and chronic isolation. Overall, these results show that isolation in zebrafish is an effective tool to study fundamental mechanisms controlling social interaction at behavioral and physiological levels.
Drug discovery in whole-organisms such as zebrafish is a promising approach for identifying biologically-relevant lead compounds. However, high content imaging of zebrafish at cellular resolution is challenging due to the difficulty in orienting larvae en masse such that the cell type of interest is in clear view. We report the development of the multi-pose imaging method, which uses 96-well round bottom plates combined with a standard liquid handler to repose the larvae within each well multiple times, such that an image in a specific orientation can be acquired. We have validated this method in a chemo-genetic zebrafish model of dopaminergic neuron degeneration. For this purpose, we have developed an analysis pipeline that identifies the larval brain in each image and then quantifies neuronal health in CellProfiler. Our method achieves a SSMD* score of 6.96 (robust Z’-factor of 0.56) and is suitable for screening libraries up to 10⁵ compounds in size.
The VAST BioImager system is a set of tools developed for zebrafish researchers who require the collection of images from a large number of 2-7 dpf zebrafish larvae. The VAST BioImager automates larval handling, positioning and orientation tasks. Color Images at about 10 micron resolution are collected from the on-board camera of the system. If images of greater resolution and detail are required, this system is mounted on an upright microscope, such as a confocal or fluorescence microscope, to utilize their capabilities. The system loads a larvae, positions it in view of the camera, determines orientation using pattern recognition analysis, and then more precisely positions to user-defined orientation for optimal imaging of any desired tissue or organ system. Multiple images of the same larva can be collected. The specific part of each larva and the desired orientation and position is identified by the researcher and an experiment defining the settings and a series of steps can be saved and repeated for imaging of subsequent larvae. The system captures images, then ejects and loads another larva from either a bulk reservoir, a well of a 96 well plate using the LP Sampler, or individually targeted larvae from a Petri dish or other container using the VAST Pipettor. Alternative manual protocols for handling larvae for image collection are tedious and time consuming. The VAST BioImager automates these steps to allow for greater throughput of assays and screens requiring high-content image collection of zebrafish larvae such as might be used in drug discovery and toxicology studies.
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.
Zebrafish exhibit remarkable alterations in behaviour and morphology as they develop from early larval stages to mature adults. In this study we compare the locomotion parameters of six common zebrafish strains from two different laboratories to determine the stability and repeatability of these behaviours. Our results demonstrate large variability in locomotion and fast swim events between strains and between laboratories across time. These data highlight the necessity for careful, strain-specific controls when analysing locomotor phenotypes and open up the possibility of standardising the quantification of zebrafish behaviour at multiple life stages.
Zebrafish are a widely utilised animal model in developmental genetics, and owing to recent advances in our understanding of zebrafish behaviour, their utility as a comparative model in behavioural neuroscience is beginning to be realised. One widely reported behavioural measure is the novel tank-diving assay, which has been often cited as a test of anxiety and stress reactivity. Despite its wide utilisation, and various validations against anxiolytic drugs, reporting of pre-test housing has been sparse in the literature. As zebrafish are a shoaling species, we predicted that housing environment would affect their stress reactivity and, as such, their response in the tank-diving procedure. In our first experiment, we tested various aspects of housing (large groups, large groups with no contact, paired, visual contact only, olfactory contact only) and found that the tank diving response was mediated by visual contact with conspecifics. We also tested the basal cortisol levels of group and individually housed fish, and found that individually housed individuals have lower basal cortisol levels. In our second experiment we found ethanol appeared to have an anxiolytic effect with individually housed fish but not those that were group housed. In our final experiment, we examined the effects of changing the fishes' water prior to tank diving as an additional acclimation procedure. We found that this had no effect on individually housed fish, but appeared to affect the typical tank diving responses of the group housed individuals. In conclusion, we demonstrate that housing represents an important factor in obtaining reliable data from this methodology, and should be considered by researchers interested in comparative models of anxiety in zebrafish in order to refine their approach and to increase the power in their experiments.
Copy number variants (CNVs) represent a substantial source of genomic variation in vertebrates and have been associated with numerous human diseases. Despite this, the extent of CNVs in the zebrafish, an important model for human disease, remains unknown. Using 80 zebrafish genomes, representing three commonly used laboratory strains and one native population, we constructed a genome-wide, high-resolution CNV map for the zebrafish comprising 6,080 CNV elements and encompassing 14.6% of the zebrafish reference genome. This amount of copy number variation is four times that previously observed in other vertebrates, including humans. Moreover, 69% of the CNV elements exhibited strain specificity, with the highest number observed for Tubingen. This variation likely arose, in part, from Tubingen's large founding size and composite population origin. Additional population genetic studies also provided important insight into the origins and substructure of these commonly used laboratory strains. This extensive variation among and within zebrafish strains may have functional effects that impact phenotype and, if not properly addressed, such extensive levels of germ-line variation and population substructure in this commonly used model organism can potentially confound studies intended for translation to human diseases.
With the arsenal of genetic tools available for zebrafish, this species has been successfully used to investigate the genetic aspects of human diseases from developmental disorders to cancer. Interest in the behavior and brain function of zebrafish is also increasing as CNS disorders may be modeled and studied with this species. Alcoholism and alcohol abuse are among the most devastating and costliest diseases. However, the mechanisms of these diseases are not fully understood. Zebrafish has been proposed as a model organism to study such mechanisms. Characterization of alcohol's effects on zebrafish is a necessary step in this research.
Here, we compare the effects of acute alcohol (EtOH) administration on the behavior of zebrafish from 4 distinct laboratory-bred populations using automated as well as observation based behavioral quantification methods.
Alcohol treatment resulted in significant dose-dependent behavioral changes but the dose-response trajectories differed among zebrafish populations.
The results demonstrate for the first time a genetic component in alcohol responses in adult zebrafish and also show the feasibility of high throughput behavioral screening. We discuss the exploration and exploitation of the genetic differences found.
Information on the economic impact of alcohol consumption can provide important evidence in supporting policies to reduce its associated harm. To date, several studies on the economic costs of alcohol consumption have been conducted worldwide. This study aims to review the economic impact of alcohol worldwide, summarizing the state of knowledge with regard to two elements: (1) cost components included in the estimation; (2) the methodologies employed in works conducted to date.
Relevant publications concerning the societal cost of alcohol consumption published during the years 1990-2007 were identified through MEDLINE. The World Health Organization's global status report on alcohol, bibliographies and expert communications were also used to identify additional relevant studies.
Twenty studies met the inclusion criteria for full review while an additional two studies were considered for partial review. Most studies employed the human capital approach and estimated the gross cost of alcohol consumption. Both direct and indirect costs were taken into account in all studies while intangible costs were incorporated in only a few studies. The economic burden of alcohol in the 12 selected countries was estimated to equate to 0.45 - 5.44% of Gross Domestic Product (GDP).
Discrepancies in the estimation method and cost components included in the analyses limit a direct comparison across studies. The findings, however, consistently confirmed that the economic burden of alcohol on society is substantial. Given the importance of this issue and the limitation in generalizing the findings across different settings, further well-designed research studies are warranted in specific countries to support the formulation of alcohol-related policies.
Zebrafish is gaining popularity in behavioral neuroscience in general and in alcohol research in particular. Alcohol is known to affect numerous molecular mechanisms depending on dose and administration regimen. Prominent among these mechanisms are several neurotransmitter systems. Here we analyze the responses of the dopaminergic and serotoninergic neurotransmitter systems of zebrafish to acute alcohol treatment (1 h long exposure of adult fish to 0.00%, 0.25%, 0.50%, or 1.00% ethyl alcohol) by testing the concentration of dopamine, its metabolite DOPAC, and serotonin and its metabolite 5-HIAA from whole brain extracts. We utilize a sensitive HPLC method and describe significant alcohol induced changes in zebrafish for the first time. We show that dopamine significantly increased in a quasi-linear dose dependent manner, DOPAC showed a smaller apparent increase which was non-significant, while both serotonin and 5-HIAA showed a significant increase only in the highest acute dose group. We discuss the methodological novelty of our work and theorize about the implications of the neurotransmitter level changes from a behavioral perspective.
The zebrafish has been in the forefront of developmental genetics for decades and has also been gaining attention in neurobehavioral genetics. It has been proposed to model alcohol-induced changes in human brain function and behavior. Here, adult zebrafish populations, AB and SF (short-fin wild type), were exposed to chronic treatment (several days in 0.00% or 0.50% alcohol v/v) and a subsequent acute treatment (1 h in 0.00%, 0.25%, 0.50% or 1.00% alcohol). Behavioral responses of zebrafish to computer-animated images, including a zebrafish shoal and a predator, were quantified using videotracking. Neurochemical changes in the dopaminergic and serotoninergic systems in the brain of the fish were measured using high-precision liquid chromatography with electrochemical detection. The results showed genetic differences in numerous aspects of alcohol-induced changes, including, for the first time, the behavioral effects of withdrawal from alcohol and neurochemical responses to alcohol. For example, withdrawal from alcohol abolished shoaling and increased dopamine and 3,4-dihydroxyphenylacetic acid in AB but not in SF fish. The findings show that, first, acute and chronic alcohol induced changes are quantifiable with automated behavioral paradigms; second, robust neurochemical changes are also detectable; and third, genetic factors influence both alcohol-induced behavioral and neurotransmitter level changes. Although the causal relationship underlying the alcohol-induced changes in behavior and neurochemistry is speculative at this point, the results suggest that zebrafish will be a useful tool for the analysis of the biological mechanisms of alcohol-induced functional changes in the adult brain.
Despite much evidence of its economic and social costs, alcohol use continues to increase. Much remains to be known as to the effects of alcohol on neurodevelopment across the lifespan and in both sexes. We provide a comprehensive overview of the methodological approaches to ethanol administration when using animal models (primarily rodent models) and their translational relevance, as well as some of the advantages and disadvantages of each approach. Special consideration is given to early developmental periods (prenatal through adolescence), as well as to the types of research questions that are best addressed by specific methodologies. The zebrafish is used increasingly in alcohol research, and how to use this model effectively as a preclinical model is reviewed as well.
The zebrafish is becoming increasingly well utilized in several fields of biology, including behavioral neuroscience. This review, based upon an Outstanding Achievement Award lecture presented by the author at an IBNS conference, explores the potential reasons for the popularity of this species. First, some theoretical questions are discussed, including why a comparative approach using more than a single laboratory animal species (the mouse) may be important. Points about evolutionary conservation are considered. Discussion on why complexity may build over time, and what it means when a species is considered “primitive” vs “advanced”, are also included. Arguments about how using a “primitive” species may work to the researcher’s advantage are made in the context of translational relevance. Most discussions center around a complex functional aspect of the brain, relational learning and memory, and use empirical examples from the author’s own laboratory. It is hoped that these examples will show how a simple vertebrate, like the zebrafish, may help the investigator address fundamental mechanistic questions about complex brain function and behavior.
The zebrafish is a premier laboratory model for developmental genetics and is used increasingly for ethology and behavioral neuroscience. A fuller appreciation of zebrafish behavior, and its underlying neurobiology, physiology, and genetics, demands an understanding of the species' ecology, past, and present. Studies of natural history are beginning to provide such context and reveal that zebrafish lives across diverse habitats, encompassing tremendous variation in temperature, water quality, altitude, and community composition. Integrating knowledge of biotic and abiotic environments with studies of behavior in the lab and field should provide new insights into the evolutionary origins and selective consequences of zebrafish behavioral phenotypes.
The zebrafish is becoming increasingly popular in behavioral neuroscience and behavior genetics as this species offers a potentially cheaper and simpler alternative to classical rodent laboratory species in the quest to model and understand the mechanisms of human CNS disorders. Anxiety-related human disorders still represent a large unmet medical need despite numerous treatment options already available. The zebrafish has been proposed as an excellent animal model for the analysis of anxiety. In this chapter, we will focus on the behavioral aspect of this research and discuss methods, e.g., mainly visual but also olfactory stimuli, with which aversive, anxiety-like, and/or fear responses may be induced in the zebrafish. We will also review some of the behavioral responses induced by such stimuli and the methods one can employ to quantify them. We also briefly discuss psychopharmacological and genetic studies as examples of modeling anxiety using the zebrafish. The chapter focuses on adult zebrafish but also mentions larval zebrafish and their use in anxiety research. The chapter is a snapshot of this fast-developing field, and its goal is to exemplify the utility of the zebrafish in modeling and the analysis of anxiety.
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.
The popularity of zebrafish is exponentially increasing. Unlike in the past, the zebrafish now is a subject of investigation by scientists of practically every subfield of biology, including psychopharmacology and behavior genetics. Simply put, the zebrafish is no longer considered a simple egg-producing tube for efficient delivery of thousands of embryos to be studied only by developmental biologists. With the broadening interest, come numerous challenges. One of the most fundamental of which is how to maintain and breed zebrafish. Here we consider some questions of zebrafish maintenance and breeding, and provide a few examples of what we argue would be important issues where some change may be required. We emphasize that systematic parametric analyses of environmental factors are needed to optimize the laboratory environment for the zebrafish. Last, we argue that understanding the ecology and ethology of the zebrafish is important for establishing proper maintenance and breeding of this species.
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.
Alcohol abuse is a major determinant of public health outcomes. Worldwide data from 2016 indicate that alcohol is the seventh leading risk factor in terms of disability-adjusted life years, an increase of more than 25% from 1990 to 2016. Understanding the epidemiology of alcoholic liver disease, including the regional variations in consumption and public policy, is an area of active research. In countries where the per capita consumption of alcohol decreases, there appears to be an associated decrease in disease burden. Given alcohol's health burden, an increased focus on alcohol control policies is needed.
Background:
Zebrafish larvae have a high potential as model system to replace rodents, especially in screening and drug discovery applications. However, an experimental setup to deliver mild electrical stimuli with simultaneous high throughput behavioural tracking has not yet been described.
New method:
A new tool was designed, making the delivery of electrical stimuli in a 96-well plate format possible. Using custom made electrode clips that can be slid over the walls of a square 96-well plate, 80 larvae could be tested simultaneously and behavioural responses recorded.
Results:
As proof of principle, two applications were tested: 1) The behavioural response after a single stimulus and the effect of buprenorphine on this response. 2) Habituation of locomotor activity to multiple stimuli and the involvement of the NMDA receptor. Reduced locomotor activity was observed after a single 5 V stimulus, however not with lower intensity stimuli. Pre-treatment with the analgesic buprenorphine prevented this response. Specificity of buprenorphine was confirmed using the antagonist naloxone. Habituation of locomotor activity was seen in response to multiple stimuli, depending on the inter stimulus interval. Treatment with the NMDA receptor antagonist memantine disrupted behavioural habituation.
Comparison with existing methods:
The equipment and setup described here are the first of its kind using a 96-well plate format, thereby increasing the potential throughput in screening applications using zebrafish larvae.
Conclusion:
The combination of the described electrode clips for stimulus delivery and behavioural tracking allows for the use of zebrafish larvae in a new array of medium to high throughput applications.
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.
Introduction:
Only a few medications are available for the treatment of alcohol use disorders (AUDs).
Areas covered:
This paper discusses approved AUD medications, including the opioid antagonists; naltrexone and nalmefene (the latter is licensed for reduction of alcohol consumption only), the putative glutamate receptor antagonist acamprosate and the aldehyde dehydrogenase inhibitor disulfiram. It also covers off-label medications of interest, including topiramate, gabapentin, ondansetron, varenicline, baclofen, sodium oxybate and antidepressants. Clinical implications, benefits and risks of treatment are discussed.
Expert opinion:
Acamprosate, naltrexone, nalmefene and disulfiram are the only approved “alcohol-specific” drugs. Acamprosate and naltrexone have been evaluated in numerous clinical trials and represent evidence-based treatments in AUDs. Nalmefene use, however, is controversial. Supervised disulfiram is a second-line treatment approach. Compounds developed and licensed for different neuropsychiatric disorders are potential alternatives. Encouraging results have been reported for topiramate, gabapentin and also varenicline, which might be useful in patients with comorbid nicotine dependence. The GABA-B receptor agonist baclofen has been studied more intensively and shown mixed results; it is currently licensed for the treatment of AUDs in France only. Gabapentin may be close to approval in the USA. Further studies of these novel treatment approaches in AUDs are needed.
The purpose of this review is to present animal research models that can be used to screen and/or repurpose medications for the treatment of alcohol abuse and dependence. The focus will be on rats and in particular selectively bred rats. Brief introductions discuss various aspects of the clinical picture, which provide characteristics of individuals with alcohol use disorders (AUDs) to model in animals. Following this, multiple selectively bred rat lines will be described and evaluated in the context of animal models used to screen medications to treat AUDs. Next, common behavioral tests for drug efficacy will be discussed particularly as they relate to stages in the addiction cycle. Tables highlighting studies that have tested the effects of compounds using the respective techniques are included. Wherever possible the Tables are organized chronologically in ascending order to describe changes in the focus of research on AUDs over time. In general, high ethanol-consuming selectively bred rats have been used to test a wide range of compounds. Older studies usually followed neurobiological findings in the selected lines that supported an association with a propensity for high ethanol intake. Most of these tests evaluated the compound's effects on the maintenance of ethanol drinking. Very few compounds have been tested during ethanol-seeking and/or relapse and fewer still have assessed their effects during the acquisition of AUDs. Overall, while a substantial number of neurotransmitter and neuromodulatory system targets have been assessed; the roles of sex- and age-of-animal, as well as the acquisition of AUDs, ethanol-seeking and relapse continue to be factors and behaviors needing further study.
Alcohol addiction is a major unmet medical and economic issue for which very few efficacious pharmacological treatment options are currently available. The development and identification of new compounds and drugs to treat alcohol addiction is hampered by the high costs and low amenability of traditional laboratory rodents to high-throughput behavioral screens. The zebrafish represents an excellent compromise between systems complexity and practical simplicity by overcoming many limitations inherent in these rodent models. In this chapter, we review current advances in the behavioral and neurochemical characterization of ethanol-induced changes in zebrafish. We also discuss the basic principles and methods of and the most recent advances in using paradigms with which one can screen for compounds altering acute and chronic ethanol-induced effects in zebrafish.
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.
Unlabelled:
The zebrafish is a relatively new model organism and has become a valuable tool in genetic, developmental, and pharmacological researches. Zebrafish larvae, compared with adult, are particularly suitable for high-throughput screening of drug effects. AB and TU are well established in-bred zebrafish strains. The behavioral responses to acute MK-801 treatments (0, 5, 20, 100, and 200μM) under illumination at 50lx were studied using zebrafish larvae of both AB and TU strains at 7dpf with ZebraLab software. Two behavioral parameters, traveling distance and activity counts, were analyzed. "Traveling distance" represents locomotor activity, whereas "activity count" is any activity including small, non-ambulatory movements. Zebrafish larvae of TU strain demonstrated inhibitory effects in both behavioral parameters in response to MK-801 treatment. Zebrafish larvae of AB strain showed lack of responses to MK-801 treatments in traveling distance, and showed increases in activity counts. Therefore, zebrafish larvae of AB and TU strains demonstrated opposite responses in activity counts towards MK-801 treatment. Differences in the level of neurotransmitters and their respective metabolites (NE and MHPG, DA and DOPAC, 5-HT and 5-HIAA) between AB and TU strain zebrafish larvae were discovered by HPLC analysis, which was related to the strain-dependent differential behavioral responses to MK-801 treatment.
Conclusion:
Under the influences of MK-801, in contrast with TU strain, AB strain zebrafish larvae demonstrated activity changes similar to previous studies on rodents. AB strain larvae are better model organisms than TU strain larvae in MK-801 related behavioral studies.
Alcohol abuse and dependence is a rapidly growing problem with few treatment options available. The zebrafish has become a popular animal model for behavioural neuroscience. This species may be appropriate for investigating the effects of alcohol on the vertebrate brain. In the current review, we examine the literature by discussing how alcohol alters behaviour in zebrafish and how it may affect biological correlates. We focus on two phenomena that are often examined in the context of alcohol-induced neuroplasticity. Alcohol tolerance (a progressive decrease in the effect of alcohol over time) is often observed following continuous (chronic) exposure to low concentrations of alcohol. Alcohol sensitization also called reverse tolerance (a progressive increase in the effect of alcohol over time) is often observed following repeated discrete exposures to higher concentrations of alcohol. These two phenomena may underlie the development and maintenance of alcohol addiction. The phenotypical characterization of these responses in zebrafish may be the first important steps in establishing this species as a tool for the analysis of the molecular and neurobiological mechanisms underlying human alcohol addiction.
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.
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.
Since the publication of our initial review of restraint stress in 1986, much work has continued with this technique, either as a tool for the investigation of other pharmacological, physiological, or pathologic phenomena or with restraint stress itself serving as the object of the study. As we noted in 1986, the major use of restraint has been for the induction of stress responses in animals and, more specifically, for the investigation of drug effects, particularly as they affect typical stress-related pathology—gastrointestinal, neuroendocrine, and immunological agents have been extensively studied. In compiling this update on restraint stress and its effects, we noted an increasing emphasis on central nervous system mechanisms in peripheral disease, especially gastrointestinal disease. In particular, many CNS-active agents have been tested for their effects on gastric and duodenal lesion formation and gastric secretion, including antidepressants, antipsychotics, anxiolytics, noradrenergic, serotonergic, dopaminergic, and peptidergic compounds. Some of these agents are especially active in the gastrointestinal tract even when administered centrally, further solidifying the concept of a brain-gut axis. The present update includes studies of: methods and procedures, prerestraint manipulations, postrestraint/healing effects, and drug effects. In addition, a current bibliography of reports that have employed restraint is included.
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.
Because psychotropic drugs affect behavior, we can use changes in behavior to discover psychotropic drugs. The original prototypes of most neuroactive medicines were discovered in humans, rodents and other model organisms. Most of these discoveries were made by chance, but the process of behavior based drug discovery can be made more systematic and efficient. Fully automated platforms for analyzing the behavior of embryonic zebrafish capture digital video recordings of animals in each individual well of a 96-well plate before, during, and after a series of stimuli. To analyze systematically the thousands of behavioral recordings obtained from a large-scale chemical screen, we transform these behavioral recordings into numerical barcodes, providing a concise and interpretable summary of the observed phenotypes in each well. Systems-level analysis of these behavioral phenotypes generate testable hypotheses about the molecular mechanisms of poorly understood drugs and behaviors. By combining the in vivo relevance of behavior-based phenotyping with the scale and automation of modern drug screening technologies, systematic behavioral barcoding represents a means of discovering psychotropic drugs and provides a powerful, systematic approach for unraveling the complexities of vertebrate behavior.
Zebrafish (Danio rerio) are rapidly emerging as a useful animal model in neurobehavioral research. Mounting evidence shows the suitability of zebrafish to model various aspects of anxiety-related states. Here, we evaluate established and novel approaches to uncover the molecular substrates, genetic pathways and neural circuits of anxiety using adult zebrafish. Experimental approaches to modeling anxiety in zebrafish include novelty-based paradigms, pharmacological and genetic manipulations, as well as innovative video-tracking, 3D-reconstructions, bioinformatics-based searchable databases and omics-based tools. Complementing traditional rodent models of anxiety, we provide a conceptual framework for the wider application of zebrafish and other aquatic models in anxiety research. This article is part of a Special Issue entitled 'Anxiety and Depression'.
Zebrafish are at the forefront of neurobiological research and have been gaining popularity as a viable and valid behavioral model in a variety of research applications (e.g., assessing drug induced behavioral changes). This model becomes even more attractive when considering the behavioral changes that follow exposure to compounds that are water-soluble. As such, several studies have implicated both acute and chronic ethanol exposure in the modulation of zebrafish behavior. Within this arena there appears to be a common trend across multiple studies. As with many drugs ethanol appears to influence behavior in a dose-dependent manner. In this review, we compare and contrast several studies that measure behavior as a result of alcohol exposure. Appended to this review are pilot data that report zebrafish blood alcohol concentrations as a function of acute exposure.
Steatosis is the most common consequence of acute alcohol abuse, such as occurs during a drinking binge. Acute alcohol induced steatosis may predispose to more severe hepatic disease. We have developed a model of alcoholic liver disease (ALD) in zebrafish larvae to provide a system in which the genes and pathways that contribute to steatosis can be rapidly identified. Zebrafish larvae represent an attractive vertebrate model for studying acute ALD because they possess the pathways to metabolize alcohol, the liver is mature by 4 days post-fertilization (dpf), and alcohol can be simply added to their water. Exposing 4 dpf zebrafish larvae to 2% ethanol (EtOH) for 32 hours achieves ∼80 mM intracellular EtOH and upregulation of hepatic cyp2e1, sod, and bip, indicating that EtOH is metabolized and provokes oxidative stress. EtOH-treated larvae develop ALD as demonstrated by hepatomegaly and steatosis. Increased lipogenesis driven by the sterol response element binding protein (SREBP) transcription factors is essential for steatosis associated with chronic alcohol ingestion but it has not been determined if the same pathway is essential for steatosis following a drinking binge. We report that several Srebp target genes are induced in the liver of zebrafish exposed to EtOH. We used fish which harbor a mutation in the gene encoding the membrane bound transcription factor protease 1 (mbtps1; also called site-1 protease) and embryos in which the Srebp cleavage activating protein (scap) is knocked down to determine the requirement of this pathway in acute ALD. We find that both means of blocking Srebp activation prevents steatosis in response to 2% EtOH. Moreover, this is accompanied by the failure to activate several Srebp target genes in response to alcohol. We conclude that Srebps are required for steatosis in response to acute alcohol exposure. Moreover, these data highlight the utility of zebrafish as a useful new vertebrate model to study ALD.
Human neuropsychiatric conditions associated with abnormally exaggerated or misdirected fear (anxiety disorders and phobias) still represent a large unmet medical need because the biological mechanisms underlying these diseases are not well understood. Animal models have been proposed to facilitate this research. Here I review the literature with a focus on zebrafish, an upcoming laboratory organism in behavioral brain research. I argue that abnormal human fear responses are likely the result of the malfunction of neurobiological mechanisms (brain areas, circuits and/or molecular mechanisms) that originally evolved to support avoidance of predators or other harm in nature. I also argue that the understanding of the normal as well as pathological functioning of such mechanisms may be best achieved if one utilizes naturalistic experimental approaches. In case of laboratory model organisms, this may entail presenting stimuli associated with predators and measuring species-specific antipredatory responses. Although zebrafish is a relatively new subject of such inquiry, I review the recently rapidly increasing number of zebrafish studies in this area, and conclude that zebrafish is a promising research tool for the analysis of the neurobiology and genetics of vertebrate fear responses.
Presently, the zebrafish is the only vertebrate model compatible with contemporary paradigms of drug discovery. Zebrafish embryos are amenable to automation necessary for high-throughput chemical screens, and optical transparency makes them potentially suited for image-based screening. However, the lack of tools for automated analysis of complex images presents an obstacle to using the zebrafish as a high-throughput screening model. We have developed an automated system for imaging and analyzing zebrafish embryos in multi-well plates regardless of embryo orientation and without user intervention. Images of fluorescent embryos were acquired on a high-content reader and analyzed using an artificial intelligence-based image analysis method termed Cognition Network Technology (CNT). CNT reliably detected transgenic fluorescent embryos (Tg(fli1:EGFP)(y1)) arrayed in 96-well plates and quantified intersegmental blood vessel development in embryos treated with small molecule inhibitors of anigiogenesis. The results demonstrate it is feasible to adapt image-based high-content screening methodology to measure complex whole organism phenotypes.
The pharmacogenetic differences among individuals in their capacity to metabolize ingested alcohol are possibly responsible for the large inter-individual and inter-ethnic variations observed in the outcome of alcohol use and misuse. Based on results of adoption, twin, and family studies it is now widely accepted that the vulnerability to alcoholism is determined by genetic factors as well as by environment. There is a constant search for biological markers and specific genes which could identify individuals genetically predisposed to alcohol abuse and alcoholism. Numerous 'candidate genes' for alcoholism have been suggested including the alcohol metabolizing enzymes, alcohol dehydrogenase (ADH) and aldehyde dehydrogenase (ALDH). Both ADH and ALDH exhibit genetic heterogeneity. An atypical form of ADH (ADH2), which contains a variant beta 2 subunit instead of the usual beta 1 subunit, differs substantially from the usual form in its kinetic properties and is found more frequently among the Japanese, Chinese and other Mongoloid populations than in Caucasoids and Negroids. A widely prevalent genetic polymorphism has been observed for ALDH; about 50% of Japanese and Chinese livers possess an inactive ALDH (ALDH2 isozyme) whereas none of the Caucasian or Negroid populations show this isozyme abnormality. These metabolic polymorphisms seem to contribute to differences in the in vivo elimination rate of ethanol and acetaldehyde, and may explain differences in alcohol-related behaviour and its disease outcome. Taken together, Orientals who possess an atypical ALDH2 gene are more sensitive to acute responses to alcohol, tend to be discouraged from drinking alcohol, and consequently are at lower risk of developing alcohol-related disorders. However, more work is needed to support these findings. Recent advances in molecular genetics have made it possible to analyze directly the human genome. This may help in a better understanding of the complex genetic and environmental factors in alcohol abuse by providing prospects for identification of gene loci which may be responsible for predisposition to, and protection from, alcoholism.
Zebrafish genes and development are being studied in a growing number of laboratories. Given that many other organisms are already being exploited by large numbers of investigators, and that our general knowledge about the zebrafish embryo and genome is at present rather sketchy, why should we now concern ourselves with how this tropical fish develops? Whereas the zebrafish embryo is similar in important ways to other vertebrate embryos, it is relatively simple and unusually accessible for both cellular and genetic analyses.