Social deficits following embryonic ethanol exposure arise in post-larval zebrafish.

Prenatal alcohol exposure is the leading cause of birth defects, collectively termed fetal alcohol spectrum disorders (FASD). In the United States and Canada, 1 in 100 children will be born with FASD. Some of the most commonly debilitating defects of FASD are in social behavior. Zebrafish are highly social animals, and embryonic ethanol exposure from 24 to 26 hours post-fertilization disrupts this social (shoaling) response in adult zebrafish. Recent findings have suggested that social behaviors are present in zebrafish larvae as young as 3 weeks, but how they relate to adult shoaling is unclear. We tested the same ethanol-exposed zebrafish for social impairments at 3 weeks then again at 16 weeks. At both ages, live conspecifics were used to elicit a social response. We did not find alcohol-induced differences in behavior in 3-week-old fish when they were able to see conspecifics. We do find evidence that control zebrafish are able to use nonvisual stimuli to detect conspecifics, and this behavior is disrupted in the alcohol-exposed fish. As adults, these fish displayed a significant decrease in social behavior when conspecifics are visible. This surprising finding demonstrates that the adult and larval social behaviors are, at least partly, separable. Future work will investigate the nature of these nonvisual cues and how the neurocircuitry differs between the larval and adult social behaviors.

Forward Genetic Screening Using Behavioral Tests in Zebrafish: A Proof of Concept Analysis of Mutants.

The zebrafish enjoys several advantages over other model organisms. It is small, easy to maintain, prolific, and numerous genetic tools are available for it. For example, forward genetic screens have allowed investigators to identify important genes potentially involved in a variety of functions from embryogenesis to cancer. However, despite its sophisticated behavioral repertoire, behavioral methods have rarely been utilized in forward genetic screens. Here, we employ a two-tiered strategy, a proof of concept study, to explore the feasibility of behavioral screens. We generated mutant lines using transposon-based insertional mutagenesis, allowing us to bias mutant selection with target genes expressed within the brain. Furthermore, we employed an efficient and fast behavioral pre-selection in which we investigated the locomotory response of 5-day post-fertilization old larval fish to hyperosmotic shock. Based on this assay, we selected five lines for our lower throughput secondary adult behavioral screen. The latter screen utilized tests in which computer animated image presentation and video-tracking-based automated quantification of behavior allowed us to compare heterozygous zebrafish with their wild-type siblings on their responses to a variety of stimuli. We found significant mutation induced adult behavioral alterations in 4 out of the 5 lines analyzed, including changes in response to social or fear inducing stimuli, to handling and novelty, or in habituation to novelty. We discuss the pros and cons of behavioral phenotyping and of the use of different forward genetic methods in biomedical research with zebrafish.

Embryonic alcohol exposure impairs the dopaminergic system and social behavioral responses in adult zebrafish.

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 postfertilization) to low doses of alcohol (0, 0.25, 0.50, 0.75, and 1% vol/vol) 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 a low concentration of alcohol (0.5% or 1% vol/vol) for 2 hours at 24 hours postfertilization 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 3,4-dihydroxyphenylacetic acid from whole brain extracts of these fish using high-pressure liquid chromatograph. 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 3,4-dihydroxyphenylacetic acid 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.

Modeling Fetal Alcohol Spectrum Disorders in Zebrafish to Characterize the Impact of an Adverse Embryonic Environment on Adult Social Behavior.

Fetal alcohol spectrum disorders (FASD) describe all alcohol-induced birth defects. Birth defects such as growth deficiencies, craniofacial, behavioral, and cognitive abnormalities are associated with FASD. Social difficulties are common behavioral abnormalities associated with FASD and often result in serious health issues. Animal models are critical to understanding the mechanisms responsible for ethanol-induced social defects. Zebrafish are social vertebrates that produce externally fertilized transparent eggs; these characteristics provide researchers with a precise yet simple procedure for creating the FASD phenotype and an innate behavior that can be leveraged to model the social deficits associated with FASD. Thus, zebrafish are ideal for characterizing the social deficits of FASD. The goal of the current protocol is to provide the user with a simple behavioral assay that can be used to characterize the consequences of a negative environment early during development and the effects it can have on social behavior in adulthood. The protocol can be used to characterize the effect mutations or teratogens have on adult social behavior. The protocol outlined here demonstrates how to characterize the social behavior of individual fish during a 20-min social assay. Furthermore, the data obtained using the current protocol provides evidence that the protocol can be used to characterize the effects of embryonic ethanol-induced social defects in adult zebrafish.

Social behavioral phenotyping of the zebrafish casper mutant following embryonic alcohol exposure.

The term Fetal Alcohol Spectrum Disorder (FASD) describes all the deleterious consequences of prenatal alcohol exposure. Impaired social behavior is a common symptom of FASD. The zebrafish has emerged as a powerful model organism with which to examine the effects of embryonic alcohol exposure on social behavior due to an innate strong behavior, called shoaling. The relative transparency of the embryo also makes zebrafish powerful for cellular analyses, such as characterizing neural circuitry. However, as zebrafish develop, pigmentation begins to obscure the brain and other tissues. Due to mutations disrupting pigmentation, the casper zebrafish strain remains relatively transparent throughout adulthood, potentially permitting researchers to image neural circuits in vivo, via epifluorescence, confocal and light sheet microscopy. Currently, however the behavioral profile of casper zebrafish post embryonic alcohol exposure has not been completed. We report that exposure to 1% alcohol from either 6 to 24, or 24 to 26 h postfertilization reduces the social behavior of adult casper zebrafish. Our findings set the stage for the use of this important zebrafish resource in studies of FASD.

Gene-environment interactions in development and disease.

Developmental geneticists continue to make substantial jumps in our understanding of the genetic pathways that regulate development. This understanding stems predominantly from analyses of genetically tractable model organisms developing in laboratory environments. This environment is vastly different from that in which human development occurs. As such, most causes of developmental defects in humans are thought to involve multifactorial gene-gene and gene-environment interactions. In this review, we discuss how gene-environment interactions with environmental teratogens may predispose embryos to structural malformations. We elaborate on the growing number of gene-ethanol interactions that might underlie susceptibility to fetal alcohol spectrum disorders. WIREs Dev Biol 2017, 6:e247. doi: 10.1002/wdev.247 For further resources related to this article, please visit the WIREs website.

Associative learning in the multichamber tank: A new learning paradigm for zebrafish.

The zebrafish has been gaining prominence in the field of behavioural brain research as this species offers a good balance between system complexity and practical simplicity. While the number of studies examining the behaviour of zebrafish has exponentially increased over the past decade, the need is still substantial for paradigms capable of assessing cognitive and mnemonic characteristics of this species. Here we describe and utilize a novel visual discrimination task with which we evaluated acquisition of CS (colour)-US (sight of conspecifics) association in adult zebrafish. We report significant acquisition of CS-US association indicated by the increased time the fish spent in and the increased frequency of visits of the target chamber during a probe trial in the absence of reward. Given the simplicity of the apparatus and procedure, we conclude that the new task may be employed to assay learning and memory in adult zebrafish in an efficient manner.

Impairment of social behaviour persists two years after embryonic alcohol exposure in zebrafish: A model of fetal alcohol spectrum disorders.

Zebrafish naturally form social groups called shoals. Previously, we have shown that submerging zebrafish eggs into low concentrations of alcohol (0.00, 0.25, 0.50, 0.75 and 1.00 vol/vol% external bath concentration) during development (24h post-fertilization) for two hours resulted in impaired shoaling response in seven month old young adult zebrafish. Here we investigate whether this embryonic alcohol exposure induced behavioural deficit persists to older age. Zebrafish embryos were exposed either to fresh system water (control) or to 1% alcohol for two hours, 24h after fertilization, and were raised in a high-density tank system. Social behaviour was tested by presenting the experimental fish with a computer animated group of zebrafish images, while automated tracking software measured their behaviour. Control fish were found to respond strongly to animated conspecific images by reducing their distanceand remaining close to the images during image presentation, embryonic alcohol treated fish did not. Our results suggest that the impaired shoaling response of the alcohol exposed fish was not due to altered motor function or visual perception, but likely to a central nervous system alteration affecting social behaviour itself. We found the effects of embryonic alcohol exposure on social behaviour not to diminish with age, a result that demonstrates the deleterious and potentially life-long consequences of exposure to even small amount of alcohol during embryonic development in vertebrates.

The effect of the number and size of animated conspecific images on shoaling responses of zebrafish.

The zebrafish is increasingly utilized in biomedical and psychopharmacological research aimed at modeling human brain disorders. Abnormal social behavior represents the core symptom of several neuropsychiatric and neurodevelopmental disorders. The zebrafish is a highly social species and has been proposed for modeling such disorders. Behavioral paradigms that can induce zebrafish social behavior are of importance. Here, we utilize a paradigm in which zebrafish are presented with computer animated images of conspecifics. We systematically varied the size of these images relative to the body size of the experimental fish and also investigated the potential effect of presenting different number of images in an attempt to optimize the paradigm. We report that images similar in size to the experimental fish induced a strong shoaling response (reduction of distance to the image presentation screen) both when the body size of the experimental fish was varied with the image size being held constant and when the image size was varied with the body size of the experimental fish being held constant. We also report that within the number range studied (from 1 to 8 conspecific stimulus fish), presentation of all animated shoals, but the image of a single conspecific stimulus fish, led to significant reduction of distance to the presentation screen. We conclude that the shoal image presentation paradigm induces robust social responses that are quantifiable in an automated manner, making the paradigm useful for screening of drugs and/or mutations.

Acute ethanol exposure induces behavioural differences in two zebrafish (Danio rerio) strains: a time course analysis.

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 vs. 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.