A simple semi-automated home-tank method and procedure to explore classical associative learning in adult zebrafish.

The zebrafish is a laboratory species that gained increasing popularity the last decade in a variety of subfields of biology, including toxicology, ecology, medicine, and the neurosciences. An important phenotype often measured in these fields is behaviour. Consequently, numerous new behavioural apparati and paradigms have been developed for the zebrafish, including methods for the analysis of learning and memory in adult zebrafish. Perhaps the biggest obstacle in these methods is that zebrafish is particularly sensitive to human handling. To overcome this confound, automated learning paradigms have been developed with varying success. In this manuscript, we present a semi-automated home tank-based learning/memory test paradigm utilizing visual cues, and show that it is capable of quantifying classical associative learning performance in zebrafish. We demonstrate that in this task, zebrafish successfully acquire the association between coloured-light and food reward. The hardware and software components of the task are easy and cheap to obtain and simple to assemble and set up. The procedures of the paradigm allow the test fish to remain completely undisturbed by the experimenter for several days in their home (test) tank, eliminating human handling or human interference induced stress. We demonstrate that the development of cheap and simple automated home-tank-based learning paradigms for the zebrafish is feasible. We argue that such tasks will allow us to better characterize numerous cognitive and mnemonic features of the zebrafish, including elemental as well as configural learning and memory, which will, in turn, also enhance our ability to study neurobiological mechanisms underlying learning and memory using this model organism.

Video playback versus live stimuli to assess quantity discrimination in angelfish (Pterophyllum scalare).

Video playback is a widely used technique for presentation of visual stimuli in animal behavior research. In the analysis of behavioral responses to social cues, presentation of video recordings of live conspecifics represents a consistently reproducible stimulus. However, video-recordings do not interact with the experimental subject, and thus this stimulus may be inferior in the social context. Here, we evaluated how angelfish (Pterophyllum scalare) respond to a video playback of conspecifics versus a live shoal of conspecifics. Using binary choice tests, subjects were presented different stimuli. Time spent close to one versus the other stimulus was considered an index of preference. We found angelfish to prefer a live shoal of conspecifics to an empty tank, and also the video playback of a shoal of conspecifics to a blank screen, although the level of preference in the latter was lower than in the former. These results indicate that video-playback of live conspecifics may be appropriate in angelfish, thus allowing manipulation of specific cues that angelfish may use in quantity discrimination. However, when we directly contrasted a live and a video recorded shoal, both having the same number of members, experimental fish preferred the live shoal. When the choice consisted of a live shoal of four conspecifics versus a video playback of a shoal of nine conspecifics no clear preference emerged. These results imply that video-playback has disadvantages in quantity discrimination studies with angelfish. Exploring procedural and/or technological parameters will verify the suitability of video-recording-based stimulus presentation for future use in angelfish.

The Tapping Assay: A Simple Method to Induce Fear Responses in Zebrafish.

The zebrafish is increasingly employed in behavioral neuroscience as a translationally relevant model organism for human central nervous system disorders. One of the most prevalent CNS disorders representing an unmet medical need is the disorder cluster defined under the umbrella term anxiety disorders. Zebrafish have been shown to respond to a variety of anxiety and fear inducing stimuli and have been suggested for modeling human anxiety. Here, we describe a simple method with which we intend to induce fear/anxiety responses in this species. The method allows us to deliver a visual and lateral line stimulus (vibration or "tapping") to the fish with the use of a moving object, a ball colliding with the side glass of the experimental tank. We describe the hardware construction of the apparatus and the procedure of the behavioral paradigm. We also present data on how zebrafish respond to the tapping. Our results demonstrate that the method induces significant fear/anxiety responses. We argue that the simplicity of the method and the efficiency of the paradigm should make it popular among those who plan to use zebrafish as a tool in anxiety research.

Early social deprivation does not affect cortisol response to acute and chronic stress in zebrafish.

Social isolation is a well-established technique for inducing early adversity but, in rodent models, the need of parental care makes it difficult to distinguish the effects of social deprivation from the consequences of nutritional deficiencies. Zebrafish do not require parental care, allowing separation of social deprivation from nutritional deprivation, and have emerged as a promising model to study ontogeny of normal and pathological behaviors relevant for human neuropsychological disorders. Previous reports of life-long isolation in zebrafish showed some consistency with mammalian literature, depicting later social deficits and locomotor hyperactivity. However, unlike reports of higher anxiety and stress behavior in isolated rodents and primates, behavioral responses were tapered in isolated fish. To examine whether life-long developmental isolation has a dampening effect on zebrafish endocrine stress response, we applied stressors to zebrafish siblings that were either isolated or socialized, and compared their whole-body cortisol levels with non-stressed control siblings kept in low-housing densities. Utilizing previously validated paradigms (exposure to novel tank and unpredictable chronic stress), we exposed separate groups (n = 9-14, mixed-sex) of social and isolated zebrafish to acute and chronic stressors and measured their cortisol levels. A univariate ANOVA and post-hoc Tukey's HSD tests confirmed that compared to socially raised control fish, developmental isolation did not increase baseline cortisol levels in zebrafish. Additionally, compared to the non-stressed condition, application of both acute and chronic stressors significantly increased cortisol levels in isolated fish and, to a similar degree, to socially raised fish. Our findings suggest that zebrafish isolation studies may help separate effects of social deprivation from nonsocial aspects of early adversity. These studies further substantiate the use of developmental isolation in zebrafish, particularly with acute and chronic stress paradigms, for modeling neuropsychological disorders.LAY SUMMARYA difficult childhood can make humans react more frequently or severely to later stress and modeling this effect in animals can help explain how and why early stress affects subsequent mental and physical health. Early social isolation does not affect later response to stressful situations in adult zebrafish, providing us with a model of psychiatric disorders that allows separation of effects of poor physical environments (lacking food, shelter, etc.) from poor social environments (lack of appropriate socialization).

Lasting alterations induced in glial cell phenotypes by short exposure to alcohol during embryonic development in zebrafish.

Despite the known teratogenic effects of alcohol (ethanol) on the developing human fetus, the prevalence of fetal alcohol spectrum disorder (FASD) is not decreasing. Appropriate treatment for this life-long disease has not been developed, and even diagnostic biomarkers are unavailable. FASD remains a large unmet medical need. Numerous animal models have been developed to mimic FASD and study potential underlying biological mechanisms. However, most of these models focused on neuronal phenotypes. Given that glial cells represent the majority of cells in the vertebrate brain, and given the increasingly appreciated roles they play in a myriad of neuronal functions as well as CNS disorders, we decided to investigate potential embryonic alcohol exposure induced changes in them. Building upon a previously introduced zebrafish model of milder and most prevalent forms of FASD, we investigated the effect of a 2-hour-long exposure to alcohol (1% vol/vol bath concentration) employed at the 24th hour postfertilization stage of development of zebrafish on a number of glial cell-related phenotypes. We studied oligodendrocyte, astrocyte as well as microglia-related phenotypes using immunohistochemistry, lipid, and enzyme activity analyses. We report significant changes in wide-spread glial cell phenotypes induced by embryonic alcohol exposure in the zebrafish brain and conclude that the zebrafish will advance our understanding of the mechanisms of this devastating disorder.

Targeted knockout of a chemokine-like gene increases anxiety and fear responses.

Emotional responses, such as fear and anxiety, are fundamentally important behavioral phenomena with strong fitness components in most animal species. Anxiety-related disorders continue to represent a major unmet medical need in our society, mostly because we still do not fully understand the mechanisms of these diseases. Animal models may speed up discovery of these mechanisms. The zebrafish is a highly promising model organism in this field. Here, we report the identification of a chemokine-like gene family, samdori (sam), and present functional characterization of one of its members, sam2 We show exclusive mRNA expression of sam2 in the CNS, predominantly in the dorsal habenula, telencephalon, and hypothalamus. We found knockout (KO) zebrafish to exhibit altered anxiety-related responses in the tank, scototaxis and shoaling assays, and increased crh mRNA expression in their hypothalamus compared with wild-type fish. To investigate generalizability of our findings to mammals, we developed a Sam2 KO mouse and compared it to wild-type littermates. Consistent with zebrafish findings, homozygous KO mice exhibited signs of elevated anxiety. We also found bath application of purified SAM2 protein to increase inhibitory postsynaptic transmission onto CRH neurons of the paraventricular nucleus. Finally, we identified a human homolog of SAM2, and were able to refine a candidate gene region encompassing SAM2, among 21 annotated genes, which is associated with intellectual disability and autism spectrum disorder in the 12q14.1 deletion syndrome. Taken together, these results suggest a crucial and evolutionarily conserved role of sam2 in regulating mechanisms associated with anxiety.

Optogenetic Manipulation of Olfactory Responses in Transgenic Zebrafish: A Neurobiological and Behavioral Study.

Olfaction is an important neural system for survival and fundamental behaviors such as predator avoidance, food finding, memory formation, reproduction, and social communication. However, the neural circuits and pathways associated with the olfactory system in various behaviors are not fully understood. Recent advances in optogenetics, high-resolution in vivo imaging, and reconstructions of neuronal circuits have created new opportunities to understand such neural circuits. Here, we generated a transgenic zebrafish to manipulate olfactory signal optically, expressing the Channelrhodopsin (ChR2) under the control of the olfactory specific promoter, omp. We observed light-induced neuronal activity of olfactory system in the transgenic fish by examining c-fos expression, and a calcium indicator suggesting that blue light stimulation caused activation of olfactory neurons in a non-invasive manner. To examine whether the photo-activation of olfactory sensory neurons affect behavior of zebrafish larvae, we devised a behavioral choice paradigm and tested how zebrafish larvae choose between two conflicting sensory cues, an aversive odor or the naturally preferred phototaxis. We found that when the conflicting cues (the preferred light and aversive odor) were presented together simultaneously, zebrafish larvae swam away from the aversive odor. However, the transgenic fish with photo-activation were insensitive to the aversive odor and exhibited olfactory desensitization upon optical stimulation of ChR2. These results show that an aversive olfactory stimulus can override phototaxis, and that olfaction is important in decision making in zebrafish. This new transgenic model will be useful for the analysis of olfaction related behaviors and for the dissection of underlying neural circuits.

Food density and preferred quantity: discrimination of small and large numbers in angelfish (Pterophyllum scalare).

Many animal species share the ability to discriminate between sets with different quantity of food items. In fish, this ability has rarely been investigated, although findings have been obtained do indicate a preference, as in other animals, for sets with large over small quantities. The role played by food item size has also been found to be important in the discrimination. However, another potentially important non-numerical variable, food density, has not been investigated. In this study, we examined the influence of density (inter-item distance) in the decision-making process of food discrimination in angelfish (Pterophyllum scalare). In a binary choice task, we kept the number and size of food items constant, but contrasted a set containing food items spaced further apart (sparse set) to another set with food items spaced more closely (dense set). We conducted this analysis with sets in the small (3 vs 3 food items) and in the large number range (5 vs 5 food items) and also varied the specific spatial arrangements of the food items in the sets. Contrary to expectations, angelfish showed a preference for the sparse sets over the dense sets in the five vs five contrasts irrespective of the specific spatial arrangement, but exhibited no preference in case of the three vs three contrasts. Subsequently, we slightly lengthened the inter-item distance in the dense sets, and found preference for the dense over the sparse sets. Last, we further examined the potential effect of spatial configuration of the items in the sets, but found no effect of this latter factor. Overall, these results indicate that higher density of the contrasted food item sets significantly influences choice in angelfish, which prefer denser sets if a clear discriminability of each individual item within the sets is provided.

Argus: An open-source and flexible software application for automated quantification of behavior during social interaction in adult zebrafish.

Zebrafish show great potential for behavioral neuroscience. Promising lines of research, however, require the development and validation of software tools that will allow automated and cost-effective behavioral analysis. Building on our previous work with the RealFishTracker (in-house-developed tracking system), we present Argus, a data extraction and analysis tool built in the open-source R language for behavioral researchers without any expertise in R. Argus includes a new, user-friendly, and efficient graphical user interface, instead of a command-line interface, and offers simplicity and flexibility in measuring complex zebrafish behavior through customizable parameters. In this article, we compare Argus with Noldus EthoVision and Noldus The Observer, to validate this new system. All three software applications were originally designed to quantify the behavior of a single subject. We first also performed an analysis of the movement of individual fish and compared the performance of the three software applications. Next we computed and quantified the behavioral variables that characterize dyadic interactions between zebrafish. We found that Argus and EthoVision extract similar absolute values and patterns of changes in these values for several behavioral measures, including speed, freezing, erratic movement, and interindividual distance. In contrast, the manual coding of behavior in The Observer showed weaker correlations with the two tracking methods (EthoVision and Argus). Thus, Argus is a novel, cost-effective, and customizable method for the analysis of adult zebrafish behavior that may be utilized for the behavioral quantification of both single and dyadic interacting subjects, but further sophistication will be needed for the proper identification of complex motor patterns, measures that a human observers can easily detect.

Lasting changes induced by mild alcohol exposure during embryonic development in BDNF, NCAM and synaptophysin-positive neurons quantified in adult zebrafish.

Fetal alcohol spectrum disorder 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 hr post-fertilization (hpf) for 2 hr and analyzed 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.

Developmental social isolation affects adult behavior, social interaction, and dopamine metabolite levels in zebrafish.

The zebrafish is a social vertebrate and an excellent translational model for a variety of human disorders. Abnormal social behavior is a hallmark of several human brain disorders. Social behavioral problems can arise as a result of adverse early social environment. Little is known about the effects of early social isolation in adult zebrafish. We compared zebrafish that were isolated for either short (7 days) or long duration (180 days) to socially housed zebrafish, testing their behavior across ontogenesis (ages 10, 30, 60, 90, 120, 180 days), and shoal cohesion and whole-brain monoamines and their metabolites in adulthood. Long social isolation increased locomotion and decreased shoal cohesion and anxiety in the open-field in adult. Additionally, both short and long social isolation reduced dopamine metabolite levels in response to social stimuli. Thus, early social isolation has lasting effects in zebrafish, and may be employed to generate zebrafish models of human neuropsychiatric conditions.

The integration of sociality, monoamines and stress neuroendocrinology in fish models: applications in the neurosciences.

Animal-focused research has been crucial for scientific advancement, but rodents are still taking a starring role. Starting as merely supporting evidence found in rodents, the use of fish models has slowly taken a more central role and expanded its overall contributions in areas such as social sciences, evolution, physiology and recently in translational medical research. In the neurosciences, zebrafish Danio rerio have been widely adopted, contributing to our understanding of the genetic control of brain processes and the effects of pharmacological manipulations. However, discussion continues regarding the paradox of function versus structure, when fishes and mammals are compared and on the potentially evolutionarily conserved nature of behaviour across fish species. From a behavioural standpoint, we explore aversive-stress and social behaviour in selected fish models and refer to the extensive contributions of stress and monoaminergic systems. We suggest that, in spite of marked neuroanatomical differences between fishes and mammals, stress and sociality are conserved at the behavioural and molecular levels. We also suggest that stress and sociality are mediated by monoamines in predictable and non-trivial ways and that monoamines could bridge the relationship between stress and social behaviour. To reconcile the level of divergence with the level of similarity, we need neuroanatomical, pharmacological, behavioural and ecological studies conducted in the laboratory and in nature. These areas need to add to each other to enhance our understanding of fish behaviour and ultimately how this all may lead to better model systems for translational studies.

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.

Zebrafish prefer larger to smaller shoals: analysis of quantity estimation in a genetically tractable model organism.

Numerical abilities have been demonstrated in a variety of non-human vertebrates. However, underlying biological mechanisms have been difficult to study due to a paucity of experimental tools. Powerful genetic and neurobiological tools already exist for the zebrafish, but numerical abilities remain scarcely explored with this species. Here, we investigate the choice made by single experimental zebrafish between numerically different shoals of conspecifics presented concurrently on opposite sides of the experimental tank. We examined this choice using the AB strain and pet store zebrafish. We found zebrafish of both populations to generally prefer the numerically larger shoal to the smaller one. This preference was significant for contrasted ratios above or equalling 2:1 (i.e. 4 vs. 0, 4 vs. 1, 8 vs. 2, 6 vs. 2 and 6 vs. 3). Interestingly, zebrafish showed no significant preference when each of the two contrasted shoals had at least 4 members, e.g. in a contrast 8 versus 4. These results confirm that zebrafish possess the ability to distinguish larger numbers of items from smaller number of items, in a shoaling context, with a potential limit above 4. Our findings confirm the utility of the zebrafish for the exploration of both the behavioural and the biological mechanisms underlying numerical abilities in vertebrates.

Quantity discrimination in angelfish (Pterophyllum scalare) is maintained after a 30-s retention interval in the large but not in the small number range.

The ability to discriminate between sets that differ in the number of elements can be useful in different contexts and may have survival and fitness consequences. As such, numerical/quantity discrimination has been demonstrated in a diversity of animal species. In the laboratory, this ability has been analyzed, for example, using binary choice tests. Furthermore, when the different number of items first presented to the subjects are subsequently obscured, i.e., are not visible at the moment of making a choice, the task requires memory for the size of the sets. In previous work, angelfish (Pterophyllum scalare) have been found to be able to discriminate shoals differing in the number of shoal members both in the small (less than 4) and the large (4 or more) number range, and they were able to perform well even when a short memory retention interval (2-15 s) was imposed. In the current study, we increased the retention interval to 30 s during which the shoals to choose between were obscured, and investigated whether angelfish could show preference for the larger shoal they saw before this interval. Subjects were faced with a discrimination between numerically small shoals (≤4 fish) and also between numerically large (≥4 fish) shoals of conspecifics. We found angelfish not to be able to remember the location of larger versus smaller shoals in the small number range, but to exhibit significant memory for the larger shoal in the large number range as long as the ratio between these shoals was at least 2:1. These results, together with prior findings, suggest the existence of two separate quantity estimation systems, the object file system for small number of items that does not work with the longer retention interval and the analogue magnitude system for larger number of items that does.

Behavioral Responses to Novelty or to a Predator Stimulus Are Not Altered in Adult Zebrafish by Early Embryonic Alcohol Exposure.

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.

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.

Embryonic alcohol exposure: Towards the development of a zebrafish model of fetal alcohol spectrum disorders.

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.

Chronic and acute alcohol administration induced neurochemical changes in the brain: comparison of distinct zebrafish populations.

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.

Subcutaneous dye injection for marking and identification of individual adult zebrafish (Danio rerio) in behavioral studies.

The zebrafish is increasingly utilized in behavioral brain research, as it offers a useful compromise between system complexity and practical simplicity. However, a potential drawback of this species in behavioral research is that individuals are difficult to distinguish. Here we describe a simple marking procedure, subcutaneous injection of color dyes, that may alleviate this problem. The procedure allowed us to successfully mark zebrafish and distinguish them for a period of more than 30 days, which is sufficiently long for most behavioral paradigms developed for this species. In addition, we also provide data suggesting that the injection-based marking does not significantly alter social interaction, as defined by the frequency of agonistic behaviors within shoals.