adgrl3.1-deficient zebrafish show noradrenaline-mediated externalizing behaviors, and altered expression of externalizing disorder-candidate genes, suggesting functional targets for treatment.

Externalizing disorders (ED) are a cause of concern for public health, and their high heritability makes genetic risk factors a priority for research. Adhesion G-Protein-Coupled Receptor L3 (ADGRL3) is strongly linked to several EDs, and loss-of-function models have shown the impacts of this gene on several core ED-related behaviors. For example, adgrl3.1-/- zebrafish show high levels of hyperactivity. However, our understanding of the mechanisms by which this gene influences behavior is incomplete. Here we characterized, for the first time, externalizing behavioral phenotypes of adgrl3.1-/- zebrafish and found them to be highly impulsive, show risk-taking in a novel environment, have attentional deficits, and show high levels of hyperactivity. All of these phenotypes were rescued by atomoxetine, demonstrating noradrenergic mediation of the externalizing effects of adgrl3.1. Transcriptomic analyses of the brains of adgrl3.1-/- vs. wild-type fish revealed several differentially expressed genes and enriched gene clusters that were independent of noradrenergic manipulation. This suggests new putative functional pathways underlying ED-related behaviors, and potential targets for the treatment of ED.

Ontogeny of working memory and behavioural flexibility in the free movement pattern (FMP) Y-maze in zebrafish.

The acquisition of executive skills such as working memory, decision-making and adaptive responding occur at different stages of central nervous system development. Zebrafish (Danio rerio) are increasingly used in behavioural neuroscience for complex behavioural tasks, and there is a critical need to understand the ontogeny of their executive functions. Zebrafish across developmental stages (4, 7, 14, 30 and 90 days post fertilisation (dpf)), were assessed to track development of working memory (WM) and behavioural flexibility (BF) using the free movement pattern Y-maze (FMP Y-maze). Several differences in both WM and BF were identified during the transition from yolk-dependent to independent feeding. Specifically, WM is evident in all age groups, even from 4 dpf. However, BF is not developed until larvae start free feeding, and show significant improvement thereafter, with young adults (90 dpf) demonstrating the most well-defined BF. We demonstrate, for the first time, objective WM processes in 4 dpf zebrafish larvae. This suggests that those wishing to study WM in zebrafish may be able to do so from 4 dpf, thus drastically increasing throughput. In addition, we show that zebrafish follow distinct stages of cognitive development and age-related changes during the early developmental period. Finally, our findings indicate distinct WM and BF mechanisms, which may be useful to study for translational purposes.

Transgenerational effects of early-life stress on anxiety behavior in zebrafish (Danio rerio).

Early-life adversity impacts on anxiety-related behaviors in adulthood. The effects of such adversity not only affect the animal itself, but can be passed on transgenerationally. Pervasive effects of experimentally-induced early-life stress (ELS) have been documented in adult zebrafish but it is not clear if this can be passed on via the germline. Here, we investigated the effects of ELS across three generations, by analyzing the responses of adult animals exposed to ELS in two different anxiety-related tasks, as well as in social behavior, memory, and cognition. Animals exposed to ELS (at 7 days-post-fertilization) showed a marked attenuation of specific anxiety-related behaviors (F0) as adults, and these alterations were maintained across two subsequent generations (F1 and F2). This suggest zebrafish may be a useful model organism to study the transgenerational effects of ELS, and how this pertains to (for example) neuropsychiatric disorders. In addition, our data may naturally provoke questions regarding consideration of the environment of laboratory-housed zebrafish at early developmental stages. In particular, more work may be necessary to determine how different environmental stressors could affect data variability across laboratories.

The larval diving response (LDR): Validation of an automated, high-throughput, ecologically relevant measure of anxiety-related behavior in larval zebrafish (Danio rerio).

BACKGROUND: Zebrafish are used in anxiety research as the species' naturalistic diving response to a new environment is a reliable and validated marker for anxiety-like behavior. One of the benefits of using zebrafish is the potential for high throughput drug screens in fish at the larval stage. However, at present, tests of anxiety in larvae and adults often measure different endpoints. NEW METHOD: Here, for the first time, we have adapted the novel tank diving response test for examining diving behavior in zebrafish larvae to assess anxiety-like behaviors at very early-stages (7 days-post-fertilization [dpf]). COMPARISON WITH EXISTING METHODS: Current methods to examine anxiety in larvae can show low reliability, and measure different endpoints as in adults, thus calling into question their translational relevance. RESULTS: We found that 7dpf zebrafish spent more time at the bottom of a small novel tank. We validated this as anxiety-like behaviors with diazepam reducing, and caffeine increasing the time spent in the bottom of the novel environment. CONCLUSIONS: This new automated and high-throughput screening tool has the potential use for screening of anxiogenic and anxiolytic compounds, and for studies aiming to better understand anxiety-like behaviors.

Modelling ADHD-Like Phenotypes in Zebrafish.

The use of multiple species to model complex human psychiatric disorders, such as ADHD, can give important insights into conserved evolutionary patterns underlying multidomain behaviors (e.g., locomotion, attention, and impulsivity). Here we discuss the advantages and challenges in modelling ADHD-like phenotypes in zebrafish (Danio rerio), a vertebrate species that has been widely used in neuroscience and behavior research. Moreover, multiple behavioral tasks can be used to model the core symptoms of ADHD and its comorbidities. We present a critical review of current ADHD studies in zebrafish, and how this species might be used to accelerate the discovery of new drug treatments for this disorder.

Using zebrafish (Danio rerio) models to understand the critical role of social interactions in mental health and wellbeing.

Social behavior represents a beneficial interaction between conspecifics that is critical for maintaining health and wellbeing. Dysfunctional or poor social interaction are associated with increased risk of physical (e.g., vascular) and psychiatric disorders (e.g., anxiety, depression, and substance abuse). Although the impact of negative and positive social interactions is well-studied, their underlying mechanisms remain poorly understood. Zebrafish have well-characterized social behavior phenotypes, high genetic homology with humans, relative experimental simplicity and the potential for high-throughput screens. Here, we discuss the use of zebrafish as a candidate model organism for studying the fundamental mechanisms underlying social interactions, as well as potential impacts of social isolation on human health and wellbeing. Overall, the growing utility of zebrafish models may improve our understanding of how the presence and absence of social interactions can differentially modulate various molecular and physiological biomarkers, as well as a wide range of other behaviors.

The zebrafish (Danio rerio) anxiety test battery: comparison of behavioral responses in the novel tank diving and light-dark tasks following exposure to anxiogenic and anxiolytic compounds.

RATIONALE: Triangulation of approaches (i.e., using several tests of the same construct) can be extremely useful for increasing the robustness of the findings being widely used when working with behavioral testing, especially when using rodents as a translational model. Although zebrafish are widely used in neuropharmacology research due to their high-throughput screening potential for new therapeutic drugs, behavioral test battery effects following pharmacological manipulations are still unknown. METHODS: Here, we tested the effects of an anxiety test battery and test time following pharmacological manipulations in zebrafish by using two behavioral tasks: the novel tank diving task (NTT) and the light-dark test (LDT). Fluoxetine and conspecific alarm substance (CAS) were chosen to induce anxiolytic and anxiogenic-like behavior, respectively. RESULTS: For non-drug-treated animals, no differences were observed for testing order (NTT → LDT or LDT → NTT) and there was a strong correlation between performances on the two behavioral tasks. However, we found that during drug treatment, NTT/LDT responses are affected by the tested order depending on the test time being fluoxetine effects higher at the second behavioral task (6 min later) and CAS effects lower across time. CONCLUSIONS: Overall, our data supports the use of baseline behavior assessment using this anxiety test battery. However, when working with drug exposure, data analysis must carefully consider time-drug-response and data variability across behavioral tasks.

The impact of water changes on stress and subject variation in a zebrafish (Danio rerio) anxiety-related task.

BACKGROUND: Zebrafish have been widely used to study anxiety-related phenotypes using the novel tank test (NTT). Although the NTT is well-characterized and commonly used by researchers, there is still a lack of information regarding how different experimental variables such as water quality can influence NTT performance. Zebrafish use different chemical cues and olfactory stimuli to communicate in water, so we predicted that water change frequency would affect cortisol, locomotion and anxiety-related parameters in the NTT. NEW METHODS: After extensive literature research, we found that only about 18% of papers using NTT report partial or complete water changes between subjects. Here, we tested multiple zebrafish in the NTT using the same water up to 9 consecutive times (with no water change) and analyzed cortisol levels, as a stressrelated marker. RESULTS: We found that when using the same water for more than 4 trials, data variability is increased and a higher number of extreme values is observed for the time spent in the top zone and immobility. Moreover, after 4 trials with no water change, increased cortisol levels are observed, indicating that animals show increased stressrelated responses with the lack of water changes. CONCLUSIONS: This study shows that lack of water change can significantly influence zebrafish stress-responses in the NTT. Altogether, behavioral experiments should avoid using the same water when testing multiple fish in the task, especially when looking at anxiety in the NTT.

Tricaine Methanesulfonate (MS222) Has Short-Term Effects on Young Adult Zebrafish (Danio rerio) Working Memory and Cognitive Flexibility, but Not on Aging Fish.

Exposure to anesthetic drugs is common in biomedical sciences being part of routine procedures in different translational species, however its impacts on memory and cognition are still debated, having different impacts depending on drug and age. The zebrafish (Danio rerio) is a translational species widely used in behavioral neuroscience, where tricaine methanesulfonate (MS222) is the most acceptable and used drug when conducting routine procedures. Based on this, we investigated the effects of MS222 (100 mg/l) in young adults and aging zebrafish 1, 2, 3, and 7 days after exposure. Animals' were submitted to the anesthetic procedure until loss of body posture, slowing of opercular movements and lack of response to tail touch with a plastic pipette were achieved, then further left in the drug for 3 min. After that, animals (6 mpf vs. 24 mpf) were transferred to a recovery tank until fully recovered and transferred back to their housing system until further testing in the free movement pattern (FMP) Y-maze, which assesses zebrafish working memory and cognitive lexibility. Young animals had significant impairment in their working memory and cognitive flexibility 1 and 2 days after the exposure to MS222, being fully recovered by day 3 and with no effects 7 days post drug exposure. Increased repetitions were also observed for animals exposed to MS222 which could indicate increased stress-related response in animals up to 2 days after drug exposure. No drug effect was observed in aging animals besides their natural decreased alternations and working memory. Overall, behavioral experiments after routine procedures using MS222 should be performed with caution and need to be delayed, at least 3 days after exposure where working memory, cognitive flexibility, and repetitive behavior are back to normal.

The impact of chronic unpredictable early-life stress (CUELS) on boldness and stress-reactivity: Differential effects of stress duration and context of testing.

Early-life stress (ELS) has been shown to result in a diverse array of long-lasting impacts; for example, increasing vulnerability to disease or building 'resilience' in adulthood. Previously, zebrafish (Danio rerio) have been used to understand the mechanisms by which ELS induces different behavioral phenotypes in adults, with alterations in both learning and anxiety observed in exposed individuals. Here, we subjected zebrafish larvae to chronic unpredictable early-life stress (CUELS) for 7 or 14 days, to investigate the impact on boldness towards a new environment and novel object, and stress-reactivity. We observed that 7 days of CUELS resulted in increased time spent in the top of a novel tank (indicating boldness) but did not alter approach to a novel object. Although CUELS did not affect stress-reactivity in terms of cortisol levels, decreased anxiety-like response to conspecific alarm substance (CAS) was observed in both ELS groups (7 and 14 days of CUELS). Therefore, for the first time, we observe a potential negative effect of CUELS by dampening the behavioral stress response following exposure to CAS. Overall, these data support the use of zebrafish as a translational model to study the broad range of ELS-induced permanent changes in behavior. It could also be used to investigate the mechanisms underlying both the positive and the negative effects of early-life adversity.

The effects of two stressors on working memory and cognitive flexibility in zebrafish (Danio rerio): The protective role of D1/D5 agonist on stress responses.

Acute stressors are recurrent in multiple species' lives and can facilitate or impair cognition. The use of zebrafish (Danio rerio) as a translational species to understand the mechanisms by which stress induces different behavioral phenotypes has been widely studied. Two acute stressors are recognized when using this species: (1) conspecific alarm substance (CAS); and (2) net chasing. Here, we tested if CAS or net chasing would affect working memory and cognitive flexibility by testing performance in the FMP Y-maze after exposure to stress. We observed that CAS altered zebrafish behavioral phenotypes by increasing repetitive behavior; meanwhile, animals showed different patterns of repetitive behavior when exposed to net chasing, depending on the chasing direction. Because D1 receptors were previously studied as a potential mechanism underlying stress responses in different species, here, we pretreated fish with a D1/D5 agonist (SKF-38393) to assess whether this system plays a role in repetitive behavior in the FMP Y-maze. The pretreatment with D1/D5 agonist significantly decreased repetitive behavior in CAS exposed animals, and cortisol levels for both stressed groups, suggesting that the dopaminergic system plays an important role in zebrafish stress-related responses.

The cognitive and behavioral effects of D-amphetamine and nicotine sensitization in adult zebrafish.

BACKGROUND: Zebrafish are growing in use as a model for understanding drug dependence and addiction. Sensitization paradigms have been a useful tool in identifying mechanisms involved in drug-induced behavioral and neurological changes, but in zebrafish have tended to focus on locomotor, rather than cognitive, endpoints. METHODS: Here, we used a novel method, the FMP Y-maze, which measures continuous performance through a series of repeated binary choices (L vs R), to establish a model for assessing parameters associated with psychostimulant-induced behavioral and cognitive sensitization in adult zebrafish. RESULTS: Repeat, intermittent exposure to d-amphetamine (AMPH) for 14 days increased alternations (LRLR) in the maze, suggesting improved working memory, which was enhanced further following drug challenge after a short withdrawal period, suggesting behavioral sensitization. However, this cognitive enhancement coincided with a reduction in the use of other exploration strategies, hypolocomotion, and inhibition of cognitive flexibility. Like AMPH, exposure to nicotine (NIC) increased alternations following drug challenge after chronic treatment. Repeat NIC exposure appeared to induce both cognitive and psychomotor sensitization, as evidenced by increased working memory performance (alternations) and locomotor activity, without negatively impacting other search strategies or cognitive flexibility. CONCLUSION: Chronic treatment with AMPH or NIC boosts cognitive performance in adult zebrafish. Cognitive sensitization occurred with both drugs, resulting in enhanced working memory; however, repeat AMPH exposure, following a withdrawal period, resulted in inhibited cognitive flexibility, an effect not evident with repeat NIC exposure. Cognitive and behavioral sensitization paradigms in zebrafish could serve as a useful tool for assessing cognitive states which result in cognitive enhancing or impairing effects of drugs.

Dopaminergic modulation of working memory and cognitive flexibility in a zebrafish model of aging-related cognitive decline.

Healthy aging is associated with a decline in memory and executive function, which have both been linked with aberrant dopaminergic signaling. We examined the relationship between cognitive performance and dopamine function of young and aging zebrafish (Danio rerio). We revealed age-related decreases in working memory and cognitive flexibility in the Free-Movement Pattern (FMP) Y-maze. An increase in drd5 gene expression in aging adults coincided with a decrease in cognitive performance. Treatment with a D1/D5 receptor agonist (SKF-38393, 35 µM) 30 minutes prior to behavioral assessment resulted in improved working memory in aging zebrafish, but no effect in younger adults. However, an "overdosing" effect caused by agonist treatment resulted in downregulation of dat expression in 6-month old, treated zebrafish. The translational relevance of these findings was tested in humans by analyzing exploratory behavior in young-adult, 18-35-year olds, and aged adults, 70+ year olds, in a virtual FMP Y-maze. Our findings revealed similar age-related decline in working memory. Thus, strongly supporting zebrafish as a translational model of aging and cognitive decline.

The Free-movement pattern Y-maze: A cross-species measure of working memory and executive function.

Numerous neurodegenerative and psychiatric disorders are associated with deficits in executive functions such as working memory and cognitive flexibility. Progress in developing effective treatments for disorders may benefit from targeting these cognitive impairments, the success of which is predicated on the development of animal models with validated behavioural assays. Zebrafish offer a promising model for studying complex brain disorders, but tasks assessing executive function are lacking. The Free-movement pattern (FMP) Y-maze combines aspects of the common Y-maze assay, which exploits the inherent motivation of an organism to explore an unknown environment, with analysis based on a series of sequential two-choice discriminations. We validate the task as a measure of working memory and executive function by comparing task performance parameters in adult zebrafish treated with a range of glutamatergic, cholinergic and dopaminergic drugs known to impair working memory and cognitive flexibility. We demonstrate the cross-species validity of the task by assessing performance parameters in adapted versions of the task for mice and Drosophila, and finally a virtual version in humans, and identify remarkable commonalities between vertebrate species' navigation of the maze. Together, our results demonstrate that the FMP Y-maze is a sensitive assay for assessing working memory and cognitive flexibility across species from invertebrates to humans, providing a simple and widely applicable behavioural assay with exceptional translational relevance.

Moderate early life stress improves adult zebrafish (Danio rerio) working memory but does not affect social and anxiety-like responses.

Early life stress (ELS) is defined as a short or chronic period of trauma, environmental or social deprivation, which can affect different neurochemical and behavioral patterns during adulthood. Zebrafish (Danio rerio) have been widely used as a model system to understand human neurodevelopmental disorders and display translationally relevant behavioral and stress-regulating systems. In this study, we aimed to investigate the effects of moderate ELS by exposing young animals (6-weeks postfertilization), for 3 consecutive days, to three stressors, and analyzing the impact of this on adult zebrafish behavior (16-week postfertilization). The ELS impact in adults was assessed through analysis of performance on tests of unconditioned memory (free movement pattern Y-maze test), exploratory and anxiety-related task (novel tank diving test), and social cohesion (shoaling test). Here, we show for the first time that moderate ELS increases the number of alternations in turn-direction compared to repetitions in the unconditioned Y-maze task, suggesting increased working memory, but has no effect on shoal cohesion, locomotor profile, or anxiety-like behavior. Overall, our data suggest that moderate ELS may be linked to adaptive flexibility which contributes to build "resilience" in adult zebrafish by improving working memory performance.

Chronic unpredictable early-life stress (CUELS) protocol: Early-life stress changes anxiety levels of adult zebrafish.

Early-life stress can lead to two different behavioral responses: (1) increased susceptibility to psychiatric disorders or (2) resilience. Here, we created a chronic unpredictable early-life stress (CUELS) protocol to assess the effects of early experiences in adult zebrafish. Animals were exposed to mild stressors twice a day and the duration was varied between groups (0, 1, 3, 7 and 14 days of stress). The stressor consisted of light/dark cycle changes; social isolation; overcrowding; water changes; water cooling; mechanical stirring; water heating; and immersion in shallow water. Behavior was assessed at young stages (21 days post-fertilization - open field analysis) and adulthood (4-months-old - novel tank diving test, light/dark task, shoaling, free movement pattern Y-maze and Pavlovian fear conditioning). Cortisol levels were assessed to evaluate the impact of CUELS in the HPI axis. Zebrafish exposed to 7 days of CUELS showed a decreased anxiety-like phenotype in two behavioral tasks, presenting increased time spent in top and decreased time spent in the dark area. Animals exposed to 14 days of CUELS showed an opposite anxious phenotype compared to 3 and 7 days of CUELS. No significant changes were observed in memory and cognition, social behavior and cortisol levels. In general, 7 days of CUELS protocol decreased anxiety in young and adult zebrafish, and could be used to understand the mechanisms underlying early-life experiences-derived alterations in neural circuits of anxiety.

The importance of pH: How aquarium water is affecting behavioural responses to drug exposure in larval zebrafish.

There has been rapid growth in the use of larval zebrafish as a complementary vertebrate model for drug discovery, abuse liability and pharmacological toxicology, resulting in a huge increase in zebrafish facilities worldwide. However, many research groups working with zebrafish do not typically report the pH of husbandry conditions in methodologies, nor are the pH of drug treatments reported in many research articles. This unknown factor can be a major contributor in the differential effects of drug treatments. Therefore, as a case study, we tested the impact of altering pH of several drugs of abuse and assessed locomotor changes associated with a single drug concentration delivered at different pHs. We found that a change of a single pH unit, within the pH ranges commonly used in zebrafish husbandry, was enough to alter locomotor activity at a fixed drug concentration. Many pharmacological agents are dependent on environmental factors, such as pH, to determine bioavailability. Efficaciousness for many classes of drug is dependent on their ionization state in which shifts towards uncharged species can influence the easy of a drug crossing biological membranes. Thus, we urge users to report pH in husbandry methods and drug treatments to improve replicability and inter-study comparisons.

Zebrafish models of impulsivity and impulse control disorders.

Impulse control disorders (ICDs) are characterized by generalized difficulty controlling emotions and behaviors. ICDs are a broad group of the central nervous system (CNS) disorders including conduct disorder, intermittent explosive, oppositional-defiant disorder, antisocial personality disorder, kleptomania, pyromania and other illnesses. Although they all share a common feature (aberrant impulsivity), their pathobiology is complex and poorly understood. There are also currently no ICD-specific therapies to treat these illnesses. Animal models are a valuable tool for studying ICD pathobiology and potential therapies. The zebrafish (Danio rerio) has become a useful model organism to study CNS disorders due to high genetic and physiological homology to mammals, and sensitivity to various pharmacological and genetic manipulations. Here, we summarize experimental models of impulsivity and ICD in zebrafish and highlight their growing translational significance. We also emphasize the need for further development of zebrafish ICD models to improve our understanding of their pathogenesis and to search for novel therapeutic treatments.

Modulation of redox and insulin signaling underlie the anti-hyperglycemic and antioxidant effects of diphenyl diselenide in zebrafish.

The organic selenium compound diphenyl diselenide (DD) has been recognized as an antioxidant and neuroprotective agent, exerting an anti-hyperglycemic effect in experimental models of diabetes. However, the precise mechanisms involved in the protection are unclear. Using the zebrafish (Danio rerio) as a model organism, here we investigated biomarkers underlying the protective effects of DD against hyperglycemia, targeting in a transcriptional approach the redox and insulin-signaling pathway. Fish were fed on a diet containing DD (3 mg/kg) for 74 days. In the last 14 days, they were exposed to a 111 mM glucose solution to induce a hyperglycemic state. DD reduced blood glucose levels as well as normalized the brain mRNA transcription of four insulin receptors-coding genes (Insra1, Insra2, Insrb1, Insrb2), which were down-regulated by glucose. DD alone caused an up-regulation of relative mRNA transcription in both Insra receptors and glucose transporter 3 genes. DD counteracted hyperglycemia-induced lipid peroxidation, protein and thiol depletion. Along with the decreased activity of antioxidant enzymes SOD and GPx, the brain of hyperglycemic fish presented a reduction in mRNA transcription of FoxO3A, FoxO3B, Nrf2, GPx3A, SOD1, and SOD2 genes. Besides normalizing the transcriptional levels, DD caused an up-regulation of relative mRNAs that encode Nrf2, FoxO1A, FOXO3A, GPx4A, PTP1B, AKT and SelP. Collectively, our findings suggest that the antioxidant and anti-hyperglycemic actions of DD in a zebrafish diabetes model are likely associated with the regulation of the oxidative stress resistance and the insulin-signaling pathway and that could be related to the modulation at mRNA level of two important transcription factors, Nrf2 and FoxO.

Role of the serotonergic system in ethanol-induced aggression and anxiety: A pharmacological approach using the zebrafish model.

Acute ethanol (EtOH) consumption exerts a biphasic effect on behavior and increases serotonin levels in the brain. However, the molecular mechanisms underlying alcohol-mediated behavioral responses still remain to be fully elucidated. Here, we investigate pharmacologically the involvement of the serotonergic pathway on acute EtOH-induced behavioral changes in zebrafish. We exposed zebrafish to 0.25, 0.5, 1.0% (v/v) EtOH for 1 h and analyzed the effects on aggression, anxiety-like behaviors, and locomotion. EtOH concentrations that changed behavioral responses were selected to the subsequent experiments. As a pharmacological approach, we used pCPA (inhibitor of tryptophan hydroxylase), WAY100135 (5-HT1A antagonist), buspirone (5-HT1A agonist), CGS12066A and CGS12066B (5-HT1B antagonist and agonist, respectively), ketanserin (5-HT2A antagonist) and (±)-DOI hydrochloride (5-HT2A agonist). All serotonergic receptors tested modulated aggression, with a key role of 5-HT2A in aggressive behavior following 0.25% EtOH exposure. Because CGS12066B mimicked 0.5% EtOH anxiolysis, which was antagonized by CGS12066A, we hypothesized that anxiolytic-like responses are possibly mediated by 5-HT1B receptors. Conversely, the depressant effects of EtOH are probably not related with direct changes on serotonergic pathway. Overall, our novel findings demonstrate a role of the serotonergic system in modulating the behavioral effects of EtOH in zebrafish. These data also reinforce the growing utility of zebrafish models in alcohol research and help elucidate the neurobiological mechanisms underlying alcohol abuse and associated complex behavioral phenotypes.