Zebrafish models to study drug abuse-related phenotypes.

Mounting evidence implicates the zebrafish (Danio rerio) as a promising model species for reward and addiction research. Modeling drug abuse-related behavior in both adult and larval zebrafish produced a wealth of clinically translatable data, also demonstrating their sensitivity to various drugs of abuse and the ability to develop tolerance. Several studies have also applied withdrawal paradigms to model the adverse effects of drug abuse in zebrafish. In this review, we summarize recent findings of a wide spectrum of zebrafish drug abuse-related behavioral and physiological phenotypes, discuss the existing challenges, and outline potential future directions of research in this field.

Behavioral effects of MDMA ('ecstasy') on adult zebrafish.

3,4-methylenedioxymethamphetamine (MDMA, 'ecstasy') is a potent psychedelic drug inducing euphoria and hypersociability in humans, as well as hyperactivity and anxiety in rodents. Adult zebrafish (Danio rerio) have become a widely used species in neurobehavioral research. Here, we explore the effects of a wide range (0.25-120 mg/l) of acute MDMA doses on zebrafish behavior in the novel tank test. Although MDMA was inactive at lower doses (0.25-10 mg/l), higher doses reduced bottom swimming and immobility (40-120 mg/l) and impaired intrasession habituation (10-120 mg/l). MDMA also elevated brain c-fos expression, collectively confirming the usage of zebrafish models for screening of hallucinogenic compounds.

Neurosteroid vitamin D system as a nontraditional drug target in neuropsychopharmacology.

Vitamin D is becoming increasingly recognized as a nontraditional drug target for different brain pathologies. Although widely known for their role in calcium metabolism, vitamin D and its receptor have been linked to several brain disorders, including cognitive decline, epilepsy, affective disorders, and schizophrenia. Here we discuss mounting evidence, and parallel recent clinical and animal behavioral, genetic and pharmacological data to emphasize the emerging role of the neurosteroid vitamin D system in brain function.

Perspectives on experimental models of serotonin syndrome in zebrafish.

Serotonin syndrome (SS) is a serious life-threatening disorder associated with elevated brain serotonergic function. With the growing use of serotonergic drugs, SS affects a large portion of general population, becoming a major biomedical concern. SS-like behaviors have also been reported in animals following administration of serotonergic drugs. Although clinical and rodent studies have provided significant insight into the etiology of SS, its exact mechanisms and risk factors remain poorly understood. The need to develop more efficient psychotropic drugs also requires extensive high-throughput screening of novel compounds using sensitive in-vivo tests. The use of zebrafish (Danio rerio) in neuroscience research is rapidly expanding due to their homology to humans, robust behavioral and physiological responses, genetic tractability, and low costs. Here we discuss the potential of zebrafish models to study SS-related phenotypes induced by selected serotonergic drugs. Overall, zebrafish exposed to serotonergic agents and their combinations exhibit a characteristic top dwelling (surfacing behavior) and hypolocomotion which may represent potential markers of SS-like states in zebrafish. This behavior in zebrafish models positively correlates with brain concentrations of serotonin, suggesting the developing utility of zebrafish (and other aquatic models) for studying SS. Future research is expected to foster high-throughput screening of drug interactions, and pharmacogenetics studies identifying zebrafish mutations implicated in pathological SS-like states.

Psychopharmacological effects of acute exposure to kynurenic acid (KYNA) in zebrafish.

A metabolite of the kynurenine pathway, kynurenic acid (KYNA) is an important endogenous neuromodulator and neuroprotector, that also exerts neurotropic effects following exogenous administration. In humans and animals, KYNA regulates affective and cognitive responses, acting mainly as an antagonist of glutamatergic receptors. However, the complete psychopharmacological profile of KYNA (which includes the activity of several neurotransmitter receptors) is poorly understood, and merit further studies. Aquatic models are rapidly emerging as useful tools in translational psychopharmacology research. Here, we exposed adult zebrafish (Danio rerio) to exogenous KYNA for 20 min, and assessed their behavior in the novel tank test. Exposure to KYNA (20 mg/L) in this paradigm evoked overt effects in fish, including decreased latency to enter the top half of the tank, increased number of top entries and longer top duration. In contrast, locomotor activity indices (swimming distance and velocity) were not affected by KYNA in this study. Overall, our results show KYNA has an anxiolytic-like pharmacological effect in zebrafish, and therefore strongly support the utility of zebrafish models in neurotropic drug screening, including drugs acting at central glutamatergic system. Robust phenotypic differences evoked by KYNA, revealed here using three-dimensional (3D) reconstructions of zebrafish locomotion in X, Y and time (Z) coordinates, confirm this notion, also demonstrating the value of 3D-based phenotyping approaches for high-throughput drug screening using zebrafish models.

Unique and potent effects of acute ibogaine on zebrafish: the developing utility of novel aquatic models for hallucinogenic drug research.

An indole alkaloid, ibogaine is the principal psychoactive component of the iboga plant, used by indigenous peoples in West Africa for centuries. Modulating multiple neurotransmitter systems, the drug is a potent hallucinogen in humans, although its psychotropic effects remain poorly understood. Expanding the range of model species is an important strategy for translational neuroscience research. Here we exposed adult zebrafish (Danio rerio) to 10 and 20mg/L of ibogaine, testing them in the novel tank, light-dark box, open field, mirror stimulation, social preference and shoaling tests. In the novel tank test, the zebrafish natural diving response (geotaxis) was reversed by ibogaine, inducing initial top swimming followed by bottom dwelling. Ibogaine also attenuated the innate preference for dark environments (scototaxis) in the light-dark box test. While it did not exert overt locomotor or thigmotaxic responses in the open field test, the drug altered spatiotemporal exploration of novel environment, inducing clear preference of some areas over others. Ibogaine also promoted 'mirror' exploration in the mirror stimulation test, disrupted group cohesion in the shoaling test, and evoked strong coloration responses due to melanophore aggregation, but did not alter brain c-fos expression or whole-body cortisol levels. Overall, our results support the complex pharmacological profile of ibogaine and its high sensitivity in zebrafish models, dose-dependently affecting multiple behavioral domains. While future investigations in zebrafish may help elucidate the mechanisms underlying these unique behavioral effects, our study strongly supports the developing utility of aquatic models in hallucinogenic drug research. High sensitivity of three-dimensional phenotyping approaches applied here to behavioral effects of ibogaine in zebrafish provides further evidence of how 3D reconstructions of zebrafish swimming paths may be useful for high-throughput pharmacological screening.

Constructing the habituome for phenotype-driven zebrafish research.

Intra-session habituation to novelty reflects spatial working memory (related to exploration and cognition), and is observed in various species, including zebrafish (Danio rerio). With the growing understanding of complex zebrafish behaviors, the extent to which they habituate remains unclear. Here we perform a large-scale characterization of zebrafish novelty-evoked (novel tank and open field) behaviors, to establish their grouping based on intra-session habituation and sensitivity to anxiolytic or anxiogenic manipulations. We also assess multiple behaviors in high- and low-anxiety sub-cohorts of a large heterogeneous zebrafish population, comparing their habituation profiles. Overall, our analyses demonstrate that anxiety responsivity and the ability to habituate show little correlation for multiple zebrafish behaviors, suggesting that they most likely represent distinct behavioral phenomena in novel environments. Using these data, we also present the habituome--a new conceptual approach to study affective and cognitive responses in zebrafish by examining a big set of their habituation phenotypes. Given marked similarity in animal novelty exploration, this approach may also be used to construct habituomes in other model organisms, including rodents and humans.

Potential translational targets revealed by linking mouse grooming behavioral phenotypes to gene expression using public databases.

Rodent self-grooming is an important, evolutionarily conserved behavior, highly sensitive to pharmacological and genetic manipulations. Mice with aberrant grooming phenotypes are currently used to model various human disorders. Therefore, it is critical to understand the biology of grooming behavior, and to assess its translational validity to humans. The present in-silico study used publicly available gene expression and behavioral data obtained from several inbred mouse strains in the open-field, light-dark box, elevated plus- and elevated zero-maze tests. As grooming duration differed between strains, our analysis revealed several candidate genes with significant correlations between gene expression in the brain and grooming duration. The Allen Brain Atlas, STRING, GoMiner and Mouse Genome Informatics databases were used to functionally map and analyze these candidate mouse genes against their human orthologs, assessing the strain ranking of their expression and the regional distribution of expression in the mouse brain. This allowed us to identify an interconnected network of candidate genes (which have expression levels that correlate with grooming behavior), display altered patterns of expression in key brain areas related to grooming, and underlie important functions in the brain. Collectively, our results demonstrate the utility of large-scale, high-throughput data-mining and in-silico modeling for linking genomic and behavioral data, as well as their potential to identify novel neural targets for complex neurobehavioral phenotypes, including grooming.

Towards a comprehensive catalog of zebrafish behavior 1.0 and beyond.

Zebrafish (Danio rerio) are rapidly gaining popularity in translational neuroscience and behavioral research. Physiological similarity to mammals, ease of genetic manipulations, sensitivity to pharmacological and genetic factors, robust behavior, low cost, and potential for high-throughput screening contribute to the growing utility of zebrafish models in this field. Understanding zebrafish behavioral phenotypes provides important insights into neural pathways, physiological biomarkers, and genetic underpinnings of normal and pathological brain function. Novel zebrafish paradigms continue to appear with an encouraging pace, thus necessitating a consistent terminology and improved understanding of the behavioral repertoire. What can zebrafish 'do', and how does their altered brain function translate into behavioral actions? To help address these questions, we have developed a detailed catalog of zebrafish behaviors (Zebrafish Behavior Catalog, ZBC) that covers both larval and adult models. Representing a beginning of creating a more comprehensive ethogram of zebrafish behavior, this effort will improve interpretation of published findings, foster cross-species behavioral modeling, and encourage new groups to apply zebrafish neurobehavioral paradigms in their research. In addition, this glossary creates a framework for developing a zebrafish neurobehavioral ontology, ultimately to become part of a unified animal neurobehavioral ontology, which collectively will contribute to better integration of biological data within and across species.

Alterations in grooming activity and syntax in heterozygous SERT and BDNF knockout mice: the utility of behavior-recognition tools to characterize mutant mouse phenotypes.

Serotonin transporter (SERT) and brain-derived neurotrophic factor (BDNF) are key modulators of molecular signaling, cognition and behavior. Although SERT and BDNF mutant mouse phenotypes have been extensively characterized, little is known about their self-grooming behavior. Grooming represents an important behavioral domain sensitive to environmental stimuli and is increasingly used as a model for repetitive behavioral syndromes, such as autism and attention deficit/hyperactivity disorder. The present study used heterozygous ((+/-)) SERT and BDNF male mutant mice on a C57BL/6J background and assessed their spontaneous self-grooming behavior applying both manual and automated techniques. Overall, SERT(+/-) mice displayed a general increase in grooming behavior, as indicated by more grooming bouts and more transitions between specific grooming stages. SERT(+/-) mice also aborted more grooming bouts, but showed generally unaltered activity levels in the observation chamber. In contrast, BDNF(+/-) mice displayed a global reduction in grooming activity, with fewer bouts and transitions between specific grooming stages, altered grooming syntax, as well as hypolocomotion and increased turning behavior. Finally, grooming data collected by manual and automated methods (HomeCageScan) significantly correlated in our experiments, confirming the utility of automated high-throughput quantification of grooming behaviors in various genetic mouse models with increased or decreased grooming phenotypes. Taken together, these findings indicate that mouse self-grooming behavior is a reliable behavioral biomarker of genetic deficits in SERT and BDNF pathways, and can be reliably measured using automated behavior-recognition technology.

A survey of the neuroscience resource landscape: perspectives from the neuroscience information framework.

The number of available neuroscience resources (databases, tools, materials, and networks) available via the Web continues to expand, particularly in light of newly implemented data sharing policies required by funding agencies and journals. However, the nature of dense, multifaceted neuroscience data and the design of classic search engine systems make efficient, reliable, and relevant discovery of such resources a significant challenge. This challenge is especially pertinent for online databases, whose dynamic content is largely opaque to contemporary search engines. The Neuroscience Information Framework was initiated to address this problem of finding and utilizing neuroscience-relevant resources. Since its first production release in 2008, NIF has been surveying the resource landscape for the neurosciences, identifying relevant resources and working to make them easily discoverable by the neuroscience community. In this chapter, we provide a survey of the resource landscape for neuroscience: what types of resources are available, how many there are, what they contain, and most importantly, ways in which these resources can be utilized by the research community to advance neuroscience research.

Behavioral and physiological effects of RDX on adult zebrafish.

1,3,5-Trinitroperhydro-1,3,5-triazine (RDX) is a nitroamine explosive, with common toxic effects including seizures. Here, we explore the behavioral effects of acute RDX exposure in adult zebrafish Danio rerio, a rapidly developing model in neuroscience and neurotoxicology research. Overall, a 30-min exposure to RDX low dose of 0.1 mM evoked behavioral activation in zebrafish, while a higher dose of 1 mM markedly reduced exploration, increased freezing and evoked seizure-like responses (i.e., bouts of hyperactivity, spasms, and corkscrew swimming). Likewise, whole-body cortisol levels were also significantly elevated in fish exposed to 1 mM (but not 0.1 mM) RDX. In line with clinical and animal data, our study demonstrates the dose-dependent behavioral activation and pro-convulsant effects of RDX in zebrafish-based models.

Automated high-throughput neurophenotyping of zebrafish social behavior.

Zebrafish (Danio rerio) are rapidly becoming an important model organism in neuroscience research, representing an excellent species to study complex social phenotypes. Zebrafish actively form shoals, which can be used to quantify their shoaling behaviors, highly sensitive to various experimental manipulations. Recent advances in video-tracking techniques have enabled simultaneous tracking of multiple subjects, previously assessed by manual scoring of animal behavior. Here we examined the effect of group-size in the shoaling paradigm (ranging from 2 to 8 fish), and evaluated the ability of novel video-tracking tools to accurately track an entire shoal, compared to traditional manual analysis of shoaling phenotypes. To further validate our approach, the effects of the psychotropic drugs lysergic acid diethylamide (LSD) and 3,4-methlenedioxymethamphetamine (MDMA), as well as exposure to alarm pheromone, previously shown to affect zebrafish shoaling, were examined. Overall, a significant difference in group size was shown in the 2-fish vs. the 3-, 4-, 5-, 6-, 7- and 8-fish groups. Moreover, both LSD and MDMA treatments reduced shoaling (assessed by increased inter-fish distance) as well as proximity (time spent together) among fish. In contrast, exposure to alarm pheromone yielded an increase in shoaling and in proximity in a time-dependent manner. Importantly, a highly significant correlation for manual vs. automated analyses was revealed across all experiments. Collectively, this study further supports the utility of zebrafish to study social behavior, also demonstrating the capacity of video-tracking technology to assess zebrafish shoaling in a high-throughput and reliable manner.

Three-dimensional neurophenotyping of adult zebrafish behavior.

The use of adult zebrafish (Danio rerio) in neurobehavioral research is rapidly expanding. The present large-scale study applied the newest video-tracking and data-mining technologies to further examine zebrafish anxiety-like phenotypes. Here, we generated temporal and spatial three-dimensional (3D) reconstructions of zebrafish locomotion, globally assessed behavioral profiles evoked by several anxiogenic and anxiolytic manipulations, mapped individual endpoints to 3D reconstructions, and performed cluster analysis to reconfirm behavioral correlates of high- and low-anxiety states. The application of 3D swim path reconstructions consolidates behavioral data (while increasing data density) and provides a novel way to examine and represent zebrafish behavior. It also enables rapid optimization of video tracking settings to improve quantification of automated parameters, and suggests that spatiotemporal organization of zebrafish swimming activity can be affected by various experimental manipulations in a manner predicted by their anxiolytic or anxiogenic nature. Our approach markedly enhances the power of zebrafish behavioral analyses, providing innovative framework for high-throughput 3D phenotyping of adult zebrafish behavior.

Acute stress disrupts performance of zebrafish in the cued and spatial memory tests: the utility of fish models to study stress-memory interplay.

The zebrafish (Danio rerio) has emerged as a promising model organism for affective or cognitive neuroscience research, and may be useful to study the interplay between memory and anxiety-related states. To assess the effects of acute psychological stress on spatial and cued memory, adult zebrafish were trained in an aquatic plus-maze for 14 days using food bait as a reward. Two ecologically relevant stressors (alarm pheromone or Indian leaf fish exposure) were applied to acutely stress zebrafish immediately prior to the final (testing) trial. Overall, acute single inescapable stress markedly impaired spatial and cued memory in zebrafish plus-maze test, reducing the number of correct arm entries and time spent in the target arm. This observation parallels rodent and clinical literature on memory-impairing effects of acute stress, strongly supporting the utility of zebrafish in neurobehavioral research.

Pharmacological modulation of anxiety-like phenotypes in adult zebrafish behavioral models.

Zebrafish (Danio rerio) are becoming increasingly popular in neurobehavioral research. Here, we summarize recent data on behavioral responses of adult zebrafish to a wide spectrum of putative anxiolytic and anxiogenic agents. Using the novel tank test as a sensitive and efficient behavioral assay, zebrafish anxiety-like behavior can be bi-directionally modulated by drugs affecting the gamma-aminobutyric acid, monoaminergic, cholinergic, glutamatergic and opioidergic systems. Complementing human and rodent data, zebrafish drug-evoked phenotypes obtained in this test support this species as a useful model for neurobehavioral and psychopharmacological research.

Homebase behavior of zebrafish in novelty-based paradigms.

Zebrafish (Danio rerio) are emerging as a promising model species in neuroscience research. Many traditional rodent behavioral paradigms may be adapted for zebrafish testing. Exposing zebrafish to three different "open field" tanks for 30 min, we showed that fish display robust homebase behavior, in which one area of the tank is chosen as a preferred point of reference during the test, which the fish frequently return to and spend a longer duration in. This phenotype strikingly resembles rodent homebase behavior, confirming that both species use homebases as "reference points" for their exploration. Our study introduces a simple method for zebrafish homebase phenotyping, and further supports the utility of these fish in neurobehavioral and cognitive research.

Qui non proficit, deficit: experimental models for 'integrative' research of affective disorders.

Experimental models are an important tool for the study of biological mechanisms of psychiatric disorders. Although encouraging progress has been made in biological psychiatry of affective disorders, there remain numerous methodological, conceptual, and translational challenges in this field. Mounting clinical data support the view that psychiatric disorders as spectra, rather than as discrete or isolated illnesses. This requires new theories as well as new animal paradigms for "integrative" modeling of psychiatric disorders and their spectra. Here we discuss recent "integrative" experimental models and concepts that promise to advance translational research of affective disorders.

Modeling seizure-related behavioral and endocrine phenotypes in adult zebrafish.

Larval zebrafish (Danio rerio) have recently been suggested as a high-throughput experimental model of epilepsy-related pathogenetic states. Here we use adult zebrafish to study behavioral symptoms associated with drug-evoked seizures. Experimental epilepsy-like states were evoked in zebrafish by exposure for 20min to three chemoconvulsant drugs: caffeine (250mg/L; 1.3mM), pentylenetetrazole (1.5g/L; 11.0mM) and picrotoxin (100mg/L; 0.17mM). Fish behavior was analyzed using manual and video-tracking methods (Noldus Ethovision XT7). Compared to their respective controls, all three drug-treated groups showed robust seizure-like responses (hyperactivity bouts, spasms, circular and corkscrew swimming) accompanied by elevated whole-body cortisol levels (assessed by ELISA). In contrast, control fish did not display seizure-like behaviors and had significantly lower cortisol levels. Paralleling behavioral and endocrine phenotypes observed in clinical and rodent studies, our data implicates adult zebrafish as an emerging experimental model for epilepsy research.