Zebrafish neurobehavioral phenomics for aquatic neuropharmacology and toxicology research.

Zebrafish (Danio rerio) are rapidly emerging as an important model organism for aquatic neuropharmacology and toxicology research. The behavioral/phenotypic complexity of zebrafish allows for thorough dissection of complex human brain disorders and drug-evoked pathological states. As numerous zebrafish models become available with a wide spectrum of behavioral, genetic, and environmental methods to test novel drugs, here we discuss recent zebrafish phenomics methods to facilitate drug discovery, particularly in the field of biological psychiatry. Additionally, behavioral, neurological, and endocrine endpoints are becoming increasingly well-characterized in zebrafish, making them an inexpensive, robust and effective model for toxicology research and pharmacological screening. We also discuss zebrafish behavioral phenotypes, experimental considerations, pharmacological candidates and relevance of zebrafish neurophenomics to other 'omics' (e.g., genomic, proteomic) approaches. Finally, we critically evaluate the limitations of utilizing this model organism, and outline future strategies of research in the field of zebrafish phenomics.

Improving treatment of neurodevelopmental disorders: recommendations based on preclinical studies.

INTRODUCTION: Neurodevelopmental disorders (NDDs) are common and severely debilitating. Their chronic nature and reliance on both genetic and environmental factors makes studying NDDs and their treatment a challenging task. AREAS COVERED: Herein, the authors discuss the neurobiological mechanisms of NDDs, and present recommendations on their translational research and therapy, outlined by the International Stress and Behavior Society. Various drugs currently prescribed to treat NDDs also represent a highly diverse group. Acting on various neurotransmitter and physiological systems, these drugs often lack specificity of action, and are commonly used to treat multiple other psychiatric conditions. There has also been relatively little progress in the development of novel medications to treat NDDs. Based on clinical, preclinical and translational models of NDDs, our recommendations cover a wide range of methodological approaches and conceptual strategies. EXPERT OPINION: To improve pharmacotherapy and drug discovery for NDDs, we need a stronger emphasis on targeting multiple endophenotypes, a better dissection of genetic/epigenetic factors or "hidden heritability," and a careful consideration of potential developmental/trophic roles of brain neurotransmitters. The validity of animal NDD models can be improved through discovery of novel (behavioral, physiological and neuroimaging) biomarkers, applying proper environmental enrichment, widening the spectrum of model organisms, targeting developmental trajectories of NDD-related behaviors and comorbid conditions beyond traditional NDDs. While these recommendations cannot be addressed all in once, our increased understanding of NDD pathobiology may trigger innovative cross-disciplinary research expanding beyond traditional methods and concepts.

Effects of LSD on grooming behavior in serotonin transporter heterozygous (Sert⁺/⁻) mice.

Serotonin (5-HT) plays a crucial role in the brain, modulating mood, cognition and reward. The serotonin transporter (SERT) is responsible for the reuptake of 5-HT from the synaptic cleft and regulates serotonin signaling in the brain. In humans, SERT genetic variance is linked to the pathogenesis of various psychiatric disorders, including anxiety, autism spectrum disorders (ASD) and obsessive-compulsive disorder (OCD). Rodent self-grooming is a complex, evolutionarily conserved patterned behavior relevant to stress, ASD and OCD. Genetic ablation of mouse Sert causes various behavioral deficits, including increased anxiety and grooming behavior. The hallucinogenic drug lysergic acid diethylamide (LSD) is a potent serotonergic agonist known to modulate human and animal behavior. Here, we examined heterozygous Sert(+/-) mouse behavior following acute administration of LSD (0.32 mg/kg). Overall, Sert(+/-) mice displayed a longer duration of self-grooming behavior regardless of LSD treatment. In contrast, LSD increased serotonin-sensitive behaviors, such as head twitching, tremors and backwards gait behaviors in both Sert(+/+) and Sert(+/-) mice. There were no significant interactions between LSD treatment and Sert gene dosage in any of the behavioral domains measured. These results suggest that Sert(+/-) mice may respond to the behavioral effects of LSD in a similar manner to wild-type mice.

Neurobiology of rodent self-grooming and its value for translational neuroscience.

Self-grooming is a complex innate behaviour with an evolutionarily conserved sequencing pattern and is one of the most frequently performed behavioural activities in rodents. In this Review, we discuss the neurobiology of rodent self-grooming, and we highlight studies of rodent models of neuropsychiatric disorders--including models of autism spectrum disorder and obsessive compulsive disorder--that have assessed self-grooming phenotypes. We suggest that rodent self-grooming may be a useful measure of repetitive behaviour in such models, and therefore of value to translational psychiatry. Assessment of rodent self-grooming may also be useful for understanding the neural circuits that are involved in complex sequential patterns of action.

Building Zebrafish Neurobehavioral Phenomics: Effects of Common Environmental Factors on Anxiety and Locomotor Activity.

Zebrafish are emerging as an important model organism for neurobehavioral phenomics research. Given the likely variation of zebrafish behavioral phenotypes between and within laboratories, in this study, we examine the influence and variability of several common environmental modifiers on adult zebrafish anxiety and locomotor activity. Utilizing the novel tank paradigm, this study assessed the role of various laboratory factors, including experimenter/handling, testing time and days, batch, and the order of testing, on the behavior of a large population of experimentally naive control fish. Although time of the day, experimenter identity, and order of testing had little effect on zebrafish anxiety and locomotor activity levels, subtle differences were found for testing days and batches. Our study establishes how zebrafish behaviors are modulated by common environmental/laboratory factors and outlines several implications for zebrafish neurobehavioral phenomics research.

A novel 3D method of locomotor analysis in adult zebrafish: Implications for automated detection of CNS drug-evoked phenotypes.

BACKGROUND: Expanding the spectrum of organisms to model human brain phenotypes is critical for our improved understanding of the pathobiology of neuropsychiatric disorders. Given the clear limitations of existing mammalian models, there is an urgent need for low-cost, high-throughput in-vivo technologies for drug and gene discovery. NEW METHOD: Here, we introduce a new automated method for generating 3D (X,Y,Z) swim trajectories in adult zebrafish (Danio rerio), to improve their neurophenotyping. RESULTS: Based on the Track3D module of EthoVision XT video tracking software (Noldus Information Technology), this tool enhances the efficient, high-throughput 3D analyses of zebrafish behavioral responses. Applied to adult zebrafish behavior, this 3D method is highly sensitive to various classes of psychotropic drugs, including selected psychostimulant and hallucinogenic agents. COMPARISON WITH EXISTING METHODS: Our present method offers a marked advance in the existing 2D and 3D methods of zebrafish behavioral phenotyping, minimizing research time and recording high-resolution, automatically synchronized videos with calculated, high-precision object positioning. CONCLUSIONS: Our novel approach brings practical simplicity and 'integrative' capacity to the often complex and error-prone quantification of zebrafish behavioral phenotypes. Illustrating the value of 3D swim path reconstructions for identifying experimentally-evoked phenotypic profiles, this method fosters innovative, ethologically relevant, and fully automated small molecule screens using adult zebrafish.

Understanding the genetic architectonics of complex CNS traits: Lost by the association, but found in the interaction?

Recent evidence supports the value of endophenotypes and genome-wide association studies in psychiatric genetics, and their importance for dissecting the neural pathways and molecular mechanisms of complex neuropsychiatric disorders. Continuing this important discussion, here we outline three new mechanisms by which novel classes of genes may facilitate CNS pathogenesis without directly worsening its individual 'established' endophenotypes. These putative genetic mechanisms can apply to other human disorders in general, and may also be used for designing novel effective CNS drug treatments.

Targeting drug sensitivity predictors: New potential strategies to improve pharmacotherapy of human brain disorders.

One of the main challenges in medicine is the lack of efficient drug therapies for common human disorders. For example, although depressed patients receive powerful antidepressants, many often remain resistant to psychopharmacotherapy. The growing recognition of complex interplay between the drug targets and the predictors of drug sensitivity requires an improved understanding of these two key aspects of drug action and their potentially shared molecular networks. Here, we apply the concept of endophenotypes and their interplay to drug action and sensitivity. Based on these analyses, we postulate that novel drugs may be developed by targeting specific molecular pathways that integrate drug targets with drug sensitivity predictors.

Developing highER-throughput zebrafish screens for in-vivo CNS drug discovery.

The high prevalence of brain disorders and the lack of their efficient treatments necessitate improved in-vivo pre-clinical models and tests. The zebrafish (Danio rerio), a vertebrate species with high genetic and physiological homology to humans, is an excellent organism for innovative central nervous system (CNS) drug discovery and small molecule screening. Here, we outline new strategies for developing higher-throughput zebrafish screens to test neuroactive drugs and predict their pharmacological mechanisms. With the growing application of automated 3D phenotyping, machine learning algorithms, movement pattern- and behavior recognition, and multi-animal video-tracking, zebrafish screens are expected to markedly improve CNS drug discovery.

The failure of anxiolytic therapies in early clinical trials: what needs to be done.

INTRODUCTION: Anxiety spectrum disorders (ASDs) are highly prevalent psychiatric illnesses that affect millions of people worldwide. Strongly associated with stress, common ASDs include generalized anxiety disorder, panic, social anxiety, phobias and drug-abuse-related anxiety. In addition to ASDs, several other prevalent psychiatric illnesses represent trauma/stressor-related disorders, such as post-traumatic stress disorder and acute stress disorder. Anxiolytic drugs, commonly prescribed to treat ASDs and trauma/stressor-related disorders, form a highly heterogenous group, modulating multiple neurotransmitters and physiological mechanisms. However, overt individual differences in efficacy and the potential for serious side-effects (including addiction and drug interaction) indicate a need for further drug development. Yet, over the past 50 years, there has been relatively little progress in the development of novel anxiolytic medications, especially when promising candidate drugs often fail in early clinical trials. AREAS COVERED: Herein, the authors present recommendations of the Task Force on Anxiolytic Drugs of the International Stress and Behavior Society on how to improve anxiolytic drug discovery. These recommendations cover a wide spectrum of aspects, ranging from methodological improvements to conceptual insights and innovation. EXPERT OPINION: In order to improve the success of anxiolytic drugs in early clinical trials, the goals of preclinical trials may need to be adjusted from a clinical perspective and better synchronized with those of clinical studies. Indeed, it is important to realize that the strategic goals and approaches must be similar if we want to have a smoother transition between phases.

Targeting dynamic interplay among disordered domains or endophenotypes to understand complex neuropsychiatric disorders: Translational lessons from preclinical models.

Contemporary biological psychiatry uses clinical and experimental (animal) models to increase our understanding of brain pathogenesis. Modeling psychiatric disorders is currently performed by targeting various key neurobehavioral clusters of phenotypic traits (domains), including affective, cognitive, social, motor and reward. Analyses of such domains and their 'smaller units' - individual endophenotypes - are critical for the study of complex brain disorders and their neural underpinnings. The spectrum nature of brain disorders and the importance of pathogenetic linkage among various disordered domains or endophenotypes have also been recognized as an important strategic direction of translational research. Here, we discuss cross-domain analyses of animal models, and focus on their value for mimicking the clinical overlap between disordered neurobehavioral domains in humans. Based on recent experimental evidence, we argue that understanding of brain pathogenesis requires modeling the clinically relevant inter-relationships between various individual endophenotypes (or their domains).

Anxiogenic-like effects of chronic nicotine exposure in zebrafish.

Nicotine is one of the most widely used and abused legal drugs. Although its pharmacological profile has been extensively investigated in humans and rodents, nicotine CNS action remains poorly understood. The importance of finding evolutionarily conserved signaling pathways, and the need to apply high-throughput in vivo screens for CNS drug discovery, necessitate novel efficient experimental models for nicotine research. Zebrafish (Danio rerio) are rapidly emerging as an excellent organism for studying drug abuse, neuropharmacology and toxicology and have recently been applied to testing nicotine. Anxiolytic, rewarding and memory-modulating effects of acute nicotine treatment in zebrafish are consistently reported in the literature. However, while nicotine abuse is more relevant to long-term exposure models, little is known about chronic effects of nicotine on zebrafish behavior. In the present study, chronic 4-day exposure to 1-2mg/L nicotine mildly increased adult zebrafish shoaling but did not alter baseline cortisol levels. We also found that chronic exposure to nicotine evokes robust anxiogenic behavioral responses in zebrafish tested in the novel tank test paradigm. Generally paralleling clinical and rodent data on anxiogenic effects of chronic nicotine, our study supports the developing utility of zebrafish for nicotine research.

Acoustic features of prairie vole (Microtus ochrogaster) ultrasonic vocalizations covary with heart rate.

Vocalizations serve as a conspecific social communication system among mammals. Modulation of acoustic features embedded within vocalizations is used by several mammalian species to signal whether it is safe or dangerous to approach conspecific and heterospecific mammals. As described by the Polyvagal Theory, the phylogenetic shift in the evolution of mammals involved an adaptive neuroanatomical link between the neural circuits regulating heart rate and the muscles involved in modulating the acoustic features of vocalizations. However, few studies have investigated the covariation between heart rate and the acoustic features of vocalizations. In the current study, we document that specific features of vocalizations covary with heart rate in a highly social and vocal mammal, the prairie vole (Microtus ochrogaster). Findings with the prairie vole illustrate that higher pitch (i.e., fundamental frequency) and less variability in acoustic features of vocalizations (i.e., less vocal prosody) are associated with elevated heart rate. The study provides the first documentation that the acoustic features of prairie vole vocalizations may function as a surrogate index of heart rate.

Cytokine and endocrine parameters in mouse chronic social defeat: implications for translational 'cross-domain' modeling of stress-related brain disorders.

Mounting clinical and experimental evidence implicates various cytokines in stress-related affective brain disorders. Here, we analyze behavioral phenotypes in C57BL/6J male mice following the chronic social defeat stress paradigm, and examine their serum cytokines and corticosterone levels. Loser mice experiencing 20 days of daily 15-min social confrontations demonstrate elevated levels of pro-inflammatory cytokines interleukin IL-7 and vascular endothelial growth factor (VEGF), as well as a trend to increase IL-6 and IL-15. We also found higher levels of an anti-inflammatory cytokine IL-10 in the winner mice, with unaltered serum IL-2, IL-4, IL-1a, MCP-1 and corticosterone levels between the groups. Overall, our results suggest that animal affective-like states correlate with specific cytokine profiles, including some cytokines (e.g., VEGF, IL-7 or IL-15) whose role in neuropsychiatric disorders is only beginning to emerge. This study emphasizes the importance of integrative analyses of neural and immune phenotypes in stress and stress-related neurobehavioral disorders. These findings may also help foster the search for new therapeutic and preventative strategies that target selected cytokines and their signaling pathways.

Developing better and more valid animal models of brain disorders.

Valid sensitive animal models are crucial for understanding the pathobiology of complex human disorders, such as anxiety, autism, depression and schizophrenia, which all have the 'spectrum' nature. Discussing new important strategic directions of research in this field, here we focus i) on cross-species validation of animal models, ii) ensuring their population (external) validity, and iii) the need to target the interplay between multiple disordered domains. We note that optimal animal models of brain disorders should target evolutionary conserved 'core' traits/domains and specifically mimic the clinically relevant inter-relationships between these domains.

Aquatic toxicology of fluoxetine: understanding the knowns and the unknowns.

Fluoxetine is one of the most prescribed psychotropic medications, and is an agent of increasing interest for environmental toxicology. Fish and other aquatic organisms are excellent models to study neuroactive small molecules like fluoxetine. However, prone to variance due to experimental factors, data obtained in these models need to be interpreted with caution, using proper experimental protocols, study designs, validated endpoints as well as well-established models and tests. Choosing the treatment protocol and dose range for fluoxetine and other serotonergic drugs is critical for obtaining valid test results and correct data interpretation. Here we discuss the value of aquatic models to study fluoxetine effects, based on prior high-quality research, and outline the directions of future translational studies in the field. We review fluoxetine-evoked phenotypes in acute vs. chronic protocols, discussing them in the contact of complex role of serotonin in behavioral regulation. We conclude that zebrafish and other aquatic models represent a useful in-vivo tool for fluoxetine pharmacology and (eco)toxicology research.

Developing zebrafish models relevant to PTSD and other trauma- and stressor-related disorders.

While post-traumatic stress disorder (PTSD) and other trauma- and stress-related disorders (TSRDs) represent a serious societal and public health concern, their pathogenesis is largely unknown. Given the clinical complexity of TSRD development and susceptibility, greater investigation into candidate biomarkers and specific genetic pathways implicated in both risk and resilience to trauma becomes critical. In line with this, numerous animal models have been extensively used to better understand the pathogenic mechanisms of PTSD and related TSRD. Here, we discuss the rapidly increasing potential of zebrafish as models of these disorders, and how their use may aid researchers in uncovering novel treatments and therapies in this field.

Zebrafish models for translational neuroscience research: from tank to bedside.

The zebrafish (Danio rerio) is emerging as a new important species for studying mechanisms of brain function and dysfunction. Focusing on selected central nervous system (CNS) disorders (brain cancer, epilepsy, and anxiety) and using them as examples, we discuss the value of zebrafish models in translational neuroscience. We further evaluate the contribution of zebrafish to neuroimaging, circuit level, and drug discovery research. Outlining the role of zebrafish in modeling a wide range of human brain disorders, we also summarize recent applications and existing challenges in this field. Finally, we emphasize the potential of zebrafish models in behavioral phenomics and high-throughput genetic/small molecule screening, which is critical for CNS drug discovery and identifying novel candidate genes.