Legal aspects of zebrafish neuropharmacology and neurotoxicology research.

Despite the growing emphasis on translational neuropharmacology and drug discovery research, the legality underlying these fields are seldom considered. The zebrafish (Danio rerio) is an increasingly utilized model organism in neuropharmacology and neurotoxicology. As the acceptance of zebrafish in biomedicine continues to grow, the legal aspects of their applications remain outpaced by this exponential growth. Therefore, there is a need to evaluate the legal aspects of zebrafish applications to CNS drug research. Here, we discuss a wide range of regulatory topics relevant to zebrafish research, such as the bioethics of experimentation (including studies of stress and pain), welfare protection laws, the recent advances in CNS drug discovery, and specific legal aspects of controlled substance research in this aquatic species. The conceptualization and understanding of the zebrafish welfare and its promise as a model in toxicology can also potentially shape environmental protection practices and inform policy making.

Zebrafish models of autism spectrum disorder.

Autism spectrum disorder (ASD) is a complex neurodevelopmental disorder characterized by motor, social and cognitive deficits that develop early during childhood. The pathogenesis of ASD is not well characterized and involves a multifaceted interaction between genetic, neurobiological and environmental factors. Animal (experimental) models possess evolutionarily conserved behaviors and molecular pathways that are highly relevant for studying ASD. The zebrafish (Danio rerio) is a relatively new animal model with promise for understanding the pathogenesis of complex brain disorders and discovering novel treatments. As a highly social and genetically tractable organism, zebrafish have recently been applied to model a variety of deficits relevant to ASD. Here, we discuss the developing utility of zebrafish models of ASD, as well as current behavioral, toxicological and genetic models of ASD, and future directions of research in this field.

Modeling consequences of prolonged strong unpredictable stress in zebrafish: Complex effects on behavior and physiology.

Chronic stress is the major pathogenetic factor of human anxiety and depression. Zebrafish (Danio rerio) have become a novel popular model species for neuroscience research and CNS drug discovery. The utility of zebrafish for mimicking human affective disorders is also rapidly growing. Here, we present a new zebrafish model of clinically relevant, prolonged unpredictable strong chronic stress (PUCS). The 5-week PUCS induced overt anxiety-like and motor retardation-like behaviors in adult zebrafish, also elevating whole-body cortisol and proinflammatory cytokines - interleukins IL-1ß and IL-6. PUCS also elevated whole-body levels of the anti-inflammatory cytokine IL-10 and increased the density of dendritic spines in zebrafish telencephalic neurons. Chronic treatment of fish with an antidepressant fluoxetine (0.1mg/L for 8days) normalized their behavioral and endocrine phenotypes, as well as corrected stress-elevated IL-1ß and IL-6 levels, similar to clinical and rodent data. The CNS expression of the bdnf gene, the two genes of its receptors (trkB, p75), and the gfap gene of glia biomarker, the glial fibrillary acidic protein, was unaltered in all three groups. However, PUCS elevated whole-body BDNF levels and the telencephalic dendritic spine density (which were corrected by fluoxetine), thereby somewhat differing from the effects of chronic stress in rodents. Together, these findings support zebrafish as a useful in-vivo model of chronic stress, also calling for further cross-species studies of both shared/overlapping and distinct neurobiological responses to chronic stress.

Comparative Analyses of Zebrafish Anxiety-Like Behavior Using Conflict-Based Novelty Tests.

Modeling of stress and anxiety in adult zebrafish (Danio rerio) is increasingly utilized in neuroscience research and central nervous system (CNS) drug discovery. Representing the most commonly used zebrafish anxiety models, the novel tank test (NTT) focuses on zebrafish diving in response to potentially threatening stimuli, whereas the light-dark test (LDT) is based on fish scototaxis (innate preference for dark vs. bright areas). Here, we systematically evaluate the utility of these two tests, combining meta-analyses of published literature with comparative in vivo behavioral and whole-body endocrine (cortisol) testing. Overall, the NTT and LDT behaviors demonstrate a generally good cross-test correlation in vivo, whereas meta-analyses of published literature show that both tests have similar sensitivity to zebrafish anxiety-like states. Finally, NTT evokes higher levels of cortisol, likely representing a more stressful procedure than LDT. Collectively, our study reappraises NTT and LDT for studying anxiety-like states in zebrafish, and emphasizes their developing utility for neurobehavioral research. These findings can help optimize drug screening procedures by choosing more appropriate models for testing anxiolytic or anxiogenic drugs.

Zebrafish models in neuropsychopharmacology and CNS drug discovery.

Despite the high prevalence of neuropsychiatric disorders, their aetiology and molecular mechanisms remain poorly understood. The zebrafish (Danio rerio) is increasingly utilized as a powerful animal model in neuropharmacology research and in vivo drug screening. Collectively, this makes zebrafish a useful tool for drug discovery and the identification of disordered molecular pathways. Here, we discuss zebrafish models of selected human neuropsychiatric disorders and drug-induced phenotypes. As well as covering a broad range of brain disorders (from anxiety and psychoses to neurodegeneration), we also summarize recent developments in zebrafish genetics and small molecule screening, which markedly enhance the disease modelling and the discovery of novel drug targets.

Alzheimer's disease in the zebrafish: where can we take it?

With the ever-growing geriatric population, research on brain diseases such as dementia is more imperative now than ever. The most prevalent of all dementias is Alzheimer's disease, a progressive neurodegenerative disease that presents with deficits in memory, cognition, motor skills, and a general decline in the quality of life. The social and economic burden associated with Alzheimer's disease is tremendous and is projected to grow even greater over the coming years. There is a specific need to elucidate and improve the treatments available, not only to alleviate the symptoms related to dementias such as Alzheimer's but also to prevent the formation of the disease. This is an effort that can be expedited and made more efficient by utilizing an animal model such as the zebrafish. This paper reviews the utility of zebrafish in Alzheimer's research by examining research on a sampling of the treatments available for the disease, specifically donepezil, memantine, and methylene blue. The human model and the shortcomings of the rodent model are also discussed.

Understanding zebrafish cognition.

Zebrafish (Danio rerio) are rapidly becoming a popular model organism in translational and cognitive neuroscience research. Both larval and adult zebrafish continue to increase our understanding of cognitive mechanisms and their genetic and pharmacological modulation. Here, we discuss the developing utility of zebrafish in understanding cognitive phenotypes and their deficits, relevant to a wide range human brain disorders. We also discuss the potential of zebrafish models for high-throughput genetic mutant and small molecule screening (e.g., amnestics, cognitive enhancers, neurodevelopmental/neurodegenerative drugs), which becomes critical for identifying novel candidate genes and molecular drug targets to treat cognitive deficits. In addition to discussing the existing challenges and future strategic directions in this field, we emphasize how zebrafish models of cognitive phenotypes continue to form an interesting and rapidly emerging new field in neuroscience.

Methylene Blue Facilitates Memory Retention in Zebrafish in a Dose-Dependent Manner.

Methylene blue (MB) is an FDA-grandfathered drug with memory-enhancing effects at low doses, but opposite effects at high doses. We investigated the effects of four MB doses (0.1, 0.5, 5.0, or 10.0 µM) on zebrafish memory retention in the T-maze task. After training fish to swim into a certain arm of the T-maze, the fish were placed into a tank containing one of the four MB doses or a control tank containing blue food dye. Subsequently, fish were placed into the T-maze for memory retention testing. Results indicated that MB produced hormetic dose-response effects on memory. Fish that received the 0.5 µM dose performed significantly better at the T-maze than those that received higher doses. Fish who received 5.0 µM did not exhibit a significant difference in performance from control fish, and the fish that received the 10.0 µM dose performed significantly worse than lower doses. These findings support the utility of zebrafish in comparative research and their potential value for testing of MB and other neuropsychopharmacological treatments in animal models of memory disorders.

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.

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.

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.

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.

Modeling PTSD in the zebrafish: are we there yet?

Post-traumatic stress disorder is an anxiety disorder that can develop following one or more traumatic events that threaten one's safety or make the victim feel helpless. Currently there are an increasing number of cases in the population in part due to the number of soldiers returning from combat. The disorder is characterized by symptoms that include hypervigilance, sleep disturbances, social and cognitive degradation, and memory flashbacks. Most of the research has been centered on the human and rodent as subjects but recently another viable contender has emerged - the zebrafish (Danio rerio). The zebrafish is a strong comparative model with the ability to exhibit a wide variety of behaviors, complex learning, and neurobiological changes that can be extrapolated to the human condition. The zebrafish is an ideal organism to study pharmacological treatments as well as the neurological underpinnings of the disorder. Here we review a sampling of the human and rodent model literature on post-traumatic stress disorder focusing on symptomology, current treatments, and stress paradigms. We also make the argument for the inclusion of the zebrafish model in future studies investigating the causes, symptoms, and treatments of post-traumatic stress disorder.

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.

Zebrafish and conditioned place preference: a translational model of drug reward.

Addiction and substance abuse are found ubiquitously throughout human society. In the United States, these disorders are responsible for amassing hundreds of billions of dollars in annual costs associated with healthcare, crime and lost productivity. Efficacious treatments remain few in number, the development of which will be facilitated by comprehension of environmental, genetic, pharmacological and neurobiological mechanisms implicated in the pathogenesis of addiction. Animal models such as the zebrafish (Danio rerio) have gained momentum within various domains of translational research, and as a model of complex brain disorders (e.g., drug abuse). Behavioral quantification within the conditioned place preference (CPP) paradigm serves as a measure of the rewarding qualities of a given substance. If the animal develops an increase in preference for the drug paired environment, it is inferred that the drug has positive-reinforcing properties. This paper discusses the utility of the zebrafish model in conjunction with the CPP paradigm and reports CPP behavior following acute exposure to 0.0%, 0.25%, 0.50%, and 1.00% alcohol, and 0 mg/L, 50 mg/L, 100 mg/L and 150 mg/L caffeine.

Back to basics: searching for a comprehensive framework for exploring individual differences in zebrafish (Danio rerio) behavior.

Individual differences (IDs) in behavior among nonhuman animals have been documented in a wide range of taxa. Although traditionally considered noise around an average, other potentially adaptive sources of phenotypic variation exist. IDs in behavior that are consistent across time and context are more recently recognized as expressions of underlying personality traits, which may even be heritable. Unfortunately, despite the rapid advances that have been made in animal personality research utilizing fish the last decade, a few have detailed the groundwork necessary to document consistency in behavior across time and context. This foundation is required, by definition, before one can draw conclusions about personality traits. Here, we examine whether IDs in behavior are consistent over time and across contexts and explore the construct validity of six commonly used behavioral assays for examining four personality traits: aggression, boldness, fear, and exploration. Thirty zebrafish (Danio rerio) were exposed twice each to a small open field, large open field, mirror, emergence, novel object, and predator response test. Results revealed consistency in most behavioral measures across both time and context. There was mixed evidence for the construct validity of these assays in capturing the targeted personality traits.

Gaining translational momentum: more zebrafish models for neuroscience research.

Zebrafish (Danio rerio) are rapidly becoming a popular model organism in translational neuroscience and biological psychiatry research. Here we discuss conceptual, practical and other related aspects of using zebrafish in this field ("from tank to bedside"), and critically evaluate both advantages and limitations of zebrafish models of human brain disorders. We emphasize the need to more actively develop zebrafish models for neuroscience research focusing on complex traits.

The utility of the zebrafish model in conditioned place preference to assess the rewarding effects of drugs.

Substance abuse is a significant public health concern both domestically and worldwide. The persistent use of substances regardless of aversive consequences forces the user to give higher priority to the drug than to normal activities and obligations. The harmful and hazardous use of psychoactive substances can lead to a dependence syndrome. In this regard, the genetic and neurobiological underpinnings of reward-seeking behavior need to be fully understood in order to develop effective pharmacotherapies and other methods of treatment. Animal models are often implemented in preclinical screening for testing the efficacy of novel treatments. Several paradigms exist that model various facets of addiction including sensitization, tolerance, withdrawal, drug seeking, extinction, and relapse. Self-administration and, most notably, conditioned place preference (CPP) are relatively simple tests that serve as indicators of the aforementioned aspects of addiction by means of behavioral quantification. CPP is a commonly used technique to evaluate the motivational effects of compounds and experiences that have been associated with a positive or negative reward, which capitalizes on the basic principles of Pavlovian conditioning. During training, the unconditioned stimulus is consistently paired with a neutral set of environmental stimuli, which obtain, during conditioning, secondary motivational properties that elicit approach behavior in the absence of the unconditioned stimulus. For over 50 years, rodents have been the primary test subjects. However, the zebrafish (Danio rerio) is gaining favor as a valuable model organism in the fields of biology, genetics, and behavioral neuroscience. This paper presents a discussion on the merits, advantages, and limitations of the zebrafish model and its utility in relation to CPP.

Alcohol-induced behavior change in zebrafish models.

Zebrafish are at the forefront of neurobiological research and have been gaining popularity as a viable and valid behavioral model in a variety of research applications (e.g., assessing drug induced behavioral changes). This model becomes even more attractive when considering the behavioral changes that follow exposure to compounds that are water-soluble. As such, several studies have implicated both acute and chronic ethanol exposure in the modulation of zebrafish behavior. Within this arena there appears to be a common trend across multiple studies. As with many drugs ethanol appears to influence behavior in a dose-dependent manner. In this review, we compare and contrast several studies that measure behavior as a result of alcohol exposure. Appended to this review are pilot data that report zebrafish blood alcohol concentrations as a function of acute exposure.

Assessing attention in the zebrafish: Are we there yet?

Traditionally, rodent sustained attention models are used for studying the neurobiological underpinnings of attention, for assessing the disruptive and interactive effects of drugs and environmental toxins and for predicting the efficacy of pharmacotherapies for attention disorders. Virtually all-major psychiatric disorders are characterized by disturbances in attention or concentration. Additionally, many psychoactive drugs produce simultaneous effects on a variety of psychological processes. Behavioral measures in tasks designed to assess cognitive processes in rodents characterize and dissociate these multiple influences. While the zebrafish (Danio rerio) has been at the vanguard of neurobiological research and is increasing in popularity as a model organism for behavioral applications, their attentional capacity has not been fully assessed. Here we review some of the more popular animal models and discuss the utility of a choice discrimination zebrafish model.