Developing Peripheral Biochemical Biomarkers of Brain Disorders: Insights from Zebrafish Models.

High prevalence of human brain disorders necessitates development of the reliable peripheral biomarkers as diagnostic and disease-monitoring tools. In addition to clinical studies, animal models markedly advance studying of non-brain abnormalities associated with brain pathogenesis. The zebrafish (Danio rerio) is becoming increasingly popular as an animal model organism in translational neuroscience. These fish share some practical advantages over mammalian models together with high genetic homology and evolutionarily conserved biochemical and neurobehavioral phenotypes, thus enabling large-scale modeling of human brain diseases. Here, we review mounting evidence on peripheral biomarkers of brain disorders in zebrafish models, focusing on altered biochemistry (lipids, carbohydrates, proteins, and other non-signal molecules, as well as metabolic reactions and activity of enzymes). Collectively, these data strongly support the utility of zebrafish (from a systems biology standpoint) to study peripheral manifestations of brain disorders, as well as highlight potential applications of biochemical biomarkers in zebrafish models to biomarker-based drug discovery and development.

Psychopharmacological characterization of an emerging drug of abuse, a synthetic opioid U-47700, in adult zebrafish.

3,4-Dichloro-N-[2-(dimethylamino)cyclohexyl]-N-methylbenzamide (U-47700) is a selective µ-opioid receptor agonist originally synthesized as a prospective analgesic drug. Several times more potent than morphine, U-47700 has high abuse potential and may cause clinical neurotoxicity, euphoria, respiratory depression and occasional mortality. U-47700 also evokes analgesia, sedation and euphoria-like states in both humans and rodents. Despite the growing use and abuse of U-47700, its psychopharmacological and toxicological profiles in vivo remain poorly understood. The zebrafish (Danio rerio) is rapidly becoming a popular aquatic model organism for central nervous system (CNS) disease modeling and drug discovery. Here, we examine acute (1, 5, 10, 25 and 50 mg/L for 20-min) and chronic (0.1, 0.5 and 1 mg/L for 14 days) effects of U-47700 in adult zebrafish. Overall, we found overt sedation evoked in fish by acute, and hyperlocomotion with an anxiolytic-like action by chronic, drug treatments. Acute treatment with 1 and 10 mg/L U-47700 also resulted in detectable amounts of this drug in the brain samples, supporting its permeability through the blood-brain barrier. Collectively, these findings emphasize complex dose- and treatment-dependent CNS effects of U-47700 following its acute and chronic administration. Our study also supports high sensitivity of zebrafish to U-47700, and suggests these aquatic models as promising in-vivo screens for probing potential CNS effects evoked by novel synthetic opioid drugs.

Decoding the role of zebrafish neuroglia in CNS disease modeling.

Neuroglia, including microglia and astrocytes, is a critical component of the central nervous system (CNS) that interacts with neurons to modulate brain activity, development, metabolism and signaling pathways. Thus, a better understanding of the role of neuroglia in the brain is critical. Complementing clinical and rodent data, the zebrafish (Danio rerio) is rapidly becoming an important model organism to probe the role of neuroglia in brain disorders. With high genetic and physiological similarity to humans and rodents, zebrafish possess some common (shared), as well as some specific molecular biomarkers and features of neuroglia development and functioning. Studying these common and zebrafish-specific aspects of neuroglia may generate important insights into key brain mechanisms, including neurodevelopmental, neurodegenerative, neuroregenerative and neurological processes. Here, we discuss the biology of neuroglia in humans, rodents and fish, its role in various CNS functions, and further directions of translational research into the role of neuroglia in CNS disorders using zebrafish models.

Abnormal repetitive behaviors in zebrafish and their relevance to human brain disorders.

Abnormal repetitive behaviors (ARBs) are a prominent symptom of numerous human brain disorders and are commonly seen in rodent models as well. While rodent studies of ARBs continue to dominate the field, mounting evidence suggests that zebrafish (Danio rerio) also display ARB-like phenotypes and may therefore be a novel model organism for ARB research. In addition to clear practical research advantages as a model species, zebrafish share high genetic and physiological homology to humans and rodents, including multiple ARB-related genes and robust behaviors relevant to ARB. Here, we discuss a wide spectrum of stereotypic repetitive behaviors in zebrafish, data on their genetic and pharmacological modulation, and the overall translational relevance of fish ARBs to modeling human brain disorders. Overall, the zebrafish is rapidly emerging as a new promising model to study ARBs and their underlying mechanisms.

When fish take a bath: Psychopharmacological characterization of the effects of a synthetic cathinone bath salt 'flakka' on adult zebrafish.

Alpha-pyrrolidinopentiophenone (α-PVP) is a synthetic cathinone which exerts robust mental and physiological effects clinically, as well as causes aberrant stereotypic behaviors and altered locomotion in rodents. Given the rich spectrum of pharmacological activity of α-PVP in rodents and humans, as well as its high abuse potential, further studies are needed to better understand the pharmacology and toxicology of this drug. The zebrafish (Danio rerio) is a relatively novel model organism in neuropharmacology and toxicology research. Here, we characterize behavioral effects of α-PVP in adult zebrafish following its acute (1, 5, 25 and 50 mg/L for 20 min) and chronic (1, 5 and 10 mg/L for 7 days) treatments. Overall, acute exposure to α-PVP evoked psychostimulant (but not anxiolytic-like) effects in zebrafish novel tank test, with characteristic stereotypic 'side-to-side' bottom swimming at 5, 25 and 50 mg/L. The high-performance liquid chromatography/high-resolution mass spectrometry (HPLC/HRMS) analyses of zebrafish brains showed detectable levels of α-PVP following its acute administration, likely underlying the observed behavioral effects. Although acute 2-day discontinuation of chronic 7-day α-PVP at 1, 5 and 10 mg/L produced no effects, hypolocomotion occurred after a 7-day chronic treatment and repeated withdrawal, resembling rodent effects of some chronic psychostimulants. Collectively, these findings support zebrafish sensitivity to α-PVP and show some parallels with its effects in mammals and humans. This study also suggests that aquatic models based on zebrafish can help further examine the CNS effects evoked by α-PVP and screen for related synthetic new psychoactive drugs.

DARK Classics in Chemical Neuroscience: α-Pyrrolidinovalerophenone ("Flakka").

Flakka (alpha-pyrrolidinovalerophenone, α-PVP) is a new psychoactive substance, chemically close to cathinone, the primary psychoactive alkaloid of khat ( Catha edulis). Like other synthetic cathinones, α-PVP is a potent inhibitor of the dopamine and norepinephrine transporters. Its robust clinical effects include hallucinations, arousal, aggression/violence, and euphoria. In animal models, α-PVP evokes hyperlocomotion and aberrant/stereotypic behaviors. Here, we discuss the history, synthesis, pharmacological mechanisms, metabolism, abuse potential, and societal impact of α-PVP. Today, α-PVP is a tightly controlled substance, currently banned in the United States and other countries worldwide. However, the growing abuse and complex central nervous system (CNS) effects of α-PVP remain poorly understood, necessitating further pharmacological and pharmacogenetic studies of this drug. Its interesting pharmacological profile (co-inhibition of dopamine and norepinephrine, but not serotonin, transporters) also calls for further studies of α-PVP in animal models, to dissect serotonergic from other monoaminergic mechanisms of action of drugs of abuse. Finally, screening α-PVP and related compounds in vivo may foster discovery of new CNS drugs, including developing novel CNS drugs and identifying their molecular targets.

Understanding zebrafish aggressive behavior.

Aggression is a common agonistic behavior affecting social life and well-being of humans and animals. However, the underlying mechanisms of aggression remain poorly understood. For decades, studies of aggression have mostly focused on laboratory rodents. The growing importance of evolutionarily relevant, cross-species disease modeling necessitates novel model organisms to study aggression and its pathobiology. The zebrafish (Danio rerio) is rapidly becoming a new experimental model organism in neurobehavioral research. Zebrafish demonstrate high genetic and physiological homology with mammals, fully sequenced genome, ease of husbandry and testing, as well as rich, robust behavioral repertoire. As zebrafish present overt aggressive behaviors, here we focus on their behavioral models and discuss their utility in probing aggression neurobiology and its genetic, pharmacological and environmental modulation. We argue that zebrafish-based models represent an excellent translational tool to understand aggressive behaviors and related pathobiological brain mechanisms.

DARK Classics in Chemical Neuroscience: Atropine, Scopolamine, and Other Anticholinergic Deliriant Hallucinogens.

Anticholinergic drugs based on tropane alkaloids, including atropine, scopolamine, and hyoscyamine, have been used for various medicinal and toxic purposes for millennia. These drugs are competitive antagonists of acetylcholine muscarinic (M-) receptors that potently modulate the central nervous system (CNS). Currently used clinically to treat vomiting, nausea, and bradycardia, as well as alongside other anesthetics to avoid vagal inhibition, these drugs also evoke potent psychotropic effects, including characteristic delirium-like states with hallucinations, altered mood, and cognitive deficits. Given the growing clinical importance of anti-M deliriant hallucinogens, here we discuss their use and abuse, clinical importance, and the growing value in preclinical (experimental) animal models relevant to modeling CNS functions and dysfunctions.

The Effects of Chronic Amitriptyline on Zebrafish Behavior and Monoamine Neurochemistry.

Amitriptyline is a commonly used tricyclic antidepressant (TCA) inhibiting serotonin and norepinephrine reuptake. The exact CNS action of TCAs remains poorly understood, necessitating new screening approaches and novel model organisms. Zebrafish (Danio rerio) are rapidly emerging as a promising tool for pharmacological research of antidepressants, including amitriptyline. Here, we examine the effects of chronic 2-week exposure to 10 and 50 µg/L amitriptyline on zebrafish behavior and monoamine neurotransmitters. Overall, the drug at 50 µg/L evoked pronounced anxiolytic-like effects in the novel tank test (assessed by more time in top, fewer transition and shorter latency to enter the top). Like other TCAs, amitriptyline reduced serotonin turnover, but also significantly elevated whole-brain norepinephrine and dopamine levels. The latter effect was not reported in this model previously, and accompanied higher brain expression of tyrosine hydroxylase (a rate-limiting enzyme of catecholamine biosynthesis), but unaltered expression of dopamine-ß-hydroxylase and monoamine oxidase (the enzymes of dopamine metabolism). This response may underlie chronic amitriptyline action on dopamine and norepinephrine neurotransmission, and contribute to the complex CNS profile of this drug observed both clinically and in animal models. Collectively, these findings also confirm the important role of monoamine modulation in the regulation of anxiety-related behavior in zebrafish, and support the utility of this organism as a promising in-vivo model for CNS drug screening.

Understanding antidepressant discontinuation syndrome (ADS) through preclinical experimental models.

Antidepressant drugs are currently one of the most prescribed medications. In addition to treatment resistance and side effects of antidepressants, their clinical use is further complicated by antidepressant discontinuation syndrome (ADS). ADS is a common problem in patients following the interruption, dose reduction, or discontinuation of antidepressant drugs. Clinically, ADS resembles a classical drug withdrawal syndrome, albeit differing from it because antidepressants generally do not induce addiction. The growing clinical importance and prevalence of ADS necessitate novel experimental (animal) models of this disorder. Currently available preclinical models of ADS are mainly rodent-based, and study mostly serotonergic antidepressants and their combinations. Here, we systematically assess clinical ADS symptoms and discuss current trends and challenges in the field of experimental (animal) models of ADS. We also outline basic mechanisms underlying ADS pathobiology, evaluate its genetic, pharmacological and environmental determinants, and emphasize how using animal models may help generate important translational insights into human ADS condition, its prevention and therapy.

Animal inflammation-based models of depression and their application to drug discovery.

INTRODUCTION: Depression, anxiety and other affective disorders are globally widespread and severely debilitating human brain diseases. Despite their high prevalence and mental health impact, affective pathogenesis is poorly understood, and often remains recurrent and resistant to treatment. The lack of efficient antidepressants and presently limited conceptual innovation necessitate novel approaches and new drug targets in the field of antidepressant therapy. Areas covered: Herein, the authors discuss the emerging role of neuro-immune interactions in affective pathogenesis, which can become useful targets for CNS drug discovery, including modulating neuroinflammatory pathways to alleviate affective pathogenesis. Expert opinion: Mounting evidence implicates microglia, polyunsaturated fatty acids (PUFAs), glucocorticoids and gut microbiota in both inflammation and depression. It is suggested that novel antidepressants can be developed based on targeting microglia-, PUFAs-, glucocorticoid- and gut microbiota-mediated cellular pathways. In addition, the authors call for a wider application of novel model organisms, such as zebrafish, in studying shared, evolutionarily conserved (and therefore, core) neuro-immune mechanisms of depression.

Acute effects of amitriptyline on adult zebrafish: Potential relevance to antidepressant drug screening and modeling human toxidromes.

The need to develop novel antidepressants is an emerging problem in biomedicine. An aquatic vertebrate species, the zebrafish (Danio rerio) may serve as a useful in-vivo screen for CNS drugs, and displays high sensitivity to a wide range of antidepressants. Amitriptyline is a commonly used tricyclic antidepressant which acts primarily as a serotonin and noradrenaline reuptake inhibitor. Here, we characterize drug-induced behavioral and neurochemical responses in adult zebrafish following their acute exposure to amitriptyline. Overall, the drug at 1 and 5mg/L significantly increased time spent in top and shortened the latency to enter it, thereby paralleling recent reports on zebrafish 'serotonin toxicity-like behavior' caused by various serotonergic agents. The 10mg/L dose of the drug also significantly decreased top entries and maximal velocity and evoked overt ataxia, likely due to emerging non-specific toxic effects. Amitriptyline at 5 and 10mg/L also dose-dependently increased serotonin turnover, but not noradrenaline levels, in zebrafish whole-brain samples. Overall, zebrafish high sensitivity to acute effects of amitriptyline can help improve our understanding of psychopharmacological profiles of this compound and the related CNS drugs, and contributes further to the development of aquatic experimental models of human toxidromes.

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.

Effects of a non-competitive N-methyl-d-aspartate (NMDA) antagonist, tiletamine, in adult zebrafish.

Tiletamine is a non-competitive N-methyl-d-aspartate (NMDA) receptor antagonist chemically related to ketamine and phencyclidine. A common veterinary anesthetic drug, tiletamine is currently a Schedule III controlled substance in USA. This compound exerts sedative effects in humans and animals, also having an abuse potential, toxicity and dissociative hallucinogenic properties clinically. However, the neurotropic profile of tiletamine remains poorly understood, necessitating novel models and in-vivo screens, including non-mammalian species. Zebrafish (Danio rerio) are rapidly becoming a popular model organism for screening various CNS drugs, including those acting at NMDA receptors. Here, we investigated acute behavioral effects of 1, 5 and 10mg/L of tiletamine on adult zebrafish. In the standard novel tank test, a 20-min immersion in 1mg/L of tiletamine produced no overt differences from control zebrafish (receiving 0.1% DMSO vehicle), except for reduced top entries. In contrast, tiletamine at 5 and 10mg/L exerted robust dose-dependent sedative effects in zebrafish (also darkening their skin coloration, similar to ketamine and PCP). Gas chromatography/mass spectrometry (GC/MS) analyses revealed no tiletamine peaks in control and 1mg/L groups, but detected tiletamine peaks in zebrafish brain samples at 5 and 10mg/L. Together, these findings demonstrate potent neurotropic effects of tiletamine in zebrafish, and their high sensitivity to this drug. Our findings also support the growing utility of fish-based aquatic screens for studying neuroactive properties of NMDA antagonists in-vivo.