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.

Forward Genetics-Based Approaches to Understanding the Systems Biology and Molecular Mechanisms of Epilepsy.

Epilepsy is a highly prevalent, severely debilitating neurological disorder characterized by seizures and neuronal hyperactivity due to an imbalanced neurotransmission. As genetic factors play a key role in epilepsy and its treatment, various genetic and genomic technologies continue to dissect the genetic causes of this disorder. However, the exact pathogenesis of epilepsy is not fully understood, necessitating further translational studies of this condition. Here, we applied a computational in silico approach to generate a comprehensive network of molecular pathways involved in epilepsy, based on known human candidate epilepsy genes and their established molecular interactors. Clustering the resulting network identified potential key interactors that may contribute to the development of epilepsy, and revealed functional molecular pathways associated with this disorder, including those related to neuronal hyperactivity, cytoskeletal and mitochondrial function, and metabolism. While traditional antiepileptic drugs often target single mechanisms associated with epilepsy, recent studies suggest targeting downstream pathways as an alternative efficient strategy. However, many potential downstream pathways have not yet been considered as promising targets for antiepileptic treatment. Our study calls for further research into the complexity of molecular mechanisms underlying epilepsy, aiming to develop more effective treatments targeting novel putative downstream pathways of this disorder.

Current State of Modeling Human Psychiatric Disorders Using Zebrafish.

Psychiatric disorders are highly prevalent brain pathologies that represent an urgent, unmet biomedical problem. Since reliable clinical diagnoses are essential for the treatment of psychiatric disorders, their animal models with robust, relevant behavioral and physiological endpoints become necessary. Zebrafish (Danio rerio) display well-defined, complex behaviors in major neurobehavioral domains which are evolutionarily conserved and strikingly parallel to those seen in rodents and humans. Although zebrafish are increasingly often used to model psychiatric disorders, there are also multiple challenges with such models as well. The field may therefore benefit from a balanced, disease-oriented discussion that considers the clinical prevalence, the pathological complexity, and societal importance of the disorders in question, and the extent of its detalization in zebrafish central nervous system (CNS) studies. Here, we critically discuss the use of zebrafish for modeling human psychiatric disorders in general, and highlight the topics for further in-depth consideration, in order to foster and (re)focus translational biological neuroscience research utilizing zebrafish. Recent developments in molecular biology research utilizing this model species have also been summarized here, collectively calling for a wider use of zebrafish in translational CNS disease modeling.

Towards Modeling Anhedonia and Its Treatment in Zebrafish.

Mood disorders, especially depression, are a major cause of human disability. The loss of pleasure (anhedonia) is a common, severely debilitating symptom of clinical depression. Experimental animal models are widely used to better understand depression pathogenesis and to develop novel antidepressant therapies. In rodents, various experimental models of anhedonia have already been developed and extensively validated. Complementing rodent studies, the zebrafish (Danio rerio) is emerging as a powerful model organism to assess pathobiological mechanisms of affective disorders, including depression. Here, we critically discuss the potential of zebrafish for modeling anhedonia and studying its molecular mechanisms and translational implications.

Can we gain translational insights into the functional roles of cerebral cortex from acortical rodent and naturally acortical zebrafish models?

Cerebral cortex is found only in mammals and is particularly prominent and developed in humans. Various rodent models with fully or partially ablated cortex are commonly used to probe the role of cortex in brain functions and its multiple subcortical projections, including pallium, thalamus and the limbic system. Various rodent models are traditionally used to study the role of cortex in brain functions. A small teleost fish, the zebrafish (Danio rerio), has gained popularity in neuroscience research, and albeit (like other fishes) lacking cortex, its brain performs well some key functions (e.g., memory, consciousness and motivation) with complex, context-specific and well-defined behaviors. Can rodent and zebrafish models help generate insights into the role of cortex in brain functions, and dissect its cortex-specific (vs. non-cortical) functions? To address this conceptual question, here we evaluate brain functionality in intact vs. decorticated rodents and further compare it in the zebrafish, a naturally occurring acortical species. Overall, comparing cortical and acortical rodent models with naturally acortical zebrafish reveals both distinct and overlapping contributions of neocortex and 'precortical' zebrafish telencephalic regions to higher brain functions. Albeit morphologically different, mammalian neocortex and fish pallium may possess more functional similarities than it is presently recognized, calling for further integrative research utilizing both cortical and decorticated/acortical vertebrate model organisms.

Experimental models of human cortical malformations: from mammals to 'acortical' zebrafish.

Human neocortex controls and integrates cognition, emotions, perception and complex behaviors. Aberrant cortical development can be triggered by multiple genetic and environmental factors, causing cortical malformations. Animal models, especially rodents, are a valuable tool to probe molecular and physiological mechanisms of cortical malformations. Complementing rodent studies, the zebrafish (Danio rerio) is an important model organism in biomedicine. Although the zebrafish (like other fishes) lacks neocortex, here we argue that this species can still be used to model various aspects and brain phenomena related to human cortical malformations. We also discuss novel perspectives in this field, covering both advantages and limitations of using mammalian and zebrafish models in cortical malformation research. Summarizing mounting evidence, we also highlight the importance of translationally-relevant insights into the pathogenesis of cortical malformations from animal models, and discuss future strategies of research in the field.

Towards experimental models of delirium utilizing zebrafish.

Delirium is an acute neuropsychiatric condition characterized by impaired behavior and cognition. Although the syndrome has been known for millennia, its CNS mechanisms and risk factors remain poorly understood. Experimental animal models, especially rodent-based, are commonly used to probe various pathogenetic aspects of delirium. Complementing rodents, the zebrafish (Danio rerio) emerges as a promising novel model organism to study delirium. Zebrafish demonstrate high genetic and physiological homology to mammals, easy maintenance, robust behaviors in various sensitive behavioral tests, and the potential to screen for pharmacological agents relevant to delirium. Here, we critically discuss recent developments in the field, and emphasize the developing utility of zebrafish models for translational studies of delirium and deliriant drugs. Overall, the zebrafish represents a valuable and promising aquatic model species whose use may help understand delirium etiology, as well as develop novel therapies for this severely debilitating disorder.

Understanding CNS Effects of Antimicrobial Drugs Using Zebrafish Models.

Antimicrobial drugs represent a diverse group of widely utilized antibiotic, antifungal, antiparasitic and antiviral agents. Their growing use and clinical importance necessitate our improved understanding of physiological effects of antimicrobial drugs, including their potential effects on the central nervous system (CNS), at molecular, cellular, and behavioral levels. In addition, antimicrobial drugs can alter the composition of gut microbiota, and hence affect the gut-microbiota-brain axis, further modulating brain and behavioral processes. Complementing rodent studies, the zebrafish (Danio rerio) emerges as a powerful model system for screening various antimicrobial drugs, including probing their putative CNS effects. Here, we critically discuss recent evidence on the effects of antimicrobial drugs on brain and behavior in zebrafish, and outline future related lines of research using this aquatic model organism.

Solfeggio-frequency music exposure reverses cognitive and endocrine deficits evoked by a 24-h light exposure in adult zebrafish.

Music therapy has long been used as a non-pharmacological intervention to improve cognitive function and mood in humans. Mounting rodent evidence also supports beneficial impact of music exposure on animal cognitive performance. The zebrafish (Danio rerio) is an important emerging aquatic animal model in translational biomedical and neuroscience research. Here, we evaluate the effects of intermittent (2-h or 6-h twice daily) and continuous (24-h) solfeggio-frequency music exposure on behavioral, cognitive and endocrine parameters in adult zebrafish whose circadian rhythm was disturbed by a 24-h light exposure. Overall, a 24-h light exposure stress evokes overt cognitive deficits in the inhibitory avoidance test and elevates zebrafish whole-body cortisol levels. However, these effects were reversed by solfeggio-frequency music exposure for 2 or 6 h twice daily, and by continuous 24-h exposure. Collectively, these findings suggest a positive modulation of cognitive and endocrine responses in adult zebrafish by environmental enrichment via the long-term exposure to music, and reinforces zebrafish as a robust, sensitive model organism for neurocognitive and neuroendocrine research.

Exploring CNS Effects of American Traditional Medicines using Zebrafish Models.

Although American traditional medicine (ATM) has been practiced for millennia, its complex multi-target mechanisms of therapeutic action remain poorly understood. Animal models are widely used to elucidate the therapeutic effects of various ATMs, including their modulation of brain and behavior. Complementing rodent models, the zebrafish (Danio rerio) is a promising novel organism in translational neuroscience and neuropharmacology research. Here, we emphasize the growing value of zebrafish for testing neurotropic effects of ATMs and outline future directions of research in this field. We also demonstrate the developing utility of zebrafish as complementary models for probing CNS mechanisms of ATM action and their potential to treat brain disorders.

Evolutionarily conserved gene expression patterns for affective disorders revealed using cross-species brain transcriptomic analyses in humans, rats and zebrafish.

Widespread, debilitating and often treatment-resistant, depression and other stress-related neuropsychiatric disorders represent an urgent unmet biomedical and societal problem. Although animal models of these disorders are commonly used to study stress pathogenesis, they are often difficult to translate across species into valuable and meaningful clinically relevant data. To address this problem, here we utilized several cross-species/cross-taxon approaches to identify potential evolutionarily conserved differentially expressed genes and their sets. We also assessed enrichment of these genes for transcription factors DNA-binding sites down- and up- stream from their genetic sequences. For this, we compared our own RNA-seq brain transcriptomic data obtained from chronically stressed rats and zebrafish with publicly available human transcriptomic data for patients with major depression and their respective healthy control groups. Utilizing these data from the three species, we next analyzed their differential gene expression, gene set enrichment and protein-protein interaction networks, combined with validated tools for data pooling. This approach allowed us to identify several key brain proteins (GRIA1, DLG1, CDH1, THRB, PLCG2, NGEF, IKZF1 and FEZF2) as promising, evolutionarily conserved and shared affective 'hub' protein targets, as well as to propose a novel gene set that may be used to further study affective pathogenesis. Overall, these approaches may advance cross-species brain transcriptomic analyses, and call for further cross-species studies into putative shared molecular mechanisms of affective pathogenesis.

Towards translational modeling of behavioral despair and its treatment in zebrafish.

Depression is a widespread and severely debilitating neuropsychiatric disorder whose key clinical symptoms include low mood, anhedonia and despair (the inability or unwillingness to overcome stressors). Experimental animal models are widely used to improve our mechanistic understanding of depression pathogenesis, and to develop novel antidepressant therapies. In rodents, various experimental models of 'behavioral despair' have already been developed and rigorously validated. Complementing rodent studies, the zebrafish (Danio rerio) is emerging as a powerful model organism to assess pathobiological mechanisms of depression and other related affective disorders. Here, we critically discuss the developing potential and important translational implications of zebrafish models for studying despair and its mechanisms, and the utility of such aquatic models for antidepressant drug screening.

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.

Artificial intelligence-driven phenotyping of zebrafish psychoactive drug responses.

Zebrafish (Danio rerio) are rapidly emerging in biomedicine as promising tools for disease modelling and drug discovery. The use of zebrafish for neuroscience research is also growing rapidly, necessitating novel reliable and unbiased methods of neurophenotypic data collection and analyses. Here, we applied the artificial intelligence (AI) neural network-based algorithms to a large dataset of adult zebrafish locomotor tracks collected previously in a series of in vivo experiments with multiple established psychotropic drugs. We first trained AI to recognize various drugs from a wide range of psychotropic agents tested, and then confirmed prediction accuracy of trained AI by comparing several agents with known similar behavioral and pharmacological profiles. Presenting a framework for innovative neurophenotyping, this proof-of-concept study aims to improve AI-driven movement pattern classification in zebrafish, thereby fostering drug discovery and development utilizing this key model organism.

Acute behavioral and Neurochemical Effects of Novel N-Benzyl-2-Phenylethylamine Derivatives in Adult Zebrafish.

Hallucinogenic drugs potently affect brain and behavior and have also recently emerged as potentially promising agents in pharmacotherapy. Complementing laboratory rodents, the zebrafish (Danio rerio) is a powerful animal model organism for screening neuroactive drugs, including hallucinogens. Here, we test a battery of ten novel N-benzyl-2-phenylethylamine (NBPEA) derivatives with the 2,4- and 3,4-dimethoxy substitutions in the phenethylamine moiety and the -OCH3, -OCF3, -F, -Cl, and -Br substitutions in the ortho position of the phenyl ring of the N-benzyl moiety, assessing their acute behavioral and neurochemical effects in the adult zebrafish. Overall, substitutions in the Overall, substitutions in the N-benzyl moiety modulate locomotion, and substitutions in the phenethylamine moiety alter zebrafish anxiety-like behavior, also affecting the brain serotonin and/or dopamine turnover. The 24H-NBOMe(F) and 34H-NBOMe(F) treatment also reduced zebrafish despair-like behavior. Computational analyses of zebrafish behavioral data by artificial intelligence identified several distinct clusters for these agents, including anxiogenic/hypolocomotor (24H-NBF, 24H-NBOMe, and 34H-NBF), behaviorally inert (34H-NBBr, 34H-NBCl, and 34H-NBOMe), anxiogenic/hallucinogenic-like (24H-NBBr, 24H-NBCl, and 24H-NBOMe(F)), and anxiolytic/hallucinogenic-like (34H-NBOMe(F)) drugs. Our computational analyses also revealed phenotypic similarity of the behavioral activity of some NBPEAs to that of selected conventional serotonergic and antiglutamatergic hallucinogens. In silico functional molecular activity modeling further supported the overlap of the drug targets for NBPEAs tested here and the conventional serotonergic and antiglutamatergic hallucinogens. Overall, these findings suggest potent neuroactive properties of several novel synthetic NBPEAs, detected in a sensitive in vivo vertebrate model system, the zebrafish, raising the possibility of their potential clinical use and abuse.

Unconventional anxiety pharmacology in zebrafish: Drugs beyond traditional anxiogenic and anxiolytic spectra.

Anxiety is the most prevalent brain disorder and a common cause of human disability. Animal models are critical for understanding anxiety pathogenesis and its pharmacotherapy. The zebrafish (Danio rerio) is increasingly utilized as a powerful model organism in anxiety research and anxiolytic drug screening. High similarity between human, rodent and zebrafish molecular targets implies shared signaling pathways involved in anxiety pathogenesis. However, mounting evidence shows that zebrafish behavior can be modulated by drugs beyond conventional anxiolytics or anxiogenics. Furthermore, these effects may differ from human and/or rodent responses, as such 'unconventional' drugs may affect zebrafish behavior despite having no such profiles (or exerting opposite effects) in humans or rodents. Here, we discuss the effects of several putative unconventional anxiotropic drugs (aspirin, lysergic acid diethylamide (LSD), nicotine, naloxone and naltrexone) and their potential mechanisms of action in zebrafish. Emphasizing the growing utility of zebrafish models in CNS drug discovery, such unconventional anxiety pharmacology may provide important, evolutionarily relevant insights into complex regulation of anxiety in biological systems. Albeit seemingly complicating direct translation from zebrafish into clinical phenotypes, this knowledge may instead foster the development of novel CNS drugs, eventually facilitating innovative treatment of patients based on novel 'unconventional' targets identified in fish models.

Understanding how stress responses and stress-related behaviors have evolved in zebrafish and mammals.

Stress response is essential for the organism to quickly restore physiological homeostasis disturbed by various environmental insults. In addition to well-established physiological cascades, stress also evokes various brain and behavioral responses. Aquatic animal models, including the zebrafish (Danio rerio), have been extensively used to probe pathobiological mechanisms of stress and stress-related brain disorders. Here, we critically discuss the use of zebrafish models for studying mechanisms of stress and modeling its disorders experimentally, with a particular cross-taxon focus on the potential evolution of stress responses from zebrafish to rodents and humans, as well as its translational implications.

Modulation of behavioral and neurochemical responses of adult zebrafish by fluoxetine, eicosapentaenoic acid and lipopolysaccharide in the prolonged chronic unpredictable stress model.

Long-term recurrent stress is a common cause of neuropsychiatric disorders. Animal models are widely used to study the pathogenesis of stress-related psychiatric disorders. The zebrafish (Danio rerio) is emerging as a powerful tool to study chronic stress and its mechanisms. Here, we developed a prolonged 11-week chronic unpredictable stress (PCUS) model in zebrafish to more fully mimic chronic stress in human populations. We also examined behavioral and neurochemical alterations in zebrafish, and attempted to modulate these states by 3-week treatment with an antidepressant fluoxetine, a neuroprotective omega-3 polyunsaturated fatty acid eicosapentaenoic acid (EPA), a pro-inflammatory endotoxin lipopolysaccharide (LPS), and their combinations. Overall, PCUS induced severe anxiety and elevated norepinephrine levels, whereas fluoxetine (alone or combined with other agents) corrected most of these behavioral deficits. While EPA and LPS alone had little effects on the zebrafish PCUS-induced anxiety behavior, both fluoxetine (alone or in combination) and EPA restored norepinephrine levels, whereas LPS + EPA increased dopamine levels. As these data support the validity of PCUS as an effective tool to study stress-related pathologies in zebrafish, further research is needed into the ability of various conventional and novel treatments to modulate behavioral and neurochemical biomarkers of chronic stress in this model organism.

The Extracellular Matrix-Derived Biomarkers for Diagnosis, Prognosis, and Personalized Therapy of Malignant Tumors.

The tumor biomarkers already have proven clinical value and have become an integral part in cancer management and modern translational oncology. The tumor tissue microenvironment (TME), which includes extracellular matrix (ECM), signaling molecules, immune and stromal cells, and adjacent non-tumorous tissue, contributes to cancer pathogenesis. Thus, TME-derived biomarkers have many clinical applications. This review is predominately based on the most recent publications (manuscripts published in a last 5 years, or seminal publications published earlier) and fills a gap in the current literature on the cancer biomarkers derived from the TME, with particular attention given to the ECM and products of its processing and degradation, ECM-associated extracellular vesicles (EVs), biomechanical characteristics of ECM, and ECM-derived biomarkers predicting response to the immunotherapy. We discuss the clinical utility of the TME-incorporating three-dimensional in vitro and ex vivo cell culture models for personalized therapy. We conclude that ECM is a critical driver of malignancies and ECM-derived biomarkers should be included in diagnostics and prognostics panels of markers in the clinic.

Rational Surface Design of Upconversion Nanoparticles with Polyethylenimine Coating for Biomedical Applications: Better Safe than Brighter?

Upconversion nanoparticles (UCNPs) coated with polyethylenimine (PEI) are popular background-free optical contrast probes and efficient drug and gene delivery agents attracting attention in science, industry, and medicine. Their unique optical properties are especially useful for subsurface nanotheranostics applications, in particular, in skin. However, high cytotoxicity of PEI limits safe use of UCNP@PEI, and this represents a major barrier for clinical translation of UCNP@PEI-based technologies. Our study aims to address this problem by exploring additional surface modifications to UCNP@PEI to create less toxic and functional nanotheranostic materials. We designed and synthesized six types of layered polymer coatings that envelop the original UCNP@PEI surface, five of which reduced the cytotoxicity to human skin keratinocytes under acute (24 h) and subacute (120 h) exposure. In parallel, we examined the photoluminescence spectra and lifetime of the surface-modified UCNP@PEI. To quantify their brightness, we developed original methodology to precisely measure the colloidal concentration to normalize the photoluminescence signal using a nondigesting mass spectrometry protocol. Our results, specified for the individual coatings, show that, despite decreasing the cytotoxicity, the external polymer coatings of UCNP@PEI quench the upconversion photoluminescence in biologically relevant aqueous environments. This trade-off between cytotoxicity and brightness for surface-coated UCNPs emphasizes the need for the combined assessment of the viability of normal cells exposed to the nanoparticles and the photophysical properties of postmodification UCNPs. We present an optimized methodology for rational surface design of UCNP@PEI in biologically relevant conditions, which is essential to facilitate the translation of such nanoparticles to the clinical applications.