Zebrafish models for studying cognitive enhancers.

Cognitive decline is commonly seen both in normal aging and in neurodegenerative and neuropsychiatric diseases. Various experimental animal models represent a valuable tool to study brain cognitive processes and their deficits. Equally important is the search for novel drugs to treat cognitive deficits and improve cognitions. Complementing rodent and clinical findings, studies utilizing zebrafish (Danio rerio) are rapidly gaining popularity in translational cognitive research and neuroactive drug screening. Here, we discuss the value of zebrafish models and assays for screening nootropic (cognitive enhancer) drugs and the discovery of novel nootropics. We also discuss the existing challenges, and outline future directions of research in this field.

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.

Acetic acid-induced pain elicits stress-, and camouflage-related responses in zebrafish: Modulatory effects of opioidergic drugs on neurobehavioral phenotypes.

While pain results from the activation of nociceptors following noxious stimuli, mounting evidence links pain- and stress-related responses in mammals. In zebrafish, the activation of hypothalamic-pituitary-interrenal (HPI) axis may also regulate body pigmentation (the camouflage response). Here, we aimed to investigate a putative relationship between pain-, stress-, and camouflage-related parameters in adult zebrafish. To answer this question, we assessed whether intraperitoneal acetic acid injection can activate the HPI axis, measuring whole-body cortisol and the camouflage response as physiological endpoints in the presence or absence of morphine or naloxone, an opioid antagonist. Acetic acid induced a stereotypic circling behavior in the top of the tank, accompanied by abdominal writhing-like response, a specific phenotype that reflects local nociceptive effect. Both whole-body cortisol levels and camouflage response increased in the acetic acid group, while morphine prevented these responses, and naloxone antagonized morphine-induced effects. Moreover, we observed positive correlations between representative behavioral, physiological and skin coloration endpoints, and a "pain index" was proposed to summarize phenotypic profile of zebrafish under different pharmacological manipulations. Collectively, these findings suggest a coordinated activation of pain, camouflage- and stress-related pathways following acetic acid injection in zebrafish. Our data also support that camouflage response represents a novel and relevant biomarker for future probing pain and stress neurobiology, with a robust sensitivity to opioidergic drugs.

Animal Inflammation-Based Models of Neuropsychiatric Disorders.

Mounting evidence links psychiatric disorders to central and systemic inflammation. Experimental (animal) models of psychiatric disorders are important tools for translational biopsychiatry research and CNS drug discovery. Current experimental models, most typically involving rodents, continue to reveal shared fundamental pathological pathways and biomarkers underlying the pathogenetic link between brain illnesses and neuroinflammation. Recent data also show that various proinflammatory factors can alter brain neurochemistry, modulating the levels of neurohormones and neurotrophins in neurons and microglia. The role of "active" glia in releasing a wide range of proinflammatory cytokines also implicates glial cells in various psychiatric disorders. Here, we discuss recent animal inflammation-related models of psychiatric disorders, focusing on their translational perspectives and the use of some novel promising model organisms (zebrafish), to better understand the evolutionally conservative role of inflammation in neuropsychiatric conditions.

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 Zebrafish Models of CNS Channelopathies.

Channelopathies are a large group of systemic disorders whose pathogenesis is associated with dysfunctional ion channels. Aberrant transmembrane transport of K+, Na+, Ca2+ and Cl- by these channels in the brain induces central nervous system (CNS) channelopathies, most commonly including epilepsy, but also migraine, as well as various movement and psychiatric disorders. Animal models are a useful tool for studying pathogenesis of a wide range of brain disorders, including channelopathies. Complementing multiple well-established rodent models, the zebrafish (Danio rerio) has become a popular translational model organism for neurobiology, psychopharmacology and toxicology research, and for probing mechanisms underlying CNS pathogenesis. Here, we discuss current prospects and challenges of developing genetic, pharmacological and other experimental models of major CNS channelopathies based on zebrafish.

Acetic acid-induced nociception modulates sociability in adult zebrafish: Influence on shoaling behavior in heterogeneous groups and social preference.

Due to the recognition of fishes as sentient beings, the zebrafish (Danio rerio) has become an emergent animal model system to investigate the biological processes of nocifensive responses. Here, we aimed to characterize the zebrafish social behavior in a nociception-based context. For this purpose, using a three-dimensional analysis of heterogeneous shoals, we investigated the main behavioral responses in two 6-min trials: before (baseline) and after a single intraperitoneal (i.p) injection of 10 µL phosphate-buffered saline (PBS) (control), acetic acid 5% (AA), morphine 2.5 mg/kg (MOR) or acetic acid 5% plus morphine 2.5 mg/kg (AA + MOR) in one subject from a four-fish shoal. The social preference of individuals for tanks with shoals of fish treated with PBS, 5 % AA, or to an empty aquarium was also tested. We verified that AA administration disrupted the shoal homogeneity by eliciting dispersion of the treated fish with simultaneous clustering of non-manipulated fish. In general, morphine coadministration protected against AA-induced behavioral changes. The social preference test revealed a clear preference to conspecifics (PBS and AA) over an empty tank. However, a prominent preference for PBS- over AA-treated shoal was verified. Overall, our novel findings show that nociception can modulate zebrafish sociability, possibly due to the visual recognition of nocifensive responses. Although future studies are needed to elucidate how nociception modulates zebrafish social behavior, our results contribute to improve the welfare assessment of zebrafish shoals under distinct experimental manipulations.

Induction of aggression and anxiety-like responses by perfluorooctanoic acid is accompanied by modulation of cholinergic- and purinergic signaling-related parameters in adult zebrafish.

Perfluorooctanoic acid (PFOA) is a contaminant of global concern owing to its prevalent occurrence in aquatic and terrestrial environments with potential hazardous impact on living organisms. Here, we investigated the influence of realistic environmental concentrations of PFOA (0, 0.25, 0.5, or 1.0 mg/L) on relevant behaviors of adult zebrafish (Danio rerio) (e.g., exploration to novelty, social preference, and aggression) and the possible role of PFOA in modulating cholinergic and purinergic signaling in the brain after exposure for 7 consecutive days. PFOA significantly increased geotaxis as well as reduced vertical exploration (a behavioral endpoint for anxiety), and increased the frequency and duration of aggressive episodes without affecting their social preference. Exposure to PFOA did not affect ADP hydrolysis, whereas ATP and AMP hydrolysis were significantly increased at the highest concentration tested. However, AChE activity was markedly decreased in all PFOA-exposed groups when compared with control. In conclusion, PFOA induces aggression and anxiety-like behavior in adult zebrafish and modulates both cholinergic and purinergic signaling biomarkers. These novel data can provide valuable insights into possible health threats related to human activities, demonstrating the utility of adult zebrafish to elucidate how PFOA affects neurobehavioral responses in aquatic organisms.

Influence of acid-sensing ion channel blocker on behavioral responses in a zebrafish model of acute visceral pain.

Acid-sensing ion channels (ASICs) play significant roles in numerous neurological and pathological conditions, including pain. Although acid-induced nociception has been characterized previously in zebrafish, the contribution of ASICs in modulating pain-like behaviors is still unknown. Here, we investigated the role of amiloride, a nonselective ASICs blocker, in the negative modulation of specific behavioral responses in a zebrafish-based model of acute visceral pain. We verified that intraperitoneal injection (i.p.) of 0.25, 0.5, 1.0, and 2.0 mg/mL amiloride alone or vehicle did not change zebrafish behavior compared to saline-treated fish. Administration of 2.5% acetic acid (i.p.) elicited writhing-like response evidenced by the abnormal body curvature and impaired locomotion and motor activity. Attenuation of acetic acid-induced pain was verified at lower amiloride doses (0.25 and 0.5 mg/mL) whereas 1.0 and 2.0 mg/mL abolished pain-like responses. The protective effect of the highest amiloride dose tested was evident in preventing writhing-like responses and impaired locomotion and vertical activity. Collectively, amiloride antagonized abdominal writhing-like phenotype and aberrant behaviors, supporting the involvement of ASICs in a zebrafish-based model of acute visceral pain.

The Use of Zebrafish as a Non-traditional Model Organism in Translational Pain Research: The Knowns and the Unknowns.

The ability of the nervous system to detect a wide range of noxious stimuli is crucial to avoid life-threatening injury and to trigger protective behavioral and physiological responses. Pain represents a complex phenomenon, including nociception associated with cognitive and emotional processing. Animal experimental models have been developed to understand the mechanisms involved in pain response, as well as to discover novel pharmacological and non-pharmacological anti-pain therapies. Due to the genetic tractability, similar physiology, low cost, and rich behavioral repertoire, the zebrafish (Danio rerio) is a powerful aquatic model for modeling pain responses. Here, we summarize the molecular machinery of zebrafish responses to painful stimuli, as well as emphasize how zebrafish-based pain models have been successfully used to understand specific molecular, physiological, and behavioral changes following different algogens and/or noxious stimuli (e.g., acetic acid, formalin, histamine, Complete Freund's Adjuvant, cinnamaldehyde, allyl isothiocyanate, and fin clipping). We also discuss recent advances in zebrafish-based studies and outline the potential advantages and limitations of the existing models to examine the mechanisms underlying pain responses from evolutionary and translational perspectives. Finally, we outline how zebrafish models can represent emergent tools to explore pain behaviors and pain-related mood disorders, as well as to facilitate analgesic therapy screening in translational pain research.

Predictable chronic stress modulates behavioral and neuroendocrine phenotypes of zebrafish: Influence of two homotypic stressors on stress-mediated responses.

The zebrafish (Danio rerio) has been considered a suitable model organism to assess the evolutionarily conserved bases of behavioral and neuroendocrine responses to stress. Depending on the nature of the stressor, prolonged stress may elicit habituation or evoke long-term changes in the central nervous systems (CNS) often associated with various neuropsychiatric disorders. Conspecific alarm substance (CAS) and net chasing (NC) constitute chemical and physical stressors, respectively, which cause aversive behaviors and physiological changes in fishes. Here, we investigate whether predictable chronic stress (PCS) using two homotypic stressors differently modulates behavioral and physiological responses in zebrafish. PCS-CAS or PCS-NC were performed for 14 days, 2-times daily, while locomotion, exploratory activity, anxiety-like behaviors, and whole-body cortisol levels were measured on day 15. PCS-CAS reduced distance traveled, the number of transitions and time in top area, as well as increased the latency to enter the top in the novel tank test. In the light/dark test, CAS-exposed fish showed decreased time spent in lit area, shorter latency to enter the dark area, and increased risk assessments. PCS-CAS also increased whole-body cortisol levels in zebrafish. Although PCS-NC reduced the latency to enter the dark area, whole-body cortisol levels did not change. Moreover, acute experiments revealed that both CAS and NC promoted anxiogenesis and increased cortisol levels, suggesting habituation to stress following PCS-NC. Overall, our novel findings demonstrate that PCS induces behavioral and physiological changes in zebrafish depending on the nature of the stressor.

Three- and bi-dimensional analyses of the shoaling behavior in zebrafish: Influence of modulators of anxiety-like responses.

Social behaviors are key components that play adaptive roles in various species, including humans. The zebrafish (Danio rerio) is a social species and the shoaling behavior can be pharmacologically manipulated either by anxiogenic or anxiolytic substances, providing translatable data in neuropsychiatric research. Here, we aimed to characterize the shoaling behavior in zebrafish under different pharmacological manipulations in a three-dimensional (3D) perspective using the spatial coordinates of the fish positions. Temporal and spatial reconstructions of shoal occupancy were performed after exposure to conspecific alarm substance (CAS) and caffeine (CAF) (anxiogenic substances) or diazepam (DZP) (a classical anxiolytic drug). Behavioral 3D analyses and spatiotemporal reconstructions of the shoaling behavior revealed that both CAS and CAF decreased the shoal volume, the average fish distance to the centoid point, and increased shoal geotaxis, but only CAS reduced the inter-fish distance when compared to control (CTRL). Conversely, DZP group showed increased shoal volume and inter-fish distance. Because substantial differences were verified when the shoaling response was analyzed in 3D and 2D perspectives, we reinforce the use of 3D reconstructions of fish positions to assess how different manipulations affect the social behavior of zebrafish. The novel procedure described here represents an easy-to-use, inexpensive, and alternative tool to perform a spatiotemporal reconstruction of the shoal occupancy under different pharmacological manipulations, complementing the existing quantification of locomotion activity of multiple fish.

Modeling psychiatric comorbid symptoms of epileptic seizures in zebrafish.

Epilepsy is a debilitating neurological disorder characterized by recurrent unprovoked seizures. Anxiety, cognitive deficits, depressive-like symptoms, and social dysfunction are psychiatric comorbidities with high prevalence in epileptic patients. Due to the genetic and behavioral tractability, the zebrafish is a promising model organism to understand the neural bases involved in epilepsy-related comorbidities. Here, we aimed to characterize some behavioral phenotypes paralleling those observed in epilepsy-related comorbidities after a single pentylenetetrazole (PTZ) exposure in zebrafish. We also analyzed the influence of whole-body cortisol levels in the behavioral responses measured. Fish were exposed to 10 mM PTZ for 20 min to induce epileptic seizures. After 24 h recovery period, locomotion and anxiety-like responses (novel tank and light-dark tests), social interaction (shoaling behavior task), and memory retention (inhibitory avoidance protocol) were assessed. Basically, PTZ impaired habituation to novelty stress, evoked anxiogenic-like behaviors, disrupted shoaling, and caused memory consolidation deficits in zebrafish without changing whole-body cortisol levels. In conclusion, our novel findings further validate the use of zebrafish as a suitable tool for modeling epilepsy-related comorbidities in translational neuropsychiatric research.

Involvement of anxiety-like behaviors and brain oxidative stress in the chronic effects of alarm reaction in zebrafish populations.

Aversive conditions elicit anxiety responses that prepare the organism to an eventual threat. Nonetheless, prolonged anxiety is a pathological condition associated with various neuropsychiatric disorders. Here, we evaluated whether the conspecific alarm substance (CAS), a chemical cue that elicits aversion, influences anxiety-like behaviors and modulates brain oxidative stress-related parameters in wild-type (WT) and leopard (leo) zebrafish following a repeated exposure protocol. CAS exposure was performed for 5 min, once daily for 7 consecutive days. In the 8th day, animals were tested in the light/dark and novel tank tests and their brains were further dissected for biochemical analyses. CAS chronically induced anxiogenic-like states in WT and leo populations when their behaviors were analyzed in the light/dark and novel tank tests. CAS also increased catalase (CAT) and glutathione S-transferase (GST) activities, as well as non-protein thiol (NPSH) content in WT and leo, but only leo had increased thiobarbituric reactive substance (TBARS) levels in the brain. At baseline conditions, leo was more 'anxious' when compared to WT, displaying lower CAT activity and carbonylated protein (CP) levels. Overall, CAS chronically triggers anxiety-like behavior in zebrafish populations, which may be associated with changes in oxidative stress-related parameters. Furthermore, the use of different zebrafish populations may serve as an interesting tool in future research aiming to investigate the neurobehavioral bases of neuropsychiatric disorders in vertebrates.

Naloxone prolongs abdominal constriction writhing-like behavior in a zebrafish-based pain model.

The ability to detect noxious stimuli is essential to survival. However, pathological pain is maladaptive and severely debilitating. Endogenous and exogenous opioids modulate pain responses via opioid receptors, reducing pain sensibility. Due to the high genetic and physiological similarities to rodents and humans, the zebrafish is a valuable tool to assess pain responses and the underlying mechanisms involved in nociception. Although morphine attenuates pain-like responses of zebrafish, there are no data showing if the antagonism of opioid receptors prolongs pain duration in the absence of an exogenous opioid. Here, we investigated whether a common opioid antagonist naloxone affects the abdominal constriction writhing-like response, recently characterized as a zebrafish-based pain behavior. Animals were injected intraperitoneally with acetic acid (5.0%), naloxone (1.25 mg/kg; 2.5 mg/kg; 5.0 mg/kg) or acetic acid with naloxone to investigate the changes in their body curvature for 1 h. Acetic acid elicited a robust pain-like response in zebrafish, as assessed by aberrant abdominal body curvature, while no effects were observed following PBS injection. Although naloxone alone did not alter the frequency and duration of this behavior, it dose-dependently prolonged acetic acid-induced abdominal curvature response. Besides reinforcing the use of the abdominal writhing-like phenotype as a behavioral endpoint to measure acute pain responses in zebrafish models, our novel data suggest a putative role of endogenous opioids in modulating the recovery from pain stimulation in zebrafish.

Understanding nociception-related phenotypes in adult zebrafish: Behavioral and pharmacological characterization using a new acetic acid model.

Pain, a severely debilitating symptom of many human disorders, is a growing, unmet biomedical problem. Although the use of zebrafish (Danio rerio) to investigate both behavioral and physiological nociception-related responses is expanding rapidly, the characterization of behavioral phenotypes that reflect injury location is limited, making the results of such studies difficult to interpret. Here, we characterize putative nociception-related behavioral phenotypes in adult zebrafish following an intraperitoneal (i.p.) administration of acetic acid, a well-established protocol for visceral pain in rodents. Acetic acid (2.5 and 5.0%) induced an abdominal constriction-like response, which was assessed by measuring a body curvature index. Moreover, all doses tested (0.5-5.0%) reduced distance traveled and vertical activity in the novel tank test. Freezing duration increased following 5.0% acetic acid, whereas fish injected with 1.0, 2.5, and 5.0% spent more time in top area of the tank. Both morphine (an opioid analgesic) and diclofenac (a nonsteroidal anti-inflammatory drug, NSAID) prevented the 5.0% acetic acid-induced changes in body curvature index, whereas naloxone blocked these effects of morphine. Overall, zebrafish exposed to a single acetic acid i.p. injection display abnormal body curvature and specific changes in behavioral parameters sensitive to anti-nociceptive pharmacological modulation. We suggest that the abdominal constriction-like response represents a novel specific nociceptive-related phenotype in zebrafish. In general, our findings support the growing utility of zebrafish in translational pain research and antinociceptive drug discovery.