Cellular and molecular mechanisms of aflatoxin B1-mediated neurotoxicity: The therapeutic role of natural bioactive compounds.

Aflatoxin B1 (AFB1), a dietary toxin from the mold Aspergillus species, is well acknowledged to elicit extra-hepatic toxicity in both animals and humans. The neurotoxicity of AFB1 has become a global public health concern. Contemporary research on how AFB1 enters the brain to elicit neuronal dysregulation leading to noxious neurological outcomes has increased greatly in recent years. The current review discusses several neurotoxic outcomes and susceptible targets of AFB1 toxicity at cellular, molecular and genetic levels. Specifically, neurotoxicity studies involving the use of brain homogenates, neuroblastoma cell line IMR-32, human brain microvascular endothelial cells, microglial cells, and astrocytes, as well as mammalian and non-mammalian models to unravel the mechanisms associated with AFB1 exposure are highlighted. Further, some naturally occurring bioactive compounds with compelling therapeutic effects on AFB1-induced neurotoxicity are reviewed. In conclusion, available data from literature highlight AFB1 as a neurotoxin and its possible pathological contribution to neurological disorders. Further mechanistic studies aimed at discovering and developing effective therapeutics for AFB1 neurotoxicity is warranted.

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.

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.

Stress increases susceptibility to pentylenetetrazole-induced seizures in adult zebrafish.

Stress is the body's reaction to any change that requires adaptive responses. In various organisms, stress is a seizure-related comorbidity. Despite the exposure to stressors eliciting aversive behaviors in zebrafish, there are no data showing whether stress potentiates epileptic seizures in this species. Here, we investigated whether a previous exposure to an intense acute stressor positively modulates the susceptibility to seizures in pentylenetetrazole (PTZ)-challenged zebrafish. The conspecific alarm substance (CAS) was used to elicit aversive responses (3.5 mL/L for 5 min), observed by increased bottom dwelling and erratic movements. Then, fish were immediately exposed to 7.5 mM PTZ for 10 min to induce seizure-like behaviors. Stress increased the seizure intensity, the number of clonic-like seizure behaviors (score 4), as well as facilitated the occurrence of score 4 episodes by decreasing the latency in which fish reached the score 4. Moreover, fish with heightened anxiety showed increased susceptibility to PTZ, since positive correlations between anxiety- and seizure-like behaviors were found. Overall, since CAS also increased whole-body cortisol levels in zebrafish, our novel findings show a prominent response to PTZ-induced seizures in previously stressed zebrafish. Moreover, we reinforce the growing utility of zebrafish models to assess seizure-related comorbidities aiming to elucidate how stress can affect epileptic seizures in vertebrates.

Taurine modulates the stress response in zebrafish.

The zebrafish (Danio rerio) is used as an emergent model organism to investigate the behavioral and physiological responses to stress. The anxiolytic-like effects of taurine in zebrafish support the existence of different mechanisms of action, which can play a role in preventing stress-related disorders (i.e., modulation of GABAA, strychnine-sensitive glycine, and NMDA receptors, as well as antioxidant properties). Herein, we investigate whether taurine modulates some behavioral and biochemical responses in zebrafish acutely submitted to chemical and mechanical stressors. We pretreated zebrafish for 1 h in beakers at 42, 150, and 400 mg/L taurine. Fish were later acutely exposed to a chemical stressor (conspecific alarm substance) or to a mechanical stressor (net chasing), which elicits escaping responses and aversive behaviors. Locomotion, exploration, and defensive-like behaviors were measured using the novel tank and the light-dark tests. Biochemical (brain oxidative stress-related parameters) and whole-body cortisol levels were also quantified. We showed that taurine prevents anxiety/fear-like behaviors and protein carbonylation and dampens the cortisol response following acute stress in zebrafish. In summary, our results demonstrate a protective role of taurine against stress-induced behavioral and biochemical changes, thereby reinforcing the growing utility of zebrafish models to investigate the neuroprotective actions of taurine in vertebrates.

Sensory ecology of ostariophysan alarm substances.

Chemical communication of predation risk has evolved multiple times in fish species, with conspecific alarm substance (CAS) being the most well understood mechanism. CAS is released after epithelial damage, usually when prey fish are captured by a predator and elicits neurobehavioural adjustments in conspecifics which increase the probability of avoiding predation. As such, CAS is a partial predator stimulus, eliciting risk assessment-like and avoidance behaviours and disrupting the predation sequence. The present paper reviews the distribution and putative composition of CAS in fish and presents a model for the neural processing of these structures by the olfactory and the brain aversive systems. Applications of CAS in the behavioural neurosciences and neuropharmacology are also presented, exploiting the potential of model fish [e.g., zebrafish Danio rerio, guppies Poecilia reticulata, minnows Phoxinus phoxinus) in neurobehavioural research.

Neurobehavioral and biochemical changes in Nauphoeta cinerea following dietary exposure to chlorpyrifos.

The present study aimed to increase our understanding about the mode of toxic action of organophosphate pesticides in insects by evaluating the biochemical and neurobehavioral characteristics in Nauphoeta cinerea exposed to chlorpyrifos (CPF)-contaminated diet. The insects were exposed for 35 consecutive days to CPF at 0.078, 0.15625, 0.3125 and 0.625µg/g feed. Locomotor behavior was assessed for a 10-min trial in a novel arena and subsequently, biochemical analyses were carried out using the cockroaches' heads. In comparison to control, CPF-exposed cockroaches showed significant decreases in the total distance traveled, body rotation, turn angle and meandering, along with significant increase in the number of falls, time and episodes of immobility. The marked decrease in the exploratory profiles of CPF-exposed cockroaches was confirmed by track plots, whereas occupancy plot analyses showed a progressive dispersion at 0.15625µg/g feed group. Moreover, the heads of CPF-exposed cockroaches showed marked decrease in acetylcholinesterase activity and antioxidant status with concomitant significant elevation in dichlorofluorescein oxidation and lipid peroxidation levels in CPF-treated cockroaches. Gas Chromatography-Mass Spectrometry analyses revealed bioaccumulation of CPF in cockroaches exposed to concentrations above 0.078µg/g feed. The findings from this investigation showed N. cinerea as a value model organism for the risk assessment of environmental organophosphate contamination in insects.

Azadirachtin, a neem-derived biopesticide, impairs behavioral and hematological parameters in carp (Cyprinus carpio).

Azadirachtin (Aza) is a promisor biopesticide used in organic production and aquaculture. Although this compound is apparently safe, there is evidence that it may have deleterious effects on fish. Behavioral and hematological tests are grouped into a set of parameters that may predict potential toxicity of chemical compounds. Here, we investigate the effects of Aza, in the commercial formulation Neenmax™ , on carp (Cyprinus carpio) by defining LC50 (96 h), and testing behavioral and hematological parameters. In our study, LC50 was estimated at 80 µL/L. We exposed carp to Aza at 20, 40, and 60 µL/L, values based on 25, 50, and 75% of LC50 , respectively. At 60 µL/L, Aza promoted significant changes in several parameters, increasing the distance traveled and absolute turn angle. In addition, the same concentration decreased the time spent immobile and the number of immobile episodes. Hematological parameters, such as hematocrit, hemoglobin, hematimetrics index, and red cell distribution, were decreased at 60 µL/L Aza exposure. In conclusion, our study demonstrates that 60 µL/L Aza altered locomotor activity, motor pattern, and hematological parameters, suggesting potential toxicity to carp after acute exposure. In addition, this is the first report that evaluates the actions of a chemical contaminant using automated behavioral tracking of carp, which may be a useful tool for assessing the potential toxicity of biopesticides in conjunction with hematological tests. © 2015 Wiley Periodicals, Inc. Environ Toxicol 31: 1381-1388, 2016.

Copper acutely impairs behavioral function and muscle acetylcholinesterase activity in zebrafish (Danio rerio).

Copper is a heavy metal found at relatively high concentrations in surface waters around the world. Copper is a micronutrient at low concentrations and is essential to several organisms. At higher concentrations copper can become toxic, which reveal the importance of studying the toxic effects of this metal on the aquatic life. Thus, the objective of this study was to evaluate the toxic effects of copper on the behavior and biochemical parameters of zebrafish (Danio rerio). Zebrafish were exposed for 24h at a concentration of 0.006 mg/L Cu. After the exposure period, behavioral profile of animals was recorded through 6 min using two different apparatuses tests: the Novel Tank and the Light-Dark test. After behavioral testing, animals were euthanized with a solution of 250 mg/L of tricaine (MS-222). Brain, muscle, liver and gills were extracted for analysis of parameters related to oxidative stress and accumulation of copper in these tissues. Acetylcholinesterase (AChE) activity was determined in brain and muscle. Results showed acute exposure to copper induces significant changes in behavioral profile of zebrafish by changing locomotion and natural tendency to avoid brightly lit area. On the other hand, there were no significant effects on parameters related to oxidative stress. AChE activity decreased significantly in zebrafish muscle, but there were no significant changes in cerebral AChE activity. Copper levels in tissues did not increase significantly compared to the controls. Taken together, these results indicate that a low concentration of copper can acutely affect behavioral profile of adult zebrafish which could be partially related to an inhibition on muscle AChE activity. These results reinforce the need of additional tests to establishment of safe copper concentrations to aquatic organisms and the importance of behavioral parameters in ecotoxicological studies.

Utility of zebrafish-based models in understanding molecular mechanisms of neurotoxicity mediated by the gut-brain axis.

The gut microbes perform several beneficial functions which impact the periphery and central nervous systems of the host. Gut microbiota dysbiosis is acknowledged as a major contributor to the development of several neuropsychiatric and neurological disorders including bipolar disorder, depression, anxiety, Parkinson's disease, Alzheimer's disease, attention deficit hyperactivity disorder, and autism spectrum disorder. Thus, elucidation of how the gut microbiota-brain axis plays a role in health and disease conditions is a potential novel approach to prevent and treat brain disorders. The zebrafish (Danio rerio) is an invaluable vertebrate model that possesses conserved brain and intestinal features with those of humans, thus making zebrafish a valued model to investigate the interplay between the gut microbiota and host health. This chapter describes current findings on the utility of zebrafish in understanding molecular mechanisms of neurotoxicity mediated via the gut microbiota-brain axis. Specifically, it highlights the utility of zebrafish as a model organism for understanding how anthropogenic chemicals, pharmaceuticals and bacteria exposure affect animals and human health via the gut-brain axis.

Towards zebrafish models to unravel translational insights of obsessive-compulsive disorder: A neurobehavioral perspective.

Obsessive-compulsive disorder (OCD) is a chronic and debilitating illness that has been considered a polygenic and multifactorial disorder, challenging effective therapeutic interventions. Although invaluable advances have been obtained from human and rodent studies, several molecular and mechanistic aspects of OCD etiology are still obscure. Thus, the use of non-traditional animal models may foster innovative approaches in this field, aiming to elucidate the underlying mechanisms of disease from an evolutionary perspective. The zebrafish (Danio rerio) has been increasingly considered a powerful organism in translational neuroscience research, especially due to the intrinsic features of the species. Here, we outline target mechanisms of OCD for translational research, and discuss how zebrafish-based models can contribute to explore neurobehavioral aspects resembling those found in OCD. We also identify possible advantages and limitations of potential zebrafish-based models, as well as highlight future directions in both etiological and therapeutic research. Lastly, we reinforce the use of zebrafish as a promising tool to unravel the biological basis of OCD, as well as novel pharmacological therapies in the field.

The influence of acute dopamine transporter inhibition on manic-, depressive-like phenotypes, and brain oxidative status in adult zebrafish.

Functional changes in dopamine transporter (DAT) are related to various psychiatric conditions, including bipolar disorder (BD) symptoms. In experimental research, the inhibition of DAT induces behavioral alterations that recapitulate symptoms found in BD patients, including mania and depressive mood. Thus, developing novel animal models that mimic BD-related conditions by pharmacologically modulating the dopaminergic signaling is relevant. The zebrafish (Danio rerio) has been considered a suitable vertebrate system for modeling BD-like responses, due to the well-characterized behavioral responses and evolutionarily conservation of the dopaminergic system of this species. Here, we investigate whether GBR 12909, a selective inhibitor of DAT, causes neurobehavioral alterations in zebrafish similar to those observed in BD patients. Behaviors were recorded after a single intraperitoneal (i.p.) administration of GBR 12909 at different doses (3.75, 7.5, 15 and 30 mg/kg). To observe temporal effects on behavior, swim path parameters were measured immediately after the administration period during 30 min. Locomotion, anxiety-like behavior, social preference, aggression, despair-like behavior, and oxidative stress-related biomarkers in the brain were measured 30 min post administration. GBR 12909 induced prominent effects on locomotor activity and vertical exploration during the 30-min period. Hyperactivity was observed in GBR 30 group after 25 min, while all doses markedly reduced vertical drifts. GBR 12909 elicited hyperlocomotion, anxiety-like behavior, decreased social preference, aggression, and induced depressive-like behavior in a behavioral despair task. Depending on the dose, GBR 12909 also decreased SOD activity and TBARS levels, as well as increased GR activity and NPSH content. Collectively, our novel findings show that a single GBR 12909 administration evokes neurobehavioral changes that recapitulate manic- and depressive-like states observed in rodents, fostering the use of zebrafish models to explore BD-like responses in translational neuroscience research.

Assessing the exploratory profile of two zebrafish populations: Influence of anxiety-like phenotypes and independent trials on homebase-related parameters and exploration.

Anxiety is a protective behavior when animals face aversive conditions. The open field test (OFT) is used to assess the spatio-temporal dynamics of exploration, in which both homebase formation and recognition of environmental cues may reflect habituation to unfamiliar conditions. Because emotional- and affective-like states influence exploration patterns and mnemonic aspects, we aimed to verify whether the exploratory behaviors of two zebrafish populations showing distinct baselines of anxiety differ in two OFT sessions. Firstly, we assessed the baseline anxiety-like responses of short fin (SF) and leopard (LEO) populations using the novel tank test (NTT) and light-dark test (LDT) in 6-min trials. Fish were later tested in two consecutive days in the OFT, where the spatial occupancy and exploratory profile were analyzed for 30 min. In general, LEO showed pronounced diving behavior and scototaxis in the NTT and LDT, respectively, in which an "anxiety index" corroborated their exacerbated anxiety-like behavior. In the OFT, the SF population spent less time to establish the homebase in the 1st trial, while only LEO showed a markedly reduction in the latency to homebase formation in the 2nd trial. Both locomotion and homebase-related activities were decreased in the 2nd trial, in which animals also revealed increased occupancy in the center area of the apparatus. Moreover, we verified a significant percentage of homebase conservation for both populations, while only SF showed reduced the number of trips and increased the average length of trips. Principal component analyses revealed that distinct factors accounted for total variances between trials for each population tested. While homebase exploration was reduced in the 2nd trial for SF, an increased occupancy in the center area and hypolocomotion were the main factors that contribute to the effects observed in LEO during re-exposure to the OFT. In conclusion, our novel data support the homebase conservation in zebrafish subjected to independent OFT sessions, as well as corroborate a population-dependent effect on specific behavioral parameters related to exploration.

Expanding the use of homebase-related parameters to investigate how distinct stressful conditions affect zebrafish behaviors.

Stress is a physiological reaction that allows the organisms to cope with challenging situations daily. Thus, elucidating the behavioral outcomes following different stressors is of great importance in translational research. Here, we aimed to characterize the main factors which explain similarities and differences of two stress protocols on zebrafish exploratory activity. To answer this point, we performed behavioral analyses aiming to simplify the data structure associated with homebase-related measurements in an integrated manner. Adult zebrafish were exposed to conspecific alarm substance for 5 min (acute stress protocol - AS) or submitted to 7 days of unpredictable chronic stress (UCS). Immediately after AS or in the subsequent day following UCS (8th day), fish were individually tested in the open field and the behaviors were recorded for 30 min to posterior identification of homebase locations. For both protocols, behavioral clustering revealed two major clusters, grouping homebase- and locomotor-related parameters, respectively. While AS increased both positive and negative correlations between exploratory and locomotor endpoints, a significant increase in negative correlations was found in UCS-challenged fish. Comparison of the principal component analyses data set revealed a reduced exploratory activity using the homebase in AS group, while decreased locomotion in the periphery and anxiety-like behaviors were evidenced in UCS fish. In conclusion, our findings revealed a different structure of behavior in zebrafish following AS and UCS protocols, supporting the existence of distinct behavioral strategies to cope with acute and chronic stress. Furthermore, we expand the use of homebase-related measurements as a valuable tool to investigate complex behavioral modulations in future translational neuropsychiatry research.

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.

Long-term proline exposure alters nucleotide catabolism and ectonucleotidase gene expression in zebrafish brain.

Hyperprolinemia is an inherited disorder of proline metabolism and hyperprolinemic patients can present neurological manifestations, such as seizures cognitive dysfunctions, and psychotic disorders. However, the underlying mechanisms of these symptoms are still unclear. Since adenine nucleotides play crucial roles in neurotransmission and neuromodulation, we evaluated the in vivo and in vitro effects of proline on ectonucleotidase activities and gene expression in zebrafish brain. For the in vivo studies, animals were exposed at two proline concentrations (1.5 and 3.0 mM) during 1 h or 7 days (short- or long-term treatments, respectively). For the in vitro assays, different proline concentrations (ranging from 3.0 to 1000 µM) were tested. Short-term proline exposure did not promote significant changes on the ectonucleotidase activities and gene expression. Long-term proline exposure significantly increased ATP catabolism in both concentrations tested (14 % and 22 %, respectively), whereas ADP and AMP hydrolysis were increased only at 3.0 mM proline (21 % and 17 %, respectively) when compared to control. Moreover, the relative gene expression of enpd3 increased in both treated groups after long-term proline, whereas enptd1 increased only at 3.0 mM proline. Proline in vitro did not promote significant changes on ectonucleotidase activities. Altogether, these data indicate that the enzymes responsible for the control of extracellular nucleotides levels might be altered after proline exposure in zebrafish, contributing to better understand the pathophysiology of this disease. Moreover, such findings might facilitate the use of the zebrafish as a complementary vertebrate model for studying inborn errors of amino acid metabolism.

A novel behavioral paradigm to measure anxiety-like behaviors in zebrafish by the concomitant assessment of geotaxis and scototaxis.

Pathological anxiety is a set of diseases characterized by specific clinical manifestations and the use of alternative models may provide novel insights in translational neurobehavioral research. In zebrafish, the separate performance of novel tank and light dark tests in different order to assess anxiety using a same animal may provide conflicting data due to the battery effect and/or time-drug-response and variability across tests. To improve data reliability, we aimed to characterize a novel behavioral paradigm to measure geotaxis and scototaxis as anxiety-like responses in the same trial. The novel apparatus consisted of four colored-compartments, with specific white- and black sections delimited in both bottom and upper areas of the tank. The main baseline responses of zebrafish in the novel apparatus were measured and animals were further exposed to modulators of anxiety. Zebrafish showed robust habituation to novelty stress during the 6-min trial with preference for the black section while exploring the top area. Fluoxetine (100 µg/L, 15 min) reduced geotaxis and scototaxis and ketamine (20 mg/L, 20 min) decreased geotaxis and increased the distance traveled in the black section while exploring the top, possibly due to the increased circling behavior. As anxiogenic modulators, conspecific alarm substance (3.5 mL/L, 5 min) exacerbated risk assessment, geotaxis, and scototaxis, whereas caffeine (10 mg/L, 15 min) increased geotaxis and exploration in the black section of the top area. Since important correlations were also found for relevant anxiety-like behaviors, our findings support the predictive validity of this novel paradigm to simultaneously assess geotaxis and scototaxis in zebrafish. Moreover, it fully adheres to the 3Rs principle of animal experimentation of reducing the number of subjects tested, execution time, also minimizing a potential battery effect.

Ketamine acutely impairs memory consolidation and repeated exposure promotes stereotyped behavior without changing anxiety- and aggression-like parameters in adult zebrafish.

Ketamine is a dissociative anesthetic in human and veterinary clinic, as well as an abuse drug that acts on several neurotransmitter systems. The use of alternative animal models, such as zebrafish, is emerging to study the effects of drugs on neurobehavioral responses. Here, we evaluated the effects of ketamine on memory consolidation (acute protocol), as well as on anxiety-, aggressive-like behavior, and whole-body cortisol levels in adult zebrafish after a repeated exposure. For the acute protocol, fish were tested in the inhibitory avoidance task (training and testing with a 24-hour interval). Immediately after the training session, fish were exposed to ketamine (0, 2, 20, or 40 mg/L) for 20 min. The exploratory activity was also measured 24 h after acute exposure to exclude the influence of impaired locomotion on memory performance. For the repeated exposure, animals were exposed to the same concentrations of ketamine for 20 min (7 days). After the last exposure (24 h later), anxiety- and aggression-like behaviors were quantified in the novel tank and mirror-induced aggression tests, respectively, as well as whole-body cortisol levels measurements were performed. The highest ketamine concentration tested (40 mg/L) acutely induced a slight memory impairment in the inhibitory avoidance task without changing locomotion and anxiety-like behaviors. Although locomotion, anxiety-, aggressive-like behaviors, and whole-body cortisol levels did not change after repeated exposure, 40 mg/L ketamine increased circling behavior. Overall, our data reinforce that ketamine acutely affects multiple behavioral domains in zebrafish, in which repeated ketamine exposure elicits stereotyped behavior, without changing locomotion, aggression, and anxiety/stress-related parameters.

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.

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.