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 outline future directions in both etiological and therapeutic research. Lastly, we reinforce the use of zebrafish as a promising tool to unravel the biological bases of OCD, as well as novel pharmacological therapies in the field.

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.

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.

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.

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.

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.

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.

Utility of cockroach as a model organism in the assessment of toxicological impacts of environmental pollutants.

Environmental pollution is a global concern because of its associated risks to human health and ecosystem. The bio-monitoring of environmental health has attracted much attention in recent years and efforts to minimize environmental contamination as well as to delineate toxicological mechanisms related to toxic exposure are essential to improve the health conditions of both humans and animals. This review aims to substantiate the need and advantages in utilizing cockroaches as a complementary, non-mammalian model to further understand the noxious impact of environmental contaminants on humans and animals. We discuss recent advances in neurotoxicology, immunotoxicology, reproductive and developmental toxicology, environmental forensic entomotoxicology, and environmental toxicology that corroborate the utility of the cockroach (Periplaneta americana, Blaptica dubia, Blattella germanica and Nauphoeta cinerea) in addressing toxicological mechanisms as well as a sensor of environmental pollution. Indeed, recent improvements in behavioural assessment and the detection of potential biomarkers allow for the recognition of phenotypic alterations in cockroaches following exposure to toxic chemicals namely saxitoxin, methylmercury, polychlorinated biphenyls, electromagnetic fields, pharmaceuticals, polycyclic aromatic hydrocarbon, chemical warfare agents and nanoparticles. The review provides a state-of-the-art update on the current utility of cockroach models in various aspects of toxicology as well as discusses the potential limitations and future perspectives.

Influence of acute and unpredictable chronic stress on spatio-temporal dynamics of exploratory activity in zebrafish with emphasis on homebase-related behaviors.

The open field is a suitable task to analyze the sequential organization of exploratory activity and the homebase formation represents an important feature of environmental recognition. Although the zebrafish can define homebase locations, there are no data reporting how stressful conditions modulate complex behaviors of this aquatic species in the open field so far. Here, we aimed to characterize the spatio-temporal exploratory activity of adult zebrafish in the open field test, as well as to verify the responsiveness of homebase-related parameters to acute stress (AS) and unpredictable chronic stress (UCS) protocols. Animals were exposed to conspecific alarm substance for 5 min or subjected to a 7-days stress protocol using distinct stressors in an unpredictable manner. Immediately after exposure to AS or 24 h after UCS, fish were individually placed in a circular tank and their behaviors were recorded for 30 min to identify the respective homebase for each animal. We observed that UCS, but not AS, increased thigmotaxis compared to the non-stressed fish. Notably, the sequential organization of exploratory activity showed robust differences depending on the stress protocol. After the first 15 min of trial, AS-challenged fish apparently used the homebase to organize briefly explorations to the environment. Conversely, the UCS group was more immobile in the homebase after periodically performing 'swimming bursts' to the periphery with a greater number of stops per trip. Physiological stress responses were confirmed by the increased whole-body cortisol in both AS and UCS groups. In conclusion, our novel findings report a different exploratory profile related to stress responses in adult zebrafish tested in the open field, supporting the sensitivity of homebase-related parameters to manipulations that modulate affective-like states.

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.

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.

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.

Zebrafish as a potential non-traditional model organism in translational bipolar disorder research: Genetic and behavioral insights.

Bipolar disorder (BD) is a severe and debilitating illness that affects 1-2% of the population worldwide. BD is characterized by recurrent and extreme mood swings, including mania/hypomania and depression. Animal experimental models have been used to elucidate the mechanisms underlying BD and different strategies have been proposed to assess BD-like symptoms. The zebrafish (Danio rerio) has been considered a suitable vertebrate system for modeling BD-like responses, due to the genetic tractability, molecular/physiological conservation, and well-characterized behavioral responses. In this review, we discuss how zebrafish-based models can be successfully used to understand molecular, biochemical, and behavioral alterations paralleling those found in BD. We also outline some advantages and limitations of this aquatic species to examine BD-like phenotypes in translational neurobehavioral research. Overall, we reinforce the use of zebrafish as a promising tool to investigate the neural basis associated with BD-like behaviors, which may foster the discovery of novel pharmacological therapies.

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.

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.

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.

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.

Understanding sex differences in zebrafish pain- and fear-related behaviors.

Sex is an important variable in translational biomedical research. While overt sex differences have been reported for pain and fear-like behaviors in humans and rodents, these differences in other popular model organisms, such as zebrafish, remain poorly understood. Here, we evaluate potential sex differences in zebrafish behavioral responses to pain (intraperitoneal administration of 5% acetic acid) and fear stimuli (exposure to alarm substance). Overall, both male and female zebrafish exposed to pain (acetic acid injection) show lesser distance traveled, fewer top entries and more writhing-like pain-related behavior vs. controls, whereas female fish more robustly (than males) altered some other pain-like behaviors (e.g., increasing freezing episodes and time in top) in this model. In contrast, zebrafish of both sexes responded equally strongly to fear evoked by acute alarm substance exposure. Collectively, these findings emphasize the growing importance of studying sex differences in zebrafish behavioral and pain models.