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.

Complexed amino acid minerals vs. bis-glycinate chelated minerals: Impact on the performance of old laying hens.

The present study was to evaluate the effect of trace minerals (Zn, Mn, and Cu) from complexed amino acid minerals (ZMCAA) and bis-glycinate chelated minerals (ZMCGly) in laying hen diets on performance, internal and external egg quality, yolk mineral deposition, intestinal morphometry, and bone characteristics. From 78 to 98 weeks of age, 400 White LSL-Lite strain laying hens were distributed in a randomized design with 4 treatments with 10 replicates per treatment. Treatments were distributed in a 2 × 2 factorial arrangement using either Zn, Mn, and Cu of ZMCAA or ZMCGly source at 2 levels: low (20, 20, and 3.5 mg/kg of Zn, Mn, and Cu, respectively) or high (40, 40, and 7 mg/kg of Zn, Mn, and Cu, respectively). The analysis of variance was performed, and in cases where differences were observed, the means were compared using Tukey's test (P < 0.05). The source and level of trace mineral supplementation had a significant impact on the performance of laying hens. Hens fed ZMCAA had higher egg production (P = 0.01), egg weight (P = 0.02), egg mass (P = 0.01), and lower feed conversion ratio (P = 0.05) compared to those fed ZMCGly. The ZMCAA supplementation showed higher albumen height (P = 0.01), albumen weight (P = 0.01), and eggshell thickness (P < 0.01). The deposition of Zn (P < 0.01), Mn (P < 0.01), and Cu (P < 0.01) in the egg yolk was greater for hens received ZMCAA. Tibia weight (P = 0.04) and bone densitometry (P < 0.01) in the tibia were higher with ZMCAA supplementation. In the small intestine, ZMCAA resulted in longer villi (P = 0.02) and shorter crypt depth (P = 0.01) in the duodenum. Jejunum and ileum measurements were influenced by the level and source of trace minerals (P < 0.05). Laying hens fed ZMCAA exhibited superior performance, egg quality, deposition of trace minerals in the egg yolk, and bone density compared to hens fed ZMCGly. In this study, older laying hens supplemented with ZMCAA at lower levels demonstrated adequate levels of supplementation.

The Impact of Phytase and Different Levels of Supplemental Amino Acid Complexed Minerals in Diets of Older Laying Hens.

A study was conducted to evaluate the effects of different sources and levels of supplemental amino acid-complexed minerals (AACM), with and without enzyme phytase (EZ). A total of 512 Dekalb White laying hens at 67 weeks of age were used in a 2 × 3 + 2 factorial arrangement of 8 treatments and 8 replications each. The main effects included EZ supplementation (10,000 FTU kg-1) and AACM inclusion level (100, 70, and 40% of inorganic mineral recommendations), plus 2 control treatments. The group of hens fed AACM-100 showed lower feed intake than the inorganic mineral (IM) group. The diet containing AACM-EZ-70 provided a higher (p < 0.05) laying percentage and a lower (p < 0.05) feed conversion ratio than both the IM and IM-EZ diets. The groups fed AACM-EZ-40, AACM-EZ-100, and AACM-70 produced heavier yolks (p < 0.05). Hens fed IM laid eggs with the lowest yolk and albumen weights (p < 0.05). Layers fed with AACM-100 and AACM-70 produced the most resistant eggshells to breakage (p < 0.05). In diets containing phytase, the optimal AACM recommendations for better performance and egg quality in older laying hens are: 42, 49, 5.6, 28, 0.175, and 0.70 mg kg-1 for Zn, Mn, Cu, Fe, Se, and I, respectively.

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.

New approaches concerning the testis of Astyanax lacustris (Characidae): immunohistochemical studies.

Astyanax lacustris, locally known as lambari-do-rabo-amarelo, is a study model for Neotropical fish. Testis of A. lacustris shows deep morphophysiological changes throughout the annual reproductive cycle. This work analyzed the distribution of claudin-1, actin, and cytokeratin as elements of the cytoskeleton in germinal epithelium and interstitium; the distribution of type I collagen, fibronectin, and laminin as extracellular matrix compounds; and the localization of androgen receptor in the testis of this species. Claudin-1, cytokeratin, and actin were present in the Sertoli cells and modified Sertoli cells, and actin was also detected in peritubular myoid cells. Type I collagen were in the interstitial tissue, laminin in the basement membrane of germinal epithelium and endothelium, but fibronectin was additionally detected in the germinal epithelium compartment. The labeling of androgen receptor was higher in peritubular myoid cells and undifferentiated spermatogonia, and weaker labeling was detected in type B spermatogonia. Therefore, the present work highlights new aspects of the biology of the testis of A. lacustris, and contribute to amplify the understanding of this organ.

Behavioral profile of adult zebrafish acutely exposed to a selective dopamine uptake inhibitor, GBR 12909.

BACKGROUND: The dopamine transporter (DAT) is the main regulator of dopamine concentration in the extrasynaptic space. The pharmacological inhibition of the DAT results in a wide spectrum of behavioral manifestations, which have been identified so far in a limited number of species, mostly in rodents. AIM: Here, we used another well-recognized model organism, the zebrafish (Danio rerio), to explore the behavioral effects of GBR 12909, a highly-affine selective DAT blocker. METHODS: We evaluated zebrafish locomotion, novelty-related exploration, spatial cognition, and social phenotypes in the novel tank, habituation and shoaling tests, following acute 20-min water immersion in GBR 12909. RESULTS: Our findings show hypolocomotion, anxiety-like state, and impaired spatial cognition in fish acutely treated with GBR 12909. This behavioral profile generally parallels that of the DAT knockout rodents and zebrafish, and it overlaps with behavioral effects of other DAT-inhibiting drugs of abuse, such as cocaine and D-amphetamine. CONCLUSION: Collectively, our data support the utility of zebrafish in translational studies on DAT targeting neuropharmacology and strongly implicate DAT aberration as an important mechanisms involved in neurological and psychiatric diseases.

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.

Disease Behavior and Treatment Response of Special Histological Types of Triple-Negative Breast Cancer.

BACKGROUND: Special histological types (SHT) of triple-negative breast cancer (TNBC) are a heterogeneous group of rare poorly understood diseases. We aimed to evaluate the clinical features, treatment, and outcomes of patients with SHT of TNBC. METHODS: We evaluated patients with a SHT of TNBC treated in a cancer center between 2009 and 2020. The endpoints were characterization of clinical and pathological features, pathologic complete response (PCR) rate after neoadjuvant chemotherapy, disease-free survival (DFS), progression-free survival, and overall survival (OS). RESULTS: The 132 patients included had the following histologies: metaplastic (n=71), medullary pattern (n=14), lobular (n=12), adenoid cystic (n=12), apocrine (n=10), and others (n=13). Metaplastic, lobular, and medullary pattern tumors had higher grade (66.6-85.7% grade 3); adenoid cystic and apocrine had mainly grade 1-2 (70-83.3%). Metaplastic and lobular carcinomas had higher disease stages (47.8% and 58.2% stages III-IV). PCR rates were 10.3% for metaplastic and 33.3% for lobular carcinomas, with 5-year DFS rates of 56% and 51.4%. Medullary pattern carcinomas had a great response to treatment, with PCR rate of 100%, and 5-year DFS rate of 92.8%. Apocrine carcinomas also had favorable prognosis, with no recurrence after early disease treatment, and 5-year DFS rate of 83.3%. Adenoid cystic carcinomas had intermediate prognosis, with 5-year DFS rate of 66.6%. CONCLUSION: SHT of TNBC encompasses heterogeneous malignancies with distinct behaviors. Lobular and metaplastic carcinomas showed high aggressiveness and poor treatment response, while medullary pattern and apocrine carcinomas had favorable outcomes. Treatment strategies focus on molecular features of each of these diseases are warranted.

Outbreak caused by the SARS-CoV-2 Omicron variant in the psychiatric ward of a general hospital in Brazil.

BACKGROUND: An outbreak of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) Omicron variant was detected in the psychiatric ward of a general hospital in Brasília, Brazil. METHODS: We report the investigation, clinical outcomes, viral sequencing, and control measures applied to outbreak containment. RESULTS: The overall attack rate was 95% (23/24) in a period of 13 days. Among the cases, 78% (18/23) were vaccinated and 17% (4/23) required intensive care. The Omicron variant was isolated from the 19 sequenced samples. CONCLUSIONS: The findings highlight the potential harm that highly transmissible variants may generate among hospitalized populations, particularly those with comorbidities.

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.

Chemical characterisation by UPLC-Q-ToF-MS/MS and antibacterial potential of Coffea arabica L. leaves: A coffee by-product.

INTRODUCTION: Coffea arabica L. leaves are considered a by-product of the coffee industry however they are sources of several bioactive compounds. OBJECTIVES: This study aimed to evaluate the chemical composition and the in vitro antibacterial activity of the lyophilised ethanol extract of arabica coffee leaves (EE-CaL). MATERIAL AND METHODS: The chemical characterisation of EE-CaL was performed using ultra-performance liquid chromatography coupled to quadrupole time-of-flight mass spectrometry (UPLC-Q-ToF-MS/MS). The in vitro antibacterial effect of EE-CaL was evaluated using the broth microdilution method and the adapted drop plate agar method to determine the minimum inhibitory concentration (MIC) and the minimum bactericidal concentration (MBC), respectively. RESULTS: The chemical analysis of EE-CaL revealed the presence of compounds from the alkaloid class, such as trigonelline and caffeine, in addition to the phenolic compounds such as quinic acid, 5-caffeoylquinic acid, caffeic acid-O-hexoside, mangiferin, (epi)catechin, (epi)catechin monoglucoside and procyanidin trimer. Regarding the antibacterial potential, EE-CaL was active against Gram-positive and Gram-negative bacteria, being more effective against Escherichia coli (ATCC 25922) (MIC = 2500 µg/mL and bactericidal effect). CONCLUSION: The results of this research suggest that coffee leaves, a by-product, possess compounds with antibacterial properties. Thus, further studies with coffee leaf extracts must be carried out to relate the compounds present in the extract with the antibacterial activity and find the mechanisms of action of this extract against bacteria.

Sensitivity of different DNA extraction methods and PCR to detect resistance in patients with leprosy stratified by the bacilloscopic index.

INTRODUCTION: Antimicrobial resistance in leprosy is an emerging problem, and the quantitative impact of low bacilloscopic indexes (BIs) on the sensitivity of molecular tests is unknown. We aimed to evaluate the sensitivity of gene sequencing for the detection of mutations related to antimicrobial resistance in Mycobacterium leprae in patients with low BIs using an analytical model. METHODS: Patients with leprosy were included and divided into two groups depending on their BIs (≥ 2+ and < 2+). The sensitivities of the two DNA extraction methods were compared after amplifying and sequencing the repetitive element (RLEP), folP1, rpoB and gyrA in M. leprae. RESULTS: We included 56 patients with leprosy: 35 had BIs less than 2+ (22 had negative slit-skin smear [SSS] results) and 21 patients had BIs greater than or equal to 2+. The sensitivity of the amplification of the RLEP target and the gene sequencing of folP1, rpoB and gyrA was 50 to 70% lower in patients with a BI less than 2+ and was significantly reduced in patients with lower BIs for all targets (p < 0.001). One patient had a mutation in the folP1 gene, and 14 patients had mutations in the gyrA gene, but no mutations related to antimicrobial resistance were found. CONCLUSIONS: We can conclude that the sensitivity of molecular tests is directly related to the BI, but these tests can still detect up to 20% of the targets in patients with BIs < 2+. New strategies to improve the sensitivity for detecting antimicrobial resistance in leprosy patients and reasonable clinical criteria for follow-up and the introduction of alternative treatments must be developed.

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.

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.

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.