Opioidergic modulation of stress-induced hyperalgesia in adult zebrafish

Opioidergic modulation of stress-induced hyperalgesia in adult zebrafish | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Opioidergic modulation of stress-induced hyperalgesia in adult zebrafish Fabiano Costa, Lana Ferreira, Lucca Lima, Julia Canzian, Allan Kalueff, and 2 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8855618/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 9 You are reading this latest preprint version Abstract Depression and pain share overlapping central neurobiological pathways that represent key pharmacological targets in neuropsychiatric and analgesic research. The relationship between these two conditions is bidirectional, with chronic pain contributing to the development or exacerbation of depressive symptoms, and depression intensifying the perception and tolerance of pain. However, the neuropharmacological mechanisms by which unpredictable chronic stress (UCS) modulates nociception remain poorly understood in translational vertebrate models. Here we pharmacologically characterized stress-induced nociceptive responses using a 7–14-day UCS protocol in zebrafish, followed by intraperitoneal administration of 1–5% (v/v) acetic acid to induce nociceptive responses. Behavioral assays were performed immediately after the injection, testing abdominal constriction (writhing-like behavior) as a pain-related endpoint, and locomotor activity levels as an additional behavioral measure related to nociception and stress. The UCS exposure elevated whole-body cortisol levels, which were attenuated by morphine but not by diclofenac, supporting the involvement of central stress–pain neuropharmacological pathways. Together, these findings establish a pharmacologically tractable zebrafish model of stress-induced hyperalgesia with translational relevance for CNS-targeted analgesic discovery, highlighting the overlap between stress-related and nociceptive pathways and supporting this species as a model to investigate stress–pain comorbidity. zebrafish chronic stress nociception opioidergic system hyperalgesia Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 1. Introduction Affective disorders and pain have long been recognized as comorbid conditions (Han and Pae 2015 ), with chronic pain worsening depressive symptoms, and depression, in turn, intensifying pain perception (Hooten 2016 ; Yao et al. 2023 ). Clinical studies suggest that individuals experiencing chronic stress are more susceptible to developing chronic pain disorders, including fibromyalgia and irritable bowel syndrome, highlighting the critical role of stress and stress-related central nervous system (CNS) deficits in pain perception (Borsook et al. 2012 ; Vachon-Presseau et al. 2013 ). Importantly, shared pathways of stress and pain involve central monoaminergic and opioidergic neurotransmission, systems that represent key pharmacological targets in neuropsychiatric and analgesic therapies and play a crucial role in modulating mood and pain (Sheng et al. 2017 ; Haase and Brown 2015 ). The overlap of these neural circuits, especially the limbic system responsible for both emotional- and pain-processing responses, further supports the integrated nature of their pathogenesis (Sheng et al. 2017 ; Meerwijk et al. 2013 ). Chronic neuroinflammation, a common feature of both depression and certain types of pain, also contributes to these overlapping pathogenetic mechanisms (Dooley et al. 2018 ; Walker et al. 2014 ; Lee and Giuliani 2019 ). Animal experimental models are essential tools for investigating the neuropharmacological mechanisms underlying stress–pain interactions (Ulrich-Lai and Herman 2009 ). A common protocol used to assess depression-like states in animal models is the exposure to chronic stress, such as the unpredictable chronic stress (UCS) (Burstein and Doron 2018 ; Piato et al. 2011 ). Based on repeated exposure to varied and unpredictable stressors, the rodent UCS model has been widely used to mimic depression-related behaviors and neurochemical changes observed in humans (Willner 2017 ). These neurobehavioral effects of UCS are associated with dysregulation of the hypothalamic-pituitary-adrenal (HPA) axis, increased pro-inflammatory cytokines, and impaired serotonergic function, all of which contribute to both depressive symptoms and heightened pain sensitivity (hyperalgesia) (Slavich and Irwin 2014 ; Burke et al. 2017 ). Complementing rodent models, the zebrafish ( Danio rerio ) provides a scalable vertebrate platform for neuropharmacological investigations of complex CNS disorders, including depression and pain (Stewart et al. 2014 ; Kysil et al. 2017 ). The UCS paradigms have successfully been adapted for zebrafish, demonstrating overt behavioral, neurochemical and genomic phenotypes that generally parallel those observed in rodent stress models (Wong et al. 2010 ; Piato et al. 2011 ). Indeed, UCS-exposed zebrafish exhibit anxiety-like behaviors, reduced exploration and altered monoaminergic neurotransmission, paralleling rodent and clinical findings (Kysil et al. 2017 ; Maximino et al. 2010 ). Since stress-induced changes affect mood-related behaviors (Piato et al. 2011 ; Oliveira et al. 2013 ), whereas mood and pain share common pathogenetic pathways (Han and Pae 2015 ; Yang and Chang 2019 ; De Ridder et al. 2021 ), stress may impact pain sensitivity, hence supporting the interplay between chronic stress, depression, and pain. Despite the growing interest in zebrafish models for studying pain and depression (Kalueff et al. 2014 ; Costa et al. 2019b ; Costa et al. 2022 ), the relationship between stress and pain responses in these fish remains poorly understood. However, it may help clarify the evolutionary conserved link between pain- and depression-like phenotypes across vertebrates. Thus, we hypothesize that unpredictable chronic stress (UCS) produces a pharmacologically tractable hyperalgesic phenotype mediated by central mechanisms in zebrafish. Understanding these mechanisms is critical for developing CNS-targeted pharmacological strategies that address stress–pain comorbidity. Here, we pharmacologically characterize a zebrafish model of stress-induced hyperalgesia using centrally and peripherally acting analgesics to dissociate underlying mechanisms. The present study applied a 7–14-day UCS paradigm to induce depression-like phenotypes in zebrafish, followed by intraperitoneal administration (i.p.) of a nociceptive agent (1–5% acetic acid, AA) and quantifying pain-like behaviors using the aquatic writhing assay. To explore the underlying mechanisms of UCS-induced hyperalgesia, we further examined the effects of morphine, a centrally acting analgesic, and diclofenac, a peripheral anti-inflammatory agent. This combined protocol allows for the concurrent assessment of depression- and pain-related responses in the same organism, providing a translationally relevant platform to investigate shared mechanisms underlying affective and nociceptive processes and to distinguish central versus peripheral modulation of stress-induced hyperalgesia. 2. Methods 2.1. Animals A total of 300 adult wild-type zebrafish (AB strain; 5–7 months old; ~50:50 male:female) were maintained in automated recirculating systems (ZebTEC, Tecniplast, Italy) with reverse-osmosis-filtered water and conditions, as in (Westerfield 1993 ), for at least 2 weeks prior to the beginning of experiments. Since no sex differences were observed in AA–induced pain in zebrafish (Costa et al., 2019a ) and their cortisol measures (Costa et al., 2023 ), male and female data were pooled to maximize statistical power and to adhere to the 3Rs principle of animal experimentation. Animals were obtained from an in-house breeding colony and maintained under standard conditions at the authors’ facility. Fish were maintained at 28℃ ± 2℃, pH 7.0–7.5, water conductivity 300–700 µS, ammonia < 0.02 mg/L, hardness 80–300 mg/L, nitrite < 1 mg/L, nitrate < 50 mg/L, and chloride 0 mg/L, under a 14 h light:10 h dark photoperiod cycle (lights on: 07:00 am) and fed with commercial flakes (TetraMin Tropical Flake Fish™) three times a day. Water pH and conductivity were monitored daily, and nitrogen compounds were measured weekly, in accordance with established zebrafish husbandry standards (Alestrom et al. 2020 ). All protocols were approved by the Institutional Animal Care Committee (CEUA, protocol number 11607) and animal experimentation fully adhered to the National Institutes of Health Guide for the Care and Use of Laboratory Animals and the guidelines of the National Animal Experimentation Control Council (CONCEA). The AB fish were selected here as a common zebrafish strain widely used in neurobehavioral assays (Bertoncello et al. 2024 ). 2.2. Chemicals The following chemicals were used in the present study: acetic acid (AA; Merck KGaA, Darmstadt, Germany, CAS No. 64-19-7), morphine sulfate (MOR; Sigma-Aldrich, St. Louis, USA, CAS No. 6211-15-0), phosphate buffered saline (PBS; Sigma-Aldrich), diclofenac sodium (DS; Novartis, São Paulo, Brazil; CAS No. 15307-79-6). 2.3. Unpredictable chronic stress protocol (UCS) Following a two-week acclimation period, the UCS was performed based on a protocol described previously, with some modifications (Piato et al. 2011 ). Briefly, fish were submitted twice a day to various stressors for 7-day/14-day, as summarized in Table 1 . UCS stressors used included Change of environments : keeping the tanks surrounded by a different color from the animal's natural environment (green, yellow, red) for 6 h; Heating : increasing the water temperature of the tank up to 33°C for 30 min; Social isolation : animals were placed individually in a 250 mL beaker for 6 h; Cooling : cooling the tank water down to 23°C for 30 min; Crowding : placing a group of 10 animals for 50 min in a 250 mL beaker; Low water level : decreasing the water level in housing tanks until the animals' dorsal body walls are exposed for 2 min; Changing water : animals were kept in the tank while the water was changed three consecutive times; Changing tanks : animals were transferred to a fresh tanks three times; Net chasing : chasing animals for 8 min with a net. Aeration and temperature were controlled during each stressor presentation (except during heating and cooling stress). To prevent habituation and maintain stress unpredictability, the time and sequence of stressor presentations were changed daily. An unstressed control group remained intact in the same room for 7 or 14 days, respectively. Table 1 Unpredictable chronic stress protocol. Week Thursday Friday Saturday Sunday Monday Tuesday Wednesday Week 1 Water change Social isolation Crowding Low water level Heating Net chasing Crowding Heating Cooling Change environment Tank change Cooling Change environments Social isolation Week 2 Low water level Tank change Heating Social isolation Net chasing Cooling Heating Cooling Crowding Water change Change environments Low water level Tank change Crowding Week 3 Behavioral analysis 2.4. Assessing the effects of acetic acid on unpredictable chronic stress protocol (UCS) Zebrafish ( n = 8–10 per group) were randomly selected from at least three housing tanks (for non-stressed control animals) or from 7-day/14-day UCS batches (for stressed animals UCS7 and UCS14, respectively). Because no differences were observed in body curvature indexes when 5 vs . 10 animals were treated with phosphate-buffered saline (PBS) or acetic acid (AA) in previous reports (Costa et al. 2019b ), and given prior analyses demonstrating sufficient statistical power (α = 0.05, power = 0.80) for detecting significant differences in behavioral endpoints, a sample size of 8–10 animals per group was considered appropriate. Moreover, this sample size is consistent with similar experimental designs that successfully minimized intra-group variability and ensured reproducible results, while adhering to the 3Rs principles of ethical animal experimentation. Animals were randomly selected from at least three housing tanks, gently handled, and anesthetized in cold water (< 5 s), as previously described (Kinkel et al. 2010 ). They were briefly immobilized using a small wet net and injected intraperitoneally through the midline between the pelvic fins with PBS (control), UCS7 and UCS14 (stress groups) and AA (1.0 and 5.0% vol/vol in PBS), as well as UCS7 or UCS14 following 1.0% AA. The i.p. injection occurred in a short period (5 s), and was preferred over other methods for i.p. administration in fish ( e.g. , chemical anesthesia with tricaine or similar agents) to avoid confounding effects of anesthesia on the pharmacological effects examined here. Importantly, this protocol allows a fast evaluation of the swimming activity after fish return to the water, minimizing potential pharmacological interference on complex behaviors ( e.g. , immobility and locomotion). Behavioral recordings started as soon as the fish regained postural equilibrium (~ 1 min post-injection), thus allowing a more accurate characterization of acute pain responses (Costa et al. 2019b ). Because no differences in body curvature indexes were observed between male and female following a 1.0% AA injection (Costa et al. 2019b ; Rambo et al. 2017 ), male and female were combined as a unisex group. All injections were performed using a BD Ultra-fine™ 30U syringe (needle size 6 x 0.25 mm) with 10 µL (shown not to impair normal zebrafish behaviors (Kinkel et al. 2010 ; Richetti et al. 2011 )). The concentrations of AA used here were selected based on previous studies (Taylor et al. 2017 ; Sneddon et al. 2003 ; Costa et al. 2019b ). 2.5. Assessing the effects of morphine and diclofenac on unpredictable chronic stress protocol (UCS) following acetic acid Zebrafish ( n = 8–10 per group) were randomly selected from at least three housing tanks (for non-stressed animals) or from UCS7 batches (for stressed animals). Animals were gently handled, anesthetized in cold water (Kinkel et al. 2010 ), and briefly immobilized using a small wet net and individually injected i.p., as described above. To assess the potential antinociceptive effects of diclofenac and morphine, animals were assigned into the PBS, morphine (MOR), and diclofenac (DS) as control groups, as well as 1.0% AA, UCS7 + 1.0% AA, UCS7 + 1.0% AA + DS, UCS7 + 1.0% AA + MOR. The UCS7 animals were pretreated with DS (40 mg/kg) 15 min prior to the test and further injected with 1.0% AA. Diclofenac was selected as a clinically approved nonsteroidal anti-inflammatory drug (Altman et al. 2015 ) widely used in experimental models to assess nociceptive-related phenotypes. Morphine was chosen here as classical, clinically approved and potent opioid analgesic drug (Pathan and Williams 2012 ) widely used in experimental models of pain (Hamann et al. 2016 ; Llorca-Torralba et al. 2018 ; Rodrigues-Filho et al. 2004 ; Bjorkman et al. 1992 ), with a known sensitivity in zebrafish (Taylor et al. 2017 ; Costa et al. 2019b ). The doses of MOR and DS used here were selected based on previous reports (Sneddon et al. 2003 ; Taylor et al. 2017 ; Costa et al. 2019b ). 2.6. Behavioral analyses Immediately after the experimental procedures, fish were individually transferred to observation tanks (15×13 x 10 cm, length x height x width) with a 10-cm water depth and behavioral activities were recorded for 6 min using a digital camera (webcam Ultra HD Logitech® 4K PRO, Lausanne, Swiss). Behavioral recordings began as soon as the fish regained equilibrium (± 1 min post-injection), to ensure accurate acute pain assessments. Because no sex differences are observed in fish behavior following AA i.p. administration (Costa et al. 2019b ), male and female zebrafish were separated from their experimental tanks and divided randomly for each cohort by a computerized random number generator ( www.random.org ). Water conditions during behavioral testing matched those of the housing tanks. Behavioral testing was performed between 12:00 and 16:00, and zebrafish were fasted on the day of experiments (prior to behavioral testing). All experiments were performed as planned, and all analyses and all endpoints assessed were included without omission. After the experimental procedures, animals were immediately anesthetized in cold water (4°C) and then euthanized by decapitation following the cessation of breathing for > 30 s. 2.7. Assessing writhing-like behavior and locomotor activity All behavioral analyses were performed offline by experimenters blinded to the treatment groups, in an automated unbiased manner using the automated video-tracking software (EthoVision® XT11.5, Noldus IT, Wageningen, Netherlands) at 30 frames/s, to quantify the distance traveled (cm) and immobility duration (s), based on the center body point coordinates. Behavioral phenotypes assessed here fully adhered to a formal classification of fish behavior provided in the comprehensive Zebrafish Behavioral Catalogue (ZBC) (Kalueff et al. 2025 ; Kalueff et al. 2013 ). A part of zebrafish pain-related behaviors (ZBC1 term 1.104), writhing-like behavior (ZBC2 term 2.121) represents an abnormal constriction of body after the administration of AA, reflecting acute discomfort, stress and pain (Kalueff et al. 2025 ). The body curvature (reflecting a typical writhing-like behavior) was used as a behavioral endpoint to measure pain responses, as previously reported (Costa et al. 2019b ; Costa et al. 2023 ; Costa et al. 2019a ). Briefly, every 30 s of recording, screenshots of the animal's sagittal plane were taken (6 min = 12 screenshots), and later analyzed using ImageJ 1.45 for Windows (Research Services Branch, the National Institute of Mental Health, National Institutes of Health, NIH)). We selected three points of each animal: frontal (in the front of the head), central (middle of the animal body – between anal and dorsal fins) and posterior (at the dorsal fin), to estimate the angle of fish body curvature. The angles obtained were subtracted from 180° and multiplied by (-1) to estimate the body curvature index. The area under the curve (AUC) was calculated to quantify the total body curvature index over time, integrating the curvature index values derived from each anatomical point. AUC provides a comprehensive measure of both the magnitude and persistence of curvature, allowing for comparisons between individuals or treatment groups. Variations in the body curvature index were examined by two trained observers blinded to the experimental condition (inter-rater reliability > 0.90). Immobility was defined as a complete immobility (< 0.59 cm/s) except small movements of fins and eyes, accompanied by fast opercular beat rates, as described elsewhere (Wiprich et al. 2020 ). A representative image illustrating differences in body curvature of treated fish is shown in Supplementary Fig. 1. Moreover, a representative heatmap of zebrafish locomotor profiles under all treatments is shown in Supplementary Fig. 2. 2.8. Whole-body cortisol measurements Immediately after the treatments, fish were euthanized and then frozen in liquid nitrogen for 20–30 s for cortisol extraction. Whole-body cortisol was extracted following the ether-based extraction protocol (Mezzomo et al., 2019) and quantified in duplicates using a commercially enzyme-linked immunosorbent assay kit (EIAgen™ Cortisol test, BioChem ImmunoSystems) (Sink et al., 2008). A strong positive correlation was observed (r2 = 0.9413), and inter- and intra-assay coefficients of variation values were low (7–10% and 5–9%, respectively). Results were expressed as ng cortisol/g tissue. 2.9. Statistical analyses and data handling Data normality and homogeneity of variances were analyzed by Kolmogorov–Smirnov and Bartlett’s tests, respectively. Changes in body curvature and behavioral activity were analyzed by one-way analysis of variance (ANOVA, factor: treatment), followed by post-hoc Tukey's test multiple comparison test for significant ANOVA data. Results were expressed as means ± standard error of the mean (S.E.M.). Immobility duration was log-transformed to meet assumptions of normality, the analyses were then performed on transformed data. Inter-rater reliability was assessed by Spearman’s rank correlation (r > 0.85). Statistical analysis was performed using the GraphPad Prism9 software (GraphPad Software, Boston, USA). All fish tested were included in the final analysis without attrition or exclusion, and all planned analyses are presented here. All behavioral and statistical analyses were performed by experimenters that remained blinded to the treatment groups. 3. Results 3.1. Acetic acid changes body curvature in zebrafish exposed to the UCS protocol Although 1.0% AA alone did not change AUC relative to controls, a significant treatment effect was observed ( F (6, 43) = 37.27; p < 0.0001), as UCS7/UCS14 following 1.0% AA injection increased AUC compared to PBS control group (Fig. 1 A). Likewise, while UCS7 and UCS14 groups and control fish showed unaltered (absent) writhing-like body curvature, AUC increased in both 5.0% AA and 1.0% AA + UCS7/14 groups (Fig. 1 A-B; F (3, 31) = 24.69; p < 0.0001). All treated groups showed a significant reduction in traveled distance compared to controls (Fig. 2 A; F (5, 49) = 31.69; p < 0.0001). Immobility duration significantly increased in the UCS7 or UCS14 groups and the 1.0% AA + UCS7/14 groups compared to controls (Fig. 2 B; F (5, 49) = 52.13; p < 0.0001). In contrast, 1.0% AA injection alone did not affect zebrafish immobility duration. Because both UCS7 and UCS14 evoked similar results in all tests performed here, we chose the UCS7 protocol for further experiments, to comply with bioethical principles aimed at reducing bioexperimentation. 3.2. Morphine, but not diclofenac, reduces UCS effects on acetic acid-induced pain Zebrafish treated with UCS7 + 1.0% AA showed significantly higher AUC compared to control fish. While diclofenac pretreatment did not reverse this increase, and morphine co-administration did not fully restore AUC to baseline, it significantly reduced AUC compared to the 1.0% AA + UCS7 and diclofenac-treated groups (Fig. 3 A; F (6, 39) = 52.48; p < 0.0001). Furthermore, diclofenac pretreatment did not reverse the AUC increase over time, whereas morphine co-administration attenuated this increase (Fig. 3 B; F (5, 32) = 56.81; p < 0.0001). Although both morphine and diclofenac reversed the reduced distance traveled in UCS7 + 1.0% AA-treated zebrafish (Fig. 4 A; F (6, 46) = 6.35; p < 0.0001), they did not reverse the increased immobility duration in UCS7 + 1.0% AA-treated zebrafish (Fig. 4 B). Additionally, a representative heatmap of adult zebrafish locomotor profiles is depicted in Supplementary Fig. 2. 3.3. Morphine, but not diclofenac, reduces the whole-body cortisol levels on zebrafish exposed to UCS following acetic acid Zebrafish exposed to unpredictable chronic stress (UCS; 7 or 14 days) or to UCS7 or UCS14 followed by 1% AA showed increased whole-body cortisol levels compared with both control (non-treated animals) and PBS groups (Fig. 5 A, F (4,30) = 66.52, p < 0.0001; Fig. 5 B, F (4,30) = 134.5, p < 0.0001). Neither injection of 1% AA nor PBS alone increased whole-body cortisol levels in adult zebrafish. While pretreatment with diclofenac does not prevent the cortisol increase in zebrafish exposed to UCS7 + 1% AA, morphine significantly reduced cortisol levels in zebrafish exposed to UCS7 + 1% AA compared with the UCS7 and 1% AA groups (Fig. 5 C; F (5,36) = 97.89, p < 0.0001). 4. Discussion Here we demonstrate a pharmacologically sensitive hyperalgesic phenotype induced by chronic stress in adult zebrafish. The link between stress and pain has been extensively examined in both rodent and clinical literature. For example, acute stress evokes pronounced stress-induced hypoalgesia (Timmers et al. 2018 ), likely representing a protective mechanism, allowing individuals to focus on escaping immediate vital threats (Ulrich-Lai and Herman 2009 ). Unlike acute stress, the effects of chronic stress exposure on pain sensitivity are less clear. For example, chronic stress has been reported to increase pain in clinical studies (Timmers et al. 2018 ) and rodent studies (Liu et al. 2019 ). Importantly, these effects are strongly modulated by centrally acting pharmacological systems, particularly opioidergic pathways that regulate both stress reactivity and nociceptive processing. In contrast to previous studies showing no effects after a single 1% acetic acid (AA) injection, we demonstrated that unpredictable chronic stress (UCS)-treated animals following 1% AA alter body curvature, and these effects were accompanied by other pain-like behavioral changes ( e.g ., reduced locomotion and increased freezing). Furthermore, the present study also demonstrated that UCS-induced hyperalgesia in zebrafish was sensitive to morphine (MOR), but not diclofenac (DS). Since the injection of 1% AA alone does not affect freezing, and animals exposed to UCS followed by 1% AA showed marked changes in locomotor behavior, these behavioral changes are largely attributable to the UCS protocol, rather than the AA challenge. Lastly, neither morphine nor diclofenac pretreatment reversed these effects. Therefore, these findings confirm the impact of UCS on pain sensitivity in adult zebrafish, with differential effects of analgesics targeting two distinct mechanisms of pain. In zebrafish, genes associated with stress responses show overlapping expression patterns in depression and pain models (de Abreu et al. 2021 ), which also show common monoaminergic mechanisms (Demin et al. 2020 ). Chronic stress disrupts several brain regions and neurotransmitters related to pain and mood responses in zebrafish. For instance, the hypothalamus plays a crucial role in stress response, regulating dopaminergic and serotonergic pathways (Martins et al. 2024 ; Corradi and Filosa 2021 ). The dorsal raphe is rich in serotonergic neurons and shows activity modulation during stress (Martins et al. 2024 ). The telencephalon , homologous to the mammalian amygdala, is involved in processing stress and anxiety-related behaviors in fish by altered glutamatergic neurotransmission (Corradi and Filosa 2021 ). The Caudal hypothalamus presents a group of dopaminergic neurons, influencing locomotor and exploratory behaviors, and potentially mediating stress-coping strategies (Corradi and Filosa 2021 ). Similar to humans, chronic stress-induced dysregulation of these neurotransmitters and brain regions may represent a potential interaction between emotional states and pain perception in zebrafish models. Because 1.0% of AA alone did not change writhing-like response in zebrafish (but not UCS-exposed), this suggests a potential interaction between chronic stress-induced depression and pain, which was also corrected by morphine, but not diclofenac. Such contrasting effects of morphine and diclofenac likely arise from distinct mechanisms by which these drugs modulate pain. This pharmacological dissociation provides functional evidence that UCS-induced hyperalgesia primarily involves central neuropharmacological mechanisms rather than peripheral inflammatory pathways. For instance, in humans, morphine primarily acts through opioid receptors, centrally blocking pain transmission, influencing emotional pain responses, and playing a key role in mood regulation (Reeves et al. 2022 ). Moreover, the µ-opioid (MOP) and δ-opioid (DOP) receptors, both activated by morphine, produce analgesic effects and evoke affective behaviors. For instance, MOP agonists induce euphoria and improve stress coping, while DOP agonists trigger anxiolytic and antidepressant effects (Lutz and Kieffer 2013 ; Valentino and Volkow 2018 ). Like humans, the zebrafish µ-opioid (zMOP) receptor is widely distributed in brain regions involved in analgesia and mood, which produces similar effects (Sivalingam et al. 2020 ). For instance, zMOP activation shows analgesic properties, as morphine prevents pain behaviors in both adult and larval zebrafish in inflammatory and visceral pain models, while also inducing addiction (Magalhaes et al. 2017 ; Taylor et al. 2017 ; Costa et al. 2019b ). Interestingly, like in humans, adverse effects can also be observed after zMOP activation with different agonists, including sedation (mitragynine and morphine) (Khor et al. 2011 ; Cachat et al. 2010 ) and, reduced gut mobility (loperamide) (Shi et al. 2014 ), suggesting evolutionarily conserved biological functions related to pain and mediated by these receptors. While morphine primarily targets zMOP, it also shows affinity for zebrafish δ-opioid (zDOP) receptor (Rodriguez et al. 2000 ) which is expressed as two functional copies (zDOPa and zDOPb) (Barrallo et al. 1998 ; Pinal-Seoane et al. 2006 ), both widely spread throughout the brain, including regions related to analgesia and mood (Pinal-Seoane et al. 2006 ). Hence, the conservation of opioidergic circuitry between zebrafish and mammals reinforces the translational value of this model for investigating CNS-targeted analgesic mechanisms. In contrast, nonsteroidal anti-inflammatory drugs (NSAIDs), such as diclofenac, inhibit both cyclooxygenase-1 (COX-1) and cyclooxygenase-2 (COX-2) enzymes, which reduces prostaglandin synthesis associated with peripheral inflammation in humans (Leathers and Rogers 2023 ). Although zebrafish possess one copy of COX-1 ( ptgs1 ) and two functional copies of COX-2 ( ptgs2a/ptgs2b ), only the latter two are related to the inflammatory and analgesic process (Leiba et al. 2023 ). Thus, both ptgs2a and ptgs2b contribute to peripheral pain signaling and represent key targets for NSAID-mediated analgesia, particularly in hyperalgesic and inflammatory conditions (Leiba et al. 2023 ). NSAIDs are effective in reducing pain by suppressing the production of pro-inflammatory prostaglandins, which are key mediators in triggering peripheral pain signaling (Leathers and Rogers 2023 ). Since the analgesic effects of diclofenac are peripheral (Leathers and Rogers 2023 ), it is expected that it is less effective in correcting UCS-induced hyperalgesia, likely involving central modulation. Therefore, the central action of morphine highlights its dual role in managing both pain and mood, unlike the diclofenac peripheral effects. In zebrafish, locomotor deficits are often associated with reduced animal welfare, which is crucial to determining mood- and pain-related behaviors (Kalueff et al. 2013 ). For instance, adult zebrafish generally present hypolocomotion following the administration of harmful substances (Costa et al. 2019a ; Taylor et al. 2017 ). However, depression-like behavior in zebrafish also manifests as hypolocomotion and increased freezing duration following UCS exposure (Demin et al. 2021 ). Here, zebrafish exposed to UCS protocol with or without 1.0% AA displayed significant differences in locomotion compared to the control group. Furthermore, administration of morphine or diclofenac reversed altered distance traveled in UCS + 1.0% AA group. Since behavioral exploratory modulation is related to central brain areas, altered immobility duration observed here is likely primarily caused by UCS. Although locomotor measures can be influenced by multiple factors, the selective pharmacological sensitivity of writhing-like behavior supports its interpretation as a nociceptive endpoint rather than a nonspecific motor effect. In contrast, although both 1.0% AA- and UCS-exposed zebrafish groups reduced distance traveled, morphine co-administration and diclofenac pretreatment prevented this effect. While this implicates central opioid mechanisms in mitigating stress-induced pain, other neurotransmitters ( e.g ., serotonin, dopamine, and glutamate) modulate affective and pain effects in zebrafish (Demin et al. 2020 ), hence calling for further studies of potential interaction between these neurotransmitters and opioid receptors ( e.g ., zMOP and zDOP) in chronic stress and pain. Here, the whole-body cortisol data supports a pain-related phenotype after UCS exposure with AA injection, which is at least partly driven by activation of the stress axis. Animals exposed to UCS showed elevated cortisol levels (with and without AA injection), which were partly reduced by morphine injection. This pattern is consistent with activation of the hypothalamic–pituitary–interrenal (HPI) axis (homologous to the mammalian hypothalamic–pituitary–adrenal (HPA) axis) exposed to UCS, which raises circulating cortisol and lowers the pain threshold. The fact that morphine reduced both pain behavior and cortisol levels suggests an opioidergic modulation of stress responsiveness that contributes to the observed sensitization. For instance, HPI activation can lead to β-endorphin (β-END) release, which negatively modulates the HPI axis and decreases cortisol levels (Gonzalez-Nunez et al. 2003 ). Like morphine, β-END is also a µ-opioid receptor agonist, which can promote analgesic effects (Zaig et al. 2021 ). Also, our data are in line with previous findings showing an inhibitory effect of endogenous opioids on the HPA axis via both µ- and κ-opioid receptors in humans, further corroborating the influence of the opioidergic system on stress-related pathways (Kreek et al. 2005 ). Therefore, the morphine injection, together with β-END released by HPI activation, may contribute to the analgesic effects observed here. In summary, the behavioral and endocrine findings converge: UCS increases stress hormones and pain sensitivity, and the opioidergic intervention attenuates both. Together, these convergent endpoints strengthen the neuropharmacological interpretation of UCS-induced hyperalgesia as a centrally mediated stress–pain interaction. Although this study provides robust behavioral evidence that UCS lowers pain threshold following AA injection in zebrafish, certain methodological considerations should be noted. While whole-body cortisol was measured in the same cohorts, future inclusion of additional neurochemical or receptor-expression endpoints could further clarify central versus peripheral contributions. Such approaches would further refine the neuropharmacological resolution of the model. While the well-established doses of morphine (2.5 mg/kg) and diclofenac (40 mg/kg) follow standard zebrafish pain protocols and ensures comparability with previous data, a dose–response curve may offer additional pharmacological resolution in future studies. Additionally, sexes were pooled based on prior reports, describing no sex differences in nociceptive or cortisol responses, a choice that enhances statistical power; however, targeted sex-specific analyses and further dissociation of locomotor from nociceptive outcomes remain valuable avenues for extending these findings. Lastly, while molecular validation of receptor-specific mechanisms would provide additional resolution, the present study was designed to establish a functional neuropharmacological phenotype suitable for behavioral pharmacology and translational screening. In addition to its translational value, the zebrafish model offers unique advantages for scalable drug discovery and high-throughput screening. Its small size, low maintenance costs, and compatibility with automated behavioral tracking enable rapid testing of multiple compounds and doses under controlled conditions (Kalueff et al., 2014 ; Stewart et al., 2014 ). The robust and quantifiable UCS-induced hyperalgesia phenotype described here, combined with validated pharmacological responsiveness to centrally acting agents and the whole-body cortisol measures, provides a reliable platform for identifying and prioritizing CNS-targeted analgesics. Such scalability underscores the potential of this model to bridge preclinical findings with early-phase drug development more efficiently than traditional mammalian systems. This scalability positions the model as a valuable intermediary between basic neuropharmacological research and preclinical analgesic development. In summary, our findings demonstrate that UCS lowers the pain threshold in adult zebrafish, supporting the interplay between UCS-induced behavioral deficits and pain and also highlighting the relevance of zebrafish models for studying the relationship between pain and depression. The modulation by morphine, but not diclofenac, suggests central rather than peripheral mechanisms of this phenotype. Beyond their neuropharmacological relevance, these results establish a robust and quantifiable stress-induced hyperalgesia phenotype suitable for preclinical drug discovery, particularly for screening CNS-targeted analgesics, and provide a tractable platform for probing the neurobiological basis of stress–pain comorbidity. Finally, our results support the use of zebrafish as a translational model for investigating depression- and pain-responses in vivo, as well as to screen novel CNS drugs that target both domains individually and/or jointly. Abbreviations AA: Acetic acid AUC: Area under the curve β-END: β-endorphin CEUA: Animal Use Ethics Committee CNS: Central Nervous System CONCEA: National Animal Experimentation Control Council COX-1: Cyclooxygenase-1 COX-2: Cyclooxygenase-2 CTRL: Control DOP: Delta-opioid receptor DS: Diclofenac sodium HPA: Hypothalamic-Pituitary-Adrenal axis HPI: Hypothalamic-Pituitary-Interrenal axis i.p.: Intraperitoneal MOP: Mu-opioid receptor NIH: National Institutes of Health NSAIDs: Nonsteroidal anti-inflammatory drugs PBS: Phosphate-buffered saline UCS: Unpredictable Chronic Stress zDOPa: Zebrafish δ-opioid receptor type a zDOPb: Zebrafish δ-opioid receptor type b zMOP: Zebrafish µ-opioid receptor ZBC: Zebrafish Behavioral Catalogue Declarations Funding This study was supported by the Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES; Finance Code 001), Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq; Proc. 402097/2023-8), Fundação de Amparo à Pesquisa do Estado do Rio Grande do Sul (FAPERGS), and Instituto Nacional de Ciência e Tecnologia para Excitotoxicidade e Neuroproteção. CDB and DBR are recipients of CNPq research productivity grants (Proc. 306115/2023-9 and Proc. 307690/2021–0). DBR is recipient of FAPERGS fellowship grants (Proc. 23/2551–0001853–5 and Proc. 24/2551–0001237–0). Declaration of Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. Ethical Statement All procedures involving animals were conducted in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals and the guidelines of the Brazilian National Council for the Control of Animal Experimentation (CONCEA). The use of laboratory animals was approved by the Institutional Animal Care and Use Committee of PUCRS (CEUA-PUCRS), under protocol number 11607. References Alestrom P, D'Angelo L, Midtlyng PJ, Schorderet DF, Schulte-Merker S, Sohm F, Warner S (2020) Zebrafish: Housing and husbandry recommendations. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-8855618","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":591702615,"identity":"b0963ff7-cc19-4376-b6d3-d6eda18dd16d","order_by":0,"name":"Fabiano Costa","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA10lEQVRIiWNgGAWjYBAC9gYILccPZhlYENbCcwBEJjAYS/aAWAYSxGtJ3HAjAcQiRotE8sOHP3/YGUvOfH51w48CCQb+9u4EAlrSjI15EpLl+KVzym72AB0mcebsBrxa7HnOsEkzJDAbS87OSbvBA9RiIJGLXwsPUIvkj4T6xA03z6Td/EOUFvYeNgmehMNA77Mfu02cLextQL+kHQcGcg7bbRkDCR6CfuFhZn748IdNNTAqjz+7+eaPjRx/ey9+Lci6DcAkscpBgP0BKapHwSgYBaNgBAEAre9DQByyHD8AAAAASUVORK5CYII=","orcid":"","institution":"Pontifical Catholic University of Rio Grande do Sul","correspondingAuthor":true,"prefix":"","firstName":"Fabiano","middleName":"","lastName":"Costa","suffix":""},{"id":591702616,"identity":"164e9310-bbf9-449a-9b7e-11129b22771f","order_by":1,"name":"Lana Ferreira","email":"","orcid":"","institution":"Pontifical Catholic University of Rio Grande do Sul","correspondingAuthor":false,"prefix":"","firstName":"Lana","middleName":"","lastName":"Ferreira","suffix":""},{"id":591702617,"identity":"02ddfcf1-d2a8-415f-bbff-55586b5d41ff","order_by":2,"name":"Lucca Lima","email":"","orcid":"","institution":"Pontifical Catholic University of Rio Grande do Sul","correspondingAuthor":false,"prefix":"","firstName":"Lucca","middleName":"","lastName":"Lima","suffix":""},{"id":591702618,"identity":"9f115064-3e46-4b90-be4a-672ddfbc1f6d","order_by":3,"name":"Julia Canzian","email":"","orcid":"","institution":"Universidade Federal de Santa Maria","correspondingAuthor":false,"prefix":"","firstName":"Julia","middleName":"","lastName":"Canzian","suffix":""},{"id":591702619,"identity":"fbbbbe30-f7df-4ea7-bdaa-1a6d34dd1e94","order_by":4,"name":"Allan Kalueff","email":"","orcid":"","institution":"Xi’an Jiaotong-Liverpool University","correspondingAuthor":false,"prefix":"","firstName":"Allan","middleName":"","lastName":"Kalueff","suffix":""},{"id":591702620,"identity":"533f8f4f-ac79-4bfc-abdf-9be2ab9584e0","order_by":5,"name":"Denis Rosemberg","email":"","orcid":"","institution":"Universidade Federal de Santa Maria","correspondingAuthor":false,"prefix":"","firstName":"Denis","middleName":"","lastName":"Rosemberg","suffix":""},{"id":591702621,"identity":"98aac2ea-4ac4-4c09-a626-f17d18961c3b","order_by":6,"name":"Carla Bonan","email":"","orcid":"","institution":"Pontifical Catholic University of Rio Grande do Sul","correspondingAuthor":false,"prefix":"","firstName":"Carla","middleName":"","lastName":"Bonan","suffix":""}],"badges":[],"createdAt":"2026-02-11 21:09:04","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-8855618/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-8855618/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":103049565,"identity":"4fdf3e22-500d-4125-9d43-bc4bde4fd81e","added_by":"auto","created_at":"2026-02-20 07:42:40","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":1301807,"visible":true,"origin":"","legend":"\u003cp\u003eEffects of 1.0% acetic acid (AA) on the body curvature in chronic stressed zebrafish (UCS). (A) Area under curve (AUC) values were calculated and expressed as arbitrary units. (B) Changes in body curvature index in PBS, 1.0% AA, 5.0% AA, 1.0% AA + UCS (7), 1.0% AA + UCS (14), UCS (7), and UCS (14) groups across time. One-way ANOVA followed by Tukey’s post-hoc test (*\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.0001 vs. PBS; \u003csup\u003e#\u003c/sup\u003e\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.0001 vs. AA 1.0%; \u003cem\u003en\u003c/em\u003e = 8-10 per group).\u003c/p\u003e","description":"","filename":"Fig.1.png","url":"https://assets-eu.researchsquare.com/files/rs-8855618/v1/98eaaee93171f5936ab63372.png"},{"id":103049950,"identity":"28854547-88b1-4967-9bd2-763ee5dffbea","added_by":"auto","created_at":"2026-02-20 07:47:25","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":718492,"visible":true,"origin":"","legend":"\u003cp\u003eEffects of unpredictable chronic stress (UCS) alone and following 1.0% acetic acid (AA)-induced changes in zebrafish locomotor behavior. Locomotor endpoints were assessed by distance traveled (A) and immobility duration (B). Data are expressed as means ± S.E.M. and analyzed by one-way ANOVA (factor: treatment), followed by Tukey's post-hoc test for significant ANOVA data (****\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.0001 vs. PBS; ####\u003cem\u003ep\u003c/em\u003e\u0026lt; 0.0001 vs. AA 1.0%; \u003cem\u003en\u003c/em\u003e = 8-10 per group).\u003c/p\u003e","description":"","filename":"Fig.2.png","url":"https://assets-eu.researchsquare.com/files/rs-8855618/v1/1944721f0eacbf1c4397de51.png"},{"id":102989660,"identity":"7f450cc7-8f9f-4f2f-96bc-be7d24ab627e","added_by":"auto","created_at":"2026-02-19 11:13:08","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":1092582,"visible":true,"origin":"","legend":"\u003cp\u003eEffects of morphine (MOR, 2.5 mg/kg, i.p.) and diclofenac sodium (DS, 40 mg/kg, i.p.) following acetic acid (AA; 1.0%) in the body curvature in chronic stressed zebrafish (UCS). (A) Area under curve (AUC) values were calculated and expressed as arbitrary units. (B) Changes in body curvature index in PBS, MOR, DS, 1.0% AA, 1.0% AA + UCS (7), 1.0% AA + UCS (7) + DS, and 1.0% AA + UCS (7) + MOR groups across time. One-way ANOVA followed by Tukey’s post-hoc test (**\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.005; ****\u003cem\u003ep\u003c/em\u003e\u0026lt; 0.001; \u003cem\u003en\u003c/em\u003e = 8-10 per group).\u003c/p\u003e","description":"","filename":"Fig.3.png","url":"https://assets-eu.researchsquare.com/files/rs-8855618/v1/a774282a96645fe4644334ac.png"},{"id":103049752,"identity":"6d7aa3a9-0b74-4e4d-81ac-411603d5a4a6","added_by":"auto","created_at":"2026-02-20 07:45:34","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":699442,"visible":true,"origin":"","legend":"\u003cp\u003eEffects of morphine (MOR, 2.5 mg/kg, i.p.) and diclofenac sodium (DS, 40 mg/kg, i.p.) on chronic stressed animals (UCS) following 1.0% acetic acid (AA)-induced changes in zebrafish locomotor behavior. Locomotor endpoints were assessed by distance traveled (A) and immobility duration (B). Data are expressed as means ± S.E.M. and analyzed by one-way ANOVA (factor: treatment), followed by Tukey's post-hoc test for significant ANOVA data (*\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05 vs. PBS; **\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.005 vs. PBS; ***\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.005 vs. PBS; ****\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.001 vs. PBS; \u003cem\u003en\u003c/em\u003e= 8-10 per group).\u003c/p\u003e","description":"","filename":"Fig.4.png","url":"https://assets-eu.researchsquare.com/files/rs-8855618/v1/edcf96961993d9cca7ab219b.png"},{"id":102989664,"identity":"82f90c55-1102-4a90-8939-fffdfd4490ab","added_by":"auto","created_at":"2026-02-19 11:13:08","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":1015097,"visible":true,"origin":"","legend":"\u003cp\u003eEffects of exposure to UCS following 1% acetic acid (AA) on whole body cortisol levels (A-B) in the presence of morphine and diclofenac (C). Data are expressed as means ± S.E.M. and analyzed by one-way ANOVA (factor: treatment), followed by Tukey’s post-hoc test for significant ANOVA data (****\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.001; \u003cem\u003en\u003c/em\u003e = 8-10 per group). CTRL – non-treated animals (naïve group), PBS - phosphate buffer saline (control group), MOR - morphine (2.5 mg/kg), DS – diclofenac (40 mg/kg), AA - 1% vol/vol.\u003c/p\u003e","description":"","filename":"Fig.5.png","url":"https://assets-eu.researchsquare.com/files/rs-8855618/v1/ed93ecf5eb36cc60104fb9e4.png"},{"id":103504280,"identity":"961cfc4e-ff75-4f84-85e8-a9b592803221","added_by":"auto","created_at":"2026-02-26 13:18:56","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":5772724,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8855618/v1/1f033456-becf-4d2f-93d4-b286060cf4ca.pdf"},{"id":103049745,"identity":"51d5b93d-2589-4041-8c5a-157d915ba5d5","added_by":"auto","created_at":"2026-02-20 07:45:25","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":1333702,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSupplementary Fig. 1. \u003c/strong\u003eRepresentative images displaying the zebrafish phenotypes after acetic acid (AA) injection, as well as the effects of AA + UCS protocol vs. the PBS group.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eSupplementary Fig. 2. \u003c/strong\u003eRepresentative heatmap of zebrafish locomotor profiles under main treatments.\u003c/p\u003e","description":"","filename":"SupplementaryInformation.docx","url":"https://assets-eu.researchsquare.com/files/rs-8855618/v1/cc2ab8761aa0a787fe97838a.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Opioidergic modulation of stress-induced hyperalgesia in adult zebrafish","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003eAffective disorders and pain have long been recognized as comorbid conditions (Han and Pae \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2015\u003c/span\u003e), with chronic pain worsening depressive symptoms, and depression, in turn, intensifying pain perception (Hooten \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2016\u003c/span\u003e; Yao et al. \u003cspan citationid=\"CR68\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). Clinical studies suggest that individuals experiencing chronic stress are more susceptible to developing chronic pain disorders, including fibromyalgia and irritable bowel syndrome, highlighting the critical role of stress and stress-related central nervous system (CNS) deficits in pain perception (Borsook et al. \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2012\u003c/span\u003e; Vachon-Presseau et al. \u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e2013\u003c/span\u003e). Importantly, shared pathways of stress and pain involve central monoaminergic and opioidergic neurotransmission, systems that represent key pharmacological targets in neuropsychiatric and analgesic therapies and play a crucial role in modulating mood and pain (Sheng et al. \u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e2017\u003c/span\u003e; Haase and Brown \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2015\u003c/span\u003e). The overlap of these neural circuits, especially the limbic system responsible for both emotional- and pain-processing responses, further supports the integrated nature of their pathogenesis (Sheng et al. \u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e2017\u003c/span\u003e; Meerwijk et al. \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e2013\u003c/span\u003e). Chronic neuroinflammation, a common feature of both depression and certain types of pain, also contributes to these overlapping pathogenetic mechanisms (Dooley et al. \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2018\u003c/span\u003e; Walker et al. \u003cspan citationid=\"CR62\" class=\"CitationRef\"\u003e2014\u003c/span\u003e; Lee and Giuliani \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e2019\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eAnimal experimental models are essential tools for investigating the neuropharmacological mechanisms underlying stress\u0026ndash;pain interactions (Ulrich-Lai and Herman \u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e2009\u003c/span\u003e). A common protocol used to assess depression-like states in animal models is the exposure to chronic stress, such as the unpredictable chronic stress (UCS) (Burstein and Doron \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2018\u003c/span\u003e; Piato et al. \u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e2011\u003c/span\u003e). Based on repeated exposure to varied and unpredictable stressors, the rodent UCS model has been widely used to mimic depression-related behaviors and neurochemical changes observed in humans (Willner \u003cspan citationid=\"CR64\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). These neurobehavioral effects of UCS are associated with dysregulation of the hypothalamic-pituitary-adrenal (HPA) axis, increased pro-inflammatory cytokines, and impaired serotonergic function, all of which contribute to both depressive symptoms and heightened pain sensitivity (hyperalgesia) (Slavich and Irwin \u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e2014\u003c/span\u003e; Burke et al. \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2017\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eComplementing rodent models, the zebrafish (\u003cem\u003eDanio rerio\u003c/em\u003e) provides a scalable vertebrate platform for neuropharmacological investigations of complex CNS disorders, including depression and pain (Stewart et al. \u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e2014\u003c/span\u003e; Kysil et al. \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). The UCS paradigms have successfully been adapted for zebrafish, demonstrating overt behavioral, neurochemical and genomic phenotypes that generally parallel those observed in rodent stress models (Wong et al. \u003cspan citationid=\"CR66\" class=\"CitationRef\"\u003e2010\u003c/span\u003e; Piato et al. \u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e2011\u003c/span\u003e). Indeed, UCS-exposed zebrafish exhibit anxiety-like behaviors, reduced exploration and altered monoaminergic neurotransmission, paralleling rodent and clinical findings (Kysil et al. \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e2017\u003c/span\u003e; Maximino et al. \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e2010\u003c/span\u003e). Since stress-induced changes affect mood-related behaviors (Piato et al. \u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e2011\u003c/span\u003e; Oliveira et al. \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e2013\u003c/span\u003e), whereas mood and pain share common pathogenetic pathways (Han and Pae \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2015\u003c/span\u003e; Yang and Chang \u003cspan citationid=\"CR67\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; De Ridder et al. \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2021\u003c/span\u003e), stress may impact pain sensitivity, hence supporting the interplay between chronic stress, depression, and pain. Despite the growing interest in zebrafish models for studying pain and depression (Kalueff et al. \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2014\u003c/span\u003e; Costa et al. \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2019b\u003c/span\u003e; Costa et al. \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2022\u003c/span\u003e), the relationship between stress and pain responses in these fish remains poorly understood. However, it may help clarify the evolutionary conserved link between pain- and depression-like phenotypes across vertebrates. Thus, we hypothesize that unpredictable chronic stress (UCS) produces a pharmacologically tractable hyperalgesic phenotype mediated by central mechanisms in zebrafish.\u003c/p\u003e \u003cp\u003eUnderstanding these mechanisms is critical for developing CNS-targeted pharmacological strategies that address stress\u0026ndash;pain comorbidity. Here, we pharmacologically characterize a zebrafish model of stress-induced hyperalgesia using centrally and peripherally acting analgesics to dissociate underlying mechanisms. The present study applied a 7\u0026ndash;14-day UCS paradigm to induce depression-like phenotypes in zebrafish, followed by intraperitoneal administration (i.p.) of a nociceptive agent (1\u0026ndash;5% acetic acid, AA) and quantifying pain-like behaviors using the aquatic writhing assay. To explore the underlying mechanisms of UCS-induced hyperalgesia, we further examined the effects of morphine, a centrally acting analgesic, and diclofenac, a peripheral anti-inflammatory agent. This combined protocol allows for the concurrent assessment of depression- and pain-related responses in the same organism, providing a translationally relevant platform to investigate shared mechanisms underlying affective and nociceptive processes and to distinguish central versus peripheral modulation of stress-induced hyperalgesia.\u003c/p\u003e"},{"header":"2. Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\n \u003ch2\u003e2.1. Animals\u003c/h2\u003e\n \u003cp\u003eA total of 300 adult wild-type zebrafish (AB strain; 5\u0026ndash;7 months old; ~50:50 male:female) were maintained in automated recirculating systems (ZebTEC, Tecniplast, Italy) with reverse-osmosis-filtered water and conditions, as in (Westerfield \u003cspan class=\"CitationRef\"\u003e1993\u003c/span\u003e), for at least 2 weeks prior to the beginning of experiments. Since no sex differences were observed in AA\u0026ndash;induced pain in zebrafish (Costa et al., \u003cspan class=\"CitationRef\"\u003e2019a\u003c/span\u003e) and their cortisol measures (Costa et al., \u003cspan class=\"CitationRef\"\u003e2023\u003c/span\u003e), male and female data were pooled to maximize statistical power and to adhere to the 3Rs principle of animal experimentation. Animals were obtained from an in-house breeding colony and maintained under standard conditions at the authors\u0026rsquo; facility. Fish were maintained at 28℃ \u0026plusmn; 2℃, pH 7.0\u0026ndash;7.5, water conductivity 300\u0026ndash;700 \u0026micro;S, ammonia\u0026thinsp;\u0026lt;\u0026thinsp;0.02 mg/L, hardness 80\u0026ndash;300 mg/L, nitrite\u0026thinsp;\u0026lt;\u0026thinsp;1 mg/L, nitrate\u0026thinsp;\u0026lt;\u0026thinsp;50 mg/L, and chloride 0 mg/L, under a 14 h light:10 h dark photoperiod cycle (lights on: 07:00 am) and fed with commercial flakes (TetraMin Tropical Flake Fish\u0026trade;) three times a day. Water pH and conductivity were monitored daily, and nitrogen compounds were measured weekly, in accordance with established zebrafish husbandry standards (Alestrom et al. \u003cspan class=\"CitationRef\"\u003e2020\u003c/span\u003e). All protocols were approved by the Institutional Animal Care Committee (CEUA, protocol number 11607) and animal experimentation fully adhered to the National Institutes of Health Guide for the Care and Use of Laboratory Animals and the guidelines of the National Animal Experimentation Control Council (CONCEA). The AB fish were selected here as a common zebrafish strain widely used in neurobehavioral assays (Bertoncello et al. \u003cspan class=\"CitationRef\"\u003e2024\u003c/span\u003e).\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec4\" class=\"Section2\"\u003e\n \u003ch2\u003e2.2. Chemicals\u003c/h2\u003e\n \u003cp\u003eThe following chemicals were used in the present study: acetic acid (AA; Merck KGaA, Darmstadt, Germany, CAS No. 64-19-7), morphine sulfate (MOR; Sigma-Aldrich, St. Louis, USA, CAS No. 6211-15-0), phosphate buffered saline (PBS; Sigma-Aldrich), diclofenac sodium (DS; Novartis, S\u0026atilde;o Paulo, Brazil; CAS No. 15307-79-6).\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec5\" class=\"Section2\"\u003e\n \u003ch2\u003e2.3. Unpredictable chronic stress protocol (UCS)\u003c/h2\u003e\n \u003cp\u003eFollowing a two-week acclimation period, the UCS was performed based on a protocol described previously, with some modifications (Piato et al. \u003cspan class=\"CitationRef\"\u003e2011\u003c/span\u003e). Briefly, fish were submitted twice a day to various stressors for 7-day/14-day, as summarized in Table \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e. UCS stressors used included \u003cem\u003eChange of environments\u003c/em\u003e: keeping the tanks surrounded by a different color from the animal\u0026apos;s natural environment (green, yellow, red) for 6 h; \u003cem\u003eHeating\u003c/em\u003e: increasing the water temperature of the tank up to 33\u0026deg;C for 30 min; \u003cem\u003eSocial isolation\u003c/em\u003e: animals were placed individually in a 250 mL beaker for 6 h; \u003cem\u003eCooling\u003c/em\u003e: cooling the tank water down to 23\u0026deg;C for 30 min; \u003cem\u003eCrowding\u003c/em\u003e: placing a group of 10 animals for 50 min in a 250 mL beaker; \u003cem\u003eLow water level\u003c/em\u003e: decreasing the water level in housing tanks until the animals\u0026apos; dorsal body walls are exposed for 2 min; \u003cem\u003eChanging water\u003c/em\u003e: animals were kept in the tank while the water was changed three consecutive times; \u003cem\u003eChanging tanks\u003c/em\u003e: animals were transferred to a fresh tanks three times; \u003cem\u003eNet chasing\u003c/em\u003e: chasing animals for 8 min with a net. Aeration and temperature were controlled during each stressor presentation (except during heating and cooling stress). To prevent habituation and maintain stress unpredictability, the time and sequence of stressor presentations were changed daily. An unstressed control group remained intact in the same room for 7 or 14 days, respectively.\u003c/p\u003e\n \u003cdiv class=\"gridtable\"\u003e\u0026nbsp;\u003ctable id=\"Tab1\" border=\"1\"\u003e\n \u003ccaption language=\"En\"\u003e\n \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e\n \u003cdiv class=\"CaptionContent\"\u003e\n \u003cp\u003eUnpredictable chronic stress protocol.\u003c/p\u003e\n \u003c/div\u003e\n \u003c/caption\u003e\n \u003ccolgroup cols=\"8\"\u003e\u003c/colgroup\u003e\n \u003cthead\u003e\n \u003ctr\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eWeek\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eThursday\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eFriday\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eSaturday\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eSunday\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eMonday\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eTuesday\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eWednesday\u003c/p\u003e\n \u003c/th\u003e\n \u003c/tr\u003e\n \u003c/thead\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003eWeek 1\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eWater change\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eSocial isolation\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eCrowding\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eLow water level\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eHeating\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eNet chasing\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eCrowding\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eHeating\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eCooling\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eChange environment\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eTank change\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eCooling\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eChange environments\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eSocial isolation\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003eWeek 2\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eLow water level\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eTank change\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eHeating\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eSocial isolation\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eNet chasing\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eCooling\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eHeating\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eCooling\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eCrowding\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eWater change\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eChange environments\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eLow water level\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eTank change\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eCrowding\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003eWeek 3\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eBehavioral analysis\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003c/table\u003e\n \u003c/div\u003e\n \u003cp\u003e\u003cbr\u003e\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec6\" class=\"Section2\"\u003e\n \u003ch2\u003e2.4. Assessing the effects of acetic acid on unpredictable chronic stress protocol (UCS)\u003c/h2\u003e\n \u003cp\u003eZebrafish (\u003cem\u003en\u003c/em\u003e\u0026thinsp;=\u0026thinsp;8\u0026ndash;10 per group) were randomly selected from at least three housing tanks (for non-stressed control animals) or from 7-day/14-day UCS batches (for stressed animals UCS7 and UCS14, respectively). Because no differences were observed in body curvature indexes when 5 \u003cem\u003evs\u003c/em\u003e. 10 animals were treated with phosphate-buffered saline (PBS) or acetic acid (AA) in previous reports (Costa et al. \u003cspan class=\"CitationRef\"\u003e2019b\u003c/span\u003e), and given prior analyses demonstrating sufficient statistical power (\u0026alpha;\u0026thinsp;=\u0026thinsp;0.05, power\u0026thinsp;=\u0026thinsp;0.80) for detecting significant differences in behavioral endpoints, a sample size of 8\u0026ndash;10 animals per group was considered appropriate. Moreover, this sample size is consistent with similar experimental designs that successfully minimized intra-group variability and ensured reproducible results, while adhering to the 3Rs principles of ethical animal experimentation.\u003c/p\u003e\n \u003cp\u003eAnimals were randomly selected from at least three housing tanks, gently handled, and anesthetized in cold water (\u0026lt;\u0026thinsp;5 s), as previously described (Kinkel et al. \u003cspan class=\"CitationRef\"\u003e2010\u003c/span\u003e). They were briefly immobilized using a small wet net and injected intraperitoneally through the midline between the pelvic fins with PBS (control), UCS7 and UCS14 (stress groups) and AA (1.0 and 5.0% vol/vol in PBS), as well as UCS7 or UCS14 following 1.0% AA. The i.p. injection occurred in a short period (5 s), and was preferred over other methods for i.p. administration in fish (\u003cem\u003ee.g.\u003c/em\u003e, chemical anesthesia with tricaine or similar agents) to avoid confounding effects of anesthesia on the pharmacological effects examined here. Importantly, this protocol allows a fast evaluation of the swimming activity after fish return to the water, minimizing potential pharmacological interference on complex behaviors (\u003cem\u003ee.g.\u003c/em\u003e, immobility and locomotion). Behavioral recordings started as soon as the fish regained postural equilibrium (~\u0026thinsp;1 min post-injection), thus allowing a more accurate characterization of acute pain responses (Costa et al. \u003cspan class=\"CitationRef\"\u003e2019b\u003c/span\u003e). Because no differences in body curvature indexes were observed between male and female following a 1.0% AA injection (Costa et al. \u003cspan class=\"CitationRef\"\u003e2019b\u003c/span\u003e; Rambo et al. \u003cspan class=\"CitationRef\"\u003e2017\u003c/span\u003e), male and female were combined as a unisex group. All injections were performed using a BD Ultra-fine\u0026trade; 30U syringe (needle size 6 x 0.25 mm) with 10 \u0026micro;L (shown not to impair normal zebrafish behaviors (Kinkel et al. \u003cspan class=\"CitationRef\"\u003e2010\u003c/span\u003e; Richetti et al. \u003cspan class=\"CitationRef\"\u003e2011\u003c/span\u003e)). The concentrations of AA used here were selected based on previous studies (Taylor et al. \u003cspan class=\"CitationRef\"\u003e2017\u003c/span\u003e; Sneddon et al. \u003cspan class=\"CitationRef\"\u003e2003\u003c/span\u003e; Costa et al. \u003cspan class=\"CitationRef\"\u003e2019b\u003c/span\u003e).\u003c/p\u003e\n \u003cp\u003e\u003cem\u003e2.5. Assessing the effects of morphine and diclofenac on unpredictable chronic stress protocol (UCS) following acetic acid\u003c/em\u003e\u003c/p\u003e\n \u003cp\u003eZebrafish (\u003cem\u003en\u003c/em\u003e\u0026thinsp;=\u0026thinsp;8\u0026ndash;10 per group) were randomly selected from at least three housing tanks (for non-stressed animals) or from UCS7 batches (for stressed animals). Animals were gently handled, anesthetized in cold water (Kinkel et al. \u003cspan class=\"CitationRef\"\u003e2010\u003c/span\u003e), and briefly immobilized using a small wet net and individually injected i.p., as described above. To assess the potential antinociceptive effects of diclofenac and morphine, animals were assigned into the PBS, morphine (MOR), and diclofenac (DS) as control groups, as well as 1.0% AA, UCS7\u0026thinsp;+\u0026thinsp;1.0% AA, UCS7\u0026thinsp;+\u0026thinsp;1.0% AA\u0026thinsp;+\u0026thinsp;DS, UCS7\u0026thinsp;+\u0026thinsp;1.0% AA\u0026thinsp;+\u0026thinsp;MOR. The UCS7 animals were pretreated with DS (40 mg/kg) 15 min prior to the test and further injected with 1.0% AA. Diclofenac was selected as a clinically approved nonsteroidal anti-inflammatory drug (Altman et al. \u003cspan class=\"CitationRef\"\u003e2015\u003c/span\u003e) widely used in experimental models to assess nociceptive-related phenotypes. Morphine was chosen here as classical, clinically approved and potent opioid analgesic drug (Pathan and Williams \u003cspan class=\"CitationRef\"\u003e2012\u003c/span\u003e) widely used in experimental models of pain (Hamann et al. \u003cspan class=\"CitationRef\"\u003e2016\u003c/span\u003e; Llorca-Torralba et al. \u003cspan class=\"CitationRef\"\u003e2018\u003c/span\u003e; Rodrigues-Filho et al. \u003cspan class=\"CitationRef\"\u003e2004\u003c/span\u003e; Bjorkman et al. \u003cspan class=\"CitationRef\"\u003e1992\u003c/span\u003e), with a known sensitivity in zebrafish (Taylor et al. \u003cspan class=\"CitationRef\"\u003e2017\u003c/span\u003e; Costa et al. \u003cspan class=\"CitationRef\"\u003e2019b\u003c/span\u003e). The doses of MOR and DS used here were selected based on previous reports (Sneddon et al. \u003cspan class=\"CitationRef\"\u003e2003\u003c/span\u003e; Taylor et al. \u003cspan class=\"CitationRef\"\u003e2017\u003c/span\u003e; Costa et al. \u003cspan class=\"CitationRef\"\u003e2019b\u003c/span\u003e).\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec7\" class=\"Section2\"\u003e\n \u003ch2\u003e2.6. Behavioral analyses\u003c/h2\u003e\n \u003cp\u003eImmediately after the experimental procedures, fish were individually transferred to observation tanks (15\u0026times;13 x 10 cm, length x height x width) with a 10-cm water depth and behavioral activities were recorded for 6 min using a digital camera (webcam Ultra HD Logitech\u0026reg; 4K PRO, Lausanne, Swiss). Behavioral recordings began as soon as the fish regained equilibrium (\u0026plusmn;\u0026thinsp;1 min post-injection), to ensure accurate acute pain assessments. Because no sex differences are observed in fish behavior following AA i.p. administration (Costa et al. \u003cspan class=\"CitationRef\"\u003e2019b\u003c/span\u003e), male and female zebrafish were separated from their experimental tanks and divided randomly for each cohort by a computerized random number generator (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ewww.random.org\u003c/span\u003e\u003c/span\u003e). Water conditions during behavioral testing matched those of the housing tanks. Behavioral testing was performed between 12:00 and 16:00, and zebrafish were fasted on the day of experiments (prior to behavioral testing). All experiments were performed as planned, and all analyses and all endpoints assessed were included without omission. After the experimental procedures, animals were immediately anesthetized in cold water (4\u0026deg;C) and then euthanized by decapitation following the cessation of breathing for \u0026gt;\u0026thinsp;30 s.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e\n \u003ch2\u003e2.7. Assessing writhing-like behavior and locomotor activity\u003c/h2\u003e\n \u003cp\u003eAll behavioral analyses were performed offline by experimenters blinded to the treatment groups, in an automated unbiased manner using the automated video-tracking software (EthoVision\u0026reg; XT11.5, Noldus IT, Wageningen, Netherlands) at 30 frames/s, to quantify the distance traveled (cm) and immobility duration (s), based on the center body point coordinates. Behavioral phenotypes assessed here fully adhered to a formal classification of fish behavior provided in the comprehensive Zebrafish Behavioral Catalogue (ZBC) (Kalueff et al. \u003cspan class=\"CitationRef\"\u003e2025\u003c/span\u003e; Kalueff et al. \u003cspan class=\"CitationRef\"\u003e2013\u003c/span\u003e). A part of zebrafish pain-related behaviors (ZBC1 term 1.104), writhing-like behavior (ZBC2 term 2.121) represents an abnormal constriction of body after the administration of AA, reflecting acute discomfort, stress and pain (Kalueff et al. \u003cspan class=\"CitationRef\"\u003e2025\u003c/span\u003e). The body curvature (reflecting a typical writhing-like behavior) was used as a behavioral endpoint to measure pain responses, as previously reported (Costa et al. \u003cspan class=\"CitationRef\"\u003e2019b\u003c/span\u003e; Costa et al. \u003cspan class=\"CitationRef\"\u003e2023\u003c/span\u003e; Costa et al. \u003cspan class=\"CitationRef\"\u003e2019a\u003c/span\u003e). Briefly, every 30 s of recording, screenshots of the animal\u0026apos;s sagittal plane were taken (6 min\u0026thinsp;=\u0026thinsp;12 screenshots), and later analyzed using ImageJ 1.45 for Windows (Research Services Branch, the National Institute of Mental Health, National Institutes of Health, NIH)). We selected three points of each animal: frontal (in the front of the head), central (middle of the animal body \u0026ndash; between anal and dorsal fins) and posterior (at the dorsal fin), to estimate the angle of fish body curvature. The angles obtained were subtracted from 180\u0026deg; and multiplied by (-1) to estimate the body curvature index. The area under the curve (AUC) was calculated to quantify the total body curvature index over time, integrating the curvature index values derived from each anatomical point. AUC provides a comprehensive measure of both the magnitude and persistence of curvature, allowing for comparisons between individuals or treatment groups. Variations in the body curvature index were examined by two trained observers blinded to the experimental condition (inter-rater reliability\u0026thinsp;\u0026gt;\u0026thinsp;0.90). Immobility was defined as a complete immobility (\u0026lt;\u0026thinsp;0.59 cm/s) except small movements of fins and eyes, accompanied by fast opercular beat rates, as described elsewhere (Wiprich et al. \u003cspan class=\"CitationRef\"\u003e2020\u003c/span\u003e). A representative image illustrating differences in body curvature of treated fish is shown in Supplementary Fig.\u0026nbsp;1. Moreover, a representative heatmap of zebrafish locomotor profiles under all treatments is shown in Supplementary Fig.\u0026nbsp;2.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec9\" class=\"Section2\"\u003e\n \u003ch2\u003e2.8. Whole-body cortisol measurements\u003c/h2\u003e\n \u003cp\u003eImmediately after the treatments, fish were euthanized and then frozen in liquid nitrogen for 20\u0026ndash;30 s for cortisol extraction. Whole-body cortisol was extracted following the ether-based extraction protocol (Mezzomo et al., 2019) and quantified in duplicates using a commercially enzyme-linked immunosorbent assay kit (EIAgen\u0026trade; Cortisol test, BioChem ImmunoSystems) (Sink et al., 2008). A strong positive correlation was observed (r2\u0026thinsp;=\u0026thinsp;0.9413), and inter- and intra-assay coefficients of variation values were low (7\u0026ndash;10% and 5\u0026ndash;9%, respectively). Results were expressed as ng cortisol/g tissue.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec10\" class=\"Section2\"\u003e\n \u003ch2\u003e2.9. Statistical analyses and data handling\u003c/h2\u003e\n \u003cp\u003eData normality and homogeneity of variances were analyzed by Kolmogorov\u0026ndash;Smirnov and Bartlett\u0026rsquo;s tests, respectively. Changes in body curvature and behavioral activity were analyzed by one-way analysis of variance (ANOVA, factor: treatment), followed by post-hoc Tukey\u0026apos;s test multiple comparison test for significant ANOVA data. Results were expressed as means\u0026thinsp;\u0026plusmn;\u0026thinsp;standard error of the mean (S.E.M.). Immobility duration was log-transformed to meet assumptions of normality, the analyses were then performed on transformed data. Inter-rater reliability was assessed by Spearman\u0026rsquo;s rank correlation (r\u0026thinsp;\u0026gt;\u0026thinsp;0.85). Statistical analysis was performed using the GraphPad Prism9 software (GraphPad Software, Boston, USA). All fish tested were included in the final analysis without attrition or exclusion, and all planned analyses are presented here. All behavioral and statistical analyses were performed by experimenters that remained blinded to the treatment groups.\u003c/p\u003e\n\u003c/div\u003e"},{"header":"3. Results","content":"\u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003e3.1. Acetic acid changes body curvature in zebrafish exposed to the UCS protocol\u003c/h2\u003e \u003cp\u003eAlthough 1.0% AA alone did not change AUC relative to controls, a significant treatment effect was observed (\u003cem\u003eF\u003c/em\u003e\u003csub\u003e(6, 43)\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;37.27; \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.0001), as UCS7/UCS14 following 1.0% AA injection increased AUC compared to PBS control group (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA). Likewise, while UCS7 and UCS14 groups and control fish showed unaltered (absent) writhing-like body curvature, AUC increased in both 5.0% AA and 1.0% AA\u0026thinsp;+\u0026thinsp;UCS7/14 groups (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA-B; \u003cem\u003eF\u003c/em\u003e\u003csub\u003e(3, 31)\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;24.69; \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.0001). All treated groups showed a significant reduction in traveled distance compared to controls (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA; \u003cem\u003eF\u003c/em\u003e\u003csub\u003e(5, 49)\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;31.69; \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.0001). Immobility duration significantly increased in the UCS7 or UCS14 groups and the 1.0% AA\u0026thinsp;+\u0026thinsp;UCS7/14 groups compared to controls (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB; \u003cem\u003eF\u003c/em\u003e\u003csub\u003e(5, 49)\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;52.13; \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.0001). In contrast, 1.0% AA injection alone did not affect zebrafish immobility duration. Because both UCS7 and UCS14 evoked similar results in all tests performed here, we chose the UCS7 protocol for further experiments, to comply with bioethical principles aimed at reducing bioexperimentation.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003e3.2. Morphine, but not diclofenac, reduces UCS effects on acetic acid-induced pain\u003c/h2\u003e \u003cp\u003eZebrafish treated with UCS7\u0026thinsp;+\u0026thinsp;1.0% AA showed significantly higher AUC compared to control fish. While diclofenac pretreatment did not reverse this increase, and morphine co-administration did not fully restore AUC to baseline, it significantly reduced AUC compared to the 1.0% AA\u0026thinsp;+\u0026thinsp;UCS7 and diclofenac-treated groups (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA; \u003cem\u003eF\u003c/em\u003e\u003csub\u003e(6, 39)\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;52.48; \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.0001). Furthermore, diclofenac pretreatment did not reverse the AUC increase over time, whereas morphine co-administration attenuated this increase (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB; \u003cem\u003eF\u003c/em\u003e\u003csub\u003e(5, 32)\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;56.81; \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.0001). Although both morphine and diclofenac reversed the reduced distance traveled in UCS7\u0026thinsp;+\u0026thinsp;1.0% AA-treated zebrafish (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA; \u003cem\u003eF\u003c/em\u003e\u003csub\u003e(6, 46)\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;6.35; \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.0001), they did not reverse the increased immobility duration in UCS7\u0026thinsp;+\u0026thinsp;1.0% AA-treated zebrafish (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB). Additionally, a representative heatmap of adult zebrafish locomotor profiles is depicted in Supplementary Fig.\u0026nbsp;2.\u003c/p\u003e \u003cp\u003e \u003cem\u003e3.3. Morphine, but not diclofenac, reduces the whole-body cortisol levels on zebrafish exposed to UCS following acetic acid\u003c/em\u003e \u003c/p\u003e \u003cp\u003eZebrafish exposed to unpredictable chronic stress (UCS; 7 or 14 days) or to UCS7 or UCS14 followed by 1% AA showed increased whole-body cortisol levels compared with both control (non-treated animals) and PBS groups (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA, F\u003csub\u003e(4,30)\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;66.52, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.0001; Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB, F\u003csub\u003e(4,30)\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;134.5, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.0001). Neither injection of 1% AA nor PBS alone increased whole-body cortisol levels in adult zebrafish. While pretreatment with diclofenac does not prevent the cortisol increase in zebrafish exposed to UCS7\u0026thinsp;+\u0026thinsp;1% AA, morphine significantly reduced cortisol levels in zebrafish exposed to UCS7\u0026thinsp;+\u0026thinsp;1% AA compared with the UCS7 and 1% AA groups (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eC; \u003cem\u003eF\u003c/em\u003e\u003csub\u003e(5,36)\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;97.89, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.0001).\u003c/p\u003e \u003c/div\u003e"},{"header":"4. Discussion","content":"\u003cp\u003eHere we demonstrate a pharmacologically sensitive hyperalgesic phenotype induced by chronic stress in adult zebrafish. The link between stress and pain has been extensively examined in both rodent and clinical literature. For example, acute stress evokes pronounced stress-induced hypoalgesia (Timmers et al. \u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e2018\u003c/span\u003e), likely representing a protective mechanism, allowing individuals to focus on escaping immediate vital threats (Ulrich-Lai and Herman \u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e2009\u003c/span\u003e). Unlike acute stress, the effects of chronic stress exposure on pain sensitivity are less clear. For example, chronic stress has been reported to increase pain in clinical studies (Timmers et al. \u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e2018\u003c/span\u003e) and rodent studies (Liu et al. \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). Importantly, these effects are strongly modulated by centrally acting pharmacological systems, particularly opioidergic pathways that regulate both stress reactivity and nociceptive processing.\u003c/p\u003e \u003cp\u003eIn contrast to previous studies showing no effects after a single 1% acetic acid (AA) injection, we demonstrated that unpredictable chronic stress (UCS)-treated animals following 1% AA alter body curvature, and these effects were accompanied by other pain-like behavioral changes (\u003cem\u003ee.g\u003c/em\u003e., reduced locomotion and increased freezing). Furthermore, the present study also demonstrated that UCS-induced hyperalgesia in zebrafish was sensitive to morphine (MOR), but not diclofenac (DS). Since the injection of 1% AA alone does not affect freezing, and animals exposed to UCS followed by 1% AA showed marked changes in locomotor behavior, these behavioral changes are largely attributable to the UCS protocol, rather than the AA challenge. Lastly, neither morphine nor diclofenac pretreatment reversed these effects. Therefore, these findings confirm the impact of UCS on pain sensitivity in adult zebrafish, with differential effects of analgesics targeting two distinct mechanisms of pain.\u003c/p\u003e \u003cp\u003eIn zebrafish, genes associated with stress responses show overlapping expression patterns in depression and pain models (de Abreu et al. \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2021\u003c/span\u003e), which also show common monoaminergic mechanisms (Demin et al. \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). Chronic stress disrupts several brain regions and neurotransmitters related to pain and mood responses in zebrafish. For instance, the \u003cem\u003ehypothalamus\u003c/em\u003e plays a crucial role in stress response, regulating dopaminergic and serotonergic pathways (Martins et al. \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e2024\u003c/span\u003e; Corradi and Filosa \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). The \u003cem\u003edorsal raphe\u003c/em\u003e is rich in serotonergic neurons and shows activity modulation during stress (Martins et al. \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). The \u003cem\u003etelencephalon\u003c/em\u003e, homologous to the mammalian amygdala, is involved in processing stress and anxiety-related behaviors in fish by altered glutamatergic neurotransmission (Corradi and Filosa \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). The \u003cem\u003eCaudal hypothalamus\u003c/em\u003e presents a group of dopaminergic neurons, influencing locomotor and exploratory behaviors, and potentially mediating stress-coping strategies (Corradi and Filosa \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). Similar to humans, chronic stress-induced dysregulation of these neurotransmitters and brain regions may represent a potential interaction between emotional states and pain perception in zebrafish models. Because 1.0% of AA alone did not change writhing-like response in zebrafish (but not UCS-exposed), this suggests a potential interaction between chronic stress-induced depression and pain, which was also corrected by morphine, but not diclofenac. Such contrasting effects of morphine and diclofenac likely arise from distinct mechanisms by which these drugs modulate pain. This pharmacological dissociation provides functional evidence that UCS-induced hyperalgesia primarily involves central neuropharmacological mechanisms rather than peripheral inflammatory pathways. For instance, in humans, morphine primarily acts through opioid receptors, centrally blocking pain transmission, influencing emotional pain responses, and playing a key role in mood regulation (Reeves et al. \u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). Moreover, the \u0026micro;-opioid (MOP) and δ-opioid (DOP) receptors, both activated by morphine, produce analgesic effects and evoke affective behaviors. For instance, MOP agonists induce euphoria and improve stress coping, while DOP agonists trigger anxiolytic and antidepressant effects (Lutz and Kieffer \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e2013\u003c/span\u003e; Valentino and Volkow \u003cspan citationid=\"CR61\" class=\"CitationRef\"\u003e2018\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eLike humans, the zebrafish \u0026micro;-opioid (zMOP) receptor is widely distributed in brain regions involved in analgesia and mood, which produces similar effects (Sivalingam et al. \u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). For instance, zMOP activation shows analgesic properties, as morphine prevents pain behaviors in both adult and larval zebrafish in inflammatory and visceral pain models, while also inducing addiction (Magalhaes et al. \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e2017\u003c/span\u003e; Taylor et al. \u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e2017\u003c/span\u003e; Costa et al. \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2019b\u003c/span\u003e). Interestingly, like in humans, adverse effects can also be observed after zMOP activation with different agonists, including sedation (mitragynine and morphine) (Khor et al. \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2011\u003c/span\u003e; Cachat et al. \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2010\u003c/span\u003e) and, reduced gut mobility (loperamide) (Shi et al. \u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e2014\u003c/span\u003e), suggesting evolutionarily conserved biological functions related to pain and mediated by these receptors. While morphine primarily targets zMOP, it also shows affinity for zebrafish δ-opioid (zDOP) receptor (Rodriguez et al. \u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e2000\u003c/span\u003e) which is expressed as two functional copies (zDOPa and zDOPb) (Barrallo et al. \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e1998\u003c/span\u003e; Pinal-Seoane et al. \u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e2006\u003c/span\u003e), both widely spread throughout the brain, including regions related to analgesia and mood (Pinal-Seoane et al. \u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e2006\u003c/span\u003e). Hence, the conservation of opioidergic circuitry between zebrafish and mammals reinforces the translational value of this model for investigating CNS-targeted analgesic mechanisms.\u003c/p\u003e \u003cp\u003eIn contrast, nonsteroidal anti-inflammatory drugs (NSAIDs), such as diclofenac, inhibit both cyclooxygenase-1 (COX-1) and cyclooxygenase-2 (COX-2) enzymes, which reduces prostaglandin synthesis associated with peripheral inflammation in humans (Leathers and Rogers \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). Although zebrafish possess one copy of COX-1 (\u003cem\u003eptgs1\u003c/em\u003e) and two functional copies of COX-2 (\u003cem\u003eptgs2a/ptgs2b\u003c/em\u003e), only the latter two are related to the inflammatory and analgesic process (Leiba et al. \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). Thus, both \u003cem\u003eptgs2a\u003c/em\u003e and \u003cem\u003eptgs2b\u003c/em\u003e contribute to peripheral pain signaling and represent key targets for NSAID-mediated analgesia, particularly in hyperalgesic and inflammatory conditions (Leiba et al. \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). NSAIDs are effective in reducing pain by suppressing the production of pro-inflammatory prostaglandins, which are key mediators in triggering peripheral pain signaling (Leathers and Rogers \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). Since the analgesic effects of diclofenac are peripheral (Leathers and Rogers \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e2023\u003c/span\u003e), it is expected that it is less effective in correcting UCS-induced hyperalgesia, likely involving central modulation. Therefore, the central action of morphine highlights its dual role in managing both pain and mood, unlike the diclofenac peripheral effects.\u003c/p\u003e \u003cp\u003eIn zebrafish, locomotor deficits are often associated with reduced animal welfare, which is crucial to determining mood- and pain-related behaviors (Kalueff et al. \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2013\u003c/span\u003e). For instance, adult zebrafish generally present hypolocomotion following the administration of harmful substances (Costa et al. \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2019a\u003c/span\u003e; Taylor et al. \u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). However, depression-like behavior in zebrafish also manifests as hypolocomotion and increased freezing duration following UCS exposure (Demin et al. \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). Here, zebrafish exposed to UCS protocol with or without 1.0% AA displayed significant differences in locomotion compared to the control group. Furthermore, administration of morphine or diclofenac reversed altered distance traveled in UCS\u0026thinsp;+\u0026thinsp;1.0% AA group. Since behavioral exploratory modulation is related to central brain areas, altered immobility duration observed here is likely primarily caused by UCS. Although locomotor measures can be influenced by multiple factors, the selective pharmacological sensitivity of writhing-like behavior supports its interpretation as a nociceptive endpoint rather than a nonspecific motor effect.\u003c/p\u003e \u003cp\u003eIn contrast, although both 1.0% AA- and UCS-exposed zebrafish groups reduced distance traveled, morphine co-administration and diclofenac pretreatment prevented this effect. While this implicates central opioid mechanisms in mitigating stress-induced pain, other neurotransmitters (\u003cem\u003ee.g\u003c/em\u003e., serotonin, dopamine, and glutamate) modulate affective and pain effects in zebrafish (Demin et al. \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2020\u003c/span\u003e), hence calling for further studies of potential interaction between these neurotransmitters and opioid receptors (\u003cem\u003ee.g\u003c/em\u003e., zMOP and zDOP) in chronic stress and pain.\u003c/p\u003e \u003cp\u003eHere, the whole-body cortisol data supports a pain-related phenotype after UCS exposure with AA injection, which is at least partly driven by activation of the stress axis. Animals exposed to UCS showed elevated cortisol levels (with and without AA injection), which were partly reduced by morphine injection. This pattern is consistent with activation of the hypothalamic\u0026ndash;pituitary\u0026ndash;interrenal (HPI) axis (homologous to the mammalian hypothalamic\u0026ndash;pituitary\u0026ndash;adrenal (HPA) axis) exposed to UCS, which raises circulating cortisol and lowers the pain threshold. The fact that morphine reduced both pain behavior and cortisol levels suggests an opioidergic modulation of stress responsiveness that contributes to the observed sensitization. For instance, HPI activation can lead to β-endorphin (β-END) release, which negatively modulates the HPI axis and decreases cortisol levels (Gonzalez-Nunez et al. \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2003\u003c/span\u003e). Like morphine, β-END is also a \u0026micro;-opioid receptor agonist, which can promote analgesic effects (Zaig et al. \u003cspan citationid=\"CR69\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). Also, our data are in line with previous findings showing an inhibitory effect of endogenous opioids on the HPA axis via both \u0026micro;- and κ-opioid receptors in humans, further corroborating the influence of the opioidergic system on stress-related pathways (Kreek et al. \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e2005\u003c/span\u003e). Therefore, the morphine injection, together with β-END released by HPI activation, may contribute to the analgesic effects observed here. In summary, the behavioral and endocrine findings converge: UCS increases stress hormones and pain sensitivity, and the opioidergic intervention attenuates both. Together, these convergent endpoints strengthen the neuropharmacological interpretation of UCS-induced hyperalgesia as a centrally mediated stress\u0026ndash;pain interaction.\u003c/p\u003e \u003cp\u003eAlthough this study provides robust behavioral evidence that UCS lowers pain threshold following AA injection in zebrafish, certain methodological considerations should be noted. While whole-body cortisol was measured in the same cohorts, future inclusion of additional neurochemical or receptor-expression endpoints could further clarify central versus peripheral contributions. Such approaches would further refine the neuropharmacological resolution of the model. While the well-established doses of morphine (2.5 mg/kg) and diclofenac (40 mg/kg) follow standard zebrafish pain protocols and ensures comparability with previous data, a dose\u0026ndash;response curve may offer additional pharmacological resolution in future studies. Additionally, sexes were pooled based on prior reports, describing no sex differences in nociceptive or cortisol responses, a choice that enhances statistical power; however, targeted sex-specific analyses and further dissociation of locomotor from nociceptive outcomes remain valuable avenues for extending these findings. Lastly, while molecular validation of receptor-specific mechanisms would provide additional resolution, the present study was designed to establish a functional neuropharmacological phenotype suitable for behavioral pharmacology and translational screening.\u003c/p\u003e \u003cp\u003eIn addition to its translational value, the zebrafish model offers unique advantages for scalable drug discovery and high-throughput screening. Its small size, low maintenance costs, and compatibility with automated behavioral tracking enable rapid testing of multiple compounds and doses under controlled conditions (Kalueff et al., \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2014\u003c/span\u003e; Stewart et al., \u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e2014\u003c/span\u003e). The robust and quantifiable UCS-induced hyperalgesia phenotype described here, combined with validated pharmacological responsiveness to centrally acting agents and the whole-body cortisol measures, provides a reliable platform for identifying and prioritizing CNS-targeted analgesics. Such scalability underscores the potential of this model to bridge preclinical findings with early-phase drug development more efficiently than traditional mammalian systems. This scalability positions the model as a valuable intermediary between basic neuropharmacological research and preclinical analgesic development.\u003c/p\u003e \u003cp\u003eIn summary, our findings demonstrate that UCS lowers the pain threshold in adult zebrafish, supporting the interplay between UCS-induced behavioral deficits and pain and also highlighting the relevance of zebrafish models for studying the relationship between pain and depression. The modulation by morphine, but not diclofenac, suggests central rather than peripheral mechanisms of this phenotype. Beyond their neuropharmacological relevance, these results establish a robust and quantifiable stress-induced hyperalgesia phenotype suitable for preclinical drug discovery, particularly for screening CNS-targeted analgesics, and provide a tractable platform for probing the neurobiological basis of stress\u0026ndash;pain comorbidity. Finally, our results support the use of zebrafish as a translational model for investigating depression- and pain-responses in vivo, as well as to screen novel CNS drugs that target both domains individually and/or jointly.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cp\u003eAA: \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;\u0026nbsp;Acetic acid\u003c/p\u003e\n\u003cp\u003eAUC: \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;\u0026nbsp;Area under the curve\u003c/p\u003e\n\u003cp\u003eβ-END: \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;\u0026nbsp;β-endorphin\u003c/p\u003e\n\u003cp\u003eCEUA: \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;Animal Use Ethics Committee\u003c/p\u003e\n\u003cp\u003eCNS: \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;\u0026nbsp;Central Nervous System\u003c/p\u003e\n\u003cp\u003eCONCEA: \u0026nbsp; \u0026nbsp; \u0026nbsp;National Animal Experimentation Control Council\u003c/p\u003e\n\u003cp\u003eCOX-1: \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;\u0026nbsp;Cyclooxygenase-1\u003c/p\u003e\n\u003cp\u003eCOX-2: \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;\u0026nbsp;Cyclooxygenase-2\u003c/p\u003e\n\u003cp\u003eCTRL: \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;\u0026nbsp;Control\u003c/p\u003e\n\u003cp\u003eDOP: \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;\u0026nbsp;Delta-opioid receptor\u003c/p\u003e\n\u003cp\u003eDS: \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;Diclofenac sodium\u003c/p\u003e\n\u003cp\u003eHPA: \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;\u0026nbsp;Hypothalamic-Pituitary-Adrenal axis\u003c/p\u003e\n\u003cp\u003eHPI: \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;\u0026nbsp;Hypothalamic-Pituitary-Interrenal axis\u003c/p\u003e\n\u003cp\u003ei.p.: \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;Intraperitoneal\u003c/p\u003e\n\u003cp\u003eMOP: \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;Mu-opioid receptor\u003c/p\u003e\n\u003cp\u003eNIH: \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;National Institutes of Health\u003c/p\u003e\n\u003cp\u003eNSAIDs: \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;\u0026nbsp;Nonsteroidal anti-inflammatory drugs\u003c/p\u003e\n\u003cp\u003ePBS: \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;Phosphate-buffered saline\u003c/p\u003e\n\u003cp\u003eUCS: \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;\u0026nbsp;Unpredictable Chronic Stress\u003c/p\u003e\n\u003cp\u003ezDOPa: \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;\u0026nbsp;Zebrafish δ-opioid receptor type a\u003c/p\u003e\n\u003cp\u003ezDOPb: \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;\u0026nbsp;Zebrafish δ-opioid receptor type b\u003c/p\u003e\n\u003cp\u003ezMOP: \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;\u0026nbsp;Zebrafish µ-opioid receptor\u003c/p\u003e\n\u003cp\u003eZBC: \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;\u0026nbsp;Zebrafish Behavioral Catalogue\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis study was supported by the Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES; Finance Code 001), Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq; Proc. 402097/2023-8), Fundação de Amparo à Pesquisa do Estado do Rio Grande do Sul (FAPERGS), and Instituto Nacional de Ciência e Tecnologia para Excitotoxicidade e Neuroproteção. CDB and DBR are recipients of CNPq research productivity grants (Proc. 306115/2023-9 and Proc. 307690/2021–0). DBR is recipient of FAPERGS fellowship grants (Proc. 23/2551–0001853–5 and Proc. 24/2551–0001237–0).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDeclaration of Interest\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthical Statement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll procedures involving animals were conducted in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals and the guidelines of the Brazilian National Council for the Control of Animal Experimentation (CONCEA). The use of laboratory animals was approved by the Institutional Animal Care and Use Committee of PUCRS (CEUA-PUCRS), under protocol number 11607.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n \u003cli\u003eAlestrom P, D\u0026apos;Angelo L, Midtlyng PJ, Schorderet DF, Schulte-Merker S, Sohm F, Warner S (2020) Zebrafish: Housing and husbandry recommendations. Lab Anim 54 (3):213-224. doi:10.1177/0023677219869037\u003c/li\u003e\n \u003cli\u003eAltman R, Bosch B, Brune K, Patrignani P, Young C (2015) Advances in NSAID development: evolution of diclofenac products using pharmaceutical technology. Drugs 75 (8):859-877. doi:10.1007/s40265-015-0392-z\u003c/li\u003e\n \u003cli\u003eBarrallo A, Gonzalez-Sarmiento R, Porteros A, Garcia-Isidoro M, Rodriguez RE (1998) Cloning, molecular characterization, and distribution of a gene homologous to delta opioid receptor from zebrafish (Danio rerio). 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Elife 10. doi:10.7554/eLife.63407\u003cstrong\u003e\u003c/strong\u003e\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":false,"email":"","identity":"journal-of-neural-transmission","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"","title":"Journal of Neural Transmission","twitterHandle":"","acdcEnabled":false,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"VoR Journals","inReviewEnabled":false,"inReviewRevisionsEnabled":false},"keywords":"zebrafish, chronic stress, nociception, opioidergic system, hyperalgesia","lastPublishedDoi":"10.21203/rs.3.rs-8855618/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-8855618/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eDepression and pain share overlapping central neurobiological pathways that represent key pharmacological targets in neuropsychiatric and analgesic research. The relationship between these two conditions is bidirectional, with chronic pain contributing to the development or exacerbation of depressive symptoms, and depression intensifying the perception and tolerance of pain. However, the neuropharmacological mechanisms by which unpredictable chronic stress (UCS) modulates nociception remain poorly understood in translational vertebrate models. Here we pharmacologically characterized stress-induced nociceptive responses using a 7\u0026ndash;14-day UCS protocol in zebrafish, followed by intraperitoneal administration of 1\u0026ndash;5% (v/v) acetic acid to induce nociceptive responses. Behavioral assays were performed immediately after the injection, testing abdominal constriction (writhing-like behavior) as a pain-related endpoint, and locomotor activity levels as an additional behavioral measure related to nociception and stress. The UCS exposure elevated whole-body cortisol levels, which were attenuated by morphine but not by diclofenac, supporting the involvement of central stress\u0026ndash;pain neuropharmacological pathways. Together, these findings establish a pharmacologically tractable zebrafish model of stress-induced hyperalgesia with translational relevance for CNS-targeted analgesic discovery, highlighting the overlap between stress-related and nociceptive pathways and supporting this species as a model to investigate stress\u0026ndash;pain comorbidity.\u003c/p\u003e","manuscriptTitle":"Opioidergic modulation of stress-induced hyperalgesia in adult zebrafish","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-02-19 11:13:02","doi":"10.21203/rs.3.rs-8855618/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2026-02-27T15:24:32+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-02-26T16:51:48+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-02-24T14:00:27+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"334215347036405633462862419685940344707","date":"2026-02-15T18:06:41+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"213366675645200387657018940159482086479","date":"2026-02-15T16:40:42+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2026-02-13T14:15:40+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2026-02-13T07:43:52+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2026-02-12T18:40:48+00:00","index":"","fulltext":""},{"type":"submitted","content":"Journal of Neural Transmission","date":"2026-02-11T21:03:52+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":false,"email":"","identity":"journal-of-neural-transmission","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"","title":"Journal of Neural Transmission","twitterHandle":"","acdcEnabled":false,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"VoR Journals","inReviewEnabled":false,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"9c3c1330-8572-42ef-8dc5-09ae453cef35","owner":[],"postedDate":"February 19th, 2026","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[],"tags":[],"updatedAt":"2026-04-10T12:39:51+00:00","versionOfRecord":[],"versionCreatedAt":"2026-02-19 11:13:02","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-8855618","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-8855618","identity":"rs-8855618","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}