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

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

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

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

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

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

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

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

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

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

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

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

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

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