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.

Environmental stressors differentially modulate anxiety-like behaviour in male and female zebrafish.

How differently male and female responds in a stressful situation is a matter of curiosity. Apart from curiosity, this opens a new arena to the synthesis of personalized/individualized medications. Here, we used zebrafish, a suitable experimental animal model to study stress and anxiety. We evaluated the differential responses in adult male and female zebrafish on the acute exposure of three different stressors: Caffeine (100 mg/L), Conspecific alarm substance (3.5 ml/L), and sight of sympatric predators (Leaf fish and Snakehead) with the help of two different behavioural paradigms (Novel tank test & Predator exposure). Behavioural responses were captured over 6 minutes and quantified using Smart 3.0. Male zebrafish were found to be more responsive to caffeine treatment. Conspecific alarm substance-challenged males and females showed robust alarm reactions whereas females were found to be more prone to it. Female zebrafish showed statistically significant aversion to the visual representation of sympatric predators. Taken together, each stressor induced differential responses in male and female zebrafish.

The zebrafish (Danio rerio) anxiety test battery: comparison of behavioral responses in the novel tank diving and light-dark tasks following exposure to anxiogenic and anxiolytic compounds.

RATIONALE: Triangulation of approaches (i.e., using several tests of the same construct) can be extremely useful for increasing the robustness of the findings being widely used when working with behavioral testing, especially when using rodents as a translational model. Although zebrafish are widely used in neuropharmacology research due to their high-throughput screening potential for new therapeutic drugs, behavioral test battery effects following pharmacological manipulations are still unknown. METHODS: Here, we tested the effects of an anxiety test battery and test time following pharmacological manipulations in zebrafish by using two behavioral tasks: the novel tank diving task (NTT) and the light-dark test (LDT). Fluoxetine and conspecific alarm substance (CAS) were chosen to induce anxiolytic and anxiogenic-like behavior, respectively. RESULTS: For non-drug-treated animals, no differences were observed for testing order (NTT → LDT or LDT → NTT) and there was a strong correlation between performances on the two behavioral tasks. However, we found that during drug treatment, NTT/LDT responses are affected by the tested order depending on the test time being fluoxetine effects higher at the second behavioral task (6 min later) and CAS effects lower across time. CONCLUSIONS: Overall, our data supports the use of baseline behavior assessment using this anxiety test battery. However, when working with drug exposure, data analysis must carefully consider time-drug-response and data variability across behavioral tasks.

The impact of chronic unpredictable early-life stress (CUELS) on boldness and stress-reactivity: Differential effects of stress duration and context of testing.

Early-life stress (ELS) has been shown to result in a diverse array of long-lasting impacts; for example, increasing vulnerability to disease or building 'resilience' in adulthood. Previously, zebrafish (Danio rerio) have been used to understand the mechanisms by which ELS induces different behavioral phenotypes in adults, with alterations in both learning and anxiety observed in exposed individuals. Here, we subjected zebrafish larvae to chronic unpredictable early-life stress (CUELS) for 7 or 14 days, to investigate the impact on boldness towards a new environment and novel object, and stress-reactivity. We observed that 7 days of CUELS resulted in increased time spent in the top of a novel tank (indicating boldness) but did not alter approach to a novel object. Although CUELS did not affect stress-reactivity in terms of cortisol levels, decreased anxiety-like response to conspecific alarm substance (CAS) was observed in both ELS groups (7 and 14 days of CUELS). Therefore, for the first time, we observe a potential negative effect of CUELS by dampening the behavioral stress response following exposure to CAS. Overall, these data support the use of zebrafish as a translational model to study the broad range of ELS-induced permanent changes in behavior. It could also be used to investigate the mechanisms underlying both the positive and the negative effects of early-life adversity.

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.

Nicotine prevents anxiety-like behavioral responses in zebrafish.

Anxiety-related disorders are severe psychiatric conditions that involve complex physiological and behavioral maladaptive responses. The use of conspecific alarm substance (CAS) for inducing anxiety-like behaviors in fish species provides important translational insights of how aversive conditions modulate neurobehavioral functions. Because nicotine may elicit anxiolytic-like responses, here we investigated whether acute nicotine exposure prevents CAS-induced anxiogenic-like behaviors in zebrafish. We used both novel tank and light-dark tests as two well-established paradigms for measuring anxiety-like phenotypes. Fish were individually exposed to 1 mg/L nicotine or non-chlorinated water for 3 min and then transferred to other tanks in the absence or presence of 3.5 mL/L CAS for 5 min. Later, the behavior of fish was tested in the novel tank test or in the light-dark preference test. As expected, CAS triggered aversive behaviors by increasing bottom-dwelling, freezing, erratic movements, scototaxis, and risk assessment episodes. Nicotine alone elicited anxiolytic-like behaviors since it increased the time spent in the top, as well as the average duration of entry in the lit compartment. Moreover, nicotine pretreatment prevented CAS-induced aversive responses without changing locomotion, suggesting that anxiolysis could play a role, at least in part, to the behavioral effects of nicotine observed here. Overall, these novel findings show the beneficial effects of nicotine on anxiogenic responses in zebrafish. We also reinforce the practical advantages of this aquatic species to explore the relieving properties of nicotine, as well as to understand the neurobiological bases involved in anxiety-related disorders and associated therapeutic targets.

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.

Nicotine increases fear responses and brain acetylcholinesterase activity in a context-dependent manner in zebrafish.

Nicotine is an alkaloid with positive effects on learning and memory processes. Exposure to conspecific alarm substance (CAS) elicits fear responses in zebrafish, but the effects of nicotine on aversive behaviors and associative learning in this species remain unclear. Here, we evaluated whether nicotine enhances contextual fear responses in zebrafish and investigated a putative involvement of brain acetylcholinesterase (AChE) in associative learning. Fish were exposed to 1 mg/L nicotine for 3 min and then kept in non-chlorinated water for 20 min. Later, animals were transferred to experimental tanks in the absence or presence of 3.5 mL/L CAS for 5 min (training session). After 24 h, fish were tested in tanks with similar or altered context in the absence of CAS (post-training session) and brain AChE activity was further assessed. At training, CAS increased freezing, erratic movements, and decreased the time spent in top area, while nicotine abolished the effects of CAS on erratic movements. Nicotine/CAS group tested in a similar context showed exacerbated freezing and reduced transitions to top area. Moreover, a decrease in distance traveled was observed in control, nicotine, and nicotine/CAS groups at post-training. Nicotine also stimulated brain AChE activity in CAS-exposed animals reintroduced in tanks with similar context. Although freezing bouts and time spent in top could serve as behavioral endpoints that reflect CAS-induced sensitization, the effects of nicotine occurred in a context-dependent manner. Collectively, our data suggest an involvement of cholinergic signaling in aversive learning, reinforcing the growing utility of zebrafish models to explore the neurobehavioral effects of nicotine in vertebrates.

Time-dependent sensitization of stress responses in zebrafish: A putative model for post-traumatic stress disorder.

Time-dependent sensitization (TDS)-the delayed increase in neurobehavioral responses to heterotypic stressors after exposure to an intense, inescapable stressor-has been proposed as an animal model for post-traumatic stress disorder (PTSD). Translationally relevant stressors used in TDS are capable of affecting more than one behavioral domain and produce interindividual variability in responsiveness. Here, conspecific alarm substance (CAS) is shown to induce TDS in zebrafish in inter- and intra-population-specific way. Exposure to CAS, an ecologically relevant stimulus which produces fear-like responses acutely, increased anxiety and arousal in zebrafish from the blue shortfin (BSF) phenotype 24h after stimulus delivery. Anxiety-like responses were differently affected immediately and 24h after stimulus delivery. Anxiety-like responses were more sensitized in zebrafish from the longfin (LOF) than in the BSF phenotype, an effect which is reminiscent of "basal" differences in anxiety-like behavior. After application of behavioral cutoff criteria, CAS was shown to produce intense TDS in ∼25% of LOF animals, while ∼20% of exposed animals showed little evidence of TDS. Overall, these results suggest that CAS induces TDS in zebrafish after a 24h "incubation" period, with inter- and intra-population variability that underlines its face and ecological validity.