Role of the serotonergic system in ethanol-induced aggression and anxiety: A pharmacological approach using the zebrafish model.

Acute ethanol (EtOH) consumption exerts a biphasic effect on behavior and increases serotonin levels in the brain. However, the molecular mechanisms underlying alcohol-mediated behavioral responses still remain to be fully elucidated. Here, we investigate pharmacologically the involvement of the serotonergic pathway on acute EtOH-induced behavioral changes in zebrafish. We exposed zebrafish to 0.25, 0.5, 1.0% (v/v) EtOH for 1 h and analyzed the effects on aggression, anxiety-like behaviors, and locomotion. EtOH concentrations that changed behavioral responses were selected to the subsequent experiments. As a pharmacological approach, we used pCPA (inhibitor of tryptophan hydroxylase), WAY100135 (5-HT1A antagonist), buspirone (5-HT1A agonist), CGS12066A and CGS12066B (5-HT1B antagonist and agonist, respectively), ketanserin (5-HT2A antagonist) and (±)-DOI hydrochloride (5-HT2A agonist). All serotonergic receptors tested modulated aggression, with a key role of 5-HT2A in aggressive behavior following 0.25% EtOH exposure. Because CGS12066B mimicked 0.5% EtOH anxiolysis, which was antagonized by CGS12066A, we hypothesized that anxiolytic-like responses are possibly mediated by 5-HT1B receptors. Conversely, the depressant effects of EtOH are probably not related with direct changes on serotonergic pathway. Overall, our novel findings demonstrate a role of the serotonergic system in modulating the behavioral effects of EtOH in zebrafish. These data also reinforce the growing utility of zebrafish models in alcohol research and help elucidate the neurobiological mechanisms underlying alcohol abuse and associated complex behavioral phenotypes.

Concomitant taurine exposure counteracts ethanol-induced changes in locomotor and anxiety-like responses in zebrafish.

Taurine (TAU) is a ß-amino sulfonic acid with pleiotropic roles in the brain, including the neuromodulatory activity via GABAergic and glycinergic agonism. This molecule is found at high concentrations in energy drinks and is often mixed with alcohol in beverages. Although TAU has a neuroprotective role in the brain, the putative risks of mixing TAU and EtOH are not fully understood. Here, we investigated whether TAU modulates locomotor and anxiety-like behavior in adult zebrafish by using the novel tank and light-dark tests following acute EtOH exposure at anxiogenic and anxiolytic concentrations. Zebrafish were individually exposed to water (control), TAU (42, 150, and 400 mg/L), and EtOH (0.25% (v/v) and 1% (v/v)) both independently and cotreated for 1 h. EtOH 0.25% and TAU produced U-shape anxiolytic-like behavior in the light-dark test, TAU 42 and 400 positively modulated EtOH effects, and TAU 150 exerted a protective effect. All TAU concentrations counteracted EtOH 1%-induced locomotion impairment, as well as the anxiogenic-like behavior. Finally, all TAU concentrations when given independently or cotreated with EtOH 0.25% and 1% decreased the risk assessment of the lit compartment. Principal component analyses revealed that exploration and anxiety-like responses were the main behaviors that contribute to the effects of TAU and EtOH. Overall, we demonstrate that TAU differently modulates EtOH-induced anxiolytic- and anxiogenic-like behaviors depending on the concentration, suggesting a complex mechanism underlying TAU and EtOH interactions.

Exploring Object Discrimination in Zebrafish: Behavioral Performance and Scopolamine-Induced Cognitive Deficits at Different Retention Intervals.

The object discrimination test allows the testing of different memory retention periods. However, few behavioral endpoints have been measured in fish species such that retention is often assessed using a single parameter (time spent in object area). Here, we aimed to explore the object discrimination test in zebrafish by assessing their behavioral performance after 1 or 24 h retention interval periods. To characterize putative interaction-like behaviors, fish were tested in the absence or presence of scopolamine (1 h before test session). Zebrafish were habituated for 3 consecutive days in the experimental tank, and training session was performed for 10 min using two identical nonpreferred objects (black cube or sphere). After the retention intervals, a familiar object was replaced by a novel object (test session, 10 min). Fish were also exposed to the novel tank diving test to assess locomotion and anxiety-like behaviors. At 1 h retention interval, animals performed more circular-like investigation near the familiar object, whereas 24 h after training session, a prominent rapid investigation was observed when animals explore the nonfamiliar object. Because scopolamine abolished these phenotypes, as well as the increased time spent in the novel object area during the test without changing locomotion and anxiety-related parameters, the behavioral responses described here may predictively reflect interaction-like behaviors involved in object discrimination memory in zebrafish models.

Taurine Protects from Pentylenetetrazole-Induced Behavioral and Neurochemical Changes in Zebrafish.

Epilepsy is a common neurological disorder characterized by recurrent unprovoked seizures, which culminate in various neurobehavioral and neurochemical changes. Taurine (TAU) is an amino sulfonic acid which acts an endogenous inhibitory neuromodulator. Moreover, TAU displays intrinsic antioxidant activity, contributing to its beneficial actions in the CNS. Here, we evaluated whether TAU pretreatment protects from pentylenetetrazole (PTZ)-induced behavioral alterations and oxidative stress-related parameters in zebrafish brain tissue. Fish were pretreated with 42, 150, and 400 mg/L TAU (40 min) and further exposed to 10 mM PTZ (20 min) to analyze the seizure-like behaviors. As a positive control, another group was previously treated with 75 µM diazepam (DZP). Afterwards, biochemical experiments were performed. All TAU concentrations tested decreased seizure intensity in the first 150 s. Importantly, 150 mg/L TAU attenuated seizure-like behavioral scores, decreased seizure intensity, reduced the frequency of clonic-like seizures (score 4), and increased the latency to score 4. TAU (150 mg/L) also prevented oxidative stress in PTZ-challenged fish by decreasing lipid peroxidation and protein carbonylation and preventing changes on nonprotein thiol levels. No significant changes were observed in MTT assay and LDH activity. Differently than observed in DZP group, TAU did not affect the overall swimming activity of fish, suggesting different mechanisms of action. Collectively, we show that TAU attenuates PTZ-induced seizure-like behaviors and brain oxidative stress in zebrafish, suggesting the involvement of antioxidant mechanisms in neuroprotection.

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.