There is growing interest in zebrafish as a model organism in behavioral pharmacology research. Several anxiety behaviors have been characterized in zebrafish, but the effect of anxiolytic drugs on these parameters has been scarcely studied. The purpose of this work was to assess the predictive validity of acute treatment with anxiolytic drugs on behavioral parameters of anxiety. In the first task we simultaneously observed behavior of adult zebrafish on four parameters: height in the tank, locomotion, color, and shoal cohesion. The second task was the assessment of light/dark preference for 5 min. The benzodiazepines clonazepam, bromazepam, diazepam, and a moderate dose of ethanol significantly reduced shoal cohesion. Buspirone specifically increased zebrafish exploration of higher portions of the tank. In the light/dark task, all benzodiazepines, buspirone, and ethanol increased time spent in the light compartment. After treatment with anxiolytics, fish typically spent more than 60s and rarely less than 40s in the light compartment whereas controls (n=45) spent 33.3±14.4s and always less than 60s in the light compartment. Propranolol had no clear effects in these tasks. These results suggest that light/dark preference in zebrafish is a practical, low-cost, and sensitive screening task for anxiolytic drugs. Height in the tank and shoal cohesion seem to be useful behavioral parameters in discriminating different classes of these drugs.

Zebrafish has been increasingly used in behavioral studies, but data can present high variability. Most studies have been performed using isolated zebrafish, despite their interactive nature and shoaling behavior. We compared adult zebrafish behavior and cortisol levels after exposure to novelty as well as sensitivity to Pentylenetetrazole (PTZ)-induced seizures in animals tested individually or in groups of three (triplets). In the exploratory behavior task, data from single fish and triplets were not significantly different, but single fish data were more disperse in latency, to enter and time spent in the tank upper part, and crossings. In the light-dark task, time in the light zone and crossings were not different between groups, but latency to enter the dark zone and data variability were. We also observed that the latency to reach stage III seizures induced by PTZ was higher in triplets, but data dispersion was not different from single fish. Finally, cortisol levels of fish individually exposed to a novel environment were higher and more variable than triplets, while both groups had higher levels than unmanipulated animals. Thus, when tested individually, zebrafish are more stressed and present more variable behavior due to disruption of their natural shoal strategies. These features can be beneficial or detrimental depending on study aims and should be considered when designing, analyzing, and interpreting zebrafish behavioral data.

Adenosine is an endogenous modulator of brain functions, which presents anticonvulsant properties. In addition, its levels can be increased during neural injury. The modulation of extracellular adenosine levels by ectonucleotidase and adenosine deaminase (ADA) activities may represent a key mechanism in the control of epileptogenesis. In the present study, we investigated the effects of acute seizure episodes and antiepileptic drug (AED) treatments on ectonucleotidases and ADA activities in adult zebrafish brain. Our data have demonstrated that pentylenetetrazole (PTZ)-induced seizures did not alter ATP, ADP, and AMP hydrolysis in brain membrane fractions. However, there was a significant increase on ecto-ADA and soluble ADA activities in PTZ-treated animals immediately after a clonus-like convulsion and loss of posture, which are typical behavioral changes observed in Stage 3. Furthermore, our results have demonstrated that AED pretreatments prevented the stimulatory effect promoted by PTZ exposure on ADA activities. The PTZ and AED treatments did not promote alterations on ADA gene expression. Interestingly, when exposed to PTZ, animals pretreated with AEDs showed longer latency to reach the clonus-like seizure status, which is an effect that matches the suppression of the increase of ADA activity promoted by the AEDs. These data suggest that the adenosine deamination could be involved in the control of seizure development in zebrafish and may be modulated by AED treatments.

The assessment of shoaling in adult zebrafish is technically difficult, but important, given their social nature. The present study aimed to characterize a new protocol using simple automated tracking software to evaluate general behavior and social interaction simultaneously. To this end, we used a single tank with a central transparent glass division and placed one zebrafish on each side for 5 min. This strategy allows fish to interact visually at the same time that individual automated evaluation of behavior can be easily performed. Our results showed that, when two fish are placed side-by-side, there is an increase in their height in the tank compared with isolated fish and they remain close to each other. The pharmacological treatments with benzodiazepines (bromazepam and clonazepam) and the serotonergic drugs buspirone, fluoxetine, and escitalopram did not affect locomotion at the concentrations tested, except for the highest concentration of buspirone. Nevertheless, benzodiazepines increased interfish distance (i.e. reduced shoaling behavior) and serotonergic drugs elevated height in the tank. These results support the use of the side-by-side exploratory test for behavioral studies with the zebrafish, including high-throughput behavioral screening for antidepressants and anxiolytics.