Resumo

Introduction: Zebrafish (Danio rerio), a fish native from India, is an emerging player in disease research and drug screening. It has been used as a model organism to study development, neuroscience, pharmacology, toxicology, behavior and teratology. The employment of this specie has many advantages when compared to other vertebrates, such as low cost, easy handling and maintenance, robust and fast reproduction, and genetic homology to humans. Objective: The purpose of this study was to develop a model of chronic stress in zebrafish. We evaluated the effects of stress on anxiety levels, memory, and corticotrophin releasing factor (CRF) and glucocorticoid receptor (GR) gene expression. Methods: Male adult zebrafish (n=15-20) were submitted to a 7-day unpredictable chronic stress (UCS) protocol, during which the fish were submitted twice a day to a variety of stressors always presented in random order. After the stress period, 3 fish at a time were tested in an appropriated tank to evaluate the behavioral parameters of height in the tank, color, shoal cohesion and locomotion. 24 hours later fish were submitted to inhibitory avoidance task to assess memory performance. Briefly, latencies to enter the dark compartment associated with a shock were measured in training and test sessions. Another group of stressed fish was used to measure CRF and GR mRNA by semi-quantitative RT-PCR. A non-stressed group kept at the same room but not submitted to stressors was submitted to the same behavioral and molecular analysis above. Protocols were approved by the Ethics Committee of PUCRS number 09/00126. Results: The UCS protocol induced an anxiety-like behavior showed by increase in time that fish spent in the tank bottom, as well as color and shoal cohesion alterations (p<0.05). Stressed fish also showed memory impairment in inhibitory avoidance task (control: 40.2 s ± 7.9; stress group: 16.3 s ± 3.3 in test session, p<0.0001). Moreover, the stress protocol caused a decrease in CRF (control: 162 ± 6.25; stress group: 129 ± 18.2, p<0.05) without altering GR expression. Data analyzed by Kruskal-Wallis/Mann-Whitney or ANOVA/Duncan. Conclusion: This study demonstrated for the first time the effects of chronic stress in zebrafish on behavioral and molecular parameters. The protocol presents adequate construct validity (a sound theoretical rationale) because the zebrafish stress response is similar to human (hypothalamo-pituitary-adrenal activation). More studies are necessary to determine face and predictive validity of this model.