Abstract
Zebrafish larvae show a rapid increase in cortisol in response to acute stressors, followed by a decline. While these responses are documented, both the duration of the refractory period to repeated stressors and the role of glucocorticoid receptors (GR) in specific phases of the glucocorticoid negative feedback are still being clarified. We explored these questions using water vortices as stressors, combined with GR blockage and measurements of whole-body cortisol in zebrafish larvae subjected to single and repeated stress protocols. Cortisol levels were elevated 10 min after stress onset and returned to baseline within 30-40 min, depending on the stressor strength. In response to homotypic stress, cortisol levels rose above baseline if the second stressor occurred 60 or 120 min after the first, but not with a 30-min interval. This suggests a rapid cortisol-mediated feedback loop with a refractory period of at least 30 min. Treatment with a GR blocker delayed the return to baseline and suppressed the refractory period, indicating GR-dependent early-phase feedback regulation. These findings are consistent with mammalian models and provide a framework for further analyses of early-life cortisol responses and feedback in zebrafish larvae, ideal for non-invasive imaging and high-throughput screening.