A Standardized Unpredictable Chronic Stress Protocol for Adult Zebrafish

The unpredictable chronic stress (UCS) protocol, originating in the 1980s, has contributed to depression research by inducing behavioral and physiological changes in rodents that resemble the symptoms observed in patients. Its translational potential led to widespread adoption, but adaptations and variations to the protocol have raised concerns about reproducibility. Over a decade ago, our laboratory adapted the UCS protocol for zebrafish (Danio rerio), aiming to bridge species-specific gaps in stress research. Since then, several studies have reported alterations in outcomes related to the clinical manifestations of stress-related disorders, providing evidence on the applicability of the model for testing drugs and other therapeutic strategies. However, there is substantial heterogeneity in protocols and research findings, hindering comparability across studies. To address these challenges, we describe a 14-day UCS protocol for adult zebrafish in detail, involving a series of varied stressors administered twice daily. This comprehensive protocol includes stressors, such as tank changes, net chasing, overcrowding, low water levels, and temperature fluctuations. Each step is designed to ensure reproducibility and to mimic the unpredictable and varied nature of stress experienced by humans. By establishing standardized procedures, we aim to enhance the consistency, reliability, and translational potential of UCS research using zebrafish, ultimately contributing to more robust and comparable findings across studies. © 2026 The Author(s). Current Protocols published by Wiley Periodicals LLC. Basic Protocol: UCS protocol for adult zebrafish.