Methods to Quantify Basal and Stress-Induced Cortisol Response in Larval Zebrafish

The zebrafish model is being increasingly used in stress and anxiety research. The homologue of the hypothalamic-pituitary-adrenal (HPA) axis has been found in fish and is referred to as the hypothalamic-pituitary-�interrenal (HPI) axis. HPA (mammalian) and HPI (fish) axes are homologous in term of the general functional organization and physiology of the stress response via similarities in anatomy, connectivity, and molecular constituents. Because of these qualities, a range of complementing assays are being developed to assess the behavioral and endocrine correlates of the stress response in both larval and adult zebrafish. The protocol presented here describes a simple methodology for measurement of whole-body cortisol levels in larval zebrafish using a cortisol radioimmunoassay (RIA) optimized for very small volume. Our protocol represents the first cortisol RIA method for larval zebrafish, thus complementing the previously characterized cortisol RIA for adult zebrafish while offering an alternative to the ELISA method. We also provide a methodology for the use of an acute stress paradigm customized for zebrafish larvae, i.e., acute mild electric shock. The mild electric shock stress paradigm developed here is similar to the foot-shock stress paradigm widely used in rodents, which has not been yet characterized in zebrafish. We show how this stress paradigm can be easily used in combination with the cortisol RIA method to rapidly reveal functionality of the HPI axis during ontogeny in larval zebrafish, especially with regard to the stress hyporesponsive period.