PROJECT SUMMARY Serotonin has been long been recognized as an important modulator of mood and behavior, yet it projection- specific dynamics are little understood. While serotonin has been well studied in anxiolytic contexts, activity of certain serotonin projections works to increase stress behaviors. In particular, serotonergic projections from dorsal raphe to the anterodorsal bed nucleus of the stria terminalis have been shown to be anxiogenic. Despite decades of targeting serotonin, a consensus on serotonin function and release dynamic is lacking. Pharmacological approaches, microdialysis, and chemogenetic manipulations have produced key insights, but none provide millisecond resolution for serotonin release and correlates of specific reactions and behaviors until recently. The Tian lab is in the final stages of developing two serotonin specific biosensors (sLight and iSeroSnFR) that open the door for new possibilities in serotonin research. I will these sensors combined with fiber-photometry to optically measure behaviorally driven serotonin release in an anxiogenic serotonin circuit with high temporal and spatial resolution. The series of experiments outlined in this proposal are significant because they will: Aim 1) rigorously demonstrate the in vivo utility of serotonin sensors in dissecting anxiety circuit, providing end users important protocols and guidelines for fast technological implementation; Aim2) map dynamics of serotonin release in anxiogenic circuits to stress behavior, and discover how activity in this circuit is impacted in anxious animals and determine the mechanistic impacts of acute vs. chronic SSRI administration on anxiogenic serotonin signaling; Aim 3) determine the developmental effects of pubertal testosterone on the maturation of anxiogenic serotonin activity and SSRI efficacy.