PROJECT SUMMARY Nucleus Accumbens (NAc) activity is critically important for response to motivated stimuli, including aversive stimuli. However, it is not known how the NAc processes and responds to these stimuli across the 24hrs cycle. We and others have found NAc shows daily changes in neuronal activity which appears to be in phase with circadian-dependent dopamine release from the primary dopamine input to the NAc, the ventral tegmental area (VTA). Therefore, response to motivated stimuli may vary depending on time-of-day. We recently discovered the circadian gene CRYPTOCHROME (CRY) modulates Gs-protein coupled with dopamine 1 (D1) receptor which may alter neuronal activity and excitability of NAc projection neurons. These data suggest circadian regulation of dopamine receptor activity in the NAc has the ability to strongly alter response to dopamine driven by aversive stimuli via the dopamine 1 receptor. We hypothesize that CRY controls circadian NAc activity through modulation of dopamine 1 receptor function. In year 1 of this proposal, we will assess the role of CRY in regulating circadian dopamine 1 receptor function ex vivo and in vivo. We will selective knockdown CRYs in the NAc and assess D1 receptor function by whole-cell patch clamp physiology by monitoring voltage-gated K+ currents ex vivo and by fiber photometry to monitor D1 receptor signal transduction dynamics in vivo. In year 2, we will use ex vivo approaches with selective knockdown of CRYs to assess DA-dependent NAc excitability. We will further determine circadian-dependent VTA dopamine release and NAc neuronal activity changes in response to aversive stimuli in vivo by monitoring calcium signaling with fiber photometry. This study will allow us to dissect mechanistic, circadian regulation of NAc neurophysiological response and dopamine receptor function. The overall aim of this project is to determine the role of CRY in regulating excitability and function of the NAc and how this regulation controls NAc response to aversive stimuli. This proposal will provide translationally relevant information about circadian regulation of basic mechanistic underpinnings of NAc dopamine function and behaviorally relevant, neurobiological response to aversive stimuli. This work will further provide a foundation for understanding how circadian-driven NAc changes underlie susceptibility to mood disorders such as depression.