PROJECT SUMMARY Substance use disorder (SUD) remains a large problem in the United States. The primary goal of the CARRS Center is to understand how sleep and circadian rhythm traits and environmental disruptions during adolescence lead to increased vulnerability for substance abuse. We predict that a combination of biological and environmental factors contribute to this increased risk. The goal of Project 3 in the Center is to provide translational studies in both human subjects and rodent models that determine mechanistic details of how circadian rhythm and sleep disruption alter reward circuitry. We will also test potential therapeutic treatments for social jet lag in adolescents that might mitigate risk for SUD. Here we will use a noninvasive method of assessing the human molecular clock using skin cells collected from hair follicles, which has been fully optimized for the measure of molecular rhythms. However, no study to date has cultured cells from adolescents for molecular rhythm measurement, associated molecular rhythms with other sleep, reward and circadian measures, or investigated the effects of potentially therapeutic compounds on these rhythms. In Project 3 we will be utilizing hair follicle samples collected in P1/2 from human subjects and combine this data with the thorough rhythm, sleep, cognition, and reward data collected in those projects. We will also determine how molecular measures in rodents correlate with behavioral and electrophysiological data collected in P4/5. Moreover, we are testing potential pharmacological interventions on molecular rhythms which will inform future clinical trials. We are also using rodents to provide detailed gene and protein expression in brain regions of interest in response to specific manipulations of sleep and circadian rhythms. The questions we want to answer are: (1) How are sleep and circadian rhythm phenotypes and addiction vulnerability related to molecular rhythms? This will be tested in both humans and rats through cell culture studies. (2) What are the molecular mechanisms by which circadian rhythm and sleep disruptions in combination or independently lead to increased vulnerability for substance abuse in adolescents? This will be tested in rodents generated in Core B that have experienced specific sleep and circadian manipulations followed by RNA sequencing and proteomics in PFC and NAc. (3) Are there pharmacological interventions that will shift and/or amplify molecular rhythms in human subjects that could be useful for adolescents with delayed chronotypes and circadian misalignment? This will be directly tested in cell culture. Taken together, these studies could point towards novel treatments for adolescents that are at risk for substance use disorders.