PROJECT SUMMARY The main goals of this training proposal is 1) to provide Li Li, MD, PhD, a practicing anesthesiologist at the University of Washington, with additional scientific and professional skills to facilitate his transition to become an independent investigator, and 2) to better understand how the locus coeruleus (LC), at the cellular and circuit levels, contributes to opioid-induced sleep disturbances. Studying sleep disturbances caused by opioids is important because opioids have enormous societal impact, causing over 46,000 overdose deaths in 2018 and almost $80 billion in economic cost annually in the United States; and because recent studies have implicated several aspects of sleep disturbances caused by opioids, including poorer cognition, decreased pain tolerance, and increased negative affect, in contributing to chronic opioid use and relapse. Interestingly, the LC and paraventricular thalamus (PVT) are two brain regions that modulate sleep-wake balance and mediate withdrawal behaviors from opioids, but their roles in opioid-induced sleep disturbances are relatively unknown. Thus, during his K99 phase, Dr. Li aims to 1) examine LC neuronal activity in relation to sleep patterns in a mouse model of acute and chronic opioid administration and 2) determine the role of local opioid receptors in the LC in opioid- induced sleep disturbances. Furthermore, he plans to receive training in 1-photon microendoscopic imaging, optogenetic manipulation of opioid receptors, and cell-specific drug targeting using drug acutely restricted by tethering (DART); and will be mentored by Prof. Michael Bruchas, a world-renowned expert in opioid neurobiology, and a committee of experts in neural circuitry analysis, sleep, and substance abuse disorders. Together, his training will help transition to his independent R00 phase, where he plans to use his trained skills 1) to examine PVT neuronal activity in relation to sleep patterns in acute and chronic opioid administration, and to determine role of local opioid receptors in PVT in opioid-induced sleep disturbances. Studies in the independent phase will prepare him for a future R01 submission to further examine arousal neural circuitry response to sedative and anesthetic drugs. Thus, this proposal aims to characterize the LC-PVT circuit in a mouse model of acute and chronic opioid administration using cutting-edge tools in neural circuit analysis and cell-specific pharmacology in order to better understand the cellular and circuit mechanism underlying opioid- induced sleep disturbances, and provides the essential training and mentorship for Dr. Li to help him achieve his career goal of starting his own lab and becoming an independent investigator.