Project Summary/Abstract Opioid Use Disorder remains a dire public health problem, but opioid agonists such as fentanyl remain a first- line therapy for several pain conditions. As in recreational use settings, prolonged use of opioid agonists in pain management can produce physical dependence and a paradoxical decrease in pain thresholds and tolerance, which increase patients’ reliance on opioids and increase the likelihood of transitioning to Opioid Use Disorder. Continual stimulation of the inhibitory µ-opioid receptor (MOR), the primary mediator of the analgesic and rewarding effects of opioid agonists, induces counter-adaptive excitatory processes and hyperexcitability in MOR-expressing neurons. To discover new treatments that leverage the benefits of opioids but mitigate aversive and life-threatening side effects of prolonged opioid use, it is critical to determine the specific cell-types and neural circuits in the brain that are susceptible to the opioid-induced cellular maladaptations that underlie dependence and OIH. MORs are densely expressed throughout ascending pain pathways, including in the parabrachial nucleus of the pons (PBNMOR). PBNMOR neurons project to the capsular region of the central amygdala (CeC), which itself contains a pronociceptive population of neurons expressing Protein Kinase C-δ (CeCPKCδ) Activation of the PBNMOR®CeC pathway decreases pain tolerance and increases aversion-related responses, but its role in driving OIH and withdrawal, and the contribution of CeCPKCδ neurons in particular, has not been investigated. The goal of the proposal is to determine the impact of fentanyl dependence on the neural activity in the PBNMOR®CeCPKCδ pathway and whether such activity drives withdrawal and OIH-related behaviors. Aim 1 will investigate the effects of fentanyl dependence on PBNMOR®CeC projections and their role in driving OIH and withdrawal behavior by using in vivo population calcium imaging and chemogenetic manipulations during nociceptive assays and withdrawal. Aim 2 will image and manipulate the CeCPKCδ population during behavior to determine its contribution to OIH and withdrawal. Successful completion of these Aims will lay the foundation for future investigations of the pathophysiology of opioid dependence. Ideally, results from this work will suggest novel therapeutic avenues for reducing dependence mechanisms within specific cell-types. Ms. Wooldridge will receive expert training in chemogenetics, in vivo calcium imaging and its analysis, viral-mediated genetic targeting, and rigorous experimental design and statistics. The addition of this training will facilitate the applicant’s current and future research goals and enable her to have continual impact on basic neuroscience research throughout a future career as an independent academic researcher.