Project Summary The proposed research is relevant to public health because opioid use is prevalent in the United States and the human and financial costs associated with tolerance and withdrawal are at crisis levels. In order to reduce opioid abuse and misuse, our long-term goal is to determine the intracellular mechanisms to lead to these clinical problems. The objective of the proposed research is to understand the molecular involvement of adenylyl cyclase signaling and potassium channels in the peripheral and central nervous system during chronic opioid exposure using rodent models. A great deal of work has been done investigating the paradoxical phenomena of hypertrophied adenylyl cyclase activity and expression that occurs during chronic opioid exposure. The central hypothesis is that increased activity adenylyl cyclase and downstream mediators decrease KATP channel activity, leading to neuronal depolarization and increased hypersensitivity and spontaneous pain. The rationale of this proposal is that its completion will identify key intracellular targets of adenylyl cyclases, including potassium channels such as ATP-sensitive potassium (KATP) channels, which will help us to classify molecules that alter neuronal excitability and may play a key role in hypersensitivity during chronic opioid exposure. Given the history of research into adenylyl cyclase and inhibitory G-protein coupled signaling in the nervous system, it is surprising that fundamental questions still exist as to how these molecules affect neurophysiology of pain processing. Our first hypothesis is that overall expression of adenylyl cyclase 1 in the dorsal root ganglia and spinal cord increases after chronic morphine exposure. Our second hypothesis is that upregulation of adenylyl cyclase 1, and consequently cAMP, protein kinase A, and Epac molecules decrease KATP channel activity in vitro. Our third hypothesis is that upregulation of KATP channel subunits in the dorsal root ganglia and spinal cord using intrathecal injection of adenovirus viral vectors will improve mechanical hypersensitivity, mobility, and nerve conduction in mice after upregulated adenylyl cyclase during chronic opioid exposure. These approaches should prove to be complementary to one another and will provide the greatest opportunity to observe changes that occur in the nervous system after chronic opioid exposure. We plan on addressing these hypotheses through an innovative combination of in situ hybridization, electrophysiology, and potassium flux assays in vitro, and genetic approaches in vivo. The proposed work is important because completion of these studies will determine if the inverse relationship between adenylyl cyclase and KATP channel functionality could ultimately underlie and promote pain signaling seen clinically during opioid tolerance and withdrawal. KATP channels present an unutilized and interesting target for the development of drugs to treat opioid abuse and misuse. These results will ha...