Project Summary Opioids such as morphine and fentanyl effectively relieve acute and post-operative pain but long-term use is problematic due to their abuse liability. Long-term opioid use causes cellular and circuit level adaptations that can lead to addiction, but the precise mechanisms are not fully understood. Furthermore, withdrawal from opioids unmasks these adaptive changes facilitating increased drug intake and promoting relapse. This proposal will use an optogenetic approach to isolate glutamate projection from both opioid sensitive (thalamic) and insensitive (cortical) terminals coming into the striatum in mice. Whole-cell electrophysiological recordings in brain slices will be used to investigate the mechanisms mediating adaptive changes following chronic opioid treatment and withdrawal. Receptor phosphorylation is a key cellular event mediating acute desensitization and long-term tolerance in cell-body specific µ-opioid receptors (MORs), but little is known about the role of phosphorylation in mediating signaling in presynaptic terminal MORs. Experiments in Aim 1 will therefore elucidate the role of phosphorylation in mediating acute sensitivity and long-term tolerance to both morphine and fentanyl after chronic treatment in terminal MORs. Furthermore, adaptations following chronic treatment go beyond the receptor level and can influence downstream second messengers like adenylyl cyclase. Adenylyl cyclase can be metabolized to adenosine in central nervous system synapses to modulate glutamate release. Experiments in Aim 2 will therefore focus on the mechanisms underlying release and regulation of adenosine in striatal synapses, specifically the role of MORs in mediating adenosine concentration after chronic opioid treatment. The overall hypothesis is that MOR phosphorylation is a key signaling event that establishes both acute sensitivity and long-term tolerance to opioids, and activation of MORs plays a critical role in mediating the concentration of adenosine in striatal synapses. Ultimately, the results from this study will address the role of phosphorylation in terminal MOR signaling and determine receptor and cellular adaptations that result from chronic opioid treatment and withdrawal. By looking at the effects of both morphine (a partial agonist) and fentanyl (a full agonist), a comprehensive understanding of the role of receptor phosphorylation on synaptic transmission after chronic opioid exposure will be established. Understanding the key receptor and cellular changes that mediate synaptic activity after chronic opioid treatment is a crucial first step in identifying novel therapeutic targets to treat opioid use disorder.