PROJECT SUMMARY Withdrawal is a major obstacle in overcoming opioid dependence and addiction. Identifying the neural circuits involved and how opioids modulate their activity is essential for developing new therapeutic strategies. The medial habenula (MHb) expresses particularly high levels of mu-opioid receptor (MOR) and emerging evidence implicates the MHb as a hotspot for the physical and affective symptoms of opioid withdrawal and relapse. Despite this, there is remarkably little information on how MOR signaling influences MHb projection neurons, or how chronic MOR signaling could induce physiological changes in MHb circuits that contribute to withdrawal. MHb neurons project principally to the interpeduncular nucleus (IPN) where they can release acetylcholine (ACh) or glutamate; and some of these neurons can co-release both glutamate and ACh. While cholinergic- and glutamatergic-defined MHb neurons have been implicated in processes underlying addiction, the respective roles and relative importance of these co-transmitters remain unclear. Based on prior literature and preliminary data we posit an important role for glutamate and ACh co-release from MHb to IPN in mediating effects of opioid dependence, including withdrawal. In this exploratory proposal we aim to test how acute and chronic MOR activation influences activity in MHb and synaptic transmission in IPN and contributes to opioid dependence.