Project Summary Opioid addiction is a devastating illness with far-reaching personal and public health consequences. The physiological tolerance and dependence that develop with repeated opioid treatment are a common precursor to addictive behaviors. This proposal is aimed at determining which midbrain circuits are altered by prolonged opioid use and, more specifically, which neurons contribute to opioid dependence. When given acutely, opioids disinhibit the release of dopamine from midbrain neurons as inhibitory opioid receptors on GABAergic neurons are activated. This leads to increases of dopamine tone downstream. With repeated exposure, a receptor- independent, homeostatic mechanism increases the activity of these GABAergic neurons to an opioid tolerant state. Tolerant GABAergic neurons are hyper active and strongly inhibit dopaminergic neurons in the absence of opioids. This is a likely cause of the decrease in dopamine tone that is associated with opioid withdrawal. These GABAergic and dopaminergic changes occur primarily in the ventral tegmental area (VTA), a midbrain region involved in both reward and aversion. Since the discovery of these opioid mechanisms, multiple distinct VTA dopamine circuits have been described and found to have different functions with respect to reward and aversion which calls into question the circuit-specificity of known opioid effects. Overall, a comprehensive characterization of acute and long-term opioid effects in midbrain sub-circuits is very much lacking. The proposed studies use patch clamp electrophysiology with retrograde tracers to determine the effects of opioids on different VTA output pathways and identify which neurons demonstrate the previously described cellular withdrawal characteristics. They also explore the relevance of different mesolimbic sub-regions to opioid dependence by recording dopamine release changes in vivo using fiber photometry with a genetically encoded dopamine sensor. Our previous research suggests that the rewarding aspects of opioid exposure and the aversive responses to opioid dependence (withdrawal) may be mechanistically separable. By separating the acute and chronic components of opioid use at the circuit level, this research will fill a gap in our understanding of how dependence develops in the brain. This work has implications both for improved treatment of opioid addiction and for development of safer analgesics with reduced risk for dependence. As a fellowship project, it will also provide a framework for training in several essential neuroscience techniques with the help of a diverse collection of experts in a highly collaborative research environment.