Fentanyl and other synthetic opioids are killing thousands of Americans each year. The overall goal of this research is to better understand the role of the primary central noradrenergic system in fentanyl withdrawal. We have known for decades that increased locus coeruleus-noradrenergic (LC-NE) system activity is critically involved in both stress-induced increases negative affect and is potently modulated by withdrawal from opioids. However, there is extensive, long-term controversy over the contribution of the LC-NE system to opioid withdrawal-related behaviors. Critically, opioid withdrawal increases glutamate efflux into the locus coeruleus. This fundamental feature of opioid withdrawal was identified and rigorously studied for many years, generating potential therapeutic interventions that never reached the clinic. Study on this topic went largely quiet after lesions studies indicated an intact LC-NE system was not necessary for the withdrawal-induced place aversion or somatic signs of withdrawal. Despite these important findings, relatively little is known about afferent glutamatergic regulation of LC-NE neurons in opioid withdrawal-induced negative affect. We will determine whether a newly identified midbrain glutamatergic input to LC-NE neurons contributes to negative affective behaviors associated with opioid withdrawal. We have now identified a largely unstudied afferent projection to the LC from excitatory neurons in the ventral tegmental area (VTA), a region critical for processing reward and motivation. While projections from these glutamatergic VTA neurons to the nucleus accumbens and lateral habenula have opposing valences on aversion in mouse models, we hypothesize that VTA glutamate projections to the LC-NE system produce negative affect and will be modulated by opioid withdrawal. Our preliminary electrophysiology data suggests that this glutamatergic input to the LC amplifies other sources of glutamate. In this proposal, we begin by seeking to determine whether withdrawal from fentanyl selectively modifies this excitatory input the LC compared to other well-established excitatory inputs. Here, we will use brain slice electrophysiology and in vivo microdialysis to test the hypothesis that excitatory VTA-LC activity is selectively enhanced in opioid withdrawal. In the second aim, we will conduct behavioral studies modulating this excitatory VTA-LC projection to determine whether it is needed for natural avoidance behaviors that guide actions away from risk. Our preliminary data shows these behaviors that are amplified following fentanyl withdrawal. Together these experiments will generate previously unattainable information about fentanyl-induced plasticity in VTA glutamate inputs to LC-NE neurons and the negative affective behaviors these neurons generate. These studies will clarify function of the excitatory VTA-LC projection by determining 1) whether fentanyl withdrawal alters their input to the LC-NE system and 2) their role ...