Pain is experienced by up to 20% of the US population at any given time. Opioid pain medications are potent analgesics commonly used to treat pain symptoms, but the misuse of opioids presents a major limitation for pain management. There is increasing support for sex differences for opioid misuse among pain patients. Although, prevalence rates for prescription opioid misuse between men and women are often found to be similar, this finding contrasts with the consistently higher prevalence of abuse in men compared to women. For example, male pain patients treated with opioids are more likely to increase intake of their opioid doses, misuse prescriptions, and die from overdose. However, little is known about sex-specific trends in long-term use of prescribed opioids for pain conditions. Sexual differences appear to play an important role in opioid misuse, but preclinical data on how pain or prior history of opioid intake contributes to sex differences in opioid abuse liability are limited. Preliminary findings from our lab have recapitulated the sex-dependent trends found in clinical data using a preclinical model. Our data show that male rats self-administer higher amounts of fentanyl than females. In addition, we found that persistent inflammatory pain increases fentanyl intake in males relative to females. This suggests that inflammatory pain conditions lead to a higher misuse liability selectively in males. In this project, we propose to investigate how pain alters neuroadaptative processes during long-term opioid use in a sex-specific manner and whether these functional changes are predictive of maladaptive patterns of fentanyl intake. The mesolimbic system is a key network node that integrates pain and reward. DA transmission in the mesolimbic system, via the ventral tegmental area (VTA) to the nucleus accumbens (NAc), has long been recognized for its role in reward processing and motivated behaviors. However, it remains unclear whether sex- dependent effects of pain on opioid use are mediated by sex differences in the patterns of VTA DA activity. Our preliminary data shows that persistent inflammatory pain reduces VTA DA neuron excitability by increasing the inhibitory drive onto VTA DA neurons from the GABAergic inputs of the rostromedial tegmental nucleus (RMTg). This suggests that under conditions of pain, higher opioid doses are necessary to trigger levels of VTA DA release comparable to those produced in non-pain conditions. However, it is unknown whether similar mechanisms underlie sex differences in the patterns of opioid misuse. Based on our preliminary findings, we hypothesize that pain exacerbates RMTg-mediated inhibition of VTA DA cell activity in males, but not females following opioid self-administration. This project aims to address this gap in our understanding by investigating the sex-specific functional consequences of pain and long-term opioid use within the RMTgVTA pathway.