Projection-defined ventral tegmental area neurons exhibit fentanyl-induced molecular and functional adaptations that differentially support drug-context associations.

Opioid use disorder (OUD) remains a major public health concern affecting millions of individuals, with potent synthetic opioids driving overdose deaths. To advance OUD treatment, it is necessary to improve understanding of how synthetic opioids impact the brain and promote persistent use. The ventral tegmental area (VTA) sends diverse projections to several regions including nucleus accumbens (NAc) and prefrontal cortex (PFC), and is critical for opioid reward. Previous work shows downstream projection target influences baseline properties and opioid-induced neuroadaptations in VTA, but most work has focused on natural opioids and single neurotransmitter-defined cell-types. As such, there is limited understanding of how diverse VTA cell-types function together to mediate drug-directed behaviors through their shared circuit outputs. Here, we investigate how projection-defined VTA neurons exhibit differential responses to the synthetic opioid fentanyl in male and female mice. We used RNA sequencing and weighted gene co-expression network analysis to show that fentanyl induces discordant transcriptional adaptations in VTA neurons that project to NAc vs PFC. We then used in vivo calcium imaging and chemogenetics to determine functional differences in how these projections encode fentanyl-related behavior. We found different timescales of activation between VTA-NAc and VTA-PFC during conditioned place preference that support distinct aspects of fentanyl-context association. Together, our data demonstrate that based on projection target alone, VTA neurons undergo vastly different adaptations in response to fentanyl. This study provides new insight into VTA encoding of opioid behaviors that advance understanding of the neural mechanisms underlying OUD.