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

Synthetic opioids like fentanyl are contributing to unprecedented overdose rates. Despite the abundance of fentanyl in the illicit drug supply, an understanding of fentanyl’s effects on the brain remains lacking. The ventral tegmental area (VTA) is an important site of fentanyl’s rewarding actions in the brain and sends diverse projections to several regions including the nucleus accumbens (NAc) and prefrontal cortex (PFC). Previous work shows downstream projection target influences baseline properties and opioid-induced neuroadaptations in VTA, but most work has focused on single neurotransmitter-defined cell types. Here, we investigate how projection-defined VTA neurons exhibit different responses to fentanyl independent of cell type. We use RNA sequencing to show that fentanyl induces discordant transcriptional adaptations in VTA neurons that project to NAc vs PFC. We then used fiber photometry and chemogenetics to characterize functional differences in 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 can undergo vastly different adaptations in response to fentanyl, providing new insight into VTA encoding of opioid behaviors.