PROJECT SUMMARY The opioid epidemic has skyrocketed in the United States. Despite the enormous public health impact, the basic mechanisms underlying the development of opioid use disorder (OUD) remain unclear. Striatal dopamine (DA) neurotransmission is critical for opioid reward-related behaviors. The nucleus accumbens (NAc) region within ventral striatum serves as a crucial node for opioid-conditioned reinforcement via dopamine receptor signaling. Furthermore, the medial shell of the NAc (mNAcSh) has increasingly been implicated in opioid reinforcement behavior in a mouse self-administration model. Opioids enhance DA release from midbrain DA neurons, whose projections converge with glutamatergic input in the NAc. 95% of the NAc neuronal population consists of GABAergic medium spiny neurons (MSNs) are largely segregated into two populations based on expression of DA D1 (D1R) versus D2 (D2R) receptors. Indeed, D1R+ and D2R+ MSNs project to different output structures in the basal ganglia circuitry which have opposite effects on motor control: D1R+ MSNs exert stimulatory effects on movement via the direct pathway whereas D2R+ MSNs exert inhibitory control over movement via the indirect pathway. Moreover, the DA receptors expressed by D1R+ versus D2R+ MSNs are coupled to different G proteins which signal via different intracellular pathways. In the context of opioids, morphine differentially acts on D1R+ versus D2R+ NAc MSNs. Specifically: 1) morphine administration increases glutamatergic synaptic strength in NAc D1R+ MSNs, while D2R+ MSNs show opposite effects; and 2) morphine exposure preferentially generates silent synapses in NAc D2R+ MSNs compared to D1R+ MSNs. Anatomic and functional evidence suggests that the composition of striatal D1R+ and D2R+ MSN populations is more heterogenous than previously thought. Until recently, the inability to selectively target D1/2R-co-expressing MSNs has made it very difficult to dissect the respective contributions of these co-expressing MSNs given their close proximity in the striatum. As a result, most studies have been in vitro and relied on ectopic receptor overexpression, leading to conflicting findings and unclear relevance in vivo. Moreover, the specific roles of D1/2R-co-expressing MSNs in opioid abuse have yet to be examined. To address this, we developed tools to dissect the functional and anatomical properties of different MSN subpopulations within the same brain. This includes use of the intersectional genetic INTRSECT2.0 system, which allows us to label and manipulate subsets of MSNs expressing D1R or D2R alone versus those co-expressing D1R and D2R with great temporal and spatial specificity. With these tools, we will determine if D1/2R-co-expressing MSNs represent a distinct MSN population, different from neurons that only express D1R or D2R. We hypothesize that D1/2R-co-expressing NAc MSNs have unique anatomic, physiologic, and behavioral functions critical for opioid actions. To address thi...