Project Summary/Abstract The mesolimbic dopamine (DA) system originating in the ventral tegmental area (VTA) plays a critical role in reward-based learning. DA neurons in the VTA display phasic burst firing in response to unexpected primary rewards. The timing of this bursting activity shifts to the onset of reward-predicting cues during repeated cue- reward pairing, where cue presentation needs to precede reward delivery for effective conditioning. DA neuron bursting gives rise to phasic DA transients lasting several seconds in the nucleus accumbens (NAc), a key site for the formation of cue-reward memory. In general, reward-evoked DA transients are thought to promote Hebbian plasticity induced by coordinated pairing of presynaptic and postsynaptic activities (pre-post pairing) during conditioning. However, this assumption leads to the following conundrum (known as the distal reward problem): how can DA, which communicates via slow intracellular signaling cascades, influence the consequence of preceding neural activities to regulate synaptic plasticity? In addressing this question, it is of note that DA neuron bursting to the cue develops during the early phase of cue-reward conditioning. This raises the possibility that DA transients elicited by the cue, not by the reward, may act to drive the learning of specific cue-reward associations as conditioning progresses. Thus, this project will explore the cellular mechanisms supporting the idea that DA transients need to precede the pre-post pairing to regulate Hebbian plasticity in the NAc. Cytosolic Ca2+ signaling dependent on the intracellular messenger inositol 1,4,5- triphosphate (IP3) can act as a coincidence detector to mediate synaptic plasticity. Medium spiny projection neurons (MSNs) in the NAc comprise two subpopulations, i.e., D1 receptor-positive and D2 receptor-positive MSNs, that play opposing roles in reward-driven behavior. Our recent study has reported differential regulation of IP3-Ca2+ signaling by preceding DA transients in these two MSN subpopulations. We hypothesize that DA will enhance long-term potentiation (LTP) of glutamatergic transmission in D1-positive MSNs while preventing LTP, or promoting long-term depression (LTD), in D2-positive MSNs through opposing regulation of IP3-Ca2+ signaling. Temporal rules governing DA action on plasticity will be examined by varying the timing of DA transients, produced by local pressure ejection of DA or optogenetic stimulation of DA fibers, relative to the pre-post pairing. The goal of this R21 project is to open a new line of research addressing the role and timing of phasic DA signals in regulating synaptic plasticity underlying reward learning.