Abstract Treating substance use disorder (SUD) remains an unmet medical need, in part, due to its high relapse rate. During drug abstinence, re-exposure to cues previously associated with drug use triggers robust drug craving and seeking, leading to drug relapse. Rodent studies reveal that the glutamatergic projection from the infralimbic prefrontal cortex (IL) to nucleus accumbens shell (NAcSh) is targeted by cocaine experience to regulate cue-associated reward seeking. IL projections form monosynaptic connections on both of the two subtypes of principal, medium spiny neurons (MSNs) within the NAcSh, one predominantly expressing D1 dopamine receptors and the other expressing D2 receptors. Increasing evidence suggests that the IL-to-D1 vs. IL-to-D2 sub-projections undergo different adaptive changes after cocaine self-administration and differentially contribute to cocaine-associated behaviors. However, it remains poorly understood whether these IL sub-projections are changed through modification of existing synapses without changing the basic circuit connectivity, or through synapse formation or elimination that results in profound rewiring of circuit connectivity. The currently proposed work will address this knowledge gap by focusing on cocaine-generated AMPA receptor (AMPAR)-silent synapses, a type of glutamatergic synapses that contains NMDA receptors without functionally stable AMPARs. Silent synapses can be generated de novo via synaptogenesis and, upon AMPAR insertion and maturation, create and stabilize new network connections. Conversely, silent synapses can also stem from pre-existing synapses through AMPAR internalization, and this initial synaptic weakening may lead to the eventual synapse elimination to reduce network connections. Preliminary studies reveal that cocaine self-administration generated silent synapses in both the IL-to-D1 and IL-to-D2 sub-projections, which anatomically and functionally correlated with synaptogenesis vs. synaptic weakening, respectively. These and other results lead to the central hypothesis that silent synapse-mediated strengthening vs. weakening are simultaneously induced within the IL-to-D1 and IL-to-D2 sub-projections, respectively, after cocaine withdrawal, and the resulting circuit remodeling differentially contributes to cue-induced cocaine seeking. To test this hypothesis, the proposed experiments will (1) characterize the anatomical and electrophysiological consequences of silent synapse-mediated remodeling of IL-to-D1 and IL-to-D2 sub-projections, (2) determine the role of each of the remodeled IL-to-NAcSh sub-projections in formation of neuronal ensembles that potentially encode cue-induced cocaine seeking, and (3) determine how each of the remodeled IL-to-NAcSh sub-projections influences cue-induced cocaine seeking and motivation to obtain cocaine after withdrawal from cocaine. These objectives are highly relevant to the mission of the NIDA, NIH, as the resulting findings will provide novel,...