SUMMARY: The ability to inhibit drinking is a significant challenge for recovering alcoholics, especially in the presence of alcohol- associated cues. Repeated alcohol exposure induces neuroadaptations that persist beyond acute withdrawal, and which increase alcohol’s incentive salience, leading to escalation of alcohol intake and aversion-resistant alcohol seeking. Alcohol use also causes deficits in cognitive functions associated with the medial prefrontal cortex (mPFC), which further fuel compulsive drinking and relapse. In rodents, alcohol seeking activates specialized networks within the ventral (infralimbic, IL) and prelimbic (PL) regions of the mPFC, which play largely opposite roles in the control of relapse behavior. While activation of the PL drives reinstatement, neurons in the IL facilitate extinction learning and inhibit drug-seeking through their projections to the Nucleus Accumbens shell, as well as the basolateral amygdala (BLA). However, the synaptic mechanisms that drive maladaptive plasticity in these circuits during the transition from controlled to compulsive alcohol- seeking remain largely unclear. The experiments in this application will provide a better understanding of network-specific mechanisms through which chronic alcohol exposure and withdrawal affect executive cognitive functions of the mPFC and diminish inhibitory control over goal-directed behavior. In Aim 1 we use patch-clamp electrophysiology and optogenetic stimulation to determine changes in long-range glutamatergic inputs from the BLA onto identified projection neurons in the IL and PL following extended access to alcohol, as well as under postdependent conditions. We will use Targeted Recombination in Active Populations (TRAP2) with Fos2A- iCreER mice to express channelrhodopsin selectively in those BLA afferents to the mPFC that are activated during withdrawal, and we will determine how alterations in these inputs develop over time (from goal-directed to compulsive alcohol- seeking). We will perform voltage-clamp recordings from retrogradely-labeled neurons that project back to the BLA, and we will determine alcohol-induced changes in postsynaptic glutamate receptor function and presynaptic release following reinstatement. Experiments in Aim 2 will use combinational retrograde Cre delivery and a Cre-dependent reporter to label the same IL and PL projection neurons for high-resolution morphometric analyses of spines and glutamate receptor expression in order to compare changes specifically in those neurons that contribute to relapse behavior (visualized via co-labeling for the activity marker phospho-CREB) and those that do not (pCREB-negative cells). In Aim 3 we will again use TRAP2 mice to test whether optogenetic manipulations of specific ensembles in the mPFC, or of inputs from the BLA to the mPFC (each again TRAPed during withdrawal) can reverse alcohol-induced cognitive deficits and reduce drug- seeking. Taken together, these studies will provide impor...