Project Summary A major problem in treating drug abuse is persistent vulnerability to relapse, even after long periods of abstinence. In the incubation of cocaine craving model of relapse, rats undergo extended access to cocaine self- administration. During withdrawal, rats exhibit a progressive intensification (incubation) of cue-induced cocaine craving. We have shown that calcium (Ca2+)-permeable AMPA receptors (CP-AMPAR), comprised exclusively of the GluA1 subunit, accumulate in the nucleus accumbens (NAc) during late withdrawal and thereafter are required for the expression of incubated cue-induced craving. Therefore, understanding mechanisms regulating CP-AMPAR accumulation and maintenance may yield novel therapeutic targets for reducing craving and prolonging abstinence. Work from our lab and others have shown that cocaine withdrawal is associated with multiple adaptations that predict reduced Ca2+ levels in NAc medium spiny neurons (MSN). However, little is known about mechanisms that may link this reduced activity during withdrawal to the subsequent insertion of CP-AMPARs. One promising candidate involves the retinoic acid (RA) signaling cascade. Studies in hippocampal neurons have demonstrated that Ca2+ levels associated with basal synaptic transmission are sufficient to suppress RA synthesis. However, following a period of inactivity, RA synthesis is disinhibited, leading to increased translation of GluA1 and synaptic insertion of homomeric GluA1 CP-AMPARs. CP-AMPAR accumulation in the NAc during incubation may likewise involve reduced activity and has been demonstrated in our unpublished studies to be associated with increased translation of GluA1. Based on these similarities to RA- induced synaptic scaling, my central hypothesis is that cocaine incubation leads to a decrease in activity in NAc neurons at baseline, increased RA synthesis, and scaling up of CP-AMPARs. Aim 1 will determine if Ca2+ levels are altered in the NAc core during incubation of cocaine craving. I will use fiber photometry to measure changes in intracellular Ca2+, a correlate of neural activity, in early and late withdrawal from extended access to cocaine (or saline) self-administration. I will measure Ca2+ at baseline and during exposure to a cocaine-associated cue. Aim 2 will determine whether RA contributes to CP-AMPAR accumulation in NAc core MSN of cocaine incubated rats. Whole-cell patch clamp recordings in brain slices from cocaine incubated or saline rats will be used to determine if pharmacological manipulation of RA signaling alters AMPAR-mediated synaptic transmission in NAc medium spiny neurons. Aim 3 will determine if RA mediates synaptic scaling in cultured NAc MSN. I will use a RA reporter system to confirm that RA expression in MSN is activity- dependent, and pharmacological methods to determine whether RA-dependent scaling of CP-AMPAR occurs in NAc neurons. While these studies are underway, I will participate in a multi-faceted training plan to de...