Project Summary A major problem for individuals suffering from addiction is the persistent vulnerability to relapse, even after long periods of abstinence. In the `incubation of cocaine craving' model of relapse, rats self-administer cocaine using an extended access procedure, and then experience a prolonged abstinence period. During abstinence, rats exhibit a progressive intensification (incubation) of cue-induced cocaine craving. We have shown that Ca2+-permeable AMPA receptors (CP-AMPAR), comprised exclusively of the GluA1 subunit, accumulate in the nucleus accumbens core (NAcc) during abstinence and thereafter are required for the expression of incubated cue-induced craving. Thus, understanding the mechanisms regulating CP-AMPAR maintenance and removal may yield novel therapeutic targets for reducing craving and prolonging abstinence. Work from our lab has shown that CP-AMPAR mediated currents in the NAcc require active protein translation, as they are blocked by general protein translation inhibitors. Also, treatment with a general protein translation inhibitor just before the cue-induced craving test reduces incubated seeking. However, little is known about the specifics of this critical protein translation. Under some conditions, inhibition of general protein translation actually increases the translation of a subset of mRNA with 5' upstream open reading frames, such as Oligophrenin-1 (OPHN1). In the hippocampus, OPHN1 is necessary for eIF2α-mediated mGluR-LTD and the removal of synaptic AMPARs. In the VTA, this pathway plays a role in bidirectional CP-AMPAR plasticity in response to i.p. cocaine exposure. Thus, in our NAcc studies, treatment with protein translation inhibitors may have increased translation of OPHN1, mimicking mGluR-LTD and removing synaptic CP-AMPARs. My hypothesis is that, following cocaine self-administration and prolonged abstinence, OPHN1 translation is low in the NAcc, permitting the accumulation and maintenance of CP-AMPARs. In addition, in incubated rats during the seeking test, OPHN1 translation is reduced due to eIF2α-dephosphorylation, enabling CP-AMPARs to stay in synapses during the test and mediate incubated seeking. Aim 1 will determine if OPHN1 translation is dysregulated in incubated rats using viral Translating Ribosome Affinity Purification (vTRAP) coupled with qRT-PCR to quantify Ophn1, Gria1 and Gria2 mRNA during incubation of cocaine craving. Changes in mRNA will be compared to changes in newly translated proteins using puromycin-labeling of nascent proteins and immunoblotting. I will also use several techniques to localize the critical translation. Aim 2 will determine the role of OPHN1 in the expression of incubated seeking. Here I will use a similar vTRAP approach to examine actively translated mRNAs before and after a seeking test in incubated rats. Also, I will experimentally knockdown OPHN1 to test if it is necessary for mGlu1-LTD-mediated removal of CP-AMPARs and normalization of incubated se...