Rapid protein translation in nucleus accumbens neurons in response to a cocaine-associated cue

Oregon Health & Science University, Portland OR

Investigators

Abstract

Project Summary Cues associated with drugs of abuse are powerful triggers of craving and relapse, and increased reactivity to such cues is a core feature of substance use disorder. To understand mechanisms that support cue-induced cocaine craving, we use the incubation of craving model, in which rats exhibit progressive intensification (incubation) of cue-induced craving during the first weeks of withdrawal from cocaine self-administration (SA); craving then remains elevated for months, modeling persistence of vulnerability in recovering cocaine users. We have shown that, after ~1 month of withdrawal, incubation depends on persistent strengthening of AMPAR synaptic transmission in nucleus accumbens core (NAcc) which in turn depends on tonic dysregulation of protein translation during withdrawal. On top of withdrawal-dependent changes, we found that additional rapid translation is evoked when `incubated' rats are re-exposed to cocaine cues, and that this additional translation is required for these cues to elicit expression of incubated craving. Nothing is known about mRNAs differentially translated during cue re-exposure, a significant gap since translation is required for the incubated craving response. Aim 1 will fill this gap by using translating ribosome affinity purification to isolate actively translating mRNAs, which will then be analyzed by RNA-seq (TRAP-seq). The two main subtypes of NAcc medium spiny neurons (MSN), expressing D1 or D2 DA receptors, will be studied using a Cre-dependent TRAP virus and D1-Cre or adenosine 2a (A2a)-Cre rats (the A2a receptor is a marker for D2 DA receptor-expressing MSN). MSN subtypes must be distinguished because they regulate behavior via complex and sometimes oppositional interactions, and because they exhibit distinct synaptic plasticity after incubation of cocaine craving. Male and female D1- or A2a- Cre rats will self-administer saline or cocaine (6 h/d x 10 d; each infusion paired with a light cue). They will then undergo a 15-min cue-induced seeking test on withdrawal day (WD) 1 (baseline craving) or WD40 (after incubation). NAcc will be collected immediately and processed for TRAP-seq to identify mRNAs that are actively translating during the period of cue exposure. Translating mRNAs specific to the cocaine WD40 group are strong candidates for mediating the incubated response to cocaine cues, particularly since we can compare to existing data on translating mRNAs from rats that did not receive a seeking test in order to distinguish seeking test- induced from withdrawal-induced changes in translation. In Aim 2, we will select ~10 such translating mRNAs for validation at the mRNA and protein levels using qPCR and Western blotting, respectively. This proposal fits the R21 mechanism, as it breaks new ground methodologically (first to combine TRAP-seq, transgenic rats, and a translationally relevant cocaine SA model) and conceptually (first exploration of rapid protein translation elicited by a drug-associated cue in any cell type).