Cocaine addicts have a long-term vulnerability to relapse during abstinence, which may be triggered by drug-associated cues. To identify therapeutic targets for prevention of relapse, the synaptic plasticity that occurs during withdrawal must be better understood. In the incubation animal model of addiction, rats show a progressive increase in cue-induced craving after withdrawal from extended access self-administration, a phenomenon termed 'incubation'. Previous work in my lab revealed that high conductance Ca2+-permeable (CP) AMPA receptors (AMPARs) accumulate at synapses in the nucleus accumbens (NAc) during prolonged withdrawal and mediate the expression of 'incubated'cue-induced craving. The accumulation of CP-AMPARs in the NAc during incubation coincided with an increase in total and cell surface levels of GluA1, but not GluA2;furthermore, co-immunoprecipitation studies indicated an increase in GluA1 homomers. These findings suggest that an increase in GluA1 protein mediates formation of CP-AMPARs during incubation, and that most of the new CP-AMPARs are GluA1 homomers. The mechanism(s) that underlie this increase in GluA1 protein levels are unknown, but major candidates include persistent changes in protein synthesis and protein degradation during withdrawal. My central hypothesis is that incubation is associated with increased GluA1 synthesis coupled with altered ubiquitin-proteasome system (UPS) dependent protein degradation. This will be tested through three aims. The first two aims will be conducted in rats after 45-55 days of withdrawal from extended access cocaine or saline self-administration, when synaptic CP-AMPAR levels are elevated in the NAc of rats that self-administered cocaine.
Aim 1 will determine if enhanced GluA1 mRNA translation and/or an increase in dendritic GluA1 mRNA levels occurs in the NAc during incubation. I will use polysome profile analysis to measure GluA1 mRNA translation rate and prepare synaptoneurosomes from NAc tissue to quantify dendritic GluA1 mRNA. These are two techniques that have not been utilized in studying cocaine- induced plasticity.
Aim 2 will determine whether persistent changes in proteasome activity occur in the NAc during incubation. There have been no prior studies on the role of the UPS in plasticity elicited by cocaine self- administration and withdrawal. I will quantify polyubiquitinated proteins targeted fo proteasomal degradation using GST pull-downs and measure proteasome activity using fluorogenic assays.
In Aim 3, I will determine whether UPS activity in the NAc is altered during the expression of 'incubated'cue-induced cocaine seeking, in other words, after memory retrieval, using the same methods as in Aim 2. This proposal will better our understanding of the plasticity that underlies enhancement of craving during withdrawal and the plasticity involved in retrieval of a memory that elicits cocaine craving. While these studies are underway, I will participate in a Training Plan that employs coursework, individual mentoring, and collaborative interactions to develop the non-bench skills needed to reach my goal of becoming a PI in an academic setting.
Environmental cues previously associated with drug use are powerful triggers for relapse in abstinent cocaine users. The proposed research is relevant to NIH's mission of reducing the burden of illness because it characterizes the involvement of novel mechanisms - alterations in protein synthesis and protein degradation - that may underlie enhanced cue-induced cocaine craving during withdrawal. By focusing on novel mechanisms, this work may reveal innovative therapeutic targets.
|Werner, Craig T; Murray, Conor H; Reimers, Jeremy M et al. (2016) Trafficking of calcium-permeable and calcium-impermeable AMPA receptors in nucleus accumbens medium spiny neurons co-cultured with prefrontal cortex neurons. Neuropharmacology :|
|Werner, Craig T; Milovanovic, Mike; Christian, Daniel T et al. (2015) Response of the Ubiquitin-Proteasome System to Memory Retrieval After Extended-Access Cocaine or Saline Self-Administration. Neuropsychopharmacology 40:3006-14|
|Otis, James M; Werner, Craig T; Mueller, Devin (2015) Noradrenergic regulation of fear and drug-associated memory reconsolidation. Neuropsychopharmacology 40:793-803|