Epac signaling and AMPA receptor plasticity during incubation of cocaine craving

与以前使用可卡因相关的环境线索是复发的有力触发因素，即使在 已经禁欲很长时间了。了解这一持续存在的漏洞的努力揭示了 在大鼠和人类中，线索诱导的渴望在戒酒期间逐渐加剧(孵化)。 可卡因自我管理。我们对孵化的研究主要集中在核内的兴奋性突触。 伏隔核(NAC)，其中含GluA2的不透钙AMPAR(CI-AMPAR)通常介导 AMPAR传输的大部分。我们发现，这些突触的加强，通过加入更高的 电导钙透性AMPAR(CP-AMPAR)，在停药约一个月后发生 广泛接触的可卡因自我给药，此后是培养的可卡因欲望表达的基础。 通过了解导致CP-AMPAR积累的级联，我们可以确定新的靶点 治疗性干预。该R21的目标是验证环状AMP的两个直接效应者--PKA(蛋白激酶A)和EPAC(由cAMP直接激活的交换蛋白)--共同工作以增加 孵化过程中CP-AMPAR对NAc内突触传递的相对贡献。具体来说，我们 假设在可卡因戒断过程中，NAc中棘神经元(MSN)中EPAC的激活导致 P38 MAPK的激活和CI-AMPAR的移除；这为CP-AMPAR进入这些突触“腾出了空间” 通过一种涉及PKA激活的机制。在目标1中，我们将使用全细胞膜片钳记录 确定突触后EPAC和p38 MAPK激活是否导致NAC突触中CI-AMPAR的移除 在药物幼稚的雄性和雌性大鼠中，以及这种作用在孵化渴求后是否被阻断。如果 在保证的情况下，我们将确定在NAC核心中敲除Epac2的shRNA是否阻止了渴望和 CP-AMPAR积累。AIM 2将检测突触后PKA激活对AMPAR传递的影响 药物幼稚大鼠和渴求孵化后的MSNS，并评估在 孵化。该项目非常适合R21机制，因为EPAC是我们实验室和 菲尔德(公开搜索可卡因和EPAC只发现了3篇论文)，因为人们对此知之甚少 突触后PKA信号在调节NAc MSN中AMPAR传递中的作用意义很高 不仅是因为需要新的治疗方法来治疗可卡因复发，而且因为我们的发现将 洞察调控兴奋性突触传递的基本机制，这是相关的 导致许多脑部疾病。最后，这项工作将为R01应用程序测试新假设奠定基础 NAC MSN中的钙信号在可卡因戒断过程中减少；最终这抑制了腺苷 环化酶5是MSN的主要异构体，导致EPAC/PKA活化和CP-AMPAR插入。

Environmental cues associated with prior cocaine use are powerful triggers for relapse, even in users who have been abstinent for a long period of time. Efforts to understand this persistent vulnerability have revealed that, in rats and humans, cue-induced craving progressively intensifies (`incubates') during abstinence from cocaine self-administration. Our studies of incubation have focused on excitatory synapses in the nucleus accumbens (NAc) core, where GluA2-containing Ca2+-impermeable AMPARs (CI-AMPARs) normally mediate the bulk of AMPAR transmission. We found that strengthening of these synapses, via incorporation of higher conductance Ca2+-permeable AMPARs (CP-AMPARs), occurs after about a month of withdrawal from extended-access cocaine self-administration and thereafter underlies expression of incubated cocaine craving. By understanding the cascade leading to CP-AMPAR accumulation, we may identify novel targets for therapeutic intervention. The goal of this R21 is to test the hypothesis that two direct effectors of cyclic AMP - PKA (protein kinase A) and Epac (exchange protein directly activated by cAMP) - work together to increase the relative contribution of CP-AMPARs to synaptic transmission in the NAc during incubation. Specifically, we hypothesize that, during cocaine withdrawal, Epac activation in NAc medium spiny neurons (MSNs) leads to p38 MAPK activation and removal of CI-AMPARs; this “makes room” for CP-AMPARs to enter these synapses via a mechanism involving PKA activation. In Aim 1, we will use whole-cell patch-clamp recordings to determine if postsynaptic Epac and p38 MAPK activation leads to removal of CI-AMPARs from NAc synapses in drug-naïve male and female rats, and whether this effect is occluded after incubation of craving. If warranted, we will determine if shRNA knockdown of Epac2 in the NAc core prevents incubation of craving and CP-AMPAR accumulation. Aim 2 will test the effects of postsynaptic PKA activation on AMPAR transmission in MSNs of drug-naïve rats and after incubation of craving, and assess the level of PKA activity in the NAc during incubation. This project is well suited to the R21 mechanism because Epac is a new focus for our lab and the field (a pubmed search on cocaine and Epac revealed only 3 papers) and because very little is known about the role of postsynaptic PKA signaling in regulating AMPAR transmission in NAc MSNs. Significance is high not only because of the need for novel therapeutic approaches to cocaine relapse but because our findings will provide insight into basic mechanisms regulating excitatory synaptic transmission in the NAc, which is relevant to many brain disorders. Finally, this work will set the stage for an R01 application testing the novel hypothesis that Ca2+ signaling in NAc MSNs decreases during cocaine withdrawal; ultimately this disinhibits adenylyl cyclase 5, the predominant isoform in MSNs, leading to Epac/PKA activation and CP-AMPAR insertion.