Epac signaling and AMPA receptor plasticity during incubation of cocaine craving

可卡因渴望孵化过程中的 Epac 信号传导和 AMPA 受体可塑性

• 批准号: 9463304
• 负责人: Marina Elizabeth Wolf
• 金额: $ 20.45万
• 依托单位: OREGON HEALTH & SCIENCE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2018
• 资助国家: 美国
• 起止时间: 2018-09-01 至 2020-08-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9463304
• 关键词: AMPA Receptors; Abstinence; Adenylate Cyclase; Biochemical; Biochemistry; Brain Diseases; Cell physiology; Cells; Cocaine; Cocaine Dependence; Cocaine Users; Cues; Cyclic AMP; Cyclic AMP-Dependent Protein Kinases; Data; Drug usage; Excision; Excitatory Synapse; Exploratory/Developmental Grant; Female; Future; Goals; Human; Incubated; Infusion procedures; Investigation; Lead; MAPK14 gene; Measures; Mediating; Methamphetamine; Mission; Modeling; Neurons; Nose; Nucleus Accumbens; Paper; Permeability; Pharmaceutical Preparations; Pharmacology; Protein Isoforms; Proteins; PubMed; Rattus; Relapse; Research; Role; Self Administration; Self-Administered; Signal Transduction; Synapses; Synaptic Transmission; Testing; Therapeutic; Therapeutic Intervention; Time; United States National Institutes of Health; Ventral Tegmental Area; Withdrawal; Work; addiction; base; burden of illness; cocaine exposure; cocaine relapse; cocaine use; craving; dopaminergic neuron; insight; knock-down; male; mitogen-activated protein kinase p38; new therapeutic target; novel; novel strategies; novel therapeutic intervention; patch clamp; postsynaptic; prevent; response; small hairpin RNA; trafficking; transmission process

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.

与先前使用可卡因相关的环境线索是复发的强大触发因素，即使在使用者中也是如此。 已经禁欲很久了为了了解这种持续存在的脆弱性， 在大鼠和人类中，在戒断期间，线索诱导的渴望逐渐加剧（“孵化”）， 可卡因自我管理我们对潜伏期的研究主要集中在细胞核中的兴奋性突触 在NAc核心中，含有GluA 2的Ca 2+不可渗透的AMPAR（CI-AMPAR）通常介导 大量的AMPAR传输。我们发现，这些突触的加强，通过纳入更高的 电导Ca 2+渗透性AMPAR（CP-AMPAR），发生在约一个月的退出后， 长期接触可卡因自我给药，此后成为潜伏可卡因渴望表达的基础。 通过了解导致CP-AMPAR积累的级联反应，我们可以确定新的靶点， 治疗干预该R21的目的是检验环AMP的两种直接效应物- PKA（蛋白激酶A）和Epac（由cAMP直接激活的交换蛋白）-共同作用以增加cAMP的表达的假设。 CP-AMPAR在孵育期间对NAc中突触传递的相对贡献。我们特别 假设，在可卡因戒断期间，NAc中棘神经元（MSN）中的Epac激活导致 p38 MAPK激活和CI-AMPAR的去除;这为CP-AMPAR进入这些突触“腾出空间” 通过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激活是否导致从NAc突触去除CI-AMPAR 在未经药物处理的雄性和雌性大鼠中，以及这种效应在渴望孵化后是否被阻断。如果 为了证明这一点，我们将确定在NAc核心中Epac 2的shRNA敲低是否可以阻止渴望的孵化， CP-AMPAR蓄积。目的2将测试突触后PKA激活对AMPAR传递的影响， 在药物未处理的大鼠的MSN中以及在渴望孵化后，评估在药物处理期间NAc中PKA活性的水平。 孵化这个项目非常适合R21机制，因为Epac是我们实验室的一个新焦点， 领域（对可卡因和Epac的pubmed搜索只显示了3篇论文），因为对可卡因和Epac的了解很少。 突触后PKA信号传导在NAc MSNs中调节AMPAR传递的作用。重要性高 这不仅是因为需要新的治疗方法来治疗可卡因复吸， 提供了对调节NAc兴奋性突触传递的基本机制的深入了解， 许多脑部疾病的症状最后，这项工作将为R 01应用程序测试新的假设奠定基础 在可卡因戒断过程中，NAc MSNs中的Ca 2+信号传导减少;最终这会抑制腺苷酸 环化酶5，MSN中的主要同种型，导致Epac/PKA激活和CP-AMPAR插入。