Plasticity in nucleus accumbens spines during incubation of cocaine craving

描述（由申请人提供）：治疗可卡因成瘾的一个主要问题是长期易复发，即使在戒掉几个月后也是如此。这种持续的脆弱性表明，长期的神经适应有助于复发行为。了解这些持续的适应对治疗剂的开发至关重要。我们的实验室使用了一种被称为潜伏期模型的大鼠可卡因成瘾模型，在该模型中，线索诱导的可卡因渴望在停止长期获取可卡因自我给药的前两个月逐渐增强（孵化）。我们的重点是伏隔核（NAc），这是一个与药物滥用的强化特性密切相关的大脑区域。我们之前的研究表明，在停药3-4周后，Ca2+渗透性AMPARs （CP-AMPARs）在NAc的中棘神经元的兴奋性突触中积累，然后持续数月。一旦它们在NAc突触中积累，这些CP-AMPARs就会介导培养的线索诱导的可卡因渴望的表达。因此，调节cp - ampar的机制是潜在的治疗靶点。虽然我们之前的工作已经收集了AMPAR在培养过程中的可塑性的详细信息，但对于NMDARs或树突棘的结构改变如何起作用知之甚少。先前检查这些参数的研究通常使用非偶然的可卡因方案，这些方案对评估可卡因渴望不直接有用。本研究的目的是在CP-AMPAR积累前（WD15）和CP-AMPAR积累后（WD35），表征NAc树突棘中脊柱形态和谷氨酸受体介导的Ca2+信号传导，以确定与CP-AMPAR积累相关的单脊柱水平的可塑性。我的主要假设是，潜伏期伴随着树突脊柱重塑，包括含有CP-AMPARs但不含NMDARs的脊柱的形成。这一假设将通过追求两个特定目标来验证：1)在可卡因渴求潜伏期表征中棘神经元（MSNs）的树突棘密度和形态。单个NAc神经元将用Lucifer黄色填充，用共聚焦显微镜成像，并用NeuronStudio软件进行分析。2)确定可卡因和对照大鼠在NAc MSN个体脊柱水平上NMDAR和AMPAR介导的Ca2+内流是否存在差异。同时电生理测量的2光子Ca2+成像将用于分析CP-AMPARs和NMDARs对生理盐水或可卡因自我给药WD15或WD35上NAc棘中Ca2+信号的贡献。笼化NMDA和AMPA化合物将用于在单脊柱水平解剖NMDARs和cp - ampar的功能贡献。这些研究将在单脊柱水平上为可卡因诱导的神经适应提供一个新的窗口，并进一步了解谷氨酸受体与脊柱可塑性之间的关系。当这项工作正在进行时，我将参加一个培训计划，该计划采用课程作业，指导和协作互动来发展非长凳技能，以达到我成为学术环境中的PI的目标。

DESCRIPTION (provided by applicant): A major problem in treating cocaine addiction is the long lasting vulnerability to relapse, even after months of abstinence. This persistent vulnerability suggests that long lasting neuroadaptations contribute to relapse behavior. Understanding these persistent adaptations is critical to the development of therapeutic agents. Our lab uses a rat model of cocaine addiction, termed the incubation model, in which cue-induced cocaine craving progressively intensifies (incubates) over the first 2 months of withdrawal from extended access cocaine self-administration. We focus on the nucleus accumbens (NAc), a brain region critically involved in the reinforcing properties of drugs of abuse. We showed previously that Ca2+-permeable AMPARs (CP-AMPARs) accumulate in excitatory synapses onto medium spiny neurons of the NAc after 3-4 weeks of withdrawal and then persist for months. Once they accumulate in NAc synapses, these CP-AMPARs mediate the expression of incubated cue-induced cocaine craving. Therefore mechanisms regulating CP-AMPARs are potential therapeutic targets. While our previous work has gathered detailed information on AMPAR plasticity during incubation, little is known about how NMDARs or structural alterations to dendritic spines contribute. Prior studies examining these parameters have typically used non-contingent cocaine regimens which are not directly useful for assessing cocaine craving. The objective of this proposal is to characterize spine morphology and glutamate receptor-mediated Ca2+ signaling in NAc dendritic spines at a withdrawal time before CP-AMPARs accumulate (WD15) and a time after CP-AMPAR accumulation (WD35), to identify plasticity at the single spine level that is associated with CP-AMPAR accumulation. My central hypothesis is that incubation is accompanied by dendritic spine remodeling that involves the formation of spines that contain CP-AMPARs but not NMDARs. This hypothesis will be tested by pursuing two specific aims: 1) Characterize dendritic spine density and morphology in medium spiny neurons (MSNs) during incubation of cocaine craving. Single NAc neurons will be filled with Lucifer yellow, imaged with confocal microscopy, and analyzed with NeuronStudio software. 2) Determine if cocaine and control rats differ in NMDAR and AMPAR mediated Ca2+ influx at the individual spine level in NAc MSN. 2-photon Ca2+ imaging with concurrent electrophysiological measurements will be used to analyze the contribution of CP-AMPARs and NMDARs to Ca2+ signaling in NAc spines on WD15 or WD35 from saline or cocaine self-administration. Caged NMDA and AMPA compounds will be used to dissect functional contributions of NMDARs and CP-AMPARs at the single spine level. These studies will provide a novel window on cocaine-induced neuroadaptations at the single spine level and further our understanding of relationships between glutamate receptor and spine plasticity. While this work is underway, I will participate in a Training Plan that employs coursework, mentoring, and collaborative interactions to develop the non-bench skills needed to reach my goal of becoming a PI in an academic setting.