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持续的复发脆弱性，即使在几个月的禁欲。这种持续的脆弱性表明，长期持久的神经适应有助于复发行为。了解这些持续的适应性对于治疗药物的开发至关重要。我们的实验室使用可卡因成瘾的大鼠模型，称为潜伏期模型，其中线索诱导的可卡因渴望在从长期获得可卡因自我给药的前2个月内逐渐加剧（潜伏期）。我们专注于神经核（NAc），一个大脑区域，在滥用药物的强化性能至关重要。我们以前表明，Ca 2+渗透AMPAR（CP-AMPAR）积累在兴奋性突触中的中型多刺神经元的NAc后3-4周的撤退，然后持续数月。一旦它们在NAc突触中积累，这些CP-AMPAR介导了孵育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。虽然我们以前的工作已经收集了关于孵化期间AMPAR可塑性的详细信息，但对NMDAR或树突棘结构改变的贡献知之甚少。以前的研究检查这些参数通常使用非偶然的可卡因方案，这是不直接用于评估可卡因的渴望。本提案的目的是表征棘形态和谷氨酸受体介导的Ca 2+信号传导在NAc树突棘在停药前CP-AMPAR积累的时间（WD 15）和CP-AMPAR积累后的时间（WD 35），以确定可塑性在单个棘水平，与CP-AMPAR积累。我的中心假设是，孵化伴随着树突棘重塑，涉及含有CP-AMPAR但不含NMDAR的棘的形成。这一假设将通过追求两个具体目标来检验：1）表征可卡因渴求孵育期间中等多刺神经元（MSN）中的树突棘密度和形态。将单个NAc神经元填充荧光黄，用共聚焦显微镜成像，并用NeuronStudio软件分析。2)确定可卡因和对照组大鼠在NAc MSN的个体脊柱水平上NMDAR和AMPAR介导的Ca 2+内流是否不同。2-光子Ca 2+成像与同时进行的电生理学测量将用于分析CP-AMPAR和NMDAR对来自盐水或可卡因自身给药的WD 15或WD 35时NAc脊髓中Ca 2+信号传导的贡献。笼状NMDA和AMPA化合物将用于在单个脊柱水平上剖析NMDAR和CP-AMPAR的功能贡献。这些研究将提供一个新的窗口可卡因诱导的神经适应在单一的脊柱水平，并进一步了解谷氨酸受体和脊柱可塑性之间的关系。虽然这项工作正在进行中，我将参加一个培训计划，采用课程，辅导和协作互动，以发展所需的非板凳技能，以达到我成为PI在学术环境的目标。

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.