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，即使在戒毒几个月后也是如此。这种持久的脆弱性表明，长期的神经适应有助于复发行为。了解这些持久的适应对于治疗药物的开发至关重要。我们的实验室使用了一种被称为孵化模型的可卡因成瘾大鼠模型，在该模型中，线索诱导的可卡因渴望在戒断长时间使用可卡因的前2个月内逐渐加剧(孵化)。我们专注于伏隔核(NAC)，这是一个与滥用药物强化特性密切相关的大脑区域。我们以前的研究表明，在戒断3-4周后，钙离子通透性AMPAR(CP-AMPAR)在兴奋性突触中聚集在NAC的中等棘神经元上，然后持续数月。一旦它们在NAC突触中积累，这些CP-AMPAR就介导了孵化线索诱导的可卡因渴求的表达。因此，CP-AMPAR的调控机制是潜在的治疗靶点。虽然我们之前的工作已经收集了关于AMPAR在孵化过程中可塑性的详细信息，但对于NMDAR或树突棘的结构变化是如何起作用的知之甚少。以前检查这些参数的研究通常使用非偶发可卡因方案，这对评估可卡因的渴望没有直接作用。本研究的目的是研究Cp-AMPAR积聚前(WD15)和Cp-AMPAR积聚后(WD35)NAC树突棘中的脊椎形态和谷氨酸受体介导的钙信号，以确定单个脊椎水平上与Cp-AMPAR积聚相关的可塑性。我的中心假设是，孵化伴随着树突棘的重塑，涉及到含有CP-AMPAR而不含NMDAR的脊椎的形成。这一假说将通过追求两个具体目标来验证：1)表征培养可卡因渴求过程中中棘神经元(MSN)中树突棘的密度和形态。将单个NAC神经元填充荧光黄，用共聚焦显微镜成像，并用NeuronStudio软件进行分析。2)确定可卡因和对照组大鼠在NAC MSN的NMDAR和AMPAR介导的单个脊髓水平的钙内流是否存在差异。我们将利用双光子钙离子成像结合同步电生理测量来分析生理盐水或可卡因自身给药对WD15或WD35上NAC棘中CP-AMPAR和NMDARs对钙信号的影响。笼式NMDA和AMPA化合物将被用来在单个脊柱水平上剖析NMDAR和CP-AMPAR的功能贡献。这些研究将为可卡因诱导的单个脊柱水平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。在这项工作进行期间，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板凳技能，以实现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.