Activity-regulated cytoskeleton-associated protein mediates nucleus accumbens function via cell type-specific action”

修改后的项目摘要/摘要部分 伏隔核(NAC)是调节情绪相关行为的关键脑区，但这一作用的细胞类型和分子仍在确定中。此外，许多与NAC相关的行为表现出性别差异，但我们缺乏对这些差异的调节机制的洞察。对NAC功能和行为性别差异的分子和细胞基础的研究将通过确定新的靶点和机制来填补关键的知识空白。该项目的长期目标是确定活动调节的细胞骨架相关蛋白(Arc)在成年NAC中作用的神经生物学机制，重点是Arc通过细胞类型特定的动作在介导性别特异性细胞和行为NAC功能中的作用。这项建议的总体目标是确定Arc在与NAC相关的行为经历(如应激、动机、新奇暴露)后的雄性和雌性小鼠NAC中的作用和调节。中心假设是Arc通过特定细胞类型的突触作用来调节NAC的功能和相关行为。这个项目的基本原理是，识别介导NAC相关行为的特定细胞类型，以及Arc在这些细胞中的功能，将指导未来关于性别差异的细胞和突触机制的假说。中心假说将以两个具体目标进行检验：1)根据NAC相关的行为经验建立诱导Arc的细胞类型，并要求Arc动作来调节行为反应；2)确定Arc在NAC中的亚细胞定位及其在突触可塑性中的作用。在第一个目标中，将使用针对Arc蛋白和遗传编码的、细胞类型特定的荧光团的免疫组织化学技术来识别响应NAC相关行为经验而诱导Arc的关键细胞群。需要Arc动作来介导NAC相关行为的细胞将在表达遗传编码的细胞类型特定cre的小鼠中得到鉴定，并将依赖于cre的shRNA病毒传递到成年NAC。在第二个目标中，将使用生化分离技术在行为经验之后立即评估Arc的亚细胞定位。此外，Arc对NAC突触功能的贡献将通过电生理技术来评估，以检测基本生理学和诱导实验人员诱导的NAC相关的、Arc介导的形式突触可塑性的能力(例如，BDNF诱导的LTP、DHPG诱导的LTD)。本申请中提出的研究具有创新性，因为它专注于研究NAC功能的分子中介，定位Arc在NAC中特定细胞类型中的作用，并将Arc的作用与细胞和行为性别差异联系起来。这项拟议的研究意义重大，因为它为单一脑区(NAC)的特定细胞中的单一分子(即Arc)确立了一个新的角色，作为NAC功能和行为性别差异的中介。这些实验为Arc在NAC介导的行为中的相关性、NAC功能和行为的性别差异提供了关键证据，并为未来的实验描述Arc的性别特异性调控和功能提供了关键信息。

Modified Project Summary/Abstract Section The Nucleus Accumbens (NAc) is a key brain region mediating mood-relevant behaviors but the cell types and molecules underlying this contribution are still being identified. Additionally, many NAc-relevant behaviors show sex differences, but we lack insight into the mechanisms mediating these differences. Studies on the molecular and cellular bases of sex differences in NAc function and behavior will fill a key knowledge gap by identifying novel targets and mechanisms. The long-term goal of this project is to determine the neurobiological mechanisms of activity-regulated cytoskeleton-associated protein (Arc) action in the adult NAc, focused on Arc’s role in mediating sex-specific cellular and behavioral NAc functions via cell type-specific action. The overall objective of this proposal is to determine the role and regulation of Arc in the NAc of male and female mice following NAc-related behavioral experiences (e.g. stress, motivation, novelty exposure). The central hypothesis is that Arc regulates NAc function and associated behaviors via cell type-specific synaptic action. The rationale for this project is that the identification of specific cell types mediating NAc-related behaviors, and Arc’s function in those cells, directs future hypotheses on the cellular and synaptic mechanisms of sex differences. The central hypothesis will be tested with two specific aims: 1) Establish the cell types inducing Arc following NAc-relevant behavioral experience and requiring Arc action to mediate behavioral responses; 2) Determine the subcellular localization of Arc and its role in synaptic plasticity in the NAc. In the first aim, key cell populations inducing Arc in response to NAc-relevant behavior experience will be identified using immunohistochemical techniques directed against Arc protein and genetically encoded, cell type-specific fluorophores. The cells requiring Arc action to mediate NAc-relevant behavior will be identified with in mice expressing genetically encoded cell type-specific cre, with cre-dependent shRNA virus delivery to the adult NAc. In the second aim, the subcellular localization of Arc will be assessed immediately following behavior experience using biochemical fractionation techniques. Additionally, Arc’s contribution to NAc synaptic function will be assessed using electrophysiological techniques to examine basal physiology and the ability to induce NAc-relevant, Arc-mediated forms of experimenter-induced synaptic plasticity (e.g. BDNF-induced LTP, DHPG induced-LTD). The research proposed in this application is innovative, as it is focused on examining a molecular mediator of NAc function, localizing roles for Arc in specific cell types within the NAc and connecting Arc action to cellular and behavioral sex differences. The proposed research is significant as it establishes a novel role for a single molecule (i.e. Arc) in specific cells of a single brain region (NAc) as mediator of sex differences in NAc function and behavior. The proposed experiments provide critical evidence for the relevance of Arc in NAc-mediated behavior, sex differences in NAc function and behavior, and critical information for future experiments to delineate the sex-specific regulation and function of Arc.