Amphetamine-Induced Transcriptional Plasticity in Striatal GABAergic Interneurons

安非他明诱导纹状体 GABA 能中间神经元的转录可塑性

• 批准号: 8322981
• 负责人: Anne Elizabeth West
• 金额: $ 19.13万
• 依托单位: DUKE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2012
• 资助国家: 美国
• 起止时间: 2012-03-01 至 2014-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8322981
• 关键词: Acute; Amphetamines; Antibodies; Behavioral; Biochemical; Brain; Brain region; Cell Nucleus; Cells; Chromatin; Chronic; Corpus striatum structure; DNA-Binding Proteins; Data; Data Set; Databases; Diffuse; Dose; Drug abuse; Enhancers; Epigenetic Process; Fluorescence-Activated Cell Sorting; Gene Expression; Genes; Genetic Transcription; Genomics; Goals; Histone Acetylation; Histone H3; Immediate-Early Genes; Injection of therapeutic agent; Interneuron function; Interneurons; Label; Lead; Mediating; Methyl-CpG-Binding Protein 2; Molecular; Molecular Profiling; Mus; Mutation; Neurons; Nucleus Accumbens; Outcome; Output; Pathology; Pharmaceutical Preparations; Phosphorylation; Physiology; Play; Population; Process; Property; Protocols documentation; RNA; RNA Sequences; Regulation; Rewards; Role; Site; Synapses; Techniques; Therapeutic Intervention; Transcriptional Regulation; Ventral Striatum; Ventral Tegmental Area; addiction; base; behavioral sensitization; brain tissue; cell type; chromatin immunoprecipitation; dopaminergic neuron; drug of abuse; experience; histone modification; insight; interest; neuroadaptation; novel; novel strategies; prevent; programs; promoter; psychostimulant; relating to nervous system; response

DESCRIPTION (provided by applicant): The mesolimbic reward circuit is comprised of dopaminergic neurons in the ventral tegmental area and their targets in the nucleus accumbens (NAc) and other associated limbic brain regions. This circuit is the major site of action for addictive drugs such as psychostimulant amphetamine (AMPH). The reinforcing properties of AMPH are mediated by changes in the physiology and synaptic conectivity of neurons in the NAc. Considerable evidence suggests that AMPH-induced changes in striatal gene expression are essential for these cellular adaptations, and chromatin regulation has been implicated as a mechanism that may contribute to the persistence of these changes in neuronal physiology. However the striatum is comprised of multiple kinds of neurons that are synaptically interconnected into functional microcircuits, and very little is known about whether or how cell-type specific differences in AMPH-regulated transcription impact striatal function. We propose to take a novel approach to this question by using a protocol we have developed for fluorescence- activated cell sorting (FACS) to characterize AMPH-induced changes in gene transcription in striatal fast- spiking GABAergic interneurons (FSIs). FSIs play a crucial role in gating striatal output, however it remains unknown whether these neurons experience AMPH-dependent adaptations. The diffuse distribution of FSIs has presented a significant barrier to biochemical analysis of their gene transcription and chromatin regulation. However we discovered that AMPH administration drives rapid and robust phosphorylation of the methyl-DNA binding protein MeCP2 at Ser421 (pMeCP2) in the NAc, and that this AMPH-induced phosphorylation occurs selectively in FSIs. On the basis of these findings we have developed FACS protocols to use the pMeCP2 antibody as a label to purify AMPH-activated FSIs from the mouse striatum. Here we propose to use this technique in order to determine whether FSIs show plasticity of transcriptional regulation in response to repeated AMPH exposure. In Aim 1 we will FACS purify FSIs from the striatum of mice that received either acute or repeated AMPH injections and profile changes in gene expression by RNA-Seq. In Aim 2 we will FACS purify FSI nuclei from the striatum of mice that received either acute or repeated AMPH injections and perform ChIP-Seq with antibodies against acetylated histone H3 as an indication of chromatin regulation. If we observe differences in gene expression or chromatin regulation, these data would provide the first evidence that this interneuron population experiences molecular adaptations in response to repeated AMPH exposure. This outcome would be exciting because it would raise the posibility that transcriptional plasticity is accompanied by AMPH-induced adaptations in FSI function. We anticipate that identifying FSI genes regulated by repeated AMPH will suggest new hypotheses of how plasticity of this important interneuron population may contribute to AMPH-induced changes in mesolimbic circuit functions. PUBLIC HEALTH RELEVANCE: Chronic drug abuse can be attributed to the ability of addictive substances to induce persistent adaptations in the reward circuits of the brain. This study will identify cell-type specific amphetamine-induced changes in neuronal gene expression that may contribute to this process. Because reward circuit adaptations are part of the pathology that leads to addiction, these genes represent important potential targets for therapeutic intervention.

描述（由申请人提供）：中脑边缘奖赏回路由腹侧被盖区的多巴胺能神经元及其在脑桥核（NAc）和其他相关边缘脑区域的靶点组成。这条神经回路是成瘾性药物如精神兴奋剂安非他明（AMPH）的主要作用部位。AMPH的增强特性是由NAc中神经元的生理学和突触连接性的变化介导的。相当多的证据表明，AMPH诱导的纹状体基因表达的变化是必不可少的这些细胞的适应，和染色质调节已牵连作为一种机制，可能有助于这些变化的持久性在神经元生理。然而，纹状体由多种神经元组成，这些神经元在突触上相互连接成功能性微电路，并且关于AMPH调节的转录中的细胞类型特异性差异是否或如何影响纹状体功能知之甚少。我们建议采取一种新的方法来解决这个问题，通过使用我们已经开发的荧光激活细胞分选（FACS）的协议来表征纹状体快速尖峰GABA能中间神经元（FSI）中AMPH诱导的基因转录变化。FSI在门控纹状体输出中起着至关重要的作用，然而这些神经元是否经历AMPH依赖性适应仍然未知。FSI的弥散分布对其基因转录和染色质调控的生化分析构成了重大障碍。然而，我们发现，AMPH管理驱动快速和强大的甲基-DNA结合蛋白MeCP 2在Ser 421（pMeCP 2）在NAc磷酸化，这种AMPH诱导的磷酸化选择性地发生在FSI。在这些发现的基础上，我们开发了FACS方案，使用pMeCP 2抗体作为标记，从小鼠纹状体纯化AMPH激活的FSI。在这里，我们建议使用这种技术，以确定是否FSI显示可塑性的转录调节响应反复AMPH曝光。在目标1中，我们将从接受急性或重复AMPH注射的小鼠纹状体中纯化FSI，并通过RNA-Seq分析基因表达的变化。在目标2中，我们将通过流式细胞仪纯化接受急性或重复注射AMPH的小鼠纹状体中的FSI核，并使用针对乙酰化组蛋白H3的抗体进行ChIP-Seq，作为染色质调节的指示。如果我们观察到基因表达或染色质调节的差异，这些数据将提供第一个证据，表明这种中间神经元群体在响应反复AMPH暴露时经历分子适应。这一结果将是令人兴奋的，因为它将提高转录可塑性伴随着AMPH诱导的FSI功能适应的可能性。我们预计，确定FSI基因调控的反复AMPH将提出新的假设，如何可塑性这一重要的中间神经元人口可能有助于AMPH诱导的变化，中脑边缘电路功能。 公共卫生相关性：慢性药物滥用可归因于成瘾物质诱导大脑奖赏回路持续适应的能力。这项研究将确定细胞类型特异性安非他明诱导的神经元基因表达的变化，可能有助于这一过程。由于奖赏回路适应是导致成瘾的病理学的一部分，这些基因代表了治疗干预的重要潜在靶点。