Amphetamine-Induced Transcriptional Plasticity in Striatal GABAergic Interneurons

描述（由申请人提供）：中边缘奖赏回路由位于腹侧被盖区的多巴胺能神经元及其在伏隔核（NAc）和其他相关的大脑边缘区域的靶点组成。这一回路是上瘾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中神经元的生理和突触连通性的变化介导的。大量证据表明，amph诱导的纹状体基因表达的变化对于这些细胞适应是必不可少的，染色质调节被认为是神经元生理学中这些变化持续存在的一种机制。然而，纹状体是由多种神经元组成的，这些神经元通过突触相互连接形成功能性微电路，对于amph调节的转录中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。我们建议采用一种新的方法来解决这个问题，即使用我们开发的荧光活化细胞分选（FACS）方案来表征amph诱导的纹状体快速尖峰gaba能中间神经元（FSIs）基因转录的变化。fsi在控制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了依赖于amph的适应尚不清楚。fsi的弥漫性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可以驱动NAc中甲基dna结合蛋白MeCP2 Ser421位点（pMeCP2）的快速和强大磷酸化，并且这种AMPH诱导的磷酸化在FSIs中选择性发生。在这些发现的基础上，我们开发了FACS方案，使用pMeCP2抗体作为标记从小鼠纹状体中纯化amph激活的FSIs。在这里，我们建议使用这种技术来确定fsi是否在反复暴露于AMPH时表现出转录调节的可塑性。在目的1中，我们将用FACS从急性或重复注射AMPH的小鼠纹状体中纯化FSIs，并通过RNA-Seq分析基因表达的变化。在Aim 2中，我们将从急性或重复注射AMPH的小鼠纹状体中纯化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控的差异，这些数据将提供第一个证据，证明这种中间神经元群体在反复暴露于AMPH时经历了分子适应。这一结果令人兴奋，因为它提高了转录可塑性伴随着amph诱导的FSI功能适应的可能性。我们预计，鉴定由重复AMPH调节的FSI基因将提出新的假设，即这种重要的中间神经元群体的可塑性如何有助于AMPH诱导的中脑边缘回路功能的变化。

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.