Amphetamine-Induced Transcriptional Plasticity in Striatal GABAergic Interneurons

中脑边缘奖赏回路由腹侧被盖区的多巴胺能神经元和它们的神经元组成。 靶点在脑桥核（NAc）和其他相关的边缘脑区域。这条线路是主要的站点 对成瘾性药物如精神兴奋剂安非他明（AMPH）的作用。的增强性能 AMPH是由NAc中神经元的生理学和突触连接性的变化介导的。 大量证据表明，AMPH诱导的纹状体基因表达的变化是必不可少的， 这些细胞的适应性和染色质调节被认为是一种机制， 这些变化在神经生理学上的持续性。然而，纹状体是由多个 这些神经元通过突触相互连接成功能性的微电路， 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。我们 我建议采用一种新的方法来解决这个问题，方法是使用我们为荧光而开发的协议- 活化细胞分选（FACS），以表征AMPH诱导的纹状体快- GABA能中间神经元（FSI）。FSI在控制纹状体输出方面起着至关重要的作用，但它仍然是 目前尚不清楚这些神经元是否经历了依赖AMPH的适应。FSI的弥散分布 对它们的基因转录和染色质调节的生化分析提出了重大障碍。 然而，我们发现，AMPH的管理驱动快速和强大的磷酸化的甲基-DNA 结合蛋白MeCP 2在Ser 421（pMeCP 2）在NAc，并认为这种AMPH诱导的磷酸化发生 选择性地在FSI中。在这些发现的基础上，我们开发了使用pMeCP 2 抗体作为标记物从小鼠纹状体纯化AMPH激活的FSI。在这里，我们建议使用这个 技术，以确定FSI是否显示转录调节的可塑性， 反复暴露于AMPH在目标1中，我们将用流式细胞仪从接受以下任一种治疗的小鼠的纹状体中纯化FSI 急性或重复AMPH注射和通过RNA-Seq的基因表达谱变化。在目标2中， FACS从接受急性或重复AMPH注射的小鼠纹状体中纯化FSI细胞核， 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， 这种中间神经元群体经历了分子适应，以响应反复的AMPH暴露。 这一结果将是令人兴奋的，因为它将提高转录可塑性的可能性， 伴随着AMPH诱导的FSI功能适应。我们预计，确定FSI基因调控， 通过反复的AMPH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可能 有助于AMPH诱导的中脑边缘电路功能的变化。

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.