REGULATION OF GENE EXPRESSION IN STRIATAL NEURONS

描述（改编自申请人摘要）： 受体介导的基因表达（刺激-转录偶联）在成熟的 CNS神经元被认为是处理适应性信息的重要组成部分， 神经生理学（神经可塑性）的变化与各种正常 和异常的神经活动纹状体作为基底神经节的重要组成部分， 是研究这种基因表达的主要中心位点之一。 我们最近的体内和体外研究表明，代谢型谷氨酸 受体（mGiuR），其在纹状体投射神经元中密集表达， 参与调节纹状体神经元中阿片基因的表达 （纹状体黑质中的前强啡肽原，和纹状体苍白球中的前脑啡肽原， 神经元）。该条例是促进性的，可以通过以下方式调解： 选择性激活I组mGluRs，其与 磷酸肌醇水解（PI），而不是II或III组mGluR，其是 与腺苷酸环化酶负偶联。基于这些令人鼓舞的结果， 本项目提出了一系列的实验来探索和表征 Ⅰ类mGluRs对纹状体阿片肽基因表达的调节 在体内和解剖细胞内信号通路负责 I组敏感的刺激-转录偶联。我们的工作 假设是激活了与代谢物偶联第I组mGluRs 上调纹状体神经元中阿片基因的表达， 转录因子敏感性级联作为信号通路的桥接表面 I组mGluR刺激核基因表达。多学科 方法，这一假设将在三个目标是测试（1） 确认和表征促进剂的药理学特征 I组选择性阿片肽对纹状体阿片基因表达的调节 激动剂/拮抗剂，并研究可能的突触前和突触后机制 作为I组调节纹状体阿片样物质表达的基础， 良好表征的体内大鼠模型，（2）区分 两种I组亚型mGluR 1和mGluR 5，在这种情况下，使用 亚型选择性激动剂/拮抗剂、反义寡核苷酸和突变小鼠 （mGluR 1/5敲除），和约L鉴定细胞内效应物（Ca 2+， CaMK、CREB、CBP和AP-1）桥接膜上的I型mGluR刺激， 体外培养原代纹状体神经元核内阿片样物质基因表达的研究 用药理学（抑制剂/激活剂）和分子（反义） 寡核苷酸、Ca 2+图像、酶测定和DNA结合活性）方法。我们 将依赖于定量原位杂交来分析mRNA表达， 体内和体外。该项目的完成将改善我们目前的 了解成熟CNS中基因表达的转录调控 神经元由于诱导型基因表达被认为参与了 心理可塑性的发展，该项目的数据将提供有价值的 深入了解各种精神疾病的细胞/分子机制（运动， 精神和认知障碍）。

DESCRIPTION(Adapted from applicant's abstract): Receptor-mediated gene expression (stimulus-transcription coupling) in matured CNS neurons is thought to be an important component in processing adaptive changes in neuronal physiology (neuroplasticity) related to a variety of normal and abnormal neural activities. As a key part of basal ganglia, striatum is among the major central sites for the investigation of such gene expression. Our recent studies both in vivo and in vitro show that metabotropic glutamate receptors (mGiuRs), which are densely expressed in striatal projection neurons, are involved in the regulation of opioid gene expression in striatal neurons (preprodynorphin in striatonigral, and preproenkephalin in striatopallidal, neurons). The regulation is facilitatory in nature and may be mediated through selective activation of group I mGluRs, which are positively coupied to phosphoinositide hydrolysis (PI), rather than group II or III mGluRs, which are negatively coupled to adenylate cyclase. Based on these encouraging hndings, a series of experiments was proposed in this project to explore and characterize the regulation of striatal opioid peptide gene expression by the group I mGluRs in vivo and to dissect intracellular signaling pathways responsible for the group I-sensitive stimulus-transcription coupling in vitro. Our working hypothesis is that activation of the investigator-coupled group I mGluRs upregulates opioid gene expression in striatal neurons and that a investigator-sensitive cascade serves as the signaling pathway bridging surface group I mGluR stimulation to nuclear gene expression. With multidisciplinary approaches, this hypothesis will be tested in three aims which are to (1) confirm and characterize pharmacological profiles of the facilitatory regulation of opioid gene expression in the striatum with the group I selective agonists/antagonists, and investigate possible pre- and postsynaptic mechanisms underlying the group I regulation of striatal opioid expression in a well-characterized in vivo rat model, (2) differentiate the relative importance of the two group I subtypes, mGluR1 and mGluR5, in this event in vivo using the subtype-selective agonists/antagonists, antisense oligos and mutant mice (mGluR1/5 knockouts), and aboutL identify the intraceliular effectors (Ca2+, CaMK, CREB, CBP and AP-1) bridging group I mGluR stimulation on the membrane to opioid gene expression in the nucleus in vitro in primary striatal neuronal cultures with pharmacological (inhibitors/activators) and molecular (antisense oligos, Ca2+ image, enzymatic assay and DNA binding activity) approaches. We will rely on quantitative in situ hybridization to analyze mRNA expression in vivo and invitro. Accomplishment of this project will improve our current understanding of transcriptional regulation of gene expression in matured CNS neurons. Since inducible gene expression is conceived to participate in the development of psychoplasticity, data from this project will provide valuable insight into celiular/molecular mechanisms for various mental illnesses (motor, psychiatric and cognitive impairments).