Metabotropic Glutamate Regulation of Amphetamine Action

描述(申请人提供)：一次注射精神刺激剂D-苯丙胺(AMPH)，增加大鼠纹状体阿片类基因的表达(分别在纹状体和纹状体苍白核投射神经元中的前强啡肽和前脑啡肽)。这些可诱导的基因组反应是由表面受体(刺激-转录偶联)介导的，被认为是与滥用药物成瘾特性相关的心理可塑性发展的基本分子步骤。最近的研究发现，代谢性谷氨酸受体(MGluRs)在调节Amph刺激的阿片基因表达中发挥着重要作用。此外，细胞内钙离子的动员对mGluRs的贡献似乎是至关重要的，这表明钙连接的I组mGluRs参与了这一事件。基于这些令人鼓舞的发现，在这个项目的继续中，提出了一系列实验，以探索和表征第一组mGluRs和钙敏感信号通路对amph刺激的阿片类药物基因表达的调节。 我们的工作假设是，I组mGluRs和相关信号通路参与了AMPH刺激的纹状体神经元阿片类基因表达的调节。通过多学科的方法，这一假说将在体内得到以下4个目标的验证：(1)通过检测选择性的I组激动剂/拮抗剂对AMPH刺激的急性AMPH刺激的纹状体阿片基因表达的影响，确定I组受体在AMPH作用中的中介作用；(2)区分两个I组亚型mGluR1和mGluR5的相对重要性，在该事件中，使用亚型选择性激动剂/拮抗剂、反义寡核苷酸和突变小鼠(mGluR1或5基因敲除)，(3)在蛋白激酶水平，通过检测amph对纹状体依赖的CaMK II磷酸化的影响以及反义寡核苷酸降低CaMK II活性对amph刺激的阿片基因表达的影响，来检测CaMK II在amph作用中的作用，以及(4)在转录水平上，通过监测桥联I组mGluR刺激细胞膜上的mGluR刺激，评估CaMK II反应转录因子cAMP反应元件结合蛋白(CREB)和Elk-1对核内阿片基因表达的重要性 AMPH刺激下纹状体神经元中这些转录因子对I/CaMK II敏感的磷酸化和DNA结合活性。我们将依靠定量原位杂交来分析体内的mRNA表达。 该项目的完成将阐明I组受体和信号通路如何调节成熟纹状体神经元中阿片基因的表达，以响应精神刺激剂的暴露。由于可诱导的基因表达被认为参与心理可塑性的发展，来自该项目的数据将为药物滥用长期作用的细胞/分子机制提供有价值的见解。

DESCRIPTION (provided by applicant): A single injection of the psychostimulant, D-amphetamine (AMPH), increases opioid gene expression in the rat striatum (preprodynorphin and preproenkephalin in striatonigral and striatopallidal projection neurons, respectively). These inducible genomic responses are mediated by surface receptors (stimulus-transcription coupling) and considered as essential molecular steps for the development of psychoplasticity related to addictive properties of drugs of abuse. From studies conducted in the last period, metabotropic glutamate receptors (mGluRs) were found to play a profound role in mediating AMPH-stimulated opioid gene expression. Moreover, intracellular Ca2+ mobilization appears to be critical for the mGluRs' contribution, indicating the participation of Ca2+-Iinked group I mGluRs in this event. Based on these encouraging findings, a series of experiments was proposed in this continuation of project to explore and characterize the regulation of AMPH-stimulated opioid gene expression by the group 1 mGluRs and the Ca2+-sensitive signaling pathway. Our working hypothesis is that the group I mGluRs and associative signaling pathways participate in the mediation of AMPH-stimulated opioid gene expression in striatal neurons. With multidisciplinary approaches, this hypothesis will be tested in vivo in the following 4 aims designed to: (1) define the role of group I receptors in the mediation of AMPH action through examining effects of the selective group I agonists/antagonists on acute AMPH-stimulated striatal opioid gene expression in a well-characterized rat model, (2) differentiate the relative importance of the two group I subtypes, mGluR1 and mGluR5, in this event using the subtype-selective agonists/antagonists, antisense oligos and mutant mice (mGluR1 or 5 knockouts), (3) at the protein kinase level, examine the contribution of CaMK II to AMPH action by testing effects of AMPH on the group I-dependent phosphorylation of CaMK II in the striatum and effects of decreased CaMK II activity by antisense oligos on AMPH-stimulated opioid gene expression, and (4) at the transcription level, evaluate the importance of the CaMK II-responsive transcription factors, cAMP-response-element-binding protein (CREB) and Elk-1, in bridging group I mGluR stimulation on the membrane to opioid gene expression in the nucleus via monitoring the group I/CamK II-sensitive phosphorylation and DNA binding activity of these transcription factors in striatal neurons in response to AMPH stimulation. We will rely on quantitative in situ hybridization to analyze mRNA expression in vivo. Accomplishment of this project will elucidate how group I receptors and signaling pathways mediate opioid gene expression in matured striatal neurons in response to psychostimulant exposure. Since inducible gene expression is conceived to participate in the development of psychoplasticity, data from this project will provide valuable insight in cellular/molecular mechanisms underlying long-term actions of drugs of abuse.