Opioids and Conditional Amygdala NMDA Receptor Knockout

描述(由申请人提供)： 提交这份修订后的申请是为了向迈克尔·格拉斯博士提供四年的分子神经药理学和体视学方法指导培训，以研究药物滥用问题，主要赞助人是查尔斯·因图里西博士，联合赞助人是弗吉尼亚·皮克尔博士。该项目将允许候选人从事全职研究，这将有助于他被任命为康奈尔大学威尔医学院终身教职助理教授。该提案中的项目将通过使用特定和多功能形式的Cre-loxP技术创建时空敲除来解决杏仁核中必要的NMDA-NR1(NR1)受体亚单位的基因表达在阿片类药物依赖的发展和表达中的作用。该Cre-loxP系统已成功应用于成年动物脊髓中NR1基因的条件性敲除(即空间敲除)(即时间敲除)。这种有条件的基因敲除方法在成年动物特定脑区实现NR1基因表达显著降低的可行性，以及它对行为、神经激活和树突棘蛋白质靶向的影响从未被确定。这一建议将整合条件基因敲除的创新方法和高分辨率定量电子显微镜方法，以及既定的行为范式，以实现以下目标：1)。产生仅限于成年小鼠杏仁核的NR1基因敲除，2)。研究杏仁核条件性NR1基因敲除对吗啡依赖和耐受的发展和表达的影响，3)。确定杏仁核条件性NR1基因敲除对阿片类药物戒断诱导的神经激活的影响，4)。研究杏仁核NR1基因敲除对与NR1在结构和功能上相关联的蛋白质的细胞靶向的影响，包括树突棘中的NMDA-R2(NR2)亚单位和神经元型一氧化氮合酶(NNOS)。这些研究将为杏仁核NR1亚单位基因表达在阿片类药物依赖形成中的作用提供重要的新信息，并为长期使用阿片类药物诱导神经可塑性的调节机制提供新的见解，对维持药物使用和复发具有重要意义。

DESCRIPTION (provided by applicant): This revised application is being submitted to provide Dr. Michael Glass, with four years of mentored training in molecular neuropharmacological and stereological methods to study drug abuse under the primary sponsorship of Dr Charles Inturissi and co-sponsorship of Dr. Virginia Pickel. This project will allow the candidate to engage in full-time research which will be facilitated by his appointment to tenure-track assistant professor at the Weill Medical College of Cornell University. The projects in this proposal will address the role of amygdala gene expression of the essential NMDA-NR1 (NR1) receptor subunit in the development and expression of opiate dependence by creating spatial-temporal knockouts using a specific and versatile form of Cre-loxP technology. This Cre-loxP system has been successfully used to achieve conditional NR1 gene knockout in spinal cord (i.e. spatial knockout) in adult animals (i.e. temporal knockout) with changes in injury-induced pain. The viability of this conditional gene knockout approach to achieve significant reductions of NR1 gene expression in a specific brain region of adult animals, and its consequences for behavior, neural activation, and protein targeting in dendritic spines has never been determined. This proposal will integrate innovative methods for conditional gene knockout and high resolution quantitative electron microscopic methods along with established behavioral paradigms to achieve the following aims: 1). produce NR1 knockout limited to the amygdala of adult mice, 2). examine the effects of conditional NR1 knockout in the amygdala on the development and expression of morphine dependence and tolerance, 3). determine the effects of conditional NR1 knockout in the amygdala on neural activation induced by opiate withdrawal, 4). examine the effects of amygdala NR1 knockout on the cellular targeting of proteins that are structurally and functionally linked to NR1, including the NMDA-R2 (NR2) subunit and neuronal nitric oxide synthase (nNOS) in dendritic spines. These studies will provide important new information on the role of amygdala NR1 subunit gene expression in the development of opiate dependence, and provide novel insights into the mechanisms mediating neural plasticity induced by chronic opiate use with implications for maintenance of drug use and relapse.