MOLECULAR CHARACTERIZATION OF GLUTAMATE RECEPTOR EXPRESSION IN BRAIN

脑中谷氨酸受体表达的分子特征

• 批准号: 2575645
• 负责人: A BUONANNO
• 金额: --
• 依托单位: EUNICE KENNEDY SHRIVER NATIONAL INSTITUTE OF CHILD HEALTH & HUMAN DEVELOPMENT
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/2575645
• 关键词: cerebellum; genetic transcription; genetically modified animals; glutamate receptor; glutamates; granule cell; innervation; laboratory mouse; neural plasticity; neurotrophic factors; receptor expression; synaptogenesis; tissue /cell culture; transfection

Activity-dependent neural plasticity during development and in the adult is largely mediated by N-methyl-D-aspartate receptor (NR) activation. Glutamate is the natural ligand for this excitatory amino acid receptor that is expressed specifically in the CNS. The functional properties of NRs have been found to change in the cerebellum and visual cortex during the development; the changes in the visual system are activity-dependent. Using in situ hybridization, we have found that the down-regulation of NR2B and increase of NR2C mRNA levels in the internal granule layer of the cerebellum closely follow the spatial-temporal pattern of synapse formation, suggesting that these NR genes are regulated by innervation. Using cerebellar slice cultures, we have found that neurotrophic factors specifically up-regulate NR2C mRNA levels with little effects on NR2B expression. On the other hand, preliminary evidence suggests that the down-regulation of NR2B in granule cells during synaptogenesis may result from activity. Thus, different NR subunits may not only function to distinctly modulate synaptic connections in response to activity, but their expression patterns may also be differentially responsive to epigenetic factors. In order to understand the complex mechanisms that direct regional-specific transcription of NR genes during neurogenesis and modulate their levels in response to synaptic activity, we are investigating the mechanisms that control expression of the NR2 subunits at the transcriptional level. Utilizing transgenic mice, we have identified different upstream regions of the NR2B gene that are required for its neural-specificity and its developmental down-regulation in the cerebellum. We have recently succeeded transfecting cultures of primary cerebellar granule cells with NR2B reporter constructs, which has enabled us to delineate sequences that regulate NR2B transcription. Deletional and mutational analyses are in progress to identify the cis-acting sequences that mediate neural-specific transcription of the NR2B gene in cultured cells and in the brain of transgenic mice. In collaboration with Dr. Kusiak, we are using transgenic mice to analyze NR 1 DNA regulatory sequences that confer neural specificity; the DNA constructs were previously shown to function in transfected PC12 cells. In preliminary experiments we found that the upstream 3.0 kb of the NR1 gene directs expression of the beta-gal transgene to most CNS neurons. The NR cis-acting elements identified in these studies will be used to isolate novel transcription factors that confer neural specificity and mediate the effects of electrical activity.

发育过程中和成年期的活动依赖性神经可塑性 主要由N-甲基-D-天冬氨酸受体（NR）激活介导。 谷氨酸是这种兴奋性氨基酸受体的天然配体 在中枢神经系统中特异性表达。的功能特性 已经发现，在大脑皮层和视皮层中， 发展;视觉系统的变化是依赖于活动的。 使用原位杂交，我们发现，下调的 NR 2B和NR 2C mRNA水平的增加， 小脑紧密地遵循突触的时空模式 形成，表明这些NR基因受神经支配。 利用小脑切片培养，我们发现神经营养因子 特异性上调NR 2C mRNA水平，而对NR 2B几乎没有影响 表情另一方面，初步证据表明， 在突触发生过程中，颗粒细胞中NR 2B的下调可能导致 从活动。因此，不同的NR亚基可能不仅起作用， 明显地调节突触连接以响应活动，但是 它们的表达模式也可能对 表观遗传因素为了理解复杂的机制， NR基因在神经发生中的直接区域特异性转录 并根据突触活动调节它们的水平， 研究控制NR 2亚单位表达的机制 在转录水平上。利用转基因小鼠， 确定了NR 2B基因的不同上游区域， 因为它的神经特异性和在发育中的下调， 小脑我们最近成功地培养了 小脑颗粒细胞与NR 2B报告结构，这使得 我们描绘调控NR 2B转录的序列。缺失型 和突变分析正在进行中，以确定顺式作用 介导NR 2B基因神经特异性转录的序列， 培养的细胞和转基因小鼠的大脑中。 合作 与库西亚克博士一起，我们正在使用转基因小鼠来分析NR 1 DNA 赋予神经特异性的调节序列; DNA构建体 先前显示在转染的PC 12细胞中起作用。在 初步实验发现，NR 1基因上游3.0 kb 将β-gal转基因的表达导向大多数CNS神经元。nr个 在这些研究中鉴定的顺式作用元件将用于分离 赋予神经特异性并介导 电活动的影响。