MOLECULAR CHARACTERIZATION OF GLUTAMATE RECEPTOR EXPRESSION IN BRAIN

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（NMDAR）激活介导; 这种兴奋性氨基酸受体的天然配体是谷氨酸。 在突触发生过程中，已经发现NMDAR在以下方面起主要作用： 稳定哺乳动物和低等动物视觉系统中的神经连接 脊椎动物，并在登记体感地图。在成年人中， 受体参与了长期的 增强，一个过程的特点是增加突触 对电刺激的反应。因此，NMDAR 为理解可雄性化特性提供了一个有趣的目标 的CNS。作为阐明分子机制的第一步， 将这些受体家族的表达靶向于特定的神经细胞， 人口在发展过程中，我们已经开始分析表达 不同受体亚基的变化，并分析了 转录调控元件调节NMDAR基因表达。 利用原位杂交组织化学，我们和其他人发现， ε亚基在神经发育过程中差异表达。到 开始阐明调节NMDAR表达的分子机制， 克隆ε 2基因的上游区，测序， 表征了多个转录起始位点通过 RNA酶保护和基于引物延伸的PCR技术。 携带氯霉素乙酰转移酶（CAT）的转基因小鼠 由ε 2上游序列驱动的构建体表达报告基因 特别是在大脑中。实验正在进行中，以描绘 顺式作用元件赋予发育和神经元特异性 转录。复制缺陷型腺病毒载体用于 已经研究了将表达构建体引入神经元。这 工作类型为将来使用epsilon2提供了基础 基因治疗实验中的调节序列。

Activity-dependent neural plasticity during development and in the adult is largely mediated by N-methyl-D-aspartate receptor (NMDAR) activation; the natural ligand for this excitatory amino acid receptor is glutamate. During synaptogenesis, NMDARs have been found to play a major role in stabilizing neural connections in the visual system of mammals and lower vertebrates, and in registering somatosensory maps. In the adult, NMDA receptors have been implicated in the induction of long-term potentiation, a process characterized by the increase of synaptic efficacy in response to electrical stimulation. For these reasons, NMDARs provide an interesting target for understanding the maleable properties of the CNS. As a first step to elucidating the molecular mechanisms that target the expression of these family of receptors to specific neural populations during development, we have begun to analyze the expression of different receptor subunits during development and to dissect the transcriptional regulatory elements that modulate NMDAR gene expression. Using in situ hybridization histochemistry, we and others found that the epsilonsubunits are differentially expressed during neurodevelopment. To begin elucidating the molecular mechanisms regulating NMDAR expression, the upstream region of the epsilon2 gene was cloned, sequenced and characterized. Multiple transcription initiation sites were mapped by RNase protection and a PCR technique based on primer extension. Transgenic mice harboring a chloramphenicol acetyltransferase (CAT) construct, driven by epsilon2 upstream sequences, expressed the reporter specifically in the brain. Experiments are in progress to delineate the cis-acting elements conferring developmental and neuron-specific transcription. The use of replication-deficient adenovirus vectors to introduce expression constructs into neurons has been investigated. This type of work provides the foundation for the future use of epsilon2 regulatory sequences in gene therapy experiments.