DEVELOPMENTAL REGULATION OF GLUTAMATE RECEPTOR FUNCTION

在发展中的中枢神经系统内局部电路的精炼依赖于 关于一种竞争性突触发生机制，通过它去极化 目标单元反馈只稳定那些有效的输入 在产生去极化过程中。据推测，突触 引起突触后钙离子和兴奋性氨基酸受体的变化， 尤其是N-甲基-D-天冬氨酸(NMDA)受体，因为它的 电压门控特性对这一发展形式至关重要 突触可塑性。初步研究表明，在发育过程中 浅层皮肤NMDA受体NMDAR1的mRNA表达 大鼠上丘(SC)的各层与 出生后视网膜和皮质输入的精细化时期 区域。此外，慢性治疗低水平AP5或MK801的SC 在缓释塑料打乱了结构的细化 视网膜睫状体投射，同时阻止发育 NMDAR1的mRNA表达增加。 拟议的实验将使用大鼠干细胞来追踪分子和 这些发现的功能含义。目标是定义 谷氨酸受体活性水平与结构的关系 突触变化与特异性谷氨酸受体的表达 已知与突触后钙变化相关的亚单位 努力确定NMDA受体亚单位的消息水平是否 被选择性地调节以在以下期间处于高水平运行 突触改变。定量Northern分析和原位杂交 将用于定义NMDA的mRNA表达的时间进程 相对于代谢性谷氨酸受体mGluR1的受体， GluR1和GluR3是非NMDA型离子亲和性受体的亚单位，GluR2是 已知的降低GluR1和GluR2钙离子通透性的亚基 与它们一起在卵母细胞中表达。正常大鼠SC的这些变化 将与长期服用AP5的大鼠的mRNA水平进行比较， LAP5、CNQX或NMDA。这些治疗将在两岁之前开始 或在NMDAR1mRNA表达急剧上升期间。基因表达的变化 年龄或治疗与电压钳的表达相关 急性心肌梗死患者的生理研究和共聚焦显微镜下的钙离子成像 SC切片用于评价NMDA和非NMDA的功能有效性 类似组织中的谷氨酸受体。如果功能之间的差异 观察检测和信使核糖核酸水平，蛋白质水平的分析 使用针对特定受体亚单位的抗体进行， 免疫细胞化学和免疫沉淀。 希望这些调查将提供对 影响突触后的谷氨酸受体的正常调节 Ca++及其功能和突触生成的影响 监管。这些发现也可能影响我们对 儿童癫痫、神经退行性疾病与康复 中枢神经系统创伤。谷氨酸受体，特别是那些改变钙离子的受体 水平，已经牵涉到所有这些神经系统的例子 功能障碍。

The refinement of local circuitry within the developing CNS is dependent on a competitive synaptogenic mechanism through which depolarizations of target cells feed back to stabilize only those inputs that are effective in producing the depolarization. It is hypothesized that synaptically induced changes in post-synaptic Ca++ and excitatory amino acid receptors, particularly the N-methyl-D-aspartate (NMDA) receptor, because of its voltage-gated property, are critical to this developmental from of synaptic plasticity. Initial studies show that a developmental increase in the expression of mRNA for the NMDA receptor NMDAR1 in the superficial layers of the rat superior colliculus (SC) is associated with the postnatal period of refinement of the retinal and cortical inputs to that area. In addition, chronic treatment of SC with low levels of AP5 or MK801 in slow release plastic disrupts the structural refinement of the retinocollicular projection and simultaneously blocks the developmental increase in mRNA for NMDAR1. Proposed experiments will use the rat SC to pursue the molecular and functional implications of these findings. The goal is to define the relationship between activity level of glutamate receptors, structural synaptic changes and the expression of particular glutamate receptor subunits known to be associated with post-synaptic Ca++ changes in an effort to determine if message levels for the NMDA receptor subunits are selectively regulated to function at high levels during the period of synaptic change. Quantitative Northern analyses, and in situ hybridization will be used to define the time course of expression of mRNA for NMDA receptors relative to that of the metabotropic glutamate receptor mGluR1, GluR1 and GluR3, subunits of nonNMDA ionotropic receptors and GluR2, the subunit known to reduce Ca++ permeability of GluR1 and GluR2 when expressed with them in oocytes. These changes in the SC of normal rats will be compared with mRNA levels in rats chronically treated with AP5, LAP5, CNQX or NMDA. These treatments will be initiated at two ages, before or during the steep rise in NMDAR1 mRNA expression. Changes in mRNA expression with age or treatment will be correlated with voltage clamp physiological studies and Ca++ imaging with confocal microscopy in acute slices of SC to assess the functional effectiveness of NMDA and non-NMDA glutamate receptors in similar tissue. If discrepancies between functional assays and mRNA levels are observed, analyses at the protein level will be conducted using antibodies to specific receptor subunits, immunocytochemistry and immunoprecipitation. It is hoped that these investigation will provide basic insights into the normal regulation of the glutamate receptors that effect post-synaptic Ca++ and into the functional and synaptogenic consequences of that regulation. The findings could also impact on our understanding of childhood seizure disorders, neurodegenerative disease and recovery from CNS trauma. Glutamate receptors, particularly those that alter Ca++ levels, have been implicated in all of these instances of nervous system dysfunction.