DEVELOPMENTAL EXPRESSION OF GABA-A RECEPTOR MRNAS

描述：(调查人员摘要)这项计划的长期目标 该项目旨在了解GABAA受体的表达是如何调控的 中枢神经系统。这种多亚单位、配体门控的离子通道是 对大脑中主要的抑制性神经递质GABA的作用。 最近的分子生物学研究证明，每个受体 亚基由一个基因家族编码，这些基因表现出不同的区域 分配。在小脑颗粒神经元中，至少有8个亚单位RNA 表达出来，其中许多在第二次出生后增加了几倍 星期。这些变化在时间上与广泛的小脑一致。 生发区(EGL)的分化和颗粒细胞迁移 到他们成熟的职位。培养颗粒神经元的研究 显示这些转录水平的增加不是预先编程的， 但依赖于小脑发育的各个方面。这些 结果提示，小脑颗粒神经元中存在GABAA受体mRNA 受出生后接收到的环境线索的影响 差异化。这项提议将检验这一假设，并确定 调节受体亚单位mRNA表达的信号。获得洞察力 小脑GABAA受体亚单位mRNAs调控机制的研究 颗粒神经元，提出了几个实验。第一，年龄和阶段 颗粒神经元在小脑成熟时的状态 和/或是否有能力表达受体mRNAs将被确定。这个 细胞年龄对亚单位转录表达的重要性将是 用RT-PCR检测在几个出生后年龄制备的培养物。 因为这样的培养物包含处于多个成熟阶段的细胞， 亚基成绩单也将在发育相同的情况下进行检查。 用抗血清免疫分离细胞制备的培养物-- 特定的标记。第二，突触前和突触后互动的作用 在调节颗粒神经元亚单位方面，将对mRNAs进行研究。这个 传入输入的影响将在脑桥的共培养中确定 核外植体和颗粒神经元的RT-PCR检测；颗粒的重要性 神经元与目标浦肯野神经元的相互作用将通过 两个小鼠浦肯野细胞突变体的原位杂交 不同阶段的退化。第三，环境的作用 调节GABAA的信号(如神经营养素或谷氨酸) 受体mRNAs将通过维持细胞在条件下的状态来确定 模仿小脑的环境。最后，两者之间的关系 亚基信使核糖核酸和多肽的表达将用亚基- 特定的抗血清。拟议的研究将产生重要的新的 关于发育性表达和调控的信息 已鉴定神经元中的GABAA受体mRNAs和多肽 人口。此外，这些研究将提供对 小脑分化与小脑发育的关系 GABAA受体系统。

DESCRIPTION: (Investigator's Abstract) The long term goal of this project is to understand how GABAA receptor expression is regulated in the CNS. This multisubunit, ligand-gated ion channel is the site of action for GABA, the main inhibitory neurotransmitter in the brain. Recent molecular biologic studies have documented that each receptor subunit is encoded by a family of genes which exhibit distinct regional distributions. In cerebellar granule neurons, at least 8 subunit RNAs are expressed, many of which rise several-fold in the second postnatal week. These changes coincide temporally with extensive cerebellar differentiation and granule cell migration from a germinal zone (the EGL) to their mature positions. Studies on cultured granule neurons have shown that increases in these transcript levels is not preprogrammed, but are dependent on various aspects of cerebellar development. These findings suggest that GABAA receptor mRNA in cerebellar granule neurons is influenced by environmental cues received during postnatal differentiation. This proposal will test this hypothesis and identify signals that regulate receptor subunit mRNA expression. To gain insight into the regulation of GABAA receptor subunit mRNAs in cerebellar granule neurons, several experiments are proposed. First, age and stage of cerebellar maturation at which granule neurons become committed and/or competent to express receptor mRNAs will be determined. The importance of cell age for subunit transcript expression will be examined using RT-PCR in cultures prepared at several postnatal ages. Because such cultures contain cells at multiple stages of maturation, subunit transcripts will also be examined in developmentally homogeneous cultures prepared by immunoisolating cells with antisera to stage- specific markers. Second, the role of pre- and postsynaptic interactions in regulating granule neuron subunit mRNAs will be investigated. The influence of afferent input will be determined in cocultures of pontine nuclei explants and granule neurons by RT-PCR; the importance of granule neuron interaction with target Purkinje neurons will be determined with in situ hybridization in two mouse mutants which undergo Purkinje cell degeneration at different stages. Third, the role of environmental signals (such as neurotrophins or glutamate) in modulating GABAA receptor mRNAs will be determined by maintaining cells in conditions mimicking the cerebellar milieu. Finally, the relationship between subunit mRNA and polypeptide expression will be examined using subunit- specific antisera. The proposed studies will yield important new information concerning the developmental expression and regulation of GABAA receptor mRNAs and polypeptides in an identified neuronal population. Moreover, these studies will provide insight into the relationship between cerebellar differentiation and the development of the GABAA receptor system.