DEVELOPMENTAL EXPRESSION OF GABA-A RECEPTOR MRNAS

描述：（研究者摘要）本研究的长期目标 该项目旨在了解GABAA受体的表达是如何调节的， CNS。 这种多亚基配体门控离子通道是 GABA是大脑中主要的抑制性神经递质。 最近的分子生物学研究表明，每种受体 亚基由一个基因家族编码，该基因家族表现出不同的区域性特征， 分布。 在小脑颗粒神经元中，至少有8个亚基RNA， 表达，其中许多在第二次出生后升高数倍， 周 这些变化与广泛的小脑 分化和颗粒细胞从胚泡区（EGL）迁移 他们成熟的位置。 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 结果表明，小脑颗粒神经元GABAA受体mRNA 受到出生后环境线索的影响 分化 本提案将检验这一假设， 调节受体亚基mRNA表达的信号。 获得洞察力 小脑GABAA受体亚单位mRNA的调节 颗粒神经元，提出了几个实验。 一、年龄与阶段 颗粒神经元在小脑成熟时 和/或表达受体mRNA的能力。 的 细胞年龄对于亚基转录本表达重要性将是 在几个出生后年龄制备的培养物中使用RT-PCR进行检查。 因为这种培养物含有处于多个成熟阶段的细胞， 亚基转录本也将在发育同质的 通过用抗血清免疫分离细胞制备的培养物， 特定的标记。 第二，突触前和突触后相互作用的作用 在调节颗粒神经元亚基mRNA中的作用。 的 将在脑桥的共培养物中确定传入输入的影响 RT-PCR检测细胞核和颗粒神经元的表达; 神经元与靶浦肯野神经元的相互作用将用 两种经历浦肯野细胞的小鼠突变体的原位杂交 不同阶段的退化。 第三，环境的作用 信号（如神经营养因子或谷氨酸）调节GABAA 受体mRNA将通过将细胞维持在 模仿小脑环境。 最后， 亚基mRNA和多肽表达将使用亚基- 特异性抗血清 拟议的研究将产生重要的新成果， 关于发育表达和调节的信息 一种经鉴定的神经元GABAA受体mRNA和多肽 人口 此外，这些研究将提供深入了解 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.