Cerebellar Inhibitory Synapses in GABAR subunits KO mice

GABAR 亚基 KO 小鼠的小脑抑制性突触

• 批准号: 6606572
• 负责人: Stefano Vicini
• 金额: $ 29.44万
• 依托单位: GEORGETOWN UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2003
• 资助国家: 美国
• 起止时间: 2003-03-01 至 2008-02-29
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6606572
• 关键词: GABA receptor; SDS polyacrylamide gel electrophoresis; benzodiazepines; biological signal transduction; cerebellum; electrophysiology; excitatory aminoacid; genetically modified animals; immunocytochemistry; laboratory mouse; molecular biology; neural inhibition; neurons; pharmacokinetics; posttranslational modifications; receptor expression; synapses; tissue /cell culture; western blottings

DESCRIPTION (provided by applicant): GABAa receptors play a critical role in anxiety, sleep and the pathogenesis of many neurological disorders. Molecular cloning of gamma-aminobutyric A (GABA)a receptors has revealed a considerable heterogeneity of constituent subunits. Different GABAa receptor isoforms in cells transfected with specific combinations of subunits have unique rates of channel transition between distinct conformational states that determines a "molecular kinetic fingerprint" of GABA responses. Using mice with specific deletion of subunit genes we will prove that this molecular fingerprint set the time course of inhibitory synaptic currents (IPSCs) a crucial factor in determining the strength of the inhibitory synapse and a target of many commonly prescribed drugs. We focus on cerebellar granular and stellate neurons where a progressive developmental decrease in IPSCs duration parallels changes in GABAa receptor subunit expression. Supported by our preliminary finding that the deletion of the alpha1 subunit of GABAa receptor prevents developmental changes in IPSCs' kinetics, our hypothesis is that distinct GABAa receptor subtypes, anatomically restricted and developmentally regulated lead to the specific functional properties of inhibitory activity that underlie behavior and neurological disorders. The outcome of our study will allow linking the heterogeneity of molecular structures to the functional heterogeneity of inhibitory synapses. Whole-cell recordings of synaptic and extrasynaptic GABA currents in neurons of the mouse cerebellum in slices and primary neuronal cultures will be complementary to recordings of GABA-activated channel currents in outside-out patches excised from these neurons. The biophysical study of native GABA channel will be integrated by studying those recorded from cells transiently transfected with specific subunits of GABAa receptors and from transgenic mice missing specific subunits. The goal is to identify native subunit combinations and ultimately link these different receptor combinations to their particular role in GABA-mediated inhibition in cerebellar neurons.

描述（由申请人提供）：GABA a受体在焦虑、睡眠和许多神经系统疾病的发病机制中起关键作用。γ-氨基丁酸A（GABA）α受体的分子克隆揭示了组成亚基的相当大的异质性。在用亚基的特定组合转染的细胞中，不同的GABAa受体同种型在不同的构象状态之间具有独特的通道转换速率，其确定GABA响应的“分子动力学指纹”。使用特定缺失亚基基因的小鼠，我们将证明这种分子指纹设置抑制性突触电流（IPSC）的时间过程，这是决定抑制性突触强度的关键因素，也是许多常用处方药的靶点。我们专注于小脑颗粒和星状神经元，其中IPSC持续时间的进行性发育减少与GABA α受体亚单位表达的变化平行。基于我们的初步发现，GABAa受体α 1亚基的缺失阻止了IPSC动力学的发育变化，我们的假设是，不同的GABAa受体亚型，解剖学上的限制和发育调节导致了行为和神经系统疾病的抑制活性的特定功能特性。 我们的研究结果将允许连接的异质性的分子结构的功能异质性的抑制性突触。全细胞记录的突触和突触外GABA电流在小鼠小脑的神经元切片和原代神经元培养将是互补的GABA激活的通道电流从这些神经元切除的外向补丁记录。天然GABA通道的生物物理学研究将通过研究从GABAa受体的特定亚基瞬时转染的细胞和从缺失特定亚基的转基因小鼠记录的那些来整合。我们的目标是确定天然亚基组合，并最终将这些不同的受体组合与它们在GABA介导的小脑神经元抑制中的特定作用联系起来。