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: 6700776
• 负责人: Stefano Vicini
• 金额: $ 30.43万
• 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/6700776
• 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）：GABAa 受体在焦虑、睡眠和许多神经系统疾病的发病机制中发挥着关键作用。 γ-氨基丁酸 A (GABA)a 受体的分子克隆揭示了组成亚基的相当大的异质性。转染特定亚基组合的细胞中的不同 GABAa 受体亚型在不同构象状态之间具有独特的通道转换速率，这决定了 GABA 反应的“分子动力学指纹”。使用具有特定亚基基因删除的小鼠，我们将证明这种分子指纹设定了抑制性突触电流（IPSC）的时间过程，这是确定抑制性突触强度的关键因素，也是许多常用处方药物的靶标。我们关注小脑颗粒和星状神经元，其中 IPSC 持续时间的逐渐发育减少与 GABAa 受体亚基表达的变化平行。我们的初步发现支持 GABAa 受体 α1 亚基的缺失会阻止 IPSC 动力学的发育变化，我们的假设是，不同的 GABAa 受体亚型、解剖学限制和发育调节导致了行为和神经系统疾病背后的抑制活性的特定功能特性。 我们的研究结果将允许将分子结构的异质性与抑制性突触的功能异质性联系起来。小鼠小脑神经元切片和原代神经元培养物中突触和突触外 GABA 电流的全细胞记录将与从这些神经元切除的外侧斑块中 GABA 激活通道电流的记录互补。天然 GABA 通道的生物物理研究将通过研究瞬时转染 GABAa 受体特定亚基的细胞和缺失特定亚基的转基因小鼠记录的数据来整合。目标是识别天然亚基组合，并最终将这些不同的受体组合与其在 GABA 介导的小脑神经元抑制中的特殊作用联系起来。