Cerebellar Inhibitory Synapses in GABAR subunits KO mice

描述(由申请人提供)：GABAA受体在焦虑、睡眠和许多神经疾病的发病机制中发挥关键作用。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。在转染亚单位特定组合的细胞中，不同的GABAA受体亚型在不同构象状态之间具有独特的通道转换率，这决定了GABA反应的“分子动力学指纹”。利用特定亚基基因缺失的小鼠，我们将证明这种分子指纹设置了抑制性突触电流(IPSCs)的时间进程，这是决定抑制性突触强度的关键因素，也是许多常用药物的靶点。我们专注于小脑颗粒和星状神经元，在这些神经元中，IPSCs持续时间的进行性发展缩短与GABAA受体亚单位表达的变化平行。我们的初步发现支持GABAA受体α1亚单位的缺失阻止了IPSCs动力学的发育变化，我们的假设是，不同的GABAA受体亚型，解剖上受限的和发育调节的，导致了抑制活性的特定功能特性，这些特性是行为和神经疾病的基础。 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。在切片和原代神经元培养中记录小鼠小脑神经元突触和突触外GABA电流的全细胞记录将补充从这些神经元切除的外向斑块中GABA激活的通道电流的记录。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的记录来整合。我们的目标是识别天然的亚单位组合，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。

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.