CNS GABA-A POSTSYNAPTIC RESPONSES--SINGLE CHANNEL BASIS

为了更好地了解最基本的机制和药理学 重要的抑制性神经递质在中枢神经系统，这项研究将 研究突触后GABA-A受体功能 生理学相关的条件。 膜片钳记录和超快 配体应用技术将用于：1）表征 GABA-A受体抑制性突触后电流的单通道基础 （IPSC）响应，以及2）识别单通道机制 GABA介导的IPSC的潜在药理学调节。 GABA-A受体由蛋白质复合物组成， 氯离子选择性通道，并含有调节结合位点 GABA和临床上重要的苯二氮卓类，巴比妥类， 类固醇和惊厥药 γ-氨基丁酸与受体的结合 突触后膜引起宏观IPSC组成的随机 由于许多单个离子通道的打开而产生的反应。 IPSC幅度和/或时间过程可以被直接改变， 调节神经元抑制。 GABA介导的单通道基础 IPSC和药物对IPSC的调节尚未得到广泛研究。 以前的研究表明，GABA和变构调节剂， GABA受体改变单通道门控（打开和关闭） 动力学性质，而不是通道电导。 但由于 受体脱敏可以改变离子的门控特性 通道，这些研究一直受到相对较慢的速度和 长时间的配体暴露于受体。 在突触处， 释放和摄取机制导致快速和可能短暂的GABA 暴露于突触后受体。 因此， 神经递质和药物的作用将更好地研究条件 更类似于在突触处发现的，例如在本研究中提出的。 本研究的目的是：1）表征动力学性质， 开发GABA介导的IPSC的单通道相关模型， 2)确定药理学基础的单通道机制， GABA介导的IPSC的调节，以及3）表征单个 通道特性的GABA受体激动剂依赖脱敏作为 它与IPSC反应的调节有关。 将进行研究 使用新开发的超快配体应用技术， 从培养的大鼠皮层神经元获得的外向补丁。 以前开发的单通道分析技术将得到扩展 并应用于调查。 本研究将建立一个模型， 了解和预测，在各种条件下，GABA介导的， 基于随机干扰的IPSC调制响应 单个GABA受体通道的活性。 长期目标是 该项目旨在更好地了解突触GABA受体 功能和临床相关的GABA受体药理学。

To better understand the basic mechanisms and pharmacology of the most important inhibitory neurotransmitter in the CNS, this study will investigate postsynaptic GABA-A receptor function under more physiologically relevant conditions. Patch clamp recording and ultrafast ligand application techniques will be utilized to: 1) characterize the single channel basis of GABA-A receptor inhibitory postsynaptic current (IPSC) responses, and 2) identify the single channel mechanisms underlying pharmacologic modulation of GABA-mediated IPSCs. The GABA-A receptor is composed of a protein complex that forms a chloride selective ion channel, and contains binding sites for regulation by GABA and by clinically important benzodiazepines, barbiturates, steroids and convulsants. Binding of GABA to receptors in the postsynaptic membrane evokes a macroscopic IPSC composed of a stochastic response resulting from the opening of many individual ion channels. IPSC amplitude and/or time course can be pharmacologically altered to modulate neuronal inhibition. The single channel basis for GABA-mediated IPSCs and for IPSC modulation by drugs has not been extensively studied. Previous studies have indicated that GABA and allosteric modulators of the GABA receptor altered single channel gating (opening and closing) kinetic properties, rather than channel conductance. However, since receptor desensitization may alter gating characteristics of the ion channel, these studies have been limited by relatively slow speed and long duration of ligand exposure to the receptor. At the synapse, release and uptake mechanisms result in rapid and probably transient GABA exposure to the postsynaptic receptor. Therefore, mechanisms of neurotransmitter and drug action would be better studied under conditions more similar to that found at the synapse such as proposed in this study. Aims of this study are: 1) to characterize the kinetic properties and to develop a model of the single channel correlate of GABA-mediated IPSCs, 2) to identify the single channel mechanisms underlying pharmacologic regulation of GABA-mediated IPSCs, and 3) to characterize the single channel properties of GABA receptor agonist dependent desensitization as it is related to modulation of IPSC responses. Studies will be performed using newly developed ultrafast ligand application techniques on excised outside-out patches obtained from rat cortical neurons in culture. Previously developed single channel analysis techniques will be extended and applied in the investigations. This study will establish a model to understand and predict, under a variety of conditions, GABA-mediated and pharmacologically modulated IPSC responses based upon the stochastic activity of single GABA receptor channels. The long-term goal of this project is to provide a better understanding of synaptic GABA receptor function and of clinically relevant GABA receptor pharmacology.