CNS GABA-A POSTSYNAPTIC RESPONSES--SINGLE CHANNEL BASIS

为了更好地了解MOST的基本机制和药理作用 中枢神经系统中重要的抑制性神经递质，本研究将 More条件下突触后GABA-A受体功能的研究 生理上相关的条件。膜片钳记录与超快 配基应用技术将被用于：1)表征 GABA-A受体抑制突触后电流的单通道基础 (IPSC)答复，以及2)确定单一渠道机制 GABA介导的ipscs的潜在药理调节。 GABA-A受体由一种蛋白质复合体组成，它形成一种 氯离子选择性离子通道，并含有调节结合部位 通过GABA和临床上重要的苯二氮卓类，巴比妥酸盐， 类固醇和惊厥药。γ-氨基丁酸与大脑皮层受体的结合 突触后膜诱发宏观的IPSC，由随机的 由于许多单独的离子通道打开而引起的响应。 IPSC幅度和/或时间进程可以通过药物改变来 调节神经元抑制。GABA介导的单通道基础 IPSC及其药物对IPSC的调节作用尚未得到广泛研究。 先前的研究表明，GABA和变构调节剂 GABA受体改变单通道门控(开启和关闭) 动力学属性，而不是通道电导。然而，由于 受体脱敏可能改变离子的门控特性 渠道，这些研究一直受到相对缓慢的速度和 长时间接触受体的配体。在突触处， 释放和摄取机制导致快速且可能是瞬时的GABA 接触突触后受体。因此，其作用机制是 神经递质和药物作用将在以下条件下得到更好的研究 更类似于在突触上发现的，如本研究中提出的。 这项研究的目的是：1)表征动力学性质和 建立GABA介导的IPSCs的单通道关联模型， 2)确定药理学基础上的单通道机制 对GABA介导的IPSCs的调节，以及3)表征单个 GABA受体激动剂依赖性脱敏AS的通道特性 它与IPSC反应的调制有关。将进行研究 利用最新开发的超快配基应用技术对已切除的 从培养的大鼠皮质神经元获得由外向外的贴片。 以前开发的单通道分析技术将得到扩展 并在调查中得到应用。这项研究将建立一个模型来 了解并预测，在各种条件下，GABA介导的和 基于随机性的药物调制的IPSC反应 单个GABA受体通道的活性。这样做的长期目标是 该项目旨在提供对突触GABA受体的更好理解 临床相关的GABA受体药理学的功能。

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.