BARBITURATE/BENZODIAZEPINE ACTIONS ON GABA C1 CHANNELS

原代分离培养的小鼠脊髓和皮层神经元 将用于研究苯二氮卓类（BDZ）受体的作用 配体和巴比妥类药物对GABA激活的全细胞电流和单个 通道电流 这些研究的一个主要目标是了解 GABA受体及其相关离子通道的组织和 临床使用的抗惊厥药物改变GABA能的机制 抑制作用 为了进行这些研究，细胞内，电压钳和 将膜片钳技术应用于细胞培养中的小鼠神经元。 BDZ受体配体和巴比妥类药物的药理学研究将在 使用细胞内记录和电压钳技术进行。 脊髓和皮层神经元将被刺穿， 将记录对应用GABA的反应。 BDZ受体 配体和巴比妥酸盐将应用于神经元使用局部灌注 技术. 将在给药前获得对照GABA反应 应用程序. BDZ受体配体或巴比妥酸盐将使用 独立控制的微量移液管。 一个主要目标将是 表征巴比妥类药物和BDZ受体配体对 GABA激活的氯电流和电压到激动剂的全范围， 拮抗剂和反向激动剂BDZ受体配体以及全系列 抗惊厥药和麻醉巴比妥类药物。 这些调查的第二阶段将涉及适用 脊髓和皮层神经元的膜片钳技术。 外切 将使用对称氯化物培养基获得外斑， 膜片钳移液管中的非渗透性阳离子。 在这种情况下， GABA激活+细胞中2-4皮安的单通道氯电流 100 mV范围。 这些通道具有10-50皮西门子的电导。 在第一部分中表征的巴比妥酸盐和BDZ受体配体 这些研究将应用于切除的外侧斑块及其 将确定对GABA激活的单氯电流的影响。 主要重点将放在获得动力学方案， 描述了巴比妥酸盐和BDZ受体配体修饰 GABA激活的氯离子通道的门控特性 水流 例如，有人提出，BDZ增强了 单通道氯电流激活的概率，但不 修改通道开放的平均持续时间。 然而，巴比妥类药物具有 被提议延长信道持续时间。 我们将检验这些假设 直接.

Mouse spinal cord and cortical neurons in primary dissociated cell culture will be used to investigate the actions of benzodiazepine (BDZ) receptor ligands and barbiturates on GABA-activated whole cell currents and single channel currents. A major goal of these studies is to understand the organization of the GABA receptor and its associated ion channel and the mechanisms whereby clinically used anticonvulsant drugs modify GABAergic inhibition. To perform these studies, intracellular, voltage clamp and patch clamp techniques will be applied to the mouse neurons in cell culture. Pharmacological studies of BDZ receptor ligands and barbiturates will be performed using intracellular recording and voltage clamp techniques. Spinal cord and cortical neurons will be impaled and the voltage or current responses to the application of GABA will be recorded. BDZ receptor ligands and barbiturates will be applied to neurons using local superfusion techniques. Control GABA responses will be obtained prior to drug application. BDZ receptor ligands or barbiturates will be applied using independently controlled micropipettes. A major goal will be to characterize the actions of barbiturates and BDZ receptor ligands on GABA-activated chloride currents and voltages to the full range of agonist, antagonist and inverse agonist BDZ receptor ligands and the full range of anticonvulsant and anesthetic barbiturates. A second phase of these investigations will involve application of the patch clamp technique to spinal cord and cortical neurons. Excised outside out patches will be obtained using symmetrical chloride medium and nonpermeant cations in the patch clamp pipette. Under these circumstances, GABA activates single channel chloride currents of 2-4 picoamps in the + 100 mV range. These channels have conductances of 10-50 picosiemens. Barbiturates and BDZ receptor ligands characterized in the first part of the studies will be applied to the excised outside out patches and their effect on the GABA-activated single chloride currents will be determined. Major emphasis will be placed upon obtaining a kinetic scheme which will describe the mechanism whereby barbiturates and BDZ receptor ligands modify the gating characteristics of the GABA-activated single channel chloride currents. For example, it has been proposed that BDZs enhance the probability for activation of single channel chloride currents but do not modify the mean duration of channel opening. Barbiturates, however, have been proposed to prolong channel duration. We will test these hypotheses directly.