Synaptic Mechanisms of General Anesthetic Action

描述(申请人提供)：这项竞争性延续方案的长期目标是了解全身麻醉药对突触传递的作用机制。了解现有全身麻醉药的治疗作用和不良反应的机制将有助于其安全的临床应用，同时能够合理开发更具体的药物，减少副作用。我们的中心假设是，全身麻醉药通过影响突触前离子通道的药物和递质特定的突触前机制来影响神经递质的释放。该项目将通过结合神经化学、电生理和生化技术来完成，具体目标如下：1)确定挥发性麻醉剂影响分离神经末梢谷氨酸和GABA释放的机制，以验证挥发性麻醉剂对谷氨酸和GABA释放的影响是通过作用于突触前离子通道的假说。2)研究挥发性麻醉药对电压门控性钠通道的电生理效应，验证临床浓度下挥发性麻醉药对电压门控性钠通道具有状态依赖性效应的假说。3)阐明挥发性麻醉药对中枢神经系统递质释放的脑区、递质和年龄依赖性的影响，以验证不同神经末梢之间递质释放机制的异质性导致对全身麻醉药的突触前敏感性不同的假设。实验将使用从中枢神经系统不同区域分离的啮齿动物神经末梢来研究突触前麻醉在亚细胞部分的作用，不受细胞间相互作用的影响，并服从药理学、电生理和生化分析。方法包括分析挥发性麻醉剂对放射性标记谷氨酸、GABA、去甲肾上腺素和多巴胺基础和诱发释放的影响；比较异氟烷和其他钠通道阻滞剂对天然、重组和细菌钠通道生物物理特性的影响；以及对分离的神经末梢标本中表达的离子通道进行免疫化学分析。尽管临床应用广泛，但我们对全麻药如何起作用的理解并不完整。更好地了解它们的机制将允许更安全地使用当前的麻醉药，并促进麻醉药的开发，减少危险的心血管和呼吸系统副作用。

DESCRIPTION (provided by applicant): The long-term goal of this competing continuation proposal is to understand the mechanisms of action of general anesthetics on synaptic transmission. Understanding the mechanisms of both the therapeutic and undesired effects of existing general anesthetics will facilitate their safe clinical use while enabling the rational development of more specific agents with reduced side-effects. Our central hypothesis is that general anesthetics affect neurotransmitter release by agent- and transmitter-specific presynaptic mechanisms involving effects on presynaptic ion channels. The project will be accomplished through a combination of neurochemical, electrophysiological and biochemical techniques via the following proposed Specific Aims: 1) Determine the mechanisms by which volatile anesthetics affect glutamate and GABA release from isolated nerve terminals to test the hypothesis that the effects of volatile anesthetics on glutamate and GABA release result from actions on presynaptic ion channels. 2) Characterize the electrophysiological effects of volatile anesthetics on voltage-gated Na+ channels to test the hypothesis that volatile anesthetics have state-dependent effects on voltage-gated Na+ channels at clinical concentrations. 3) Elucidate brain region-, transmitter- and age-dependent effects of volatile anesthetics on transmitter release in the CNS to test the hypothesis that heterogeneity in transmitter release mechanisms between various nerve terminal types results in differential presynaptic sensitivities to general anesthetics. Experiments will employ rodent nerve terminals isolated from various CNS regions to study presynaptic anesthetic effects in a subcellular fraction free of intercellular interactions and amenable to pharmacological, electrophysiological and biochemical analysis. Methods will include analysis of volatile anesthetic effects on basal and evoked release of radiolabeled glutamate, GABA, norepinephrine, and dopamine; comparison of the effects of isoflurane and other Na+ channel blockers on native, recombinant and bacterial Na+ channel biophysical properties; and immunochemical analysis of ion channels expressed in isolated nerve terminal preparations. Despite widespread clinical use, our understanding of how general anesthetics work is incomplete. Better understanding of their mechanisms will allow safer use of current anesthetics and facilitate development of anesthetics with fewer dangerous cardiovascular and respiratory side-effects.