Synaptic Mechanisms of General Anesthetic Action

描述（由申请人提供）：该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和不良反应的机制将有助于其安全的临床使用，同时能够合理开发更特异性的药物，减少副作用。我们的中心假设是，全身麻醉剂影响神经递质释放的代理和递质特异性突触前机制，涉及对突触前离子通道的影响。本课题将通过神经化学、电生理和生物化学技术相结合的方法完成，具体目标如下：1）确定挥发性麻醉药影响离体神经末梢谷氨酸和GABA释放的机制，以验证挥发性麻醉药对谷氨酸和GABA释放的影响是通过作用于突触前离子通道而产生的假设。2)表征挥发性麻醉剂对电压门控Na+通道的电生理学效应，以检验挥发性麻醉剂在临床浓度下对电压门控Na+通道具有状态依赖性效应的假设。3)阐明挥发性麻醉剂对CNS中递质释放的脑区域、递质和年龄依赖性影响，以检验不同神经末梢类型之间递质释放机制的异质性导致对全身麻醉剂的突触前敏感性差异的假设。实验将采用从各种CNS区域分离的啮齿动物神经末梢，以研究无细胞间相互作用的亚细胞部分中的突触前麻醉作用，并进行药理学、电生理学和生物化学分析。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的基础和诱发释放的影响;比较异氟烷和其他Na+通道阻滞剂对天然、重组和细菌Na+通道生物物理特性的影响;以及对分离神经末梢制剂中表达的离子通道进行免疫化学分析。尽管广泛的临床应用，我们对全身麻醉剂如何工作的理解是不完整的。更好地了解它们的机制将允许更安全地使用当前的麻醉剂，并促进开发具有更少危险的心血管和呼吸系统副作用的麻醉剂。

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.