Synaptic Mechanisms of General Anesthetic Action

描述(申请人提供)：对于如此广泛使用和医学上重要的一类药物来说，全麻药的药理学和毒理学是非常不完整的，这些药物被用于越来越年长和病情越来越重的患者。对麻醉作用机制的了解不足以解释任何麻醉剂如何导致失忆、意识丧失或静止(随着剂量的增加)，这些都是全身麻醉的基本特征。麻醉药对突触传递具有强大而特异的作用，包括突触前对神经递质释放的作用和突触后对受体的作用。这项研究的主要目的是通过实验将麻醉剂对神经递质释放的影响从更好地理解突触后动作中分离出来，以了解这些效应的突触前机制。突触前活动可能与麻醉药的治疗作用(意识丧失、健忘、静止)和/或其毒性作用(神经毒性、呼吸抑制、心血管抑制)有关。了解麻醉药的突触机制对于开发副作用改善的麻醉药和优化高危患者的现有麻醉技术是至关重要的。我们已经证明，全麻药通过突触前机制抑制谷氨酸的释放，这些作用是递质特异性的，涉及对特定Na+通道亚型的区域特异性抑制。为了更全面地了解挥发性麻醉药的突触前作用，我们现在建议将重点放在挥发性麻醉药的区域和递质特异性作用以及Na+通道阻断机制上。我们的中心假设是，全身麻醉药通过影响突触前离子通道，通过突触特异性机制影响神经递质的释放。我们将采用综合和协作的多学科方法来验证这一假说，具体目的如下：目的1-确定挥发性麻醉剂对分离神经末梢神经递质释放的不同影响机制，以检验由于突触前机制的不同，它们对递质释放具有突触特异性影响的假说；目的2-确定挥发性麻醉剂对完整神经元胞吐的神经递质专一性作用和机制，以验证它们通过神经递质专一性和离子通道依赖性机制差异性抑制突触小泡吐出的假说；目的3-确定挥发性麻醉剂对电压门控Na+通道的作用机制和调节，以验证其通过状态依赖机制抑制Na+通道亚型的假说。辅助方法包括分析麻醉剂对完整神经末梢递质释放的影响，单个培养海马神经元的突触小泡吐出，以及特定Na+通道亚型的生物物理性质。这些研究对于从分子水平理解突触前麻醉机制，以及平衡兴奋性和抑制性突触传递中所需的麻醉效果和潜在的毒性作用是至关重要的。 公共卫生相关性：麻醉剂导致健忘、意识丧失和静止的分子和细胞机制是全身麻醉的基本特征，尽管它们在现代药典中起着关键作用，但尚不清楚。我们已经证明，吸入麻醉剂通过抑制Na+通道来抑制神经末梢的神经递质释放，涉及的机制随特定麻醉剂、神经递质和中枢神经系统区域的不同而不同。我们现在建议详细研究吸入麻醉药的特定突触前作用和钠离子通道阻断机制，以更全面地了解全麻药的作用机制，以便开发出更少不良副作用的未来麻醉药，并更安全地使用现有麻醉药。

DESCRIPTION (provided by applicant): The pharmacology and toxicology of general anesthetics are remarkably incomplete for such a widely used and medically important class of drugs that are administered to increasingly older and sicker patients. Knowledge of the mechanisms of anesthetic action is insufficient to explain how any anesthetic produces amnesia, unconsciousness or immobilization (with increasing doses), the cardinal features of general anesthesia. Anesthetics have potent and specific effects on synaptic transmission, including both presynaptic actions on the release of neurotransmitters and postsynaptic actions on receptors. The principal objective of this research proposal is to understand the presynaptic mechanisms of anesthetic effects on neurotransmitter release by experimentally isolating these effects from their better understood postsynaptic actions. Presynaptic actions could be involved in therapeutic effects (unconsciousness, amnesia, immobility) and/or their toxic effects (neurotoxicity, respiratory depression, cardiovascular depression) of anesthetics. Understanding synaptic mechanisms of anesthetics is essential for development of anesthetics with improved side-effect profiles and for optimization of current anesthetic techniques in high-risk patients. We have shown that general anesthetics inhibit glutamate release by presynaptic mechanisms and that these effects are transmitter-specific and involve region-specific inhibition of specific Na+ channel subtypes. We now propose to focus on the region- and transmitter-specific actions and Na+ channel blocking mechanisms of volatile anesthetics in order to more fully understand their presynaptic actions. Our central hypothesis is that general anesthetics affect neurotransmitter release by synapse-specific mechanisms due to effects on presynaptic ion channels. We will test this hypothesis using an integrative and collaborative multidisciplinary approach by the following Specific Aims: Aim 1-Determine the mechanisms by which volatile anesthetics differentially affect neurotransmitter release from isolated nerve terminals to test the hypothesis that they have synapse-specific effects on transmitter release due to differences in presynaptic mechanisms; Aim 2-Determine the neurotransmitter-specific effects and mechanisms of volatile anesthetics on exocytosis in intact neurons to test the hypothesis that they differentially inhibit synaptic vesicle exocytosis by neurotransmitter-specific and ion channel-dependent mechanisms; and Aim 3-Determine the mechanisms and regulation of volatile anesthetic effects on voltage- gated Na+ channels to test the hypothesis that they inhibit Na+ channel subtypes by state-dependent mechanisms. Complementary approaches include analysis of anesthetic effects on transmitter release from intact nerve terminals, synaptic vesicle exocytosis from single cultured hippocampal neurons, and biophysical properties of specific Na+ channel subtypes. Such studies are essential to a molecular understanding of presynaptic anesthetic mechanisms and the balance between desirable and potentially toxic anesthetic effects on excitatory and inhibitory synaptic transmission. PUBLIC HEALTH RELEVANCE: The molecular and cellular mechanisms by which anesthetics produce amnesia, unconsciousness and immobilization, the cardinal features of general anesthesia, are unknown despite their critical role in the modern pharmacopoeia. We have shown that inhaled anesthetics inhibit neurotransmitter release from nerve terminals by inhibition of Na+ channels involving mechanisms that vary with the specific anesthetic, neurotransmitter, and region of the central nervous system. We now propose to investigate the specific presynaptic actions and Na+ channel blocking mechanisms of inhaled anesthetics in detail to more fully understand how general anesthetics act so that future anesthetics can be developed with reduced undesirable side-effects and current anesthetics can be used more safely.