Anesthetic Action: Channel Substrates & Molecular Mechanisms

描述（由申请人提供）：大约150年前，在外科手术中发现了对病人进行麻醉的方法，这是临床医学的一个重大进步。从那时起，尽管麻醉药典有了许多重要的发展，但大多数全身麻醉药物介导临床重要作用的机制仍然是谜。在这方面，细胞和分子研究已经导致了一种流行的观点，即膜离子通道代表了最相关的麻醉靶点，最近来自遗传小鼠模型的行为分析表明，GABAA受体通道在一些（尽管不是全部）静脉麻醉剂的作用中是卓越的。GABAA受体可能对吸入麻醉剂的作用不那么关键，因为其他离子通道靶点正在寻找中。在这项应用中，我们提出用小鼠敲除模型进行分子、细胞和行为实验，以检验两种可选的麻醉敏感离子通道——TASK背景钾通道和HCN起搏器阳离子通道——在固定和催眠麻醉作用中的作用。我们的工作模型是，麻醉调节这两个通道中的一个或两个有助于：减少运动神经元的兴奋性，这与固定有关；通过丘脑皮层回路的活动诱导类似睡眠的催眠状态；脑干胺能神经元的抑制与张力和睡眠有关。在Specific Aim 1中，我们使用了我们实验室开发的TASK-1和TASK-3的常规和条件小鼠敲除；在Specific Aim 2中，我们使用了先前描述的常规HCN1和HCN2敲除小鼠。为了这两个目的，敲除小鼠模型首先通过分子、免疫化学和行为方法进行验证。我们使用膜片钳记录大脑切片，以确定通道敲除对我们工作模型中确定的关键神经元件（即运动神经元、丘脑皮质中继神经元、皮质锥体神经元和脑干胺能神经元）的电生理特性的影响。最后，我们使用固定和催眠的既定行为分析来测试基因敲除动物是否改变了对吸入和静脉麻醉药物的敏感性。提出的研究提供了TASK和HCN通道亚基对麻醉药细胞作用的贡献的关键测试，以及它们在这些临床重要药物的行为相关作用中的作用。鉴定麻醉的分子和神经底物可能导致发现更安全、更有效的麻醉化合物，这是麻醉研究的根本目标。此外，这些研究可能为觉醒的神经机制提供新的见解。

DESCRIPTION (provided by applicant): The discovery of a means to anesthetize patients for surgical procedures, some 150 years ago, represented a major advance in clinical medicine. Since that time, despite numerous important developments in the anesthetic pharmacopoeia, the mechanisms by which most general anesthetic drugs mediate clinically important actions have remained enigmatic. In this regard, cellular and molecular studies have led to a prevailing current view that membrane ion channels represent the most relevant anesthetic targets, and recent behavioral assays from genetic mouse models implicate GABAA receptor channels as preeminent for some, though not all, actions of intravenous anesthetics. The GABAA receptors may not be as critical for actions of inhalational anesthetics, for which other ion channel targets are sought. In this application, we propose molecular, cellular and behavioral experiments with mouse knockout models to examine a role for two types of alternative anesthetic-sensitive ion channels - TASK background potassium channels and HCN pacemaker cation channels - in immobilizing and hypnotic anesthetic actions. Our working model is that anesthetic modulation of either or both these channels contributes: to decreased excitability in motoneurons that is associated with immobilization; to induction of sleep-like hypnotic states through actions in thalamocortical.circuits; and to inhibition of brainstem aminergic neurons that is associated with both atonia and sleep. In Specific Aim 1, we use conventional and conditional mouse knockouts of TASK-1 and TASK-3 that are developed in our laboratory; and in Specific Aim 2, we use previously described conventional HCN1 and HCN2 knockout mice. For both aims, the knockout mouse models are first validated by molecular, immunochemical and behavioral approaches. We use patch clamp recordings from brain slices to determine effects of channel knockout on electrophysiological properties of the key neural elements identified in our working model (i.e., motoneurons, thalamocortical relay neurons, cortical pyramidal neurons, and brainstem aminergic neurons). Finally, we test if knockout animals have altered sensitivity to actions of inhaled and intravenous anesthetic agents using established behavioral assays of immobilization and hypnosis. The proposed studies provide a critical test of TASK and HCN channel subunit contributions to cellular actions of anesthetics, and of their role in behaviorally relevant actions of these clinically important drugs. Identification of molecular and neural substrates for anesthesia may lead to discovery of safer, more effective anesthetic compounds, a fundamental goal of anesthesia research. In addition, the studies may provide new insights into neural mechanisms of arousal.