Anesthetic Action: Channel Substrates & Molecular Mechanisms

描述（由申请人提供）：大约150年前，麻醉患者进行外科手术的方法的发现代表了临床医学的重大进步。从那时起，尽管麻醉药药典中有许多重要的发展，但大多数全身麻醉药介导临床重要作用的机制仍然是个谜。在这方面，细胞和分子研究已经导致了一个流行的观点，即膜离子通道代表最相关的麻醉剂靶点，最近的遗传小鼠模型的行为测定表明GABAA受体通道对静脉麻醉剂的一些（尽管不是全部）作用是突出的。GABAA受体可能对吸入麻醉剂的作用不那么关键，因此寻找其他离子通道靶点。在这个应用中，我们提出了分子，细胞和行为实验与小鼠基因敲除模型，以检查两种类型的替代麻醉敏感离子通道的作用-任务背景钾通道和HCN起搏器阳离子通道-在固定和催眠麻醉行动。我们的工作模型是，麻醉剂对这两个通道中的一个或两个通道的调节有助于：降低运动神经元的兴奋性，这与固定有关;通过丘脑皮层回路的作用诱导睡眠样催眠状态;抑制脑干胺能神经元，这与张力不足和睡眠有关。在特定目标1中，我们使用我们实验室开发的常规和条件性的Task-I和Task-3敲除小鼠;在特定目标2中，我们使用先前描述的常规HCN 1和HCN 2敲除小鼠。对于这两个目标，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。我们使用来自脑切片的膜片钳记录来确定通道敲除对在我们的工作模型中识别的关键神经元件的电生理特性的影响（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）。最后，我们测试基因敲除动物是否改变了敏感性的行动吸入和静脉麻醉剂使用既定的行为测定固定和催眠。拟议的研究提供了一个关键的测试的任务和HCN通道亚基的贡献麻醉剂的细胞行动，以及他们的作用，这些临床上重要的药物的行为相关的行动。麻醉的分子和神经底物的鉴定可能会导致发现更安全，更有效的麻醉化合物，麻醉研究的基本目标。此外，这些研究可能为唤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.