Anesthetic Action: Channel Substrates & Molecular Mechanisms

描述(申请人提供)：大约150年前，发现了一种麻醉病人进行外科手术的方法，代表了临床医学的重大进步。从那时起，尽管麻醉药典中有许多重要的进展，但大多数全身麻醉药调节临床重要作用的机制仍然是个谜。在这方面，细胞和分子研究导致了目前流行的观点，即膜离子通道是最相关的麻醉靶点，最近来自遗传小鼠模型的行为分析表明，GABAA受体通道对静脉麻醉剂的某些(尽管不是全部)作用具有优势。GABAA受体对吸入麻醉剂的作用可能不那么关键，人们正在寻找其他离子通道靶点。在这一应用中，我们提出了用小鼠基因敲除模型进行分子、细胞和行为实验，以考察两种可选的麻醉敏感离子通道-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：与制动相关的运动神经元兴奋性降低；通过丘脑皮质回路的作用诱导类似睡眠的催眠状态；以及与紧张症和睡眠相关的脑干胺能神经元的抑制。在特定目标1中，我们使用我们实验室开发的TASK-1和TASK-3的常规和条件敲除小鼠；在特定目标2中，我们使用先前描述的常规HCN1和HCN2基因敲除小鼠。出于这两个目的，基因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。我们使用脑片的膜片钳记录来确定通道敲除对我们工作模型中识别的关键神经元件(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，我们使用已建立的固定和催眠行为分析来测试基因敲除动物是否改变了对吸入麻醉剂和静脉麻醉剂的反应的敏感性。建议的研究提供了一个关键的测试任务和HCN通道亚单位对麻醉药的细胞活动的贡献，以及它们在这些临床重要药物的行为相关作用中的作用。识别麻醉的分子和神经底物可能会导致发现更安全、更有效的麻醉剂化合物，这是麻醉研究的基本目标。此外，这些研究可能会对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.