Anesthetic Action: Molecular Substrates & Neural Mechanisms

描述（由申请人提供）：麻醉师通常从多种化学成分中选择，使患者失去知觉和知觉，使手术过程无痛。然而，值得注意的是，大多数麻醉药物介导其临床重要作用的分子和神经元机制仍然不确定。在新的小鼠遗传模型中，候选麻醉靶点可以被禁用，无论是在整体上还是在特定的细胞类型中，都开始揭示不同分子靶点和细胞群对特定麻醉作用的相对贡献。在这个应用中，我们使用这样的模型来检查两个阈下麻醉敏感离子通道——HCN超极化激活的阳离子通道和TASK背景钾通道——在介导临床重要的麻醉作用中的贡献。基于我们实验室最近发表的结果，指导Specific Aim 1的假设是，皮质锥体神经元中树突状HCN1通道的选择性抑制有助于麻醉诱导的催眠。拟议的实验主要关注氯胺酮介导的HCN1作用，氯胺酮是一种解离麻醉剂，在常规HCN1敲除小鼠中获得了催眠敏感性的显著降低。我们的目的是：通过使用前脑选择性HCN1敲除模型来检查锥体神经元在催眠麻醉作用中的作用；表征其他解离性麻醉剂对HCN通道和麻醉诱导催眠的影响；确定氯胺酮对HCN1通道亚单位选择性作用的分子决定因素；并开发/测试敲入小鼠模型，其中HCN1通道完好无损，但对氯胺酮不敏感。基于新的初步数据的Specific Aim 2的工作模型是局部麻醉剂对TASK通道的抑制有助于其有害作用。我们的目的是：评估常规TASK-1-/-:TASK-3-/-敲除小鼠全身给药的局麻药对惊厥前中枢神经系统的影响；表征TASK通道对不同局部麻醉剂的相对敏感性；确定TASK通道对兴奋性丘脑皮质回路神经元局部麻醉作用的贡献；并通过细胞特异性条件TASK敲除小鼠，专门研究这些神经元在局部麻醉剂TASK通道介导的中枢神经系统效应中的作用。为了实现这两个目标，我们使用了多种方法，包括通道诱变和来自转染细胞的膜片钳记录；新型条件小鼠模型的分子和免疫化学验证野生型和突变型小鼠关键神经元的体细胞和树突记录；以及对这些小鼠麻醉敏感性的行为评估。拟议的研究提供了HCN和TASK通道亚基对全身和局部麻醉剂的特定神经元和行为行为的贡献的测试。对这些分子和神经机制的识别可能会导致发现更安全、更有效的麻醉化合物，这是麻醉研究的一个重要目标。

DESCRIPTION (provided by applicant): Anesthesiologists routinely select from a host of chemically diverse compounds to render patients unconscious and insentient, allowing painless performance of surgical procedures. Remarkably, however, the molecular and neuronal mechanisms by which most anesthetic drugs mediate their clinically important actions still remain uncertain. The development of new mouse genetic models in which candidate anesthetic targets can be disabled, either globally or in specific cell types, is beginning to reveal relative contributions of different molecular targets and cell groups to particular anesthetic actions. In this application, we employ such models to examine contributions of two subthreshold anesthetic-sensitive ion channels - HCN hyperpolarization- activated cation channels and TASK background potassium channels - in mediating clinically important anesthetic actions. The hypothesis guiding Specific Aim 1, which builds on recently published results from our laboratory, is that selective inhibition of dendritic HCN1 channels in cortical pyramidal neurons contributes to anesthetic-induced hypnosis. Proposed experiments focus primarily on HCN1-mediated actions of ketamine, a dissociative anesthetic for which a robust decrease in hypnotic sensitivity was obtained in conventional HCN1 knockout mice. Our aims are to: examine the role of pyramidal neurons in hypnotic anesthetic actions by using a forebrain-selective HCN1 knockout model; characterize effects of other dissociative anesthetics on HCN channels and anesthetic-induced hypnosis; identify molecular determinants for subunit-selective effects of ketamine on HCN1 channels; and develop/test a knock-in mouse model in which HCN1 channels are intact, but rendered insensitive to ketamine. The working model underlying Specific Aim 2, which derives from new preliminary data, is that inhibition of TASK channels by local anesthetics contributes to their deleterious effects. Our aims are to: evaluate pro-convulsive CNS effects of systemically administered local anesthetics in conventional TASK-1-/-:TASK-3-/- knockout mice; characterize relative sensitivity of TASK channels to different local anesthetics that vary in systemic toxicity; determine TASK channel contributions to local anesthetic action on excitatory thalamocortical circuit neurons; and examine specifically the role of those neurons in TASK channel-mediated CNS effects of local anesthetics by using cell- specific conditional TASK knockout mice. For both aims, we use a variety of approaches, including channel mutagenesis and patch clamp recordings from transfected cells; molecular and immunochemical validation of novel conditional mouse models; somatic and dendritic recordings from key neurons in wild type and mutant mice; and behavioral assessments of anesthetic sensitivity in those mice. The proposed studies provide a test of HCN and TASK channel subunit contributions to specific neuronal and behavioral actions of general and local anesthetics. Identification of these molecular and neural mechanisms may lead to discovery of safer, more effective anesthetic compounds, an important goal of anesthesia research. PUBLIC HEALTH RELEVANCE: General and local anesthetics remain among the most widely used and clinically useful drugs. Despite prevalent use and extensive clinical experience with these compounds, the molecular and neural mechanisms by which they mediate their desirable and untoward actions remain uncertain. The research undertaken in this proposal seeks to clarify those mechanisms, and thus to provide insights that may lead to development of more effective and safer anesthetic agents.