Anesthetic Action: Molecular Substrates & Neural Mechanisms

描述(由申请人提供)：麻醉师通常从一系列化学成分不同的化合物中进行选择，使患者失去知觉和知觉，从而实现无痛手术。然而，值得注意的是，大多数麻醉药调节其临床重要作用的分子和神经机制仍然不确定。在新的小鼠遗传模型中，候选麻醉靶点可以全局或在特定细胞类型中被禁用，这一发展开始揭示不同分子靶点和细胞组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值下麻醉敏感离子通道-HCN超极化激活的阳离子通道和任务背景钾通道-在介导临床上重要的麻醉作用中的作用。指导特定目标1的假设建立在我们实验室最近发表的结果基础上，即选择性抑制皮质锥体神经元中的树突状HCN1通道有助于麻醉诱导的催眠。拟议的实验主要集中在HCN1介导的氯胺酮的作用，氯胺酮是一种解离麻醉剂，在传统的HCN1基因敲除小鼠中，催眠敏感度显著降低。我们的目标是：利用前脑选择性的HCN1基因敲除模型，研究锥体神经元在催眠麻醉作用中的作用；表征其他解离麻醉剂对HCN通道和麻醉诱导的催眠的影响；识别氯胺酮对HCN1通道亚单位选择性影响的分子决定因素；以及建立/测试敲入的小鼠模型，在该模型中，HCN1通道是完整的，但对氯胺酮不敏感。根据新的初步数据得出的特定目标2背后的工作模式是，局部麻醉剂对任务通道的抑制导致了它们的有害影响。我们的目标是：在常规TASK-1-/-：TASK-3-/-基因敲除小鼠中评估全身局部麻醉药的促惊厥中枢神经系统效应；表征任务通道对不同全身毒性的局部麻醉剂的相对敏感性；确定任务通道对兴奋性丘脑皮质环路神经元局部麻醉作用的贡献；并利用细胞特异性条件任务基因敲除小鼠来具体检测这些神经元在任务通道介导的局部麻醉剂中枢神经系统效应中的作用。为了这两个目标，我们使用了各种方法，包括从转基因细胞进行通道突变和膜片钳记录；对新的条件小鼠模型进行分子和免疫化学验证；从野生型和突变小鼠的关键神经元进行体细胞和树突记录；以及对这些小鼠的麻醉敏感性进行行为评估。所提出的研究提供了一种测试HCN和任务通道亚单位对全麻药和局麻药的特定神经和行为行为的贡献。识别这些分子和神经机制可能导致发现更安全、更有效的麻醉剂化合物，这是麻醉研究的重要目标。 公共卫生相关性：全身麻醉药和局部麻醉药仍然是最广泛使用和临床有用的药物之一。尽管这些化合物的广泛应用和丰富的临床经验，它们调节其可取和不愉快行为的分子和神经机制仍然不确定。本提案中进行的研究旨在阐明这些机制，从而提供可能导致开发更有效和更安全的麻醉剂的见解。

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.