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选择，使患者失去知觉和无知觉，从而实现无痛手术。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机制仍然不确定。新的小鼠遗传模型的发展，其中候选麻醉靶点可以被禁用，无论是全球或特定的细胞类型，开始揭示不同的分子靶点和细胞群的相对贡献特定的麻醉行动。在这个应用中，我们采用这样的模型来研究两个亚阈值麻醉敏感离子通道- HCN超极化激活的阳离子通道和ASK背景钾通道-在介导临床上重要的麻醉作用的贡献。基于我们实验室最近发表的结果，指导特异性目标1的假设是选择性抑制皮质锥体神经元中的树突状HCN 1通道有助于麻醉诱导的催眠。拟议的实验主要集中在HCN 1介导的氯胺酮，一种解离性麻醉剂，其中一个强大的催眠敏感性下降，在传统的HCN 1基因敲除小鼠获得的行动。我们的目标是：通过使用前脑选择性HCN 1敲除模型检查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;确定氯胺酮对HCN 1通道的亚单位选择性影响的分子决定因素;并开发/测试敲入小鼠模型，其中HCN 1通道是完整的，但对氯胺酮不敏感。从新的初步数据中推导出的特定目标2的工作模型是，局部麻醉剂对ASK通道的抑制有助于其有害作用。我们的目标是：在常规的TASK-1-/-：TASK-3-/-敲除小鼠中评价全身施用的局部麻醉剂的促惊厥CNS作用;表征TASK通道对不同的局部麻醉剂的相对敏感性，所述不同的局部麻醉剂在全身毒性方面不同;确定TASK通道对局部麻醉剂对兴奋性丘脑皮质回路神经元的作用的贡献;并通过使用细胞特异性条件性ASK基因敲除小鼠来具体检查这些神经元在局部麻醉剂的ASK通道介导的CNS效应中的作用。对于这两个目标，我们使用了各种方法，包括从转染细胞的通道诱变和膜片钳记录;新的条件小鼠模型的分子和免疫化学验证;从野生型和突变小鼠的关键神经元的体细胞和树突状记录;和这些小鼠的麻醉敏感性的行为评估。 拟议的研究提供了一个测试的HCN和任务通道亚基的贡献特定的神经元和行为的行动，全身和局部麻醉药。这些分子和神经机制的鉴定可能会导致发现更安全，更有效的麻醉化合物，麻醉研究的一个重要目标。 公共卫生相关性：全身和局部麻醉药仍然是最广泛使用和临床有用的药物。尽管这些化合物的普遍使用和广泛的临床经验，但它们介导其理想和不良作用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.