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.
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