General anesthesia by inhalation agents has been an indispensable part of patient care for over 150 years, but to an extent unique among modern pharmacologic agents we do not understand how anesthetics work. Two properties common to a state of general anesthesia are immobility in response to a noxious stimulus and amnesia. Immobility is a function of anesthetic actions in the spinal cord. This project will test two hypotheses: 1. Volatile anesthetics produce immobility by acting directly on motor neurons in spinal cord. 2. Volatile anesthetics produce immobility by combinations of actions on multiple ligand-gated ion channels including the major subtypes of glutamate receptors (AMPA, kainate, NMDA) as well as inhibitory GABAA and glycine receptors. These are two complementary specific aims. 1. We will identify postsynaptic actions on motor neurons by comparing responses evoked by dorsal root stimulation to those evoked by direct application of glutamate, using patch clamp recording in visually identified motor neurons in mouse spinal cord slices. These studies will use receptor-specific antagonists to isolate anesthetic actions on glutamate AMPA, kainate, NMDA, GABAA and glycine receptors. 2. We will estimate the importance of specific receptors by studying genetically engineered mice in which glutamate, GABAA or glycine receptors have been altered either by deletion of a subunit or by over-expression of a subunit mutated to be resistant to anesthetic agents. These studies will measure evoked potentials in isolated intact spinal cords and motor neuron responses in spinal cord slices to determine which pathways are altered by the mutations and how anesthetic sensitivity (Eger/Sonner) and define the receptor properties at the molecular level (Harris). This project will provide information to guide the studies of the Eger/Sonner and Harris projects. This project focuses on anesthetic actions in an integrated part of the nervous system that contains all the important molecular targets of anesthetic action actin in concerting in their native milieu. The project thus targets the level between behavioral studies in intact animals and molecular studies on receptors expressed in non-neural cells. The results of this study will identify for the first time a cellular locus for the anesthetic endpoint of immobility in response to a noxious stimulus, and will define the contributions of the important subtypes of amino acid- gated channels to this endpoint.
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