The long-term goal of this competing continuation proposal is to understand the mechanisms of action of general anesthetics on synaptic transmission. Understanding the mechanisms of both the therapeutic and undesired effects of existing general anesthetics will facilitate their safe clinical use while enabling the rational development of more specific agents with reduced side-effects. Our central hypothesis is that general anesthetics affect neurotransmitter release by agent- and transmitter-specific presynaptic mechanisms involving effects on presynaptic ion channels. The project will be accomplished through a combination of neurochemical, electrophysiological and biochemical techniques via the following proposed Specific Aims: 1) Determine the mechanisms by which volatile anesthetics affect glutamate and GABA release from isolated nerve terminals to test the hypothesis that the effects of volatile anesthetics on glutamate and GABA release result from actions on presynaptic ion channels. 2) Characterize the electrophysiological effects of volatile anesthetics on voltage-gated Na+ channels to test the hypothesis that volatile anesthetics have state-dependent effects on voltage-gated Na+ channels at clinical concentrations. 3) Elucidate brain region-, transmitter- and age-dependent effects of volatile anesthetics on transmitter release in the CNS to test the hypothesis that heterogeneity in transmitter release mechanisms between various nerve terminal types results in differential presynaptic sensitivities to general anesthetics. Experiments will employ rodent nerve terminals isolated from various CNS regions to study presynaptic anesthetic effects in a subcellular fraction free of intercellular interactions and amenable to pharmacological, electrophysiological and biochemical analysis. Methods will include analysis of volatile anesthetic effects on basal and evoked release of radiolabeled glutamate, GABA, norepinephrine, and dopamine;comparison of the effects of isoflurane and other Na+ channel blockers on native, recombinant and bacterial Na+ channel biophysical properties;and immunochemical analysis of ion channels expressed in isolated nerve terminal preparations. Despite widespread clinical use, our understanding of how general anesthetics work is incomplete. Better understanding of their mechanisms will allow safer use of current anesthetics and facilitate development of anesthetics with fewer dangerous cardiovascular and respiratory side-effects.
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