The broad, long-term objective of this proposal is to understand at a molecular level the mechanisms of action of general anesthetics on synaptic transmission in the central nervous system. Identification of the mechanisms involved in the therapeutic and toxic effects of existing anesthetic agents will facilitate the development of more specific agents with fewer adverse effects. The hypothesis to be evaluated is that general anesthetics affect transmitter release by agent- and transmitter-specific presynaptic mechanisms.
The Specific Aims are to 1) Determine the effects of general anesthetics on synaptosomal neurotransmitter release; 2) Determine the mechanisms of general anesthetic effects on neurotransmitter release; and 3) Investigate the effects of general anesthetics on neuronal Na+ channels (which mediate anesthetic inhibition of glutamate release). The experimental design is to identify the effects of general anesthetics on neurotransmitter release in a subcellular preparation (synaptosomes) that is free of cellular interactions and amenable to pharmacological analysis, and then to characterize the mechanism(s) of the anesthetic effects by analyzing associated changes in presynaptic ion channel function, intracellular ion concentrations, membrane potential, presynaptic receptor function and second messenger systems. The methods to be used include neurochemical analysis of anesthetic effects on spontaneous and evoked glutamate, gamma-aminobutyric acid, norepinephrine, dopamine and cholecystokinin-8 release from rat brain synaptosomes; spectrofluorimetric assays of synaptosomal Na+, Ca2+, and C1-concentrations, membrane potential and pH; pharmacologic analysis of the roles of specific ion channels and protein kinases in the effects of anesthetics on transmitter release; and patch-clamp recording of presynaptic Na+ currents in vesicles made by fusing synaptosomes. Determination of the presynaptic effects of general anesthetics on transmitter release, and the mechanisms of these effects, is essential in linking the molecular and cellular actions of anesthetics on neuronal function and thus in understanding the mechanisms of action of this clinically important, but poorly understood, class of drugs.
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