The goal of the proposed experiments is a detailed characterization of the membrane chemosensitivity of mammalian cerebral cortical neurons to glutamate, a probable cortical excitatory neurotransmitter. Previous studies of glutamate action in intact systems have suggested a variety of effects on membrane potential and conductance, but include unknown contributions from several factors, including local increases in extracellular K+ during glutamate action, electrogenic uptake of glutamate into terminals, and electrotonically distorted effects of glutamate on """"""""remote"""""""" dendritic sites. It is proposed to study glutamate action in a sparse cortical cell culture system where these factors can be reduced or eliminated. In addition, care will be taken to block glutamate-evoked release of other neurotransmitters from presynaptic terminals, another potential complicating factor. Mouse cortical neurons will be plated in sparse cell culture and impaled with an intracellular electrode for recording or current injection. Glutamate will be applied to the soma or proximal dendrites under direct visual guidance, either by iontophoresis, or at known concentration by pressure ejection from blunt micropipeties. Effects of glutamate on membrane potential and conductance will be defined, with attention to underlying membrane I-V non-linearities, and difficulties associated with finite length constants. K+ sensitive electrodes will be used to measure neighborhood extracellular K+ accumulation, which is expected to be small in sparse culture. Subsequent quantitative experiments will define the glutamate dose-response relationship, and the pharmacology of glutamate antagonists. Glutamate chemosensitivity on individual neurons will be mapped out by focal iontophoresis, looking for evidence of either multiple response types, or """"""""hot spots"""""""". Population studies will look for differences in the glutamate chemosensitivity of morphologically defined subclasses of cortical neurons. The proposed experiments will provide information critical to subsequent studies of glutamate neurotransmission, and will also provide a necessary background for more detailed studies of the glutamate channel. The proposed pharmacological characterization of cortical glutamate receptors could have eventual relevance to the therapy of certain human cortical diseases, including degenerative diseases, hepatic encephalopathy, and epilepsy.
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