The excitatory amino acids are probably the most important class of excitatory transmitters in the brain, but in excess they cause the death of neurons. There are at least three types of excitatory amino acid receptors, defined by the """"""""specific"""""""" agonists N-methyl-Daspartate (NMDA), quisqualate and kainate, and each can cause excitotoxicity, although kainate is the most toxic. In spite of recent advance the mechanisms of excitotoxicity are unclear and may not be the same at the three types of receptors. The present proposal is for continued support for studies on excitotoxicity. We will test four possible mechanisms of toxicity, including a) accumulation of intracellular calcium beyond the ability of the cell to buffer it, with the calcium entering through either agonist-activated channels or voltage-dependent calcium channels; b) osmotic and concentration gradient disruption secondary to excessive entry of sodium and chloride; c) free radical formation, possibly secondary to calcium accumulation leading to activation of proteases; and d) lack of receptor desensitization, especially of the kainate receptor, leads to damage secondary to one or more of the above factors. We propose to perform whole cell patch recordings from acute dissociated piriform and hippocampal neurons, and study electrophysiologic indicators of toxicity in piriform cortex slices, using intracellular and population response recordings with bath application of agonists. We will study the three specific agonists and BMAA and BOAA, two unique amino acids associated with human disease. In the patch studies we will compare trypsin and mechanically dissociated neurons for differences in desensitization and current-voltage relations, then use mechanically dissociated neurons to study the role of divalent cations in carrying or blocking agonist-activated currents, analyze the properties of desensitization if it occurs and characterize receptors for the two unique amino acids. In the slice studies we will evaluate the effect of calcium, sodium and chloride concentrations and inhibitors of free radical production or scavengers on loss of excitability for each of the give agonists. The proposed studies have the potential to both contribute to the fundamental knowledge of the actions of the excitatory amino acids, and to lead to increased understanding of the variety of mechanisms responsible for excitotoxicity.
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