MECHANISMS OF EXCITOTOXIC DAMAGE IN CENTRAL NEURONS

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In recent years it has become clear that elevations of intracellular Ca2+ are involved both in synaptic plasticity in neurons and, when excessive, in neuronal cell death. Glutamate-induced "excitotoxic" neuronal damage is likely to be involved in stroke, head trauma, and status epilepticus. This delayed damage seems to offer an opportunity for intervention, and indeed a number of laboratories have offered evidence that blockade of Ca2+ entry can limit neuronal cell death. Much has been learned of the important routes of Ca2+ entry. Attention has focused on the N-methyl-D-aspartate (NMDA) class of glutamate receptors because of their high Ca2+ permeability. Recently it has been discovered that some non-NMDDA glutamate receptors are also permeable to Ca2+, and combinations of the cloned non-NMDA glutamate receptor subunits which are Ca2+-permeable as well as Ca2+-impermeable have been found. However, the relationships between the functional physiology of non--NMDA receptors in neurons and the underlying patterns of subunit expression have not been established with certainty. Little is known of the physiology of the secondary mediators of Ca2+-induced structural changes, such as Ca2+-activated proteases, lipases, and kinases. This project is intended to define in molecular terms the role of non- NMDDA glutamate receptor subunits in Ca2+ entry in central neurons, and further, to begin to explore the mediators of Ca2+-induced injury such as the Ca2+-activated protease calpain I. For the first of these tasks, physiological measurements of a neuron's activity, such as whole cell voltage clamp or [Ca2+] microfluorimetric measurements, must be employed in conjunction with assays of the subunit expression pattern in the same cell, such as subunit specific immunocytochemistry, in situ hybridization, or single cell polymerase chain reaction. Such methods can relate the functional receptors in a given cell to the set of receptor subunits expressed, and illuminate the appropriate molecular targets for blockade of toxic Ca2+ influx. To undertake the second task, the study of calpain I activation in normal as well as excitotoxic cellular processes, requires a means to monitor calpain activity in single cells; this can be accomplished through the use of intracellular fluorogenic substrates in fluorimetric and imaging experiments. By the study of the mechanisms of excitotoxicity, it will be possible to more effectively direct therapeutic interventions to the processes which cause irreversible neuronal injury.