Nerve cell death resulting from stroke, head injury and a range of degenerative disorders is a major cause of mortality and also has devastating effects on the lives of survivors. Understanding the mechanisms of this cell death is an important goal, since development of successful treatments could greatly minimize the subsequent social cost of these conditions. A common triggering event in neurodegeneration is widely held to be the excessive release of the excitatory transmitter glutamate, and subsequent massive Ca2+ influx. This process has been termed """"""""excitotoxicity."""""""" We have investigated excitotoxic mechanisms in restricted regions of nerve dendrites, and shown that glutamate receptor agonists initiate sustained Ca2+ elevations at these sites, and that these """"""""secondary Ca2+ responses"""""""" then spread very slowly throughout neurons and lead to cell death. Our hypothesis is that fine dendrites, being richly endowed with Ca2+ -permeable glutamate receptors and channels, act as initiation sites of excitotoxic Ca2+ signals, and that procedures to prevent the initiation and/or spread of secondary Ca2+ responses will provide important new approaches to limiting cell death. We have exploited differences in excitotoxic vulnerability between inbred murine strains (C57B1/6 and C57B1/10) to clearly demonstrate the involvement of these responses in excitotoxic cell death in intact slice preparations. As well as implicating secondary responses as key determinants of excitotoxic vulnerability, this work has resulted in novel preparations that permit the first rigorous investigations of 1) mechanisms underlying secondary response initiation, 2) mechanisms by which secondary responses spread through dendritic processes and 3) how secondary responses trigger cellular damage. Acute murine hippocampal slices will be studied using combined electrophysiological and single-cell Ca2+ imaging approaches. Secondary responses will be initiated using glutamate receptor agonists, and pharmacological approaches used to identify the routes of Ca2+ entry involved in triggering, and progression of these events. Studies to determine the basis for murine strain differences in secondary response generation will be complemented by immunohistochemical approaches. A further set of mechanistic studies will test the hypothesis that mitochondrial Ca2+ loading is a key event linking secondary responses to cellular damage, using imaging methods to assess mitochondrial function, Ca2+ loading and production of reactive oxygen species. In the long term, results from these studies should provide a basis for novel interventions designed at preventing cell death following excitotoxic stimulation at sites remote from neuronal somata. such as might occur in a very wide range of neurological disorders.
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