Depolarization is a trophic stimulus for neurons, i.e., prevents neuronal death. Spiral ganglion neurons (SGNs) die following the loss of hair cells. Electrical stimulation promotes survival of deafferented SGNs in vivo, raising the possibility of using electrical stimulation to maintain survival of SGNs in humans deaf as a result of loss of hair cell function. In particular, the efficacy of electronic cochlear implants would be greatly augmented if SGN death was prevented. To this end, we propose studies directed towards determining the mechanism by which depolarization promotes survival of SGNs. These studies take advantage of an in vitro model of the regulation of SGN survival by neurotrophic stimuli. Using this model, we find that depolarization, functioning by increasing cytosolic Ca2+, is a stronger trophic stimulus than neurotrophic factors. There are three general objectives: The first is to further characterize the in vitro model of the maintenance of SGN survival by depolarization and neurotrophic factors: (1) Molecular criteria derived from studies of neuronal apoptosis in other systems will be applied to the death of SGNs to determine if it is apoptotic. (2) Are there different subpopulations of SGNs that differ in their ability to be supported by depolarization and neurotrophic factors? (3) How long after withdrawal of trophic support do SGNs become committed to a cell death fate? The second objective is to initiate studies of the mechanism by which depolarization prevents the death of SGNs become committed to a cell death fate? The second objective is to initiate studies of the mechanism by which depolarization prevents the death of SGNs in vitro: (1) Does depolarization promote survival by inducing autotrophic mechanisms in the spiral ganglion cells? A variety of techniques will be used to detect induction of neurotrophic factors, neurotrophic factor receptors, and apoptosis-inhibiting genes by depolarization in neuronal and non-neuronal cells in the culture. (2) What intracellular signal pathway(s) does depolarization use to accomplish trophic signaling? These studies proposed here focus on signaling through MAP kinases and on signaling through CREB family transcription factors because these are known to be activated by neurotrophic factors and by cytosolic Ca2+. Molecular genetic and pharmacologic techniques will be used to activate or inhibit specific molecules in these pathways to assess their involvement in neurotrophic signaling . The third objective is to relate these studies of trophic stimulation by depolarization in vitro to the inhibition of SGN death in vivo by electrical stimulation. (1) Is the death of deafferented SGNs in vivo apoptotic? (2) Does electrical stimulation of deafferented SGNs in vivo result in the same molecular events that are induced in vivo by depolarization: The studies included in the first two objective will identify particular depolarization-induced events relevant to cell survival, e.g., induction of neurotrophic factors. Electrically stimulated cochlea from deafened rats will be examined for expression of these same events in vivo.
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