The aim of this proposal is to continue our work on volume regulation in astrocytes. We will examine the causes, regulation and consequences of astrocytic swelling, a feature common to many pathological states. As a model system we use a well- characterized in vitro model of astrocytes; primary astrocyte cultures prepared from neonatal rat brain, and we swell them using hypotonic medium or addition of putative endogenous effectors. In this manner we can isolate the swelling and volume regulation processes and examine the direct effects of activators or inhibitors without the problems and limitations associated with in situ studies. We will use tracer methods or an extracellular electrical impedance system to study the magnitude, and high voltage electron microscopy to study as [3H] taurine, [3H] glutamate and [3H] D-aspartate of high pressure liquid chromatography to study the authentic processes will be done. Electrophysiology, specifically membrane potential and whole cell voltage clamp will also be performed. Fluorescent probe methods will be used to study intracellular Ca2 and pH during swelling and immunocytochemistry and protein chemistry to study any possible cytoskeletal changes. Finally, effects of hypotonic medium on efflux of glutamate and taurine from brain slices will also be done. The overall hypothesis to be tested is that astrocytic swelling in pathological states is deleterious and by preventing such swelling we can prevent neurological deficit or death in conditions such as trauma, ischemia and hypoxia in which astroglial swelling is known to occur. One mechanism we wish to test is that astrocytic swelling leads to the release of potential neurotoxic compounds such as L-glutamate present in astrocytes in relatively high amounts and that a class of inhibitors, already shown by us and others to be effective, in improving or preventing death from experimental head trauma or a model of Reye's syndrome, acts by preventing massive efflux of glutamate from swollen astrocytes.
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