Prolonged stimulation of excitatory amino acid receptors has been implicated in pathological states such as ischemia, hypoglycemia, anoxia, epilepsy and several neurodegenerative disorders. The developing brain may be particularly susceptible to glutamate neurotoxicity associated with disease states such as hypoxia-ischemia and prolonged seizures. The overall goal of the proposed studies is to investigate mechanisms of glutamate neurotoxicity in the developing rat cortex and to better understand developmental regulation of calcium transport mechanisms. The effects of glutamate on intracellular Ca++ level in neocortical pyramidal neurons will be examined. Spatial images of Ca++ changes will be obtained using digital fluorescence imaging techniques. This will indicate if Ca++ sensitive neuronal compartments are present which may contribute to cell death. It will also be determined if the increased number of synaptic NMDA receptors present in the developing neocortex is associated with an increased influx of Ca++ through NMDA channels. The contribution of calcium sensitive intracellular calcium pools will be assessed. To determine how homeostatic functions of glia cells contribute to neurotoxicity, path clamp recordings from cultured glial cells will be used to examine the time of expression of NA+/glutamate uptake systems in glia during development. It will also be determined if the ability of astrocytes to spatially buffer extracellular K+ is developmentally regulated. In order to study the anatomical distribution and developmental regulation of calcium uptake and release systems in the neocortex, sections of rat neocortex will be examined by 45/CA++ autoradiography to visualize Ca++ uptake and release systems. This will provide information about steady-state calcium regulation, including the relative affinity and capacity of the plasma membrane and endoplasmic reticulum components of the extrusion/sequestration/release machinery. Finally, developmental regulation of individual calcium transport mechanisms will be studied using in situ hybridization with cDNA probes to the plasma membrane and endoplasmic reticulum Ca++ ATPase pumps. Analysis of the expression of these mRNAs will allow a comparison of the relative levels of these calcium regulatory mechanisms during development. The proposed studies will increase our understanding of alterations in Ca++ homeostases in glutamate-induced neurotoxicity in the developing brain.
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