Recent data from this lab together with emerging results from other labs suggest that glutamate plays an important and widespread role in hypothalamic and neuroendocrine regulation. Despite the importance of hypothalamic glutamate, it has received relatively little attention compared with the attention given to other neuroactive substances also found in the medial hypothalamus. The proposed experiments focus on the excitatory amino acid glutamate, its receptors, and cellular responses to glutamate in the hypothalamus, using the rat as an experimental model. We will use immunocytochemistry and in situ hybridization to study the expression of glutamate receptor genes in the hypothalamus, using cDNA coding for AMPA and kainate types of glutamate receptors, and antisera against the receptor protein in tissue culture and in ultrathin sections from the hypothalamus. The excitotoxic role of glutamate will be examined with a silver stain which demonstrates the cell body and dendritic arbors of neurons soon after injury by glutamate. Digital Ca2+ imaging will be used to examine long-lasting intracellular Ca2+ rises after stimulation with glutamate agonists, particularly relative to possible cytotoxic injury. To test the hypothesis that the presence of glutamate during development of the neuroendocrine hypothalamus influences the subtype of receptor expressed, we will use in situ hybridization and patch clamp recording. To examine glutamate involvement in local circuitry of the hypothalamus, we will combine intracellular electrophysiology, dye-filling, and ultrastructural immunocytochemistry with glutamate antisera. These experiments should provide a better understanding of glutamate involvement in hypothalamic function. They will also address some basic questions from a cellular level, and will examine some clinically relevant experiments with glutamate toxicity. The hypothalamus is involved in the regulation of endocrine and metabolic events and glutamate may play a critical role in the health and disease of these and other systems.
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