Pyramidal neurons in the hippocampus are intimately involved in the processes of learning and memory, and are vulnerable to neurodegeneration in several disorders including Alzheimer's disease and stroke. The proposed research employs a hippocampal cell culture paradigm to examine the molecular mechanisms that regulate the cellular architecture of hippocampal circuitry. The experiments are designed to test the hypothesis that basic fibroblast growth factor (bFGF) and the excitatory amino acid glutamate regulate neuroarchitecture by affecting the expression of neural development-associated proteins (NDAPs) and intracellular calcium levels. The recent cloning of the receptors for bFGF and the kainate type glutamate receptor will allow us to directly examine: the cellular localization of these receptors; dynamic aspects of receptor expression during the development and degeneration of neural circuitry; functional roles for the receptors in different neuroarchitectural processes (i.e., neurite outgrowth, synaptogenesis, and selective neuronal death). Receptor expression and localization will be evaluated by immunocytochemistry and in situ hybridization. Roles for the receptors in the actions of endogenous FGF and glutamate will be determined by blocking receptor expression and activation with antisense oligonucleotides and receptor antibodies, respectively. The intracellular mechanisms involved in the actions of bFGF and glutamate will then be studied using a battery of modern technical approaches. Immunocytochemistry, in situ hybridization, and biochemical techniques will be used to examine spatial and dynamic aspects of the expression of the NDAPs GAP43, tau, and MAP2. Roles for NDAPs in the regulation of neuroarchitecture will be directly tested by blocking their production with antisense oligonucleotides. Fluorescence ratio imaging of intracellular calcium levels using the calcium indicator dye fura-2 will be used to test the hypothesis that intracellular calcium plays a central role in coordinating the regulation of neuroarchitecture by bFGF, glutamate, and NDAPs. Taken together, these experiments will provide important insight into how different inter- and intracellular signals are integrated to regulate the formation and plasticity of functional neural circuitry. This work will identify regulatory systems likely to be involved in the aberrant events leading to age-associated deterioration of neuroarchitecture.
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