This project has the primary overall goal of seeking changes in calcium regulatory mRNA and protein expression in hippocampus that are common to both aging and glucocorticoid (GC) activation. Those studies test the hypothesis that changes in Ca/2+ regulation common to aging and GC activation are important in neuronal vulnerability. In the prior period we developed extensive ribonuclease protection assay (RPA) approaches to examine effects of aging on hippocampal mRNA expression of 11 calcium- related genes (e.g., the alpha/1D subunit, alpha/1C subunit and beta subunit of the L-type calcium channel, plasma membrane calcium membrane calcium ATPase (PMCA) isoforms 1 and 2, calmodulin II, calcineurin cyclophilin D, glucocorticoid receptor (GR), mineralocorticoid receptor (MR) and as a control, glyceraldehyde phosphate dehydrogenase, (GAPDH)). The equivalent proteins were measured in aging for most of these genes. Surprisingly few changes common to aging and GC activation were found. However, both aging and GCs increased 3 genes: cycophilin A, calmodulin II, and the alpha/1D subunit of the L-type calcium channel. In situ hybridization showed that A/1D changes in aging were most pronounced in field CA1. Protein measurements (westerns) did not show clear effects of aging, but required more tissue and are mess sensitive than RPAs. The need to homogenize tissues for both RPAs and Westerns appears to be a major obscuring factor. In the next period, therefore, it is focused these studies at the single cell and/or regional level, and to introduce direct collections with functional measure (electrophysiology, calcium imaging, collaboration with Project 3) to pursue these leads and more accurately test the main hypotheses. To achieve this improved accuracy and localization, the next phase will utilize single-cell RT-PCR method methods in neurons from which electrophysiological recordings are obtained, and extensive in situ hybridization in aged or adrenalectomized rats versus controls. Proteins assays will be enhanced by performing immunoautoradiography (IAR) and regional dissections prior to western blotting analyses. These studies will more accurately be able to test the test that increased expression of the mRNA for the L-type calcium channel, for calmodulin or for cyclophilin induced by aging or hormonal modulation alters calcium channel activity and/or calcium homeostasis and modifies neuronal vulnerability.
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