EXPRESSION PROFILE OF ANTISENSE OLIGODEOXYNUCLEOTIDE (ODN) ACTIVITY

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We have previously characterized coordinate changes in the expression of multiple genes that occur during the ageing process in single live neurons from the aged rat hippocampi. In an effort to correlate the glucocorticoid model of ageing with actual aged neurons we have shown that coordinate changes in mRNA abundance occur in the sub-regions of the hippocampus in response to glucocorticoid challenge in a time-dependent manner. Of the changes that have been observed there are several which are consistent between the two experimental systems - including changes in the excitatory to inhibitory response potential of the hippocampus. We have further examined the effects of glucocorticoids on the expression profiles of individual hippocampal pyramidal cells in primary culture. Based upon these preliminary expression profiles we propose to expand upon these studies by performing add-back experiments in which we attempt to alter the expression of particular mRNAs by using antisense oligonucleotides to manipulate the production of mRNA and functional protein. This will be accomplished in three ways 1) expression profiling of individual dispersed neurons (from hippocampi isolated from glucocorticoid treated rats) which have been inhected with the particular antisense oligonucleotides combined with electrophysiological recordings of these same cells 2) isolation of novel mRNAs whose abundances are altered by antisense manipulation using cDNA enrichment techniques and 3) protein profiling of individual cells using immuno-aRNA to determine whether particular antisense oligonucleotides alter the amount of detectable protein for the targeted mRNA. These particular experiments are biologically extremely selective and sensitive because of the specificity of the starting cDNA and protein, i.e. that from a single cell. These experiments will investigate the mechanisms of antisense function by determine whether administration of oligonucleotides into the nucleus or cytoplasm of cells differentially affects the expression profile. The hypothesis to be examined is that coordinate changes in mRNA levels which provide a fingerprint of glucocorticoid challenged neurons can be manipulated in a predictable manner by addition of antisense oligonucleotides resulting in the alteration of levels of specific mRNAs and proteins within these cells. These data will likely have therapeutic implications for long term steroid treatments.