The studies of gene expression in development have been extended to include aspects of injury, neuroprotection and repair. To this end, we have used the model of kainate-induced seizures in rats which is accompanied by selective excitotoxic cell death in hippocampal regions. Using the quantitative RT-PCR method previously established in this laboratory, we have discovered that the developmental forms of GAD, G67I80 and G67I86 (alternatively spliced forms of GAD67 coding for two truncated proteins) are selectively and reexpressed in the hippocampus following kainate treatment. Together with evidence showing the limited expression of G67I80/86 mRNA to neurogenesis of the developing hippocampus, we have provided an indication for the recapitulation of developmental programs. In situ hybridization using a probe specific for G67I80/86 shows a pattern of kainate-induction which is restricted to the dentate gyrus, which is largely protected from cell death. Does this observation signify that the G67I80/86 counteracts excitotoxicity through production of the inhibitory transmitter GABA, or could this be the "tip of the iceberg" of reexpressed developmental genes that exercise a cumulative neuroprotective function? Analogously, we have employed our previously established expressed gene survey strategy, MAPP (multiplex arbitrarily primed PCR) to identify changes in gene expression in dentate gyrus following kainate treatment. We have been able to identify several differentially expressed genes, ranging from heat shock proteins, T- complex protein, the nACh receptor alpha-7 subunit to 6 novel genes. The state of maturation of fetal CNS cells directly correlates to their buoyant density, allowing the separation of cell populations using a Percoll density gradient. This is reflected in the differential expression of neurofilament and nestin mRNAs as developmental markers in different buoyant fractions. To reduce the template requirement, we have developed a direct RT-PCR technique which permits RT-PCR analysis of single 500 cell sample. By combining this technique with continuous percoll separation of naturally banding fractions of cells, we are characterizing the expression of a Multitude of genes to unequivocally define the cell types that constitute the developing CNS. We have conducted a 45-gene mRNA expression survey in the spinal cord from E12 to adult of genes which belong to the groups of (a) growth factors and their receptors (cumulatively restricted to early development); (b) neurotransmitter synthesizing enzymes and receptors (cumulatively transiently overexpressed during neurogenesis); (c) intracellular signaling genes (diverse expression patterns); and (d) developmental markers (controls). Our results show that genes in development are regulated in groups and phases, suggesting that gene expression clusters behave as units. These units are an important key to understanding function in a context that approaches the reality of cross-linked, redundant and self-stabilizing genetic networks. Robotic tools for the expression analysis of gene families in distinct cell populations are currently being established in this group and are being further explored in cooperation with the Technology Transfer Office in a CRADA.