Abstracts

Rationale: Injury to the CA1 but not CA3 hippocampal subregion of juvenile animals (P20) following a single injection of kainic acid (KA) (1 KA) is attenuated if they have a history of two sustained neonatal seizures on P6 and P9 (3 KA). The underlying mechanisms of this spatially distinct neuroprotection and the responsible major signaling cascades remain unknown. In order to identify gene candidates involved in the spatially protective effects, we previously profiled transcriptomes of the isolated CA1 subregion after 1 KA and 3 KA. The present study isolated genes of the CA3 subregion under the same conditions. Methods: The CA3 was isolated and total RNA was extracted, subjected to RT-PCR, and hybridized with a rat microarray platform to identify genes involved in the spatially protective effects produced by multiple early-life seizures. Several transcripts were validated with QPCR and immunohistochemistry.Results: A large number of genes (8,183) were regulated: 1 KA (30.6%) vs. 3 KA (69.4%). Many were uniquely controlled: 830 transcripts were commonly upregulated and 290 were commonly downregulated. Although many autophagy genes were triggered within the CA3, many protective genes were also differentially upregulated, particularly after 3 KA, but with different expression profiles. These included but were not limited to Ca++ modulated proteins (e.g. annexins, calmodulin), apoptosis inhibitors, adaptor-related protein complexes, ADAM metalloproteinases, adaptor ATG autophagy genes, caspase cascade activators, ATP-mediated gliotransmitters, GTP binding proteins, cyclins, F-box proteins, growth factors, interleukins, heat shock proteins, certain ionotropic and metabotropic glutamatergic and GABAergic neurotransmitter receptors and synthesizing enzymes. Differential downregulation between the groups included ankyrin-repeat proteins, adenosine receptors, ATP synthases, caspases as well as their recruitment domains, Ca++ channels, certain heat shock proteins, NFKB activating proteins, synaptosomal associated proteins, potassium voltage gated channels, and zinc finger domains. More genes were upregulated after 3 KA compared to 1 KA. Moreover, many more genes were downregulated in the CA3 compared to the CA1 after 3 KA (1393 vs. 41). Several transcripts were validated with immunohistochemistry further revealing differential regional stability. For example, the CA3 showed calm 1 and calm 3 but not calm 2 transcripts were regulated in the microarray and QPCR assays, whereas calm 2 was steady and only regulated in the CA1 subregion. After 3 KA, immunohistochemistry revealed opposite patterns of calm 2 protein expression ( CA1, CA3). Cox 1 and casp 6 were relatively stable in the CA3 but differentially altered in the CA1.Conclusions: Results suggest that regional differences in gene expression are responsible for early life seizure-induced preconditioning and age-dependent differences in cell viability. Resistance to insult may occur by region specific attenuation of glutamate stimulated Ca++ currents, reduced apoptosis, and induction of survival signaling pathways.