Abstracts

Early childhood convulsions have been correlated with memory impairment and hippocampal neuron loss in patients with intractable temporal lobe epilepsy. Animals subjected to kainate(KA) seizures at postnatal day (P)15 sustain more extensive hippocampal neuronal injury after second KA in adulthood. To study the molecular basis of this priming effect of early-life seizures, we probed over 8,000 genes using high-density oligonucleotide arrays and identified gene expression patterns in the hippocampus at various times following KA seizures at P15.
At 1h, 24h, and 240h after KA (3 mg/kg, [italic]i.p[/italic].) or PBS injection at P15, animals were anesthetized with isoflurane, decapitated and the hippocampi were dissected. Total RNA was isolated from each animal individually, and equal amounts of RNA from 4 animals were pooled for each Affymetrix Genechip[reg]. Three independent hybridizations were performed per condition per time point (total of 18 U34A profiles). Microarray Suite[reg] was used for the probe-level and GeneSpring[reg], for gene-level data analysis. KA samples were normalized against controls and genes with expression levels close to the background were removed. We selected genes showing at least two-fold changes, and used Welch[apos]s approximate t-test, one-way ANOVA and K-means clustering.
Among 40 known genes differentially regulated at 1h, 32 were upregulated in the rats treated with KA. Over 80% were IEGs/transcription factors including ania2,4,6a,7, egr1,2,4, nr4a1, 2,3,c-fos, junB, homer1a, arc, narp, btg2, C/EBP and CREM. Microtubule-associated-protein2 and ubiquitin-conjugating enzyme were among 8 downregulated at 1h. All but 8 genes returned to baseline by 24h, and a new set of 152 genes (2/3 up, 1/3 down) were identified at 24h. Noteworthy were induction of inflammation-related genes (19) such as TNF[alpha], MHCclassII proteins, interferon-induced protein, CD9, c1q, and cytokineA (Sigje). Downregulated were receptors (9) and enzymes (19, including kinases (CaMKII, CaMKK, IP3K, PKC)and phosphatases); a notable exception was glutathion peroxidase(up3-fold). At 240h, only 19 genes were differentially regulated. A K-means clutering (specifying 9 groups) of the 129 transcripts significantly changed over time revealed the following prominent 5 patterns: (1) increase at 24h (2) increase at 1h;decrease at 24h (3) increase at 24h; decrease at 240h (4) decrease at 24h (5) increase at 1h; normalize by 24h.
Complex and dynamic patterns of hippocampal gene expression were induced by early-life seizures. Conspicuously absent were the genes involved in the cell death pathway, consistent with absence of cell death after KA at P15. Instead, we found a change in expression of survival-promoting and inflammation-related genes. In stark contrast to adults, returning of the majority of genes in our study to control levels by 240h suggests plastic, homeostatic response of immature brain to excessive, dysregulating stimulation.
[Supported by: K08NS02068 and the NINDS/NIMH Microarray Consortium.]