SRF modulates seizure occurrence, activity induced gene transcription and hippocampal circuit reorganization in the mouse pilocarpine epilepsy model

The transcription factor SRF (serum response factor) mediates epilepsy mediated gene expression Seizures were induced by administration of pilocarpine, a muscarinic cholinergic agonist [50, 51]. In brief, 20 minutes before pilocarpine administration, animals were injected with a low dose of the cholinergic antagonist methyl scopolamine nitrate (1 mg/kg, s.c.; Sigma, Germany) to reduce peripheral cholinergic effects. Animals received an intra-peritoneal injection of pilocarpine hydrochloride (335 or 350 mg/kg; Sigma, Germany) to induce an SE. Littermate control mice were injected with Ringer solution. Diazepam (4 mg/kg, s.c.; Ratiopharm, Germany) was administered to the animals 40 min after the onset of SE to terminate seizures. All drugs were dissolved in Ringer solution. 100 ng total RNA was used as starting material and 5.5 µg ssDNA per hybridization (GeneChip Fluidics Station 450; Affymetrix, Santa Clara, CA). The total RNAs were amplified and labeled following the Whole Transcript (WT) Sense Target Labeling Assay (http://www.affymetrix.com). Labeled ssDNA was hybridized to Mouse Gene 1.0 ST Affymetrix GeneChip arrays (Affymetrix, Santa Clara, CA). The chips were scanned with an Affymetrix GeneChip Scanner 3000 and subsequent images analyzed using Affymetrix® Expression Console™ Software (Affymetrix). Raw feature data were normalized and intensity expression summary values for each probe set were calculated using robust multiarray average [53]. Microarrays were analyzed with microarray Я US and Limma 2way Anova with p-Value and FDR cutoffs at 0.001 and 2 fold change cut-off.