Abstracts

More than 30% of epilepsy patients do not respond completely to AEDs and are considered are medically intractable. Understanding the molecular mechanisms of antiepileptic drugs may begin to elucidate reasons for AED resistance. There is evidence that the effects of some of these drugs extend beyond ion channel blockade and second messengers to cause changes in gene transcription. Valproate causes changes in gene expression in mammalian cells that may relate to its mechanism of action. We first studied changes in gene expression in whole cultures following treatment with valproate to gain a broad view of altered transcription. Next we studied a subset of the candidate genes that are particularly relevant to epilepsy in single cells that were immunohistochemically identified as excitatory neurons, inhibitory neurons, or glial cells. After 14 days in vitro, dissociated rat embryonic hippocampal cultures were exposed for 24 hours to sodium valproate 1mM or sodium chloride 1mM. Whole cultures were lysed with Trizol and total RNA was extracted. mRNA was amplified from those samples, and the resulting radiolabeled mRNA was used to probe macroarrays containing epilepsy related candidate cDNAs. Expression of several mRNAs was altered in the VPA treated cultures thus warranting a cell specific examination of these genes. Similar cultures were fixed with 4% paraformaldehyde and immunostained for specific protein markers: vesicular glutamate transporter (VGLUT2) to identify excitatory neurons; vesicular GABA transporter (VGAT) to identify inhibitory neurons, and glial fibrillary protein (GFAP) to identify glial cells. Single cells were aspirated from cover slips using microscalpal. The mRNA was amplified from each of these cells and the resulting radiolabeled aRNA was used to probe cDNA macroarrays that contained the genes identified in the first stage. The first set of experiments yielded a solid confirmation that mRNA expression of some epilepsy related genes are altered in vitro by VPA. Several ion channel subunit exhibited differential expression after exposure to VPA including: GABA[sub]A[/sub][alpha]5, GABA[sub]A [/sub][beta]1, GABA[sub]A [/sub][beta]2 all of which increased; and GluR5 which decreased. In addition, GAD 65 more than doubled in expression. Immunohistochemistry confirmed the presence of inhibitory neurons, excitatory neurons, and glial cells on the cover slips. Single cell microscalpal aspiration reveals differential expression of epilepsy related genes in each cell type studied. Valproate caused alteration in the expression of several epilepsy related genes. The single cell gene expression studies confirmed differential changes in gene expression in inhibitory neurons, excitatory neurons, and glial cells. The most profound changes occurred in levels of ion channel subunit expression. These studies suggest that pharmacogenomic changes caused by valproic acid may contribute to its mechanism of antiepileptic action. (Supported by National Epifellows Foundation.)