Abstracts

Malformations of cortical development are a leading cause of medication resistant epilepsy. Methylazoxymethanol (MAM) treated rats develop neuroanatomical abnormalities including neuronal nodular heterotopias that exhibit similar characteristics to those found in human epilepsy surgery pathology specimens. These animals are more susceptible to induced seizures and hippocampal heterotopias are capable of generating seizure-like activity in vitro. Hippocampal heterotopic neurons exhibit altered levels of a potassium channel subunit, Kv4.2 (Castro et al. 2001) and decreased expression of GABA reuptake proteins (Calcagnotto et al. 2002). Here, we hypothesize that these changes are part of a larger pattern of alteration in excitatory/inhibitory systems as evidenced by alterations in gene transcription. Pregnant S-D rats were injected with MAM on embryonic day 15. Slides of prenatally-MAM treated rat pup hippocampus were immunolabeled with murine neuronal nuclear antigen (NeuN) antibody and then in situ transcription was performed. Single cell dissection was used to isolate contents of heterotopic neurons and normotopic CA2 neurons and then mRNA amplification was used to amplify mRNA signal from each cell. Resulting p32-CTP labeled mRNA was used to probe macroarrays consisting of full length cDNA transcripts of neurotransmitter related candidate genes. Hybridization intensity was determined using a phosphoimager and results were analyzed using a one-way ANOVA. Expression of mRNA for candidate genes including transcription factors, neurotransmitter receptor subunits, ion channels, and select cell signaling molecules were examined from single MAM-induced NeuN labeled hippocampal heterotopic neurons. These were compared to normotopic NeuN labeled CA2 hippocampal cells also from MAM treated animals. The most significant differences in mRNA expression were found in Calcium and Calmodulin regulated Kinase II alpha, where the MAM nodule neurons exhibited more than twice the expression found in control neurons (P[lt]0.05). While differences were found between heterotopic and normotopic neuron mRNA expression of several genes, the most profound difference was the finding that heterotopic hippocampal neurons have double the amount of CaMKII alpha. This finding is consistent with published neurophysiologic and immunohistochemical data suggesting that both excitatory and inhibitory systems are altered in MAM induced hippocampal heterotopias. Further analysis of gene transcription changes in this animal model of cortical malformations could provide valuable insights to the clinical condition. (Supported by NINDS R010405, R21-NS39938, R21NS40231, Parents Against Childhood Epilepsy (PACE) to P.B.C and NIH R01-NS40272 to S.C.B.)