Abstracts

Our previous studies utilized quantitative trait loci mapping in seizure resistant C57BL/6 (B6) and seizure sensitive DBA/2 (D2) mice to document linkage to a gene(s) on mouse chr 1 with a large effect on seizure susceptibility. We identified the inward rectifying potassium ion channel gene Kcnj10 as our primary candidate based on its function and location. In addition we demonstrated a Kcnj10 coding region variation that differentiates B6 from D2 (Ferraro et al., Mam Gen 2004 15:239-251) and a coding region variation in the human KCNJ10 that is associated with epilepsy (Buono et al., Epi Res 2004 58:175-183). Since the coding region variations alter the amino acid sequence of the Kir4.1 protein in mice (T262S) and humans (R271C), we hypothesize that altered protein function is related to seizure susceptibility. However, no detailed studies of Kir4.1 expression in the brain have been reported. Since differences in expression could underlie the seizure sensitivity difference in the inbred strains, we used immuno-histochemistry and image analyses to localize the Kir4.1 protein in the brains of B6 and D2 mice and to compare expression levels between the two strains. Six male mice of each strain were used. Brains were fixed by perfusion and immersion, paraffin embedded, sectioned in the sagital plane and stained with anti Kir4.1 (Alamone Labs Inc.). Antibody was visualized with Vecatstain reagents, photographed and analyzed (Image-Pro Plus, v 4.0). Our results demonstrate that the Kir4.1 staining was prominent in tectum, tegmentum, olfactory bulb, hypothalamus, thalamus, fornix, septum, and brainstem with weaker staining in hippocampus, cortex and cerebellum. In contrast to all previous reports of Kir4.1 localization being restricted to glial cells, we find clear evidence of immunoreactivity in cortical pyramidal neurons and Purkinje cells. Image analyses comparing intensity of stain between B6 (n=6) and D2 (n=6) mice was performed. Six regions were systematically scanned (brainstem, cerebellum, olfactory bulb, hippocampus, thalamus, hypothalamus, frontal cortex) and showed no statistically significant differences between B6 and D2 mice. We conclude that Kir4.1 is expressed in both neurons and glial cells and that brain expression levels are not different between B6 and D2 mice. (Supported by Grants R01NS40396 to RJB and R01NS40554 to TNF.)