Abstracts

RATIONALE: Since alterations of mitochondrial function have been shown to be involved in epileptogenesis we investigated putative alterations of the mitochondrial membrane potential in rat hippocampal slices using the pilocarpine model of chronic epilepsy.
METHODS: We used the mitochondrial membrane potential sensitive fluorescent dye rhodamine 123 to follow alteration of the mitochondrial membrane potential in pyramidal neurons in living 150 [mu]m thick rat hippocampal slices using fluorescence microscopy. To demonstrate the usefulness of this method for the quantitative measurement of mitochondrial membrane potential we used rhodamine 123 fluorescence spectroscopy of digitonin-treated hippocampal homogenates.
RESULTS: Applying a potassium diffusion potential calibration procedure this methods allows in digitonin-treated hippocampal homogenates a quantitative determination of alterations of mitochondrial membrane potential. In hippocampal slices the rhodamine 123 fluorescence signal decreased if pyruvate (10 mM) was added to the glucose-containing slice perfusion medium. This is an indication for the substrate dependent increase of the mitochondrial membrane potential. The stimulation of oxidative phosphorylation by KCl (10 mM) or the uncoupler TTFB (10 [mu]M) resulted in a dramatic fluorescence increase indicating mitochondrial depolarization. In the presence of glucose or pyruvate we observed in slices of pilocarpine-treated chronic epileptic rats in the CA3 and CA1 hippocampal subfields neurons with elevated rhodamine 123 fluorescence which did not further increase after the addition of KCl or TTFB to the slice perfusion medium. This is an indication for mitochondrial depolarization in these neurons pointing to either insufficient substrate supply or dysfunction of mitochondrial oxidative phosphorylation. Since we were able to detect decreased activities of NADH:CoQ oxidoreductase and cytochrome c oxidase at control activities of succinate dehydrogenase and citrate synthase in the CA3 and CA1 hippocampal subfields we suggest a dysfunction of mitochondrial oxidative phosphorylation in pyramidal neurons of chronic epileptic rats.
CONCLUSIONS: Our findings confirm the presence of defects of mitochondrial oxidative phosphorylation in hippocampal pyramidal neurons of chronic epileptic rats. These data strongly suggest the involvement of mitochondria in epileptogenesis.
[Supported by: a grant from Deutsche Forschungsgemeinschaft (Ku 911/11-1).]