Abstract

Emerging evidence suggests that mitochondrial dysfunction contributes to the pathophysiology of epilepsy. Recurrent mitochondrial Ca²⁺ ion load during seizures might act on mitochondrial membrane potential (ΔΨm) and proton motive force. By using electrophysiology and confocal laser-scanning microscopy, we investigated the effects of epileptiform activity, as induced by low-Mg²⁺ ion perfusion in hippocampal slice cultures, on changes in ΔΨm and in mitochondrial Ca²⁺ ion concentration ([Ca²⁺]_m).

The mitochondrial compartment was identified by monitoring ΔΨm in the soma and dendrites of patched CA3 pyramidal cells using the mitochondria-specific voltage-sensitive dye rhodamine-123 (Rh-123). Interictal activity was accompanied by localized mitochondrial depolarization that was restricted to a few mitochondria in small dendrites. In contrast, robust Rh-123 release into the cytosol was observed during seizure-like events (SLEs), indicating simultaneous depolarization of mitochondria. This was critically dependent on Ca²⁺ ion uptake and extrusion, because inhibition of the mitochondrial Ca²⁺ ion uniporter by Ru360 and the mitochondrial Na⁺/Ca²⁺ ion exchanger by 7-chloro-5-(2-chlorophenyl)-1,5-dihydro-4,1-benzothiazepin-2(3H)-one but not the inhibitor of mitochondrial permeability transition pore, cyclosporin A, decreased the SLE-associated mitochondrial depolarization.

The Ca²⁺ ion dependence of simultaneous mitochondrial depolarization suggested enhanced Ca²⁺ ion cycling across mitochondrial membranes during epileptiform activity. Indeed, [Ca²⁺]_m fluctuated during interictal activity in single dendrites, and these fluctuations spread over the entire mitochondrial compartment during SLEs, as revealed using mitochondria-specific dyes (rhod-2 and rhod-ff) and spatial frequency-based image analysis. These findings strengthen the hypothesis that epileptic activity results in Ca²⁺ ion-dependent changes in mitochondrial function that might contribute to the neuronal injury during epilepsy.