Glutamate-induced mitochondrial depolarisation and perturbation of calcium homeostasis in cultured rat hippocampal neurones

1.. The objective of this study was to clarify the relationships between loss of mitochondrial potential and the perturbation of neuronal Ca2+ homeostasis induced by a toxic glutamate challenge. Digital fluorescence imaging techniques were employed to monitor simultaneously changes in cytoplasmic Ca2+ concentration ([Ca2+](i)) and mitochondrial potential (Delta Psi(m)) in individual hippocampal neurones in culture coloaded with fura-2 AM or fura-2FF BM and rhodamine 123 (Rh 123). 2. In most cells (96%) at 6-7 days in, vitro (DIV) and in a small proportion of cells (29%) at 11-17 DIV the [Ca2+](1) increase induced by exposure to 100 mu M glutamate for 10 min was associated with a small mitochondrial depolarisation, followed by mitochondrial repolarisation, and a degree of recovery of [Ca2+](i) following glutamate washout. In the majority of neurones at 11-17 DIV (71%), exposure to glutamate for 10 mill induced a profound mono- or biphasic mitochondrial depolarisation, which was clearly correlated with a sustained [Ca2+](i) plateau despite the removal of glutamate. 3. Addition of glutamate receptor antagonists (15 mu M MK-801 plus 75 mu M 6-cyano-7-nitro-quinoxaline-2,3-dione (CNQX)) to the washout solution did not affect the post-glutamate [Ca2+](i) plateau in neurones exhibiting a profound mitochondrial depolarisation but greatly improved [Ca2+](i) recovery in those neurones undergoing only a small mitochondrial depolarisation, suggesting that the release of endogenous glutamate delays [Ca2+](i) recovery in the postglutamate period. 4. Cyclosporin A (500 nM) or N-methyl Val-4-cyclosporin A (200 nM) delayed or even prevented the development of the second phase of mitochondrial depolarisation in cells at 11-17 DIV and increased the proportion of neurones exhibiting a small monophasic mitochondrial depolarisation and [Ca2+](i) recovery upon glutamate removal. 5. We have thus described a striking correlation between mitochondrial depolarisation and the failure of cells to restore [Ca2+](i) following a toxic glutamate challenge. These data suggest that; mitochondrial dysfunction plays a major role in the deregulation of [Ca2+](i) associated with glutamate toxicity.