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

Olga Vergun, Julie Keelan, Boris I. Khodorov * and Michael R. Duchen

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 (_m) in individual hippocampal neurones in culture coloaded with fura-2 AM or fura-2FF AM and rhodamine 123 (Rh 123).

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⁺]_i increase induced by exposure to 100 µM 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 min induced a profound mono- or biphasic mitochondrial depolarisation, which was clearly correlated with a sustained [Ca²⁺]_i plateau despite the removal of glutamate.

Addition of glutamate receptor antagonists (15 µM MK-801 plus 75 µM 6-cyano-7-nitroquinoxaline-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.

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.

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.

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