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 (ΔΨm) 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.

No abstract

Delayed neuronal death following prolonged (10-15 min) stimulation of Glu receptors is known to depend on sustained elevation of cytosolic Ca(2+) concentration ([Ca(2+)](i)) which may persist far beyond the termination of Glu exposure. Mitochondrial depolarization (MD) plays a central role in this Ca(2+) deregulation: it inhibits the uniporter-mediated Ca(2+) uptake and reverses ATP synthetase which enhances greatly ATP consumption during Glu exposure. MD-induced inhibition of Ca(2+) uptake in the face of continued Ca(2+) influx through Glu-activated channels leads to a secondary increase of [Ca(2+)](i) which, in its turn, enhances MD and thus [Ca(2+)](i). Antioxidants fail to suppress this pathological regenerative process which indicates that reactive oxygen species are not involved in its development. In mature nerve cells (>11 DIV), the post-glutamate [Ca(2+)](i) plateau associated with profound MD usually appears after 10-15 min Glu (100 microM) exposure. In contrast, in young cells (<9 DIV) delayed Ca(2+) deregulation (DCD) occurs only after 30-60 min Glu exposure. This difference is apparently determined by a dramatic increase in the susceptibility of mitochondia to Ca(2+) overload during nerve cells maturation. The exact mechanisms of Glu-induced profound MD and its coupling with the impairment of Ca(2+) extrusion following toxic Glu challenge is not clarified yet. Their elucidation demands a study of dynamic changes in local concentrations of ATP, Ca(2+), H(+), Na(+) and protein kinase C using novel methodological approaches.

No abstract

Exposure of hippocampal neurones to glutamate at toxic levels is associated with a profound collapse of mitochondrial potential and deregulation of calcium homeostasis. We have explored the contributions of reactive oxygen species (ROS) to these events, considered to represent the first steps in the progression to cell death. Digital imaging techniques were used to monitor changes in cytosolic Ca2+ concentration ([Ca2+]c; fura‐2FF) and mitochondrial potential (Δψm; rhodamine 123); rates of ROS generation were assessed using hydroethidium (HEt); and membrane currents were measured with the whole‐cell configuration of the patch clamp technique. Inhibitors of lipid peroxidation (trolox plus ascorbate) and scavengers of superoxide or hydrogen peroxide (manganese(III) tetrakis(4‐benzoic acid) porphyrin (MnTBAP) and TEMPO plus catalase), had only minimal impact on the mitochondrial depolarisation and the sustained increase in [Ca2+]c during and following a 10 min exposure to glutamate. The antioxidants completely suppressed ROS generated by xanthine with xanthine oxidase. No significant increase in ROS production was detected with HEt during a 10 min glutamate exposure. A combination of antioxidants (TEMPO, catalase, trolox and ascorbate) delayed but did not prevent the glutamate‐induced mitochondrial depolarisation and the secondary [Ca2+]c rise. However, this was attributable to a transient inhibition of the NMDA current by the antioxidants. Despite their inability to attenuate the glutamate‐induced collapse of Δψm and destabilisation of [Ca2+]c homeostasis, the antioxidants conferred significant protection in assays of cell viability at 24 h after a 10 min excitotoxic challenge. The data obtained suggest that antioxidants exert their protective effect against glutamate‐induced neuronal death through steps downstream of a sustained increase in [Ca2+]c associated with the collapse of Δψm.