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.

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.

Glutathione (GSH) is a major antioxidant system in the mammalian central nervous system (CNS). Abnormalities of GSH metabolism have been associated with many disorders of the CNS, including Parkinson's, Alzheimer's, and Huntingdon's diseases and ischaemic/reperfusion injury. Investigation of GSH levels in the CNS generally relies on biochemical assays from cultures enriched for different cell types. Because glia influence neuronal metabolism, we have studied cultures in which neurons and glia are cocultured. This approach demands fluorescence imaging to differentiate between the different cell types in the culture, permitted by the use of monochlorobimane (MCB), which reacts with GSH to produce a fluorescent product. We have defined the conditions required to ensure steady-state MCB loading and show the specificity of MCB for GSH through a reaction catalysed by glutathione-S-transferase (GST). [GSH] was consistently higher in glia than in neurons, and [GSH] in both cell types decreased with time in culture. Inhibition of GSH synthesis by buthionine sulfoximine (BSO) caused a greater proportional depletion of GSH in glia than in neurons. The depletion of GSH induced by BSO was significantly greater in cells cultured for >10 days. Furthermore, release of GSH from glia and its breakdown by the ectoenzyme gamma-glutamyltranspeptidase (gammaGT) maintains [GSH] in neurons. In older cultures, inhibition of gammaGT by acivicin caused significant depletion of neuronal GSH. After inhibition of GSH synthesis by BSO, inhibition of the glia-neuron trafficking pathway by acivicin caused widespread neuronal death. Such neurotoxicity was independent of the endogenous glutamate and nitric oxide synthase, suggesting that it is not due to secondary excitotoxicity.

Glutamate neurotoxicity has been attributed to cellular Ca2+ overload. As mitochondrial depolarisation may represent a pivotal step in the progression to cell death, we have used digital imaging techniques to examine the relationship between cytosolic Ca2+ concentration ([Ca2+]c) and mitochondrial potential (ΔΨm) during glutamate toxicity, and to define the mechanisms underlying mitochondrial dysfunction. In cells of > 11 days in vitro (DIV), exposure to 50 mM potassium or 100 μM glutamate had different consequences for ΔΨm. KCl caused a small transient loss of ΔΨm but in response to glutamate there was a profound loss of ΔΨm. In cells of 7–10 DIV, glutamate caused only a modest and reversible drop in ΔΨm. Using fura‐2 to measure [Ca2+]c, responses to KCl and glutamate did not appear significantly different. However, use of the low affinity indicator fura‐2FF revealed a difference in the [Ca2+]c responses to KCl and glutamate, which clearly correlated with the loss of ΔΨm. Neurons exhibiting a profound mitochondrial depolarisation also showed a large secondary increase in the fura‐2FF ratio. The glutamate‐induced loss of ΔΨm was dependent on Ca2+ influx. However, inhibition of nitric oxide synthase (NOS) by L‐NAME significantly attenuated the loss of ΔΨm. Furthermore, photolysis of caged NO at levels that had no effect alone promoted a profound mitochondrial depolarisation when combined with high [Ca2+]c, either in response to KCl or to glutamate in cultures at 7–10 DIV. In cells that showed only modest mitochondrial responses to glutamate, induction of a mitochondrial depolarisation by the addition of NO was followed by a secondary rise in [Ca2+]c. These data suggest that [Ca2+]c and nitric oxide act synergistically to cause mitochondrial dysfunction and impaired [Ca2+]c homeostasis during glutamate toxicity.