Exploration of the role of reactive oxygen species in glutamate neurotoxicity in rat hippocampal neurones in culture

1. 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. 2. Digital imaging techniques were used to monitor changes in cytosolic Ca(2+) concentration ([Ca(2+)](c); fura-2FF) and mitochondrial potential (Delta psi (m); 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. 3. 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 [Ca(2+)](c) during and following a 10 min exposure to glutamate. 4. 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. 5. 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. 6. Despite their inability to attenuate the glutamate-induced collapse of Delta psi (m) and destabilisation of [Ca(2+)](c) homeostasis, the antioxidants conferred significant protection in assays of cell viability at 24 h after a 10 mill 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 [Ca(2+)](c) associated with the collapse of Delta psi (m).