Excitotoxic mitochondrial depolarisation requires both calcium and nitric oxide in rat hippocampal neurons

1. 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 (Delta Psi(m)) during glutamate toxicity, and to define the mechanisms underlying mitochondrial dysfunction. 2. In cells of > 11 days in vitro (DIV), exposure to 50 mM potassium or 100 mu m glutamate had different consequences for Delta Psi(m). KCl caused a small transient loss of Delta Psi(m) but in response to glutamate there was a profound loss of Delta Psi(m). In cells of 7-10 DIV, glutamate caused only a modest and reversible drop in Delta Psi(m). 3. 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 Delta Psi(m). Neurons exhibiting a profound mitochondrial depolarisation also showed a large secondary increase in the fura-2FF ratio. 4. The glutamate-induced loss of Delta Psi(m) was dependent on Ca2+ influx. However, inhibition of nitric oxide synthase (NOX) by L-NAME significantly attenuated the loss of Delta Psi(m). 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 KCI or to glutamate in cultures at 7-10 DIV. 5. 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.