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

Glutamate neurotoxicity has been attributed to cellular Ca 2+ 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 Ca 2+ concentration ([Ca 2+ ] c ) and mitochondrial potential (ΔΨ m ) 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 ΔΨ m . KCl caused a small transient loss of ΔΨ m but in response to glutamate there was a profound loss of ΔΨ m . In cells of 7–10 DIV, glutamate caused only a modest and reversible drop in ΔΨ m . Using fura-2 to measure [Ca 2+ ] 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 [Ca 2+ ] c responses to KCl and glutamate, which clearly correlated with the loss of ΔΨ m . Neurons exhibiting a profound mitochondrial depolarisation also showed a large secondary increase in the fura-2FF ratio. The glutamate-induced loss of ΔΨ m was dependent on Ca 2+ influx. However, inhibition of nitric oxide synthase (NOS) by L-NAME significantly attenuated the loss of ΔΨ m . Furthermore, photolysis of caged NO at levels that had no effect alone promoted a profound mitochondrial depolarisation when combined with high [Ca 2+ ] 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 [Ca 2+ ] c . These data suggest that [Ca 2+ ] c and nitric oxide act synergistically to cause mitochondrial dysfunction and impaired [Ca 2+ ] c homeostasis during glutamate toxicity.