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

Julie Keelan, Olga Vergun and Michael R. Duchen

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 (_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 _m. KCl caused a small transient loss of _m but in response to glutamate there was a profound loss of _m. In cells of 7-10 DIV, glutamate caused only a modest and reversible drop in _m.

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 _m. Neurons exhibiting a profound mitochondrial depolarisation also showed a large secondary increase in the fura-2FF ratio.

The glutamate-induced loss of _m was dependent on Ca²⁺ influx. However, inhibition of nitric oxide synthase (NOS) by L-NAME significantly attenuated the loss of _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 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.

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