Quantitative evaluation of mitochondrial calcium content in rat cortical neurones following a glutamate stimulus

• Published in: The Journal of physiology 2001-03, Vol.531 (3), p.793-805
• Main Authors: Brocard, Jacques B., Tassetto, Michel, Reynolds, Ian J.
• Format: Article
• Language: English
• Subjects: Animals; Calcium - metabolism; Cells, Cultured; Cerebral Cortex - cytology; Cerebral Cortex - drug effects; Cerebral Cortex - metabolism; Glutamic Acid - pharmacology; Mitochondria - metabolism; Neurons - metabolism; Original; Osmolar Concentration; Rats; Rats, Sprague-Dawley; Time Factors
• ISSN: 0022-3751; 1469-7793
• DOI: 10.1111/j.1469-7793.2001.0793h.x

Recent observations showed that a mitochondrial Ca 2+ increase is necessary for an NMDA receptor stimulus to be toxic to cortical neurones. In an attempt to determine the magnitude of the Ca 2+ fluxes involved in this phenomenon, we used carbonylcyanide- p -(trifluoromethoxy)phenylhydrazone (FCCP), a mitochondrial proton gradient uncoupler, to release mitochondrial free calcium ([Ca 2+ ] m ) during and following a glutamate stimulus, and magfura-2 to monitor cytoplasmic free calcium ([Ca 2+ ] c ). FCCP treatment of previously unstimulated neurones barely changed [Ca 2+ ] c whereas when added after a glutamate stimulus it elevated [Ca 2+ ] c to a much greater extent than did exposure to glutamate, suggesting a very large accumulation of Ca 2+ in the mitochondria. Mitochondrial Ca 2+ uptake was dependent on glutamate concentration, whereas the changes in the overall quantity of Ca 2+ entering the cell, obtained by simultaneously treating neurones with glutamate and FCCP, showed a response that was essentially all-or-none. Mitochondrial Ca 2+ uptake was also dependent on the nature and duration of a given stimulus as shown by comparing [Ca 2+ ] m associated with depolarization and treatment with kainate, NMDA or glutamate. Large mitochondrial Ca 2+ accumulation only occurred after a glutamate or NMDA stimulus. These studies provide a method of estimating the accumulation of Ca 2+ in the mitochondria of neurones, and suggest that millimolar concentrations of Ca 2+ may be reached following intense glutamate stimulation. It was shown that substantially more Ca 2+ enters neurones following glutamate receptor activation than is reflected by [Ca 2+ ] c increases.