title: Glutamate-induced mitochondrial depolarisation and perturbation of calcium homeostasis in cultured rat hippocampal neurones.
abstract: 1. The objective of this study was to clarify the relationships between loss of mitochondrial potential and the perturbation of neuronal Ca2+ homeostasis induced by a toxic glutamate challenge. Digital fluorescence imaging techniques were employed to monitor simultaneously changes in cytoplasmic Ca2+ concentration ([Ca2+]i) and mitochondrial potential (DeltaPsim) in individual hippocampal neurones in culture coloaded with fura-2 AM or fura-2FF AM and rhodamine 123 (Rh 123). 2. In most cells (96 %) at 6-7 days in vitro (DIV) and in a small proportion of cells (29 %) at 11-17 DIV the [Ca2+]i increase induced by exposure to 100 microM glutamate for 10 min was associated with a small mitochondrial depolarisation, followed by mitochondrial repolarisation, and a degree of recovery of [Ca2+]i following glutamate washout. In the majority of neurones at 11-17 DIV (71 %), exposure to glutamate for 10 min induced a profound mono- or biphasic mitochondrial depolarisation, which was clearly correlated with a sustained [Ca2+]i plateau despite the removal of glutamate. 3. Addition of glutamate receptor antagonists (15 microM MK-801 plus 75 microM 6-cyano-7-nitroquinoxaline-2, 3-dione (CNQX)) to the washout solution did not affect the post-glutamate [Ca2+]i plateau in neurones exhibiting a profound mitochondrial depolarisation but greatly improved [Ca2+]i recovery in those neurones undergoing only a small mitochondrial depolarisation, suggesting that the release of endogenous glutamate delays [Ca2+]i recovery in the postglutamate period. 4. Cyclosporin A (500 nM) or N-methyl Val-4-cyclosporin A (200 nM) delayed or even prevented the development of the second phase of mitochondrial depolarisation in cells at 11-17 DIV and increased the proportion of neurones exhibiting a small monophasic mitochondrial depolarisation and [Ca2+]i recovery upon glutamate removal. 5. We have thus described a striking correlation between mitochondrial depolarisation and the failure of cells to restore [Ca2+]i following a toxic glutamate challenge. These data suggest that mitochondrial dysfunction plays a major role in the deregulation of [Ca2+]i associated with glutamate toxicity.
authors.affiliation: Department of Physiology, University College London, Gower Street, London WC1E 6BT, UK. o.vergun@ucl.ac.uk
authors.forename: O
authors.initials: O
authors.lastname: Vergun
authors.forename: J
authors.initials: J
authors.lastname: Keelan
authors.forename: B I
authors.initials: BI
authors.lastname: Khodorov
authors.forename: M R
authors.initials: MR
authors.lastname: Duchen
mesh_terms: D000255:Adenosine Triphosphate / Q000502:physiology; D000818:Animals; D002118:Calcium / Q000502:physiology*; D002478:Cells, Cultured; D002586:Cesium / Q000494:pharmacology; D004594:Electrophysiology; D018698:Glutamic Acid / Q000633:toxicity*; D006624:Hippocampus / Q000166:cytology / Q000187:drug effects / Q000502:physiology*; D006706:Homeostasis / Q000187:drug effects*; D007091:Image Processing, Computer-Assisted; D007700:Kinetics; D008928:Mitochondria / Q000187:drug effects / Q000502:physiology*; D009474:Neurons / Q000187:drug effects / Q000502:physiology*; D051381:Rats
chemical_list: D002586:Cesium; D018698:Glutamic Acid; D000255:Adenosine Triphosphate; D002118:Calcium