Mitochondrial membrane potential and glutamate excitotoxicity in cultured cerebellar granule cells

J Neurosci. 2000 Oct 1;20(19):7208-19. doi: 10.1523/JNEUROSCI.20-19-07208.2000.


The relationship between changes in mitochondrial membrane potential (Deltapsi(m)) and the failure of cytoplasmic Ca(2+) homeostasis, delayed Ca(2+)deregulation (DCD), is investigated for cultured rat cerebellar granule cells exposed to glutamate. To interpret the single-cell fluorescence response of cells loaded with tetramethylrhodamine methyl ester (TMRM(+)) or rhodamine-123, we devised and validated a mathematical simulation with well characterized effectors of Deltapsi(m) and plasma membrane potential (Deltapsi(P)). Glutamate usually caused an immediate decrease in Deltapsi(m) of <10 mV, attributable to Ca(2+) accumulation rather than enhanced ATP demand, and these cells continued to generate ATP by oxidative phosphorylation until DCD. Cells for which the mitochondria showed a larger initial depolarization deregulated more rapidly. The mitochondria in a subpopulation of glutamate-exposed cells that failed to extrude Ca(2+) that was released from the matrix after protonophore addition were bioenergetically competent. The onset of DCD during continuous glutamate exposure in the presence or absence of oligomycin was associated with a slowly developing mitochondrial depolarization, but cause and effect could not be established readily. In contrast, the slowly developing mitochondrial depolarization after transient NMDA receptor activation occurs before cytoplasmic free Ca(2+) ([Ca(2+)](c)) has risen to the set point at which mitochondria retain Ca(2+). In the presence of oligomycin no increase in [Ca(2+)](c) occurs during this depolarization. We conclude that transient Ca(2+) loading of mitochondria as a consequence of NMDA receptor activation initiates oxidative damage to both plasma membrane Ca(2+) extrusion pathways and the inhibition of mitochondrial respiration. Depending on experimental conditions, one of these factors becomes rate-limiting and precipitates DCD.

MeSH terms

  • Action Potentials / drug effects
  • Adenosine Triphosphate / metabolism
  • Animals
  • Anti-Bacterial Agents / pharmacology
  • Antimycin A / pharmacology
  • Calcium / metabolism
  • Cells, Cultured
  • Cerebellum / cytology
  • Cerebellum / drug effects
  • Cerebellum / metabolism*
  • Cytoplasmic Granules*
  • Enzyme Inhibitors / pharmacology
  • Fluorescent Dyes
  • Glutamic Acid / metabolism*
  • Glutamic Acid / pharmacology
  • Intracellular Membranes / metabolism*
  • Membrane Potentials / drug effects
  • Mitochondria / metabolism*
  • Potassium Chloride / metabolism
  • Potassium Chloride / pharmacology
  • Rats
  • Rats, Wistar
  • Receptors, Glutamate / metabolism
  • Rhodamines
  • Uncoupling Agents / pharmacology


  • Anti-Bacterial Agents
  • Enzyme Inhibitors
  • Fluorescent Dyes
  • Receptors, Glutamate
  • Rhodamines
  • Uncoupling Agents
  • tetramethylrhodamine methyl ester
  • Glutamic Acid
  • Antimycin A
  • Potassium Chloride
  • Adenosine Triphosphate
  • Calcium