Spontaneous Changes in Mitochondrial Membrane Potential in Single Isolated Brain Mitochondria

In this study we measured ΔΨm in single isolated brain mitochondria using rhodamine 123. Mitochondria were attached to coverslips and superfused with K +-based HEPES-buffer medium supplemented with malate and glutamate. In ∼70% of energized mitochondria we observed large amplitude spontaneous fluctuations in ΔΨm with a time course comparable to that observed previously in mitochondria of intact cells. The other 30% of mitochondria maintained a stable ΔΨm. Some of the “stable” mitochondria began to fluctuate spontaneously during the recording period. However, none of the initially fluctuating mitochondria became stable. Upon the removal of substrates from the medium or application of small amounts of Ca 2+, rhodamine 123 fluorescence rapidly dropped to background values in fluctuating mitochondria, while nonfluctuating mitochondria depolarized with a delay and often began to fluctuate before complete depolarization. The changes in ΔΨm were not connected to oxidant production since reducing illumination or the addition of antioxidants had no effect on ΔΨm. Fluctuating mitochondria did not lose calcein, nor was there any effect of cyclosporin A on ΔΨm, which ruled out a contribution of permeability transition. We conclude that the fluctuations in ΔΨm reflect an intermediate, unstable state of mitochondria that may lead to or reflect mitochondrial dysfunction.