Spontaneous Changes in Mitochondrial Membrane Potential in Cultured Neurons

Abstract

Using the mitochondrial membrane potential (ΔΨ_m)-sensitive fluorescent dyes 5,5′,6,6′-tetrachloro-1,1′,3,3′-tetraethylbenzimidazolocarbocyanine iodide (JC-1) and tetramethylrhodamine methyl ester (TMRM), we have observed spontaneous changes in the ΔΨ_m of cultured forebrain neurons. These fluctuations in ΔΨ_m appear to represent partial, transient depolarizations of individual mitochondria. The frequency of these ΔΨ_mfluctuations can be significantly lowered by exposure to a photo-induced oxidant burden, an ATP synthase inhibitor, or a glutamate-induced sodium load, without changing overall JC-1 fluorescence intensity. These spontaneous fluctuations in JC-1 signal were not inhibited by altering plasma membrane activity with tetrodotoxin or MK-801 or by blocking the mitochondrial permeability transition pore (PTP) with cyclosporin A. Neurons loaded with TMRM showed similar, low-amplitude, spontaneous fluctuations in ΔΨ_m. We hypothesize that these ΔΨ_mfluctuations are dependent on the proper functioning of the mitochondria and reflect mitochondria alternating between the active and inactive states of oxidative phosphorylation.