Ratiometric high-resolution imaging of JC-1 fluorescence reveals the subcellular heterogeneity of astrocytic mitochondria

Abstract

Using the mitochondrial potential (ΔΨm) marker JC-1 (5,5′,6,6′-tetrachloro-1,1′,3,3′-tetraethylbenzimidazolylcarbocyanine iodide) and high-resolution imaging, we functionally analyzed mitochondria in cultured rat hippocampal astrocytes. Ratiometric detection of JC-1 fluorescence identified mitochondria with high and low ΔΨm. Mitochondrial density was highest in the perinuclear region, whereas ΔΨm tended to be higher in peripheral mitochondria. Spontaneous ΔΨm fluctuations, representing episodes of increased energization, appeared in individual mitochondria or synchronized in mitochondrial clusters. They continued upon withdrawal of extracellular Ca2+, but were antagonized by dantrolene or 2-aminoethoxydiphenylborate (2-APB). Fluo-3 imaging revealed local cytosolic Ca2+ transients with similar kinetics that also were depressed by dantrolene and 2-APB. Massive cellular Ca2+ load or metabolic impairment abolished ΔΨm fluctuations, occasionally evoking heterogeneous mitochondrial depolarizations. The detected diversity and ΔΨm heterogeneity of mitochondria confirms that even in less structurally polarized cells, such as astrocytes, specialized mitochondrial subpopulations coexist. We conclude that ΔΨm fluctuations are an indication of mitochondrial viability and are triggered by local Ca2+ release from the endoplasmic reticulum. This spatially confined organelle crosstalk contributes to the functional heterogeneity of mitochondria and may serve to adapt the metabolism of glial cells to the activity and metabolic demand of complex neuronal networks. The established ratiometric JC-1 imaging—especially combined with two-photon microscopy—enables quantitative functional analyses of individual mitochondria as well as the comparison of mitochondrial heterogeneity in different preparations and/or treatment conditions.