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

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

Potassium Ion Homeostasis and Mitochondrial Redox Activity in Brain: Relative Changes as Indicators of Hypoxia

This study was directed at relating ion transport and mitochondrial redox activity during hypoxia, as a step toward definition of brain oxygen sufficiency. To accomplish this, extracellular potassium ion activity (K+o) was recorded by ion-selective microelectrodes while reduction/oxidation (redox) ratios of cytochrome oxidase (cytochrome a,a3) were monitored by reflection spectrophotometry in cerebral cortex of rats anesthetized with pentobarbital. In normoxia, neuronal activation by direct cortical stimulation produced transient oxidation of cytochrome a,a3 and elevation of K+o. Moderate hypoxia (Pao2 above 50 mm Hg) resulted in reduction of cytochrome a,a3 but only slight elevation of K+o. At this level of hypoxia, cytochrome a,a3 continued to respond to neuronal activation with transient shifts toward oxidation and rates of K+o reaccumulation were unchanged from control. When Pao2 was further decreased below a critical threshold, stimulus-provoked oxidative responses of mitochondrial reactants were replaced by shifts toward reduction, but rates of reaccumulation of K+, spilled into the extracellular space by neuronal activation, remained unchanged. Only during severe hypoxia (Pao2 less than 20 mm Hg) was it possible in some animals to record a slowing in the reaccumulation of K+o without provocation of spreading cortical depression. These data indicate that ion transport activity in cerebral cortex is more refractory to hypoxia than is mitochondrial redox functioning. They suggest an in vivo parallel to the “cushioning” effect of mitochondria in vitro, in which oxygen consumption remains constant despite fluctuations in oxygenation and redox ratios, and also that there may be a greater anaerobic capacity to provide energy for ion transport in mammalian brain than has previously been appreciated