Spectral imaging microscopy demonstrates cytoplasmic pH oscillations in glial cells

Glial cells exhibit distinct cellular domains, somata, and filopodia. Thus the cytoplasmic pH (pH_cyt) and/or the behavior of the fluorescent ion indicator might be different in these cellular domains because of distinct microenvironments. To address these issues, we loaded C6 glial cells with carboxyseminaphthorhodafluor (SNARF)-1 and evaluated pH_cyt using spectral imaging microscopy. This approach allowed us to study pH_cyt in discrete cellular domains with high temporal, spatial, and spectral resolution. Because there are differences in the cell microenvironment that may affect the behavior of SNARF-1, we performed in situ titrations in discrete cellular regions of single cells encompassing the somata and filopodia. The in situ titration parameters apparent acid-base dissociation constant (pK'_a), maximum ratio (R_max), and minimum ratio (R_min) had a mean coefficient of variation approximately six times greater than those measured in vitro. Therefore, the individual in situ titration parameters obtained from specific cellular domains were used to estimate the pH_cyt of each region. These studies indicated that glial cells exhibit pH_cyt heterogeneities and pH_cyt oscillations in both the absence and presence of physiological HCO₃^–. The amplitude and frequency of the pH_cyt oscillations were affected by alkalosis, by acidosis, and by inhibitors of the ubiquitous Na⁺/H⁺ exchanger- and HCO₃^–-based H⁺-transporting mechanisms. Optical imaging approaches used in conjunction with BCECF as a pH probe corroborated the existence of pH_cyt oscillations in glial cells. proton gradients; proton waves; carboxyseminaphthorhodafluor-1; 2',7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein