Determination of intracellular pH in the in vitro hippocampal slice preparation by transillumination spectrophotometry of Neutral red

A method is presented for measuring intracellular pH (pHi) spectrophotometrically in hippocampal slices using the pH dye indicator, Neutral red (NR). Measurements of pHi by NR were compared directly with the creatine kinase (CK) equilibrium method. Slices were bathed in artificial cerebrospinal fluid buffered with bicarbonate/CO₂. Intracellular pH in hippocampal slices was found to be more alkaline (approximately 0.3 pH units) than buffer pH and far more alkaline (0.5 pH units) than extracellular pH. Resting intracellular alkalinity was observed by using both the NR and the CK equilibrium methods. The method may be useful for studies of pH regulation in intact functioning tissues in vitro where rapid and repeated measurements are necessary or where cell size precludes measurements with pH-sensitive microelectrodes.