SUMMARYThe intrinsic proton buffering power (/1) of individual rat hippocampal and neocortical neurones maintained in culture has been investigated using the fluorescent dye 2',7'-bis(carboxymethyl)-5,6-carboxyfluorescein) (BCECF). The steady-state intracellular pH (pH,) was estimated to be 7.03 + 0.04 (n = 22) in Hepes-buffered media and /3i estimated from the addition and removal of weak bases was ca 10 mM (pH unit)-' at pHi values near to 7. Estimates of /#j made from butyric acid challenges were inconsistent with estimates made at the same pH, using NH4C1 withdrawal.However, estimating /?j with butyrate in the presence of the monocarboxylate ion transport inhibitor a-cyano-hydroxy-cinnamate (CHC) yielded /, values commensurate with those measured using NH4Cl. Application of CHC alone lead to a rapid fall in pH,, suggesting a significant contribution of the monocarboxylate transporter to pHi regulation. /3i was also estimated from a step increase in extracellular PCo2* This yielded a value of 11 mM at an average pH, of 7 1, which is similar to that of the other estimates reported here. /3j was found to increase with decreasing pH,: each unit drop in pH, increased buffering power by about 60 %. Blockade of pHi regulation did not significantly affect estimates of #3. The change in buffering power with pH could be closely modelled from the known concentrations of free amino acids and organic phosphates.

SUMMARYThe role of bicarbonate as a hydrogen ion buffer has been investigated using the fluorescent dye BCECF in individual rat cerebellar, hippocampal and neocortical neurones maintained in culture.The steady-state intracellular pH (pHi) was estimated to be 7-07 + 0 05 (n = 22) in CO2-HCO3 -buffered media. Buffering power (,8) estimated from the addition and removal of weak bases was ca 10 mM (pH unit)-' and was found to be similar in both CO2-HCO3 -and Hepes-bufered media. The membrane-permeant carbonic anhydrase inhibitor, acetazolamide (10-20,UM), did not affect estimates of ,. The results indicate that CO2-HCO3-does not act as an open buffer system in these neurones.

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