Determination of extracellular bicarbonate and carbon dioxide concentrations in brain slices using carbonate and...

The extracellular pH of the brain is subject to shifts during neural activity. To understand these pH changes, it is necessary to measure [H+], [HCO3-], [CO3(2-)] and [CO2]. In principle, this can be accomplished using CO3(2-) and pH-sensitive microelectrodes; however, interference from HCO3- and Cl-, and physiological changes in [HCO3-], complicate measurements with CO3(2-) electrodes. Calibration requires knowledge of slope response, interference constants and corrections for [HCO3-] shifts. We show that when [HCO3-] is altered at constant [CO2] in the absence of Cl-, the HCO3- interference cancels and the Nikolsky equation reduces to the Nernst equation for CO3(2-). Measurement of CO3(2-) slope response by this method yielded a value of 28.5 +/- 0.72 mV per decade change in [CO3(2-)]. In Cl(-)-containing solutions, interference coefficient for HCO3- and Cl- were determined by altering [HCO3-] at constant [CO2], changing [CO2] at constant [HCO3-], then solving the simultaneous Nikolsky equations for each transition. The mean interference constants corresponded to selectivity ratios of 245:1 and 1150:1 for CO3(2-) over HCO3- and Cl- respectively. To correct for possible changes in [HCO3-], the equilibrium relation between CO3(2-) and HCO3- was substituted into the Nikolsky equation to yield an equation in [CO3(2-)] and [H+]. By simultaneously measuring shifts in [H+] with a pH microelectrode, this equation is readily solved for [CO3(2-)]. These methods were tested by measuring [HCO3-] and [CO2] in experimental solutions, and in the extracellular fluid of rat hippocampal slices.