A new approach for analyzing pHi recoveries from NH4+‐induced acid loads, applied to rat hippocampal neurons and astrocytes

Abstract

Regulation of intracellular pH (pHi) in brain cells (neurons and neuroglia) is essential for optimal cell function and primarily maintained by acid/base transporters in the SLC9 and SLC4 families. Large deviations in pHi can lead to disrupted cellular function or even cell death, whereas small changes have been proposed as a signaling method due to the pH‐sensitive residues within protein structure. In the hippocampus (HC), tight regulation of pHi underlies the cascading reactions and impacts the structure of proteins necessary for learning and memory formation. The present work provides a detailed examination of the methods used in the analysis of steady‐state pHi and the recovery from NH4/NH3 induced acidosis by the SLC9s in the absence of CO2/HCO3−, when SLC4 (HCO3−‐sensitive) acid/base transporters are largely inactive. In the present study, we use the pH‐sensitive dye BCECF to examine HC neurons and astrocytes, co‐cultured from embryonic (E18–20) Sprague Dawley rats. In the protocol, two sequential NH4/NH3 pulses are delivered and the steady‐state pHi values is identified before (checkpoint C) and after (E for pulse 1 and F for pulse 2) the NH4/NH3 pulse. Typically, steady‐state pHi values at C were more alkaline than E and F, most likely due to the inactivity of HCO3−‐dependent acid loaders in the SLC4 family. The recovery from acidosis is fit with a double exponential (DExp) which we replot as dpHi/dt vs pHi. With this traditional approach, dpHi/dt, as it approaches the asymptotic pHi, becomes slightly non‐linear. To exploit the mainly linear portion of the dpHi/dt vs. pHi plot (from the DExp fit), we fit these dpHi/dt vs. pHi points with a single exponential (SExp) to produce a quasi–single‐exponential rate constant. This analysis—when transformed to the pHi vs. time domain—generally produces a very good fit to the original pHi vs. time data. We summarize the twin pHi recoveries from individual experiments in thumbnails in which we display the quasi–single‐exponential dpHi/dt line segments that represent the pHi recoveries from the first and second NH3/NH4+ pulses. For each line segment in the thumbnail, the slope represents the quasi–single‐exponential rate constant, and the projected pHi‐axis asymptote represents the predicted new steady‐state pHi. The thumbnails permit a quick assessment of the pHi recoveries from acidosis in many cells, including a sense of the variability between the first and second pulses, or the variability among cells. The advantage to this approach is that our method of analysis allows an entire population of cells to be examined individually but quickly.Support or Funding InformationThis work was supported by the NIH National Heart, Lung, and Blood Institute. Grant # R01 NS018400