JP‐RL‐2023‐285396: Reply to ‘Letter to the Editor’ (JP‐LE‐2023‐285385)

We thankDrsGourine and Theparambil for their interest in our recent work (Li et al., 2023) and especially for pointing out the error in the title, which has been corrected in the final version of the paper. In this paper, we sought to eliminate the NBCe1 Na+–HCO3− cotransporter specifically from astrocytes and thereby test its hypothesized role in CO2-stimulated breathing. To this end, we obtained mice with floxed Scl4a4 alleles and crossed them into two different astrocyte-selective Cre mouse lines, GFAP-Cre and Aldh1l1-CreERT2; for the latter, Cre+ adult mice were treated with tamoxifen to trigger the gene recombination. The results were clear; there was no discernible difference in CO2-stimulated breathing between control groups from each mouse line and their corresponding conditional knockout littermates. In their letter, Drs Gourine and Theparambil make several observations with respect to the experimental results and our description of those findings. These comments can be summarized essentially as the rote criticisms that accompany studies using conditional knockout models, especially in cases when ‘negative’ results fail to implicate the gene targeted for elimination. Thus, they raise concerns that deletion of NBCe1 was incomplete and/or that some astrocytes might have retained residual transporter function. Of course, we recognize these common issues with conditional mouse models and the general difficulties of ‘proving the negative’, and therefore acknowledged these caveats and limitations explicitly in our own discussion of the results. In short, we concede that it is not possible to conclude that there was a complete loss of NBCe1 function in all brainstem astrocytes in either of the mouse models we used; we think this should be obvious from the text. That said, we provide multiple lines of evidence – molecular and electrophysiological – for NBCe1 depletion in astrocytes, and the accompanying letter takes issue with specific experimental findings that we will address here. First, they note that we stated, but did not show evidence for, a “more widespread deletion of NBCe1 in brainstem astrocytes by using tamoxifen-treated Aldh1l1-Cre/ERT2;Slc4a4fl/fl mice”. Here, we present RNAscope data for Slc4a4 expression in two brainstem regions from control and tamoxifen-treated Cre-positive Aldh1l1-Cre/ERT2;Slc4a4fl/fl mice (Fig. 1). As evident in these images, Slc4a4 transcripts were essentially eliminated in astrocytes of the Aldh1l1-Cre conditional knockout mice; this finding was representative of the brainstem more broadly and supports the widespread astrocytic NBCe1 deletion we reported. We trust these data will allay this concern. We also directly recorded HCO3−-activated whole-cell currents from astrocytes in the region of the retrotrapezoid nucleus in brainstem slices obtained from the GFAP-Cre line and attributed those to NBCe1 based on their sensitivity to S0859. In the letter, this S0859 sensitivity was deemed unconvincing by virtue of the timing of our drug application and a comparison to the time course of decrementing HCO3−-activated currents in recordings from cultured rat astrocytes obtained by a different laboratory in different experimental conditions. We cannot account for the characteristics of NBCe1 currents obtained by that other group, but we found both smaller peak HCO3−-activated currents and smaller S0859-sensitive currents in Cre-positive mice (Li et al., 2023: see our fig. 1K). Furthermore, even if we accept the argument that large and decrementing HCO3−-activated currents are expected from NBCe1 (in the absence of S0859), we note that those decrementing currents were also not observed in astrocyte recordings from Cre+ mice (Li et al., 2023: see our fig. 1J, right), again consistent with reduced NBCe1 expression. The letter also suggests that measures of intracellular pH might have provided another useful readout of NBCe1 function. Although that is certainly true, those data could be influenced by the many other factors that serve to regulate intracellular pH and are thus less direct than the S0859-sensitive NBCe1 currents we obtained. We do not find these specific concerns compelling. Finally, the letter ends by bringing in a discussion of a related paper that appeared almost simultaneously with our own and which nicely used global deletion of NBCe1-B/C isoforms (in so-called KOb/c mice) to examine effects on ventilatory and renal responses to a metabolic acidosis (MAc) challenge (Brady et al., 2023). Most importantly, and as the letter acknowledges, that paper did not directly test CO2-stimulated breathing, making direct comparisons difficult with respect to our key observations. Moreover, Brady et al. (2023) also caution, correctly, that the modest deficit in stimulation of ventilation (V̇E) for the global KOb/c mice, observed during the first day of an MAc challenge, could reflect disturbances ‘at other sites’. Brady et al. (2023) noted that KOb/c mice had higher baseline V̇E that was attributable entirely to elevated tidal volume (VT), as we also observed for Aldh1l1-Cre conditional knockoutmice inwhichNBCe1was deleted from the kidney. In our case, the increased VT could be attributed to a chronic baseline MAc that was compensated, in part, by a respiratory alkalosis, as demonstrated with arterial blood gas measurements. In the KOb/c mice, assessment of venous, not arterial, blood gases suggested a fully compensated MAc, with higher baseline V̇E and lower baseline PCO2. The compensatory development of respiratory alkalosis implies functional respiratory chemoreception in both the conditional and global knockout NBCe1 models. We submit that the impaired ventilatory response to a superimposed MAc challenge in the KOb/c mice, observed in the context of a compensated MAc at baseline, might have been attributable to a ceiling effect for MAc stimulation of VT and V̇E rather than to some disruption in respiratory chemosensitivity per se. [Parenthetically, in their letter, Drs Gourine and Theparambil evidently favour the conclusions from the paper by Brady et al. (2023) and do not seem particularly concerned about the absence of any functional validation presented at the cellular level of the NBCe1 knockout in astrocytes of the KOb/c mice. ] In summary, we acknowledge (again) the general caveats and limitations of our work with these conditional mouse models and recognize the difficulty in obtaining definitive proof for a ‘negative result’ despite our numerous validation studies. Beyond our study, we take some additional reassurance from recent work with yet another conditional astrocyte NBCe1 knockout model, based on a