Immunocytochemical techniques identify Na + ‐coupled HCO 3 − transporters (NCBTs) in chemosensitive neurons of the Medullary Raphé

The medullary raphé of the medulla oblongata contains chemosensitive neurons that sense Δ[CO 2 ]. Serotonergic (5HT) neurons increase their firing rate in response to increases in arterial [CO 2 ]. The GABAergic response is a decreased firing rate. Both contribute to increased pulmonary ventilation. The neuronal electroneutral Na/HCO 3 − cotransporters (nNCBTs) respond to intracellular acid loads (e.g. caused by increased [CO 2 ]) by actively moving HCO 3 − into cells to restore the intracellular pH. We hypothesized that nNCBTs are less abundant in serotonergic and GABAergic neurons than in non‐chemosensitive neurons. A reduced nNCBT presence would be expected to slow pH i recovery, sustaining the fall in pH i and maintaining altered firing rates in 5HT and GABA neurons. An immunocytochemistry protocol was developed to distinguish cell types using monoclonal primary antibodies against MAP2, a neuronal marker; TPH, a 5HT neuronal marker; and GAD2, a GABA neuronal marker. The cell type–specific staining was colocalized with polyclonal antibodies against the nNCBTs: NBCn1, NBCn2, and NDCBE. Quantitative image analysis techniques were applied to distinguish positively stained cells from the background signals. Contrary to the hypothesis, the fraction of nNCBT‐positive cells was >80% in all cell populations for all 3 nNCBTs, and not substantially different for TPH + vs TPH − neurons or GAD2 + vs GAD2 − .