Immunocytochemical techniques identify Na+‐coupled HCO3− transporters (NCBTs) in chemosensitive neurons of the Medullary Raphé

Abstract

The medullary raphé of the medulla oblongata contains chemosensitive neurons that sense Δ[CO2]. Serotonergic (5HT) neurons increase their firing rate in response to increases in arterial [CO2]. The GABAergic response is a decreased firing rate. Both contribute to increased pulmonary ventilation. The neuronal electroneutral Na/HCO3− cotransporters (nNCBTs) respond to intracellular acid loads (e.g. caused by increased [CO2]) by actively moving HCO3− 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 pHi recovery, sustaining the fall in pHi 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−.