Immunocytochemical identification of electroneutral Na⁺-coupled HCO₃⁻ transporters in freshly dissociated mouse medullary raphé neurons.

The medullary raphé (MR) of the medulla oblongata contains chemosensitive neurons that respond to increases in arterial [CO₂], by altering firing rate, with increases being associated with serotonergic (5-hydroxytryptamine [5HT]) neurons and decreases, with GABAergic neurons. Both types of neurons contribute to increased alveolar ventilation. Decreases in intracellular pH are thought to link the rise in [CO₂] to increased ventilation. Because electroneutral Na(+)-coupled HCO₃(-) transporters (nNCBTs), which help protect cells from intracellular acidosis, are expressed robustly in the neurons of the central nervous system, a key question is whether these transporters are present in chemosensitive neurons. Therefore, we used an immunocytochemistry approach to identify neurons (using a microtubule associated protein-2 monoclonal antibody) and specifically 5HT neurons (TPH monoclonal antibody) or GABAergic neurons (GAD2 monoclonal antibody) in freshly dissociated cells from the mouse MR. We also co-labeled with polyclonal antibodies against the three nNCBTs: NBCn1, NDCBE, and NBCn2. We exploited ePet-EYFP (enhanced yellow fluorescent protein) mice (with EYFP-labeled 5HT neurons) as well as mice genetically deficient in each of the three nNCBTs. Quantitative image analysis distinguished positively stained cells from background signals. We found that >80% of GAD2(+) cells also were positive for NDCBE, and >90% of the TPH(+) and GAD2(+) cells were positive for the other nNCBTs. Assuming that the transporters are independently distributed among neurons, we can conclude that virtually all chemosensitive MR neurons contain at least one nNCBT. Published by Elsevier Ltd.