Neuromedin B Expression Defines the Mouse Retrotrapezoid Nucleus.

The retrotrapezoid nucleus (RTN) consists, by definition, of Phox2b-expressing, glutamatergic, non-catecholaminergic, noncholinergic neurons located in the parafacial region of the medulla oblongata. An unknown proportion of RTN neurons are central respiratory chemoreceptors and there is mounting evidence for biochemical diversity among these cells. Here, we used multiplexed hybridization and single-cell RNA-Seq in male and female mice to provide a more comprehensive view of the phenotypic diversity of RTN neurons. We now demonstrate that the RTN of mice can be identified with a single and specific marker, mRNA (). Most (∼75%) RTN neurons express low-to-moderate levels of and display chemoreceptor properties. Namely they are activated by hypercapnia, but not by hypoxia, and express proton sensors, TASK-2 and Gpr4. These -low RTN neurons also express varying levels of transcripts for , , and , and receptors for substance P, orexin, serotonin, and ATP. A subset of RTN neurons (∼20-25%), typically larger than average, express very high levels of mRNA. These -high RTN neurons do not express after hypercapnia and have low-to-undetectable levels of or transcripts; they also express , but are essentially devoid of and transcripts. In male rats, is also a marker of the RTN but, unlike in mice, this gene is expressed by other types of nearby neurons located within the ventromedial medulla. In sum, is a selective marker of the RTN in rodents; -low neurons, the vast majority, are central respiratory chemoreceptors, whereas -high neurons likely have other functions. Central respiratory chemoreceptors regulate arterial PCO by adjusting lung ventilation. Such cells have recently been identified within the retrotrapezoid nucleus (RTN), a brainstem nucleus defined by genetic lineage and a cumbersome combination of markers. Using single-cell RNA-Seq and multiplexed hybridization, we show here that a single marker, mRNA (), identifies RTN neurons in rodents. We also suggest that >75% of these neurons are chemoreceptors because they are strongly activated by hypercapnia and express high levels of proton sensors ( and ). The other RTN neurons express very high levels of , but low levels of , and do not respond to hypercapnia. Their function is unknown.