Patch‐Seq Analysis of Dbx1‐derived Respiratory Neurons of the preBötzinger Complex in Neonatal Mice

Abstract

Inspiratory breathing movements depend on neurons in the preBötzinger complex (preBötC) whose progenitors express the transcription factor Dbx1 (henceforth Dbx1 neurons). These Dbx1 preBötC neurons generate both the rhythm and the rudimentary motor output pattern. Electrophysiological properties may differentiate rhythmogenic and pattern-related neurons, but whether two discrete functional classes of Dbx1 preBötC neurons exist remains to be demonstrated. Here, we use the Patch-Seq technique to measure the transcriptomes of individual Dbx1 preBötC neurons. We aim to develop a comprehensive understanding of the molecular components of the inspiratory core oscillator and to investigate whether discrete neuron classes exist therein. We recorded Dbx1 inspiratory neurons in whole-cell patch-clamp mode to assess their electrophysiological properties and then aspirated their cellular contents into the patch electrode for single-cell RNA-sequencing. We sequenced a total of 18 samples: 7 putative rhythm generators, 9 putative output neurons, and 1 that we could not definitely characterize physiologically. We obtained an average of 11,371,215 uniquely aligned paired-end reads per sample with an average length of 151 bp. More than a dozen differentially expressed genes distinguish putatively rhythmogenic and pattern-related Dbx1 preBötC neurons. These data make testable predictions about the composition and function of the mammalian preBötC that advance understanding at the molecular genetic level.