Transcriptomes of electrophysiologically recorded Dbx1-derived respiratory neurons of the preBötzinger complex in neonatal mice

Abstract

Breathing depends on interneurons in the preBötzinger complex (preBötC) derived from Dbx1-expressing precursors. Here we investigate whether rhythm- and pattern-generating functions reside in discrete classes of Dbx1 preBötC neurons. In a slice model of breathing with ~ 5 s cycle period, putatively rhythmogenic Type-1 Dbx1 preBötC neurons activate 100–300 ms prior to Type-2 neurons, putatively specialized for output pattern, and 300–500 ms prior to the inspiratory motor output. We sequenced Type-1 and Type-2 transcriptomes and identified differential expression of 123 genes including ionotropic receptors (Gria3, Gabra1) that may explain their preinspiratory activation profiles and Ca2+ signaling (Cracr2a, Sgk1) involved in inspiratory and sigh bursts. Surprisingly, neuropeptide receptors that influence breathing (e.g., µ-opioid and bombesin-like peptide receptors) were only sparsely expressed, which suggests that cognate peptides and opioid drugs exert their profound effects on a small fraction of the preBötC core. These data in the public domain help explain the neural origins of breathing.

Single cell transcriptome sequencing of inspiratory neurons of the preBötzinger complex in neonatal mice

“…3 Box plots showing expression levels of the 8,916 genes expressed in 19 transcriptomes of preBötC neurons from either Kallurkar et al ., 2022 (ref. 25 ) or the 10 samples in the present report. The Kallurkar 2022 data sets are samples 1–9 and 11–18 (grey box plots).…”

“…5 Histogram showing probability of gene expression level for the 8,916 genes expressed in at least 19 transcriptomes of preBötC neurons from either Kallurkar et al ., 2022 (ref. 25 ) (grey solid line) or the 10 samples in the present report (broken black line for samples 1–4 and solid black line for samples 5–10). The heights of the first bin (0.13, 0.05, and 0.09) correspond to the fraction of mm10 genes not expressed in the Kallurkar 2022 and present data sets.…”

“…We tested for A-type K + current ( I A ), characteristic of Type-1 preBötC neurons by applying suprathreshold depolarizing current step commands of 500 ms duration from a holding potential of –70 mV. Type-1 preBötC neurons expressing I A exhibited delayed excitation of 120–220 ms whereas Type-2 preBötC neurons without I A expression do not show delayed excitation in response to similar depolarizing current step commands 16 , 17 , 25 (compare Fig. 2a , middle vs. b, middle).…”

“…Mapping statistics for Kallurkar 2022 are associated with ref. 25 . In brief, 0.85 ± 0.04 represents the fraction of sequences from the present data set mapped uniquely to mm10, 0.05 ± 0.02 represents the multi-mapped fraction, and 0.10 ± 0.03 represents the unmapped fraction.…”

“…It would be difficult to draw conclusions about differential expression between Type-1 and Type-2 Dbx1 preBötC neurons on the basis of a single Type-2 transcriptome. Given the minimal batch effects (see above), one could analyze the Type-2 transcriptome here with the transcriptomes previously disseminated in Kallurkar et al .’s report 25 .…”