Transcriptomes of electrophysiologically recorded Dbx1-derived respiratory neurons of the preB\xf6tzinger complex in neonatal mice

Prajkta S Kallurkar,Yae K Sugimura,Christopher A Del Negro,Gregory D Conradi Smith,Maria Cristina D Picardo,Margaret S Saha

doi:10.1038/s41598-022-06834-z

Prajkta S Kallurkar, Yae K Sugimura + Show 4 more

Open Access

https://doi.org/10.1038/s41598-022-06834-z

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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.

Highlights

Inspiration, the preeminent active phase of breathing, originates in the preBötzinger complex of the lower brainstem[1,2]
We analyzed Dbx[1] preBötzinger complex (preBötC) neurons using Patch-Seq[18], which entails whole-cell patch-clamp recording followed by next-generation sequencing (Supplementary Fig. 1A) and bioinformatics (Supplementary Fig. 1B)
We focused on measuring the intrinsic membrane properties i.e., delayed excitation and sag potentials, at the expense of recording fewer inspiratory burst cycles

Summary

Introduction

Inspiration, the preeminent active phase of breathing, originates in the preBötzinger complex (preBötC) of the lower brainstem[1,2]. Inspiration begins with a low amplitude preinspiratory phase attributable solely to rhythmogenic neurons. As their activity crosses threshold, preinspiration leads to an inexorable high amplitude inspiratory burst, which recruits an additional class of pattern-related neurons that drive motor output[2,11,12,13]. SSTexpressing (SST+) preBötC neurons discharge during inspiration and postinspiration, i.e., the output phases of the inspiratory breathing cycle, rather than during the rhythmogenic preinspiratory phase. Photostimulation in the preBötC of adult mice that express channelrhodopsin (ChR2) in S ST+ neurons preferentially affects inspiratory motor pattern[6]. Type-1 neurons express A-type transient K+ current (IA) whose blockade perturbs preinspiratory activity and destabilizes the inspiratory rhythm in vitro[16]. Type-2 neurons express hyperpolarization-activated cationic current (Ih)[14] whose blockade profoundly affects motor output with mild effects on rhythm[17]

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