The preBötzinger complex (preBötC) generates the rhythm and rudimentary motor pattern for inspiratory breathing movements. Here, we test "burstlet" theory (Kam et al., 2013a), which posits that low amplitude burstlets, subthreshold from the standpoint of inspiratory bursts, reflect the fundamental oscillator of the preBötC. In turn, a discrete suprathreshold process transforms burstlets into full amplitude inspiratory bursts that drive motor output, measurable via hypoglossal nerve (XII) discharge in vitro. We recap observations by Kam and Feldman in neonatal mouse slice preparations: field recordings from preBötC demonstrate bursts and concurrent XII motor output intermingled with lower amplitude burstlets that do not produce XII motor output. Manipulations of excitability affect the relative prevalence of bursts and burstlets and modulate their frequency. Whole-cell and photonic recordings of preBötC neurons suggest that burstlets involve inconstant subsets of rhythmogenic interneurons. We conclude that discrete rhythm-and pattern-generating mechanisms coexist in the preBötC and that burstlets reflect its fundamental rhythmogenic nature.

Inspiratory breathing movements depend on pre-Bötzinger complex (preBötC) interneurons that express calcium (Ca 2+ )-activated nonselective cationic current ( I CAN ) to generate robust neural bursts. Hypothesized to be rhythmogenic, reducing I CAN is predicted to slow down or stop breathing; its contributions to motor pattern would be reflected in the magnitude of movements (output). We tested the role(s) of I CAN using reverse genetic techniques to diminish its putative ion channels Trpm4 or Trpc3 in preBötC neurons in vivo. Adult mice transduced with Trpm4-targeted short hairpin RNA (shRNA) progressively decreased the tidal volume of breaths yet surprisingly increased breathing frequency, often followed by gasping and fatal respiratory failure. Mice transduced with Trpc3-targeted shRNA survived with no changes in breathing. Patch-clamp and field recordings from the preBötC in mouse slices also showed an increase in the frequency and a decrease in the magnitude of preBötC neural bursts in the presence of Trpm4 antagonist 9-phenanthrol, whereas the Trpc3 antagonist pyrazole-3 (pyr-3) showed inconsistent effects on magnitude and no effect on frequency. These data suggest that Trpm4 mediates I CAN , whose influence on frequency contradicts a direct role in rhythm generation. We conclude that Trpm4-mediated I CAN is indispensable for motor output but not the rhythmogenic core mechanism of the breathing central pattern generator.

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.