To understand the neural origins of rhythmic behavior one must characterize the central pattern generator circuit and quantify the population size needed to sustain functionality. Breathing-related interneurons of the brainstem pre-Bötzinger complex (preBötC) that putatively comprise the core respiratory rhythm generator in mammals are derived from Dbx1-expressing precursors. Here, we show that selective photonic destruction of Dbx1 preBötC neurons in neonatal mouse slices impairs respiratory rhythm but surprisingly also the magnitude of motor output; respiratory hypoglossal nerve discharge decreased and its frequency steadily diminished until rhythm stopped irreversibly after 85±20 (mean ± SEM) cellular ablations, which corresponds to ∼15% of the estimated population. These results demonstrate that a single canonical interneuron class generates respiratory rhythm and contributes in a premotor capacity, whereas these functions are normally attributed to discrete populations. We also establish quantitative cellular parameters that govern network viability, which may have ramifications for respiratory pathology in disease states.DOI: http://dx.doi.org/10.7554/eLife.03427.001

Key points• The transcription factor Dbx1 gives rise to putatively respiratory rhythm-generating neurons in the pre-Bötzinger complex. Comparative analysis of Dbx1-derived (Dbx1 + ) and non-Dbx1-derived (Dbx1 − ) neurons can help elucidate the cellular bases of respiratory rhythm generation. • In vitro, Dbx1+ neurons activate earlier in the respiratory cycle, discharge larger magnitude inspiratory bursts and exhibit a lower rheobase compared with Dbx1 − neurons.+ neurons tend to express the intrinsic currents I A (transient outward A-current) and I h (hyperpolarization-activated current) in diametric opposition, which may facilitate temporal summation of excitatory synaptic inputs, whereas the Dbx1 − neurons show no significant pattern of expression regarding I A and I h .• The Dbx1 + neurons exhibit smooth, spineless dendrites that project in the transverse plane, whereas the Dbx1 − neurons are confined to the transverse plane to a lesser extent and sometimes exhibit spines.• The properties of Dbx1 + neurons that may contribute to respiratory rhythmogenesis include a high level of excitability linked to ongoing network activity and dendritic properties that may facilitate synaptic integration.Abstract Breathing in mammals depends on an inspiratory-related rhythm that is generated by glutamatergic neurons in the pre-Bötzinger complex (preBötC) of the lower brainstem. A substantial subset of putative rhythm-generating preBötC neurons derive from a single genetic line that expresses the transcription factor Dbx1, but the cellular mechanisms of rhythmogenesis remain incompletely understood. To elucidate these mechanisms, we carried out a comparative analysis of Dbx1-expressing neurons (Dbx1 + ) and non-Dbx1-derived (Dbx1 − ) neurons in the preBötC. Whole-cell recordings in rhythmically active newborn mouse slice preparations showed that Dbx1 + neurons activate earlier in the respiratory cycle and discharge greater magnitude inspiratory bursts compared with Dbx1 − neurons. Furthermore, Dbx1 + neurons required less input current to discharge spikes (rheobase) in the context of network activity. The expression of intrinsic membrane properties indicative of A-current (I A ) and hyperpolarization-activated current (I h ) tended to be mutually exclusive in Dbx1 + neurons. In contrast, there was no such relationship in the expression of currents I A and I h in Dbx1 − neurons. Confocal imaging and digital morphological reconstruction of recorded neurons revealed dendritic spines on Dbx1 in part, to a higher level of intrinsic excitability in the context of network synaptic activity. Furthermore, Dbx1 + neuronal morphology may facilitate temporal summation and integration of local synaptic inputs from other Dbx1 + neurons, taking place largely in the dendrites, which could be important for initiating and maintaining bursts and synchronizing activity during the inspiratory phase.

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.