Trpm4 ion channels in pre-Bötzinger complex interneurons are essential for breathing motor pattern but not rhythm.

Inspiratory breathing movements depend on pre-Bötzinger complex (preBötC) interneurons that express calcium (Ca2+)-activated nonselective cationic current (ICAN) to generate robust neural bursts. Hypothesized to be rhythmogenic, reducing ICAN 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 ICAN 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 ICAN, whose influence on frequency contradicts a direct role in rhythm generation. We conclude that Trpm4-mediated ICAN is indispensable for motor output but not the rhythmogenic core mechanism of the breathing central pattern generator.

Morphology of Dbx1 respiratory neurons in the preBötzinger complex and reticular formation of neonatal mice.

The relationship between neuron morphology and function is a perennial issue in neuroscience. Information about synaptic integration, network connectivity, and the specific roles of neuronal subpopulations can be obtained through morphological analysis of key neurons within a microcircuit. Here we present morphologies of two classes of brainstem respiratory neurons. First, interneurons derived from Dbx1-expressing precursors (Dbx1 neurons) in the preBötzinger complex (preBötC) of the ventral medulla that generate the rhythm for inspiratory breathing movements. Second, Dbx1 neurons of the intermediate reticular formation that influence the motor pattern of pharyngeal and lingual movements during the inspiratory phase of the breathing cycle. We describe the image acquisition and subsequent digitization of morphologies of respiratory Dbx1 neurons from the preBötC and the intermediate reticular formation that were first recorded in vitro. These data can be analyzed comparatively to examine how morphology influences the roles of Dbx1 preBötC and Dbx1 reticular interneurons in respiration and can also be utilized to create morphologically accurate compartmental models for simulation and modeling of respiratory circuits.

Dbx1 precursor cells are a source of inspiratory XII premotoneurons.

All behaviors require coordinated activation of motoneurons from central command and premotor networks. The genetic identities of premotoneurons providing behaviorally relevant excitation to any pool of respiratory motoneurons remain unknown. Recently, we established in vitro that Dbx1-derived pre-Bötzinger complex neurons are critical for rhythm generation and that a subpopulation serves a premotor function (Wang et al., 2014). Here, we further show that a subpopulation of Dbx1-derived intermediate reticular (IRt) neurons are rhythmically active during inspiration and project to the hypoglossal (XII) nucleus that contains motoneurons important for maintaining airway patency. Laser ablation of Dbx1 IRt neurons, 57% of which are glutamatergic, decreased ipsilateral inspiratory motor output without affecting frequency. We conclude that a subset of Dbx1 IRt neurons is a source of premotor excitatory drive, contributing to the inspiratory behavior of XII motoneurons, as well as a key component of the airway control network whose dysfunction contributes to sleep apnea.

Laser ablation of Dbx1 neurons in the pre-Bötzinger complex stops inspiratory rhythm and impairs output in neonatal mice.

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.

Physiological and morphological properties of Dbx1-derived respiratory neurons in the pre-Botzinger complex of neonatal mice.

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 (IA) and hyperpolarization-activated current (Ih) tended to be mutually exclusive in Dbx1(+) neurons. In contrast, there was no such relationship in the expression of currents IA and Ih in Dbx1(-) neurons. Confocal imaging and digital morphological reconstruction of recorded neurons revealed dendritic spines on Dbx1(-) neurons, but Dbx1(+) neurons were spineless. The morphology of Dbx1(+) neurons was largely confined to the transverse plane, whereas Dbx1(-) neurons projected dendrites to a greater extent in the parasagittal plane. The putative rhythmogenic nature of Dbx1(+) neurons may be attributable, 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.