Inspiratory and sigh breathing rhythms depend on distinct cellular signalling mechanisms in the preBötzinger complex.

Breathing behaviour involves the generation of normal breaths (eupnoea) on a timescale of seconds and sigh breaths on the order of minutes. Both rhythms emerge in tandem from a single brainstem site, but whether and how a single cell population can generate two disparate rhythms remains unclear. We posit that recurrent synaptic excitation in concert with synaptic depression and cellular refractoriness gives rise to the eupnoea rhythm, whereas an intracellular calcium oscillation that is slower by orders of magnitude gives rise to the sigh rhythm. A mathematical model capturing these dynamics simultaneously generates eupnoea and sigh rhythms with disparate frequencies, which can be separately regulated by physiological parameters. We experimentally validated key model predictions regarding intracellular calcium signalling. All vertebrate brains feature a network oscillator that drives the breathing pump for regular respiration. However, in air-breathing mammals with compliant lungs susceptible to collapse, the breathing rhythmogenic network may have refashioned ubiquitous intracellular signalling systems to produce a second slower rhythm (for sighs) that prevents atelectasis without impeding eupnoea. KEY POINTS: A simplified activity-based model of the preBötC generates inspiratory and sigh rhythms from a single neuron population. Inspiration is attributable to a canonical excitatory network oscillator mechanism. Sigh emerges from intracellular calcium signalling. The model predicts that perturbations of calcium uptake and release across the endoplasmic reticulum counterintuitively accelerate and decelerate sigh rhythmicity, respectively, which was experimentally validated. Vertebrate evolution may have adapted existing intracellular signalling mechanisms to produce slow oscillations needed to optimize pulmonary function in mammals.

Role of NaV1.6-mediated persistent sodium current and bursting-pacemaker properties in breathing rhythm generation.

Inspiration is the inexorable active phase of breathing. The brainstem pre-Bötzinger complex (preBötC) gives rise to inspiratory neural rhythm, but its underlying cellular and ionic bases remain unclear. The long-standing "pacemaker hypothesis" posits that the persistent Na+ current (INaP) that gives rise to bursting-pacemaker properties in preBötC interneurons is essential for rhythmogenesis. We tested the pacemaker hypothesis by conditionally knocking out and knocking down the Scn8a (Nav1.6 [voltage-gated sodium channel 1.6]) gene in core rhythmogenic preBötC neurons. Deleting Scn8a substantially decreases the INaP and abolishes bursting-pacemaker activity, which slows inspiratory rhythm in vitro and negatively impacts the postnatal development of ventilation. Diminishing Scn8a via genetic interference has no impact on breathing in adult mice. We argue that the Scn8a-mediated INaP is not obligatory but that it influences the development and rhythmic function of the preBötC. The ubiquity of the INaP in respiratory brainstem interneurons could underlie breathing-related behaviors such as neonatal phonation or rhythmogenesis in different physiological conditions.

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

Neurons in the brainstem preBötzinger complex (preBötC) generate the rhythm and rudimentary motor pattern for inspiratory breathing movements. We performed whole-cell patch-clamp recordings from inspiratory neurons in the preBötC of neonatal mouse slices that retain breathing-related rhythmicity in vitro. We classified neurons based on their electrophysiological properties and genetic background, and then aspirated their cellular contents for single-cell RNA sequencing (scRNA-seq). This data set provides the raw nucleotide sequences (FASTQ files) and annotated files of nucleotide sequences mapped to the mouse genome (mm10 from Ensembl), which includes the fragment counts, gene lengths, and fragments per kilobase of transcript per million mapped reads (FPKM). These data reflect the transcriptomes of the neurons that generate the rhythm and pattern for inspiratory breathing movements.

Breathing Rhythm and Pattern and Their Influence on Emotion.

Breathing is a vital rhythmic motor behavior with a surprisingly broad influence on the brain and body. The apparent simplicity of breathing belies a complex neural control system, the breathing central pattern generator (bCPG), that exhibits diverse operational modes to regulate gas exchange and coordinate breathing with an array of behaviors. In this review, we focus on selected advances in our understanding of the bCPG. At the core of the bCPG is the preBötzinger complex (preBötC), which drives inspiratory rhythm via an unexpectedly sophisticated emergent mechanism. Synchronization dynamics underlying preBötC rhythmogenesis imbue the system with robustness and lability. These dynamics are modulated by inputs from throughout the brain and generate rhythmic, patterned activity that is widely distributed. The connectivity and an emerging literature support a link between breathing, emotion, and cognition that is becoming experimentally tractable. These advances bring great potential for elucidating function and dysfunction in breathing and other mammalian neural circuits.

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

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.

KCNQ Current Contributes to Inspiratory Burst Termination in the Pre-Bötzinger Complex of Neonatal Rats in vitro.

The pre-Bötzinger complex (preBötC) of the ventral medulla generates the mammalian inspiratory breathing rhythm. When isolated in explants and deprived of synaptic inhibition, the preBötC continues to generate inspiratory-related rhythm. Mechanisms underlying burst generation have been investigated for decades, but cellular and synaptic mechanisms responsible for burst termination have received less attention. KCNQ-mediated K+ currents contribute to burst termination in other systems, and their transcripts are expressed in preBötC neurons. Therefore, we tested the hypothesis that KCNQ channels also contribute to burst termination in the preBötC. We recorded KCNQ-like currents in preBötC inspiratory neurons in neonatal rat slices that retain respiratory rhythmicity. Blocking KCNQ channels with XE991 or linopirdine (applied via superfusion or locally) increased inspiratory burst duration by 2- to 3-fold. By contrast, activation of KCNQ with retigabine decreased inspiratory burst duration by ~35%. These data from reduced preparations suggest that the KCNQ current in preBötC neurons contributes to inspiratory burst termination.

Role of Synaptic Inhibition in the Coupling of the Respiratory Rhythms that Underlie Eupnea and Sigh Behaviors.

The preBötzinger complex (preBötC) gives rise to two types of breathing behavior under normal physiological conditions: eupnea and sighing. Here, we examine the neural mechanisms that couple their underlying rhythms. We measured breathing in awake intact adult mice and recorded inspiratory rhythms from the preBötC in neonatal mouse brainstem slice preparations. We show previously undocumented variability in the temporal relationship between sigh breaths or bursts and their preceding eupneic breaths or inspiratory bursts. Investigating the synaptic mechanisms for this variability in vitro, we further show that pharmacological blockade of chloride-mediated synaptic inhibition strengthens inspiratory-to-sigh temporal coupling. These findings contrast with previous literature, which suggested glycinergic inhibition linked sigh bursts to their preceding inspiratory bursts with minimal time intervals. Furthermore, we verify that pharmacological disinhibition did not alter the duration of the prolonged interval that follows a sigh burst before resumption of the inspiratory rhythm. These results demonstrate that synaptic inhibition does not enhance coupling between sighs and preceding inspiratory events or contribute to post-sigh apneas. Instead, we conclude that excitatory synaptic mechanisms coordinate inspiratory (eupnea) and sigh rhythms.

Evaluating the Burstlet Theory of Inspiratory Rhythm and Pattern Generation.

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.

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.

Breathing matters.

Breathing is a well-described, vital and surprisingly complex behaviour, with behavioural and physiological outputs that are easy to directly measure. Key neural elements for generating breathing pattern are distinct, compact and form a network amenable to detailed interrogation, promising the imminent discovery of molecular, cellular, synaptic and network mechanisms that give rise to the behaviour. Coupled oscillatory microcircuits make up the rhythmic core of the breathing network. Primary among these is the preBötzinger Complex (preBötC), which is composed of excitatory rhythmogenic interneurons and excitatory and inhibitory pattern-forming interneurons that together produce the essential periodic drive for inspiration. The preBötC coordinates all phases of the breathing cycle, coordinates breathing with orofacial behaviours and strongly influences, and is influenced by, emotion and cognition. Here, we review progress towards cracking the inner workings of this vital core.

Dbx1 Pre-Bötzinger Complex Interneurons Comprise the Core Inspiratory Oscillator for Breathing in Unanesthetized Adult Mice.

The brainstem pre-Bötzinger complex (preBötC) generates inspiratory breathing rhythms, but which neurons comprise its rhythmogenic core? Dbx1-derived neurons may play the preeminent role in rhythm generation, an idea well founded at perinatal stages of development but incompletely evaluated in adulthood. We expressed archaerhodopsin or channelrhodopsin in Dbx1 preBötC neurons in intact adult mice to interrogate their function. Prolonged photoinhibition slowed down or stopped breathing, whereas prolonged photostimulation sped up breathing. Brief inspiratory-phase photoinhibition evoked the next breath earlier than expected, whereas brief expiratory-phase photoinhibition delayed the subsequent breath. Conversely, brief inspiratory-phase photostimulation increased inspiratory duration and delayed the subsequent breath, whereas brief expiratory-phase photostimulation evoked the next breath earlier than expected. Because they govern the frequency and precise timing of breaths in awake adult mice with sensorimotor feedback intact, Dbx1 preBötC neurons constitute an essential core component of the inspiratory oscillator, knowledge directly relevant to human health and physiology.

Dendritic A-Current in Rhythmically Active PreBötzinger Complex Neurons in Organotypic Cultures from Newborn Mice.

The brainstem preBötzinger complex (preBötC) generates the inspiratory rhythm for breathing. The onset of neural activity that precipitates the inspiratory phase of the respiratory cycle may depend on the activity of type-1 preBötC neurons, which exhibit a transient outward K+ current, IA Inspiratory rhythm generation can be studied ex vivo because the preBötC remains rhythmically active in vitro, both in acute brainstem slices and organotypic cultures. Advantageous optical conditions in organotypic slice cultures from newborn mice of either sex allowed us to investigate how IA impacts Ca2+ transients occurring in the dendrites of rhythmically active type-1 preBötC neurons. The amplitude of dendritic Ca2+ transients evoked via voltage increases originating from the soma significantly increased after an IA antagonist, 4-aminopyridine (4-AP), was applied to the perfusion bath or to local dendritic regions. Similarly, glutamate-evoked postsynaptic depolarizations recorded at the soma increased in amplitude when 4-AP was coapplied with glutamate at distal dendritic locations. We conclude that IA is expressed on type-1 preBötC neuron dendrites. We propose that IA filters synaptic input, shunting sparse excitation, while enabling temporally summated events to pass more readily as a result of IA inactivation. Dendritic IA in rhythmically active preBötC neurons could thus ensure that inspiratory motor activity does not occur until excitatory synaptic drive is synchronized and well coordinated among cellular constituents of the preBötC during inspiratory rhythmogenesis. The biophysical properties of dendritic IA might thus promote robustness and regularity of breathing rhythms.SIGNIFICANCE STATEMENT Brainstem neurons in the preBötC generate the oscillatory activity that underlies breathing. PreBötC neurons express voltage-dependent currents that can influence inspiratory activity, among which is a transient potassium current (IA) previously identified in a rhythmogenic excitatory subset of type-1 preBötC neurons. We sought to determine whether IA is expressed in the dendrites of preBötC. We found that dendrites of type-1 preBötC neurons indeed express IA, which may aid in shunting sparse non-summating synaptic inputs, while enabling strong summating excitatory inputs to readily pass and thus influence somatic membrane potential trajectory. The subcellular distribution of IA in rhythmically active neurons of the preBötC may thus be critical for producing well coordinated ensemble activity during inspiratory burst formation.

Transcriptome of neonatal preBötzinger complex neurones in Dbx1 reporter mice.

We sequenced the transcriptome of brainstem interneurons in the specialized respiratory rhythmogenic site dubbed preBötzinger Complex (preBötC) from newborn mice. To distinguish molecular characteristics of the core oscillator we compared preBötC neurons derived from Dbx1-expressing progenitors that are respiratory rhythmogenic to neighbouring non-Dbx1-derived neurons, which support other respiratory and non-respiratory functions. Results in three categories are particularly salient. First, Dbx1 preBötC neurons express κ-opioid receptors in addition to µ-opioid receptors that heretofore have been associated with opiate respiratory depression, which may have clinical applications. Second, Dbx1 preBötC neurons express the hypoxia-inducible transcription factor Hif1a at levels three-times higher than non-Dbx1 neurons, which links core rhythmogenic microcircuits to O2-related chemosensation for the first time. Third, we detected a suite of transcription factors including Hoxa4 whose expression pattern may define the rostral preBötC border, Pbx3 that may influence ipsilateral connectivity, and Pax8 that may pertain to a ventrally-derived subset of Dbx1 preBötC neurons. These data establish the transcriptomic signature of the core respiratory oscillator at a perinatal stage of development.

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.

Fate mapping neurons and glia derived from Dbx1-expressing progenitors in mouse preBötzinger complex.

The brainstem preBötzinger complex (preBötC) generates the inspiratory breathing rhythm, and its core rhythmogenic interneurons are derived from Dbx1-expressing progenitors. To study the neural bases of breathing, tamoxifen-inducible Cre-driver mice and Cre-dependent reporters are used to identify, record, and perturb Dbx1 preBötC neurons. However, the relationship between tamoxifen administration and reporter protein expression in preBötC neurons and glia has not been quantified. To address this problem, we crossed mice that express tamoxifen-inducible Cre recombinase under the control of the Dbx1 gene (Dbx1CreERT2) with Cre-dependent fluorescent reporter mice (Rosa26tdTomato), administered tamoxifen at different times during development, and analyzed tdTomato expression in the preBötC of their offspring. We also crossed Rosa26tdTomato reporters with mice that constitutively express Cre driven by Dbx1 (Dbx1Cre) and analyzed tdTomato expression in the preBötC of their offspring for comparison. We show that Dbx1-expressing progenitors give rise to preBötC neurons and glia. Peak neuronal tdTomato expression occurs when tamoxifen is administered at embryonic day 9.5 (E9.5), whereas tdTomato expression in glia shows no clear relationship with tamoxifen timing. These results can be used to bias reporter protein expression in neurons (or glia). Tamoxifen administration at E9.5 labels 91% of Dbx1-derived neurons in the preBötC, yet only 48% of Dbx1-derived glia. By fate mapping Dbx1-expressing progenitors, this study illustrates the developmental assemblage of Dbx1-derived cells in preBötC, which can be used to design intersectional Cre/lox experiments that interrogate its cellular composition, structure, and function.

Transient Suppression of Dbx1 PreBötzinger Interneurons Disrupts Breathing in Adult Mice.

Interneurons derived from Dbx1-expressing precursors located in the brainstem preBötzinger complex (preBötC) putatively form the core oscillator for inspiratory breathing movements. We tested this Dbx1 core hypothesis by expressing archaerhodopsin in Dbx1-derived interneurons and then transiently hyperpolarizing these neurons while measuring respiratory rhythm in vitro or breathing in vagus-intact adult mice. Transient illumination of the preBötC interrupted inspiratory rhythm in both slice preparations and sedated mice. In awake mice, light application reduced breathing frequency and prolonged the inspiratory duration. Support for the Dbx1 core hypothesis previously came from embryonic and perinatal mouse experiments, but these data suggest that Dbx1-derived preBötC interneurons are rhythmogenic in adult mice too. The neural origins of breathing behavior can be attributed to a localized and genetically well-defined interneuron population.

Functional Interactions between Mammalian Respiratory Rhythmogenic and Premotor Circuitry.

UNLABELLED: Breathing in mammals depends on rhythms that originate from the preBötzinger complex (preBötC) of the ventral medulla and a network of brainstem and spinal premotor neurons. The rhythm-generating core of the preBötC, as well as some premotor circuits, consist of interneurons derived from Dbx1-expressing precursors (Dbx1 neurons), but the structure and function of these networks remain incompletely understood. We previously developed a cell-specific detection and laser ablation system to interrogate respiratory network structure and function in a slice model of breathing that retains the preBötC, the respiratory-related hypoglossal (XII) motor nucleus and XII premotor circuits. In spontaneously rhythmic slices, cumulative ablation of Dbx1 preBötC neurons decreased XII motor output by ∼50% after ∼15 cell deletions, and then decelerated and terminated rhythmic function altogether as the tally increased to ∼85 neurons. In contrast, cumulatively deleting Dbx1 XII premotor neurons decreased motor output monotonically but did not affect frequency nor stop XII output regardless of the ablation tally. Here, we couple an existing preBötC model with a premotor population in several topological configurations to investigate which one may replicate the laser ablation experiments best. If the XII premotor population is a "small-world" network (rich in local connections with sparse long-range connections among constituent premotor neurons) and connected with the preBötC such that the total number of incoming synapses remains fixed, then the in silico system successfully replicates the in vitro laser ablation experiments. This study proposes a feasible configuration for circuits consisting of Dbx1-derived interneurons that generate inspiratory rhythm and motor pattern. SIGNIFICANCE STATEMENT: To produce a breathing-related motor pattern, a brainstem core oscillator circuit projects to a population of premotor interneurons, but the assemblage of this network remains incompletely understood. Here we applied network modeling and numerical simulation to discover respiratory circuit configurations that successfully replicate photonic cell ablation experiments targeting either the core oscillator or premotor network, respectively. If premotor neurons are interconnected in a so-called "small-world" network with a fixed number of incoming synapses balanced between premotor and rhythmogenic neurons, then our simulations match their experimental benchmarks. These results provide a framework of experimentally testable predictions regarding the rudimentary structure and function of respiratory rhythm- and pattern-generating circuits in the brainstem of mammals.

Organotypic slice cultures containing the preBötzinger complex generate respiratory-like rhythms.

Study of acute brain stem slice preparations in vitro has advanced our understanding of the cellular and synaptic mechanisms of respiratory rhythm generation, but their inherent limitations preclude long-term manipulation and recording experiments. In the current study, we have developed an organotypic slice culture preparation containing the preBötzinger complex (preBötC), the core inspiratory rhythm generator of the ventrolateral brain stem. We measured bilateral synchronous network oscillations, using calcium-sensitive fluorescent dyes, in both ventrolateral (presumably the preBötC) and dorsomedial regions of slice cultures at 7-43 days in vitro. These calcium oscillations appear to be driven by periodic bursts of inspiratory neuronal activity, because whole cell recordings from ventrolateral neurons in culture revealed inspiratory-like drive potentials, and no oscillatory activity was detected from glial fibrillary associated protein-expressing astrocytes in cultures. Acute slices showed a burst frequency of 10.9 ± 4.2 bursts/min, which was not different from that of brain stem slice cultures (13.7 ± 10.6 bursts/min). However, slice cocultures that include two cerebellar explants placed along the dorsolateral border of the brainstem displayed up to 193% faster burst frequency (22.4 ± 8.3 bursts/min) and higher signal amplitude (340%) compared with acute slices. We conclude that preBötC-containing slice cultures retain inspiratory-like rhythmic function and therefore may facilitate lines of experimentation that involve extended incubation (e.g., genetic transfection or chronic drug exposure) while simultaneously being amenable to imaging and electrophysiology at cellular, synaptic, and network levels.

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.

Mechanisms Leading to Rhythm Cessation in the Respiratory PreBötzinger Complex Due to Piecewise Cumulative Neuronal Deletions

The mammalian breathing rhythm putatively originates from Dbx1-derived interneurons in the preBötzinger complex (preBötC) of the ventral medulla. Cumulative deletion of ∼15% of Dbx1 preBötC neurons in an in vitro breathing model stops rhythmic bursts of respiratory-related motor output. Here we assemble in silico models of preBötC networks using random graphs for structure, and ordinary differential equations for dynamics, to examine the mechanisms responsible for the loss of spontaneous respiratory rhythm and motor output measured experimentally in vitro. Model networks subjected to cellular ablations similarly discontinue functionality. However, our analyses indicate that model preBötC networks remain topologically intact even after rhythm cessation, suggesting that dynamics coupled with structural properties of the underlying network are responsible for rhythm cessation. Simulations show that cumulative cellular ablations diminish the number of neurons that can be recruited to spike per unit time. When the recruitment rate drops below 1 neuron/ms the network stops spontaneous rhythmic activity. Neurons that play pre-eminent roles in rhythmogenesis include those that commence spiking during the quiescent phase between respiratory bursts and those with a high number of incoming synapses, which both play key roles in recruitment, i.e., recurrent excitation leading to network bursts. Selectively ablating neurons with many incoming synapses impairs recurrent excitation and stops spontaneous rhythmic activity and motor output with lower ablation tallies compared with random deletions. This study provides a theoretical framework for the operating mechanism of mammalian central pattern generator networks and their susceptibility to loss-of-function in the case of disease or neurodegeneration.