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.

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.

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.

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.

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.

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.

Understanding the rhythm of breathing: so near, yet so far.

Breathing is an essential behavior that presents a unique opportunity to understand how the nervous system functions normally, how it balances inherent robustness with a highly regulated lability, how it adapts to both rapidly and slowly changing conditions, and how particular dysfunctions result in disease. We focus on recent advancements related to two essential sites for respiratory rhythmogenesis: (a) the preBötzinger Complex (preBötC) as the site for the generation of inspiratory rhythm and (b) the retrotrapezoid nucleus/parafacial respiratory group (RTN/pFRG) as the site for the generation of active expiration.

Synaptic Depression Influences Inspiratory-Expiratory Phase Transition in Dbx1 Interneurons of the preBötzinger Complex in Neonatal Mice.

The brainstem preBötzinger complex (preBötC) generates the rhythm underlying inspiratory breathing movements and its core interneurons are derived from Dbx1-expressing precursors. Recurrent synaptic excitation is required to initiate inspiratory bursts, but whether excitatory synaptic mechanisms also contribute to inspiratory-expiratory phase transition is unknown. Here, we examined the role of short-term synaptic depression using a rhythmically active neonatal mouse brainstem slice preparation. We show that afferent axonal projections to Dbx1 preBötC neurons undergo activity-dependent depression and we identify a refractory period (∼2 s) after endogenous inspiratory bursts that precludes light-evoked bursts in channelrhodopsin-expressing Dbx1 preBötC neurons. We demonstrate that the duration of the refractory period-but neither the cycle period nor the magnitude of endogenous inspiratory bursts-is sensitive to changes in extracellular Ca(2+). Further, we show that postsynaptic factors are unlikely to explain the refractory period or its modulation by Ca(2+). Our findings are consistent with the hypothesis that short-term synaptic depression in Dbx1 preBötC neurons influences the inspiratory-expiratory phase transition during respiratory rhythmogenesis. SIGNIFICANCE STATEMENT: Theories of breathing's neural origins have heretofore focused on intrinsically bursting "pacemaker" cells operating in conjunction with synaptic inhibition for phase transition and cycle timing. However, contemporary studies falsify an obligatory role for pacemaker-like neurons and synaptic inhibition, giving credence to burst-generating mechanisms based on recurrent excitation among glutamatergic interneurons of the respiratory kernel. Here, we investigated the role of short-term synaptic depression in inspiratory-expiratory phase transition. Until now, this role remained an untested prediction of mathematical models. The present data emphasize that synaptic properties of excitatory interneurons of the respiratory rhythmogenic kernel, derived from Dbx1-expressing precursors, may provide the core logic underlying the rhythm for breathing.

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.

Cumulative lesioning of respiratory interneurons disrupts and precludes motor rhythms in vitro.

How brain functions degenerate in the face of progressive cell loss is an important issue that pertains to neurodegenerative diseases and basic properties of neural networks. We developed an automated system that uses two-photon microscopy to detect rhythmic neurons from calcium activity, and then individually laser ablates the targets while monitoring network function in real time. We applied this system to the mammalian respiratory oscillator located in the pre-Bötzinger Complex (preBötC) of the ventral medulla, which spontaneously generates breathing-related motor activity in vitro. Here, we show that cumulatively deleting preBötC neurons progressively decreases respiratory frequency and the amplitude of motor output. On average, the deletion of 120 ± 45 neurons stopped spontaneous respiratory rhythm, and our data suggest ≈82% of the rhythm-generating neurons remain unlesioned. Cumulative ablations in other medullary respiratory regions did not affect frequency but diminished the amplitude of motor output to a lesser degree. These results suggest that the preBötC can sustain insults that destroy no more than ≈18% of its constituent interneurons, which may have implications for the onset of respiratory pathologies in disease states.

Axon targeting of the alpha 7 nicotinic receptor in developing hippocampal neurons by Gprin1 regulates growth.

Cholinergic signaling plays an important role in regulating the growth and regeneration of axons in the nervous system. The α7 nicotinic receptor (α7) can drive synaptic development and plasticity in the hippocampus. Here, we show that activation of α7 significantly reduces axon growth in hippocampal neurons by coupling to G protein-regulated inducer of neurite outgrowth 1 (Gprin1), which targets it to the growth cone. Knockdown of Gprin1 expression using RNAi is found sufficient to abolish the localization and calcium signaling of α7 at the growth cone. In addition, an α7/Gprin1 interaction appears intimately linked to a Gαo, growth-associated protein 43, and CDC42 cytoskeletal regulatory pathway within the developing axon. These findings demonstrate that α7 regulates axon growth in hippocampal neurons, thereby likely contributing to synaptic formation in the developing brain.

Automated cell-specific laser detection and ablation of neural circuits in neonatal brain tissue.

A key feature of neurodegenerative disease is the pathological loss of neurons that participate in generating behaviour. To investigate network properties of neural circuits and provide a complementary tool to study neurodegeneration in vitro or in situ, we developed an automated cell-specific laser detection and ablation system. The instrument consists of a two-photon and visible-wavelength confocal imaging setup, controlled by executive software, that identifies neurons in preparations based on genetically encoded fluorescent proteins or Ca(2+) imaging, and then sequentially ablates cell targets while monitoring network function concurrently. Pathological changes in network function can be directly attributed to ablated cells, which are logged in real time. Here, we investigated brainstem respiratory circuits to demonstrate single-cell precision in ablation during physiological network activity, but the technique could be applied to interrogate network properties in neural systems that retain network functionality in reduced preparations in vitro or in situ.

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.

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.

Dendritic calcium activity precedes inspiratory bursts in preBotzinger complex neurons.

Medullary interneurons of the preBötzinger complex assemble excitatory networks that produce inspiratory-related neural rhythms, but the importance of somatodendritic conductances in rhythm generation is still incompletely understood. Synaptic input may cause Ca(2+) accumulation postsynaptically to evoke a Ca(2+)-activated inward current that contributes to inspiratory burst generation. We measured Ca(2+) transients by two-photon imaging dendrites while recording neuronal somata electrophysiologically. Dendritic Ca(2+) accumulation frequently precedes inspiratory bursts, particularly at recording sites 50-300 µm distal from the soma. Preinspiratory Ca(2+) transients occur in hotspots, not ubiquitously, in dendrites. Ca(2+) activity propagates orthodromically toward the soma (and antidromically to more distal regions of the dendrite) at rapid rates (300-700 µm/s). These high propagation rates suggest that dendritic Ca(2+) activates an inward current to electrotonically depolarize the soma, rather than propagate as a regenerative Ca(2+) wave. These data provide new evidence that respiratory rhythmogenesis may depend on dendritic burst-generating conductances activated in the context of network activity.

Calcium-activated nonspecific cation current and synaptic depression promote network-dependent burst oscillations.

Central pattern generators (CPGs) produce neural-motor rhythms that often depend on specialized cellular or synaptic properties such as pacemaker neurons or alternating phases of synaptic inhibition. Motivated by experimental evidence suggesting that activity in the mammalian respiratory CPG, the preBötzinger complex, does not require either of these components, we present and analyze a mathematical model demonstrating an unconventional mechanism of rhythm generation in which glutamatergic synapses and the short-term depression of excitatory transmission play key rhythmogenic roles. Recurrent synaptic excitation triggers postsynaptic Ca(2+)-activated nonspecific cation current (I(CAN)) to initiate a network-wide burst. Robust depolarization due to I(CAN) also causes voltage-dependent spike inactivation, which diminishes recurrent excitation and thus attenuates postsynaptic Ca(2+) accumulation. Consequently, activity-dependent outward currents-produced by Na/K ATPase pumps or other ionic mechanisms-can terminate the burst and cause a transient quiescent state in the network. The recovery of sporadic spiking activity rekindles excitatory interactions and initiates a new cycle. Because synaptic inputs gate postsynaptic burst-generating conductances, this rhythm-generating mechanism represents a new paradigm that can be dubbed a 'group pacemaker' in which the basic rhythmogenic unit encompasses a fully interdependent ensemble of synaptic and intrinsic components. This conceptual framework should be considered as an alternative to traditional models when analyzing CPGs for which mechanistic details have not yet been elucidated.

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.

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.

Developmental origin of preBötzinger complex respiratory neurons.

A subset of preBötzinger Complex (preBötC) neurokinin 1 receptor (NK1R) and somatostatin peptide (SST)-expressing neurons are necessary for breathing in adult rats, in vivo. Their developmental origins and relationship to other preBötC glutamatergic neurons are unknown. Here we show, in mice, that the "core" of preBötC SST(+)/NK1R(+)/SST 2a receptor(+) (SST2aR) neurons, are derived from Dbx1-expressing progenitors. We also show that Dbx1-derived neurons heterogeneously coexpress NK1R and SST2aR within and beyond the borders of preBötC. More striking, we find that nearly all non-catecholaminergic glutamatergic neurons of the ventrolateral medulla (VLM) are also Dbx1 derived. PreBötC SST(+) neurons are born between E9.5 and E11.5 in the same proportion as non-SST-expressing neurons. Additionally, preBötC Dbx1 neurons are respiratory modulated and show an early inspiratory phase of firing in rhythmically active slice preparations. Loss of Dbx1 eliminates all glutamatergic neurons from the respiratory VLM including preBötC NK1R(+)/SST(+) neurons. Dbx1 mutant mice do not express any spontaneous respiratory behaviors in vivo. Moreover, they do not generate rhythmic inspiratory activity in isolated en bloc preparations even after acidic or serotonergic stimulation. These data indicate that preBötC core neurons represent a subset of a larger, more heterogeneous population of VLM Dbx1-derived neurons. These data indicate that Dbx1-derived neurons are essential for the expression and, we hypothesize, are responsible for the generation of respiratory behavior both in vitro and in vivo.

Interactions of persistent sodium and calcium-activated nonspecific cationic currents yield dynamically distinct bursting regimes in a model of respiratory neurons.

The preBötzinger complex (preBötC) is a heterogeneous neuronal network within the mammalian brainstem that has been experimentally found to generate robust, synchronous bursts that drive the inspiratory phase of the respiratory rhythm. The persistent sodium (NaP) current is observed in every preBötC neuron, and significant modeling effort has characterized its contribution to square-wave bursting in the preBötC. Recent experimental work demonstrated that neurons within the preBötC are endowed with a calcium-activated nonspecific cationic (CAN) current that is activated by a signaling cascade initiated by glutamate. In a preBötC model, the CAN current was shown to promote robust bursts that experience depolarization block (DB bursts). We consider a self-coupled model neuron, which we represent as a single compartment based on our experimental finding of electrotonic compactness, under variation of g (NaP), the conductance of the NaP current, and g (CAN), the conductance of the CAN current. Varying these two conductances yields a spectrum of activity patterns, including quiescence, tonic activity, square-wave bursting, DB bursting, and a novel mixture of square-wave and DB bursts, which match well with activity that we observe in experimental preparations. We elucidate the mechanisms underlying these dynamics, as well as the transitions between these regimes and the occurrence of bistability, by applying the mathematical tools of bifurcation analysis and slow-fast decomposition. Based on the prevalence of NaP and CAN currents, we expect that the generalizable framework for modeling their interactions that we present may be relevant to the rhythmicity of other brain areas beyond the preBötC as well.