Abstract
Airway integrity must be continuously maintained throughout life. Sensory neurons guard against airway obstruction and, on a moment-by-moment basis, enact vital reflexes to maintain respiratory function1,2. Decreased lung capacity is common and life-threatening across many respiratory diseases, and lung collapse can be acutely evoked by chest wall trauma, pneumothorax or airway compression. Here we characterize a neuronal reflex of the vagus nerve evoked by airway closure that leads to gasping. In vivo vagal ganglion imaging revealed dedicated sensory neurons that detect airway compression but not airway stretch. Vagal neurons expressing PVALB mediate airway closure responses and innervate clusters of lung epithelial cells called neuroepithelial bodies (NEBs). Stimulating NEBs or vagal PVALB neurons evoked gasping in the absence of airway threats, whereas ablating NEBs or vagal PVALB neurons eliminated gasping in response to airway closure. Single-cell RNA sequencing revealed that NEBs uniformly express the mechanoreceptor PIEZO2, and targeted knockout of Piezo2 in NEBs eliminated responses to airway closure. NEBs were dispensable for the Hering–Breuer inspiratory reflex, which indicated that discrete terminal structures detect airway closure and inflation. Similar to the involvement of Merkel cells in touch sensation3,4, NEBs are PIEZO2-expressing epithelial cells and, moreover, are crucial for an aspect of lung mechanosensation. These findings expand our understanding of neuronal diversity in the airways and reveal a dedicated vagal pathway that detects airway closure to help preserve respiratory function.
Main
Sensory neurons that constitute the interoceptive nervous system relay information to the brain from vital organs in the body5. Within the airways, sensory neurons provide essential feedback to control breathing, promote gas exchange, protect the airways through cough and laryngeal guarding reflexes, detect pathogens to induce sickness, and elicit perceptions of breathlessness, also known as dyspnoea or air hunger1,2,6,7,8. Neuronal surveillance of the airways enables the detection of life-threatening inefficiencies in gas exchange, which can be a characteristic of cardiopulmonary disease. In response, neural circuits trigger compensatory reflexes such as gasps (quick and deep inspirations also called augmented breaths or sighs) to reopen closed airways, bring air into the lungs and relieve respiratory distress9,10,11,12,13. Gasps are triggered by many airway threats, including hypoxia, bronchospasm, pulmonary congestion and thoracic compression, precede voluntary behaviours such as speaking and singing, and are often the first and last breaths of life. Neural circuits in the brain have been identified that coordinate the gasping motor response13, but sensory pathways that lead to gasping, and their mechanisms of action, require additional exploration.
The vagus nerve provides the major sensory innervation of the airways, and classical studies have described airway neurons as rapidly adapting mechanoreceptors, slowly adapting mechanoreceptors or chemosensitive C fibres1,2,5. Recent single-cell expression profiling and genetic approaches have revealed a richer diversity of vagal neurons in the larynx, trachea and lungs6,14,15,16,17. Notably, the sensory properties and functions of many vagal neurons with particular transcriptional signatures and/or airway terminal morphologies are undefined2,6,14,16, which suggests that additional airway-to-brain reflexes remain uncharted.
Mechanoreceptors that detect lung stretch are perhaps the best-studied airway sensory neurons. In 1868, Hering and Breuer reported that mechanical inflation of the airways causes a reflexive inhibition of breathing or apnoea18, now termed the Hering–Breuer inspiratory reflex. Airway stretch receptors are slowly adapting mechanoreceptors activated with each inspiration during tidal breathing and directly sense lung distension through the mechanosensory ion channel PIEZO2 (refs. 6,19,20,21). Hering and Breuer also reported physiological responses to decreases in airway volume18, and Adrian later observed lung deflation responses in single unit recordings of vagal afferents19. Deflation responses were not observed during normal expiration and were subsequently ascribed to polymodal nociceptors, including trachea-enriched C fibres, which also detect cough-evoking irritants, juxtacapillary fibres in the pulmonary vasculature (so-called J fibres) and/or rapidly adapting mechanoreceptors also activated by high threshold stretch1,22,23,24. It has remained unclear whether dedicated receptors for airway closure exist in the conducting airways of the lung. If so, their functions and mechanisms of action have remained unknown.
Responses to airway closure in the mouse
We first sought to characterize physiological responses to airway closure in the mouse, a tractable model system that enables genetic experiments for mechanistic study. In other animals, airway closure triggers gasping and reflexive increases in inspiration, with some variability across species and paradigm reported25,26,27,28. We used several methods to decrease functional airway volume in the mouse: (1) airway compression by inflating a cuff surrounding the thorax; (2) airway suction by applying negative pressure within the trachea; and (3) bronchoconstriction by tracheal delivery of nebulized methacholine (Fig. 1a). Physiological changes were recorded by measuring tracheal pressure (which reports the frequency and magnitude of each inspiration and exhalation), oesophageal pressure (as a proxy for intrathoracic pressure) and heart rate. Each method of airway closure triggered reflexive gasps (Fig. 1b), with a stereotypical pattern characterized by a powerful, deep inspiration and subsequent rapid exhalation (defined by a >50% increase in expiration compared with the previous and subsequent breath). Gasps were also associated with increased activity of intercostal muscles and the diaphragm (Extended Data Fig. 1a,b), as measured by electromyography. The frequency of gasping depended on the depth of anaesthesia, and under urethane anaesthesia, gasping was rarely observed at baseline but was routinely observed following airway compression (Extended Data Fig. 1c). Gasping was also triggered by hypoxia (10–12% O2), to a lesser extent by nebulized citric acid and not by other stimuli, including inhaled particulates (microbeads), hypercapnia or hyperoxia (Fig. 1c). Mechanical stimuli evoked a higher gasp frequency than chemical stimuli tested, and this is probably due, at least partially, to a shorter time delay to first gasp after introduction of the stimulus (Extended Data Fig. 1d). We observed that thoracic compression, under these conditions, promoted additional changes in lung mechanics, including decreased lung compliance through chest wall restriction (Extended Data Fig. 1e). Onset of airway compression reduced airway pressure and respiratory rate (Extended Data Fig. 1f), presumably because increased intrathoracic pressure provides dominant suppression over any compensatory inspiratory drive, but had no effect on blood oxygen saturation (Extended Data Fig. 1f–h).
Gasping responses to thoracic compression persisted following transection of several vagus nerve branches, including the superior laryngeal nerve (SLN), the recurrent laryngeal nerve (RLN) and the glossopharyngeal nerve. Responses were instead abolished after transection of the vagus nerve trunk below the SLN departure point (Fig. 1d), a procedure that eliminates vagal fibres below the larynx, including those to the lung. By comparison, gasps and increases in tidal volume induced by hypoxia persisted after transection of the vagus nerve trunk below the SLN, but instead were lost after transection of the glossopharyngeal nerve, consistent with a role for carotid body chemosensation29. Together, these findings indicate that airway closure, induced by airway compression, suction or bronchoconstriction, induces a gasping reflex in the mouse through the vagus nerve.
Vagal activity during airway closure
Vagal fibre recording studies have produced conflicting data about whether airway deflation decreases activity of airway stretch neurons, activates dedicated neurons and/or activates polymodal neurons that also detect other stimuli, such as chemical threats or high-threshold airway stretch19,22,23,30. Here we used in vivo calcium imaging within vagal ganglia6,20 to investigate vagal responses to airway closure. Vagal ganglion imaging enables a parallel analysis of real-time responses in >100 individual neurons per experiment. We used the progeny of lsl-SALSA mice mice, which express the calcium reporter GCaMP6f-tdTomato (also called SALSA31) from a Cre-dependent allele, crossed with Vglut2-ires-cre mice, in which Cre recombinase is expressed in all vagal sensory neurons. We imaged vagal ganglia while connections to the lungs were preserved (Fig. 2). As we previously observed20, airway stretch evoked calcium transients in a small group of vagal sensory neurons (7.1%, 93 out of 1,303 neurons, 7 mice) that express PIEZO2 (refs. 6,20,21). Compressing the airways by inflating a thoracic cuff triggered acute calcium responses in 9.2% of vagal sensory neurons (120 out of 1,303 neurons, 7 mice). Most airway closure receptors (75.8%, 91 out of 120) did not respond to airway stretch, but some (24.2%, 29 out of 120) responded weakly. Airway suction activated a smaller group of vagal sensory neurons (5.1%, 25 out of 495), most of which also responded to airway compression. Nebulized methacholine also stimulated some vagal neurons (Extended Data Fig. 2c,d), which largely overlapped with compression-sensing neurons (60.7%, 54 out of 89) but not neurons selective for lung inflation (33 out of 89 responded to compression but not inflation, 21 out of 89 responded to both compression and inflation and 3 out of 89 responded to inflation but not compression) (Extended Data Fig. 2d and Supplementary Video 1). The percentages of neurons that responded to different stimuli across trials were generally conserved across mice (Extended Data Fig. 2a,b). Together, these findings indicate that airway compression acutely activates a subset of vagal sensory neurons, with the major cohort unresponsive to airway stretch, which suggests that there is a dedicated vagal pathway for detecting airway closure.
Vagal PVALB neurons mediate gasping
Vagal ganglia contain dozens of molecularly distinct sensory neuron types6,15,17,32,33, so we asked which sensory neurons mediate airway-closure-induced gasping. First, we used optogenetics to activate various vagal sensory neurons and measured reflexive gasping behaviour. We expressed the light-activated ion channel channelrhodopsin-2 from a Cre-dependent allele (lsl-ChR2) in different vagal sensory neuron types using Cre driver mice, including P2ry1-ires-cre, Pvalb-t2a-cre, Crhr2-ires-cre, Npy2r-ires-cre, Gpr65-ires-cre and Vglut2-ires-cre mice. As previously reported6,14,20,34, we then stimulated vagal sensory neurons by shining light on the vagus nerve trunk, ganglion or particular nerve branches (Fig. 3a). Activating all vagal sensory neurons in Vglut2-ires-cre;lsl-ChR2 mice evoked reflexive gasps, with a frequency of 6.0 gasps per min of illumination, whereas activating CRHR2, NPY2R, GPR65 or other neuron types did not (Fig. 3b). Optogenetic stimulation of PVALB or P2RY1 neurons similarly caused fictive gasping, with PVALB predominantly expressed in subsets of vagal P2RY1 neurons. Activation of P2RY1 but not PVALB neurons also caused swallowing, as previously reported6 (Extended Data Fig. 3a,b,c). In Pvalb-t2a-cre;lsl-ChR2 mice, gasping was evoked by illumination of vagal ganglia or the vagal trunk distal to the SLN departure point, consistent with a role for lung afferents (Extended Data Fig. 3d). Gasps were evoked by ganglion illumination after transection of the trunk, which indicated a role for sensory neurons that transmit the information to the brain (Extended Data Fig. 3d). Pvalb is enriched in three clusters of vagal sensory neurons, only one of which also expresses Olfr78. We generated Olfr78-p2a-cre mice, and optogenetic stimulation of vagal OLFR78 sensory neurons also evoked gasping behaviour (Extended Data Fig. 3e). These experiments pinpoint transcriptome-defined vagal neuron subtypes that mediate gasping (Fig. 3c), and these neurons are distinct from P2RY1 neurons that mediate swallowing6….
