Illuminating Airway Nerve Structure and Function in Chronic Cough.

Airway nerves regulate vital airway functions including bronchoconstriction, cough, and control of respiration. Dysregulation of airway nerves underlies the development and manifestations of airway diseases such as chronic cough, where sensitization of neural pathways leads to excessive cough triggering. Nerves are heterogeneous in both expression and function. Recent advances in confocal imaging and in targeted genetic manipulation of airway nerves have expanded our ability to visualize neural organization, study neuro-immune interactions, and selectively modulate nerve activation. As a result, we have an unprecedented ability to quantitatively assess neural remodeling and its role in the development of airway disease. This review highlights our existing understanding of neural heterogeneity and how advances in methodology have illuminated airway nerve morphology and function in health and disease.

Infectious and Inflammatory Pathways to Cough.

Coughing is a dynamic physiological process resulting from input of vagal sensory neurons innervating the airways and perceived airway irritation. Although cough serves to protect and clear the airways, it can also be exploited by respiratory pathogens to facilitate disease transmission. Microbial components or infection-induced inflammatory mediators can directly interact with sensory nerve receptors to induce a cough response. Analysis of cough-generated aerosols and transmission studies have further demonstrated how infectious disease is spread through coughing. This review summarizes the neurophysiology of cough, cough induction by respiratory pathogens and inflammation, and cough-mediated disease transmission.

Neural control of the lower airways: Role in cough and airway inflammatory disease.

Airway function is under constant neurophysiological control, in order to maximize airflow and gas exchange and to protect the airways from aspiration, damage, and infection. There are multiple sensory nerve subtypes, whose disparate functions provide a wide array of sensory information into the CNS. Activation of these subtypes triggers specific reflexes, including cough and alterations in autonomic efferent control of airway smooth muscle, secretory cells, and vasculature. Importantly, every aspect of these reflex arcs can be impacted and altered by local inflammation caused by chronic lung disease such as asthma, bronchitis, and infections. Excessive and inappropriate activity in sensory and autonomic nerves within the airways is thought to contribute to the morbidity and symptoms associated with lung disease.

Modeling and simulation of vagal afferent input of the cough reflex.

We employed computational modeling to investigate previously conducted experiments of the effect of vagal afferent modulation on the cough reflex in an anesthetized cat animal model. Specifically, we simulated unilateral cooling of the vagus nerve and analyzed characteristics of coughs produced by a computational model of brainstem cough/respiratory neuronal network. Unilateral vagal cooling was simulated by a reduction of cough afferent input (corresponding to unilateral vagal cooling) to the cough network. All these attempts resulted in only mild decreases in investigated cough characteristics such as cough number, amplitudes of inspiratory and expiratory cough efforts in comparison with experimental data. Multifactorial alterations of model characteristics during cough simulations were required to approximate cough motor patterns that were observed during unilateral vagal cooling in vivo. The results support the plausibility of a more complex NTS processing system for cough afferent information than has been proposed.

Influence of intrathoracic vagotomy on the cough reflex in the anesthetized cat.

Recurrent laryngeal afferent fibers are primarily responsible for cough in response to mechanical or chemical stimulation of the upper trachea and larynx in the guinea pig. Lower airway slowly adapting receptors have been proposed to have a permissive effect on the cough reflex. We hypothesized that vagotomy below the recurrent laryngeal nerve branch would depress mechanically or chemically induced cough. In anesthetized, bilaterally thoracotomized, artificially ventilated cats, thoracic vagotomy nearly eliminated cough induced by mechanical stimulation of the intrathoracic airway, significantly depressed mechanically stimulated laryngeal cough, and eliminated capsaicin-induced cough. These results support an important role of lower airway sensory feedback in the production of tracheobronchial and laryngeal cough in the cat. Further, at least some of this feedback is due to excitation from pulmonary volume-sensitive sensory receptors.

Sensory modulation of airways immunity.

The airways are constantly exposed to a multitude of inhaled particles and, as such, require a finely tuned discrimination between harmful or potentially threatening stimuli, and discrete responses to maintain homeostasis. Both the immune and nervous systems have the ability to sense environmental (and internal) signals, to integrate the obtained information and to initiate a protective reaction. Lung immunity and innervation are known to be individually involved in these processes, but it is becoming clear that they can also influence one another via a multitude of complex mechanisms. Here, we specifically describe how sensory innervation affects airways immunity with a focus on pathological conditions such as asthma or infections, describing cellular and molecular mechanisms, and highlighting potentially novel therapeutic targets.

The role of astrocytes in the nucleus tractus solitarii in maintaining central control of autonomic function.

The nucleus tractus solitarii (nTS) is the first central site for the termination and integration of autonomic and respiratory sensory information. Sensory afferents terminating in the nTS as well as the embedded nTS neurocircuitry release and utilize glutamate that is critical for maintenance of baseline cardiorespiratory parameters and initiating cardiorespiratory reflexes, including those activated by bouts of hypoxia. nTS astrocytes contribute to synaptic and neuronal activity through a variety of mechanisms, including gliotransmission and regulation of glutamate in the extracellular space via membrane-bound transporters. Here, we aim to highlight recent evidence for the role of astrocytes within the nTS and their regulation of autonomic and cardiorespiratory processes under normal and hypoxic conditions.

Mini Review: Central Organization of Airway Afferent Nerve Circuits.

Airway afferents monitor the local chemical and physical micro-environments in the airway wall and lungs and send this information centrally to regulate neural circuits involved in setting autonomic tone, evoking reflex and volitional respiratory motor outflows, encoding perceivable sensations and contributing to higher order cognitive processing. In this mini-review we present a current overview of the central wiring of airway afferent circuits in the brainstem and brain, highlighting recent discoveries that augment our understanding of airway sensory processing. We additionally explore how advances in describing the molecular diversity of airway afferents may influence future research efforts aimed at defining central mesoscale connectivity of airway afferent pathways. A refined understanding of how functionally distinct airway afferent pathways are organized in the brain will provide deeper insight into the physiology of airway afferent-evoked responses and may foster opportunities for targeted modulation of specific pathways involved in disease.

Reciprocal connectivity of the periaqueductal gray with the ponto-medullary respiratory network in rat.

Synaptic activities of the periaqueductal gray (PAG) can modulate or appropriate the respiratory motor activities in the context of behavior and emotion via descending projections to nucleus retroambiguus. However, alternative anatomical pathways for the mediation of PAG-evoked respiratory modulation via core nuclei of the brainstem respiratory network remains only partially described. We injected the retrograde tracer Cholera toxin subunit B (CT-B) in the pontine Kölliker-Fuse nucleus (KFn, n = 5), medullary Bötzinger (BötC, n = 3) and pre-Bötzinger complexes (pre-BötC; n = 3), and the caudal raphé nuclei (n = 3), and quantified the descending connectivity of the PAG targeting these brainstem respiratory regions. CT-B injections in the KFn, pre-BötC, and caudal raphé, but not in the BötC, resulted in CT-B-labeled neurons that were predominantly located in the lateral and ventrolateral PAG columns. In turn, CT-B injections in the lateral and ventrolateral PAG columns (n = 4) produced the highest numbers of CT-B-labeled neurons in the KFn and far fewer numbers of labeled neurons in the pre-BötC, BötC, and caudal raphé. Analysis of the relative projection strength revealed that the KFn shares the densest reciprocal connectivity with the PAG (ventrolateral and lateral columns, in particular). Overall, our data imply that the PAG may engage a distributed respiratory rhythm and pattern generating network beyond the nucleus retroambiguus to mediate downstream modulation of breathing. However, the reciprocal connectivity of the KFn and PAG suggests specific roles for synaptic interaction between these two nuclei that are most likely related to the regulation of upper airway patency during vocalization or other volitional orofacial behaviors.

Interfering with airway nerves in cough associated with asthma.

Cough is a troublesome and often refractory symptom of asthma, which is associated with poor control of disease. The pathogenesis of asthmatic cough has mainly been attributed to bronchoconstriction, but recent evidence indicate that cough reflex hypersensitivity or neuronal dysfunction is a feature of asthma, even in those with mild stable disease. This is likely resistant to the mainstay treatment ICS/LABA which inhibits classic asthmatic response. Such refractory cough might manifest more predominantly in the day-time rather than night-time. Treatment options of such refractory cough or cough reflex hypersensitivity in asthma targeting the nerves (LTRAs, tiotropium, and potentially bronchial thermoplasty) are discussed.

Effects of persistent sodium current blockade in respiratory circuits depend on the pharmacological mechanism of action and network dynamics.

The mechanism(s) of action of most commonly used pharmacological blockers of voltage-gated ion channels are well understood; however, this knowledge is rarely considered when interpreting experimental data. Effects of blockade are often assumed to be equivalent, regardless of the mechanism of the blocker involved. Using computer simulations, we demonstrate that this assumption may not always be correct. We simulate the blockade of a persistent sodium current (INaP), proposed to underlie rhythm generation in pre-Bötzinger complex (pre-BötC) respiratory neurons, via two distinct pharmacological mechanisms: (1) pore obstruction mediated by tetrodotoxin and (2) altered inactivation dynamics mediated by riluzole. The reported effects of experimental application of tetrodotoxin and riluzole in respiratory circuits are diverse and seemingly contradictory and have led to considerable debate within the field as to the specific role of INaP in respiratory circuits. The results of our simulations match a wide array of experimental data spanning from the level of isolated pre-BötC neurons to the level of the intact respiratory network and also generate a series of experimentally testable predictions. Specifically, in this study we: (1) provide a mechanistic explanation for seemingly contradictory experimental results from in vitro studies of INaP block, (2) show that the effects of INaP block in in vitro preparations are not necessarily equivalent to those in more intact preparations, (3) demonstrate and explain why riluzole application may fail to effectively block INaP in the intact respiratory network, and (4) derive the prediction that effective block of INaP by low concentration tetrodotoxin will stop respiratory rhythm generation in the intact respiratory network. These simulations support a critical role for INaP in respiratory rhythmogenesis in vivo and illustrate the importance of considering mechanism when interpreting and simulating data relating to pharmacological blockade.

Structural and functional defects of the respiratory neural system in the medulla and spinal cord of Pax6 mutant rats.

Pax6 is an important transcription factor expressed in several discrete domains of the developing central nervous system. It has been reported that Pax6 is involved in the specification of subtypes of hindbrain motor neurons. Pax6 homozygous mutant (rSey2/rSey2) rats die soon after birth, probably due to impaired respiratory movement. To determine whether the respiratory center in the medulla functions normally, we analyzed the histological and neurophysiological properties of the medulla and spinal cord in fetal rats with this mutation. First, the medulla of rSey2/rSey2 at embryonic (E) 21.5-E22.5 tended to be smaller than those from heterozygous mutant (rSey2/+) and wild-type (+/+) littermates. Through immunohistochemical analysis, we confirmed normal distribution of Phox2b-expressing cells in the parafacial respiratory group (pFRG) of rSey2/rSey2 rats. Expression of neurokinin-1 receptor (NK-1R) was weak and dispersed in rSey2/rSey2 rats. In addition, rSey2/rSey2 rats have a defect of the hypoglossal nerve root. Electrophysiological analysis using brainstem-spinal cord preparations (E21.5-E22.5) revealed that rSey2/rSey2 rats showed larger fluctuation of the amplitude of inspiratory activity monitored from the fourth cervical root although there was no significant difference in the respiratory rate among rSey2/rSey2, rSey2/+, and +/+ littermates. The response of respiratory rhythm to high CO2 was similar among all genotypes. Optical recordings of neuronal activity revealed that the activity of the pFRG tended to be weaker and inspiratory activity appeared in more scattered areas in the caudal ventral medulla in the rSey2/rSey2 rats. These results suggest that the basal activity of the respiratory system was preserved with mild impairment of the inspiratory activity in the rSey2/rSey2 rats and that the Pax6 gene is involved in the functional development of the neuronal system producing effective inspiratory motor outputs for survival.

The serotonergic system and the control of breathing during development.

Serotonin (5-hydroxytryptamine 5-HT) was first discovered in the late 1940's as an endogenous bioactive amine capable of inducing vasoconstriction, and in the mid-1950's was found in the brain. It was in these early years that some of the first demonstrations were made regarding a role for brain 5-HT in neurological function and behavior, including data implicating reduced brain levels of 5-HT in clinical depression. Since that time, advances in molecular biology and physiological approaches in basic science research have intensely focused on 5-HT in the brain, and the many facets of its role during embryonic development, post-natal maturation, and neural function in adulthood continues to be established. This review focuses on what is known about the developmental roles for the 5-HT system, which we define as the neurons producing 5-HT along with pre-and post-synaptic receptors, in a vital homeostatic motor behavior - the control of breathing. We will cover what is known about the embryonic origins and fate specification of 5-HT neurons, and how the 5-HT system influences pre- and post-natal maturation of the ventilatory control system. In addition, we will focus on the role of the 5-HT system in specific respiratory behaviors during fetal, neonatal and postnatal development, and the relevance of dysfunction in this system in respiratory-related human pathologies including Sudden Infant Death Syndrome (SIDS).

The Role of the Cerebellum in Control of Swallow: Evidence of Inspiratory Activity During Swallow.

Anatomical connections are reported between the cerebellum and brainstem nuclei involved in swallow such as the nucleus tractus solitarius, nucleus ambiguus, and Kölliker-fuse nuclei. Despite these connections, a functional role of the cerebellum during swallow has not been elucidated. Therefore, we examined the effects of cerebellectomy on swallow muscle recruitment and swallow-breathing coordination in anesthetized freely breathing cats. Electromyograms were recorded from upper airway, pharyngeal, laryngeal, diaphragm, and chest wall muscles before and after complete cerebellectomy. Removal of the cerebellum reduced the excitability of swallow (i.e., swallow number), and muscle recruitment of the geniohyoid, thyroarytenoid, parasternal (chestwall), and diaphragm muscles, but did not disrupt swallow-breathing coordination. Additionally, diaphragm and parasternal muscle activity during swallow is reduced after cerebellectomy, while no changes were observed during breathing. These findings suggest the cerebellum modulates muscle excitability during recruitment, but not pattern or coordination of swallow with breathing.

Regional brain stem activations during capsaicin inhalation using functional magnetic resonance imaging in humans.

Coughing is an airway protective behavior elicited by airway irritation. Animal studies show that airway sensory information is relayed via vagal sensory fibers to termination sites within dorsal caudal brain stem and thereafter relayed to more rostral sites. Using functional magnetic resonance imaging (fMRI) in humans, we previously reported that inhalation of the tussigenic stimulus capsaicin evokes a perception of airway irritation ("urge to cough") accompanied by activations in a widely distributed brain network including the primary sensorimotor, insular, prefrontal, and posterior parietal cortices. Here we refine our imaging approach to provide a directed survey of brain stem areas activated by airway irritation. In 15 healthy participants, inhalation of capsaicin at a maximal dose that elicits a strong urge to cough without behavioral coughing was associated with activation of medullary regions overlapping with the nucleus of the solitary tract, paratrigeminal nucleus, spinal trigeminal nucleus and tract, cardiorespiratory regulatory areas homologous to the ventrolateral medulla in animals, and the midline raphe. Interestingly, the magnitude of activation within two cardiorespiratory regulatory areas was positively correlated ( r2 = 0.47, 0.48) with participants' subjective ratings of their urge to cough. Capsaicin-related activations were also observed within the pons and midbrain. The current results add to knowledge of the representation and processing of information regarding airway irritation in the human brain, which is pertinent to the pursuit of novel cough therapies. NEW & NOTEWORTHY Functional brain imaging in humans was optimized for the brain stem. We provide the first detailed description of brain stem sites activated in response to airway irritation. The results are consistent with findings in animal studies and extend our foundational knowledge of brain processing of airway irritation in humans.

New aspects of neuroinflammation and neuroimmune crosstalk in the airways.

Neuroimmune interaction has long been discussed in the pathogenesis of allergic airway diseases, such as allergic asthma. Mediators released during inflammation can alter the function of both sensory and parasympathetic neurons innervating the airways. Evidence has been provided that the inflammatory response can be altered by various mediators that are released by sensory and parasympathetic neurons and vice versa. Our aim is to demonstrate recent developments in the reciprocal neuroimmune interaction and to include, if available, data from in vivo and clinical studies.

The Neuroplastic and Therapeutic Potential of Spinal Interneurons in the Injured Spinal Cord.

The central nervous system is not a static, hard-wired organ. Examples of neuroplasticity, whether at the level of the synapse, the cell, or within and between circuits, can be found during development, throughout the progression of disease, or after injury. One essential component of the molecular, anatomical, and functional changes associated with neuroplasticity is the spinal interneuron (SpIN). Here, we draw on recent multidisciplinary studies to identify and interrogate subsets of SpINs and their roles in locomotor and respiratory circuits. We highlight some of the recent progress that elucidates the importance of SpINs in circuits affected by spinal cord injury (SCI), especially those within respiratory networks; we also discuss potential ways that spinal neuroplasticity can be therapeutically harnessed for recovery.

How important is the CO2 chemoreflex for the control of breathing? Environmental and evolutionary considerations.

Haldane and Priestley (1905) discovered that the ventilatory control system is highly sensitive to CO2. This "CO2 chemoreflex" has been interpreted to dominate control of resting arterial PCO2/pH (PaCO2/pHa) by monitoring PaCO2/pHa and altering ventilation through negative feedback. However, PaCO2/pHa varies little in mammals as ventilation tightly couples to metabolic demands, which may minimize chemoreflex control of PaCO2. The purpose of this synthesis is to (1) interpret data from experimental models with meager CO2 chemoreflexes to infer their role in ventilatory control of steady-state PaCO2, and (2) identify physiological causes of respiratory acidosis occurring normally across vertebrate classes. Interestingly, multiple rodent and amphibian models with minimal/absent CO2 chemoreflexes exhibit normal ventilation, gas exchange, and PaCO2/pHa. The chemoreflex, therefore, plays at most a minor role in ventilatory control at rest; however, the chemoreflex may be critical for recovering PaCO2 following acute respiratory acidosis induced by breath-holding and activity in many ectothermic vertebrates. An apparently small role for CO2 feedback in the genesis of normal breathing contradicts the prevailing view that central CO2/pH chemoreceptors increased in importance throughout vertebrate evolution. Since the CO2 chemoreflex contributes minimally to resting ventilation, these CO2 chemoreceptors may have instead decreased importance throughout tetrapod evolution, particularly with the onset and refinement of neural innovations that improved the matching of ventilation to tissue metabolic demands. This distinct and elusive "metabolic ventilatory drive" likely underlies steady-state PaCO2 in air-breathers. Uncovering the mechanisms and evolution of the metabolic ventilatory drive presents a challenge to clinically-oriented and comparative respiratory physiologists alike.

Ozone-induced eosinophil recruitment to airways is altered by antigen sensitization and tumor necrosis factor-α blockade.

Ozone is an atmospheric pollutant that causes lung inflammation and airway hyperresponsiveness. Ozone's effects occur in two distinct phases that are mediated by different populations of eosinophils. In the acute phase 1 day after exposure, mature airway-resident eosinophils alter parasympathetic nerve function that results in airway hyperresponsiveness. At this time point, the severity of hyperresponsiveness correlates with the number of eosinophils in close proximity to airway nerves, but not with eosinophils in bronchoalveolar lavage. Three days later, newly divided eosinophils are recruited to airways by a tumor necrosis factor-α-dependent mechanism. These new eosinophils paradoxically attenuate ozone-induced airway hyperresponsiveness. Ozone's effects on airway tissue eosinophils and nerve-associated eosinophils 3 days after exposure are unknown. Thus, we tested ozone's effects on eosinophils in airway subepithelium and around airway nerves 1 and 3 days after ozone in nonsensitized and ovalbumin-sensitized guinea pigs with or without the tumor necrosis factor-α antagonist, etanercept, and compared changes in eosinophils with ozone-induced airway hyperresponsiveness. More eosinophils were present in small, noncartilaginous airways and along small airway nerves compared to large cartilaginous airways in all treatment groups. The number of airway and nerve-associated eosinophils were unaffected 1 day after ozone exposure, whereas significantly fewer airway eosinophils were present 3 days later. Airway and nerve-associated eosinophils were also decreased in small airways 3 days after ozone in sensitized animals. These changes were blocked by etanercept. Airway eosinophils, but not nerve-associated or bronchoalveolar lavage eosinophils correlated with airway hyperresponsiveness 3 days after ozone. Our findings indicate ozone causes persistent alterations in airway eosinophils and reinforce the importance of characterizing eosinophils' effects within distinct airway compartments.

Andreas Vesalius' 500th Anniversary: Initial Integral Understanding of Voice Production.

BACKGROUND: Voice production relies on the integrated functioning of a three-part system: respiration, phonation and resonance, and articulation. To commemorate the 500th anniversary of the great anatomist Andreas Vesalius (1515-1564), we report on his understanding of this integral system. METHODS: The text of Vesalius' masterpiece De Humani Corporis Fabrica Libri Septum and an eyewitness report of the public dissection of three corpses by Vesalius in Bologna, Italy, in 1540, were searched for references to the voice-producing anatomical structures and their function. We clustered the traced, separate parts for the first time. RESULTS: We found that Vesalius recognized the importance for voice production of many details of the respiratory system, the voice box, and various structures of resonance and articulation. He stressed that voice production was a cerebral function and extensively recorded the innervation of the voice-producing organs by the cranial nerves. CONCLUSIONS: Vesalius was the first to publicly record the concept of voice production as an integrated and cerebrally directed function of respiration, phonation and resonance, and articulation. In doing so nearly 500 years ago, he laid a firm basis for the understanding of the physiology of voice production and speech and its management as we know it today.