Nanako Mori

Identification of the tracheal and laryngeal afferent neurones mediating cough in anaesthetized guinea‐pigs

We have identified the tracheal and laryngeal afferent nerves regulating cough in anaesthetized guinea‐pigs. Cough was evoked by electrical or mechanical stimulation of the tracheal or laryngeal mucosa, or by citric acid applied topically to the trachea or larynx. By contrast, neither capsaicin nor bradykinin challenges to the trachea or larynx evoked cough. Bradykinin and histamine administered intravenously also failed to evoke cough. Electrophysiological studies revealed that the majority of capsaicin‐sensitive afferent neurones (both Aδ‐ and C‐fibres) innervating the rostral trachea and larynx have their cell bodies in the jugular ganglia and project to the airways via the superior laryngeal nerves. Capsaicin‐insensitive afferent neurones with cell bodies in the nodose ganglia projected to the rostral trachea and larynx via the recurrent laryngeal nerves. Severing the recurrent nerves abolished coughing evoked from the trachea and larynx whereas severing the superior laryngeal nerves was without effect on coughing. The data indicate that the tracheal and laryngeal afferent neurones regulating cough are polymodal Aδ‐fibres that arise from the nodose ganglia. These afferent neurones are activated by punctate mechanical stimulation and acid but are unresponsive to capsaicin, bradykinin, smooth muscle contraction, longitudinal or transverse stretching of the airways, or distension. Comparing these physiological properties with those of intrapulmonary mechanoreceptors indicates that the afferent neurones mediating cough are quite distinct from the well‐defined rapidly and slowly adapting stretch receptors innervating the airways and lungs. We propose that these airway afferent neurones represent a distinct subtype and that their primary function is regulation of the cough reflex.

Synergistic interactions between airway afferent nerve subtypes regulating the cough reflex in guinea-pigs

Cough initiated from the trachea and larynx in anaesthetized guinea‐pigs is mediated by capsaicin‐insensitive, mechanically sensitive vagal afferent neurones. Tachykinin‐containing, capsaicin‐sensitive C‐fibres also innervate the airways and have been implicated in the cough reflex. Capsaicin‐sensitive nerves act centrally and synergistically to modify reflex bronchospasm initiated by airway mechanoreceptor stimulation. The hypothesis that polymodal mechanoreceptors and capsaicin‐sensitive afferent nerves similarly interact centrally to regulate coughing was addressed in this study. Cough was evoked from the tracheal mucosa either electrically (16 Hz, 10 s trains, 1–10 V) or by citric acid (0.001–2 m). Neither capsaicin nor bradykinin evoked a cough when applied to the trachea of anaesthetized guinea‐pigs, but they substantially reduced the electrical threshold for initiating the cough reflex. The TRPV1 receptor antagonist capsazepine prevented the increased cough sensitivity induced by capsaicin. These effects of topically applied capsaicin and bradykinin were not due to interactions between afferent nerve subtypes within the tracheal wall or a direct effect on the cough receptors, as they were mimicked by nebulizing 1 mg ml−1 bradykinin into the lower airways and by microinjecting 0.5 nmol capsaicin into nucleus of the solitary tract (nTS). Citric acid‐induced coughing was also potentiated by inhalation of bradykinin. The effects of tracheal capsaicin challenge on cough were mimicked by microinjecting substance P (0.5–5 nmol) into the nTS and prevented by intracerebroventricular administration (20 nmol h−1) of the neurokinin receptor antagonists CP99994 or SB223412. Tracheal application of these antagonists was without effect. C‐fibre activation may thus sensitize the cough reflex via central mechanisms.

Selective Expression of a Sodium Pump Isozyme by Cough Receptors and Evidence for Its Essential Role in Regulating Cough

We have identified a distinct subtype of airway vagal afferent nerve that plays an essential role in regulating the cough reflex. These afferents are exquisitely sensitive to punctate mechanical stimuli, acid, and decreases in extracellular chloride concentrations, but are insensitive to capsaicin, bradykinin, histamine, adenosine, serotonin, or changes in airway intraluminal pressures. In this study we used intravital imaging, retrograde neuronal tracing, and electrophysiological analyses to characterize the structural basis for their peculiar mechanical sensitivity and to further characterize the regulation of their excitability. In completing these experiments, we uncovered evidence for an essential role of an isozyme of Na+-K+ ATPase in regulating cough. These vagal sensory neurons arise bilaterally from the nodose ganglia and are selectively and brilliantly stained intravitally with the styryl dye FM2-10. Cough receptor terminations are confined and adherent to the extracellular matrix separating the airway epithelium and smooth muscle layers, a site of extensive remodeling in asthma and chronic obstructive pulmonary disease. The cough receptor terminals uniquely express the α3 subunit of Na+-K+ ATPase. Intravital staining of cough receptors by FM2-10, cough receptor excitability in vitro, and coughing in vivo are potently and selectively inhibited by the sodium pump inhibitor ouabain. These data provide the first detailed morphological description of the peripheral terminals of the sensory nerves regulating cough and identify a selective molecular target for their modulation.

Differential effects of airway afferent nerve subtypes on cough and respiration in anesthetized guinea pigs

The hypothesis that respiratory reflexes, such as cough, reflect the net and often opposing effects of activation of multiple afferent nerve subpopulations throughout the airways was evaluated. Laryngeal and tracheal mucosal challenge with either citric acid or mechanical probing reliably evoked coughing in anesthetized guinea pigs. No other stimulus reliably evoked coughing in these animals, regardless of route of administration and despite some profound effects on respiration. Selectively activating vagal C-fibers arising from the nodose ganglia with either adenosine or 2-methyl-5-HT evoked only tachypnea. Selectively activating vagal afferents arising from the jugular ganglia induced respiratory slowing and apnea. Nasal afferent nerve activation by capsaicin, citric acid, hypertonic saline, or histamine evoked only respiratory slowing. Histamine, which activates intrapulmonary rapidly adapting receptors but not airway or lung C-fibers or tracheal bronchial cough receptors induced bronchospasm and tachypnea, but no coughing. The results indicate that the reflexes initiated by stimuli thought to be selective for some afferent nerve subtypes will likely depend on the net and potentially opposing effects of multiple afferent nerve subpopulations throughout the airways. The data also provide further evidence that the afferent nerves regulating cough in anesthetized guinea pigs are distinct from either C-fibers or intrapulmonary rapidly adapting receptors.

Encoding of the cough reflex in anesthetized guinea pigs

We have previously described the physiological and morphological properties of the cough receptors and their sites of termination in the airways and centrally in the nucleus tractus solitarius (nTS). In the present study, we have addressed the hypothesis that the primary central synapses of the cough receptors subserve an essential role in the encoding of cough. We found that cough requires sustained, high-frequency (≥8-Hz) afferent nerve activation. We also found evidence for processes that both facilitate (summation, sensitization) and inhibit the initiation of cough. Sensitization of cough occurs with repetitive subthreshold activation of the cough receptors or by coincident activation of C-fibers and/or nTS neurokinin receptor activation. Desensitization of cough evoked by repetitive and/or continuous afferent nerve activation has a rapid onset (<60 s) and does not differentiate between tussive stimuli, suggesting a central nervous system-dependent process. The cough reflex can also be actively inhibited upon activation of other airway afferent nerve subtypes, including slowly adapting receptors and pulmonary C-fibers. The sensitization and desensitization of cough are likely attributable to the prominent, primary, and unique role of N-methyl-d-aspartate receptor-dependent signaling at the central synapses of the cough receptors. These attributes may have direct relevance to the presentation of cough in disease and for the effectiveness of antitussive therapies.

An essential component to brainstem cough gating identified in anesthetized guinea pigs.

Coughing protects and clears the airways and lungs of inhaled irritants, particulates, pathogens, and accumulated secretions. An initial urge to cough, and an almost binary output suggests gating mechanisms that encode and modulate this defensive reflex. Whether this “gate” has a physical location for the physiological barrier it poses to cough is unknown. Here we describe a critical component to cough gating, the central terminations of the cough receptors. A novel microinjection strategy defined coordinates for microinjection of glutamate receptor antagonists that nearly abolished cough evoked from the trachea and larynx in anesthetized guinea pigs while having no effect on basal respiratory rate and little or no effect on reflexes attributed to activating other afferent nerve subtypes. Comparable microinjections in adjacent brainstem locations (0.5–2 mm distal) were without effect on coughing. Subsequent transganglionic and dual tracing studies confirmed that the central terminations of the cough receptors and their primary relay neurons are found bilaterally within nucleus tractus solitarius (nTS), lateral to the commissural subnucleus and perhaps in the medial subnuclei. These synapses possess the physiological characteristics of a cough gate. Their localization should facilitate more mechanistic studies of the encoding and gating of cough.—Canning, B. J., Nanako Mori. An essential component to brainstem cough gating identified in anesthetized guinea pigs. FASEB J. 24, 3916–3926 (2010). www.fasebj.org

The role of trigeminal nasal TRPM8-expressing afferent neurons in the antitussive effects of menthol

The cold-sensitive cation channel TRPM8 is a target for menthol, which is used routinely as a cough suppressant and as an additive to tobacco and food products. Given that cold temperatures and menthol activate neurons through gating of TRPM8, it is unclear how menthol actively suppresses cough. In this study we describe the antitussive effects of (-)-menthol in conscious and anesthetized guinea pigs. In anesthetized guinea pigs, cough evoked by citric acid applied topically to the tracheal mucosa was suppressed by menthol only when it was selectively administered as vapors to the upper airways. Menthol applied topically to the tracheal mucosa prior to and during citric acid application or administered continuously as vapors or as an aerosol to the lower airways was without effect on cough. These actions of upper airway menthol treatment were mimicked by cold air delivered to the upper airways but not by (+)-menthol, the inactive isomer of menthol, or by the TRPM8/TRPA1 agonist icilin administered directly to the trachea. Subsequent molecular analyses confirmed the expression of TRPM8 in a subset of nasal trigeminal afferent neurons that do not coincidently express TRPA1 or TRPV1. We conclude that menthol suppresses cough evoked in the lower airways primarily through a reflex initiated from the nose.

Sympathetic nerve-dependent regulation of mucosal vascular tone modifies airway smooth muscle reactivity

The airways contain a dense subepithelial microvascular plexus that is involved in the supply and clearance of substances to and from the airway wall. We set out to test the hypothesis that airway smooth muscle reactivity to bronchoconstricting agents may be dependent on airway mucosal blood flow. Immunohistochemical staining identified vasoconstrictor and vasodilator nerve fibers associated with subepithelial blood vessels in the guinea pig airways. Intravital microscopy of the tracheal mucosal microvasculature in anesthetized guinea pigs revealed that blockade of α-adrenergic receptors increased baseline arteriole diameter by ~40%, whereas the α-adrenergic receptor agonist phenylephrine produced a modest (5%) vasoconstriction in excess of the baseline tone. In subsequent in vivo experiments, tracheal contractions evoked by topically applied histamine were significantly reduced (P < 0.05) and enhanced by α-adrenergic receptor blockade and activation, respectively. α-Adrenergic ligands produced similar significant (P < 0.05) effects on airway smooth muscle contractions evoked by topically administered capsaicin, intravenously administered neurokinin A, inhaled histamine, and topically administered antigen in sensitized animals. These responses were independent of any direct effect of α-adrenergic ligands on the airway smooth muscle tone. The data suggest that changes in blood flow in the vessels supplying the airways regulate the reactivity of the underlying airway smooth muscle to locally released and exogenously administered agents by regulating their clearance. We speculate that changes in mucosal vascular function or changes in neuronal regulation of the airway vasculature may contribute to airways responsiveness in disease.

Gastroesophageal reflux disease in children with asthma: Treatment implications

An association between asthma and gastroesophageal reflux disease (GERD) has long been recognized both mechanistically and epidemiologically. The clinical relevance of this interplay continues to be explored, with special interest given to the role of GERD in the worsening of asthma. The effect of GERD is most frequently contemplated in patients with asthma that is difficult to control.Medical and surgical anti-reflux trials attempting to alter asthma symptoms have reported mixed but generally underwhelming results, although asthma symptom scores are generally improved following effective treatment of GERD. Many of the pharmaceutical studies can be criticised for having too short a duration or for likely incomplete acid suppression.Few trials have specifically studied pediatric populations. Because GERD is a common condition, particularly in young children, the role reflux plays in the worsening of asthma symptoms and the potential benefit on asthma of anti-reflux therapy warrants further exploration. Whether or not treating symptomatic GERD reduces the symptoms and severity of asthma in children, GERD coexisting with asthma should be aggressively treated. GERD symptoms in most patients with or without asthma can be controlled medically with continuous use of proton pump inhibitors such as omeprazole and lansoprazole and to a lesser extent by histamine H2 receptor antagonists such as famotidine and cimetidine.

Fluorescent styryl dyes FM1-43 and FM2-10 are muscarinic receptor antagonists: intravital visualization of receptor occupancy

The fluorescent styryl dyes FM1‐43 and FM2‐10 have been used to visualize the endocytic and exocytic processes involved in neurotransmission in a variety of central and peripheral nerve preparations. Their utility is limited to some extent by a poorly understood vesicular‐independent labelling of cells and tissues. We show here that one likely cause of this troublesome background labelling is that FM1‐43 and FM2‐10 are selective and competitive antagonists at both cloned and endogenously expressed muscarinic acetylcholine receptors. In radioligand binding studies, FM1‐43 and FM2‐10 bound with moderate affinity (23–220 nm) to membranes of Chinese hamster ovary (CHO) cells expressing cloned human muscarinic receptors (M1–M5). In functional studies in vitro, FM1‐43 and FM2‐10 inhibited electrical field stimulation (EFS) and acetylcholine‐induced cholinergic contractions of guinea‐pig tracheal strips (IC50: FM1‐43, 0.4 ± 0.1; FM2‐10, 1.6 ± 0.1 μm; concentration of antagonist producing a 2‐fold leftward shift in the acetylcholine concentration–response curve (Kb): FM1‐43, 0.3 ± 0.1; FM2‐10, 15.8 ± 10.1 μm). Neither compound inhibited EFS‐evoked, non‐adrenergic non‐cholinergic nerve‐mediated relaxations or contractions of the airways, or contractions mediated by histamine H1 receptor or tachykinin NK2 receptor activation. Incubating freshly excised tracheal whole‐mount preparations with 5 μm FM1‐43 resulted in intense fluorescence labelling of the smooth muscle that was reduced by up to 90% in the presence of selective M2 and M3 receptor antagonists. The potency of the FM dyes as muscarinic receptor antagonists is within the concentration range used to study vesicular cycling at nerve terminals. Given that muscarinic receptors play a key role in the regulation of neurotransmitter release from a variety of neurones, the anticholinergic properties of FM dyes may have important implications when studying vesicular events in the nervous system. In addition, these dyes may provide a novel tool for visualizing muscarinic receptor occupancy in living tissue or cell preparations.