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.

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.

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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 ϩ -K ϩ 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 ␣ 3 subunit of Na ϩ -K ϩ 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.

ments carried out in conscious guinea pigs suggest that citric acidevoked coughing is partly mediated by transient receptor potential vanilloid type 1 (TRPV1) receptor-dependent activation of tachykinin-containing, capsaicin-sensitive C fibers. In vitro electrophysiological analyses indicate, however, that acid also activates capsaicinsensitive and -insensitive vagal afferent nerves by a TRPV1-independent mechanism, and studies in anesthetized guinea pigs show that coughing evoked by acid is mediated by activation of capsaicininsensitive vagal afferent nerves. In the present study, we have characterized the mechanisms of citric acid-evoked coughing in anesthetized guinea pigs. Drugs were administered directly to the Krebs buffer perfusing the extrathoracic trachea. Citric acid was applied topically to the tracheal mucosa, directly into the tracheal perfusate in increasing concentrations and at 1-min intervals. Citric acid dose dependently evoked coughing in anesthetized guinea pigs. This was mimicked by hydrochloric acid but not by sodium citrate. The coughing evoked by acid was nearly or completely abolished by TTX or by cutting the recurrent laryngeal nerves. Perfusing the trachea with a low Cl Ϫ buffer potentiated the acid-induced cough reflex. In contrast, prior capsaicin desensitization, 10 M capsazepine, Ca 2ϩ -free perfusate, 0.1 M iberiotoxin, 1 M atropine, 10 M isoproterenol, 10 M albuterol, 3 M indomethacin, 0.1 M HOE-140, a combination of neurokinin 1 (NK1; CP-99994), NK2 (SR-48968), and NK3 (SB-223412) receptor antagonists (0.1 M each), a combination of histamine H 1 (3 M pyrilamine) and cysLT1 (1 M ICI-198615) receptor antagonists, superior laryngeal nerve transection, or epithelium removal did not inhibit citric acid-evoked coughing. These and other data indicate that citric acid-evoked coughing in anesthetized guinea pigs is mediated by direct activation of capsaicin-insensitive vagal afferent nerves, perhaps through sequential activation of acid-sensing ion channels and chloride channels. TRPV1 ; capsaicin; rapidly adapting receptor CITRIC ACID AND TARTARIC ACID are used routinely in clinical and preclinical studies of the cough reflex (15,27,35). In conscious guinea pigs, citric acid-evoked coughing is inhibited by the capsaicin receptor transient receptor potential vanilloid type 1 (TRPV1) antagonists capsazepine and iodo-resiniferatoxin or the TRPV1 channel blocker Ruthenium Red, and can also be inhibited by neurokinin 1 (NK 1 ), NK 2 , and/or NK 3 receptor antagonists (1,2,7,18,34,54,55). The acid-evoked coughing is mimicked by inhalation of the TRPV1 receptor agonists capsaicin, resiniferatoxin, and anandamide in both animals and human subjects, and prior capsaicin desensitization abolishes citric acid-evoked coughing in awake guinea pigs (10,25,35,50). Because the only tachykinin-containing nerves innervating the airways of normal, uninflamed guinea pigs are capsaicinsensitive C fibers (21, 32 49, 56), these data provide overwhelming evidence that acid-evoked coughing in conscious guinea pi...