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

Abstract

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 Adelta- 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 Adelta-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.

Figure 1. Schematic diagrams of the tracheal innervation, the preparation used to study cough, and a representative trace of a cough evoked in an anaesthetized guinea-pig

A, diagram of the extrinsic innervation of the trachea, including the recurrent (RLN) and superior (SLN) laryngeal nerves. B, schematic diagram of the preparation used to study cough. Pressure changes at the tracheal cannula (PT) are used to monitor respiration and cough. C, representative trace of coughing initiated by mechanically probing the tracheal mucosa. Cough is defined visually by the experimenter and based on the timing (< 1 s for the entire manoeuvre) and magnitude of the inspiratory (appearing as a downward deflection in the pressure trace) and expiratory (>500% of expiratory pressure during tidal breathing) efforts.

Figure 2. The effects of anaesthesia on bradykinin-evoked coughing in guinea-pigs

Conscious (A, n = 12) or anaesthetized (B, n = 5) guinea-pigs were challenged for 10 min in a flow through chamber with nebulized bradykinin (10 mg ml⁻¹). Pressure changes within the chamber were used to monitor respiration and coughing. Coughs evoked by bradykinin were counted and the average number of coughs is presented in C.

Figure 3. The effect of cutting the superior (SLNs) or recurrent (RLNs) laryngeal nerves on coughing evoked by mechanically probing or electrically stimulating the tracheal or laryngeal mucosa of anaesthetized guinea-pigs

Results are graphed as the percentage of animals coughing in response to mechanical (von Frey filament (4.7 mN), A) and electrical (16 Hz, 10 s, 12 V, 1 ms pulse duration, B) stimuli. The numbers above each bar indicate the number of animals that coughed/the number of animals that were challenged.

Figure 4. Representative recordings from tracheal afferent neurones originating in the nodose (A) and jugular (B) ganglia of guinea-pigs

Three subtypes of vagal afferent neurones innervate the trachea, larynx and mainstem bronchi of guinea-pigs: jugular ganglia neurones (both Aδ- and C-fibres) that are activated by acid, punctate mechanical stimuli, capsaicin and bradykinin, and nodose ganglia neurones conducting action potentials in the Aδ range that are activated by acid and punctate mechanical stimuli. Neither capsaicin nor bradykinin activates or sensitizes nodose ganglia neurones for activation. See text and Tables 2 and 3 for further details.

Figure 5. Reflex responses initiated by citric acid, capsaicin and bradykinin applied topically to the tracheal mucosa of anaesthetized guinea-pigs

A, representative trace of coughing evoked by 0.1 m citric acid applied to the tracheal mucosa. Respiration and coughing were monitored by recording tracheal pressure (PT) through a side port in the tracheal cannula (see Fig. 1). Citric acid (0.01–2 m) evoked cough when applied in 100 μl aliquots. At low concentrations, cough was not accompanied by marked changes in respiratory rate. At high concentrations of citric acid (0.3–2 m), however, prolonged (2–3 min) decreases in respiratory rate or apnoea occurred (not shown). B, unlike citric acid, capsaicin applied in 100 μl aliquots (3–10 μm) or continuously superfused as shown failed to evoke cough in anaesthetized guinea-pigs. Rather, capsaicin acutely and profoundly slowed or stopped respiration entirely, an effect that gradually reversed. At low concentrations of capsaicin (0.1–1 μm), after an initial slowing of rate, respiration eventually increased to above baseline rates. Note gasping initiated at the end of the sustained apnoeas. Tracheal challenge with bradykinin induced responses comparable to those induced by capsaicin. C, percentage of animals coughing in response to challenge with capsaicin (n = 15), bradykinin (n = 14) or citric acid (n = 19).

Figure 6. Representative traces of the responses of intrapulmonary stretch receptors to sustained increases in distending pressures in vitro

The upper traces in each panel are extracellular recordings from the cell bodies of vagal afferent neurones innervating the intrapulmonary airways. The lower traces in each panel are measurements of tracheal perfusion pressure (see text for further details of the experimental design). As expected, two subtypes of intrapulmonary stretch receptors were identified, slowly adapting (A), with a low threshold for activation by stretch (10–20 cmH₂O) and an adaptation index of <20%, and rapidly adapting (B), also with a generally low threshold for activation by stretch (10–30 cmH₂O) but an adaptation index of >80%. The rapidly adapting receptors remained rapidly adapting even when distending pressures were increased to 4 times (4X) threshold.

Figure 7. Conduction velocity of airway and lung mechanoreceptors

Graphic presentation of the distribution of conduction velocities of putative cough receptors innervating the trachea, larynx and bronchi (n = 136), and rapidly adapting stretch receptors (RARs) innervating the intrapulmonary airways and lungs (n = 14).

Figure 8. Activation of intrapulmonary rapidly adapting receptors (RARs) by methacholine and by ATP receptor agonists in vitro

A, coincident with the increases in tracheal perfusion pressure (depicted in the lower traces of each panel) evoked by pulmonary arterial administration of methacholine, RARs are activated robustly and in a non-adapting fashion (the upper traces in A and B are extracellular recordings from intrapulmonary RARs). This effect of methacholine can be mimicked by histamine and prevented by isoproterenol, indicating that airway smooth muscle contraction is the likely cause of this activation. B, in contrast to methacholine and histamine, ATP and the non-hydrolysable form of ATP, α,β-methylene ATP, have no effect on tracheal perfusion pressure but robustly activate airway stretch receptors. This effect of the purinergic receptor agonists is prevented by the P2X receptor antagonist PPADS. RARs and SARs responded identically to methacholine, histamine and to the ATP receptor agonists (not shown). C, mean ±s.e.m. action potentials initiated in intrapulmonary RARs and the putative cough receptors innervating the larynx, trachea and bronchi of guinea-pigs by challenges with methacholine (1–10 μm) and ATP (10–100 μm). See text for further details. Each bar is the mean ±s.e.m. of 4–8 separate experiments.