A Model of the Central Regulatory System for Cough Reflex

Abstract

Cough is an important defensive reflex that eliminates particles and secretions from the airways and protects the lower airways from the aspiration of foreign materials. Although the classical cough center is thought to be situated in or around the nucleus tractus solitarius (NTS) of the brainstem, our understanding of its profile is still incomplete. Accumulating evidence suggests a new concept of the central regulatory system for cough reflex. The cough pattern generator in the brainstem appears to be identical to the respiratory pattern generator and to function by reshaping of the discharge pattern of respiratory neurons. The generated cough motor task is transmitted to spinal motoneurons through the descending respiratory pathways. The cough-gating mechanism receives the peripheral tussigenic information through the relay neurons in the NTS and activates such a functionally flexible pattern generator by producing triggering signals. This review focuses on the cough-gating neurons that constitute the gating mechanism and play a crucial role in the generation of cough reflex.

1. Introduction

Coughing is a sudden and often repetitively occurring protective reflex and can happen both voluntarily and involuntarily. It promotes the removal of mucus or inhaled particles from the airways with large airflows generated by abdominal muscle contractions.^1–4) The cough reflex consists of three phases⁵⁾; a large inhalation (an inspiratory phase of cough), a forced exhalation against a closed glottis (a compressive phase of cough), and an intense airflow from the lungs following opening of the glottis, usually accompanied by a peculiar sound (an expulsive phase of cough). The multifunctional respiratory pattern generator in the brainstem, which is presumably the same network as the cough pattern generator, undergoes reconfiguration to produce cough.^6–8) Although coughing and breathing are generated by a common respiratory muscular system, these behaviors differ significantly in their mechanical features and regulation. Coughing occurs in a discontinuous, threshold-dependent manner, while breathing occurs spontaneously and regularly. The distinctive occurrence implies the existence of specific mechanism that regulates the forcefulness of each cough effort and number of coughs.^9,10) The functional cough-gating mechanism is thought to activate the cough/respiratory pattern generator, leading to control the excitability of this airway defensive behavior, while it is not involved in the regulation of breathing.

2. Tussigenic Afferents

The cell bodies of sensory fibers innervating the airways arise from either the nodose or jugular ganglia.¹¹⁾ Stimuli that initiate the cough reflex stimulate sensory nerve fibers that have been divided broadly into three main groups; slowly adapting stretch receptors (SARs), rapidly adapting receptors (irritant receptors) and nociceptive receptor fibers (C-fibers). Retrograde tracing studies combined with electrophysiological analyses of nerve activities in guinea pigs provide evidence in support of the hypothesis that irritant receptor fibers innervating the trachea and bronchus arise from neurons situated in the nodose ganglia, whereas the vast majority of C-fibers arise from neurons in the jugular ganglia.¹²⁾ As reviewed by Bolser,¹³⁾ Canning,¹⁴⁾ and Reynolds et al.,¹⁵⁾ the lower airway contains specific cough-producing receptors/fibers such as SARs, irritant receptors and pulmonary C-fibers. Also, laryngeal irritant receptors and C-fibers presented in the upper airway participate in cough reflexes. Among these, pulmonary and laryngeal irritant receptors are the main afferents most readily associated with the cough reflex.^3,16,17) An important role of C-fibers in cough has been proposed because cough can be induced by chemical stimuli such as citric acid and capsaicin, which are known to stimulate the C-fibers. There is also evidence that C-fibers do not evoke cough and might even inhibit cough.¹⁸⁾ The SAR afferent inputs are not directly involved in cough and might play a permissive role in production of cough reflex.¹⁹⁾ Additionally, Canning and Mori^10,20) suggest ‘cough receptors’ that are differentiated from SARs, irritant receptors and C-fibers.

3. Cough Reflex in Experimental Animals

Practically, cough reflex is initiated by three different methods; mechanical and chemical (such as substance P, bradykinin and acid) stimulation of the intratracheal mucosa, and direct activation of afferent nerves.^11,21,22) Mechanical stimulation activates laryngeal irritant receptors but not C-fibers, and chemical stimulation activates both irritant receptors and C-fiber endings.^16,23) Electrical stimulation of the superior laryngeal nerve (SLN) afferents may excite myelinated and unmyelinated fibers that include laryngeal irritant fibers and C-fibers, respectively.^4,17) Overall, mechanical stimulation is more selective and appropriate to activate irritant receptors. By contrast, chemical and electrical stimuli can activate the combinations of receptors to induce the cough reflex. The latter prevails over the former for assessment of the reflex pathways as well as the sites of antitussive action, since chemical stimulation often induces various respiratory reflexes unrelated to cough and since repeated applications of tussigenic chemicals quickly induce “tachyphylaxis” phenomena.²⁴⁾

Cough reflex is experimentally recorded from unanesthetized and spontaneously breathing animals including guinea pigs,^24–26) rats^27,28) and mice.^29,30) A whole-body plethysmographical recording of guinea pigs shows constant and stable inspiratory-expiratory waveforms reflecting regular breathing (Fig. 1A). Microinjection of citric acid into the larynx elicits several times of coughing, characterized by a specific waveform pattern having a deep inspiration immediately followed by a rapid large expiration simultaneously with sound. Similarly, inhalation of chemical substance by aerosol is able to induce cough reflex.^25,26,28,29) However, inhalation of irritants occasionally produces various respiratory-related reflexes other than cough, such as apnea, sneeze, sigh, expiratory reflex and augmented breath.^1,17,24) Recordings of tracheal airflows or electromyograms from respiratory muscles are performed to monitor cough reflex in anesthetized animals.^{10,20,25,26,31–34)} A characteristic large inspiratory–expiratory airflow or electromyogram occurs during cough induced by mechanical or chemical stimulation. Basal anesthesia influences the induction of cough reflex.³⁵⁾ It does not prevent the airway afferent nerve activation and fails to prevent cough evoked by mechanical or acid stimulation of the airways, but completely prevents cough evoked by capsaicin and bradykinin. Deep anesthesia prevents cough induced by any stimulation.

Fig. 1. Typical Cough Reflex and Fictive Cough Response

(A1) Plethysmographical recording of cough responses in an unanesthetized and unrestrained guinea pig. Cough reflex was evoked by intratracheal application of citric acid (7.5%, 20 µL). The waveform represents inspiration (upward) and expiration (downward). Large flow deflections in the waveform represent cough reflexes (indicated by asterisks). (A2) Magnified view of a cough response together with sound, determined by the waveform pattern with deep inspiration followed by rapid large expiration. (B1) Fictive cough response was evoked by electrical stimulation of the SLN in a decerebrate cat. Responses of the phrenic (PN) and iliohypogastric nerves (IHN) during fictive cough induced by repetitive stimulation of the SLN (0.3 mA intensity, 0.1 ms pulse width, 5 Hz, indicated by a horizontal bar). The S2C response is indicted by an arrow. (B2) Fast recordings of PN and IHN discharges during fictive cough. There is an overlapping period (70–200 ms) between the PN and IHN discharges. S1C; the stage 1 of fictive cough, S2C; the stage 2 of fictive cough.

4. Fictive Cough in Experimental Animals

Fictive cough is evoked in guinea pigs³⁶⁾ and cats,^8,37–41) but hardly in rats.³⁶⁾ It is monitored from reflex discharges of the inspiratory (phrenic) and expiratory (iliohypogastric) efferent nerves (Fig. 1B). The phrenic nerve regulates the diaphragm contraction and the iliohypogastric nerve controls contraction of the abdominal muscles. In normal respiration,^42–44) the phrenic nerve displays an augmenting discharge during inspiration, small decrementing discharge during postinspiration (stage 1 of expiration) and complete silence during late expiration (stage 2 of expiration). The iliohypogastric nerve displays no discharge during inspiration and a small augmenting or tonic discharge during expiration. During fictive cough induced by repetitive stimulation of the SLN^6,39,40) or mechanical stimulation of the intratracheal mucosa,^5,41) the phrenic nerve shows an increased inspiratory discharge that is immediately followed by a rapid, large spindle-shaped discharge in the iliohypogastric nerve. The phase of phrenic nerve discharge is called the stage 1 of fictive cough (S1C) and the phase of iliohypogastric nerve discharge is called the stage 2 of fictive cough (S2C). There is a short overlapping period between the phrenic and iliohypogastric discharges. The S2C is more critical than S1C for achievement of cough reflex.

Fictive cough is peripherally initiated by electrical stimulation of the afferent nerves such as SLN and vagus nerve, mechanical or chemical stimulation of the intratracheal mucosa in paralyzed and artificially ventilated animals.^{13,36,37,39,41)} It is also evoked centrally by repetitive electrical microstimulation of the nucleus tractus solitarius (NTS) in cats^40,45) and in guinea pigs.⁴⁶⁾ The NTS stimulation activates directly the tussigenic afferent terminals or the kernel of the cough network that is comparable with the so-called cough center.^47–49) Increasing the stimulus intensity reinforces the cough-related discharges of both nerves. Sites where microstimulation can induce a fictive cough are confined to the ventrolateral region of the NTS and adjusting reticular formation, indicating that fictive cough occurs in a region- and intensity-dependent manner (see Fig. 1 in ref. 40). However, the possibility always remains that electrical stimulation of the medulla affects not only neuronal somata but also intramedullary cough-related pathways.⁷⁾

5. Central Cough Reflex Pathway

Occurrence of cough is regulated at the level of the brainstem through integration of tussigenic afferent inputs. Bolser and Davenport⁹⁾ and Shannon et al.^5,41) proposed a model of the central cough reflex pathway. In this model, the tracheobronchial and laryngeal tussigenic afferents converge on the gating mechanism via relay neurons in the NTS, and then the gated signals activate the central cough pattern generator. Finally, the produced cough motor task is transmitted to the inspiratory and expiratory muscles through activation of respiratory motoneurons. The cough pattern generator appears to be identical to the respiratory pattern generator and to function by reshaping of the discharge pattern of respiratory neurons.^38,41) The gating mechanism may exist between the relay neurons and the cough pattern generator, and play an essential role in generation of cough reflex.

6. Cough-Gating Neurons

It is unclear whether cough-gating comprises a physical structure with specific neurons filtering and then encoding the epileptiform changes in motor output, or whether gating is the physiological sum of multiple regulatory elements that influence both breathing and coughing. Recently, we have found the cough-gating neurons that may constitute the gating mechanism.⁴⁰⁾ The criteria for this neuron are (1) excitation during both the S1C and S2C, (2) pauci-synaptic inputs from afferent nerves, (3) localization in the ventrolateral NTS and surrounding reticular formation, and (4) vulnerability of the cough-related depolarization to codeine. Such characteristics are different from those of typical respiratory neurons and 2nd order relay neurons.

6.1. Membrane Potential during Fictive Cough

Membrane potentials of the cough-gating neurons can be recorded in the decerebrate and unanesthetized cat (Fig. 2A). The cough-gating neurons excite during fictive cough; the depolarization starts at the onset of the S1C, peaks at the onset of the S2C, and repolarizes along with the decline of the iliohypogastric nerve discharge. The membrane response in each stage of the fictive cough corresponds to discharges of the phrenic or iliohypogastric nerve. This characteristic being active during both the S1C and S2C is cough-specific. The cough-gating neurons display three types of membrane potential fluctuations during normal respiration⁴⁰⁾; (1) phase-spanning respiration-modulated fluctuations (Figs. 2A, 5), (2) no respiration-related membrane potential changes (Fig. 2B), and (3) weak inspiratory-modulated fluctuations (Fig. 3A1). They have either random, phase-spanning or no spiking during respiration.^8,40)

Fig. 2. Typical Membrane Potential Trajectories in Cough-Related Neurons

(A) Membrane potential in a cough-gating neuron and effects of intravenous dizocilpine in a decerebrate cat. Fictive cough was induced by repetitive stimulation of the SLN. The S2C responses are indicted by arrows. Traces were obtained before (Before) and 15 min after injection of dizocilpine (Dizocilpine 0.1 mg/kg, i.v.). Dizocilpine suppressed the membrane depolarization during both the S1C and S2C, suggesting that the NMDA receptor mechanisms contribute to the cough-related depolarization. MP; membrane potential, PN; phrenic nerve, IHN; iliohypogastric nerve. (B) Membrane potentials in 2nd order relay, cough-gating, augmenting inspiratory bulbospinal (aug-I BS) and augmenting expiratory bulbospinal (aug-E BS) neurons are shown together with the discharges of PN and IHN during fictive cough. The cough-gating neurons are specifically recruited during both the S1C and S2C.

Fig. 3. Typical Short-Latency Waves of Postsynaptic Potentials in Cough-Related Neurons

(A1) Membrane potential changes (MP) during fictive cough induced by repetitive stimulation of the SLN in a cough-gating neuron of a decerebrate cat. The S2C responses are indicted by arrows. (A2) Short-latency waves of EPSPs induced by single stimulation of the SLN (0.3 mA intensity, 0.1 ms pulse width) were taken before (Before) and during iontophoresis of an antagonist of AMPA receptors, NBQX (50 nA) or an antagonist of NMDA receptors, dizocilpine (50 nA) in the same neuron shown in A1. The EPSP wave is inhibited by iontophoresis of NBQX, but not by dizocilpine. (B) Short-latency waves of EPSPs and IPSPs induced by single stimulation of the SLN in 2nd order relay, cough-gating, aug-I BS and aug-E BS neurons are shown along with the PN discharge. Horizontal dot lines indicate the reference membrane potential.

Furthermore, intravenous dizocilpine, an antagonist of N-methyl-d-aspartate (NMDA) receptor, completely blocks the cough-related depolarization in the cough-gating neurons and also suppresses the occurrence of cough-related discharges in the phrenic and iliohypogastric nerves in the cat (Fig. 2A). Dizocilpine had neither effect on respiration-related membrane potential fluctuations in the neuron nor discharges in the phrenic and iliohypogastric nerves. This suggests that the NMDA receptor mechanism participates in cough-related membrane depolarization in the cough-gating neurons. It has been also demonstrated that cough reflexes are inhibited by antagonists of NMDA receptors in rats,²⁷⁾ rabbits⁵⁰⁾ and guinea pigs.¹⁰⁾

Membrane potential changes in other cough-related neurons including the 2nd order relay, augmenting inspiratory bulbospinal (aug-I BS), and augmenting expiratory bulbospinal (aug-E BS) neurons are illustrated together with burst discharges in the phrenic and iliohypogastric nerves during fictive cough (Fig. 2B). The 2nd order relay neurons are characterized by neither respiration-related membrane fluctuation nor cough-related membrane potential change. They display either no, random or tonic spiking. Aug-I BS neurons, which are the presynaptic sources for inspiratory motoneurons in the spinal cord,^42–44) depolarize during the S1C and hyperpolarize during the S2C.⁴⁰⁾ The S1C depolarization is concerted with the augmented phrenic discharge. Aug-E BS neurons, which are the presynaptic sources for expiratory motoneurons in the spinal cord,^42–44) show a rapid, large depolarization and burst discharges during the S2C.³⁹⁾ Tight synchronization occurs between membrane depolarization and the burst discharge of the iliohypogastric nerve.

6.2. Short-Latency Postsynaptic Potentials Induced by Tussigenic Afferents

Single pulse stimulation of the SLN afferents evokes a short-latency (<10 ms) wave of excitatory postsynaptic potentials (EPSPs) in the cough-gating neurons of the cat (Fig. 3A2). The EPSP waves are decreased by iontophoresis of NBQX (2,3-dioxo-6-nitro-1,2,3,4-tetrahydrobenzo[f]quinoxaline-7-sulfonamide) onto the recorded neuron but not by dizocilpine, suggesting that the SLN-induced, short-latency EPSP waves in cough-related neurons are mediated by the AMPA (α-amino-3-hydroxy-5-methylisoxazole-4-propionic acid) or non-NMDA receptor mechanism but not by the NMDA receptor mechanism.

The short-latency waves of EPSPs and inhibitory postsynaptic potentials (IPSPs) induced in the 2nd order relay, aug-I BS, and aug-E BS neurons by single stimulation of the SLN are shown together with the phrenic nerve discharges (Fig. 3B). The 2nd order relay neurons respond with a long-lasting EPSP wave. Aug-I BS neurons respond with a wave of IPSPs, which is preceded by a short-lasting EPSP wave. Aug-E BS neurons show an EPSP wave, which is sometimes followed by an IPSP wave. This indicates that all cough-related neurons receive the pauci-synaptic excitatory inputs from the laryngeal tussigenic afferents.^4,39,51)

6.3. Distribution in Nucleus Tractus Solitarius

The cough-gating neurons are intermingled with 2nd order relay neurons in the ventrolateral part of the NTS and surrounding reticular formation (between 1.5 mm caudal and 2.0 mm rostral to the obex) where fictive cough is induced by microstimulation of the NTS (Fig. 4).⁴⁰⁾ Fos-like proteins are expressed in such area after laryngeal-induced cough.^38,52) It is suggested that the ventrolateral division of the NTS is discrete regions of peripheral afferent termination and of localization of the cough-gating neurons. This agrees with the recent results from guinea pigs that an essential component to the brainstem cough gating may be localized in the restricted region of NTS which is lateral to the commissural subnucleus.^10,53)

Fig. 4. Localization of Cough-Gating Neurons

The cough-gating and 2nd order relay neurons are displayed at the reference planes of the cat brainstem (P 13.5, P 14.7 and P 16.0).⁵⁷⁾ Note that distribution of cough-gating neurons overlaps with cough-inducible sites (indicated by gray areas). Abbreviations: CU; cuneate nucleus, CX; external cuneate nucleus, FTL; lateral tegmental field, LR; lateral reticular nucleus, NA; nucleus ambiguous, NTS; nucleus tractus solitarius, 5SL; laminar spinal trigeminal nucleus, 5ST; spinal trigeminal nucleus, 12; hypoglossal nucleus.

6.4. Suppression of Cough-Related Activity by Codeine

As described above, the cough-gating neurons display membrane depolarization during fictive cough induced by repetitive stimulation of the SLN (Fig. 5). Intravenously injected codeine inhibits both the S1C and S2C depolarization in concert with depression of the serial response of phrenic and iliohypogastric nerves. These results lead a conclusion that the cough-gating neurons in the NTS are the target cells for the narcotic antitussive agents.^13,32,48) This is supported by our previous reports⁴⁶⁾ that intravenous injection of codeine inhibits the induction of fictive cough by microstimulation of the NTS and that microinjection of codeine into the NTS inhibits the induction of fictive cough by repetitive stimulation of the ipsilateral SLN. In the previous reports,^54–56) two medullary areas that have received the most attention regarding the actions of antitussive drugs are the NTS and the caudal ventral respiratory group (VRG). The pontine respiratory group (PRG) and rostral VRG also contain neurons that may participate in the production of cough and could represent potential sites of action of antitussive agents. However, the basal activities in the cough-gating neuron during eupneic respiration are not affected by codeine (Fig. 5). Furthermore, codeine has no effect on the respiration-related discharges in both phrenic and iliohypogastric nerves. This suggests that the main network including the VRG and PRG for maintaining the eupneic respiratory motor pattern may be excluded from the primary site of action of the central antitussive drugs.

Fig. 5. Effects of Intravenous Codeine on a Cough-Gating Neuron in a Decerebrate Cat

Traces were obtained before (Before) and 10 min after injection of codeine (Codeine 1.0 mg/kg, i.v.). Codeine suppressed the membrane depolarization during both the S1C and S2C. The S2C responses are indicted by arrows. MP; membrane potential, PN; phrenic nerve, IHN; iliohypogastric nerve.

7. Conclusion

The feature of cough reflex characterized by a discontinuous and threshold-dependent manner leads to postulate existence of the gating mechanism that regulates the encoding of cough numbers and forcefulness. Several lines of evidence suggest the cough-gating neurons that play a specific role in the generation of cough in the NTS.^8,40) Here, a model of the central regulatory system for cough reflex is proposed (Fig. 6). The model features; (1) the 2nd order relay neurons receive the peripheral tussigenic inputs, (2) the cough-gating neurons integrate afferent inputs and regulate the burst timing for cough (number and intensity of cough), (3) the core cough/respiratory pattern generator generates the cough motor pattern for activating the aug-I BS and aug-E BS neurons, and (4) the respiratory efferent nerves conduct the final motor task that controls inspiratory and expiratory muscle contractions.

Fig. 6. A Model of the Central Regulatory System for Cough Reflex

The model depicts: (1) The 2nd order relay neurons in the NTS receive tussigenic afferent information. (2) The cough-gating neurons constituting the gating mechanism receive outputs from the 2nd order relay neurons. (3) The cough/respiratory pattern generator receives the gated signals produced by the cough-gating neurons and the cough motor pattern is transmitted to the aug-I BS and aug-E BS neurons. (4) The PN and IHN are activated during the corresponding phase of cough reflex to contract the diaphragm and abdominal muscles, respectively.

In summary, the ventrolateral NTS is an essential part of the cough reflex pathway. The cough-gating neurons that depolarize during both S1C and S2C may play an important role in production of the cough reflex. The NMDA receptor mechanisms are involved in the cough-related depolarization of the cough-gating neurons. The cough-gating neurons are the target cells for the centrally acting antitussive agents.^15,25,56)

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