GABA
A- and glycine-mediated inhibitory modulation of the cough reflex in
the caudal nucleus tractus solitarii of the rabbit
Elenia Cinelli, Ludovica Iovino, Fulvia Bongianni, Tito Pantaleo, and Donatella Mutolo
Dipartimento di Medicina Sperimentale e Clinica, Sezione Scienze Fisiologiche, Universita` degli Studi di Firenze, Florence, Italy
Submitted 20 May 2016; accepted in final form 5 July 2016 Cinelli E, Iovino L, Bongianni F, Pantaleo T, Mutolo D.
GABAA- and glycine-mediated inhibitory modulation of the cough
reflex in the caudal nucleus tractus solitarii of the rabbit. Am J Physiol Lung Cell Mol Physiol 311: L570 –L580, 2016. First published July 8, 2016; doi:10.1152/ajplung.00205.2016.—Cough-related sensory in-puts from rapidly adapting receptors (RARs) and C fibers are pro-cessed by second-order neurons mainly located in the caudal nucleus
tractus solitarii (NTS). Both GABAAand glycine receptors have been
proven to be involved in the inhibitory control of second-order cells receiving RAR projections. We investigated the role of these receptors within the caudal NTS in the modulation of the cough reflex induced by either mechanical or chemical stimulation of the tracheobronchial tree in pentobarbital sodium-anesthetized, spontaneously breathing rabbits. Bilateral microinjections (30 –50 nl) of the receptor antago-nists bicuculline and strychnine as well as of the receptor agoantago-nists muscimol and glycine were performed. Bicuculline (0.1 mM) and strychnine (1 mM) caused decreases in peak abdominal activity and marked increases in respiratory frequency due to decreases in both inspiratory time (TI) and expiratory time (TE), without concomitant changes in arterial blood pressure. Noticeably, these microinjections induced potentiation of the cough reflex consisting of increases in the cough number associated with decreases either in cough-related TI
after bicuculline or in both cough-related TIand TEafter strychnine.
The effects caused by muscimol (0.1 mM) and glycine (10 mM) were in the opposite direction to those produced by the corresponding
antagonists. The results show that both GABAAand glycine receptors
within the caudal NTS mediate a potent inhibitory modulation of the pattern of breathing and cough reflex responses. They strongly suggest that disinhibition is one important mechanism underlying cough regulation and possibly provide new hints for novel effective antitus-sive strategies.
GABAA receptors; glycine receptors; control of breathing; cough
reflex; nucleus tractus solitarii
COUGH PLAYS A VERY IMPORTANTrole in airway protection (40). Related sensory inputs are processed by second-order interneu-rons located in the nucleus tractus solitarii (NTS) and, espe-cially, in the commissural subnucleus (27, 41, 42, 47, 55). Receptors mainly located in the trachea, carina, and main bronchi belong to the large family of pulmonary A␦ rapidly adapting receptors (RARs; for a review see Ref. 68), including the so-called “cough receptors” described in the guinea pig airways (14, 16). They are well known to be involved in cough mediation. RARs are a very heterogeneous population of polymodal receptors; however, each of them does not respond to all stimuli that have been described as effective for their activation. Some of them are in particular sensitive to
mechan-ical stimulation of airway mucosa and to chemmechan-ical irritant stimuli such as acid, cigarette smoke, and ammonia and when activated by such stimuli cause cough reflex responses. Exper-imental evidence suggests that these receptors are located in the large extrapulmonary airways (68). On the other hand, RARs located in the intrapulmonary airways evoke other types of reflexes (hyperpnea/tachypnea, augmented breaths, bron-choconstriction, laryngeal closure, etc.) and, only very seldom, cough (68). Cough-related afferent inputs may also originate from bronchopulmonary C fibers, although their role seems to be more complex (see Refs. 15, 16, 18, 22, 43, 68, 80 for further references).
Cough is one of the most common and troubling symptoms for which patients seek medical attention. Cough is purposeful and useful under many circumstances but in case of persistent or chronic cough (24) is without an apparent aim or benefit and can result in a wide range of physical and psychological complications that considerably impair the quality of life. Available antitussive therapies have limited efficacy and un-acceptable side effects. Thus further research is needed to find better antitussive drugs (24). We believe that not only studies on animal models characterized by cough hypersensitivity but also investigations on the basic neural mechanisms subserving the cough reflex may provide useful hints for the development of novel antitussive therapies.
Our knowledge on the central organization of the cough reflex is far from complete. In particular, the role of the NTS in the genesis of the cough motor pattern appears to be more extensive than that of a mere relay station (8). On the other hand, it seems definitively ascertained that at least two med-ullary structures, i.e., the caudal NTS and the caudal ventral respiratory group (VRG), are important sites of action of centrally active antitussive drugs and probably components of the central neural circuit involved in cough regulation (8, 9, 21, 51, 54, 55, 57, 64, 66). In particular, the caudal NTS has been considered for its synaptic organization a strategic site where excitatory and inhibitory neurotransmitters contribute to the control of this airway defensive reflex (e.g., Refs. 31, 41). Second-order neurons possibly related to the cough afferent pathway (RAR cells) located in the caudal NTS have been proven to receive both phasic glycinergic and tonic GABAer-gic (GABAA) inhibition (28 –31). Accordingly, both GABA and glycine terminals are widely expressed in the NTS includ-ing its caudal aspects (see e.g., Refs. 2, 25 for further refer-ences). While the activation of GABAB receptors within the caudal NTS has been shown to be involved in the downregu-lation of the cough reflex (51; see also Ref. 18), no information is available, to our knowledge, on the role of GABAA and glycine receptors.
Address for reprint requests and other correspondence: D. Mutolo, Diparti-mento di Medicina Sperimentale e Clinica, Sezione Scienze Fisiologiche, Universita` degli Studi di Firenze, Viale G.B. Morgagni 63, 50134 Firenze, Italy (e-mail: donatella.mutolo@unifi.it).
First published July 8, 2016; doi:10.1152/ajplung.00205.2016.
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The present study was undertaken on pentobarbital sodium-anesthetized, spontaneously breathing rabbits with the main purpose of investigating the role of GABAA and glycine receptors within the caudal NTS in the modulation of the cough reflex induced by either mechanical or chemical stimulation of the tracheobronchial tree. We addressed this issue by using GABAAand glycine receptor agonists and antagonists micro-injected into the caudal NTS. The results provide evidence that a potent GABAA- and glycine-mediated inhibition is exerted at this level on both baseline respiratory activity and cough reflex responses.
MATERIALS AND METHODS
Ethical approval. Animal care and experimental procedures were conducted in accordance with the Italian legislation and the official regulations of the European Community Council on the use of laboratory animals (Directive 86/609/EEC and 2010/63/UE). The study was approved by the Animal Care and Use Committee of the University of Florence. The number of animals used and their suffer-ing was reduced as much as possible. Details about the methods employed have previously been described in our previous studies on the NTS region and will be only concisely reported here (21, 51–53, 55, 57).
Animal preparation and recording procedures. Experiments were carried out on 24 male New Zealand White rabbits (2.8 –3.4 kg) anesthetized with pentobarbital sodium (40 mg/kg iv, supplemented by 2– 4 mg/kg every 30 min; Sigma-Aldrich, St. Louis, MO). Atropine (0.15 mg/kg im) was administered to reduce mucosal secretion in the airways. The adequacy of anesthesia was assessed by the absence of reflex withdrawal of the hindlimb in response to noxious pinching of the hindpaw. Additional criteria were the presence of a stable and regular pattern of phrenic bursts and the absence of fluctuations in arterial blood pressure. The trachea was cannulated and polyethylene catheters were inserted into a femoral artery and vein for monitoring
arterial blood pressure and drug delivery, respectively. The C3or C5
phrenic root on one side was prepared for recordings. The animal was placed in a prone position and fixed by a stereotaxic head holder and vertebral clamps. The head was ventroflexed for optimal exposure of the dorsal surface of the medulla by occipital craniotomy. Body temperature was maintained at 38.5–39°C by a heating blanket con-trolled by a rectal thermistor probe.
Efferent phrenic nerve activity was recorded with bipolar platinum electrodes. Abdominal muscle electromyographic (EMG) activity was recorded by wire electrodes. Phrenic and abdominal activities were amplified, full-wave rectified, and “integrated” (low-pass resistor-capacitor filter, time constant 100 ms). Arterial blood pressure was
recorded by a strain-gauge manometer and end-tidal CO2 partial
pressure by an infrared CO2 analyzer (Capnocheck Plus, Smiths
Medical PM, Waukesha, WI). Cardiorespiratory variables were ana-lyzed using a personal computer, supplied with an appropriate inter-face (Digidata 1440, Molecular Devices, Sunnyvale, CA) and soft-ware (Axoscope, Molecular Devices).
Microinjection procedures. Bilateral microinjections were per-formed at two different sites along the rostrocaudal extent of the caudal NTS into the lateral commissural subnucleus. The first was at the level of the caudal-most end of the area postrema, 0.6 – 0.8 mm lateral to the midline and 0.7– 0.8 mm below the dorsal medullary surface. The second was 0.5 mm more caudal, 0.4 – 0.5 mm lateral to the midline and 0.7– 0.8 mm below the dorsal medullary surface. The stereotaxic coordinates were selected according to the atlas of Mees-sen and Olszewski (48).
Microinjections (30 –50 nl) were performed as described in our
previous reports via a glass micropipette (tip diameter 10 –25 m).
The volume of the injectate was measured directly by monitoring the movement of the fluid meniscus in the pipette barrel with a dissecting
microscope equipped with a fine reticule. The following drugs were
used: 0.1 mM bicuculline methiodide (a GABAAreceptor antagonist,
Sigma-Aldrich), 0.1 mM muscimol (a GABAA receptor agonist,
Tocris Bioscience, Bristol, UK), 1 mM strychnine hydrochloride (a glycine receptor antagonist, Sigma-Aldrich), and 10 mM glycine (Tocris Bioscience). Each drug was dissolved in 0.9% NaCl solution. Drug concentrations were in the same range as those previously used in in vivo preparations (10, 17, 37, 63). Control injections of equal volumes of the vehicle solution at the responsive sites were also performed. The localization of injection sites is diagrammatically illustrated in Fig. 1 and was confirmed by injecting green fluorescent latex microspheres (LumaFluor, New City, NY) added to the drug solution in some preparations (three for bicuculline and three for strychnine).
Stimulation procedures. Mechanical stimulation was delivered by a custom-built device recently described and validated (57) using a 0.5-mm diameter nylon fiber with a smoothed tip inserted through a lateral port of the tracheal cannula. The device allowed to set the number of forth-and-back movements or cycles (1–3 cycles), shaft velocity (10 –20 mm/s), and shaft displacement (10 –20 mm). Me-chanical stimulation was set at 1 cycle, 15 mm/s velocity, and 15-mm displacement to produce a bout of two to four coughs. The stimulation protocol comprised three stimulation trials performed in succession
(at ⬃1-min intervals) before drug administration, repeated ⬃5 min
after the completion of all the microinjections and at appropriate intervals (at least 5 min) to follow the recovery process for a maximum of 3 h.
Chemical stimulation of the tracheobronchial tree was performed by means of citric acid inhalation (for details see Ref. 53). Citric acid (1 M, Sigma-Aldrich) was freshly dissolved in 0.9% NaCl solution and nebulized. The opening of the tracheal cannula, through which the rabbits were spontaneously breathing, was exposed to a steady stream
of the nebulized citric acid solution for⬃3 s. Chemical stimulation
was always applied 2–3 min after mechanically induced cough and caused a bout of several coughs usually immediately followed by a tachypneic response. As a rule, chemical stimulation was performed
both before and⬃10 min after the completion of the injections and
repeated at appropriate intervals (ⱖ10 min) to follow the recovery
process. Owing to the very short-lasting effects (⬍5 min) evoked by
10 mM glycine microinjections, changes in cough reflex responses
were assessed within⬃2–3 min after the completion of the injections.
To this purpose, glycine microinjections were executed in succession
in the same preparation with an interval of⬃15 min to test the effects
on the cough reflex in response to either mechanical (three trials with different glycine microinjections) or chemical (a single trial) stimu-lation. The 15-min interval was considered more than sufficient to
ensure complete recovery (seeRESULTS). All stimulation procedures
were performed at least 5– 6 min after each supplemental dose of pentobarbital to avoid its possible immediate influence on the re-corded variables.
Histology. The histological control of pipette tracks and injection sites was performed as previously described (for details, see Refs. 52,
55). In particular, frozen 20-m coronal sections stained with cresyl
violet were used. Coronal sections of the medulla in which injection sites were marked by fluorescent microspheres were examined in a light and epifluorescence microscope (Eclipse E400, Nikon, Japan) equipped with the Nikon Intensilight C-HGFI mercury-fiber illumi-nator. A Nikon DS-Fi1digital camera was used to take photomicro-graphs. Illustrations were prepared in Adobe Photoshop CS3 (Adobe Systems, San Jose, CA).
Data collection and analysis. Respiratory variables were measured during eupneic breathing and reflex responses. The inspiratory (TI) and expiratory (TE) times, as well as the total duration of the respiratory cycle (TT), were measured on recordings of raw phrenic nerve activity (57, 65). The respiratory frequency was subsequently calculated (breaths/min). Peak amplitude (arbitrary units) of the phrenic nerve activity and abdominal EMG activity were measured on
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integrated traces and normalized by expressing them in relative units (RU), i.e., as a fraction (or percentage) of the highest achievable amplitude observed in each animal. Breathing pattern variables were measured for an average of five consecutive breaths prior to and following drug microinjections. Furthermore, systolic and diastolic blood pressures were measured at 2-s intervals and mean arterial pressure was calculated as the diastolic pressure plus one-third of the pulse pressure. The measurement periods of cardiorespiratory vari-ables were the same selected for cough-related varivari-ables (see below). Owing to the small variations in respiratory and cardiovascular variables within each measurement period, average values were taken as single measurements for the purpose of analysis.
The cough motor pattern in response to mechanical or chemical stimulation of the tracheobronchial tree is characterized by repeated coughs. Cough-related variables included the cough-related TT, TI, and TE, peak phrenic amplitude (RU), peak abdominal activity (RU) and the cough number, i.e., the number of coughs following each stimulation. Cough-related variables were measured and averaged before and after drug administration at the time when the maximum response and the complete recovery were observed (three trials for mechanical stimulation and a single trial for citric acid inhalation). The average values of cough-related variables were taken as single measurements for subsequent statistical analysis (GraphPad Prism 5, La Jolla, CA). An expiration reflex could occur as the first motor event in a cough epoch (40, 78; for further details and comments see Refs. 21, 51–53, 55, 71). Expiration reflexes were not considered for data analysis. Owing to the absence of cough responses with muscimol and
in one case with glycine (seeRESULTS), only quantitative data related
to the cough number have been reported since no reliable results could be obtained for the other cough-related variables expressed as mean values of all trials and included in the statistical analysis.
The effects of each individual treatment were evaluated by using one-way repeated-measures ANOVA followed by Student-Newman-Keuls tests. When appropriate, changes in respiratory variables were also expressed as percentage variations of control values. Reported
values are means⫾ SE; P ⬍ 0.05 was taken as significant.
RESULTS
Role of GABAAreceptors. Bilateral microinjections (n⫽ 6)
of 0.1 mM bicuculline (30 –50 nl; 3–5 pmol) at the two selected caudal NTS sites caused within 1 min the appearance of a high-frequency, irregular breathing pattern superimposed on tonic phrenic nerve activity and accompanied by strong reduc-tions or complete suppression of abdominal muscle activity. Respiratory activity evaluated after ⬃10 min, i.e., when the maximum change in cough response occurred, was character-ized by moderate increases in respiratory frequency (from 51.5 ⫾ 3.2 to 69.9 ⫾ 3.9 breaths/min, ⫹38.1 ⫾ 9.9%; P ⬍ 0.01) due to decreases in both TI and TE (Table 1; see also control recordings before reflex responses in Fig. 2). Changes in ongoing respiratory activity displayed a progressive recov-ery that was complete within 60 min. No significant changes in arterial blood pressure were observed (Table 1). Small but Fig. 1. Localization of injection sites. Left: diagrammatic representation of a dorsal view of the medulla oblongata of the rabbit showing the sites where bilateral microinjections (Œ) of different drugs have been performed into the caudal aspect of the nucleus tractus solitarii (NTS). AP, area postrema; DRG, dorsal respiratory group. Right: diagram of a coronal section of the medulla oblongata at the level indicated in the left panel (dashed line) showing the location of representative sites where the microinjections have been performed. To simplify the presentation of results, only the distribution of sites where 0.1 mM bicuculline () was injected has been reported. The location of injection sites () into some control regions where bicuculline and strychnine did not cause appreciable changes in the pattern of breathing and cough responses is also shown. Representative sites of control drug microinjections at locations⬎0.8 mm caudal to the responsive NTS sites have not been illustrated. NCM, nucleus cuneatus medialis; NDV, nucleus dorsalis nervi vagi; NG, nucleus gracilis; NOI, nucleus olivaris inferior; NV, nucleus tractus spinalis nervi trigemini; NXII; nucleus nervi hypoglossi; P, tractus piramidalis. The atlas of Meessen and Olszewski (48) and the atlas of Shek et al. (69) were used for comparison. An example of the location of fluorescent beads microinjected into the caudal NTS (green) is reported in the photomicrograph that represents a histological image of the rectangular area depicted on the diagram of the coronal section. The histological section is counterstained with cresyl violet. Light-field and fluorescent photomicrographs have been superimposed.
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consistent decreases in end-tidal CO2 partial pressure (from 32.9 ⫾ 0.5 to 31.3 ⫾ 0.4 mmHg; P ⬍ 0.05) were observed. Changes in cough-related variables reached a maximum within ⬃10 min and are reported in Table 2. Cough responses caused
by mechanical and chemical stimulations of the tracheobron-chial tree displayed increases in the cough number associated with decreases in the cough-related TT mainly due to reduc-tions in TI (Table 2 and Fig. 2). Cough-related variables Table 1. Cardiorespiratory variables during eupneic breathing before and approximately 10 min after bilateral
microinjections of bicuculline and strychnine into the caudal NTS
TT, s TI, s TE, s PPA, RU PAA, RU MAP, mmHg
Bicuculline (n⫽ 6) Control 1.19⫾ 0.08 0.36⫾ 0.02 0.83⫾ 0.02 0.53⫾ 0.02 0.05⫾ 0.01 100.1⫾ 5.3 0.1 mM 0.87⫾ 0.05** 0.30⫾ 0.01* 0.57⫾ 0.05* 0.52⫾ 0.02 0.01⫾ 0.008** 99.5⫾ 4.5 Recovery 1.18⫾ 0.06 0.37⫾ 0.02 0.82⫾ 0.06 0.53⫾ 0.02 0.04⫾ 0.01 99.7⫾ 4.8 Strychnine (n⫽ 6) Control 1.10⫾ 0.06 0.38⫾ 0.02 0.72⫾ 0.06 0.54⫾ 0.01 0.05⫾ 0.01 98.9⫾ 5.8 1 mM 0.78⫾ 0.07** 0.33⫾ 0.01* 0.46⫾ 0.07* 0.52⫾ 0.02 0.01⫾ 0.005** 98.6⫾ 6.1 Recovery 1.09⫾ 0.05 0.39⫾ 0.02 0.71⫾ 0.06 0.54⫾ 0.02 0.05⫾ 0.01 99.2⫾ 5.7
Values are means⫾ SE; n, number of animals; TT, cycle duration; TI, inspiratory time; TE, expiratory time; PPA, peak phrenic activity in relative units (RU); PAA, peak abdominal activity in relative units (RU); MAP, mean arterial blood pressure. *P⬍ 0.05, **P ⬍ 0.01 compared with controls as well as with recovery.
Fig. 2. Potentiating effects on cough reflex responses induced by 0.1 mM bicuculline microinjected into the caudal NTS in 1 anesthetized spontaneously breathing rabbit. Increases in cough responses⬃10 min after bilateral microinjections of bicuculline, i.e., when the maximum effect occurred. Recovery of cough responses was taken⬃60 min after the injections. The onset of mechanical stimulation is indicated by arrows while chemical stimulation is marked by solid bars. Phr IN, phrenic integrated neurogram; Phr N, phrenic neurogram; Abd IEMG, abdominal integrated electromyographic activity; Abd EMG, abdominal electromyo-graphic activity. Time (5 s) and voltage (0.1 and 0.5 mV) calibrations of original recordings are reported.
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displayed a progressive recovery and resumed control values within 60 min.
To corroborate the results on GABAA receptor-mediated modulation of the cough reflex, bilateral microinjections (n⫽ 4) of 0.1 mM muscimol (30 –50 nl; 3–5 pmol) at the same medullary sites were performed. They induced a progressive depression of cough responses that were completely sup-pressed within⬃10 min after the microinjections (Table 3 and Fig. 3). Despite an initial decrease in respiratory frequency, at this time no obvious or consistent change in cardiorespiratory variables during baseline eupneic breathing were present (sta-tistical data not shown; see also control recordings before reflex responses in Fig. 3, and for a general assessment of cardiorespiratory variables under control conditions see Table 1 and Refs. 51, 54, 55). Cough responses recovered to a great extent within⬃3 h.
Role of glycine receptors. In another series of experiments,
the effects of bilateral microinjections (n ⫽ 6) of 1 mM strychnine (30 –50 nl; 30 –50 pmol) into the caudal NTS were investigated. Similarly to bicuculline, strychnine induced within⬃2 min a high-frequency, irregular pattern of breathing superimposed on tonic phrenic nerve activity with the concom-itant reduction or complete abolition of abdominal activity.
Respiratory activity was evaluated⬃10 min after the injections at the time when the maximum effect on the cough reflex occurred. Marked increases in the respiratory frequency (from 55.7 ⫾ 2.9 to 81.9 ⫾ 4.9 breaths/min, ⫹ 47.1 ⫾ 5.2%; P ⬍ 0.01) due to decreases in both TIand TEwere observed (Table 1; see also control recordings before reflex responses in Fig. 4). Changes in the pattern of breathing progressively recovered within 60 min. No significant changes in arterial blood pressure were observed (Table 1). End-tidal CO2 partial pressure showed small decreases (from 31.6 ⫾ 0.6 to 29.2 ⫾ 0.6 mmHg; P ⬍ 0.05). Changes in cough-related variables ⬃10 min after bilateral microinjections of 1 mM strychnine are reported in Table 2. Cough responses caused by mechanical and chemical stimulations of the tracheobronchial tree showed increases in the cough number associated with decreases in the cough-related TTdue to reductions in both TIand TE(Table 2 and Fig. 4). Cough reflex responses displayed a progressive recovery that was complete within 60 min.
The glycine receptor-mediated modulation of the cough reflex was further investigated by bilateral microinjections (n⫽ 4) of 10 mM glycine (30–50 nl; 300–500 pmol) into the caudal NTS. Consistent and pronounced reductions or even the complete abolition of cough responses (one case) to both mechanical and chemical stimulation of the tracheobronchial tree were induced within 2–3 min after the completion of the injections (Table 3 and Fig. 5). With the exception of a very transient decrease in respiratory frequency immediately after the microinjections, no concomitant significant changes in cardiorespiratory variables during eupneic control breathing were observed (statistical data not shown; see also control recordings before reflex responses in Fig. 5). The recovery of cough responses was complete within a few minutes (5–7 min).
Controls. In four additional preparations, bilateral control
microinjections of 0.1 mM bicuculline (4 trials) or 1 mM strychnine (4 trials) were performed at different medullary locations sufficiently far from the responsive sites (see, e.g., Refs. 21, 44, 52, 55, 57, 59, 70). Control microinjections of Table 2. Cough-related variables before and approximately 10 min after bilateral microinjections of bicuculline and
strychnine into the caudal NTS
CN TT, s TI, s TE, s PPA, RU PAA, RU Bicuculline (n⫽ 6) Mechanical stimulation Control 2.59⫾ 0.38 0.55⫾ 0.03 0.38⫾ 0.03 0.17⫾ 0.02 0.63⫾ 0.02 0.55⫾ 0.03 0.1 mM 4.17⫾ 0.46** 0.49⫾ 0.02* 0.32⫾ 0.01* 0.17⫾ 0.01 0.55⫾ 0.04 0.50⫾ 0.06 Recovery 2.53⫾ 0.33 0.55⫾ 0.03 0.37⫾ 0.03 0.18⫾ 0.02 0.64⫾ 0.02 0.54⫾ 0.02
Citric acid inhalation
Control 2.88⫾ 0.32 0.55⫾ 0.03 0.36⫾ 0.02 0.19⫾ 0.01 0.61⫾ 0.01 0.54⫾ 0.02 0.1 mM 5.25⫾ 0.40** 0.48⫾ 0.02* 0.31⫾ 0.01* 0.17⫾ 0.01 0.54⫾ 0.03 0.46⫾ 0.04 Recovery 3.00⫾ 0.44 0.53⫾ 0.02 0.34⫾ 0.02 0.19⫾ 0.01 0.59⫾ 0.03 0.55⫾ 0.01 Strychnine (n⫽ 6) Mechanical stimulation Control 2.90⫾ 0.27 0.55⫾ 0.02 0.36⫾ 0.01 0.19⫾ 0.02 0.64⫾ 0.01 0.60⫾ 0.01 1 mM 4.67⫾ 0.23** 0.46⫾ 0.01* 0.33⫾ 0.01* 0.13⫾ 0.01* 0.53⫾ 0.04 0.42⫾ 0.02* Recovery 2.88⫾ 0.31 0.56⫾ 0.03 0.36⫾ 0.01 0.20⫾ 0.02 0.61⫾ 0.01 0.56⫾ 0.01
Citric acid inhalation
Control 3.01⫾ 0.51 0.54⫾ 0.02 0.34⫾ 0.01 0.20⫾ 0.02 0.64⫾ 0.01 0.57⫾ 0.02
1 mM 4.75⫾ 0.54** 0.46⫾ 0.01* 0.31⫾ 0.01* 0.15⫾ 0.01* 0.55⫾ 0.05 0.41⫾ 0.01*
Recovery 3.00⫾ 0.52 0.53⫾ 0.02 0.34⫾ 0.01 0.19⫾ 0.01 0.62⫾ 0.02 0.58⫾ 0.02
Values are means⫾ SE; n, number of animals; CN, cough number; TT, cycle duration; TI, inspiratory time; TE, expiratory time; PPA, peak phrenic activity in relative units (RU); PAA, peak abdominal activity in relative units (RU). *P⬍ 0.05, **P ⬍ 0.01 compared with control cough as well as with recovery.
Table 3. Effects on the cough number induced by muscimol
and glycine microinjections into the caudal NTS
Muscimol (n⫽ 4)
Mechanical stimulation Citric acid inhalation
Control 2.87⫾ 0.43 Control 3.10⫾ 0.59
0.1 mM 0.1 mM
Recovery 2.82⫾ 0.39 Recovery 3.12⫾ 0.62
Glycine (n⫽ 4)
Mechanical stimulation Citric acid inhalation
Control 3.12⫾ 0.51 Control 3.20⫾ 0.27
10 mM 0.81⫾ 0.42** 10 mM 0.87⫾ 0.43**
Recovery 3.17⫾ 0.54 Recovery 3.22⫾ 0.28 Values are means⫾ SE; n, number of animals. **P ⬍ 0.01 compared with control cough as well as with recovery.
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each drug were executed into each of the following neural structures (see Refs. 48, 69): the nucleus cuneatus medialis, the nucleus tractus spinalis nervi trigemini, and the region located ⬎0.8 mm caudal to the responsive NTS sites. In all instances, these injections failed to induce the characteristic effects re-ported above. As in our previous studies on the NTS, control injections of equal volumes of the vehicle solution at the responsive sites (3 trials performed in 3 different preparations before drug administration) were ineffective.
The localization of all injection sites was confirmed by the histological control performed either by the observation of pipette tracks and injection sites or, in some instances, micro-sphere fluorescence (see Histology underMATERIALS AND METH -ODSfor references). An example of the location of fluorescent beads microinjected into the caudal NTS is reported in Fig. 1. The localization of injection sites on a dorsal view of the medulla oblongata as well as the distribution of injection sites within the caudal NTS and some control regions are also
illustrated. For simplicity, only the distribution of sites where 0.1 mM bicuculline was injected has been reported.
DISCUSSION
This study provides relevant contributions to the present knowledge on the control of the cough reflex and respiratory activity exerted at the level of the caudal NTS. It shows for the first time that both GABAA and glycine receptors mediate a potent inhibitory modulation of the pattern of breathing and, particularly, of cough reflex responses, thus supporting the notion that the NTS region is an important neural component involved in the central regulation of both respiration and airway defensive reflexes. The results also indicate that GABAergic and glycinergic inhibition is already present under basal conditions. Interestingly, the specificity of the inhibitory modulation of the cough reflex is confirmed by the effects obtained in response to microinjections of the specific agonists Fig. 3. Depressant effects of 0.1 mM muscimol microinjected into the caudal NTS on cough reflex responses in 1 anesthetized spontaneously breathing rabbit. Complete suppression of cough responses⬃10 min after bilateral microinjections. Recovery ⬃3 h after the injections. Onset of mechanical stimulation indicated by arrows. Chemical stimulation marked by solid bars. Phr IN, phrenic integrated neurogram; Phr N, phrenic neurogram; Abd IEMG, abdominal integrated electromyographic activity; Abd EMG, abdominal electromyographic activity. Time (5 s) and voltage (0.1 and 0.5 mV) calibrations of original recordings are reported.
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that were in the opposite direction to those produced by bicuculline and strychnine.
Methodological consideration and general remarks.
Micro-injection procedures, their reliability, and the spread of the injectate have been extensively discussed in previous reports (21, 51–55; see also Refs. 44, 59, 70). Injection sites were selected by using stereotaxic coordinates according to the atlas of Meessen and Olszewski (48). Their localization was con-firmed by the histological control. The specificity of drug-induced effects is supported by the absence of changes in the breathing pattern and cough reflex responses following drug microinjections at sites sufficiently far from the responsive sites as well as following vehicle microinjections into the caudal NTS. The concentrations of bicuculline, muscimol, and strychnine were selected in preliminary trials to ensure their effectiveness on the cough reflex without inducing too dra-matic changes in the ongoing respiratory activity. Given the very short-lasting inhibitory effects of glycine (due not only to
a very rapid diffusion, but also to physiological mechanisms such as neurotransmitter reuptake) and the consequent rapid recovery observed in preliminary trials, we employed micro-injections with relatively high concentrations of this drug to allow sufficient time for stimulation trials.
Changes in the pattern of breathing. We will deal with this
subject only briefly since our attention was mainly focused on the cough reflex. However, we believe that the changes ob-served in the ongoing respiratory activity are of interest. The results disclose a tonic inhibitory control of this activity at the level of the caudal NTS and strongly suggest that one impor-tant mechanism of respiratory frequency modulation may rely on a disinhibitory process exerted on GABAA and glycine receptors. Our results show that GABAAand glycine receptor antagonists cause marked changes in respiratory timing that are typically associated with the function of a rhythm-generating mechanism (8, 32, 76), thus suggesting the presence of ele-ments contributing to the respiratory rhythmogenesis in the Fig. 4. Potentiating effects on cough reflex responses caused by 1 mM strychnine microinjected into the caudal NTS in 1 anesthetized spontaneously breathing rabbit. Increases in cough responses⬃10 min after bilateral microinjections of strychnine (maximum effect). Recovery ⬃60 min after the injections. The onset of mechanical stimulation is indicated by arrows while chemical stimulation periods are marked by solid bars. Phr IN, phrenic integrated neurogram; Phr N, phrenic neurogram; Abd IEMG, abdominal integrated electromyographic activity; Abd EMG, abdominal electromyographic activity. Time (5 s) and voltage (0.1 and 0.5 mV) calibrations of original recordings are reported.
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caudal NTS. Although it has been shown that the dorsal part of the medulla does not have a primary role in respiratory rhythm generation (e.g., Refs. 1, 36), it cannot be excluded that neurons in the caudal NTS, either related to the vagal input or not, are embedded in a pontomedullary circuit implicated in the control of the respiratory timing (5, 12, 76). The possibility also exists that in the medulla, including the caudal NTS, there are multiple coupled oscillators, each of which can drive a different behavior, such as breathing, coughing, and swallow-ing (8). In this context, it seems appropriate to mention that recent reports have proposed that, at variance with previous studies in the rat, NTS region has an important role in the neural control of breathing (3, 38). It should be also taken into consideration that the effects on respiratory timing could be, at least in part, related to the removal of the input arising from second-order sensory neurons in the cough afferent pathway either activated by RARs (RAR cells) or by C-fiber endings (C cells). Activation of these interneurons is well known to cause decreases in TEand increases in respiratory frequency (68).
The results also show that disinhibition of neurons in the caudal NTS strongly decreases expiratory activity in abdomi-nal muscles. These findings recall previous studies (13) show-ing that the caudal portion of NTS, correspondshow-ing to the caudal dorsal respiratory group, has a clear role in the regulation of the expiratory motor output: the blockade or functional ablation of this area by focal cooling causes obvious increases in rhythmic expiratory activity.
Changes in the cough reflex responses. As above reported
for the ongoing pattern of breathing, the results show that the cough reflex is subject to an intense GABAergic and glycin-ergic inhibitory control and indicate that the disinhibition is an important mechanism in the modulation of the cough reflex. Obviously, disinhibition phenomena may lead to changes in the excitability and discharge of caudal NTS neurons. As already mentioned, evidence has been provided that inhibitory inputs impinge on GABAAand glycine receptors of RAR cells (28, 30, 31, 45). These latter are, in our opinion, plausible second-order neurons in the cough afferent pathway. In fact, Fig. 5. Decreases in cough reflex responses induced by 10 mM glycine microinjected into the caudal NTS in 1 anesthetized spontaneously breathing rabbit. Very marked reduction in cough responses 2 min after bilateral microinjections of glycine. The recovery of cough responses was taken⬃10 min after the injections. Onset of mechanical stimulation indicated by arrows. Chemical stimulation marked by solid bars. Phr IN, phrenic integrated neurogram; Phr N, phrenic neurogram; Abd IEMG, abdominal integrated electromyographic activity; Abd EMG, abdominal electromyographic activity. Time (5 s) and voltage (0.1 and 0.5 mV) calibrations of original recordings are reported.
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described RAR cells were sensitive to chemical stimuli, such as inhalation of ammonia vapor, that can elicit the cough reflex in response to the stimulation of receptors located in the upper extrapulmonary airways in different animal species including the rabbit (see, e.g., Refs. 40, 46). In addition, recorded RAR cells were located caudal to the obex and especially in the lateral commissural subnucleus of NTS (e.g., Refs. 41, 42, 45), i.e., in a location closely corresponding to that where lesions or ionotropic glutamate receptor blockades suppress the cough reflex without affecting the Breuer-Hering inflation reflex or the pulmonary chemoreflex (42, 55). Both a tonic and a phasic inhibition is conveyed to RAR cells. While the origin of tonic inputs has not been determined, phasic inhibition (29) appears to be mainly due to second-order neurons in the pulmonary stretch receptor pathway, the so-called P cells (4, 23, 62). These neurons have been shown to be GABAergic and some of them may corelease GABA and glycine (29). These interneu-rons, in turn, have been reported to be a site of convergence and complex integration of several excitatory and inhibitory influences (49). No information is at present available, to our knowledge, on the inhibitory inputs impinging on second-order C cells, but a role of these interneurons in the observed disinhibition phenomena could also be taken into consider-ation. The caudal NTS and, in particular, RAR cells may receive inhibitory inputs from components of the brain stem respiratory network and send to them extensive projections (27, 31, 41, 61 for further references). Among the targets of these projections there are neurons involved in rhythm gener-ation and pattern formgener-ation. Furthermore, the cough reflex, and conceivably the caudal NTS, are also under the inhibitory control of higher brain structures as demonstrated, for instance, by the effects exerted on this reflex by voluntary suppression and mindfulness (see, e.g., Ref. 81 for further references). Inhibitory influences on the cough reflex possibly at the level of the caudal NTS can be brought into action by the stimulation of lung stretch receptors that project to P cells (e.g., Ref. 15) and of pulmonary C fibers (e.g., Refs. 72, 73; but see Ref. 50). In addition, evidence has been provided that inhibitory inputs to the central cough-generating mechanism may arise from nasal trigeminal receptors under particular circumstances such as the local application of a TRPA1 agonist in guinea pigs (6) or of a TRPM8 agonist in humans (11). As to the receptors involved in cough production in the present study, we suggest that they are similar to the cough receptors described in the guinea pig (14, 16). This type of receptors is possibly present also in the rabbit (see, e.g., present results and Ref. 58) as well as in other animal species. However, to our knowledge, there is no evidence that these receptors in all animal species are sensitive exclusively to punctate mechanical stimuli and acid as demonstrated for guinea pig cough receptors (14, 16).
The results show that one of the most significant changes following bicuculline or strychnine microinjections is the in-crease in the cough number. Like in a previous report on the role of GABAAreceptors in the caudal VRG (20), increases in the cough number could appear to be related to increases in the respiratory frequency. Both these outcomes, however, depend on bicuculline or strychnine microinjections and it is not possible to infer a relationship of cause and effect between them. In this context, it should be recalled that the cough number has been suggested to depend mainly on the activation of a central gate mechanism (7, 9). Interestingly, we have
provided evidence (50) suggesting that the timing of the cough motor pattern depends, to some extent, on the timing of baseline respiratory activity. However, changes in the cough-related timing variables do not, in the present study, parallel exactly the changes in the timing of the ongoing respiratory activity (see RESULTS). The slight differences in the effects on cough-related variables (Table 2) observed in response to bicuculline and strychnine are difficult to explain. They could be related to differences in the concentration of each antagonist as well as in the target of each inhibitory neurotransmitter or in the strength of the related synapses. Furthermore, it seems unlikely that the small changes observed in end-tidal CO2 partial pressure could have affected the characteristics of the cough motor pattern (for review see Refs. 35, 79). Finally, it should be mentioned that drug-induced changes in arterial blood pressure that could affect the intensity of cough re-sponses (65) were not observed.
In a recent study, Bolser et al. (8) have proposed that rhythmic circuits exist in the NTS for coughing and swallowing. This hypothesis was supported by changes in cycle duration in re-sponse to microinjections of cough suppressants performed in the rabbit (21, 57) as well as by variations in the ongoing respiratory activity and cough responses following bilateral microinjections of kynurenic acid in the cat NTS (see Ref. 8). The changes in timing observed in the present and previous studies by us seem to be consistent with this hypothesis. The proposed model gets further insights into the neural mechanisms generating cough and swallowing as well as into the coordination between these airway defensive reflexes. Furthermore, it appears to include at least part of the neuronal connections concerning the caudal NTS above reported by us (for details see Ref. 8).
Interestingly, Canning and colleagues have reported in an earlier study (47) that cough receptor afferents in the guinea pig terminate in the commissural subnucleus. However, more recently (15) they have reported that cough afferents from the extrathoracic trachea terminate in NTS locations rostral and lateral to the obex and to the commissural subnucleus, i.e., sites more rostral than those investigated in the present study. We cannot exclude that these regions display responses to micro-injections of agonists and antagonists of GABAAand glycine receptors similar to those shown in the caudal NTS.
Conclusions and perspectives. One of the main conclusions of
this study is that the cough reflex and possibly also other airway defensive reflexes are modulated by reducing the potent inhibitory control exerted at the level of the caudal NTS. A similar modu-lation can occur at the level of the caudal VRG (20). This mode of action appears to be very effective in preventing under basal conditions reflex actions (such as cough, sneeze, and swallowing) that could heavily interfere with usual activities. However, it allows defense reactions to occur when triggered by appropriate stimuli. Of course, this is not the sole mechanism of cough reflex regulation present. Presynaptic control of neurotransmitter release and excitatory pathways may exist as well (47, 55). The present findings also encourage further investigations on second-order neurons involved in the cough afferent pathway and, in particular, on C cells devoted to disclose their synaptic connections and responses to excitatory and inhibitory inputs. Recently, the atten-tion of researchers (see Refs. 19, 21, 24, 34, 52, 57, 60 for further references) has been focused on the similarities between the mechanisms underlying the cough reflex and those subserving nociception and pain sensation. Actually, a parallel should be
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more appropriately made between the cough reflex and nocicep-tive reflexes as well as between the urge to cough and pain sensation (see also, e.g., Ref. 34). The presence of the potent inhibitory control demonstrated in the present study may suggest that both chronic cough and chronic pain could be due, at least to some extent, to an impairment of GABAergic and glycinergic inhibition. Accordingly, neuropathic pain has been reported to be related to an impairment of the activation of GABAAand glycine receptors (39, 67, 74, 82). Here, we have provided evidence suggesting the possibility that cough hypersensitivity can be produced as a consequence of disinhibition. In this context, it can be of interest to mention that alterations in the sensitivity of NTS neurons to the activation of GABA receptors have been recently shown in hypertensive rats, thus suggesting that an impairment of inhibitory mechanisms may contribute to a visceral pathology (for review see Ref. 83). We propose that improvements in the knowledge of basic mechanisms underlying the defensive re-flexes, in particular the cough reflex, could possibly lead to the development of novel and more effective therapeutic approaches for chronic pathological conditions for instance with the employ-ment of specific drugs acting on subunits of GABAAor glycine receptors. In addition, present results as well as those of previous studies on cough-enhancing effects of substance P (15, 47, 55 for further references) and lisinopril (56) microinjected into the cau-dal NTS indicate that central neural mechanisms involved in the upregulation of the cough reflex do exist. They could provide hints for investigations on therapeutic strategies for neurological diseases characterized by impaired airway protective reflexes, including cough and swallowing, such as Parkinson’s disease (e.g., Refs. 26, 33, 75, 77).
GRANTS
This study was supported by grants from the Ministry of Education, University and Research of Italy. E. Cinelli was supported by a postdoctoral fellowship from the Fondazione Internazionale Menarini.
DISCLOSURES
No conflicts of interest, financial or otherwise, are declared by the author(s). AUTHOR CONTRIBUTIONS
E.C., L.I., F.B., T.P., and D.M. conception and design of research; E.C., L.I., F.B., and D.M. performed experiments; E.C., L.I., F.B., and D.M. analyzed data; E.C., L.I., F.B., T.P., and D.M. interpreted results of experi-ments; E.C. and L.I. prepared figures; E.C. and D.M. drafted manuscript; E.C., F.B., T.P., and D.M. edited and revised manuscript; E.C., L.I., F.B., T.P., and D.M. approved final version of manuscript.
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