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1 The State Key Laboratory of Medical Neurobiology, Fudan University Shanghai Medical College, PR China 2 College of Life Sciences, Tongji University, PR China 3 Department of Surgery, Fudan University Zhongshan Hospital, PR China
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(Received 10 October 2006;
accepted after revision 1 November 2006; first published online 10 November 2006)
Corresponding author J. Wang: The State Key Laboratory of Medical Neurobiology, Fudan University Shanghai Medical College, 138 Yi-Xue-Yuan Road, Shanghai 200032, PR China. Email: wangjj{at}shmu.edu.cn
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Introduction |
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Currently, our knowledge about the central distribution of the airway-related preganglionic parasympathetic motoneurones (APPMs) comes mainly from anatomical tracing studies, which have shown that APPMs are primarily located in three sites in the medulla: the compact portion of the nucleus ambiguus (cNA); the area ventral/ventrolateral to cNA (vNA); and the dorsal motor nucleus of the vagus. Unfortunately, tracer methodologies have met great difficulties in elucidating whether different airway segments, and whether different tissue structures within the same airway segment, are represented by specific central neurones. The main difficulty resides not only with localizing the tracer injections accurately in the desired tissue but also with preventing the injected material from diffusing to adjacent areas (Fox & Powley, 1989). Even so, there has been some evidence to indicate that APPMs controlling different segments of the airway might be differentially located in the ventrolateral medulla. Airway-related preganglionic parasympathetic motoneurones labelled by application of tracers into the laryngeal muscles (Waldbaum et al. 2001), the trachealis muscles (Haxhiu et al. 1993) and the lung parenchyma (Hadziefendic & Haxhiu, 1999) were similar in distribution and were in both cNA and vNA in the ventrolateral medulla; however, those APPMs labelled by finely controlled application of tracers directly to the laryngeal nerve were confined within cNA (Irnaten et al. 2001a,b; Barazzoni et al. 2005; Okano et al. 2006), suggesting that tracer application directly to the laryngeal nerve might be more specific in labelling APPMs projecting to laryngeal nerves.
The respiratory-related synaptic control of APPMs has not been investigated, and whether differently targeted APPMs are modulated differentially by central respiratory activity is not known. In living medullary slices in which APPMs have been precisely localized by anatomical tracing method, examination of the respiratory-related synaptic control of APPMs would reveal the possible differences of these neurones from their functional aspect. In this study, we have combined fluorescent tracing method and patch-clamp method to study putative APPMs in brainstem slices with respiratory rhythm in newborn rats. The results indicate that those putative APPMs labelled by tracer application directly to the recurrent laryngeal nerve (RLN) and those putative APPMs labelled by tracer application to the tracheal wall had different distributions in the ventrolateral medulla and different respiratory-related modulation of their synaptic inputs. These results provide evidence that APPMs controlling different segments of the airway might be dissociated in the ventrolateral medulla both anatomically and in functional control.
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Methods |
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Halothane (0.5 ml) was dripped into a glass box (5 cm x 5 cm x 5 cm) with a lid and cotton pad on the bottom. Three- to 4-day-old Sprague–Dawley rats (Shanghai Institute for Family Planning) were put into the box for 30 s with the lid on. This procedure anaesthetized the rats but kept their breathing at a relatively normal level. When the rat lost responsiveness to a needle prick stimulus to the limbs, the body was surrounded by ice-filled bags to decrease the body temperature and slow the heart. Once spontaneous breathing stopped (usually within 2 min), the animal was put on an ice-filled bag in a supine posture, and a ventral mid-line incision was made in the neck to expose the extrathoracic trachea. Rhodamine (XRITC, Molecular Probes, 1% solution, 0.5 µl) was injected into the tracheal wall between the fourth and the eighth tracheal cartilage ring using a glass pipette (tip diameter 30 µm), which was attached to a syringe through polyethylene tubing. In some animals, the RLN was dissected unilaterally or bilaterally and isolated from the surrounding tissues with Parafilm, and rhodamine (about 0.1 µl) was applied to a 1 mm nerve segment before the nerve was surrounded by a glutin sponge. As a control, in three animals the tracer (0.5 µl) was injected into the oesophageal wall at the same level as the tracheal wall injection. The incision was sutured, and the animals were heated with a thermostatically controlled pad (30°C) to help recovery. During the whole surgery period (about 5 min), the body temperature of the animal was below 10°C and the animal exhibited no spontaneous breathing or struggling. After the surgery, the animals usually started spontaneous breathing within 3 min and started moving freely within another 5 min. The animals were allowed 48 h to recover.
Slice preparation
48-52 hours after the surgery the animal was anaesthetized deeply with halothane applied as before and decapitated at the supracollicular level. The brain was submerged in cold (4°C) artificial cerebral spinal fluid (ACSF) of the following composition (mM): NaCl, 124; KCl, 3.0; KH2PO4, 1.2; CaCl2, 2.4; MgSO4, 1.3; NaHCO3, 26; and D-glucose, 10; constantly bubbled with 95% O2–5% CO2, pH 7.4. The cerebellum was removed and the hindbrain was isolated using a dissection microscope. The brainstem was then secured in the slicing chamber of a vibratome (Leica VT 1000S) filled with the same ACSF. The rostral end of the brainstem was set upwards and the dorsal surface was glued to an agar block facing the razor. The brainstem was sectioned serially at variable thickness in the transverse plane. Once the nucleus ambiguus was visible under the microscope, a single medulla slice 500–800 µm thick, of which one to two hypoglossal rootlets in each side were retained, was taken for experimentation. The thick medullary slice preparation generates rhythmic inspiratory-related discharge in hypoglossal rootlets (Smith et al. 1991). The slice was transferred into the recording chamber and submerged in flowing ACSF (flow rate, 8–11 ml min–1). The rostral cutting plane of the slice was set upwards to allow fluorescent identification and patch-clamp recording of the putative APPMs in the ventrolateral medulla. The temperature was maintained at 23 ± 0.5°C, and the concentration of KCl in the ACSF was increased to 10 mM to allow steady recording of the respiratory rhythm.
Electrophysiological recording
Individual putative APPMs were identified by the presence of the fluorescent tracer using an Olympus upright microscope with a x40 water immersion objective. The patch pipettes (2.0–5.0 M
) were normally filled with a solution consisting of (mM): potassium gluconate, 130; Hepes, 10; EGTA, 10; CaCl2, 1; and MgCl2, 1; pH 7.3 (briefly, the potassium gluconate-dominated pipette solution). With this pipette solution, the Cl–-mediated GABAergic and glycinergic inhibitory synaptic currents were minimized at a holding potential of –80 mV and only the glutamatergic excitatory synaptic currents (inward) could be recorded. However, at a holding potential of –50 mV both the inhibitory (outward) and the excitatory (inward) synaptic events could be recorded. In some experiments, the patch pipettes were filled with a solution consisting of (mM): KCl, 150; MgCl2, 2; Hepes, 10; EGTA, 2; and Mg-ATP, 2; pH 7.35 (briefly, the KCl-dominated pipette solution), and the neurones were voltage clamped at –80 mV. With this pipette solution and holding voltage, both the excitatory and the inhibitory synaptic events were recorded as inward currents. The pipette resistance and capacitance were not compensated either before or after gaining intracellular access.
The patch-clamp signal was amplified with an Axopatch 200B amplifier (10 kHz sampling frequency; 1 kHz filter frequency), digitized with 1322A digidata, and collected with Clampex 9.0 software (Axon Instruments, USA). The activity of the hypoglossal rootlets was recorded using a suction electrode and was amplified with a BMA-931 bioamplifier (5 kHz sampling frequency; 10–1000 Hz bandpass; 50 000 times), electronically integrated (time constant, 
= 50 ms) with MA-1000 Moving Averager (CWE Inc., PA, Ardmore, USA) before feeding into the computer. All animal procedures were performed in compliance with the institutional guidelines at Fudan University, and were in accordance with the internationally accepted principles in the care and use of experimental animals.
Drug application
Drugs were always applied to the bath. Strychnine (10 µM) and picrotoxin (10 µM) were used to block the glycinergic and the GABAergic receptors, respectively. 6-Cyano-7-nitroquinoxaline-2,3-dione (CNQX, 50 µM) was used to block non-NMDA glutamate receptors. All drugs were purchased from Sigma-Aldrich (St Louis, MO, USA).
Data analysis
Spontaneous synaptic activity and the respiratory-related changes of the synaptic activity were analysed with MiniAnalysis (Synaptosoft, version 4.3.1) with minimal acceptable amplitude at 10 pA. Results are presented as means ± S.E.M. and statistically compared with Student's paired t test. Significant difference was set at P < 0.05.
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Results |
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Fluorescently labelled putative APPMs were examined only in the ventrolateral portion of the rostral cutting plane of the slices. After tracer injections into the tracheal wall, fluorescenctly labelled putative APPMs were found in both cNA and vNA in nine of the 16 slices examined and were found only in vNA in seven slices. After tracer application to unilateral (in two animals) or bilateral (in three animals) RLN, fluorescently labelled putative APPMs were exclusively found within cNA, and only in ipsilateral cNA were fluorescently labelled putative APPMs found if the tracer was applied unilaterally. The fluorescently labelled putative APPMs within cNA were not visually different in size and shape compared with those unlabelled in this site, and were round, oval, triangular or multiangular (Fig. 1A–C). The fluorescently labelled putative APPMs in vNA were larger in size and were spindly, triangular or multipolar (Fig. 1D and E). These vNA neurones were either scattered or clustered, spread from the close ventral/ventrolateral vicinity of cNA to more ventral/ventrolateral sites.
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Respiratory modulation of the fluorescently labelled putative APPMs within cNA
Under our recording conditions, rhythmic respiratory-like hypoglossal bursts were obtained in all the 19 slices examined, and the frequency ranged from 4.1 to 13.4 bursts min–1 (11.36 ± 2.11 bursts min–1).
With the patch pipettes filled with the potassium gluconate-dominated pipette solution and the neurones voltage clamped at –50 mV, the fluorescently labelled putative APPMs examined within cNA (23 neurones examined) showed rich inhibitory (outward) and very scarce excitatory (inward) synaptic activity. In 14 of these neurones, the inhibitory synaptic activity showed an inspiratory-related decrease (Fig. 2A). In nine of these neurones, the inhibitory synaptic activity showed an inspiratory-related increase, and the baseline current usually showed an inspiratory-related upward shift (Fig. 2B). All these 23 putative APPMs were neurones that had been labelled by tracer injection into the tracheal wall. Since the inhibitory and the excitatory synaptic activity are recorded in opposite directions at a holding potential of –50 mV, some of the excitatory synaptic activity might have been counteracted by the much more frequent inhibitory synaptic activity. To better record the excitatory synaptic activity, all these 23 neurones were switched to a holding potential of –80 mV. At this holding potential, the excitatory synaptic activity of these neurones was still extremely scarce (range, 0–0.51 Hz; 0.14 ± 0.04 Hz; n = 23) and did not show any respiratory-related change (not shown).
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The fluorescently labelled putative APPMs in vNA showed inspiratory-related augmentation of either excitatory or inhibitory synaptic activity
With the patch pipette filled with the potassium gluconate-dominated pipette solution and the neurones clamped at –50 mV, all the putative APPMs in vNA showed an inspiratory-related increase of either the excitatory or the inhibitory synaptic activity.
Some putative APPMs in vNA (type I; 11 neurones identified) showed simultaneous increases in both the frequency and the amplitude of the excitatory synaptic activity during central inspiratory bursts (Fig. 5A, D and E). The baseline frequency of the excitatory (inward) synaptic activity of this type of putative APPMs ranged from 2.33 to 7.54 Hz (4.60 ± 0.77 Hz; examined in 11 neurones). In a majority (9 of 11) of this type of neurones, the inhibitory (outward) synaptic activity was either absent or extremely scarce (< 0.1 Hz); however, in two of these 11 neurones the frequency of the inhibitory synaptic activity was much more frequent (4.11 and 3.22 Hz, respectively), as is also shown in Fig. 5A. Bath application of CNQX (50 µM) reversibly abolished the inspiratory-like hypoglossal bursts (not shown) and reversibly blocked all the excitatory synaptic activity (Fig. 5B). In the two neurones that had relatively more frequent inhibitory synaptic activity, picrotoxin (10 µM) or strychnine (10 µM) each blocked a proportion, and combined application of these two drugs abolished all, of the inhibitory synaptic activity (Fig. 5C). In addition, three of these 11 neurones were also recorded in the cell-attached mode before gaining intracellular access, and these neurones generated an inspiratory-related discharge (not shown).
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Putative oesophageal motoneurones showed no inspiratory-related change of the synaptic inputs
Five neurones that had been labelled by tracer application into the oesophageal wall were examined. Comparable with those putative APPMs within can, these putative oesophageal motoneurones also received rich inhibitory and scarce excitatory synaptic inputs. However, in none of these five neurones did the synaptic activity show any inspiratory-related change (not shown).
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Discussion |
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Airway-related preganglionic parasympathetic motoneurones projecting to the RLN might be exclusively located within cNA. This hypothesis is supported by this study, and is also supported by previous studies showing that finely controlled application of tracers directly to the superior laryngeal nerve only retrogradely labelled APPMs within cNA (Irnaten et al. 2001a,b; Barazzoni et al. 2005; Okano et al. 2006). Regarding the central distribution of APPMs projecting to the trachea, this study has suggested two possibilities. One is that these neurones were only located in vNA. The tracer injected into the trachea wall might have diffused rostrally to the laryx or laterally to the RLN, thereby resulting in labelling of motoneurones in cNA that control laryngeal muscles. The other is that these neurones were located in both cNA and vNA.
Previous studies (Barillot et al. 1990; Ono et al. 2006) had identified both inspiratory- and expiratory-related laryngeal motoneurones in the nucleus ambiguus, which showed phasic fluctuation of the membrane potential in the respiratory cycle. Both the excitatory and the inhibitory inputs had also been indicated in these neurones (Hayakawa et al. 2000; Ono et al. 2006). However, how the laryngeal motoneurones generate inspiratory-related and/or expiratory-related discharge has not been understood at the synaptic level. In this study, some fluorescently labelled putative APPMs within cNA might be more likely to discharge during the inspiratory phase of respiration and might be inpiratory related. Some other fluorescently labelled putative APPMs within cNA might be more likely to discharge during the expiratory phase of respiration and might be expiratory related.
The RLN is known to innervate the oesophagus in addition to the airway (Liebermann-Meffert et al. 1999). Oesophageal motoneurones, however, had been reported to receive no respiratory-related synaptic inputs in rats in a previous study (Kruszewska et al. 1994). In this study, four putative APPMs within cNA showed no respiratory-related change of the synaptic activity, and these four neurones were among those that were labelled by tracer application to the RLN. These four neurones might therefore be oesophageal motoneurones. In contrast, those putative APPMs in cNA that were labelled by tracer application to the RLN, of which the inhibitory synaptic activity showed inspiratory-related changes, might be related to the control of laryngeal muscles.
It is known that the RLN innervates laryngeal constrictors (adductors), which dilate the glottis during inspiration to decrease airway resistance, and innervates laryngeal dilating muscles (abductors), which slightly narrow the glottis during the early period of expiration, i.e. postinspiration, to brake expiratory airflow and so limit collapse of the lung (Bartlett, 1986). In the present study, in cNA the inspiratory-related putative APPMs might project to the laryngeal adductors and the expiratory-related putative APPMs might project to the laryngeal abductors.
In collaboration with the superior laryngeal nerve, the RLN also plays important roles in controlling the larynx during vocalization, straining and several airway defensive reflexes including swallowing, coughing and sneezing (Shiba et al. 1997). In brainstem slices, the synaptic inputs of the putative APPMs within cNA have lost many tonic and functionally related activities, and the respiratory-related modulation of the inhibitory synaptic inputs might simply reflect one aspect of the respiratory modulation of these neurones.
In vNA, the putative APPMs of type I might be inspiratory-related motoneurones. Some (4 of 8) putative APPMs of type II might be expiratory-related motoneurones, and others (4 of 8) might be postinspiratory-related motoneurones. At least some putative APPMs in vNA might be tracheal related, since they were labelled by tracer injection into the tracheal wall. The respiratory-related modulation of the putative APPMs in vNA in this study is in consistent with a previous study that had identified both inspiratory and expiratory neurones in the tracheal ganglion (Baker, 1986). The inspiratory-related motoneurones in vNA are likely to project to the tracheal ganglion, synapse on the inspiratory neurones and cause inspiratory contraction of tracheal muscle. The expiratory-related and the postinspiratory-related motoneurones in vNA are also likely to project to the tracheal ganglion and to synapse on the expiratory/postinspiratory neurones, but their functions are hard to predict at present.
A previous study found that injection of tracers into the pericardial sac, which was assumed to retrogradely label preganglionic cardiac vagal motoneurones, had in fact labelled mostly oesophageal-related and few cardiac-related vagal motoneurones in and around cNA (Grkovic et al. 2005). Mislabelling has been raised as a severe problem regarding retrograde labelling of central neurones thereafter. In the present study, labelling of putative oesophageal motoneurones could not be prevented technically when the tracer was applied to the RLN, since this nerve innervates both the oesophagus and the airway. There should, however, have been no mislabelling of oesophageal motoneurones when the tracer was injected into the tracheal wall because all the putative APPMs labelled by this tracing method showed inspiratory-related changes of synaptic activity.
It has been noticed that the respiratory neuronal network in rats experiences developmental changes during the late fatal and the neonatal periods (Paton et al. 1994; Denavit-Saubie et al. 1994; Onimaru & Homma, 2002). The respiratory modulation of APPMs might not be the same in adult rats as that in newborn rats. Unfortunately, in adult rats, although both the excitatory and the inhibitory inputs of the laryngeal motoneurones have been identified by many previous studies, little is known about how these inputs are organized during the respiratory cycle. Regarding tracheal motoneurones, nothing was previously known about their synaptic inputs. Therefore, it is not known whether the respiratory-related modulation of APPMs observed in newborn rats in this study is also applicable to adult rats. Further study is necessary regarding this issue.
Overall, this study has, for the first time, characterized the synaptic inputs of putative APPMs and has revealed the synaptic mechanisms for the respiratory-related functional control of these neurones. The results provide evidence that APPMs controlling different segments of the airway might be dissociated in the ventrolateral medulla both anatomically and in functional control.
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References |
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XII, integrated hypoglossal activity.
