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. 2001 Apr 15;532(Pt 2):535–548. doi: 10.1111/j.1469-7793.2001.0535f.x

Respiratory activity in glossopharyngeal, vagus and accessory nerves and pharyngeal constrictors in newborn rat in vitro

Makito Iizuka 1
PMCID: PMC2278554  PMID: 11306670

Abstract

  1. Previously, in a brainstem-spinal cord-rib preparation from neonatal rats we demonstrated that a decrement in extracellular pH (from about 7.4 to 7.1) caused expiratory activity in an internal intercostal muscle (IIM) during the first half of the expiratory phase (Ea). As the initial step in finding nerves or muscles firing during the second half of the expiratory phase (Eb), the patterns of activity in the glossopharyngeal, vagus and accessory nerves were examined in the present study.

  2. Since the emerging motor rootlets of these three nerves (> 20; collected into about 10 bundles before the jugular foramen) are distributed in a continuous fashion from rostral to caudal levels of the brainstem, visual identification was impossible. Therefore, antidromic compound action potentials evoked by stimulation of the glossopharyngeal nerve (IX), the pharyngeal branch of the vagus nerve (PhX), the superior laryngeal nerve (SLN), the cervical vagus nerve (CX) and the accessory nerve (XI) were recorded from the peripheral stumps of the various rootlets. Nerve rootlets could be categorised into rostral, intermediate and caudal groups (rostIX-XI, intIX-XI, caudIX-XI). The rostIX-XI rootlets showed their largest potential on IX stimulation, while the intIX-XI and caudIX-XI rootlets showed their largest potentials on CX stimulation. The intIX-XI rootlets showed larger potentials on PhX and SLN stimulation than the caudIX-XI rootlets.

  3. Activity was recorded simultaneously from the central stumps of the rootlets in the above three groups. Most rootlets showed inspiratory bursts. Under low pH conditions, all representatives of group rostIX-XI, most of intIX-XI and about half of caudIX-XI showed additional bursts during the Ea phase. Groups intIX-XI and caudIX-XI but not rostIX-XI also showed discrete bursts during the Eb phase in some preparations. In general, expiratory activity was prominent in intIX-XI. The spinal branch of XI showed no consistent respiratory activity.

  4. Since the intIX-XI rootlets showed Eb bursts and large antidromic potentials on stimulation of PhX and SLN (which innervate the inferior pharyngeal constrictor muscle (IPC)), electromyograms were recorded from the rostral and caudal parts of IPC (rIPC and cIPC). Under low pH conditions, cIPC showed bursts during the Ea and Eb phases, while rIPC showed bursts predominantly during the Eb phase.

  5. These results indicate that recording from rIPC would be a useful way of examining the neuronal mechanisms responsible for Eb phase activity.

Respiration involves complex movements that require numerous motoneurones distributed along the neuraxis to fire with spatio-temporally correct timing. Although the respiratory network in the brainstem is intrinsically capable of providing the correct timing for muscle activation, the organisation of this network is still not completely understood (for review, see Bianchi et al. 1995). In the cat, the activity in the phrenic and internal intercostal nerves reveals that the respiratory rhythm consists essentially of three phases: inspiratory, post-inspiratory and expiratory (Richter, 1982). The post-inspiratory phase comes after the inspiratory phase and corresponds to the period of passive expiration, during which the thoracic and abdominal expiratory muscles do not contract. In this period, the expiratory airflow is controlled by the resistive action of the activated adductor muscles in the larynx (Bartlett et al. 1973; Gautier et al. 1973). However, some studies have reported that in decerebrate cats, the abdominal expiratory nerve shows its peak activity during the post-inspiratory phase (Fregosi et al. 1987; Fregosi & Bartlett, 1988). Moreover, unit recording from the abdominal nerve showed that the firing of some motor units was limited to the post-inspiratory phase (Fregosi et al. 1992). Therefore, whether the post-inspiratory phase is or is not completely passive may depend on the conditions. During the expiratory phase, which follows the post-inspiratory phase, the expiratory muscles of the rib cage produce active expiratory airflow. This period of active expiration may not be present during quiet breathing (Richter, 1982). In recent studies in the decerebrate rat, membrane potential trajectories of most ventral respiratory group neurones also displayed three phases, as in the cat (for review, see Duffin et al. 2000). Although the phase switches and the durations of the various phases are assumed to be dependent both on the synaptic interactions between/within the subgroups of medullary respiratory neurones and on intrinsic membrane properties, the precise details of the neuronal mechanisms are still the subject of speculation (Bianchi et al. 1995).

For investigations of the respiratory network, the in vitro brainstem-spinal cord preparation obtained from the neonatal rat (Suzue, 1984) appears to be a useful experimental model (for review, see Onimaru et al. 1997). This in vitro preparation has several advantages over an in vivo preparation, including a more precise control of the extracellular environment. Our recent study indicated that the neuronal mechanisms required for intercostal expiratory activity are preserved in this in vitro preparation (Iizuka, 1999). When the central chemoreceptor was activated by a decrement in the extracellular pH, an expiratory burst in the internal intercostal muscles, one of the main muscles producing expiratory airflow, appeared in the first half of the expiratory phase (Ea). Likewise, in the vagotomised, spontaneously breathing neonatal rat, intercostal expiratory activity has been shown to peak during the Ea phase (Janczewski & Aoki, 1999). Hence, in an in vitro preparation under low pH conditions, it should be possible to discriminate three sub-phases within the respiratory cycle: inspiratory, Ea and the second half of the expiratory phase (Eb). The Ea phase would seem to be the period of active expiratory airflow in the rat, while the Eb phase seems to be either passive or the second part of the active expiratory phase. Although the neuronal mechanisms regulating the duration of the Eb phase would be expected to play a crucial role in determining respiratory frequency in the rat, there have been no studies of the Eb phase in this species. One reason is that the identity of the nerves or muscles active specifically during the Eb phase is not known in the rat. To help clarify the neuronal mechanisms involved in Eb-phase determination, we thought it important to find out which muscles and nerves exhibit activity specifically during the Eb phase.

It is known that some of the motoneurones innervating upper airway muscles are active during either the post-inspiratory or expiratory phase in the cat (Bianchi & Barillot, 1975; Grélot et al. 1989; Barillot et al. 1990). In the present study, therefore, we examined the effects of a decrement in the extracellular pH on the pattern of respiratory activity in the glossopharyngeal, vagus and accessory nerves, which contain motor nerve fibres innervating the upper airway and neck muscles, using isolated brainstem-spinal cord-rib preparations obtained from neonatal rats. In the second part of the study, we developed a brainstem-spinal cord-rib-pharynx preparation and obtained electromyograms from the inferior pharyngeal constrictor muscles. We found that under low pH conditions, the rostral portion of the inferior pharyngeal constrictor muscle is active specifically during the Eb phase. Preliminary reports of the results described here have been published in abstract form (Iizuka et al. 1999; Iizuka, 2000).

METHODS

Three different kinds of preparation were used in the present study: a brainstem-spinal cord preparation, a brainstem-spinal cord-rib preparation and a brainstem-spinal cord-rib-pharynx preparation.

Brainstem-spinal cord preparation

Five neonatal Wistar rats aged 2-4 days were used. Under deep ether anaesthesia, the scalp over the coronal suture was removed and the brainstem was transected caudal to bregma by insertion of a razor blade. The skin over the head and back was removed together with the forelimbs and the whole body was transected at the lumbar level. The preparation was then transferred to a dissection chamber filled with cold modified Krebs solution (13-18 °C; for composition, see below). The skin over the neck and chest was removed, as were the superficial glands of the neck and the infrahyoid muscles. After the sternum had been removed, all viscera located caudal to the trachea were removed together with the diaphragm. The glossopharyngeal nerve, the superior laryngeal nerve and the pharyngeal branch of the vagus nerve were all cut on the right side. Then, the right stylohyoid and stylopharyngeus muscles were removed. The pharynx was isolated from the skull base, turned dorsal side up together with the trachea and oesophagus, then displaced to the left side, special care being taken not to damage the glossopharyngeal nerve, pharyngeal branch of the vagus nerve, or superior laryngeal nerve on the left side. The ventral surface of the brainstem and spinal cord was exposed by removing the skull base and the ventral vertebral body. The hypoglossal nerve was cut at the surface of the brainstem. The spinal cord was transected at the upper thoracic level and all ventral and dorsal roots were cut. The superior cervical ganglion and sympathetic trunk were removed. The common carotid artery was separated from the cervical vagus nerve and removed. The glossopharyngeal nerve, the superior laryngeal nerve and the pharyngeal branch of the vagus nerve were cut proximal to their ramification. The brainstem was transected just rostral to the trigeminal nerve root (Fig. 1A). After the glossopharyngeal nerve, pharyngeal branch of the vagus nerve, superior laryngeal nerve, cervical vagus nerve and accessory nerve and ganglion had been freed from as much of the surrounding connective tissues as possible, the brainstem-spinal cord was isolated together with these nerves (Fig. 1). To avoid damaging the nerves, the piece of occipital bone surrounding the jugular foramen was allowed to remain in two out of five preparations (asterisk in Fig. 1A).

Figure 1. Experimental arrangement.

Figure 1

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A, low-power magnification photograph of the brainstem-spinal cord preparation in the experimental chamber. Five suction electrodes for electrical stimulation are already set up. A piece of the occipital bone remained in this particular preparation (*). B, high-power magnification photograph of the boxed region in A. To make discrimination among motor rootlets easier, the sensory rootlets of the glossopharyngeal and vagus nerves were cut (filled and open circle, respectively). At least seven relatively large nerve bundles (a-g) can be seen in this preparation. After these seven rootlets had been cut carefully near the brainstem, their peripheral stumps were incorporated into suction electrodes for recording. Antidromic action potentials evoked by stimulation of the glossopharyngeal nerve (IX), the pharyngeal branch of the vagus nerve (PhX), the superior laryngeal nerve (SLN), the cervical vagus nerve (CX) and the accessory nerve (XI) were recorded from the seven rootlets (Fig. 2). SpXI, spinal branch of the accessory nerve.

Brainstem-spinal cord-rib preparation

Twenty-one neonatal Wistar rats aged 0-3 days were used. The procedure was similar to one described previously (Iizuka, 1999) except that a part of the occipital bone surrounding the jugular foramen remained attached to the preparation. This bone was displaced to the lateral side and pinned onto the silicone rubber floor of the chamber. This manipulation increased the space between the bone and the brainstem and aided the discrimination of the cranial nerve rootlets.

Brainstem-spinal cord-rib-pharynx preparation

Twelve neonatal Wistar rats aged 0-3 days were used. The procedure was similar to one described previously (Iizuka, 1999) except that the innervation of the pharyngeal constrictor muscles was left intact in the present study. As described in the previous section, the pharynx, trachea and oesophagus were turned dorsal side up and displaced to the left, with special care being taken not to damage the glossopharyngeal nerve and pharyngeal branch of the vagus nerve on the left side. As much of the surrounding connective tissue as possible was removed. To improve perfusion, the ventral part of the bone surrounding the jugular foramen was removed.

Each of the three kinds of preparation was placed in a recording chamber (11 ml) and pinned onto the silicone rubber floor with the ventral side uppermost (see Fig. 1 in Iizuka, 1999, and Fig. 1A). The preparation was continuously perfused at 25 ± 1 °C with modified Krebs solution containing (mm): 124 NaCl, 5 KCl, 2.4 CaCl2, 1.3 MgSO4, 26 NaHCO3, 1.2 KH2PO4 and 30 d-glucose (pH adjusted to 7.4 by bubbling with 95 % O2 and 5 % CO2). The flow rate was adjusted to 2.5-5.0 ml min−1. When a perfusate of lower pH was required, [HCO3−] was decreased from 26 to 10 mm by equimolar substitution with [Cl−] prior to equilibration with 95 % O2 and 5 % CO2 (pH 7.1).

Recording and stimulation

A glass suction electrode was used for recording from, or stimulation of, nerves. A bipolar Teflon-coated platinum-iridium electrode (75 or 113 μm outer diameter, insulated except for the tip) was used for electromyographic recording. In experiments using the brainstem-spinal cord-rib preparation or the brainstem-spinal cord-rib-pharynx preparation, inspiratory motor activity was recorded from the proximal cut end of the fourth cervical (C4) ventral root. In the case of the internal intercostal muscle (IIM), electromyograms were obtained from an area between the chondrocostal junctions of the ninth to eleventh ribs (see Fig. 1 in Iizuka, 1999). The ribs near the recording site were pinned firmly to prevent movement.

In 10 brainstem-spinal cord-rib preparations, nerve discharges were recorded simultaneously from the proximal cut end of three groups of cranial nerve rootlets categorised as the rostral, intermediate and caudal groups (see Results). To assess the noise level in the recording system, neuronal activity was inactivated by bath application of high potassium solution (40 mm KCl) in these 10 preparations. Nerve discharges were recorded from the ascending bundle of fibres representing the spinal branch of the accessory nerve (see Results) in another 11 brainstem-spinal cord-rib preparations (with all cranial branches of the accessory nerve rootlets that merged with the spinal branch of the accessory nerve cut). To be sure that the spinal branch of the accessory nerve remained uninjured following such manipulations as dissection or incorporation into the suction electrode, we confirmed in eight preparations that an overt nerve discharge was evoked by electrical stimulation (10-30 V, 1.0 ms duration) of the contralateral C4 lateral funiculus (using an electrode similar to that used for the recording of the electromyograms). Spontaneous discharges in this nerve were occasionally observed at rest in three preparations.

In experiments using the brainstem-spinal cord-rib-pharynx preparation, electromyograms were obtained from the surface of the rostral and caudal parts of the inferior pharyngeal constrictor muscles (boxed regions in Fig. 7; see also Fig. 1 in Bieger & Hopkins, 1987). The signals so obtained were amplified (gain, 500-10 000; band-pass filter, 15-3000 Hz) using a high-gain AC-coupled amplifier (AB-610J; Nihon Kohden). Recordings were stored in a pulse-code modulation data recorder (PC208A; Sony) for further off-line analysis, digitised at 2000 Hz (MacLab/8s; ADInstruments) and analysed using attached software (Chart v3.6s, ADInstruments).

Figure 7. Photograph of pharyngeal constrictor muscles.

Figure 7

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Electromyograms were recorded from the two enclosed regions, which correspond to the rostral and caudal parts of the inferior pharyngeal constrictor muscle (rIPC, cIPC). IX, glossopharyngeal nerve: PhX, pharyngeal branch of the vagus nerve: SLN, superior laryngeal nerve: CX, cervical vagus nerve.

Antidromic compound action potentials - evoked by electrical stimulation of the whole glossopharyngeal nerve, the pharyngeal branch of the vagus nerve, the superior laryngeal nerve, the cervical vagus nerve or the accessory nerve (rectangular pulse, 0.5 Hz, 10 V, 0.3 ms pulse duration; see Fig. 1A for the configuration of the stimulating electrodes) - were obtained from the peripheral stumps of the cut cranial nerve rootlets near the surface of the brainstem in experiments using the brainstem-spinal cord preparation (gain: 500-5000, band-pass filter: 0.08-3000 Hz). When the amplitude of the compound action potential was larger than 0.1 mV, we confirmed that the amplitude was unchanged when the stimulus intensity was increased from 10 V to 20-30 V in all five preparations. Twenty amplified signals evoked by stimulation of one peripheral nerve were digitised at 2000 Hz (MacLab/8s; ADInstruments) and averaged using attached software (Scope v3.6s; ADInstruments). The amplitude of the averaged response was taken as the difference between the base line and the peak. For each rootlet, the averaged record showing the largest response was taken as the ‘standard’. Then, the amplitude of the averaged responses evoked by stimulation of each of the other peripheral nerves was expressed relative to this standard. To verify the position of the recorded rootlet, a photograph was taken using a camera (PM-10AK; Olympus) attached to a stereo microscope (SZ6045TRPT; Olympus) before and/or after electrical potentials had been recorded from that rootlet.

Definition of the respiratory phases

As in previous studies (Smith et al. 1990; Iizuka 1999), the inspiratory phase (I) and expiratory phase (E) were defined as the period during a C4 burst and the intervening silent period between C4 bursts, respectively, under normal pH conditions. Previously, in a brainstem-spinal cord-rib preparation from neonatal rats we demonstrated that when the extracellular pH was lowered (from about 7.4 to 7.1), expiratory activity was seen in IIM during the first half of the expiratory phase (Iizuka, 1999). In the present study, therefore, the inspiratory phase (I), the first half of the expiratory phase (Ea) and the second half of the expiratory phase (Eb) were defined, respectively, as the period from the onset of a C4 burst to that of an IIM burst, the period during an IIM burst and the period from the end of an IIM burst to the onset of the following C4 burst (all under low pH conditions).

The experiments were performed with the approval of the Animal Research Committee of the Ibaraki Prefectural University of Health Sciences, which operates in accordance with Japanese Governmental Law (No. 105).

RESULTS

Antidromic compound action potentials in cranial nerve rootlets near the brainstem following peripheral nerve stimulation

Discrimination between the glossopharyngeal nerve, the vagus nerve and the cranial branch of the accessory nerve at the surface of the brainstem is difficult in the rat, since their emerging rootlets are distributed in a continuous fashion from rostral to caudal levels of the brainstem (Fig. 1B). Therefore, to establish the projection of motor fibres in each nerve rootlet, antidromic compound action potentials evoked by stimulation of the glossopharyngeal nerve (IX), the pharyngeal branch of the vagus nerve (PhX), the superior laryngeal nerve (SLN), the cervical vagus nerve (CX) or the accessory nerve (XI) were recorded from the peripheral stump of each motor nerve rootlet in five brainstem-spinal cord preparations.

More than 20 motor rootlets could be seen emerging from the surface of the brainstem. These were loosely collected into about 10 bundles before the entrance to the jugular foramen. Fine nerve rootlets (less than about 30 μm in diameter) were neglected in this experiment. In the preparation shown in Fig. 1, there were at least seven bundles of motor nerve rootlets (Fig. 1B a-g). The nerve potentials recorded from the peripheral stumps of a-g are shown in Fig. 2a-g, respectively. In the most rostral rootlets (Fig. 2a), IX stimulation evoked a larger antidromic compound action potential than stimulation of the other four nerves. Small responses were also evoked by CX stimulation. In b-g, CX stimulation evoked the largest response. PhX and SLN stimulation evoked larger responses in b-d than in e-g. A few fine rootlets emerged from a position in the medulla caudal to the rootlets shown as g in Fig. 1B. These most caudal medullary rootlets, which properly belong to the cranial branch of XI, travelled laterally to join the ascending bundle of fibres representing the spinal branch of XI (labelled SpXI in Fig. 1B; see details below). In Fig. 1B, the close proximity of SpXI to the lateral surface of the medulla just caudal to g means that it hides these rootlets from view. We ignored rootlets less than about 30 μm in diameter (which were actually the majority) and recorded from only five rootlets in three preparations. The largest response in these five rootlets was always evoked by CX stimulation, although small potentials were also evoked by XI stimulation (amplitude 2.7, 3.2, 6.6, 6.7 or 22.6 % of the response to CX stimulation).

Figure 2. Antidromic compound action potentials evoked by peripheral nerve stimulation.

Figure 2

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Mean electrical potentials (n= 20 potentials in each case) in each nerve rootlet evoked by stimulation of the glossopharyngeal nerve (IX), the pharyngeal branch of the vagus nerve (PhX), the superior laryngeal nerve (SLN), the cervical vagus nerve (CX) and the accessory nerve (XI). Data are from the preparation shown in Fig. 1. Records in a-g were obtained from the nerve rootlets shown as a-g, respectively, in Fig. 1B. Records in h and i were obtained from the spinal branch of the accessory nerve (SpXI) when the ascending bundle representing this nerve was incorporated into the suction electrode up to the C1 or C3 level, respectively (the same electrode being used in each case). The number at the right end of each trace indicates the factor by which the value shown against the ordinate scale bar (see ‘standard’ trace in same vertical column (a-i)) should be divided (e.g. a bar of the length representing 0.4 mV for IX in a would represent 0.067 mV for PhX in a).

In the next experiment, we recorded from the spinal branch of the accessory nerve (SpXI), which emerges from the lateral side of the cervical cord as many fine rootlets. These converge and ascend as a bundle along the lateral side of the cervical cord and medulla (SpXI in Fig. 1B) eventually to enter the jugular foramen. We wanted to record from the ascending bundle itself. So, with the rootlets emerging from each cervical segment cut, the suction electrode was advanced from the caudal end of the bundle up to a given spinal level so that it eventually contained all the fibres emerging from that level and the more caudal segments. In this experiment, the electrode was advanced as far as either C1 or C3 (see Fig. 2h and i, respectively). When recording was made in this way from fibres emerging from C3 and the more caudal segments, XI stimulation evoked by far the largest response (Fig. 2i) and no measurable response was evoked by CX stimulation. When the electrode was advanced as far as C1 - effectively adding the C1 and C2 rootlets to the recording - CX stimulation evoked a small potential (in one preparation, 1.0 % of the amplitude of the response to XI stimulation; in another, 1.8 %) (Figs 2h, and 3D and E). This result indicates that it is the rootlets emerging from the C1 and C2 segments that contain the fibres passing to CX. This conclusion was supported by recordings made from the rootlets emerging from individual segments (see below).

Figure 3. Relation between the position of the rootlet and the amplitude of the compound action potential.

Figure 3

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Ordinates in plots A-E (each obtained from a different preparation) show the relative amplitude of the potential in each nerve rootlet evoked by IX, PhX, SLN, CX and XI stimulation. The peak amplitude of the largest evoked potential in a given rootlet was used as the standard (i.e. 1.0) for the other evoked potentials in the same rootlet. On two occasions in A and B, a rootlet, when cut, divided into two strands that could not be incorporated into a single suction electrode. Recordings were therefore made separately from the two strands. The data obtained from the two strands making up a single rootlet are plotted close together. Recordings obtained from SpXI are plotted on the right side of B-E. C1, C2, C4: rootlets of SpXI emerging from C1, C2 and C4 segments, respectively. a(C1), a(C3): ascending bundle representing SpXI nerve incorporated into the electrode up to segmental level C1 or C3, respectively.

Figure 3 summarises the above experiments. The left-hand parts relate to experiments on cranial rootlets. In the most rostral rootlets, stimulation of nerve IX elicited the largest response in all five preparations. Small potentials (2.7-11.2 % of ‘standard’ in amplitude, n= 5) were also evoked by CX stimulation in these most rostral rootlets. In most of the other rootlets, the largest response was evoked by CX stimulation. Although there was considerable variation among preparations, the responses evoked by PhX and SLN stimulation were larger in amplitude in the second to fourth or sixth rootlets from the rostral end than in the caudal three rootlets. These results indicated that the cranial nerve rootlets can be categorised into three groups on the basis of the level at which they emerge. Thus, the rostral group (rostIX-XI; most rostral rootlets) contains fibres mainly passing to IX, the intermediate group (intIX-XI; the second to fourth or sixth rootlets) contains those passing mainly to CX but also to PhX and SLN, while the caudal group (caudIX-XI; the most caudal three rootlets) contains those passing mainly to CX.

The right-hand parts in Fig. 3 relate to experiments on SpXI (recordings from the individual segments C1, C2 or C4 or from the ascending bundle up to C1 or C3). In SpXI, the largest response was always evoked by XI stimulation (Fig. 3B-E). As shown in Fig. 3B and C, the potential evoked by CX stimulation was larger in the nerve rootlets emerging from the C1 segment than in those emerging from the C2 or C4 segments.

Effect of a decrement in extracellular pH on respiratory activity in the glossopharyngeal, vagus and accessory nerves

The previous experiment showed that near the brainstem, the nerve rootlets can be categorised into three groups (rostIX-XI, intIX-XI and caudIX-XI). In the next experiment, nerve discharges were simultaneously recorded from the proximal cut ends of these three groups of nerve rootlets in 10 brainstem-spinal cord-rib preparations.

All rootlets examined, except one caudIX-XI, showed bursts during the I phase. Figure 4A and B displays recordings obtained from rostIX-XI, intIX-XI and caudIX-XI rootlets under normal and low pH conditions (about pH 7.4 and 7.1, respectively) in two different preparations. As shown in the left panel in Fig. 4A and B, few discharges occurred during the E phase in rostIX-XI under normal pH conditions. When low pH solution was applied, discernible burst discharges occurred during the Ea phase in rostIX-XI, while no discernible bursts occurred during the Eb phase (Fig. 4A and B, middle panel). The intIX-XI rootlet whose activity is illustrated in Fig. 4A showed spike-like and weak tonic discharges during the E phase under normal pH conditions. Under low pH conditions, this rootlet showed bursts during the Ea phase but no discernible bursts during the Eb phase (in which only tonic activity was observed). The representative record from intIX-XI in Fig. 4B shows clear bursts during the Eb phase. Bursts during the Ea phase were also discernible in intIX-XI in some respiratory cycles (asterisk in Fig. 4B). The caudIX-XI rootlets showed weak tonic activity during the E phase (Fig. 4A and B, left panel). In these rootlets, activity was greater during the Ea and Eb phases than during the E phase but the enhancement was less marked than that seen in intIX-XI (Fig. 4A and B). Only small bursts can be seen during the Ea phase in caudIX-XI in Fig. 4A. No bursts are discernible during either the Ea or Eb phase in caudIX-XI in Fig. 4B.

Figure 4. Respiratory-related discharges under normal and low pH conditions.

Figure 4

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A and B, records of discharges in the C4 ventral root (C4), internal intercostal muscle (IIM), the most rostral group of rootlets (rostIX-XI) and the intermediate and caudal groups of rootlets (intIX-XI and caudIX-XI). A and B were obtained from two different preparations. The left, middle and right panels show records obtained under normal pH conditions, low pH conditions and high potassium conditions, respectively. Vertical broken lines in B indicate boundaries of the respiratory phases. I, E, Ea and Eb indicate the following phases: inspiratory, expiratory, first half of expiratory and second half of expiratory, respectively. The intIX-XI rootlet in B showed a discernible burst during the Ea phase in some respiratory cycles (*).

Figure 5 summarises the patterns of activity obtained from the three groups of rootlets during the E, Ea and Eb phases. No group showed consistent burst activity during the E phase under normal pH conditions. However, about half the intIX-XI rootlets showed clear tonic activity. In the Ea phase (under low pH conditions), only 5 out of 10 caudIX-XI showed consistent bursts, while all rostIX-XI and 9 out of 10 intIX-XI showed Ea bursts. Bursts during the Eb phase were not observed in any rostIX-XI. Although clear bursts during the Eb phase were observed in only three intIX-XI, the other seven all seemed to show tonic activity during this phase. In fact, in intIX-XI the type of activity present in the Eb phase was often difficult to determine. Since all 10 intIX-XI showed large bursts throughout the I phase and nine throughout the Ea phase, it was sometimes impossible to discriminate between (a) tonic low-amplitude activity occurring throughout the cycle but visible only during the Eb phase, and (b) a low-amplitude Eb burst extending from the end of the preceding Ea to the start of the next I phase. In general, expiratory activity was prominent in intIX-XI. Four caudIX-XI showed bursts during the Eb phase. These results suggest that some intIX-XI and caudIX-XI rootlets contain motor fibres firing specifically during the Eb phase.

Figure 5. Summary of the pattern of activity.

Figure 5

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The histograms show the percentage of nerve rootlets that showed the specified type of activity in rostIX-XI, intIX-XI and caudIX-XI during E (at normal pH), and Ea and Eb (at low pH) phases. The patterns of activity were categorised into the following five types. Burst, bursts always occurred; Tonic, tonic activity without any clear peak and with an amplitude more than two times the noise level; WeakT, tonic activity with an amplitude less than two times the noise level; Spike, large spike-like potentials less than 10 times per respiratory cycle; None, no discernible activity. Data were obtained from 10 preparations.

In 11 brainstem-spinal cord-rib preparations, nerve discharges were recorded from the central stump of the ascending bundle of the spinal accessory nerve (SpXI). The original recordings in Fig. 6 show no respiratory activity in SpXI under normal or low pH conditions. To be sure that SpXI had not been damaged during such manipulations as dissection or incorporation into the suction electrode, electrical stimulation was applied to the C4 lateral funiculus on the contralateral side. This stimulation evoked burst discharges in SpXI (Fig. 6B). This result (discharge only on C4 stimulation) was obtained in five preparations. In another six preparations, although some spontaneous expiratory-related bursts were observed, these bursts did not occur consistently at either level of pH or consistently in either the first or second half of the expiratory phase.

Figure 6. Motor activity in the spinal branch of the accessory nerve under normal and low pH conditions.

Figure 6

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A and B, records of discharges in the C4 ventral root (C4), an internal intercostal muscle (IIM) and the spinal branch of the accessory nerve (SpXI) under normal (A) and low (B) pH conditions. Arrow in B indicates the time electrical stimulation was applied to the contralateral C4 lateral funiculus (c C4LF).

Effect of a decrement in extracellular pH on respiratory activity in pharyngeal constrictor muscles

The results described in the previous section showed that expiratory activity was prominent in intIX-XI, and that some intIX-XI showed burst activity during the Eb phase. As indicated in the first section, a characteristic of intIX-XI is that it contains axons running to SLN and/or PhX. Since it is known that SLN and PhX innervate the pharyngeal constrictor muscles (Bieger & Hopkins, 1987), electromyograms were recorded from the inferior pharyngeal constrictor muscle using the brainstem-spinal cord-rib-pharynx preparation. As indicated in Fig. 7, electromyograms were recorded from two boxed regions corresponding to the rostral and caudal parts of the inferior pharyngeal constrictor muscles in the rat (rIPC and cIPC, respectively).

Figure 8 shows representative records obtained from rIPC and cIPC. The record from cIPC shows a plateau-like discharge pattern throughout the E phase under normal pH conditions (Fig. 8A). By contrast, rIPC showed only weak activity during the E phase. In accordance with this result, we saw visual evidence of phasic contractions of cIPC throughout the E phase, while rIPC showed no contractions at all under normal pH conditions. Thus, it seems that the deep layer of cIPC or the muscles in the cervical oesophagus close to its junction with the pharynx contracted more actively than those represented by rIPC. When low pH solution was applied, burst activity during the Eb phase was induced in rIPC (Fig. 8B). Respiratory activity in cIPC was also increased and discrete bursts were observed during the Ea and Eb phases (Fig. 8B).

Figure 8. Electromyograms recorded from the inferior pharyngeal constrictor muscle.

Figure 8

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A and B, records obtained from C4, IIM, rIPC and cIPC under normal and low pH conditions, respectively.

In all 12 preparations, there was no consistent respiratory activity in rIPC under normal pH conditions. Under low pH conditions, rIPC showed burst activity restricted to the Eb phase in 10 out of 12 preparations, while weak sporadic activity occurred in the other two preparations. Under normal pH conditions, cIPC showed a plateau-like discharge pattern throughout the E phase in 8 out of 12 preparations, tonic activity throughout the respiratory cycle in two preparations and intermittent burst activity in the remaining two preparations (restricted to the E phase in one but not in the other). When low pH solution was applied, the activity in cIPC during the Ea and Eb phases was enhanced in all preparations examined. In 8 out of 12 preparations, cIPC showed discrete burst activity during both the Ea and Eb phases. In three preparations, discrete bursts were observed only during the Ea phase. In these preparations, although a discrete burst was not discernible during the Eb phase, the activity during the Eb phase was clear and a decrement in activity was seen during the I phase. In the remaining preparation (in which cIPC showed intermittent burst activity under normal pH conditions), cIPC showed a plateau-like discharge pattern throughout the Ea and Eb phases under low pH conditions.

DISCUSSION

Respiratory activity in the glossopharyngeal and vagus nerves

The present study is the first to examine the effects of extracellular pH on respiratory activity in the glossopharyngeal and vagus nerves in an isolated brainstem-spinal cord preparation from neonatal rats. The rootlets of these cranial nerves showed consistent burst discharges during the Ea and/or Eb phases under low pH conditions but no discernible expiratory bursts during either the first or second half of the E phase under normal pH conditions. It has been reported that a late expiratory and/or early expiratory discharge is present (bilaterally) in the glossopharyngeal and vagus nerves in most brainstem-spinal cord preparations from neonatal rats under normal pH conditions (Smith et al. 1990). These authors also mentioned that the expiratory activity in a given nerve occurred intermittently in any preparation and showed considerable cycle-to-cycle variation in discharge amplitude. In our previous study (Iizuka, 1999), we occasionally observed expiratory rib movements or intercostal expiratory activity before or just after the recording was started under normal pH conditions. However, such expiratory activity disappeared within 1 h (Iizuka, 1999). A similar phenomenon was observed in the present study. During the disappearance of the intercostal expiratory activity, the nerve rootlets of the rostIX-XI, intIX-XI or caudIX-XI groups showed a respiratory pattern similar to that described by Smith et al. (1990). When the intercostal expiratory activity had completely disappeared, however, discrete and consistent bursts were not discernible during either the first or second half of the E phase.

It is known that in adult rats the glossopharyngeal nerve exhibits respiratory burst activity that starts in the late expiratory phase (which would correspond to the Eb phase in the present study) and terminates at the end of the inspiratory phase (Fukuda, 1992; Frugiére & Barillot, 1994; Fukuda et al. 1995). Since the largest antidromic compound action potential in rostIX-XI was always evoked by glossopharyngeal nerve stimulation, the respiratory activity in rostIX-XI was thought to be similar to that in the glossopharyngeal nerve. In the present study, however, there was no discernible phase lag between the onset of inspiratory activity in C4 and that of the activity in rostIX-XI under normal pH conditions (Fig. 4). Furthermore, all rostIX-XI showed bursts during the Ea phase under low pH conditions (Figs 4 and 5). The explanation for these discrepancies between the data from adult rats in vivo and neonatal rats in vitro remains unclear. In the present study, a small number of nerve fibres in the rostIX-XI rootlets projected to the cervical vagus nerve (CX). It is known that in the decerebrate adult rat, some motoneurones that project to CX exhibit an abrupt and maximal depolarisation soon after phrenic activity ends; this is followed by a progressive repolarisation of the membrane potential (Zheng et al. 1991). Since this pattern of membrane potential changes would cause a discharge during the Ea phase, fibres projecting to CX in rostIX-XI may have caused the Ea burst.

The present study suggested that the intIX-XI rootlets contained fibres passing mainly to CX but also to the pharyngeal branch of the vagus nerve (PhX) and the superior laryngeal nerve (SLN), while the caudIX-XI rootlets projected mainly to CX. It is known that CX contains nerve fibres projecting to the recurrent laryngeal nerve, which innervates the laryngeal muscles. In the anaesthetised cat, the majority of fibres isolated from the recurrent laryngeal nerve show impulse activity during inspiration, although many have been found that discharge impulses during expiration (Green & Neil, 1955; Eyzaguirre & Taylor, 1963). Similarly, inspiratory activity was more prominent than expiratory activity in most intIX-XI and caudIX-XI rootlets in the present study.

A fairly recent study using an in vivo neonatal rat preparation showed that CX exhibits burst discharges peaking in the inspiratory phase and the post-inspiratory phase (which would correspond to the Ea phase in the present study; Wang et al. 1996). In anaesthetised or decerebrate adult rats, electroneurograms from most PhX have been found to show their peak discharge during the inspiratory and/or post-inspiratory phases, although some were continuously active without any respiratory modulation (Frugiére & Barillot, 1994). It is known that in the adult rat in vivo, the respiratory discharges in SLN begin in the late expiratory phase and cease at the end of the inspiratory phase (Fukuda & Honda, 1982a,b). As far as we know, fibres that exhibit discharges restricted to the Eb phase have not been reported in CX, PhX or SLN in the rat, although three kinds of expiratory motor units (firing either during the post-inspiratory or late expiratory phases or throughout the expiratory phase) have been observed in PhX in decerebrate cats (Grélot et al. 1989). Overall, these results obtained from the in vivo preparation are in good agreement with the present results showing that all groups of intIX-XI and caudIX-XI rootlets, except one, showed burst activity during the I phase and many of them showed bursts during the Ea phase under low pH conditions.

Activity in the spinal branch of the accessory nerve

The present study is the first to indicate that the spinal branch of the accessory nerve (SpXI) shows no consistent respiratory activity in the rat, even when central respiratory drive is enhanced by central chemoreceptor activation. SpXI projects mainly to the accessory nerve, although a small part of this nerve, especially the rostral part, contains fibres projecting to the cervical vagus nerve (Figs 2h and i, and 3). It is known that the accessory nerve innervates the sternomastoid, cleidomastoid, cleidotrapezius and acromiotrapezius muscles in the rat (Brichta et al. 1987). The above results suggest that in the rat, these neck muscles play little role in the respiratory movement of the rib cage. In the dog, similarly, the sternomastoid muscle failed to show any respiratory activity when the respiratory drive was increased (either by airway occlusion or by hypercapnia) (De Troyer et al. 1994). However, it is known that normal human subjects invariably contract the sternomastoid muscle during inspiration (Raper et al. 1966; De Troyer & Estenne, 1984). The reason for this species difference is uncertain but it suggests that in the dog and rat, neck-muscle contraction may be ineffective as far as movement of the rib cage is concerned, unlike the situation in humans (De Troyer & Estenne, 1984).

Respiratory activity in the pharyngeal constrictor muscles

Electroneurograms from the pharyngeal constrictor muscles of the rat were obtained for the first time in the present study. The results indicated that the rostral and caudal parts of the inferior pharyngeal constrictor muscles (rIPC and cIPC) showed quite different patterns both of respiratory activity at normal pH and of recruitment by central chemoreceptor activation. Under normal pH conditions, cIPC showed bursts throughout the expiratory phase, while rIPC showed weak activity or no activity at all. Under low pH conditions, cIPC showed two discrete bursts during the Ea and Eb phases, while rIPC showed bursts predominantly during the Eb phase. Although the functional significance of the difference in respiratory activity between rIPC and cIPC remains unknown, it would seem that the respiratory centre exerts a differential control over these two muscle regions.

In the anaesthetised or decerebrate cat, the middle pharyngeal constrictor (MPC) muscle shows bursts during the expiratory phase under normocapnia (Sherry & Megirian, 1975; Kuna & Vanoye, 1997). Although IPC does not exhibit respiratory-related activity under normocapnia, progressive hypercapnia did evoke expiratory activity (Kuna & Vanoye, 1997). In humans, although superior PC, MPC and IPC muscles all showed respiratory activity infrequently during quiet breathing, all three showed expiratory activity under hypercapnia (Kuna et al. 1997). These and the present results indicate the presence of species differences in the respiratory activity of the PC muscles, although in general it would seem that an enhancement of respiratory drive leads to expiratory activity in these muscles. Sherry & Megirian (1975) speculated that contraction of PC muscles might reduce dead-space during hypercapnia, thereby promoting the exodus of CO2.

Respiratory phases in the in vitro preparation

Under low pH conditions, the internal intercostal muscle (IIM) showed burst activity during the Ea phase, while rIPC showed bursts during the Eb phase in our brainstem-spinal cord-rib preparation. By contrast, under normal pH conditions burst activity limited to the first or second half of the E phase was not observed in IIM or in any of the cranial nerves or muscles examined, although cIPC showed burst activity throughout the E phase. These results raise two possibilities concerning the composition of the respiratory phase in this in vitro preparation. The first is that the basic respiratory phase consists of three sub-phases (I, Ea and Eb) under both normal and low pH conditions. Under normal pH conditions, however, the Ea and Eb phases were not apparent because the motoneurones that innervate IIM or rIPC could not fire due to the presence of a weak excitatory synaptic input. The second possibility is that the increase in respiratory drive evoked via the central chemoreceptor switches the composition of the respiratory phase between two (I and E) and three sub-phases at the level of the motor output.

Using the isolated brainstem-spinal cord preparation, a number of studies have examined the respiratory neurones in the ventrolateral medulla under normal pH conditions (Smith et al. 1990; Kawai et al. 1996; for review, see Ballanyi et al. 1999). At present, three types of expiratory neurone - tonic expiratory, post-inspiratory and late expiratory - are recognised (Smith et al. 1990; Kawai et al. 1996; Arata et al. 1998). The existence of the post-inspiratory neurone and late expiratory neurone might imply that the respiratory phase consists of three phases under normal pH conditions. However, since the activity of the post-inspiratory neurone was found to be depressed by a decrement in pH (Kawai et al. 1996), this neurone is unlikely to the source of the excitatory inputs to the IIM motoneurone. The lack of an enhancement of firing in the post-inspiratory neurones under low pH conditions (Kawai et al. 1996) suggests that a new group of neurones responsible for the intercostal Ea burst is recruited by central chemoreceptor activation. When Iizuka (1999) evoked an intercostal Ea burst by central chemoreceptor activation, the respiratory rhythm was unstable until a new, stable phase relationship was established. This result also supports the idea of recruitment of a new group of neurones firing in the Ea phase and suggests that central chemoreceptor activation switches the composition of the respiratory phase from two to three sub-phases. Although various types of respiratory neurones have been described under normal pH conditions (Smith et al. 1990) and their firing or membrane potential patterns indicate the existence of four respiratory phases (Arata et al. 1998), it is still unclear whether and how their patterns of activity are reflected by the subdivisions of the respiratory phase at the level of the motor output.

In the present study, bath application of low pH solution evoked respiratory bursts in rIPC predominantly during the Eb phase. Hence, there would be expected to be a group of neurones that starts firing or shows enhanced firing during the Eb phase on central chemoreceptor activation. Although a late expiratory neurone has been identified under normal pH conditions (Smith et al. 1990), it remains unknown whether enhancement of its late expiratory activity or recruitment of a new group of Eb-firing neurones is responsible for the appearance of the Eb burst in rIPC under low pH conditions. Electromyograms from rIPC should provide clues to help us solve this problem.

Neuronal substrate for intercostal Ea-burst generation

Our previous study showed that on bath application of strychnine, a glycine receptor antagonist, the C4 inspiratory and intercostal Ea bursts largely overlap in a brainstem-spinal cord-rib preparation from neonatal rats (Iizuka, 1999). However, the durations of their bursts were not affected. On the basis of these results, we suggested the existence of discrete burst-generating mechanisms for inspiratory and expiratory (i.e. Ea) motor activity. It is known that the neuronal mechanisms for inspiratory-rhythm generation exist in slice preparations of the brainstem (Smith et al. 1991). In such preparations, inspiratory activity could be recorded from the hypoglossal nerve rootlets under high-potassium conditions. The presence of not only inspiratory but also post-inspiratory neurones has been demonstrated within medullary slices obtained from young mice (Ramirez et al. 1997). In such slices, bath application of strychnine prolonged the firing period of the post-inspiratory neurones into the second half of the expiratory phase (via a depression of the inhibitory postsynaptic potential); however, it did not cause this firing to overlap the inspiratory burst (Ramirez et al. 1997). Since these effects of strychnine on the firing pattern of post-inspiratory neurones were quite different from its effects on the intercostal Ea bursts in our previous study on the brainstem-spinal cord-rib preparation from neonatal rats (Iizuka, 1999), it seems unlikely that the Ea-burst generator was present in the regions included within the slices used by Ramirez et al. (1997). However, a recent study by Lieske et al. (2000) showed that anoxic stimulation changed the firing pattern of the post-inspiratory neurone to a pattern in phase with the activity of the inspiratory-related population situated among the ventral respiratory group of neurones within the medullary slice. A group of these post-inspiratory neurones is one of the candidates for the neuronal substrate responsible for Ea-burst generation. The present study has demonstrated that under low pH conditions, many of the cranial nerve rootlets emerging from the brainstem show bursts during the Ea phase. Since a slice preparation that retains these cranial rootlets could easily be prepared, it should be possible to use such a preparation to determine the region responsible for Ea-burst generation.

Acknowledgments

The author wishes to thank Drs Sei-Ichi Sasaki and Norio Kudo for valuable comments on the manuscript. The author also wishes to thank Ms Eriko Suzuki and Ms Chiho Notomo for their contribution to the preliminary experiments. The study was supported by Grants-in-Aid for Scientific Research from the Ministry of Education, Science and Culture of Japan.

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