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. 1999 Oct 1;520(Pt 1):293–302. doi: 10.1111/j.1469-7793.1999.00293.x

Intercostal expiratory activity in an in vitro brainstem-spinal cord-rib preparation from the neonatal rat

Makito Iizuka 1
PMCID: PMC2269573  PMID: 10517820

Abstract

  1. We examined whether expiratory activity can be observed when central chemoreceptors are activated by a decrement in the extracellular pH in an isolated brainstem-spinal cord-rib preparation from 0- to 3-day-old rats. Expiratory activity was defined as the burst activity that occurs in an internal intercostal muscle (IIM) during the silent period between the periodic inspiratory bursts in the C4 ventral root (which contains phrenic motor axons).

  2. During perfusion with modified Krebs solution (26 mm HCO3−, 5% CO2, pH 7.4), there was no consistent activity in IIM, though rhythmic inspiratory motor activity always appeared in the C4 ventral root.

  3. When the pH of the perfusate was lowered from about 7.4 to 7.1 by reducing [HCO3−] from 26 to 10 mm, the frequency of the C4 inspiratory rhythm increased, and rhythmic activity appeared in IIM. In most cases, the rhythmic burst in IIM started just after the cessation of the C4 inspiratory burst and coincided with movement of the ribs in a caudal direction. This intercostal expiratory burst was limited to the first half of the expiratory phase.

  4. The coordinated reciprocal motor activity between the C4 ventral root and IIM changed to a largely overlapping pattern when strychnine (5–10 μm), a glycine receptor antagonist, was added to the perfusate.

  5. These results suggest (i) that the neuronal mechanisms responsible for expiratory motor activity are preserved in this in vitro preparation and (ii) that the glycinergic inhibitory system plays an important role in the coordination between inspiratory and expiratory motor activity during respiration.

The in vitro brainstem-spinal cord preparation obtained from the neonatal rat (Suzue, 1984) appears to be a useful experimental model for investigating the neural substrate underlying respiratory rhythm generation and central chemosensitivity (for review, see Onimaru et al. 1997). The in vitro preparation has several advantages over the in vivo preparation, including a more precise control of the extracellular environment. In most studies using an in vitro preparation, however, a neurographic recording from only the phrenic or hypoglossal nerve was used to represent the respiratory motor output. Thus, expiratory motor activity was not monitored in these studies.

In the in vitro preparation, the phasic activity in ventral roots containing phrenic motor axons (C3-C6) (Kuzuhara & Chou, 1980; Goshgarian & Rafols, 1981) is coincident with the discharge recorded from all thoracic (T1-T13) ventral roots (Smith et al. 1990). The thoracic ventral root contains not only axons innervating the inspiratory muscles (external intercostal, levator costae) but also axons innervating the expiratory muscles (internal intercostal, triangularis sterni). Smith et al. (1990) reported that although an occasional expiratory discharge occurred in caudal thoracic spinal nerves in some in vitro preparations, there was no expiratory motor activity at all in most preparations of that type. Furthermore, only rhythmic and synchronized upward movements of the ribs have been observed in vitro (Suzue, 1984; Hamada et al. 1992). On the basis of this apparent absence of expiratory motor activity and the stereotyped ‘rapidly peaking-slowly decreasing’ pattern of activity seen in the cervical roots, some investigators have raised the possibility that the rhythmic activity of the in vitro neonatal rat preparation corresponds to gasping rather than the eupnoea seen in vivo (for review, see St John, 1996). For this reason, we thought it important to know whether the neuronal mechanisms responsible for generating the expiratory motor pattern are functionally preserved in the in vitro preparation.

It is well established that the ventral medulla plays a major role in the ventilatory response to changes in blood pH and PCO2 (for review, see Loeschcke, 1982). The change in tidal volume evoked by central chemoreceptor stimulation correlates well with the change in integrated phrenic nerve activity (Eldridge, 1971). Moreover, an increment in end-tidal CO2 evoked either by the addition of CO2 to the inspired air or by CO2 rebreathing causes an increment in the amplitude of bursts in expiratory intercostal muscle activity in decerebrate cats and anaesthetized dogs (Bainton et al. 1978; Oliven & Kelsen, 1989). Central chemosensitivity seems to be preserved in in vitro preparations obtained from neonatal rats (Suzue, 1984; Harada et al. 1985), since a decrement in extracellular pH causes an increase in the frequency of the inspiratory rhythm. Recently, it was suggested that extracellular pH, rather than PCO2, is the primary stimulating factor in central chemosensitivity (Voipio & Ballanyi, 1997). In all these studies using the in vitro preparation, no recordings were obtained from expiratory motor nerves or muscles. In the present study, therefore, we examined whether a decrement in pH would elicit overt expiratory motor activity in the in vitro preparation.

Blockade of glycine receptors has no clear effect on the respiratory rhythm per se in the in vitro preparation (Murakoshi & Otsuka, 1985; Onimaru et al. 1990). On the other hand, it is known that glycinergic inhibition plays a crucial role in the establishment of an alternate pattern of activity in extensor and flexor hindlimb muscles during chemically induced locomotion in in vitro preparations obtained from neonatal or fetal rats (Cowley & Schmidt, 1995; Iizuka et al. 1998). To establish the involvement, if any, of glycinergic inhibition in the coordination between inspiratory and expiratory motor activity in our in vitro preparation, the effects of strychnine, a glycine receptor antagonist, were examined. A preliminary report of the results described here has been published in abstract form (Iizuka, 1998).

METHODS

Preparation and solutions

Nineteen neonatal Wistar rats aged 0–3 days were used. Under deep ether anaesthesia, the scalp over the coronal suture was removed and the brainstem was transected caudal to bregma. The skin over the head and back was removed together with the forelimbs and the preparation was transferred to a dissection chamber filled with cold modified Krebs solution (13-18°C; for composition, see below). The skin over the chest was removed and, after the sternum had been excised, all viscera were removed together with the diaphragm. The ventral surface of the brainstem and spinal cord was exposed by removing the base of the skull and the ventral vertebral body. All ventral and dorsal roots from first cervical to first or second thoracic level were cut. The medulla oblongata was transected at a level just caudal to the sixth cranial nerve roots, so that the pons was not present in this preparation. The brainstem-spinal cord-rib preparation was placed in a recording chamber (volume, 11 ml) and pinned onto the silicone rubber floor with the ventral side uppermost (Fig. 1). The preparation was continuously perfused, via a perfusion tube placed just above the surface of the brainstem (Fig. 1), with modified Krebs solution at 25 ± 1°C. This contained (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 perfusion rate was adjusted to 2.5–3.0 ml min−1. To reduce the pH of the perfusate, [HCO3−] was decreased from 26 to 10 mm by equimolar substitution with Cl−, this perfusate also being equilibrated with 95 % O2 and 5 % CO2 (pH 7.1).

Figure 1. Experimental arrangement.

Figure 1

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Photograph of the experimental chamber from the top showing the preparation pinned ventral side uppermost. Scale bar represents 5 mm. Recordings from the internal intercostal muscle were made within the hatched region.

Recordings

Inspiratory motor activity was recorded via a glass suction electrode applied to the proximal cut end of the fourth cervical (C4) ventral root. An electromyogram was obtained from an internal intercostal muscle using a bipolar Teflon-coated platinum-iridium electrode (75 or 113 μm outer diameter, insulated except for the tip). Recordings from the internal intercostal muscle were obtained from an area between the chondrocostal junctions of the ninth to eleventh ribs (the hatched region in Fig. 1). The ribs near the recording site were pinned rigidly to prevent movement. In all seven experiments in which we examined the effect of a glycine receptor antagonist, strychnine sulphate (Wako, Japan), motor discharges were also monitored from one of the third to fifth lumbar (L3-L5) ventral roots by way of a glass suction electrode. Signals were amplified (gain: 1000-10 000; bandpass filter: 15-3000 Hz) using a high-gain AC-coupled amplifier (AB-610J; Nihon Kohden, Japan). All recordings were printed on thermosensitive paper (RTA-1200; Nihon Kohden) and stored on a pulse-code modulation data recorder (PC208A; Sony, Japan) for further off-line analysis.

Analysis

As in a previous study (Smith et al. 1990), the inspiratory and expiratory phases were defined as the period during a C4 burst and the intervening silent period between C4 bursts, respectively. The following two parameters were measured to enable us to examine the effect of a decrement in extracellular pH on respiratory motor activity: cycle period (the period from the onset of one burst in the C4 ventral root to the onset of the next burst) and inspiratory burst duration (the period from the onset to the end of a C4 inspiratory burst). The period between the end of a given C4 burst and the onset of an intercostal expiratory burst was also measured in order to examine the extent of the reciprocity. In experiments to examine the effect of strychnine, two additional parameters were measured: expiratory burst duration (the period from the onset to the end of an expiratory burst in the internal intercostal muscle) and Ionset-Eonset (the period from the onset of a given C4 inspiratory burst to the onset of the corresponding intercostal expiratory burst). The parameter Ionset-Eonset was given a positive value when the C4 burst preceded the intercostal expiratory burst. The amplified signals were digitized at 2000 Hz per channel (MacLab/8s, ADInstruments, Australia) and analysed using attached software (Chart v3.5.2/s, ADInstruments). The unit of measurement used for the cycle period was 50 ms. For the other four parameters, 5 ms was used as the unit of measurement. All parameters were measured over ten consecutive respiratory cycles and are presented as means ±s.d. The Wilcoxon signed rank test or the Mann-Whitney U test was used to test for the significance of differences in paired or unpaired data, respectively. The level of statistical significance was set at P < 0.05.

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

Effect of a decrement in the pH of the perfusate on expiratory muscle activity

We occasionally observed expiratory rib movements or intercostal expiratory activity before or just after the recording was started under normal pH conditions (26 mm HCO3−, 5 % CO2, pH 7.4). However, such expiratory activity disappeared within 1 h after the perfusate was warmed from 13-18°C to 25 ± 1°C. During perfusion with normal pH solution, the cycle period and the duration of the C4 inspiratory burst were 22.0 ± 5.3 s and 1.51 ± 0.36 s (n = 10), respectively.

Figures 2 and 3 display representative recordings showing the effect of a decrement in extracellular pH. When the low pH solution (10 mm HCO3−, 5 % CO2, pH 7.1) was applied, the frequency of the rhythm in the C4 ventral root initially increased (Fig 2A-C, Fig 3A and B). The latency to onset of this increase in frequency was between about 1 and 8 min after bath application of the low pH solution in the nineteen preparations examined (see ○ in Fig 2A and Fig 3B). To examine the effect of a decrement in extracellular pH on the respiratory rhythm in more detail, the cycle period and inspiratory burst duration were measured in ten preparations before and after the low pH solution was applied; the results are summarized in Fig. 4. The cycle period was significantly decreased by application of low pH solution in all ten preparations (from 22.0 ± 5.3 to 11.9 ± 1.3 s; P < 0.01; n = 10). Inspiratory burst duration was not changed consistently and the group means showed no significant change overall (normal pH solution, 1.51 ± 0.36 s; low pH solution, 1.33 ± 0.28 s; P = 0.24; n = 10).

Figure 2. Effect of a decrement in pH on respiratory motor activity.

Figure 2

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A, records of discharges in the C4 ventral root (C4) and an internal intercostal muscle (IIM). The pH of the perfusate was decreased by reducing [HCO3−] from 26 to 10 mm at the beginning of the hatched bar. B-F, the parts of recording A above the horizontal bars displayed on an expanded time scale. G and H, the parts of recording E above the horizontal bars displayed on an expanded time scale. ○ in A indicates onset of the increment in frequency of the inspiratory rhythm. ⋄ in D indicates an inspiratory C4 burst without a corresponding intercostal expiratory burst. ♦ indicates an expiratory burst occurring before expiratory bursts began appearing at regular intervals (•). ⋆ and ▴ indicate expiratory bursts occurring before and after a C4 inspiratory burst, respectively. In the period after ▵, an expiratory burst consistently occurred after the termination of a C4 inspiratory burst.

Figure 3. Establishment of a stable phase relationship after bath application of a low pH solution.

Figure 3

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A, records of discharges in the C4 ventral root and an internal intercostal muscle. The pH of the perfusate was decreased by reducing [HCO3−] from 26 to 10 mm at the beginning of the hatched bar. B-D, the parts of recording A above the horizontal bars displayed on an expanded time scale. ○, •, ▵, ▴ and ⋆ have the same meanings as in Fig. 2. ⋆ indicates an expiratory burst without a corresponding inspiratory C4 burst.

Figure 4. Effect of a decrement in pH on cycle period and inspiratory burst duration.

Figure 4

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Each symbol indicates the mean of the data obtained from a given preparation. A line is drawn between data points obtained from one and the same preparation under normal and low pH conditions. Where data obtained under low pH conditions showed a significant (P < 0.05) increment or decrement relative to the corresponding data obtained under normal pH conditions, this is indicated by + or -, respectively. * Significant difference between the groups, P < 0.05.

The appearance of regular expiratory bursts followed the increment in inspiratory frequency by about 0–10 min (3.2 ± 3.1 min, n = 15; see • in Fig 2D and Fig 3B). Before the stable phase relationship was established, some respiratory cycles exhibited an expiratory burst (♦) but some did not (⋄). The expiratory burst occurred sometimes before and sometimes after the C4 inspiratory burst (⋆ and ▴ in Fig 2D and Fig 3C, respectively). Furthermore, intercostal expiratory bursts sometimes occurred with no corresponding inspiratory C4 burst (⋆ in Fig. 3C). At and after the point indicated by ▵ in Fig 2D and Fig 3C, the expiratory bursts consistently occurred after termination of the corresponding C4 inspiratory burst (Fig 2E, F and Fig 3D) and were accompanied by movements of the ribs in a caudal direction. This pattern of respiratory motor activity was observed in sixteen out of nineteen preparations. Overall, the intercostal expiratory burst appeared 0.31 ± 0.19 s after the end of the C4 inspiratory burst (n = 16; range 0.08–0.83 s).

Expiratory activity of short duration (< 0.5 s) in the internal intercostal muscles often preceded the C4 inspiratory burst (Fig. 2G). Although such pre-inspiratory activity did not occur consistently (Fig. 2H), it was observed (preceding about 50 % of inspiratory bursts) in eleven out of sixteen preparations. The pre-inspiratory activity occasionally continued right up to the beginning of the C4 inspiratory burst (preceding about 20 % of inspiratory bursts) in these preparations. In three other preparations, weak intercostal activity occurred at the beginning of the C4 inspiratory burst (in about 70 % of inspiratory bursts). Since these types of activity were similar in amplitude to the expiratory bursts seen after the termination of the C4 inspiratory burst and were accompanied by brief movements of the ribs in a caudal direction, the activity is unlikely to represent contamination from inspiratory muscles. These types of activity were not observed in the remaining two of the sixteen preparations.

In three out of the four preparations that showed a stable respiratory motor pattern more than 30 min after the start of perfusion with low pH solution, both the amplitude and duration of the expiratory bursts in the internal intercostal muscle declined gradually over time even though the low pH solution was still being applied (as shown in Fig. 2E and F). The decline in the expiratory bursts was especially apparent about 5–10 min after the expiratory bursts first appeared, as shown in Fig. 2A. The expiratory bursts continued to occur for more than 10 min after the onset of rhythmic expiratory bursts in twelve out of sixteen preparations. In all the preparations examined, re-perfusion with low pH solution after washing for about 30 min with normal pH solution caused expiratory bursts to occur again.

Another pattern of respiratory motor activity was dominant in three out of nineteen preparations. In this pattern, as shown by ▪ in Fig. 5C, several inspiratory C4 bursts occurred in a cluster. The number of inspiratory bursts per cluster was always less than eight. Intercostal expiratory activity appeared mainly in the pre-inspiratory phase in these three preparations (Fig. 5D). As indicated by the arrows in Fig. 5C, sustained expiratory bursts often appeared between the clusters. Such sustained bursts occurred after about 25, 90 or 100 % of clusters in the three preparations. These sustained bursts were similar in duration to the expiratory bursts shown in Fig 2 and Fig 3. In the preparation illustrated in Fig. 5, a respiratory rhythm similar to that shown in Fig. 2 and Fig. 3D was observed 10 min after application of the low pH solution (Fig. 5A and B). This was also observed in one other preparation. In two out of these three preparations, neither the amplitude nor the duration of the pre-inspiratory bursts in the internal intercostal muscle declined over time during perfusion with low pH solution lasting more than 30 min.

Figure 5. Pattern of expiratory activity seen when inspiratory bursts occurred in a cluster.

Figure 5

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Records of discharges in the C4 ventral root and an internal intercostal muscle. B and D, the parts of recordings A and C above the horizontal bars, respectively, displayed on an expanded time scale. Data were obtained from the same preparation at 10 min (A) and 85 min (C) after bath application of low pH solution. In C, ▪ indicates a cluster of inspiratory bursts. Arrows indicate expiratory bursts of long duration.

Effect of strychnine on inspiratory-expiratory motor coordination

To clarify the involvement of glycinergic inhibition in the production of coordinated respiratory motor activity, the effects of strychnine (5–10 μm; a glycine receptor antagonist) were examined in seven preparations during perfusion with our usual low pH solution. Motor activity was also recorded from one of the L3 to L5 ventral roots to help us discriminate seizure-like discharges from respiratory activity. A representative example is shown in Fig. 6. Bath application of 10 μm strychnine had no clear effect on the periodic respiratory activity, although a seizure-like discharge often disturbed the respiratory activity (Fig. 6A). About 10 min after the application of strychnine, the bursts of activity in the internal intercostal muscle largely overlapped with those in the C4 ventral root (compare Fig. 6B with 6C). However, the duration of the C4 bursts was still different from that of the intercostal bursts (Fig. 6C). After prolonged seizure-like discharges, the burst activity in the internal intercostal muscle was often weak (arrows in Fig. 6A), while those in the C4 ventral root were less affected. Similar results were obtained in all preparations examined. The effects of strychnine on each measured parameter (see Methods) are summarized in Fig. 7. The reduction in the Ionset-Eonset parameter was very large and statistically significant in all preparations. Overall, Ionset-Eonset was significantly decreased from 1.73 ± 0.45 s to 0.10 ± 0.01 s by strychnine (n = 7, P < 0.05). As exemplified by Fig. 6D, the onset of the C4 burst always preceded that of the corresponding intercostal burst under strychnine in all preparations. The other three parameters were less affected by strychnine. The cycle period tended to be shortened but overall it did not change significantly (control: 13.1 ± 2.0 s; strychnine: 11.6 ± 2.4 s; n = 7; P = 0.06). The duration of the expiratory burst tended to be decreased by strychnine. However, overall there was no significant difference in expiratory burst duration (control: 2.44 ± 0.57 s; strychnine: 2.00 ± 0.31 s; n = 7; P = 0.06). The effect of strychnine on inspiratory burst duration varied between preparations and overall it did not change significantly (control: 1.47 ± 0.39 s; strychnine: 1.37 ± 0.36 s; n = 7; P = 0.61).

Figure 6. Effect of strychnine.

Figure 6

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A, records of discharges in the C4 ventral root, internal intercostal muscle (IIM) and L4 ventral root. Strychnine (10 μm) was applied at the beginning of the hatched bar. Seizure-like activity can be distinguished from respiratory activity by the large synchronous discharges in the L4 ventral root. The respiratory burst in IIM after each seizure-like discharge was weak, as shown by the arrows. B and C, the parts of recording A above the horizontal bars displayed on an expanded time scale. D, upper trace shows superimposed records of the three successive respiratory C4 bursts indicated by horizontal bars in C. Lower traces are the corresponding recordings from IIM. The vertical dashed line indicates the start of the C4 bursts. ▴ indicates the onset of burst activity in IIM. The vertical gain of the recordings from IIM shown in D was increased by a factor of two compared to the records in A-C.

Figure 7. Summary of the effects of strychnine on various parameters.

Figure 7

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Each symbol indicates the mean of the data obtained from a given preparation. A line is drawn between data points obtained from one and the same preparation before and after the application of strychnine. Where data obtained under strychnine showed a significant (P < 0.05) increment or decrement relative to the corresponding data obtained under control conditions, this is indicated by + or -, respectively. * Significant difference between the groups, P < 0.05.

DISCUSSION

By using central chemoreceptor stimulation, we have shown for the first time that the neuronal mechanisms necessary for expiratory motor activity are functionally preserved in the in vitro brainstem-spinal cord-rib preparation from neonatal rats. The present results suggest (a) that our preparation is capable of producing a pattern of respiratory motor activity resembling that seen during eupnoeic respiration and (b) that the glycinergic inhibitory system plays an important role in the coordination of inspiratory and expiratory motor activity. The results will be discussed under the following headings: (i) influence of experimental conditions; (ii) intercostal expiratory activity; (iii) involvement of glycinergic inhibition in the coordination of expiratory and inspiratory motor activity.

Influence of experimental conditions

The expiratory burst in the internal intercostal muscle showed sparser discharges than the inspiratory burst in the C4 ventral root (e.g. Fig. 2G and H). In the present study, the distance between the two platinum-iridium wires used for electromyographic recording was less than 1 mm. Furthermore, the electrode was placed just on the surface of the muscle in order to minimize the contamination by electrical activity from other muscles. Consequently, the bipolar electrode would record electrical activity from a small number of muscle fibres. On the other hand, the suction electrode would record activity from a large number of C4 phrenic motor axons, since the suction electrode incorporated the whole C4 ventral root. In fact, the appearance of the expiratory burst was similar to that of the C4 inspiratory burst (except for the phase difference), when activity was recorded from the whole thoracic ventral root under our usual low pH conditions (M. Iizuka, unpublished observation). Since the thoracic ventral root also contains inspiratory motor axons (Smith et al. 1990), however, the respiratory burst in the thoracic ventral root showed two peaks corresponding to inspiratory and expiratory activity.

It is known that expiratory motor discharges occur in the internal intercostal muscles during eupnoea in the rat, cat and dog in vivo (Fregosi & Bartlett, 1989; Oliven & Kelsen, 1989; Janczewski & Aoki, 1999). On the other hand, there were no expiratory bursts in the internal intercostal muscles during perfusion with normal pH solution in the present in vitro preparations obtained from neonatal rats. The extracellular pH within the medulla, which is assumed to include the central chemoreceptive fields, is about 7.1–7.2 in anaesthetized, spontaneously breathing cats (see Fig. 3 in Arita et al. 1989). Since the normal pH solution (pH 7.4) was directly applied to the surface of the brainstem in the present study, it may be that the pH of the chemoreceptive field was more alkaline than it is in vivo. This may have been responsible for the absence of expiratory motor activity.

The intercostal expiratory bursts gradually weakened during bath application of the low pH solution in most preparations. The explanation for this decline remains unclear at present. Since re-perfusion with low pH solution after washing for about 30 min with normal pH solution caused expiratory bursts to occur again, it is evident that the neuronal mechanisms responsible for the expiratory motor activity were not inactivated irreversibly. Possibly, repeated application of low pH solution will be necessary to evoke a prolonged period of expiratory motor activity in the in vitro preparation.

The phrenic activity of the in vitro preparation rises rapidly to a peak and then gradually declines (e.g. Suzue, 1984; Onimaru & Homma, 1987; Smith et al. 1990). This pattern of activity resembles that seen during gasping rather than eupnoea in adult and neonatal rats in vivo (Fung et al. 1994; Wang et al. 1996). However, there are some differences between the respiratory activity seen in vitro and gasping in vivo. Though neither the frequency nor the intensity of gasps is altered by hypercapnia in adult cats in vivo (St John & Knuth, 1981), the frequency of the respiratory rhythm increased when the pH of the perfusate was decreased in in vitro preparations from neonatal rats (Suzue, 1984; Harada et al. 1985). During gasping in the adult mammal, expiratory motor activity, especially in the intercostal and abdominal nerves, is much weaker than that seen during eupnoea; indeed, it may even be absent (St John et al. 1989). The present study has shown that low pH induces expiratory motor activity in the internal intercostal muscle in the in vitro neonatal rat preparation. These responses to pH are similar to those observed in in vivo experiments during eupnoea (Bainton et al. 1978; Fregosi & Bartlett, 1989; Oliven & Kelsen, 1989). Although further experiments will be necessary for a full comparison to be made between the in vivo and in vitro preparations, the present in vitro preparation is a fascinating experimental model enabling us to explore the generation not only of the respiratory rhythm but also of the respiratory motor pattern.

Intercostal expiratory activity

Expiratory neural activity in thoracic and cranial nerves is not consistently recorded in in vitro preparations obtained from neonatal rats (Smith et al. 1990). Although this situation prevailed during perfusion with normal pH solution in the present study, use of the low pH solution caused an increase in the frequency of the inspiratory rhythm and the appearance of expiratory motor activity in the internal intercostal muscle. Thus, the present study is the first to demonstrate that the neuronal mechanisms necessary for expiratory motor activity can be functionally preserved in an in vitro preparation derived from neonates.

Expiratory intercostal muscles are active either throughout the expiratory phase or during its latter half in the adult cat in vivo (e.g. Bainton et al. 1978; Fung & St John, 1995; Duron & Rose, 1997). In contrast, intercostal expiratory activity was limited to the first half of the expiratory phase in the present study. Very recently, it was reported that intercostal expiratory activity actually peaked during the first half of the expiratory phase and then declined until the next inspiration during eupnoea in the neonatal rat in vivo (Janczewski & Aoki, 1999). Therefore, the difference in the phase in which expiratory intercostal muscles are active may derive from a difference in species or in the developmental stage of the animals used. Although in one report expiratory activity could be recorded from about 40 % of electrodes placed within the intercostal space of adult rats under halothane anaesthesia, the pattern of the expiratory discharges was not mentioned in detail (Peláková & Palecek, 1985). Further experiments will be needed to clarify the phase in which expiratory intercostal muscles are active in adult rats in vivo.

Involvement of glycinergic inhibition in the coordination of expiratory and inspiratory motor activity

The present study suggests that glycinergic inhibition plays a crucial role in coordinated pattern generation involving alternate expiratory and inspiratory motor activity in the neonatal rat. Under strychnine, the C4 inspiratory burst and the intercostal expiratory burst largely overlapped. On the other hand, the burst duration in the intercostal muscle still differed from that in the C4 ventral root (Fig. 6C) and was not significantly affected by strychnine. These results are the first evidence suggesting the existence of discrete burst-generating mechanisms for inspiratory and expiratory motor activity in the neonatal rat. Furthermore, they also suggest that termination of neither type of burst depends on glycinergic inhibition, at least in the in vitro preparation obtained from the neonatal rat.

It is known that strychnine, a glycine receptor antagonist, changes alternating motor activity (between the left and right ventral roots or between extensor and flexor muscle nerves) into synchronous patterns during chemically induced locomotion in in vitro preparations obtained from neonatal or fetal rats (Cowley & Schmidt, 1995; Iizuka et al. 1998). Similar results were also obtained from the developing wallaby (Ho, 1997) and lamprey (Cohen & Harris-Warrick, 1984). All these studies indicated that glycinergic inhibition within the spinal locomotor networks plays a crucial role in the establishment of an alternating pattern of motor activity. In the present study, the respiratory reciprocal motor activity between the C4 ventral root and internal intercostal muscle changed to a largely overlapping pattern under strychnine. Taken together, the above observations suggest that both the locomotor networks in the spinal cord and the respiratory networks in the brainstem use glycinergic inhibition in the generation of an alternating motor activity, even though they are quite different in their functions and anatomical locations.

It was recently reported that bilateral injection of strychnine into the pre-Bötzinger complex either greatly reduced phrenic burst amplitude or irreversibly blocked rhythmic phrenic discharges in the anaesthetized adult cat in vivo (Pierrefiche et al. 1998). Similarly, a reduction in synaptic inhibition produced by reducing the [Cl−] of artificial blood alters and eventually abolishes the respiratory rhythm in an in situ arterially perfused adult rat preparation (Hayashi & Lipski, 1992). Thus, in adult mammals the generation of alternating expiratory and inspiratory activity cannot be isolated from respiratory rhythm generation by a blockade of glycinergic inhibition. On the other hand, strychnine has no clear effect on the inspiratory rhythm itself in in vitro preparations from neonatal rats (Murakoshi & Otsuka, 1985; Onimaru et al. 1990). This was confirmed in the present study. A few years ago, it was shown that strychnine abolishes inspiratory rhythm in the adult mouse but is ineffective in the neonatal mouse both in vivo and in vitro (Paton & Richter, 1995). These studies and the present study suggest that while glycinergic inhibition plays little role in inspiratory rhythm generation in the neonate, it has a crucial role to play in the generation of a coordinated respiratory motor pattern at this developmental stage.

Acknowledgments

The author wishes to thank Drs S.-I. Sasaki and N. Kudo for valuable comments on the manuscript.

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