The depolarization of I neurones and hyperpolarization of PI neurones at the spontaneous onset of inspiration was in most animals (12/16) associated with a transient burst of phrenic activity resembling phase 3 described in the present study. This phrenic burst was not affected by dizocilpine (not shown, but see Fig. 4 in Haji et al. 1996b). The membrane potential of I and PI neurones was also recorded in one animal in which vagal stimulation reproducibly triggered an inspiratory 'on-switch' before and after dizocilpine administration (Fig. 6). In this animal the evoked phase 3 phrenic burst starting inspiration was comparable with the burst of phrenic activity preceding the 'ramp' discharge and accompanied by hyperpolarization of PI neurones and depolarization of I neurones observed during spontaneous inspiratory on-switch. Phase 1 and phase 2 PSPs evoked by the stimulus have no equivalent during spontaneous on-switch. In contrast, phase 3 responses appear to be a higher-order network-generated event which have an equivalent during spontaneous inspiratory on-switch. In our experimental conditions, the short burst of phrenic activity elicited by SLN stimulation at mid-expiration was never followed by a resumption of inspiration.
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Figure 6. PSPs in PI and I neurones during on-switch of inspiration evoked by vagal stimulation
In both neurones the stimulus was applied in expiration after dizocilpine administration. It evoked an EPSP in a post-inspiratory laryngeal motoneurone (A) and concomitantly an IPSP in an inspiratory laryngeal motoneurone (B). The late burst of phrenic activity (phase 3) was followed by a decrease of activity, then by resumption of discharge as the PI neurone hyperpolarized and the I neurone depolarized.
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DISCUSSION |
The present results suggest that synaptic responses to stimulation of the SLN or vagus nerve exhibit three successive phases. The early excitatory PSP (phase 1) was independent of the type of neurone, I or PI, whereas during phase 2 (phrenic inhibition) and phase 3 (late phrenic burst), the PSPs had opposite directions in I and PI neurones. These responses were not significantly affected by dizocilpine, which indicates that the pathways involved in the generation of these three successive phases might be distinct from the pathways of the centrally controlled IOS, which have been shown to act through activation of NMDA receptors (Pierrefiche et al. 1992). Furthermore, the dizocilpine-insensitive late burst of phrenic activity (phase 3) is analysed and proposed to be the first stage of inspiratory on-switch.
Different phases of postsynaptic responses evoked by SLN stimulation on respiratory neurones
Stimulation of SLN afferents evoked a rather uniform PSP response in I and PI neurones, always starting with an early and short lasting phase 1 EPSP. This EPSP was evoked in both inspiratory and expiratory phases with latencies which were not significantly different in I and PI neurones, and may represent a relatively direct oligosynaptic projection of SLN afferents on both types of respiratory neurones. Our data are therefore in agreement with other groups which observed this early excitation in I and PI neurones and on the whole phrenic nerve activity contralateral to the stimulation (Berger & Mitchell, 1976; Iscoe et al. 1979; Sica, Cohen, Donnelly & Zhang, 1984; Bellingham, Lipski & Voss, 1989; Donnelly, Sica, Cohen & Zhang, 1989; Jiang & Lipski, 1992). In contrast, the early PSP response is inhibitory in expiratory neurones of the VRG and Bötzinger Complex (Czyzyk-Krzeska & Lawson, 1991; Jiang & Lipski, 1992). Phase 2 IPSPs started approximately 10 ms after the stimulus in I neurones while EPSPs continued in PI neurones in accordance with previous observations (Czyzyk-Krzeska & Lawson, 1991) and reciprocal inhibition between the different types of respiratory neurones (Bianchi et al. 1995). During the inspiratory phase, while the initial EPSP reached its peak in PI neurones, a phase 2 IPSP occurred in I neurones and the phrenic nerve ceased to discharge. These PSPs probably originated from distinct groups of respiratory neurones producing excitation and inhibition in response to early excitation by SLN afferents (Czyzyk-Krzeska & Lawson, 1991).
Peripherally evoked on-switch processes
The period of phrenic silence evoked by SLN or vagal stimulation was often followed by a short burst of activity. Its onset coincided with the onset of an IPSP in PI neurones and an EPSP in I neurones (phase 3). During inspiration, this burst was often followed by the resumption of phrenic activity. During expiration the same stimulus evoked the same phase 3 PSP sequence and transient burst of phrenic activity with a similar latency to that during inspiration, after which the evoked phrenic burst could also be followed by a re-setting of inspiration. A similar short burst of activity initiates the phrenic discharge during the 'spontaneous' on-switch and, like the evoked phrenic burst, is unaffected by dizocilpine. This suggests that the resumption of inspiratory activity when the transient phrenic inhibition fails to evolve into an IOS might be due to activation of an on-switch process triggered by the stimulus after an initial phase 1-2 reversible off-switch. Recent work by Oku & Dick (1992) and Oku, Dick & Cherniack (1993) indeed supports the idea that phase 3 burst of phrenic nerve discharge previously observed by several authors (Berger & Mitchell, 1976; Iscoe et al. 1979; McCrimmon et al. 1987) might be viewed as an abortive 'on-switch'. Although SLN stimulation during the expiratory phase generally prolongs the phase and delays the next inspiration, a phase-resetting characteristic of SLN stimulation has been demonstrated in cats with small pontine lesions (Oku & Dick, 1992) and in a few non-lesioned animals (Oku et al. 1993). In these animals, SLN stimulation applied during late expiration consistently terminated expiration and prematurely advanced the onset of the next inspiration. Oku & Dick (1995) proposed a model explaining the paradoxical phase-resetting response to SLN stimulation by an activation by SLN afferents of E-decrementing and I-decrementing (i.e. early-I) neurones. The present recordings of I and PI neurones support this hypothesis, although we did not record early-I neurones. Considering synaptic relationships between I, PI and early-I neurones (Bianchi et al. 1995), it is likely that the phase 3 IPSP observed in PI neurones during the burst of phrenic activity might originate from early-I neurones (Ezure, Manabe & Otake, 1989).
Taken together, the data show that stimulation of peripheral afferents evoked rather uniform polyphasic responses of the respiratory neurones and of the phrenic nerve throughout the respiratory cycle. How the respiratory network evolves after these responses might depend essentially on the state of the network at the time when the stimulus is delivered, leading to either resumption of phrenic inspiratory activity, IOS, resumption of expiration, or on-switch.
Respiratory-related discharge of PI neurones produces 'irreversible' off-switch
The discharge of PI neurones defines one of the two phases of expiration (Champagnat, Denavit-Saubié, Moyanova & Rondouin, 1982; Richter, 1982; Richter, Ballantyne & Remmers, 1986; St John & Zhou, 1989; Pierrefiche et al. 1992; Oku et al. 1993). Although many neurones with a post-inspiratory discharge are undoubtedly motoneurones (Zheng, Barillot & Bianchi, 1991), indirect evidence based on the patterns of chloride-mediated IPSPs and glycinergic postsynaptic inhibition in other neurones (Richter et al. 1986; Schmid, Foutz & Denavit-Saubié, 1996) supports the idea that PI neurones exert a widespread inhibition on other respiratory neurones, particularly I neurones (Richter et al. 1986). Thus the IPSP observed on I neurones after the initial EPSP might originate from PI neurones. Furthermore, the timing of PI discharge just after inspiratory arrest, suggests that these neurones carry out the 'irreversible' phase of the IOS process, preventing the re-activation of phrenic discharge (Richter et al. 1986). However, intervention of other neurones (i.e. late-inspiratory) in the evoked IOS cannot be excluded.
SLN stimulation evokes a premature discharge of PI neurones
During spontaneous IOS, PI neurones started to depolarize 30-50 ms before the arrest of the phrenic activity, giving rise to an 'off-switch potential' (Fig. 3B, the slow slope in the window), whereas SLN stimulation evoked a massive, fast and synchronous synaptic activation of PI neurones which preceded the transient phrenic inhibition or the IOS. Thus, stimulation of peripheral afferents changed the normally slow respiratory-related depolarization of PI neurones into a fast and steep depolarization reaching quickly the threshold for action potentials. This is in keeping with previous works which showed that SLN stimulation activates PI neurones and tyroarytenoid motoneurones on a similar time scale (Remmers et al. 1986; St John & Zhou 1989), and that a brief activation of the tyroarytenoid nerve precedes the reactivation of the phrenic nerve when reversible phrenic inhibition is triggered by a short train of stimuli applied to the SLN (Oku et al. 1993). A strong activation of the discharge of PI neurones by various peripheral inputs during the post-inspiratory phase might be the mechanism through which these inputs modulate the respiratory rhythm (Remmers et al. 1986; Lawson, Richter, Ballantyne & Lalley, 1989). Therefore, we conclude that in normal conditions the IOS process starts by a slow 'off-switch potential' whereas during stimulation, synchronized activation of the inputs triggers the discharge of PI neurones which prematurely block inspiratory discharges within a few milliseconds, well before the onset of the 'off-switch potential'. SLN stimulation activated PI motoneurones and non-antidromically responding PI neurones in a similar fashion. This is in keeping with previous results which did not reveal any difference in the pre-synaptic drive (cycle-related changes in membrane resistance or excitability) of motoneurones and presumed propriobulbar neurones with same patterns of cycle-related discharge (Pierrefiche et al. 1992; Haji et al. 1996b).
Evoked responses are not mediated by NMDA receptors
Dizocilpine administration decreases respiratory-related EPSPs and IPSPs throughout the respiratory cycle (Feldman, Windhorst, Anders & Richter, 1992, Haji et al. 1996b). This might be the main reason why the transient inhibition of phrenic activity evoked by SLN stimulation was prolonged after dizocilpine, because the EPSPs and IPSPs evoked in I and PI neurones showed no significant change. This ineffectiveness of systemically administered dizocilpine on the peripherally evoked PSPs supports the idea that NMDA receptors are not significantly involved in the pathways between the primary SLN afferents and the target I or PI neurones. Indeed our microiontophoretic study (Haji et al. 1996a) has provided evidence that the EPSPs evoked by peripheral afferent stimulation are mediated by glutamatergic synapses acting through non-NMDA receptors on the target neurones, and the IPSPs are mediated by GABAA receptors. The circuits and/or synapses of the evoked PSPs might thus not be directly involved in the spontaneous IOS, the occurrence of which is delayed by dizocilpine (Pierrefiche et al. 1992; Feldman et al. 1992; Haji et al. 1996b). Moreover, relay neurones in the NTS responsible for the short-latency excitation of phrenic motor output and subsequent IOS also involve activation of non-NMDA receptors (Karius, Ling & Speck, 1993, 1994; Karius & Speck, 1995). These observations suggest that the most likely target of dizocilpine action might be a presynaptic source or modulatory control of the respiratory neuronal network but not of the pathways responsible for the peripherally evoked PSPs. Such presynaptic control might originate from the pons (Ling, Karius & Speck, 1994), within a population of early-I neurones (Dick, Bellingham & Richter, 1994), whose accommodation of discharge might be blocked by dizocilpine (Pierrefiche et al. 1992; Pierrefiche, Champagnat & Richter, 1995).
We had previously shown that dizocilpine, while increasing inspiratory time, decreases the respiratory-related discharge and fluctuations of the membrane potential of PI neurones, but not their progressive depolarization preceding a naturally occurring IOS (Pierrefiche et al. 1992; Haji et al. 1996b). The present results show that NMDA receptor blockade does not impair their activation by peripheral inputs.
Conclusion
The present results are consistent with the hypothesis that stimulation of SLN afferents interrupts the inspiratory discharge by producing a synchronized, premature excitation of PI neurones. Dizocilpine depresses the respiratory-related potentials but not the evoked potentials of PI neurones, which might allow afferent inputs from peripheral afferents to terminate inspiration through activation of these neurones. Peripheral stimulation also triggered a long-latency brief excitation of phrenic nerve discharge and related PSPs on respiratory neurones, which may represent the first step of the inspiratory on-switch process, and was unaffected by NMDA receptor blockade.
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Acknowledgements
This work was supported by CNRS, DRET 95/091 (France) and a Grant-in-Aid for Scientific Research from the Ministry of Education, Science, Sports and Culture of Japan (no. 05680672) and a Grant for International Scientific Research Program (no. 07044238) of Japan.
Corresponding author
M. Denavit-Saubié: Institut Alfred Fessard, Biologie Fonctionnelle du Neurone, CNRS, 91198 Gif-sur-Yvette, France.
Email: saubie{at}iaf.cnrs-gif.fr
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Top
Introduction
as measured in brain tissue. The type of respiratory neurone was identified by the pattern of membrane potential fluctuations in relation to phrenic nerve activity. To identify the axonal projections of recorded neurones, the central ends of the ipsilateral vagus nerve and SLN were stimulated with a 0·1 ms pulse of 0·1-0·3 mA intensity, using bipolar silver electrodes (Haji, Pierrefiche, Foutz, Champagnat, Denavit-Saubié & Takeda, 1996a). The spinal axons were identified by stimulation with an array of five concentric stimulating electrodes inserted into the ventrolateral part of the C2-C3 spinal cord, using 0·2 ms pulses of 0·3-0·5 mA.
