Respiratory interneurons of the lower cervical (C4-C5) cord ...

Pfliigers Arch (1993) 425:313-320

E i ~176 Journal of Physiology 9 Springer-Verlag 1993

Respiratory interneurons of the lower cervical (C4-C5) cord: membrane potential changes during fictive coughing, vomiting, and swallowing in the decerebrate cat Laurent Gr~lot, St~phane Milano, F~d~rico Portillo*, Alan D. Miller** Dtpartement de Physiologie et Neurophysiologie, Laboratoire de Neurobiologie de ta Respiration, Case 351, CNRS URA 205, Facult6 des Sciences et Techniques Saint Jtrtme, F-13397 Marseille Ctdex 20, France Received October 15, 1992/Received after revision May 24, 1993/Accepted June 25, 1993

Abstract. The possible roles of interneurons in the C4C5 cervical spinal cord in conveying central drives to phrenic motoneurons during different behaviour patterns were investigated using intracellular recordings in decerebrate, paralysed, artificially ventilated cats. Eleven cells were tentatively classified as respiratory interneurons since they: (i) could not be antidromically activated from the ipsilateral whole intrathoracic phrenic nerve, and (ii) exhibited large membrane potential changes during eupnea (7.3 mV---3.6, range 2 - 1 3 . 5 m V ) or non-respiratory behaviour patterns. Six neurons depolarized in phase with phrenic discharge; four others depolarized during the expiratory phase; one neuron exhibited depolarization during the end of both expiration and inspiration. A variety of responses was observed during fictive coughing, vomiting, and swallowing. The results are consistent with C4-C5 expiratory interneurons conveying inhibition to phrenic motoneurons during different behaviour patterns. The responses of inspiratory and multiphasic neurons suggest that the roles of these interneurons are mode complex than simply relaying central excitatory or inhibitory drive to phrenic motoneurons.

Key words: C4-C5 cervical respiratory interneurons Coughing - Vomiting - Swallowing - Decerebrate cat - Membrane potentials - Central pattern generators

interneurons as well as being monosynaptically transmitted from bulbospinal neurons [9, 27]. Respiratory interneurons in the thoracic spinal cord have recently been characterized in some detail [22, 23] ; however, less information is available about respiratory interneurons in the region of the phrenic motor pool [4, 34]. Inspiratory drive to phrenic motoneurons originates from medullary dorsal and ventral respiratory group (DRG, VRG respectively) inspiratory neurons [33]. These bulbospinal inspiratory neurons are also active during coughing [17, 20] and swallowing [17], but are inhibited and mainly silent during vomiting [6, 32]. In the present study, we undertook the first investigation of the possible roles of respiratory neurons at the level of the phrenic motor pool in conveying central drive to phrenic motoneurons during different non-respiratory behaviour patterns involving respiratory muscles. Since stable intracellular recording requires the use of paralysed animals, we studied the responses of these interneurons during such fictive behaviour patterns such as coughing [8, 13], vomiting [31] and swallowing [14], the occurrences of which were determined by recording motor nerve activity patterns characteristic of these behaviour patterns in non-paralysed animals. A preliminary report of some of this work has been presented [16]. All recordings from respiratory neurons of this series were made in conjunction with a study of the membrane potential changes of phrenic motoneurons during fictive non-respiratory behaviour patterns [18].

Introduction In the locomotor system, spinal interneurons help both in conveying central drives to spinal motoneurons and in integrating central and peripheral inputs [21]. In the respiratory system, central excitatory drive to spinal respiratory motoneurons is channelled through spinal * Present address: Dpt. de Neurociencias, Catedra de Fisiologia, Facultad de Medicina, Plaza Fragela S/N, E-Cadiz, Spain ** Present address: The Rockefeller University, 1230 New York Avenue, New York NY 10021, USA Correspondence to: L. Grtlot

Materials and methods Experiments were conducted on nine adult cats of either sex that were decerebrated, paralysed, and artificially ventilated, using previously described procedures [6, 15, 18]. Briefly, the animals were initially anaesthetized with Saffan (Glaxovet, 1.5 ml - kg -I, i.v.) and maintained at a surgical level of anaesthesia using halothane (Fluothan, Coopers). The trachea, femoral veins and artery, and bladder were cannulated. Animals were decerebrated at the mid-collicular level following ligation of the external carotid arteries. Anaesthesia was then discontinued,

Animal preparation.

314 and the animals were paralysed using gallamine triethiodide (2 mg 9 kg -~ - h -1 i.v., supplemented as required) and artificially ventilated (end tidal pCO2 = 4.5-5.5 %). Since the vagi were left intact to induce vomiting (see below), tidal volume and pump frequency were set (typically 2 0 - 3 0 ml and 2 5 - 3 0 per rain, respectively) to prevent a 1 : 1 synchronization of the central respiratory rhythm with that of lung inflation. Tracheal and blood pressures were monitored and displayed on the chart recorder. Mean blood pressure was maintained above 90 mm Hg (12 kPa) using, if necessary, i.v. administration of metaraminol bitartrate (Aramine; Merck, Sharp and Dohme). Rectal temperature was maintained between 36-38~ using a servo-controlled heating pad.

Recording and stimulation. The activities of the intact right C5 phrenic, Lt or L2 abdominal, pharyngeal branch of the vagus, and hypoglossal nerves were recorded using bipolar silver electrodes. The signals were amplified and filtered (bandpass 0.01-10 kHz). Stimulating electrodes were placed on supradiaphragmatic (thoracic) phrenic and vagus nerves and on the superior laryngeal nerves (SLN). To expose the first two nerves, a pneumothorax was made in the sixth or seventh intercostal space. The lung were retracted, and two pairs of electrodes were placed around the dorsal and ventral vagi just rostral to the diaphragm. An additional pair was placed on the right intrathoracic phrenic nerve just above the diaphragm. Since all animals underwent a bilateral pneumothorax, a positive end-expiratory pressure of 3 - 5 cm H20 (0.30.5 kPa) was added to prevent atelectasis. The C5 phrenic motor pool was located using 5 - to 10-MO glass micropipettes filled with 3.0-3.5 M NaCt while stimulating the whole ipsilateral intrathoracic phrenic nerve (0.t- to 0.2-ms pulses, 5 - 1 5 V, 1 Hz) to evoke an antidromic field potential. This intensity was 3 - 4 times stronger than that required to evoke a maximal volley on the C5 rootlet. Once the phrenic nucleus was located, recordings of intracellular potentials were made with 10to 25-Mf2 micropipettes filled with 3.0-3.5 M KC1. This type of micropipette was used to try to reveal the presence of inhibitory postsynaptic potentials (IPSPs) by injecting negative current through the electrode. Signals were amplified by a high impedance circuit incorporating capacity compensation, DC offset and a bridge circuit for current injection across the recording microelectrode (Transidyne 1600). Membrane potentials were defined as the difference between intracellular and extracellular potentials, using as a reference a grounded silver-silver chloride electrode inserted into the neck muscles. All measurements were corrected, if necessary, by measuring the juxtra-cellular potentials after the microelectrode was withdrawn from the cell. Fictive vomiting was produced by electrical stimulation of the thoracic vagus nerves (typically continuous trains of 0.8-ms pulses, 2 5 - 3 3 Hz, 10-50V). Fictive coughing and swallowing were evoked by electrical stimulation of a superior laryngeal nerve (0.1to 0.2-ms pulses, 2 - 5 V, 2 - 5 Hz for coughing or 1 0 - 3 0 Hz for swallowing)9 Signals were simultaneously displayed on a chart recorder (Gould TA 2000) and oscilloscopes and stored on tape (Neuro Data DR 890). Chart recordings at high speed (typically 100 mm/s) were made for measurements of membrane potential changes during different fictive behaviour patterns. Five successive respiratory cycles were used to obtain the mean respiratoryrelated membrane depolarization. For the non-respiratory behaviour patterns, mean values were calculated using all depolarizations that were produced during repeated episodes of a given behaviour. Since the animals were paralysed in this experimental series, the terms "coughing", "vomiting'and "swallowing" refer to fictive coughing, vomiting and swallowing, respectively9

Results

The intracellular activities o f 11 respiratory-related neurons (6 inspiratory, 4 expiratory, 1 multiphasic) were recorded in the region o f the phrenic m o t o r nucleus. These

Table 1. Numbers of inspiratory, expiratory, and multiphasic interneurons studied during various behaviours Interneuron

Behaviour pattern C

V

S

C+V

C+S

V+S

C+V +S

Total

I

2

1

1

0

2

0

0

6

E Multi Total

2 0 4

0 0 1

1 0 2

0 1 1

0 0 2

1 0 1

0 0 0

4 1 11

I, Inspiratory; E, expiratory; Multi, multiphasic, C, coughing, V, vomiting, S, swallowing, C + V + S, numbers of intemeurons studied during all 3 non-respiratory behaviours; C + V, C + S and V + S, numbers of those studied during two indicated behaviours together

cells were considered to be interneurons since they failed to exhibit antidromic action potentials in response to suprathreshold thoracic phrenic nerve stimulation. Moreover, some o f these cells (i. e. expiratory and multiphasic) exhibited inappropriate m e m b r a n e potential changes to be mistaken for phrenic or spinal accessory motoneurons. These intracellular recordings were considered to be f r o m the cell bodies o f neurons, rather than f r o m axons, on the basis o f the presence o f m e m b r a n e potential changes that occurred during eupnea (7.3 m V _+ 3.6, SD, range 2 - 1 3 . 5 mV) and non-respiratory behaviour patterns. These respiratory neurons were probably not R e n s h a w cells since none responded synaptically to single shocks o f the phrenic nerve [19, 24]. T h e resting m e m b r a n e potentials o f the six inspiratory neurons averaged - 7 7 _+ 6 m V (range - 6 8 to - 8 6 m V ) and those o f the four expiratory neurons averaged - 6 8 . 2 +- 4 m V (range - 6 3 to - 7 2 mV). Seven neurons were studied during coughing, three during vomiting and five during swallowing. Two neurons were recorded f r o m during both c o u g h i n g and swallowing, one during c o u g h i n g and vomiting, and one during vomiting and swallowing (Table 1). Nine neurons were located in C5; two inspiratory neurons were in the caudalmost aspect o f C4.

Eupnea All six inspiratory neurons exhibited a ramp depolarization (11.3 _ + 2 . 2 m V , range 8 . 7 - 1 3 . 5 m V ) during phrenic discharge similar to that o f phrenic motoneurons (Fig. 1 A) [5, 18]. At the termination o f inspiration, the m e m b r a n e potentials repolarized abruptly, one neuron exhibiting a clear but w e a k transient hyperpolarization (1.5 _+ 0A m V ) (Fig. 3 A1). Four inspiratory neurons fired spontaneously, with the m e a n discharge frequency being 42 + 19 spikes/s (range 2 5 - 6 6 spikes/s). The two remaining cells were quiescent under the experimental conditions. Injection o f continuous negative current perf o r m e d only into one inspiratory neuron caused the reversal o f IPSPs into waves o f depolarization during the later part o f expiration.

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Fig. 1 A-D. Membrane potential changes recorded from an in-

spiratory (A) and three expiratory(B-D) C4-C5 interneuronsduring normal ventilation. In this and following figures, traces from top to bottom represent neuron's membrane potentials (MP), C5 phrenic (Phr) and abdominal (Abd-L1) nerve activities. The expiratory neurons exhibited platean-like depolarizations, with weakly augmenting(B, C) or decrementing(D) slopes; arrows in B and D indicate the maximum depolarization

(13.3 _+ 2.3 mV, range 12.9-15.8 mV), causing neuronal discharge (50 _+ 22 spikes/s, range 2 5 - 7 0 spikes/s) (Fig. 2 A). At the end of Scl the membrane potentials of two cells exhibited a rapid, transient hyperpolarization, which was immediately followed by a single short-lasting (250 _+ 5ms, range 2 2 0 - 2 9 0 m s ) depolarization that caused a low frequency discharge consisting of four to eight action potentials (Fig. 2 A). The membrane potentials of two other inspiratory cells rapidly repolarized at the end of Scl and then slowly depolarized (8 + 1.8 mV) throughout Sc2. The expiratory and multiphasic neurons depolarized (9.6 _+ 7.4mV, range 4 . 9 18.2 mV) and fired during Sc2 (Fig. 2 B2). In addition to inducing fictive coughing, superior laryngeal nerve stimulation also produced short latency excitatory postsynaptic potentials (EPSPs) in the expiratory (40 _+ 12ms, range 2 0 - 8 0 m s , n = 7) and multiphasic (32 + 13 ms, range 1 6 - 6 0 ms, n = 11) neurons both prior to and during SCa of coughing (Fig. 2 B2). Vomiting

Recordings were made from four expiratory neurons during eupneic conditions. They exhibited a clear depolarization (5.25 + 1.2mV, range 4 - 7 m V ) between phrenic bursts. The waveform of the depolarization was plateau-like with either weakly augmenting (n = 2) (Fig. 1 B, C) or decrementing (n = 2) patterns (Fig. 1 D). Near the onset of the phrenic burst, these cells rapidly repolarized. In three of them, this initial repolarization was followed by a second stage of a more gradual repolarization during the phrenic burst (Fig. 1 B, D). During eupneic conditions, three expiratory neurons were quiescent (Fig. I C, D); the remaining cell fired throughout expiration (Fig. 1 B). One neuron, silent during normal ventilation, was classified as a multiphasic respiratory neuron since its membrane was depolarized (typically 2 - 3 mv) during the end of both the expiratory and inspiratory phases (Fig. 2 B1). Since a KC1 filled microelectrode was used to record from this interneuron, at least one of these two waves of depolarization could correspond to a reversal of IPSPs due to leakage of chloride ions into the cell from the recording micropipette. However, this possibility seems unlikely since this multiphasic pattern of depolarization was observed immediately after the cell impalement and moreover did not evolve during the whole duration of recording.

Coughing

The activities of four inspiratory, two expiratory and one multiphasic neuron were recorded during fictive coughing, which was characterized by a large phrenic discharge (Stage 1 of coughing, Scl) followed immediately by a strong abdominal nerve activation (Stage 2, Sc2) consisting of either a single long-lasting discharge or a series of short-lasting bursts [13]. During Scl, all inspiratory interneurons exhibited ramp-like depolarizations

The activities of one inspiratory, one expiratory and one multiphasic neuron were recorded during fictive vomiting, which was characterized by a series of synchronous bursts of phrenic and abdominal motor nerves, culminating in an expulsion phase in which abdominal discharge was prolonged both with respect to phrenic discharge and to abdominal discharge during the preceding retching phase [31]. At the onset of each burst of phrenic and abdominal coactivation during retching, the inspiratory and multiphasic neurons abruptly depolarized to membrane potentials less negative than during normal ventilation (Fig. 3 A2, B). The membrane potentials of both cells repolarized rapidly at the end of each retch and during the inter-retch period maintained levels similar to those observed during expiration. During the expulsion phase, the inspiratory neuron exhibited a pattern of membrane potential change that was initially the same as that observed during retching. However, during the period when phrenic discharge ceased, but abdominal activity continued, this neuron exhibited an additional depolarization which evolved in two stages and resulted in neuronal discharge that continued after the end of the expulsion phase (Fig. 3 A2). The expiratory neuron, in contrast, depolarized only during the inter-retch period (Fig. 3 C). These depolarizations were too small to reach the spiking threshold. However, after the end of the expulsion phase, the cell received a stronger depolarization that caused it to fire. Swallowing

The activities of three inspiratory and two expiratory neurons were recorded during the laryngeal-induced buccopharyngeal stage of fictive swallowing, which was characterized by brief bursts of activity in the hypoglossal and pharyngeal vagus nerves [18]. All three inspiratory neurons exhibited a short-lasting (530

316 COUGHING !rlii

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A

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Scl

Phr SC 2

Abd-L1 SLN St.

B1

B2

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_+ 40 ms, range 4 8 0 - 5 6 0 ms) swallowing-related depolarization (Fig. 4 A ; amplitude 11.4 _+ 1.5mV, range 9 . 8 - 1 2 . 7 mV) that always caused a sustained discharge (mean frequency, 57 _+ 26 spikes/s, range 2 7 - 6 9 spikes/ s). The onset of this neuronal discharge occurred just after the end of the weak swallowing-related phrenic breakthrough occurring during periods of apnea. Both expiratory neurons, in contrast, exhibited a short-lasting (410 -+ 260 ms, range 2 2 0 - 6 0 0 ms) weak hyperpolarization (3.6 _+ 1.1 mV, range 4 . 5 - 2 . 8 mV) during swallowing (Fig. 4 B).

Discussion The existence of respiratory-related interneurons in the lower cervical spinal cord (C4-C6), suspected for many years [3, 7, 10], has been recently documented. Investigations based on extracellular recordings in both rabbit and cat indicate the presence of respiratory-related (i.e.

ii

1 sec

Fig. 2 A, B. Changes in membrane potentials of two respiratory (C4-C5) interneurons during fictive coughing induced by electrical stimulation of the superior laryngeal nerves (horizontal thin bar; SLN St.). Fictive coughing is characterized by a large phrenic discharge (Scl), immediately followed by strong abdominal nerve activation (Sc2). A Membrane potential trajectory of an inspiratory interneuron during fictive coughing, the eupneic activity of which is shown in Fig. 1 A. At the onset of Sc2, this interneuron exhibited a rapid and transient hyperpolarization, which was immediately followed by a single short-lasting depolarization resulting in two action potentials (arrow). B1 Recording of a multiphasic [i. e, depolarized at the end of both inspiration and expiration (*)] interneuron during eupneic conditions. B2 During fictive coughing, this multiphasic interneuron exhibited a strong depolarization causing intense neuronal discharge throughout Sc2. Arrows indicate the excitatory postsynaptic potentials (EPSPs) evoked by SLN stimulation

inspiratory, expiratory and post-inspiratory) neurons in the vicinity of the phrenic nucleus [4, 34]. Identification of these units as interneurons was based on: (i) their lack of response to phrenic nerve (i. e. C5 rootlet) stimulation; (ii) the shape and duration of the action potential; and (iii) retention of activity when the recording electrode was moved over a relatively long distance. The present study, on the basis of intracellular recordings in the cervical spinal cord of the decerebrate cat, provides strong evidence for the existence of such interneurons in the C4-C5 segments. These neurons had membrane potential changes in phase with central respiratory activity indicating that they might receive a drive from medullary respiratory neurons and/or from upper cervical respiratory propriospinal neurons projecting to the C5 segment [2]. These respiratory-related neurons are not Renshaw interneurons, already described in this part of the spinal cord [19, 24], since they were not monosynaptically excited by antidromic stimulation of the ipsilateral whole thoracic phrenic nerve.

317 SWALLOWING

VOMITING

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Fig. 3 A-C. Changes in membrane potentials of three respiratory (C4-C5) interneurons during fictive vomiting evoked by supradiaphragmatic vagal stimulation (horizontal thin bar, X Th St.). Vomiting consists of synchronous phrenic and abdominal nerve activities during the retching phase (R) followed, in the expulsion phase (E), by prolonged activity in the abdominal nerve. A1 During eupnea, this inspiratory interneuron exhibited a ramp-like depolarization during phrenic discharge, followed by a weak, transient hyperpolarization (arrows). A2 During the retching phase (R), this same cell depolarized abruptly and reached MP levels less negative than during eupnea (compare with panel A1). Then, the membrane repolarized rapidly at the end of the retching burst and exhibited, during the inter-retch period, levels similar to that observed during expiration. Note that the onset of its firing activity was synchronized with that of Abd-L1. During the expulsion phase, the membrane potential trajectory was initially similar to that observed during retching. However, note the depolarization producing action potentials after cessation of the abdominal phase of expulsion, a period when both nerves are silent. B Membrane potential trajectory of the multiphasic interneuron during retching, the eupneic pattern of which is shown in Fig. 2 B1. The quality of impalement of this neuron degraded from the time when coughing was induced (Fig. 2 B2). No data were obtained from this neuron during expulsion. C Membrane potential trajectory of the weakly decrementing expiratory interneuron during vomiting, the eupneic pattern of which is shown in Fig. 1 D. The MP depolarized between retches but the cell did not fire before the end of the abdominal phase of expulsion

Definition of cells as interneurons

We and others (e.g. [22]) defined an interneuron as a neuron in the ventral horn of the spinal cord that is not a motoneuron. Hence, all the cells described in the present report are presumably interneurons since they did not exhibit antidromic action potentials in response to electrical shocks applied to the ipsilateral thoracic phrenic nerve with an intensity ( 5 - 1 5 V) 3 - 4 times stronger than that required to evoke a maximal volley on the C5 rootlet. The efficacy o f this stimulation was confirmed

Fig. 4 A, B. Changes in membrane potentials of two respiratory (C4-C5) interneurons during buccopharyngeal stages of fictive swallowing induced by electrical stimulation of the superior laryngeal nerves (horizontal thin bar, SLN St.). Rhythmic fictive swallowing is characterized by brief bursts of activity in the hypoglassal nerve (XII). A Membrane potential trajectory of an inspiratory interneuron during swallowing, the eupneic pattern of which is shown in Fig. 1 A. This cell exhibited short-lasting depolarizations causing strong firing activities. Note that the onset of spiking discharges occurred just after the end of weak swallowing-related phrenic activations (arrows; s). B Membrane potential trajectory of an expiratory interneuron during swaUowing, the eupneic activity of which is shown in Fig. 1 B. The MP wealdy hyperpolarized during rhythmic swallowing (arrows; s). Note that these hyperpolarizations are weaker than that observed during normal inspiration (*)

throughout the experiment by recording in the same track or region antidromically identified phrenic motoneurons. Furthermore, no motoneurons exhibiting either expiratory or multiphasic respiratory patterns have been described in this region of the cervical spinal cord. In addition, most of these cells, including inspiratoryrelated ones, exhibited patterns of activities during the non-respiratory behaviour patterns clearly different from those already described for phrenic motoneurons [18, 28] or for accessory respiratory motoneurons (i.e. trapezius; see Fig. 5 in [12]). In the present study, no attempt was made to use antidromic stimulation to identify trapezius motoneutons, which are the only spinal accessory motoneurons with somata lying close to phrenic motoneurons [35]. However, we believe that since most (4/6) of the inspiratory cells described in the present report discharged spontaneously, they are unlikely to be trapezius motoneurons. Under our experimental conditions, trapezius motoneurons do not exhibit any spontaneous inspiratory activity during quiet breathing even though they can be easily activated during non-respiratory behaviour patterns such as vomiting [12] and coughing (L. Grrlot and S. Milano, unpublished observations). Inspiratory interneurons

During normal ventilation, descending inspiratory drive to phrenic motoneurons originates from inspiratory bulbospinal neurons of the DRG and VRG [33]. Inspiratory C4-C5 interneurons exhibited a ramp-like m e m b r a n e de-

318 polarization similar to that exhibited by both inspiratory bulbospinal neurons and motoneurons. These observations are consistent with the work of Davies et al. [9] indicating that central excitatory drive from DRG and VRG bulbospinal neurons to phrenic motoneurons is mainly relayed via spinal interneurons rather than by monosynaptic connections. Chloride ion injection into the soma of an inspiratory interneuron reversed waves of IPSPs in late expiration demonstrating that the neuron's membrane potential was also shaped by inhibitory mechanisms arising either from expiratory bulbospinal cells or from spinal (segmental or propriospinal) expiratory interneurons. Inspiratory C4-C5 interneurons of the present report might also contribute to the distribution of excitatory drives reaching phrenic motoneurons during coughing, since they exhibited membrane potential changes consistent with those of motoneurons [18] and inspiratory bulbospinal neurons [17]. In contrast, during swallowing, the inspiratory interneurons only exhibited depolarization and firing after the cessation of weak phrenic discharge, a pattern clearly different from that observed in inspiratory bulbospinal neurons [17]. Furthermore, during vomiting, bulbospinal inspiratory neurons of both the DRG and VRG are inhibited and mainly silent [6, 32]. On the contrary, the single inspiratory interneuron from which recordings were made during vomiting exhibited a membrane potential trajectory similar to that of phrenic motoneurons during retches and the first part of expulsion. However, unlike phrenic motoneurons, which repolarize during the second (i.e. abdominal) part of expulsion [18], this inspiratory interneuron depolarized again and fired a burst of action potentials. Such a pattern of membrane potential changes, the function of which remains unclear, reveals that some of the inspiratory interneurons of the C4-C5 spinal cord receive a different input than phrenic motoneurons. Thus, on the basis of their behaviour during both swallowing and vomiting, inspiratory C4-C5 interneurons are not simple relay neurons interposed between medullary respiratory neurons and phrenic motoneurons.

Expiratory interneurons The existence of expiratory-related units in the C4-C5 segments has been described earlier in both cats [4] and rabbits [34]. It is unclear how these cells are driven during expiration, since so far no excitatory projections from medullary expiratory neurons to the phrenic nucleus have been reported [25, 26]. One possible role for these expiratory interneurons is to mediate inhibition of phrenic motoneurons during normal ventilation and non-respiratory behaviour patterns. During normal ventilation, it was classically accepted that phrenic motoneurons receive a wave of IPSPs during the late part of expiration [5] originating from inhibitory bulbospinal augmenting expiratory neurons of the Bttzinger complex [26]. More recently, we described that the inhibition of phrenic motoneurons is

more complex since they also receive a wave of IPSPs during the early part of expiration [29]. It is most unlikely that this early expiratory inhibition originates from medullary expiratory neurons projecting onto phrenic motoneurons [29]. Hence, C4-C5 expiratory interneurons might be responsible for producing such an early expiratory inhibition of phrenic motoneurons. Similarly, during the Sc2 stage (abdominal) of coughing, phrenic motoneurons receive chloride-dependent IPSPs [18]. The expiratory interneurons described in the present study may mediate this inhibition. During vomiting, phrenic motoneurons receive both excitation and inhibition [18]. The inhibition occurs during both the inter-retches and the later part of the expulsion phase. Inhibitory augmenting expiratory bulbospinal neurons of the Bttzinger complex fire during the inter-retch period and could inhibit phrenic motoneurons during this phase [30]. In addition, C4-C5 expiratory interneurons may also contribute to this inhibition since the one expiratory interneuron studied during fictive vomiting depolarized throughout the inter-retch period. This interneuron also fired immediately after the end of the expulsion phase and thus could maintain, but not initiate, the inhibition of phrenic motoneurons that starts earlier during the expulsion phase.

MuItiphasic interneuron One interneuron, silent during normal ventilation, was classified as a multiphasic respiratory-related neuron since it depolarized during the end of both the expiratory and inspiratory phases. This uncommon diphasic membrane depolarization was apparently not related to the ventilator (i. e. lung inflation). To our knowledge, such a pattern of membrane potentials has not been described for lower cervical respiratory-related interneurons and motoneurons. However, multiphasic respiratory-related cells have been reported in thoracic segments in the vicinity of intercostal motoneurons [22]. At the present time, the respiratory function, if any, of multiphasic interneurons remains obscure. They might be involved in the control of spinal respiratory-related motoneurons during non-respiratory behaviour patterns. Indeed, the single multiphasic neuron described in the present report exhibited a large depolarization and fired intensely during the second stage of coughing, a period during which phrenic motoneurons receive potent waves of chloridedependent IPSPs [18]. However, this cell also exhibited an intense activation during each retch of vomiting, when phrenic motoneurons apparently do not receive any chloride-dependent inhibition [18]. Hence, these contradictory observations do not allow us to conclude whether excitatory or inhibitory connections exist between multiphasic interneurons and spinal respiratoryrelated motoneurons.

Additional functional considerations and conclusions In the present report, using an intracellular recording technique, we confirmed the existence of interneurons

319 exhibiting various types of respiratory membrane potentins, in close proximity to phrenic motoneurons. Furthermore, our results revealed for the first time that these respiratory-related interneurons are also active during non-respiratory behaviour patterns involving the thoraco-abdominal respiratory-related muscles (e.g. vomiring and coughing). However, as previously suggested by others [23], the precise role played by these interneutons in transmitting medullary drive to phrenic or spinal accessory motoneurons or possibly to thoraco-lumbar motoneurons needs to be further investigated. One way to elucidate this role would be to determine the spinal axonal projection of these interneurons and to examine, with spike-triggered averaging, the nature (excitatory or inhibitory) of focal synaptic potentials resulting from action potentials in individual interneurons. Using this experimental approach, significant advances have been made recently in the understanding of the function of thoracic respiratory interneurons [23]. These authors clearly demonstrated that thoracic interneurons with phasic activity most strongly modulated by the respiratory cycle provide inhibition to unidentified target neurons. However, extrapolation of these results to the lower cervical cord would be too speculative since the thoracic spinal segments, but not the lower cervical ones, contain both expiratory and inspiratory respiratory-related motoneurons. Finally, the respiratory neuronal circuitry of the lower cervical cord [4, 34, present results] presents striking similarities in terms of the patterns of activity with those more intensely studied in both the thoracic cord [22] and the brain stem [11]. Under normal physiological conditions, the spinal respiratory network is thought to be under the control of the brain stem circuitry; however, after high spinal cord transection and under special circumstances, this spinal network can generate a phasic motor output [1, 36]. Thus, one m a y consider the respiratory network of the lower cervical cord as a primitive oscillator, the function of which is no longer to generate a rhythm, but more likely to integrate various central (e.g. from the coughing, vomiting or swallowing neuronal medullary networks) and segmental (e.g. proprioceptive afferents) drives to elicit the most appropriate pattern of activity on phrenic or spinal accessory motoneurons critical for accomplishing complex vital behaviour patterns.

Acknowledgements. We are grateful to Prof. Armand L. Bianchi for helpful discussions and critical evaluation of the manuscript. This study was supported by grants from the Centre National de la Recherche Scientifique (CNRS, URA 205), France, the Direction des Recherches, Etudes et Techniques (DRET, 90/108), France, and the National Institute of Neurological Disorders and Stroke, USA (NS20585).

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