JOURNALOF NEUROPHYSIOLOGY Vol. 68, No. 6, December 1992. Print&

in U.S.A.

Membrane Potential Changes of Phrenic Motoneurons During Fictive Vomiting, Coughing, and Swallowing in the Decer&mk Cat LAURENT GRELOT, STEPHANE AND ARMAND L. BIANCHI

MILANO,

FEDERICO

PORTILLO,

ALAN

D. MILLER,

Dkpartement de Physiologie et Neurophysiologie, Centre National de la Recherche Scientzjique Unite’ de Recherche Associke 205, Facultk des Sciences et Techniques Saint Je’ro^me, 13397 Marseille ckdex 13, France

SUMMARY

AND

CONCLUSIONS

1. The patterns of membrane potential changes of phrenic motoneurons were compared during fictive vomiting, fictive coughing, and fictive swallowing in decerebrate, paralyzed cats. These fictive behaviors were identified by motor nerve discharge patterns similar to those recorded from the muscles of nonparalyzed animals. Phrenic motoneurons (~2 = 54) were identified by antidromic activation from the thoracic phrenic nerve. Intracellular recordings were obtained from 27 motoneurons during fictive vomiting, 40 during fictive coughing, and 27 during fictive swallowing. Sixteen motoneurons were recorded during both fictive coughing and fictive swallowing, eight during both fictive coughing and fictive vomiting, and two during both fictive vomiting and fictive swallowing. Seven motoneurons were studied during all three behaviors. 2. Fictive vomiting, typically evoked by electrical stimulation of abdominal vagal afferents, was characterized by a series of bursts of coactivation 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. During fictive vomiting, phrenic motoneurons depolarized abruptly, and the amplitude of depolarization was significantly greater than during control inspirations. They then repolarized slowly throughout the phrenic burst, rapidly repolarizing at the end of each phrenic burst during retching and reaching a level similar to that observed during expiration. During the expulsion phase, the pattern was initially the same. However, after the cessation of phrenic discharge, the membrane potential repolarized slowly until the end of the abdominal burst, exhibiting greater synaptic noise than during expiration. One phrenic motoneuron, presumably innervating the periesophageal region of the diaphragm, received a strong hyperpolarization just before the onset of the emetic episode and fired for shorter periods during fictive vomiting than did other phrenic motoneurons. Reversal of inhibitory postsynaptic potentials (IPSPs) by chloride ion and/ or current injections into six motoneurons revealed the presence of inhibition during the period between phrenic bursts during fictive vomiting and also during the final phase of expulsion when phrenic discharge ceased but abdominal discharge continued. 3. Fictive coughing, evoked by repetitive electrical stimulation of superior laryngeal nerve afferents, was characterized by a large phrenic discharge followed immediately by a large abdominal nerve discharge. During fictive coughing, phrenic motoneurons retained their ramplike depolarizations throughout phrenic discharge; however, the amplitude of depolarization was greater than during inspiration. During the subsequent abdominal nerve discharge, the phrenic membrane potential usually underwent an initial rapid, transient hyperpolarization followed by a gradual repolarization associated with increased synaptic noise. Reversal 2110

of IPSPs in one motoneuron revealed a strong inhibition during the period of abdominal nerve discharge. 4. The buccopharyngeal stage of fictive swallowing was characterized by brief bursts of activity in the pharyngeal vagus and hypoglossal nerves and was usually observed both after an episode of fictive vomiting and in response to repetitive electrical stimulation of superior laryngeal afferents. During fictive swallowing, phrenic motoneurons exhibited a brief depolarization that was half the amplitude of that observed during inspiration. No IPSPs were observed either before, during, or after the depolarization. 5. In conclusion, the descending central drives to phrenic motoneurons, as revealed by the patterns of membrane potential changes, differ markedly during fictive vomiting, coughing, and swallowing. In addition to excitation, inhibitory inputs are involved in shaping phrenic motoneuronal discharge during vomiting and coughing.

INTRODUCTION

The diaphragm and abdominal muscles are the main muscles of inspiration and expiration, respectively. However, these muscles are also important in numerous additional motor activities, including common expulsive behaviors such as micturition, defecation, parturition, coughing, and vomiting. Vomiting results in the expulsion of material from the stomach and upper part of the small intestine, whereas coughing servesto clear the upper airway. During vomiting, the diaphragm discharges in phase with the abdominal muscles (McCarthy and Borison 1974; Miller et al. 1987; Monges et al. 1978) whereas during coughing abdominal discharge occurs immediately after diaphragmatic discharge with little or no overlap between the two (Bolser 1991; Grelot and Milan0 199 1; Tomori and Widdicombe 1969) . The discharge patterns of individual phrenic motor axons during these two behaviors have recently been described (Milan0 et al. 1992). In the present seriesof experiments, we have monitored the somal membrane potential changesof phrenic motoneurons to determine the nature of the descending central drives to them during these two different expulsive behaviors. In addition, we include in this report an analysis of phrenic activity during swallowing, which usually occurs both after an episode of vomiting (Grelot et al. 1990a) and during laryngeal afferent stimulation used to induce coughing. Because stable recording from phrenic motoneurons requires the use of paralyzed animals, the occurrences of fic-

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PHRENIC

MOTONEURONS

IN

tive vomiting (Grelot et al. 1990a; Miller et al. 1987), fictive coughing (Bolser 199 1; Grelot and Milan0 199 1) and fictive swallowing (Grelot 1989; Grelot et al. 1989) were determined by recording the activity patterns of the motor nerves to the muscles that produce these behaviors in nonparalyzed animals. A preliminary report of some of this work has been presented (Grelot et al. 1990b).

NONRESPIRATORY

A

2111

BEHAVIORS

Fictive

vomiting

and

swallowing

Phr

Abd

Ll

PH-X METHODS

Animal preparation

I I

Experiments were conducted using 19 adult cats of either sex that were decerebrated, paralyzed, and artificially ventilated. The various experimental procedures have been previously described in detail (Bianchi and Grelot 1989; G&lot et al. 1989-199 1). Briefly, the animals were initially anesthetized with Saffan (Glaxovet, 1.5 ml/kg) and then maintained at a surgical level of anesthesia using a mixture of room air and l-2.5% halothane (Fluothan, Coopers). The trachea, femoral veins and artery, and urinary bladder were cannulated. Animals were decerebrated at the midcollicular level after ligation of the external carotid arteries. Anesthesia was then discontinued, and the animals were paralyzed using gallamine triethiodide (Flaxedil, Specia; 2 mg kg-’ . h-’ iv, supplemented as required) and artificially ventilated (end-tidal CO2 = 4-6%). Mean blood pressure was maintained >90 mmHg, using, if necessary, intravenous administration of metaraminol bitartrate (Aramine; Merck, Sharp & Dohme). Rectal temperature was maintained between 36 and 38°C using a servo-controlled heating pad.

r.XII

X Th

B

’ ’

I

-v

Fictive

I ,

St.

coughing

and swallowing

SC1

s /” I-I / /I I,

/” III I II

/” IIr II II

/” I,r I/ II

I ,, ,,, ,

J”

l

SLN

Recording and stimulation

IiZ

1. Patterns of various respiratory-related nerve activities during fictive vomiting (A ), coughing (B), and swallowing (A and B). A : fictive vomiting was elicited by intrathoracic vagal stimulation (X Th St.), which started before the onset of the traces in A and was terminated after the onset of fictive vomiting, as indicated by the horizontal line. Fictive vomiting was characterized by phrenic (Phr) and abdominal ( Abd Ll ) nerve coactivations. B: fictive coughing, which consists of a large phrenic burst (SC 1) followed by a strong abdominal nerve discharge ( Sc2), was elicited by superior laryngeal nerve stimulation ( SLN St.), the duration of which is indicated by the horizontal line. Note that fictive swallowing, characterized by bursts of activity in the respiratory-related oropharyngeal nerves (PH-X, 1.X11, and r.XII) and indicated by arrows and “s”, occurs both after fictive vomiting (A ) and in response to superior laryngeal nerve stimu“breakthroughs” observed during the laryngeal-inlation (B). Phrenic duced apnea coincide with the discharges of the oropharyngeal nerves ) . Traces in both panels, from top to bottom : Phr, C, or C6 phrenic ( --nerve; Abd Ll , L, abdominal nerve; Ph-X, pharyngeal branch of the vagus nerve; LXII and r.XII, left and right hypoglossal nerves. FIG.

Nerve activities were recorded from the intact C5 phrenic, L1 or L, abdominal, pharyngeal branch of the vagus, and hypoglossal nerves using bipolar silver electrodes. The same type of electrode was also used to stimulate the right thoracic phrenic nerve, both superior laryngeal nerves, and both lower thoracic (supradiaphragmatic) vagus nerves. We located the C,-C, phrenic motor pool using 5- to lo-MQ glass micropipettes filled with a saturated (3.0-3.5 M) NaCl solution while stimulating the phrenic nerve in the thorax just above the diaphragm to evoke an antidromic field potential. Once the phrenic nucleus was located, the recording electrode was switched to a lo- to 25-MQ pipette filled with saturated (3.0-3.5 M) KC1 for intracellular recording. Phrenic motoneurons were identified by antidromic activation from the phrenic nerve. Intracellular potentials were amplified through a high-impedance circuit incorporating capacity compensation, DC offset, and a bridge circuit for 1. Distribution of earZyand late PMs during various nonrespiratorybehaviors

TABLE

Nonrespiratory

PMs Early Late Total

St.

Behaviors

c

v

s

c+v

c+s

v+s

c+v+s

Total

5 4 9

3 7 10

2 0 2

0 8 8

4 12 16

0 2 2

4 3 7

18 36 54

PMs, phrenic motoneurons; C, coughing; V, vomiting; S, swallowing. C, V, and S indicate the number of PMs studied solely duringone nonrespiratory behavior. C+V, C+S, and V+S indicate the number of those studied during two behaviors. C+V+S indicates the number of PMs studied during all three behaviors.

current injection across the recording microelectrode (Transidyne 1600). For each phrenic motoneuron, the membrane potential was defined as the difference between intracellular and extracellular potentials, using as reference a single grounded silver-silver chloride electrode inserted into the neck muscles. All measurements were corrected, if necessary, by measuring the extracellular potential in the close vicinity of the recorded neuron after the microelectrode was withdrawn from the cell. To reveal the presence of inhibitory postsynaptic potentials ( IPSPs) that might hyperpolarize phrenic motoneurons during particular phases of different behaviors, we attempted to reverse postsynaptic hyperpolarizations to depolarizations by intracellular application of continuous negative currents (5-25 nA, 5-20 min). Nerve activities were amplified and filtered (bandpass 0.0 l- 10 kHz). Data were simultaneously displayed on a chart recorder (Gould TA 2000) and oscilloscopes and were stored on tape (Neuro-Corder DR-890).

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GRgLOT

2112

ET AL.

Fictive

Eupnea

vomiting

MP

-42 mV -57

XthSt.

-60,

1 set 2. Membrane potential (MP) changes of 2 phrenic motoneurons during normal ventilation (Eupnea) and vomiting. On the basis of their recruitment times in eupnea, the motoneuron depicted in A was classified as “early” and that in C as “late.” During the retching phase of fictive vomiting, both early (B) and late (D) motoneurons were strongly depolarized during each coactivation of phrenic and abdominal nerve activity (e.g., initial pairs of vertical hatched lines in B and 0) and were recruited at the start of the burst. During the expulsion phase, after the cessation of phrenic activity (right vertical hatched line), both motoneurons repolarized to their expiratory (resting) levels, either after a prolonged depolarization (B) or more rapidly (0). During the later part of the expulsion phase, the early motoneuron exhibited an atypical firing activity and the late motoneuron increased synaptic noise (II, -+ ) . Time calibration bars 1 s. Other abbreviations as in Fig. 1. FIG.

Fictive vomiting was usually produced by electrical stimulation of the thoracic vagus nerves (continuous trains of 0.8-ms pulses, 25-33 Hz, IO-50 V). In some cases, vomiting was elicited by administration of the emetic drugs lobeline sulfate (Sigma, l-2 mg/ kg) (Laffan and Borison 1957) and naloxone (Research Biochemicals, l-2 mg/ kg) (Costello and Borison 1977). Fictive coughing and swallowing were evoked by electrical stimulation of the superior laryngeal nerves [ 0. I- to 0.2-ms pulses, 2-5 V, 2-5 Hz (coughing) or lo-30 Hz (swallowing)].

Data analysis Chart recordings at high speed (typically 100 mm/s) were made for measurements of membrane potential changes during the different fictive motor activities. For each phrenic motoneuron, the

amplitude and duration of depolarization were measured during each behavior. Five successive respiratory cycles were used to obtain the mean inspiratory-related membrane depolarization. For the other motor activities, mean values were calculated using all depolarizations that were produced during repeated episodes of a given behavior. Statistical comparisons of the mean depolarizations during different behaviors were performed using a repeatedmeasure analysis of variance ( ANOVA / Student-Newman-Keuls test). Values are expressed as means ~frSD. RESULTS

Intracellular recordings were obtained from 54 phrenic motoneurons in 19 cats (range l-8 motoneurons per exper-

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PHRENIC MOTONEURONS

Eupnea

B

IN NONRESPIRATORY

Fictive

vomiting

BEHAVIORS

2113

C

Coactivations

MP -51 mV -63 I

Abd-Ll

I

-

PH-X

Phr -

2 set 3. Membrane potential (MP) changes of a phrenic motoneuron, presumably innervating the periesophageal region of the diaphragm, during normal ventilation (A ), fictive vomiting (B), and isolated coactivations of the phrenic and abdominal nerves. Asterisks in B and C: membrane hyperpolarizations of the motoneuron at the onset of fictive vomiting (B) and at the onset of what appeared to be the beginning of an episode of fictive vomiting that failed to develop ( C) . Arrows: weak depolarizations during the 2nd half of each phrenic burst during fictive vomiting. Abbreviations as in previous figures. FIG.

iment ). Resting membrane potentials ranged between -92 and -47 mV in different cells and averaged -69 t 7.9 (SD) mV. Twenty-seven motoneurons were studied during fictive vomiting, 40 during fictive coughing, and 27 during fictive swallowing. Sixteen motoneurons were recorded during both fictive coughing and fictive swallowing, eight during both fictive coughing and fictive vomiting, and two during both fictive vomiting and fictive swallowing. Seven motoneurons were studied during all three behaviors (Table 1). Most motoneurons were recorded in the C, portion of the phrenic nucleus. Fictive vomiting Fictive vomiting was characterized by a series of bursts of coactivation 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 ( Fig. 1A ) ( Miller et al. 1987 ) . During fictive vomiting, phrenic motoneurons always depolarized abruptly, and the amplitude of depolarization was typically greater than during eupnea (Fig. 2). They then repolarized slowly, their firing rates decreasing throughout the phrenic burst. Subsequently, the membrane potential repolarized at the end of each phrenic burst during retching and usually reached a level similar to that observed during expiration. During the expulsion phase, the pattern was initially the same. However, after the cessation of phrenic discharge, the membrane potential repolarized slowly until the end of the

abdominal burst and exhibited greater synaptic noise than during expiration (Fig. 2 D, -+ ) . The mean membrane depolarization during retching (19.5 t 7.5 mV, range 9.7-34.9 mV) was significantly larger (P < 0.05) than the inspiratory-related depolarization (12.5 t 3.9 mV, range 4.7-19.5 mV). In 20 motoneurons studied during both the retching and expulsion phases of fictive vomiting, there was a small but significant reduction (P < 0.05) in the amplitude of depolarization during expulsion ( 18.5 t 6.4 mV, range 8.2-29 mV) compared with that observed during retching (20.6 t 8.2 mV, range 8.6-34.4 mV). The duration of phrenic depolarization during retching (0.8 t 0.4 s, range 0.5-2.0 s) did not differ significantly (P > 0.2) from that observed during inspiration (0.8 t 0.2 s, range 0.4-l .8 s), but more than doubled during expulsion ( 1.8 t 0.8 s, range 0.3-3.4 s) (P < 0.05). One phrenic motoneuron (Fig. 3) was hyperpolarized ( 3 mV) at the onset of fictive vomiting (Fig. 3 B, asterisk) and at the onset of what appeared to be the beginning of an emetic episode that failed to develop (Fig. 3C, asterisks). Then its membrane was abruptly depolarized during approximately the first half of each phrenic burst during the fictive emetic episode. During the second half of these phrenic bursts, the membrane exhibited a weaker depolarization (Fig. 3 B, arrows) insufficient to reach firing threshold. The behavior of this motoneuron during fictive vomiting suggests that it might innervate the periesophageal region of the diaphragm (Miller et al. 1988; Monges et al. 1978). Chloride iontophoresis into six phrenic motoneurons revealed chloride-dependent IPSPs during the interretch pe-

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GReLOT

2114

ET AL.

VOMITING

EUPNEA A

CONTROL

B

Phr

C

E

REVERSAL

(1)

REVERSAL

(2)

D

F

G

FIG. 4. Membrane potential ( MP) changes of a phrenic motoneuron during normal ventilation (A, C, and E) and fictive vomiting (B, D, F, and G) before (A and B, CONTROL) and after intracellular chloride iontophoresis ( C-G, REVERSAL 1 and 2). Reversals of inhibitory postsynaptic potentials ( IPSPs) during eupnea and fictive vomiting were obtained after 10 ( C and D) and 20 (E-G) min of intracellular chloride iontophoresis (5-20 nA, negative current); current was off during recordings. Between retches, the reversed waves of IPSPs (D, F, and G) were similar in amplitude to those developed during control expirations (C and E). During the late part of the expulsion phase, beginning after the cessation of phrenic activity (vertical hatched lines in B, D, F, and G), the motoneuron received a strong wave of IPSPs reversed by 20 min of chloride injection (F and G, arrows). Dotted lines (F and G) indicate the motoneuronal membrane potential trajectory after 10 min of chloride injection (0). Time bars, 2 s. Abbreviations as in previous figures.

riods and during the later part of expulsion when the phrenic motoneurons stopped firing (Fig. 4, F and G, arrows).

Phrenic motoneurons were classified as early or late depending on whether the motoneuron started firing before or after 10% of the duration between the onset and peak of the

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PHRENIC MOTONEURONS

IN NONRESPIRATORY

BEHAVIORS

Eupnea

2115

Fictive

coughing

MP -48 mV -60

Abd-Ll Phr SLN St.

C

i

Fictive

Eupnea I

coughing

II

-67 mV -77

SLN St.

SLN St.

-

2 set

5. Membrane potential (MP) changes of 2 phrenic motoneurons during eupnea and fictive coughing induced by electrical stimulation of laryngeal afferents. One motoneuron (A and B) was classified as early, the other ( C-Z) as late. During fictive coughing (B, D, and E) both motoneurons were strongly depolarized during phrenic activity (SC, ) and then exhibited a membrane potential trajectory consisting of a single (B) or a series (D and E, o ) of depolarizations synchronized with the bursts of abdominal nerve activity (SC,). During the latter phase, both motoneurons exhibited increased synaptic noise (B, + ) . Asterisks (B and E) : weak firing activity during SC,. Time bars, 2 s. Abbreviations as in the previous figures. FIG.

phrenic During earlier similar

burst during eupnea (Hilaire and Monteau 1979). fictive vomiting, all late motoneurons began to fire in the phrenic burst (Fig. 2, C and D), a pattern to that previously described (Milan0 et al. 1992).

Fictive coughing

Fictive coughing was characterized by a large phrenic discharge [ Stage 1 of coughing ( SC1 )] followed immediately by a huge burst of abdominal nerve activity [Stage 2 of coughing (SC~)] consisting of either a single long-lasting discharge or a series of short-lasting bursts (Fig. 1B) (Bolser 199 1; Grelot and Milan0 199 1). During fictive coughing, all 40 phrenic motoneurons retained their ramplike depolarizations with increasing firing throughout Scl. The duration of the depolarization during SC1 ( 1.5 t 0.5 s, range

0.9-3.8 s) was approximately twice that observed during inspiration (0.8 t 0.3 s, range 0.5-1.8 s) (P < 0.05). The amplitude of the SC1 depolarization ( 14.4 t 5 8 mV, range 7.3-29.5 mV) was significantly greater (P < 0.05) than the inspiratory-related depolarization ( 12.4 t 4.6 mV, range 4.7-25.3 mV) (Fig. 5, D and E). In the 15 phrenic motoneurons activated during both coughing and vomiting, the amplitude of the Scl depolarization during fictive coughing was significantly less (P < 0.05) ( 12.6 t 4 mV, range 7.6-22 mV) than that observed during the retching phase of fictive vomiting ( 16.6 t 7.2 mV, range 8.6-34.9 mV). During Sc2, phrenic motoneurons remained depolarized. This Sc2 depolarization was in most ( n = 38 ) cases preceded by a rapid, transient hyperpolarization (Fig. 5, D and E). In nine phrenic motoneurons, the Sc2 depolarization was responsible for a weak firing activity consisting of

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GRfiLOT

2116

A

Eupnea

ET AL.

Fictive

coughing

IMP -51 mV -63

Abd-Ll

Phr

2 set SLN St. 6. Membrane potential (MP) changes of a phrenic motoneuron, presumably innervating the periesophageal region of the diaphragm, during eupnea (A) and fictive coughing (B) induced by electrical stimulation of laryngeal afferents. Asterisk in B: membrane hyperpolarization during the abdominal component of fictive coughing (SC,). The same motoneuron is depicted in Fig. 3. Abbreviations as in previous figures. FIG.

1- 14 action potentials (Fig. 5, B and E, asterisks). In 3 1 comparison with that during the normal Sc2 (Fig. 5, B and E), a higher discharge frequency (Fig. 7). motoneurons, Sc2 consisted of a single long-lasting depolarization ( 1.1 t 0.8 s, range 0.3-3.7 s; amplitude, 8 t 0.5 mV, range l-2 1 mV) with increased synaptic noise. In eight neu- Fictive swallowing rons, a synchronized series of depolarizations was also observed when abdominal nerve activity during Sc2 consisted The buccopharyngeal stage of fictive swallowing, elicited of 2-4 bursts (Fig. 5, D and E, 0). These brief (0.5 t 0.3 s, by electrical stimulation of the superior laryngeal nerves, range 0.2- 1.3 s) depolarizations kept the membrane depo- was characterized by a single brief burst of activity in the larized for a total of 1.5 t 0.6 s (range 0.7-2.6 s). pharyngeal vagus and hypoglossal nerves (Grelot 1989; Unlike all other motoneurons, which were depolarized Grelot et al. 1989, 1990a). During prolonged stimulation, rhythmic fictive swallowing was evident as repetitive bursts during Sc2, the one motoneuron that presumably innervated the periesophageal region exhibited a potent ( 5.7 of activity in these nerves (the buccopharyngeal stage of mV) hyperpolarization lasting 1.1 s (Fig. 6 B). swallowing) (Fig. 1 B). A similar pattern of swallowing was Intracellular chloride iontophoresis into one late motousually observed just after the end of the expulsion phase of neuron caused the reversal of chloride-dependent IPSPs fictive vomiting (Fig. 1A). During fictive swallowing, all during Sc2. This resulted in a huge depolarization and, in (n = 27) phrenic motoneurons, including the one presumDownloaded from www.physiology.org/journal/jn at Midwestern Univ Lib (132.174.254.157) on February 12, 2019.

PHRENIC

MOTONEURONS

Fictive Reversal

A

IN NONRESPIRATORY

2117

BEHAVIORS

coughing

(I)

Reversal

R

(2)

Phr

SLN St.

SLN St.

1 set

FIG. 7. Membrane potential (MP) changes of a phrenic motoneuron during fictive coughing after intracellular chloride iontophoresis for 10 (A ) and 12 (B) min ( 5-20 nA, negative current) ; current was on during recordings. Note that during the abdominal component of fictive coughing (SC,), the motoneuron exhibited large depolarizations (+ ) corresponding to reversals of waves of inhibitory postsynaptic potentials ( IPSPs) larger than those developed in late expiration ( *). Abbreviations as in previous figures.

able crural motoneuron, exhibited a short-lasting swallowing-related depolarization (0.5 t 0.1 s, range 0.2-0.8 s; amplitude, 6 t 3.4 mV, range 1. I- 17.7 ma/r), which was significantly smaller (P < 0.05) compared with the inspiratory-related depolarization (amplitude 12.9 t 4.4 mV, range 6.8-25.3 mV). Only 15 (55%) motoneurons exhibited weak firing activity, consisting typically of three (range 1- 11) action potentials during at least one of the depolarizations during rhythmic swallowing (Fig. 8). Fictive swallowing was produced in one motoneuron during periods of IPSP reversals during ventilation; however, no reversed Rhythmic

fictive

swallowing

IPSPs were observed either immediately before, during, or after phrenic depolarization during swallowing. Early and late motoneurons exhibited similar behavior during fictive swallowing. DISCUSSION

To better understand various descending central drives to phrenic motoneurons, we have examined the membrane potential changes of phrenic motoneurons during different nonrespiratory fictive motor activities requiring the activation of the diaphragm and compared these with those observed during respiration. Fictive vomiting

MP -50 mV -62

Abd L XII Phr FIG. 8. Membrane potential (MP) changes of a phrenic motoneuron during fictive rhythmic swallowing. Concomitantly with each burst of hypoglossal nerve (XII) activity, the phrenic motoneuron membrane was weakly depolarized. During some of these brief depolarizations, the motoneuron exhibited a low-frequency discharge consisting of l-3 action potentials. Abbreviations as in previous figures.

During rhythmic coactivations of phrenic and abdominal nerves, characteristic of vomiting (Miller et al. 1987), phrenic motoneurons depolarized abruptly. These rapid changes clearly differ from the ramplike depolarization observed during normal ventilation (see also Berger 1979). In addition, the amplitude of depolarization increased during fictive vomiting as compared with inspiration. The brain stem neurons that provide the powerful excitation to phrenic motoneurons during fictive vomiting remain unknown. Bulbospinal inspiratory neurons in the medullary dorsal and ventral respiratory groups (DRG, VRG), which transmit central respiratory drive potentials ( Sears 1964) to phrenic motoneurons during normal breathing (Monteau and Hilaire 199 1 ), are inhibited ( Bianchi and Grelot 1989) and usually silent during fictive vomiting (Miller et al. 1990). In contrast, - 50% of inspiratory propriospinal neurons in the upper cervical spinal cord ( C,--C3) are active

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2118

GRfiLOT

during phrenic discharge during fictive vomiting (Nonaka and Miller 199 1) . Upper cervical inspiratory neurons project mainly to the thoracic spinal cord and, to a lesser extent, to the C,-C6 gray matter (Lipski and Duffin 1986). Although no monosynaptic connections have been demonstrated between upper cervical inspiratory neurons and phrenic motoneurons ( Lipski and Duffin 1986 ), these propriospinal neurons may affect phrenic motoneurons through oligosynaptic pathways involving spinal interneurons. During the interretch phase, the membrane potentials of phrenic motoneurons repolarized to a level similar to that during normal expiration. This suggests that throughout the retching phase, phrenic motoneurons did not receive waves of IPSPs stronger than those observed during normal breathing. After intracellular chloride iontophoresis or negative current injection into phrenic motoneurons, IPSPs were reversed into waves of depolarization, clearly demonstrating the presence of IPSPs during the interretch phase. This inhibition is probably mediated by augmenting expiratory bulbospinal neurons located in the rostra1 part of the ventral respiratory group near the retrofacial nucleus (the so-called B&zinger complex). These cells monosynaptitally inhibit phrenic motoneurons during normal breathing (Merrill and Fedorko 1984) and discharge during the interretch periods during fictive vomiting (Miller and Nonaka 1990). Functionally, inhibition of phrenic motoneurons during the interretch period would prevent any additional motoneuronal activation by inspiratory DRG and VRG bulbospinal neurons, some of which fire near the end of the phrenic burst and during the early interretch period, likely because of an inhibitory rebound mechanism (Bianchi and Grelot 1989; Miller et al. 1990). Phrenic motoneurons also receive chloride-dependent IPSPs during the later part of the expulsion phase when phrenic discharge has ceased but abdominal discharge continues. Augmenting expiratory neurons located near the retrofacial nucleus (B&zinger complex) are the only bulbospinal neurons that have been shown to inhibit phrenic motoneurons (Merrill and Fedorko 1984). However, very few (2 of 19) bulbospinal Botzinger neurons fire when phrenic motoneurons are inhibited during the final portion of expulsion (Miller and Nonaka 1990), suggesting that additional unknown inputs play an important role in producing this inhibition. The region of the diaphragm that surrounds the esophagus relaxes during vomiting, especially during expulsion, presumably facilitating rostra1 movement of gastric contents (Miller et al. 1988, Monges et al. 1978). We observed one phrenic motoneuron whose discharge pattern during fictive vomiting indicated that it might innervate the periesophageal region. Unlike all other motoneurons, this motoneuron received a hyperpolarization at the onset of fictive vomiting, the origin of which remains unknown. In addition, this motoneuron was depolarized to firing levels only during the first part of the phrenic bursts during retching and expulsion. These observations demonstrate that the descending drives to phrenic motoneurons from the medullary circuitry that coordinates vomiting are complex, consisting of both excitatory and inhibitory inputs, and differ for moto-

ET AL.

neurons that innervate the periesophageal region versus the rest of the diaphragm. Fictive coughing Phrenic motoneurons receive a depolarization during the first (inspiratory) stage of coughing that exceeds the amplitude of that observed during inspiration. During the second (expiratory) stage of coughing, phrenic motoneurons are weakly depolarized and receive chloride-dependent IPSPs. The excitation during Scl could be mediated by bulbospinal DRG inspiratory neurons, which fire only during this period of coughing (Grelot et al. 199 1). VRG inspiratory neurons, with uncharacterized projections, are also active during coughing ( JakuS et al. 1985 ) . The cells responsible for the synaptic inhibition observed during Sc2 are unknown. No recordings have yet been made from inhibitory augmenting B&zinger expiratory bulbospinal neurons during coughing, although VRG expiratory neurons, with unknown axonal projections, are known to be active during cough (JakuS et al. 1985). The inhibition of phrenic motoneurons may also be mediated by propriospinal interneurons receiving collateral projections from the axons of medullary neurons providing excitation to abdominal motoneuronal pools. Fictive swallowing During the buccopharyngeal stage of swallowing, there is a marked reduction in phrenic discharge and, simultaneously, a strong activation of the glottal adductors (Doty and Bosma 1956; G&lot 1989). During the later, esophageal stage of swallowing, the periesophageal portion of the diaphragm relaxes, presumably facilitating transit of the swallowed bolus, while the activity of the remainder of the diaphragm returns to normal (Altschuler et al. 1987). The relaxation of the periesophageal diaphragm is abolished by vagotomy, which severs the afferent limb of a reflex in which extension of the esophagus results in periesophageal relaxation (Altschuler et al. 1985; Duron 1975). The periesophageal relaxation associated with swallowing and esophageal distension does not appear to result from a reduction in the activity of DRG and VRG inspiratory neurons (Altschuler et al. 1987). In contrast, the periesophageal relaxation associated with vomiting is part of the central motor program for vomiting and does not depend on a reflex arising from esophageal distension (Miller et al. 1988). In the present study, we observed that during the buccopharyngeal stage of fictive swallowing, phrenic motoneurons exhibited a weak depolarization that resulted in few action potentials. This excitation may have been mediated by DRG bulbospinal inspiratory neurons, many of which are active during fictive swallowing (Grelot et al. 199 1). No IPSPs were observed in association with this small phrenic depolarization. The mechanical significance of the weak diaphragmatic activation during the buccopharyngeal stage of swallowing remains to be clarified. In summary, different trajectories of phrenic motoneuronal membrane potentials were observed during respiration, vomiting, coughing, and swallowing. In addition to excitation, inhibitory inputs play an important role in shaping phrenic discharge during these behaviors.

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MOTONEURONS

IN NONRESPIRATORY

We thank Dr. Steve Iscoe for helpful editorial suggestions. This study was supported by grants from the Centre National de la Recherche Scientifique (URA 205)) France, the Direction des Recherches, Etudes et Techniques (DRET, 90/ 108), France, and the National Institute of Neurological and Communicative Disorders and Stroke (NS-20585). Present addresses: F. Portillo, Dpt. de Fisiologia (Catedra de Fisiologia) , Facultad de Medicina, Plaza Fragela S/N, Cadiz, Spain; A. D. Miller, The Rockefeller University, 1230 York Ave., New York, NY 1002 1. Address for reprint requests: L. Grelot, Departement de Physiologie et Neurophysiologie, Laboratoire de Neurobiologie et Neurophysiologie Fonctionnelles, Case 35 1, Faculte des Sciences Saint Jerome, 13397 Marseille cedex 13, France. Received 20 March 1992; accepted in final form 20 August 1992. REFERENCES ALTSCHULER,~. M., BOYLE, J.T., NIXON, T.E., PACK, A.I., ANDCOHEN, S. Simultaneous reflex inhibition of lower esophageal sphincter and crural diaphragm in cats. Am. J. Physiol. 249 (Gastrointest. Liver Physiol. 12): G586-G591, 1985. ALTSCHULER, S. M., DAVIES, R. O., AND PACK, A. I. Role ofmedullary inspiratory neurones in the control of the diaphragm during oesophageal stimulation in cats. J. Physiol. Land. 39 1: 289-298, 1987. BERGER,A. J. Phrenic motoneurons in the cat: subpopulations and nature of respiratory drive potentials. J. Neurophysiol. 42: 76-90, 1979. BIANCHI, A. L. AND GR~LOT, L. Converse motor output of inspiratory bulbospinal premotoneurones during vomiting. Neurosci. Lett. 104: 298-302,

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