Medullary axonal projetions of respiratory neurons of pontile pneumotaxic center

Respiration Physiology (1982)48, 357-373 Elsevier Biomedical Press

357

M E D U L L A R Y A X O N A L P R O J E C T I O N S OF R E S P I R A T O R Y N E U R O N S OF P O N T I L E P N E U M O T A X I C C E N T E R

ARMAND

L. B I A N C H I

and WALTER

M . St. J O H N ~

D~partement de Physiologic et Neurophysiologie, Facult~ des Sciences et Techniques St.-Jdr6me, rue Henri Poincar~, 13397 Marseille Cedex 13, France

Abstract. In decerebrate, cerebellectomized, vagotomized, paralyzed and ventilated cats, activities were recorded from the phrenic nerve and single respiratory neurons in the area of the nucleus parabrachialis medialis and K611ike~Fuse nucleus. Stimuli were delivered in the medulla and cervical spinal cord to elicit antidromic action potentials for these neurons and, hence, establish their axonal projections. Antidromic activation was obtained for 18 of 193 neurons following medullary stimulations. Following spinal stimulations, only two respiratory neurons exhibited some responses characteristic of antidromic activation. In the same pontile areas, a number of neurons with no respiratory-modulated or spontaneous activities were antidromically activated by medullary or spinal stimulations. Results are considered in the context of neuroanatomical studies which have established possible interconnections within the brainstem respiratory control system, and hypotheses for functions of the pontile pneumotaxic center in ventilatory control. Antidromic activation Cat Control of ventilation Medulla

Pontobulbar respiratory neurons Pneumotaxic center Spinal cord

In 1923, L u m s d e n d e m o n s t r a t e d t h a t t h e r e s p i r a t o r y p a t t e r n w a s a l t e r e d f r o m e u p n e a to a p n e u s i s f o l l o w i n g a m i d - p o n t i l e b r a i n s t e m t r a n s e c t i o n c a u d a l to t h e ' p n e u m o t a x i c c e n t e r ' . B a s e d p r i m a r i l y u p o n t h e w o r k o f B e r t r a n d a n d H u g e l i n (1971), the p n e u m o t a x i c c e n t e r is n o w a c c e p t e d as s y n o n y m o u s w i t h the n u c l e u s p a r a b r a c h i a l i s m e d i a l i s a n d K 6 1 1 i k e ~ F u s e n u c l e u s . S i n c e lesions in t h e s e n u c l e i p r o d u c e a p n e u s i s in d e c e r e b r a t e , v a g o t o m i z e d a n i m a l s (e.g. St. J o h n , 1979), it is e v i d e n t t h a t respir a t o r y n e u r o n s o f t h e p n e u m o t a x i c c e n t e r m u s t h a v e a x o n a l p r o j e c t i o n s to m o r e c a u d a l r e s p i r a t o r y n e u r o n s . T w o lines o f e v i d e n c e i m p l y t h a t t h e s e p r o j e c t i o n s m a y AcceptedJbr publication 3 March 1982 I On leave from Department of Physiology, Dartmouth Medical School, Hanover, N.H., U.S.A.

0034-5687/82/0000-0000/$2.75 © Elsevier Biomedical Press

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impinge upon respiratory neurons of the medulla or cervical spinal cord by a pathway having few, or only one, synapse. First, electrical stimulation of the pneumotaxic center induces changes in activities of medullary respiratory neurons and the phrenic nerve within a brief latency, suggesting a ~paucisynaptic' pathway (Cohen, 1970; Bertrand and Hugelin, 1971; Cohen, 1971). Second, following injections of horseradish peroxidase into medullary regions which include the dorsal and ventral respiratory nuclei or into the cervical spinal cord, labelled cells were found in both the nucleus parabrachialis medialis and K611ike~Fuse nucleus (Kuypers and Maisky, 1975; Denavit-Saubi6 and Riche, 1977; Bystrzycka, 1980; Takeuchi et al., 1980; Kalia, 1981). However, there is no evidence if the neurons which were labelled were respiratory-modulated or non-respiratory since the nucleus parabrachialis and K611ike~Fuse nucleus contain both of these types of neurons (see Bertrand et al., 1973; table 1). Using the technique ofantidromic invasion, we have investigated if rostral pontile respiratory-modulated neurons do have axonal projections to the medulla or spinal cord. Our results demonstrate that there is a limited number of pontobulbar respiratory neurons. The existence of pontospinal respiratory neurons could only be tentatively established. Finally, we found that relatively large numbers of rostral pontile neurons, which discharged with no discernible respiratory modulation and whose activities were monitored at loci close to respiratory-modulated units, did exhibit antidromic action potentials following stimulations in the medulla or spinal cord.

.Methods

I. G E N E R A L

Eighteen adult cats of either sex were used. The surgical preparation of these animals was similar to that previously described (St. John, 1979; Bianchi and St. John, 1981). Thus, under anesthesia with Alfatdsine and halothane, the following procedures were performed: (1) cannulations of the trachea, both femoral veins and a femoral artery, (2) midcollicular decerebration, (3) cerebellectomy, (4) laminectomy from C~ to C7, (5) bilateral midcervical vagotomy, (6) isolation and section of C5 and/or C6 phrenic nerve rootlets unilaterally. Exposed surfaces of the brainstem and spinal cord were covered with warm paraffin oil. Following these procedures, halothane anesthesia was discontinued, the animals were paralyzed with gallamine triethiodide and artificial ventilation was administered by a respirator. A bilateral pneumothorax was then performed and a positive endexpiratory pressure of approximately 2 cm H20 was applied. Recordings of pontile neuronal activities did not begin until at least one hour after the discontinuation of anesthesia. Rectal temperature was maintained at 36 39°C by heating pads.

PONTOBULBAR R E S P I R A T O R Y N E U R O N S

359

II. M O N I T O R I N G OF R E S P I R A T O R Y A N D C A R D I O V A S C U L A R VARIABLES

Efferent phrenic nerve activity was recorded by a bipolar electrode which was immersed in paraffin oil. This activity was amplified, filtered (5.0-1.0 x 104 Hz) and viewed on an oscilloscope. The end-tidal fractional concentration of CO2 was continuously monitored and was maintained at 0.055-0.075 by delivery of a hyperoxic-hypercapnic gas mixture to the respirator. This hypercapnia was used to increase the number of respiratory-modulated neurons which would be active (Cohen, 1968). Arterial blood pressure was monitored by attaching the femoral artery cannula to a pressure transducer amplifier system. Mean arterial pressure was at least 80 m m Hg during all experiments and solutions of dextran and/or metaraminol bitartrate were infused intravenously when necessary.

111. SITES OF S T I M U L A T I O N .

In order to identify the axonal projections of pontile respiratory neurons, arrays of concentric bipolar electrodes (Rhodes Medical Instruments, SNE-100, contact diameter, center: 0.1 mm, outer: 0.25 mm, 50 k• impedance measured in saline with direct current) were inserted into the medulla and the cervical spinal cord. For the spinal cord, ten electrodes were positioned bilaterally over a longitudinal distance of 20 ram, extending from approximately C3 to C6. The most rostral electrodes were 1.0 m m lateral to the midline. Every 5.00 mm caudal, electrodes were placed 0.75 m m more lateral. The vertical position of this array was initially close to the ventral surface but, in various trials, was elevated to more dorsal levels. In the medulla, seven electrodes were aligned in two rows approximately 0 and 2 m m rostral to the obex. The latter row of three electrodes were 0.0 and 3.0 m m lateral to the midline on both sides. At the obex, electrodes were placed 2.0 and 4.0 m m lateral to the midline bilaterally. As required, the array was moved from just below the brainstem surface to the base of the skull. In addition to these arrays, a single concentric bipolar electrode of the same type was used to explore other loci in the rostral medulla and pons. Stimuli to all electrodes were delivered via a W-P Instruments 830 series stimulator with isolation units. Insertion of the electrodes described above caused a transient increase in respiratory frequency and, for the spinal array, a transient decrease in arterial pressure in some animals. For a few cats, these changes were irreversible and data were not collected.

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IV. MONITORING OF RESPIRATORY UNIT ACTIVITIES Single units, whose patterns o f activity were linked to the phasic periods o f phrenic nerve activity were sought in rostral pontile regions which included both the nucleus parabrachialis medialis and K611iker-Fuse nucleus (Bertrand and Hugelin, 1971). Thus, explorations extended from the caudal edge o f the inferior colliculus to 3.0 m m caudal, from 4.0 to 5.5 m m lateral to the midline and from 0.0 to 6.0 m m below the brainstem surface. In this same area, activities were also recorded from nonrespiratory neurons, whose patterns of discharge bore no discernible relationship to the periods of phrenic nerve activity. Unit activities were monitored with tungsten microelectrodes (impedance = 9-12 Mff~ at l k H z ; F. Haer Co.) and amplified by a Transidyne General Corp. Model 1600 amplifier. A second stage of amplification and filtering (30-1.0 x 104 Hz) was also used. Unit activities were viewed on an oscilloscope.

V. EXPERIMENTAL PROTOCOL U p o n obtaining the activity of a single pontile respiratory or non-respiratory neuron, stimuli, at approximately 30 V, 1Hz and 0.1 msec duration, were sequentially delivered through the electrodes described in section III. The aim was to elicit an action potential having the same form as that for the spontaneous discharge of the unit. When such an action potential was elicited, the voltage of stimulation was changed to a level just above threshold (typically less than 10 V), and stimuli were delivered to ascertain if the action potential resulted from antidromic activation. Criteria for antidromic activation have been described in detail previously (Bianchi, 1971 ; Bianchi and St. John, 1981). The two most imortant of these were: (1) each stimulus, even those delivered at high frequency (100 Hz), elicited a single action potential; (2) 'collision' of the orthodromic action potential with that resulting from stimulation. For a few neuronal activities, the 'critical delay' for antidromic activation was determined as the time after a spontaneous action potential at which approximately 500/0 of the stimuli elicited antidromic action potentials. Activities of neurons and the phrenic nerve were recorded on photographic film and/or on magnetic tape. The distance between the stimulating and the recording electrodes was measured and divided by the latency of the antidromic response to obtain the axonal conduction velocity.

vI. VERIFICATION OF ELECTRODE PLACEMENTS; STATISTICAL

Evaluations of data. The positions of the recording and stimulating electrodes were defined with reference to structures on the surface of the brainstem or spinal

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PONTOBULBAR RESPIRATORY NEURONS

cord. Some sites of recording and stimulation were marked by electrocoagulation and verified from serial frozen sections. Data are expressed as mean values _+ standard error. Statistical evaluations were performed by the non-parametric Mann Whitney test (two-sided). By significantly different is meant that the probability was less than 0.05 that the means of the two populations were equal.

Results

Activities of 193 respiratory neurons were recorded close to or within the nucleus parabrachialis medialis and K611iker-Fuse nucleus. The largest proportion of these had phase-spanning patterns of activity (table 1). These neurons either discharged phasically across portions of both the periods of phrenic nerve activity and inactivity (43 neurons), or discharged tonically with an increase in the discharge frequency during one portion of the respiratory cycle (91 neurons) (fig. 1). Lesser numbers of neuronal activities, which were limited to the phasic periods of activity of the phrenic nerve (inspiratory neurons) or to the periods between the phrenic bursts (expiratory neurons), were also recorded (table 1 ; fig. 2).

1. P O N T O B U L B A R R E S P I R A T O R Y N E U R O N S

Eighteen of the respiratory neurons exhibited antidromic action potentials following stimulations in the medulla and, hence, were designated as pontobulbar. The collision of the orthodromic action potential with that elicited by stimulation provides evidence of this antidromic activation (fig. 3). Except for two possible pontospinal neurons, we were not able to activate antidromically any of the remaining respiratory neurons even by stimulations several millimeters caudal to the recording microelectrode. As reported in table 1, pontobulbar neurons constituted approximately I 0 ~ of the phase-spanning population. Four of these had phasic discharge patterns; the TABLE 1 Types of pontile neuronal activities recorded. PB = p o n t o b u l b a r ; PS = pontospinal; N A A = not antidromically activated; N = number of units; ? = doubt as to antidromic activation. Respiratory

(N =

193)

lnspiratory

Expiratory

Phase-spanning

(N = 42)

(N = tT)

(N = 134)

PB 3

PS 1?

NAA 38

PB 1

PS 1?

NAA 15

PB 14

PS 0

NAA 120

Non-respiratory (N = 17)

Non-active (N = 37)

PB 11

PB 17

PS 6

PS 19

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A.L. BIANCH1 AND W.M. St. JOHN

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Fig. 1. Examples of discharge patterns of rostral pontile respiratory neurons having phase-spanning patterns of activity. Note that neurons (Ne) A, C, D and E exhibited augmentations of discharge during the period of the phrenic burst (Ph) whereas unit B increased its discharge frequency during the period between bursts. Drawing shows loci. 12 mm rostral to the obex (+ 12), at which activities were recorded. BC = brachium conjunctivum; BP = brachium pontis; FTG = gigantocellular tegmental field; P = pyramidal tract. Note: in all figures, upward deflections indicate negative potentials.

rest d i s c h a r g e d t o n i c a l l y (fig. 3). P o n t o b u l b a r units f o r m e d lesser p r o p o r t i o n s o f the p h a s i c i n s p i r a t o r y a n d e x p i r a t o r y p o p u l a t i o n s (table 1). M e d u l l a r y loci in which stimuli elicited a n t i d r o m i c a c t i o n p o t e n t i a l s are shown in figs. 4 a n d 5. S o m e o f these loci c o i n c i d e d with the d o r s a l a n d ventral r e s p i r a t o r y nuclei (Bianchi, 1971). F o r different neurons, the effective sites o f s t i m u l a t i o n were either ipsilateral or c o n t r a l a t e r a l to the r e c o r d i n g site (fig. 5). H o w e v e r , a n t i d r o m i c a c t i o n p o t e n t i a l s were never elicited f r o m b o t h ipsilateral a n d c o n t r a l a t e r a l stimulations. T h e distance between the s t i m u l a t i n g a n d r e c o r d i n g electrodes a n d the latencies o f the a n t i d r o m i c r e s p o n s e s (range = 1.8 10 msec; m e a n = 6.42 +_ 0.57 msec) yielded a x o n a l c o n d u c t i o n velocities which r a n g e d from 1.0 to 10 m/sec (mean = 2.32 + 0.38 m/sec). F o r three o f the p o n t o b u l b a r neurons, h a v i n g relatively slow a x o n a l c o n d u c t i o n velocities (i.e. less t h a n 2.5 m/sec), there were seemingly r a n d o m f l u c t u a t i o n s o f 0.9 1.0 msec in the latencies o f the a n t i d r o m i c responses. The latencies o f a n t i d r o m i c

363

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action potentials for the other pontobulbar respiratory neurons varied by less than 0.5 msec.

II. P O N T O S P 1 N A L R E S P I R A T O R Y N E U R O N S

Activities of only one phasic inspiratory and one phasic expiratory neuron were recorded which might be tentatively considered as having exhibited antidromic action potentials following spinal stimulations (table 1). For both neurons, the

364

A . L . B I A N C H i A N D W. M. St. J O H N

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365

PONTOBULBAR RESPIRATORY NEURONS

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Fig. 4. Loci of recordings of activities of pontobulbar and pontospinal neurons and of medullary stimulations for pontobulbar neurons. The sites at which activities were recorded from respiratory pontobulbar neurons ( 0 ) , non-respiratory and non-active pontobulbar neurons (©) and non-respiratory and non-active pontospinal neurons (A) have been projected on the closest sagittal section to those illustrated. 'Lateral' is the distance from the mid-sagittal plane. The area within which activities were recorded from not-antidromically activated respiratory neurons is shown in outline. Sites of stimulation are designated by x for respiratory neurons and ® for non-respiratory and non-active neurons. Note that the number of loci for stimulation is less than that for recordings due to the elicitation of antidromic action potentials for a number of neurons from a single medullary site. Some of the sites of stimulation and recording were confirmed by histology. AMB = nucleus ambiguus; BC = brachium conjunctivum, BP = brachium pontis; C N F = cuneiform nucleus; FTL = lateral tegmental field; IC = inferior colliculus; IOP = principal nucleus of inferior olive: K F = Kolliker-Fuse nucleus; LLD = dorsal nucleus of lateral lemniscus; NPB = nucleus parabrachialis; P G R = pontine gray, rostroventral division; R F N = retrofacial nucleus; S = solitary tract; TB = trapezoid body; VIN = inferior vestibular nucleus; VLV = lateral vestibular nucleus; 5M = motor trigeminal nucleus; 5SM = alaminar spinal trigeminal nucleus; 5ST = spinal trigeminal tract. (Redrawn from Berman, 1968.)

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A. L, B I A N C H I A N D W. M. St. J O H N

m" Fig. 5, Ipsilateral or contralateral projections of pontobulba] ~ respiratory neurons. Sites of recording (Q) and stimulation ( + ) are projected on a dorsal view of the caudal brainstem. Note that the number of sites illustrated is less than the total number of activities which were recorded (table 1). This reflects recordings in a single vertical track and/or elicitation of antidromic action potentials from a single medullary locus.

effective site of stimulation was lpsilateral to the recording site. While the action potentials which were elicited had the same forms as for the spontaneous discharges of the units, the activity of the expiratory neuron could not be recor~led for a sufficient period to observe a collision. For the inspiratory neuron, only a smaller action potential, and not a true collision, was recorded (fig. 6A). These two neurons cannot, therefore, be definitively considered as pontospinal. Despite the delivery of stimuli in numerous spinal loci, no other respiratory neurons exhibited responses characteristic of antidromic activation.

III. P O N T O B U L B A R A N D P O N T O S P I N A L N O N - R E S P I R A T O R Y N E U R O N S

Collisions of orthodromic action potentials of non-respiratory neurons with action potentials elicited by medullary or spinal stimulations are illustrated in figs. 6 and 7. For the non-respiratory pontobulbar neurons, the effective sites of stimulation overlapped with those for the respiratory pontobulbar neurons (fig. 4). The axonal conduction velocities (14.6 _+ 3.75 m/sec) were significantly higher than those of the respiratory pontobulbar neurons (2.32 _+ 0.38 m/sec). For the non-respiratory pontospinal neurons, loci for eliciting antidromic action potentials were ipsilateral, in the ventral portion of the spinal cord. The axonal conduction velocities of these pontospinal neurons were 34.8 _+ 8.92 m/sec. It must be emphasized that, whereas the limited number of respiratory ponto-

PONTOBULBAR RESPIRATORY NEURONS

A

367

/

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8

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lsec Fig. 6. Examples of activities of pontospinal neurons. Bar below upper tracing indicates 5.0 msec; bar below lower pair of tracings indicates 1.0 sec. Times of stimulation in the ipsilateral spinal cord are designated by dots. Recording sites are shown in the drawing. Tracings in panel A are of a respiratory neuronal activity for which no 'true' collision (arrow) was obtained (see text). Records of panel B are for the activity of a non-respiratory neuron. Panels C and D illustrate the activations of previously silent neurons by spinal stimulations. In C, the action potentials which were elicited were different from those of the spontaneously active neuron and, hence, there was no collision. See figs. 1 and 3 for further explanation.

bulbar and pontospinal neurons were the end result of stimulations at many medullary and spinal loci, a systematic search for the axons of the non-respiratory neurons was not performed. Thus, it is probable that the number of non-respiratory pontobulbar and pontospinal neurons represents only a small proportion of the population.

IV. PONTOBULBAR AND PONTOSPINAL NEURONS WITHOUT SPONTANEOUS ACTIVITY During

the recordings of activities of pontile neurons,

we occasionally observed

that stimulations elicited action potentials which were different from those of the spontaneously

a c t i v e n e u r o n s . T h e s e r e s p o n s e s (figs. 6 a n d 7) w e r e n o t e v o k e d field

A. L. BLANCH1 A N D W. M. St. J O H N

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PONTOBULBAR RESPIRATORY NEURONS

369

potentials since they were not altered by increases in voltage or changes in the durations of stimuli. Moreover, since these responses followed high frequency stimulation with a constant latency, we concluded that these action potentials resulted from antidromic, rather than synaptic, activations (see Bianchi and St. John, 1981). As shown in fig. 4, sites at which these antidromic action potentials were recorded were very close to those at which pontile respiratory unit activities were monitored. Critical sites of stimulations were similar for the non-active, the non-respiratory, and the respiratory neurons. The axonal conduction velocities of the non-active pontobulbar units (7.52 +_ 1.31 m/sec) were not significantly different from those of the non-respiratory pontobulbar units (14.6 _+ 3.75 m/sec) but were significantly higher than those of the respiratory pontobulbar neurons (2.32 _+ 0.38 m/sec). Axonal conduction velocities of the non-active (54.8 + 3.58 m/sec) and the non-respiratory pontospinal units (34.8 _+ 8.92 m/sec) were not significantly different.

Discussion

The major conclusion of this study is that there are direct axonal projections from rostral pontile respiratory neurons to loci coinciding with the dorsal and ventral medullary respiratory nuclei. While we have no evidence concerning the terminals of these axons, it is probable that these must be either directly upon respiratory neurons of these two nuclei or upon interneurons which are both anatomically and synaptically close to the medullary respiratory neurons. The primary evidence supporting this conclusion is that, following stimulations in the nucleus parabrachialis medialis, changes in the discharge frequencies of medullary respiratory neurons occur with mean latencies of 4.5-9.4 msec (Cohen, 1970; Bertrand and Hugelin, 1971; Bassal et al., 1981). If it is assumed that these changes partially reflect influences from pontobulbar respiratory neurons, then the mean latency of 6.42 msec for antidromic activation of these neurons implies that there cannot be numerous synapses interposed between most pontobulbar and medullary respiratory neurons. However, even if only one synapse is present, it is difficult to conclude that all respiratory responses from stimulations of the pneumotaxic center are modulated via these pontobulbar neurons. In the studies of Bertrand and Hugelin (1971) and Cohen (1971), considered above, it was reported that latencies for inhibitions of medullary respiratory neuronal and phrenic activities could be as short as 1.8 msec and & 6 msec, respectively, following stimulations of the pneumotaxic center. The population of pontobulbar respiratory neurons could not mediate these inhibitory responses since only two of these neurons had antidromic latencies of less than 4 msec. Thus, the question arises as to the neural mechanisms responsible for these inhibitions. We believe that there are four possible mechanisms. The first possibility is that these inhibitory responses are served by respiratory neurons which synapse immediately caudal to the nucleus parabrachialis medialis

370

A.L. BIANCHI A N D W. M. St. J O H N

and/or K611iker Fuse nucleus. Second, the non-respiratory or non-active pontobulbar and pontospinal neurons in the area of the pneumotaxic center may convey these inhibitions. The relatively fast axonal conduction velocities of these neurons, noted in Results, support this possible role. A third, related possibility is that these inhibitions are due to the stimulation of axons of neurons which are not generally considered as part of the respiratory control system. This is supported by the finding of Bassal and Bianchi (1981) that an inhibition of phrenic discharge, at a latency approximating that following stimulation in the nucleus parabrachialis medialis, is obtained following stimulations of the superior colliculus, the pontile reticular formation or the red nucleus. The final possibility is that a number of the non-respiratory or non-active pontobulbar and/or pontospinal neurons can acquire a respiratory-modulated pattern of activity in a different experimental preparation from the decerebrate animal. A capacity for neurons within the nucleus parabrachialis medialis to change their discharge patterns from non-respiratory to respiratorymodulated with a change in the experimental preparation has been demonstrated by Feldman et al. (1976). Although inherent to the above considerations of the short latency inhibitory responses, it is important to emphasize that both pontile respiratory and nonrespiratory neurons had axonal projections to similar medullary loci. Moreover, only for the latter group could spinal axons be clearly established. These findings demonstrate, as did our previous results (Bianchi and St. John, 1981), that neuroanatomical studies, such as those cited in the introduction, cannot establish connections among components of the brainstem respiratory control system. Our inability to activate antidromically a greater number of rostral pontile neurons could be due to the impossibility of stimulating all loci. Also, our arrays of stimulating electrodes might have sectioned the axons of some neurons. We believe, however, that the latter cannot be considered as a significant factor for, in a previous study in which a similar spinal array was used, 7 0 ~ of the inspiratory neurons of the dorsal medullary respiratory nucleus were antidromically activated by spinal stimulations (Bianchi and St. John, 1981). Thus, we must conclude that the great majority of rostral pontile respiratory neurons have axons which either terminate immediately caudal to the nucleus parabrachialis medialis and K611iker-Fuse nucleus, project to more rostral loci, or end upon other respiratory neurons within the pneumotaxic center. There is some evidence in support of each of these possibilities. In 1977, Hugelin described a 'pathway', extending from the rostral pons to the level of the facial nucleus, in which lesions resulted in apneusis in vagotomized cats. Although we stimulated areas which included this pathway, we never observed antidromic activations. We therefore conclude that this pathway contains one or more synapses with the first synapse occurring immediately caudal to the nucleus parabrachialis medialis and/or K611iker-Fuse nucleus. The concept that neurons of the pneumotaxic center may have axonal projections to more rostral structures is supported by the observation that, following injections of horseradish peroxidase into the thalamus and hypothalamus, labelled cells were

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found in the same pontile areas in which we recorded respiratory neuronal activities (McBride and Sutin, 1976). However, there is, of course, no indication if the cells which were labelled were respiratory neurons. The possibility that the axons of many respiratory neurons remain within the nucleus parabrachialis medialis and K611ike>Fuse nucleus has been proposed by Bertrand et al. (1974). While we have no evidence in support of this proposal, the high percentage of not-antidromically activated respiratory neurons implies that these interconnections are possible. The proposal of Bertrand et al. (1974) for interconnections among respiratory neurons of the pneumotaxic center was based, in part, upon the high density of such neuronal activities. Indeed, these investigators report that the proportion of respiratory neuronal activities in the nucleus parabrachialis medialis and K611iker-Fuse nucleus is equal to or above that within the areas of the dorsal and ventral medullary respiratory nuclei (Bertrand et al., 1973; Vibert el al., 1976). Compared to our previous studies using decerebrate animals (St. John and Wang, 1977 ; Kirsten et al., 1978; St. John, 1981; Bianchi and St. John, 1981), we did not find a concentration of respiratory-modulated neuronal activities in the rostral pons which was as great as that in the medulla. This quantitative difference could be due to numerous factors including, most importantly, different procedures of microelectrode recording and different experimental preparations. A final point remains to be discussed. This concerns our observations that some of the pontobulbar respiratory neurons exhibited variations as great as 1.0 msec in the latencies for antidromic activations. Such variations differed from those previously reported for bulbospinal, laryngeal and bulbopontile respiratory neurons (Bianchi, 1971 ; Merrill, 1974; Barillot and Bianchi, 1979; Lipski et al., 1979; Bianchi and St. John, 1981) by being of greater magnitude and not related to the periods of the respiratory cycle. While this observation is novel for the respiratory control system, similar variations have been noted for neurons in other portions of the mammalian central nervous system. These variations depend upon the previous history of activity of the neuron (see Swadlow and Waxman, 1975, for review). In s u m m a r y , results of the present and our previous study (Bianchi and St. John, 1981) confirm a long-standing hypothesis for the organization of the respiratory control system, namely, that there is a loop interconnecting the 'medullary respiratory centers' and the pontile pneumotaxic center (Pitts et al., 1939). Thus, we have demonstrated that some respiratory neurons in both the dorsal and ventral medullary respiratory nuclei have axons which project to regions approximating the nucleus parabrachialis medialis and K611iker-Fuse nucleus (Bianchi and St. John, 1981). Since the discharge patterns of these bulbopontile respiratory neurons, taken in total, cover all of the respiratory cycle, we have proposed that neurons within the pneumotaxic center receive information from medullary neurons throughout all of inspiration and expiration. In this same context, the discharge patterns of the pontobulbar respiratory neurons allow for the possibility that medullary respiratory neurons receive information from respiratory neurons of the pneumotaxic center

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t h r o u g h o u t t h e e n t i r e r e s p i r a t o r y cycle. H e n c e , o u r s t u d i e s p r o v i d e a f r a m e w o r k to e x p l a i n the c o o r d i n a t i o n o f r e s p i r a t o r y - r e l a t e d n e u r o n a l a c t i v i t i e s o f the m e d u l l a a n d ports. A s has b e e n e v i d e n t since the w o r k o f L u m s d e n in 1923, such a c o o r d i n a t i o n is n e c e s s a r y for t h e m a n i f e s t a t i o n o f the e u p n e i c r e s p i r a t o r y cycle.

Acknowledgments This work was supported by grants D,G.R.S.T (France). W.M.

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a n d f r o m t h e P e r k i n s F u n d o f the A m e r i c a l P h y s i o l o g i c a l S o c i e t y . T h e a u t h o r s e x p r e s s t h e i r a p p r e c i a t i o n to M r . J . P . w i t h e l e c t r o n i c e q u i p m e n t a n d to M r s . J. R o m a n

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