ACTA NEUHOBIOL. EXP. 1981, 41: 237-242

Short communication

RESPIRATORY ACTIVITY GENERATED BY A SPLIT BRAINSTEM PREPARATION OF THE RABBIT

H. GROMYSZ and W. A. KARCZEWSKI

Department of Neurophysiology, Medical Research Centre, Polish Academy of Sciences, Dworkowa 3, 00-784 Warsaw, Poland

Key words: control of breathing, split-brainstem, symmetry of respiratory "cent- res"

Abstract. Mid-sagittal incisions of the brainstem were performed in anaesthetized, vagotomised, paralysed and artificially ventilated rabbits. The activity of both phrenic nerves, blood pressure and end-tidal COzO/o were continuously rhonitored. The results fell into two groups: in one of them a relatively small separation of both halves of the medulla extending from the obex to at least 4 mm rostrally elicited asynchro- nous firing in both phrenic nerves ("split respiratory centre"). In the other group, in which the incisions were placed either more caudally or more rostrally, or when small strands of the nervous tissue were left to provide connections between both sides of the brainstem, this. phenomenon did not appear. It is concluded that there are two symme- trical respiratory networks in both halves of. the brainstem and that their synchronous firing depends upon intact connections extending from the obex to the caudal end of nucleus of VII nerve.

It is not clear whether the brainstem neurones that are responsible for respiratory rhythmogenesis form an integral, extensively intercon- nected population, or rather two symmetrical networks active in both halves of the ponto-medullary complex and synchronized by a more specialized pathway (or pathways). Also the problem whether, to what extent, and at what level the bulbo-spinal axons (i.e., the fibres descen- ding from the proper respiratory neurones to phrenic and intercostal motoneurones) are crossing has been the matter of controversy. Salmo-

iraghi and Burns (7) have shown in cats that respiratory movements are irreversibly arrested by a mid-sagittal incision extending from 2 mm caudal to 2 mm rostra1 to the obex. On the other hand, Kahn and Wang (3) demonstrated that although a similar incision does affect the breathing pattern, it does not abolish rhythmic respiration. More re- eently, Sears (8) and Bainton et al. (1) concluded that a mid-sagittal incision in the region of obex interrupts the inspiratory bulbo-spinal .axons, thus producing a functional state called "expiratory apneusis" (8), i.e., a tonic, C02 - dependent activity of expiratory neurones. None -of these authors attempted, however, to compare the behaviour of the separated halves of the respiratory complex at various stages of splitting, and only Langendorff et al. (4) suggested years ago that two halves of the rabbit's diaphragm may perform independent contractions after splitting the medulla at the same level which has been claimed as crucial for respiratory movements by some of the more recent authors (1, 7, 8).

Although even one well-documented experiment, that would une- ~quivocally demonstrate that breathing is not arrested in the split brain- stem animal would provide sufficient evidence that there are two respiratory networks whose mutual conections are not crucial for the rhythmogenesis itself (and that their descending axons do not predo- minantly cross), we decided to re-investigate thoroughly the problem using the simultaneously recorded activity of both phrenic nerves as monitors of the central respiratory rhythmicity.

The experiments were performed on 13 animals. The rabbits were first given intravenous neuroleptic analgesia (fentanyl and dehydro- benzperidol, 0.01 and 0.5 mg/kg, respectively) and then, after tracheo- stomy, halothane (0.7 volO/o in a 1 : 1 mixture of air with oxygen). After paralysis with d-tubocurarine (5 mgkg) the animals were arti- ficially ventilated at eupnoeic level (tested by frequent estimations of .arterial Po,, Pcoz and pH and continuous monitoring of the end-tidal COz concentration which was kept between 3.5 and 4.5 volO/o). Both vagus nerves and both phrenic C3 roots were dissected in the neck and cut. The skin incision was then tightly sewn, the animal reversed from supine to prone position and placed in a stereotaxic apparatus. An occipital craniotomy was made and the medulla widely exposed. The phrenic roots were now reached through the dorsal approach, placed $on two pairs of bipolar silver electrodes and covered with warm mineral !oil. The activity of both phrenic roots was amplified and recorded in a conventional manner and as an "integrated" signal (time constant 130 ms) on a Honeywell 4408A seven channel Visicorder along with end-tidal C02 and arterial blood pressure. After recording these para- meters in control conditions, mid-sagittal incisions of the medulla (and

Fig. 1. Microscapic cross-section of the brainstem showing the extent of midline incision in experiment 1 in Fig. 3A. A, level of the obex; B, C , D, E and F, 1, 2, 3, 4 and 4.5 mm rostrally to obex, respectively. Anatomical str.uctures (according to Messen a.nd Olszewski): 1. Nucleus tractus so.lita~rii; 2. Nucleus reticularis parvacellularis; 3. Nucleus reti,cularis ventralis; 4. Nu- cleus olivaris; 5. Subnucleus tr.alctus spinalis n. trigemini; 6. Nucleus reti- cularis lateralis; 7. Nucleus ambiguus; 8. Nucleus reticularis gigantocellu- laris; 9. Nucleus retrofacialis; 10. Nucleus facialis; 11. Nucleus triangularis.

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Fig. 2. Effect of a mid-sagittal section of the brainstem from obex to 4 mm rostra11 (sectim.1 in Fig. 3-4). Traces finom top to bottom: A and B, "integrated" ,activities of the left and right phrenic nerve, re~~pectively; C, direct record of the left phrenic n. activity; D, reference line for end-tidal COI (4 volOlo); E, direct record of the right phrenic n. activity; F, arterial blood pressu8re; G , end-tidal C02 record. Horizontal bar, 1 s. Spi,kes retouched. I, Cont~ol; 11, 10 min after the incision. Note that two diffe-

rent rhythms and patterns are recorded frcm both phrenic nerves.

on some occ~sions, pons) were performed with a 3 mm segment of a razor blade mounte'd in the holder of the stereotaxic frame's micro- manipulator. The blade was lowered towards the base of the skull and this procedure was repeated until a cut of desired length was obtained. The cerebellum was not removed. The sections extended from 3 mm caudal to 9 mm rostral to the obex, the smallest being 4 mm and the largest 12 mm long. They were made in varying sequences, the physio- logical variables being recorded immediately after and then 5 and 10 min after each incision. Experiments in which brain oedema, excessive bleeding or a rapid fall in'blocd pressure developed after an incision were discontinued. Each experiment was completed by fixing the brain in a 10°/o formaldehyde solution. After 3 days frozen sections were made (50 pm) and examined under a microscope to check the extent and com- pleteness of 'the incisions.

Mid-sagittal separations of the brainstem did in no instance arrest the rhythmic activity of the phrenic nerves. The most striking effect was that a relatively small separation of two halves of the medulla at the level from obex to 4 mm rostrally (Fig. 1) elicited a loss of syn- chronization between the rhythmic volleys conducted along the right and left phrenic nerves. As a result, both phrenic nerves discharged at two different frequencies and with two different patterns (Fig. 2). This result was reproduced in seven experiments in which incisions of differ- ent length, but always separating structures on either side of the obex and rostrally to the caudal end of Nucleus n. facialis (N. VII) were per- formed (Fig. 3A). In the remaining six experiments this phenomenon did not appear and both phrenic 'nerves discharged synchronously (Fig. 4). The incisions were, however, placed either more caudally or more rostrally to the aforementioned shortest section, or small strands of nervous tissue were left to provide connections of both sides of the brainstem (Fig. 3B).

We conclude that there are two relatively independent respiratory networks in both halves of the medulla and that their synchronous act- ion depends upon intact mutual connections at the level from obex to the caudal end of N. VII. This would be in good agreement with our recent observation (2) that unilateral microinjections of lignocain into structures a t the level between the obex and th'e rostral end of N. retro- facialis (6) alter the inspiratory discharge in the contralateral phrenic nerve, thus suggesting the existence of an important functional connect- ion at this level. We are not able to confirm the view that inspiratory bulbo-spinal axons cross at the level described by Salmoiraghi and Burns (7) and that their section elicits "expiratory apneusis" (8), since in 0v.r experiments even more extensive sections did not greatly affect

8 - Acta Neurobiol. Exp. 2/81

the rhythmic inspiratory discharge in the phrenic motoneurones. The old observation of Langendorff et al. (4) - which has been apparently forgotten by some authors and misquoted by others (7) - has been therefore confirmed. One can argue that species differences (i.e., rabbit vs cat) might be responsible for the differences, but in view of Kahn and Wang's results (3) this does not seem to be the case. Moreover, cats

Fig. 3. Schematic representation of mid-sagittal incisions which elicited asyn- ch ronx~s firing of both phrenic nerves (A) and those that failed to do so (B). Left halves of the diagrams show some structures of the brainstem for orien- tation: N. V r u t , motor nucleus of the Vth nerve; N. VII, Nucleus n. facialis; N.rVI1,-Nucleus retrofacilaliis; A. c, Nucleus armbiguus (see 6). Right halves present the sxtent of mid-sa@ttal separations of both halves of the lower brainstem, based upon histological examln~&ion. Vertical scale, mm rostrd and caudal to

obex (V); numbers on the honizontal axis, individual inoisiools.

wit11 chronically split brainstem were shown to breathe normally after recovery froni surgery (5). We therefore conclude that a split brainstem preparation is capable of producing a virtually normal respiratory rhythm in each of its halves and that normally the respiratory output is syn- chronized at the level extending from the obex to the caudal end of N , facialis.

The authors are greatly indebted to Miss K. Srmzyhska and to Mrs K. kusz - czyk for their skillful technical1 asslistaince, to Mrs T. Warnawin for gmometric estimations and Mrs B. Sudziarska for preparing the manuscript. This investigat- ion was supported by Project 10.4.2 of the Pdish Academy of Sciences.

Fig. 4. Effect of mid-sagittal section of the brainstem from 2.5 mm caudal to 2.5 mm rostra1 to obex (No. 5 in Fig. 3B). 'Traces firam b p to bottom: A, "integrated" and B, direc- tly recorded activity of the left phrenic n.; C, directly recorded; D, "integrated" activity of the right phrenic nerve; E, end-tidal C02010 F, arterial blood pressure. I, Control; 11, 10 min after the incision. Note synchronous

discharge in both nerves.

1. BAINTON, C. R., KIRKWOOD, P. A. and SEARS, T. A. 1978. O n the trans- mission of the stimulating effects of carbon dioxide to the muscles of respiration. J. Physial. 280: 249-272.

2. GROMYSZ, H.? KARCZEWSKI, W. A., NASLORSKA, E., RUSZCZYK, K. a n d SROCZYNSKA, K. 1980. Effects of reversible elimination of some bulbar structures on t h e generation o f respiratory pat tern i n rabbits. Acta Neuro- b i d . Exp. 40: 507-514.

3. KAHN, N. and WANG, S. C. 1965. Descending respiratory pathways i n t h e medulla oblongata of the cat. Am. J. Physiol. 209: 599-603.

4. LANGENDORFF, O., NITSCHMANN, R. and WITZACK, H. 1881. Studien iiber die 1,n.nervation der Athernbeiwegungen. Ueber ungleichzeitige Thiitigkeit beider Zwerchfellshalften. Arch. Anat. Phys id . Physiol. Abt., Leipzig, 78-81.

5. MANCIA, M. 1969. Electraphys~iolagica1 and behavioural changes w i n g to split- ing of the brainstem in cats. Electroencephalogr. Clin. Neurophysiol. 27: 487-502.

6. MESSEN, H. and OLSZEWSKI, J. 1949. A cytoarchitectonic atlas of t h e rhom- bencephalon of the ,rabbit. S. Karger, Basel, 52 ,p.

7. SALM'OIRAGHI, G. C. a n d BURNS, B. D. 1960. Notes on mechanisw of rhytmic respiration. J. Neurqhysiol . 23: 14-26.

8. SEARS, T. A. 1977. The respiratory motoneuron and apneusis. Fed. Proc. 36: 2412-2420.

Accepted 5 November 1980