Respiratory responses to brain stem stimulation

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Respiratory Responses to Brain Stem Stimulation F. B E R G M A N N , U. LEIBOWITZ AND A. D. K O R C Z Y N Department of Pharmacology, The Hebrew Unn,erslO', Hadassah Medtcal School, Jerusalem (Israel)

INTRODUCTION

The localisation of the mechanisms regulating respiration has been attempted by numerous investigators. In addition to the 'respiratory centres' in the medulla, a 'pneumotaxic centre' was located in the anterior ports (PITTS et al. 1939) and an 'apneustic centre' in its posterior part (LuMSDEN 1923; WANG et al. 1957). Other studies have implicated also more rostral structures such as the hypothalamus, because it was observed that local lesions in this region reduced the rate of spontaneous breathing (REDGATE AND GELLnORN 1957) and that local applications of thiopentone depressed the polypnoea following sciatic stimulation (REDGATE 1963). The experiments of earlier investigators were usually confined to anaesthetlsed or decerebrate animals (BEATONAND MAGOUN 1941 ; BROOKHART1940; PITTS et al. 1939; ROBSON et al. 1963 ; WANG et al. 1957; Wvss 1954) and therefore our knowledge about the role of the anterior brain structures in respiratory regulation is meagre. However, recent studies in this laboratory have evinced the importance of anterior parts of the brain, as transectlons eliminating the rostra1 structures or application of anaesthetics depressed the spontaneous breathing and altered the response to peripheral nerve stimulation (KoRcZYN et al. 1965; LEIBOWITZ et al. 1965). Hence it is necessary to base central locahsations on experiments with intact, wakeful ammals. The present study ~s concerned with the physiological mechanisms regulating the rate of breathing. In order to determine the &stribution of acceleratory and deceleratory points, electrodes were systematically implanted along the whole brain stem. After the responses of the conscious ammal had been established, the effect of increasing doses of pentobarbltone was explored, since previous studies had shown that the barbiturate usually suppresses the actwity of acceleratory neurons before it blocks their deceleratory antagonists. Comparison of respiratory reactions m the same animal before and after application of the anaesthetic may therefore reveal the presence of neurons which cannot be recognlsed in either condition alone. It has been suggested previously that the ratio of the antagomstic neurons varies along the brain stem, the acceleratory components being predominant m the more rostral parts (LEIBOWlTZ et al. 1965). On the basis of our earlier experiments it was, however, not possible to ascertain the presence of deceleratory neurons in anterior J neurol. Set. (1966) 3:217-228

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F. BERGMANN, U. LEIBOWITZ, A. D. KORCZYN

brain structures. As will be shown, stimulations in the brain stem before and after barbiturisation yield direct information on the regional distribution of both types of neurons and thereby help in the elucidation of the mechanisms regulating the rate of breathing. MATERIAL AND METHODS

Fifty-five rabbits of either sex, weighing between 1.8 and 3.5 kg were used. Under ether anaesthesia, the trachea was cannulated and connected to a rubber tambour for recording respiratory movements on a kymograph. By means of a stereotaxic socket (HESs 1932), bipolar concentric electrodes were introduced as described previously (GUTMAN et al. 1963). The stainless steel electrodes consisted of an inner wire of 0.2 mm diameter and an outer tube of 0.7 mm. Both were insulated over their whole length except at the tip, the inner wire protruding 0.5-1.0 mm. After the operation, the wounds were infiltrated w~th xylocaine 2 9/ooand ether was discontinued. The animal was placed in a hammock which permitted free movement of head and legs. Stimulations were started not earlier than one hour after cessation of general anaesthesia. It has been shown previously (K-oRCZYN et al. 1965) that this interval is sufficient for exhalation of the ether. Square wave pulses were delivered from a Tektronix type 162 stimulation unit and were monitored continuously on a Tektronix oscilloscope. The following parameters were used: pulse duration 2 msec, frequency 5-200 c/s, intensity 0.5-4 V, applying always as low a voltage as possible. Each stimulation period extended over 10 sec. A new volley was started only after the respiration had regained control rate. Similarly, when barbiturate was given, the stimulation experiments were not resumed before the respiration had again reached a constant level. A 2 ~ solution of pentobarbitone in saline was injected through a vein m the ear, in repeated doses of 5-15 mg/kg, until the animal died. Then both carotid arteries were perfused with 10~o formalin and the brain fixed in 4 ~ formalin. For histological

A

B

C

D

ir

F

O

H

I

(

d

K

9

,,>

rM1, o

. \~

~

15

,,,,,,,,~

Fig. 1. Paramedian longitudinal section through the rabbit's brmn (according to the atlas of MONNIER AND GANGLOFF1961), for identification of the planes shown in Fig. 2. Abbreviations: CA, commissura anterior; Cb, cerebellum; CF, columna fornicis; CM, corpus mammillare; Col, colliculus inferior; CoS, colliculus superior; MO, medulla oblongata; NC, nucleus caudatus; NR, nueleus ruber; P, pons; TH, thalamus; TMT, tractus mammillo-thalamvcus. J. neurol. Sci. (1966) 3:217-228

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control o f the stimulated points, the frozen brain was cut into sections o f 90# thickness which were stained with gallocyanine (EINARSON 1932). The electrode positions were plotted into charts o f the rabbit's brain according to the atlas o f MONNIER AND GANGLOFF (1961). F o r medullary points, charts were drawn according to the atlas o f POI"TER (1911), using an extension o f the coordinates o f MONNIER AND GANGLOFF (plane H - K , see Fig. 1).

RESULTS

Patterns of Respiratory Responses Evoked by Central Stimulation of the Nonanaesthetised Rabbit In 55 animals a total o f 211 points was stimulated. As shown in Fig. 2, these points were &spersed over the whole brain stem, from the pre-optic area (plane A in Fig. 1)

I K

H

aA.°

Fig. 2. Transverse sections through the rabbit's brain, showing distribution of the various types of respiratory responses. Although electrodes were placed on both sides of the brain stem, all stimulated points were projected onto one half of the brain The dots (O) mark acceleratory points, stimulation of which increased the rate of breathing. The triangles Ok) indicate mixed points, where both tachypnoeic and bradypnoe~c responses were evoked. The squares ([]) show deceleratory points, stimulation of which caused bradypnoea or apnoea. The plus (+) signs mark silent points. to the posterior medulla (plane K) (see also Table 1). In the non-anaesthetised rabbzt, the points could be classified in four groups depending on the pattern o f the evoked respiratory response:

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F. BERGMANN, U. LEIBOWITZ,A. D. KORCZYN A

B

C

Fig. 3. Acceleratory response to brain stem stimulation. Male rabbit, 2.2 kg. Electrodes m plane B, about 7 mm to the right of the mldline, near ventral border of brain, at 18 mm depth from surface of the skull. Stimulation at 3 V. Frequencies marked on lower pictures. Upper record, respiration (respiration downwards); lower line, stimulation signal of 10 sec. A : wakeful ammal. Note acceleration of breathing at all frequencies. B: after 30 mg/kg pentobarbltone ~.v. Tachypnoe~creactions diminished. C. after 60 mg/kg. Respiratory responses to electrical stimulation disappeared without reversal.

Group 1. Stimulation over the whole frequency range quickened respiration (63 % of all points), the maximal effect appearing usually between 40-100 c/s (Figs. 3A and 4A). This type of response resembled the reaction to sciatic stimulation in the animal in the waking state, where acceleration was maximal in the same frequency range

(KORCZY~ et al. 1965). Group 2. 'Mixed' responses. Here the respiratory reaction varied with the rate of stimulation. Low frequencies quickened the breathing, while high frequencies slowed it down (15% of all points) (Fig. 5A). The extent of acceleration or deceleration, under comparable conditions of stimulation, was not the same in each animal. Similarly, transition from accelerahon into deceleratory reactions took place at different rates of stimulation. In some cases, biphaslc responses were encountered m which the deceleratory phase always preceded the tachypnoeic period. The pattern of mixed J. neurol Sci (1966) 3" 217-228

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TABLE 1 DISTRIBUTION

OF 'RESPIRATORY

P O I N T S ~ I N T H E B R A I N S T E M O F T H E NO N- A NA E S TH E TI ZE D R A B B I T

plane*

Acceleratory points (Group 1)

'Mixed' points (Group 2)

Deceleratory pomts (Group 3)

Total reactive pomt~

A

II

0

0

11

1

B

8

0

0

8

3

C D

10 19

2 0

l 2

13 21

1 1

E

14

2

3

19

2

F G

12 16

3 3

2 6

17 25

1 2

H

19

4

1

24

4

I

14

2

2

18

4

J K

7 4

6 9

4 2

17 15

2 2

134

31

23

188

23

Transverse

Total

'Sdent' points (Group 4)

* Coordinates of the atlas of MONNIERANDGANGLOFF(1961) (see Fig. 1). Planes A-D correspond to the diencephalon, E represents the mesencephalon, F-G the pons and H-K the medulla oblongata.

responses is thus similar to the effect o f vagal stimulation in the conscious animal (KoRCZYN et al. 1965). Group 3. Deceleratory reactions at all rates o f stimulation (11 ~ o f all points). The degree o f slowing depended on the frequency used, the maximal effect varying between slight retardation and complete respiratory arrest. Usually at 100 c/s a climax was reached, the deceleratory action declining at still higher frequencies. It should be noted that bradypnoeic responses over the whole frequency range have never been encountered in experiments with peripheral nerve stimulation, in the absence o f anaesthetics. Group 4. I n 23 points (11 ~o), electrical excitation had no effect on breathing under any conditions. However, as will be shown later in this paper, in 4 o f these 'silent' points stimulation after injection o f pentobarbitone gave a definite respiratory effect (see Fig. 6).

TABLE 2 DISTRIBUTION

OF A C C E L E R A T O R Y

Region

Dlencephalon (plane A-D) Mesencephalon (plane E) Pons (plane F-G) Medulla (plane H-K)

POINTS ALONG

Reactive points

53 19 42 74

THE BRAIN STEM OF THE CONSCIOUS RABBIT

Pure accelerator), points

48 14 28 44

Acceleratory points (%)

91 74 67 59

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F. BERGMANN, U. LEIBOWITZ, A. D. KORCZYN

D&trtbution of Respirator), Responses m the Brain Stem T h e d i s t r i b u t i o n o f the points, f r o m which the different types o f responses were evoked, changes a l o n g the brain stem (Table 2). Thus the p r o p o r t i o n o f pure a c c e l e r a t o r y p o i n t s declines a n d t h a t o f mixed a n d d e c e l e r a t o r y p o i n t s increases in caudal direction.

Effect of Pentobarbitone on Respiratory Responses to Central Stimulation T h e b a r b i t u r a t e caused a slowing d o w n o f s p o n t a n e o u s b r e a t h i n g a n d d i m i n i s h e d g r a d u a l l y the n u m b e r o f reactive points, in relation to the dosage used (Table 3). T h e m o s t c o n s p i c u o u s effect o f the d r u g on evoked r e s p i r a t o r y responses consisted in the c o n v e r s i o n o f a c c e l e r a t o r y into d e c e l e r a t o r y reactions. Out o f 91 p o i n t s which in the n o n - a n a e s t h e t i s e d a m m a l caused only a n increase in r e s p i r a t o r y rate, 29 (32 %)

Fig. 4.

Fig. 5

Fig. 4. Barbiturate-reduced reversal of the respiratory reaction to brain stem stlmulauon. Fern',de rabbit, 1.9 kg. Electrodes m substantla nigra, 16 mm below surface of the skull (plane E, about 1.5 mm to the right of the midline); stirrtulatlon with 3 V. Markings as in Fig. 3. A: pure acceleratory responses to all frequencies in the wakeful animal. B: reversal into apnoeic reactions after intravenous injection of 20 mg/kg pentobarbitone Fig. 5. 'Mixed' response to brain stem stimulation. Male rabbit, 2.3 kg. Stimulation near ventral border of posterior pons (left part of plane G), 19 mm below surface of skull, with 2 V. Markings as in Fig. 3. A : wakeful animal. Note tachypnoeic response at low and deceleration at high frequencies of stimulauon. B: after 10 mg/kg pentobarbitone i.v. While the response to 5 c/s is still ac-~leratory, stimulation wtth 20 c/s causes a biphasic reactton and with 100 c/s complete apnoea.

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gave rise to bradypnoea when stimulated again after barbiturate (Fig. 4). The response was inverted in steps in accordance with the dosage applied. The amount of drug required to reverse the effect of high frequency stimulation was smaller than the one necessary for reversal of the reactions, evoked at low frequenoes. Accordingly, acceleratory points were usually converted first into 'm~xed' ones and became purely deceleratory only after higher doses of the barbiturate. Biphasic responses, appearing after pentobarbitone, exhibited the same character as the corresponding reactions m the wakeful ammal, i.e. the deceleratory phase always preceded the tachypnoelc period (Fig. 5B).

A

B

C

D

...... I

5o/s

.......

so/s toq/s

Fig. 6. 'Silent point'. Female rabbit, 2.3 kg Stimulation In substantm retlcularls of thalamus (at plane D about 1.5 mm to the right of the mldhne), 15 mm below surface of the skull, with 2 V. A: wakeful animal. B, C and D: after 15, 30 and 45 mg/kg pentobarbitone respectively. At A, note ineffectiveness of stimulus at all frequencies used. At C, the same stimuli caused complete apnoea, while at D the deceleratory response to 100 c/s is already diminished.

Different points in the same a m m a l showed widely divergent drug susceptibihty. Some of the acceleratory points underwent reversal already after a dose of 5 mg/kg, while others required 40 mg/kg or more. However, m 68 ~ of all acceleratory points the drug did not cause inversion, but diminished gradually the effect of stimulation tall its complete disappearance (Fig. 3B, C). In m~xed and purely deceleratory points, small doses of pentobarbitone reinforced the bradypnoeic effect. In 4 of the 23 silent points encountered, a deceleratory response could be elicited after barbiturisation (Fig. 6). Since the tachypnoeic reactions were reversed or abolished by doses of drugs smaller than those affecting the deceleratory J. neurol. Sci. (1966) 3:217-228

224

F. BERGMANN, U. LEIBOWITZ, A. D. KORCZYN TABLE 3

INCREASE OF

DECELERATORY REACTIONS TO

BRAIN STEM STIMULATION AFTER GRADED

DOSES

OF

PENTOBARBITONE

Pentobarbttone (mg/kg ) 0 10 20 30 40 50 60-70

Total reacttre pomt~

Mtxed and deceleratory point~

Mtxed and decelerator3' points (o~)

188 105 86 52 27 2O 19

54 32 39 32 18 14 12

29 30 45 62 67 7O 63

Note that above 30 mg/kg of pentobarbltone no significant change in the percentage of the antagonistic responses was encountered, while the number of reactive points decreased steadily,

points, the percentage of the latter increased gradually with cumulative applications of pentobarbitone (Table 3). Sufficiently high doses ultimately suppressed all types of response although the total amount required varied from one point to the other. Thus some points were silenced already by 10 mg/kg, while others remained active even after injection of 70 mg/kg, i.e. a respiratory response could be evoked from them as long as the animal maintained spontaneous breathing. Barbiturate-resistant tachypnoeic reactions were characterized by the low frequency necessary for their activation; conversely, for refractory bradypnoeic responses high frequency stimulation (100 c/s or more) was typical.

DISCUSSION

In the present study, respiratory reactions were evoked by stimulation along the whole brain stem. This raises the question whether the responses obtained express specific respiratory functions It has become customary to attribute a specific role in the regulation of breathing to certain defined anatomical structures. So for instance, the vagus nerve can produce either tachypnoea or bradypnoea (and apnoea), depending on the frequency of electrical stimulation (Wvss 1954). In contrast, stimulation of somatic nerves, e.g. the sciatic, invariably accelerates breathing. The distinction between these two types of nerves is further stressed by the different effect of anaesthetics. In the case of the vagus the drugs block first the acceleratory pathways so that stimulation at any frequency produces only bradypnoea; in the case of the sciatic, stimulation becomes less effective in the anaesthetized cat, but does not cause deceleration (KoRczvN et al. 1965). Subsequent transection experiments revealed, however, that the divergencies between vagal and somatic stimulation are only of a quantitative nature (LEtaOWITZ et al. 1965). After mid-collicular decerebration, excitation of the sciatic nerve also evokes bradypnoeic responses. Thus botb. nerves can exert a dual effect on respiration; J neurol. Scl. (1966) 3:217-228

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for the vagus this property may be demonstrated already in the intact animal, while sciatic stimulation produces bradypnoea only after elimination of anterior brain structures. It was therefore concluded that the 'specific' influence of the vagus on breathing is merely due to its association with pulmonary stretch receptors and not to a particular central mechanism. It is well known that regular breathing persists after bilateral vagotomy. Recently it was suggested that the normal respiratory rate is the result of summation and integration of the total sensory input (SALMOIRAGHI1963). Many kinds of stimuli, such as pain, temperature changes or chemical stimuli, influence the rate of breathing. SERGIEVSKn AND IVANOV (1958) have shown that with progressive de-afferentatlon the dog becomes less sensitive to CO2 In the hght of these observations, the question of the 'specificity' of the nervous elements influencing the rate of breathing loses much of its significance. The same respiratory effects which are obtained by peripheral stimulation, appear also after central excitation and m both cases anaesthetics modify the reaction in a similar way. This suggests th.at the same neurons respond to both peripheral and central stimulation and further that the elements excited also pamcipate in the physiological regulation of breathing. The regulatory mechanisms are, however, not rigid and can adjust themselves to varying external and internal conditions (GESELLet al. 1937). Thus, in the present experiments, the respiratory response changed its character with varlanons in frequency of stimulation or in depth of anaesthesia. Moreover, some points which were silent in the conscious animal became active only after application of barNturate. Thus it appears that not all potential pamcipants of the system are functioning at all times. SALMOIRAGHIAND BURNS (1960) and ROBSON (1963) have established the existence of mutually inhibitory networks of inspiratory and expiratory neurons. It follows from their results that the rate-controlling mechanisms, explored in the present study, must influence respiration by affecting both types of rhythmically active respiratory neurons, i.e. they must be superimposed upon them. Thus, quickening of breathing requires that a given type of neuron, once it has been activated, should also be rapidly silenced by the inhibitory effect of its antagonist. The opposite is true for the slowing down of breathing, i.e. bradypnoea (or apnoea) is caused by the lack of rapid alternation of activation and inhibition of the antagonistic respiratory neurons (ROBSON et al. 1963). It has previously been reported that brain transections, progressing in caudal direction, gradually slow down the rate of spontaneous breathing (LEIBOWITZet al. 1965). This observation can be explained by the present finding that in rostral brain structures acceleratory neurons are relatively more abundant. They are, however, everywhere intermingled with deceleratory elements, the percentage of the latter increasing in caudal direction (Table 2). Therefore, one cannot speak of a localized 'pneumotaxic c e n t r e ' (WANG et al. 1957), but only of a pneumotaxlc (-- acceleratory) mechanism, in accordance with the statement of SALMOIRAGHI(1963) that the term 'respiratory centre carries a functional rather than a precise anatomical connotation'. The two antagonistic elements influencing respiratory rate are also interwoven in the medulla (see Fig. 2, H-K). Here again, no circumscribed area was found stlmuJ. neurol. Sct (1966) 3:217-228

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F. BERGMANN, U. LEIBOWITZ,A. D. KORCZYN

latlon of which in the conscious ammal yields only deceleratory responses, as could be expected if separate inspiratory and expiratory centres would exist. The apparent divergence between our present findings and the classical results of P~vrs et al (1939) and PITTS (1941) may be ascribed to the following facts. First, these and other investigators (see e.g. BEATON AND MAGOUN 1941)dehberately neglected responses in which the rate of breathing increased, as they were only concerned with apnoea. Furthermore, they usually worked with decerebrate or heavily anaesthetized animals, i.e. under conditions that may convert reactions which are acceleratory in the wakeful, intact ammal, into deceleratory or apnoeic responses. In addition, localization of inspiratory and expiratory centres was usually based on stimulation with high tYequency and/or high voltage; e.g. PITTS et al. (1939) applied in their experiments 240 c/s and 8 V. However, Fig. 5 demonstrates that a mere increase m frequency may change a tachypnoeic reaction into a bradypnoetc one Likewise, unpublished results from our laboratory show that elevation of current intensity has effects similar to those of increasing the frequency. Thus, our findings are in line with those of BROOKHART (1940) who, while working with decorticate (not decerebrate) unanaesthetized dogs and using low parameters of stimulation, failed to observe separate expiratory and inspiratory centres. The present experiments demonstrate again some characteristic features which distinguish acceleratory reactions from their deceleratory counterparts, lhe ~brmer are more susceptible to blockade by anaesthetics and exhibit a longer latency --- two properties typical for polysynaptic pathways (FRENCH et al. 1953). Similar differences in the electrophysiological properties of acceleratory and deceleratory mechamsms were encountered in our earher experiments with vagal and somatic nerve stimulations (LEIBOWITZ et al. 1965; KORCZVN et al. 1965). It is thus easily understood why - under the influence of anaesthetics - - points of Group I may produce mixed or deceleratory reactions. Nevertheless the number of synapses involved m acceleratory pathways varies from one point to the other. This is evident from the great difference in drug sensitivity, some acceleratory points being resistant to very large amounts of barbiturate, nearly the lethal dose. However, on the average deceleratory circmts involve fewer synapses. Both our experiments with peripheral nerve stimulation and with central excitation reveal that under deep anaesthesia as well as after transections the deceleratory neurons exhibit an independent activity; therefore these elements cannot be merely inhibitors o f their acceleratory counterparts. On the other hand, it cannot be ruled out that the acceleratory elements may act by inhibiting their deceleratory antagonists. Such a relationship would be consistent with the higher concentration of acceleratory neurons in the rostral parts of the brain and with the fact that the acceleratory pathways comprise more synapses. An answer to this problem could be obtained by the use of drugs which can block selectively the deceleratory elements. ACKNOWLEDGEMENTS This research was supported by a generous grant from the Joshua Green Fellowship J. neurol. Set. (1966) 3:217-228

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T r u s t t o AMOS D . KORCZYN a n d f o r m s p a r t o f a n M . S c . t h e s i s , s u b m i t t e d t o t h e F a c u l ty o f M e d i c i n e , T h e H e b r e w

University, Jerusalem.

The authors

wish to thank

MR. R. KNAFO f o r t h e p r e p a r a t i o n o f t h e d r a w i n g s .

SUMMARY

Electrical stimulation in the brain stem of the rabbit can evoke four different types of respiratory responses. Pure acceleratory and pure deceleratory reactions at all parameters of stimulation; mixed responses, i.e. tachypnoea at low and bradypnoea at high rates of stimulation; and absence of reactions in ~silent points'. Throughout the brain stem the various points are intermingled. However, the proportion of acceleratory points declines and that of mixed and deceleratory points increases in caudal direction. Anaesthesia can convert tachypnoeic into mixed or bradypnoeic responses, even some silent points becoming deceleratory. Sufficiently high doses of anaesthetics suppress all types of respiratory responses, while a small number of points is refractory to doses of barbiturate, which are nearly lethal. In the intact, conscious animal it is impossible to define a circumscribed pneumotaxic centre. Simdarly, in the medulla no locahzed area was found stimulation of which would yield only bradypnoeic responses. These observations challenge the theory of separate medullary inspiratory and expiratory centres.

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