J. Phy8zol. (1979), 287, pp. 467-491 With 8 text-figure. Printed in Great Britain

467

DIFFERENTIAL ALTERATION BY HYPERCAPNIA AND HYPOXIA OF THE APNEUSTIC RESPIRATORY PATTERN IN DECEREBRATE CATS

BY WALTER M. ST JOHN From the Department of Physiology, Dartmouth Medical School, Hanover, New Hampshire 03755, US.A. (Received 15 May 1978) SUMMARY

1. A combination of bilateral lesions within the nucleus parabrachialis medialis complex (n.p.b.m.) and bilateral vagotomy typically resulted in an apneustic respiratory pattern in decerebrate and paralysed cats. Integrated efferent phrenic nerve activity was recorded as an index of the respiratory rhythm. 2. Changes in components of this apneustic breathing cycle were evaluated in response to steady-state hypercapnia and hypoxia. The components evaluated were (a) the period of phrenic discharge (inspiratory time, TI), (b) the period of no detectable phrenic activity (expiratory time, TE), (c) the total duration of the apneustic respiratory cycle (TTOT, the sum of T, and TE), and (d) the average height of the integrated phrenic nerve activity (apneustic depth). 3. Elevations of PA, co. from values below 45 torr to 50-60 torr, under both hyperoxic and normoxic conditions, resulted in significant elevations of TI, TE, TTOT and depth. Further PA, CO, elevations to approximately 70 torr caused no change, or frequently, a decrease in TI, TE and TTOT; the apneustic depth increased in most animals. 4. Diminutions in PA, 0, from normoxic to hypoxic levels at isocapnia typically caused an increase in apneustic depth and, concomitantly, significant decreases in TI, TE and TTOT. 5. Pharmacological stimulation of the carotid chemoreceptors by intracarotid administration of 1*0-20 ,sg NaCN produced a premature onset of phrenic nerve activity if delivered during the expiratory period. Such NaCN administrations, delivered during the inspiratory phase, resulted in an augmentation of the integrated phrenic discharge and a premature termination of phrenic activity. Carotid sinus nerve section eliminated the response to NaCN administration. 6. In experimental animals having bilateral carotid sinus nerve section, normoxic hypercapnia caused similar changes in the apneustic breathing pattern to those recorded in cats having intact carotid chemoreceptors. However, isocapnic hypoxia induced time-dependent changes in the pattern of phrenic discharge including diminutions in depth, an onset of gasping-type activity, or expiratory apnea. 7. In a few animals, bilateral n.p.b.m. lesions and bilateral vagotomy resulted in expiratory apnea which was continuous as long as ventilation with air was maintained. This expiratory apnea was replaced by an apneustic breathing pattern

W. M. ST JOHN following diminutions of PA, 0° below 90 torr. This establishment of an apneustic breathing pattern by hypoxia was observed both in animals having intact, as well as sectioned, carotid sinus nerves. This expiratory apnea could also be terminated by a single apneustic inspiration following general somatic stimulation or, in cats having intact carotid chemoreceptors, following intracarotid NaCN administration. 8. It is concluded that hypercapnia and hypoxia produce differential alterations of the apneustic breathing pattern in decerebrate cats. Further, the hypoxia-induced changes are considered to represent the net result of carotid chemoreceptor stimulation and brain stem depression. The results of this study are considered in the context of proposed mechanisms for phase-switching of the respiratory cycle. 468

TNTRODUCTION

In a series of articles which appeared in this journal in 1938 and 1939, Stella conclusively demonstrated that apneusis results from a combination of midpontile brain stem transaction and suppression of vagal activity in cats. It is now well recognized that, in this cat preparation, the inspiratory apnea of apneusis is usually not permanent but is ultimately succeeded by an expiratory phase (e.g. Breckenridge & Hoff, 1950; Wang, Ngai & Frumin, 1957). The latter expiratory phase is followed, in turn, by another prolonged inspiratory period and so forth as a cyclical pattern of apneustic breathing is established. Moreover, recent studies have shown that punctate lesions within the nucleus parabrachialis medialis complex (i.e. the pneumotaxic centre) combined with vagotomy are sufficient for apneusis production in anaesthetized (Euler, Marttila, Remmers & Trippenbach, 1976; St John, 1975), decerebrate (Euler et al. 1976; St John, Bond & Pasley, 1975; St John & Wang, 1977a; Tang, 1967) or encephale isole cats (Bertrand & Hugelin, 1971). Beginning with the work of Stella noted above, various authors have examined changes in the pattern of apneusis resulting from hypercapnia. There is general agreement that increasing hypercapnia causes a progressive increase in the inspiratory depth of apneusis (Stella, 1938b; Ngai, 1957; Tang, 1967; Euler et al. 1976). However, results concerning hypercapnia-induced alterations of inspiratory and expiratory durations are quite conflicting. Thus, whereas Ngai (1957) and Euler et al. (1976) report that hypercapnia caused a shortening or no change of the inspiratory duration, Tang (1967) found a prolongation of this phase. Stella (1938a, b, 1939) did not define the inspiratory duration in his studies but reversibly cold blocked the vagal nerves for a relatively fixed interval. The expiratory duration during apneustic breathing is stated to be unchanged (Euler et al. 1976) or shortened by hypercapnia (Ngai, 1957). While Tang (1967) does not explicitly comment on changes in expiratory duration, it appears, from his published experimental records (pp. 359, 361), that this duration increased or remained constant as C02 was elevated to various levels. The influence of hypoxia upon apneusis was examined in a 1939 report by Stella. He observed that, in cats having mid-pontile brain stem transactions, the depth of apneusis which ensued upon cold blocking the vagi was greater during hypoxia than normoxia. As noted above, Stella did not determine the inspiratory or expiratory durations in his reports. These hypoxia-induced changes have been only briefly considered in two studies. However, results of these reports strongly imply that the

469 APNEUSTIC RESPIRATORY PATTERNS effect of hypoxia upon the pattern of apneusis may differ from that of hypercapnia. Thus, while Tang (1967) emphasized primarily that inhalation of 10% 02 caused the appearance of gasping during apneusis, he did note a 'diminished inspiratory spasm' during hypoxia in some portions of his experimental records. Similarly, Euler et al. (1976) found that in progressive asphyxia, resulting from a cessation of artificial ventilation to paralysed experimental animals, the inspiratory duration of apneusis progressively diminished. Neither the study of Tang (1967) nor that of Euler et al. (1976) considers the site (i.e. carotid chemoreceptors or central nervous system) responsible for these hypoxia-induced changes. However, there is rather consistent experimental evidence that direct pharmacological stimulation of these carotid chemoreceptors by cyanide can cause both a premature development of an apneustic inspiratory spasm and/or a premature termination of this inspiratory phase (Stella, 1939; Ngai, 1957). Considering the incomplete and conflicting nature of these results discussed above, it was believed that a detailed examination of hypercapnia- and hypoxia-induced alterations of the apneustic breathing pattern was of importance. In studies described herein, it has been found that hypercapnia and hypoxia differentially alter the pattern of apneusis. Moreover, both carotid chemoreceptor and central nervous system mechanisms contribute significantly to the hypoxia-induced changes. METHODS

I. General The experimental protocol of this study was directed at obtaining a preparation in which the influence of various levels of peripheral and central chemoreceptor stimulation upon the pattern of apneusis might be evaluated. In order to filfil this experimental aim, the following procedures were performed in all animals: (1) anaesthetization, (2) decerebration, (3) bilateral pneumotaxic centre ablation, (4) bilateral vagal section, (5) recording of efferent phrenic nerve activity, and (6) paralyzing the decerebrate animal and concomitant administration of artificial ventilation. Additionally, (7) the carotid sinus nerves were bilaterally sectioned in some experimental animals. Although anaesthesia induction preceded the other procedures (nos. 2-7) noted above, the sequence in which these remaining experimental steps were performed was varied as required by the design of the particular experiment. A description of each experimental procedure is detailed below. II. Surgical procedures Twenty adult cats of either sex, weighing between 2-0 and 5 0 kg, were included in these experiments. All animals were anaesthetized with halothane (Fluothane, Ayerst Laboratories). The trachea was cannulated at a mid-cervical level and cannulae were placed in a femoral artery and vein. The external carotid arteries were ligated caudal to the lingual artery branch (Wang, et al. 1957; Crouch, 1969) in eleven experimental animals. In the remaining cats, the external carotid arteries were ligated rostral to the lingual arteries; these latter vessels were cannulated with the cannulae being advanced caudally into the external carotid arteries. The vagus nerves were isolated bilaterally at the mid-cervical level and were either sectioned immediately or were surrounded by loose ligatures and sectioned later. The animals were then positioned in a Kopf stereotaxic apparatus and the brain stem transacted at an intercollicular level (Wang et al. 1957). Halothane anaesthesia was discontinued following decerebration. The rectal temperature was continuously monitored (Yellow Springs Instrument Co., Telethermometer) and was maintained at 37 5-39-5 0C by a heating pad and/or heat from electric lamps. The animals were paralysed by i.v. administration of gallamine triethiodine (Flaxedil, American Cyanamid Co., 5.0 mg/kg initially and 2-5 mg/kg approximately every 30 min thereafter). Subsequent to gallamine administration, artificial ventilation was administered by a Harvard 661 respirator.

470

W. M. ST JOHN

III. Evaluation of carotid chemoreceptor function; denervation of the carotid chemoreceptor8 In those animals in which external carotid artery cannulae were placed, the carotid chemoreceptors were pharmacologically stimulated by injection of 1 0-20 jug NaCN (in 0-1 ml. isotonic saline) into these cannulae. Records of phrenic nerve activity (see below) or breathing pattern, monitored by pneumotachograph methods (St John, 1975), were obtained before and after the NaCN ainistrations. Bilateral carotid sinus nerve section was performed as described previously (St John & Wang, 1977b). Intra-arterial NaCN injections were repeated following the carotid sinus nerve sections.

IV. Isolation and monitoring of phrenic rootlet activity Cs and/or a, phrenic nerve rootlets were exposed in the neck via a lateral approach as described previously (St John & Wang, 1977a, b). Phrenic nerve activity, monitored by a bipolar electrode (Palmer), was amplified and electronically filtered (bandpass 10-10,000 Hz) by two Grass DP9B differential amplifiers arranged in series. An additional bandpass filter (600-8000 Hz) was inserted in the post-amplification stage. Signals from the amplifier-bandpass system were led to a Textronic R561B oscilloscope for visual observation, to a Grass AM4A audio monitor, and to a 'leaky' integrator circuit (time constant 0-02 sec). V. Pneumotazc centre ablation Bilateral lesions of the dorsolateral pontile tegmentum, sufficient to ablate the nucleus parabrachialis medialis complex (n.p.b.m.), were placed in all animals using previously detailed stereotaxic co-ordinates (St John et al. 1971; St John & Wang, 1977a). Radiofrequency power (60-75 0C) derived from a Kopf RFG4 lesion generator and delivered through a Kopf K1308Z calibrated electrode was used for lesion production. Subsequent to unilateral pneumotaxic centre ablation, a minimum period of 30 min was taken prior to the placement of the contralateral lesion. VI. Respiratory and cardiovascular variable measurements During apneusis, as d in terms of integrated phrenic nerve activity, the following variables have been determined: (a) T1 - the temporal period of the phrenic discharge, corresponding to the period of inspiratory activity, (b) Tz - the interval between consecutive periods of phrenic activity, corresponding to the expiratory phase, (c) TTOT - the total duration of the respiratory cycle which is equal to the sum of T1 and TR, and (d) depth - the average excursion of the integrated phrenic discharge. The end-tidal C02 and 0. partial pressures (PA, co and P,oO,) were continuously monitored using a Beckman LB-2 C02 analyzer and a Beckman OM-11 0O analyser as described previously (St John & Wang, 1977a, b). Additionally, the arterial blood pressure was monitored by connecting the femoral artery cannula to a Statham P23AC pressure transducer. The outputs from this Statham transducer, as well as those from the Beckman analysers and the 'leaky integrator circuit' noted above, were recorded, after suitable amplification, on a Grass 5D polygraph. Changes in the respiratory and cardiovascular variables were determined in response to the following test stimuli: (a) hypercapnia at normoxia (PA, CO = 27-70 torr, PA, 0 = 116-142 torr), (b) hypercapnia at hyperoxia (PA,co, = 31-70 torr, PA,Og> 500 torr), (c) hypercapnia at hypoxia (PA, co, = 28-70 torr, PAAO3 = 75 or 60 torr), (d) hypoxia at isocapnia (PA.O. = 142-52 torr, PAcO,= constant in a given experiment), and (e) hyperoxia at isocapnia (PA. = 142 to > 500 torr, PA.co, = constant in a given experiment). Changes in PA,COs and/or P.AOS levels were obtained, as described previously (St John & Wang, 1977 a, b), by altering the gas composition delivered to the intake port of the Harvard respirator. Values of respiratory or cardiovascular variables reported herein represent, with certain explicitely noted exceptions, values obtained after PA,CO, and PiAOX had been maintained at a given level for a minimum of 7-10 min. Exposure to the various test stimuli groups (a-e) was in a random order. Likewise, the variable stimuli within a group were presented randomly (e.g. hypercapnia at normoxia, PA, co, randomly changed as Pa O, maintained constant). Not all experimental animals were exposed to all test stimuli. In addition to these stimuli noted above, the carotid chemoreceptors were pharmacologically stimulated by intra-arterial NaCN injections in some experiments (section III above).

APNEUSTIC RESPIRATORY PATTERNS

471

VII. Histologica verification of lesion placmen; statttical evaluation of data At the termination of each experiment, the brain stem was removed and immediately placed in a 10 % buffered formalin solution. Serial brain stem sections, embedded in paraffin and stained with haematoxylin-eosin, were obtained. Data were evaluated by the non-parametric Wilcoxon matched-pairs signed-rank test (twosided). By significantly different in this report is meant that the probability was less than 0-05 that the means of the two populations were equal. RESULTS

Subsequent to the completion of the pneumotaxic centre ablation and vagotomy procedures, an apneustic respiratory pattern was established in most animals. However, following these procedures in three cats, the initial period of sustained phrenic discharge (inspiratory phase) was succeeded by a continuous expiratory phase (i.e. TE greater than 30 min) as long as ventilation with air was maintained. In two other animals, only the continuous expiratory period was observed. Results concerning these five animals exhibiting continuous expiratory activity will be described below (section II). TABTrn

1. Alterations of apneusis by elevations of end-tidal CO2 partial pressure under normoxic or hyperoxic conditions in cats having intact carotid sinus nerves. Mean values + s.E. are presented for TI, TB, TTOT and depth of apneusis and for mean arterial blood pressure (B.P.) at the appropriate PA0o, and PA, 0. levels. n is the number of experimental animals examined. a = P < 0 05 compared with values at PA,co2 of 36 or 40 torr, b = P < 0-05 compared to values at PAco of 52 or 54 torr (Wilcoxon test, two-sided)

PAXC02

PA. 0g

Mean

36 2-7 52a 1-6 69ab

133 5-71 127a 2-60 124a

5.E.

1.0

2*20

Mean

40 2-3

629

54a 03

601 19.5 602 8-48

(torr)

Mean 8.E. Mean

5.E.

(torr)

TR T1 TTOT (sec) (sec) (sec) Normoxic hypercapnia 46 20 26 4.9 4-6 4-2

Depth (units)

B.P.

15 0.1 38a 4-2

(mmHg) n

423a 350 200a

143 66-1 87a

566a 330 288a

42a

121 8-77 141a 9.01 135

140

35

128

5.8

8-02

13

112 4-06 119 5-46 123 5-13

6 6

6

Hyperoxic hypercapnia

5.E. Mean S.E.

Mean S.E.

6711 0.8

7'17

23 8-6 64a 37 202a 160

37 14 40

9.7 38 8-5

61 18 104 44 240a 164

3.3 18a

4.5 32ab 4-1

6 6 6

I. Animals exhibiting apneu8tic breathing A. Response to PA, CO, elevations under normoxia or hyperoxia (1) Animals having intact carotid chemoreceptors. Changes in the PA, Co, level under normoxic conditions produced a profound alteration in the apneustic pattern. Thus, as reported in Table 1, there were statistically significant increases in TI, TE, TTOT, and the depth of apneusis when PA, CO, was elevated from 36 to 52 or

W. M. ST JOHN

472

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473 APNEUSTIC RESPIRATORY PATTERNS 69 torr. These variables did not change significantly as PA CO, was altered from 52 to 69 torr. Presentation of these mean values does not reveal an important characteristic of the hypercapnia-induced changes in the apneustic pattern. This characteristic is illustrated in Fig. 1 in which an experimental record is presented 100 _ x

E 50

-

0-

100_ lOOJ x

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-

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x

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PA Co (torr) Fig. 2. Values of TI, TB, TTOT and depth during apneusis at the specified levels of PCOg for each individual animal having intact carotid sinus nerves. P^ACO was altered under nornoxic conditions. TI, T]g, TTOT and depth values are expressed as a percentage of the maximum value obtained (% max.). The number assigned to each animal (i.e. 1-9) does not indicate an identical animal to that cat having the same number in any subsequent Figure.

illustrating the pattern of apneusis at various levels of end-tidal CO2. Compared to values at FA, co, approximating 003 (panel F), TI, TE, TTOT and the depth of apneusis were all greater at FA, CO, of 005-0-06 (panels C and D). However,

474

W. M. ST JOHN

not all of these variables were increased further at higher FA' CO. levels; rather, as illustrated in panel A, T, values at FA, CO, of 0*09 were less than those obtained at FA, Co, of 0-05-0-06. These results illustrated in Fig. 1 were typical of those obtained for each experimental animal as evidenced by data presented in Fig. 2. In this Figure, values of TI, TE, TTOT and depth of apneusis are expressed as a percentage of the maximum value obtained. Note that, in the very great majority of animals, elevations of PA, CO, from the minimum value examined (left-most points on each curve) caused increases in TI, TE, TTOT and depth of apneusis. However, it is also important to observe, as was exemplified in Fig. 1, that, for many animals, the maximum value level. Rather, in these of these variables did not occur at the maximum PA, cats, elevations of PA, c02 from 45-58 to 65-72 torr caused a decline of TI, (nos. 1, 2, 4, 7, 9), TE (nos. 1, 6, 7), TTOT (nos. 1, 2, 5, 7) and/or depth (nos. 4, 5). For two animals of Fig. 2 (nos. 3, 9) only values of T, and depth are reported. These cats exhibited continuous inspiratory activity (TI > 15 min) at one or more P, CO, levels; hence, no values of TE or TTOT could be obtained. Data from these cats, as well as from cat no. 8 which was examined at only two PA, CO. levels, were not included in the statistical data pool of Table 1. Two additional points must be defined concerning these data. The first is the extreme magnitude of T1 and TE values which could be obtained. As noted above, cat no. 3 exhibited a T, in excess of 15 min at one PA, co, level (i.e. 54 torr). However, elevation of PA, CO to 68 torr did not result in a cessation of the phrenic discharge which continued unabated for a second 15 min interval. The T, of this animal at a PA, co, of 41 torr was 44 sec. In a similar fashion, TE values in excess of 10 min were not uncommon in these experimental animals. A second point is the marked variability in T1, TE and TTOT values between animals. This variability is evidenced by the large s.E. values for these measurements reported in Table 1. Similar results to those described above for normoxic hypercapnia were also obtained with PA, Co, elevations under hyperoxic conditions. Thus, elevations of PA, Co, from minimum values below 50 torr caused elevations of T1, depth and, with the exception of one animal, elevations of TE and TTOT also. As with normoxichypercapnic stimuli, the maximum values of TI, TE, TTOT and depth did not always occur at the maximum PA, Co, level examined. Statistical treatment of data obtained in response to hyperoxic-hypercapnic stimuli revealed that the changes in T, and the depth of apneusis were significant as PA, CO, was elevated from a mean level of 40 torr to 54 or 67 torr (Table 1). Changes in TTOT were significant only at the highest PA, co, levels. TE changes were not significant. This lack of significance for TE and TTOT alterations was due to one animal, noted above, in which TE and TTOT declined as PA, CO, was increased. The mean arterial blood pressure of each animal usually rose as PA, co, was elevated both under normoxic and hyperoxic conditions. This mean blood pressure was in excess of 90 mmHg for each animal at all PA, CO, levels. (2) Animalt having bilateral carotid sinus nerve section. Under normoxic conditions, hypercapnia-induced changes in TI, TE, TTOT, and the depth of apneusis of cats having carotid sinus nerve sections were qualitatively similar to those of animals having intact carotid chemoreceptors. Thus, as reported in Table 2, TI, TTOT and

Co,

APNEUSTIC RESPIRATORY PATTERNS 475 depth significantly increased as PA co0 was elevated from 40 to 53 or 65 torr. Likewise, in individual cats, elevations of PA, C02 from minimal levels of 37-44 to 51-54 torr resulted in elevations of T, (five out of five animals), TE (four out of five animals), TTOT (four out of five cats) and depth (five out of five cats). As was observed for cats having intact carotid chemoreceptors, the maximum values of these variables did not always occur at the maximum PA, co, level. Rather, as Po, co. was elevated from approximately 50 torr, TI, TE, TTOT and depth either remained constant or declined in most animals. TABLE 2. Alterations of apneusis by elevations of end-tidal CO2 partial pressure under normoxic or hyperoxic conditions in cats having bilateral carotid sinus nerve section. Mean values+s +.E. are presented for TI, TE, TTOT and depth of apneusis and for mean arterial blood pressure (B.P.) at the appropriate PACO2 and PA,O, levels. n is the number of experimental animals examined. a = P < 0-05 compared to values at PA, CO, of 40 or 37 torr, b = P < 0-05 compared to values at PA, cc, of 53 or 52 torr (Wilcoxon test, two-sided)

PA, CO2 (torr)

PA. 02

(torr)

Mean 5.E. Mean 5.E. Mean

53a

134

0-6 65ab

5.E.

0-5

1-58 130b 1-58

Mean S.E. Mean

37 52a

660 4-64 642a

5.E. Mean 5.E.

1-0

3-66

65ab

631b

0-3

4-25

40

134

1-4

1-05

1-7

T,T1 TTOT (sec) (sec) (sec) Normoxic hypercapnia 84 43 128 44 14 58-8 212a 68 281" 106 30 132 113a 67 180a 28 72-2 44-4 Hyperoxic hypercapnia 45 25 124 19 7-0 74-2 58 70 128 20 34 51-1 56 64 120 24 38 48-1

Depth (units)

B.P.

(mmHg) n

13

110

3-0

15-1

32a

109

4-0

15-2

37a

105

3-3

15-8

17

105

4-7

13-5

29a

108a

3-3

8-4

32

109 11-8

3-0

5

5

5

6 6 6

In contrast to these results with normoxic-hypercapnic stimuli, elevations of P 0, under hyperoxic conditions (Table 2) resulted in no significant changes in TI, TE or TTOT of apneusis; the apneustic depth did increase significantly as PA, Co, was raised from 37 to 52 torr. This absence of statistically significant changes reflected the non-systematic changes in TI, TE and TTOT of the six experimental animals (Fig. 3). B. Response to PA, 0, dimunitions at isocapnia (1) Animals having intact carotid chemoreceptors. Changes in PA, 0 from hyperoxia to normoxia at isocapnia (Table 3) resulted in no statistically significant changes in T1, TTOT or depth; TE declined significantly. Further diminutions in PA, °, produced a profound alteration of apneusis. Thus, as reported in Table 3, a change in PA, 0, from normoxic to hypoxic levels caused significant diminutions in TI, TE and TTOT. The absence of significant changes in the apneustic depth reflected a decrease in depth by one animal as PA, 02 was lowered to both hypoxic levels; a second animal also showed a decrease in depth at the lowest PA, O,. The mean

W. M. ST JOHN 476 arterial blood pressure was not significantly altered by exposure to either hypoxic level. A typical experimental record of hypoxia-induced changes in apneusis appear in Fig. 4. (2) Animals having bilateral carotid sinus nerve section. Diminutions in PA, 0, from hyperoxic to normoxic levels at isocapnia produced no significant changes in 100 _ -7 x

E 0-0

50

r

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50

100

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40

50

60

70

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Fig. 3. Values of TI, TE, TTOT and depth during apneusis at the specified levels of PA,co, for each individual animal having bilateral carotid sinus nerve section. PAco, was altered under hyperoxic conditions. TI, TB, TTOT and depth are expressed as a percentage of the maximum value obtained (% max.). See also legend for Fig. 2.

As shown in Fig. 5, further decreases in PA, 0 to approximately 75 torr resulted in a decrease in apneustic depth and variable changes in TI, TE and TTOT for four of the five experimental animals

TI, TE, TTOT or the depth of apneusis.

APNEUSTIC RESPIRATORY PATTERNS 477 examined. Values for the fifth experimental animal are not presented for a PA, 0. level approximating 75 torr. This cat (no. 5) exhibited an alteration in the respiratory pattern from apneusis to gasping subsequent to hypoxic exposure. This latter respiratory pattern was qualitatively similar to that which previous investigators have also described as 'gasping' (e.g. Lumsden, 1923, fig. 3; Wang et al. 1957, fig. 3). Subsequent to diminutions of PA 0, to 60 torr, profound alterations of the apneustic respiratory pattern were obtained in all experimental animals. Similar changes were also produced by repeated diminutions in PA 0, to 76-73 torr at differing PA, co, levels. These changes were of three types: (1) a diminution in the depth of apneusis with variable changes in T, and TE (e.g. Fig. 6), (2) an onset of gasping-type activity, and (3) expiratory apnea. As shown in Fig. 6, these changes TABLE 3. Alterations of apneusis by diminutions of end-tidal 02 partial pressure in cats having intact carotid sinus nerves under isocapnic conditions. Mean values+s.E. are presented for TI, Tx, TTOT and depth of apneusis and for mean arterial blood pressure (B.P.) at the appropriate PACO, and PA,O2 levels. Panel A values reflect PAO, changes from hyperoxia to normoxia. Changes obtained subsequent to normoxic-hypoxic transitions are reported in panels B and C. n is the number of experimental animals examined. a = P < 0 05 compared to hyperoxic (A) or normoxic (B, C) values (Wilcoxon test, two-sided)

PAc,C02 Mean

5.E.

Mean

5.E.

PAO

T1

(torr)

(torr)

(sec)

60 3.5 59 4-1

606 8-63

198 160 182 143

131a 4*39

TB

TTOT

Depth

(sec)

(units)

240 163 209 141

28 59 28 3.2

122 9.73 132

23 4.1 30 3.4

135 7.56 140 9.47

8

69.2

332 96.6 166a 81X3

332 157

467 205

24 3.4

136 8.16

7

119 13*1

7

(sec) A 42

7*2 27a

6*0 B 253

Mean

56

122

5.E.

2-3 56 2.2

5*31

0*9

78 17 57 16

5.E.

55 2-2

124 2*76

134 55.1

Mean 5.E.

55

59a

50a

51a

101a

30

2.1

1.4

19

22

28

3.4

Mean 8.E.

76"

90*7

109"

B.P. (mmHg) n 6

6

11-6

8

C Mean

in respiratory pattern were time dependent after the onset of hypoxia. Frequently, within a short interval (< 30 see), the gasping and/or apneic periods succeeded the altered apneustic patterns. Elevations in PA 0, could result in the re-establishment of the 'normal' apneusis (Fig. 6); however, in some animals, the gasping-type respirations continued. Thus, it was not possible to obtain accurate steady-state determinations of the changes in apneusis resulting from diminitions in PA, 0, in these animals having carotid sinus nerve sections. C, Response to P A, co, elevations under hypoxic conditions (1) Animals having intact carotid chemoreceptors. Elevations of PA, CO, under hypoxic conditions (PA 02 approx. 75 torr) resulted in no significant changes in

W. M. ST JOHN TI, TE, or TTOT; the apneustic depth did increase significantly (Table 4). This lack of significant changes in the timing of apneustic breaths reflected non-systematic alterations of TI, TE and TTOT in each individual animal. A similar lack of significant changes was noted for PA co, elevations at PA, 0 levels approximating 60 torr (Table 4). The mean arterial blood pressure of each animal usually rose with these PcoA elevations.

478

A

Phr

0-10

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(2) Anim~als having bilateral carotid tinus nerve 8ectionB. Animals having bilateral carotid sinus nerve section exhibited time-dependent changes in the respiratory pattern after the onset of hypoxia (see Section I-B-2 above). Thus, no steady-state determinations of changes in phrenic activity which resulted from hypoxic hypercapnic stimuli could be obtained in these cats.

APNEUSTIC RESPIRATORY PATTERNS

479

II. Animals exhibiting continuous expiratory apnea As noted above, five experimental animals exhibited a prolonged expiratory period (TE > 30 min) subsequent to pneumotaxic centre ablation and bilateral vagotomy. The carotid chemoreceptors were intact in three of these cats whereas the other two animals had bilateral carotid sinus nerve section. It was found that 150

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480

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100 torr). Finally, an examination of the procedures used by Tang (1967) leads to the conclusion that animals in his study which exhibited increases in apneustic T, in response to hypercapnia represented determinations on that portion of the TI-PA, co0 curve at which, as reported herein, T, increases with PA, CO2 elevations. In contrast to these results of Ngai (1957), Tang (1967) and St John & Wang (1977 a) which, I believe, are confirmed by the present report, results detailed herein are in conflict with those of Euler et al. (1976). These investigators report that, in pentobarbital anaesthetized or decerebrate cats, 'changes in PCO2 were not accompanied by major or consistent changes in the duration of the apneustic inspiration ' (Euler et al. 1976, p. 335). The experimental procedures utilized by Euler et al. differed from those of the previous reports noted above and the present study in at

486

W. M. ST JOHN

least two potentially significant aspects. First, although small bilateral lesions within the nucleus parabrachialis medialis complex were usually placed in the Euler et al. study, in several experiments n.p.b.m. lesions were only unilateral. Secondly, in that study, the vagi were apparently only sectioned in some experiments. In the remaining paralysed animals, phasic afferent activity from pulmonary stretch receptors was eliminated by a cessation of artificial ventilation. It is not clear from the data of Euler et al. (1975) how many, if any, cats having unilateral n.p.b.m. lesions and/or functional rather than surgical, vagotomies were included in the evaluations of hypercapnia-induced changes in T, and TE. Yet, if any such cats were included, it is possible that activity from the intact n.p.b.m. (Euler & Trippenbach, 1975) or the fall in PA 0, during cessation of ventilation (see, Euler et al. 1975, p. 330) might have contributed to a premature termination of the apneustic inspiratory activity. In any case, the above discussion provides considerable support for the recent contention of Sears (1977) that such terms as apneusis should only be used 'to describe the phenomenons under consideration coupled with the relevant anatomical descriptions of the location of the experimental procedures used to study them'. Although the data of the present study, discussed above, are conflicting with those of Euler et al. (1975), other theoretical and experimental results from the Euler laboratory are confirmed and extended by the present findings. In 1975, Bradley et al. presented 'a model of the central and reflex inhibition of inspiration in the cat'. This model, which has been extensively considered in many contemporary papers (e.g. Cohen & Feldman, 1977; Euler, 1977) will be considered herein only as to those salient features which are directly applicable to the present report. Briefly, therefore, this model considers that inspiratory activity (CIE) is generated at some undefined site within the brain stem respiratory controller. This inspiratory activity proceeds to the respiratory motoneurones of the spinal cord and also, in parallel, to a 'pool' wherein it is summated with activity from pulmonary vagal afferent fibres. Efferent activity from this pool of summated inspiratory and vagal activity is conveyed to a 'switch neurone pool'. When the threshold of these switch neurones is reached, the inspiratory activity generation is inhibited and inspiration terminated (inspiratory off-switch, I-E switching). An early observation from Euler's laboratory, which indeed preceded the publication of the above model, was that the magnitude of pulmonary inflation required to terminate inspiration decreased with time after inspiratory commencement (Clark & Euler, 1972). Early in inspiration, CIE would be minimal and, hence, larger inflations would be required to reach the threshold of the switch neurone pool than would be needed in late inspiration when such activity would be high. The threshold of this switch neuronal pool is considered to be influenced by a variety of factors. For the purposes of the present report, two of these factors are of primary importance: efferents from the n.p.b.m., acting mainly to lower the threshold (Euler & Trippenbach, 1975), and elevations of CO2 (presumably increased central chemoreceptor activity) causing an elevation of threshold (Bradley, Euler, Marttila & Roos, 1975; Euler, 1977). The characteristics of switching from expiration to inspiration expiratoryy offswitch, E-I switching) have not been examined in as great detail as the I-E switching

APNEUSTIC RESPIRATORY PATTERNS

487 discussed above. Nevertheless, as with I-E switching, it has been found that the intensity of the stimulus required to produce a premature inspiration decreases with time after the beginning of the expiratory phase. Stimuli which are capable of promoting an E-I transition in this manner include electrical stimulation of the dorsolateral pontile tegmentum (Cohen, 1971), somatic afferent stimulation (Iscoe & Polosa, 1976) or lung deflation (Knox, 1973). Subsequent to the combined processes of pneumotaxic centre ablation and vagotomy, the threshold of the I-E switch neuronal pool would be elevated and, given a constant level of inspiratory activity, the activity impinging upon these switch neurones from the pool of summated inspiratory and vagal activities would, quite obviously, be reduced. It is thus quite easy to visualize how, as Euler et al. (1975) describe, if inspiratory activity were to saturate before the elevated threshold of the switch neuronal pool is reached, inspiration would be rather continuous and apneusis would result. The mechanisms whereby the inspiratory off-switch is finally activated to terminate an apneutic inspiration are largely undefined. However, in studies of animals having n.p.b.m. lesions, Euler et al. (1975) found that the volume of lung inflation which was sufficient to terminate inspiration decreased to a constant level as the time of inspiration increased. This constant volume threshold ultimately decreased towards zero close to the time for the spontaneous termination of the apneustic inspiration. Comparable studies of the threshold for expiratory off-switch have been performed in animals having bilateral 'apneusis-producing' pontile lesions (Knox & King, 1976). In these animals, it was found that the volume of deflation necessary to elicit a premature inspiration was equal to that of unlesioned cats early in expiration; later in expiration, this volume threshold decayed more slowly in cats having the pontile lesions. However, within 3 0 sec after the beginning of expiration, the volume threshold for E-I switching was close to zero (Knox & King, 1976). Unfortunately, longer expiratory periods were not examined and, hence, the possibility of a constant, but small, threshold for E-I switching, similar to that described above for I-E switching, must remain as a viable possibility. Results of the present study would certainly support the concept that the threshold of the inspiratory off-switch mechanism is elevated following bilateral n.p.b.m. lesions and bilateral vagotomy. Moreover, results herein also imply that the threshold of the expiratory off-switch mechanism is similarly elevated or, at least, decays much more slowly. As noted in Results, T1 and TE values in excess of 600 sec were not unusual in animals exhibiting a spontaneous, cyclical apneustic breathing pattern. If it is assumed that the duration of the inspiratory and expiratory phases reflect, in some manner, the degree of elevation of the thresholds for respiratory phase-switching, then it is perhaps not surprising, given the very long T1 and TE values noted above, that, in some animals, the thresholds of the inspiratory and/or expiratory off-switch mechanisms would never be attained. Hence, in these animals, inspiration or expiration would be continuous. In addition to these elevations of threshold which occur per se subsequent to n.p.b.m. lesions and vagal section, results herein also demonstrate that these threshold levels may be altered by hypercapnia and hypoxia. According to the model of Bradley et al. (1975), elevations of PA, CO, produce

W. M. ST JOHN both an increase in CIE and an increase in the threshold of the inspiratory off-switch mechanism. Results of the present study are consistent with this hypothesis for, 488

subsequent to elevations of PA, Co. from control levels, both the apneustic depth (i.e. increased inspiratory activity) and the inspiratory duration (increased off-switch threshold) were augmented. These results moreover imply that the hypercapniainduced increase in inspiratory activity does not match the change in threshold. If such a match were obtained, it would be expected that the duration of the apneustic inspiration would remain constant as the depth increased. In this context, it is believed that the decreases in T, observed in many animals subsequent toPA, CO, elevations above approx. 50 torr may represent another indication of a mismatch between changes in CIE and those of the inspiratory off-switch threshold. This alteration of TI, occurring in many animals concomitant with a further increase in apneustic depth, may imply a saturation of the off-switch threshold, but not of inspiratory activity, at high PA, Co, levels. In contrast to these hypercapnia-induced changes, results obtained subsequent to PA, °2 diminutions imply that hypoxia simultaneously causes an increase in inspiratory activity and a reduction in the inspiratory and expiratory off-switch thresholds. Further, I believe that the data of this report are consistent with the conclusion that hypoxia-induced alterations of apneusis are the net result of carotid chemoreceptor stimulation and brain stem respiratory controller depression. In support of this conclusion, it was noted that a hypoxia-induced increase in apneustic depth, reflective of an increase in inspiratory activity, was obtained only in those animals having intact carotid chemoreceptors. A direct pharmacological stimulation of these carotid chemoreceptors by NaCN administration also increased this apneustic depth. The ability of these cyanide injections to terminate an ongoing apneustic inspiration is again explicable if it is considered that carotid chemoreceptor stimulation increases inspiratory activity. Thus, it follows directly from the model of Bradley et al. (1975) that a marked rise in such activity, occurring at a constant threshold of the switch neuronal pool, would greatly increase the probability of inspiratory off-switch activation. In contrast to these results obtained with animals having intact carotid chemoreceptors, hypoxia caused a time-dependent decrease in apneustic depth in animals having bilateral carotid sinus nerve sections. However, diminutions in PA °8 caused a decrease in T, and TE in animals having intact carotid sinus nerves and moreover, terminated expiratory apnea and established a cyclical pattern of apneustic breathing both in these cats and those having carotid sinus nerve sections. These results are most consistent with the conclusion that generalized central nervous system hypoxia decreases inspiratory activity and also the thresholds for both the inspiratory and expiratory off-switch mechanisms. The ability of general somatic stimulation or carotid chemoreceptor stimulation to similarly cause a phase switch of the apneustic respiratory cycle from expiration to inspiration may not be due, I believe, to a similar mechanism as that caused by central nervous system hypoxia. As noted above, the threshold level of the expiratory off-switch mechanism seemingly decays with time (see, Euler, 1977) since the stimulus intensity required to promote an E-I transition decreases as expiration progresses. It is thus possible that during a sustained expiratory period, only a minimal stimulus, promoting a

APNEUSTIC RESPIRATORY PATTERNS

489

rise in inspiratory activity, would be required to terminate expiration and produce a full inspiratory period. If this hypothesis is correct, it would be expected that the stimulus intensity required to promote an E-I transition should decrease with time after the termination of an apneustic inspiratory period or,, correspondingly, an equivalent stimulus should be more effective in promoting an E-I transition the later in the expiratory phase in which it was delivered. In this context, it was observed in several experiments that 'touch' was ineffective in promoting an E-I transition is delivered immediately after inspiratory termination; thus, somatic stimulation was effective only after a finite expiratory period. In contrast to these time-dependent effects of somatic stimulation, NaCN administration produced an E-I transition regardless of the time of injection during the expiratory phase. However, no systematic dose-response curve for NaCN was constructed and it is thus possible that the doses used in this study, while very small (i.e. 1 0-20 jug), still produced a rise in inspiratory activity which was supramaximal for E-I transition. In this context, it must be noted that Eldridge (1972) found that a variety of chemical stimuli to the carotid chemoreceptors (including NaCN) caused no inspiratory movements if delivered during the expiratory phase. However, in some animals, this carotid chemoreceptor stimulation 'was associated with a small brust of tiny phrenic nerve action potentials' (Eldridge, 1972). While there is no obvious explanation for this difference in results, yet this difference might imply that carotid chemoreceptor stimulation is a more efficacious ventilatory stimulus in animals deprived of pneumotaxic and vagal mechanisms. Two additional observations of this study require some consideration. The first of these was the absence of systematic changes in TI, TE or TTOT of cats having carotid sinus nerve sections upon PA, C2 elevations under hyperoxic conditions. While there is no firm basis to explain this absence of statistically significant changes, yet it was observed that large, spontaneous fluctuations in arterial blood pressure occurred in these animals, especially when PA, CO, was elevated to approximately 70 torr. Thus, it is entirely possible that the relative brain stem oxygen levels may have changed with time even under steady-state conditions of PA, co, and PA, o, The second unusual observation was the positive response of a single animal to saline injected into the external carotid artery cannulae. Systemic saline injections produced no effect upon ventilation in this animal and, moreover, carotid sinus nerve section eliminated the response to intra-arterial saline and also NaCN injections. It is thus only possible to speculate that, for some undefined reason, the carotid chemoreceptor mechanisms of this animal were sensitive to saline. In summary, it is believed that the results of this study are of interest when considered in the context of the 'apneustic breathing pattern' which has been frequently described in spontaneously breathing animals. Thus, e.g. Breckenridge & Hoff (1950), Ngai (1957), and Wang et al. (1957), all report that the apneustic breathing pattern was characterized by a long period of inspiratory apnea terminated by a spontaneous expiration. One or more brief respiratory excursions then occurred after which another prolonged inspiratory phase was noted. Results of the present study imply that progressive brain stem hypoxia might have been a contributing factor to the spontaneous termination of the prolonged inspiratory period. In view of the finite circulation time from the lungs to the brain stem, it would be expected

W. M. ST JOHN that the subsequent inspiratory excursion would reach a saturation level while the inspiratory and expiratory off-switch thresholds were still depressed by the brain stem hypoxia. Thus, relatively brief inspiratory and expiratory periods would be expected. Only when brain stem oxygenation had risen sufficiently to elevate these off-switch thresholds would another prolonged inspiratory period be noted. Despite the results of the present study, which demonstrate and characterize the differential effects of hypercapnia and hypoxia upon apneusis, we still have no firm indication as to those neural mechanisms directly responsible for spontaneous commencement or termination of an apneustic breath. This statement is most provocative when it is considered that such a spontaneous respiratory-phase switching can occur after 30 min of continuous inspiration or expiration. Elucidation of the neurophysiological basis of these I-E and E-I switching mechanisms is inherent to a major and central unexplained problem of respiratory control. This problem is, of course, the neurophysiological mechanisms responsible for the genesis of the respiratory cycle. 490

The author expresses his appreciation to Dr Donald Bartlett for his review of this manuscript, and assistance in the preparation thereof. The technical and/or secretarial assistance of Mrs Gail Lyman and Mr Kurt Knuth is likewise gratefully acknowledged. This work was supported by grants from the National Heart, Lung, and Blood Institute - National Institutes of Health (research grant 20574, Research Career Development Award 00346) and from the Parker B. Francis Foundation. REFERENCES

BERTRAND, F. & HUGELrN, A. (1971). Respiratory synchronizing function of nucleus parabrachialis medialis: pneumotaxic mechanism. J. Neurophyaiol. 34, 189-207. BRADLEY, G. W., EULER, C. VON, MEARLA, I. & Roos, B. (1975). A model of the central and reflex inhibition of inspiration in the cat. Biol. Cybernetic8 19, 105-116. BRECKENRIDGE, C. G. & HoFT, H. E. (1950). Pontine and medullary regulation of respiration in the cat. Am. J. Phyfiol. 160, 385-394. COiRK, F. J. & EULER, C. VON (1972). On the regulation of depth and rate of breathing.

J. Physiol. 222, 267-295. CoHEN, M. I. (1971). Switching of the respiratory phases and evoked phrenic responses produced by rostral pontine electrical stimulation. J. Physiol. 217, 133-158. COHEN, M. I. & FELDMAN, J. L. (1977). Models of respiratory phase-switching. Fedn Proc. 36, 2367-2374.

CROUCH, J. E. (1969). Text Atla8 of Cat Anatomy, pp. 212-214. Philadelphia: Lea & Febiger. ELDRIDGE, F. L. (1972). The importance of timing on the respiratory effects of intermittent carotid body chemoreceptor stimulation. J. Phy8iol. 222, 319-333. EuLER, C. VON. (1977). The functional organization of the respiratory phase-switching mechanisms. Fedn Proc. 36, 2375-2380. EuMER, C. VON, MARTarLA, I., REmMERs, J. E. & TRIPPENBACH, T. (1976). Effects of lesions in the parabrachial nucleus on the mechanisms for central and reflex termination of inspiration in the cat. Acta physiol. 8cand. 96, 324-337. EuLER, C. VON & TRIPPENBACH, T. (1976). Excitability changes of the inspiratory 'off-switch' mechanism tested by electrical stimulation in nucleus parabrachialis in the cat. Aaa phy8iol. 8cand. 97, 175-188. FITZGERALD, R. S. (1973). Relationships between tidal volume and phrenic nerve activity during hypercapnia and hypoxia. Acta neurobiol. exp. 33, 419-425. GAuT' , H. (1976). Pattern of breathing during hypoxia or hypercapnia of the awake or anesthetized cat. Resp. Physiol. 27, 193-206. HALDANE, J. S., N8, J. C. & PRIESTLEY, J. G. (1919). The respiratory response to anoxaemia. J. Phy8iol. 52, 420-432.

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ISCOE, S. & POLOSA, C. (1976). Synchronization of respiratory frequency by somatic afferent stimulation. J. apple. Phy8iol. 40, 138-148. KNox, C. K. (1973). Characteristics of inflation and deflation reflexes during expiration in the cat. J. Neurop-hy8ai. 36, 284-295. KNox, C. K. & KMIG, G. W. (1976). Changes in the Breuer-Hering reflexes following rostral pontine lesion. Re.&p. Phy~sol. 28, 189-206. LuM5DEN, T. (1923). Observations on the respiratory centres in the cat. J. Phy8iol. 57, 153-160. Nag, S. H. (1957). Pulmonary ventilation studies on pontile and medullary cats. Changes in 02 consumption, in arterial blood pH, CO2 tension and 02 saturation, and in response to CO, and cyanide. Am. J. Phyiol. 190, 356-360. ST JOHN, W. M., GLAssER, R. L. & KYNG, R. A. (1971). Apneustic breathing after vagotomy in cats with chronic pneumotaxic center lesions. Rewp. Phy8iol. 12, 239-250. ST JOHN, W. M. (1975). Differing responses to hypercapnia and hypoxia following pneumotaxic center ablation. Re&p. Phy9iol. 23, 1-9. ST JOHN, W. M., BoiD, G. C. & PASLEY, J. N. (1975). Integration of chemoreceptor stimuli by rostral brainstem respiratory areas. J. apple. Phyaiol. 39, 209-214. ST JOHN, W. M. & WANG, S. C. (1976). Integration of chemoreceptor stimuli by caudal pontile and rostral medullary sites. J. apple. Phy8iol. 41, 612-622. ST JOHN, W. M. (1977). Integration of peripheral and central chemoreceptor stimuli by pontine and medullary respiratory centers. Fedn Proc. 36, 2421-2427. ST JOHN, W. M. & WANG, S. C. (1977a). Alteration from apneusis to more regular rhythmic respiration in decerebrate cats. Reap. Phyuiol. 31, 91-106. ST JOEM, W. M. & WANG, S. C. (1977b). Response of medullary respiratory neurons to hypercapnia and isocapnic hypoxia. J. appi. Phyaiol. 43, 812-822. SEUAR, T. A. (1977). The respiratory motoneuron and apneusis. Fedn Proc. 36, 2412-2420. STELLA, G. (1938a). On the mechanism of production and the physiological significance of ' apneusis'. J. Phy8iol. 93, 10-23. STELLA, G. (1938b). The dependency of the activity of the 'apneustic' centre on the carbon dioxide of the arterial blood. J. Phy8iol. 93, 263-275. STELLA, G. (1939). The reflex response of the 'apneustic centre' to stimulation of the chemoreceptors of the carotid sinus. J. Physiol. 95, 365-372. TANG, P. C. (1967). Brain stem control of respiratory depth and rate in the cat. Resp. Phy8wl. 3, 349-3 66. WANG, S. C., NGM, S. H. & Fiumr, M. J. (1957). Organization of central respiratory mechanisms in the brain stem of the cat: genesis of normal respiratory rhythmicity. Am. J. Physaiol. 190, 333-342.