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Importance of sympathetic mechanism on cardiac arrhythmias induced by picrotoxin
EXPERIMENTAL
NEUROLOGY
Importance
of
36, 389-398
(1972)
Sympathetic
Arrhythmias
Mechanism
Induced
by
on
Cardiac
Picrotoxin
T. M. LEE, K. L. YANG, J. S. Kuo, AND C. Y. CHAI l Departnzertt of Biofihysics and Kohlberg Memorial Medical Research Laboratory, National Defense Medical Center and Department of Medical Research, Veterans Gerlrral Hospital, Taipei Mmicipality, Republic of China Rcceiked
.4pril
17, 1972
The effects of picrotoxin administered via various routes on cardiac arrhythmias were studied in chloralose anesthetized cats. It was found that the efficiency of arrhythmias production in term of dose required and the latency of onset decreased from the order of intracerebral, intracerebroventricular (fourth ventricle) and intravenous injection. Intracardiac injection was ineffective. Among the sites of intracerebral injection, the pressor region of the medulla oblongata was most effective. The pressor region of the posterior hypothalamus came next. Direct injection of picrotoxin into the depressor region of the anterior hypothalamus and the vagal areas of the medulla, i.e., nucleus and tractus solitarius, ambiguus nucleus, did not produce arrhythmias. Topical application of picrotoxin on spinal cord (T,-,) produced cardiac arrhythmias in half of the animals tested. Vagotomy did not affect the arrhythmias induced by intracerebral and spinal injection. The picrotoxin-induced arrhythmias were not affected by midcollicular and additional transection of the brain stem unless the level of lower medulla was reached. The data suggest that picrotoxin induced cardiac arrhythmias by activation of the sympathetic mechanism of the central nervous system, mainly of the medulla. The integrity of the vagal mechanism is not indispensable. Introduction
On comparison of the dose-route relationship of intravenous, subarachnoid (cortical) and intracerebroventricular administration of picrotoxin, Bircher Kanai and Wang (1) observed that the fourth ventricular route was most effective in inducing cardiac arrhythmias. These investigators therefore, suggested that a mechanism sensitive to picrotoxin possibly resides in the structures of the fourth ventricle for arrhythmias production. No attempt was made, however, to verify this contention in decerebrate animals and to identify the specific region in the medulla involved. Bircher, 1 This project was supported in part by Grants from the China Medical Board of New York, Inc. and the International Foundation of U. S. A. The investigators thank Drs. C. T. Loo and T. M. Peng for their encouragement and support throughout the course of this investigation. 389 Q 1972 by Academic Press, Inc. All rights of reproduction in any form
reserved.
390
LEE
ET
AL.
Kanai and Wang (2) observed that both parasympathetic and sympathetic pathways were involved in picrotoxin-induced cardiac arrhythmias (PIA), but the parasympathetic mechanism was more prominent. Vagotomy or intravenous administration of anticholinergic agents prevented .W-80% of the cardiac arrhythmias induced by intracerebroventricularly injected picrotoxin. After total sympathectomy, low spinal cord transection or reserpine treatment, picrotoxin still induced marked bradycardic arrhythmias and A-V block (2). However, many experimental data have proved that centrogenic cardiac arrhythmias can be produced by activation of the sympathetic system alone (3, 4, 9). Therefore the importance of sympathetic mechanismon PIA cannot be neglected. The present investigation was undertaken to determine whether the medulla is essential for PIA and whether the sympathetic or the parasympathetic mechanismin the brain stem is more sensitive to intracerebrally injected picrotoxin. Methods
Seventy-two cats of both sexes, weighing Z&3.2 kg, under chloralose were used. They were immobilized when necessary with w/k, ip) succinylcholine chloride (0.2-0.5 mg, iv). Rectal temperature was maintained at 3% throughout the course of experiment by irradiation with infrared light. The trachea was cannulated for artificial ventilation and the femoral artery was catheterized and blood pressure monitored with a Statham P23AC transducer. Heart rate was monitored with a Grass 5P4 tachometer triggered by arterial pulses. Electrocardiogram lead I or II was monitored bv a Grass 5P4 preamplifier. The femoral vein was cannulated for intravenous injection. For intracardiac injection, the left ventricle was cannulated with a polyethylene tubing through the right subclavian artery. For intracerebroventricular or intracerebral injections, the animal was placed in a prone position and the head was fixed in a David Kopf stereotaxic instrument. A spinal needle (G26) was positioned into the fourth ventricle or into the desired region of the hypothalamus or medulla oblongata. The needle used for intracerebral injection was insulated except for 2 mm at the tip. So it might also be used as a unipolar electrode for effecting electrical stimulation by passing through d-c current derived from a Grass S4 stimulator. Decerebration was completed by removal of all the brain tissue rostra1 to the bony tentorium, as was described ( 17). The animal was killed at the end of experiment, and the head was perfused with physiological saline solution followed by 10% formalin. The correct position of the intracerebroventricular cannula was examined grossly and the site of intracerebral injection was identified by histological sections stained with Weil’s method. (40
CARDIAC
ARRHYTHMIAS
391
Results
The results of picrotoxin administered via various routes with regard to dose of administration, vasomotor and cardiac changes, incidence of onset and duration of PIA and the effects of vagotomy are summarized in Table 1. I&ravenozu (iv) and Intvacerebroventricular (ivt) Injection. Picrotoxin 4 mg/kg iv or 60-100 pg ivt produced cardiac arrhythmias in all four animals tested concomitant with an increase of the mean blood pressure (avg 52 and 59 mm Hg) and decrease of heart rate (avg 85 and 100 beat/min). The arrhythmias consistedof atria1 tachycardia, supraventricular and ventricular premature contractions and nodal and sinus premature systoles. The latency of onset was faster in ivt than iv injection. In eight other animals (four iv and four ivt) the effects of vagotomy on the arrhythmias were tested. Vagotomy abolished the PIA in threefourths of the animals after iv and half of the animals after ivt injection. Eight animals (four iv and four ivt) not included in Table 1 were subjected to midcollicular decerebration and to additional transection at the brain stem after PIA had developed. In these, decerebration did not affect the arrhythmias, nor did additional transection of the brain stem at the caudal pons or midmedulla. The PIA were abruptly terminated by subsequent transection at the caudal medulla (Fig. 1). Intracardiac Injection. Picrotoxin, ZOO-300 pg. directly injected into the left ventricle of the heart produced slight change of the blood pressure and the heart rate. Further increase of the dose to 900 /lg caused muscular twitching or even convulsion but cardiac arrhythmias still were not observed (four animals). Intracerebral Injection of Picrotoxin. The effects of intracerebral injection depended on the site of injection: Picrotoxin, 30-45 pg, injected bilaterally (four animals) in the lateral or posterior hypothalamus, in which electrical stimulation elicited moderate to marked pressor response and cardioacceleration, produced similar increase of blood pressure (avg 64 mm Hg) and heart rate (30 beat/min) and cardiac arrhythmias. Unilateral injection (four animals) produced similar effects, but the latency of onset of arrhythmias was longer 8.7 against 7 min). Unlike that of ivt administration, intrahypothalamic picrotoxin never produced reflex bradycardia although the systolic blood pressure had increased to 250 mm Hg or higher. Vagotomy neither could prevent nor terminate these arrhythmias (another 6 animals). In three of the four animals, bilateral administration of picrotoxin, 3045 pg, into the anterior hypothalamus, in which electrical stimulation produced moderate depressor and bradycardic effect, only caused a slight increase of blood pressure (avg 14 mm Hg) and heart rate (average 16 beat/min) ; PI.4 were observed only in one animal who developed marked
cord
u Asterisk(*) h Mean f
indicates SE.
Medulla oblongata Pressor (bilateral) Pressor (unilateral) Nucleus and tractus solitarius or ambiguus nucleus (bilateral)
I ntracerebral Hypothalamus Pressor (bilateral) Pressor (unilateral) Depressor (bilateral)
Spinal
another
16-24 16-24 1624
30-4.5 30-45 30-4.5
group
of experiment.
4/4 818 0.‘6
4/4 l/4
4/a
214
800-1200
O/4
414
of animals tested
4/4
No.
No. of observation
\?A VAKIOUS CAKDIOVASCULAH
60-100
200-300
Intracardiac
IntracerebroventricuIar
8000-12000
Dose (I.%)
OF PICKOTQXIN OTHER
Intravenous
Route of administration
EFFECTS
0.3 0.35
(0.2-0.5) (0.2-0.7)
7 (S-8) (7710)
9 (7-12!
8 (S-12)
12 (8-15)
Onset average (range) (mm)
8.5
1
8 (7-9) 4 (2-S)
(3-6)
(15-22)
10 (8-12) 5 (4-9)
4.5
17.6
18 (15-24)
Duration average (range) (min)
OF ADMINISTKATION RESIWNSIB INDUCED
PIA
ROUTES
TABLE
No.
o/4+ o/4*
O/6* o/4* l/l
64 f SO f 14 f
20.9 16.7 4.4
8.1
13.6
2.4
21.3
+154 =t 36.3 +142 f 39.8 + 23 f 18.6
+ + +
+39zt
o/2
59 f
8f
+ +
52 f
+
2/4*
3/4*
of PIA
AND
Average change of mean blood pressure (mm Hd
AKKHYTHMIAS
No. of abolition after vagotomy a
ON CAKDIAC RY PKK~TOXIN
30 + 28 f 16 f
26 f
85 f
14 f
f
15.1 6.4 8.3
7.4
14.8
6.2
25.4
+ 71 31 24.7 $- 64+22.1 + 30 f 14.4
+ + +
+
-
+
-100
Average change of heart rate h (beat/mm)
s
2
w E
CARDIAC
393
ARRHYTHMIAS
hypertension and tachycardia and the PIA were terminated by vagotomy. Picrotoxin, 16-24 pg, was injected bilaterally (four animals) into pressor loci of the medulla oblongata, in which electrical stimulation elicited marked pressor and cardioaccelerator effects. The blood pressure and heart rate instanteously increased to 154 mm Hg and 71 beat/min in average and PIA occurred in less than 30 set after the injection. Unilateral injection (eight animals) was similarly effective but the PIA lasted shorter (Table 1 ; Fig. 2). Vagotomy did not affect these PIA. Convulsion or muscular twitching was not observed. In six animals picrotoxin, 16-24 pg, was injected into the nucleus and tractus solitarius or the ambiguus nucleus of both sides, in which electrical stimulation elicited marked decrease of blood pressure and heart rate. The injection produced an initial bradycardia for about lo-15 set after with a slight elevation of the mean blood pressure 23 mm Hg and of the heart rate 30 beat/min in average. PIA were not observed in all these animals (Fig. 3). Spinal Cord. Picrotoxin, 800-1200 pg, injected to the subarachnoid space of the spinal cord at T,.? through an implanted polyethylene tubing, produced an increase of blood pressure (avg 39 mm Hg) and heart rate
2 150 9
*
b
c
b
C
wi-
d /
1. Effects on ECG, blood pressure, and heart rate of midcollicular decerebraand additional transection at the lower brain stem on cardiac arrhythmias induced by picrotoxin. a. Ten minutes after picrotoxin, 4 mg/kg, iv. Note the hypertension and marked bradycardia (control blood pressure and heart rate were 110/75 mm Hg and 210 beats/min). b. Twelve minutes after picrotoxin ventricular premature contraction occurred. c. Cardiac arrhythmias still persisted after midcollicular decerebration, and additional transection at the level of the lower pons (d), and middle medulla (e). f. The arrhythmias were abruptly terminated after transection at the lower medulla. FIG.
tion
FIG. 2. Effects of vagotomy on cardiac arrhythmias induced by injection of picrotoxin into one side of the pressor region of the medulla oblongata. The left panels show the pressor responses upon electrical stimulation of the pressor region. a. Control. b. Right after the injection of picrotoxin, 20 pg, into the pressor region (first arrow), blood pressure was markedly increased concomitant with reflex bradycardia. Twenty seconds later cardiac arrhythmias appeared. c-e. Cardiac arrhythmias were still evident after bilateral vagotomy (second arrow.) f. Sinus tachycardia remained through out the rest of the course. Calibration lines: 20 set, and 1 sec.
(avg 26 beat/mm) ; PIA which were not affected by vagotomy occurred in two of the four animals tested. Muscular twitching or convulsion was evident in all the animals. Discussion
Cardiac arrhythmias induced by picrotoxin are central in origin and are possibly induced by activation of the medullary mechanism (1, 2). Indeed, injection of picrotoxin into the left ventricle of the heart did not produce cardiac arrhythmias or apparent changes of blood pressure and heart rate despite the dose had been so increased as to cause muscular twitching. Intravenously or intracerebroventricularly injected picrotoxin induced cardiac arrhythmias and the arrhythmias were not affected by midcollicular decerebration, with or without additional transection at the
---.
*
-.-
FIG. 3. Cardiovascular changes induced by injection of picrotoxin into the nucleus and tractus of solitarius on both sides of the medulla oblongata. The two panels on the left side show the depressor responses upon electrical stimulation of the vagal loci. a. Control. b and c. Within 8 min after the administration of picrotoxin. 20 pg, into the vagal loci. Note the insignificant changes of blood pressure and heart rate. Arrow : picrotoxin injection. Calibration lines : 20 set, 20 set, and 1 sec.
CARDIAC
FIG.
4. Sites
of injections
giving
395
ARRHYTHMIAS
results
shown
in Fig.
2 (right)
and
Fig.
3 (left).
upper and middle medulla. Abolition of the arrhythmias were evident only when transection was made at the lower medulla. However, the medulla is not the exclusive structure responsible for PIA. Intracerebral injection of picrotoxin into the hypothalamus also produced cardiac arrhythmias although the dose of picrotoxin required was bigger and the latency of onset longer. Moreover, topical application of picrotoxin onto the spinal cord also produced cardiac arrhythmias. The relative importance of the sympathetic and parasympathetic mechanism to cardiac arrhythmias induced by activation of the central nervous system has been the subject of interest. The importance of the vagus mechanism has been emphasized. Stimulation of the pressor regions of the lateral and posterior hypothalamus more readily produced cardiac arrhythmias after the stimulation had terminated and when the elevation of blood pressure was still at the plateau (6, 10). Therefore, reflex activation of the vagus system contributes partially to these poststimulatory arrhythmias (13, 15). That the genesis of cardiac arrhythmias depends on the interaction between sympathetic and parasympathetic mechanisms also has been documented. Subliminal stimulation of either the reticular formation of the brain stem or the vagus nerve alone did not produce cardiac arrhythmias while simultaneous combination of both stimulations did (14). Neither isolated stimulation of the vagus nerve nor of the stellate ganglion induced cardiac arrhythmias. When the same vagal stimulation was imposed with continuous stimulation of the stellate ganglion multiple ventricular premature contraction occurred ( 10). On the other hand, there are many experimental evidences indicating the importance of sympathetic mechanism on the centrogenic arrhythmias. Cardiac arrhythmias have been produced during bilateral carotid occlusion in vagotomized cats (4) and during stimulation of the pressor regions of the lower brain stem in the vagotomized monkey (9). Cardiac arrhythmias or aberrant complex in ECG have been observed in vagotomized
396
LEE
ET
AL.
animals during stimulation of the ventromedial hypothalamus (6) and the mesencephalon (6, 11). Extrasystoles upon stimulation of the lateral and posterior hypothalamus became even more prominent following vagotomy despite the elimination of the poststimulatory arrhythmias (8). Vagotomy or blockade of the parasympathetic system did not affect the cardiac arrhythmias induced by haloprane and haloprane-carbon dioxide (7). The present investigation observed that activation of the parasympathetic mechanism is not indispensible for PIA in as much as selective injection of picrotoxin into the parasympathetc regions of the medulla, i.e., the tractus and nucleus solitarius or ambiguus nucleus, never produced cardiac arrhythmias. Activation of the depressor mechanism of the anterior hypothalamus is not important either as this area produced cardiac arrhythmias only in one of the four animals tested. In this animal, marked hypertension and tachycardia were evident suggesting the diffusion of picrotoxin into the pressor mechanism. On the contrary, injection of picrotoxin into the sympathetic pressor regions of the lateral or posterior hypothalamus and medulla oblongata produced cardiac arrhythmias concomitant with marked hypertension and tachycardia. Reflex bradycardia was not evident despite of a very high blood pressure (over 250 mm Hg). Furthermore, activation of the spinal (sympathetic) mechanism also induced arrhythmias. The induced arrhythmias were not affected by vagotomy. These suggest that activation of the sympathetic mechanism alone without the integrity of the vagal mechanism can produce PIA. The above contention is at variance with a report of Bircher, Kanai and Wang (2) regarding the predominance of vagal mechanism on the PIA which was based on observation of intravenous and intracerebroventricular injection (1, 2). When picrotoxin was injected via these routes both sympathetic and parasympathetic mechanisms were activated and naturally vagotomy would affect the induced arrhythmias. Indeed, in the present investigation we also observed that of PIA after intravenous (three of four) and intracerebroventricular (two of four) injections were abolished by vagotomy. However, when picrotoxin is injected directly to the sympathetic region, the action is highly selective and the vagal mechanism is excited to a least extent. Moreover, such injection may even possibly activate the inhibitory pathways to the vagal mechanism and causes inhibition of the latter. The existence of such inhibitory mechanisms has been suggested during the activation of the baroceptor reflex upon carotid occlusion (3, 4). In addition, Bircher, Kanai and Wang (1, 2) used unanesthetized but paralysed dogs. Careful observation of the tracing of their experiments
CARDIAC
397
ARRHYTHMIAS
(1, 2, 15) revealed that sinus arrhythmias were very apparent, suggestive of high vagal tonicity (5, 12). Therefore the effect of vagotomy on the induced arrhythmias would be more prominent. In the present investigation light anesthetized cats were used and sinus arrhythmias were never observed. This may also contribute to the difference in results. References 1. BIRCHER, R. P., T. KANAI, and S. C. WANG. 1962. Intravenous, cortical and intraventricular dose-effect relationship of pentylenetetrazol, picrotoxin and deslanoside in dogs. Elrctroct~ccpkalogr. Clirz. Newophysiol. 14: 25fX67. 2. BIRCHER. R. P., T. KAKAI, and S. C. WANG. 1963. Mechanism of cardiac arrhythmias and blood pressure changes induced in dogs by pentylenetetrazol, picrotoxin and deslanoside. /. Phannacol. E.rp. They. 141 : 6-14. 3. CHAI, C. Y., and S. C. WANG. 1968. Integration of sympathetic cardiovascular mechanisms in medulla oblongata of the cat. Awzer. J. Physiol. 215: 1310-1315. 4. CHAI, C. Y., T. F. HUANG, and S. C. WANG. 1968. Mechanisms of cardiac arrhythmias induced by baroceptor reflexes in cats. Amer. J. Physiol. 215: 13161323. 5. HAMLIN, R. L., C. R. S~IITH, and D. L. SMETZER. 1966. Sinus arrhythmias in the dog. Amr. J. Physiol. 210 : 321-328. 6. HOCICMAN, C. H., H. P. MAUCK, JR., and E. C. HOFF. 1966. ECG changes resultII. A spectrum of ventricular arrhythmias of ing from cerebral stimulation. sympathetic origin. rltner. Heart I. 71: 695-700. 7. KATZ, R. L. 1966. Neural factors affecting cardiac arrhythmias induced by hatopropane. J. Pharrr~acol. Exp. Ther. 152: B-94. 8. KORTEWEG, G. C. J., J. TH. F. BOELES, and J. T. CATE. 1957. Influence of stimulation of some subcortical areas on electrocardiogram. J. Nezwophysiol 20: lOO107. 9. Kuo, J. S., C. Y. CHAI, T. M. LEE, C. N. LIU, and R. K. S. LIM. 1970. Localization of central cardiovascular control mechanism in the brain stem of the monkey. Exp. Neural. 29 : 131-141. 10. MANNING, J. W., and M. D. COTTON. 1962. Mechanism of cardiac arrhythmias induced by diencephalic stimulation. Aww. J. Physiol. 203 : 11.20-1124. 11. MAUCK, H. P. JR., C. H. HOCKMAN, and E. C. HOFF. 1964. ECG changes after cerebral stimulation. I. Anomalous atrioventricular excitation elicited by electrical stimulation of the mesencephalic reticular formation. Amer. Heart J. 66: 98-101. 12. MCCRADY, J. D., C. VALLBONA, and H. E. HOFF. 1966. Neural origin of the respiratory-heart rate response. Amer. J. Physiol. 211: 323-328. 13.
PARKER,
I. T., C. G. GUNN, and T. arrhythmias. Cli+l. Res. 10 : 179-179.
14.
PVRPURA,
15.
RII~ER,
N.
LYNN.
1962.
Experimental
centrogenic
D. P., J. L. POOL, E. M. HOUSEPIAN, M. GIRADO, S. A. JACOBSON, and R. J. SEYMOUR. 1958. Hypothermic potentiation of centrally induced cardiac irregularities. Altesthesiology 19 : 27-37. W. F., F. DEPIERRE, J. ROBERTS, nephrine and hydrocarbon-epinephrine Exli. Ther. 114 : l-9.
B. B. BOY, and J. REILLY. disturbance in the cat.
1955. The epiJ. Pharmacol.
398
LEE
ET
AL.
16. ROZEAR. M., R. P. BIRCHER, C. Y. CHAI, and S. C. WANG. 1968. Effects of intracerebroventricular l-hyoscyamine, ethybenztropine and procaine on cardiac arrhythmias induced in dogs by pentylenetetrazol, picrotoxin or deslanoside. 1~. J. Newoplzarmacol. 7 : l-6. 17. WANG, S. C., and C. Y. CHAI. 1962. Central control of sympathetic cardioacceleration in medulla oblongata of the cat. Amer. J. Physiol. 202: 31-34.
NEUROLOGY
Importance
of
36, 389-398
(1972)
Sympathetic
Arrhythmias
Mechanism
Induced
by
on
Cardiac
Picrotoxin
T. M. LEE, K. L. YANG, J. S. Kuo, AND C. Y. CHAI l Departnzertt of Biofihysics and Kohlberg Memorial Medical Research Laboratory, National Defense Medical Center and Department of Medical Research, Veterans Gerlrral Hospital, Taipei Mmicipality, Republic of China Rcceiked
.4pril
17, 1972
The effects of picrotoxin administered via various routes on cardiac arrhythmias were studied in chloralose anesthetized cats. It was found that the efficiency of arrhythmias production in term of dose required and the latency of onset decreased from the order of intracerebral, intracerebroventricular (fourth ventricle) and intravenous injection. Intracardiac injection was ineffective. Among the sites of intracerebral injection, the pressor region of the medulla oblongata was most effective. The pressor region of the posterior hypothalamus came next. Direct injection of picrotoxin into the depressor region of the anterior hypothalamus and the vagal areas of the medulla, i.e., nucleus and tractus solitarius, ambiguus nucleus, did not produce arrhythmias. Topical application of picrotoxin on spinal cord (T,-,) produced cardiac arrhythmias in half of the animals tested. Vagotomy did not affect the arrhythmias induced by intracerebral and spinal injection. The picrotoxin-induced arrhythmias were not affected by midcollicular and additional transection of the brain stem unless the level of lower medulla was reached. The data suggest that picrotoxin induced cardiac arrhythmias by activation of the sympathetic mechanism of the central nervous system, mainly of the medulla. The integrity of the vagal mechanism is not indispensable. Introduction
On comparison of the dose-route relationship of intravenous, subarachnoid (cortical) and intracerebroventricular administration of picrotoxin, Bircher Kanai and Wang (1) observed that the fourth ventricular route was most effective in inducing cardiac arrhythmias. These investigators therefore, suggested that a mechanism sensitive to picrotoxin possibly resides in the structures of the fourth ventricle for arrhythmias production. No attempt was made, however, to verify this contention in decerebrate animals and to identify the specific region in the medulla involved. Bircher, 1 This project was supported in part by Grants from the China Medical Board of New York, Inc. and the International Foundation of U. S. A. The investigators thank Drs. C. T. Loo and T. M. Peng for their encouragement and support throughout the course of this investigation. 389 Q 1972 by Academic Press, Inc. All rights of reproduction in any form
reserved.
390
LEE
ET
AL.
Kanai and Wang (2) observed that both parasympathetic and sympathetic pathways were involved in picrotoxin-induced cardiac arrhythmias (PIA), but the parasympathetic mechanism was more prominent. Vagotomy or intravenous administration of anticholinergic agents prevented .W-80% of the cardiac arrhythmias induced by intracerebroventricularly injected picrotoxin. After total sympathectomy, low spinal cord transection or reserpine treatment, picrotoxin still induced marked bradycardic arrhythmias and A-V block (2). However, many experimental data have proved that centrogenic cardiac arrhythmias can be produced by activation of the sympathetic system alone (3, 4, 9). Therefore the importance of sympathetic mechanismon PIA cannot be neglected. The present investigation was undertaken to determine whether the medulla is essential for PIA and whether the sympathetic or the parasympathetic mechanismin the brain stem is more sensitive to intracerebrally injected picrotoxin. Methods
Seventy-two cats of both sexes, weighing Z&3.2 kg, under chloralose were used. They were immobilized when necessary with w/k, ip) succinylcholine chloride (0.2-0.5 mg, iv). Rectal temperature was maintained at 3% throughout the course of experiment by irradiation with infrared light. The trachea was cannulated for artificial ventilation and the femoral artery was catheterized and blood pressure monitored with a Statham P23AC transducer. Heart rate was monitored with a Grass 5P4 tachometer triggered by arterial pulses. Electrocardiogram lead I or II was monitored bv a Grass 5P4 preamplifier. The femoral vein was cannulated for intravenous injection. For intracardiac injection, the left ventricle was cannulated with a polyethylene tubing through the right subclavian artery. For intracerebroventricular or intracerebral injections, the animal was placed in a prone position and the head was fixed in a David Kopf stereotaxic instrument. A spinal needle (G26) was positioned into the fourth ventricle or into the desired region of the hypothalamus or medulla oblongata. The needle used for intracerebral injection was insulated except for 2 mm at the tip. So it might also be used as a unipolar electrode for effecting electrical stimulation by passing through d-c current derived from a Grass S4 stimulator. Decerebration was completed by removal of all the brain tissue rostra1 to the bony tentorium, as was described ( 17). The animal was killed at the end of experiment, and the head was perfused with physiological saline solution followed by 10% formalin. The correct position of the intracerebroventricular cannula was examined grossly and the site of intracerebral injection was identified by histological sections stained with Weil’s method. (40
CARDIAC
ARRHYTHMIAS
391
Results
The results of picrotoxin administered via various routes with regard to dose of administration, vasomotor and cardiac changes, incidence of onset and duration of PIA and the effects of vagotomy are summarized in Table 1. I&ravenozu (iv) and Intvacerebroventricular (ivt) Injection. Picrotoxin 4 mg/kg iv or 60-100 pg ivt produced cardiac arrhythmias in all four animals tested concomitant with an increase of the mean blood pressure (avg 52 and 59 mm Hg) and decrease of heart rate (avg 85 and 100 beat/min). The arrhythmias consistedof atria1 tachycardia, supraventricular and ventricular premature contractions and nodal and sinus premature systoles. The latency of onset was faster in ivt than iv injection. In eight other animals (four iv and four ivt) the effects of vagotomy on the arrhythmias were tested. Vagotomy abolished the PIA in threefourths of the animals after iv and half of the animals after ivt injection. Eight animals (four iv and four ivt) not included in Table 1 were subjected to midcollicular decerebration and to additional transection at the brain stem after PIA had developed. In these, decerebration did not affect the arrhythmias, nor did additional transection of the brain stem at the caudal pons or midmedulla. The PIA were abruptly terminated by subsequent transection at the caudal medulla (Fig. 1). Intracardiac Injection. Picrotoxin, ZOO-300 pg. directly injected into the left ventricle of the heart produced slight change of the blood pressure and the heart rate. Further increase of the dose to 900 /lg caused muscular twitching or even convulsion but cardiac arrhythmias still were not observed (four animals). Intracerebral Injection of Picrotoxin. The effects of intracerebral injection depended on the site of injection: Picrotoxin, 30-45 pg, injected bilaterally (four animals) in the lateral or posterior hypothalamus, in which electrical stimulation elicited moderate to marked pressor response and cardioacceleration, produced similar increase of blood pressure (avg 64 mm Hg) and heart rate (30 beat/min) and cardiac arrhythmias. Unilateral injection (four animals) produced similar effects, but the latency of onset of arrhythmias was longer 8.7 against 7 min). Unlike that of ivt administration, intrahypothalamic picrotoxin never produced reflex bradycardia although the systolic blood pressure had increased to 250 mm Hg or higher. Vagotomy neither could prevent nor terminate these arrhythmias (another 6 animals). In three of the four animals, bilateral administration of picrotoxin, 3045 pg, into the anterior hypothalamus, in which electrical stimulation produced moderate depressor and bradycardic effect, only caused a slight increase of blood pressure (avg 14 mm Hg) and heart rate (average 16 beat/min) ; PI.4 were observed only in one animal who developed marked
cord
u Asterisk(*) h Mean f
indicates SE.
Medulla oblongata Pressor (bilateral) Pressor (unilateral) Nucleus and tractus solitarius or ambiguus nucleus (bilateral)
I ntracerebral Hypothalamus Pressor (bilateral) Pressor (unilateral) Depressor (bilateral)
Spinal
another
16-24 16-24 1624
30-4.5 30-45 30-4.5
group
of experiment.
4/4 818 0.‘6
4/4 l/4
4/a
214
800-1200
O/4
414
of animals tested
4/4
No.
No. of observation
\?A VAKIOUS CAKDIOVASCULAH
60-100
200-300
Intracardiac
IntracerebroventricuIar
8000-12000
Dose (I.%)
OF PICKOTQXIN OTHER
Intravenous
Route of administration
EFFECTS
0.3 0.35
(0.2-0.5) (0.2-0.7)
7 (S-8) (7710)
9 (7-12!
8 (S-12)
12 (8-15)
Onset average (range) (mm)
8.5
1
8 (7-9) 4 (2-S)
(3-6)
(15-22)
10 (8-12) 5 (4-9)
4.5
17.6
18 (15-24)
Duration average (range) (min)
OF ADMINISTKATION RESIWNSIB INDUCED
PIA
ROUTES
TABLE
No.
o/4+ o/4*
O/6* o/4* l/l
64 f SO f 14 f
20.9 16.7 4.4
8.1
13.6
2.4
21.3
+154 =t 36.3 +142 f 39.8 + 23 f 18.6
+ + +
+39zt
o/2
59 f
8f
+ +
52 f
+
2/4*
3/4*
of PIA
AND
Average change of mean blood pressure (mm Hd
AKKHYTHMIAS
No. of abolition after vagotomy a
ON CAKDIAC RY PKK~TOXIN
30 + 28 f 16 f
26 f
85 f
14 f
f
15.1 6.4 8.3
7.4
14.8
6.2
25.4
+ 71 31 24.7 $- 64+22.1 + 30 f 14.4
+ + +
+
-
+
-100
Average change of heart rate h (beat/mm)
s
2
w E
CARDIAC
393
ARRHYTHMIAS
hypertension and tachycardia and the PIA were terminated by vagotomy. Picrotoxin, 16-24 pg, was injected bilaterally (four animals) into pressor loci of the medulla oblongata, in which electrical stimulation elicited marked pressor and cardioaccelerator effects. The blood pressure and heart rate instanteously increased to 154 mm Hg and 71 beat/min in average and PIA occurred in less than 30 set after the injection. Unilateral injection (eight animals) was similarly effective but the PIA lasted shorter (Table 1 ; Fig. 2). Vagotomy did not affect these PIA. Convulsion or muscular twitching was not observed. In six animals picrotoxin, 16-24 pg, was injected into the nucleus and tractus solitarius or the ambiguus nucleus of both sides, in which electrical stimulation elicited marked decrease of blood pressure and heart rate. The injection produced an initial bradycardia for about lo-15 set after with a slight elevation of the mean blood pressure 23 mm Hg and of the heart rate 30 beat/min in average. PIA were not observed in all these animals (Fig. 3). Spinal Cord. Picrotoxin, 800-1200 pg, injected to the subarachnoid space of the spinal cord at T,.? through an implanted polyethylene tubing, produced an increase of blood pressure (avg 39 mm Hg) and heart rate
2 150 9
*
b
c
b
C
wi-
d /
1. Effects on ECG, blood pressure, and heart rate of midcollicular decerebraand additional transection at the lower brain stem on cardiac arrhythmias induced by picrotoxin. a. Ten minutes after picrotoxin, 4 mg/kg, iv. Note the hypertension and marked bradycardia (control blood pressure and heart rate were 110/75 mm Hg and 210 beats/min). b. Twelve minutes after picrotoxin ventricular premature contraction occurred. c. Cardiac arrhythmias still persisted after midcollicular decerebration, and additional transection at the level of the lower pons (d), and middle medulla (e). f. The arrhythmias were abruptly terminated after transection at the lower medulla. FIG.
tion
FIG. 2. Effects of vagotomy on cardiac arrhythmias induced by injection of picrotoxin into one side of the pressor region of the medulla oblongata. The left panels show the pressor responses upon electrical stimulation of the pressor region. a. Control. b. Right after the injection of picrotoxin, 20 pg, into the pressor region (first arrow), blood pressure was markedly increased concomitant with reflex bradycardia. Twenty seconds later cardiac arrhythmias appeared. c-e. Cardiac arrhythmias were still evident after bilateral vagotomy (second arrow.) f. Sinus tachycardia remained through out the rest of the course. Calibration lines: 20 set, and 1 sec.
(avg 26 beat/mm) ; PIA which were not affected by vagotomy occurred in two of the four animals tested. Muscular twitching or convulsion was evident in all the animals. Discussion
Cardiac arrhythmias induced by picrotoxin are central in origin and are possibly induced by activation of the medullary mechanism (1, 2). Indeed, injection of picrotoxin into the left ventricle of the heart did not produce cardiac arrhythmias or apparent changes of blood pressure and heart rate despite the dose had been so increased as to cause muscular twitching. Intravenously or intracerebroventricularly injected picrotoxin induced cardiac arrhythmias and the arrhythmias were not affected by midcollicular decerebration, with or without additional transection at the
---.
*
-.-
FIG. 3. Cardiovascular changes induced by injection of picrotoxin into the nucleus and tractus of solitarius on both sides of the medulla oblongata. The two panels on the left side show the depressor responses upon electrical stimulation of the vagal loci. a. Control. b and c. Within 8 min after the administration of picrotoxin. 20 pg, into the vagal loci. Note the insignificant changes of blood pressure and heart rate. Arrow : picrotoxin injection. Calibration lines : 20 set, 20 set, and 1 sec.
CARDIAC
FIG.
4. Sites
of injections
giving
395
ARRHYTHMIAS
results
shown
in Fig.
2 (right)
and
Fig.
3 (left).
upper and middle medulla. Abolition of the arrhythmias were evident only when transection was made at the lower medulla. However, the medulla is not the exclusive structure responsible for PIA. Intracerebral injection of picrotoxin into the hypothalamus also produced cardiac arrhythmias although the dose of picrotoxin required was bigger and the latency of onset longer. Moreover, topical application of picrotoxin onto the spinal cord also produced cardiac arrhythmias. The relative importance of the sympathetic and parasympathetic mechanism to cardiac arrhythmias induced by activation of the central nervous system has been the subject of interest. The importance of the vagus mechanism has been emphasized. Stimulation of the pressor regions of the lateral and posterior hypothalamus more readily produced cardiac arrhythmias after the stimulation had terminated and when the elevation of blood pressure was still at the plateau (6, 10). Therefore, reflex activation of the vagus system contributes partially to these poststimulatory arrhythmias (13, 15). That the genesis of cardiac arrhythmias depends on the interaction between sympathetic and parasympathetic mechanisms also has been documented. Subliminal stimulation of either the reticular formation of the brain stem or the vagus nerve alone did not produce cardiac arrhythmias while simultaneous combination of both stimulations did (14). Neither isolated stimulation of the vagus nerve nor of the stellate ganglion induced cardiac arrhythmias. When the same vagal stimulation was imposed with continuous stimulation of the stellate ganglion multiple ventricular premature contraction occurred ( 10). On the other hand, there are many experimental evidences indicating the importance of sympathetic mechanism on the centrogenic arrhythmias. Cardiac arrhythmias have been produced during bilateral carotid occlusion in vagotomized cats (4) and during stimulation of the pressor regions of the lower brain stem in the vagotomized monkey (9). Cardiac arrhythmias or aberrant complex in ECG have been observed in vagotomized
396
LEE
ET
AL.
animals during stimulation of the ventromedial hypothalamus (6) and the mesencephalon (6, 11). Extrasystoles upon stimulation of the lateral and posterior hypothalamus became even more prominent following vagotomy despite the elimination of the poststimulatory arrhythmias (8). Vagotomy or blockade of the parasympathetic system did not affect the cardiac arrhythmias induced by haloprane and haloprane-carbon dioxide (7). The present investigation observed that activation of the parasympathetic mechanism is not indispensible for PIA in as much as selective injection of picrotoxin into the parasympathetc regions of the medulla, i.e., the tractus and nucleus solitarius or ambiguus nucleus, never produced cardiac arrhythmias. Activation of the depressor mechanism of the anterior hypothalamus is not important either as this area produced cardiac arrhythmias only in one of the four animals tested. In this animal, marked hypertension and tachycardia were evident suggesting the diffusion of picrotoxin into the pressor mechanism. On the contrary, injection of picrotoxin into the sympathetic pressor regions of the lateral or posterior hypothalamus and medulla oblongata produced cardiac arrhythmias concomitant with marked hypertension and tachycardia. Reflex bradycardia was not evident despite of a very high blood pressure (over 250 mm Hg). Furthermore, activation of the spinal (sympathetic) mechanism also induced arrhythmias. The induced arrhythmias were not affected by vagotomy. These suggest that activation of the sympathetic mechanism alone without the integrity of the vagal mechanism can produce PIA. The above contention is at variance with a report of Bircher, Kanai and Wang (2) regarding the predominance of vagal mechanism on the PIA which was based on observation of intravenous and intracerebroventricular injection (1, 2). When picrotoxin was injected via these routes both sympathetic and parasympathetic mechanisms were activated and naturally vagotomy would affect the induced arrhythmias. Indeed, in the present investigation we also observed that of PIA after intravenous (three of four) and intracerebroventricular (two of four) injections were abolished by vagotomy. However, when picrotoxin is injected directly to the sympathetic region, the action is highly selective and the vagal mechanism is excited to a least extent. Moreover, such injection may even possibly activate the inhibitory pathways to the vagal mechanism and causes inhibition of the latter. The existence of such inhibitory mechanisms has been suggested during the activation of the baroceptor reflex upon carotid occlusion (3, 4). In addition, Bircher, Kanai and Wang (1, 2) used unanesthetized but paralysed dogs. Careful observation of the tracing of their experiments
CARDIAC
397
ARRHYTHMIAS
(1, 2, 15) revealed that sinus arrhythmias were very apparent, suggestive of high vagal tonicity (5, 12). Therefore the effect of vagotomy on the induced arrhythmias would be more prominent. In the present investigation light anesthetized cats were used and sinus arrhythmias were never observed. This may also contribute to the difference in results. References 1. BIRCHER, R. P., T. KANAI, and S. C. WANG. 1962. Intravenous, cortical and intraventricular dose-effect relationship of pentylenetetrazol, picrotoxin and deslanoside in dogs. Elrctroct~ccpkalogr. Clirz. Newophysiol. 14: 25fX67. 2. BIRCHER. R. P., T. KAKAI, and S. C. WANG. 1963. Mechanism of cardiac arrhythmias and blood pressure changes induced in dogs by pentylenetetrazol, picrotoxin and deslanoside. /. Phannacol. E.rp. They. 141 : 6-14. 3. CHAI, C. Y., and S. C. WANG. 1968. Integration of sympathetic cardiovascular mechanisms in medulla oblongata of the cat. Awzer. J. Physiol. 215: 1310-1315. 4. CHAI, C. Y., T. F. HUANG, and S. C. WANG. 1968. Mechanisms of cardiac arrhythmias induced by baroceptor reflexes in cats. Amer. J. Physiol. 215: 13161323. 5. HAMLIN, R. L., C. R. S~IITH, and D. L. SMETZER. 1966. Sinus arrhythmias in the dog. Amr. J. Physiol. 210 : 321-328. 6. HOCICMAN, C. H., H. P. MAUCK, JR., and E. C. HOFF. 1966. ECG changes resultII. A spectrum of ventricular arrhythmias of ing from cerebral stimulation. sympathetic origin. rltner. Heart I. 71: 695-700. 7. KATZ, R. L. 1966. Neural factors affecting cardiac arrhythmias induced by hatopropane. J. Pharrr~acol. Exp. Ther. 152: B-94. 8. KORTEWEG, G. C. J., J. TH. F. BOELES, and J. T. CATE. 1957. Influence of stimulation of some subcortical areas on electrocardiogram. J. Nezwophysiol 20: lOO107. 9. Kuo, J. S., C. Y. CHAI, T. M. LEE, C. N. LIU, and R. K. S. LIM. 1970. Localization of central cardiovascular control mechanism in the brain stem of the monkey. Exp. Neural. 29 : 131-141. 10. MANNING, J. W., and M. D. COTTON. 1962. Mechanism of cardiac arrhythmias induced by diencephalic stimulation. Aww. J. Physiol. 203 : 11.20-1124. 11. MAUCK, H. P. JR., C. H. HOCKMAN, and E. C. HOFF. 1964. ECG changes after cerebral stimulation. I. Anomalous atrioventricular excitation elicited by electrical stimulation of the mesencephalic reticular formation. Amer. Heart J. 66: 98-101. 12. MCCRADY, J. D., C. VALLBONA, and H. E. HOFF. 1966. Neural origin of the respiratory-heart rate response. Amer. J. Physiol. 211: 323-328. 13.
PARKER,
I. T., C. G. GUNN, and T. arrhythmias. Cli+l. Res. 10 : 179-179.
14.
PVRPURA,
15.
RII~ER,
N.
LYNN.
1962.
Experimental
centrogenic
D. P., J. L. POOL, E. M. HOUSEPIAN, M. GIRADO, S. A. JACOBSON, and R. J. SEYMOUR. 1958. Hypothermic potentiation of centrally induced cardiac irregularities. Altesthesiology 19 : 27-37. W. F., F. DEPIERRE, J. ROBERTS, nephrine and hydrocarbon-epinephrine Exli. Ther. 114 : l-9.
B. B. BOY, and J. REILLY. disturbance in the cat.
1955. The epiJ. Pharmacol.
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ET
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16. ROZEAR. M., R. P. BIRCHER, C. Y. CHAI, and S. C. WANG. 1968. Effects of intracerebroventricular l-hyoscyamine, ethybenztropine and procaine on cardiac arrhythmias induced in dogs by pentylenetetrazol, picrotoxin or deslanoside. 1~. J. Newoplzarmacol. 7 : l-6. 17. WANG, S. C., and C. Y. CHAI. 1962. Central control of sympathetic cardioacceleration in medulla oblongata of the cat. Amer. J. Physiol. 202: 31-34.