Effects of cardiac glycosides on spontaneous efferent activity in vagus and sympathetic nerves of cats

Int. J. Neuropharmac., 1969,s. 379-387

Pergamon Press.

Printed inGt.Britain.

EFFECTS OF CARDIAC GLYCOSIDES ON SPONTANEOUS EFFERENT ACTIVITY IN VAGUS AND SYMPATHETIC NERVES OF CATS* Department

of Pharmacology,

P. L. MCLAIN School of Medicine, University of Pittsburgh,

Pittsburgh, Pa.

(Accepted 22 January 1969) Summary-Efferent neural traffic over filaments from the cervical and thoracic vagi, and over postganglionic filaments emerging from the stellate ganglion, was studied in cats subjected to progressive, fatal intoxication with digitoxin or ouabain. Vagal efferent activity was increased by glycoside administration in 71% of trials. Depression was never observed. No specific effect on sympathetic activity was noted prior to the onset of cardiac arrhythmia. In the later stages of glycoside intoxication, however, enhancement of sympathetic activity was uniformly observed. Except as an almost terminal event, neither vagal nor sympathetic activity was consistently related to heart rate or level of systemic blood pressure. A reciprocal relationship between the two autonomic systems was not revealed. THE IDEA of

a nervous component in the pharmacology of the digitalis glycosides is more than a century old (TRAUBE, 1851, quoted by CUSHNY,1897). Glycoside enhancement of vagal activity is strongly indicated by an impressive series of observations and has become widely accepted (MODELL, 1961). For the greater part, the evidence has involved the elimination or amelioration of glycoside bradycardia by vagotomy (ROBINSON and WILSON, 1918), section of carotid sinus and aortic nerves (HEYMANS et al., 1932), or atropinization (CUSHNY et al., 1912; GOLD et al., 1939). The results, while preponderantly favorable to the stated conclusion, have not been entirely so (JAMESand NADEAU, 1961; DE BECO, 1931; WELLS et al., 1943 ; MCLAIN et al., 1959) and have been variously interpreted (MODELL, 1961). Reports implicating the sympathetic system (DAGGETT and WEISFELDT,1965) were similarly indirect. While such studies are very valuable, cardiovascular measurements in situ are always resultants of a large number of intrinsic cardiac and reflex effects which, in a given set of circumstances, may be so complex as to defy rigorous analysis. For this reason, a study of impulse traffic in nervous pathways which are known to be involved in the regulation of cardiac activity seemed desirable. The autonomic efferent systems, as the established effector pathways for nervous control of the heart and blood vessels, afforded a logical starting point. The complexity of the vagus nerve could conceivably be circumvented by study of small, cardio-inhibitory branches close to the heart (GREEN, 1959). As for the sympathetic system, the well-known accelerator function of filaments from the stellate ganglion (LIDDELL and SHERRINGTON, 1929) suggested these as favorable for sampling. The possibility of a peripheral but still nerve-related site of action for the glycosides was recognized (WELLS et al., 1943 ; FRANKE, 1951) but not explored. *Supportedby USPHS Grant No. HE 08059. 379

380

P. L. MCLAIN

In the work here reported, efferent impulse traffic was monitored on filaments from the cervical and thoracic vagi, and on postganglionic filaments emerging from the stellate ganglion, in cats subjected to progressive intoxication with digitoxin or ouabain. Multifiber rather than single-unit preparations were employed since they afforded representative samples of activity in the respective systems and permitted a study of central discharge patterns. While vagal and sympathetic activity were usually observed in separate animals, a series of experiments was performed in which activity in the two systems was recorded simultaneously. Neurophysiological data were correlated with blood pressure, the electrocardiogram, and in some instances respiration, simultaneously recorded. METHODS All experiments were performed on cats, l-3-4.5 kg in weight (mean 2.3 kg) without regard to age or sex. Animals were anesthetized by intraperitoneal injections of alphachloralose, 80 mg/kg, or by ether inhalation. The tracheawascannulated toinsurean adequate airway and to permit artificial ventilation when needed. In open-chest preparations, which included all except experiments on the cervical vagus, positive-pressure ventilation was supplied by means of a reciprocal air pump. Body temperature, monitored by rectal thermometry, was maintained with heat lamps. Drugs were administered by catheter into a femoral vein. Arterial pressure was monitored from a femoral or common carotid artery, respiration by recording chest movement or intra-tracheal pressure changes. Lead II of the electrocardiogram was recorded routinely. A Grass Model 7 polygraph, with appropriate accessories, was employed. For studies on the vagus, the right nerve was used exclusively. In the case of the cervical vagus, the vagosympathetic trunk was exposed through a midline incision in the neck and separated from the carotid artery by blunt dissection for a distance of about 2cm. Thenerve was then split along the line separating the sympathetic from the vagus division. The former was left intact. Filaments were then teased from the vagus division, tested for cardioinhibitory activity by artificial electrical stimulation, sectioned distally at a convenient level, and prepared for recording as indicated below. The thoracic branch of the vagus most often employed was one which leaves the main vagus trunk about 1 cm cephalad to the root of the right lung and runs caudally and medially beneath the azygos vein to join the cardiac plexus. This nerve was almost always strongly cardio-inhibitory when stimulated artificially. Occasionally another, higher, branch was chosen which had the desired characteristics. After testing, the branch was cut distally and prepared for recording. The main trunk of the thoracic vagus was always cut caudal to the point of emergence of the branch used for recording. A pocket to contain mineral oil was made by suspending from an overhead frame the incised edges of the parietal pleura in the vicinity of the nerve. In all experiments involving the thoracic vagus alone, the right stellate ganglion was excised to reduce the sympathetic outflow which may be expected in nearly all vagus branches (MARGUTHet al., 1951). The sympathetic elements used for recording were postganglionic filaments emerging medially from the right stellate ganglion. These fibers usually arched caudally to join the right vagus trunk but occasionally crossed the vagus to enter the cardiac plexus. When only sympathetic activity was to be recorded, the vagus was not disturbed. Sympathetic fibers were prepared for recording in the manner outlined below. When simultaneous recording of vagal and sympathetic activity was undertaken, details of preparation were essentially those just described for the separate nerves. In such cases,

Effects of

cardiac glycosideson spontaneousefferent activity in vagusand sympatheticnerves of cats 381

of course, the stellate ganglion was not excised, and the thoracic vagus trunk was sectioned below the level of the branch under study. Duplicate amplification and recording channels were provided. Nerve filaments were prepared for the recording of action potentials by cutting as far as possible distally and desheathing under a dissecting microscope. The filaments were then immersed in warm mineral oil and mounted on silver electrodes connected to the amplifying system. Differential amplification was usually employed. A grounded electrode, positioned proximally to the recording electrodes or in contact with tissue in the vicinity, was found to minimize undesired potentials from heart, respiratory movements, or other sources of interference. The preamplifiers were Grass Model PSCR equipped with high impedance probes. Preamplifier input was filtered to favor a frequency band of 35-100 counts/set. The amplified signal activated a Tetronix Type RM 565 oscilloscope through Tetronix 2A6 1 preamplifiers run at low gain. A 60 counts/set notch filter was used at this point. From the oscilloscope, the signals were transferred to a magnetic tape recorder (Honeywell 8100) through a differential preamplifier. An audio monitor was also included in the system. The animal was enclosed in a grounded, copper-screen cage. Data were processed by photographing the oscilloscope trace at appropriate intervals and by integrating the electrical activity as a function of time. Two integrating systems were employed. The average of ongoing neural activity was obtained by means of a Grass 7P3A preamplifier and integrator, usually operated on-line. This system yielded short-epoch integration, and was well adapted to revealing transients, such as bursts of activity. True integration, cumulative over epochs of 30-60 set, was performed by a Grass 7PlOA integrator, operated in connection with the 7P3A, the signal being obtained usually from the magnetic tape record. The integrated records were continuous, covering an entire experiment. Thus, photographic samples of action potentials were supplemented by a running record of the level of neural activity which could be assessed on a moment-to-moment basis over any convenient time interval. For interpretation of results, each animal served as its own control. Quantitative comparisons among animals were not practicable. The typical experimental plan was as follows. Appropriate surgery was performed and the selected nerve or nerves mounted for recording of action potentials. The presence of spontaneous activity was verified during a stabilization period which lasted from a few minutes to half an hour. Then followed a period during which various preliminary tests might be performed. Sham injections, if employed, were included in this period. Alternatively, the control period was completely devoid of experimental interference in order that spontaneous variations in activity might be observed. Next, the glycoside was administered in accordance with one of the following schedules. Digitoxin (Crystodigin, Lilly) was administered intravenously either as a single dose of 0*16-0~20 mg/kg or in divided doses of 0.04 mg/kg every 5 min until death. Ouabain (Ouabain Injection USP, Lilly) was administered only in divided doses, 5-20 pg/kg by vein, every 5 min until death. RESULTS

All of the observations reported here were on multifiber preparations. No attempt was made to record single units, but single-unit activity was sometimes discernible. While the direction of impulse conduction was controlled by nerve section, there was no certainty as to the source or destination of the activity recorded. This was especially true of the vagus filaments, discussed more completely below. Because of the nature of the data, results were finally expressed in qualitative terms.

P. L. MCLAIN

382

Characteristics 1. Efirent

of spontaneous activity vagus activity. Twenty-nine successful experiments were performed utilizing

the thoracic branch of the right vagus nerve, and nineteen involving filaments from the right cervical vagus. Included were 11 experiments in which postganglionic sympathetic activity was recorded simultaneously with vagal activity. Filaments which showed marked inspiratory rhythm were avoided since they were not considered to be cardio-inhibitory (JEWETT,1964).

Spontaneous efferent activity appeared in various forms (Fig. 1). The most common pattern consisted of bursts of asynchronous firing which occurred at irregular and unpredictable intervals from several per set to 1 per min. The bursts varied in duration from a few msec to several sec. Usually, with the differential method of recording employed, the signal-to-noise ratio was low. The bursting pattern was only roughly characteristic for a given animal, and often changed during an experiment. Waxing and waning of activity were unrelated to any other observed physiological event. Attempts to identify cardio-inhibitory firing by applying the criteria of JEWETT(1964) were unconvincing. Bilateral carotid occlusion reduced the observed activity in 67% of trials. Epinephrine hypertension produced an enhancement in 50 % of trials, but correlation between arterial pressure and vagal activity was poor. Clear-cut pulse modulation of vagal firing was never seen. Other investigators (e.g., CALARESUand PEARCE,1965) have had similar experiences, although several have reported a cardiac rhythm in vagus fibers (JEWETT,1964 ; OKADA et al., 1961). Cardiac rate showed no consistent relationship to the level of vagal activity. Thus, while the vagus filaments employed in these experiments were shown by previous electrical stimulation to contain cardio-inhibitory elements, there emerged no direct evidence that the activity recorded was generated by such elements. 2. E&rent sympathetic activity. Spontaneous activity from postganglionic filaments of the right stellate ganglion was recorded in twenty-six animals. In general, sympathetic activity was much more abundant than vagal activity, and much more consistent in pattern. Characteristic of this system was the occurrence of short bursts, appearing with irregular frequency of the order of l-5 bursts per set (Fig. 1). Burst duration varied widely, even in the same nerve, and might be as brief as O-1set or ten times as long. The bursts nearly always occurred in groups or clusters, often with several set between successive groups. This spacing also, was highly variable even for a given preparation. These characteristics were usually unrelated to the general level of arterial pressure, to pulse rate, or to respiration under control conditions. The only exception was the occasional appearance of pulse-related bursts when the arterial pressure was very low. In such cases, firing corresponded to the diastolic phase of the heart cycle. Cardiac rhythm of sympathetic discharge has also been observed by others (BRONK et al., 1934). When activity was monitored simultaneously on vagus and sympathetic filaments (Fig. l), the differences between the two systems were confirmed directly and convincingly. Results of sham injections In a majority of the experiments, each dose of drug was washed into thecirculationwitha small amount (usually 2 ml) of 0.9 % sodium chloride solution. In view of the observations of OKADA et al. (1961), sham injections (one to five in number, at 5 min intervals in imitation of the pattern of glycoside administration) were included in the pre-glycoside control periods. Further, sham injections of 50% ethanol (generally O-4 ml) were employed as a control against a possible solvent effect in the case of digitoxin. Alternatively, the experiments

SYMP

VAGUS

I

I I SEC

FIG. 1. Patterns of efferent spontaneous sympathetic and vagal activity, recorded simultaneously. Upper trace was recorded from a postganglionic filament from the right stellate ganglion; lower trace was from a small, cardio-inhibitory branch of the right thoracic vagus nerve. No drug.

TOTAL OUABAIN mcg/ kg

VAGUS

0

PULSE

60

VAGUS

PULSE

VAGUS PULSE I

I I SEC FIG. 2. Effect of ouabain on efferent vagal activity. In each panel, the upper trace is activity recorded from a small branch of the right thoracic vagus nerve; the lower trace is right femoral arterial pressure pulse. Ouabain administered intrav~ously, 20 pg/kg per 5 min. Neuro. f.p. 382

TOTAL OUABAIN

SYMP.

PULSE

85

SYMP.

8 MCV

PULSE I

I SEC FIG. 4. Late effect of ouabain on efferent sympathetic activity. In each panel, the upper trace is activity recorded from a filament from the right stellate ganglion; the lower trace is right femoral arterial pressure pulse. Ouabain administered intravenously, 8.5 pg/kg per 5 min. In lower panel, note modulation of sympathetic firing by changes in general level of arterial pressure. Mean arterial pressure at this point varied between 75 and 85 mm Hg.

OUABAIN 92 mcq/kg

SYMP.

12 MCV C PULSE

I SEC FIG. 5. Late effect of ouabain on efferent sympathetic activity. Upper trace: activity recorded from a filament from the right stellate ganglion. Lower trace: right femoral arterial pressure pulse. Note pulse modulation of neural traffic, with greatest activity in diastolic phase. Ouabain administered intravenously, 11.5pg/kg per 5 min.

Effects of cardiac glycosides on spontaneous

efferent activity in vagus and sympathetic

nerves of cats 383

included control periods without injections of any kind, lasting from 5 to 40 min. significant variations in spontaneous activity of both vagus and sympathetic filaments certainly occurred during these control periods. However, no consistent patterns of change emerged, and the risk of confusing a glycoside effect with a saline or ethanol effect was considered to be small. Results of glycoside administration 1. Vagm e&rent activity. Digitoxin was administered to twenty-three animals, ouabain

to twenty-five. Results did not justify a distinction between the two glycosides, between cervical and thoracic filaments of the vagus, or among the various dose schedules employed. All are combined, therefore, in the first section of Table 1. Enhancement of efferent vagal

Nerve

No. of animals

Increase

Frequency of effect Decrease No change

Doubtful

Vagus Cervical Thoracic Total vagus

19 29 48

11 23 34*

0 0 0

8 1 9

0 5 5

Sympa#etic Early: Late

26 26

2:+

8 1

8 1

3 1

* 71 ‘A; binomial, 9.5*Aconfidence limits, S-t33 %. t 89 %; binomial, 95 % confidence limits, 70-98 %. $ Prior to onset of cardiac arrhythmia.

activity by glycoside administration was clearly demonstrated in 71% of forty-eight experiments (Fig. 2). Generally, burst frequency and amplitude were increased, and cumulative electrical activity, derived from a continuous tape record, was elevated. No instances of vagal depression were noted. The results categorized in the table as “doubtful” showed sufficient spontaneous variation to preclude a clear interpretation of enhancement but might have been scored as such with less conservative criteria. In multiple-dose experiments, increased vagal activity was frequently observed with the first dose of glycoside, while maximum activity usually developed after several additional doses, followed by a decline in the severely intoxicated animal (Fig. 3). Detailed examination of dose-effect relationships was not an objective in this study. No consistent correlation was observed between vagal efferent firing patterns and either heart rate or arterial pressure. Glycoside bradycardia was not prominent in these experiments. 2. Sympathetic e&rent activity. Ouabain was the only glycoside employed in studies on sympathetic activity. It was found necessary to distin~ish between early and late effects, as indicated in Table 1. In the period prior to the onset of cardiac arrhythmia, the glycoside apparently had no specific influence on the level of efferent sympathetic traffic. Roughly coincident with the development of cardiac arrhythmia, however, sympathetic activity became dramatically enhanced (Fig. 4). The later stages of glycoside intoxication are often characterized by hypertension, frequently alternating with marked hypotension, resulting in wide, cyclic swings in arterial pressure. During this stage, sympathetic activity also

P. L. MCLAIN

384 UNITS

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TIME DOSE

(MIN.1 0 NO. OFOUABAIN

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30 3

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FIG. 3. Effect of ouabain on cumulative electrical activity in vagus and sympathetic nerves. Spontaneous efferent activity, in arbitrary units, was averaged for each 5 min period. Solid line: cardio-inhibitory branch of right thoracic vagus nerve. Broken line: postganglionic filament from right stellate ganglion. Sinus rate (open circles) and mean femoral arterial pressure (filled circles) are also shown.

fluctuated widely, and in quite precise reciprocal relationship to the systemic blood pressure. Neural activity was markedly diminished or completely absent at the pressure peaks and greatly intensified at low pressure (Fig. 4). In addition, there developed in many animals a pressure-pulse modulation of sympathetic outflow such that each cardiac cycle was reflected by a burst of firing, most intense in diastole (Fig. 5). The terminal fall in blood pressure was regularly associated with continuous, violent firing which lasted many minutes after the circulation had stopped. 3. Simultaneous recording of vagus and sympathetic activity. In eleven animals, simultaneous monitoring of vagus and sympathetic efferent traffic was undertaken to determine whether the glycoside might modify the autonomic outflows reciprocally. Activity was so modified (vagal enhancement, sympathetic depression) in only three experiments. In three more, enhancement of both systems occurred, but not synchronously. For the entire series, the results were indistinguishable from those obtained when the vagus and sympathetic nerves were studied in separate animals.

DISCUSSION

The results here reported certainly support the conventional view that cardiac glycosides augment efferent vagal function (CUSHNY et al., 1912; CUSHNY, 1918; HEYMANSet al., 1932; GOLD et al., 1939). What is lacking, so far as the cardiac pharmacology of the glycosides is concerned, is unequivocal evidence that the augmentation observed involved cardioinhibitory nerve fibers. The question pertains especially to filaments from the cervical vagus, but applies also to the small thoracic branch employed, since there are probably few if

Effectsof cardiac glycosideson spontaneousefferent activity in vagusand sympatheticnerves of cats 385 any purely inhibitory branches available (MARGUTHet al., 1951). Indeed, there is evidence (IRIUCHIJIMA and KUMADA,1964 ; CALARESIand PEARCE,1965) that the number of cardioinhibitory fibers in the vagus may not be very great. The elegant criteria of JEWETT(1964) were not very helpful for identification of cardio-inhibitory activity, possibly because population rather than single-unit activity was being observed. However, filaments showing any trace of inspiratory rhythmicity were avoided. It can only be stated with certainty that the vagus filaments used in these experiments contained cardio-inhibitory elements; artificial stimulation of them slowed the heart. In the present experiments, glycoside administration resulted almost uniformly in a significant elevation of systemic arterial pressure. Mean arterial pressure during the control period (i.e. after operative preparation and before drug administration) was somewhat low, averaging 5 1 mm Hg. Reflex vagal augmentation from baroreceptor stimulation was therefore a possibility. During the early phases of glycoside intoxication, blood pressure and vagal activity often increased roughly in parallel (e.g. Fig. 3). One could not, of course, expect a strict quantitative relationship with population firing. However, even qualitatively, the correlation between glycoside-induced vagal enhancement and alterations in blood pressure was not sufficiently consistent to ascribe the former to a pressure reflex. One illustration of the sort of discrepancy encountered is shown in Fig. 3, where, after thefifthdoseofouabain, blood pressure and vagus activity were clearly unrelated. Failure to observe a correlation between vagal activity and cardiac rate was a disappointing aspect of the results, since one of the original objectives of the study was to assess the role of the vagus in glycoside bradycardia (MCLAIN et al., 1959). CALARESU and PEARCE(1965) have reported a similar experience. Glycoside bradycardia was noted only occasionally in these experiments. The reasons are not clear but may involve the prolonged operative preparation of the animals or the schedule of gylcoside administration. Further investigation of this matter is projected. It may be stated, however, that experience which extends far beyond the present work has demonstrated the hazard of taking cardiac rate as an index of any single neural function. With regard to sympathetic traffic, one might have expected the glycosides to cause a direct or indirect depression. DAGGETTand WEISFELDT(1965) concluded that sympathetic tone was depressed by cardiac glycosides because, in dogs with normal hearts, acetylstrophanthidin produced positive inotropism and increased peripheral resistance only when the vascular reflexes had been chemically or surgically obtunded. ABIKO et al. (1965) reported depression of sympathetic discharge from the left stellate ganglion in the early stages of glycoside intoxication in cats, provided that either the vagi or the carotid sinus nerves were left intact, but not when both sets of nerves had been severed. In contrast, Table 1 shows that, prior to the appearance of overt cardiac toxicity, sympathetic efferent activity was diminished in only 31% of the fibers examined, and that an almost equal number showed enhancement or no change as a result of glycoside administration. Sometimes the response appeared to be diphasic, as in Fig. 3. Since in these experiments the carotid sinus nerves and at least one vagus were invariably undisturbed, the difference from the results of ABIKO et al. (1965) is not readily explained. It is possible that the intact preparations of DAGGETT and WEISFELDT (1965) had an initial reflex balance different from those reported here. However, strong sympathetic tone was always demonstrated prior to glycoside administration. The later increase in sympathetic firing, and its relationship to fluctuations in blood pressure, have been described above but require further comment. First, it was observed that the sympathetic enhancement usually preceded the appearance of cardiac arrhythmia,

386

P. L. MCLAIN

but developed at about the same dose level of glycoside. The difference in time was generally less than 5 min. Secondly, the pressure-related fluctuations in firing were rarely seen in the early stages of the intoxication, but were quite characteristic of the period of cardiac arrhythmia. When blood pressure fell or rose precipitously, sympathetic activity showed reciprocal fluctuations greatly in excess of expectations corresponding to a more stable circulation. With regard to the first point, it is difficult to avoid the conclusion that sympathetic enhancement resulted specifically from glycoside administration in a manner grossly resembling the vagal effect, but out of phase with the latter and, perhaps, more powerful. The close proximity in time between the appearance of sympathetic enhancement and cardiac arrhythmia suggested a possible relationship between these events. If catecholamine release is part of the cardiac pharmacology of the glycosides (TANZ, 1964; DHALLA and MCLAIN, 1966), increase in sympathetic activity might well account for at least some of it, possibly in this way contributing to the onset of arrhythmia, especially in the presence of vagal augmentation (RIKER et al., 1955; ROBERTS and BAER, 1960). A relationship of sympathetic activity to the development of ventricular arrhythmias is also indicated by the studies of HAN and MOE (1964) and HAN et al. (1964), who related ventricular vulnerability to temporal dispersion of recovery of excitability and demonstrated that the latter was increased by stimulation of sympathetic nerves and, inter alia, by ouabain (HAN and MOE, 1964). The fact that the fibrillation threshold was elevated (following a brief decrease) by catecholamine injection yet decreased by sympathetic stimulation (HAN et al., 1964) may be understood on the basis of the widespread yet highly discrete distribution of sympathetic fibers to ventricular muscle (RANDALL et al., 1968). Thus, excessive sympathetic activity could lead to ventricular chaos. Bursts of violent sympathetic activity in association with acute hypotensive episodes could easily have been non-specific reflex responses to circulatory emergency. It was not surprising, for example, to encounter sustained and vigorous firing when the circulation stopped as a result of ventricular fibrillation. However, certain aspects of such events suggested that physiological responses may have been modified by the presence of the glycoside. Sympathetic activity was markedly depressed (and often completely inhibited) in the very earliest stages of recovery from an episode of hypotension, often with the first strong heart contraction. Also, sympathetic responses to minor (but precipitous) fluctuations in blood pressure seemed to be grossly exaggerated. While strictly comparable circulatory phenomena did not occur prior to the late stages of glycoside intoxication, the impression certainly was that of an exquisite sensitivity of the reflex mechanism controlling the sympathetic discharge. Nothing of the sort was ever observed in vagus filaments. Since the glycosides are reported to sensitize baroreceptors (HEYMANS et al., 1932), these observations may reflect the sympathetic expression of such activity. If so, perhaps insufficient attention has been given in the past to the sympathetic

component

of baroreceptor

reflexes (butsee BRONK

et al,, 1934). Glycoside effects on chemoreceptors, either vascular or cardiac, must also be considered. It is obvious that, for a more complete understanding of the nervous component of glycoside pharmacology, afferent as well as efferent systems must be studied. Such observations have been made and will be reported separately. Acknowledgements-The author is grateful to Drs. A. S. MARRAZZI a.nd E. Ross HART for encouragement, assistance. and facilities in the early stages of this investigation. Dr. THOMASF. REDICKwas an enthusiastic collaborator until he accepted a position at another institution. Mrs. JANETB. MCCRYSTALand Mrs. FRANCES PLUMMERboth contributed more than their excellent technical assistance.

Effects of cardiac glycosides on spontaneous

efferent activity in vagus and sympathetic nerves of cats 387 REFERENCES

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JAMES,T. N. and NADEAU,R. A. (1961). Chronotropic etfects of digitalis studied by direct perfusion of the sinus node. J. Lab. clin. Med. 58: 83 I. JEWEIT, D. L. (1964). Activity of single efferent fibers in the cervical vagus nerves of the dog with special reference to cardio-inhibitorv fibers. J. Phvsiol.. Lond. 175: 321-357. LIDDELL,E. G. T. and SHERRIN~TON,C. (1929). Mammalian Physiology. Clarendon Press, Oxford. 160 pp. MCLAIN, P. L., KRUSE, T. K. and REDICK, T. F. (1959). The effect of atropine on digitoxin bradycardia in cats. J. Pharmac. exp. Ther. 126: 76-81. MARGUTH, H., RAULE, W. and SCHAEFER,H. (1951). Action potentials in centrifugal heart nerves. Pfliigers Arch. ges. Physiol. 245: 224-245. MODELL.W. (1961). Clinical nharmacofoev of digitalis materials. Clin. Pharmac. Ther. 2: 177-190. OKADA, ‘H., GKA~OTO, K. and NISIDA,?. (1961). The activity of the cardio-regulatory and abdominal sympathetic nerves of the cat in the Bainbridge reflex. Jup. J. Physiol. 11: 520-529. RANDALL, W. C., SZENTIVANYI,M., PACE, J. B., WECHSLER,J. S. and KAYE, M. P. (1968). Patterns of sympathetic nerve projections onto the canine heart. Circ. Res. 22: 315-323. RIKER, W. F., DEPIERRE,F., ROBERTS,J., ROY, B. B. and REILLY,J. (1955). The epinephrine and hydrocarbonepinephrine disturbance in the cat. J. Pharmac. exp. Ther. 114: l-9. ROBERTS,J. and BAER,R. (1960). A method for the evaluation of depressants of subatrial rhythmic function of the heart in the intact animal. J. Pharmac. exp. Ther. 129: 36-41. ROBINSON,C. G. and WILSON,F. N. (1918). A quantitative study of the effect of digitalis on the heart of the cat. J. Pharmac. exp. Ther. 10: 491-507. TANZ, R. D. (1964). The action of ouabain on cardiac muscle treated with reserpineand dichloroisoproterenol. J. Pharmac. exp. Ther. 144: 205-213. WELLS, J. A., DRAGSTEDT,C. A., RALL, J. E. and RUCIE,D. A. (1943). Inhibition of the cardio-inhibitory action of acetylcholine by digitalis. Fedn Proc. 2: 93-94.