AMERICAN Vol. 230,
JOURNAL No. 4, April
OF PHYSIOLOGY 1976. Printed
in U.S.A.
Effect of carotid sinus nerve stimulation on cardiorespiratory responses
pattern
MATTHEW N. LEVY AND HARRISON ZIESKE (With the Technical Assistance of Frank Walters) Department of Investigative Medicine, Mount Sinai Hospital, and Department of Physiology, Case Western Reserve University, Cleveland, Ohio 44106
LEVY, MATTHEW N., AND HARRISON ZIESKE. Effect of carotid sinus nerve stimulation pattern on cardiorespiratory responses. Am. J. Physiol. 230(4): 951-958. 1976.-The reflex responses to steady and intermittent stimulation of the carotid sinus nerve (CSN) were compared in anesthetized dogs. Intermittent stimulation was less effective than steady stimulation in reducing the arterial blood pressure, and the disparity was exaggerated after acute sinoaortic denervation. With the sinoaortic nerves intact, at low mean stimulation frequencies the heart rate responses were greater during intermittent than during steady CSN stimulation. At higher mean stimulation frequencies, however, steady CSN stimuli were more effective than were the intermittent type. After sinoaortic denervation, steady stimuli evoked greater heart rate responses than did intermittent stimuli over the entire mean frequency range studied. Reflex changes in respiratory depth and frequency were also greater during steady than during intermittent CSN stimulation. The greater efficacy of steady than of intermittent stimulation in evoking the observed reflex cardiovascular and respiratory changes is probably ascribable to the pronounced frequency limitation at the first synapse of the baroreceptor reflex in the brain. arterial blood pressure; autonomic nervous system; baroreceptor reflex; chemoreceptor reflex; chloralose anesthesia; heart rate; pulmonary ventilation; sinoaortic reflexes; vagus nerves
THE NATURAL EXCITATION of the baroreceptors by the arterial pulse, the afferent impulses in the sinoaortic nerves are not distributed uniformly in time, but they are clustered in groups within each cardiac cycle (3, 6, 16, 28). Also there are periodic alterations in baroreceptor impulse frequency as the blood pressure varies with respiration (28). Furthermore, small cyclic changes in chemoreceptor impulse frequency have been recorded during the respiratory cycle (1, 8, 9). Since the sinoaortic reflexes constitute a nonlinear system (7, 17, 18, 20, 23), it may be anticipated that the average steady-state responses to a periodically varying sensory input will differ from those to a constant input of the same mean amplitude. The reflex responses to steady and to oscillatory pressures in the isolated carotid sinuses have been compared, and considerable differences have been observed (6, 24). From such studies, however, it is difficult to distinguish the role of the sensory receptors per se from that of the central and efferent portions of the system. Electrical stimulation of the sinoaortic nerves does permit the investigator to DURING
bypass the sensory receptor portion of the reflex arc and provides the opportunity for precise control of the stimulation parameters. However, it does introduce certain difficulties, notably that of separating the effects of simultaneous stimulation of baroreceptor and chemoreceptor fibers. There have been only a few studies of the reflex effects of variations in the pattern of stimulation of the sinoaortic nerves (2, 5, 10, 12, 14, 19, 29). The stimulation parameters in most of these studies have been limited, and the results have been variable. The present study was undertaken in an attempt to acquire more quantitative data over a broader range of stimulation parameters. METHODS
Mongrel dogs were anesthetized with chloralose, 75 mg/kg iv, after premeditation with morphine sulfate, 1 mg/kg im. A constant light level of anesthesia was sustained by means of a slow intravenous drip of 2% chloralose in isotonic saline. A tracheal cannula was inserted via a midline cervical incision. Both carotid sinus nerves (CSN) were isolated, and bipolar platinum hook electrodes were applied to the right nerve. Liquid paraffin was poured into the region of the right CSN to prevent drying of the nerve and to minimize current spread from the electrodes to the surrounding tissues. Arterial blood pressure was recorded from a femoral artery by means of a pressure transducer (Statham P23AA). The rate and depth of respiration were assessed by using a second pressure transducer to register intratracheal pressure. The amplitude of the intratracheal pressure change was used as an index of respiratory depth. The arterial and intratracheal. pressures were recorded on an ink-writing oscillograph (Brush Mark 200) and on magnetic tape (Honeywell 7600). A bipolar electrode catheter was passed into the right ventricle via the right external jugular vein to permit registration of the ventricular electrogram. An analog computer (Electronic Associates, Inc. 580) was used to compute the R-R interval from the ventricular electrogram signal on a beat-by-beat basis; the output was recorded on the ink-writing oscillograph. Also, at appropriate points in the experiment, the ventricular electrogram and arterial blood pressure signals were transmitted to a clock channel and to an analog/digital converter channel, respectively, of a digital computer (Digital Equip-
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M. N. LEVY
952
ment Corp. PDP 12). The means, standard deviations, and standard errors of the mean for the R-R interval and arterial blood pressure were computed for a preselected number of beats, usually 50 or 100. Two groups of experiments were conducted, depending on the duration of a so-called basic period. In the first group, consisting of nine dogs, a basic period of 1 s was employed to simulate the approximate duration of the cardiac cycle in a resting, unanesthetized animal. In the second group, consisting of 10 animals, a longer basic period of 5 or 10 s was used to approximate the duration of a respiratory cycle. In the first group, constant numbers of stimuli were delivered to the right CSN during each basic period of 1 s, but the pattern of delivery of those stimuli was varto ied. For example, if n stimuli were being delivered the CSN each second, they were delivered equally spaced over the following time intervals: 1, 0.5, 0.25, and 0.125 s. The corresponding stimulation patterns will be referred to as l/l, l/2, l/4, and l/8 duty cycles, respectively. Figure 1 illustrates the stimulation pattern for a mean frequency of 16 Hz, with duty cycles of l/l, l/2, and l/4. In all experiments, supramaximal voltages were used (usually 5 V) at two pulse widths (0.1 and 1.0 ms) and at three mean frequencies (16, 32, and 64 Hz). The stimulation parameters were monitored on a cathode-ray oscilloscope (Tektronix, Inc., model 564). The permutations of pulse width and mean frequency were applied in a random sequence in each experiment. For a given combination of pulse width and mean frequency, the order of application of the various duty cycles was randomized according to the following scheme. First, control data were collected in the absence of CSN stimulation. Then, a duty cycle of l/l (constant frequency spread out evenly over the entire second) was applied, and data were collected after the responses had attained a steady state (usually l-2 min). Next, one of the duty cycles less than l/l (either l/Z, l/4, or l/S) was employed. After data had again been collected during the new steady state, the l/l stimulus pattern was reapplied. Then, another duty cycle less than l/l was used, followed once more by the l/l stimulus pattern. Finally, the third of the duty cycles less than l/l was used, followed once more by the l/l pattern. Thus, in each case, cycles of l/2, l/4, and l/8 were preceded and followed by a period of l/l stimulation. Before a new combination of pulse width and mean frequency was applied, CSN stimulation was discontinued, and new control observations were obtained. The stimulatic n sequences described above were carwhile both CSN and both ried out in each experiment
I I I I I I I I I I I I I I I I I I I III
IIIlllllIIIIIllI IIIIIIIIIIIIIIII
I 0.0
0.2
0.4
I
I 0.6 TIME,
I
I 0.8
I
I 1.0
I
I 1.2
I
I 1.4
I
I 1.6
SEC
1. Patterns of stimulation with a mean frequency of 16 Hz, for duty cycles of l/l, I/2, and l/4; duty cycle of l/8 is not included in figure. FIG.
AND
H. ZIESKE
vagosympathetic trunks were intact. The vagi were then securely ligated, and both CSN were tied close to the carotid sinuses. The ligature on the right CSN was located between the carotid sinus and the stimulating electrodes. After a new steady state had been attained, a similar set of experiments was conducted, using a randomization scheme similar to that described above. In most experiments, only a stimulus pulse width of 0.1 ms was used after ligation of the CSN and vagi. In a second group of 10 animals, basic periods of 5 and 10 s were used. All stimulation voltages were supramaximal at a pulse width of 1.0 ms, and the mean frequencies used were 2, 4, and 8 Hz. These low frequencies were selected in an attempt to simulate the gentle respiratory modulation of the prevailing background level of sympathetic activity. The permutations of frequencies and basic periods were applied in a random sequence. The scheme for randomizing the duty cycles was similar to that used in the first series of experiments, i.e., a random sequence of the l/2, l/4, and l/8 duty cycles, each preceded and followed by a period of l/ 1 duty cycle. RESULTS
Basic
Period,
1s 0.1 nzs. The changes
in arterial blood pressure (PA), cardiac cycle duration (R-R interval), and respiratory activity produced by CSN stimulation in a representative experiment in which the vagi were intact as shown in Fig. 2. At the two ends of the record are shown the control values of these variables; the duty cycle ratio of O/l signifies the absence of CSN stimulation. After the first control observation was obtained, the right CSN was stimulated at several duty cycles at a mean frequency of 32 Hz. The duty cycle ratio of l/l was employed during four alternate periods of about 80 s each; only the first three appear in the figure. At this duty cycle, a steady frequency of 32 Hz was applied throughout each successive basic period of 1 s. Alternating with these periods of steady stimulation were periods with duty cycles of l/4, l/8, and l/2, respectively. It is apparent from the figure that the reductions in PA produced by steady CSN stimulation (duty cycle = l/l) were considerably greater than those evoked by intermittent stimulation (duty cycle < l/l). Also, steady CSN stimulation elicited greater increases in R-R interval and in the rate and depth of respiration than did intermittent stimulation. The effects of steady CSN stimulation on PA, R-R interval, and respiratory depth and frequency are presented in Table 1 for the nine animals in which the basic period was 1 s. It is apparent that at pulse widths of both 0.1 and 1.0 ms, frequencies from 16 to 64 Hz were equally effective in lowering the arterial pressure when the vagi and CSN were intact. With the vagi and CSN tied, CSN stimulation central to the ligature produced a much greater decline in P, than when the vagi and CSN were intact, and the effects were more pronounced at frequencies of 32 and 64 Hz than at 16 Hz. Also, with the vagi and CSN intact, CSN stimulation at 16 Hz produced a greater increase in the R-R interval than at the Puke
width,
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953
- .A&‘%--
I0I 1
FIG. 2. Changes in arterial blood pressure, R-R interval, and respiratory activity in response to different patterns of stimulation of right CSN in a representative experiment. In left and right panels, duty cycle of O/l denotes absence of CSN stimulation. In middle panel, CSN was stimulated at 5 V, 0.1 ms, at a mean frequency of 32 Hz. Duty cycles of l/l, l/2, l/4, and l/8 signify that each second 32 stimuli were delivered within l/l, l/2, l/4, and l/8 s, respectively. At top of record, time marker indicates 4- and 40-s intervals.
TABLE I. Arterial blood pressure, R-R interval, and respiratory depth and frequency in absence of carotid sinus nerve stimulation and during steady stimulation of right carotid sinus nerve in nine dogs Vagi
and
Pulse
width,
0
16
32
64
230
122 k 16
122 228
122 226
411 +153
569 2358
450 2132
100
133 +71
138 232
9.0 22.7
14.1 25.3
22.3
means
2 SD.
In absence
Stimulation
CSN
Intact
0.1 ms
Vagi (Pulse
Pulse
width,
1.0 ms
0
16
32
64
153 +25
115 -t 18
116 223
118 227
512 +280
395 2116
623 2286
492 -t164
169 268
100
483 2215
and CSN Tied width, 0.1 ms)
0
16
32
64
204 +33
114 *14
94 234
95 234
448 2144
267 216
292 218
301 230
289 ~27
316 2145
147 230
100
20.6 27.7
16.2 28.4
19.3 *4.0
9.3 23.5
sinus
nerve
frequency, Hz Arterial
156
pressure, mmHg R-R interval, ms Respiratory depth, ?+ of control Respiratory frev-w, min Values
13.7
16.0 25.3
11.0 ~4.5
lt7
111
139 149 ~62 229
13.: 26.4
8.4 9.5 ~2.7 54.4
’ are
ofcarotid
stimulation,
frequency
= OHz.
higher stimulation frequencies. With the vagi and CSN tied, the changes in R-R interval were smaller and did not vary much over the frequency range employed. The respiratory effects of CSN stimulation were considerably more pronounced when the pulse width was 1.0 ms than when it was only 0.1 ms. Also, with the greater pulse width, the effects on respiratory depth were less pronounced as the stimulation frequency was increased. However, the changes in respiratory rate were not significantly affected by the frequency of stimulation over the range from 16 to 64 Hz. The effects of intermittent CSN stimulation on the and respiratory depth (RD) changes in PA, R-R interval, are shown in Fig. 3. The three-dimensional graphs included in this figure represent the percent changes in these responses from their corresponding values during steady CSN stimulation (l/l duty cycle). The graphs represent the composite data from the nine animals and
were constructed from multinomial regression equations (4) of the form: y = b,, + b,D + b,F + b,,D2 + b,F’ + b,DF, where y is the percent change in the response variable, D is the duty cycle, F is the mean stimulation frequency, and bi are the regression coefficients. The quadratic terms D2 and F2 were included to permit a better fit if the response variable did not change linearly with duty cycle or stimulation frequency. The crossproduct term, DF, was incorporated to provide a better fit in the event of a significant interaction between duty cycle and frequency. In Fig. 3, A and B, the vertical axis represents the percent change (APA) from the P, during steady CSN stimulation (l/l duty cycle). Since all points on the response surfaces represent positive values of APA, the values of P, during cycles of l/2, l/4, and l/8 were greater than those during steady stimulation. In other words, steady CSN stimulation produced a greater reduction in P, than did intermittent stimulation at any of the duty cycles that were employed; an example is clearly seen in the upper tracing of Fig. 2. It is also evident from Fig. 3, A and B, that AP* increases as the value of the duty cycle is diminished. Hence, the lower the value of the duty cycle, the less effective the stimulus in reducing PA. For each of the regression equations represented by the response surfaces in Fig. 3, the multiple regression coefficient, R, was computed. The square of that coefficient measures the extent to which the variance of the response is accounted for by the independent variables in the regression equation (4). For the response surfaces depicted in Fig. 3, A and B, R2 was 0.985 and 0.938, respectively; i. e., 98.5 and 93.8 percent of the observed variances in PA were accounted for by the respective regression equations. Hence, the regression equations (and the response surfaces constructed from them) closely fit the experimental data. The values of R” and of the regression coefficients for all the response surfaces in Figs. 3-5 are compiled in Table 2. After the vagi and CSN had been ligated, steady CSN stimulation with a pulse width of 0.1 ms produced a greater reduction in P, than it did when the vagi and CSN were intact (Table 1). Comparison of Fig. 3, A and B, shows that the differences between steady and intermittent CSN stimulation were more pronounced after the vagi and CSN were tied than when they were intact. With the vagi and CSN tied (Fig. 3B), PA was about 10% greater at a duty cycle of l/2 than during steady stimulation, over the frequency range of 16-64 Hz. At smaller duty cycles, the differences between the effects of steady and intermittent stimulation were considerably more pronounced, and this difference was exaggerated at higher stimulation frequencies. For example, at a duty cycle of l/8, PA was about 25% greater than during steady stimulation when a mean frequency of 16 Hz was employed, and the difference increased to about 35% at a frequency of 64 Hz. The changes in R-R interval produced by intermittent CSN stimulation at a pulse width of 0.1 ms were not markedly different from the effects of steady stimulabetween the tion (Fig. 3, C and D); the differences responses to intermittent and steady stimulation were
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M. N. LEVY A Pa
TABLE
multiple equations
____----Response Surface ---
2. Regression correlation portrayed h
(I
h -..I-
coefficients coefficient by graphs h,
AND
H. ZIESKE
(bi) and squares (Rz) for regression in Fig. 3 to 5
of
---~
h.1
h,
h,
K?
-
3A 3B 3c 30 3E 3F
19.5 40.0 -26.2 -6.4 -25.8 -20.2
.131 .200 1.177 - .030 - 1.857 .269
-62.4 -146.5 45.5 22.6 89.5 - 171.6
-.002 55.8 .OOO 174.2 -.013 -50.1 .ooo -29.5 .024 11.4 .007 366.6
4A 4B
29.6 -57.5
.088 2.436
-101.9 91.8
-.004 -.023
5A 5B
- 10.5 - 12.4
- 1.632 -1.880
59.1 47.5
83.9 -94.9
.161 - -391 -.123 .055 -.813 -1.657
,985 .938 .975 .977 .933 .792
.586 -.634
.932 .978
-.176 -67.6 3.339 ,958 2.904 .996 -.225 -30.6 ~~___~ The regression coefficients are for the equationy = b,, + b,D + b,F + h,,D” + h ,F” + h-,DF, where-v is the response variable, D is the duty cycle, and F is the mean stimulation frequency.
FIG. 3. Response surfaces for 9 dogs in which right CSN was stimulated over a range of mean frequencies and duty cycles, using a basic period of 1 s and a stimulus pulse width of 0.1 ms. Responses (APA, ARR, and ARD) represent percent change in arterial pressure, R-R interval, and respiratory depth, respectively, evoked by intermittent CSN stimulation relative to values obtained during steady CSN stimulation at same mean frequency. In left panels, vagi and CSN were tightly ligated; ligature on right CSN was tied between carotid sinus and stimulating electrodes.
within 27%. With the vagi intact (Fig. 30, the direction of the effect varied with the mean frequency of stimulation. At 16 Hz, the change in the R-R interval during intermittent stimulation was less than with steady stimulation. However, at a mean frequency of 64 Hz, intermittent stimulation produced a greater effect on the R-R interval than did steady stimulation. After the vagi and CSN were tied, on the other hand, the changes in R-R interval were consistently less with intermittent than with steady CSN stimulation; i.e., all values of ARR were negative (Fig. 30). With the vagi and CSN intact, intermittent CSN stimulation was considerably less effective in enhancing the depth of respiration than was steady stimulation. At a mean frequency of 16 HZ, a duty cycle of l/2 was 10% less effective than steady stimulation, but the difference amounted to about 40% at a duty cycle of l/8 (Fig. 3E; note that the upward direction along the vertical scale indicates increasing negativity). The differences in response between intermittent and steady stimulation were more pronounced at a mean frequency of 32 Hz than at the higher and lower frequencies used in these experiments. With the vagi and CSN tied, intermittent CSN stimulation also had less effect on respiratory depth than did steady stimulation (Fig. 3F). At a duty cycle of l/2, the difference did not vary much with frequency, whereas at a duty cycle of l/8, the difference was more pronounced the lower the mean frequency. The changes in respiratory frequency were directionally similar to those in respiratory depth. With the vagi intact, the differences between steady and intermittent CSN stimulation were greater the lower the duty cycle ratio. On the other hand, after the vagi were tied, the effects of changes in duty cycle were minimal, but the differences were greater the less the mean frequency of CSN stimulation. Pulse width, 1 .O ms. Steady stimulation of the CSN with a wider pulse width (1.0 ms) had a considerably greater effect on respiration and a somewhat greater influence on R-R interval than did steady stimulation with a pulse width VIO as wide (Table 1). Also, the effects of intermittent stimulation were directionally
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CAROTID
SINUS
similar to those at the narrower pulse width, but the magnitudes of the effects were different, as may be seen by comparing Figs. 3 and 4. Intermittent CSN stimulation produced less depression of PA than did steady stimulation (Fig. 4A). At a duty cycle of l/Z, PA was about 5% greater during intermittent than during steady stimulation, and the effect was virtually independent of mean frequency. The difference was more pronounced at lower duty cycles and at lower mean frequencies. The differences in the heart rate responses between intermittent and steady CSN stimulation were more pronounced with a pulse width of 1.0 ms than with one of 0.1 ms. At the narrower pulse width (Fig. 3C), the differences between intermittent and steady stimulation were confined largely to a range of *5%, whereas at the wider pulse width (Fig. 4B), the differences varied from -15 to slightly over +liI%. The percentage differences in respiratory activity between steady and intermittent stimulation at a pulse width of 1.0 ms were quite similar to those obtained with a pulse width of 0.1 ms. In both cases, the differences between the changes in respiratory activity during steady and intermittent stimulation became more pronounced the lower the duty cycle ratio. Basic Periods,
955
STIMULATION
5 and 10 s
For the 10 dogs in which basic periods of 5 and 10 s were used, the changes in PA and R-R interval produced by steady CSN stimulation are shown in Table 3. As expected, the changes in P, evoked by stimulation at 2 and 4 Hz were minimal, but the reduction elicited at 8 Hz was more substantial. The relative changes in R-R interval were greater than those in P,; at 8 Hz, the R-R interval was about twice the control level (0 Hz). From Table 1, in the animals in which steady stimulation of the CSN was accomplished at a pulse width of 1.0 ms over a frequency range of 16-64 Hz, the maximum change in R-R interval occurred at 16 Hz. From the data in Tables 1 and 3, therefore, it appears that the maxi-
FIG. 4. Response surfaces for arterial pressure and R-R interval for same 9 dogs as in Fig. 3, but for which pulse width of CSN stimuli was 1.0 ms. Basic period, 1 s; vagi and CSN intact.
TABLE
interval carotid
3. Changes in arterial blood pressure during steady stimulation of right sinus nerve in 10 dogs Basic
Stimulation frequency 9 HZ Arterial pressure, mmHg R-R inverval, ms Values
Period:
5s
Basic
and R-R
Period:
10s
0
2
4
8
0
2
4
8
117 kl7
114 +20
110 218
102 +18
142 +32
134 227
131 228
117 520
492 2146
616 5161
643 2203
920 2364
420 2108
534 +131
593 2116
769 2201
are means
+ SD.
mum effect on R-R interval during steady CSN stimulation at a pulse width of 1.0 ms occurred at a frequency of about 8-16 Hz. Over the frequency range of 2-8 Hz, intermittent CSN stimulation with basic periods of either 5 or 10 s had only negligible effects on PA. The differences in the PA responses produced by intermittent and steady stimulation amounted to only about 1 or 2%. With respect to heart rate, however, the effects of intermittent and steady CSN stimulation were appreciable (Fig. 5). At a duty cycle of l/8, for example, CSN stimulation at a mean frequency of 2 Hz was about 10% less effective than was steady stimulation in prolonging the R-R interval. At the same duty cycle but at a mean frequency of 8 Hz, the disparities were even more pronounced; the difference was about 25% when the basic period was 5 s (Fig. 5A) and about 35% when the basic period was 10 s (Fig. 5B). The regression coefficients for these response surfaces are included in Table 2. DISCUSSION
If the neural activity in the baroreceptor fibers in the CSN was proportional to the instantaneous level of pressure in the carotid sinus, changing the duty cycle of CSN stimulation at a given mean frequency in the present experiments would be equivalent to altering the amplitude of the intraluminal pressure oscillations at a constant mean pressure level. Decreasing the duty cycle ratio would be analogous to increasing the amplitude of the pressure oscillation but narrowing the pressure pulse duration proportionately. With respect to the systemic arterial pressure (P,) response, decreasing the duty cycle of CSN stimulation in the present series of experiments caused less reduction in PA than did steady stimulation. On the other hand, it is well established (6, 15, 23, 25) that pulsatile pressures in the carotid sinus have a greater depressor effect than do steady pressures at the same mean pressure level, except perhaps at excessively high intrasinusal pressures. The differences in the responses to intrasinusal pressure variations in those previous studies and to electrical stimulation of the CSN in the present study must lie largely in the transducing properties of the arterial baroreceptors. Pulsatile pressures in the lower and middle pressure
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956
FIG. 5. Response surfaces for changes in R-R interval for 10 dogs in which basic periods of 5 and 10 s were used. Vertical scale (ARR) refers to percent change in R-R interval evoked by intermittent right CSN stimulation relative to that obtained during steady stimulation.
ranges have been shown to evoke a greater total number of impulses in afferent baroreceptor fibers than do steady pressures at the same mean level (15, 20, 23, 25). In the present study, on the other hand, supramaximal electrical stimuli were applied to the CSN. Therefore, the total number of impulses must have remained constant, irrespective of the duty cycle. Several groups of investigators (2, 5, 10, 12, 14, 19. 29) have employed supramaximal electrical stimulation of the sinoaortic nerves in order to study the effect of changes in the pattern of afferent activity per se. Such studies have been conducted under a wide variety of experimental conditions, and the results have been variable. The first detailed study was conducted by Douglas et al. (5) in 1956. They found that, in general, the pattern of stimulation of the aortic nerves in rabbits had little influence on the reflex blood pressure response. At high mean frequencies, however, the responses were usually greater with steady than with intermittent stimulation. More recently, however, Zerbst et al. (29) reported that intermittent stimulation of the aortic nerves in rabbits had a greater depressor effect than did steady stimulation at the same mean frequency. Kendrick et al. (14) observed that submaximal stimulation of the CSN in anesthetized cats had a much greater depressor effect when the stimuli were delivered in an intermittent rather than in a steady pattern. Presumably this effect was mediated via the larger, lower threshold baroreceptor fibers in the CSN. However, when supramaximal stimuli were used, the differences between steady and intermittent stimulation were small. Richter et al. (19) found that in dogs anesthetized with chloralose and urethan, the changes in efferent sympathetic activity produced by CSN stimulation showed much less adaptation in response to intermittent than to steady excitation. In unanesthetized dogs (12) and in dogs anesthetized with chloralose (lo), it was noted by Jonzon et al. (10) that the depressor effects of steady and intermittent CSN stimulation did not differ apprecia-
M. N. LEVY
AND
H. ZIESKE
bly. However, intermittent stimulation had a considerably greater effect on heart rate than did steady stimulation. Thus, most of the previous investigators have reported that the depressor responses to intermittent stimulation of the sinoaortic nerves are either not appreciably different from or are somewhat greater than those to steady stimulation. The results of our experiments do differ from those of the preceding studies in that we found the depressor effects of steady CSN stimulation to be greater than those of intermittent stimulation. As shown in Figs. 3, A and B, and 4A, the differences were more marked the less the duty cycle; i.e., the more pronounced the intermittency. The mean arterial pressures produced by CSN stimulation, using a pulse width of 0.1 ms, were only 5-15% different with steady than with intermittent stimulation when the vagi were intact (Fig. 3A). However, after bilateral vagotomy (Fig. 3B) or with wider pulse widths (Fig. 4A), the differences were significantly greater. The reasons for the discrepancies between our results and those of previous investigators have not been determined. The differences between our results and certain of the preceding studies may be related to differences in species, anesthesia, stimulation parameters, or selection of sinoaortic nerve (i.e., CSN rather than aortic). The greater efficacy of steady than of intermittent stimulation in lowering blood pressure in our experiments is probably ascribable mainly to the pronounced frequency limitation in this reflex. Seller and Illert (22) recorded evoked potentials in neurons located in the nucleus tractus solitarius of anesthetized cats in response to CSN stimulation. These neurons probably represent secondary neurons in the baroreflex path. They found that at CSN stimulation frequencies of only 10 Hz, the amplitude of the evoked potentials was about half the amplitude at 1 Hz, and at 40 Hz, it was about one-fifth that at 1 Hz. Yet the compound action potential of the CSN itself was unchanged at frequencies as high as 120 Hz. The progressively greater reflex response with increases in steady CSN stimulation frequencies, up to the optimum frequency, probably reflects the preponderant influence of the increasing number of impulses per second over that of the reduction in amplitude of the evoked potential at the first synapse. Conversely, the reduction in the reflex response as frequency is further increased probably signifies the predominant effect of the attenuation of the evoked potential over that of the total number of impulses per second arriving at that synapse. In the experiments described in this paper, the numbers of stimuli delivered to the CSN at a given mean frequency were the same during steady and intermittent stimulation. However, the instantaneous frequencies varied inversely as the duty cycles; i.e., the lower the duty cycle, the greater the impulse frequency. Hence, it may be presumed that the lower the duty cycle the less the amplitude of the evoked potentials at the first synapse in the reflex arc regulating the arterial blood pressure. This probably accounts for the progressively greater disparities between the responses to steady and intermittent CSN stimulation as the duty
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CAROTID
SINUS
957
STIMULATION
cycle is reduced from l/2 to l/4 to l/8 in Fig. 3, A and B, and4A. At the combined higher frequencies and lower dut#y cycles (e . g . 9 64 Hz and l/8 duty cycle), there probably was also a frequency limitation for conduction in the afferent fibers of the CSN. With respect to heart rate, the differences in response CSN stimulation were not to steady and intermittent large, an d they depended on the mean frequency of stimulation (Fig. 3, C and D, and 4B). As shown in Table 1, when the vagi were intact, CSN stimulation was most effective in increasing the R-R interval at a frequency of 16 Hz, and the steady-state response became progressively less as frequency was increased. With the vagi tied, the changes in R-R interval were small and not appreciably different over the frequency range from 16 to 64 Hz. As shown in Figs. 3C and 4B, when the vagi were intact, intermittent stimulation at a mean frequency of 16 Hz produced a smaller reflex effect on the R-R interval than did steady stimulation at the same frequency. of 64 Conversely, at a mean frequency Hz, intermittent stimulation had a grea ter reflex effect on the R-R interval than did steady stimulation at the same mean frequency. Comparison of Fig. 3C with 4B indicates that these differences are about twice as prominent with pulse width of 1.0 ms than with a pulse because of the incl usion of width of 0. ms, probably more chemoreceptor fibers at the wider pulse width (13). The reasons for the greater efficacy of intermittent than of steady CSN stimulation on the R-R interval at the higher mean frequencies and for the converse effects at lower mean frequencies (Figs. 3C and 4B) have not been established. Over the frequency range in question (16-64 Hz), th e 1owest frequencies had the greatest steady-state effects on R-R interval (Table 1). Perhaps
1”
at the optimal frequency of 16 Hz, the frequency limitation of the reflex is the principal determinant in accounting for the greater efficacy of steady than of intermittent stimulation. Conversely, at higher frequencies (e.g., 64 Hz), the tendency for less adaptation (19) during intermittent than during steady stimulation may be responsible for the greater efficacy of the intermittent stimulation .. A .daptation is a prominent feature of the heart rate response to baroreceptor stimulation (11, 21, 26, 27). In Fig. 2, for example, at the time of initiation of the second and third l/l duty cycles, the change in R-R interval was great initially, and then it adapted to a much lower steady-state value. This type of adaptation involves vagal efferent pathways predominantly (26, 27). With the vagal pathways abrogated (Fig. 3D), the response surface was entirely different; at all frequenties, the R-R interval response was less pronounced during intermittent than during steady CSN stimulation, i.e., all values of ARR were negative. It is also likely that frequency limitation is pri ncipally responsible for the differences in respi ratory responses to steady and intermittent CSN stimulation. During steady stimulation at a pulse width of 1.0 ms, the maximal change in respiratory depth occurred at 16 with Hz, and the response dimi nished substa ntially greater frequ .encies (Ta .ble 1) . At thi s same pulse width, respiratory rate did not vary much with increasing stimulation frequency. Therefore, as the frequency was increased, the efficacy of each stimulus must have decreased proportionately. This 15758. Received
work
was
supported
for publication
by Public
Health
Service
Grant
HL
21 May 1975.
REFERENCES 1. BISCOE, T. J., AND M. J. PURVES. Observations on the rhythmic variation in the cat carotid body chemoreceptor activity which is the same period as respiration. J. PhysioZ., London 190: 389-412, 1967. 2. BRATTSTR~M, A., AND H. WARZEL. Influence of continuous and intermittent (R-Wave Triggered) electrical stimulation of the carotid sinus nerve on the static characteristic of the circulatory regulator. Experientia 28: 414-416, 1972. 3. BRONK, D. W., AND G. STELLA. Afferent impulses in the carotid sinus nerve. J. CeZZuZar Comp. Physiol. 1: 113-130, 1932. 4. COOLEY, W. W., AND P. R. LOHNES. Multivariate Data Analysis. New York: Wiley, 1971, p. 49-95. 5. DOUGLAS, W. W., J. M. RITCHIE, AND W. SCHAUMANN. A study of the effect of the pattern of electrical stimulation of the aortic nerve on the reflex depressor responses. J. Physiol., London 133: 232-242, 1956. 6. EAD, H. W., J. H. GREEN, AND E. NEIL. A comparison of the effects of pulsatile and non-pulsatile blood flow through the carotid sinus on the reflexogenic activity of the sinus baroreceptors in the cat. J. Physiol., London 118: 509-519, 1952. 7. FRANZ, G. N., A. M. SCHER, AND C. S. ITO. Small signal characteristics of carotid sinus baroreceptors of rabbits. J. AppZ. PhysioZ. 30: 527-535, 1971. 8. GEHRICH, J. L., AND G. P. MOORE. Statistical analysis of cyclic variations in carotid body chemoreceptor activity. J. AppZ. PhysioZ. 35: 642-648, 1973. 9. HORNBEIN, T. F., 2. J. GRIFFO, AND A. Roos. Quantitation of chemoreceptor activity: interrelation of hypoxia and hypercapnia. J. Neurophysiol. 24: 561-568, 1961. 10. JONZON, A., P. A. OBERG, G. SEDIN, AND U. SJ~STRAND. Studies of blood-pressure regulation. IV. The effects of impulse train
11.
12.
13.
14.
15.
16. 17.
18. 19.
stimulation of the carotid-sinus nerves. Acta PhysioZ. Stand. 85: 323-342, 1972. JONZON, A., P. A. OBERG, G. SEDIN, AND U. SJOSTRAND. Studies of blood-pressure in the unanaesthetized dog. I. The effects of constant-frequency stimulation of the carotid-sinus nerves. Pfluegers Arch. 340: 211-228, 1973. JONZON, A., P. A. OBERG, G. SEDIN, AND U. SJOSTRAND. Studies of blood-pressure regulation in the unanaesthetized dog. II. The effects of impulse train stimulation of the carotid-sinus nerves. Pfluegers Arch. 340: 229-249, 1973. KENDRICK, J. E., AND G. L. MATSON. Depressor and pressor afferent fibers in the carotid sinus nerve of the dog. Proc. Sot. ExptZ. BioZ. Med. 138: 175-177, 1971. KENDRICK, J. E., G. L. MATSON, B. OBERG, AND G. WENNERGREN. The effect of stimulus pattern on the pressure response to electrical stimulation of the carotid sinus nerve of cats. Proc. Sot. Exptl. BioZ. Med. 144: 412-416, 1973. KOUSHANPOUR, E., AND J. P. MCGEE. Effect of mean pressure on carotid sinus baroceptor response to pulsatile pressure. Am. J. PhysioZ. 216: 599-603, 1969. LANDGREN, S. On the excitation mechanism of the carotid baroceptors. Acta Physiol. Stand. 26: l-34, 1952. LEVISON, W. H., G. 0. BARNETT, AND W. D. JACKSON. Nonlinear analysis of the baroreceptor reflex system. CircuZation Res. 18: 673-682, 1966. ~BERG, P. A., AND U. SJOSTRAND. Studies of blood-pressure regulation III. Dynamics of arterial blood pressure on carotid-sinus nerve stimulation. Acta PhysioZ. Stand. 81: 96-109, 1971. RICHTER, D. W., W. KECK, AND H. SELLER. The course of inhibition of sympathetic activity durin g various patterns of car0 tid sinus nerve stimulation. Pfluegers Arch. 317: 110-123, 1970.
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958 20. SCHER, A. M., AND A. C. YOUNG. Servoanalysis of carotid sinus reflex effects on peripheral resistance. Circulation Res. 12: l52162, 1963. 21. SCHNEYER, K. Der Pressorezeptorische Herzreflex. 2. Kreislaufforsch. 27: 217-226, 1935. 22. SELLER, H., AND M. ILLERT. The localization of the first synapse in the carotid sinus baroreceptor reflex pathway and its alteration of the afferent input. PfZuegers Arch. 306: 1-19, 1969. 23. SPICKLER, J. W., AND P. KEZDI. Dynamic response characteristics of carotid sinus baroreceptors. Am. J. PhysioL. 212: 472-476, 1967. 24. STEGEMANN, J., AND U. TIBES. Der Einfluss von Amplitude, Frequenz und Mittelwert sinusformiger Reizdrucke an den Pressoreceptoren auf den arteriellen Mitteldruck des Hundes. Pfluegers Arch. 305: 219-228, 1969. 25. TRANK, 3. W., AND M. B. VISSCHER. Carotid sinus baroceptor
M. N. LEVY
26
27
28
29
AND
H. ZIESKE
modifications associated with endotoxin shock. Am. J. Physiol. 202: 971-977, 1962. VATNER, S. F., D. FRANKLIN, AND E. BRAUNWALD. Effects of anesthesia and sleep on circulatory response to carotid sinus nerve stimulation. Am. J. Physiol. 220: 1249-1255, 1971. WANG, S. C., AND H. L. BORISON. An analysis of the carotid sinus cardiovascular reflex mechanism. Am. J. Physiol. 150: 712-721, 1947. WIEMER, W., AND P. KIWULL. Zur gleichzeitigen Registrierung der Potentialaktivittit im Sunusnerven und ihrer Reflexwirkung auf den Bhtdruck. 2. Kreislnufforsch. 55: 1182-1188, 1966. ZERBST, E., K.-H. DITTBERNER, AND V. K~TTER. Studies on bloodpressure regulation by the use of a baroreceptor analog. Pfluepers Arch. 315: 232-249. 1970.
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JOURNAL No. 4, April
OF PHYSIOLOGY 1976. Printed
in U.S.A.
Effect of carotid sinus nerve stimulation on cardiorespiratory responses
pattern
MATTHEW N. LEVY AND HARRISON ZIESKE (With the Technical Assistance of Frank Walters) Department of Investigative Medicine, Mount Sinai Hospital, and Department of Physiology, Case Western Reserve University, Cleveland, Ohio 44106
LEVY, MATTHEW N., AND HARRISON ZIESKE. Effect of carotid sinus nerve stimulation pattern on cardiorespiratory responses. Am. J. Physiol. 230(4): 951-958. 1976.-The reflex responses to steady and intermittent stimulation of the carotid sinus nerve (CSN) were compared in anesthetized dogs. Intermittent stimulation was less effective than steady stimulation in reducing the arterial blood pressure, and the disparity was exaggerated after acute sinoaortic denervation. With the sinoaortic nerves intact, at low mean stimulation frequencies the heart rate responses were greater during intermittent than during steady CSN stimulation. At higher mean stimulation frequencies, however, steady CSN stimuli were more effective than were the intermittent type. After sinoaortic denervation, steady stimuli evoked greater heart rate responses than did intermittent stimuli over the entire mean frequency range studied. Reflex changes in respiratory depth and frequency were also greater during steady than during intermittent CSN stimulation. The greater efficacy of steady than of intermittent stimulation in evoking the observed reflex cardiovascular and respiratory changes is probably ascribable to the pronounced frequency limitation at the first synapse of the baroreceptor reflex in the brain. arterial blood pressure; autonomic nervous system; baroreceptor reflex; chemoreceptor reflex; chloralose anesthesia; heart rate; pulmonary ventilation; sinoaortic reflexes; vagus nerves
THE NATURAL EXCITATION of the baroreceptors by the arterial pulse, the afferent impulses in the sinoaortic nerves are not distributed uniformly in time, but they are clustered in groups within each cardiac cycle (3, 6, 16, 28). Also there are periodic alterations in baroreceptor impulse frequency as the blood pressure varies with respiration (28). Furthermore, small cyclic changes in chemoreceptor impulse frequency have been recorded during the respiratory cycle (1, 8, 9). Since the sinoaortic reflexes constitute a nonlinear system (7, 17, 18, 20, 23), it may be anticipated that the average steady-state responses to a periodically varying sensory input will differ from those to a constant input of the same mean amplitude. The reflex responses to steady and to oscillatory pressures in the isolated carotid sinuses have been compared, and considerable differences have been observed (6, 24). From such studies, however, it is difficult to distinguish the role of the sensory receptors per se from that of the central and efferent portions of the system. Electrical stimulation of the sinoaortic nerves does permit the investigator to DURING
bypass the sensory receptor portion of the reflex arc and provides the opportunity for precise control of the stimulation parameters. However, it does introduce certain difficulties, notably that of separating the effects of simultaneous stimulation of baroreceptor and chemoreceptor fibers. There have been only a few studies of the reflex effects of variations in the pattern of stimulation of the sinoaortic nerves (2, 5, 10, 12, 14, 19, 29). The stimulation parameters in most of these studies have been limited, and the results have been variable. The present study was undertaken in an attempt to acquire more quantitative data over a broader range of stimulation parameters. METHODS
Mongrel dogs were anesthetized with chloralose, 75 mg/kg iv, after premeditation with morphine sulfate, 1 mg/kg im. A constant light level of anesthesia was sustained by means of a slow intravenous drip of 2% chloralose in isotonic saline. A tracheal cannula was inserted via a midline cervical incision. Both carotid sinus nerves (CSN) were isolated, and bipolar platinum hook electrodes were applied to the right nerve. Liquid paraffin was poured into the region of the right CSN to prevent drying of the nerve and to minimize current spread from the electrodes to the surrounding tissues. Arterial blood pressure was recorded from a femoral artery by means of a pressure transducer (Statham P23AA). The rate and depth of respiration were assessed by using a second pressure transducer to register intratracheal pressure. The amplitude of the intratracheal pressure change was used as an index of respiratory depth. The arterial and intratracheal. pressures were recorded on an ink-writing oscillograph (Brush Mark 200) and on magnetic tape (Honeywell 7600). A bipolar electrode catheter was passed into the right ventricle via the right external jugular vein to permit registration of the ventricular electrogram. An analog computer (Electronic Associates, Inc. 580) was used to compute the R-R interval from the ventricular electrogram signal on a beat-by-beat basis; the output was recorded on the ink-writing oscillograph. Also, at appropriate points in the experiment, the ventricular electrogram and arterial blood pressure signals were transmitted to a clock channel and to an analog/digital converter channel, respectively, of a digital computer (Digital Equip-
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M. N. LEVY
952
ment Corp. PDP 12). The means, standard deviations, and standard errors of the mean for the R-R interval and arterial blood pressure were computed for a preselected number of beats, usually 50 or 100. Two groups of experiments were conducted, depending on the duration of a so-called basic period. In the first group, consisting of nine dogs, a basic period of 1 s was employed to simulate the approximate duration of the cardiac cycle in a resting, unanesthetized animal. In the second group, consisting of 10 animals, a longer basic period of 5 or 10 s was used to approximate the duration of a respiratory cycle. In the first group, constant numbers of stimuli were delivered to the right CSN during each basic period of 1 s, but the pattern of delivery of those stimuli was varto ied. For example, if n stimuli were being delivered the CSN each second, they were delivered equally spaced over the following time intervals: 1, 0.5, 0.25, and 0.125 s. The corresponding stimulation patterns will be referred to as l/l, l/2, l/4, and l/8 duty cycles, respectively. Figure 1 illustrates the stimulation pattern for a mean frequency of 16 Hz, with duty cycles of l/l, l/2, and l/4. In all experiments, supramaximal voltages were used (usually 5 V) at two pulse widths (0.1 and 1.0 ms) and at three mean frequencies (16, 32, and 64 Hz). The stimulation parameters were monitored on a cathode-ray oscilloscope (Tektronix, Inc., model 564). The permutations of pulse width and mean frequency were applied in a random sequence in each experiment. For a given combination of pulse width and mean frequency, the order of application of the various duty cycles was randomized according to the following scheme. First, control data were collected in the absence of CSN stimulation. Then, a duty cycle of l/l (constant frequency spread out evenly over the entire second) was applied, and data were collected after the responses had attained a steady state (usually l-2 min). Next, one of the duty cycles less than l/l (either l/Z, l/4, or l/S) was employed. After data had again been collected during the new steady state, the l/l stimulus pattern was reapplied. Then, another duty cycle less than l/l was used, followed once more by the l/l stimulus pattern. Finally, the third of the duty cycles less than l/l was used, followed once more by the l/l pattern. Thus, in each case, cycles of l/2, l/4, and l/8 were preceded and followed by a period of l/l stimulation. Before a new combination of pulse width and mean frequency was applied, CSN stimulation was discontinued, and new control observations were obtained. The stimulatic n sequences described above were carwhile both CSN and both ried out in each experiment
I I I I I I I I I I I I I I I I I I I III
IIIlllllIIIIIllI IIIIIIIIIIIIIIII
I 0.0
0.2
0.4
I
I 0.6 TIME,
I
I 0.8
I
I 1.0
I
I 1.2
I
I 1.4
I
I 1.6
SEC
1. Patterns of stimulation with a mean frequency of 16 Hz, for duty cycles of l/l, I/2, and l/4; duty cycle of l/8 is not included in figure. FIG.
AND
H. ZIESKE
vagosympathetic trunks were intact. The vagi were then securely ligated, and both CSN were tied close to the carotid sinuses. The ligature on the right CSN was located between the carotid sinus and the stimulating electrodes. After a new steady state had been attained, a similar set of experiments was conducted, using a randomization scheme similar to that described above. In most experiments, only a stimulus pulse width of 0.1 ms was used after ligation of the CSN and vagi. In a second group of 10 animals, basic periods of 5 and 10 s were used. All stimulation voltages were supramaximal at a pulse width of 1.0 ms, and the mean frequencies used were 2, 4, and 8 Hz. These low frequencies were selected in an attempt to simulate the gentle respiratory modulation of the prevailing background level of sympathetic activity. The permutations of frequencies and basic periods were applied in a random sequence. The scheme for randomizing the duty cycles was similar to that used in the first series of experiments, i.e., a random sequence of the l/2, l/4, and l/8 duty cycles, each preceded and followed by a period of l/ 1 duty cycle. RESULTS
Basic
Period,
1s 0.1 nzs. The changes
in arterial blood pressure (PA), cardiac cycle duration (R-R interval), and respiratory activity produced by CSN stimulation in a representative experiment in which the vagi were intact as shown in Fig. 2. At the two ends of the record are shown the control values of these variables; the duty cycle ratio of O/l signifies the absence of CSN stimulation. After the first control observation was obtained, the right CSN was stimulated at several duty cycles at a mean frequency of 32 Hz. The duty cycle ratio of l/l was employed during four alternate periods of about 80 s each; only the first three appear in the figure. At this duty cycle, a steady frequency of 32 Hz was applied throughout each successive basic period of 1 s. Alternating with these periods of steady stimulation were periods with duty cycles of l/4, l/8, and l/2, respectively. It is apparent from the figure that the reductions in PA produced by steady CSN stimulation (duty cycle = l/l) were considerably greater than those evoked by intermittent stimulation (duty cycle < l/l). Also, steady CSN stimulation elicited greater increases in R-R interval and in the rate and depth of respiration than did intermittent stimulation. The effects of steady CSN stimulation on PA, R-R interval, and respiratory depth and frequency are presented in Table 1 for the nine animals in which the basic period was 1 s. It is apparent that at pulse widths of both 0.1 and 1.0 ms, frequencies from 16 to 64 Hz were equally effective in lowering the arterial pressure when the vagi and CSN were intact. With the vagi and CSN tied, CSN stimulation central to the ligature produced a much greater decline in P, than when the vagi and CSN were intact, and the effects were more pronounced at frequencies of 32 and 64 Hz than at 16 Hz. Also, with the vagi and CSN intact, CSN stimulation at 16 Hz produced a greater increase in the R-R interval than at the Puke
width,
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953
- .A&‘%--
I0I 1
FIG. 2. Changes in arterial blood pressure, R-R interval, and respiratory activity in response to different patterns of stimulation of right CSN in a representative experiment. In left and right panels, duty cycle of O/l denotes absence of CSN stimulation. In middle panel, CSN was stimulated at 5 V, 0.1 ms, at a mean frequency of 32 Hz. Duty cycles of l/l, l/2, l/4, and l/8 signify that each second 32 stimuli were delivered within l/l, l/2, l/4, and l/8 s, respectively. At top of record, time marker indicates 4- and 40-s intervals.
TABLE I. Arterial blood pressure, R-R interval, and respiratory depth and frequency in absence of carotid sinus nerve stimulation and during steady stimulation of right carotid sinus nerve in nine dogs Vagi
and
Pulse
width,
0
16
32
64
230
122 k 16
122 228
122 226
411 +153
569 2358
450 2132
100
133 +71
138 232
9.0 22.7
14.1 25.3
22.3
means
2 SD.
In absence
Stimulation
CSN
Intact
0.1 ms
Vagi (Pulse
Pulse
width,
1.0 ms
0
16
32
64
153 +25
115 -t 18
116 223
118 227
512 +280
395 2116
623 2286
492 -t164
169 268
100
483 2215
and CSN Tied width, 0.1 ms)
0
16
32
64
204 +33
114 *14
94 234
95 234
448 2144
267 216
292 218
301 230
289 ~27
316 2145
147 230
100
20.6 27.7
16.2 28.4
19.3 *4.0
9.3 23.5
sinus
nerve
frequency, Hz Arterial
156
pressure, mmHg R-R interval, ms Respiratory depth, ?+ of control Respiratory frev-w, min Values
13.7
16.0 25.3
11.0 ~4.5
lt7
111
139 149 ~62 229
13.: 26.4
8.4 9.5 ~2.7 54.4
’ are
ofcarotid
stimulation,
frequency
= OHz.
higher stimulation frequencies. With the vagi and CSN tied, the changes in R-R interval were smaller and did not vary much over the frequency range employed. The respiratory effects of CSN stimulation were considerably more pronounced when the pulse width was 1.0 ms than when it was only 0.1 ms. Also, with the greater pulse width, the effects on respiratory depth were less pronounced as the stimulation frequency was increased. However, the changes in respiratory rate were not significantly affected by the frequency of stimulation over the range from 16 to 64 Hz. The effects of intermittent CSN stimulation on the and respiratory depth (RD) changes in PA, R-R interval, are shown in Fig. 3. The three-dimensional graphs included in this figure represent the percent changes in these responses from their corresponding values during steady CSN stimulation (l/l duty cycle). The graphs represent the composite data from the nine animals and
were constructed from multinomial regression equations (4) of the form: y = b,, + b,D + b,F + b,,D2 + b,F’ + b,DF, where y is the percent change in the response variable, D is the duty cycle, F is the mean stimulation frequency, and bi are the regression coefficients. The quadratic terms D2 and F2 were included to permit a better fit if the response variable did not change linearly with duty cycle or stimulation frequency. The crossproduct term, DF, was incorporated to provide a better fit in the event of a significant interaction between duty cycle and frequency. In Fig. 3, A and B, the vertical axis represents the percent change (APA) from the P, during steady CSN stimulation (l/l duty cycle). Since all points on the response surfaces represent positive values of APA, the values of P, during cycles of l/2, l/4, and l/8 were greater than those during steady stimulation. In other words, steady CSN stimulation produced a greater reduction in P, than did intermittent stimulation at any of the duty cycles that were employed; an example is clearly seen in the upper tracing of Fig. 2. It is also evident from Fig. 3, A and B, that AP* increases as the value of the duty cycle is diminished. Hence, the lower the value of the duty cycle, the less effective the stimulus in reducing PA. For each of the regression equations represented by the response surfaces in Fig. 3, the multiple regression coefficient, R, was computed. The square of that coefficient measures the extent to which the variance of the response is accounted for by the independent variables in the regression equation (4). For the response surfaces depicted in Fig. 3, A and B, R2 was 0.985 and 0.938, respectively; i. e., 98.5 and 93.8 percent of the observed variances in PA were accounted for by the respective regression equations. Hence, the regression equations (and the response surfaces constructed from them) closely fit the experimental data. The values of R” and of the regression coefficients for all the response surfaces in Figs. 3-5 are compiled in Table 2. After the vagi and CSN had been ligated, steady CSN stimulation with a pulse width of 0.1 ms produced a greater reduction in P, than it did when the vagi and CSN were intact (Table 1). Comparison of Fig. 3, A and B, shows that the differences between steady and intermittent CSN stimulation were more pronounced after the vagi and CSN were tied than when they were intact. With the vagi and CSN tied (Fig. 3B), PA was about 10% greater at a duty cycle of l/2 than during steady stimulation, over the frequency range of 16-64 Hz. At smaller duty cycles, the differences between the effects of steady and intermittent stimulation were considerably more pronounced, and this difference was exaggerated at higher stimulation frequencies. For example, at a duty cycle of l/8, PA was about 25% greater than during steady stimulation when a mean frequency of 16 Hz was employed, and the difference increased to about 35% at a frequency of 64 Hz. The changes in R-R interval produced by intermittent CSN stimulation at a pulse width of 0.1 ms were not markedly different from the effects of steady stimulabetween the tion (Fig. 3, C and D); the differences responses to intermittent and steady stimulation were
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M. N. LEVY A Pa
TABLE
multiple equations
____----Response Surface ---
2. Regression correlation portrayed h
(I
h -..I-
coefficients coefficient by graphs h,
AND
H. ZIESKE
(bi) and squares (Rz) for regression in Fig. 3 to 5
of
---~
h.1
h,
h,
K?
-
3A 3B 3c 30 3E 3F
19.5 40.0 -26.2 -6.4 -25.8 -20.2
.131 .200 1.177 - .030 - 1.857 .269
-62.4 -146.5 45.5 22.6 89.5 - 171.6
-.002 55.8 .OOO 174.2 -.013 -50.1 .ooo -29.5 .024 11.4 .007 366.6
4A 4B
29.6 -57.5
.088 2.436
-101.9 91.8
-.004 -.023
5A 5B
- 10.5 - 12.4
- 1.632 -1.880
59.1 47.5
83.9 -94.9
.161 - -391 -.123 .055 -.813 -1.657
,985 .938 .975 .977 .933 .792
.586 -.634
.932 .978
-.176 -67.6 3.339 ,958 2.904 .996 -.225 -30.6 ~~___~ The regression coefficients are for the equationy = b,, + b,D + b,F + h,,D” + h ,F” + h-,DF, where-v is the response variable, D is the duty cycle, and F is the mean stimulation frequency.
FIG. 3. Response surfaces for 9 dogs in which right CSN was stimulated over a range of mean frequencies and duty cycles, using a basic period of 1 s and a stimulus pulse width of 0.1 ms. Responses (APA, ARR, and ARD) represent percent change in arterial pressure, R-R interval, and respiratory depth, respectively, evoked by intermittent CSN stimulation relative to values obtained during steady CSN stimulation at same mean frequency. In left panels, vagi and CSN were tightly ligated; ligature on right CSN was tied between carotid sinus and stimulating electrodes.
within 27%. With the vagi intact (Fig. 30, the direction of the effect varied with the mean frequency of stimulation. At 16 Hz, the change in the R-R interval during intermittent stimulation was less than with steady stimulation. However, at a mean frequency of 64 Hz, intermittent stimulation produced a greater effect on the R-R interval than did steady stimulation. After the vagi and CSN were tied, on the other hand, the changes in R-R interval were consistently less with intermittent than with steady CSN stimulation; i.e., all values of ARR were negative (Fig. 30). With the vagi and CSN intact, intermittent CSN stimulation was considerably less effective in enhancing the depth of respiration than was steady stimulation. At a mean frequency of 16 HZ, a duty cycle of l/2 was 10% less effective than steady stimulation, but the difference amounted to about 40% at a duty cycle of l/8 (Fig. 3E; note that the upward direction along the vertical scale indicates increasing negativity). The differences in response between intermittent and steady stimulation were more pronounced at a mean frequency of 32 Hz than at the higher and lower frequencies used in these experiments. With the vagi and CSN tied, intermittent CSN stimulation also had less effect on respiratory depth than did steady stimulation (Fig. 3F). At a duty cycle of l/2, the difference did not vary much with frequency, whereas at a duty cycle of l/8, the difference was more pronounced the lower the mean frequency. The changes in respiratory frequency were directionally similar to those in respiratory depth. With the vagi intact, the differences between steady and intermittent CSN stimulation were greater the lower the duty cycle ratio. On the other hand, after the vagi were tied, the effects of changes in duty cycle were minimal, but the differences were greater the less the mean frequency of CSN stimulation. Pulse width, 1 .O ms. Steady stimulation of the CSN with a wider pulse width (1.0 ms) had a considerably greater effect on respiration and a somewhat greater influence on R-R interval than did steady stimulation with a pulse width VIO as wide (Table 1). Also, the effects of intermittent stimulation were directionally
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CAROTID
SINUS
similar to those at the narrower pulse width, but the magnitudes of the effects were different, as may be seen by comparing Figs. 3 and 4. Intermittent CSN stimulation produced less depression of PA than did steady stimulation (Fig. 4A). At a duty cycle of l/Z, PA was about 5% greater during intermittent than during steady stimulation, and the effect was virtually independent of mean frequency. The difference was more pronounced at lower duty cycles and at lower mean frequencies. The differences in the heart rate responses between intermittent and steady CSN stimulation were more pronounced with a pulse width of 1.0 ms than with one of 0.1 ms. At the narrower pulse width (Fig. 3C), the differences between intermittent and steady stimulation were confined largely to a range of *5%, whereas at the wider pulse width (Fig. 4B), the differences varied from -15 to slightly over +liI%. The percentage differences in respiratory activity between steady and intermittent stimulation at a pulse width of 1.0 ms were quite similar to those obtained with a pulse width of 0.1 ms. In both cases, the differences between the changes in respiratory activity during steady and intermittent stimulation became more pronounced the lower the duty cycle ratio. Basic Periods,
955
STIMULATION
5 and 10 s
For the 10 dogs in which basic periods of 5 and 10 s were used, the changes in PA and R-R interval produced by steady CSN stimulation are shown in Table 3. As expected, the changes in P, evoked by stimulation at 2 and 4 Hz were minimal, but the reduction elicited at 8 Hz was more substantial. The relative changes in R-R interval were greater than those in P,; at 8 Hz, the R-R interval was about twice the control level (0 Hz). From Table 1, in the animals in which steady stimulation of the CSN was accomplished at a pulse width of 1.0 ms over a frequency range of 16-64 Hz, the maximum change in R-R interval occurred at 16 Hz. From the data in Tables 1 and 3, therefore, it appears that the maxi-
FIG. 4. Response surfaces for arterial pressure and R-R interval for same 9 dogs as in Fig. 3, but for which pulse width of CSN stimuli was 1.0 ms. Basic period, 1 s; vagi and CSN intact.
TABLE
interval carotid
3. Changes in arterial blood pressure during steady stimulation of right sinus nerve in 10 dogs Basic
Stimulation frequency 9 HZ Arterial pressure, mmHg R-R inverval, ms Values
Period:
5s
Basic
and R-R
Period:
10s
0
2
4
8
0
2
4
8
117 kl7
114 +20
110 218
102 +18
142 +32
134 227
131 228
117 520
492 2146
616 5161
643 2203
920 2364
420 2108
534 +131
593 2116
769 2201
are means
+ SD.
mum effect on R-R interval during steady CSN stimulation at a pulse width of 1.0 ms occurred at a frequency of about 8-16 Hz. Over the frequency range of 2-8 Hz, intermittent CSN stimulation with basic periods of either 5 or 10 s had only negligible effects on PA. The differences in the PA responses produced by intermittent and steady stimulation amounted to only about 1 or 2%. With respect to heart rate, however, the effects of intermittent and steady CSN stimulation were appreciable (Fig. 5). At a duty cycle of l/8, for example, CSN stimulation at a mean frequency of 2 Hz was about 10% less effective than was steady stimulation in prolonging the R-R interval. At the same duty cycle but at a mean frequency of 8 Hz, the disparities were even more pronounced; the difference was about 25% when the basic period was 5 s (Fig. 5A) and about 35% when the basic period was 10 s (Fig. 5B). The regression coefficients for these response surfaces are included in Table 2. DISCUSSION
If the neural activity in the baroreceptor fibers in the CSN was proportional to the instantaneous level of pressure in the carotid sinus, changing the duty cycle of CSN stimulation at a given mean frequency in the present experiments would be equivalent to altering the amplitude of the intraluminal pressure oscillations at a constant mean pressure level. Decreasing the duty cycle ratio would be analogous to increasing the amplitude of the pressure oscillation but narrowing the pressure pulse duration proportionately. With respect to the systemic arterial pressure (P,) response, decreasing the duty cycle of CSN stimulation in the present series of experiments caused less reduction in PA than did steady stimulation. On the other hand, it is well established (6, 15, 23, 25) that pulsatile pressures in the carotid sinus have a greater depressor effect than do steady pressures at the same mean pressure level, except perhaps at excessively high intrasinusal pressures. The differences in the responses to intrasinusal pressure variations in those previous studies and to electrical stimulation of the CSN in the present study must lie largely in the transducing properties of the arterial baroreceptors. Pulsatile pressures in the lower and middle pressure
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956
FIG. 5. Response surfaces for changes in R-R interval for 10 dogs in which basic periods of 5 and 10 s were used. Vertical scale (ARR) refers to percent change in R-R interval evoked by intermittent right CSN stimulation relative to that obtained during steady stimulation.
ranges have been shown to evoke a greater total number of impulses in afferent baroreceptor fibers than do steady pressures at the same mean level (15, 20, 23, 25). In the present study, on the other hand, supramaximal electrical stimuli were applied to the CSN. Therefore, the total number of impulses must have remained constant, irrespective of the duty cycle. Several groups of investigators (2, 5, 10, 12, 14, 19. 29) have employed supramaximal electrical stimulation of the sinoaortic nerves in order to study the effect of changes in the pattern of afferent activity per se. Such studies have been conducted under a wide variety of experimental conditions, and the results have been variable. The first detailed study was conducted by Douglas et al. (5) in 1956. They found that, in general, the pattern of stimulation of the aortic nerves in rabbits had little influence on the reflex blood pressure response. At high mean frequencies, however, the responses were usually greater with steady than with intermittent stimulation. More recently, however, Zerbst et al. (29) reported that intermittent stimulation of the aortic nerves in rabbits had a greater depressor effect than did steady stimulation at the same mean frequency. Kendrick et al. (14) observed that submaximal stimulation of the CSN in anesthetized cats had a much greater depressor effect when the stimuli were delivered in an intermittent rather than in a steady pattern. Presumably this effect was mediated via the larger, lower threshold baroreceptor fibers in the CSN. However, when supramaximal stimuli were used, the differences between steady and intermittent stimulation were small. Richter et al. (19) found that in dogs anesthetized with chloralose and urethan, the changes in efferent sympathetic activity produced by CSN stimulation showed much less adaptation in response to intermittent than to steady excitation. In unanesthetized dogs (12) and in dogs anesthetized with chloralose (lo), it was noted by Jonzon et al. (10) that the depressor effects of steady and intermittent CSN stimulation did not differ apprecia-
M. N. LEVY
AND
H. ZIESKE
bly. However, intermittent stimulation had a considerably greater effect on heart rate than did steady stimulation. Thus, most of the previous investigators have reported that the depressor responses to intermittent stimulation of the sinoaortic nerves are either not appreciably different from or are somewhat greater than those to steady stimulation. The results of our experiments do differ from those of the preceding studies in that we found the depressor effects of steady CSN stimulation to be greater than those of intermittent stimulation. As shown in Figs. 3, A and B, and 4A, the differences were more marked the less the duty cycle; i.e., the more pronounced the intermittency. The mean arterial pressures produced by CSN stimulation, using a pulse width of 0.1 ms, were only 5-15% different with steady than with intermittent stimulation when the vagi were intact (Fig. 3A). However, after bilateral vagotomy (Fig. 3B) or with wider pulse widths (Fig. 4A), the differences were significantly greater. The reasons for the discrepancies between our results and those of previous investigators have not been determined. The differences between our results and certain of the preceding studies may be related to differences in species, anesthesia, stimulation parameters, or selection of sinoaortic nerve (i.e., CSN rather than aortic). The greater efficacy of steady than of intermittent stimulation in lowering blood pressure in our experiments is probably ascribable mainly to the pronounced frequency limitation in this reflex. Seller and Illert (22) recorded evoked potentials in neurons located in the nucleus tractus solitarius of anesthetized cats in response to CSN stimulation. These neurons probably represent secondary neurons in the baroreflex path. They found that at CSN stimulation frequencies of only 10 Hz, the amplitude of the evoked potentials was about half the amplitude at 1 Hz, and at 40 Hz, it was about one-fifth that at 1 Hz. Yet the compound action potential of the CSN itself was unchanged at frequencies as high as 120 Hz. The progressively greater reflex response with increases in steady CSN stimulation frequencies, up to the optimum frequency, probably reflects the preponderant influence of the increasing number of impulses per second over that of the reduction in amplitude of the evoked potential at the first synapse. Conversely, the reduction in the reflex response as frequency is further increased probably signifies the predominant effect of the attenuation of the evoked potential over that of the total number of impulses per second arriving at that synapse. In the experiments described in this paper, the numbers of stimuli delivered to the CSN at a given mean frequency were the same during steady and intermittent stimulation. However, the instantaneous frequencies varied inversely as the duty cycles; i.e., the lower the duty cycle, the greater the impulse frequency. Hence, it may be presumed that the lower the duty cycle the less the amplitude of the evoked potentials at the first synapse in the reflex arc regulating the arterial blood pressure. This probably accounts for the progressively greater disparities between the responses to steady and intermittent CSN stimulation as the duty
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CAROTID
SINUS
957
STIMULATION
cycle is reduced from l/2 to l/4 to l/8 in Fig. 3, A and B, and4A. At the combined higher frequencies and lower dut#y cycles (e . g . 9 64 Hz and l/8 duty cycle), there probably was also a frequency limitation for conduction in the afferent fibers of the CSN. With respect to heart rate, the differences in response CSN stimulation were not to steady and intermittent large, an d they depended on the mean frequency of stimulation (Fig. 3, C and D, and 4B). As shown in Table 1, when the vagi were intact, CSN stimulation was most effective in increasing the R-R interval at a frequency of 16 Hz, and the steady-state response became progressively less as frequency was increased. With the vagi tied, the changes in R-R interval were small and not appreciably different over the frequency range from 16 to 64 Hz. As shown in Figs. 3C and 4B, when the vagi were intact, intermittent stimulation at a mean frequency of 16 Hz produced a smaller reflex effect on the R-R interval than did steady stimulation at the same frequency. of 64 Conversely, at a mean frequency Hz, intermittent stimulation had a grea ter reflex effect on the R-R interval than did steady stimulation at the same mean frequency. Comparison of Fig. 3C with 4B indicates that these differences are about twice as prominent with pulse width of 1.0 ms than with a pulse because of the incl usion of width of 0. ms, probably more chemoreceptor fibers at the wider pulse width (13). The reasons for the greater efficacy of intermittent than of steady CSN stimulation on the R-R interval at the higher mean frequencies and for the converse effects at lower mean frequencies (Figs. 3C and 4B) have not been established. Over the frequency range in question (16-64 Hz), th e 1owest frequencies had the greatest steady-state effects on R-R interval (Table 1). Perhaps
1”
at the optimal frequency of 16 Hz, the frequency limitation of the reflex is the principal determinant in accounting for the greater efficacy of steady than of intermittent stimulation. Conversely, at higher frequencies (e.g., 64 Hz), the tendency for less adaptation (19) during intermittent than during steady stimulation may be responsible for the greater efficacy of the intermittent stimulation .. A .daptation is a prominent feature of the heart rate response to baroreceptor stimulation (11, 21, 26, 27). In Fig. 2, for example, at the time of initiation of the second and third l/l duty cycles, the change in R-R interval was great initially, and then it adapted to a much lower steady-state value. This type of adaptation involves vagal efferent pathways predominantly (26, 27). With the vagal pathways abrogated (Fig. 3D), the response surface was entirely different; at all frequenties, the R-R interval response was less pronounced during intermittent than during steady CSN stimulation, i.e., all values of ARR were negative. It is also likely that frequency limitation is pri ncipally responsible for the differences in respi ratory responses to steady and intermittent CSN stimulation. During steady stimulation at a pulse width of 1.0 ms, the maximal change in respiratory depth occurred at 16 with Hz, and the response dimi nished substa ntially greater frequ .encies (Ta .ble 1) . At thi s same pulse width, respiratory rate did not vary much with increasing stimulation frequency. Therefore, as the frequency was increased, the efficacy of each stimulus must have decreased proportionately. This 15758. Received
work
was
supported
for publication
by Public
Health
Service
Grant
HL
21 May 1975.
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