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Origin of cardiac-related synchronized cardiac sympathetic nerve activity in anaesthetized cats
Journal of the Autonomic Nervous System, 47 (1994) 131-140
131
© 1994 Elsevier Science B.V. All rights reserved 0165-1838/94/$07.00 JANS 01498
Origin of cardiac-related synchronized cardiac sympathetic nerve activity in anaesthetized cats Antti E. H e d m a n a, Kanji Matsukawa a and Ishio Ninomiya b,. a Department of Cardiac Physiology, National Cardiovascular Center, Research Institute, Osaka, Japan, and b Department of Physiology, Institute of Health Sciences, School of Medicine, Hiroshima University, 1-2-3 Kasumi-cho, Minami-ku Hiroshima 734, Japan (Received 12 July 1993) (Revision received and accepted 31 August 1993)
Key words: Baroreceptor input; Cardiac sympathetic nerve activity; Cardiac pacing; Cardiac-related rhythm Abstract To study the origin of cardiac-related rhythm in cardiac sympathetic nerve activity (CSNA), ECG, aortic pressure and CSNA were recorded when cardiac interval was changed by artificial pacing, or when the aortic nerve was stimulated after baroreceptor denervation in anaesthetized cats. CSNA was averaged by using the R-wave of ECG, or stimulus pulse as a trigger. Delay times from arterial pulse or stimulus pulse to the onset and half amplitude of inhibition and to the maximal inhibition were measured from the averaged data. The delay of inhibition in CSNA was constant and independent of pacing interval. Stimulation of the aortic nerve with single shocks caused an inhibition in averaged CSNA. The delay of inhibition was constant and independent of stimulus frequency. These results indicate that the cardiac-related rhythm in CSNA is produced reflexly by inhibiting the transmission of the fundamental rhythmicity due to periodic baroreceptor input.
Introduction
Synchronized discharges in postganglionic sympathetic nerves in awake [14,15], decerebrate [1,2,17] and anaesthetized [2,8-12] animals show a rhythmicity related to cardiac cycle at frequency of 2-6 Hz and a faster rhythmicity of 8-12 Hz. It has been agreed that the 8-12 Hz rhythm is of central origin [1,2,15,17]. Whereas there are contrasting hypotheses how cardiac-related synchronized discharges are produced.
* Corresponding author. Tel: +81 082 246 7825; Fax: +81 082 241 0508.
SSDI 0 1 6 5 - 1 8 3 8 ( 9 3 ) E 0 1 15-L
It has been stated that an irregular 2-6 Hz rhythm is generated in brain stem by multiple oscillators with a shifting lead [7]. This free running rhythm would then be entrained in a 1:1 relation to cardiac cycle by pulsatile barorecept0rs input. This concept is based mainly on two observations: phase relation between baroreceptor activity and sympathetic nerve discharges can be shifted by changing heart rate and persistence of irregular 2-6 Hz rhythmicity after baroreceptor denervation [7]. Recently, however, we have presented a new model of a fundamental rhythm oscillator and gate operators. The transmission of the fundamental rhythm (8-12 Hz) is controlled by closing the gate operators by the periodic
132
baroreceptor input and thus a cardiac-related rhythm in CSNA is produced [15]. This is seen as a reflex-inhibition in sympathetic nerve activity. According to the entrainment hypothesis, the point of maximum CSNA occurring in early or middiastole at high heart rates should shift backwards to peak systole and then into late diastole of the preceding cardiac cycle when heart rate is progressively lowered [5]. Instead, according to our hypothesis constant delay of the inhibition in sympathetic nerve activity should be expected when heart rate is changed. In the present study, we focused on this fundamental discrepancy between these two hypotheses. To reveal the mechanism producing the cardiac-related rhythm in synchronized CSNA, time relations between periodic baroreceptor input and synchronized CSNA were examined, when cardiac interval was changed by artificial pacing the heart.
Materials and Methods
Preparation of animals Fifteen cats of either sex weighing between 2.2 and 4.5 kg were anaesthetized with sodium pentobarbital (30-40 mg/kg, i.p.) and immobilized with pancuronium sodium (0.7 mg/kg, i.m.). The trachea was intubated and the lungs ventilated with intermittent positive pressure (Shinano SN480-5 respirator). The femoral artery and vein were cannulated with polyethylene catheters. Additional doses of anaesthetic (5 mg/kg, i.v.) were injected once an hour to maintain surgical anaesthesia. Arterial blood gases were measured prior to measurements of nerve activity, and if necessary the ventilation rate and tidal volume of the respirator was adjusted to ensure blood gases were within the normal range (PaO 2 = 100 + 5 mmHg, PaCO 2 = 33 _+ 3 mmHg and pH = 7.35 _+ 0.05). Body temperature was maintained throughout the experiment by warming.
branch (1-1.5 cm) of the nerve was separated using dissecting microscope (Olympus OME) and a mineral oil pool was prepared around the nerve. The efferent nerve activity was recorded at the central end of the cut nerve by a pair of silver electrodes. The signal was amplified by a biophysical amplifier ( M E G 110, Nihon Kohden) with a high cut-off frequency of 3000 Hz and a low cut-off frequency of 50 Hz. This was then passed through a full-wave rectifier circuit and integrated with a resistance-capacitance integrator with a time constant of 20 ms; this produced a positive deflection proportional to the frequency discharge in the original neurogram and it was defined as integrated CSNA (CSNA). The signals were monitored on a four channel digital storage oscilloscope (Nihon Kohden, VC-10). In all the cats, the ECG and aortic pressure (AP) were measured simultaneously. The AP was measured with a pressure transducer (Gould 5000) attached to the end of a polyethylene catheter inserted to the aorta through the right femoral artery. The signal was amplified with a strain amplifier (6 M 53, Sanei Instrument Co., Ltd.). The original neural signal, CSNA, AP and ECG were displayed on an eight-channel heat-pen recorder (Sanei, Rectigraph 8K) and stored on a seven-channel magnetic tape recorder (TEAC, SR-31) for later analysis.
Experimental protocol Data were collected under following conditions: Protocol 1. Spontaneous heart rate was slowed by locally cooling the sinoatrial node by a device consisting of a copper pipe connected to a water bath cooler; the heart was paced by silver electrodes sutured on the right atrium and the pacing signal generated with a pulse generator (Nihon Kohden Electronic Stimulator, Isolator SS-302J). The pulse width was 6-8 ms and voltage was adjusted to a minimum to pace the heart. The heart was paced at intervals from 250 ms to 600
Experimental measurements
ms.
Cardiac sympathetic nerve activity was recorded from the left inferior cardiac nerve. After the tios of 2-4th ribs on the left were removed, a
Protocol 2. After cutting bilaterally carotid sinus and vago-aortic sympathetic nerves, AP was increased with norepinephrine (1 /xg/kg, i.v.).
133
There was no decrease in CSNA, thus ensuring that all subsequent data was collected in the baroreceptor denervated state. The previous protocol was repeated. Protocol 3. In carotid sinus and vagoaortic denervated cats the central end of the cut left aortic nerve was identified. Then the nerve was stimulated by a bipolar stimulus electrode with single shocks (0.5 ms, supramaximal stimulus, Nihon Kohden Electronic Stimulator, Isolator SS302J) at intervals from 200 ms to 1000 ms independently of cardiac cycle. Marker pulses of stimulus pulses were recorded simultaneously with the original neurogram, CSNA, AP and ECG. At the end of experiments, hexamethonium bromide (l mg/kg, i.v.) was administrated. The majority of CSNA was inhibited and decreased to near-noise level, thus ensuring that the activity was recorded mainly from a postganglionic sympathetic nerve.
Data analysis The time relations between CSNA and AP, or electrical stimuli applied to the left aortic nerve, were analyzed by averaging the data (Averager DAT 110, Nihon Kohden), because cardiac-related discharges in CSNA varied from beat to
A AP mmHg
beat as shown in Fig. 1A. Sweeps of 1 s were initiated by R-waves of the ECG or by stimulus pulses and data were sampled at 1 ms intervals. Fifty sweeps of CSNA and AP or stimulus marker signals were summated; displayed in analog form on an digital storage oscilloscope (Hitachi, VC6025) and recorded by a x-y plotter (Hitachi, Graph-Plotter 681). Three delay times were measured from the averaged AP and CSNA tracings. First, from the onset of rapid rise in AP to the peak amplitude in CSNA (Fig. 1B-1). The onset of rapid rise in AP was assumed to be close to the beginning of baroreceptor activation. The peak CSNA reflects the onset of inhibition. Second, from the half amplitude of rise in AP to the half amplitude of inhibition in CSNA (Fig. 1B-2). The half amplitudes of these traces could be determined more precisely than the peaks. Third, from the peak pressure to the minimum in CSNA (Fig. 1B-3). We assumed that the maximal inhibition occurs when CSNA reaches the bottom. As already mentioned, we used a resistance-capacitance integrator with time constant of 20 ms for analysis of CSNA. Therefore, same delay was included in three delay times. It did not affect comparisons between the delay times at different intervals.
B 2
:i
i
1
" t --J
200-
AP
\
mmHg
Original CSNA
lOO_
gV
CSNA
i •
HV
i 25
CSNA ,v
' i
i
25-o2 0 0 ms
4 0 0 ms
Fig. 1. (A) Recording of aortic pressure (AP), original CSNA and integrated CSNA (CSNA) during control period in one cat. (B) Corresponding averaged (50 sweeps of 1 s, triggered by the R-wave of ECG) AP and CSNA. Delay times to the onset of inhibition in CSNA (1), to the half amplitude of inhibition in CSNA (2) and to the maximal inhibition in CSNA (3), the peak amplitude (P) and bottom amplitude (B) of CSNA.
134
B
A
Stimulus pulse
Original CSNA
~ i! ¸¸
i
" ! i!~
~i
Stimulus pulse
~ "
i
-~
1¢
pV
10-
CSNA
CSNA
~!~
i i
pV
O-
,i
--2-
O - " ~¢ij ......
-
200 ms 3-
4 0 0 ms
Fig. 2. (A) Recording of marker pulses of stimulus pulses, original CSNA and integrated CSNA (CSNA) during stimulation of the left aortic nerve with single shocks (0.5 ms, supramaximal voltage) at 500 ms intervals. (B) Corresponding averaged data (triggered by marker pulses of stimulus pulses). Delay times to the onset of inhibition (1), to the half amplitude of inhibition (2) and to the maximal inhibition (3).
The amplitude of synchronized C S N A reflects the number of active fibers. Therefore peak and bottom amplitudes of synchronized discharges were measured from averaged data (Fig. 1B). C S N A was averaged during electrical stimulation of the left aortic nerve with single stimulus pulses (Fig. 2A). Delay times from the stimulus pulse to the onset (Fig. 2B-l), to the half ampli-
tude of inhibition (Fig. 2B-2) and to the maximal inhibition (Fig. 2B-3) were measured.
Statistics Data are expressed as mean + S.E.M. An analysis of variance and paired t-test with Bonferroni correction with multiple comparison was used. A
TABLE I
Cardiac interval, systolic (SAP) and diastolic (DAP) aortic pressure, delay-times to the onset and half amplitude of inhibition and to the maximal inhibition of CSNA and the peak and bottom amplitude of discharge in CSNA (mean 5: S.E.M.) during control period and during different pacing intervals. Cardiac interval (ms)
SAP (mmHg)
DAP (mmHg)
Delay to onset of inhib,
Delay to half ampl. of inhib,
Delay to maximal inhib.
(ms)
(ms)
(ms)
Peak amplitude (/~V)
Bottom amplitude (/zV)
Control
315 + 9
155 5 : 6
108 5:7
164 5 : 7
195 5 : 7 *
233 5 : 9
6.2 + 1.9
1.4 5:0.8
I0
Pacing Pacing Pacing Pacing Pacing Pacing mean
250 300 350 500 600
152 5 : 9 152 5 : 9 149 ± 8 1515:11 159 5 : 9
114 5:8 111 + 8 107 + 7 97+7 102 5:8
162 5 : 6 160 + 7 164 5 : 8 1605:5 153 5:10
194 + 9 196 5:10 207 5:12 1925:11 214 + 21
225 5 : 8 229 5:11 245 5:10 2045:13 207 + 18
4.5 + 1.6 5.0 5:1.9 5.3 5:2.1 8.55:3.5 10.7 5:4.0
1.4 + 0.7 1.1 5:0.7 1.0 5:0.6 0.35:0.1 0.8 5:0.6
10 10 10 7 5
369_+18
151 5 : 4
107 5:4
160 5 : 3
198+ 6 *
225± 5 *
6.2+1.1
1.05:0.3
42
* Significant difference compared to the delay time to the onset of inhibition.
135 P value of less than 0.05 was considered to be statistically significant.
A 200AP
Results
mmHg 1
0
0
-
~
Control period Cardiac interval, systolic aortic pressure (SAP) and diastolic aortic pressure (DAP) were 315 + 9 ms, 155 + 6 mmHg and 108 _ 7 mmHg, respectively (Table I). Delay times to the onset of inhibition, to the half amplitude of inhibition and to the maximal inhibition were 164 + 7 ms, 195 + 7 ms and 233 + 9 ms, respectively. The delay time to the half amplitude of inhibition in CSNA and to the maximal inhibition in CSNA were significantly longer than the delay time to the onset of inhibition.
Change in cardiac interval by artificial pacing in baroreceptor intact state The heart was paced artificially with 250 ms, 300 ms, 350 ms, 500 ms and 600 ms intervals. Two examples of averaged data obtained under 250 ms and 500 ms pacing intervals are shown in Fig. 3. Delay times of the inhibition in CSNA were almost the same. In Fig. 4, relationships between delay times and cardiac interval obtained from all the cats are summarized. Delay time to the onset of inhibition (D1) varied from 153 _+ 10 ms to 164 + 8 ms, but there were no significant differences among the delay times at different pacing intervals. Delay time to the half amplitude of inhibition (D2) varied from 192 + 11 ms to 214 5: 21 ms, but no significant differences was found. Delay time to the maximal inhibition (D3) varied from 204 _+ 13 ms to 245 +_ 10 ms, but no statistically significant differences was found. The delay times to the half amplitude of inhibition (D2) in CSNA and to the maximal inhibition (D3) in CSNA were significantly longer than the delay time to the onset of inhibition (D1) (Table I). The delay times during artificial cardiac pacing did not differ significantly from those during spontaneous beating at control period (Table I). During artificial cardiac pacing with different intervals, SAP did not changed significantly but
CSNA 2 5 - ~ ~ pv
~ / f ~ ~
o-
B 200Ap
¸
mmHg1
0
CSNA 2 5 gv 0-
0
~
-
~ 200ms
Fig. 3. AveragedAP and CSNA during artificial pacing of the heart at 250 ms (A) and 500 ms intervals(B) in one cat.
DAP decreased when cardiac interval increased (Fig. 5A, Table I). The amplitude of averaged CSNA at different pacing intervals was not constant (Fig. 3). The peak amplitude tended to increase. Whereas the bottom amplitude tended to decrease when cardiac interval increased (Fig. 5B). When the interval was shortest (250 ms) the difference between peak and bottom amplitude was significantly smaller than if the cardiac interval was longer.
Baroreceptor denervation After the sino-aortic-vagal baroreceptor denervation the cardiac related rhythmicity was completely lost and faster rhythmicities were prevailing. The averaged CSNA showed small fluctua-
136
300
E E >,
200
A o r t i c nerve stimulation under baroreceptor denert~,ation In b a r o r e c e p t o r d e n e r v a t e d state the left aortic n e r v e w a s s t i m u l a t e d w i t h s i n g l e p u l s e s (0.5
D2
ms, s u p r a m a x i m a l voltage) i n d e p e n d e n t l y f r o m c a r d i a c cycle. A v e r a g e d C S N A r e s p o n s e s o b -
100
t a i n e d at s t i m u l u s i n t e r v a l s o f 250 m s , 350 m s a n d
O I
I
I
200
400
600
A 200
Cardiacinterval(ms) Fig. 4. Delay times to the onset of inhibition (D1), to the half amplitude of inhibition (D2) and to the maximal inhibition (D3) (mean + S.E.M.) in all the cats during pacing of the heart at 250 ms, 300 ms, 350 ms, 500 ms and 600 ms intervals. There were no statistically significant differences in delay times among different pacing intervals.
~ "r" E E
~
100
DAP
L , - - J m _ , ___._1
n
tions with various frequencies and as expected, artificial pacing had no effect to the rhythmicity in the CSNA (Fig. 6).
SAP
0
0
! 200
I 400
I 600
Cardiac interval (ms)
B
TABLE II
Stimulation interval and delay times to the onset of inhibition, to the half amplitude of inhibition and to the maximal inhibition in CSNA (mean -+ S.E.M.) during stimulation of the left aortic nerve
A
>
15
"-1
Stimulus interval (ms)
Dclay to onset of inhib, (ms)
Delay to half ampl. inhib, (ms)
Delay to maximal inhib. (ms)
200 250 300 350 400 500 600 800 1000
150± 7 145± 6 149± 8 147±11 152±11 145±12 149±11 138 _+16 151 _+13
196± 8 193± 7 194_+12 187+11 192+12 186±14 195_+13 191 _+13 196 ± 12
256_+10 263+14 264± 15 260+10 273_+ 7 269_+ 9 266±17 286 _+16 283 ± 10
mean
147_+ 3
193+ 4 *
267± 7 *
"O
105
~
Peak
0
=~
Bottom
(3.
5 5 5 5 5 5 5 5 5 45
There were no significant changes among the delay times at different stimulus intervals. * Significant difference in delay times compared to the delay time to the onset of inhibition.
E
<
0
200
400
600
Cardiac interval (ms) Fig. 5. (A) Systolic (SAP) and diastolic (DAP) pressure during pacing the heart at 250 ms, 300 ms, 350 ms, 500 ms and 600 ms intervals (mean±S.E.M.). * Significant ( P < 0 . 0 5 ) decrease in DAP when pacing interval was increased. (B) The peak amplitude (Peak) and the lowest amplitude (Bottom) in cardiac-related CSNA when the heart was paced.
137
A
tude of inhibition and to the maximal inhibition varied from 138 + 16 ms to 152-t-11 ms, from 186 _+ 14 ms to 196 _+ 12 ms and from 260 +_ 10 ms to 286 _+ 16 ms, respectively. There were no significant differences among the delay times at different stimulus intervals (Table II). T h e d e l a y s to the half amplitude of inhibition (193 + 4 ms) and the maximal inhibition (267 _+ 7 ms) were significantly longer that to the onset of inhibition (147 + 3 ms).
AP mmHg
2oo-
100-
CSNA uv
250--
A
B AP rnmHg
CSNA
Stimulus
200-
CSNA iJV
if-
B
25-
Stimulus 200
ms
Fig. 6. Averaged AP and CSNA during artificial pacing of the heart at 250 ms (A) and 500 ms (B) intervals in baroreccptor dcncrvatcd state in one cat.
CSNA uv
C 500 ms are shown in Fig. 7. In all the examples significant inhibition in averaged CSNA responses was produced by the stimulus. The averaged CSNA showed an inhibitory rhythm related to stimulus frequency. As described in the methods, delay times to the onset, half amplitude of inhibition and to the maximal inhibition in averaged CSNA were measured at nine stimulus intervals (Table II). Delay times obtained from all the cats were plotted as a function of stimulus interval in Fig. 8. Delay times to onset of the inhibition, to the half ampli-
Stimulus
CSNA uv
~
1
, rL_
0
-
~
0200 ms
Fig. 7. Averaged CSNA (50 sweeps of 1 s) triggered by the marker pu|ses of stimulus pulse ( * ) during stimulation of left aortic nerve at intervals of 250 ms (A), 350 ms (B) and 500 ms (C) after barorcceptor denervatJon in one cat.
138
Discussion
We studied the origin of cardiac-related CSNA by artificially pacing the heart in anaesthetized cats. Our results indicate that cardiac-related rhythm in CSNA is produced reflexly by inhibiting the transmission of fundamental rhythmicity by periodic baroreceptor input [15], not by entraining a central free running 2-6 Hz rhythm to cardiac cycle [5-7]. Time relations between inhibition in CSNA and cardiac cycle In our study, the delay of inhibition in CSNA was almost constant and independent of the length of cardiac interval during pacing of the heart at different intervals. In agreement with earlier studies [6,8,16], cardiac-related rhythm in CSNA was lost after baroreceptor denervation (Fig. 6). Stimulation of the left aortic nerve with single shocks at different intervals in baroreceptor denervated state, produced an inhibitory rhythm in CSNA with almost constant delay time. Thus, baroreceptor input was entirely responsible of producing cardiac-related inhibitory rhythm in CSNA. Constant delay times indicate that the cardiac-related rhythm, or the inhibitory rhythm related to the stimulus frequency of the aortic 30O
E 200 E loo 0
D3 ~ ~ T c ~ - ~
- ~
D1 ~
~
~
~
I
I
I
I
I
200
4o0
600
800
1000
S t i m u l u s interval (ms) Fig. 8. Delay times from the stimulus pulse to the onset of inhibition (D1), to the half amplitude of inhibition (D2) and to the maximal inhibition (D3) in CSNA (mean+S.E.M.) during stimulation of left aortic depressor nerve at intervals from 200 ms to 1000 ms after baroreceptor denervation. There were no significant differences in delay times at different stimulus intervals.
A AP
200-
mmHg
100-
CSNA
10-
gV
l
200ms
B Stimulus
h
CSNAw 1
J~ r~ n 0
-
~
0200 ms Fig. 9. (A) Averaged AP and CSNA during pacing the heart at intervals of 250 ms, 350 ms and 500 ms superimposed to the same figure in one cat. (B) Averaged CSNA during stimulation of left aortic depressor nerve at intervals of 250 ms, 350 ms and 500 ms superimposed to the same figure in other cat. The averager was triggered by the R-wave of ECG in A and by the marker pulses of the stimulus pulses ( * ) in B. The bar shows the time from the onset of inhibition to the maximal inhibition in CSNA.
nerve, were produced reflexly by periodic baroreceptor input due to pulsatile pressure or electrical stimulation. The time course of inhibition from the onset to the maximal inhibition was almost constant independently of cardiac interval when the heart was paced at different intervals (Fig. 9A). The duration of inhibition in averaged CSNA increased when cardiac interval was increased. This may be caused by increased baroreceptor activation due to increased pulsatile pressure [4]. When single
139 stimulus pulses was applied to the left aortic nerve the time course of averaged CSNA responses was almost constant independently of stimulus frequency (Fig. 9B). This suggests that the stimulus-related rhythm was caused by reflex inhibition of tonic activity in sympathetic nerve. According to our model, the tonic activity originates from an oscillator with 8-12 Hz fundamental rhythm and cardiac-related rhythm is produced by blocking the transmission of this fundamental rhythm in gate operators [8,9,15]. This is seen as a reflex-inhibition in sympathetic nerve activity. In a closed-loop condition the cardiac-related inhibitory rhythm is independent of the fundamental rhythm but related to another oscillator, the sino atrial node. Previously Gebber proposed the entrainment hypothesis based on the data in Fig. 8 in his report [5]. If we assume that the minimum activity in averaged data reflects maximum inhibition, the delay time from the peak of femoral artery pressure to minimum activity (maximal inhibition) reflects the delay time in his data. The delay time was about 145 ms at three different cardiac intervals. It is not possible to explain our data obtained during stimulation of the left aortic nerve (Figs. 7 and 9B) by entrainment hypothesis. The delay times were constant and independent of stimulus frequency and there was no entrainment in averaged activity.
Delay times of inhibition in CSNA In our study, the delay time to the onset of inhibition in CSNA (164 + 7 ms) is in accordance with that of previously studies (164 + 31 ms) [13]. Also the delay to maximal inhibition (233 + 9 ms) is at the range of the maximal inhibition response of baroreflex (125-330 ms) in CSNA after rise in carotid sinus pressure [3]. The delay time to the maximal inhibition was significantly longer than the delay time to the onset of inhibition when the aortic nerve was stimulated with single shocks. Because of the supramaximal stimulation all afferent fibers should have been activated simultaneously by the stimulus pulse. The finding that the delay time to the maximal inhibition was 120 ms longer than to the onset of inhibition probably reflects differ-
ences in reflex times among individual nerve fibers. In averaged data, inhibition starts when the fastest fibers are inhibited and reach maximum when most of the fibers are inhibited. Half amplitudes of averaged AP and CSNA tracings could be determined most precisely. Although the amplitude of averaged CSNA altered, the time course from the onset to the maximal inhibition was almost constant during artificial pacing and during stimulation of aortic nerve. The delay time to half amplitude of inhibition in CSNA during artificial pacing (198 + 6 ms) and during stimulation of aortic nerve (193 + 4 ms) were at the same range. Thus, the delay time from the half amplitude of rapid rise in AP to the half amplitude of inhibition in CSNA might be the most suitable indirect measure of baroreflex time in inhibition of CSNA. In conclusion, our results indicate that cardiac-related rhythm in CSNA is produced reflexly by inhibiting the transmission of the fundamental rhythmicity by periodic baroreceptor input, not by entraining a central free running 2 - 6 Hz rhythm to cardiac cycle.
Acknowledgements We thank prof. Tada from Osaka University, who kindly supported A.H. in this study. This study was supported by a Grant for Scientific Research from the Ministry of Education, Science and Culture and by a Research Grant for Cardiovascular Diseases from the Ministry of Health and Welfare of Japan. A.H. is a Japan Ministry of Education, Science and Culture Fellow.
References 1 Barman, S.M., Gebber, G.L. and Zhong, S., The 10-Hz rhythm in sympathetic nerve discharge, Am. J. Physiol., 262 (1992) R1006-R1014. 2 Cohen, M.I. and Gootman, P.M., Periodicities in efferent discharge of splanchnic nerve of the cat, Am. J. Physiol., 218 (1970) 1092-1101. 3 Coote, J.H. and Macleod, V.H., Evidence for the involvement in the baroreceptor reflex of a descending inhibitory pathway, J. Physiol., 241 (1974) 477-496.
140 4 Ead, H.W., Green, J.H. and Neil, E., A comparison of the effects of pulsatile and nonpulsatile blood flow through the carotid sinus on the reflexogenic activity of the sinus baroceptors in the cat, J. Physiol., 188 (1952) 509-519. 5 Gebber, G.L. Basis for phase relations between baroreceptor and sympathetic nervous discharge, Am. J. Physiol., 230 (1976) 263-270. 6 Gebber, G.L., Central oscillators responsible for sympathetic nerve discharge, Am. J. Physiol., 239 (1980) H143H155. 7 Gebber, G.L., Central determinants of sympathetic nerve discharge, In A.D. Loewy and K.M. Spyer (Eds.) Central Regulation of Autonomic Functions, Oxford Univ. Press, New York, 1990, pp. 126-144. 8 Malpas, S.C. and Ninomiya, I., Fundamental rhythm of renal sympathetic nerve activity in anaesthetized cats, J. Auton. Nerv. Syst., 37 (1992) 11-18. 9 Malpas, S.C. and Ninomiya, I., Effect of chemoreceptor stimulation on the periodicity of renal sympathetic nerve activity in anaesthetized cats, J. Auton. Nerv. Syst. 37, (1992) 19-28. 10 Malpas, S.C. and Ninomiya, I., The amplitude and periodicity of synchronized renal sympathetic nerve discharge in anaesthetized cats: differential effect of baroreceptor activity, J. Auton. Nerv. Syst., 40 (1992) 189-198.
11 Malpas, S.C. and Ninomiya, I., Effect of asphyxia on the frequency and amplitude modulation of synchronized renal nerve activity in the cat, J. Auton. Nerv. Syst. 40 (1992) 199-206. 12 Malpas, S.C. and Ninomiya, I., A new approach to analysis of synchronized sympathetic nerve activity, Am. J. Physiol., 263 (1992) H1311-H1317. 13 Ninomiya, I., Nishimaru, N. and Irisawa, H., Sympathetic nerve activity to the spleen, kidney, and heart in response to baroreceptor input, Am. J. Physiol., 221 (1971) 13461351. 14 Ninomiya, I., Nishiura, N., Matsukawa, K. and Akiyama, T., Fundamental rhythm of cardiac sympathetic nerve activity in awake cats at rest and during body movement, Jpn. J. Physiol., 39 (1989) 743-753. 15 Ninomiya, I., Akiyama, T. and Nishiura, N., Mechanism of cardiac related sympathetic nerve activity in awake cats, Am. J. Physiol., 259 (1990) R499-R506. 16 Taylor, D.G. and Gebber, G.L., Baroreceptor mechanisms controlling sympathetic nervous rhythms of central origin, Am. J. Physiol., 228, (1975) 1002-1013. 17 Zhong, S., Barman, S.M. and Gebber, G.L., Effects of brain stem lesions on 10-Hz and 2-to 6-Hz rhythms in sympathetic nerve discharge, Am. J. Physiol., 262 (1992) R1015-R1024.
131
© 1994 Elsevier Science B.V. All rights reserved 0165-1838/94/$07.00 JANS 01498
Origin of cardiac-related synchronized cardiac sympathetic nerve activity in anaesthetized cats Antti E. H e d m a n a, Kanji Matsukawa a and Ishio Ninomiya b,. a Department of Cardiac Physiology, National Cardiovascular Center, Research Institute, Osaka, Japan, and b Department of Physiology, Institute of Health Sciences, School of Medicine, Hiroshima University, 1-2-3 Kasumi-cho, Minami-ku Hiroshima 734, Japan (Received 12 July 1993) (Revision received and accepted 31 August 1993)
Key words: Baroreceptor input; Cardiac sympathetic nerve activity; Cardiac pacing; Cardiac-related rhythm Abstract To study the origin of cardiac-related rhythm in cardiac sympathetic nerve activity (CSNA), ECG, aortic pressure and CSNA were recorded when cardiac interval was changed by artificial pacing, or when the aortic nerve was stimulated after baroreceptor denervation in anaesthetized cats. CSNA was averaged by using the R-wave of ECG, or stimulus pulse as a trigger. Delay times from arterial pulse or stimulus pulse to the onset and half amplitude of inhibition and to the maximal inhibition were measured from the averaged data. The delay of inhibition in CSNA was constant and independent of pacing interval. Stimulation of the aortic nerve with single shocks caused an inhibition in averaged CSNA. The delay of inhibition was constant and independent of stimulus frequency. These results indicate that the cardiac-related rhythm in CSNA is produced reflexly by inhibiting the transmission of the fundamental rhythmicity due to periodic baroreceptor input.
Introduction
Synchronized discharges in postganglionic sympathetic nerves in awake [14,15], decerebrate [1,2,17] and anaesthetized [2,8-12] animals show a rhythmicity related to cardiac cycle at frequency of 2-6 Hz and a faster rhythmicity of 8-12 Hz. It has been agreed that the 8-12 Hz rhythm is of central origin [1,2,15,17]. Whereas there are contrasting hypotheses how cardiac-related synchronized discharges are produced.
* Corresponding author. Tel: +81 082 246 7825; Fax: +81 082 241 0508.
SSDI 0 1 6 5 - 1 8 3 8 ( 9 3 ) E 0 1 15-L
It has been stated that an irregular 2-6 Hz rhythm is generated in brain stem by multiple oscillators with a shifting lead [7]. This free running rhythm would then be entrained in a 1:1 relation to cardiac cycle by pulsatile barorecept0rs input. This concept is based mainly on two observations: phase relation between baroreceptor activity and sympathetic nerve discharges can be shifted by changing heart rate and persistence of irregular 2-6 Hz rhythmicity after baroreceptor denervation [7]. Recently, however, we have presented a new model of a fundamental rhythm oscillator and gate operators. The transmission of the fundamental rhythm (8-12 Hz) is controlled by closing the gate operators by the periodic
132
baroreceptor input and thus a cardiac-related rhythm in CSNA is produced [15]. This is seen as a reflex-inhibition in sympathetic nerve activity. According to the entrainment hypothesis, the point of maximum CSNA occurring in early or middiastole at high heart rates should shift backwards to peak systole and then into late diastole of the preceding cardiac cycle when heart rate is progressively lowered [5]. Instead, according to our hypothesis constant delay of the inhibition in sympathetic nerve activity should be expected when heart rate is changed. In the present study, we focused on this fundamental discrepancy between these two hypotheses. To reveal the mechanism producing the cardiac-related rhythm in synchronized CSNA, time relations between periodic baroreceptor input and synchronized CSNA were examined, when cardiac interval was changed by artificial pacing the heart.
Materials and Methods
Preparation of animals Fifteen cats of either sex weighing between 2.2 and 4.5 kg were anaesthetized with sodium pentobarbital (30-40 mg/kg, i.p.) and immobilized with pancuronium sodium (0.7 mg/kg, i.m.). The trachea was intubated and the lungs ventilated with intermittent positive pressure (Shinano SN480-5 respirator). The femoral artery and vein were cannulated with polyethylene catheters. Additional doses of anaesthetic (5 mg/kg, i.v.) were injected once an hour to maintain surgical anaesthesia. Arterial blood gases were measured prior to measurements of nerve activity, and if necessary the ventilation rate and tidal volume of the respirator was adjusted to ensure blood gases were within the normal range (PaO 2 = 100 + 5 mmHg, PaCO 2 = 33 _+ 3 mmHg and pH = 7.35 _+ 0.05). Body temperature was maintained throughout the experiment by warming.
branch (1-1.5 cm) of the nerve was separated using dissecting microscope (Olympus OME) and a mineral oil pool was prepared around the nerve. The efferent nerve activity was recorded at the central end of the cut nerve by a pair of silver electrodes. The signal was amplified by a biophysical amplifier ( M E G 110, Nihon Kohden) with a high cut-off frequency of 3000 Hz and a low cut-off frequency of 50 Hz. This was then passed through a full-wave rectifier circuit and integrated with a resistance-capacitance integrator with a time constant of 20 ms; this produced a positive deflection proportional to the frequency discharge in the original neurogram and it was defined as integrated CSNA (CSNA). The signals were monitored on a four channel digital storage oscilloscope (Nihon Kohden, VC-10). In all the cats, the ECG and aortic pressure (AP) were measured simultaneously. The AP was measured with a pressure transducer (Gould 5000) attached to the end of a polyethylene catheter inserted to the aorta through the right femoral artery. The signal was amplified with a strain amplifier (6 M 53, Sanei Instrument Co., Ltd.). The original neural signal, CSNA, AP and ECG were displayed on an eight-channel heat-pen recorder (Sanei, Rectigraph 8K) and stored on a seven-channel magnetic tape recorder (TEAC, SR-31) for later analysis.
Experimental protocol Data were collected under following conditions: Protocol 1. Spontaneous heart rate was slowed by locally cooling the sinoatrial node by a device consisting of a copper pipe connected to a water bath cooler; the heart was paced by silver electrodes sutured on the right atrium and the pacing signal generated with a pulse generator (Nihon Kohden Electronic Stimulator, Isolator SS-302J). The pulse width was 6-8 ms and voltage was adjusted to a minimum to pace the heart. The heart was paced at intervals from 250 ms to 600
Experimental measurements
ms.
Cardiac sympathetic nerve activity was recorded from the left inferior cardiac nerve. After the tios of 2-4th ribs on the left were removed, a
Protocol 2. After cutting bilaterally carotid sinus and vago-aortic sympathetic nerves, AP was increased with norepinephrine (1 /xg/kg, i.v.).
133
There was no decrease in CSNA, thus ensuring that all subsequent data was collected in the baroreceptor denervated state. The previous protocol was repeated. Protocol 3. In carotid sinus and vagoaortic denervated cats the central end of the cut left aortic nerve was identified. Then the nerve was stimulated by a bipolar stimulus electrode with single shocks (0.5 ms, supramaximal stimulus, Nihon Kohden Electronic Stimulator, Isolator SS302J) at intervals from 200 ms to 1000 ms independently of cardiac cycle. Marker pulses of stimulus pulses were recorded simultaneously with the original neurogram, CSNA, AP and ECG. At the end of experiments, hexamethonium bromide (l mg/kg, i.v.) was administrated. The majority of CSNA was inhibited and decreased to near-noise level, thus ensuring that the activity was recorded mainly from a postganglionic sympathetic nerve.
Data analysis The time relations between CSNA and AP, or electrical stimuli applied to the left aortic nerve, were analyzed by averaging the data (Averager DAT 110, Nihon Kohden), because cardiac-related discharges in CSNA varied from beat to
A AP mmHg
beat as shown in Fig. 1A. Sweeps of 1 s were initiated by R-waves of the ECG or by stimulus pulses and data were sampled at 1 ms intervals. Fifty sweeps of CSNA and AP or stimulus marker signals were summated; displayed in analog form on an digital storage oscilloscope (Hitachi, VC6025) and recorded by a x-y plotter (Hitachi, Graph-Plotter 681). Three delay times were measured from the averaged AP and CSNA tracings. First, from the onset of rapid rise in AP to the peak amplitude in CSNA (Fig. 1B-1). The onset of rapid rise in AP was assumed to be close to the beginning of baroreceptor activation. The peak CSNA reflects the onset of inhibition. Second, from the half amplitude of rise in AP to the half amplitude of inhibition in CSNA (Fig. 1B-2). The half amplitudes of these traces could be determined more precisely than the peaks. Third, from the peak pressure to the minimum in CSNA (Fig. 1B-3). We assumed that the maximal inhibition occurs when CSNA reaches the bottom. As already mentioned, we used a resistance-capacitance integrator with time constant of 20 ms for analysis of CSNA. Therefore, same delay was included in three delay times. It did not affect comparisons between the delay times at different intervals.
B 2
:i
i
1
" t --J
200-
AP
\
mmHg
Original CSNA
lOO_
gV
CSNA
i •
HV
i 25
CSNA ,v
' i
i
25-o2 0 0 ms
4 0 0 ms
Fig. 1. (A) Recording of aortic pressure (AP), original CSNA and integrated CSNA (CSNA) during control period in one cat. (B) Corresponding averaged (50 sweeps of 1 s, triggered by the R-wave of ECG) AP and CSNA. Delay times to the onset of inhibition in CSNA (1), to the half amplitude of inhibition in CSNA (2) and to the maximal inhibition in CSNA (3), the peak amplitude (P) and bottom amplitude (B) of CSNA.
134
B
A
Stimulus pulse
Original CSNA
~ i! ¸¸
i
" ! i!~
~i
Stimulus pulse
~ "
i
-~
1¢
pV
10-
CSNA
CSNA
~!~
i i
pV
O-
,i
--2-
O - " ~¢ij ......
-
200 ms 3-
4 0 0 ms
Fig. 2. (A) Recording of marker pulses of stimulus pulses, original CSNA and integrated CSNA (CSNA) during stimulation of the left aortic nerve with single shocks (0.5 ms, supramaximal voltage) at 500 ms intervals. (B) Corresponding averaged data (triggered by marker pulses of stimulus pulses). Delay times to the onset of inhibition (1), to the half amplitude of inhibition (2) and to the maximal inhibition (3).
The amplitude of synchronized C S N A reflects the number of active fibers. Therefore peak and bottom amplitudes of synchronized discharges were measured from averaged data (Fig. 1B). C S N A was averaged during electrical stimulation of the left aortic nerve with single stimulus pulses (Fig. 2A). Delay times from the stimulus pulse to the onset (Fig. 2B-l), to the half ampli-
tude of inhibition (Fig. 2B-2) and to the maximal inhibition (Fig. 2B-3) were measured.
Statistics Data are expressed as mean + S.E.M. An analysis of variance and paired t-test with Bonferroni correction with multiple comparison was used. A
TABLE I
Cardiac interval, systolic (SAP) and diastolic (DAP) aortic pressure, delay-times to the onset and half amplitude of inhibition and to the maximal inhibition of CSNA and the peak and bottom amplitude of discharge in CSNA (mean 5: S.E.M.) during control period and during different pacing intervals. Cardiac interval (ms)
SAP (mmHg)
DAP (mmHg)
Delay to onset of inhib,
Delay to half ampl. of inhib,
Delay to maximal inhib.
(ms)
(ms)
(ms)
Peak amplitude (/~V)
Bottom amplitude (/zV)
Control
315 + 9
155 5 : 6
108 5:7
164 5 : 7
195 5 : 7 *
233 5 : 9
6.2 + 1.9
1.4 5:0.8
I0
Pacing Pacing Pacing Pacing Pacing Pacing mean
250 300 350 500 600
152 5 : 9 152 5 : 9 149 ± 8 1515:11 159 5 : 9
114 5:8 111 + 8 107 + 7 97+7 102 5:8
162 5 : 6 160 + 7 164 5 : 8 1605:5 153 5:10
194 + 9 196 5:10 207 5:12 1925:11 214 + 21
225 5 : 8 229 5:11 245 5:10 2045:13 207 + 18
4.5 + 1.6 5.0 5:1.9 5.3 5:2.1 8.55:3.5 10.7 5:4.0
1.4 + 0.7 1.1 5:0.7 1.0 5:0.6 0.35:0.1 0.8 5:0.6
10 10 10 7 5
369_+18
151 5 : 4
107 5:4
160 5 : 3
198+ 6 *
225± 5 *
6.2+1.1
1.05:0.3
42
* Significant difference compared to the delay time to the onset of inhibition.
135 P value of less than 0.05 was considered to be statistically significant.
A 200AP
Results
mmHg 1
0
0
-
~
Control period Cardiac interval, systolic aortic pressure (SAP) and diastolic aortic pressure (DAP) were 315 + 9 ms, 155 + 6 mmHg and 108 _ 7 mmHg, respectively (Table I). Delay times to the onset of inhibition, to the half amplitude of inhibition and to the maximal inhibition were 164 + 7 ms, 195 + 7 ms and 233 + 9 ms, respectively. The delay time to the half amplitude of inhibition in CSNA and to the maximal inhibition in CSNA were significantly longer than the delay time to the onset of inhibition.
Change in cardiac interval by artificial pacing in baroreceptor intact state The heart was paced artificially with 250 ms, 300 ms, 350 ms, 500 ms and 600 ms intervals. Two examples of averaged data obtained under 250 ms and 500 ms pacing intervals are shown in Fig. 3. Delay times of the inhibition in CSNA were almost the same. In Fig. 4, relationships between delay times and cardiac interval obtained from all the cats are summarized. Delay time to the onset of inhibition (D1) varied from 153 _+ 10 ms to 164 + 8 ms, but there were no significant differences among the delay times at different pacing intervals. Delay time to the half amplitude of inhibition (D2) varied from 192 + 11 ms to 214 5: 21 ms, but no significant differences was found. Delay time to the maximal inhibition (D3) varied from 204 _+ 13 ms to 245 +_ 10 ms, but no statistically significant differences was found. The delay times to the half amplitude of inhibition (D2) in CSNA and to the maximal inhibition (D3) in CSNA were significantly longer than the delay time to the onset of inhibition (D1) (Table I). The delay times during artificial cardiac pacing did not differ significantly from those during spontaneous beating at control period (Table I). During artificial cardiac pacing with different intervals, SAP did not changed significantly but
CSNA 2 5 - ~ ~ pv
~ / f ~ ~
o-
B 200Ap
¸
mmHg1
0
CSNA 2 5 gv 0-
0
~
-
~ 200ms
Fig. 3. AveragedAP and CSNA during artificial pacing of the heart at 250 ms (A) and 500 ms intervals(B) in one cat.
DAP decreased when cardiac interval increased (Fig. 5A, Table I). The amplitude of averaged CSNA at different pacing intervals was not constant (Fig. 3). The peak amplitude tended to increase. Whereas the bottom amplitude tended to decrease when cardiac interval increased (Fig. 5B). When the interval was shortest (250 ms) the difference between peak and bottom amplitude was significantly smaller than if the cardiac interval was longer.
Baroreceptor denervation After the sino-aortic-vagal baroreceptor denervation the cardiac related rhythmicity was completely lost and faster rhythmicities were prevailing. The averaged CSNA showed small fluctua-
136
300
E E >,
200
A o r t i c nerve stimulation under baroreceptor denert~,ation In b a r o r e c e p t o r d e n e r v a t e d state the left aortic n e r v e w a s s t i m u l a t e d w i t h s i n g l e p u l s e s (0.5
D2
ms, s u p r a m a x i m a l voltage) i n d e p e n d e n t l y f r o m c a r d i a c cycle. A v e r a g e d C S N A r e s p o n s e s o b -
100
t a i n e d at s t i m u l u s i n t e r v a l s o f 250 m s , 350 m s a n d
O I
I
I
200
400
600
A 200
Cardiacinterval(ms) Fig. 4. Delay times to the onset of inhibition (D1), to the half amplitude of inhibition (D2) and to the maximal inhibition (D3) (mean + S.E.M.) in all the cats during pacing of the heart at 250 ms, 300 ms, 350 ms, 500 ms and 600 ms intervals. There were no statistically significant differences in delay times among different pacing intervals.
~ "r" E E
~
100
DAP
L , - - J m _ , ___._1
n
tions with various frequencies and as expected, artificial pacing had no effect to the rhythmicity in the CSNA (Fig. 6).
SAP
0
0
! 200
I 400
I 600
Cardiac interval (ms)
B
TABLE II
Stimulation interval and delay times to the onset of inhibition, to the half amplitude of inhibition and to the maximal inhibition in CSNA (mean -+ S.E.M.) during stimulation of the left aortic nerve
A
>
15
"-1
Stimulus interval (ms)
Dclay to onset of inhib, (ms)
Delay to half ampl. inhib, (ms)
Delay to maximal inhib. (ms)
200 250 300 350 400 500 600 800 1000
150± 7 145± 6 149± 8 147±11 152±11 145±12 149±11 138 _+16 151 _+13
196± 8 193± 7 194_+12 187+11 192+12 186±14 195_+13 191 _+13 196 ± 12
256_+10 263+14 264± 15 260+10 273_+ 7 269_+ 9 266±17 286 _+16 283 ± 10
mean
147_+ 3
193+ 4 *
267± 7 *
"O
105
~
Peak
0
=~
Bottom
(3.
5 5 5 5 5 5 5 5 5 45
There were no significant changes among the delay times at different stimulus intervals. * Significant difference in delay times compared to the delay time to the onset of inhibition.
E
<
0
200
400
600
Cardiac interval (ms) Fig. 5. (A) Systolic (SAP) and diastolic (DAP) pressure during pacing the heart at 250 ms, 300 ms, 350 ms, 500 ms and 600 ms intervals (mean±S.E.M.). * Significant ( P < 0 . 0 5 ) decrease in DAP when pacing interval was increased. (B) The peak amplitude (Peak) and the lowest amplitude (Bottom) in cardiac-related CSNA when the heart was paced.
137
A
tude of inhibition and to the maximal inhibition varied from 138 + 16 ms to 152-t-11 ms, from 186 _+ 14 ms to 196 _+ 12 ms and from 260 +_ 10 ms to 286 _+ 16 ms, respectively. There were no significant differences among the delay times at different stimulus intervals (Table II). T h e d e l a y s to the half amplitude of inhibition (193 + 4 ms) and the maximal inhibition (267 _+ 7 ms) were significantly longer that to the onset of inhibition (147 + 3 ms).
AP mmHg
2oo-
100-
CSNA uv
250--
A
B AP rnmHg
CSNA
Stimulus
200-
CSNA iJV
if-
B
25-
Stimulus 200
ms
Fig. 6. Averaged AP and CSNA during artificial pacing of the heart at 250 ms (A) and 500 ms (B) intervals in baroreccptor dcncrvatcd state in one cat.
CSNA uv
C 500 ms are shown in Fig. 7. In all the examples significant inhibition in averaged CSNA responses was produced by the stimulus. The averaged CSNA showed an inhibitory rhythm related to stimulus frequency. As described in the methods, delay times to the onset, half amplitude of inhibition and to the maximal inhibition in averaged CSNA were measured at nine stimulus intervals (Table II). Delay times obtained from all the cats were plotted as a function of stimulus interval in Fig. 8. Delay times to onset of the inhibition, to the half ampli-
Stimulus
CSNA uv
~
1
, rL_
0
-
~
0200 ms
Fig. 7. Averaged CSNA (50 sweeps of 1 s) triggered by the marker pu|ses of stimulus pulse ( * ) during stimulation of left aortic nerve at intervals of 250 ms (A), 350 ms (B) and 500 ms (C) after barorcceptor denervatJon in one cat.
138
Discussion
We studied the origin of cardiac-related CSNA by artificially pacing the heart in anaesthetized cats. Our results indicate that cardiac-related rhythm in CSNA is produced reflexly by inhibiting the transmission of fundamental rhythmicity by periodic baroreceptor input [15], not by entraining a central free running 2-6 Hz rhythm to cardiac cycle [5-7]. Time relations between inhibition in CSNA and cardiac cycle In our study, the delay of inhibition in CSNA was almost constant and independent of the length of cardiac interval during pacing of the heart at different intervals. In agreement with earlier studies [6,8,16], cardiac-related rhythm in CSNA was lost after baroreceptor denervation (Fig. 6). Stimulation of the left aortic nerve with single shocks at different intervals in baroreceptor denervated state, produced an inhibitory rhythm in CSNA with almost constant delay time. Thus, baroreceptor input was entirely responsible of producing cardiac-related inhibitory rhythm in CSNA. Constant delay times indicate that the cardiac-related rhythm, or the inhibitory rhythm related to the stimulus frequency of the aortic 30O
E 200 E loo 0
D3 ~ ~ T c ~ - ~
- ~
D1 ~
~
~
~
I
I
I
I
I
200
4o0
600
800
1000
S t i m u l u s interval (ms) Fig. 8. Delay times from the stimulus pulse to the onset of inhibition (D1), to the half amplitude of inhibition (D2) and to the maximal inhibition (D3) in CSNA (mean+S.E.M.) during stimulation of left aortic depressor nerve at intervals from 200 ms to 1000 ms after baroreceptor denervation. There were no significant differences in delay times at different stimulus intervals.
A AP
200-
mmHg
100-
CSNA
10-
gV
l
200ms
B Stimulus
h
CSNAw 1
J~ r~ n 0
-
~
0200 ms Fig. 9. (A) Averaged AP and CSNA during pacing the heart at intervals of 250 ms, 350 ms and 500 ms superimposed to the same figure in one cat. (B) Averaged CSNA during stimulation of left aortic depressor nerve at intervals of 250 ms, 350 ms and 500 ms superimposed to the same figure in other cat. The averager was triggered by the R-wave of ECG in A and by the marker pulses of the stimulus pulses ( * ) in B. The bar shows the time from the onset of inhibition to the maximal inhibition in CSNA.
nerve, were produced reflexly by periodic baroreceptor input due to pulsatile pressure or electrical stimulation. The time course of inhibition from the onset to the maximal inhibition was almost constant independently of cardiac interval when the heart was paced at different intervals (Fig. 9A). The duration of inhibition in averaged CSNA increased when cardiac interval was increased. This may be caused by increased baroreceptor activation due to increased pulsatile pressure [4]. When single
139 stimulus pulses was applied to the left aortic nerve the time course of averaged CSNA responses was almost constant independently of stimulus frequency (Fig. 9B). This suggests that the stimulus-related rhythm was caused by reflex inhibition of tonic activity in sympathetic nerve. According to our model, the tonic activity originates from an oscillator with 8-12 Hz fundamental rhythm and cardiac-related rhythm is produced by blocking the transmission of this fundamental rhythm in gate operators [8,9,15]. This is seen as a reflex-inhibition in sympathetic nerve activity. In a closed-loop condition the cardiac-related inhibitory rhythm is independent of the fundamental rhythm but related to another oscillator, the sino atrial node. Previously Gebber proposed the entrainment hypothesis based on the data in Fig. 8 in his report [5]. If we assume that the minimum activity in averaged data reflects maximum inhibition, the delay time from the peak of femoral artery pressure to minimum activity (maximal inhibition) reflects the delay time in his data. The delay time was about 145 ms at three different cardiac intervals. It is not possible to explain our data obtained during stimulation of the left aortic nerve (Figs. 7 and 9B) by entrainment hypothesis. The delay times were constant and independent of stimulus frequency and there was no entrainment in averaged activity.
Delay times of inhibition in CSNA In our study, the delay time to the onset of inhibition in CSNA (164 + 7 ms) is in accordance with that of previously studies (164 + 31 ms) [13]. Also the delay to maximal inhibition (233 + 9 ms) is at the range of the maximal inhibition response of baroreflex (125-330 ms) in CSNA after rise in carotid sinus pressure [3]. The delay time to the maximal inhibition was significantly longer than the delay time to the onset of inhibition when the aortic nerve was stimulated with single shocks. Because of the supramaximal stimulation all afferent fibers should have been activated simultaneously by the stimulus pulse. The finding that the delay time to the maximal inhibition was 120 ms longer than to the onset of inhibition probably reflects differ-
ences in reflex times among individual nerve fibers. In averaged data, inhibition starts when the fastest fibers are inhibited and reach maximum when most of the fibers are inhibited. Half amplitudes of averaged AP and CSNA tracings could be determined most precisely. Although the amplitude of averaged CSNA altered, the time course from the onset to the maximal inhibition was almost constant during artificial pacing and during stimulation of aortic nerve. The delay time to half amplitude of inhibition in CSNA during artificial pacing (198 + 6 ms) and during stimulation of aortic nerve (193 + 4 ms) were at the same range. Thus, the delay time from the half amplitude of rapid rise in AP to the half amplitude of inhibition in CSNA might be the most suitable indirect measure of baroreflex time in inhibition of CSNA. In conclusion, our results indicate that cardiac-related rhythm in CSNA is produced reflexly by inhibiting the transmission of the fundamental rhythmicity by periodic baroreceptor input, not by entraining a central free running 2 - 6 Hz rhythm to cardiac cycle.
Acknowledgements We thank prof. Tada from Osaka University, who kindly supported A.H. in this study. This study was supported by a Grant for Scientific Research from the Ministry of Education, Science and Culture and by a Research Grant for Cardiovascular Diseases from the Ministry of Health and Welfare of Japan. A.H. is a Japan Ministry of Education, Science and Culture Fellow.
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140 4 Ead, H.W., Green, J.H. and Neil, E., A comparison of the effects of pulsatile and nonpulsatile blood flow through the carotid sinus on the reflexogenic activity of the sinus baroceptors in the cat, J. Physiol., 188 (1952) 509-519. 5 Gebber, G.L. Basis for phase relations between baroreceptor and sympathetic nervous discharge, Am. J. Physiol., 230 (1976) 263-270. 6 Gebber, G.L., Central oscillators responsible for sympathetic nerve discharge, Am. J. Physiol., 239 (1980) H143H155. 7 Gebber, G.L., Central determinants of sympathetic nerve discharge, In A.D. Loewy and K.M. Spyer (Eds.) Central Regulation of Autonomic Functions, Oxford Univ. Press, New York, 1990, pp. 126-144. 8 Malpas, S.C. and Ninomiya, I., Fundamental rhythm of renal sympathetic nerve activity in anaesthetized cats, J. Auton. Nerv. Syst., 37 (1992) 11-18. 9 Malpas, S.C. and Ninomiya, I., Effect of chemoreceptor stimulation on the periodicity of renal sympathetic nerve activity in anaesthetized cats, J. Auton. Nerv. Syst. 37, (1992) 19-28. 10 Malpas, S.C. and Ninomiya, I., The amplitude and periodicity of synchronized renal sympathetic nerve discharge in anaesthetized cats: differential effect of baroreceptor activity, J. Auton. Nerv. Syst., 40 (1992) 189-198.
11 Malpas, S.C. and Ninomiya, I., Effect of asphyxia on the frequency and amplitude modulation of synchronized renal nerve activity in the cat, J. Auton. Nerv. Syst. 40 (1992) 199-206. 12 Malpas, S.C. and Ninomiya, I., A new approach to analysis of synchronized sympathetic nerve activity, Am. J. Physiol., 263 (1992) H1311-H1317. 13 Ninomiya, I., Nishimaru, N. and Irisawa, H., Sympathetic nerve activity to the spleen, kidney, and heart in response to baroreceptor input, Am. J. Physiol., 221 (1971) 13461351. 14 Ninomiya, I., Nishiura, N., Matsukawa, K. and Akiyama, T., Fundamental rhythm of cardiac sympathetic nerve activity in awake cats at rest and during body movement, Jpn. J. Physiol., 39 (1989) 743-753. 15 Ninomiya, I., Akiyama, T. and Nishiura, N., Mechanism of cardiac related sympathetic nerve activity in awake cats, Am. J. Physiol., 259 (1990) R499-R506. 16 Taylor, D.G. and Gebber, G.L., Baroreceptor mechanisms controlling sympathetic nervous rhythms of central origin, Am. J. Physiol., 228, (1975) 1002-1013. 17 Zhong, S., Barman, S.M. and Gebber, G.L., Effects of brain stem lesions on 10-Hz and 2-to 6-Hz rhythms in sympathetic nerve discharge, Am. J. Physiol., 262 (1992) R1015-R1024.