Diminished sympathetic silent period in spontaneously hypertensive rats LAWRENCE P. SCHRAMM AND GEORGE N. BARTON Department of Biomedical Engineering, The Johns Hopkins Baltimore, Maryland 21205 SCHRAMM, LAWRENCE P., AND GEORGE N. BARTON. Diminished sympathetic silent period in spontaneouslyhypertensive rats. Am. J. Physiol. 236(3): R147-152, 1979 or Am. J. Physiol.: Regulatory Integrative Camp. Physiol. 5(2): R147-152, 1979.To determine if elevated sympathetic activity occurs in spontaneous hypertension, the silent period induced in splanchnit nerves following electrical stimulation of dorsal medullary sympathoexcitatory sites was compared in anesthetized normotensive Wistar Kyoto rats (WKYs) and Okamoto spontaneously hypertensive rats (SHRs). The strength of silent periods was defined as the degree of inhibition of responses to testing stimuli delivered at various latencies following conditioning trains, and it was assumed to be inversely related to the level of sympathetic activity. Weanling SHRs exhibited weaker silent periods than weanling WKYs although, at that age, the arterial pressures of the strains were not significantly different. Silent periods were also weaker in adult SHRs than in adult WKYs. This difference persisted after arterial pressures, which fell under anesthesia, were raised by phenylephrine infusions to the respective “normal” levels in each strain. These results support the hypothesis that elevated sympathetic activity exists during both the development and maintenance of spontaneous hypertension in rats. spontaneous hypertension; tonic sympathetic activity
sympathetic
preganglionic
neurons;
HYPERACTIVITY in the etiology of elevated arterial pressure in the Okamoto strain of spontaneously hypertensive rat (SHR) remains controversial. Although cardiovascular (4)) biochemical (I I, l&17), and morphological (13) studies show sympathetic hyperactivity during the development and/or maintenance of spontaneous hypertension other studies appear to refute these findings (9, 10). Direct recordings of sympathetic activity in SHRs and normotensive rats have differed in methodology and have produced conflicting results (6, 9, 10, 19). In the present study, we compared in SHRs and normotensive Wistar Kyoto rats (WKYs) a dynamic property of sympathetic neural processing, the sympathetic silent period. The silent period, first described by Pitts and Bronk in 1942 (2l), is an episode of reduced sympathetic excitability that follows periods of spontaneous or evoked sympathoexcitation. The degree of postexcitatory inhibition, the strength of the silent period, varies inversely as a function of resting sympathetic activity (8, 21, 25). Thus a weaker silent period in SHRs would manifest heightened sympathetic activity in this strain. THE ROLE OF SYMPATHETIC
0363-6119/79/0000-0000$01.25
Copyright
0 1979 the American
Physiological
University
School of Medicine,
The purpose of the present study was to measure the strength of the silent periods in weanling and adult SHRs and WKYs to test the hypothesis that SHRs exhibit abnormally high sympathetic activity during the development and maintenance of hypertension. METHODS
Surgicalpreparation. Male rats of the Okamoto spontaneously hypertensive strain and of the Wistar Kyoto normotensive strain were used. Weanling rats, 26-31 days old, and adult rats, 100 and 120 days old, were purchased from Charles River, Inc. and Taconic Farms, Inc., respectively. Similar anesthesias could not be used in both weanling and adult rats; 450 mg/kg chloral hydrate in saline intraperitoneally, supplemented intravenously when necessary, proved satisfactory in weanling rats, but it resulted in erratic levels of anesthesia and sympathetic activity in adult rats even when supplemental doses or constant sustaining infusions were administered. Intraperitoneal allobarbital-urethan (60 and 240 mg/kg, respectively) provided a very stable anesthesia in adult rats, but it led to steadily declining arterial pressure and premature death in weanling rats. The right carotid artery, trachea, and right femoral vein were cannulated for measurement of arterial pressure, maintenance of artificial respiration, and infusion of drugs, respectively. Core temperature was thermostatically maintained at 37°C. Rats were mounted in a Kopf stereotaxic apparatus. The dorsal surface of the medulla was exposed through an occipital craniotomy. After a left flank incision, the left greater splanchnic nerve was exposed by ventral retraction of the left kidney and intestines. The nerve was carefully dissected between the celiac ganglion and its emergence from the diaphragm, cut distally, and placed on bipolar, platinum-iridium, hook electrodes. The nerve and electrodes were protected by the application of a strand of silicone valve seal (Dow Corning). Upon completion of surgery, rats were paralyzed with 15 mg/kg gallamine triethiodide (Flaxedil) and artificially respired. In preliminary experiments, we determined tidal volumes and respiratory rates that resulted in end-tidal CO2 concentrations of 5% measured by a Beckman infrared gas analyzer. Estimation of “normal” arterial pressures in SHRs and VV..Ys. Anesthesia reduces arterial pressures of weanling and adult SHRs and WKYs. During recovery Society
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from ether anesthesia, arterial pressure stabilizes at values characteristic of unanesthetized rats upon recovery of voluntary movements of the head and righting reflexes. In the present study, the arterial pressure of unanesthetized rats was estimated by the following procedure. Under ether anesthesia, a cannula was placed in the right femoral artery. Arterial pressure was continuously recorded. Rats were then permitted to breathe room air until voluntary movement and righting reflexes commenced and arterial pressure stabilized at normal values. The cannula was removed after reanesthetization. Arterial pressures of 22 weanling SHRs (91 t 4 Torr), 22 weanling WKYs (84 t 3 Torr), 8 adult SHRs (169 t 4 Torr), and 7 adult WKYs (117 t 3 Torr) were measured. Rats used for these measurements were carefully matched to those used in the neurophysiology experiments with respect to age, sex, and supplier. The values thus obtained are referred to below as normal arterial pressures for the respective ages and strains. Adjustment of arterial pressure with phenylephrine. In experiments assessing the relationship between baroreceptor activation and the strength of the silent period, arterial pressure was adjusted by intravenous infusions of phenylephrine hydrochloride (Neo-Synephrine, Winthrop, 125 mg/ml). Arterial pressure was fixed at each desired level by an infusion rate between 0.05 and 0.3 nil/ h for at least 5 min before electrophysiological studies were begun. Histological procedures. Each rat was killed at the end of the experiment by intracardiac perfusion with saline followed by 10% formalin. Frozen sections of the medulla were cut at a thickness of 40 pm, mounted, and stained by the thionin technique. Electrode tracks and sites of stimulation were located microscopically. Stimulation sites in all rats were restricted to a small portion of the medial solitary nucleus and the nucleus commissuralis. Electrical stimulation and recording. Pulsatile electrical stimulation of uniform intensity (500 PA) and duration (0.5 ms) was delivered through monopolar, stainless steel electrodes with the indifferent electrode placed on the muscle of the neck. Because the necessary stimulation sequences (see below) could not be produced by commercially available stimulators, they were generated by a microcomputer (KIM-l; MOS Technology, Inc.), which, in turn, triggered a Grass stimulator and constant current coupler. Appropriate stimulation sites were located by mapping the region just lateral to the obex for large increases in sympathetic activity and brisk pressor responses to repetitive stimulation at 50 Hz. Stimulation of these sites at l-5 Hz evoked depressor responses due to the profound silent periods that followed each stimulus-locked increase in sympathetic activity. Spontaneous sympathetic nerve activity and evoked potentials from the splanchnic nerve were amplified by a Grass P15B preamplifier whose upper and lower 3 dB points were set at 10 and 3,000 Hz, respectively. Signals were further amplified and displayed by a Tektronix D11 storage oscilloscope. Responses were averaged by a Computer of Average Transients (CAT), which was driven in external sweep mode by a microcomputer (KIM-l; MOS Technology, Inc.) to permit a complete
AND
G. N. BARTON
stimulus paradigm to be presented with adequate resolution during a single sweep. Characterization of the silent period. In all experiments, the silent period was quantitatively characterized by a modification of the conditioning-testing paradigm. The strength of the silent period was defined as the degree of inhibition exhibited by responses to “test” pulses delivered at various latencies after “conditioning” trains. As shown in Fig. 1, a conditioning train of one to five pulses (6-ms interpulse interval) was delivered to the sympathoexcitatory region at intervals of from 100 to 1,200 ms before delivery of a test pulse through the same electrode. Sympathetic responses to the test pulse were reduced during the silent period following the conditioning stimulus. The degree of postconditioning recovery of sympathetic excitability, at a given conditioning-testing interval, was determined by comparing the amplitude of the response to the test pulse to the amplitude of the response to a control pulse delivered 2.5 s before the conditioning train (see Fig. 1). The use of the control pulse in each stimulus presentation permitted comparisons of silent periods obtained when electrode movement, unavoidable in long experiments, altered the absolute magnitudes of the evoked responses. Although rats of both ages and both strains exhibited a profound postexcitatory sympathoinhibition, the strength of this inhibition was variable from one stimulus presentation to another. We were unable to decrease this variability by synchronizing the onset of stimulation with either the QRS complex of the electrocardiogram or the phase of the respiratory cycle. Therefore, we averaged at least five stimulus presentations at each conditioningtesting latency. In several experiments, the silent period was observed more traditionally as a period of postexcitatory inhibition of spontaneous sympathetic activity. In these experiments, spontaneous activity was amplified, full-wave rectified, and passed through a filter with a lo-ms time constant. At least 12 occurrences of evoked potentials and the subsequent spontaneous sympathetic activity were averaged to make each of these observations. Statistical analysis. All results are expressed as means t SE. Student’s t test (26) was used to compare groups when each was represented by a single measure. Twoway analysis of variance (two-way ANOVA) was used to compare groups when each was represented by multiple measures (26). RESULTS
General characteristics of the silent period. Splanchnit-evoked responses were invariably followed by a period of decreased spontaneous sympathetic activity and increased threshold to subsequent stimulation, a sympathetic silent period. Stimulation paradigms were, on occasion, accompanied by pressor responses. Silent periods were not caused by reflex baroreceptor inhibition of sympathetic activity because silent periods had onset latencies of less than 100 ms and durations of less than 2 s whereas pressor responses had onset latencies of at least 500 ms and durations of 4-5 s. Further, silent periods were not different in rats that did and did not exhibit
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SYMPATHETIC
SILENT
PERIOD
CONTROL PULSE 2.5
SEC~4(1-l00~
CONTROL INTERVAL
IN
SHRs
AND
R149
WKYs
CONDITION
TEST
TRAIN
PULSE 1200 CONDITI ON-T MT ERVAL
mSEC-
4
FIG. 1. Stimulus presentation of silent period. An interpulse all conditioning trains.
EST
pressor responses to the five-pulse stimulus paradigm (one control pulse, three conditioning pulses, and one test pulse). Although postexcitatory depression of spontaneous activity was not used as a measure of the silent period, it was characterized in five weanling WKY rats to ensure that its time course was similar to that of the inhibition of test responses observed in the conditioning-testing paradigm. Figure ZA illustrates a representative average of 12 splanchnic-evoked potentials and the subsequent spontaneous activity processed as described in METHODS. A large biphasic response (which appears bimodal due to rectification) was followed by approximately 400 ms of reduced sympathetic activity. Figure 2B illustrates a similar time course of elevated threshold to subsequent stimulation after a conditioning stimulus. Each of the three traces represents averages of 12 responses to control, conditioning, and test stimuli (see METHODS). The conditioning-testing paradigm was used throughout this study to characterize the silent period because it provided more easily quantifiable measurements than those obtained from spontaneous nerve activity. Figure 3 illustrates the effect of increasing the number of pulses in the conditioning train from one to five over a wide range of conditioning-testing intervals in a representative adult normotensive rat. Maximal inhibition, in this and many other rats, did not occur until 200-400 ms after the conditioning stimulus. The length of the conditioning train profoundly influenced both the magnitude and the duration of the subsequent inhibition. Although a single conditioning pulse was followed by a significant silent period, the stronger silent periods elicited by multiple conditioning pulses in both SHRs and WKYs were more easily quantified. Therefore, conditioning trains of three pulses were used in all of the studies described below. Comparison of silent periods in weanling SHRs and WKYs. The strength of the sympathetic silent period was measured in 13 weanling SHRs and 8 weanling WKYs. At ages of 26-31 days, arterial pressures of SHRs and WKYs did not differ under either normal conditions (seeMETHODS), t 4 (n = 22)vs.84 t 3 Torr (n = 22), respectively, or at surgical levels of chloral hydrate anesthesia, 61 t 5 (n. = 13) vs, 59 t 3 Torr (n. = 8), respectively. However, chloral hydrate did significantly lower arterial pressure in both strains (P < 0.01 for both strains). Figure 4 shows that silent periods were significantly weaker in SHRs (F&22) = 5.9, P < 0.05). Comparison of silent periods in adult SHRs and WKYs. The strength of the silent periods in seven anesthetized adult SHRs and seven anesthetized adult WKYs was compared by measuring the time course of recovery of responses to test stimuli presented after conditioning trains of three pulses. Figure 5 shows that the silent
used to determine duration interval of 6 ms was used for
I-+ 500
ms
200
ms
CONDITION-TEST INTERVAL
400ms
CONTROL PULSE
COND TRAIN
C-T
INT.
PULSE
2. Sympathetic silent periods in rat characterized by A, postexcitatory decrease in spontaneous sympathetic activity and B, postconditioning inhibition of test response. FIG,
period was much weaker in SHRs (F&12) = 17.5, P < 0.01). Interpretation of these results met with a complication not encountered in the weanling rats. Allobarbital-urethan anesthesia lowered arterial pressure in both strains of adult rats, However, in contrast to our observations in weanling rats, arterial pressures were significantly higher in adult SHRs than in adult WKYs both under normal conditions (see METHODS), 169 k 4 (n = 8) vs. 117 t 3 Torr (n = 7), respectively, (P < O.Ol), and under allobarbital-urethan anesthesia, 120 t 3 (n = 8) vs. 88 -+ 5 Torr (?t = 7), respectively, (P < O.Ol), Previous reports have shown that the strength of the sympathetic silent period is directly related to baroreceptor input to the medulla (8, 25), Although allobarbital-urethan anesthesia produced similar percentage decreases in arterial pressures in SHRs and WKYs (compared to normal arterial pressures in rats of these strains), it was not possible to conclude that similar percent decreases in the strengths of the silent periods would result. Thus, the possibility existed that the reduction of arterial pressure by anesthesia might either exaggerate or reduce the difference in strength of silent periods between SHRs and WKYs. To determine silent periods at pressure levels approximating those existing normally, additional experiments
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R150
L. P. SCHRAMM
FIG.
tioning
0
100
200
800
400
CONDITION
TEST
-
INTERVAL
AND
G. N. BARTON
3. Effect of 1 (A), 3 (o), and 5 (e) pulses train on recovery of test response.
in condi-
IZCC
(ms
would be responsible for the marked difference in the strengths of the silent periods in SHRs and WKYs. The data from Fig. 6 were used to compare the strengths of the silent periods of WKYs and SHRs at or near estimated normal arterial pressures for these strains, 117 and 169 Torr, respectively (see METHODS). The percent recovery of the test response observed in WKYs at 120 Torr, 5 t 4% (n = 6), was much less than the percent recovery of test responses observed in SHRs at either 160 Torr, 51 t 10% (n = 12, P c 0.01) or 180 Torr, 34 t 11% (n = 12, P < 0.05). Thus, when the effects of anesthesia on arterial pressure were compensated, adult SHRs exhibited weaker silent periods than adult WKYs. DISCUSSION
CONDITION
-
TEST
tNTERVAL
( ms)
of recovery of test responses elicited after a 3pulse conditioning train in 13 weanling SHRs (A) and 8 weanling WKYs (0). Values represent means * SE. FIG.
4. Comparison
were done in which the silent periods of 10 additional adult SHRs and 6 additional adult WKYs were characterized at the reduced arterial pressures resulting from allobarbital-urethan anesthesia and at arterial pressures which were raised, by phenylephrine infusion (see METHODS), to and beyond normal values for each strain. The strength of the silent period, characterized by measuring the percent recovery of a test response elicited 800 ms after a three pulse conditioning stimulus, increased in both strains as arterial pressure was increased (Fig. 6). The slope of the relationship would probably have been somewhat greater had one carotid sinus not been excluded by cannulation. However, there is no reason to believe that the uniform exclusion of one carotid sinus
Postexcitatory inhibition is characteristic of systems processing sympathetic activity at both medullary (5, 7) and spinal (2223) levels. We made no attempt to localize the site(s) of generation of silent periods. However, care was taken to ensure that the phenomenon observed in these experiments possessed characteristics of sympathetic silent periods described previously by others. The onset latencies and durations of the silent periods observed in the present study were well within ranges previously reported (7, 8, 12,21-23, 25,27). The strength of sympathetic silent periods increased with increasing stimulus intensity of stimulus duration as described by Pitts and Bronk (21) and others (5, 23, 25). Finally, as previously shown in several laboratories (8, 25) the strength of the sympathetic silent period increased as baroreceptor input increased. Previous studies in this laboratory have shown that the sympathetic preganglionic neurons of weanling Sprague-Dawley rats, WKYs, and SHRs are morphologically immature despite the morphological maturity of somatic motoneurons at similar ages (24). At the outset of these experiments, we hypothesized that the very profound adolescent morphogenesis of sympathetic preganglionic neurons (24) might manifest the evolution of
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SYMPATHETIC
SILENT
PERIOD
IN
SHRs
AND
R151
WKYs
FIG. 5. Comparison of recovery after a S-pulse conditioning (A) and 7 adult WKYs (0). Values
ited
CONDITION
-
TEST
INTERVAL
t ms
of test responses elictrain in 7 adult SHRs represent means f SE.
1
elicited 800 ms FIG. 6. Percent recovery of test responses after a 3- pulse conditioning train at different arterial pressures in IO adult SHRs (A) and 6 adult WKYs (0). Values represent means k SE.
4RfERIAL
PRESSURE
(mm
Hg)
silent period mechanisms. Although differences in anesthetics and animal sources preclude quantitative comparisons between silent periods in weanling and adult rats, the presence of silent periods in weanling rats disproves this hypothesis. Our ability to infer that the sympathetic nervous system is chronically hyperactive in SHRs depends on the reliability of the relationship between the strength of the sympathetic silent period and tonic levels of sympathetic activity. It is not yet clear whether the silent period determines tonic sympathetic activity or whether its strength is a function of tonic sympathetic activity (1, 12). Nevertheless, the strength of the silent period and the level of tonic sympathetic activity appear to be closely related under a variety of conditions. Cats subjected to general anesthetics that decrease tonic sympathetic activity exhibit stronger silent periods (27). Inhibition of tonic sympathetic activity by increased baroreceptor input is accompanied by stronger silent periods (8, 25). Finally, cats treated with tetanus toxin exhibit
both sympathetic hyperactivity and weakened silent periods (20). We conclude that the diminished silent periods observed in SHRs are manifestations of sympathetic hyperactivity in this strain. The evidence for sympathetic hyperactivity during the development of spontaneous hypertension in young rats in conflicting. Neonatal destruction of the sympathetic nervous system with anti-nerve growth factor prevents or minimizes development of hypertension in SHRs (2, 3). Dopamine P-hydroxylase, an indicator of postganglionic sympathetic activity, is elevated in serum and tissues of SHRs (15, 16). However, measurements of preganglionic sympathetic activity, based on ganglionic or adrenal concentrations of choline acetyltransferase, have demonstrated both normal (9) and elevated (17) levels. The present study provides the first measurement of sympathetic activity in weanling SHRs from directly recorded nerve activity. The results show that the sympathetic nervous system is hyperactive in SHRs before arterial pressure is significantly elevated.
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L. P. SCHRAMM
As early as 1967, Okamoto et al. (19) provided evidence for elevated sympathetic activity in the greater splanchnit nerve of adult SHRs. More recently, Judy et al. (6) found elevated activity in the cervical sympathetic, greater splanchnic, least splanchnic, splenic, and renal nerves of adult SHRs. On the other hand, Lais et al. (lo), recording on the lumbar sympathetic chain, did not observe elevated levels of sympathetic activity in SHRs. Biochemical indexes of sympathetic activity in adult SHRs have provided evidence for both elevated (14) and normal (9, 15) levels, The diminished sympathetic silent
AND
G. N. BARTON
periods which we observed in 100-day-old SHRs confirmation that sympathetic hyperexcitability in adults.
provide persists
The expert assistance of Mr. Kevin McKenna is gratefully acknowledged. The advice of Dr. Drew Carlson during the course of these experiments and in the preparation of the manuscript was particularly valuable. We thank Mr. Eric Howland for preparing the figures and Ms. Evelyn McCann for typing the manuscript. This research was supported by Grant HL-16315 from the National Heart, Lung, and Blood Institute. Received
31 October
1977; accepted
in final
form
13 September
1978.
REFERENCES 1. CALARESU, F. R., A. A. FAIERS, AND C. J. MOGENSON. Central neural regulation of heart and blood vessels in mammals. Prog. NeurobioZ. 5: l-35, 1975. 2. CLARK, D. W. J. Effects of immunosympathectomy on the development of high blood pressure in genetically hypertensive rats+ Circ, Res. 28: 330-336, 1971. 3. CUTXLLETTA, A. F,, L. ERINOFF, A. HELLER, J. Low, AND S. OPARIL. Development of left ventricular hypertrophy in young spontaneously hypertensive rats after peripheral sympathectomy. Circ. Res. 40: 428-434, 1977. 4. IRIUCHIJIMA, J. Arterial pressure in spontaneously hypertensive rats after high spinal cord section. Jpn. Circ. J, 40: 887-888, 1976. 5, TWAMURA, Y., Y. UCHINO, S. OZAWA, AND N. KUDO, Excitatory and inhibitory components of somato-sympathetic reflex. Brain Res. 16: 351-358, 1969. 6. JUDY, W. V., A. M. WATANABE, D. P. HENRY, H. R. BESCH, JR., W. R, MURPHY, AND G. J. HOCKEL, Sympathetic nerve activity: Role in regulation of blood pressure in the spontaneously hypertensive rat. Circ. Res. 38, Suppl. 2: 21-29, 1976. 7. KOIZUMI, K., AND C. McC. BROOKS. The integration of autonomic system reactions: A discussion of autonomic reflexes, their control and their association with somatic reactions, Ergeb. Physiol. Biol. Chem. Exp. Pharmakol. 67: l-68, 1972. 8. KO~ZUMI, K., H, SELLER, A. KAUFMAN, AND C. McC, BROOKS. Pattern of sympathetic discharges and their relation to baroreceptor and respiratory activities. Brain Res. 27: 281-294, 1971. 9. LAIS, L. T., M. J. BRODY, R. BHATNEGAR, AND R. ROSKOWSI. Evidence that hypertension appears in SHR in the absence of altered sympathetic nervous system activity or development. In: Spontaneous Hypertension: Its Pathogenesis and Complications. Proc. Second Int, Symp. Spontaneously Hypertensive Rat. Washington, DC: US Gov, Printing Off., 1977, p. 237-246. 10. LAIS, L. T., R. A. SHAFFER, AND M. J. BRODY. Neurogenic and humoral factors controlling vascular resistance in the spontaneously hypertensive rat. Circ. Res. 35: 764-774, 1974. 11. LEW, G. M, Developmental changes in catecholamines in organs and brain regions of genetically hypertensive rats. Camp. Gen. Pharmacol. 6: 115-120, 1975. 12. MANNARD, A., R. RAJCHGOT, AND C. POLOSA. Effect of post-impulse depression on background firing of sympathetic preganglionic neurons. Brain Res. 126: 243-262, 1977. 13. MATSUMOTO, M. Morphological studies on the autonomic nervous
14. 15,
16.
17.
18.
19.
20.
21.
22. 23.
24.
25. 26. 27.
system of hypertensive rats. II. Enzyme histochemical study on the superior cervical sympathetic ganglion of spontaneously hypertensive rats. Jpn, Circ. J. 31: 1187-1196, 1967. MORI, K. Participation of the sympathetic nervous system in spontaneously hypertensive rats. Jpn. Circ. J. 37: 609-618, 1973. NAGATSU, T., K. IKUTA, Y. NUMATA, T. KATO, M. SANO, I. NAGATSU, H. WMEZAWA, M. MATSUZAKI, AND T. TAKEUCHI. Vascular and brain dopamine P-hydroxylase activity in young spontaneously hypertensive rats. Science 194: 290-291, 1976. NAGATSU, T., T. KATO, Y. NUMATA, K. IKUTA, H. UMEZAWA, M. MATSUZAKI, AND T, TAKEUCHI. Serum dopamine P-hydroxylase activity in developing hypertensive rats. Nature London 251: 630631, 1974. NAKAMURA, K., AND K. NAKAMURA. Selective activation of sympathetic ganglia in young spontaneously hypertensive rats, Nature London 226: 265, 1977, NOSAKA, S., AND S. C. WANG. Carotid sinus baroceptor functions in the spontaneously hypertensive rat. Am. J. Physiol. 222: 10791084, 1972. OKAMOTO, K., S. NOSAKA, Y. YAMORI, AND M, MATSUMOTO. Participation of neural factor in the pathogenesis of hypertension in the spontaneously hypertensive rat. Jpn. Heart J. 8: 168-180,1967. PAAR, G. H., AND H. H. WELLH~NER. The action of tetanus toxin on preganglionic sympathetic reflex discharges. Naunyn-Schmiederbergs Arch. Pharmakol. 276: 437-445, 1973. PITTS, R. F., AND D. W. BRONK. Excitability cycle of the hypothalamus-sympathetic neurone system. Am. J. Physiol. 135: 504-522, 1942. POLOSA, C. The silent period of sympathetic preganglionic neurons. Can. J. Physiol. Pharmacol. 45: 1033-1045, 1967. SCHMIDT, R. F., AND K. SCH~NFUSS. An analysis of the reflex activity in the cervical sympathetic trunk induced by myelinated somatic afferents. Pfluegers Arch. 314: 175-194, 1970. SCHRAMM, L. P., J. M. STRIBLING, AND J. R. ADAIR. Developmental reorientation of sympathetic preganglionic neurons. Brain Res. 106: 166-171, 1976. SHERRER, H. Postexcitatory inhibition of sympathetic discharge. Exp. NeuroL. 7: 343-354, 1963. SNEDECOR, G. W., AND W. C. COCHRAN. StatisticaLMethods. Ames, IA: Iowa State Univ. Press, 1967. TSYRLIN, V. A. Origin of the silent period in somato- and viscerosympathetic responses. Neurofiziologiya 4: 501-509, 1972,
Downloaded from www.physiology.org/journal/ajpregu at Midwestern Univ Lib (132.174.254.157) on February 14, 2019.
sympathetic
preganglionic
neurons;
HYPERACTIVITY in the etiology of elevated arterial pressure in the Okamoto strain of spontaneously hypertensive rat (SHR) remains controversial. Although cardiovascular (4)) biochemical (I I, l&17), and morphological (13) studies show sympathetic hyperactivity during the development and/or maintenance of spontaneous hypertension other studies appear to refute these findings (9, 10). Direct recordings of sympathetic activity in SHRs and normotensive rats have differed in methodology and have produced conflicting results (6, 9, 10, 19). In the present study, we compared in SHRs and normotensive Wistar Kyoto rats (WKYs) a dynamic property of sympathetic neural processing, the sympathetic silent period. The silent period, first described by Pitts and Bronk in 1942 (2l), is an episode of reduced sympathetic excitability that follows periods of spontaneous or evoked sympathoexcitation. The degree of postexcitatory inhibition, the strength of the silent period, varies inversely as a function of resting sympathetic activity (8, 21, 25). Thus a weaker silent period in SHRs would manifest heightened sympathetic activity in this strain. THE ROLE OF SYMPATHETIC
0363-6119/79/0000-0000$01.25
Copyright
0 1979 the American
Physiological
University
School of Medicine,
The purpose of the present study was to measure the strength of the silent periods in weanling and adult SHRs and WKYs to test the hypothesis that SHRs exhibit abnormally high sympathetic activity during the development and maintenance of hypertension. METHODS
Surgicalpreparation. Male rats of the Okamoto spontaneously hypertensive strain and of the Wistar Kyoto normotensive strain were used. Weanling rats, 26-31 days old, and adult rats, 100 and 120 days old, were purchased from Charles River, Inc. and Taconic Farms, Inc., respectively. Similar anesthesias could not be used in both weanling and adult rats; 450 mg/kg chloral hydrate in saline intraperitoneally, supplemented intravenously when necessary, proved satisfactory in weanling rats, but it resulted in erratic levels of anesthesia and sympathetic activity in adult rats even when supplemental doses or constant sustaining infusions were administered. Intraperitoneal allobarbital-urethan (60 and 240 mg/kg, respectively) provided a very stable anesthesia in adult rats, but it led to steadily declining arterial pressure and premature death in weanling rats. The right carotid artery, trachea, and right femoral vein were cannulated for measurement of arterial pressure, maintenance of artificial respiration, and infusion of drugs, respectively. Core temperature was thermostatically maintained at 37°C. Rats were mounted in a Kopf stereotaxic apparatus. The dorsal surface of the medulla was exposed through an occipital craniotomy. After a left flank incision, the left greater splanchnic nerve was exposed by ventral retraction of the left kidney and intestines. The nerve was carefully dissected between the celiac ganglion and its emergence from the diaphragm, cut distally, and placed on bipolar, platinum-iridium, hook electrodes. The nerve and electrodes were protected by the application of a strand of silicone valve seal (Dow Corning). Upon completion of surgery, rats were paralyzed with 15 mg/kg gallamine triethiodide (Flaxedil) and artificially respired. In preliminary experiments, we determined tidal volumes and respiratory rates that resulted in end-tidal CO2 concentrations of 5% measured by a Beckman infrared gas analyzer. Estimation of “normal” arterial pressures in SHRs and VV..Ys. Anesthesia reduces arterial pressures of weanling and adult SHRs and WKYs. During recovery Society
Downloaded from www.physiology.org/journal/ajpregu at Midwestern Univ Lib (132.174.254.157) on February 14, 2019.
K147
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L. P. SCHRAMM
from ether anesthesia, arterial pressure stabilizes at values characteristic of unanesthetized rats upon recovery of voluntary movements of the head and righting reflexes. In the present study, the arterial pressure of unanesthetized rats was estimated by the following procedure. Under ether anesthesia, a cannula was placed in the right femoral artery. Arterial pressure was continuously recorded. Rats were then permitted to breathe room air until voluntary movement and righting reflexes commenced and arterial pressure stabilized at normal values. The cannula was removed after reanesthetization. Arterial pressures of 22 weanling SHRs (91 t 4 Torr), 22 weanling WKYs (84 t 3 Torr), 8 adult SHRs (169 t 4 Torr), and 7 adult WKYs (117 t 3 Torr) were measured. Rats used for these measurements were carefully matched to those used in the neurophysiology experiments with respect to age, sex, and supplier. The values thus obtained are referred to below as normal arterial pressures for the respective ages and strains. Adjustment of arterial pressure with phenylephrine. In experiments assessing the relationship between baroreceptor activation and the strength of the silent period, arterial pressure was adjusted by intravenous infusions of phenylephrine hydrochloride (Neo-Synephrine, Winthrop, 125 mg/ml). Arterial pressure was fixed at each desired level by an infusion rate between 0.05 and 0.3 nil/ h for at least 5 min before electrophysiological studies were begun. Histological procedures. Each rat was killed at the end of the experiment by intracardiac perfusion with saline followed by 10% formalin. Frozen sections of the medulla were cut at a thickness of 40 pm, mounted, and stained by the thionin technique. Electrode tracks and sites of stimulation were located microscopically. Stimulation sites in all rats were restricted to a small portion of the medial solitary nucleus and the nucleus commissuralis. Electrical stimulation and recording. Pulsatile electrical stimulation of uniform intensity (500 PA) and duration (0.5 ms) was delivered through monopolar, stainless steel electrodes with the indifferent electrode placed on the muscle of the neck. Because the necessary stimulation sequences (see below) could not be produced by commercially available stimulators, they were generated by a microcomputer (KIM-l; MOS Technology, Inc.), which, in turn, triggered a Grass stimulator and constant current coupler. Appropriate stimulation sites were located by mapping the region just lateral to the obex for large increases in sympathetic activity and brisk pressor responses to repetitive stimulation at 50 Hz. Stimulation of these sites at l-5 Hz evoked depressor responses due to the profound silent periods that followed each stimulus-locked increase in sympathetic activity. Spontaneous sympathetic nerve activity and evoked potentials from the splanchnic nerve were amplified by a Grass P15B preamplifier whose upper and lower 3 dB points were set at 10 and 3,000 Hz, respectively. Signals were further amplified and displayed by a Tektronix D11 storage oscilloscope. Responses were averaged by a Computer of Average Transients (CAT), which was driven in external sweep mode by a microcomputer (KIM-l; MOS Technology, Inc.) to permit a complete
AND
G. N. BARTON
stimulus paradigm to be presented with adequate resolution during a single sweep. Characterization of the silent period. In all experiments, the silent period was quantitatively characterized by a modification of the conditioning-testing paradigm. The strength of the silent period was defined as the degree of inhibition exhibited by responses to “test” pulses delivered at various latencies after “conditioning” trains. As shown in Fig. 1, a conditioning train of one to five pulses (6-ms interpulse interval) was delivered to the sympathoexcitatory region at intervals of from 100 to 1,200 ms before delivery of a test pulse through the same electrode. Sympathetic responses to the test pulse were reduced during the silent period following the conditioning stimulus. The degree of postconditioning recovery of sympathetic excitability, at a given conditioning-testing interval, was determined by comparing the amplitude of the response to the test pulse to the amplitude of the response to a control pulse delivered 2.5 s before the conditioning train (see Fig. 1). The use of the control pulse in each stimulus presentation permitted comparisons of silent periods obtained when electrode movement, unavoidable in long experiments, altered the absolute magnitudes of the evoked responses. Although rats of both ages and both strains exhibited a profound postexcitatory sympathoinhibition, the strength of this inhibition was variable from one stimulus presentation to another. We were unable to decrease this variability by synchronizing the onset of stimulation with either the QRS complex of the electrocardiogram or the phase of the respiratory cycle. Therefore, we averaged at least five stimulus presentations at each conditioningtesting latency. In several experiments, the silent period was observed more traditionally as a period of postexcitatory inhibition of spontaneous sympathetic activity. In these experiments, spontaneous activity was amplified, full-wave rectified, and passed through a filter with a lo-ms time constant. At least 12 occurrences of evoked potentials and the subsequent spontaneous sympathetic activity were averaged to make each of these observations. Statistical analysis. All results are expressed as means t SE. Student’s t test (26) was used to compare groups when each was represented by a single measure. Twoway analysis of variance (two-way ANOVA) was used to compare groups when each was represented by multiple measures (26). RESULTS
General characteristics of the silent period. Splanchnit-evoked responses were invariably followed by a period of decreased spontaneous sympathetic activity and increased threshold to subsequent stimulation, a sympathetic silent period. Stimulation paradigms were, on occasion, accompanied by pressor responses. Silent periods were not caused by reflex baroreceptor inhibition of sympathetic activity because silent periods had onset latencies of less than 100 ms and durations of less than 2 s whereas pressor responses had onset latencies of at least 500 ms and durations of 4-5 s. Further, silent periods were not different in rats that did and did not exhibit
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SYMPATHETIC
SILENT
PERIOD
CONTROL PULSE 2.5
SEC~4(1-l00~
CONTROL INTERVAL
IN
SHRs
AND
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WKYs
CONDITION
TEST
TRAIN
PULSE 1200 CONDITI ON-T MT ERVAL
mSEC-
4
FIG. 1. Stimulus presentation of silent period. An interpulse all conditioning trains.
EST
pressor responses to the five-pulse stimulus paradigm (one control pulse, three conditioning pulses, and one test pulse). Although postexcitatory depression of spontaneous activity was not used as a measure of the silent period, it was characterized in five weanling WKY rats to ensure that its time course was similar to that of the inhibition of test responses observed in the conditioning-testing paradigm. Figure ZA illustrates a representative average of 12 splanchnic-evoked potentials and the subsequent spontaneous activity processed as described in METHODS. A large biphasic response (which appears bimodal due to rectification) was followed by approximately 400 ms of reduced sympathetic activity. Figure 2B illustrates a similar time course of elevated threshold to subsequent stimulation after a conditioning stimulus. Each of the three traces represents averages of 12 responses to control, conditioning, and test stimuli (see METHODS). The conditioning-testing paradigm was used throughout this study to characterize the silent period because it provided more easily quantifiable measurements than those obtained from spontaneous nerve activity. Figure 3 illustrates the effect of increasing the number of pulses in the conditioning train from one to five over a wide range of conditioning-testing intervals in a representative adult normotensive rat. Maximal inhibition, in this and many other rats, did not occur until 200-400 ms after the conditioning stimulus. The length of the conditioning train profoundly influenced both the magnitude and the duration of the subsequent inhibition. Although a single conditioning pulse was followed by a significant silent period, the stronger silent periods elicited by multiple conditioning pulses in both SHRs and WKYs were more easily quantified. Therefore, conditioning trains of three pulses were used in all of the studies described below. Comparison of silent periods in weanling SHRs and WKYs. The strength of the sympathetic silent period was measured in 13 weanling SHRs and 8 weanling WKYs. At ages of 26-31 days, arterial pressures of SHRs and WKYs did not differ under either normal conditions (seeMETHODS), t 4 (n = 22)vs.84 t 3 Torr (n = 22), respectively, or at surgical levels of chloral hydrate anesthesia, 61 t 5 (n. = 13) vs, 59 t 3 Torr (n. = 8), respectively. However, chloral hydrate did significantly lower arterial pressure in both strains (P < 0.01 for both strains). Figure 4 shows that silent periods were significantly weaker in SHRs (F&22) = 5.9, P < 0.05). Comparison of silent periods in adult SHRs and WKYs. The strength of the silent periods in seven anesthetized adult SHRs and seven anesthetized adult WKYs was compared by measuring the time course of recovery of responses to test stimuli presented after conditioning trains of three pulses. Figure 5 shows that the silent
used to determine duration interval of 6 ms was used for
I-+ 500
ms
200
ms
CONDITION-TEST INTERVAL
400ms
CONTROL PULSE
COND TRAIN
C-T
INT.
PULSE
2. Sympathetic silent periods in rat characterized by A, postexcitatory decrease in spontaneous sympathetic activity and B, postconditioning inhibition of test response. FIG,
period was much weaker in SHRs (F&12) = 17.5, P < 0.01). Interpretation of these results met with a complication not encountered in the weanling rats. Allobarbital-urethan anesthesia lowered arterial pressure in both strains of adult rats, However, in contrast to our observations in weanling rats, arterial pressures were significantly higher in adult SHRs than in adult WKYs both under normal conditions (see METHODS), 169 k 4 (n = 8) vs. 117 t 3 Torr (n = 7), respectively, (P < O.Ol), and under allobarbital-urethan anesthesia, 120 t 3 (n = 8) vs. 88 -+ 5 Torr (?t = 7), respectively, (P < O.Ol), Previous reports have shown that the strength of the sympathetic silent period is directly related to baroreceptor input to the medulla (8, 25), Although allobarbital-urethan anesthesia produced similar percentage decreases in arterial pressures in SHRs and WKYs (compared to normal arterial pressures in rats of these strains), it was not possible to conclude that similar percent decreases in the strengths of the silent periods would result. Thus, the possibility existed that the reduction of arterial pressure by anesthesia might either exaggerate or reduce the difference in strength of silent periods between SHRs and WKYs. To determine silent periods at pressure levels approximating those existing normally, additional experiments
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FIG.
tioning
0
100
200
800
400
CONDITION
TEST
-
INTERVAL
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G. N. BARTON
3. Effect of 1 (A), 3 (o), and 5 (e) pulses train on recovery of test response.
in condi-
IZCC
(ms
would be responsible for the marked difference in the strengths of the silent periods in SHRs and WKYs. The data from Fig. 6 were used to compare the strengths of the silent periods of WKYs and SHRs at or near estimated normal arterial pressures for these strains, 117 and 169 Torr, respectively (see METHODS). The percent recovery of the test response observed in WKYs at 120 Torr, 5 t 4% (n = 6), was much less than the percent recovery of test responses observed in SHRs at either 160 Torr, 51 t 10% (n = 12, P c 0.01) or 180 Torr, 34 t 11% (n = 12, P < 0.05). Thus, when the effects of anesthesia on arterial pressure were compensated, adult SHRs exhibited weaker silent periods than adult WKYs. DISCUSSION
CONDITION
-
TEST
tNTERVAL
( ms)
of recovery of test responses elicited after a 3pulse conditioning train in 13 weanling SHRs (A) and 8 weanling WKYs (0). Values represent means * SE. FIG.
4. Comparison
were done in which the silent periods of 10 additional adult SHRs and 6 additional adult WKYs were characterized at the reduced arterial pressures resulting from allobarbital-urethan anesthesia and at arterial pressures which were raised, by phenylephrine infusion (see METHODS), to and beyond normal values for each strain. The strength of the silent period, characterized by measuring the percent recovery of a test response elicited 800 ms after a three pulse conditioning stimulus, increased in both strains as arterial pressure was increased (Fig. 6). The slope of the relationship would probably have been somewhat greater had one carotid sinus not been excluded by cannulation. However, there is no reason to believe that the uniform exclusion of one carotid sinus
Postexcitatory inhibition is characteristic of systems processing sympathetic activity at both medullary (5, 7) and spinal (2223) levels. We made no attempt to localize the site(s) of generation of silent periods. However, care was taken to ensure that the phenomenon observed in these experiments possessed characteristics of sympathetic silent periods described previously by others. The onset latencies and durations of the silent periods observed in the present study were well within ranges previously reported (7, 8, 12,21-23, 25,27). The strength of sympathetic silent periods increased with increasing stimulus intensity of stimulus duration as described by Pitts and Bronk (21) and others (5, 23, 25). Finally, as previously shown in several laboratories (8, 25) the strength of the sympathetic silent period increased as baroreceptor input increased. Previous studies in this laboratory have shown that the sympathetic preganglionic neurons of weanling Sprague-Dawley rats, WKYs, and SHRs are morphologically immature despite the morphological maturity of somatic motoneurons at similar ages (24). At the outset of these experiments, we hypothesized that the very profound adolescent morphogenesis of sympathetic preganglionic neurons (24) might manifest the evolution of
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SYMPATHETIC
SILENT
PERIOD
IN
SHRs
AND
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WKYs
FIG. 5. Comparison of recovery after a S-pulse conditioning (A) and 7 adult WKYs (0). Values
ited
CONDITION
-
TEST
INTERVAL
t ms
of test responses elictrain in 7 adult SHRs represent means f SE.
1
elicited 800 ms FIG. 6. Percent recovery of test responses after a 3- pulse conditioning train at different arterial pressures in IO adult SHRs (A) and 6 adult WKYs (0). Values represent means k SE.
4RfERIAL
PRESSURE
(mm
Hg)
silent period mechanisms. Although differences in anesthetics and animal sources preclude quantitative comparisons between silent periods in weanling and adult rats, the presence of silent periods in weanling rats disproves this hypothesis. Our ability to infer that the sympathetic nervous system is chronically hyperactive in SHRs depends on the reliability of the relationship between the strength of the sympathetic silent period and tonic levels of sympathetic activity. It is not yet clear whether the silent period determines tonic sympathetic activity or whether its strength is a function of tonic sympathetic activity (1, 12). Nevertheless, the strength of the silent period and the level of tonic sympathetic activity appear to be closely related under a variety of conditions. Cats subjected to general anesthetics that decrease tonic sympathetic activity exhibit stronger silent periods (27). Inhibition of tonic sympathetic activity by increased baroreceptor input is accompanied by stronger silent periods (8, 25). Finally, cats treated with tetanus toxin exhibit
both sympathetic hyperactivity and weakened silent periods (20). We conclude that the diminished silent periods observed in SHRs are manifestations of sympathetic hyperactivity in this strain. The evidence for sympathetic hyperactivity during the development of spontaneous hypertension in young rats in conflicting. Neonatal destruction of the sympathetic nervous system with anti-nerve growth factor prevents or minimizes development of hypertension in SHRs (2, 3). Dopamine P-hydroxylase, an indicator of postganglionic sympathetic activity, is elevated in serum and tissues of SHRs (15, 16). However, measurements of preganglionic sympathetic activity, based on ganglionic or adrenal concentrations of choline acetyltransferase, have demonstrated both normal (9) and elevated (17) levels. The present study provides the first measurement of sympathetic activity in weanling SHRs from directly recorded nerve activity. The results show that the sympathetic nervous system is hyperactive in SHRs before arterial pressure is significantly elevated.
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As early as 1967, Okamoto et al. (19) provided evidence for elevated sympathetic activity in the greater splanchnit nerve of adult SHRs. More recently, Judy et al. (6) found elevated activity in the cervical sympathetic, greater splanchnic, least splanchnic, splenic, and renal nerves of adult SHRs. On the other hand, Lais et al. (lo), recording on the lumbar sympathetic chain, did not observe elevated levels of sympathetic activity in SHRs. Biochemical indexes of sympathetic activity in adult SHRs have provided evidence for both elevated (14) and normal (9, 15) levels, The diminished sympathetic silent
AND
G. N. BARTON
periods which we observed in 100-day-old SHRs confirmation that sympathetic hyperexcitability in adults.
provide persists
The expert assistance of Mr. Kevin McKenna is gratefully acknowledged. The advice of Dr. Drew Carlson during the course of these experiments and in the preparation of the manuscript was particularly valuable. We thank Mr. Eric Howland for preparing the figures and Ms. Evelyn McCann for typing the manuscript. This research was supported by Grant HL-16315 from the National Heart, Lung, and Blood Institute. Received
31 October
1977; accepted
in final
form
13 September
1978.
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