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(Circulation. 1997;96:1667-1674.)
© 1997 American Heart Association, Inc.

Articles

Circadian Rhythms of Cardiovascular Functions Are Modulated by the Baroreflex and the Autonomic Nervous System in the Rat

Mitsutaka Makino, MD; Hiroshi Hayashi, MD; Hiroto Takezawa, MD; Makoto Hirai, MD; Hidehiko Saito, MD; ; Shizufumi Ebihara, PhD

From the Division of Cardiology, First Department of Internal Medicine, Nagoya University School of Medicine, and the Department of Animal Physiology, School of Agricultural Sciences (S.E.), Nagoya University, Japan.

Correspondence to Hiroshi Hayashi, MD, Division of Cardiology, First Department of Internal Medicine, Nagoya University School of Medicine, 65 Tsurumai, Showa-ku, Nagoya, 466, Japan.

	Abstract

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Background We assessed the hypothesis that the baroreflex and the autonomic nervous system are important in the control of the circadian rhythms of cardiovascular functions.

Methods and Results We continuously measured blood pressure (BP), heart rate (HR), and locomotor activity in sinoaortic denervated (SAD), sympathectomized, and atropine-injected rats by use of a radiotelemetry system. The circadian rhythm of mean blood pressure (MBP) was selectively disrupted in SAD rats under 12-hour light-dark (LD12:12) cycles as a result of an increase in MBP during the light period and disappeared under constant darkness (DD). The locomotor activity and HR were not remarkably affected by SAD. The circadian rhythm of MBP was suppressed in sympathectomized rats by a decrease in the MBP during the dark period, and the abrupt changes in MBP when the lighting was altered were not seen under LD. Under DD, an MBP rhythm similar to that observed under LD was obtained. Sympathectomized rats also showed lower HR levels during the dark period than intact rats under LD cycles. In atropine-injected rats, the MBP and HR increased, especially during the light period, resulting in a reduction of light-dark differences in MBP and HR. The locomotor activity showed an apparent 24-hour variation in the sympathectomized and atropine-injected rats.

Conclusions The disruption of the baroreflex selectively eliminates the circadian rhythm of BP, and the circadian rhythms of BP and HR are modulated by the autonomic nervous system in rats. The circadian rhythms of BP and HR are regulated by different mechanisms involving the autonomic nervous system.

Key Words: circadian rhythm • baroreceptors • blood pressure • nervous system, autonomic

	Introduction

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It is well accepted that various functions of the cardiovascular system change with a 24-hour cycle. For example, blood pressure, heart rate, cardiac output, and stroke volume are higher in the active phase than in the rest phase.^{1 2} Also, in humans, the incidence of cardiovascular events such as myocardial ischemia,³ myocardial infarction,⁴ sudden cardiac death,⁵ and ischemic stroke shows 24-hour variations.⁶ The abrupt increase of blood pressure that occurs in the early morning is implicated in precipitating these cardiovascular events,^{1 7} and it is reported that target-organ damage occurs with a higher frequency in some hypertensive patients whose blood pressure remains elevated throughout the night.^{8 9 10} In the search for a way to prevent these cardiovascular events, it is important to clarify the mechanisms of such 24-hour variations.

The autonomic nervous system, together with local autoregulatory mechanisms and circulatory hormones, directly influences cardiovascular functions.¹¹ The baroreceptor reflex system is also involved in stabilizing blood pressure and controlling the output of the autonomic nervous system.¹² Although recent studies using power spectral analysis have provided some additional information, the detailed mechanisms of the autonomic regulation of BP and HR remain unclear. Traditionally, neural control of the circulation has been explored mainly through the study of reflexes because of the difficulties of long-term recording of cardiovascular functions. Recently developed radiotelemetry systems, however, have made it possible to measure hemodynamic variables for a long period of time with accuracy and stability from freely moving animals.

It has been shown that the rhythms of BP and HR are controlled by an endogenous circadian oscillating system^{13 14} and that the suprachiasmatic nucleus is responsible for their rhythmicities in rats.^{15 16 17 18} However, how the circadian information from the suprachiasmatic nucleus regulates the 24-hour rhythms of BP and HR is unknown. Knowledge explaining the association of the autonomic nervous system with circadian regulation of BP and HR is scarce. Therefore, the present study was performed to investigate the role of the autonomic nervous system, including the baroreceptor reflex system, in the control of the circadian rhythms of BP and HR. This was done by measuring hemodynamic and activity variables in SAD, sympathectomized, and atropine-injected rats by use of an implantable radiotelemetry system. In addition, the correlations between hemodynamic variables and locomotor activity were assessed.

	Methods

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Female Wistar-Imamichi rats (11 to 12 weeks old; weight, 250 g) were used. They were housed individually on wood shavings in standard polyethylene cages (40.0x25.0x25.0 cm) that were placed in a light-tight box (55x210x62 cm) with ad libitum access to food and water. Light was provided by fluorescent lamps. The intensity was 500 lux at the floor of the cage in the light phase. Temperature (24±1°C) and humidity (50%) were controlled throughout the experiment.

Sinoaortic Denervation
SADs were performed in 6 rats according to the method of Krieger.¹⁹ Briefly, under ether anesthesia, a midline incision was made in the ventral neck region, and the sternocleidomastoid muscles were reflected laterally to expose the common carotid arteries, the external and internal carotid arteries, the vagi, and the cervical sympathetic trunks. The sympathetic trunk, superior laryngeal nerve, and aortic depressor nerve were bilaterally sectioned under a surgical microscope. The bifurcation and all carotid branches were stripped of fibers and connective tissues and painted with a small amount of 10% phenol. Sham operations in which the neck incision was made and the appropriate nerves were exposed were performed in 3 rats.

Chemical Sympathectomy
Chronic chemical sympathectomy was performed in 6 Wistar-Imamichi rats by the method of Julien et al.²⁰ Starting on day 7 after birth, subcutaneous injections of guanethidine sulfate (CIBA-Geigy) were given at doses of 60 mg/kg daily from day 7 to 25, 30 mg/kg daily from day 26 to 70, and 30 mg/kg every other day from day 71 to 90. Three rats were injected with the same volume of saline as controls.

Parasympathetic Blockade
In 6 rats, 2- to 3-cm incisions were made in the dorsal shoulder blades, and a subcutaneous pocket was formed. After an osmotic minipump (model 2MI2; Alza Corp) filled with atropine (Sigma Chemical Co) was inserted into the pocket, the incision was closed with metal clips. The dose of atropine automatically dispensed was 15 mg · kg^-1 · d^-1 for 14 days.

Implanting the Transmitter
Under ether anesthesia, a central abdominal incision was made and the radiotelemetry device (model TA11PA-C40 or TL11M2-C50-PXT, Physiotel) was secured to the intraperitoneal space. The sensing catheter of this device was inserted into the descending aorta distal to the renal artery against blood flow. The information received by the device was processed and digitized as radiofrequency data, which were recorded and stored in a computer in cyclic runs of 5 minutes with the Dataquest IV system (Data Sciences International). This data acquisition system has been previously described in detail.¹³ The measured parameters were SBP, DBP, MBP, HR, and locomotor activity. Locomotor activity was monitored as changes in transmitter signal strength due to transmitter locomotion. The PP was represented as the value of the difference between the SBP and DBP.

Experimental Protocol
Measurements were started 2 weeks after the implantation of transmitters (intact, n=6; SAD, n=6; sham SAD, n=3; sympathectomy, n=6; control for sympathectomy, n=3). Rats were examined under LD and then transferred to DD conditions. The period of the recording for each light condition was at least 16 days. At the end of the experiment, the SAD or sympathectomized rats were tested to confirm the success of the operations as follows: under ether anesthesia, a polyethylene catheter was inserted via the internal jugular vein into the superior vena cava for drug administration. Then, in the SAD rats, the cardiac baroreflex sensitivity, computed as the slope of the change in interbeat interval as a function of increasing SBP, was measured after several 2- to 3-µg/kg injections of phenylephrine (Kowa). This dose raises SBP by 30 mm Hg. In the sympathectomized rats, the pressure response to 500 µg/kg tyramine (Sigma) was examined as a functional index of sympathectomy. Norepinephrine levels in the heart tissue, the kidney, and the adrenal gland were confirmed by high-performance liquid chromatography with electrochemical detection. The detection was performed under a constant applied potential of 450 mV and a flow rate of 1 mL/min in the column (Eicompak MA-ODS; EICOM).

Data from 6 atropine-injected rats were also collected 2 weeks after the implantation of transmitters. The recording continued for at least 16 days under LD (pretreatment), at which time an osmotic minipump was implanted and observed for an additional 14 days (posttreatment). The data obtained for 2 days after the implantation of this osmotic minipump, beginning at the offset of the first light period, were used as the posttreatment values.

Data Analysis
Data are expressed as mean±SEM. Mean values were calculated for 1-hour intervals for each group of rats. Differences in hemodynamic and activity variables between SAD, sympathectomized, and intact rats were evaluated for statistical significance by one-way ANOVA with repeated measures. Post hoc analysis of significant effects was performed with a Fisher protected least significant difference test. Differences in baroreflex sensitivity between SAD and intact rats and in BP response to tyramine and tissue norepinephrine levels between sympathectomized and intact rats were analyzed by unpaired t test. A level of P<.05 was accepted as statistically significant. The correlations between hemodynamic variables and locomotor activity were assessed with Pearson's correlation coefficients. Values for 96 data points (data taken every hour for 4 days) were analyzed. A ² periodogram with 99.9% confidence levels was used to detect circadian periodicity. The ² periodogram was first described by Sokolove et al.²¹ It is based on the theory that if the time series did not have a periodic component, the difference between each data point and the mean of the time series became small. Qp represents normalized reliabilities of the rhythms. It is given by the following formula: Qp=k(Y-x)²/x, where k is the number of rows, x is the mean of the time series, and Y is each value of time series.

	Results

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Cardiac Baroreflex Sensitivity
The intravenous injection of phenylephrine caused little or no reflex bradycardia in SAD rats. The baroreflex sensitivity in these animals (0.62±0.26 ms/mm Hg, n=6) was significantly lower than that in intact rats (2.46±0.44 ms/mm Hg, n=6, P<.05).

Indices of Sympathectomy
Chronic treatment with guanethidine abolished the pressure response to tyramine injection. The change in SBP in the sympathectomized rats (4.2±1.1 mm Hg, n=6) was significantly less than that in intact rats (69.4±0.7 mm Hg, n=6, P<.01). Sympathectomy also significantly reduced the tissue norepinephrine levels in the heart (9.0±0.7 µg/g, 6 intact rats; 1.4±1.0 µg/g, 6 treated rats, P<.01) and the kidney (3.6±2.6 µg/g, 6 intact rats; 0.6±0.5 µg/g, 6 treated rats, P<.05), but it did not significantly alter those in the adrenal gland (93.5±3.7 µg/g, 6 intact rats; 76.6±2.2 µg/g, 6 treated rats).

Hemodynamic Effects of SAD
The levels of BP, HR, locomotor activity, and their rhythmicities in 3 sham-operated rats resembled those in 6 intact rats (data not shown). In intact rats, the locomotor activity, MBP, and HR were all entrained to photic cycles showing clear 24-hour variations, with higher levels occurring during the dark and lower levels occurring during the light period (Fig 1). ² periodograms exhibited significant 24-hour peaks in MBP and HR of intact rats under LD cycles (Fig 2, P<.001). In SAD rats, however, the rhythmicity in MBP was obviously suppressed, although the locomotor activity and HR were not remarkably affected (Figs 2 and 3). Only 1 of 6 SAD rats had a weak but significant 24-hour peak in MBP under LD cycles (P<.001). The temporal pattern of MBP in SAD rats was bimodal, with one peak occurring during the light period and the other during the dark period. The most dramatic change in MBP was its increase during the light period, which led to no light-dark differences in MBP in SAD rats (Fig 3 and Table). The 12-hour average of DBP in SAD rats during the light period was significantly increased (Table, P<.05). Although the HR levels in SAD rats were similar to those in intact rats during both the light and the dark periods, no marked peaking of HR was observed in SAD rats during the early dark period (Fig 3). The level and pattern of locomotor activity rhythms were not changed by SAD. After being transferred from the LD to the DD setting, all parameters in intact rats showed free-running rhythms (endogenous circadian rhythm under constant condition). In SAD rats, however, the circadian rhythmicity in MBP was almost abolished (none of 6 SAD rats showed significant circadian peaks in the periodogram analysis; data not shown), although the circadian rhythms of HR and locomotor activity persisted under DD conditions (Fig 4).

View larger version (50K): [in this window] [in a new window]   Figure 1. Hourly means and SEMs of locomotor activity, MBP, and HR in 6 intact rats under LD cycles. Parameters were measured continuously over 24 hours for 12 consecutive days. Mean values from 4 consecutive days were obtained for each rat. Data for locomotor activity were normalized so that average value for 12 days in each rat is 1. Shading illustrates actual time of darkness.

View larger version (23K): [in this window] [in a new window]   Figure 2. 2 periodograms of MBP and HR in intact, SAD, and chemically sympathectomized rats under LD cycles. Data from 12 consecutive days were analyzed for each rat. Oblique lines indicate significance (P=.001). Qp values (see text) above line are statistically significant (P<.001). Numbers in parentheses are numbers of rats that have significant 24-hour peak.

View larger version (48K): [in this window] [in a new window]   Figure 3. Hourly means and SEMs of locomotor activity, MBP, and HR in 6 SAD rats under LD cycles. Parameters were measured continuously over 24 hours for 12 consecutive days. Data for locomotor activity were normalized so that average value for 12 days in each rat is 1. Shading illustrates actual time of darkness.

View this table: [in this window] [in a new window]   Table 1. Hemodynamic and Activity Variables Under LD Cycles

View larger version (70K): [in this window] [in a new window]   Figure 4. Circadian variations of locomotor activity, MBP, and HR under DD conditions in an SAD rat. Data were collected continuously over 24 hours for 12 consecutive days. Because circadian period in DD was 24.4 hours in this case, means and SEMs of 1.017 hours (24.4/24 hours) were plotted. Circadian time 12 was defined as onset of activity where mean value of locomotor activity exceeded 20 counts/5 min.

Hemodynamic Effects of Sympathectomy
Hemodynamic variables and variabilities in the saline-injected rats did not differ from those of intact rats (data not shown). In sympathectomized rats, the locomotor activity showed a clear 24-hour variation, as also seen in the intact rats (Fig 5). However, the 24-hour rhythmicity in MBP was apparently affected. The characteristics observed in intact rats, that is, the abrupt rise in MBP when the light was turned off and its sharp fall when the light was turned on, were not seen in the sympathectomized rats. The MBP in sympathectomized rats gradually increased during the dark period, with a peak in the mid to late period, and gradually decreased during the light period. Compared with the 12-hour mean average for MBP, the MBP values in sympathectomized rats during the dark period were lower than in intact rats, although the difference was not significant (Fig 5 and Table). Because the MBP during the light period in sympathectomized rats was similar to that in intact rats, the light-dark differences in MBP became small, resulting in no significant 24-hour rhythmicity, as seen in the ² periodogram (Fig 2). A significant peak was seen in only one of 6 sympathectomized rats under LD cycles (P<.001). The 12-hour average of SBP during the dark period was significantly decreased, and the 12-hour averages of PP both during the light and dark periods were also significantly decreased in sympathectomized rats (Table, P<.01). Although the HR level during the dark period was significantly lower in sympathectomized rats than in intact rats (Table, P<.05), 24-hour rhythmicity in HR persisted under LD cycles (Figs 2 and 5). A significant 24-hour peak was observed by periodogram analysis in all 6 sympathectomized rats (P<.001). The HR rhythm patterns were similar to those in the SAD rats (no abrupt peak during the early dark period). In contrast to the changes observed in MBP and HR, the rhythm of locomotor activity was not markedly affected by sympathectomy under LD cycles. Under DD conditions, all three parameters showed a free-running rhythm, and the pattern of MBP variation resembled that observed under LD cycles (Fig 6). Upon periodogram analysis, 3 of 6 sympathectomized rats showed a significant circadian peak in MBP (P<.001), and 3 did not (data not shown).

View larger version (48K): [in this window] [in a new window]   Figure 5. Hourly means and SEMs of locomotor activity, MBP, and HR in 6 chemically sympathectomized rats under LD cycles. Parameters were measured continuously over 24 hours for 12 consecutive days. Data for locomotor activity were normalized so that average value for 12 days in each rat is 1. Shading illustrates actual time of darkness.

View larger version (72K): [in this window] [in a new window]   Figure 6. Circadian variations of locomotor activity, MBP, and HR under DD conditions in a chemically sympathectomized rat. Parameters were measured continuously over 24 hours for 12 consecutive days. Because circadian period in DD was 24.3 hours in this case, means and SEMs of 1.013 hours (24.3/24 hours) were plotted. Circadian time 12 was defined as onset of activity where mean value of locomotor activity exceeded 20 counts/5 min.

Hemodynamic Effects of Parasympathetic Blockade
Because the effects of atropine administration on the hemodynamic parameters lasted 4 to 5 days, the data for 2 consecutive days after the implantation of an osmotic minipump were used in the analyses. Before atropine administration, a distinct 24-hour rhythmicity was observed for all three parameters (Fig 7). However, the MBP and HR increased after atropine treatment, especially during the light period, although they gradually returned to pretreatment levels. The 12-hour mean average for the MBP during the light period significantly exceeded the pretreatment level (Table, P<.05). The HR was also significantly increased during both the light (P<.01) and the dark periods (P<.05). The increase in HR during the light period was so large that the light-dark difference in HR became small. The locomotor activity during the dark period was significantly suppressed compared with the pretreatment level (P<.01).

View larger version (64K): [in this window] [in a new window]   Figure 7. Hourly means and SEMs of locomotor activity, MBP, and HR in 6 atropine-injected rats under LD cycles. Raw data for locomotor activity were normalized so that average value for 4 days before atropine treatment in each rat is 1. Shading illustrates actual time of darkness. Atropine (arrows) indicates implantation of osmotic minipump containing atropine.

Correlation Between Hemodynamic and Activity Variables
Under LD cycles, the HR (r=.84, P<.0001) and MBP (r=.88, P<.0001) were highly correlated with the locomotor activity in intact rats (Fig 8). In SAD rats, the correlation coefficient between MBP and locomotor activity (r=.26, P=.009) was lower than in intact rats, although the coefficient between HR and locomotor activity was high (r=.89, P<.0001). In sympathectomized rats, the correlation coefficient between HR and locomotor activity (r=.84, P<.0001) was identical to that of intact rats, but the slope of the regression line was smaller than that in intact rats. The coefficient between MBP and locomotor activity (r=.41, P<.0001) was lower in sympathectomized rats than in intact rats. The HR and MBP were well correlated in intact rats (r=.94, P<.0001), but the correlations between these parameters almost disappeared both in SAD (r=.21, P=.04) and sympathectomized rats (r=.11, P=.27).

View larger version (29K): [in this window] [in a new window]   Figure 8. Scatterplot of HR and MBP against locomotor activity (ACT) and HR against MBP in intact, SAD, and chemically sympathectomized rats under LD cycles. Values for 96 data points taken every hour for 4 days were analyzed. Solid line is regression line.

	Discussion

Top Abstract Introduction Methods Results Discussion References

 
Although circadian rhythms of motor activity, drinking behavior, and body temperature have been studied repeatedly under various conditions, because of technical difficulties only a few studies have investigated those of cardiovascular functions. The advent of radiotelemetry has made it possible to collect accurate and stable data on BP and HR from freely moving animals over long periods of time. By using a telemetry system, we previously demonstrated that the BP and HR in female rats show endogenous circadian and estrous cycle–dependent variations¹³ and that the suprachiasmatic nucleus is responsible for these circadian rhythms.¹⁸ In the present study, we tried to elucidate the putative involvement of the peripheral neural control system in the circadian rhythms of cardiovascular functions. Because any treated rats did not exhibit apparent estrous cycle–dependent variations, this factor was not addressed in this study. Although the autonomic nervous system may be implicated in the expression of these variations, it is considered that this factor did not detract from the interpretation of the results, because the data were averaged for 4 days, which is the estrous cycle of rats, and the patterns of the daily rhythm did not differ from each other.

Circadian Rhythms of Cardiovascular Functions in SAD Rats
Although SAD has been reported to produce sustained hypertension,^{19 22} subsequent studies suggest that this hypertension depends on the methods of measurement, the duration of measurement, and the recording environment.^{23 24} Several studies in freely moving animals have demonstrated that BP levels return to normal or near- normal levels within days after the SAD operation.^{24 25} We therefore continuously measured BP for extended periods and found that the MBP under LD, particularly during the light period, was significantly elevated. As a result, the 24-hour rhythmicity in MBP was suppressed and a bimodal pattern was observed in SAD rats under LD cycles, although the HR and locomotor activity were not dramatically affected. This bimodal pattern of MBP was not observed under DD conditions, suggesting that photic inputs are responsible for this pattern. Although the BP variation is reportedly affected by physical activity,²⁶ the MBP variation did not necessarily depend on locomotor activity, as shown by the correlation coefficient. These results indicate that the disruption of the baroreflex elevates the BP in rats during the light period, eliminating the 24-hour rhythmicity.

To the best of our knowledge, no investigations of the circadian rhythms of BP and HR in SAD rats have previously been performed. Although the BP and HR in SAD and NTS-lesioned rats were recorded for 24 hours in the study of Buchholz et al,²⁵ circadian rhythmicity was not assessed. Cowley et al^{23 26} performed 24-hour BP recordings in SAD dogs but reported that SAD dogs exhibit exaggerated diurnal variations, with higher-than-normal pressures during the day and lower-than-normal levels at night.

Although the exact circuitry is equivocal, one model proposes a polysynaptic pathway from the NTS in the brain stem, a major integrating center that receives information from the primary afferent baroreceptor, to the rostral ventral lateral medulla through the caudal ventral lateral medulla.^{11 27 28} In this pathway, SAD intercepts afferent information from the baroreceptors to the NTS. The precise mechanisms for the selective elimination of the circadian rhythm of MBP in SAD rats could not be clarified from our results. One possibility is to assume that SAD may increase peripheral resistance through the peripheral sympathetic tone, and its increase may be relatively greater during the light period when baseline peripheral resistance is low. In fact, the DBP, which reflects peripheral resistance, was significantly elevated in SAD rats during the light period. Another possibility is to assume that the set point of the baroreceptor reflex system may fluctuate with a 24-hour cycle and the set point of BP may not be maintained because of the SAD procedure. The baroreflex sensitivity reportedly exhibits a circadian rhythm.²⁹ Although the baroreceptor reflex system is thought not to be capable of long-term regulation of BP,²⁶ our results suggest that the baroreceptors are important for regulating at least the 24-hour variations in BP.

The BP reportedly remains elevated throughout the night in some hypertensive patients ("nondippers").^{8 10} Because baroreflex sensitivity is reported to be disturbed as hypertension progresses,^{12 30} it can be inferred that the suppressed 24-hour variation in those nondippers may be due to the impaired function of their baroreceptor reflex system. However, further studies are needed to clarify this hypothesis.

Circadian Rhythms of Cardiovascular Functions in Sympathectomized Rats
Chemical sympathectomy has been reported by some researchers to reduce the BP.^{31 32} Other studies have demonstrated that it does not significantly reduce the BP of freely moving animals.^{20 33 34} These inconsistencies may be due to the techniques used for measuring BP. In the present study, the 12-hour mean average for MBP during the dark period tended to be lower in the sympathectomized than in the intact rats, although the average of MBP during the light period was similar between these groups. These results suggest that light-dark differences in BP should be considered in evaluation of the effects of sympathectomy on BP.

Chemical sympathectomy with guanethidine may activate the influence of the parasympathetic nervous system on the heart. The possible reasons for the decrease of MBP during the dark period seem to be the elimination of the sympathetic nervous system or the activation of the parasympathetic nervous system, or both. Because the SBP and PP in sympathectomized rats during the dark period were suppressed, the decrease of stroke volume, which is reflected in SBP or PP, may be responsible for the decrease of MBP during the dark period. It seems definitive, therefore, that the autonomic nervous system, particularly the sympathetic nervous system, is important for the manifestation of 24-hour rhythms of BP and HR in rats.

Knowledge explaining the association of the autonomic nervous system with the circadian rhythms of BP and HR is scarce. Warren et al¹⁷ demonstrated that the circadian rhythm of HR is selectively affected by sympathectomy with guanethidine in rats. However, we observed a distinct 24-hour variation in HR in sympathectomized rats, even though the level during the dark period was significantly decreased relative to the intact rats. This discrepancy may be due to the method of performing sympathectomy. In the study by Warren et al, guanethidine was injected into adult rats daily for 8 days, and the HR was analyzed from the data obtained during the injection.

The daily variations in MBP in intact rats, in which an abrupt rise was seen when the light was turned off and a sharp fall in MBP was seen when the light was turned on, were not observed in sympathectomized rats. These results suggest that a change in autonomic nervous tone, the majority being the sympathetic nervous tone, is required for the abrupt changes in BP at the time when the lighting is altered. In humans, BP often rises abruptly around the time of awakening.¹ If this early morning surge in BP were due to the same mechanisms as in rats, a change in autonomic nervous tone would be important for the formation of this morning surge in humans. In fact, -sympathetic vasoconstrictor activity is reportedly related to this early morning surge.⁷

Furthermore, the autonomic nervous system may play some role in the high correlation between the HR and locomotor activity, because the slope of the regression line was decreased in sympathectomized rats. The correlation between the MBP and locomotor activity was disrupted by both SAD and chemical sympathectomy. The autonomic nervous system, including the baroreflex, therefore seems to be important for this correlation. Because the correlations between the HR and MBP were almost abolished in both SAD and sympathectomized rats, the present study confirms earlier observations that in heart transplant patients³⁵ and patients dependent on ventricular-demand pacemakers,³⁶ the circadian rhythms of BP and HR are regulated by different mechanisms involving the autonomic nervous system.

Circadian Rhythms of Cardiovascular Functions Under Parasympathetic Blockade
In our experiments, the effects of atropine on MBP and HR persisted for only 4 to 5 days. Subsequently, the pattern of MBP and HR variation returned to the pretreatment pattern, although atropine was infused at a constant rate for 14 days. These changes in the pattern of MBP and HR were not observed in rats implanted with a Silastic tube in our previous study.¹³ Also, it has been reported that atropine administration increases total muscarinic receptor density by 20% to 30%.³⁷ Therefore, it is thought that the recovery of MBP and HR from elevated levels is due to such receptor upregulation. Thus, we used only 2 days of data after the implantation of the osmotic minipump for the analyses. As a result, the BP and HR increased during both the light and dark periods. Elevations in both parameters were greatest during the light period; as a result, the light-dark differences in BP and HR became small. In contrast to sympathectomy, which decreased the levels of BP and HR during the dark period, parasympathetic blockade increased these levels during the light period. It is known that activation of the parasympathetic nervous system suppresses the HR and that blockade of the parasympathetic nervous system not only enhances the activity of the sympathetic nervous system but also abolishes baroreflex control of HR. In this study, the MBP and HR levels during the light period were not affected in the sympathectomized rats, and the HR levels during both the light and dark periods were not changed in SAD rats. This implies that the elevation of the MBP and HR levels during the light period is largely due to parasympathetic blockade. Several studies using power spectral analysis have demonstrated that high-frequency power, an index of parasympathetic nervous tone, occurs at higher levels during the rest phase and lower levels during the active phase.^{38 39 40} It is reasonable to assume from these results that the parasympathetic nervous system, which is upregulated during the light period, is important for the manifestation of 24-hour rhythms of BP and HR in rats. From this point of view, it can be said that the decrease of MBP during the dark period in sympathectomized rats is mainly due to the elimination of the sympathetic nervous system. However, central oscillations are complex determinants of the spontaneous changes in the cardiovascular performance and reflex responsiveness, and neural maneuvers do not necessarily provide direct information. Because atropine crosses the blood-brain barrier, the effects of atropine on the central nervous system should be considered. Further investigations are needed to clarify these problems.

Although the autonomic nervous system is thought to play a dominant role in controlling the circadian rhythm of HR, it is possible that such hormonal factors as the renin-angiotensin system, vasopressin, and circulating catecholamines are also involved, because the circadian rhythm of HR reportedly persists in patients after heart transplantation.³⁵

Summary
The present study demonstrated that disruption of the baroreceptor reflex system selectively eliminates the circadian rhythm of BP as a result of an increase in BP during the light period under LD cycles in rats and that the circadian rhythms of BP and HR are regulated by different mechanisms, including the autonomic nervous system. Results also suggest that elimination of the sympathetic nervous system suppresses the circadian rhythms of BP and HR by decreasing BP and HR during the dark period, and parasympathetic blockade reduces the circadian rhythms of BP and HR by increasing BP and HR during the light period under LD cycles in rats. The abrupt increase and decrease in BP observed at the time when the lighting conditions are changed are eliminated by chemical sympathectomy.

	Selected Abbreviations and Acronyms

 

BP = blood pressure DBP = diastolic blood pressure DD = constant dark HR = heart rate LD = 12-hour light-dark cycle MBP = mean blood pressure NTS = nucleus tractus solitarii PP = pulse pressure SAD = sinoaortic denervation, denervated SBP = systolic blood pressure

	Acknowledgments

 
This study was supported in part by the Hibino Memorial Medical Research Fund. We would like to thank Prof Ishio Ninomiya for invaluable comments regarding our study and Prof Kiyoshi Shimada for the opportunity to perform the experiments in this study.

Received November 26, 1996; revision received March 5, 1997; accepted March 7, 1997.

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