Cardiac Neural Regulation Oscillates with the Estrous Cycle in Freely ...

GENERAL

ENDOCRINOLOGY

Cardiac Neural Regulation Oscillates with the Estrous Cycle in Freely Moving Female Rats: The Role of Endogenous Estrogens Terry B. J. Kuo, C. T. Lai, Fu-Chun Hsu, Yi-Jhan Tseng, Jia-Yi Li, Kun R. Shieh, Shih-Chih Tsai, and Cheryl C. H. Yang Sleep Research Center (T.B.J.K., C.T.L., J.-Y.L., C.C.H.Y.), Institute of Brain Science (T.B.J.K., J.-Y.L., C.C.H.Y.), National Yang-Ming University, Taipei 11221, Taiwan; Department of Education and Research (T.B.J.K., C.T.L., S.-C.T., C.C.H.Y.), Taipei City Hospital, Taipei 10341, Taiwan; Division of Neurology (F.-C.H.), The Children’s Hospital of Philadelphia, Philadelphia, Pennsylvania 19104; and Institute of Neuroscience (Y.-J.T., K.R.S.), Tzu Chi University, Hualien 97004, Taiwan

Both estrogens levels and sleep/wakefulness states have been separately reported to affect cardiac autonomic regulation. In this study, we examined the integrated effects of the estrous and sleep cycles on cardiac autonomic activity in freely moving adult female rats. Cardiac autonomic activities were measured by analyzing the power spectrum of heart rate variability. High-frequency power (HF) and low-frequency power to HF ratio are closely correlated with cardiac parasympathetic and sympathetic activity, respectively. Ten days after electrodes were implanted, electroencephalogram, electromyogram, and electrocardiogram were recorded 6 h daily for 12 consecutive days to cover at least two estrous cycles. Estrous-cycle stages were determined using vaginal smears. Sleep cycle-related heart rate variability parameter oscillations were seen in all rats. However, the estrous cyclicity and estrous-cycle-related changes were only observed in the control rats and not in ovariectomized or the estrogen receptor antagonist, tamoxifen, treatment rats. A significantly higher HF was observed in estrous rats compared with diestrous rats or ovariectomized rats no matter whether the rats were asleep or awake. However, a significantly low-frequency power to HF ratio was only observed in quiet sleep (QS) during estrus. All these differences disappeared after treatment with tamoxifen. Our results suggest that estrous-cycle-related changes in cardiac neural regulations can be mainly attributed to endogenous estrogens, and these effects are most obviously manifest during QS. Estrous rats during QS would be equivalent to the late follicular phase of the women menstrual cycle and involve strong vagal tone but weak sympathetic activity. (Endocrinology 151: 2613–2621, 2010)

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ardiac sympathovagal imbalance has been implicated in coronary artery disease and sudden cardiac death (1, 2), and these cardiovascular diseases are the leading cause of death in women especially after menopause (3). Therefore, a lack of endogenous estrogens has been suggested as a major contributing factor to cardiovascular disease among women (4, 5). Endogenous estrogens are pulsatile, and their releases are synchronized with the menstrual cycle in humans or with the estrous cycle in

other mammals. The pulsatile release of the endogenous estrogens leads to a pulsatile change in autonomic functions among women, which can induce clinical symptoms that are strongly associated with alterations in autonomic functions (6, 7). Estrogens affect cardiac neural regulation (8, 9), which is highly correlated with cardiac mortality (2, 10, 11). Estrogens increase vagal activity and decrease sympathetic activity in both pre- and postmenopausal women (12–15). Therefore, modulation of au-

ISSN Print 0013-7227 ISSN Online 1945-7170 Printed in U.S.A. Copyright © 2010 by The Endocrine Society doi: 10.1210/en.2009-1410 Received December 8, 2009. Accepted March 16, 2010. First Published Online April 14, 2010

Abbreviations: AW, Active waking; ECG, electrocardiogram; EEG, electroencephalogram; EMG, electromyogram; HF, high-frequency power; ⌬HF, HRV HF difference; HPSD, power spectral density of the HRV; HRV, heart rate variability; LF, low-frequency power; LF/HF, LF-to-HF ratio; ⌬LF/HF, difference in HRV LF/HF; MPF, mean power frequency; OVX, ovariectomized; PS, paradoxical sleep; QS, quiet sleep; RR, R-R interval; TEMG, threshold of the EMG power; TMPF, threshold of the MPF.

Endocrinology, June 2010, 151(6):2613–2621

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Cardiac Autonomic Nervous System with the Estrous Cycle

tonomic function by estrogens has been suggested to act as one of the cardiovascular protective mechanisms in women (8, 15, 16). In animal studies, exogenous administration of estradiol increases vagal and decreases sympathetic activity in anesthetized rats, from both male and female (17, 18). Sleep also has been shown to influence autonomic nervous system regulation (19, 20) and affect body function, disease status, and life expectancy (21–23). During sleep, the connection between the central nervous system and the outside environment is attenuated, which results in minimal influences originating from the external environment. Therefore, a laboratory examination during sleep ought to provide a more precise measurement of body function at a time when outside stimulations/noises are reduced (24). Whether estrous-cycle-related effects on autonomic activity are manifest more during quiet sleep (QS) is interesting and warrants further explorations. To date, the detailed mechanisms associated with the impact of different female cycle stages on cardiac neural regulations are still under debate. Many studies have reported a predominantly sympathetic regulation in the luteal phase (25–28), but this is still inconclusive (29). The follicular phase has been reported to predominantly (25, 26) or equivocally (28, 29) involve cardiac vagal regulation. In this study, to detect the autonomic functions continuously, as well as to avoid interfering with normal physiological cycles and autonomic functions, heart rate variability (HRV) analysis was applied to quantify cardiac autonomic regulation noninvasively. Using rats, the sleep/wakefulness stages were determined using electroencephalogram (EEG) recording, and the estrous-cycle state was examined daily for 12 d using vaginal smears, which is long enough to cover two estrous cycles. Thus, we were able to determine the interactive and integrated effects on the cardiac neural regulation between the sleep/ wakefulness cycle and the estrous-cycle state.

Materials and Methods


Electrode implantation The detailed electrode implantation procedures have been described previously (19, 24, 30, 31). In brief, rats were implanted with electrodes for parietal EEG, electrocardiogram (ECG), and nuchal electromyogram (EMG) recording under pentobarbital anesthesia (50 mg/kg, ip) using a standard stereotaxic apparatus. After the dorsal surface of the skull was exposed and cleaned, two stainless steel screws were driven bilaterally into the skull overlaying the parietal (2.0 mm posterior to and 2.0 mm lateral to the bregma) regions of the cortex. A reference electrode was implanted 2 mm caudal to the ␭. The electrodes did not penetrate the underlying dura. The ECG was recorded via a pair of microwires placed under the skin of the dorsal part of the body (one was between the cervical and thoracic levels, the other at the lumbar level). Two seven-strand stainless steel microwires were bilaterally inserted into the dorsal neck muscles to record EMG. All the signals were relayed to a recorder by wires through a common connector that was fixed onto the head (32).

Protocols After surgery, each rat was housed individually for 10 d to allow recovery. Started from d 11, a vaginal smear was used to determine the estrous-cycle state (between 0900 and 2200) and a 6-h daily electrophysiological recording (from 1030 to 1630) was performed in a sound-attenuated room for 12 consecutive days. The estrous cycle comprises four phases, namely diestrus, proestrus, estrus, and metestrus. Each phase was characterized by the specific morphology of the cells in the vaginal fluid using light microscopy (33). The estrous cycle lasted 4.4 ⫾ 0.6 d (mean ⫾ SD) in this study. Therefore, at least two estrous cycles were detected in our 12 consecutive days of experimental study, and the data from the second estrous cycle were used in the subsequent statistical analyses. Importantly, it should be noted that rats with induced pseudopregnancy during the 12-d protocol were excluded from the study. To determine the effects of estrous-cycle status, as well as the ovary contribution to cardiac neural regulation, the rats were divided into a sham-operation group (n ⫽ 30) and an ovariectomized (OVX) group (n ⫽ 30). The surgical operation was done in the same time of the electrode implantation (2 months old). In addition, tamoxifen, an estrogen receptor antagonist (34), was used to determine the role of endogenous estrogens on cardiac neural regulation. Another 14 sham-operation rats were divided into a vehicle group (n ⫽ 6) and a tamoxifen group (n ⫽ 8). These rats were sc injected with vehicle (sesame oil, 0.2 ml) or tamoxifen (10 mg/kg) on four consecutive days starting from the metestrous stage of the second estrous cycle of recording.

Animals

Electrophysiological recordings

The experiments were carried out on adult female Wistar Kyoto rats (2–3 months old and n ⫽ 74). The rats were obtained from the National Laboratory Animal Center in Taiwan where their breeding and care was carried out following the guidelines of the American Heart Association on Research Animal Use. They were housed in a sound-attenuated room with a 12-h light, 12-h dark cycle (light on 0600 –1800 h) with the room temperature of 22 ⫾ 2 C and the humidity of 40 –70%. All experimental protocols were approved by the Institutional Animal Care and Use Committee of National Yang-Ming University.

The bioelectrical signals were recorded using a miniature physiological data recorder (KY1C; K&Y Lab, Taipei, Taiwan). In brief, the EEG, EMG, and ECG signals were amplified 1000fold. The EEG signal was filtered in the 0.16- to 48-Hz range, the EMG signal was filtered in the 34- to 103-Hz range, and the ECG signal was filtered in the 0.72- to 103-Hz range (35). The EEG, EMG, and ECG signals were synchronously digitized using a 12-bit analog-digital converter with different sampling rates (125, 500, and 500 Hz, respectively). The digitized dataset was stored on a flash memory card for subsequent off-line analysis.


Sleep analysis The sleep analysis of the rats was performed according to a semiautomatic computing procedure that has been described in detail previously (19, 24, 31). The states of sleep and wakefulness were classified into active waking (AW), QS, and paradoxical sleep (PS). To clearly differentiate the different sleep states, power spectral analysis was continuously applied to the EEG and EMG signals to detect the sleep/wakefulness states through mean power frequency (MPF) of the EEG and power magnitude of the EMG. Data from each 16-sec window were analyzed. However, to increase the temporal resolution of the data, the signal were shifted to give a 50% overlap of the epochs. The rat’s sleep/ wakefulness state was defined as AW if the corresponding MPF was greater than the threshold of the MPF (TMPF) and the EMG power was greater than the threshold of the EMG power (TEMG). The TMPF and TEMG of each animal were defined manually by the investigator and were constant throughout each 6-h recording period. The time series of the MPF first underwent a histogram analysis, from which two separate populations relating to the AW/PS complex and QS could be clearly identified. Thus, TMPF could be set to discriminate these two populations. The histogram of the EMG time series also had two populations, but these were related to AW and the QS/PS complex. Therefore, TEMG could be set to discriminate these two populations. QS was defined if the corresponding MPF was less than TMPF and the EMG power was less than TEMG, and PS was defined if the corresponding MPF was greater than TMPF and the EMG power was less than TEMG. If the corresponding MPF was less than TMPF and the EMG power was greater than TEMG, the sleep/ wakefulness state was undefined and the data for this epoch were excluded from the subsequent statistical analyses. QS is also known as a slow-wave sleep, whereas PS is equivalent to a rapideye-movement sleep (36).

HRV analysis The detailed analytical procedures used for HRV have been previously described (24, 30, 37). Preprocessing of the ECG signals was designed according the recommended procedures for HRV analysis (38). The computer algorithm first identified each QRS complex and rejected each ventricular premature complex or noise according its likelihood in a standard QRS template. The parameters of the template included Q-R amplitude, R-S amplitude, and R-R interval (RR). Successive stationary RRs were resampled and interpolated to provide continuity in the time domain. To obtain enough data points for multiple oscillations of the ECG signal, 16-sec time segments were analyzed but with 50% (8 sec) overlap to increase the temporal resolution. A Hamming window was applied to each time segment to attenuate the leakage effect of the power spectrograms. An algorithm was then used to estimate the power density of the spectral components using on the fast Fourier transformation (39). The resulting power spectrum was corrected for attenuation resulting from the sampling and the application of the Hamming window. For each 16-sec time segment, high-frequency power (HF) (0.6 –2.4 Hz), low-frequency power (LF) (0.06 – 0.6 Hz) of the RR spectrogram were quantified by the method of integration, i.e. calculation of the area of power spectral density between the specified frequencies, and are expressed as milliseconds squared (38). The LFto-HF ratio (LF/HF) was also calculated. Previous studies have indicated that the frequency range of the cardiac vagal modulation extend from LF to HF, but the frequency range of the cardiac


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sympathetic modulation is limited in LF (40). Thus, LF/HF and HF were used as the indices of cardiac sympathetic modulation and cardiac vagal activity, respectively. LF represents the combined effect of both sympathetic and vagal modulations (38, 41– 43). Each 6-h sleep section produced a large amount of HRV data, and therefore, the mean of each HRV parameter under the different sleep/wakefulness states (AW, QS, and PS) was calculated to present the data in a simplified form for the current study.

Statistical analysis The distributions of the HF, LF, and LF/HF were not normal but were skewed to the right; therefore, the HF, LF, and LF/HF were logarithmically transformed to correct the distribution skewness (37). To study the effects of the sleep/wakefulness stages and estrous-cycle status on the HRV parameters, we calculated the differences between QS and AW and between PS and AW to further manifest the estrous-cycle-related changes. Similarly, we calculated the difference between estrus and diestrus to further manifest the sleep/wakefulness-related changes. Significant differences between the values and zero were confirmed with a 95% confidence interval analysis. Significant differences across groups were assessed using ANOVA for repeated measures. When indicated by a significant F statistic, regional differences were isolated using post hoc comparisons by the Fisher’s least significant difference test. Statistical significance was assumed for P ⬍ 0.05. The results are presented as mean ⫾ SEM.

Results In the current study, we have demonstrated that HRV parameter alterations were associated with both the estrous cycle and sleep/wakefulness states. Continuous analysis of the 2-h EEG and HRV parameters indicated that cardiac neural regulation was altered during the various parts of the estrous cycle and between the sleep/wakefulness states (Fig. 1). Rats had the most prominent HF during estrus compared with OVX and diestrus as demonstrated by both the power spectral density of the HRV (HPSD) and the quantified HF channel. In addition, compared with both AW and PS, QS was always accompanied by a lower LF or LF/HF. Also, larger RR and HF were observed during PS. Means of the HRV parameters were strongly correlated with both the estrous-cycle and the sleep/wakefulness states Fourteen sets of the EEG, EMG, and ECG electrodes (seven in sham and seven in OVX rats) survived three complete estrous cycles (Fig. 2). The means of the HRV parameters from the 6-h daily recordings indicate that, compared with the OVX rats, the sham rats had obvious estrous-cycle-related HF changes during all of the animal’s sleep/wakefulness states, but the effect was more prominent during QS and PS. In the OVX rats, however, the estrous cyclicity, as well as the cycle-related HF changes,

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further calculated and analyzed (Fig. 4). The HRV parameters from diestrus were subtracted from the HRV parameters from estrus in the same rat to offset the effects caused by estrous cycle. Compared with AW, both QS and PS had significant higher HRV HF differences (⌬HF), suggesting that both states of sleep had significantly higher cardiac neural vagal regulation than the AW state (P ⬍ 0.05, n ⫽ 30). In addition, compared with AW, the difference in HRV LF/HF (⌬LF/HF) was significantly decreased for both QS and PS, indicating that both states of sleep had significantly lower cardiac neural sympathetic regulation than the AW state (P ⬍ 0.05, n ⫽ 30). It should also be noted that the positive value of the HRV FIG. 1. Representative traces and results from continuous and simultaneous analysis of sleep and HRV. The 2-h recordings are from one OVX rat and one sham-operated rat during ⌬HF between estrus and diestrus indidiestrus (Sham-D) and during estrus (Sham-E). The sleep/wakefulness states, including AW, cated that there was higher neural vagal QS, and PS, are shown. The mean of the RR of each epoch, corresponding three-dimensional regulation during estrus compared with HPSD, HF, LF, and LF/HF, are likewise shown. A darker signal in the HPSD indicates a larger power spectral density with respect to time and frequency. The frequency range for HF and diestrus, and this could be observed for LF are denoted on right side of the HPSD. ln, Natural logarithm. all three sleep/wakefulness states. A negative value for the HRV (⌬LF/HF) disappeared. The results for the HRV parameters from 30 was observed in QS (P ⬍ 0.05, n ⫽ 30), which indicated sham or 30 OVX rats were pooled together and compared that neural sympathetic regulation was less during estrus (Fig. 3). Compared with the OVX group, the HF values of than diestrus. the sham group during both diestrus and estrus were significantly higher in all sleep/wakefulness states. In addition, the HF values in estrus were significantly higher than HRV parameters were affected by the those in diestrus during all sleep/wakefulness states. Fur- estrous-cycle state To manifest the component of HRV parameters afthermore, the LF/HF value of the estrous group was significantly lower than that of the OVX group only during fected by the estrous-cycle state, the HRV parameter difQS (Fig. 3). Compared with AW, QS had a significant ferences among different sleep/wakefulness states were longer RR and a higher HF, suggesting an increase in car- further calculated and analyzed (Fig. 5). The HRV paramdiac vagal neural modulation. In addition, LF and the eters from AW were subtracted by the HRV parameters LF/HF ratio were significantly reduced to a minimum dur- from either QS or PS in the same rat to offset the effects ing QS, indicating a dramatic decrease in cardiac sympa- caused by sleep/wakefulness state. Compared with OVX thetic neural modulation. During PS, the increases in RR and diestrous, estrous rats had a significantly higher HRV and HF became even more evident compared with AW, ⌬HF, which suggests a significant higher cardiac neural suggesting a further increase in cardiac vagal neural mod- vagal regulation during estrus (P ⬍ 0.05, n ⫽ 30). Reulation in this sleep stage. Compared with AW, a decrease garding the HRV ⌬LF/HF, a decreased value was only in LF/HF was also observed in PS; however, the reduction observed in estrus compared with OVX during QS (Fig. 5, was less than with QS, suggesting that neural sympathetic lower panel left), suggesting lower neural sympathetic regregulation was decreased in PS but not to the same ex- ulation in QS during estrus (P ⬍ 0.05, n ⫽ 30). It should also be noted that the positive value of HRV ⌬HF between tent as QS. QS and AW, as well as between PS and AW, indicated that there was higher neural vagal regulation during these two HRV parameters were affected by sleep/wake types of sleeps compared with the active awaking state; states To manifest that the component of the HRV parame- this was observed for all three different hormone states. ters were affected by sleep/wakefulness cycle, the HRV The negative values of the HRV (⌬LF/HF) that can be seen parameter differences between estrus and diestrus were between QS and AW, as well as between PS and AW (P ⬍



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AW and these differences disappeared after tamoxifen treatment. In addition, the significantly smaller ⌬LF/HF observed in QS compared with AW also disappeared after tamoxifen treatment (Fig. 6). These results suggest that sleep/ wakefulness state-related alterations from both neural vagal and sympathetic regulation were inhibited by the tamoxifen treatment. Estrogen receptor antagonist treatment abolished the estrous cyclicity and prevented the estrous-cycle state associated HRV parameter alterations The HRV parameter differences among different sleep/wakefulness states were further calculated and analyzed (Fig. 7). The HRV parameters from AW for either group were subtracted from the HRV parameters from QS or PS in the same rat to offset the effects caused by sleep/wakefulness states. After tamoxifen treatment for 4 d (10 mg/kg 䡠 d), the observed differences in ⌬HF disappeared between estrus and diestrus in both types of sleep vs. the AW state. These results suggest that the estrus vs. diestrus differences were prevented by FIG. 2. The HRV parameters fluctuated along with estrous cycle and changed during the different sleep/wakefulness states. The data of HF and LF/HF of the HRV were analyzed over a treatment with the estrogen receptor total of 6-h AW, QS, and PS and were plotted over time in OVX and sham-operated rats. The blocker tamoxifen. In addition, the values for AW were subtracted from those of QS (QS-AW) and from those of PS (PS-AW) ⌬HF was reduced after tamoxifen treatfrom the same rats. ln, Natural logarithm; D, diestrus; P, proestrus; E, estrus; M, metestrus. ment in estrus but not in diestrus in both The results are expressed as mean ⫾ SEM for seven rats/group. *, P ⬍ 0.05 vs. corresponding OVX; †, P ⬍ 0.05 vs. latest diestrus; #, P ⬍ 0.05 vs. latest proestrus; ‡, P ⬍ 0.05 vs. latest types of sleeps vs. the AW state, which estrus by least significant difference test. suggests that neural vagal autonomic regulation was inhibited by estrogen 0.05, n ⫽ 30 in each group), thus indicate that neural sym- receptor blocker only during estrus. It should also be pathetic regulation was significantly less during both types noted that the negative value of the ⌬LF/HF was reof sleep compared with the active awake state. duced after tamoxifen treatment during estrus in QS vs. AW, indicating that the reduction in neural sympathetic Estrogen receptor antagonist treatment abolished autonomic regulation during QS was decreased after the estrous cyclicity and prevented the tamoxifen treatment. sleep/wakefulness associated HRV parameter alteration To determine the role of endogenous estrogens on the Discussion HRV parameter changes across the different sleep/wakefulness states, rats were treated with either tamoxifen (10 In the current study, we found that both the estrous cycle mg/kg 䡠 d), an estrogen receptor antagonist, or vehicle for and the sleep/wake state of the rats had a significant imfour consecutive days (Fig. 6). The estrous-cycle-associ- pact on autonomic functional regulation in these freely ated effects were offset by subtracting the HRV parame- moving rats. Furthermore, the estrous cycle, as well as the ters during diestrus. Our results demonstrate a signifi- sleep/wakefulness state associated oscillations of autocantly larger ⌬HF during both QS and PS compared with nomic functional regulation, was prevented or attenuated

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FIG. 4. The HRV parameters were changed during the different sleep/ wakefulness states. ⌬HF and ⌬LF/HF of the HRV were analyzed. The results were calculated by subtracting the values during diestrus from those during estrus (Estrus-Diestrus) in the same rat. ln, Natural logarithm. The results are expressed as mean ⫾ SEM for 30 rats/group. *, P ⬍ 0.05 vs. 0 by 95% confidence interval analysis; †, P ⬍ 0.05 vs. AW by least significant difference test.

oophorectomy in premenopausal women (14). These results have established a definite role for the ovaries in elevating vagal functioning and attenuating sympathetic functioning. Our results in freely moving rats are consistent with these findings; after OVX, there was decreased FIG. 3. The HRV parameters were significantly altered in both the different estrous-cycle stages and the different sleep/wakefulness states. The data of RR, HF, LF, and LF/HF of the HRV were analyzed over a total of 6-h AW, QS, and PS in the OVX group during the sixth recording day and the sham-operated group during second diestrus and estrus. ln, Natural logarithm. The results are expressed as mean ⫾ SEM for 30 rats/group. *, P ⬍ 0.05 vs. OVX; †, P ⬍ 0.05 vs. diestrus; #, P ⬍ 0.05 vs. AW; ‡, P ⬍ 0.05 vs. QS by least significant difference test.

by treatment with the estrogen antagonist tamoxifen, which suggests an important role for endogenous estrogens in the regulation of autonomic functions. Our results suggest that estrous-cycle-related changes in cardiac neural regulations can be mainly attributed to endogenous estrogens, and these effects are manifest more during QS than during rapid-eye-movement sleep or awake states. Previous reports have shown that postmenopausal women have weaker cardiac vagal modulation and stronger cardiac sympathetic modulation compared with agematched premenopausal women (13), and these alternations can be restored after estrogen replacement therapy (13). In addition, autonomic functions are altered after

FIG. 5. The HRV parameters in rats during estrus were different from those during diestrus and from the OVX rats. ⌬HF and ⌬LF/HF of the HRV were analyzed. The results were calculated by subtracting the values of AW from those of QS (QS-AW) (left panel) and from those of PS (PS-AW) (right panel) in the same rat. ln, Natural logarithm. The results are expressed as mean ⫾ SEM for 30 rats/group. *, P ⬍ 0.05 vs. 0 by 95% confidence interval analysis; †, P ⬍ 0.05 vs. OVX; #, P ⬍ 0.05 vs. diestrus by least significant difference test.


FIG. 6. Estrogen receptor blocker treatment prevented the sleep/wakefulness state associated alterations in HRV parameters. The estrogen receptor blocker tamoxifen (10 mg/kg 䡠 d) or vehicle was given to the rats for four consecutive days. The ⌬HF and ⌬LF/HF of the HRV were recorded during the different sleep/wakefulness states. The results are calculated by subtracting the values during diestrus from those during estrus (Estrus-Diestrus) in the same rat after vehicle or tamoxifen treatment. ln, Natural logarithm. The results are expressed as mean ⫾ SEM for six to eight rats/group. *, P ⬍ 0.05 vs. 0 by 95% confidence interval analysis; †, P ⬍ 0.05 vs. vehicle by least significant difference test.

HRV HF in all three sleep/wakefulness states, indicating decreased neural vagal functioning, and increased HRV LF/HF during QS, indicating increased sympathetic functioning. In animal studies, 17␤-estradiol has been shown to significantly elevate vagal activity or attenuated sympathetic activity in anesthetized rats (17, 18). In addition, exogenous estradiol has been demonstrated to have a suppressive effect on pressor agent-induced sympathoexcitation (44) in freely moving rats. Our results are also consistent with these findings in that rats during estrus (when there is a high level of estrogens) had higher HRV HF than both rats in diestrus (when there is a lower level of estrogens) and rats after OVX. The sleep/wakefulness state associated and estrous-cycle state associated autonomic function alterations were isolated by calculating the differences in HRV parameters between estrus and diestrus (Estrus-Diestrus), as well as between the sleep states and wakefulness (QS-AW and PS-AW), respectively. We found that the Estrus-Diestrus difference showed a significant increase in ⌬HF and a significant decrease in ⌬LF/HF in QS and PS compared AW, and these effects were abrogated by treatment with the


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FIG. 7. Estrogen receptor blocker treatment prevented the estrous cycle and related alterations in HRV parameters. Estrogen receptor blocker tamoxifen (10 mg/kg 䡠 d) or vehicle was given to the rats for four consecutive days. The ⌬HF and ⌬LF/HF of the HRV were recorded during both diestrus and estrus. The results are calculated by subtracting the values for AW from those for QS (QS-AW) (left panel) and from those for PS (PS-AW) (right panel) in the same rat after vehicle or tamoxifen treatment. ln, Natural logarithm. The results are expressed as mean ⫾ SEM for six to eight rats/group. *, P ⬍ 0.05 vs. 0 by 95% confidence interval analysis; †, P ⬍ 0.05 vs. diestrus; #, P ⬍ 0.05 vs. vehicle by least significant difference test.

estrogen receptor antagonist tamoxifen. In addition, the QS-AW difference showed a significant increase in ⌬HF and a significant decrease in ⌬LF/HF during estrus compared with both OVX group and the rats during diestrus. These differences were also abrogated by tamoxifen treatment. Our results support the findings in a human study that a high level of estrogens may increase parasympathetic activity during the follicular phase of the menstrual cycle (26). Our results also indicated a cyclic pattern for the cardiac vagal-related parameters (HF) that is associated with the estrous cycle; this cyclic HF oscillation disappeared in the OVX rats. These results suggest that the ovaries of rats also play a facilitating role in cardiac vagal regulation, which is similar to humans. Based on the fact that these cyclic alterations of autonomic function are eliminated by the estrogen receptor antagonist tamoxifen, this finding further highlights the role for endogenous estrogens rather than other ovarian hormones in these estrous-cycle-related vagal changes. Taking the above into account, we conclude that both sleep/wakefulness associated changes and estrous-cycle-related changes in cardiac neural regulations can be mainly attributed to endogenous estrogens, especially during QS in estrus, which has the strongest vagal regulation and the weakest sympathetic regulation in rats. Our results suggest that the dynamic levels of endogenous estrogens found across the various different estrous-cycle states may underlie cyclic changes in vagal and sympathetic activity and that this may con-

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tribute to the cardioprotective effects suggested by previous studies (9, 13, 45). The noninvasive HRV technique used here, a technique also extensively used in humans, has allowed us to continuously monitor autonomic functions over a long period of time (such as several estrous cycles). Because autonomic functions are very sensitive to external stimuli when animals are awake, a recording during QS will have the minimum interference from outside environment and should mainly reflect the autonomic functions from the internal visceral environment. Our recent advances in analytical techniques have permitted the quantitative study of the sleep-related autonomic functioning (24, 30, 31). This sleep-related autonomic function analysis provides a more accurate way to examine the properties of drugs that affect autonomic function. For example, the estrous-cycle-related changes in cardiac neural regulations can be best demonstrated during QS and the sympathetic suppressive effect of estrus can be only detected during QS. Our results highlight the importance of endogenous estrogens to autonomic regulation in female rats. A highly significant estrous-cycle-related vagal and sympathetic fluctuation was observed during QS, and this was demonstrated to be dependent on endogenous estrogens. The autonomic functional changes that accompany the menstrual cycle and menopause are problematic, and they profoundly impact both the life expectancy of women and their quality of life. Most of these problems can be relieved by endogenous estrogens secretion during the follicular phase or by exogenous estrogen replacement therapy. Recent reports of the adverse effects of estrogen replacement therapy have limited the application of this treatment. The effects of endogenous estrogens on autonomic functional regulation, however, are still noteworthy. Our findings provide us with one of the mechanisms underlying the cardiovascular symptoms related to menstrual cycle and menopause in women. This will benefit the development of drugs targeting autonomic regulation and hopefully lower the cardiovascular risk not only of women but also of men.

Acknowledgments We thank Ms. Ying-Hua Huang for her administrative work on manuscript production. Address all correspondence and requests for reprints to: Cheryl C. H. Yang, Sleep Research Center and Institute of Brain Science, National Yang-Ming University, No. 155, Section 2, Linong Street, Taipei 11221, Taiwan. E-mail: [email protected]. This work was supported by the Ministry of Education, Aim for the Top University Plan Grant YM-97A-C-P506, the Na-


tional Science Council (Taiwan) Grant NSC-95-2320-B-010069, and the Taipei City Hospital Grant 96002-62-053. Disclosure Summary: The authors have nothing to disclose.

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