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INTRODUCTION

SLEEP AND THE DEPRIVATION OF SLEEP ARE BECOM-ING IMPORTANT ISSUES IN EVERYDAY LIFE. SLEEP DE-PRIVATION HAS CONSEQUENCES FOR PUBLIC safety and health. Some obvious problems concern attention, vigilance, and fatigue,1 but less obvious ones are cardiovascular problems and obesity.2 Sleep deprivation is known to cause molecular, physi-ological, and behavioral changes. It changes the expression of im-mediate early genes, transcription factors, and genes related to energy metabolism in the brain3 and influences leptin and grehlin levels causing increased hunger and high calorie food cravings.4 Also the functioning of the brain, measured by magnetic reso-nance imaging5 or electroencephalogram (EEG) recordings6, is changed after sleep deprivation.

Sleep wake cycles are regulated by the interplay of a homeo-static mechanism, which regulates sleep depth, and the circadian pacemaker of the suprachiasmatic nucleus (SCN), which regu-lates timing of sleep.7 Deprivation of sleep has major effects on the homeostatic sleep regulatory mechanisms but is assumed to have minor or no effects on the circadian pacemaker. The effects on sleep homeostasis explain why animals, ranging from insects8 to mammals9 sleep more deeply after sleep deprivation, and sleep occurs outside the normal rest period after sleep deprivation.10,11 In humans, sleepiness is increased during the day following sleep

deprivation.12 The timing of sleep outside the rest phase is surpris-ing given the assumption that the circadian pacemaker is largely unaffected by sleep deprivation. The findings can be explained in 2 ways; either the homeostatic mechanism overrules the circadian clock, or the functioning of the circadian pacemaker is altered by sleep deprivation. We investigated the latter option by recording SCN electrical activity patterns during and after a 6-h sleep de-privation period, and we observed a long term suppression of the electrical activity rhythm of the SCN.

METHODS

All experiments were performed under the approval of the Animal Experiments Ethical Committee of the Leiden Univer-sity Medical Center. Male Wistar rats (n=6), 300 grams at time of surgery, were implanted under deep anaesthesia with electrodes (stainless steel, diameter 0.125 mm; Plastic One) to record SCN neuronal activity. One electrode was aimed at the SCN while the other electrode, with the insulation completely removed, was placed in the cortex for reference. For the EEG recordings, elec-trodes (screw electrodes, Plastic Ones) were screwed through the skull on the dura over the right parietal cortex and the cerebel-lum. For the EMG recordings, 2 wires with suture patches (Plas-tic Ones) were inserted between the skin and the neck muscle tissue.

In vivo SCN neuronal activity, EEG, and EMG recording tech-niques were as described previously.13,14 Briefly, SCN neuronal activity was recorded online (amplification factor ~ 50,000, band pass filtered between 500-5,000 Hz, -40 dB/decade). A window discriminator converted the recorded action potentials to electron-ic pulses. A second window discriminator was set at a higher level to be able to exclude artifacts caused by the animal’s movements. The EEG and EMG were continuously recorded and amplified (amplification factor ~2000), band-pass filtered (between 0.5-30 Hz, -40 dB/decade), and subjected to analog-to-digital conversion (sampling rate 100 Hz). All data were recorded simultaneously in

Long Term Effects of Sleep Deprivation on The Mammalian Circadian PacemakerTom Deboer PhD1; László Détári PhD2; Johanna H. Meijer PhD1

1Laboratory for Neurophysiology, Department of Molecular Cell Biology, Leiden University Medical Center, Leiden, The Netherlands; 2Department of Physiology and Neurobiology, Eötvös Loránd University, Budapest, Hungary

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Disclosure StatementThis is not an industry supported study. Drs. Deboer, Détári, and Meijer have indicated no conflicts of interest.

Submitted for publication July 19, 2006Accepted for publication November 1, 2006Address correspondence to: T. de Boer, PhD, Laboratory for Neurophysiol-ogy, Department of Molecular Cell Biology, LUMC S-05-P, PO Box 9600, 2300 RC Leiden, The Netherlands, E-Mail: Tom.de_Boer@lumc.nl

Study Objectives: In mammals, sleep is controlled by a homeostatic pro-cess, which regulates depth of sleep, and by the circadian clock of the suprachiasmatic nucleus (SCN), which regulates 24-h rhythms in timing of sleep. Sleep deprivation is known to cause molecular and physiological changes and results in an alteration in the timing of sleep. It is gener-ally assumed that following sleep deprivation, homeostatic mechanisms overrule the circadian clock, allowing animals to sleep during their active phase. However, recent evidence indicates that sleep states have direct access to the circadian pacemaker of the SCN. We questioned therefore whether sleep deprivation may have long-term effects on the circadian pacemaker, which may explain altered sleep patterns following sleep de-privation. Design: To test this hypothesis, we combined SCN recordings of electri-cal impulse frequency through stationary implanted electrodes in freely moving rats with electroencephalogram recordings in the same animal

before, during, and after a mild 6-h sleep deprivation. Measurements and Results: Following sleep deprivation, SCN neuronal activity was significantly reduced to about 60% of baseline levels. The decrements in SCN activity were most obvious during NREM sleep and REM sleep and lasted for 6-7 hours. Conclusions: The data show that sleep deprivation influences not only sleep homeostatic mechanisms, but also SCN electrical activity, resulting in a strong reduction in circadian amplitude in the major output signal from the SCN. Keywords: Sleep deprivation, circadian rhythms, suprachiasmatic nucle-us, electroencephalogram, electrophysiology, sleep regulationCitation: Deboer T; Détári L; Meijer JH. Long term effects of sleep depri-vation on the mammalian circadian pacemaker. SLEEP 2007;30(3):257-262.

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10-s epochs and stored on a computer hard disk.The animals were connected to the recording system by a flexi-

ble cable and a counterbalanced swivel system, and then remained on the cable for at least one week (7-15 days) before the start of the recording. During that time and during the experimental recordings, the animals were maintained in continuous darkness to exclude the excitatory influence of light on SCN neuronal ac-tivity.13 Drinking rhythms were continuously recorded and an es-timate of circadian phase was obtained by visual inspection of drinking onset. Under constant conditions, a 24-h baseline day was recorded. Subsequently the animals were sleep deprived for 6 h, starting at rest onset (CT 0), followed by 18 (n=6) to 30 h (n=5) of recovery.

During the 6-h sleep deprivation, the animals were observed with an infrared camera in addition to the online EEG record-ing. Whenever the animals appeared drowsy or the EEG exhib-ited slow waves, they were mildly disturbed by moderate noise, by the experimentator entering the room, and, if necessary, by introducing fresh food, water, or nesting material into the cage.

The animals were never touched or disturbed during feeding and drinking.

At the end of the experiments the animals were sacrificed to verify the SCN recording sites. To mark the electrode position, a current was passed and the brain was perfused with a potassium ferrocyanide containing solution, resulting in a blue spot at the electrode tip. Recording sites were found in the dorsal SCN (n = 2), in the ventral SCN (n=3), and in the lateral SCN (n=1).

Offline EEG power density spectra were calculated in 10-s ep-ochs corresponding to the 10-s epochs of SCN neuronal activity, with a Fast Fourier Transform routine within the frequency range of 0.25-25.0 Hz in 0.1 Hz bins. EMG signals were integrated over 10-s epochs. Three vigilance states, waking, NREM sleep, and REM sleep, were determined on the basis of standardized EEG and EMG criteria for rats.14,15 Epochs containing artifacts in SCN neuronal activity or in the EEG signal were excluded from analy-sis.

All SCN neuronal activity and EEG power density data were standardized relative to the mean 24-h baseline value in NREM sleep. This enabled calculation of mean values for all animals. To analyse changes in EEG slow wave activity (SWA; mean EEG power density between 1-4 Hz) and changes in SCN neuronal activity at transitions from NREM sleep to REM sleep, 4-minute intervals free of artifacts were selected by criteria published pre-viously.14,16,17

RESULTS

In all baseline recordings, SCN neuronal activity values were high during the subjective day and low (50% of the daytime activ-ity peak) during the subjective night (Figures 1 and 2). Variability in SCN neuronal activity increased in the dark period, which may be attributable to a decrease in sleep consolidation. During sleep deprivation, electrical activity decreased to 87% of baseline. Fol-lowing the 6-h sleep deprivation, a significant reduction in SCN electrical activity was observed, which lasted 7 hours (Figure 2). During this period, the electrical activity of the SCN decreased to about 65% of the baseline values (Figure 3; P<0.05). We ana-lyzed the changes in SCN electrical activity separately for NREM sleep, REM sleep, and waking. The largest suppression of SCN neuronal activity was observed in NREM sleep (Figure 3), where electrical activity decreased to 62.8% of baseline (P<0.05) in the first 6 h after sleep deprivation. During REM sleep, activity decreased to 72.8% of baseline levels (P<0.05). During waking,

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Table 1—Amount of vigilance states in 6-h values

Circadian Waking NREM sleep REM sleep time % (SE) % (SE) % (SE)Baseline 1-6 h 31.2 (3.2) 56.9 (2.8) 11.9 (1.5) 7-12 h 25.2 (1.6) 60.2 (1.7) 14.5 (0.9) 13-18 h 72.0 (5.5) 24.6 (4.2) 3.4 (1.3) 19-24 h 65.1 (5.5) 30.8 (4.7) 4.0 (1.2)Deprivation 1-6 h 94.0 (0.9) 6.0 (0.9) 0.0 (0.0)Recovery 7-12 h 15.5 (3.2)b 66.3 (2.8)a 18.1 (1.5)b 13-18 h 52.3 (3.8)b 39.7 (3.3)b 8.1 (0.6)b 19-24 h 60.4 (3.7) 34.9 (3.7) 4.8 (0.6)

Significant differences between corresponding intervals of baseline and recovery are indicated by a(P<0.05) and b(P<0.01, two-tailed paired t-test after significant ANOVA factor ‘day’).

Figure 1—Top. A 48-h record of suprachiasmatic nuclei (SCN) neuronal activity, slow wave activity (SWA, mean electroencephalogram power density between 1-4 Hz) and vigilance states (W, waking; N, NREM sleep; R, REM sleep) of an individual animal. The first 24 h is the baseline recording starting at rest onset, followed by 6-h sleep deprivation (SD) and 18-h recovery. Each data point is the mean of thirty 10-s epochs (5 min). SWA and SCN neuronal activity are plotted as percentage of the mean activity during NREM sleep over the first 24 h. Data during sleep deprivation are depicted in white, and a vertical line at CT 6 is drawn in the baseline day corresponding to the time of the end of sleep deprivation in the experimental day. Note the decrease in electrical activ-ity between CT 6-12 on the experimental day compared with the baseline control day. Bottom. A representative example of oscilloscope traces with multiunit activity recorded during NREM sleep.

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SCN electrical activity decreased to 85.3% of baseline, but these values did not reach significance.

The reduction in SCN electrical activity was paralleled by an increase in NREM sleep SWA, visible in the 1-h values (Figure 2), and in the first 6 hours after sleep deprivation, when SWA reached 176.7 % (SE 11.3) above corresponding baseline level (P<0.0001). NREM and REM sleep were increased above base-line for several 1-h intervals throughout the first 15-h of recovery after sleep deprivation (Figure 2). The increase in NREM sleep was mainly visible during the subjective night, whereas REM sleep was increased above baseline during the subjective day as well as the subjective night (Figure 2). The 1st and 2nd 6-h interval

after sleep deprivation showed significant increments in NREM and REM sleep, compared with baseline levels (Table 1).

During the baseline recording, SCN neuronal activity was highest during waking and REM sleep; it differed significantly from neuronal activity during NREM sleep within each 6-h time interval of the circadian cycle (P<0.05 after significant ANOVA). The difference in SCN activity between NREM sleep and REM sleep remained intact after sleep deprivation despite the general reduction in SCN neuronal activity. This was clearly visible at the transition between NREM sleep and REM sleep (Fig. 4). On the 2nd day following sleep deprivation the levels of SCN electrical activity and SWA in NREM sleep were back to baseline; vigi-lance states were also back to baseline (Figure. 2).

DISCUSSION

A well-described response after sleep deprivation is an in-crease in the activity of the slow waves in the NREM sleep EEG. This response is visible in the present data and has been observed in all mammalian species tested so far. We also found a slight increase in NREM sleep and REM sleep in the first 12 hours fol-lowing sleep deprivation. The increase in SWA, and the changes in NREM sleep and REM sleep correspond well with those pre-viously reported in response to a 6-h sleep deprivation period in the rat.18,19

Elaborate studies in, among others, rats, hamsters, and humans, showed that EEG SWA in NREM sleep increases as a function of prior waking duration.20-25 These studies confirmed the notion that SWA in NREM sleep reflects a homeostatic process that keeps track of sleep need. We recently showed that spontaneous altera-tions between REM and NREM sleep during undisturbed sleep are paralleled by changes in SCN electrical activity and that SCN

Figure 2—Time course over 60 h of NREM sleep, REM sleep, SWA, and SCN neuronal activity (mean ± SEM, n=6, after hour 48 n=5) in NREM sleep across the baseline day and after 6-h sleep deprivation. SWA and SCN neuronal activity are plotted as percentage of the mean activity during NREM sleep over the first 24 h. The baseline area of the standard errors is redrawn in gray during sleep deprivation and recov-ery. The subjective day and subjective night are indicated by white and black bars (top). Stars indicate for all variables where recovery differed significantly from baseline (P<0.05, paired t-test after significant ANOVA).

Figure 3—SCN neuronal activity after sleep deprivation during waking NREM sleep and REM sleep. The bars rep-resent the mean difference in percentage from corresponding 6-h baseline intervals. Significant difference from baseline are indicated by stars (P<0.05, paired t-test after significant ANOVA).

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neuronal activity showed a negative correlation with EEG SWA.14 SCN electrical activity is elevated during REM sleep and sup-pressed during NREM sleep.14 The present results show that, at a larger time scale, the electrical activity in the SCN, one of the direct outputs of the circadian clock, is suppressed for periods up to 6-7 hours following a 6-h sleep deprivation period. This indi-cates that total sleep deprivation influences the electrical output of the circadian clock during subsequent recovery sleep. We believe that this finding is of importance for mathematical simulations of the effects of sleep deprivation. Mathematical models predicting sleepiness and alertness on the basis of homeostatic and circadian regulation of sleep assume that sleep deprivation affects homeo-static but not circadian regulation of sleep.26,27 Our results suggest that sleep deprivation influences not only sleep homeostatic re-sponses, but also changes circadian regulation.

Neuronal activity in the SCN was decreased after sleep depri-vation during both sleep states to 60%-70% of baseline. These effects are substantial, since the trough of SCN neuronal activ-ity during the night is approximately 50% of daytime peak ac-tivity. During waking, electrical activity decreased as well, but these values did not reach significance. The lack of significance was not attributable to a larger variability (Fig. 3), and seems to be caused by a reduced suppression during waking, suggesting vigilance state specific effects of sleep deprivation on subsequent SCN neuronal activity.

The decrease in SCN neuronal activity for periods of about 6 h paralleled the time course of the increase in SWA during NREM sleep. In the cortical EEG, effects of sleep deprivation are most apparent during NREM sleep and less visible during REM sleep

or waking.7 In contrast, in the SCN, sleep deprivation altered neu-ronal activity both during NREM and REM sleep. The decrease in electrical activity during REM sleep was significant for the first 6-h period following sleep deprivation. Over the next 6-h period (6-12 hours following sleep deprivation), SCN activity during REM sleep was suppressed to 80% of baseline, but was not significantly different from it. The results show that effects of sleep deprivation on SCN neuronal activity exist also outside NREM sleep, indicating that neuronal activity in deeper brain ar-eas can show clear effects of sleep loss when the cortical EEG does not.

While the general level of neuronal activity is lowered after sleep deprivation, the difference in SCN neuronal activity be-tween NREM and REM sleep is intact. This indicates that the effects of sleep deprivation and the effects of vigilance state tran-sitions on SCN neuronal activity may be additive. Different brain areas involved in vigilance state regulation have connections with the SCN. The nucleus basalis, the pedunculopontine tegmental (PPT) nucleus, and the laterodorsal tegmental (LDT) nucleus are involved in REM sleep regulation28 and provide cholinergic input to the SCN.29 Other potential pathways are serotonergic projec-tions from the raphe dorsalis,30 an area involved in NREM-REM sleep cycling.28 It is unlikely that short lasting total sleep depriva-tion affects the cholinergic system, as chronic sleep deprivation for 10 days results in only small changes in acetylcholine receptor binding.31 In contrast, brain serotonin turnover in hamsters32 and rats33 is increased after 4- or 6-h sleep deprivation, respectively. It is possible, therefore, that changes in serotonergic activity under-lie the observed changes after sleep deprivation.

Figure 4—Time course of SWA and SCN neuronal activity at the transition from NREM to REM sleep in the 2 min before and after the transition. The curves connect 10-s mean values calculated over the first 6-h after the end of the sleep deprivation (recovery curve) and the corresponding baseline period, and are plotted as percentage of the mean activity during NREM sleep over the first 24 h. Lines above the abscissa indicate where recovery and baseline data differ significantly (P<0.05, paired t-test after significant ANOVA). Note that SWA is significantly higher after sleep deprivation during NREM sleep, but reaches baseline level during REM sleep (left panel), whereas SCN neuronal activity is lower during recovery both during NREM sleep and REM sleep, although the increase from NREM to REM sleep is still intact (right panel).

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Hypocretin is a potential regulator of sleep and wakefulness; it shows high levels during the day and low levels during the night in rats.34,35 The circadian regulation of hypocretin is critically de-pendent on the integrity of the SCN, as SCN lesioned animals show a complete absence of fluctuating hypocretin levels.35,36 Hypocretin levels are roughly in antiphase to SCN electrical ac-tivity rhythms; they are low during the resting phase and high during the active phase in the nocturnal rat. Hypocretin increases in response to sleep deprivation34,35 which may be required to con-solidate wakefulness.35 Our results show that sleep deprivation induces a decrease in electrical activity, and confirm that SCN electrical activity and hypocretin levels are mirror images in the rat, under both perturbed and unperturbed situations.

In summary, our results indicate that an important marker of the circadian clock is influenced by sleep loss. This influence was significant during both sleep states, causing a general reduction in neuronal activity during sleep after sleep deprivation. The data show an influence of sleep loss on the pattern of the electrical rhythm of the SCN, which is a primary output signal regulating circadian rhythms in sleepiness and alertness.

ACKNOWLEDGEMENT

The study was supported by the European Union (Grant LSHM-CT-2005-518189) and NWO (Grant 425-204-02).

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