Evidence for a Circadian Distribution of Eye Movement Density During REM Sleep in Humans

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1 Sleep Research Online 4(2): 59-66, Printed in the USA. All rights reserved WebSciences Evidence for a Circadian Distribution of Eye Movement Density During REM Sleep in Humans Clemens Witzenhausen, Frederik W. Bes and Hartmut Schulz D e p a rtment of Neuro l o g y, HELIOS Klinikum Erfurt, Erfurt, Germany M e d c a re Automation, A m s t e rdam, The Netherlands The aim of the present study was to assess the circadian variation of rapid eye movement density (REMD) in night sleep and daytime naps of young males. Daytime naps were scheduled in 12 healthy young males at 2-h intervals between 0800 and 2400 h. Each subject performed nine naps which were scheduled in randomized order across nine nonconsecutive days. A double-nap strategy was applied with 30 min of sleep in Nap A and one complete REM sleep episode in Nap B. Naps A and B were separated by a 10-min break. All night sleep recordings were performed for each subject at the beginning and the end of the series of naps. REM density was calculated for REM sleep episodes in Nap B and for each REM episode of the two nights. REM density was defined as the ratio of the number of 3-sec epochs of REM sleep with at least one rapid eye movement and the total number of all 3-sec epochs with REM sleep. REM density showed a steady decrease from morning to late evening. During night sleep, REM density increased from the first to the second and last third of the night. REM density paralleled the circadian distribution of tonic REM sleep parameters, which is roughly inverse to that of body temperature. These results show that not only tonic but also phasic parameters of REM sleep display a circadian distribution. CURRENT CLAIM: Rapid eye movements, which are the most prominent phasic event of REM sleep, display a circadian distribution which runs in parallel with the time course of tonic REM sleep parameters and is roughly inverse to the rhythm of body temperature. The rapid eye movements are a major phasic event of REM sleep. The ratio of rapid eye movements per time in REM sleep is called REM density (REMD). While the tonic REM sleep parameters, such as REM sleep duration and REM latency, display a circadian distribution, roughly inverse to that of deep body temperature (Czeisler et al., 1980; Zulley, 1980), the temporal distribution of phasic REM events, like REMD has not been studied systematically. Aserinsky (1969, 1971) was the first to suggest that REMD might be an index of sleep need or satiety. During prolonged periods of sleep, REMD increased in the first 10 h after sleep initiation and remained at a high plateau in the extended sleep condition. REMD was roughly related to the accumulated sleep duration. Later Borbély and Wirz-Justice (1982), Lucidi et al. (1996), and recently De Gennaro et al. (2000) assumed an inverse relationship between REMD and the amount of slow wave sleep. The hypothesis by Borbély and coworkers was based on their two-process model of sleep regulation. These authors tried to explain typical variations of REM sleep parameters of depressed patients, like increased REMD of the first REM sleep episode, as a consequence of a dampened and altered time course of slow wave sleep (SWS) (Borbély and Wirz-Justice, 1982). Lucidi et al. (1996) chose a sleep curtailment paradigm to evaluate the relationship of REMD and the amount of previous slow wave sleep. Following a gradual sleep restriction, obtained by postponing sleep onset time while maintaining final awakening time constant, REMD in the ensuing recovery nights decreased linearly with the amount of prior sleep curtailment. Feinberg and coworkers (Feinberg et al., 1987, 1988) found, in the recovery nights after complete and partial sleep deprivation, a reduction of REMD in the second and third REM episode in comparison with baseline values. Thus, they supposed an inverse relationship between sleep depth, measured as slow wave activity, and REM density. The increase of REMD across the course of the night (Benoit et al., 1974; Schneider, 1978) may also support the notion of a negative relationship between REMD and sleep depth. In contrast to these hypotheses, which assume that the regulation of REMD is sleep dependent, Kobayashi et al. (1980) concluded, from a study with scheduled naps after a night of total sleep deprivation, that REMD follows a circadian distribution which parallels that of the tonic REM sleep parameters. To our best knowledge, no other data about the temporal distribution of REMD across the day-night cycle have been published. To test the hypothesis of a circadian distribution of REMD, we examined its 24-h distribution in systematically scheduled daytime naps and in night sleep. The data stem from a previously published study on the diurnal variation of sleep propensity (Bes et al., 1996). METHODS Subjects Twelve clinically healthy male volunteers without sleep complaints, mean age 26.8±3.2 years, participated in the double nap study. Their sleep habits and the absence of sleep disorders were established by a clinical interview and by sleep questionnaires (Horne and Östberg, 1976; Görtelmeyer, 1986). Correspondence: Dr. Clemens Witzenhausen, Department of Neurology, HELIOS Klinikum Erfurt GmbH, Nordhäuserstr. 74, Erfurt, Germany, Tel: , Fax: , Clemens.Witzenhausen@t-online.de.

2 6 0 WITZENHAUSEN ET A L. None of the subjects napped habitually nor were there extreme morning or evening types. The subjects were asked to keep a regular sleep schedule for the entire experimental period, and to be in bed from 2400 to 0700 h on nights preceding a nap in the lab. This was controlled by actigraphy for the 24 h preceding each nap sleep recording. The ingestion of any drugs was forbidden throughout the study. Consumption of alcohol and coffee was not allowed on the days of nap or night sleep recordings. Design Each subject napped with polysomnographic recordings at nine different day times. Additionally, a full night sleep recording was performed at the beginning and at the end of the study period. The experimental naps were randomly allocated and equidistantly distributed at 2-h intervals between 0800 and 2400 h. On each experimental day only one nap per person was recorded, thus each subject came to the lab on nine different occasions at weekly intervals. The subjects had to be in the lab three hours before the start of a nap, in order to adapt to the situation. For this reason the subjects spent the night preceding the naps, starting at 0800 and at 1000 h in the lab. All other nights preceding naps were spent at home. Nap sleep was recorded in a double-nap format. Each nap was divided into two parts, A and B, with a deliberate break of 10 min of wakefulness between Naps A and B (Figure 1). The reason for the double-nap procedure was to allow the release of slow wave sleep mainly in Nap A, and thus give REM sleep a greater chance to occur at any time after the resumption of sleep in Nap B. In part A of the double nap, sleep was restricted to 30 min after sleep onset (defined as the first 30-sec epoch of Stage 2 sleep). If a subject did not fall asleep within 120 min after lights-out, the session was ended and the data were declared as missing. At the end of the 30-min sleep episode, subjects were awakened and performed two short cognitive tests (visualization and number facility [Moran and Mefferd, 1959]) while they were out of bed. Immediately after the break Nap B started. It should contain one complete REM episode and by consequence, its length was variable. The recording was finished 15 min after the last 30-sec epoch of REM sleep. If REM sleep or any other sleep stage did not occur within two hours after the beginning of Nap B, the session was ended. REM sleep parameters, including REMD, were determined from polygraphic recordings during Nap B. Polygraphic Recordings EEG (C3-A2 and C4-A1), EOG, mental and submental EMG, and rectal temperature were recorded continuously during all sessions. A 70 Hz low pass and 0.3 Hz high pass filter were used for the EEG recordings. Sleep biosignals were recorded with a 16-channel mingograph (Siemens-Elema) on paper for manual sleep scoring in 30-sec epochs according to the standard criteria by Rechtschaffen and Kales (1968). A d d i t i o n a l l y, data were stored in digital form (sampling frequency 200 Hz) on optical disc for later analysis. For the recording of eye movements in the horizontal and vertical plane, gold electrodes were fixed bilaterally at the outer canthi of both eyes and above and below one eye. Horizontal eye movements were recorded by the common mode rejection technique (Hord, 1975). The electrodes for vertical eye movements were fixed one centimeter above and below the ramus supra- and infraorbitalis of one eye. The gain was 200 µv/10 mm; time constant 5 sec; paper speed 10 mm/sec. Before each recording session a biological calibration procedure was performed. The subject moved his eyes, according to the instructions of the lab assistant, to the right, left, above and below. In parallel the amplitude on paper was calibrated. Eye Movement Analysis REMD was analyzed visually and documented together with sleep stage scores (Rechtschaffen and Kales, 1968). Double Nap Test Nap A Nap B 30 Sleep 10 Sleep of Variable Length Lights-out Awake Awakening Figure 1: Design of the Double-nap Procedure. Sleep in Nap A was terminated 30 min after sleep onset, which was defined as the first epoch of sleep. After forced awakening subjects were out of bed for 10 min and performed two short cognitive tests (addition and visualization test). Nap B, which was of variable duration, was terminated 15 min after the end of the first REM sleep period. If a subject did not fall asleep or had no REM sleep in part B, the trial ended 120 min after lights-out. The REM density was calculated from REM sleep in Nap B.

3 CIRCADIAN DISTRIBUTION OF REM DENSITY 61 REMD was calculated as the ratio of the number of 3-sec epochs containing at least one rapid eye movement and the total number of all 3-sec epochs of REM sleep. REMD thus varied between zero (no 3-sec mini epoch) and ten (all ten 3- sec mini epochs with at least one rapid eye movement). There is a high correlation between the present definition of REMD and the total number of REMs. In an unpublished doctoral dissertation from our laboratory, Trojan (1980) has checked the relationship between visually scored REMD in 3- sec epochs and the number of visually scored single rapid eye movements for one subject with 14 consecutive recording nights. The correlation between the two measures of REMD was r=0.87 for the first, r=0.89 for the second, and r=0.91 for the third REM episode of a night. While the correlation is linear in the lower and middle frequency range, there is a ceiling effect with an underestimation of the eye movement density in the upper frequency range by the 3-sec interval scoring method. Hauri and Hawkins (1971) also found a high correlation between REMD calculated as "eye movement index" in 2.5-sec epochs and the total number of REMs. F i n a l l y, Mc Partland et al. (1978) described a positive correlation between visually scored REM density measures and computer-derived density measures of REM frequency (r=0.80) and percent phasic activity (r=0.81). Because not all subjects entered REM sleep in all naps (Table 1), REMD scored data were computed for 6-h blocks, lumping together data from three consecutive nap sessions at daytime. As a result, REMD scores were computed for the intervals h, h, and h. To adjust for the appreciable interindividual differences in REMD, the absolute REMD scores were transformed into relative REMD scores for statistical analysis. This was done by expressing REMD of a given 6-h block as the percent deviation from the individual daily mean of REMD (Table 2). For nighttime data only nights with at least one REM episode in each third of the night were included. With this restriction REMD of night sleep could be calculated in five subjects on one night and in another five subjects for two nights. Two subjects were excluded, having thirds of the night without REM sleep in both nights. For night sleep, REMD was expressed as the percentage deviation of the three nighttime Table 1 REM Density (%) of the 12 Subjects in REM Episodes of Nap B of the Double-nap Procedure at Daytime Time Subject =no sleep in Nap A; =subject not fallen asleep in Nap B; =subject without REM sleep in Nap B Time Subject Median Table 2 Relative REM Density (%) at Daytime h h h Time Subject Median Table 3 Relative REM Density (%) During Nighttime h values of 1 night; values from 2 nights h h

4 6 2 WITZENHAUSEN ET A L. REMD scores from the individual mean REMD (Table 3). Finally, the 24-h distribution of REMD was computed by combining nap sleep and night sleep data. The resulting five REM density values in the course of 24 h were again expressed as relative values in relation to the mean (Figure 4). Statistical Analysis The overall variation of REMD across time was tested nonparametrically with the Friedman and the Wilcoxon test. To control for any dependence of REMD from REM duration, Spearman rank order correlations were computed for each time block. For statistical analysis the SYSTAT program package, version MS Windows 7.0, was used. RESULTS Daytime Naps The tendency to enter REM sleep varied systematically between 0800 and 2400 h. While all 12 subjects entered REM sleep at 0800 h and 11 subjects at 2400 h, this was the case for only four subjects at 1800 and 2000 h (Table 1). Since one subject (05) entered REM sleep only three times, namely at 0800, 1000 and 2400 h, the computation of the time course of REMD across the day was based on 11 subjects only. During daytime the relative REMD showed a steady decrease from morning to late evening. In the morning sessions the relative REMD reached its highest values. In comparison with the afternoon ( h) and the evening ( h), there was a decrease in nine and an increase in two subjects during the daytime (Table 2). The decrease of REMD from the morning (mean relative REMD=115.3%) to the afternoon (mean relative REMD=92.5%) and to the evening (mean relative REMD=86.4%) was statistically significant (p<0.05, Wilcoxon-Test; Figure 2), while the difference of REMD between the afternoon and the evening hours was not significant. Nighttime Data Ten out of 12 subjects had enough REM sleep episodes to compute REMD scores for thirds of the night. Subjects 01 and 03 could not be included since they had only two REM sleep episodes each. The median of the relative REMD increased steadily during the nighttime (Table 3, Figure 3). The rise from the first (median REMD=73.7%) to the second (median REMD=110.5%) and from the first to the third time block (median REMD=121.1%) was statistically significant (p<0.05, Wilcoxon-Test) Time Blocks (hours) Figure 2: Time Course of Relative REM Density (%) in Nap Sleep. Naps were grouped in 4-h blocks. Double naps which started either at 0800 h, 1000 h or 1200 h were comprised in block h, naps which started either at 1400 h, 1600 h or 1800 h were comprised in block h, and naps which started either at 2000 h, 2200 h or 2400 h were comprised in block h. Individual data are shown for 11 subjects with REM sleep in each time block. The medians are connected by a red line. p<0.05; p<0.01.

5 CIRCADIAN DISTRIBUTION OF REM DENSITY Time Blocks (hours) Figure 3: Time Course of Relative REM Density (%) in Night Sleep. REM density was computed for time blocks of 2-h durations between 2400 and 0600 h. Individual data are shown for ten subjects with REM sleep in each time block. The medians are connected by a red line. p<0.05; p< Time Blocks (hours) Figure 4: Relative REM Density (%) Across 24 Hours. Data from scheduled naps and night sleep were combined for this graph. For a detailed description of the 4-h blocks of nap sleep see legend of Figure 2. Individual data are shown for 9 subjects with REM sleep in each time block. The medians are connected by a red line. p<0.05; p<0.01.

6 6 4 WITZENHAUSEN ET A L SOREM 83% SOREM 20% SOREM 0% Time Blocks (hours) 35.5 Figure 5: Variation of Median REM Duration (min) and Mean Body Temperature ( C) During 24 Hours for 9 out of 12 Subjects (missing REM sleep data for subject 1, 3 and 5). The percentage of sleep onset REM episodes (SOREM, defined by REM latencies <25 min) is given for the three daytime blocks on the bottom of the Figure. 24-hour Distribution In order to have time blocks of similar duration for the 24-h distribution of REMD, the night was divided in two parts. Thus, four time blocks of six hours each were formed. Subjects 01, 03, and 05 could not be included into this analysis since they had missing REMD scores in one or more time blocks. Figure 4 shows, for the remaining subjects, a clear circadian distribution of REMD. From morning to afternoon the median of the relative REMD decreased from 104.9% to 77.8%, while it increased from 81.7% in the evening to 99.7% in the first half of the night, and to 135.1% in the second half of the night. Statistically significant changes are shown in Figure 4. Body Temperature The data for deep body temperature from the nine naps were combined with those from both all-night recordings, to estimate the average time course across 24 h. Means were calculated across the measures which were available at a given time point, and this was repeated across 24 h for time points 15 min apart. The time course of body temperature displays a circadian variation, with a minimum of 36.27±0.08 C at 0545 h and a maximum of 36.92±0.07 C at 1830 h (circadian range 0.8 C). REM sleep duration and the percentage of sleep onset REM episodes (REM latency <20 min) decreased continuously between 0800 and 2000 h and increased thereafter. The time course of REM sleep duration was inversely related to the one of body temperature. The variations of REM sleep duration, SOREM and body temperature are shown in Figure 5. Correlation of REM Sleep Duration and REM Density In order to examine the dependency of REMD from REM sleep duration, Spearman rank order coefficients were calculated for each REM sleep episode (Table 4). For the total of 77 REM episodes, which occurred during daytime naps, no statistical significant correlation between REM sleep duration and REMD was found (r s =0,36, p 0.05). Correlations for the nine single time points of daytime naps were mostly positive, but only those at 1400 and 2400 h were statistically significant Table 4 Spearman Rank Order Correlations (r s ) of REM Density and REM Duration During Daytime Nap Sessions Time of Nap Number of REM Episodes r s p-value < <0.01 =not significant

7 CIRCADIAN DISTRIBUTION OF REM DENSITY 65 (p 0.05). For night sleep the correlations between REM duration and REMD were not significant, neither for the total of all 45 REM sleep episodes (r s =0.01, p 0.05), nor for thirds of the night with 15 REM sleep episodes each (1 st third: r s = -0.05, 2 nd third: r s = -0.47, 3 rd third: r s = -0.43, p 0.05). DISCUSSION The combined data from daytime naps and night sleep show a significant circadian modulation of REMD. This finding is consistent with the Kobayashi et al. (1980) finding from scheduled daytime naps after a night of total sleep deprivation. Kobayashi et al. (1980) observed the highest REMD in the morning Nap And a decrease of REMD in the afternoon and evening naps. However, their experimental design did not allow them to decide whether the decrease of REMD across the day resulted from an increased duration of prior wakefulness or represented a circadian distribution of REMD. Although the present data support the assumption of a circadian distribution of REMD, the duration of prior wakefulness cannot be ruled out as a critical factor. A fully satisfying separation of the two factors, (a) circadian (or time of day), and (b) prior wakefulness, has to await the analysis of REMD data from experiments with forced desynchronization (Wyatt et al., 1999). In the Kobayashi et al. (1980) study, as in the present study, the circadian variation of REMD run in parallel with tonic REM sleep parameters. In the present study the peak of REMD occurred around the nadir of the curve of body temperature, while REMD had its lowest level near the maximum of the body temperature curve. The assumption of a circadian distribution of REMD is not in contradiction with the results of previous studies on the intrasleep regulation of REMD. Changes of REMD after sleep deprivation, extended sleep, or in normal night sleep were interpreted as the expression of a sleep dependent process in the regulation of REMD (Aserinsky, 1969, 1971; Feinberg et al., 1987, 1988; Barbato et al., 1994; Lucidi et al., 1996). Based on two studies with prolonged periods of sleep during 30 and 54 h of continuous bed rest (Aserinsky, 1969, 1971), Aserinsky suggested that REMD might be an index of sleep satiety. Because of the widely scattered distribution of REM sleep, the author formed time intervals with varying duration between 2.5 and 12 h. In the 54-h bed rest study only five out of 11 subjects had at least one REM episode in the 12 time blocks. Nevertheless one REMD value for the whole group was calculated for each time block. In the 30-h bed rest study, the number and time course of REM sleep episodes was not presented for individual subjects. In regard to these analytical and statistical limitations, the results have to be interpreted with appropriate caution. Feinberg and coworkers observed a decrease of REMD in the second and third REM sleep episode during recovery sleep after one night of total sleep deprivation (TSD) in comparison to baseline sleep (Feinberg et al., 1987). A similar but less pronounced decrease of REMD occurred after a night with partial sleep deprivation (PSD) (Feinberg et al., 1988; Travis et al., 1991). In the TSD experiment REMD increased in the baseline night and the recovery night after TSD, but this increase was smaller for the recovery night (cf. Table 1 of Feinberg et al., 1987). It is possible that the amplitude of the diurnal time course of REMD, with lowest values at the beginning of the night, is dampened by sleep deprivation. The PSD studies with 3-4 h and 100 min night sleep only, showed no increase of REM density in the course of the night, neither at baseline nor in recovery sleep (Feinberg et al., 1988; Travis et al., 1991). Perhaps the unusual course of REMD at baseline was the reason for failing the postulated REMD variation. In all three studies of the Feinberg group (Feinberg et al., 1987, 1988; Travis et al., 1991) the reduction in REMD during recovery sleep was associated with no increase of delta EEG in the corresponding sleep cycle. Barbato et al. (1994) studied the time course of REMD in eight consecutive sleep cycles of subjects living for four weeks in a winter-type photoperiod with 10 h day and 14 h (from ) night time (L:D=10:14). Under these conditions REMD increased in consecutive sleep cycles early in sleep while it decreased toward the end of sleep, suggesting a systematic variation over time (cf. Table 3 of Barbato et al., 1994). These data are not in contradiction with our assumption of a circadian modulation of REMD but show the additional influence of an extended dark period on the shape of the suggested rhythm of phasic REM sleep activity. To our best knowledge there is only one report on the distribution of REMD under conditions of free running sleep-wake cycles in the literature. Zimmerman et al. (1980) examined REM sleep parameters of three subjects while free running. As in normal night sleep, REMD showed a steady increase in consecutive REM episodes of the main sleep phase, while the first REM episode was slightly phase advanced and its duration increased. The subjects had near 24-h sleep-wake cycles and went to bed at about the same circadian phase while free running. The results suggest that in free running sleep-wake cycles there is a certain disinhibition of REM sleep early in sleep, and that the rhythms of tonic and phasic REM sleep parameters may be coupled differently under these conditions. To get a more complete picture of the phase relationship between tonic and phasic REM sleep parameters, it would be of interest to examine REMD together with tonic REM sleep parameters under conditions of either spontaneous or forced desynchronization, i.e., in subjects with varying phase positions between the sleep-wake cycle and the cycle of body temperature. The present study has clearly different limitations. First, the number of subjects (n=12) was small, although this is true also for the other studies with sample sizes between a minimum of four subjects (Kobayashi et al., 1980) and a maximum of 13 subjects (Travis et al., 1991). Second, large time blocks had to be applied to compute REMD scores for all segments of the diurnal cycle. In our opinion, the circadian distribution of sleep propensity and REM sleep is such a strong and consistent phenomenon (e.g., Lavie, 1986; Strogatz et al., 1987; Schulz et al., 1998) that larger samples of subjects would hardly overcome the problem of few REM sleep episodes around the

8 6 6 WITZENHAUSEN ET A L. low point of sleep propensity, and as a result, missing REMD values at this circadian time. Third, REMD scores were computed from nap and night sleep data with different amounts of prior wakefulness. As already said, it would be desirable to analyze REMD from sleep data which were recorded under conditions of forced desynchrony. In regard of these limitations, the present results are preliminary and need replication. Finally, the data presented here reflect REMD for only the first REM sleep episode at daytime, and so we can draw conclusions only for the initiation of the REM process at daytime. For night sleep, the data confirmed the known increase of REMD in consecutive REM sleep episodes (Benoit et al., 1974; Schneider 1978). In conclusion, the present results suggest a circadian modulation of phasic REM sleep variables, which runs in parallel to the tonic REM sleep variables, and is inverse to the time course of deep body temperature. 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