Defining and determining the properties of the human sleep homeostat Zavada, Andrei

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1 University of Groningen Defining and determining the properties of the human sleep homeostat Zavada, Andrei IMPORTANT NOTE: You are advised to consult the publisher's version (publisher's PDF) if you wish to cite from it. Please check the document version below. Document Version Publisher's PDF, also known as Version of record Publication date: 2007 Link to publication in University of Groningen/UMCG research database Citation for published version (APA): Zavada, A. (2007). Defining and determining the properties of the human sleep homeostat. s.n. Copyright Other than for strictly personal use, it is not permitted to download or to forward/distribute the text or part of it without the consent of the author(s) and/or copyright holder(s), unless the work is under an open content license (like Creative Commons). Take-down policy If you believe that this document breaches copyright please contact us providing details, and we will remove access to the work immediately and investigate your claim. Downloaded from the University of Groningen/UMCG research database (Pure): For technical reasons the number of authors shown on this cover page is limited to 10 maximum. Download date:

2 Chapter 3 Circadian Period and Light Responses in Early and Late Chronotypes Marijke C. M. Gordijn, Andrei Zavada, Domien G. M. Beersma, B. A. E. v.d. Pol, M. Merrow, T. Roenneberg and Serge Daan 29

3 30 CHAPTER 3. PERIOD AND LIGHT SENSITIVITY Abstract Humans predominantly sleep at night, but not all at the same time. Profound differences between chronotypes exist resulting in sleep phases that in the most extreme cases do not overlap. Based on the theory of entrainment, the existence of chronotypes may arise from differences in circadian period, in sensitivity to the zeitgeber signal, in zeitgeber strength, or even from different coupling qualities of the output rhythm to the pacemaker. The present study selected extreme chronotypes to test the first two possibilities: Do extreme chronotypes differ in their endogenous circadian period and/or in their responses to light? Nine early chronotypes and 8 late chronotypes (average habitual sleep phase 22:42 7:17h and 3:01 10:54h, resp.) spent 2.5 days in a temporal isolation facility on 3 occasions. The period of the melatonin rhythms-calculation based on the drift over 3 nights-under constant dim light differed significantly between the early (24.35 h) and late (24.65 h) chronotypes. Single 4-h light pulses of 600 lux were given in the individual s evening or morning. Neither the response of the rise of melatonin, nor the response of the decline in melatonin differed significantly between early and late chronotypes. Irrespective of chronotype, the effect of evening light was greater for the melatonin onset than melatonin decline. The larger phase advances of melatonin decline relative to melatonin onset after morning light did not reach significance. We conclude that in this group, it is apparently not the response to light that is the affected characteristic underlying their circadian behaviour, but rather the endogenous period of the coupled oscillator.

4 3.1. INTRODUCTION Introduction Humans tune their sleep largely to coincide with the night, but profound variation exists between individuals in the exact timing of sleep (Horne and Östberg, 1976; Kerkhof, 1991; Kerkhof and Van Dongen, 1996; Roenneberg et al., 2003, 2004a). A survey of the general population in the Netherlands showed an average sleeping time of 00:46 to 9:09 h on free days. In contrast, extreme early and late chronotypes regularly sleep from 22:00 to 6:00 (mid-sleep 2:00, early types) or from 4:00 to 12:00 (mid-sleep 8:00, late types) on their free days (Zavada et al., 2005). Physiological variables, psychological functioning, performance, and mood show variations in circadian phase position in close association with variation in sleep timing (Kerkhof, 1991; Kerkhof and Van Dongen, 1996; Baehr et al., 2000; Duffy et al., 2001; Foret et al., 1985; Horne et al., 1980). The sleep wake cycle is the most prominent representation of human circadian behaviour. It is very easy to measure despite its complex regulation. This timing of sleep is controlled by two processes, a homeostatic sleep process that regulates sleep need and a circadian process that gates the periodic occurrence of sleep (Borbély, 1982; Daan et al., 1984). Interaction between these two processes results in a stable 24-h sleep wake cycle with deep sleep at the beginning of the night and a decay in deep sleep-associated slow-wave activity over the night. Differences in sleep timing under a normal light-dark cycle may be the consequence of functional differences in the internal circadian pacemaker gating sleep and/or of differences in the homeostatic sleep process. Previous work has produced evidence for variation between chronotypes in the homeostatic component of sleep regulation (Kerkhof, 1991; Mongrain et al., 2006a,b). In the present study we focus on the role of the internal circadian pacemaker in determining sleep phase in early and late chronotypes. The circadian control of behaviour, in this case of sleep, in entrained conditions is theoretically determined by both the intrinsic period of the pacemaker and the period and strength of the zeitgeber, i.e., the light dark cycle (Aschoff, 1962; Aschoff and Wever, 1981; Daan and Aschoff, 2001). Evidence for a correlation between sleep phase preference and intrinsic period has indeed been observed in humans in a retrospective analysis (Duffy et al., 1999, 2001; Duffy and Czeisler, 2002). The results provide support for a role of intrinsic period in human sleep phase preference. Nonetheless, there are also

5 32 CHAPTER 3. PERIOD AND LIGHT SENSITIVITY other potential sources of variation in phase position besides variation in endogenous period. A potentially relevant source of variance concerns the sensitivity to the entraining zeitgeber, i.e., light. A circadian oscillator that is perturbed or entrained with differing zeitgeber strengths shows different phase responses and potentially different stable entrained phases. Whether extreme chronotypes differ in their phase responses to light, has not been investigated. Light in the morning might for instance cause a larger phase advance in early types than in late types. Conversely, light in the evening might cause a larger phase delay in late types than in early types. There is a suggestion of such a difference in sensitivity to light in subjects suffering from Delayed Sleep Phase Syndrome (DSPS) compared to healthy controls (Aoki et al., 2001). Phenomenologically, DSPS is a syndrome that might be viewed as the extreme late form of chronotypes. Aoki et al. (2001) report increased melatonin suppression in DSPS patients compared to healthy controls in response to light (1000lux) presented 2h prior to the melatonin peak. A correlation between melatonin suppression and phase shifting effects of light in normal volunteers has been shown (Rüger et al., 2005) and these results therefore may apply to a difference in sensitivity to phase-shifting effects of light in DSPS patients. For these reasons, we have set out to study both the endogenous period and the response to light in early and late chronotypes. In the analysis of the resulting data we pay particular attention to the idea that a differentiated Evening/Morning dual oscillator system may underlie the expression of the sleep wake rhythm and the melatonin rhythm (cf. Pittendrigh and Daan (1976b); Wehr et al. (2001)). 3.2 Experimental procedures Subjects Eighteen subjects were selected as extreme early or late chronotypes out of a database of 5138 subjects in the age range of 18 60y (Fig. 3.1) who completed the Dutch version of the Munich Chronotype Questionnaire (MCTQ) on the Internet. Selection was based on the individual s mid-sleep phase on free days (MSF) (Roenneberg et al., 2003; Zavada et al., 2005), corrected for systematic changes in chronotype according to age (MSF ac ). The age correction was developed (Roenneberg,

6 3.2. EXPERIMENTAL PROCEDURES 33 Figure 3.1 Distribution of mid-sleep on free days corrected for age (see text) in 5138 Dutch residents between the ages 18 and 60 y, who completed the Munich Chronotype Questionnaire on the Internet. Symbols show the MSF of the individuals who were included in the present study. pers. comm) from a largely western European/predominantly German database of 30,000 subjects. For subjects over 20 y.o.: MSF ac = MSF ( (age 30) (age 2 900)), and for ages 20 years, an intermediate term was computed: MSF 20 = MSF ( (age 20) (age 2 400)), MSF ac = MSF To be selected in the present study, MSF ac had to be <3:38a.m. for early, and >5:58 a.m. for late chronotypes, corresponding with the 15% earliest and 15% latest in the database. Figure 3.1 shows the distribution of MSF ac in the database and the selection of the subjects. Subjects had to be free of current medication or psychiatric illness. Depression scores on the Beck Depression Inventory (Beck et al., 1988) had to be lower than 8. The experimental design included 3 protocols: a DIM light condition (DIM), an evening light condition (EL) and

7 34 CHAPTER 3. PERIOD AND LIGHT SENSITIVITY a morning light condition (ML). One early type withdrew from the study during her DIM protocol because of extreme tiredness. The DIM condition was completed by 17 extreme chronotypes; 9 early (all females), and 8 late types (2 males, 6 females). Ages differed between the two groups, but all subjects belonged to the 15% most extreme chronotypes for their age group. One early type and one late type cancelled participation after completing the DIM condition resulting in 8 early and 7 late types for the EL condition, one additional early type completed the DIM and the EL condition, but not the ML condition. This led to 7 early and 7 late types for the ML condition. Basic data on age and chronotype for the individuals is presented in Table 3.1. Subjects gave written informed consent and were paid for their participation. The medical ethics committee of University Medical Center Groningen approved the protocol Experimental design After an individual had been selected, he/she wore an Actiwatch-L R (Cambridge Technology, UK) during 3 weeks at home to measure the habitual rest activity cycle. Habitual sleep phase was estimated based on the Actiwatch R recordings by the algorithm of the sleep analysis software (Cambridge Technology, UK) in combination with a 3 week sleep diary. Based on habitual sleep phase a protocol was designed for each individual for 3 conditions to be carried out in a temporal isolation unit. Figure 3.2 shows the habitual sleep-wake patterns of 2 selected subjects and the design of their experimental conditions under temporal isolation. The isolation facility consists of four separate sleeping rooms, 3 living rooms, sanitary facilities, and a kitchen. There are no windows in any of the rooms and no information on time of day is available in any way. In the present experiment, either one or two subjects were studied simultaneously. If two, then they lived in separate rooms without contact with each other and followed protocols that were out of phase with each other. Each subject started with the DIM light condition, followed by either the Morning Light (ML) or Evening Light (EL) condition. There was always at least one week in between conditions. Four of the early types had EL first, four started with ML. Of the late types, 3 started with EL, four with ML. Each condition started 11h before habitual mid-sleep (that is, before time point 0), calculated on the basis of

8 3.2. EXPERIMENTAL PROCEDURES 35 Figure 3.2 Scheme of the experimental design, including two examples of actual sleep timing in one early and one late type included in the study obtained with Actiwatch. The study consisted of 3 weeks at home, and 3 sessions of 3 days in a time isolation facility.

9 36 CHAPTER 3. PERIOD AND LIGHT SENSITIVITY the Actiwatch R recorded sleep wake rhythms (Fig. 3.2). In an extension of the definition of internal time 0 as being the midpoint of the subjective dark phase (Daan et al., 2002), here we define habitual mid-sleep as internal time 0, or InT 0. This takes into account the fact that humans mostly generate a dark phase which more or less matches their sleep phase. Subjects were kept awake under dim red light except for the time of the light pulse and for four sleep episodes (complete darkness). Dim illumination was provided by TLD 36W/54 tubes (Philips Eindhoven, the Netherlands) with an intensity < 10 lux ambient, < 50lux maximum = 17.4µW/cm 2, shown to have no suppressing effect on melatonin concentration (Gordijn et al., 2005; Hanifin et al., 2006). The first sleep episode was scheduled from InT to InT 4.25 (hence centering at InT 0 and 8.5h long), two 3-h naps were scheduled from InT 10.5 to InT 13.5 on day 1 and again 24h later on day 2. The last sleep episode started at InT 1 on day 3 and lasted until the subjects reported wakening, or were awakened at their desired wake time (not earlier than at InT 6 on day 3). A number of late types already finished the protocol before it became clear that early types had extreme difficulty staying awake during the night of day 2, between InT 13.5 on day 1 and InT 10.5 on day 2. Since we were anxious not to lose these data we decided to allow early types to start napping 2 h earlier on day 2. As a result, all early types had their second nap between InT 8.5 and InT 13.5 on day 2 (for a total of 5h rather than 3h). In the DIM light condition subjects were exposed to the dim red light continuously while awake. In the EL and ML condition a light pulse was administered, during which the subjects remained seated looking at a TV-set at a distance of 3m, with two light boxes (Philips Bright Light R, Philips, Eindhoven, The Netherlands) placed left and right of the subject at an angle of 30 degrees at a distance of 1.5m. The intensity of the light was 600lux (204.6µW/cm 2 ) measured at eye level in the direction of gaze and this was checked repeatedly during the light pulse. In the EL condition the 4-h light pulse started for each individual at the Dim Light Melatonin Onset (DLMO) calculated on the basis of the second day under DIM light. In the ML condition the 4-h light pulse started 9h after the calculated DLMO in the second day under DIM light. These times were chosen to correspond with the phase of the maximal phase delay and maximal phase advance respectively, both on the basis of the PRC for single light pulses (Khalsa et al., 2003). The duration of 4h was chosen because of our experience

10 3.2. EXPERIMENTAL PROCEDURES 37 with 4-h light pulses in previous experiments, a quantity that consistently resulted in significant phase shifts within one day (Rüger et al., 2003, 2005). The intensity of 600lux was chosen so as to be able to induce a phase shift but not to saturate the response in both the early and late chronotypes (Laakso et al., 1993; Boivin et al., 1996) Measurements Body temperature Continuous core body temperature (CBT) data were obtained by a disposable rectal probe connected to a Temperature Puck system (Ambulatory Monitoring Inc., Ardsley NY, USA). This system consists of a small transmitter fixed to the subject s garments and connected to the rectal probe and a receiver connected to a PC. Body temperature data were recorded every minute. During brief episodes with artefacts, or when the probe was disconnected during showers etc. CBT was linearly interpolated. CBT data were smoothed by calculating the 6-h running mean (360 values) to obtain the overall circadian pattern and circadian phase markers. Melatonin In order to measure melatonin concentration in each of the three conditions, saliva samples were taken hourly when subjects were awake. Saliva was collected using Sarstedt Salivettes R with a cotton swab (Sarstedt BV, Etten-Leur, The Netherlands). When subjects were asleep, they were awakened once every two hours to produce the sample while staying in bed. When subjects were awake, they remained seated during 15 minutes prior to the collection of saliva. Eating and drinking was only allowed immediately after collection of saliva and was followed by rinsing the mouth with water. Eating and drinking, brushing teeth, and smoking was not allowed during 30 minutes prior to the collection of saliva. No coffee, black tea or chocolate was allowed at any time. Saliva samples were centrifuged immediately and stored at 20 C. Melatonin concentration was determined by radioimmunoassay using a kit (Bühlmann Laboratories AG, DPC Netherlands BV, Breda, Netherlands). All samples from one individual were analyzed within the same series. The melatonin data were used to estimate circadian phase and period, and for scheduling the timing of the light pulse. The

11 38 CHAPTER 3. PERIOD AND LIGHT SENSITIVITY melatonin curves were first normalized for each individual per condition. A 3-harmonic regression was fitted through the raw melatonin values of each individual over the first 24-h period. All values were then expressed as a fraction (%) of the fitted maximum (100%). Dim light melatonin onset (DLMO) was defined as the moment at which a straight line connecting the consecutive raw data points crossed the 25% level. Phase markers for the rise and drop of the melatonin rhythm were chosen at 50% onset (Mel-on) and 50% drop (Mel-off). These three phase markers were calculated for all 3 days in the DIM condition and for the first and third day of the EL and ML condition. The calculated DLMO on the second day under DIM light, based on the regression line through the 3 consecutive DLMO values, was used as a reference point for the scheduled light pulses in the EL and ML condition. All clock times are Central European Time, with daylight saving time option. 3.3 Results Sleep timing Age-corrected free-day mid-sleep (MSF ac ) of the 9 early chronotype subjects varied between 1:37 to 3:35h. The MSF ac of late chronotypes ranged from 6:01 to 9:30h. (Table 3.1). Habitual mid-sleep, based on 3 weeks of Actiwatch recordings was 3.96h earlier in early than in late chronotypes. Habitual sleep phase is marked with a horizontal bar in the upper part of Fig. 3.3A for both groups. The subjects in the present study completed the Horne Östberg morningness-eveningness questionnaire (Horne and Östberg, 1976) before the start of the experiment. Morningness-eveningness ratings (MEQ scores) in Table 3.1 show the expected differences in preference between early and late chronotypes. The variation in MEQ score is larger in early types than in late types Circadian period Body temperature The rhythm of the smoothed core body temperature under DIM light shows a circadian pattern with two peaks and approximately 3 troughs (Fig. 3.3B). Markers for circadian phase of the body temperature

12 3.3. RESULTS 39 Figure 3.3 (A) The average courses of melatonin during the DIM condition in 9 early and 8 late chronotypes. Melatonin concentration was normalized with respect to the fitted maximum of a 3-harmonic regression through the first 24-h of each condition and plotted at the average clock time for that group relative to its habitual sleep phase (see horizontal bars in the figure); i.e., InT 0 for the early types was at 2:59 ± 24min (s.e.m.), InT 0 for the late group was at 6:57 ± 22min (s.e.m.). (B) The average courses of 6-h running mean of body temperature during the DIM condition in 8 early and 6 late types. Time axis the same as in (A).

13 40 CHAPTER 3. PERIOD AND LIGHT SENSITIVITY Table 3.1 Subject characteristics and sleep timing in the early and late chronotypes; MSF is mid-sleep on free days, MSF ac is mid-sleep on free days corrected for age, MEQ is morningness-eveningness score on Horne Östberg rating scale. Habitual sleep times as recorded by Actiwatch. MSF and Actiwatch data are clock times, s.d. values in minutes; age, in years. See text for details. Group Gender Age (y) MSF MSF ac Habit. sleep times Onset Midsleep End MEQ early F 21 2:47 2:08 23:35 3:59 8:24 53 early F 34 3:05 3:19 23:24 3:52 8:20 51 early F 58 0:34 1:37 18:39 0:01 5:23 74 early F 43 2:45 3:23 00:00 3:55 7:51 41 early F 48 2:45 3:35 22:55 2:45 6:35 68 early F 40 2:45 3:17 22:57 2:43 6:30 65 early F 39 3:01 3:31 23:23 2:53 6:23 63 early F 22 3:20 2:41 22:57 3:11 7:25 63 early F 20 3:35 2:55 22:26 3:33 8:41 60 Average :44 2:56 22:42 2:59 7: ±s.d. ±13.1 ±54 ±42 ±96 ±72 ±66 ±10 late F 23 10:00 9:30 3:43 8:30 13:18 25 late M 21 7:38 6:58 3:39 7:24 11:09 27 late F 26 7:38 7:21 2:59 6:26 9:54 27 late F 23 8:38 8:08 4:27 8:24 12:22 25 late F 18 6:35 6:01 3:05 6:27 9:50 24 late M 20 9:17 8:33 2:28 6:22 10:16 31 late F 22 6:53 6:18 2:20 6:37 10:55 26 late F 22 7:33 7:04 1:28 5:28 09:28 27 Average :01 7:29 3:01 6:57 10: ±s.d. ±2.4 ±72 ±72 ±54 ±66 ±78 ±2.1

14 3.3. RESULTS 41 rhythm, rise and decline, were the consecutive upward and downward crossing points between the smoothed curve of an individual and the average temperature over the circadian period. The midpoint between decline and rise of the body temperature of each cycle was calculated and used as the phase of that cycle. Phase (defined as the CBT midpoint) on nights 1 and 2 in the early types (n = 9) was 2:32 (s.e.m. 20min) and 3:42 (s.e.m. 35min), respectively. Phases in the late types (n = 6) were 6:49 (s.e.m. 37min) and 8:10 (s.e.m. 34min). Circadian period was calculated as the difference between consecutive midpoints. The periods of CBT rhythms were not different between the early and late types (25.2h s.e.m. 0.5h, and 25.3h s.e.m. 0.7 h, respectively; Mann Whitney U-test). The apparent difference in mean CBT level (Fig. 3.3B) between early and late chronotypes was not significant (37.1 s.e.m. 0.1 C, and 37.2 s.e.m. 0.1 C, respectively). Melatonin Salivary melatonin concentration showed a pronounced circadian rhythm with three cycles (Fig. 3.3B). In the early types, average consecutive DLMO times in cycle 1, 2, and 3 were 21:25 (s.e.m. 43min), 22:03 (s.e.m. 39 min), and 22:07 (s.e.m. 43 min), respectively. In the late types these values were 23:38 (s.e.m. 36 min), 0:07 (s.e.m. 27 min), and 0:56 (s.e.m. 31min), respectively. The period of the endogenous circadian melatonin rhythm was defined on the basis of the drift in DLMO over the three cycles. In contrast to the period determined by the CBT rhythms, those calculated based on melatonin showed a slightly but significantly shorter period (Mann Whitney U-test, p < 0.05) for the group of early chronotypes (24.35 s.e.m. 0.06h) compared to the group of late types (24.65 s.e.m. 0.17h) Phase shifts The CBT curves showed considerable inter-individual variance and were severely masked by the fixed sleep episodes. For calculating phase shifts within one day, exact estimates of endogenous circadian phase are needed on the days before and after the light pulses. Thus, the CBT-based phase markers did not reach sufficient resolution for this analysis. Phase shifts, were, therefore, based on the analysis of the melatonin rhythms.

15 42 CHAPTER 3. PERIOD AND LIGHT SENSITIVITY Figure 3.4 The average shifts in the 50% onset of Melatonin (Melon50%) and 50 % drop of Melatonin (Meloff50%) after a 4-h evening light pulse of 600 lux (EL) and a 4-h morning light pulse of 600 lux (ML) in early and late chronotypes. EL started at DLMO of each individual, ML started 9h after DLMO. Phase shifts of the melatonin rhythm after morning light (ML) and evening light (EL) were calculated as the difference between Mel-on/Mel-off at day 3 and Mel-on/Mel-off at day 1, corrected for the drift under DIM light between day 1 and 3. The effects of EL on the melatonin rhythm could be calculated in 8 early and 7 late types; the effects of ML, in 7 early and 6 late types. Figure 3.4 shows all changes in the rise (Mel-on) and drop of melatonin (Mel-off) in early and late types, both after morning and after evening light. On the rise of the melatonin rhythm (Fig. 3.4), ML induced on average phase advances in both early types and late types that were statistically indistinguishable. EL induced clear and indistinguishable phase delays on Mel-on (Fig. 3.4) in early types and in late types. Similar results were observed for the effect of light on Mel-off. Neither the effect of ML, nor the effect of EL differed between early and late types (Fig. 3.4B).

16 3.4. DISCUSSION 43 Regardless of the chronotype, the overall effects of EL and ML on Mel-on differ from the effects on Mel-off (Repeated measures ANOVA F 1,11 = 5.0, p < 0.05). If tested separately, the delaying effects of EL were larger on Mel-on than on Mel-off (ANOVA, F 1,13 = 4.8, p < 0.05). The advancing effect of ML was on average larger on Mel-off than on Mel-on, but this may be attributable to chance variation (ANOVA, F 1,11 = 2.8, p > 0.1). 3.4 Discussion Circadian period The significant difference between the periods of the melatonin rhythms in early and late human chronotypes is indicative of a relationship between endogenous circadian period and circadian timing of human behaviour in daily life (chronotype), confirming an earlier study (Duffy et al., 1999, 2001). In that study, period was calculated (based on CBT curves) in a group of 17 subjects under a forced desynchronization protocol with either a 28-h or a 20-h day. Subjects had not been selected on their chronotype characteristics, but completed the Horne Östberg morningness-eveningness (ME) preference questionnaire as part of their pre-study screening. Diurnal preference ratings in this group varied from 31 to 63 (Fig. 1 in Duffy et al. (1999, 2001)), thus, no extreme morning or evening types were included. In the present study extreme chronotypes were selected on the basis of their actual sleep wake cycle and not on diurnal preference. Although a close correlation exists between the phase marker used (mid-sleep on free days) and Horne Östberg ME-score (Zavada et al., 2005), the selection criteria are not identical. In the present study, variation in ME-score is so large in the early types that it almost becomes indiscriminating: subjective ME-rating of only one subject fell into the category of definitely morning types, five fell into the category of moderately morning type, two assessed no preference, and one subject even scored on the category border of the moderately evening types. The variation in diurnal preference rating in the late types, on the other hand, is small: one scored for moderately evening type, the other 7 subjects for definitely evening type preference. Despite these differences in preference of behaviour as measured with the Horne Östberg morningness-eveningness questionnaire, all selected subjects where clearly either extreme early or extreme late chronotypes. Within

17 44 CHAPTER 3. PERIOD AND LIGHT SENSITIVITY this selection, we find the differences in circadian period and conclude that the differences in chronotype may, at least in part, be the result of these differences in the period. One study that selected its subject on the basis of an early sleep phase calculated the endogenous period of a single subject on the basis of free-running sleep wake and CBT rhythms in temporal isolation (Jones et al., 1999). This subject suffered from the so-called Advanced Sleep Phase Syndrome and showed an endogenous period of 23.3 h. The endogenous period of the melatonin rhythm in our early chronotypes (24.35 h) was close to the average endogenous period found in normal subjects under forced desynchrony conditions (Czeisler et al., 1999; Hiddinga et al., 1997; Koorengevel et al., 2002). The late types showed a significantly longer period (24.65 h). A difference of 0.3h as found between early and late chronotypes is a plausible explanation for differences in phase position under entrainment (Pittendrigh and Daan, 1976a). The finding of relatively long periods in both groups may be caused by the method used. A period calculated from the drift of DLMO over three days has been proven to correlate strongly with although not to match completely the period under forced desynchronization (Wright et al., 2005); for each 6-min change in circadian period under forced desynchronization a 16-min drift in DLMO was found in a constant routine on 2 consecutive days. It is impossible to tell which measure resembles the period of the master circadian oscillator best. Obviously, melatonin and core body temperature are downstream output measures of the clock. The drift over the first three days in constant conditions may show too much variance or after-effects to estimate the endogenous period accurately. On the other hand, modifications of the sleep wake cycle, as required for forced desynchrony, have been shown to induce small effects on the phase of melatonin rhythms (Gordijn et al., 1999; Danilenko et al., 2003; Burgess and Eastman, 2004). The conclusion of a difference in endogenous period between early and late chronotypes is based on the melatonin rhythm and was not supported by a difference in the CBT rhythm. A possible explanation for the lack of difference in CBT rhythm is that our study was not a constant routine, since subjects were allowed to have some activity and to take the scheduled naps. The masking effects of these behaviours may have induced too much variance in the CBT data to establish the underlying period accurately. Melatonin rhythms are more robust and less sensitive to behavioural factors under our experimental conditions.

18 3.4. DISCUSSION 45 Our early and late chronotypes differed in average age. An effect of age on the difference in endogenous period can therefore not be excluded. Others, however, have shown that the average circadian period of healthy older people does not differ from young adults (Czeisler et al., 1999). Our subjects were further selected on the basis of their early sleep phase corrected for age differences (Roenneberg et al., 2004a). Therefore, the reported shorter period in the early chronotypes is related directly to the chronotype rather than to their somewhat older age Responses to light Theoretically, chronotype depends on the balance between period of the pacemaker, zeitgeber strength, sensitivity to the zeitgeber signal, and the coupling qualities of the output rhythm to the pacemaker. In many discussions on the possible background of chronotypes the option remained open that a difference in the phase response curve to light, probably in addition to differences in intrinsic endogenous period, may underlie their existence. Our study appears to be the first to test this hypothesis. We found no differences whatsoever in responses of the circadian pacemaker to light in the subjective early morning or late evening, as measured by phase shifts of the melatonin rhythm, indicating that those parts of the phase response curve are similar between early and late chronotypes. A limitation of the present study is the small number of subjects. Although our extreme chronotypes were carefully selected, they represent only a small part of the huge, heterogeneous population. The possibility can not be ruled out that the cause of extreme chronotype behaviour may be the result of different pacemaker characteristics in different people Dual oscillator model Depending on the timing of light, the effects are different for the rising part and the decline of melatonin production. Irrespective of chronotype, the effects of evening light are larger for the melatonin onset than for the melatonin drop. The larger phase advances of melatonin decline than of melatonin onset after morning light did not reach significance. These results are partly in accordance with previous work in both rat pineal N-acetyltransferase (NAT), a critical enzyme in the production of melatonin, and in human melatonin (Buresova et al., 1991; War-

19 46 CHAPTER 3. PERIOD AND LIGHT SENSITIVITY man et al., 2003; Illnerova and Sumova, 1997; Illnerova and Vanecek, 1982). The differential responses of the rise and fall of melatonin to light in the evening and morning were first described for NAT in rats. It was explained by the hypothesized existence of two clock components, an Evening oscillator (E-oscillator) and a Morning oscillator (M-oscillator), controlling the NAT rhythm (Illnerova and Vanecek, 1982). The E M oscillator model (Pittendrigh and Daan, 1976b) postulates the central circadian pacemaker to consist of two mutually coupled oscillators: an M- and an E-oscillator, which tend to follow dawn and dusk, respectively, as natural day length changes. Properties of these oscillators differ: under constant darkness E has a short period and light pulses will induce phase delays, while M has a long period and light pulses will induce phase advances. A molecular model for the E M oscillator has more recently been proposed (Daan et al., 2001) and its predictions have been thoroughly tested in several animal studies (Steinlechner et al., 2002; Spoelstra et al., 2004; Oster et al., 2003). The model has been extensively used also to interpret human circadian data (Wehr et al., 2001). The original idea of the circadian gene Per2 as part of the E oscillator is in line with the unique finding of an hper2 mutation in some extreme sleep phase-advanced humans, i.e., some members of a family suffering from Advanced Sleep Phase syndrome (Toh et al., 2001). This mutation should affect the function of the E oscillator, resulting in the early sleep phase in affected subjects and in the short period found under forced desynchronization in one of these subjects (Jones et al., 1999). The finding of a single mutation in a clock gene resulting in Advanced Sleep Phase Syndrome is unique in circadian rhythm sleep disturbances, and it is more likely that the phenotype of early and late chronotypes in the population is based on a complex genetic background (Katzenberg et al., 1998; Iwase et al., 2002; Ebisawa et al., 2001; Archer et al., 2003; Katzenberg et al., 1999; Takano et al., 2004). This complexity may result in different pacemaker characteristics in people leading to differences in circadian behaviour. From the present data we conclude that if an E-M oscillator structure underlies human sleep timing, it is apparently not the response to light of one of these oscillators that is the affected characteristic underlying chronotypes, but rather the endogenous period of the coupled oscillator.

20 3.5. ACKNOWLEDGEMENTS Acknowledgements Financial support for this study was obtained from the 5th European Framework project BrainTime (QLG3-CT ), and the 6th European Framework project EUCLOCK (018741). We would like to thank in particular Ellis Mulder and Bonnie de Vries for technical assistance in the endocrinology lab and Sielle Gramser, Titia Rosien, Katrien Bijl and Bram van Bunnik for assistance during the experiments.

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