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HOW TO MEASURE CIRCADIAN RHYTHMS IN HUMANS
by A. Wirz-Justice, Switzerland
he scientific study of human circadian rhythms years of research, they laid the basis for the formal began when a curious sleep researcher asked properties of the human circadian system analo- Tclever questions and went ahead to test them gous to that developed by Pittendrigh and Daan for on himself. Nathaniel Kleitman spent a month in a rodents.3 dark underground cave in 1938, having developed The timing and structure of sleep and waking is an “apparatus for determining and for recording considered to arise from interactions between the motility and rectal temperature during sleep.”1 biological clock or circadian pacemaker (designat- Kleitman’s monitoring of bed movements by means ed “process C”) and a sleep homeostatic process of a primitive polygraph to produce a continuous Anna WIRZ-JUSTICE, PhD dependent on duration of prior time awake (“pro- Centre for Chronobiology 4 readout of motor activity anticipated measurement Psychiatric University Clinics cess S”) (Figure 1). This 2-process model is appli- techniques that have only recently become prac- Basel, SWITZERLAND cable not only to the sleep-wake cycle, but also to tical thanks to advances in microelectronics. He the understanding of the temporal patterns of near- clearly demonstrated that under constant dim en- ly every neuroendocrine, physiological, and psycho- vironmental conditions sleep did not retain its 24- logical function. The model has proved extremely hour pattern, but shifted later day by day. He also useful for understanding a variety of sleep distur- tried to live on a “28-hour day,” as a test of whether bances, and can be used to interpret apparent rhyth- the usual 24-hour cycle might simply be a reaction mic abnormalities in depression, as well as provid- to the outside world. The “28-hour day” is a tech- ing a framework for specific therapeutic approaches. nique now used to separate the sleep-wake cycle (which can more or less follow this long day) from Characteristics of the circadian clock the endogenous circadian cycle (which cannot). Two decades later, Jürgen Aschoff and Rütger Biological clocks help us keep time on this rotating Wever created a more comfortable underground planet. The advantage of an internal clock to regu- “bunker” for human temporal isolation experiments, late sleep and wakefulness within the appropriate measured motility (by means of sensors in the bed and floors), rectal temperature, urine output, and SELECTED ABBREVIATIONS AND ACRONYMS many other physiological and behavioral variables, and concluded that humans have endogenous cir- DLMO dim light melatonin onset cadian cycles like plants and mice and flies.2 They MCTQ Munich Chronotype Questionnaire also placed subjects on days of varying lengths, and MEQ Morning-Eveningness Questionnaire tested to what extent the biological clock could syn- SCN suprachiasmatic nuclei chronize to the given periodicity. In more than 25
he biological clock drives all circadian rhythms in humans, provide a reasonable estimate of circadian phase. Chronobiology whether relative to neurobehavioral function, hormones, requires long-term measurement over at least one 24-hour cycle, T physiology, or behavior. The most obvious rhythm is the and new microchip technologies permit noninvasive and contin- sleep-wake cycle, which differs in timing across individuals uous data collection over many days and weeks (eg, actimetry). (“chronotype”—from early-morning larks to late-night owls). The next decade of research will surely yield further insights into However, not all changes in sleep-wake cycle behavior are a con- human circadian clock function and its pathologies. sequence of abnormal clock function. Knowledge of the formal Medicographia. 2007;29:84-90. (see French abstract on page 90) properties of the circadian system, the role of zeitgebers for ad- equate synchronization to the 24-hour day, and how sleep is reg- Keywords: human circadian system; sleep regulation; forced ulated, has led to the development of stringent protocols to in- desynchrony; constant routine; ambulatory monitoring; vestigate the characteristics of circadian rhythms and sleep. These melatonin studies have provided gold standards for estimating circadian amplitude and phase, and have identified the most useful phys- Address for correspondence: Professor Anna Wirz-Justice, Centre for Chronobiology, iological or hormonal markers. We are now at the second stage Psychiatric University Clinics Basel, Wilhelm Klein Strasse 27, CH-4025 Basel, of trying to develop simpler markers for ambulatory use, which Switzerland (e-mail: [email protected])
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phases is that physiology and behavior can antic- er can entrain, and where endogenous rhythmicity ipate transitions between day and night and not retains its freerunning period (as originally shown merely react to them. A circadian clock not only by Kleitman, Aschoff, and Wever). generates a cycle to match the solar day, it must It should be noted that this natural periodicity, τ, also maintain an appropriate phase relation to it. is not only a genetically determined characteristic: This process of optimal synchronization with the τ is subject to “after-effects,” ie, is changed by what- environment is called entrainment, and is mediat- ever environmental light pattern and intensity the ed by periodic stimuli (“zeitgebers”) acting on the subject was exposed to prior to the study,2 such as clock. The endogenous period of the circadian pace- maker under time-free conditions (as in a cave or a bunker) is known as τ, and the phase relation be- Zeitgebers tween rhythm and zeitgeber during stable entrain- ment is defined as ψ (eg, the difference between the phase of a given circadian rhythm such as sleep Circadian onset and the phase of a zeitgeber such as dusk or sleep-wake cycle, 2 3 dawn) (Figure 2). LIGHT 1 2 neurobehavioral Individual differences in τ may lead to different 4 Output performance, mood, ψ—the best known example is a person with short cortisol, melatonin, τ being a “lark” chronotype, and someone with long temperature, heart rate, etc. τ being an “owl” chronotype.5 However, τ is not the only factor that influences phase: sensitivity to light or zeitgeber strength (when and how long a person is exposed to what wavelengths and intensities of Figure 1. Circadian and homeostatic regulation of sleep. Two major pro- light), and amplitude of the circadian pacemaker, cesses are involved in driving the circadian sleep-wake cycle as well as are also determinants. all other behavioral and neuroendocrine outputs: their known anatomical The most important zeitgeber is light, providing correlates are schematically represented: (1) retina; (2) suprachiasmatic the photic signal for day and night as well as the nuclei (SCN); (3) hypothalamus: anterior (ventrolateral preoptic nucle- seasons. The master circadian clock in the suprachi- us—sleep-promoting); posterior (tuberomammillar nucleus—histamine; orexin [A/B]-producing neurons [wake-promoting]); (4) midbrain and asmatic nuclei (SCN) consists of two coupled os- pons (locus coeruleus [NE]; raphe nuclei [5-HT]; pedonculopontine cillatory systems that respond to dawn and dusk.6 tegmentum and laterodorsal tegmentum [ACh]). The change in daylength with seasons is mimicked Abbreviations: 5-HT, serotonin; ACh, acetylcholine; NE, norepinephrine. in many species by changes in the duration of ac- tivity and rest (α:ρ). Three main steps are impor- tant for biological clock function (Figure 1): input Light Dark Sleep (zeitgebers, retina) ==> SCN circadian pacemaker α ρ (eg, clock genes, neurotransmitters/peptides) ==> α:ρ (activity:rest duration) output (pineal melatonin synthesis, thermoregu- lation, etc). These factors then interact with the sleep-wake homeostat to regulate, continuously in time, sleep propensity and sleep architecture, and ψ (entrained) influence phenomena as different as mood and per- Successive days formance or hormonal output.
Which circadian clock characteristics do we want to measure? τ (free run)
A graphic representation of the various character- istics of the circadian system is shown in Figure 2. First, under entrained conditions, the sleep period Phase angle sleep midpoint to the remains at a stable phase angle with respect to the LD cycle light-dark cycle (ψ), with a given activity-rest ratio DLMO (α:ρ); and, second, in the absence of time cues (zeit- gebers), the sleep period shifts later and later each Phase angle DLMO day following the frequency of the endogenous to the LD cycle pacemaker (τ). Phase angle DLMO Freerunning period (τ) to sleep midpoint The freerunning period ( ) is a characteristic of the τ 12 15 18 21 24 03 06 09 12 circadian pacemaker that can only be measured in humans using very elaborate protocols: either in a Time of day (h) time-isolation environment in dim light,2 whereby the endogenous rhythmicity reveals itself in a “free Figure 2. Schematic characteristics of the sleep-wake cycle. The sleep 7 period (green bars) is plotted on consecutive days with respect to external run,” or in a “forced desynchrony” protocol where time (the light:dark [LD] cycle) and internal time (the circadian phase the given sleep-wake cycle is much longer or short- marker dim light melatonin onset [DLMO], green circles). See text for er than the range to which the circadian pacemak- details.
How to measure circadian rhythms in humans – Wirz-Justice MEDICOGRAPHIA, VOL 29, No. 1, 2007 85 U PDATE
the duration of the photoperiod (τ is longer in win- urine) are collected under dim light and controlled ter than in summer8). Thus, a measured τ could re- posture conditions, the melatonin rhythm provides flect behavioral differences with respect to light ex- an optimal marker of circadian phase in humans. posure, rather than just a genetic difference. “Dim light melatonin onset” (DLMO) is the easiest Novel techniques now in development can mea- marker of body clock time we have, because it can sure the τ of clock gene expression in tissue cultures be feasibly measured in saliva before a person goes from skin biopsies (this yields a τ for “peripheral” to sleep.12 If an entire 24-hour rhythm is measured, clocks, whose exact relationship to the τ expressed phase can be defined as required—at the peak, the by the central clock in the SCN is not yet known).9 midline crossing point, offset of secretion, etc. How- ever, many studies measure only part of the rhythm, Phase angle (ψ) taking evening samples under controlled conditions The simplest way to find out about anyone’s pre- before sleep to measure when melatonin synthesis ferred phase position is to ask their preferred sleep begins (DLMO). To understand putative abnormal- times on free days. Habitual bedtime and wake-up ities, one can measure the phase angle of the sleep midpoint to external time (in Figure 2, dusk; but dawn is equally valid), or the phase angle of inter- 80 nal time as measured by DLMO to the LD cycle or to sleep midpoint. 70 However, melatonin assays are not (yet) a rapid Morning chronotype or an easily available method for everyday diagnos- 60 tic use. A large, carefully controlled study of mela- tonin rhythms and sleep timing in the same sub- 50 Intermediate chronotype jects has found a close phase-relationship of this
MEQ score 40 marker of internal time to the sleep midpoint (Fig- 13 Evening chronotype ure 3). An algorithm was developed that is of great 30 clinical utility, since sleep midpoint can be used as a reasonable (indirect) estimate of circadian phase 20 (this is valid only if sleep is not too disturbed14). In 19 2021 22 2324 1 addition, the algorithm allows calculation of when DLMO time (h) bright light therapy should be applied with respect to internal (ie, circadian) and not external (clock) Figure 3. Correlation of internal circadian time with chronotype in winter time. This is the first example of applying circadi- depression. The timing of dim light melatonin onset (DLMO, threshold an principles to determine individual timing of a of 3 pg/mL) is strongly correlated with the morningness-eveningness score treatment: the important therapeutic consequences on the Morning-Eveningness Questionnaire (MEQ) in patients with winter are higher remission rates to light therapy than depression (r=--0.81, N=35, P<0.001). The wide spread of chronotype is when prescribing the same clock time for everyone. similar to that found in the general population. Redrawn from unpub- lished data related to reference 13 with permission from M. Terman. We thus can use an everyday, straightforward de- termination of sleep midpoint (calculated from the time on free days are obviously chosen because this MEQ, MCTQ, or a week of sleep logs) to provide a is the comfortable ψ for that individual. The sleep rough estimate of an individual’s internal clock time. pattern gives a first simple estimate of a person’s There are provocative indications that the same phase, commonly known as chronotype, which phase-advancing strategy with early morning light, ranges from extreme early birds to extreme late- known to be beneficial for treatment of winter de- night owls. The most reliable phase marker with pression is key to sustained improvement in non- respect to sleep timing is the sleep midpoint (sleep seasonal depression. Intriguing data from a pre- onset time -- wakeup time / 2). Questionnaires about liminary study in depressed bipolar patients (on sleep preferences have long been used to better lithium) treated by sleep deprivation, indicates that quantify this characteristic. The classic Horne-Ost- morning light therapy individually timed to max- berg Morning-Eveningness Questionnaire (MEQ)10 imize a circadian rhythm phase advance not only is now available as an online self-assessment (Au- sustained the rapid sleep deprivation response, but toMEQ) with personalized feedback (www.cet.org). patients continued to improve (not found with light Recently, the Munich Chronotype Questionnaire given at 11 AM to all) (F. Benedetti, personal com- (MCTQ) has been developed,5 validated against sleep munication). logs, and also automated (www.imp-muenchen.de); it is available so far in English, German, Spanish, Amplitude Italian, French, and Dutch. Objective measurement The amplitude of a circadian oscillation is an im- of rest-activity cycle timing can be made with ac- portant characteristic. When amplitude is low, a timetry.11 zeitgeber can theoretically elicit larger phase shifts To go a step further, beyond sleep phase to inter- than when amplitude is high. Measuring amplitude nal clock phase, we need a good output of the circa- of clock function is, however, rather difficult in dian clock that can be reliably measured. The pineal practice, and there is only indirect evidence from hormone melatonin fulfils this role admirably.12 All circadian rhythms of melatonin or temperature that species that secrete melatonin do so at night; light amplitude can be diminished (by very specific tim- immediately suppresses its synthesis. It has been ing of a light pulse) or augmented (by increasing well established that if samples (saliva or blood or light intensity/duration).
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wakefulness. Thus, actimetry may be used to doc- ument changes in entrainment state related to ef- ficacy of a given treatment (whether pharmaceuti- cal or not). Indeed, a recent study in patients with seasonal affective disorder showed a delayed rest- activity cycle and low activity when depressed, with increased activity and better synchronization fol- lowing clinical improvement with light therapy.15 There has not yet been very much long-term activ- ity monitoring in major depression. These patients may not show a single or consistent abnormality, but rather, large interindividual differences in their rest-activity cycle patterns. This is what we are see- ing in an ongoing study of 6 weeks actimetry in ma- Figure 4. Long-term activity jor depression during pregnancy (Figure 4). This
Successive days monitoring in depression. An unstable rest-activity cycle is indicative of poor cir- example of an activity moni- cadian entrainment, and may contribute to some tor, the size of a wristwatch aspects of the illness. (Cambridge Neurotechnolo- gy®), is shown being worn Over the years, validation of actimetry by sleep on the nondominant hand. EEG in healthy subjects led to the development of Movements are collected at analysis programs for actimetric “sleep” that pro- 1- to 2-minute intervals and vide an estimate of a number of classic parameters stored until readout. The circadian rest-activity cycle (sleep onset, wake-up time, wake bouts, sleep effi- is double plotted, ie, day 1 ciency, etc). Analysis programs for circadian vari- and 2 on one line, day 2 and ables (of which there are a variety) can provide an 3 on the next line, etc. This estimate of, eg, relative amplitude (maximum-min- double plot makes visualiza- imum), intradaily stability (estimates of strength of tion of shifted and irregular rhythms easier. On the ver- coupling to zeitgebers), and interdaily variability tical axis of each line is the (degree of fragmentation).16 amount of activity per unit In conclusion, actimetry is an easy, noninvasive, time: the higher the bar the and relatively inexpensive tool that deserves more greater the activity (blacker). Upper panel: 6 weeks’ record- use in the clinic. It provides objective verification of ing of a control pregnant chronotype (time of going to bed and waking up), woman with regular sleep and documents changes in sleep-wake patterns dur- patterns. Middle and lower ing illness and following treatment. A minimum panels: examples of irregular of 1 week’s recording is recommended to compare rest-activity cycles and dis- 24 1224 12 24 turbed nights in pregnant the pattern of work and free days and to reduce vari- women suffering from major ability. Time of day (h) depression (Wirz-Justice, un- published data). Why circadian clock characteristics are not easy to see How can we measure the circadian rest-activity cycle? Measuring the rest-activity cycle is a first step in looking at 24-h patterns of behavior. However, to Actimetry is a noninvasive technique for ambula- dig deeper and look at endogenous circadian pace- tory monitoring of rest-activity (which is not neces- maker characteristics requires special techniques sarily always congruous with sleep-wake) cycles.11 and validated markers. It has long been recognized It is the equivalent of the running wheel for ham- that the overtly measured rhythm of a given vari- sters and mice in human circadian biology, with the able over 24 hours is not only determined by the same advantages of measurement over longer pe- biological clock and sleep homeostat, but also by riods of time than in sleep research (1 to 2 nights’ zeitgebers such as light, or behavior, which can polysomnography). Additionally, 24-hour monitor- have direct or indirect effects on many functions, ing can reveal unusual patterns of rest and activi- so-called “masking.”17 Some examples are shown in ty that provide information quite different from the Figure 5 (next page).18-20 sleep EEG (eg, timing and duration of daytime naps). Masking modifies the pattern of daily rhythms Familiarity with the animal literature on abnormal that are measured in naturalistic environments. As rest-activity cycles and the formal properties of the shown in Figure 5, postural changes rapidly affect mammalian circadian system (eg, τ , ψ, see above)3 thermoregulation,18 as does sleep,19 thus giving a can help interpret the observed phenomena. How- false estimate of amplitude and phase when look- ever, it must be clearly recognized that actimetry ing at the complete 24-hour measured curves. Light does not necessarily reflect the underlying circadi- in the evening can suppress melatonin or delay its an clock characteristics. onset, even at lower intensities (>100 lux).20 Thus, in A tenet of human chronobiology is that adequate order to elucidate the characteristics of the endoge- entrainment means better sleep and higher alert- nous circadian pacemaker, it has been necessary to ness, and better cognitive state and mood during develop protocols that control for masking effects.
How to measure circadian rhythms in humans – Wirz-Justice MEDICOGRAPHIA, VOL 29, No. 1, 2007 87 U PDATE