U PDATE 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]) 84 MEDICOGRAPHIA, VOL 29, No. 1, 2007 How to measure circadian rhythms in humans – Wirz-Justice U PDATE 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 N 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