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Overview of Circadian
Rhythms
Martha Hotz Vitaterna, Ph.D., Joseph S. Takahashi, Ph.D.,
and Fred W. Turek, Ph.D.
The daily light-dark cycle governs rhythmic changes in the behavior and/or physiology of most species. Studies have found that these changes are governed by a biological clock, which in mammals is located in two brain areas called the suprachiasmatic nuclei. The circadian cycles established by this clock occur throughout nature and have a period of approximately 24 hours. In addition, these circadian cycles can be synchronized to external time signals but also can persist in the absence of such signals. Studies have found that the internal clock consists of an array of genes and the protein products they encode, which regulate various physiological processes throughout the body. Disruptions of the biological rhythms can impair the health and well-being of the organism. KEY WORDS: circadian rhythm; time of day; biological regulation; biological adaptation; temperature; light; hypothalamus; neural cell; gene expression; mutagenesis; sleep disorder; physiological AODE (effects of alcohol or other drug use, abuse, and dependence)
O
ne of the most dramatic features of the world in which we live is the cycle of day and night. Cor respondingly, almost all species exhibit daily changes in their behavior and/or physiol ogy. These daily rhythms are not simply a response to the 24-hour changes in the physical environment imposed by the earth turning on its axis but, instead, arise from a timekeeping system within the organism. This timekeeping system, or biological “clock,” allows the organism to anticipate and prepare for the changes in the physi cal environment that are associated with day and night, thereby ensuring that the organism will “do the right thing” at the right time of the day. The biological clock also provides internal temporal organiza tion and ensures that internal changes take place in coordination with one another. The synchrony of an organism with both its external and internal environ ments is critical to the organism’s well- being and survival; a lack of synchrony
between the organism and the external environment may lead to the individ ual’s immediate demise. For example, if a nocturnal rodent were to venture from its burrow during broad daylight, the rodent would be exceptionally easy prey for other animals. Similarly, a lack of synchrony within the internal envi ronment might lead to health problems in the individual, such as those associated with jet lag, shift work, and the accom panying sleep loss (e.g., impaired cog nitive function, altered hormonal func tion, and gastrointestinal complaints). The mechanisms underlying the biological timekeeping systems and the potential consequences of their failure are among the issues addressed by researchers in the field of chronobiology.^1 In its broadest sense, chronobiology encompasses all research areas focusing on biological timing, including high- frequency cycles (e.g., hormone secre tion occurring in distinct pulses through-
MARTHA HOTZ VITATERNA, PH.D., is a senior research associate in the Center for Functional Genomics, Northwestern University, Evanston, Illinois.
JOSEPH S. TAKAHASHI, PH.D., is the director of the Center for Functional Genomics, the Walter and Mary E. Glass Professor in the Department of Neurobiology and Physiology, and an investigator at the Howard Hughes Medical Institute, Northwestern University, Evanston, Illinois.
FRED W. TUREK, PH.D., is the director of the Center for Sleep and Circadian Biology and is the Charles T. and Emma H. Morrison Professor in the Department of Neurobiology and Physiology, Northwestern University, Evanston, Illinois.
(^1) For a definition of this and other technical terms used in this article and throughout this issue of the journal, please see glossary, p. 92.
Vol. 25, No. 2, 2001 85
out the day), daily cycles (e.g., activity and rest cycles), and monthly or annual cycles (e.g., reproductive cycles in some species). Among these interrelated areas of chronobiology, this article focuses on one frequency domain—the daily cycles known as circadian rhythms. (The term “circadian” derives from the Latin phrase “circa diem,” which means “about a day.”) Although virtually all life forms— including bacteria, fungi, plants, fruit flies, fish, mice, and humans—exhibit circadian rhythms, this review is primar ily limited to the mammalian system. Other animals are discussed only in cases in which they have contributed to the understanding of the mammalian system, particularly in studies of the molecular genetic makeup of the time- keeping system. (For comparative dis cussions of other nonmammalian model systems that have contributed to the depth of understanding of circadian rhythmicity in mammals, the reader is referred to Wager-Smith and Kay 2000.) Overall, this article has the following major objectives: (1) to provide a highly selective historical overview of the field, (2) to review characteristic properties of circadian rhythms, (3) to define the structural components and the molecu lar genetic mechanisms comprising the biological clock, and (4) to explore the health effects of biological rhythms.
Historical Overview of Chronobiology
Researchers began studying biological rhythms approximately 50 years ago. Although no single experiment serves as the defining event from which to date the beginning of modern research in chronobiology, studies conducted in the 1950s on circadian rhythmicity in fruit flies by Colin Pittendrigh and in humans by Jürgen Aschoff can be con sidered its foundation. The area of sleep research, which also is subsumed under the field of chronobiology, evolved some- what independently, with the identifi cation of various sleep stages by Nathaniel Kleitman around the same time (Dement 2000). The legacies of these pioneers continue today with the advancement of the fields they founded.
The roots of the study of biological rhythms, however, reach back even fur ther, to the 1700s and the work of the French scientist de Mairan, who pub lished a monograph describing the daily leaf movements of a plant. De Mairan observed that the daily raising and low-
Virtually all
life forms—
including bacteria,
fungi, plants, fruit
flies, fish, mice,
and humans—
exhibit circadian
rhythms.
ering of the leaves continued even when the plant was placed in an interior room and thus was not exposed to sunlight. This finding suggested that the move ments represented something more than a simple response to the sun and were controlled by an internal clock.
Characteristic Properties of Circadian Rhythms
De Mairan’s apt observations illustrate one critical feature of circadian rhythms— their self-sustained nature. Thus, almost all diurnal rhythms that occur under natural conditions continue to cycle under laboratory conditions devoid of any external time-giving cues from the physical environment (e.g., under constant light or constant darkness). Circadian rhythms that are expressed in the absence of any 24-hour signals from the external environment are called free running. This means that the rhythm is not synchronized by any cyclic change in the physical environment. Strictly speaking, a diurnal rhythm should not be called circadian until it has been shown to persist under constant envi ronmental conditions and thereby can be distinguished from those rhythms
that are simply a response to 24-hour environmental changes. For practical purposes, however, there is little reason to distinguish between diurnal and cir cadian rhythms, because almost all diurnal rhythms are found to be circa dian. Nor is a terminology distinction made among circadian rhythms based on the type of environmental stimulus that synchronizes the cycle. The persistence of rhythms in the absence of a dark-light cycle or other exogenous time signal (i.e., a Zeitgeber) clearly seems to indicate the existence of some kind of internal timekeeping mechanism, or biological clock. How- ever, some investigators have pointed out that the persistence of rhythmicity does not necessarily exclude the possibility that other, uncontrolled cycles gener ated by the Earth’s revolution on its axis might be driving the rhythm (see Aschoff 1960). The hypothesis that such uncon trolled geomagnetic cues might play a role in the persistence of rhythmicity can be refuted by a second characteris tic feature of circadian rhythms: These cycles persist with a period of close to, but not exactly, 24 hours. If the rhythms were exogenously driven, they should persist with a period of exactly 24 hours. The seeming imprecision is an impor tant feature of rhythmicity, however. As Pittendrigh (1960) demonstrated, the deviation from a 24-hour cycle actually provides a means for the internal time- keeping system to be continuously aligned by and aligned to the light-dark environment. This continuous adjust ment results in greater precision in controlling the timing, or phase, of the expressed rhythms, because little drift is allowed to occur before the rhythm is “reset” to the correct phase. A third characteristic property of circadian rhythms is their ability to be synchronized, or entrained, by external time cues, such as the light-dark cycle. Thus, although circadian rhythms can persist in the absence of external time cues (meaning that they are not driven by the environment), normally such cues are present and the rhythms are aligned to them. Accordingly, if a shift in external cues occurs (e.g., following travel across time zones), the rhythms
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in that a change in temperature can affect the phase of a cycle without sub stantially altering the rate of cycling. This means that the cycle may start at an earlier or later-than-normal time but still have the same length. On the one hand, this ability of the internal clock’s pacemaker to compensate for changes in temperature is critical to its ability to predict and adapt to environmental changes, because a clock that speeds up and slows down as the temperature changes would not be useful. On the other hand, temperature compensation also is rather puzzling, because most kinds of biological processes (e.g., bio chemical reactions in the body) are accel erated or slowed by temperature changes. Ultimately, this riddle has provided a clue to the nature of the internal clock— that is, the fact that circadian rhythms have a genetic basis. Such a program of gene expression would be more resistant to temperature alteration than, for exam ple, a simple biochemical reaction. Two final properties of circadian rhythms also provide important hints of the rhythms’ makeup. One of these properties is the rhythms’ ubiquity in nature: Circadian rhythms exist in a broad array of biological processes and organisms, with similar properties and even similar phase-response curves to light. The other property is that circa dian rhythms appear to be generated at the cellular level, because the rhythms of unicellular organisms (e.g., algae or the dinoflagellate Gonyaulax ) are much the same as rhythms of highly complex mammals. Both of these observations suggest that a cycle in the activation (i.e., expression) of certain genes might underlie the timekeeping mechanism.
The Anatomical Organization of the Internal Clock
Although studies of unicellular organisms point to the cellular nature of the system generating circadian rhythms, the circa dian pacemaker in higher organisms is located in cells of specific structures of the organism. These structures include certain regions of the brain (i.e., the optic and cerebral lobes) in insects; the eyes
in certain invertebrates and vertebrates; and the pineal gland, which is located within the brain, in nonmammalian verte brates. In mammals, the circadian clock resides in two clusters of nerve cells called the suprachiasmatic nuclei (SCN), which are located in a region at the base of the brain called the anterior hypothalamus.
In mammals,
the circadian clock
resides in two
clusters of nerve
cells called the
suprachiasmatic
nuclei (SCN).
The role of the SCN was demon strated by the landmark discovery in the early 1970s that by damaging (i.e., lesioning) the SCN in rats, researchers could disrupt and abolish endocrine and behavioral circadian rhythms (for a review, see Klein et al. 1991). Further- more, by transplanting the SCN from other animals into the animals with the lesioned SCN, investigators could restore some of the circadian rhythms. Finally, the SCN’s role as a master pacemaker regulating other rhythmic systems was confirmed by similar studies in hamsters, which demonstrated that the restored rhythms exhibited the clock properties (i.e., the period, or phase, of the rhythm) of the donor rather than of the host (Ralph et al. 1990). The discovery that the SCN is the site of primary regulation of circadian rhythmicity in mammals gave researchers a focal point for their research: if one wanted to understand 24-hour timekeeping, one needed to study the clock in the SCN. Recently, however, researchers have been surprised to find that circadian rhythms could persist in isolated lungs, livers, and other tissues grown in a cul ture dish (i.e., in vitro) that were not under the control of the SCN (Yamazaki
et al. 2000). These observations indi cate that most cells and tissues of the body may be capable of modulating their activity on a circadian basis. Such findings do not, however, diminish the central role played by the SCN as the master circadian pacemaker that some- how coordinates the entire 24-hour temporal organization of cells, tissues, and the whole organism. The physio logical mechanisms underlying this coordination include signals emitted by the SCN that act on other nerve cells (i.e., neural signals) or which are also distributed through the blood to other organs (i.e., neurohormonal signals). To date, however, the characteristics of the circadian signal itself—that is, the specific manner in which the SCN “talks” to the rest of the body—remain unknown (see Stokkan et al. 2001). Although the effects of SCN lesions on numerous rhythms have been eluci dated, their effects on sleep are less clear. Thus, SCN lesions clearly disrupt the consolidation and pattern of sleep in rats but have only minimal effects on the animals’ amount of sleep or sleep need (Mistlberger et al. 1987). For this and other reasons, researchers have pos tulated that sleep is subject to two essentially independent control mecha nisms: (1) the circadian clock that mod ulates the propensity for sleep and (2) a homeostatic control that reflects the duration of prior waking (i.e., “sleep debt”). Recently, however, studies in squirrel monkeys found that SCN lesions can affect the amount of sleep. Moreover, sleep studies in mice carrying changes (i.e., mutations) in two of the genes influencing circadian cycles (i.e., the DBP and Clock genes) indicated that these mutations resulted in changes in sleep regulation (Naylor et al. 2000; Franken et al. 2000). Both of these observations raise the intriguing possi bility that the homeostatic and circa dian controls may be more interrelated than researchers previously thought.
Molecular Genetics of Circadian Rhythms
As discussed previously, the properties of circadian clocks suggested cyclic
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Overview of Circadian Rhythms
changes in the expression of certain genes as a possible mechanism underlying the internal pacemaker. This hypothe sis was supported by the demonstration in a number of species that the expression of genes and the production of proteins encoded by those genes were required for normal clock function. Nevertheless, a completely different experimental approach ultimately led to the identifi cation of molecular circadian clock components. Researchers used chemical agents to introduce numerous, random
mutations into the DNAs of the fruit fly, Drosophila melanogaster, and of the filamentous fungus Neurospora. The resulting mutant organisms then were screened for rhythm abnormalities. This mutagenesis approach led to the identi fication of the first circadian clock mutants, which were called period ( per ) and frequency ( frq , pronounced “freak”). The genes that carried the mutations in these organisms were cloned in the 1980s (for a review, see Wager-Smith and Kay 2000). However, considerable
frustration ensued as researchers sought to isolate the equivalent genes in mam mals (i.e., mammalian homologs). Finally, in the early 1990s, researchers began a similar mutagenesis screening approach in the mouse and described the first mouse circadian mutation, called Clock, in 1994 (see King and Takahashi 2000). In 1997 the gene affected by this mutation became the first mammalian circadian clock gene to be cloned (King and Takahashi 2000). Like the mutants of the Per and Frq genes, the altered Clock gene both affected the free-running rhythm period (i.e., lengthened the period) and caused a loss of persistence of circadian rhythms under constant environmental conditions. Both the
Table 1 Mammalian Circadian Clock Genes; the Corresponding Genes in the Fruit Fly, Drosophila ; and the Effects of Changes (i.e., Mutations) in Those Genes on the Behavior (i.e., Phenotype) of the Affected Animals
Mouse Drosophila Gene Alias Gene Mutant Phenotype
- Clock dClock Lengthened period; loss of persistent rhythmicity in constant conditions
mPer1 period Reduced amplitude, shortened period, or loss of rhythm
mPer2 period Shortened period, loss of rhythm
mPer3 period Modest shortening of period
CK Ιε tau Shortened period in (hamster) hamster mutants
mCry1 dcry Animals lacking the mCry2 mCry1 gene (i.e., mCry knockouts) have short ened period; mCry knockouts have length ened period; animals lacking both genes (i.e., double knockouts) have a loss of rhythm
BMAL1 MOP3 cycle Loss of rhythm
? mTim timeless Role in mammals is not clear
? DBP Modest lengthening of period
NOTE: Asterisk (*) indicates that a key role for the gene in timekeeping has been demonstrated by the pheno type of a mutant.
doubletime
C lock mutant in mice and the Per mutant in flies were the first animals of their respective species identified using such a mutagenesis approach in which the mutation manifested as altered behavior rather than an altered physiological process. Since the discovery of the Clock gene in mice, the list of circadian clock genes identified in mammals has grown in a remarkably short period of time (see table 1). For example, researchers have identified not one, but three mam malian genes that correspond to the per gene in both their structure (i.e., nucleotide sequence) and their function (King and Takahashi 2000; Lowrey and Takahashi 2000). Some of the pro- posed circadian clock genes have been identified solely based on their similarity in sequence to Drosophila clock genes and have not been confirmed to have clock function based on an examination of the behavior of the corresponding mutants. Nevertheless, the findings to date clearly indicate the outline of a pacemaker that is based on a feedback cycle of gene expression (see figure 2).
Importance of the Circadian Clock for Human Health and Well-Being
Nearly all physiological and behavioral functions in humans occur on a rhythmic basis, which in turn leads to dramatic diurnal rhythms in human performance
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Overview of Circadian Rhythms
sues. To further elucidate the regulation of circadian rhythms, researchers need a better understanding of the nature of circadian signal output from the SCN and of how these output signals may be modified once they reach their target systems. Such an enhanced understanding also would allow for a better delineation of the importance of normal temporal organization for human health and dis ease. The finding that two major causes of death—heart attacks and strokes— show time-of-day variation in their occurrence is a case in point. If scien tists knew more about the mechanisms responsible for the rhythmicity of these disorders, they might be able to iden tify more rational therapeutic strategies to influence these events. Finally, given that dramatic changes occur in the cir cadian clock system with advanced age, these changes may underlie, or at least exacerbate, the age-related deterioration in the physical and mental capabilities of older adults.
Conclusions
Although researchers in just the past few years have made great advances in understanding the molecular basis of circadian rhythmicity, this progress
builds on extensive research carried out in many laboratories during the past 50 years. Within the same period, other researchers in numerous laboratories have elucidated the critical role played by the SCN in the regulation of circa dian rhythmicity in mammals and per- haps other vertebrates. (For more infor mation on these findings and their rele vance, the reader can refer to a variety of resources on the World Wide Web, some of which are listed in table 2.) Most animals are content to obey their SCN and let it orchestrate the expression of a multitude of circadian rhythms. Humans, however, have a mind of their own and often use this mind to disobey their “internal clock”—for example, with an increasing tendency toward 24-hour availability for busi ness. The potential consequences of such an increasingly 24-hour on-call lifestyle are unknown at this point, but the evidence does not bode well. The challenge for researchers and clinicians now is to determine not only the cause but also the consequences for human health and disease of disruptions in the temporal organization of the cir cadian system. These issues also include the question of what role alcohol may play in the disruption of normal circa dian rhythms and the biological clock.
Table 2 Chronobiological Resources on the World Wide Web
Web Site Description http://www.nwu.edu/ccbm/ Web site of Northwestern University’s Center for Sleep and Circadian Biology
http://www.sleepquest.com/ Information site of William Dement’s Sleep Research Center
http://www.med.stanford.edu/school/ Narcolepsy site created by Emmanuel Psychiatry/narcolepsy Mignot at Stanford University
http://www.sleepfoundation.org/ Web site of the National Sleep Foundation
http://www.srbr.org/ Web site of the Society for Research on Biological Rhythms
http://www.cbt.virginia.edu/ Web site of the Center for Biological Timing at the University of Virginia
http://www.hhmi.org/grants/lectures Web site providing Howard Hughes Medical Institute Holiday Lectures
This question is addressed in more detail in this special issue of Alcohol Research & Health. Drs. Wasielewski and Holloway review ways in which alcohol and the body’s circadian rhythm interact, using body temperature as an index of circa dian rhythm function. The sleep-wake cycle, which constitutes a central aspect of circadian rhythms in particular, is sub ject to modification by alcohol; alcohol’s effects on the sleep of nonalcoholics and alcoholics are discussed by Drs. Roehrs and Roth and by Dr. Brower, respectively. As indicated in this article, disturbances of the normal circadian rhythmicity can result in serious health consequences, including psychiatric disorders, such as depression. At the same time, psychoac tive drugs, such as antidepressants, also have chronobiological effects. Dr. Rosenwasser explores those associations and discusses alcohol’s effects in human and animal models of depression. Other influences of alcohol on the biological clock may be even more subtle and remain rather speculative, such as the consequences of prenatal alcohol expo- sure, which is discussed by Drs. Earnest, Chen, and West. Finally, not only may alcohol consumption affect circadian rhythms, but circadian factors, such as the light-dark cycle, may also influence alcohol consumption. This topic is dis cussed by Drs. Hiller-Sturmhöfel and Kulkosky. Together, these articles offer readers insight into the interesting and complex interactions that exist between alcohol and the circadian rhythms that govern much of the behavior and well- being of all organisms, including humans. ■
References
ASCHOFF, J. Exogenous and endogenous compo nents in circadian rhythms. Cold Spring Harbor Symposia on Quantitative Biology: Volume XXV. Biological Clocks. New York: Cold Spring Harbor Press, 1960. pp. 11–28. BRUNELLO, N.; ARMITAGE, R.; FEINBERG, I.; ET AL. Depression and sleep disorders: Clinical relevance, economic burden and pharmacological treatment. Neuropsychobiology 42:107–119, 2000. DEMENT, W.C. History of sleep physiology and medicine. In: Kryer, M.H.; Roth, T.; and Dement, W.C., eds. Principles and Practice of Sleep Medicine. 3d ed. Philadelphia: W.B. Saunders, 2000.
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G L O S S A R Y
Every scientific field has its specific terminology; the scientific area of biological rhythms and sleep is no exception. This glossary defines some of the terms that readers may encounter in this article and throughout this special issue of Alcohol Research & Health.
Chronobiology: A subdiscipline of biology concerned with the timing of biological events, especially repetitive or cyclical phenomena, in individual organisms. Circadian: A term derived from the Latin phrase “circa diem,” meaning “about a day”; refers to biological variations or rhythms with a cycle of approximately 24 hours. Circadian rhythms are self-sustaining (i.e., free running ), meaning that they will persist when the organism is placed in an environ ment devoid of time cues, such as constant light or constant darkness. For comparison, see diurnal, infradian, and ultradian. Circadian time (CT): A standardized 24-hour notation of the phase in a circadian cycle that represents an estimation of the organism’s subjective time. CT 0 indicates the begin ning of a subjective day, and CT 12 is the beginning of a subjective night. For example, for a nocturnal rodent, the beginning of a subjective night (i.e., CT 12) begins with the onset of activity, whereas for a diurnal species, CT 0 would be the beginning of activity. For comparison, see Zeitgeber time. DD: A conventional notation for an environment kept in con tinuous darkness (as opposed to a light-dark cycle). For comparison, see LD. Diurnal: Varying with time of day. Diurnal rhythms may per sist when the organism is placed in an environment devoid of time cues, such as constant light or constant darkness. Therefore, diurnal variations can be either light driven or clock driven. For comparison, see circadian. Entrainment: The process of synchronization of a timekeeping mechanism to the environment, such as to a light-dark cycle, or LD. For comparison, see free running. Free running: The state of an organism (or rhythm) in the absence of any entraining stimuli. Typically, subjects are kept in constant dim light or constant darkness to assess their free-running rhythms. For comparison, see entrainment. Infradian: A term derived from the Latin phrase “infra diem,” meaning “less than a day”; refers to biological cycles that last more than 1 day and, therefore, have a frequency of less than one per day. For comparison, see circadian and ultradian. LD: Conventional notation for a light-dark environmental cycle; the numbers of hours of light and dark are typically presented separated by a colon. For example, LD 16: denotes a cycle consisting of 16 hours of light and 8 hours of dark. For comparison, see DD. Masking: The obscuring of the “true” state of a rhythm by conditions that prevent its usual expression. Usually, the phase of an entrained rhythm or the absence of entrainment
(e.g., in an animal that is unable to entrain because of some defect) is said to be masked by a light cycle. For example, the aversion of a nocturnal rodent to bright light results in its activity onset appearing to coincide with the absence of light, or “lights off,” when the animal actually has been awake for hours. For comparison, see entrainment. Nonrapid eye movement (NREM) sleep: Sleep stages that include the “deeper” stages of sleep in which dreaming typi cally does not occur. Also referred to as slow-wave sleep. For comparison, see rapid eye movement sleep. Phase shift: A change in the phase of a rhythm. This change can be measured by observing a change in the timing of a phase reference point (e.g., activity onset or the nocturnal rise in the release of the hormone melatonin) from the tim ing expected based on previous, free-running cycles. Phase shifts may be either advances (i.e., the phase reference point occurs earlier than normal) or delays (i.e., the phase refer ence point occurs later than normal). Phase-response curve (PRC): A graphical summary of the phase shifts produced by a particular manipulation, such as a light pulse or a pharmacological treatment, as a function of the phase (i.e., circadian time ) at which the manipulation occurs. Defining the PRC to light has enabled researchers to understand and predict how entrainment to light cycles is accomplished. Rapid eye movement (REM) sleep: A stage of light sleep char acterized by rapid eye movements and associated with dreaming. Also called paradoxical sleep. For comparison, see nonrapid eye movement sleep. Suprachiasmatic nucleus or nuclei (SCN): A cluster of nerve cells located in the brain region called the hypothalamus that is responsible for generating and coordinating circadian rhythmicity in mammals. Ultradian: A term derived from the Latin phrase “ultra diem,” meaning “more than a day”; refers to biological cycles that last less than 1 day and, therefore, have a frequency of more than one per day. For comparison, see circadian and infradian. Zeitgeber: A German word literally meaning “time-giver.” A time cue capable of entraining circadian rhythms. Light rep resents the most important Zeitgeber. Zeitgeber time (ZT): A standardized 24-hour notation of the phase in an entrained circadian cycle in which ZT 0 indi cates the beginning of day, or the light phase, and ZT 12 is the beginning of night, or the dark phase. For comparison, see circadian time.
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