Circadian and Circannual Rhythms and Hormones

Circadian and Circannual Rhythms and Hormones L. Ruggiero and R. Silver, Barnard College and Columbia University, New York, NY, USA ã 2010 Elsevier Ltd. All rights reserved.

Introduction and Definitions What are Circadian Rhythms? Circadian rhythms are daily cycles in behavior and physiology, driven by an internal time-keeping system. These rhythms are important because they allow organisms to coordinate their activities with regularly recurring events in the environment, such as lighting, the timing of food availability, the presence of predators, and or the yearly cycles of changing seasons. Circadian rhythms are different from externally driven rhythms in that they persist even in the absence of environmental cues. For example, mice kept in constant darkness display rhythmic locomotor behavior on the basis of the timing of their internal clocks. Circadian rhythms are also present in physiology as seen in melatonin secretion by the mammalian pineal gland. If an animal goes into a deep cave with no cues to time of day, the daily cycle of changes in melatonin persists, with peaks starting at about the time that was previously night. This persisting rhythm is said to be freerunning and has the periodicity of the internal clock. While circadian rhythms occur in the absence of external cues, they are synchronized to the local environment; the process of synchronization is called entrainment. In order for synchronization to occur, the internal timekeeper, or clock, must be set to local time. The pacemaker’s signal for local time can be one of a number of environmental cues, called zeitgebers, and include light, food availability, and social cues. The output signal from this clock can synchronize other tissues of the brain and body. This process can be seen in the example of melatonin secretion. In mammals, light (input) is transmitted to a brain clock (the timekeeper), which sends information about the light–dark cycle to the pineal gland. In the presence of light in the environment, the secretion of melatonin (output) is suppressed and the rhythm of melatonin is shifted. While we have focused on pineal melatonin secretion in this example, circadian rhythms can be seen in almost any number of physiological and behavioral processes one cares to measure. Circadian rhythms in behavior are observed by examining activity under constant conditions and in response to environmental cues. In order for entrainment to occur, the timing of physiological and behavioral rhythms must be shifted. Some animals have biological clocks that freerun with a period longer than 24 h. For example, the human endogenous clock tends to be slightly longer than 24 h. This is analogous to a watch that runs too slowly. It has to shift forward each day to stay entrained to local time.

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Other animals, such as mice, have an internal clock with a period shorter than 24 h, like a watch that runs too fast. These clocks must shift back each day to stay entrained to local time. In both cases, the internal clocks must adjust to local time on a daily basis. The resetting of the clock by an external cue is known as phase-shifting and occurs when a cue alters the temporal relationship between the timing of the stimulus and the phase of the endogenous rhythm. The degree and direction of the shift depends on both the time at which the stimulus is presented and its strength. Examples of circadian rhythms of behavior in the absence and presence of external cues can be seen in Figure 1. Criteria for Identification of Endogenous Rhythms To evaluate whether any particular response is under circadian control, three criteria must be met. First, the response must persist under constant conditions, that is, without any external cues from the environment, such as the example given earlier, in which an animal in a cave continued rhythmic secretion of melatonin. Second, the phase of the response must be reset by exposure to an external stimulus, or zeitgeber. Third, it must exhibit temperature compensation such that the rhythm is stable over a range of temperatures. This is important as many chemical processes are faster at high temperatures and slower in the cold. The circadian timekeeping mechanism compensates for fluctuations in temperature and circadian rhythms are not changed by thermal fluctuations. Internal clocks are important not only for synchronizing the organism to daily time, but also for annual cycles, thereby helping to anticipate seasonal changes. Circannual rhythms are biological activities that occur each year and are synchronized with the seasons. Such seasonal activities include the timing of mating in fall or spring, optimized so that birth coincides with the greatest availability of food, and hibernation in the winter, which occurs as a way to conserve energy. This seasonal clock, like the circadian clock, functions in constant conditions; however, it too needs to be reset by environmental cues. Similar to circadian clocks that run close to 24 h, circannual clocks run close to 12 months. For example, ground squirrels have endogenous circannual clocks that control reproductive processes. In the absence of external cues, this clock has a period of about 10.5 months. Because it is slightly shorter than 12 months, it relies on external cues, such as day length, for resetting.

Circadian and Circannual Rhythms and Hormones

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Figure 1 Hypothetical actograms depicting wheel running activity in a nocturnal animal. Black lines indicate wheel revolutions and signify periods of activity. In constant conditions (complete darkness), an animal displays free-running behavior based on its endogenous period. In this example, the animal’s period is shorter than 24 h and so it begins its activity slightly earlier each day (left). When exposed to a pulse of light (white circle) an animal will shift its activity so that its next cycle of activity will begin based on the time of light exposure (center). An animal will entrain to a light/dark cycle so that it is active when the lights are off (black bar) and inactive when the lights are on (white bar) (right).

Evolution and phylogeny of circadian rhythms

Almost all living organisms, from single-celled bacteria to primates, display rhythmic cycles of biological activity with a period close to 24 h. This system is of great importance as it controls a number of biological functions including, bioluminescence in cyanobacteria, pupal eclosion in insects, and hormone production and secretion and the sleep/wake cycle in mammals. The simplest clock mechanisms are found in prokaryotic cyanobacteria and the fungus Neurospora. Clock machinery is found in the fruit fly, Drosophila melanogaster, and more complex components exist in vertebrates. Though differences in exist in complexity across species, all organisms have an internal pacemaker that detects and utilizes cues from the environment to synchronize physiological and behavioral outputs. Circadian rhythms evolved to allow an organism to optimize its biological processes on the basis of predictable, recurring, daily, and seasonal events. For example, circadian rhythms in the earliest cells protected cellular processes, such as DNA replication, against the damaging effects of UV light during the day. In plants and some prokaryotes, an internal time-keeping system was needed to temporally separate the processes of photosynthesis, which relies on light from nitrogen fixation, which occurs during the night. In higher order animals, rhythms such as the sleep/wake cycle have likely evolved as way of conserving energy. Also, rhythms in organ systems, such as the digestive system, might have evolved to use and obtain nutrients most efficiently.

Mechanisms underlying rhythms in behavior and physiology

Rhythms in behavior and physiology are synchronized to daily and seasonal environmental changes. In some cases, animals rely solely on environmental cues to drive seasonal changes. For example, when days become longer, Syrian hamsters exhibit enlarged gonads and are reproductively competent. When the days shorten (predicting winter and decreased resources), the gonads regress and

reproductive hormone levels decrease. For this regression to occur, the Syrian hamster must first experience a period of long days. This suggests that the change in day length acts as a cue to the prevailing conditions for reproduction. While Syrian hamsters rely on changes in the environment to predict the seasons, other animals rely on an endogenous clock. Temporal regulation of hormones also allows an organism to detect seasonal changes in the absence of environmental cues. While expressed as an endogenous rhythm, melatonin levels are synchronized with changes in day length; as the duration of night decreases, so does the duration of melatonin production. This mechanism enables seasonally breeding animals to anticipate seasonal changes and to time reproductive processes. By timing their breeding to in accordance with changes in day length, animals are able to ensure that offspring will be born at a time when the environment provides the best chances for survival. For example, shortening of days stimulates breeding in sheep, but inhibits breeding in Syrian hamsters. Sheep breed in the fall months, while hamsters breed in the spring, though both give birth in the spring during a time when the environment is favorable for their young.

The brain has a circadian clock and the eye provides entraining cues

The phenomena associated with daily and seasonal cycles suggest that there is an internal clock that is synchronized to the local environment by light. In mammals, this master clock is located in the suprachiasmatic nuclei (SCN) of the hypothalamus. The SCN is a bilateral structure that lies above the optic chiasm, comprising about 10 000 neurons in each side of the nucleus. The SCN is a heterogeneous structure made of different types of cells. Circadian rhythms in activity occur within individual cells in the dorsal or shell area of the SCN. Even if these cells are dispersed and thus disconnected from each other, they still show circadian rhythms of electrical activity. In order for the SCN tissue as a whole to produce a coordinated output, however, the individual cells need to be

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synchronized to each other. Synchrony is achieved by several mechanisms acting simultaneously, including synaptic connections, gap junctions, and perhaps diffusible signals. Cells in the ventral or core part of the nucleus receive photic input directly from the retina, and then communicate this information to the oscillators of the dorsal SCN. Once synchronized to each other and to the environment, the cells in the dorsal SCN produce a coordinated output signal to other brain regions (Figure 2). The evidence that the SCN serves as the body’s ‘master clock’ is very robust as it comes from many converging lines of evidence. Destruction of the SCN and blockade of its output by applying the sodium channel blocker tetrodotoxin eliminates all rhythms including drinking, locomotor activity, body temperature, and hormone secretion. The intrinsic physiological properties of SCN neurons are responsible for generating circadian oscillations because electrophysiological recordings from cultured SCN neurons and slice preparations show that oscillations in firing rates persist for several days in vitro. Finally, when SCN neurons are transplanted into the brains of animals whose SCN have been ablated, behavioral rhythms are restored with a period corresponding to that of the donor animal. The free-running rhythm of the SCN is reset or entrained by signals from the environment and internal signals from the body. In mammals, the most potent entraining stimulus is light. Light enters the retina and the information is transmitted to the SCN, which is then synchronized to the day–night cycle. Information about light–dark cycles reaches the SCN directly by way of projections from the retina via the retinohypothalamic tract (RHT) and indirectly via the geniculohypothalamic tract (GHT). It was long thought that in mammals, the only light-sensitive cells in the body were those of the rhodopsin-containing rod and iodopsin-containing cone photoreceptors located in the outer retina. However, this notion was overthrown with the discovery of a novel opsin, termed melanopsin, a photopigment expressed in Xenopus oocytes and later found in mammalian retinal ganglion cells. Within the mammalian retina, ganglion cells receive light information from rod and cone driven pathways Inputs

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Diffusible Figure 2 The suprachiasmatic nuclei (SCN). Cells in the ventral SCN receive light input from the retina and transmit it to cells of the dorsal SCN. These rhythmic cells are coordinated by input from the ventral cells and then produce output to other areas of the hypothalamus.

and provide output from the retina to light-responsive parts of the brain. Melanopsin is found in 1–2% of retinal ganglion cells and renders these neurons intrinsically photosensitive. Melanopsin-containing retinal ganglion cells project directly to the SCN via the RHT, and in this way, they transmit light information to the brain along with rods and cones. In fact, the rods and cones synapse onto melanopsin-containing retinal ganglion cells and thereby exert their effects on the SCN through these cells. Though present in vertebrates and invertebrates, melanopsin is closely related to invertebrate opsins, suggesting that this photopigment was conserved throughout evolution. Although the eye is the only light-sensitive organ in the body of mammals, nonmammalian vertebrates have additional sources of photic input. These include deep brain photoreceptors, which are located within the hypothalamus and the pineal gland, which lies between the forebrain and the cerebellum. The deep brain photoreceptors are composed of neurons that contact cerebrospinal fluid and transmit information about environmental lighting to the median eminence region of the hypothalamus and from there to the pituitary gland. These cells express specific opsin-like photopigments and are found within fish, amphibians, reptiles, and birds. They play important roles in circadian rhythmicity. Light penetrates the skull, reaches the brain, and enables entrainment. In some bird species, reception of light information by deep brain photoreceptors leads to testicular growth, implicating a role of these neurons in seasonal reproductive processes. In addition, illumination of the hypothalamus induces migratory behavior in birds, indicating that deep brain photoreceptors also plays a role in this aspect of circannual rhythms. In nonmammalian vertebrates, the pineal gland provides an additional source of light sensitivity for photoentrainment. In mammals, the pineal does not respond to light directly, and information about the light: dark cycle reaches this gland only via the SCN. In nonmammalian vertebrates, the pineal itself is photosensitive and responds directly to light. In both mammalian and nonmammalian vertebrates, the pineal secretes melatonin during the night. The nightly secretion of melatonin occurs in proportion to the duration of darkness, while environmental light suppresses the secretion of pineal melatonin. In many nonmammalian species, including zebrafish, house sparrows, and chickens, the pineal organ has oscillator cells and is also capable of generating circadian rhythms and thereby supporting both photoreception and endogenous rhythm generation. By way of its role in tracking the duration of light and in producing melatonin, the pineal organ is important for regulating both daily and seasonal rhythms. While light is the most salient, temperature is also an effective entraining cue. Small daily fluctuations in internal body temperature appear to directly entrain the mammalian SCN. Temperature sensitivity has also been demonstrated


in isolated SCN in vitro; the cultured SCN shifts its rhythmicity following temperature pulses. This suggests that changes in hypothalamic temperature can entrain the master clock. Similarly, changes in body temperature can entrain peripheral oscillators. This is true of mice kept in different ambient temperatures show entrainment of clock genes in liver in response to changes in environmental temperature. Molecular clocks

The discovery of molecular mechanisms underlying the intrinsic rhythmicity of individual cells has had a tremendous impact on our understanding of the circadian timing system and has enabled our exploration of the fundamental nature of these mechanisms. In 1971, Konopka and Benzer identified a gene in Drosophila that led to changes in the fly’s endogenous rhythms. Mutations in this gene caused three phenotypes: a shortened period, a lengthened period, and arrhythmicity. This was a landmark finding in that it demonstrated a direct relationship between changes at the level of genes and behavior. This genetic basis for behavior led to a clearer understanding that the endogenous rhythms exhibited in the behavior of animals were actually gene-driven. The gene was termed period and was the first gene isolated that displayed a circadian phenotype. Additional so-called, ‘clock genes’ were later discovered and shown to be important not only in flies but also in mammals. Clock genes and their protein products comprise a transcription-translation loop with positive and negative feedback regulation. The rhythmic expression of clock genes is important for maintaining a

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functioning circadian clock, and disruption of these genes causes arrhythmicity (Figure 3). Cellular clocks occur not only in the SCN but also in other brain areas and in peripheral tissues. Circadian clock gene expression in mammals has been detected in the liver, heart, muscle, kidney, pancreas, adipose tissue, and lung. The function of the SCN is to coordinate tissuespecific rhythms in the rest of the brain and the body, with each other and with external stimuli. When isolated from the SCN, individual peripheral cells drift out of phase with each other and the overall rhythm of the tissue as a whole dampens. When the intracellular processes within these cells are synchronized by the SCN, peripheral tissues are able to produce coherent rhythmic outputs.

Development of circadian rhythms

A number of rhythmic processes have been detected in utero, a time when regulation of nutrients and hormones may be important for normal growth and development. These processes include heart rate and breathing. While these rhythms are endogenous to the fetus, they are entrainable by maternal signals such as body temperature, metabolic pathways, uterine contractions, and hormones, including melatonin. The purpose of in utero entrainment is not clear; however, it probably occurs in order to prepare the fetus for its novel environmental conditions. Interestingly, even after birth, newborns are still entrained by signals from the mother. This has been demonstrated in rats in which lesioning the SCN of the mother causes desynchronization of rhythms among pups within the litter.

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Figure 3 Diagram of the circadian clock mechanism. (1) The clock proteins, CLOCK (yellow) and BMAL1 (pink), drive the expression of clock genes (Per, Cry and Rev-erba) in the nucleus. (2) PER (red) and CRY (light green) proteins in the nucleus inhibit CLOCK/BMAL1 action through negative feedback. They also down-regulate Rev-erba (dark green). (3) When REV-ERBa protein is absent, Bmal1 (and possibly also Clock) genes are disinhibited and transcribed to produce new CLOCK/BMAL1 transcription factors that initiate a new cycle. Adapted from Albrecht U and Eichele G (2003) The mammalian circadian clock. Current Opinion in Genetics & Development 13: 271–277.

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While entrainment and circadian rhythmicity are seen before birth, full maturation of the circadian system occurs after birth. For example, rhythms in within the SCN are detectable before birth. In children, a regularsleep-wake rhythm at about 3 months, though after this stage infants often have disrupted sleep because of hunger or teething discomfort (Figure 4). This time course of rhythm maturation is likely attributable to the development of connections from neurons of the SCN to target sites in the brain, and maturation of connections among SCN neurons themselves, thus, the networks that allow for synchronization of rhythmic cells are also not established until later in development. The functions of circadian rhythms

The circadian timing system controls a vast number of physiological and behavioral processes including the

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sleep–wake cycle, feeding, core body temperature, and hormone production. A key problem in understanding the function of the circadian system is the determination of how the rhythmicity is coordinated among various brain regions and how this affects other organ systems. Many breakthroughs in research occurred with the discovery of clock cells, in particular, that they were expressed throughout the entire organism. This led to the understanding that the SCN was not actually the driver of rhythms, but that the rhythms were generated within each cell so that it could display rhythmicity on its own. It then became clear that the role of the master clock in the SCN was to set the phase of these peripheral clocks, thereby synchronizing them. Peripheral tissues can also be entrained without affecting the SCN phase. The brain clock provides timing information to the rest of the body to activate certain behaviors, such as when to eat. The nutrients provided by food stimulate the production of hormones and enzymes that act to set the phase of oscillators in peripheral tissues such as the liver. This phase setting, however, does not impact the phase of the SCN but only acts at the level of peripheral tissues (Figure 5). Outputs of the SCN project to other parts of the hypothalamus, where they synchronize oscillators found in extra-SCN brain sites thereby modulating the timing of synthesis and/or secretion of neurotransmitters and neurohormones. The SCN communicates by direct synaptic connections with neurosecretory cells, such as gonadotropin-secreting neurons, and indirectly via multisynaptic autonomic pathways to endocrine glands, such as the adrenal gland through hormonal release (Figure 5). In this way, the SCN exerts neural and hormonal control over the physiology of the body. This is important for many processes. The circadian control of hormone production is important for reproductive processes including ovulation, estrus, fertilization, and pregnancy. Thus, the SCN projection to gonadotropin-releasing hormone (GnRH) neurons stimulates production of gonadotropins, such as luteinizing hormone (LH) and folliclestimulating hormone (FSH). A close temporal regulation of LH is necessary for ovulation. Also, after ovulation, prolactin production is needed at a particular time to maintain pregnancy and promote lactation. In addition, in mammals, levels of corticosterone are under circadian control, expression rises prior to waking. It is thought that this rise allows animals to prepare for activity onset.

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Time of day Figure 4 Sleep rhythms in infants. Dark bands are sleep periods of an infant over a 24-h day followed for several months. Adapted from Kleitman N and Engelmann T (1953) Sleep characteristics of infants. Journal of Applied Physiology 6: 269–282.

Looking Ahead: Applications of Research in Circadian Rhythms Alterations in circadian rhythms have profound effects on the health of an individual, as a number of disorders are associated with circadian dysfunction. Phase shifting is seen in individuals experiencing jet lag. In this case, one’s


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Temporally organized processes and behavior Figure 5 SCN and peripheral tissues. The circadian system is controlled by neural and diffusible signals originating from the SCN. Peripheral tissues may be differentially regulated by SCN signals via local clocks allowing for more specific responsiveness based upon local needs and time of day.

rhythms must undergo phase adjustment to account for the change in local time. During the period of transition to the new conditions, the SCN and peripheral organs become desynchronized, and one experiences the fatigue, irritability, and insomnia of jet lag. Eventually, the various bodily rhythms entrain to the new phase, and return to appropriate synchrony with each other. People who work night shifts also need to phase adjust, and the consequences appear to be more severe than for jet lag sufferers. Shift worker’s schedules are not in phase with the light–dark cycle and they are therefore exposed to environmental light at the wrong time. When shift workers conform to the schedule of a typical day–night cycle on their days off, the rotation can lead to major disruptions of the circadian time. This leads to sleep deprivation and in more serious cases, cardiovascular dysfunction, altered metabolism, and mood disorders. Studies have shown that the incidence of on-the-job mistakes and industrial accidents is dramatically increased during night shifts, compared to day shifts. Because of the growing need in our society for around-the-clock work, understanding how to address the issues of circadian dysfunction is becoming increasingly important. Light and drug therapy have been used to treat circadian-based sleep disorders, seen in advanced and delayed sleep phase syndrome, jet lag, shift work and aging. Treatment protocols require careful

attention to circadian phase of light or drug application since these variables impact on the effectiveness of the treatment. The disruption of circadian rhythms has widespread effects on the health of all organisms and therefore it is important for current and future research to address the potential impact of changing environmental conditions. This may be especially relevant in an age of global warming, where temperature conditions and resource availability are changing rapidly. While light is the most potent entraining stimulus in mammals, temperature affects clock gene expression and modulates the effects of photoperiod on the reproductive axis in birds. A better understanding of the effects of temperature on reproduction through effects on circadian and seasonal rhythms will be useful. See also: Amphibia: Orientation and Migration; Bat Migration; Bats: Orientation, Navigation and Homing; Bird Migration; Fish Migration; Hibernation, Daily Torpor and Estivation in Mammals and Birds: Behavioral Aspects; Insect Migration; Insect Navigation; Irruptive Migration; Magnetic Compasses in Insects; Mammalian Female Sexual Behavior and Hormones; Maps and Compasses; Memory, Learning, Hormones and Behavior; Migratory Connectivity; Neural Control of Sexual Behavior; Parental Behavior and Hormones in Mammals;

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Parental Behavior and Hormones in Non-Mammalian Vertebrates; Pigeon Homing as a Model Case of GoalOriented Navigation; Reproductive Skew, Cooperative Breeding, and Eusociality in Vertebrates: Hormones; Sea Turtles: Navigation and Orientation; Seasonality: Hormones and Behavior; Sleep and Hormones; Time: What Animals Know; Vertical Migration of Aquatic Animals.

Further Reading Albrecht U and Eichele G (2003) The mammalian circadian clock. Current Opinion in Genetics & Development 13: 271–277. Antle M and Silver R (2005) Orchestrating time: Arrangements of the brain circadian clock. Trends in Neurosciences 28: 145–151. Dunlap JC (1999) Molecular bases for circadian clocks. Cell 96: 271–290. Dunlap JC, Loros JJ, and DeCoursey PJ (eds.) (2004) Chronobiology Biological Timekeeping. Sunderland, MA: Sinauer Associates. Hastings MH, Herbert J, Martensz ND, and Roberts AC (1985) Annual reproductive rhythms in mammals: Mechanisms of light synchronization. Annals of the New York Academy of Sciences 453: 182–204. Hazelerigg DG and Wagner GC (2006) Seasonal photoperiodism in vertebrates: From coincidence to amplitude. Trends in Endocrinology and Metabolism 3: 83–91.

Klein DH, Moore RY, and Reppert SM (eds.) (1991) Suprachiasmatic Nucleus the Mind’s Clock. New York, NY: Oxford University Press. Kleitmann N and Engelmann T (1953) Sleep characteristics of infants. Journal of Applied Physiology 6: 269–282. Koukkari WL and Sothern RB (2006) Introducing Biological Rhythms: A Primer on the Temporal Organization of Life, with Implications for Health, Society, Reproduction, and the Natural Environment. New York, NY: Springer. Kriegsfeld LJ and Silver R (2006) The regulation of neuroendocrine function: Timing is everything. Hormones and Behavior 49: 557–574. Paul MJ, Zucker I, and Scwartz WJ (2008) Tracking the seasons: The internal calendars of vertebrates. Philosophical Transactions of the Royal Society B 363: 341–361. Seron-Ferre M, Valenzuela GJ, and Torres-Farfan C (2007) Circadian clocks during embryonic and fetal development. Birth Defects Research 81: 204–214. Yan L, Karatsoreos I, Lesauter J, et al. (2007) Exploring spatiotemporal organization of SCN circuits. Cold Spring Harbor Symposia on Quantitative Biology 72: 527–541.

Relevant Websites http://www.srbr.org – This is the web site for the society for research in Biological Rhythms. Here one can find information about meetings, web sites for laboratories, and news in the field.