Circadian and Ultradian Clocks/Rhythmsq EW Lamont, Carleton University, Ottawa, ON, Canada S Amir, Concordia University, Montreal, QC, Canada

Introduction 1 Properties of Biological Clocks 2 Ultradian Rhythms 2 Circadian Rhythms 3 Molecular Mechanisms of Circadian Rhythms 3 Relationship of Circadian Rhythms to Physical and Mental Health 4 Relationship Between Ultradian and Circadian Clocks 4 Conclusion 5 References 5

Glossary Circadian biological rhythms of approximately 24 h in NREM non-rapid eye movement sleep, made up of several duration. sleep stages that are deﬁned based on the frequency of Clock something that is used to measure time. brain electrical activity. Chronotherapeutics treatment protocol for cancer Oscillator a series of components with a combined action whereby the time of day that chemotherapy is that varies between extreme values over a precise period of administered is chosen in order to be maximally effective time. in treating the tumor, but have minimal effects on the Period the duration of a single oscillation measured from patient. peak to peak or from one reference point to the next ie, Entrainment the process by which an environmental from midnight to midnight on a 24-h clock. stimulus regulates the period and phase of an Phase the momentary state of an oscillation. oscillator. REM rapid eye movement sleep. Infradian rhythms with a period of days, weeks, or years. Ultradian rhythms with a period of less than 1 day.

Introduction

The term biological rhythm can be used to describe any molecular, physiological, or behavioral event or process that is recurring, and is a deﬁning characteristic of all living organisms (Lloyd and Murray, 2005). Rhythmicity can be observed over the entire range of time scales from fractions of a second to years, but can be broadly grouped into three categories: ultradian, circadian, and infradian. Ultradian rhythms, such as cell division or the human rapid eye movement (REM)-non-REM (NREM) sleep cycle, can have a period, of seconds, minutes, or hours and occur multiple times per day. Circadian rhythms, including photosynthesis, the rest– activity cycle, and the plasma concentration of numerous circulating hormones, repeat approximately every 24-h. Infradian rhythms have periods of longer than 24 h, ranging from days to weeks, months, or even years. Examples include the human menstrual cycle, hibernation, and the 17-year emergence cycle of certain North American cicadas, respectively (see Fig. 1). The coordination of parallel and serial processes necessitates rhythmicity at all levels of biological organization. Ultradian rhythms likely arose in order to separate in time those mutually incompatible chemical reactions that could not be physically sepa- rated within the space of a single cell. In more complex systems, ultradian rhythmicity provides a metabolically inexpensive mechanism for efﬁcient signal transmission. For example, it may be energetically expensive or even toxic for an organism to have constantly high levels of a hormone in circulation, when the same signal can be transmitted by pulsatile hormone release (Lahav, 2004). Temporal separation of incompatible chemical reactions is also observed at a circadian time scale, such as the daily rhythm of photosynthesis. Circadian rhythmicity is also critical for the synchronization of multiple biological processes with the external environment and allows the organism to anticipate the environmental demands of the solar cycle.

q Change History: January 2016. Elaine Waddington Lamont and Shimon Amir updated their afﬁliations, revised abstract, made some changes to the text, updated ﬁgures, replaced further reading with reference list and in-text citations.

Reference Module in Neuroscience and Biobehavioral Psychology http://dx.doi.org/10.1016/B978-0-12-809324-5.00283-2 1 2 Circadian and Ultradian Clocks/Rhythms

Figure 1 Time scale of biological rhythmicity, with examples. REM, Rapid eye movement; NREM, non rapid eye movement; GH, growth hormone; GnRH, gonadotropin-releasing hormone; LH, luteinizing hormone; FSH, follicle-stimulating hormone; TH, thyroid hormone.

Properties of Biological Clocks

In their most basic form, biological rhythms consist of a negative feedback loop with a time delay (Tiana et al., 2007). For example, there are numerous proteins that repress transcription of their own genes, such as the PERIOD (PER) and CRYPTOCHROME (CRY) proteins, essential components of the mammalian circadian clock (described in more detail below, and see Fig. 2). At least one negative feedback loop is necessary for an oscillation. Most biological clocks are made up of a series of weakly coupled oscillators, which make them more robust with respect to perturbations by outside stimuli. Greater complexity also tends to allow for timing of longer intervals. The most important characteristic, and hallmark of a true biological clock, is temperature compensation: the period of the oscillation must be very similar over a wide range of biological temperatures. This has been studied extensively in circadian rhythms, but is also a characteristic of ultradian clocks. Intrinsic rhythmicity and a free running period under constant conditions are also necessary properties for biological clocks.

Ultradian Rhythms

Because the time scale of ultradian rhythms is so broad, from fractions of a second to hours, a large number of biological clocks fall into this category and a comprehensive discussion is not possible in this entry. However, an example a of a well-studied ultradian rhythm is the approximately 40-min cycle of cellular respiration observed under continuous aerobic culture conditions in the yeast

Figure 2 Molecular mechanisms of the mammalian circadian clock. Clock gene mRNAs are indicated by italics, proteins are in bold. Fine arrows indicate transcription, block arrows indicate binding or interaction, the gray arrow indicates translocation, and the open arrow indicates post- transcriptional modiﬁcation. Excitation and inhibition are indicated by plus (þ) and minus () signs respectively. C, CLOCK protein; N, NPAS2 protein; B, BMAL1 protein. Circadian and Ultradian Clocks/Rhythms 3

Saccharomyces cerevisiae (Causton et al., 2015). Interestingly, the majority of the individual organisms within a culture will become synchronized with one another, such that the O2 concentration and intracellular pH of billions of cells become perfectly coordi- nated. This ultradian oscillator meets all of the requirements of a true clock. It is intrinsically rhythmic, temperature compensated, and maintains a 40-min oscillation for months under constant culture conditions (Causton et al., 2015). The signaling mechanism responsible for the mass inter-cellular synchronization has not yet been established, but two diffusible chemicals, H2S and acetal- dehyde, are possible candidates, as they show rhythmic changes in concentration and can shift the phase of the ultradian metabolic clock (Lloyd and Murray, 2005). The molecular mechanisms of this clock are under investigation and a putative clock genes include GTS1 (Ouyang et al., 2011; Wang et al., 2001), while PER-ARNT-SIM (PAS) domain containing PSK2 acts a nutrient sensing protein kinase that modulates the yeast ultradian clock (Ouyang et al., 2011). One of the ﬁrst ultradian rhythms to be studied in humans was the REM-NREM sleep cycle, which has a period of about 90-min, and occurs 3–5 times in the average sleep episode (Moszczynski and Murray, 2012). This rhythm is composed of the synchronous activity of a number of different processes including occulomotor activity, muscle tone, dominance of the autonomic nervous system, brain electrical activity and energy utilization. Like many other biological processes, sleep shows rhythmicity at multiple time scales. In addition to a 90-min ultradian rhythm, a circadian rhythm is also evident (Dijk and Czeisler, 1995).

Circadian Rhythms

With the exception of extreme environments like the deep ocean floor, all organisms are exposed to the daily solar cycle and have an internal clock that cycles about once a day (Dunlap et al., 2004). For example, the unicellular marine algae Gonyaulax polyedra, moves up to the higher levels of the ocean to absorb sunlight and CO2 for photosynthesis during the day, but descends to the nutrient rich lower layers during the night to absorb nitrogen and phosphorous. In multicellular organisms, the molecular machinery for circadian rhythms is present in nearly every cell. These clocks perform functions specific to each individual tissue and are synchronized with the external environment by a master circadian pacemaker (Rosenwasser and Turek, 2015). In mammals, this master circadian clock is located deep in the brain in the suprachiasmatic nucleus (SCN) of the hypothalamus. The SCN has all of the defining attributes of a biological oscillator: intrinsic rhythmicity, a free running period under constant conditions, and temperature compensation. Substantial evidence indicates that the SCN is the master circadian clock, as SCN lesions abolish circadian rhythmicity of locomotor activity, feeding, drinking, and hormone release. Further- more, surgical transplantation of SCN cells can restore circadian rhythms of locomotor activity to recipient animals with SCN ablation. Hamsters with the tau mutation have an endogenous circadian rhythm with a period shorter than 24 h. Animals homozygous or heterozygous for the mutation have periods of 20 and 22 h respectively, whereas wild-type animals have an endogenous period of about 24 h. SCN grafts taken from a homozygous tau mutant donor and placed in a wild-type recipient will restore locomotor activity rhythms, with the same period as the donor animal, demonstrating that the circadian period is conferred by the donor SCN. The mechanism responsible for the shortening of the period in tau mutant hamsters is casein kinase I epsilon (CKIε), described below (Molecular Mechanisms of Circadian Rhythms), and in Fig. 2. The SCN receives photic information directly from the eye via the retinohypothalamic tract. The synchronization of the master clock occurs by a process known as photic entrainment. The primary circadian photoreceptor melanopsin is found in the soma region of most retinal ganglion cells that innervate the SCN (Provencio et al., 2000). Melanopsin containing retinal ganglion cells are relatively insensitive to brief light exposure, but gradually become more activated in response to constant illumination of an intensity similar to that of the dawn sky. Non-photic cues have relatively little impact on the SCN master clock, but nevertheless, profoundly influence circadian behavior by altering individual peripheral clocks. The non-photic stimuli known to affect circadian rhythms are numerous and include olfac- tory cues, food, pathogens, social interactions, emotional state, and conditioned stimuli (Amir and Stewart, 1998). Perhaps the most important of these is feeding. Feeding is a potent synchronizer of behavior. Nocturnal rodents will consume the majority of their daily caloric requirement during the dark portion of the light–dark cycle. If they are placed on a 24-h feeding schedule that limits food access to a short period during the daytime, nocturnal rodents will show a reliable increase in activity several hours prior to food availability (Stephan, 2002). This food anticipatory activity only occurs when the feeding schedule is in the circadian range, and rodents are unable to anticipate feeding schedules that are much shorter or longer than 24 h. The semi-hierarchical organization of circadian oscillators in mammals allows peripheral oscillators to respond to relevant stimuli, but remain synchronized with each other and the external environment via the light-specific master clock. The SCN can synchronize peripheral oscillators either by direct neural projections, or by a diffusible or blood borne signal. Although entrainment is generally top down, there is also some evidence that peripheral clocks can influence the master circadian clock. For example, under conditions of constant bright light, the rodent SCN becomes arrhythmic, but can be entrained by scheduled meals delivered once every 24 h (Lamont et al., 2005).

Molecular Mechanisms of Circadian Rhythms

Circadian rhythmicity at the molecular level is the result of both positive and negative feedback loops and includes mRNA transcription, protein translation, protein/protein interactions and post-transcriptional protein regulation (Rosenwasser and Turek, 2015) 4 Circadian and Ultradian Clocks/Rhythms

(see Fig. 2). Three eukaryotic organisms have been fairly well characterized: Neurospora, Drosophila, and rodents (Brown et al., 2012). Only the later will be described here, but there is a high degree of similarity among all three. In mammals, genes Clock and Bmal1 encode two basic helix-loop-helix (bHLH)-PAS domain-containing transcription factors called CLOCK and BMAL1 (brain and muscle ARNT-like protein 1; also known as MOP3). CLOCK and BMAL1 dimerize and bind to E-Box enhancers, activating transcription of genes Period 1, 2, and 3 (Per1, Per2, Per3), Cryptochrome 1 and 2 (Cry1, Cry2), Rora, Rorb, and Rev-Erba. RORa and RORb activate BMAL1 transcription, while REV-ERBa inhibits it, providing positive and negative feedback on the clock, respectively. The main negative feedback loop is formed by clock proteins PER and CRY, which associate and are translocated back into the nucleus, where they inhibit their own transcription by interacting with the CLOCK/BMAL1 heterodimer. Posttranslational modiﬁcations of PER and CRY are critical for the period and phase of the molecular clock. The rate of accumulation, degradation, association and translocation of PER and CRY are controlled by enzymes casein kinase I epsilon (CKIε) and delta (CKId), F-box proteins FBXL3 and bTrCP2, the Drosophila shaggy homolog glycogen synthase kinase 3 (GSK3) (Besing et al., 2015), and several other recently identiﬁed substances (Brown et al., 2012; Rosenwasser and Turek, 2015). While the commonly held view has been that the stability of the circadian negative feedback loop proteins determines the length of the circadian period, recent work by Larrondo and colleagues suggests that phosphorylation of circadian protein FREQUENCY (FRQ) controls period length in Neurospora crassa (Larrondo et al., 2015). Experiments in other species will be needed to verify if the timing of phosphorylation events, rather than accumulation of proteins is a universal feature of circadian clocks. A number of other clock components have been studied. NPAS2, an alternate dimerization partner for BMAL1, seems to be important for clock function in cells outside the master circadian clock, but is virtually absent from the SCN. Other proteins such as DEC1 and DEC2 can act as transcriptional repressors by interacting with E-Box binding sites. The roles of these and numerous other genes, proteins, and enzymes in clock function are still being delineated.

Relationship of Circadian Rhythms to Physical and Mental Health

The essential circadian clock genes are ultimately responsible for circadian rhythms of physiology and behavior, and disruptions of circadian rhythms may play a role in pathology. For example, shift workers, who habitually work during the late evening and night hours, have a higher risk of developing long-term diseases, are more likely to be hospitalized, and consult more frequently with health-care providers than individuals working a more typical day-oriented nine to five schedule. Shift work is associated with an increased of risk a number of serious health conditions including cardiovascular disease, non-insulin-dependent diabetes, gastro-intestinal illness, cancer, spontaneous abortion, premature birth, infertility, and psychological distress (Shields, 2002). Also at risk are transcontinental flight personnel, who experience a chronic asynchrony between their internal circadian rhythms and the external environment. The link between circadian rhythms and cancer may be due to the direct role of the molecular clock in the regulation of the cell cycle (Okamura, 2004). Experiments causing disruptions of the circadian system in animals, such as alterations of the light–dark cycle, SCN lesions, or clock gene mutations result in an increased risk of cancer and faster growth of pre-existing tumors. Mice lack- ing the Per2 gene are more likely to develop tumors than wild-type mice, and do not show the typical induction of essential clock genes by gamma radiation, suggesting that in wild type mice, circadian clocks respond to cellular damage in order to activate apoptosis (programmed cell death) and suppress tumors (Fu and Lee, 2003). A practical application of this research is the advent of chronotherapeutics, which aims to take advantage of the observed asynchrony in cell division and metabolic rhythms between healthy tissues and the tumor. Thus treatment can be prescribed for a time of day where chemotherapy will be maximally toxic for the tumor, but have minimal effects on the patient. The human molecular clock also has been implicated in certain sleep disorders. Mutations of the PER2 (Toh et al., 2001) and CKId (Xu et al., 2005) genes were found to be the cause of two familial cases of advance sleep phase disorder (bedtime ¼ 6–9 pm, wake ¼ 1–3 am) while the opposite condition, delayed sleep phase disorder (bedtime ¼ 3–6 am, wake ¼ 1–3 pm) was found to be associated with a polymorphism of human PER3 (Ebisawa et al., 2001). Circadian clock genes have also been implicated in mental health (Lamont et al., 2010, 2007a,b; Videnovic and Zee, 2015). For example, genes CLOCK, PER3 and TIMELESS have been associated with schizophrenia/schizoaffective disorder and bipolar disorder. Support for a role of CLOCK mutations in bipolar disorder has recently come from the animal literature, with evidence that the Clock mutant mouse might constitute an animal model of mania (Logan and Mcclung, 2015; Roybal et al., 2007). There is also evidence that the therapeutic action of the mood-stabilizing agent lithium may be related to direct effects on the circadian clock component GSK3. Even more interesting are findings that inhibition of GSK3 may be common to other mood stabilizing and antidepressant therapies including drugs which target the serotonergic and dopaminergic systems as well as electroconvulsive therapy (Gould and Manji, 2005).

Relationship Between Ultradian and Circadian Clocks

There is strong evidence that ultradian and circadian clocks are interrelated. In complex organisms, the master circadian clock synchronizes ultradian clocks with the external light–dark cycle and many processes have both ultradian and circadian rhythmic components. For example, the cell cycle has an ultradian rhythm that varies by the organism and type of cell, but cell populations Circadian and Ultradian Clocks/Rhythms 5 tend to enter into the cell cycle at the same time each day (Okamura, 2004). Another example is the secretion of numerous hormones that have both pulsatile and circadian secretion patterns. Commonalities also are found at the molecular level. Indeed posttranslational modiﬁcation by two kinases, CK1 and GSK3, seems to be essential for both circadian rhythmicity and the yeast respiratory. CK1ε and d are clock components that alter the period of the mouse circadian clock (see Fig. 2). Similarly, in yeast, inhibition of CK1 increases the period of the yeast respiratory oscillation in a dose dependent manner (Causton et al., 2015). GSK3 is also a common element in circadian and ultradian oscillations. The mood stabilizing agent lithium has been shown to lengthen the period of both ultradian and circadian clocks. Lithium lengthens the period of circadian rhythms in rodents and can lengthen the period of neuronal ﬁring of cultured SCN neurons in a dose dependent manner, via the inhibition of the core circadian clock component GSK3 (Gould and Manji, 2005). The period of the ultradian metabolic clock of the yeast S. cerevisiae is also lengthened by lithium (Salgado et al., 2002). This suggests that there are common molecular mechanisms that underlie both of these clocks. Some ultradian rhythms may actually share molecular components with the circadian clock. Like mammals, a core component of the Drosophila molecular clock is the gene period (Roche et al., 1998). A null mutation of per abolishes the circadian clock, while other mutations can lengthen or shorten its period. These mutations have the same effect on the ultradian mating song cycle. The short form of per, called pers, shortens the period of the circadian clock, but also shortens the period of the ultradian mating song cycle. Similarly, perL1 lengthens both circadian rhythms and the song cycle, while the null mutation abolishes both rhythms completely. As the molecular mechanisms of other ultradian rhythms become known, other points of commonality between circadian and ultradian rhythms will likely emerge.

Conclusion

Rhythms are a ubiquitous property of all living organisms, and not merely an anomalous phenomenon occurring in a few isolated systems. They are observable at all time scales from fractions of a second to years and are necessary for the coordination of the multi- tude of biological systems that characterize healthy cells, tissues, systems, and organisms.

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