After studying this chapter, the student should be able to:
Understand how circadian rhythms are ubiquitous throughout all life and in virtually all cells in most species.
Know how circadian rhythms are derived from intrinsic clock mechanisms.
Understand how light sensed by some cells within an organism synchronizes the circadian rhythms of the entire organism.
Understand the difference between synchronization and entrainment of circadian clocks.
Understand how circadian rhythms work in terms of sleep/activity epochs.
Understand the molecular basis of intrinsic circadian rhythms and its robustness with respect to temperature and other perturbations.
Recount the role of the suprachiasmatic nucleus (SCN) and retinal output in circadian rhythms.
Be aware of the importance of vasoactive intestinal polypeptide (VIP) in circadian rhythms.
Understand how the SCN influences the rest of the body’s circadian rhythms.
Understand dysfunctions in circadian rhythms such as seasonal affective disorder and sleep problems.
The result of billions of years of evolution, the innate biological clock is a nearly ubiquitous feature of life on Earth. The conservation of its basic function—the maintenance of a stable relationship between the organism’s internal physiologic processes and the environmental light cycle—across such a wide swath of species speaks volumes about its importance. This timekeeping mechanism is not simply a response to changing light, but rather an innate clock that responds slowly and predictably to changes in the architecture of daily light cycles. Behaviorally, this clock allows organisms to predict daily changes in the environment and to react accordingly. Physiologically, the clock serves as a master regulator of many processes, providing a temporal pattern for internal organization and output. In humans and other mammals, the master circadian clock is located in the suprachiasmatic nuclei of the hypothalamus—a pair of densely packed nuclei receiving direct light input from specialized cells in the retina and other indirect timing and physiologic information from a slew of other areas in the nervous system and body. In this chapter, we will explore the basic properties of circadian and seasonal rhythms, the more complex molecular constituents of the clock, and finally, their impact on human health.
At the formation of the solar system, the planetary nursery in the accretion disk had a natural motion, orbiting our nascent Sun and serving as the birthplace of dozens of planetoids colliding with each other like a game of cosmic pinball. These planetoids had a natural rotation parallel with the orbit of the accretion disk, but these planetary rotations tended to vary wildly as their axes wobbled in space or they slowed until they were tidally locked with their star. For Earth, the planet’s rotation was cemented and stabilized approximately 4.4 billion years ago when a Mars-sized planetoid named Theia crashed into it and formed our moon. The Moon stabilized Earth’s axis, slightly off perpendicular to the planet’s orbit around the Sun. The Moon also gave us tides once the planet had cooled enough from the impact that liquid water oceans could form. It is through geologic evidence of these powerful tides—when first formed, the Moon was 6 to 10 times closer than present—that we know that the early Earth spun at the absolutely blistering pace of 4 hours per day. Over time, as the radius of the Moon’s orbit drifted outward to its current position, the drag of the lunar tide slowed Earth’s rotation to the 24 hours we now know. Because circadian clocks can be found in life-forms as ancient as cyanobacteria and in most living things today, including humans, one cannot help but appreciate that they must have given early life an advantage. In fact, a TimeTree of Life analysis would indicate that the most recent common ancestor of cyanobacteria and humans lived some 4.1 billion years ago (with estimates as early as approximately 4.3 billion years ago), when we know that the Earth’s rotation was still much accelerated. Along with the extraordinary discovery of 4.1 billion-year-old organic carbon in 2015, this evidence suggests that the earliest life on Earth not only had to deal with its harsh and downright inhospitable environs, but also had to be able to synch their daily cycle with a period of only several hours. Yet, genes used by animals to control parts of their clocks are exaptations of older, more ancient genes that can be found in bacteria and are being used for such processes as DNA repair.
Actigraphy: A type of activity monitoring that uses accelerometry to record amount of activity (usually wrist movement) over time and plots it on an actigraph (or actogram). Algorithms can be applied to determine the amount and quality of sleep.
Activity bout (α): The segmented time of day at which an organism is active.
Aftereffect: A transient reorganization of the circadian rhythm evident in constant conditions, particularly after a stimulus or perturbation.
Chronotherapy: Therapeutic strategy in which knowledge of the internal circadian clock is used to treat patients with strategies aimed at realigning circadian rhythms or giving medication at a particular time of day to improve treatment response.
Chronotype: The time of day at which a person prefers to sleep and/or be awake in reference to the outside environment. Early types are sometimes referred to as “early birds,” and late types are sometimes referred to as “night owls.”
Circadian: Of or about a day in length; in biology, a circadian rhythm is a self-sustaining physiologic rhythm of nearly 24 hours in length that continues to oscillate in the absence of external factors or stimuli.
Circadian time (CT): All the temporal phases of a circadian rhythm that occur in 1 cycle under constant conditions. That cycle is arbitrarily divided into 24 circadian hours regardless of local time, where CT 0 is the onset of activity in diurnal animals and CT 12 is the onset of behavioral activity in nocturnal animals. Any use of CT in vitro is based on an extrapolation from a measurable behavior.
Constant darkness: A photoperiod in which light is completely absent and organisms are allowed to free-run based on their individual circadian properties.
Cyanobacteria: An ancient photosynthetic bacterial phylum that exhibits robust circadian rhythms in culture.
Desynchronization: Process by which an oscillatory network with many coupled subcomponents becomes uncoupled due to inconsistent changes in the time interval between the subcomponents so that the phase relationships among components are lost.
Diurnal: Of or due to the partitioning of an organism’s main bout of behavioral activity to the daytime.
Entrain: To achieve entrainment; the act of an environmental zeitgeber setting the phase of the clock.
Entrainment: The state of a circadian clock in which the rhythm is at a stable phase angle with the environment and is due to an entraining stimulus from that environment, called a zeitgeber.
Free-running period (FRP or τ or period): The temporal length of a single rhythmic waveform. Circadian rhythms generally have a τ around but not exactly equal to 24 hours.
Light therapy: A type of antidepressant and/or circadian (chrono-)therapy using daily white or blue (488-nm) light to artificially entrain the clock. See chronotherapy.
Nocturnal: Of or due to the partitioning of an organism’s main bout of behavioral activity to the nighttime.
Periodicity: A rhythm, especially with relatively stable characteristics from 1 cycle to the next.
Phase (Φ or φ): A specific temporal point in a rhythm. The phase of an environmental rhythm (the zeitgeber) is denoted as Φ, whereas the phase of a circadian rhythm is denoted as φ.
Phase advance (+Δφ): The amount of time that the phase of a rhythm happens earlier than was predicted by that rhythm’s free-running period (τ). If light is the zeitgeber, a phase advance is usually achieved when the light pulse is given in the late subjective night. Also, the act of achieving an earlier phase than predicted.
Phase angle (ψ): The stable temporal relationship between a point in a circadian rhythm and an environmental stimulus (zeitgeber). This is arguably the most important evolutionary characteristic of a circadian clock.
Phase delay (–Δφ): The amount of time that the phase of a rhythm happens later than was predicted by that rhythm’s free-running period (τ). If light is the zeitgeber, a phase delay is usually achieved when the light pulse is given in the early subjective night. Also, the act of achieving a later phase than predicted.
Phase response curve: A type of graph that illustrates magnitude of phase shifts (y-axis; phase advances are positive, whereas phase delays are negative) to pulses of light as a function of circadian time (x-axis). This type of graph is useful in determining how capable an organism is of entraining to the varying length of daylight.
Photoperiod: The proportion of time within each day that is allotted to either day or night, usually leading to the expression of seasonal or photoperiodic traits in living organisms.
Photoperiodic: Of or having any physiologic change due to the environmental photoperiod; having the condition of photoperiodism; a more exaggerated form of seasonality.
Rapid eye movement sleep: A phase of sleep wherein the brain is highly active and the body is in a deeply relaxed state (sleep paralysis).
Seasonality: Condition in which animals experience behavioral changes in response to environmental season but which does not wholly constitute photoperiodism.
Subjective day/night: The time of day according to the circadian clock regardless of the external local time.
Suprachiasmatic nucleus (SCN): A pair of nuclei (paradoxically referred to in the field as a single nucleus) in the anteroventral hypothalamus, just dorsal to the optic chiasm. These nuclei house the central mammalian circadian pacemaker with some 20,000 densely packed and generally GABAergic neurons, although many produce an array of other neuropeptides.
T-cycle: Any photoperiod whose light and dark portions add up to a day length other than 24 hours.
Zeitgeber: German meaning “time giver”; a phase-setting environmental stimulus (for circadian rhythms, usually light) that serves to align the environmental rhythm to the biological clock.
Zeitgeber time (ZT): All the temporal phases of a rhythm that occur in 1 cycle under the influence of a rhythmic zeitgeber. Assuming that the length of that cycle is 24 hours (a non–24-hour environmental cycle is called a “T-cycle”), it is divided into hours where ZT 0 is defined as the onset of activity in diurnal animals, and ZT 12 is defined as the onset of behavioral activity in nocturnal animals.
As such, the day–night cycle is a critical environmental factor shaping the evolution of life on Earth. In Earth’s tumultuous history, extraterrestrial impacts such as the Moon-forming event, along with geologic upheaval and vulcanism, were not uncommon. Although early life evolved in the water, these factors wreaked havoc on the ability of the Sun’s light to reach the oceans, at times darkening the planet for many years. In at least 3 extreme instances in the planet’s history, the entire globe was covered with a sheet of white ice that was miles thick in some places—the so-called “Snowball Earth.” The last of these deep ice ages lasted millions of years 800 to 600 million years ago, bouncing most of the Sun’s light back into space and trapping early life in a dark ocean. Yet we find in the present that the endogenous circadian clock is an almost ubiquitous feature of life on Earth, suggesting that it would have offered some increased fitness, even then. How did circadian rhythms become the rule rather than the exception? It is thought that the ability to anticipate day/night transitions and coordinate internal metabolic processes was advantageous for the earliest life on the planet. The evolution of photosynthesis led to large amounts of oxygen in the atmosphere and an ozone to filter damaging solar radiation. Before that time, it is thought that organisms used their circadian clocks to judge surface safety by sinking to deeper water during the day and surface feeding at night—the so-called “escape from light” hypothesis proposed by Colin Pittendrigh, who was a founding father of the chronobiology field. By tracking light–dark transitions, later photosynthetic organisms could capture the Sun’s light and fix carbon on a regular schedule. Organisms that fed on these photosynthesizers shared the ancestral characteristic and used their own clocks to time their feeding behavior. Regardless of the predator and prey relationship, an endogenous circadian rhythm allowed an organism to maintain a fairly consistent relationship (or phase angle) with the external day–night cycle, despite changes in the primary time cues (or zeitgebers). Circadian clocks could be entrained, rather than purely driven or synchronized, allowing the organismal clock to generally keep time despite irregularities in weather and seasons and/or the absence of light, such as due to volcanic ash, asteroid impact, or even Snowball Earth. This stable phase angle provided a mechanism for adaptive flexibility to the ever-changing geophysical day: the ability to predict time of day persistently, even in periods of bad weather, geologic upheaval, or severe climate change. Thus, entrainment offered some fitness that allowed feeding or photosynthesis to occur on a generally regular schedule. This last point explains why completely divergent species such as cyanobacteria and humans evolved the ability to produce circadian rhythms, despite the fact that their last common ancestor lived some 4.1 billion years ago when the length of the day was far shorter than 24 hours. Either circadian rhythms were so important to early evolving life that they persist in most species today, or circadian rhythms have become so important that convergent evolution has made them a general property of life on Earth.
Seminal work by Carl Johnson and colleagues elegantly demonstrated in cyanobacteria that circadian clocks do offer competitive fitness when the endogenous rhythm is resonant with the external environment. In mammals, where such direct competition experiments are difficult, Pat DeCoursey made a fortuitous observation in captive ground squirrels that having a functional circadian clock did indeed decrease mortality rates: While the squirrels could not get out of a pen, a predatory weasel somehow got in and disproportionately picked off clock-lesioned squirrels while at the same time providing invaluable evidence for the importance of a behavioral circadian rhythm in mammals. This incident inspired DeCoursey and her team to monitor the behavior of wild-type and clock-lesioned ground squirrels using Global Positioning System tags in the wild, further bolstering the initial results. In addition to compromised survival due to predator–prey relationships, the circadian clock can also offer fitness due to overall health of the organism. Recent work in rodent species shows that certain genetic mutations can change the speed of the clock. For example, mice with a mutation that causes a faster clock with a shorter period (ie, 22-hour cycle) die from heart and kidney failure at earlier ages when housed in conditions that provide a standard, 24-hour, light:dark (LD) 12:12 photoperiod. Remarkably, these mutant mice appear to age and live normally when housed in a 22-hour T-cycle with an LD 11:11 photoperiod.
Thus, the mammalian biological circadian clock, located in the hypothalamus of the brain (see later section on the suprachiasmatic nucleus), is an indispensable component of life in the wild. It would follow, then, that understanding how mutations in circadian genes disrupt the function of behavioral and physiologic rhythmicity is important not only on the merits of scientific interest, but also for its implications to human health.
PROPERTIES OF CIRCADIAN RHYTHMS
By definition, circadian rhythms are the physiologic processes whereby an organism regulates its activity and coordinates other physiologic processes on a self-sustained, daily cycle. The word circadian comes from the Latin words circa, which means “about,” and diem, which means “day.” Behavioral rhythms govern several characteristics in animals: feeding/fasting, drinking, waking/sleeping, attention, cognitive performance, torpor, and body temperature, among others. Physiologic circadian rhythms can be found in hormone release, immune system function, and even cell division. Presently, a circadian rhythm has 3 fundamental properties: (1) the rhythm is self-sustained and approximately 24 hours in length; (2) the rhythm is temperature compensated; and (3) the rhythm is entrained by external factors such as light.
The first property of circadian clocks was initially observed by a French scientist named Jean-Jacques d’Ortous de Mairan, who was fascinated with the daily leaf movements of the heliotropic plant, Mimosa pudica, and so he examined whether his observations were simply a direct response to the sunlight or something more ingrained in the plant. After placing the plant in a dark cabinet, he observed that daily leaf movement persisted, thus illustrating the first characteristic of a circadian clock: persistence of the rhythm in constant conditions (generally light and temperature) at a near 24-hour period. In rodent models, this endogenous period is most often assessed by behavioral monitoring—such as activity recorded by wheel running (Figure 23–1, left), infrared motion detection (Figure 23–1, right), laser beam break, or water drinking—in constant darkness. Mice, for example, are the nocturnal species that exhibit activity during the dark of night. If placed in a monitored cage in constant darkness, mice will generally exhibit a free-running period (FRP or τ) of slightly less than 24 hours and continue to run only during what they perceive as “night.” The same form of activity monitoring (known as actigraphy) may be used to assess behavioral rhythms of humans, but the FRP of diurnal humans tends to run slightly longer than 24 hours. Even in humans, wakefulness occurs during what they perceive as “day” in the absence of photic or social cues in constant routine conditions in which people are kept awake with a constant posture in continual dim ambient light, with hourly meals. The imperfect, non–24-hour clock timing found across the animal kingdom has a purpose: The closer an animal’s FRP is to 24 hours, the more difficult it is to entrain to the 24-hour, light–dark cycle.
Representative double-plotted actograms illustrate mouse behavioral circadian characteristics in response to changing lighting conditions. Left, actogram of wheel-running activity; right, actogram of the same mouse (left) as monitored by infrared (IR) motion detection. Black ticks represent activity in 5-minute bins; yellow background denotes lights on; gray background denotes lights off.
The second property of circadian rhythms is rather remarkable: Unlike other basic biochemical reactions that increase their reactivity as the temperature increases—usually 2- to 3-fold for every 10°C increase—biological clocks are seemingly unaffected by temperature. In other words, the clock continues to run at a normal periodicity regardless of ambient environmental temperature. How the clock manages to accomplish this feat is rather a mystery, but it may have something to do with redundancy built into the molecular feedback loop (see later section titled “Molecular Architecture of the Circadian Clock”). Regardless, as discussed on the topic of clock evolution, the clock must be able to maintain its innate circadian rhythm at or near its native period and be able to process incoming signals from the environment. That latter property is the third and final characteristic of a circadian rhythm.
The light–dark cycle created by the Earth’s rotation is a natural, robust, and reliable oscillation. In the modern world, each day is exactly 24 hours. In a seminal series of papers on mammalian rhythms by Colin Pittendrigh and Serge Daan, the fourth paper suggests that conservation of the phase relationship between the master oscillator (Earth’s rotation—Φ) and its slave oscillator (the circadian biological clock—φ) is the essence of entrainment. This relationship is called phase angle (ψ) and is arguably the most important aspect of a circadian rhythm. A zeitgeber (German for “time giver”) is an external cue that sets or aligns the internal biological clock to the external 24-hour light–dark cycle. The most ancient and influential of all zeitgebers is light, which readily adjusts the phase of the biological clock such that the internal circadian rhythm is synchronized to the environmental stimulus. In humans, entrainment manifests as a behavioral period equal to the period of the 24-hour entraining stimulus with a stable phase relationship (ie, phase angle) between the circadian rhythm and the environment. There are 2 proposed models of entrainment: the continuous (parametric) model and the discrete (nonparametric) model. Both models have been championed by founders of the field, and both have merits and drawbacks. In the continuous model, Jurgen Aschoff suggested that the intensity of light proportionally changed the speed of the biological clock in a phase-dependent manner, thus squeezing or stretching the internal circadian clock period to fit into the actual environmental day. In contrast, Colin Pittendrigh suggested that a discrete model could explain entrainment of the circadian clock by simply shifting the phase (Δφ) or timing of each cycle by a specific amount. This model used a phase response curve (PRC) to explain phase-dependent shifts to discrete pulses of light that, alone or in combination, added up to the period of the environmental day (Figure 23–2). Thus, in a 24-hour light–dark cycle, the internal circadian period (τ) is equal to the external 24-hour cycle of light and darkness (T).
Phase shifts to discrete pulses of light and the phase response curve (PRC) of wild-type mice (Mus musculus). A. Idealized constant darkness (DD) actograms showing that a 30-minute pulse of light (yellow) on day 0 shifts the phase of activity onset (black bars are activity) on subsequent days in a phase-dependent manner. Red line is activity onset based on free-running period; green line is the new activity onset after the light pulse; gold bar represents the total phase delay; and blue bar represents the total phase advance. B. Idealized PRC (black line) tracing the phase shifts (in hours) from the top figure over circadian time. Gold area of the graph represents the phase delay zone, and blue area of the graph represents the phase advance zone. Letters A to E in the top figure represent the shift responsible for the point in the PRC (bottom) represented by the same letters.
In Pittendrigh’s discrete model, a zeitgeber adjusts clock phase by an amount of time that corrects for the difference between the period of the master oscillator and the internal circadian clock. Light phase shifts a particular rhythm (eg, hormone levels, neuronal activity, behavioral activity levels) by an amount that depends on when the light is presented with respect to the animal’s subjective time, as defined by the animal’s internal circadian clock. With regard to light, the largest phase delays occur when presented during the early subjective night, and the largest phase advances occur when light is presented during the late subjective night, just before dawn. The functional result of a phase delay is that the rhythm peaks later than it would have if there had been no stimulus given. When a phase advance occurs, the rhythm peaks earlier than it would have if there had been no light stimulus given. The relationship between when a light pulse is given during the animal’s subjective day or night and the resulting phase shift can be plotted using a PRC, in which the time of the light presentation (with respect to the internal circadian day) is on the x-axis and the size of the phase shift is plotted on the y-axis (see Figure 23–2).
The ability to entrain to the geophysical day with a constant phase angle is of paramount importance, especially when considering that the amount of time between dawn and dusk changes with the seasons. These changes in photoperiod, or the amount of daytime relative to nighttime over a 24-hour period, may perturb the phase relationship between the master and slave oscillators. Further work on mammalian entrainment by Pittendrigh and Daan sought to address how phase angle is conserved across changes in photoperiod due to season (see later section titled “Seasonality”). They proposed that the circadian clock consists of 2 coupled oscillators—a morning and an evening oscillator. They suggested that each oscillator is entrained to a light–dark transition (either dawn or dusk) and that the relationship between these oscillators accounts for photoperiodic encoding. For diurnal species such as humans, where the majority of activity is relegated to the daytime, dawn is referred to as zeitgeber time 0, or ZT 0, regardless of the local time at which dawn occurs. Likewise, the onset of activity in diurnal animals is referred to as circadian time 0, or CT 0, when the individuals are left to free-run in constant conditions. Rather confusingly, dusk is referred to as ZT 12 regardless of what the local time is for primarily nocturnal species, whose activity is generally isolated to nighttime. The onset of activity for nocturnal species is therefore referred to as CT 12 when the animal is left to free-run in constant conditions (constant darkness).
These distinctions between how we refer to the passage of time on a circadian scale relative to the local time are difficult but important to understand. For example, consider 2 nocturnal mice with different internal circadian periods and therefore different onsets of activity relative to the local time. If a person in Bangor, Maine, were to check on the behavior of these 2 mice at local sunset on the first day of summer (ie, 8:25 pm Eastern Daylight Time [EDT] or 20:25 EDT), that would correspond to ZT 15:35 for that person, but the internal time for the 2 mice could be very different, depending on their native period and how long they have been in the dark (eg, CT 3 or CT 16). In other words, it’s complicated. The further away from the equator one goes, the bigger is the photoperiodic disparity between seasons: In the continental United States, its northernmost city of Lynden, Washington, has a summer photoperiod of LD 16.2:7.8 but only goes through a period of astronomical twilight rather than “night,” whereas the southernmost incorporated place in the United States, Key West, Florida, has a summer photoperiod of only LD 13.7:10.3 with full night and day. It is difficult enough to keep track of the nuances of seasonally changing and latitude-dependent photoperiods to even consider how the circadian clock actually processes the information. How seasonality and photoperiodism are encoded in mammals will be discussed in the later section titled “Seasonality,” along with the rules outlined by Pittendrigh and others concerning how entrainment and the conservation of phase angle persist at a tissue or cellular level.
SUPRACHIASMATIC NUCLEUS: THE CENTRAL MAMMALIAN PACEMAKER
In the latter half of the 20th century, the nascent field of chronobiology picked up speed on the mammalian front with the publication of classic and brilliant works by the field’s founding fathers. Colin Pittendrigh and Serge Daan published a masterpiece on the properties of rodent behavioral rhythms. Jurgen Aschoff furthered our understanding of human rhythms and the effects of light on the speed of the clock, postulating what Pittendrigh would later call Aschoff’s Rules, and Franz Halberg was finding rhythms in all types of organisms. What eluded these investigators, however, was the specific location of the mammalian biological clock. In 1967, Curt Richter published a study suggesting that ablation of the hypothalamus led to circadian behavioral arrhythmicity. Exactly who should receive credit for the subsequent discovery of the central pacemaker is still hotly debated, but in 1972, 2 groups independently identified the suprachiasmatic nucleus (SCN) of the hypothalamus as the biological clock in mammals. Robert Moore and Nicholas Lenn identified the retinohypothalamic tract projecting from the retina—long thought to be the primary sensor of circadian light in mammals—to the SCN. Simultaneously, Friedrich Stephan and Irving Zucker followed Richter’s lead and concluded that the SCN was the pacemaker by electrolytic lesioning of specific nuclei in the hypothalamus. Only lesioning of the SCN produced behavioral arrhythmicity. Progress in the field quickened as the SCN was found to exhibit rhythmicity of neuronal firing rates in vivo and in vitro. The SCN was firmly established as the master circadian pacemaker in mammals by transplant studies that rescued behavioral rhythmicity in SCN-lesioned animals and even conveyed upon the transplant recipient the donor’s circadian period.
The SCN is medially located in the anterior hypothalamus, dorsal to the optic chiasm and inferolateral to the third ventricle in mice (Figure 23–3A). Each nucleus is approximately 200-µm wide at its widest point on the coronal plane, 200 to 250 µm dorsoventrally, and approximately 500 to 600 µm from the rostral to caudal extremes. It is densely packed with about 10,000 small (approximately 10-µm wide) neurons, most of which are γ-aminobutyric acid (GABA)-ergic. Due to their density, these SCN neurons are readily identifiable in a coronal mouse brain section as translucent bulbs above a darker optic chiasm (Figure 23–3B). These neurons increase their spike frequency during the circadian day and upregulate nighttime firing rate in response to phase-shifting light pulses.
Murine suprachiasmatic nucleus (SCN) of the hypothalamus. A. Left, in this ventral sketch of the mouse brain, the SCN is located in the hypothalamus, just dorsal to the caudal extreme of the optic chiasm (red arrow). Right, in this sagittal view of the mouse brain, coronal SCN slice cultures are typically made on the vertical black line. Horizontal scale bar represents 1 cm. B. Dim red backlighting of a coronal hypothalamic slice culture (left); the right half of the image is inverted and grayscale to view detail as seen under regular illumination. The general shape of the nucleus is outlined in red with core/shell demarcated by blue. The ventrolateral core containing retinorecipient cells synapsing with intrinsically photosensitive retinal ganglion cells is labeled. The dorsomedial shell is also labeled, along with the chiasm. V, third ventricle. Horizontal scale bar represents 100 µm. (Part A, reproduced with permission from Ciarleglio CM, Resuehr HE, McMahon DG. Interactions of the serotonin and circadian systems: nature and nurture in rhythms and blues, Neuroscience. 2011 Dec 1;197:8-16.)
SCN cells have several characteristics that make them unique from other cells. One distinguishing feature is that SCN cells receive photic information directly from the retina, allowing them to remain entrained to the 24-hour light–dark cycle. The SCN sends efferent projections to other hypothalamic nuclei, ultimately influencing pineal gland function and melatonin release. Second, SCN cells have network properties that allow them to synchronize their activity with one another due to neuronal firing, chemical synapses, and gap junctions. Third, these small compact neurons are capable of regeneratively firing action potentials (eg, pacemaking). The frequency of these electrical events exhibits a 24-hour rhythm at the population and single-cell level, even when neurons are maintained in a low-density culture that minimizes synaptic communication.
The SCN Is Innervated by the Retina & Is Characterized by Anatomically Localized Neuropeptides
As mentioned earlier, the mammalian circadian clock is entrained to the external environment primarily by light through phase delays in the early night and phase advances in the late night. This phase-shifting process begins with photic activation of conventional photoreceptors and specialized melanopsin ganglion cells within the retina. These intrinsically photosensitive ganglion cells then signal photic information directly to the SCN via glutamatergic innervation by the retinohypothalamic tract. Glutamate and pituitary adenylate cyclase–activating peptide (PACAP) released from retinal terminals activate postsynaptic N-methyl-D-aspartate (NMDA) receptors (as well as VPAC1, VPAC2, and PAC1 receptors), resulting in an influx of calcium, membrane depolarization, and an increase in spontaneous action potential firing. Early work suggested that these retinorecipient neurons are located in the ventrolateral core of the SCN (see Figure 23–3B). However, more recent studies suggest that projections from melanopsin ganglion cells actually innervate the entire nucleus more extensively than previously thought. There are slight species-specific differences in the pattern of innervation, but most rodent models follow a general layout. Studies on light-induced expression of the immediate-early gene c-Fos suggest that many retinorecipient cells express vasoactive intestinal polypeptide (VIP) and gastrin-releasing peptide (GRP). A conserved anatomic pattern of neuropeptide expression exists in the SCN from multiple rodent models. In addition to the retinorecipient core, the SCN is also characterized by a dorsomedial shell that produces arginine vasopressin, met-enkephalin, and angiotensin II. Core neurons innervate not only each other, but also the shell of the same nucleus. There are also internuclear connections between the 2 cores and the 2 shells.
VIP Plays a Key Role in Circadian Entrainment & Behavior
Prepro-VIP is the precursor from which VIP and peptide histidine isoleucine are derived, and both are structurally similar to PACAP. VIP is a secreted peptide in the glucagon family that is expressed rhythmically in the core cells of the SCN, with a peak during the night in 12:12 LD conditions, but not in constant lighting conditions (constant darkness [DD] and constant light [LL]). All 3 share binding affinity for 3 receptor types in the brain—VPAC1, VPAC2, and PAC1—but VIP has a higher specificity for the VPAC receptor subtypes. VPAC2 (gene name Vipr2) is a G-protein–coupled receptor that is highly expressed in the mammalian SCN, and its mRNA shows a biphasic pattern of expression in LD and DD. Signaling through this receptor activates adenylyl cyclase to increase the concentration of cyclic adenosine monophosphate (cAMP), and it has been shown that rhythms in firing activity in the SCN are responsive to the phase-dependent phase-shifting effects of VIP and VPAC2 agonists via protein kinase A and mitogen-activated protein kinase (MAPK) in vitro. Exposure to constant light has been shown to significantly depress VIP concentrations in the rat SCN in a light dose–dependent manner. This result is supported by in vitro data suggesting that NMDA phase-delays neuronal firing activity in rats and causes a drop in the VIP content of core cells. These same cells respond to pulses of light during the dark period by upregulating transcription of the core circadian clock genes (Per1 and Per2; see later section titled “Molecular Architecture of the Circadian Clock”), producing a phase shift. Previous studies have shown that VIP is sufficient to phase-advance and alter electrical activity when applied alone at CT 20 to 24 in vivo and moderately sufficient to phase-delay when applied alone at CT 12 to 14 in hamsters. Altogether, these results strongly suggest a key role for VIP in photic entrainment and for VPAC2 as the essential VIP receptor in the mammalian circadian clock.
In addition to VIP, GRP and GABA also contribute to SCN coupling. For example, mice lacking VIP receptors (Vipr2–/–) and VIP (VIP–/–) have arrhythmic SCN electrical activity, along with arrhythmic or severely disrupted wheel running activity. Furthermore, daily application of a VPAC2 agonist restores molecular rhythmicity and synchrony of firing rate rhythms to VIP–/– SCN neurons, indicating that VIP is mediating SCN coupling and behavioral rhythms. When GRP is applied to SCN cultures from Vipr2–/– mice, SCN synchronization is also restored, suggesting that other neuropeptides are important in this network. Finally, daily GABA application to 2 dissociated neurons from the same culture results in resynchronization of firing rate, indicating that GABA is sufficient for SCN coupling. However, in the intact SCN network, GABAA receptor activation slows resynchronization after SCN organotypic cultures are forced into a state in which 2 populations in a single nucleus are uncoupled. Ironically, decoupling induced by VPAC2 antagonists can be reversed by blockade of GABAA receptors, suggesting that GABA signaling also mediates VIP-induced synchronization.
The Mammalian Circadian Clock Can Be Set by Nonphotic Stimuli
In addition to light, nonphotic zeitgebers or stimuli are also able to entrain the circadian clock. Such nonphotic stimuli include arousal-producing stimuli and exercise such as sleep deprivation, novel wheel running activity, saline injection, and intermittent shaking of cages. When these stimuli are presented during the subjective day, rodents will advance the onset of their activity on following days. The presence of a nonphotic stimulus is conveyed to the SCN through the following 2 main pathways: (1) a geniculohypothalamic tract (GHT) and (2) the median raphe nuclei pathway. The GHT originates from the thalamic intergeniculate leaflet (IGL) and uses neuropeptide Y (NPY), GABA, and endorphins as neurotransmitters, whereas the dorsal and median raphe nuclei pathway, which projects to the IGL, is primarily serotonergic and projects to the SCN either directly (median raphe) or indirectly (dorsal raphe) via the IGL.
The large phase shifts that occur during the subjective day when animals are presented with nonphotic stimuli are mediated, at least in part, by NPY release into the SCN from the IGL. During the subjective day, NPY itself can produce large phase advances in vivo when injected into the SCN region or applied in vitro directly to SCN slices. Furthermore, confining hamsters to a small box could not block the phase advances that were caused by NPY injections into the SCN during the subjective day, demonstrating that activity or exercise is not needed to induce a phase advance when the SCN is injected with NPY. In a second experiment within the same study, hamsters were also exposed to a novel wheel for 3 hours, with the experimental group being injected with NPY antiserum into the SCN and the control group being injected with normal rabbit serum into the SCN. Hamsters injected with normal serum exhibited phase advances similar to those of the unoperated hamsters, suggesting that NPY conveys nonphotic information to the SCN, whereas the hamsters injected with the antiserum to NPY shifted their activity by <15 minutes.
Early studies showed that VIPergic neurons are under the influence of serotonergic innervation and that treatment with the serotonin-depleting reagents para-chlorophenylalanine or 5,6-dihydroxytryptamine causes a marked decrease in VIP immunoreactivity—an effect that can be reversed with a 5-hydroxytryptamine (5-HT) receptor 1B agonist. This depletion initiates the rhythmic property of VIP mRNA expression in DD and suggests that serotonin works antagonistically to light. The increase of available serotonin by treatment with monoamine oxidase (MAO) inhibitors or by knocking out the MAOA gene increases VIP expression in rats and mice, respectively. 5-HT1B receptors are localized to the retinohypothalamic tract, suggesting an indirect effect on VIP expression by modulation of glutamate release. Similarly, 5-HT1A or 5-HT7 receptors are located on the core neurons of the SCN and allow serotonin to directly modulate VIP release and neuronal activity. Because serotonin plays a role in nonphotic entrainment and modulates the SCN directly, mice with VIP signaling deficiency—and thus lacking robust responses to photic stimuli—may have enhanced responses to nonphotic stimuli.
The Efferent Projections of the Mammalian Circadian Clock Include Both Paracrine & Synaptic Signals
As mentioned earlier, the SCN is both necessary and sufficient for driving 24-hour rhythms in physiology and behavior, including rhythms in sleep–wake activity, energy homeostasis, heart rate/blood pressure, body temperature, and hormone release. Thus, it is not surprising that the SCN projects to a wide array of brain regions and impacts the entire brain through both direct and indirect neural pathways. A classic study showed that blocking the SCN output signal (ie, sodium-dependent action potentials) with tetrodotoxin (TTX) will dissociate the “hands” of the clock, while the “gears” keep on turning. Specifically, hamsters received chronic delivery of TTX to the SCN for several weeks while housed in running wheels in DD. During the TTX delivery, 24-hour locomotor rhythms were eliminated; however, they were reinstated after TTX was discontinued. Remarkably, the phase of the reinstated locomotor rhythm was predicted by the pre-TTX phase, indicating that the internal clock had been running all along, even though the outward manifestation of the clock was disrupted.
Numerous brain regions receive direct synaptic connections from the SCN, including the median preoptic area, the bed nucleus of the stria terminalis, the IGL, the paraventricular nucleus of the hypothalamus, the dorsal medial nucleus of the hypothalamus, and the arcuate nucleus. A primary direct efferent pathway from the SCN is the subparaventricular zone, which is also a putative “switch” for diurnality. Indirect pathways to the pineal gland and pituitary are critical for 24-hour rhythmic release of hormones such as melatonin and corticosterones, respectively. The pathway from the paraventricular nucleus is important for 24-hour control of the autonomic nervous system. Altogether, this system produces 24-hour rhythms in nutrients (eg, glucose, fatty acids) and hormones (eg, insulin, glucagon, leptin, adiponectin, ghrelin).
Hormones, regulation of body temperature, and feeding behavior have all been implicated as possible mediators of the efferent resetting signal from the SCN to downstream neural and peripheral targets. Humoral output of the SCN may also be a mediator of peripheral tissue synchronization. A classic study lesioned the SCN in hamsters and then restored locomotor rhythms by transplanting fetal SCN “micropunches” into the ventricles of arrhythmic animals. Furthermore, to determine whether restoration of rhythmicity was dependent upon neural outgrowth or humoral signaling, the SCN tissue was contained within a semi-permeable capsule before transplantation, and the same result was observed. However, it is important to note that not all circadian rhythms are restored by SCN fetal transplants; specifically, rhythmic release of glucocorticoids, melatonin, and luteinizing hormone fails to recover. Taken together, these results indicate that both humoral and neuronal signals are important for synchronization of peripheral clocks. Several candidate releasable factors proposed are transforming growth factor-α, prokineticin 2 (PK2), and cardiotrophin-like cytokine. For example, PK2 expression peaks during the subjective day in the SCN, and injection of PK2 to the SCN region suppresses locomotor activity in rats at night but results in an increase in activity during the day.
CIRCADIAN MULTIOSCILLATOR SYSTEM
So far, we have introduced the circadian system as relying on a single, primary master oscillator. However, research over the past 2 decades has revealed a much more complex system in which individual cells and tissues, both neural and peripheral, have the ability to generate 24-hour rhythms in isolation from the SCN. Rhythmicity of these secondary oscillators frequently damps out after several cycles due to reduction of both cellular clock gene expression amplitude and tissue-level amplitude, due to the individual cellular oscillators drifting out of phase with one another. Thus, the SCN provide an important daily resetting signal to the secondary pacemakers in the brain as well as in tissues throughout the body such as the lung, liver, heart, and spleen. The hierarchical structure of the circadian system is also evident during resynchronization following a large shift in the light–dark cycle—simulating jet lag. Using a bioluminescent reporter of clock gene activity (see later section titled “Molecular Architecture of the Circadian Clock”), researchers have been able to track ex vivo rhythms of various tissue clocks in real time. Explants of numerous peripheral and neural tissues have been cultured following a 6-hour phase advance of the light–dark cycle at 1, 3, and 6 days following the shift. Several studies have now shown that the SCN rather quickly reentrains to the new light–dark cycle, whereas other tissues take much longer. Locomotor behavior generally takes 1 day for each hour of jet lag to adjust, so a 6-hour phase shift would take at least 6 days for full reentrainment. These ex vivo clock gene reporter studies show that for some tissues, such as the liver, 6 days is not sufficient for reentrainment.
The first autonomous neural oscillator outside of the SCN to be discovered was the retina. The retinal clock is critical for adaptation to changes in environmental lighting, including dark adaptation (via melatonin) and light adaptation (via dopamine). Not surprisingly, these neurotransmitters can also directly regulate retinal circadian phase. Since then, other extra-SCN brain regions have been identified as having daily oscillations in clock gene expression. These include nuclei in the thalamus, hypothalamus, amygdala, olfactory bulb, cerebellum, hippocampus, and many others (Figure 23–4). Outside of the SCN, the olfactory bulb has been shown to maintain the most robust rhythms ex vivo in clock gene expression reported using bioluminescence. In situ hybridization studies of the rat brain have reported rhythmic clock gene expression in numerous regions of the forebrain (prefrontal cortex, rostral agranular insula, paraventricular nucleus, amygdala, and hippocampus). The majority of the extra-SCN oscillators have circadian phases that are delayed by 4 to 12 hours from the SCN.
Multiple oscillators in the rodent brain contribute to circadian behavioral output and physiology. Medial parasagittal view; major loci are indicated by color and label. The amygdala is more lateral than can be viewed in this section and is denoted by the dashed line. PFC, prefrontal cortex. (Adapted with permission from Ciarleglio CM, Resuehr HE, McMahon DG. Interactions of the serotonin and circadian systems: nature and nurture in rhythms and blues, Neuroscience. 2011 Dec 1;197:8-16.)
Among the forebrain regions mentioned earlier, the hippocampal clock has been a primary focus of research in the past few decades because of documented 24-hour rhythmicity in neurogenesis, long-term potentiation, and signaling cascades that are important for memory formation (eg, MAPK and cAMP). Rhythms in these processes contribute to day–night differences in several hippocampal-dependent tasks including acquisition, recall, and extinction. For example, spatial working memory and novel location memory are augmented during the awake phase (night for nocturnal rodents), and these day–night differences persist when mice are housed under DD (and, thus, are under circadian clock control). The molecular clock in the forebrain appears to be necessary for these rhythms in memory since knocking out the positive limb of the molecular clock (see next section) results in poor memory at both times of day. The local clock in the hippocampus is entrained by the light–dark cycle (via the SCN) as well by other external factors. For example, food availability is critical for resetting the phase of the hippocampal clock, and synchronization of the feeding–fasting cycle with the light–dark cycle is necessary for memory and long-term potentiation. Mice that are fed during the day (rest phase) show a shifted rhythm of clock gene expression in the hippocampus as well as poor performance in novel object recognition and reduced long-term potentiation. Like the hippocampus, it is likely that the local circadian clocks in other extra-SCN oscillators also play important roles in neural function and behavior.
MOLECULAR ARCHITECTURE OF THE CIRCADIAN CLOCK
The Transcription/Translation Feedback Loop
Several clock genes take part in an interlocking transcriptional and translational feedback loop to activate and repress each other in a manner that results in an approximate 24-hour rhythm of gene expression (Figure 23–5). The key circadian genes in mammals include the activators Bmal1 and Clock (or also Npas2), which activate the E-box promoter for the Period genes Per1 and Per2 and the repressor Cryptochrome genes Cry1 and Cry2. In mammals, Clock is constitutively expressed, and Bmal1 peaks in expression during the subjective circadian night, or “lights off.” The transcription factor RORa serves to activate Bmal1 transcription and, in addition, competes with Rev-Erbα for binding to the promoter of Bmal1. CLOCK and BMAL1 dimerize in the cytoplasm after translation, are phosphorylated by casein kinase (CK)-2α, and then translocate back into the nucleus to bind to E-boxes that upregulate Per, Cry, and Rev-Erbα expression, forming the “positive arm” of the negative feedback loop.
Genetic mechanisms of the circadian neuron. Mammalian neuronal circadian rhythms are governed by an intricate gene–protein feedback loop known as the transcription/translation feedback loop (TTFL). Black dashed arrows represent movement of transcripts out of the nucleus for translation. Green arrows represent the positive loop where the resultant proteins activate transcription of Per, Cry, and Rev-Erbα. Red lines represent the negative loop where the resultant proteins inhibit their own transcription, in the case of PER and CRY, or inactivate Bmal1 transcription by competing with RORa, as in the case with REV-ERBα. ECF, extracellular fluid.
After translation, either PER1 and CRY1 or PER2 and CRY2 dimerize and then are phosphorylated by CK-1ε/δ. This serves to either degrade the proteins or to repress their own transcription in the “negative arm” of the feedback loop by translocating back into the nucleus and inhibiting their own promoter’s activation by BMAL1/CLOCK. Concurrently, Rev-Erbα inhibits Bmal1 transcription by competing with RORa for promoter binding in a second negative feedback loop. Because the Per genes are highly transcribed during the circadian day, they are a useful measure of circadian period ex vivo (Figure 23–6).
Circadian gene expression in mammals. A. Gene expression profiles of 7 key circadian genes over 4.5 days, demonstrating the self-sustaining oscillations of molecular rhythms in the presence and absence of a zeitgeber (light). Genes are denoted by colored lines (see key to right). B. Representative example of the circadian expression of Per1::GFP in suprachiasmatic nucleus (SCN) neurons over 36 hours ex vivo in projected zeitgeber time (ZT). A single brain slice from a mouse carrying the mPer1::GFP was cultured and the gene expression dynamics of the SCN were assayed via time-lapse confocal microscopy. D, dark; DD, constant darkness; GFP, green fluorescent protein; L, light. (Part B, reproduced with permission from Ciarleglio CM, Resuehr HE, McMahon DG. Interactions of the serotonin and circadian systems: nature and nurture in rhythms and blues, Neuroscience. 2011 Dec 1;197:8-16.)
Altogether, both positive and negative arms of the feedback loop result in rhythmic expression, with PER and CRY expression levels being highest during the late day and lowest during the middle of the night within the SCN. In particular, posttranslational modification has been shown to play a major role in determining period and phase. Phosphorylation is the main posttranslational modification being considered. In addition to CK2-α, CK1-ε, and CK1-δ, other kinases such as CK2-β and glycogen synthase kinase 3 (GSK3)-β have been shown to phosphorylate BMAL1, CRY1, CRY2, PER1, and PER2, which either allows the transcription factors to enter the nucleus or to be degraded by the proteasome.
Nearly every cell in the body (and many brain cells) rhythmically expresses these core circadian clock genes. The molecular clock drives transcription of a plethora of other clock-controlled genes (43% of all protein-coding genes in humans); however, which genes are rhythmically transcribed depends on the cell or tissue type. One important resource for exploring and comparing clock-controlled genes among rhythmic tissue is CircaDB (http://circadb.hogeneschlab.org). This website reports >3000 cycling genes from cell lines as well as peripheral and neural (brainstem, cerebellum, hypothalamus, and SCN) tissues. The relationship between the central clock in the SCN and the local molecular clocks in peripheral tissues is somewhat complex. One way to study this relationship is to disrupt the molecular clock throughout the body and then restore the clock only in the brain (restoring SCN output). Several years ago, the Hogenesch laboratory followed this approach and found that restoration of the molecular clock in the brains (only) of ClockΔ19-mutant mice rescued behavioral arrhythmicity and clock gene rhythmicity in the liver. These results suggest that SCN output is sufficient to drive the local clock in peripheral organs. However, clock-controlled genes specific to the liver were reduced in number and had lower amplitude rhythms. Thus, the local clock in peripheral tissues is important for functions specific to that cell type. This approach has yet to be applied to SCN–extra-SCN relationships. The results of such a study would reveal the importance of the SCN signal versus the local molecular clock in secondary oscillators in the brain.
After the Moon-forming impact, the new Earth was left spinning on an axis slightly off perpendicular to the planet’s orbit around the Sun. While this is not thought to be unusual, planets that lack a proportionally large moon either proceed with a floating axis or will right their axis to perpendicular (with respect to its orbit around a star) with time. The gravitational influence of the nascent Moon served to stabilize this off-kilter axis, creating reliable seasons. The amount of seasonal daily light, or photoperiod, varies from the most light on the first day of summer to the least light on the first day of winter. The range of light per day depends on latitude, with the most extreme latitudes (the poles) receiving the most extreme photoperiods. As life flourished on the planet, strategies for thriving with seasonal changes in photoperiod developed and are maintained today. The ability to predict oncoming hardship in the winter or abundance in the summer is thought to increase fitness in plants and animals alike. For plants such as trees, the shorter daytime indicates a decrease of higher energy short-wavelength light and the coming colder temperatures. The trees lose their foliage at this time of year and go relatively dormant. As daytime length increases, photoperiod signals the oncoming warmth and a transition to photosynthesis, and an increase in foliage and gamete production occurs.
In animals, change in photoperiod indicates a necessity for behavioral change. For nocturnal animals, summer photoperiod means that all activity has to be squeezed into a shorter night, and with the abundance of growing plant life, it is the prime time of year to breed, and thus, some male animals experience gonadal growth while females become receptive to mating. As winter approaches, the photoperiod indicates a time for hibernation and/or pelage change—lighter color and thicker coat to prepare for winter cold.
Not all animals experience these physiologic changes, but those that do are said to be photoperiodic. Laboratory mice (Mus musculus) are thought not to be photoperiodic, but they do experience a behavioral reorganization called seasonality following changes in photoperiod. In their seminal paper on entrainment, Pittendrigh and Daan demonstrated that rodents compress their main activity bout (α) in long photoperiods. In addition, this compression of activity length results in an aftereffect, or transient reorganization of the circadian rhythm that is evident in constant conditions. Specifically, the compression or shortening of the primary activity bout that is evident in long photoperiods correlates with a decreased FRP in mice—a faster clock.
The reorganization of the clock and how this accounts for behavioral changes had remained a mystery until recently, with the development of in vivo electrophysiologic and ex vivo circadian gene reporter techniques. In situ hybridization experiments showed that gene expression in response to a light pulse is different in animals taken from short (LD 8:16) and long (LD 16:8) photoperiods, specifically the immediate early gene c-Fos and pineal N-acetyltransferase, the enzyme used to produce the neurohormone melatonin. In fact, the tissue-level architecture of in vivo electrical activity of mouse SCN is dramatically different between photoperiods, initially suggesting that seasonality is encoded in the SCN by changes in the phase distribution of individual neuronal clocks.
It turns out that how seasonality is encoded in the SCN is a bit more complicated, but in an interesting way: How the SCN encodes season is not just dependent on the photoperiod that the organism inhabits presently, but also on what photoperiod they had been exposed to early in perinatal development. Several different labs demonstrated that for animals reared in an equinox photoperiod (LD 12:12), the phase relationship of neurons relative to each other is the major factor behind how the SCN encodes season and, therefore, how an animal subsequently behaves. In other words, if one were to record the circadian rhythm from a single neuron using something like the Period gene reporter in Figure 23–6B, one would find that the rhythms from many neurons in the SCN would line up (synchronize) in phase if they were recorded from an animal that had just experienced a short, winter photoperiod (LD 8:16) before the experiment. Contrarily, one would find that the rhythms from neurons derived from an animal that had just experienced a long, summer photoperiod (LD 16:8) would be much more asynchronous, peaking across the circadian “daytime.” Reasonably, SCNs harvested from equinox photoperiod demonstrate an intermediate phenotype. Therefore, in equinox-developed animals, season is encoded by neuronal circadian phase relationships. But this is only part of the story.
How the SCN encodes season, it turns out, depends on what photoperiod the animal experienced around birth. Although the phase distribution of neurons within the SCN is due to proximal photoperiod, the resultant tissue-level integration in response to seasonal change is profoundly different based on one’s season of development. Summer-developed mice exhibit remarkably stable molecular clock rhythms regarding the phase at which SCN neurons peak in Per1 expression, very close to dusk (ψ). Winter-developed mouse SCN, in contrast, varies phase angle widely, depending on the proximal season, with Per1 expression peaking more than an hour after dusk in animals harvested from LD 8:16 or peaking 2 hours before dusk in animals harvested from LD 16:8. Equinox-developed animals display an intermediate phenotype. The ethologic implications alone are stunning: If the peak of Per1 expression correlated with time of activity onset, imagine how this result might impact Dr. DeCoursey’s study involving ground squirrels discussed earlier! Furthermore, the tissue-level dynamics are not just a result of changing neuronal phase distributions, as mentioned in the previous paragraph, but also altered neuronal rhythms themselves, with the shape of the distribution changing drastically by broadening or narrowing between summer and winter photoperiods, but only if the animal was developed in a short, winter photoperiod. In addition, neuronal period decreases in long photoperiod, but only if the animal was developed in a long photoperiod. Taken together, it is fairly clear that seasonal encoding in the SCN occurs not only at the tissue level, but also at the neuronal level, and because the environment has such a profound and lasting effect on the animal’s physiology, seasonal encoding may be an example of an epigenetic modification.
These fascinating results on what should otherwise be a mundane topic are underscored by further work on the behavioral ramifications of seasonal birth, seasonality, and photoperiodism. A series of studies on seasonally developed hamsters and mice have demonstrated correlates of anxiety and depression dependent not only on the proximal photoperiod (ie, a tendency to be sadder in short photoperiod), but also on developmental photoperiod (ie, long photoperiod–developed mice are generally more anxious, and short photoperiod–developed mice are generally more “depressed”). With that in mind, one is forced to wonder what effect seasonality and seasonal birth have on humans. Homo sapiens are, in nature, seasonal breeders, with most births happening in the summer to early fall. In an industrialized society, we lose that natural behavior because we can provide for infants year-round, so an understanding of the seasonal effects of light is critical in explaining aberrations in human health.
Changes in seasonal photoperiod have been associated with mood disorders and mental disease in humans. Seasonal affective disorder, for example, is a mood disorder that affects between 0.4% and 2.7% of the US population every year and is treated with bright light therapy or selective serotonin reuptake inhibitors. A 5% to 8% winter-spring excess of births has been reported for both schizophrenia and mania/bipolar disorder. There is a spring and summer excess of births for autism and a winter-spring excess of births for neurosis. Winter-born humans have been reported to show both stronger morning preference than the other seasons on the Morningness-Eveningness Questionnaire and significantly lower (sadder) Global Seasonality scores than individuals born in the summer. Many of these disorders revolve around the serotonergic system, and for good reason: A recent study in mice reported that photoperiod at birth programs the intrinsic electrical and receptive properties of neurons in the dorsal raphe nuclei—the midbrain region that is responsible for serotonergic signaling to higher order areas of the brain. These studies suggest that birth season—or perhaps the seasonality of late gestation—imprints the organization of the brain systems that control cognition, emotion, and/or circadian rhythmicity.
In all, seasonality is an important aspect of circadian rhythmicity on a planet with changing light cycles. Its impact on human health is profound, but it is important to reiterate 1 particular aspect of seasonal physiology, and that is on sleep properties. In humans, summer sleep is very different from winter sleep. Summer sleep is usually consolidated into a single bout and compressed into a shorter night than other times of year. Winter sleep, on the other hand, is wholly different: It is decompressed, spread over a much longer night, and usually occurs in 2 or more bouts. This compression and decompression of sleep, which correlates with duration of melatonin release, can contribute to the aforementioned disorders of human health, especially in those who may be prone to the disorders because of seasonal birth.
An in-depth discussion of sleep is covered in Chapter 34, but a brief description is included here to describe the interrelationship between sleep and circadian systems. During waking, electroencephalogram (EEG) recordings show 2 different types of brainwave activity: alpha activity (at 8 to 12 Hz) and beta activity (at 13 to 30 Hz). Alpha activity occurs when a person is resting quietly and not engaging in any strenuous activity. Beta activity has a lower amplitude because many different neural circuits are being activated at once.
Sleep has been divided into 2 main types: rapid eye movement (REM) and non–rapid eye movement (NREM). The original scoring guidelines include 4 stages of NREM sleep and 1 stage of REM sleep. The new system developed by the American Academy of Sleep Medicine identifies 3 stages of NREM sleep (NREM stages 1, 2, and 3) and 1 REM sleep stage. Once an individual falls asleep, he or she will progress through stages 1 to 3 of NREM sleep (with stage 3 being the deepest level of sleep) and then ascend back up through the stages until the REM sleep stage is reached. One cycles lasts approximately 70 to 90 minutes. A person experiences approximately 4 or 5 of these cycles per night (Figure 23–7).
Sleep hypnogram demonstrating changes in sleep architecture due to age (23 years vs 68 years). REM, rapid eye movement. (Reproduced with permission from Jameson JL, Fauci AS, Kasper DL, et al: Harrison’s Principles of Internal Medicine, 20th ed. New York, NY: McGraw Hill; 2018.)
Stage 1 represents the transition from wakefulness to sleep and consists of theta activity (3.5 to 7.5 Hz), indicating neuronal firing is becoming more synchronized. At this point, a person may experience hypnic jerks, which are sleep twitches caused by muscle contractions followed immediately by relaxation. This may also be experienced as a falling sensation. Upon entering stage 2 sleep, EEG activity is characterized by sleep spindles and K complexes (Chapter 34). A sleep spindle is a burst of waves (12 to 14 Hz) that lasts about half a second. K complexes are sharp waves that are associated with inhibition of neuronal firing. Stage 3 EEG activity is then characterized by synchronized, high-amplitude, delta waves (<3.5 Hz). Heart rate, breathing rate, and brain activity also decrease at this stage, which is also known as slow-wave sleep.
After stage 3, EEG activity during REM sleep is characterized by irregular, low-voltage, fast waves that mimic those during stage 1 sleep. Using this criterion, REM sleep is very light. Eyes also begin to move rapidly back and forth while eyelids remain closed. At this point, postural muscles become very relaxed. This is referred to as sleep paralysis. Using this criterion, REM sleep is deep. This is why REM sleep is sometimes known as paradoxical sleep. Heart rate, blood pressure, and breathing also tend to vary more in REM sleep than in stages 2 to 3. People who are woken up out of REM sleep also tend to report that they had been dreaming, whereas someone woken up out of slow-wave sleep would deny that they had been dreaming.
Two-Process Model of Sleep
Sleep is regulated by 2 different processes: the circadian clock (process C) and the sleep homeostatic process (process S) (Figure 23–8). Homeostatic sleep builds up exponentially during time awake and declines exponentially during sleep. At the same time, the circadian clock drives a rhythm in attention and arousal that is independent of the duration of prior wakefulness. The distance between the 2 processes adds up to account for total sleep pressure. During a 24-hour period of sleep deprivation, many people feel a “second wind” on the following morning as process C kicks back in, overcoming the continual increase in process S.
Two-process model of sleep. Schematic demonstrating how sleep timing is controlled by 2 different processes that together contribute to overall sleep pressure. Process C (green line) is driven by the internal circadian clock and persists as a 24-hour rhythm of arousal in constant conditions, even under 40 hours of wakefulness (shown between 48 and 88 hours). Process S (black line) demonstrates the sleep homeostat that builds exponentially with increased wakefulness and decreases exponentially during sleep, resetting the homeostat. The difference between the 2 lines is the total sleep pressure. Night is represented by gray bars, and day is represented by white bars. The upper row of numbers represents zeitgeber time (ZT), whereas the lower row of numbers represents the number of consecutive hours displayed. AU, arbitrary units.
The 2-process model of sleep can also help explain dips in performance often experienced as an afternoon slump after lunch or as an early morning fog. Process C drives a 24-hour rhythm in performance that peaks in the evening for most people. Process S results in peak performance in the morning after recovering from the initial wake-up period (approximately 1 to 4 hours), referred to as sleep inertia. After that, performance driven by process S slowly declines throughout the day, but overall performance results from a combination of both process C and process S. Thus, performance is fairly high during the early part of the day due to process S and fairly high during the evening hours due to process C. That leaves the afternoon as a time when process S is declining but process C has yet to reach a full peak, causing a reduction in performance at this time of day. Of course, during the overnight hours, performance is low due to reductions in both processes. As a result, jobs that require workers to be awake and alert in the middle of the night often struggle with balancing productivity, performance, and worker safety.
For millennia, people and scientists have wondered about the purpose of sleep—even Aristotle recognized its importance for preservation of life because sleep allows the body’s organs to rest and recover. A related hypothesis states that the purpose of sleep is to conserve energy. During sleep, energy is conserved due to decreased body temperature. As a result, animals increase sleep duration when food is in short supply, thus reducing energy expenditure. Sleep improves memory. In humans, task learning improves following a night of sleep. REM sleep specifically aids in consolidation of nondeclarative (procedural) memory, whereas NREM sleep aids in consolidation of declarative memories. Similarly, natural immune response is modified by partial sleep deprivation. After just 1 night of sleep deprivation, the activity of natural killer cells, a type of white blood cell, is reduced by 28%. Sleep loss can also result in a reduction of circulating immune complexes, secondary antibody responses, and antigen uptake. Finally, recent research has highlighted the importance of sleep for proper function of the glymphatic system, in which glial cells regulate the flow of cerebrospinal fluid by shrinking and swelling. The results showed that the flow of fluid slows and the space between cells decreases during waking and increases during sleep by as much as 60%! This mechanism of sleep is very important for removing toxins from the brain, including β-amyloid, a protein implicated in Alzheimer disease.
As described earlier, circadian rhythms impact a variety of physiologic functions. Therefore, it is no surprise that circadian disruption can impair memory, reduce mental and physical reaction times, and exacerbate or increase the risk of human disease such as depression, insomnia, diabetes, obesity, immune dysfunction and cancer. In the laboratory, mice are often used to model the impact of circadian disruption on human disease since they exhibit many of the same circadian characteristics as humans, including 24-hour rhythms in temperature, melatonin, cortisol, attention, and activity. Like mice, the SCN serves as the central circadian pacemaker and is entrained by photic and nonphotic input. One notable difference is temporal niche, such that humans are diurnal and active in the daytime, whereas mice are nocturnal and active at night. Despite this major behavioral difference, clock genes are also expressed in a circadian rhythm in human tissues including white blood cells and buccal cells. As with all genes, circadian genes are prone to heritable mutations over time. These conserved variants have been identified in many of the circadian genes, with significant phenotypic consequences that have driven the field of circadian geneticists to create mouse models of the genetic change and further our understanding of circadian protein roles, function, and dysfunction.
Much effort has been made to identify genetic causes for circadian sleep disorders, which have been defined by the International Classification of Sleep Disorders (ICSD) as “a persistent or recurrent pattern of sleep disturbance due primarily to alterations in the circadian timekeeping system or a misalignment between the endogenous circadian rhythm and exogenous factors that affect the timing or duration of sleep.” Six disorders have been recognized as true circadian rhythm sleep disorder by the ICSD; these include advanced sleep phase syndrome (or familial advanced sleep phase syndrome [FASPS]), delayed sleep phase syndrome (DSPS), non–24-hour sleep–wake disorder (N-24; also known as free-running disorder), irregular sleep–wake rhythm/phase (ISWR), jet lag, and shift work.
To date, FASPS has been the only circadian disorder found to display true Mendelian inheritance. This disorder is characterized by an approximate 4-hour advanced phase angle of sleep onset every day. These people wake up in the very early morning (1 to 4 am) and fall asleep very early as well (6 to 7 pm), even in the presence of social pressure to stay awake. Although this disorder is likely to have many genetic sources, 2 such sources have been identified as either of 2 complementary mutations: a S662G mutation in the hPer2 phosphorylation site or a T44A mutation in the phosphate transfer domain of CK1-δ.
The etiology of DSPS has not been as straightforward. DSPS is the most common of the CRSDs and is characterized by extremely late bedtimes (2 to 6 am) and midday wake times (10 am to 2 pm). Although there is 1 reported case of inherited DSPS where the genetic defect is unknown, a length polymorphism located in exon 18 of the Per3 gene has shown the most promising association with the phenotype. A single nucleotide polymorphism in the Clock gene (3111 T>C) has also been a promising genetic lead, with people carrying the C/C genotype exhibiting strong evening preference, although not DSPS per se. Like FASPS, however, there are likely many genetic causes for the phenotype that have yet to be identified. Numerous clinical trials have attempted to treat delayed circadian phase with light therapy. Even delayed sleep phase associated with mental illness such as attention-deficit/hyperactivity disorder (ADHD) was successfully treated in 1 pilot study using early morning light therapy. Moreover, the reduction in ADHD symptoms was correlated with the size of the phase advance induced by light therapy.
N-24 is common in blind people and is a result of a lack of perception of photic entraining stimuli, which then leads to the person free-running through the geophysical days. N-24, therefore, does not necessarily have a primary genetic component. ISWR is characterized by the absence of circadian sleep–wake, and its cause is unknown. Jet lag is the unpleasant physiologic and psychological aftereffects of traveling across multiple time zones, and its severity is proportional to the number of time zones crossed, direction of travel (west to east is considered more severe for humans), sleep deprivation, presence of zeitgebers at the destination, and individual tolerance. While the cause of jetlag is known to be the misalignment of circadian phase with the geophysical phase at the destination (or disruption/misalignment of multiple internal endogenous oscillators), much work has been done to alleviate the symptoms and elucidate their root causes. To date, no major genetic studies have been undertaken in humans regarding N-24, ISWR, or jet lag.
The final recognized circadian rhythm sleep disorder, shift work disorder, is caused by circumstances similar to jet lag, in that misalignment of circadian phase with the zeitgebers (light, social pressure, stimulants) during the work shift causes unpleasant aftereffects or sleepiness, especially in people on a rotating schedule. Studies in individuals involved in shift work (estimated at 1 in 5 American workers) have discovered how genetic polymorphisms in circadian genes associate with shift work tolerance, sleep strategy, and other CRSDs. For example, multilocus models of polymorphisms in several clock genes are associated with alcohol or caffeine consumption and sleepiness of hospital shift work nurses. One contributing factor to sleepiness and adaptation to shift work is the decision of when to sleep on days off. For example, many shift workers switch back to sleeping at night on days off. Different sleep strategies are used by hospital shift work nurses, who frequently work several 12-hour night shifts in a row followed by several days off. The most maladaptive strategies are ones in which nurses take long, daily naps on or stay awake for >24 hours on at least 1 day per week. Nurses using these strategies reported greater sleep disturbance, an earlier chronotype, and more cardiovascular problems.
Characteristics of human mental health display an intriguing association with circadian rhythms. The symptoms of jet lag and shift work briefly described earlier offer evidence that disruption of circadian phase angle can lead not only to side effects, but also the possibility of more serious conditions. Patients with mood disorders often exhibit altered circadian phase in body temperature, cortisol, and even melatonin. It has been suggested that the connection between mood disorders and circadian rhythms may involve the literal connection between the serotonergic raphe nuclei of the brainstem and the SCN, both directly and indirectly via the IGL of the thalamus. Numerous 5-HT receptor subtypes are expressed in the SCN, and the interplay between the circadian system and the serotonergic system is thought to be extensive at both the molecular and tissue levels. In combination with the extant data on the role of clock genes in normal and abnormal human circadian rhythmicity, these data suggest not only that circadian genes could play a potentially huge role in mental health, but also that human circadian neural organization is an appropriate starting point for research into novel treatments for mental disorders. For example, some medications may have improved efficacy or reduced side effects when taken at a particular time of day. This approach is often referred to as chronotherapy.
Circadian rhythms are 24-hour oscillations in behavior and physiology that arose out of necessity to survive in a world with regularly occurring light and dark periods. In mammals, these rhythms are controlled by a primary clock located in the SCN of the hypothalamus that orchestrates secondary clocks throughout the rest of the brain and body. The SCN is necessary and sufficient for driving 24-hour rhythms in sleep/wake, locomotor activity, feeding/fasting, hormone release, heart rate/blood pressure, and so on. SCN neurons and nearly every cell in the body can generate 24-hour rhythms in transcription due to a molecular clock composed of interlocking feedback loops. However, SCN neurons have an additional feature of being able to couple into a network that allows animals to shift their clock phase to match the environmental cycle of light and dark, food availability, and even the presence of predators. This network also allows adaptation to changes in the length of the light period as the seasons change across an annual cycle. The core features of an internal clock—persistence in constant conditions, entrainment to the environment, and temperature compensation—are evident in nearly all life on Earth, including humans. The circadian clock is important for human health, and disruption of circadian rhythms (as in jet lag and shift work) can exacerbate and/or increase the risk of disease. Translation of the findings from animal models and human laboratory studies may lead to novel chronotherapeutic treatments for disease.
Circadian rhythms are 24-hour oscillations in behavior and physiology that arose out of necessity to survive in a world with regularly occurring light and dark periods.
Mammalian circadian rhythms are controlled by a primary clock located in SCN of the hypothalamus that orchestrates secondary clocks throughout the rest of the brain and body.
Nearly every cell in the body can generate 24-hour rhythms in transcription due to a molecular clock composed of interlocking feedback loops.
The SCN is necessary and sufficient for driving 24-hour rhythms in, for example, sleep/wake, locomotor activity, feeding/fasting, hormone release, heart rate, and blood pressure, and SCN neurons form a coupled network that allows animals to shift their clock phase to match the environmental cycle of light and dark, food availability, and even the presence of predators.
The circadian network allows adaptation to changes in the length of the light period as the seasons change across an annual cycle.
The core features of an internal clock—persistence in constant conditions, entrainment to the environment, and temperature compensation—are evident in nearly all life on Earth, including humans.
The circadian clock is important for human health, and disruption of circadian rhythms (as in jet lag and shift work) can exacerbate and/or increase the risk of disease.
Translation of the findings from animal models and human laboratory studies may lead to novel chronotherapeutic treatments for disease.