By Yuka Fukuda
Component of Neuroscience of Sleep

1.0: Introduction to Circadian Rhythm

Recognizing and adapting to the changes in the environment contributes to successful growth and survival, and ultimately to successful reproduction.As a response to the sunlight exposure variability resulting from the rotation of the Earth, organisms exhibit endogenous cyclic behavioural, physiological and cognitive changes that peak every 24 hours. These rhythms persist in constant environments; therefore are considered to be endogenous. The rhythms are called circadian rhythms (Latin word circa means “about”, and dies means “day”), because the cycles are close to, but not equal to, 24 hours. Most of the living things on the planet possess circadian rhythm – from the higher order animals to lower order animals like plants, fungi, roundworm c.elegans and bacterias.The study of circadian rhythm falls under chronobiology which studies the changes in organisms in daily, tidal, weekly, seasonal and annual rhythms; circadian rhythm represents the field of study of the changes in a day.
Circadian endogenous rhythm persists in environments such as darkness, though it is possible to reset the onset of the rhythm with a cue, which flexibility helps organisms to adapt. The synchronization, called entrenchment, occurs with cues of the environment called zeitgebers (German meaning of “time giver”). Light sources usually become the zeitgebers, but any cues that infer the time-of-day will entrench the internal circadian clock. The circadian rhythm is generated in Suprachiasmatic nucleus (SCN) in the hypothalamus, as well as in individual cells called peripheral oscillators. The SCN is known as the master clock, and organizes the cycle of the peripheral oscillators throughout the body, but both the SCN and the peripheral oscillators are capable of affecting each other. The rhythm is most easily noted by the behavioural changes such as eating and sleeping, and also within individual cells, such as protein expression changes. Cells can utilize this information and able to create a prediction about future, e.g. time memory (see section 4.4: Time Memory).
In this page, circadian rhythm and its implications, especially that of sleep, melatonin and memory formation, are discussed using examples from variety of organisms, mainly rats (Rattus norvegicus) and mice (Mus musculus), but also sea slugs (Aplysia californica), zebrafish (Danio rerio), and fruit flies (Drosophila melanogaster) to study molecular mechanisms of circadian rhythm.
Some of the terminologies that are repeatedly used in this page are below: Light-dark cycle, (LD), dark-dark cycle (DD), suprachiasmatic nucleus (SCN), melatonin (MT), CLOCK protein (CLK), CYCLE protein (CYC), period gene (per), timeless gene (tim), muscle aryl hydrocarbon receptor nuclear translocator-like protein (BMAL), cryptochrome gene (cry), mitogen-activated protein kinase (MAPK), cAMP-responsive element (CRE)-binding protein (CREB), N-Methyl-D-aspartic acid (NMDA), brain-derived neurotrophic factor (BDNF), dentate gyrus of hippocampus (DG), hippocampus (HPC), CA1 area of hippocampus (CA1), long term sensitization (LTS), long term memory (LTM), short term memory (STM), spontaneous firing rate (SFR), resting membrane potential (RMP), crepuscular (meaning active at transition times between light and dark – fruit flies are crepuscular)

1.1: The Origin of Circadian Rhythm - The Neural Substrates and Mechanisms

Circadian rhythm is established from the protein expression feedback loop within cells in the body. This individual rhythm is called peripheral clock or oscillators, and this is coordinated by the master clock SCN in the hypothalamus.
Because it is difficult to study genetic and cellular interaction in humans, fruit flies and mice are often used to study molecular mechanisms of circadian rhythms. These two organisms are genetically similar to humans and are simpler to study with. Scientists generally assume that systems that exist in lower order animals are also present across phylogeny, and the basic mechanism is expected to persist in higher order animals.

1.1a: Protein Feedback Cycle

The first evidence in circadian protein-gene interaction was found in fruit flies (Konopka & Benzer, 1971). In fruit flies, when proteins CLOCK (CLK) and CYCLE (CYC) heterodimerize, they enter the nucleus and bind to the area of the gene called E-box elements within the promoter of the period (per) and timeless (tim) genes. Per gene length is known to cause pleiotropy in long (perL), short (perS), and arrhythmic (per0) phenotypes, which are persistent in darkness (Panda, et. al., 2002). The genes get transcripted, and produce PER and TIM proteins outside the nucleus. PER and TIM are further heterodimerized and enter the nucleus, at which point are phosphorylated (P) by proteins such as doubletime (DBT) or casein kinase II (CKII). Once phosphorylated, TIM and PER repress the transcriptional activity of CLK–CYC (see Fig. 1) (Gerstner & Yin, 2010). Therefore the amount of PER and TIM are cyclic. This process returns to its original state in about 24 hours.
In mammals, the circadian feedback network is similar to that of fruit flies, except the network involves a protein called brain and muscle aryl hydrocarbon receptor nuclear translocator-like protein (BMAL) in place of CYC. Also, there is a gene cryptochrome (cry) instead of tim. Resulting proteins are PER and CRY, and they are phosphorylated by casein kinase Iɛ, and later enter the nucleus to inhibit CLOCK–BMAL transcriptional activation. As well, Mitogen-activated protein kinase (MAPK) phosphorylates BMAL, repressing BMAL–CLOCK activity (Gerstner & Yin, 2010) (see Fig. 1).
Fig. 1: Circadian rhythm protein feedback loop. Left hand side shows the mechanism of fruit fly, and right shows that of mouse. The left diagram shows the possible interaction of melatonin and these proteins.

1.2: The Entrenchment - the Adaptive Strategy

In this section, mechanisms that allow organisms to adapt to zeitgebers are described.

1.2a: The SCN, the Master Clock

Suprachiasmatic Nucleus (SCN) exists in each hemisphere of hypothalamus, just next to the midline. It is located above the optic chiasm, hence the name. It is a congregation of many cells, and since each cell has a rhythm, the SCN rhythm is the sum of all the rhythms as a whole.
Across phylogeny, SCN – or an equivalent mechanism – receive photic input to modulate circadian rhythm to allow environmental adaptation (Gerstner & Yin, 2010). In mammals, photoreceptors containing melanopsin project to SCN via retinohypothalamic tract (RHT). SCN then projects to subparaventricular zone (sPVz), then to the rest of the hypothalamus, including ventrolateral preoptica area (VLPO), the lateral hypothalamus (LH), and the paraventricular nucleus (PVN), to control hormone, temperature, feeding and sleep-arousal cycles. VPLO also connects to tuberomammilary nucleus (TMN), which is important for slee-wake rhythms. SCN also projects to limbic systems like hippocampus and amygdala, as well as hypocretin-expressing cells in the lateral hypothalamus, and to pineal gland (see the page on Neural Mechanism of Sleep) (see Fig. 2). These pathways influence hippocampus (HPC), and influence long and short term memories (see section 4.0: The Memory).

1.2b: The Peripheral Clock

The expressions of circadian rhythms in cells are called peripheral oscillators, and they provide feed-forward responses to the SCN. Reciprocal relationships among cellular peripheral clocks and SCN are coordinated by clock genes operating in each tissue.
Adaptation of the peripheral clocks is not well known; however, recent researches are considering the possibility of the peripheral clock rhythm shift in time memory formulation (see 4.2: Time Memory).

Fig. 2: SCN projections in fruit fly and mouse brains.

1.3: The Rhythm Within -Intracellular Protein

Aside from the circadian feedback proteins that generate the rhythm, proteins associated with synaptic plasticity in cells also exhibit rhythm.

1.3a: CREB

One of the cyclic proteins in each cell is cAMP-responsive element (CRE)-binding protein (CREB). It is an important protein that facilitates plasticity and learning. When NMDA receptors open at an activated synapse, intracellular CA2+ concentration increases, then Ca2+/calmodulin-dependent protein kinases II (CaMKII) activates P13 kinase, and then activates MAPK. MAPK then enters the nucleus and phosphorylates CREB. PhosCREB then attaches to CRE in DNA, and increases BDNF expression and remodels the synapse by altering cFOS and JIF. CREB is associated with memory; it was shown that HPC cells that encode memory increased CREB, and eradicating cells that increased CREB removed the encoded memory (Josselyn, et. al., 2004). CREB may also be associated with circadian rhythm, since CRE site that binds to CREB was found in the promoter per gene of mouse, suggesting a relationship between circadian rhythm and CREB and PER mediated transcriptional regulation (Travnickova-Bendova, et. al., 2002).

1.4: The Memory - Rhythm Manifestation in the Form of Memory

Given the fact that the proteins related to plasticity (e.g. CREB) have circadian rhythms, it is logical to assume that memory, the artifact of plasticity, is also influenced by circadian rhythm. Memory is established upon long term potentiation (LTP), plasticity and metaplasticity. LTP refers to the fact that frequent firing of a pathway strengthens it. Plasticity is the term for the ability and the flexibility of neurons to fire and to connect with others, and metaplasticity is for the ability and the extent of cells to become plastic. This is often caused by the increase of spontaneous firing rate (SFR). LTP is considered the mechanical process of learning, and metaplasticity, a relatively new topic, helps describe learning appropriately (Abraham, 1996; Barnes, 1977; Gerstner & Yin, 2010; Nishikawa, et. al., 1995). The knowledge as to what time-of-day learning is enhanced helps scientists to choose the best time to conduct studies.
There are evidences that suggest the independence of per gene in regulating memory (e.g. Sakai and colleagues found in 2004 that per0 flies can’t make long term memory, whereas over-expression enhances it). The sections below will discuss other mechanisms that circadian rhythm exerts its power over memory regulation.

1.4a: Melatonin, and Other Hormones on Memory

The exact connection between the sleep hormone melatonin (MT) and circadian rhythm is yet to be concluded, but there are two putative pathways. The first pathway is the repression of adenylate cyclase (AC) and protein kinase A (PKA), which influence cAMP-responsive element (CRE)-binding protein (CREB) activation. The second mechanism is the activation of the MAPK–CREB cascade through Ca2+ influx, which causes transcriptional activity through CRE elements in per promoters (Travnickova-Bendova, et. al., 2002). These mechanisms are described in the fruit fly diagram of Figure 1.
MT is also known to influence metaplasticity and LTP in many regions of the brain. MT rhythmic release is seen in many animals such as humans, zebrafish, sea slugs, mice and flies. It seems that MT lowers memory performance, and this is true for nocturnal mice too (Chaudhury & Colwell, 2002). One of the classic studies done in zebrafish is by Rawashdeh and colleagues in 2007, where active-avoidance conditioning of dark chamber with electric shock prompted the fish to choose the light chamber with no shock. Acquisition and memory formation was best during the active (light) time, which suggested the endogenous time-of-day effects that persisted even in DD. Melatonin treated fish had Long Term Memory (LTM) formation decline with no effects on acquisition. This effect was prevented if MT receptor antagonists were injected. However, C57Bl/6J mice, which lack MT, exhibit time-of-day-dependent changes in memory formation (Chaudhury & Colwell, 2002). If the period of inactivity exerts less MT, the peak of memory performance could occur then. This means that the circadian effect on memory is not only MT- mediated.
There are evidences that suggest possible effect of other hormones, such as those produced by the adrenal gland, on circadian rhythmicity of metaplasticity (Dana & Martinez, 1984). Therefore, circulating hormones could operate as a zeitgeber, setting the clock in hippocampal cells (Gerstner & Yin, 2010).

1.4b: Time-of-Day Effect on Metaplasticity

Synaptic efficacy, and therefore LTP, is thought to depend on time-of-day in rodents. In 1977, Barnes and colleagues found a rhythmic metaplasticity of rat Dentage Gyrus (DG) and area CA1 in hippocampus; metaplasticity was enhanced during the dark phase in DG, and during the light phase in CA1. The findings left some controversial thoughts, however, because the tissues were harvested during the opposite time-of-day from when the electrophysiology was completed. In mouse, when CA1 tissue was harvested during the light period and tested in the dark period, metaplasticity was enhanced, whereas when the tissue was harvested and tested in the light period, metaplasticity lowered; this result revealed that metaplasticity was greater when tissue was examined in the dark period (Chaudhury, et. al., 2005). This suggested that the time-of-day effects on metaplasticity depend on the time of testing, and not the time of harvest.

1.4c: Time-of-Day Effect on Memory

For researches on sleep and memory consolidation, please refer to other pages on the Neuroscience of Sleep.

One of the most celebrated studies on time-of-day effect on memory performance is done on sea slug Aplysia californica by Fernandes and colleagues in 2003, where non-associative learning of Long Term Sensitization (LTS) revealed the acquisition, not the recall, was best at aplysia’s active time in both light-dark (LD) and constant dark (DD) conditions. The experimenters measured the amount of siphon withdraw reflex in response to the electric shock to the tail of aplysias, after sensitizing them with the electrical shock to the side. Baseline withdraw response was not time-of-day-dependent, signifying that the learning, and not the natural withdraw response, was rhythmic.
Time-of-day effect on LTM on mice was studied by Chaudhury and colleagues in 2002. They found that mice display optimal memory recall and acquisition during the 'inactive phase' (the light period) for contextual, which is HPC dependent, and cued fear conditioning, which is amygdala dependent. Findings also suggested that the peak time of memory recall is under time-of-day-dependent control, and is independent of the time of training.

1.4d: Time-of-Day Effect on CREB and MAPK

The proteins involved in synaptic plasticity have rhythmic functionality; thus there are optimal times that they establish plasticity and learning. In mice, circadian cycling of cAMP and MAPK phosphorylation in the HPC paralleled time-of-day-dependent oscillations in RAS activity. It was found that the disruption of the circadian rhythm of this cAMP–MAPK–CREB cascade in the HPC, pharmacologically or optically (constant light exposure), impaired memory formation (Eckel-Mahan, et. al., 2008).
In Aplysia, LTS training during different times of day produces different levels of activated MAPK. The training in the day time increased phosphorylation of MAPK more than the training at night. This phosphorylation correlates with patterns of LTM enhancement. With agents that activates MAPK activity or MAPK-dependent transcription, phosphorylation and LTM increased at night (Lyons, 2006).
The data therefore convey that the cAMP–MAPK–CREB is a phylogenetically conserved pathway mediating the time-of-day effect on memory.

1.4e: Time Memory

Time memory, or time-stamp memory, is the memory about certain time-of-day which an event occurred (Hut & van der Zee, 2011). Time memory and the time-of-day effect on memory are different, as the former records the time that event took place, and the latter records the event better at certain times. Circadian rhythm gave organisms the senses of time in relation to the sun, and therefore to predict the regular and significant environmental conditions. An example of such use of circadian rhythm is when preys avoid predators. An initial encounter with the predator becomes information about the predator’s habits and routines, and specific locations and time can be identified and be avoided to reduce the chance of further encounter. A biological clock therefore manifests in time memory, which provides the organism with the ability to anticipate and adjust for short term changes in conditions. By encoding the time-of-day of which an event occurred, organisms are able to anticipate it the next day at the same time.
There are evidences to suggest that time memory requires not only the SCN but also the peripheral clocks (Gerstner & Yin, 2010). Rhythms of passive avoidance conditioning in rats require an intact SCN, supporting a role for central circadian pacemaker cells in the regulation of time-of-day effects on memory (Stephan & Kovacevic,1978). However, SCN ablation does not prevent expression of the time memory in a conditioned place-avoidance task of hamsters (Cain & Ralph, 2009). As well, time-of-day learning in animals with SCN lesions has been observed (Ko, 2003), which leads to a speculation that transitional changes in the environment are learned only temporally. Peripheral oscillators are therefore useful to learn the timing of specific conditions without compromising the function of the central clock that is tied to regular predictable changes of light and dark.
Based on the previous work done on fear conditioning in the amygdala, where HPC cells that react to the stimulus expressed high CREB (Josselyn, et. al., 2004), it can be speculated that those high-CREB cells are the ones that express high metaplasticity at the time of the learning (see section 3.1: CREB). More research on this field is to be expected.

1.5: Inquiry - Method to Study Circadian Rhythm

In this section, examples of methods to study circadian rhythm are introduced. There are different methods suitable for different organisms, so depending on the purpose and the goal, choosing suitable organism becomes important.

Behavioural and Biological Measures

l Wrist actigraphy – wearing a device that record the movement, it measures the sleep cycles as they are characterized by differentiated muscle tones.
l EEG – Electroencephalogram, measures the electric brain waves from the skull via scalp.
l Wheel-running activity – suitable for caged animals like mice, it measures and differentiates the active and inactive period for the duration of the recording.
Molecular Measures
l Proteomics – by comparing the amount of proteins across time, the change in protein expression can be analyzed.
Neuronal Level
l Per:luc reaction – using firefly glowing chemicals, luciferase and luciferin, the per rhythm can be measured. Luciferase gene is inserted and made to be turned on by upper-stream per promoter. The amount of luciferase and PER therefore correlates, and when luciferin is introduced in vitro, the number of photons can be counted to measure the per activity.

1.6: Beyond - Improving Health

Circadian research can provide tremendous benefits to the health of the population, because circadian rhythm disturbance can be a cause and/or a result of a disorder. The usefulness of understanding circadian rhythm ranges from memory to longevity. Since circadian rhythm is a fundamental function for organisms, its breakdown may affect memory, sleep, eating, and many other basic rhythms. In this section, such significant implications of circadian research are being discussed.

1.6a: Melatonin, Cell Resistance and Sleep Deficiency

Melatonin imbalance reduces the quality of sleep and prevents proper body restoration. It also makes the body susceptive to stress, due to the decrease of the efficacy of melatonin to regulate cortisol and blood pressure, and the decreased cell resistance and immunity (Reiter & Robinson, 1995). Reduced immunity increases the risk of cancer and further disrupts the sleep-wake cycle (Doghramji et al. 2010). The decrease in cell resistance interferes with neuronal network and neurotransmitter balance. Various transcriptomes and therefore gene expression is also affected, which leads to changes in cellular functions and plasticity (Summa & Turek, 2010). Other chemicals such as growth hormone and brain-devided neurotrophic factors (BDNF) will be produced less as well (Reiter & Robinson, 1995).
Sleep deficiency as a cause of disrupted circadian rhythm may decrease cognitive performance and attention. In addition, reduced carbohydrate tolerance, insulin response, secretion of thyrotropin, and immune response may cause (Doghramji et al. 2010). In this way, both circadian rhythm and sleep deficiency impair health.

Jet Lag

1.6aa: Jet Lag

Jet lag is a state of drowsiness and fatigue that result from traveling across time zones. The internal clock becomes inaccurate in a foreign land and is required to shift to the local time. The circadian clock can be delayed with the photic input from the retina; however, there is no mechanism to fast-forward the clock.
The true problem lies in the fact that circadian shift is effortful and unnatural to the body. Frequent shifting results in unstable chemical balances, reduced alertness and sleep. Repeated jet lag is detrimental as it may lead to arrays of psychiatric conditions, such as depression and Alzheimer Disease (Vosko et. al., 2010). For disorders caused by sleep deprivation and disruption, please refer to other pages in the Neuroscience of Sleep.

1.6b: Developmental Disorders

In infants, solidifying circadian rhythm is done in the first three months, but it is possible that children with disorders are inhibited from establishing proper circadian rhythm. In order to alleviate harmful symptoms that arise from circadian disturbances, circadian treatment can be established. For example, the effect of rhythm consolidation via skin ship at night and in the morning in infants with addiction is being experimentally examined at Ralph lab at the University of Toronto. This study is designed to find the connection between the disrupted rhythm and longevity.

1.6c: Childhood Depression

Childhood depression is thought to be partially mediated by the temporary endogenous delay in circadian rhythm at age 11-13 (Crowley, 2007). School start time, geared towards working parents and does not think about the children, completely ignores this natural shift in cycle. Not only do early adolescents must endure shift in their clock, which is difficult to adapt to, but they also need to force themselves to wake up in time for school. This causes the decline in attention, mood and wakefulness, which ultimately lead to decline in grades (see other pages under Neuroscience of Sleep).


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