Introduction
The circadian clock and sleep homeostasis are two dynamically associated physiological systems. Both are fundamental for normal physiology and contribute to many biological processes. Sleep is defined as a state of quiescence characterised by reduced responsiveness to sensory stimulation and with a lack of awareness of the external environment (1). The key to sleep is that it is a reversible state. We transition between wake and sleep states every day. This reversibility is also rhythmic and consists of sleep-wake cycles. The sleep-wake cycle is the alternating states of sleep and wake over a 24 hour period.
Much research has been done to identify the neural circuitry underlying not just sleep and wake states but also their temporal distribution and transitions.
In simple terms, sleep and wakefulness are governed by central neuronal circuitry mediated by several neural pathways and transmitters (neuro-humoral). Wakefulness is controlled by the monoaminergic systems of the brainstem, the cholinergic neurons in the brainstem and basal forebrain as well as the hyprocretin/orexin cells of the hypothalamus. Sleep, on the other hand, is regulated by groups of GABAergic neurons both in the preoptic area and the brainstem (2).
A system also exists that enables the sharp transitions between sleep and wake states. This is known as the flip-flop model of sleep. The flip-flop switch prevents the existence of intermediate states between sleep and wakefulness. This is achieved through direct mutual inhibition between the GABAergic neurons and the monoaminergic systems responsible for sleep and wake respectively (3).
The sleep-wake cycles are then governed by the interplay between 2 processes, the sleep dependant homeostatic mechanism called “Process S” and the sleep independent circadian mechanism “Process C”. The circadian pacemaker is located in the suprachiasmatic nuclei of the hypothalamus while the sleep homeostatic mechanism has not been conclusively defined (4).
In this essay, we will attempt to detail the neurophysiological mechanisms mentioned above in greater depth. We will also seek to establish some of the functional implications in clinical, social, financial and research terms.
Neural circuitry of wake and sleep
Neuro-humoral systems of wake
Ishimori in 1909 pioneered the development of humoral theories of sleep. He observed that the cerebrospinal fluid extracted from sleep-deprived dogs could induce sleep in a normal dog. This led to the hypothesis that sleep was regulated by neurotransmitter substances (5). In the 1930s, Frederick Bremer, transected the cat brainstem at 2 levels. Sleep wake cycles were maintained after a low medullary transection (encehale isole) but a higher transection between the pons and midbrain (cerveau isole) caused chronic drowsiness (6). Then in 1949, Moruzzi and Magoun showed that electrical stimulation of the midbrain reticular formation in cats produced alerting of the cortex and an awake state (7). These studies led to the hypothesis that the forebrain was kept awake by activity in the reticular formation. This led to the subsequent discovery of a neural system, known as the ascending reticular activating system (ARAS) which is the neural projection from the brainstem to the cortex and subcortical structures. The biochemical and neuro-humoral pathways of the ARAS were subsequently worked out.
The cell groups of the ARAS that promote wake states include:
Cholinergic (ACh) laterodorsal and pedunculopontine tegmental cells. These groups have inputs to the thalamus, the lateral hypothalamus, the basal forebrain, and the cortex. During wake states and during rapid eye movement (REM) sleep, these cell groups have high firing rates. The firing rates are reduced during slow wave sleep (8).
Noradrenergic (NE) locus coeruleus, serotonergic (5-HT) cells in the dorsal raphe nuclei, and histaminergic (HA) cells in the tubero-mammilary nucleus of posterior hypothalamus. These have projections to the thalamus, the basal forebrain, and the cortex. These monoaminergic neurons fire during wakefulness, are reduced during slow wave sleep and do not fire during REM sleep.
Dopaminergic (DA) ventral tegmental area, substantia nigra and periaqueductal gray projections. These promote wake states by stimulating the cortex directly. They also work indirectly through the thalamus, hypothalamus and basal forebrain (9).
Cholinergic (ACh) basal forebrain projections to the cortex. These include the cell groups of the medial septum, the vertical and horizontal diagonal bands of Broca, the preoptic area and the substantia innominata. These neurons fire during waking and REM sleep and are quiet during non-rapid eye movement (NREM) sleep.
The hypothalamic orexin/hypocretin system. These neurons activate the cortex and project to the wake-promoting areas of brainstem, hypothalamic and basal forebrain. They help to maintain long and stable wake states. (10)
In summary, wakefulness, in various aspects, is promoted by neurons producing acetylcholine and monoamines such as noradrenaline, serotonin, dopamine, and histamine. Orexins/hypocretins assist in sustaining long and stable periods of wakefulness.
Neuro-humoral systems of sleep
In 1930, neuropathologist von Economo identified that lesions in different areas of the hypothalamus was associated with different effects on sleep and wake. He found that the anterior hypothalamic area was sleep-promoting and lesions in this area led to chronic insomnia. The posterior hypothalamus on the other hand was wake-promoting and lesions in this area led to excessive sleepiness and coma (11).
The ventrolateral preoptic area (VLPO) and median preoptic area of the anterior hypothalamus are known to be involved in promoting sleep. The neurons of VLPO promote NREM sleep via GABA and the dorsomedial extension of the VLPO is involved in REM sleep regulation (12). The preoptic area is also involved in regulating sleep via thermoregulation, though the mechanisms are not completely understood (13). Lesions in the preoptic areas have also been shown to reduce both REM and NREM sleep states (14). Electrophysiological and lesional studies have shown that GABAnergic VLPO and median preoptic area project to and inhibit wake cell groups (15,16).
REM sleep is generated by the dorsolateral pons via cholinergic neurons (17). These are known as “REM on” cells. “REM on” cells switch on and “REM off” cells switch off REM sleep respectively. This allows for NREM-REM alternations. Other “REM on “ cells that have been discovered include GABAnergic neurons located in the laterodorsal tegmental nucleus (18). “REM off” cells that have been indentified include non-cholinergic mesopontine neurons.
In summary, NREM sleep is largely regulated by GABAnergic neurons in the ventrolateral preoptic area and other preoptic regions of the anterior hypothalamus. REM sleep is driven by neurons in the pons that make acetylcholine and GABA .
Sleep-wake dynamics
The wake and sleep centers as described above have a reciprocal inhibitory relationship (19). As we cannot exist in both sleep and wake states simultaneously, this switching between states is commonly known as the “flip-flop” switch model. The flip-flop model also prevents the existence of intermediate states between sleep and awake, and enables sharp transitions.
During wake states, the wake promoting cell groups inhibit the sleep-promoting ventrolateral preoptic area. Sleep, which is promoted by ventrolateral preoptic area neurons, inhibits the wake promoting cell groups of the ARAS and in turn block their own inhibition. When one set of neurons fire, either during sleep or wake, they inhibit the reciprocal promoting cell group, thereby reinforcing their own activity. This increases the stability of either state (20).
We now have a basic understanding of sleep and wake states and the underlying mechanisms that govern either state and their switching mechanisms. What then determines the temporal nature of sleep and wake?
A process exists that governs the timing of sleep and wakefulness. In this area of sleep-wake timing, 2 processes are at play, the circadian system and homeostatic sleep regulatory mechanism (21). Borbely proposed that there are 2 separate processes underlying sleep regulation; a sleep-dependent homeostatic process known as “Process S”. This rises during waking and declines during sleep. Process S represents sleep debt or propensity and this begins to build up at the onset of wakefulness and declines during sleep. The longer we stay awake, the more somnolent we become. Sleep deprivation studies show results in reduced neurocognitve abilities, impaired physiological functions and ultimately to cellular decline and death (22). Process S is postulated to be based on a neurochemical mechanism.
While the homeostatic “Process S” governs the sleep need, the other process,“Process C”, is independent of the amount of sleep or wakefulness preceding. Process C is an internal circadian clock or pacemaker that controls the timing of sleep, a biological 24 hour clock system that synchronizes with the natural day-night cycle. This circadian process also regulates the other biological mechanisms such as feeding patterns, core body temperatures and hormone production. The circadian regulatory mechanism lies in the suprachiasmatic nucleus and is vital for the sleep onset and sleep maintenance as well as the stability of wakefulness during the activity phase.
Though both circadian and the homeostatic processes appear to be two independent mechanisms, any alterations in the circadian or sleep homeostatic process would affect the timing, intensity, and structure, implicating the interdependence between the two processes in sleep–wake regulations. Clock gene expression studies highlight such interdependencies between the two process, as the clock genes, besides setting the internal timing of day, are sensitive to changes in sleep homeostasis (23). Similarly, endogenous somnogens like adenosine potentiate the dynamics of sleep by integrating various functions including circadian rhythm, though we are yet to understand the physiological mechanisms.
Circadian regulation of sleep
The circadian clock, apart from its sleep regulatory role, controls many other aspects of biology. These include core body temperature, hormonal cycles, blood pressure variations, cellular maintenance and regeneration, metabolism and cognitive behaviour and performance. All of these function in a 24-hour periodicity (25, 26). This circadian system has two fundamental characteristics. The first is an endogenous rhythmicity with a periodicity of 24 h that functions independently external environmental factors. The second is the adaptibility to have this rhthmicity shifted by external factors such as light, temperature and feeding. A consequence of the circadian rhythm is that it creates a system of alternating states, that of a biological day and night. These then cyclically alternate. Each will exhibit differences in hormonal, electrophysiological, and behavioural profiles. Circadian systems are important. This is evidenced by observational studies that show that circadian rhythm disorders lead to sleep disorders, metabolic syndromes, obesity and overall decreased life expectancy (42, 43).
The circadian clock consists of a central clock and peripheral tissue clocks. The central clock is located in the suprachiasmatic nuclei (SCN) of the hypothalamus. The suprachiasmatic nucleus receives light input from the retina in the morning, this is the pathway by which the circadian clock is constantly reset. It functions as the master pacemaker in the body. It synchronizes the other physiological rhythms in a 24 hour cycle that is governed by the day-night cycle of the earth. The central clock operates to synchronize the clocks in peripheral tissues (27).
As the primary circadian oscillator in the brain, the SCN is composed of many small interconnected neurons that vary in both the rate of action potential firing and gene expression (28). The retinohypothalamic tract, which are retinal projections terminating in the SCN, allow direct synchronisation of the circadian oscillator with that of the variations in light through the day-night cycle (29). Therefore the SCN is entrained to the day-night cycle via the retinohypothalamic tract and variations in day-cycles would correspondingly affect the circadian pacemaker. Retinal ganglion cells, now recently discovered as the third photoreceptor in the retina (non-rod, non-cone), also project to the SCN and express their effect via melanopsin (30,31).
In SCN ablation studies, destroying the SCN completely led to circadian arrhythmia in animals (32). The autonomous circadian oscillations of the SCN neurons are generated by a set of clock genes that form interlocking transcription/translation feedback loops. These loops function together to generate high amplitude of circadian rhythms of gene expression. At the molecular level, 4 integral clock proteins form the core of the SCN clock. These are either positive (activator) or negative (repressor) elements that regulate the rhythmicity of gene expression. The 2 activator elements are BMAL1 and CLOCK. They dimerize in the cytoplasm of the SCN neurons, translocate to the nucleus and bind to the enhancer and promoter sequences of the Period (Per1 and Per2) genes and Cryptochrome (Cry1 and Cry2) genes and facilitate the transcription of these genes. The resultant protein products PER and CRY then build up, and acts as repressor elements on CLOCK:BMAL1. This leads to the downreglation of their own transcription. Once PER and CRY degarde, repression on CLOCK:BMAL1 reduces and the cycle is repeated. This forms a negative feedback loop that generates the rhythmicity of circadian clocks (27). Another interlocking feedback loop is also involved. It is composed of the activator ROR and the repressor REV-ERBα. This feedback loop strengthens and stabilizes the SCN oscillations and also assists in the maintenance of accurate circadian timings (33).
Oscillations in the SCN neural activity, hallmarked by fluctuations of spontaneous action potentials, represents the functional output of these clock genes. The output is highest during the day (6-10Hz) and is lowest at night (<1 Hz) (34). The autonomous human clock is slightly longer than 24 hours (this is observed under conditions of constant darkness), but it is entrained to be 24 hours by synchronization to environmental factors that vary over a 24 hour period. (35). The major environmental factor at play is that of light. We know that the retinohypothalamic tract (RHT) links photoreceptors of the retina directly to the SCN. Light/dark cycles across the day therefore provides information to the SCN circadian oscillator. Entrainment of the SCN to the day/night cycle would synchronise the period of the endogenous/autonomous SCN circadian oscillation to equal that of the light/dark cycle of the environment (36). Entrainment allows for an appropriate and predictable relationship between the endogenous circadian clock and the day night cycle and this enables the adjustment to local time (37). Entrainment must be distinguished from immediate synchronisation as the endogenous pacemaker generates rhythm actively and is not driven by external cues. This process is adaptive and plastic to the needs of the organism (28).
The SCN clock is reset daily based on the day/night, light/dark environmental cycles. Adequate light intensity of more than 2,500 lux is required to entrain the human circadian clock. This observation has led to the use of light therapy in the treatment of disorders associated with circadian disturbances (38). The response of the SCN to light pulses is phase dependent. This means that if light exposure occurs during periods of expected light exposure (times when the subject would normally be exposed to light), there would be little or no effect on the phase of the circadian clock. However light exposure during periods of expected darkness would be produce phase shifts in circadian rhythms These shift vary according to the timing of exposure. Light exposure during sunset or early in the subjective night produces phase delays to the circadian rhythm and light exposure during sunrise or late in the subjective night produces phase advancements (39). The exact mechanisms by which the SCN gates its biphasic response in to light exposure are unclear (40).
Besides light, there are other external environmental time-giving cues, also known as a zeitgebers, that set the phase of circadian rhythms. These cues coordinate internal physiology with the external environment via the circadian pacemaker to increase survivability (41). Other examples of zeigebers are temperature, social interaction and feeding patterns.
Homeostatic regulation of sleep
Sleep homeostasis represents a simple concept, that a period of wakefulness is followed by a corresponding period of sleep. Therefore, the longer the state of wakefulness, the longer the duration and the more intense the sleep. EEG studies show that during NREM sleep, slow wave activity in the delta bandwidth (0.5-4.0 Hz) increases after a prolonged period of wakefulness. This is also known as NREM delta power (NRD) or EEG slow wave activity (SWA) and is dependent on prior wakefulness or sleep (24). Prolonged wakefulness increase NRD and sleep reduces NRD. NRD can be viewed as the electrophysiological manifestation of cortical recovery from prior wakefulness. It is used as a measure of sleep propensity during sleep. Theta activity is used as a measure of propensity during wake (46,47)
While the circadian rhythm system regulating sleep has been fairly well worked out, a similar process for sleep homeostasis has been more difficult to identify. Traditionally, most sleep homeostasis experiments have been conducted through sleep deprivation studies. Prolonging the period of wakefulness and studying its effects on biological processes have revealed changes ranging from gene expression to metabolism and behavior (44). These studies have identified several possible factors related in sleep homeostasis. However, questions that remain to be answered are: is there a molecular factor involved in sleep homeostasis or sleep propensity? What are the anatomical sites that participate in sleep homeostasis (45)?
Neuronal activity during wakefulness is the main factor driving sleep homeostasis. A concept of energy metabolism as the driving force behind sleep homeostasis. Areas of the brain that are active during wakefulness will develop greater slow wave activity during subsequent sleep (48,49). It is assumed that slow wave activity allows for restoration from neuronal activity. Neuronal activity during wakefulness consumes energy and sleep allows energy replenishment (50). A theory is that energy consumption due to increased neuronal activity during wakefulness promotes adenosine-triphosphate (ATP) release to the extracellular space, increasing extracellular adenosine concentration. Adenosine, which acts as an inhibitory neuromodulator, then decreases neuronal activity of wake-active neurons to induce sleep. There are studies that support this theory. Kalinchuk et al showed that simulating energy depletion by preventing ATP synthesis in the basal forebrain of rats, extracellular adenosine and NREM sleep was subsequently increased (52). Gass N et al showed that by blocking adenosine A1 receptors in the basal forebrain of rats, recovery sleep amount and NRD was reduced (53). Nitric oxide (NO) is another molecule that has been found to increase in concentration in the BF during prolonged wakefulness. This is brought about by inducible nitric oxide synthase and it precedes adenosine increase (54). Interventions that inhibit the increase in NO or adenosine levels inhibit recovery sleep. (55) Therefore, adenosine is accepted as an important regulatory molecule in sleep homeostasis.
Another theory of sleep homeostasis is the synaptic homeostasis theory (56). The hypothesis is that during wake, more synapses are formed and during sleep,
they are down regulated (56). This is to allow and maintain for neural plasticity for optimal functioning. Therefore it follows that with wakefulness, there is synaptic potentiation in several cortical circuits. This synaptic potentiation required for homeostatic regulation of slow-wave activity. Slow-wave activity is associated with synaptic downscaling and this in turn is tied to the beneficial effects of sleep on performance.
A third theory of sleep homeostasis is that sleep is a cellular defence mechanism mediated by the immune system (57). The hypothesis is that prolonged wake states present a threat to the body (presumed to be depleting energy). This activates the immune system and the release of cytokines. Studies have described a role of cytokines in the regulation of sleep. An inactivation of cytokines during spontaneous sleep decreases sleep (58).
Functional significance
Sleep is now commonly viewed as a 2 process model. The propensity to sleep and the circadian rhythm coordinating sleep are constantly in balance and altering each other. It is a useful to organize thoughts about sleep regulation along these 2 lines. This brings the aspects of sleep and circadian rhythm research together. These concepts help to shape the design of experiments and the evaluation of data collected.
Based on recent and current research, the understanding of process C and process S has undergone significant changes. The SCN is now seen to be coordinating and integrating and integrating circadian rhythms rather than simply generating and driving them. Homeostasis of sleep and brain function is now widely accepted as inter-related. Sleep homeostasis research is now at the heart of research into the function of sleep. It is believed that the homestasic mechanism of sleep is generated by mechanisms of energy repletion and depletion, protection or neuronal maintenance. Process S may have an underlying neuronal oscillator outside the SCN or by a behavioural relaxation oscillator as conceived by Borbely. As this has not been fully elucidated, research continues on this. The detection of local, use-dependent changes of the sleep EEG including localised SWS activity, has provided regional and functional specificity of the effect of waking on sleep and the restorative effects of sleep. Recent neurophysiological and morphological studies have also focused upon local sleep-related changes at the synaptic level and their functional implications. It is important to be aware that synapticplasticity, stability and regulation in the brain is modified by both homeostatic and circadian factors.
This two-process model also allows a simple construct to differentiate sleep disturbances appropriately and thereby initiate specific treatments. These are related to sleep deprivation and forced dysynchrony. Sleep and biological systems are tightly integrated, and disruptions of their synchrony may have adverse health consequences. Sleep deprivation has been linked with increased risk of disorders such as depression, cardiovascular disease, metabolic derangements and reduced longevity. In a sleep restriction study conducted by Doran et al, the reduction of sleep time to 6 hours had no overt effect on mental functioning on the first day, but the influence rose significantly with the number of days of restricted sleep (59), reflecting the effect of accruement of sleep debt. Deprivation of sleep results in a cumulative decrease in psychomotor vigilance. Psychomotor vigilance changes in a circadian fashion, suggesting that both the circadian clock and sleep are important for cognitive performance. Fatigue from sleep deprivation is a significant risk factor contributing to performance decreases, and as consequence, accidents often occurs at times when people are normally asleep.
Forced dysynchrony has led to disorders such as jet lag, work shift syndrome, delayed phase sleep disorder, advanced sleep phase disorder. Disruption of circadian rhythms affects physical and mental health. Sleep disorders, cardiovascular disease, diabetes, obesity, cancers, inflammatory disorders, and mood disorders (depression, schizophrenia, and attention deficit) may result from dysfunction of circadian rhythms. Jet lag and shift work cause disruption of circadian rhythms and sleep and can cause symptoms including daytime anergia, alternating complaints of insomnia and hypersomnia, emotional disturbances, and gastrointestinal distress. Long-term shift work may lead to even more severe symptoms, such as obesity, metabolic syndromes, cardiovascular disease, and even increased cancer risk (62). The social, medical and economic costs of these issues are significant (60). Genetic mutations and environmental desynchronization are the major causes of circadian rhythm disorder (61). If sleep occurs in accordance to circadian phase, the temporal separation of ‘sleep/fast-type’ phase from ‘wake/feed-type’ phase is maintained. Sleep ensures an optimal internal environment for processes occurring during the circadian resting phase.
Conclusion
In this essay, we have attempted to briefly outline the mechanisms of sleep, and the regulatory process that control it and their functional significance . There are numerous areas in these processes that remain unknown and opportunities in research to elucidate them.
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