Introduction

One of the oldest theories about why people get sleepy is that a fatigue or toxin
substance gets accumulated in the body. Initially, it was thought that this substance
should reside in the blood. However, the fact that conjoined twins with a common
circulatory system sleep independently argues against a common sleep-inducing
factor in the blood.

But then scientists thought: maybe this putative sleep factor is not in the blood
but in the brain. In order to test this idea, Legrende and Pieron in 1913 kept dogs
awake for several days. They extracted cerebrospinal fluid from these animals and
were able to induce sleep by injecting the fluid into the ventricular system of non-
sleep deprived dogs. So that indicated a sleep inducing factor was in the brain system.
The word hypnotoxin was used to describe this unknown factor. The toxin use
reflected the idea that sleep was a negative, something bad.

For centuries many thought of sleep as a purely passive process while waking was
active and being awake was like using a muscle which had to be rested during sleep.

Later physicians and scientists observed sleepiness and tiredness have a

circadian rhythmicity which cannot be explained by the simple accumulation of a
sleep-inducing factor, so the chemical theory of sleep was discarded. Today we know
there are many sleep-inducing neurochemicals.

It should be worth mentioning that sleep is not a passive state that we enter in
order to not do anything. It is more of an active process, a different stage of our
metabolism in the context of the circadian cycle. Scientists have never found brain
cells that lack energy and need to sleep. Nor have they found neurons that run-out
of neurotransmitters during the waking state and need to sleep to replenish them. It is
true that the longer a neuronal assembly has been in the waking state the more likely
it will flip to the sleeping state, but at the biochemical level, there is no imperative
that it do so because of exhaustion.

There is no universal decline in neuron firing rate during sleep. Neurons in

some areas decrease their activity during sleep while neurons in other areas actually
increase their firing rate. This is true both during NREM as well as REM period sleep
and this has been observed both through EEG scalp readings and by functional
imaging studies of the human brain.

Its not the absence of sensory stimulation that causes sleep. The organism as a
whole and individual organs and even clusters of cells undergo circadian cycles. Sleep
is something we do, not the absence of doing anything.

The regulatory mechanisms of sleep

Sleep is controlled by two powerful processes: circadian and homeostatic.
During waking hours, the sleep drive gradually increases until it reaches a critical
threshold. This drive is referred to as homeostatic. Circadian rhythm, on the other
hand, is a signal generated by the master clock, the suprachiasmatic nucleus (SCN)
located in the anterior hypothalamus. Circadian rhythm, derived from the Latin
term circa diem which literally means approximately one day is the bodys
internal clock. This clock is set at slightly over 24 hours (according to the latest
researches it comes to about 24.5 hours). It controls sleep as well as most biological
processes, including hormone production, metabolism, core body temperature
variations, and cell regeneration among others. This clock is normally highly
synchronized to environmental cues (Zeitgebers, German for time giver), the
strongest of them being the light-dark cycle. In most humans the alertness pattern
shows a biphasic distribution, with a mid-day decrease in alertness around 24pm,
followed by an increased alertness during mid to late evening, and finally declining to
its lowest levels during the night. Almost all physiological systems in humans run
slightly over a 24 hour cycle. Disturbance of this well-regulated circadian rhythm and
homeostatic drive (circadian misalignment) can lead to various sleep disorders
collectively known as circadian rhythm sleep disorders.
Compared to the circadian drive for arousal, the actual mechanism of sleep-
wake homeostasis is relatively poorly understood, despite years of research. But what
we do know is that an endogenous (i.e. naturally produced by the body itself) sleep-
regulating substance, or substances, builds up in the bodys cerebrospinal fluid during
our waking hours, which has the effect of increasing the pressure to sleep the more it
accumulates. This pressure is only released by the act of sleeping itself, during which
the levels of the sleep-regulating substance in the body rapidly declines.
This model, first posited by the Swiss sleep researcher Alexander Borbly in
the early 1980s, is often referred to as the two-process model of sleep-wake
regulation. They both counter each other, the wake-sleep state being the dominance of
one over the other.
While homeostatic sleep drive typically increases throughout the day,
effectively making a person more and more sleepy as the day goes on, it is countered
and moderated by the circadian drive for arousal, at least until late evening, when
the circadian clock slackens off its alerting system and begins sleep-
inducing melatonin production instead. This opens the so-called sleep gate (marked
by the point in the diagram above where the homeostatic sleep drive is at its greatest
distance above the circadian drive for arousal). The exact way in which this occurs is
still not fully understood, but the recent neuronal group theory of sleep theorizes that
individual groups of neurons in the brain enter into a state of sleep after a certain
threshold of activity has been reached, and that, once enough groups of neurons are in
this sleep state, the whole organism falls asleep.
During the night, while sleep is actually being experienced, the homeostatic
sleep drive rapidly dissipates, and circadian-regulated melatonin production
continues. In the early morning, melatonin secretion stops and the circadian alerting
system begins to increase its activity again. Eventually, the point is reached where
the circadian drive for arousal begins to overcome the homeostatic sleep
drive (marked by the point in the diagram above where the two curves meet),
triggering awakening, and the process begins all over again.

The circadian mechanism

The brains circadian clock regulates sleeping and feeding patterns, alertness,
core body temperature, brain wave activity, hormone production, regulation of
glucose and insulin levels, urine production, cell regeneration, and many other
biological activities. The most important hormones affected by the circadian clock, at
least insofar as they affect sleep, are melatonin (which is produced in the pineal
gland in the brain, and which chemically causes drowsiness and lowers body
temperature) and cortisol (produced in the adrenal gland, and used to form glucose or
blood sugar and to enable anti-stress and anti-inflammatory functions in the body).
The circadian clock checks its accuracy each day using external Zeitgebers,
principally the light-dark cycle. Exposure to natural daylight stimulates a nerve
pathway from special photoreceptive ganglion cells in the retina of the eye, cells that
are totally separate from the rods and cones our eyes use to generate our everyday
image of the world. These cells contain a unique light-sensitive pigment
called melanopsin, and are most sensitive to short wavelength blue light. Even
many blind people can respond to these light-dark cues, as the photoreceptive cells in
their eyes can usually recognize daylight, even through closed eyelids. The light-dark
signals are sent via the optic nerve to the suprachiasmatic nucleus, which uses them to
reset its own circadian clock each day. Melatopsin will eventually stimulate the
secretion of melatonin by the pineal gland.
The biosynthesis of melatonin is initiated by the uptake of the essential amino acid
tryptophan into pineal parenchymal cells. Tryptophan is the least abundant of
essential amino acids in normal diets. It is converted to another amino acid, 5-
hydroxytryptophan, through the action of the enzyme tryptopahn hydroxylase and
then to 5-hydroxytryptamine (serotonin) by the enzyme aromatic amino acid
decarboxylase. Serotonin concentrations are higher in the pineal than in any other
organ or in any brain region. They exhibit a striking diurnal rhythm remaining at a
maximum level during the daylight hours and falling by more than 80% soon after the
onset of darkness as the serotonin is converted to melatonin, 5-hydroxytryptophol and
other methoxyindoles. Serotonin's conversion to melatonin involves two enzymes that
are characteristic of the pineal : SNAT (serotonin-N-acetyltransferase) which converts
the serotonin to N-acetylserotonin, and HIOMT (hydroxyindole-O-methyltrasferase)
which trasfers a methyl group from S-adenosylmethionine to the 5-hydroxyl of the N-
acetylserotonin. The activities of both enzymes rise soon after the onset of
darkness because of the enhanced release of norepinephrine from sympathetic
neurons terminating on the pineal parenchymal cells.
Another portion of the serotonin liberated from pineal cells after the
onset of darkness is deaminated by the enzyme monoamine oxidase (MAO) and then
either oxidized to form 5-hydroxyindole acetic acid or reduced to form 5-
hydroxytryptophol (Fig.1). Both of these compounds are also substrates for HIOMT
and can thus be converted in the pineal to 5-methoxyindole acetic acid 5-
methoxytryptophol (Fig.1). The level of this latter indole, like that of melatonin, rises
markedly in the pineal with the onset of darkness.
 Melatonin has a remarkably vast array of actions, inhibiting the activity of
certain cells while stimulating other pathways. It does so through its different
receptors which in turn will follow a certain enzymatic cascade, as shown in the
image below.
The metabolism of sleep
Human sleep comprises of nonrapid eye movement sleep (NREM) and REM
sleep. NREM is further comprised of three stages (stages N1, N2, and N3). N3, also
referred to as slow wave sleep, is considered deep sleep with the body being least
metabolically active during this period. REM sleep is characterized by vivid dreams,
loss of muscle tone, and rapid eye movements. The EEG pattern of REM sleep closely
mimics that of wakefulness marked by a high-frequency and low-voltage wave
pattern. NREM and REM sleep occur alternatively in cycles of around 90 minutes
throughout the night. The first half of the night is predominantly NREM, and the
second half is predominantly REM sleep. Sleep architecture, though, is heavily
influenced by genetic and environmental factors including sex, race, socioeconomic
status and culture among others. Sleep duration in mammals generally depends on the
size of the animal. Elephants require only 3 hours of sleep while rats and cats can
spend up to 18 hours in sleep. It is postulated that this may be due to differences in
metabolism. Smaller animals have higher metabolic rate and higher body and brain
temperatures compared to larger animals.
It is believed that during normal sleep the metabolic rate reduces by around
15% and reaches a minimum in the morning in a standard circadian pattern. Only a
15% reduction in metabolic rate appears counter-intuitive considering the prolonged
state of physical inactivity. However, the basal metabolic rate constitutes 80% of the
metabolism needed to maintain all cellular processes in the body. Glucose utilization
in normal subjects is highest during wakeful state and lowest in NREM sleep and
intermediate in REM sleep.

Growth hormone and cortisol are two hormones that have an impact on glucose
regulation. Growth hormone is typically elevated at onset of sleep with highest levels
during slow wave sleep (SWS) while cortisol levels are greatly increased during the
second half of the sleep, predominantly in REM sleep. Studies on normal subjects
with constant glucose infusion during sleep (to suppress endogenous glucose
production) have revealed that a fall in brain glucose metabolism contributed to a
two-thirds fall in systemic glucose utilization during sleep despite increase in glucose
and insulin levels. Reduced muscle tone and anti-insulin like effect of growth
hormone surge during the first half of sleep contributes to the rest of fall in glucose
utilization. Hence there is a relative state of insulin resistance during early phases of
sleep.

During the latter part of sleep the glucose and insulin levels fall despite
continuous infusion of glucose. Other studies have shown similar findings suggesting
increased glucose utilization during REM phase of the sleep and increased glucose
levels in the evening with reduced insulin sensitivity. In addition, studies have shown
an increase in cortisol levels in evening after just one night of sleep deprivation
contributes to glucose dysregulation.

Sleep-wake homeostasis
Homeostasis, in general, is any internal biochemical system that regulates the
body's internal environment, with a view to maintaining properties such as
temperature, acidity, etc, in a stable and relatively constant condition. Sleep-wake
homeostasis, in particular, can be thought of as a kind of internal timer or counter that
generates a homeostatic sleep drive or pressure for sleep as a function of the amount
of time elapsed since the last adequate sleep episode. It is quite intuitive in its
operation: the longer we have been awake, the stronger the desire and need
to sleep becomes, and the greater the likelihood of falling asleep; the longer we have
been asleep, the more the pressure to sleep dissipates, and the greater the likelihood
of awakening.
For a substance to be classified as a putative SRS several criteria need to be
met. These include: 1) the substance and/or its receptor oscillates with sleep
propensity; 2) sleep is increased or decreased with administration of the substance; 3)
blocking the action or inhibiting the production of the substance changes sleep; 4)
disease states, e.g., infection, associated with altered sleep also change levels of the
putative SRS; and finally 5) the substance acts on known sleep regulatory circuits.
While many substances meet some of these criteria including microRNAs,
metabolites, hormones, growth factors, transcription factors, and various proteins and
their receptors, only a few meet all the required characteristics to be considered an
SRS.
The best known of these sleep-regulating substances is adenosine.
Adenosine operates as a neuromodulator in the brain, and has the effect
of inhibiting many of the bodily processes associated with wakefulness, particularly
those involving the neurotransmitters norepinephrine, acetylcholine and serotonin.
Adenosine levels in the basal forebrain rise as sleep debt builds up, and then
fall rapidly during the subsequent sleep period. Adenosine is created over the course
of the day, as a natural by-product of using up our internal energy stores (it forms the
core of adenosine triphosphate (ATP), the energy-storage molecule that powers most
of the biochemical reactions inside cells). This supports the theory that the body's
regular desire for sleep stems, at least in part, from the brain's periodic need
to replenish low stores of energy: in 1995, Craig Heller and Joel Benington proposed
this theory, based on the observation that, as the brains glycogen energy stores are
depleted throughout the day, extra-cellular adenosine builds up, and then,
during sleep, the adenosine is removed and replaced by new glycogen.
 Experiments have definitively shown that high levels of adenosine lead to
sleepiness. Studies in animals have shown that blocking adenosine's actions in the
brain increases alertness, while injections
of adenosine or similar compounds induce
apparently normal sleep.
Also, adenosine concentrations in the
brain shoot up dramatically in animals forced to stay awake. Commonly
used stimulants, like the caffeine in coffee, tea, cola and energy drinks (as well as
the theophylline in tea and chocolate), work as adenosineantagonists or receptor
blockers, inhibiting or dampening its sleepiness effect, and thereby maintaining
alertness.

Adenosine is not the only sleep regulator. Others include IL1 and TNF. IL1 and
TNF are cytokines. They are released in response to many stimuli including sleep
loss, tissue injury, and infection. Cytokines act via juxtacrine, autocrine, and paracrine
signaling but can also serve as endocrines. IL1 and TNF are pleiotropic, serving both
physiological and pathological functions including modulation of memory, mood,
inflammation and sleep. Sleep loss and altered cytokine levels are associated with
enhanced sensitivity to pain and kindling stimuli, fatigue, sleepiness and rebound
sleep, metabolic syndrome including type-2 diabetes, and impaired cognition and
memory. These sleep loss-associated symptoms can be elicited by injections of TNF
or IL1.

TNF or IL1 induces sleep in humans and animals when administered centrally
or systemically, even when given in low non-pyrogenic doses. At lower doses the
increases in sleep are restricted to NREMS. As the dose is raised, NREMS increases
further at the expense of decreased REMS and fever may occur. At high doses both
NREMS and REMS are inhibited. For example, a single intraperitoneal injection of
TNF in mice induces an additional 90 min of sleep during the first 9 h post-injection.
Microinjection of TNF or IL1 into or near hypothalamic and other sleep regulatory
circuits enhances NREMS.

The brain tracks prior wakefulness via extracellular adenosine triphosphate (ATP)
released during neuro- and glio-transmission. The transient dynamic changes in ATP
released into the extracellular space stimulate the production and release of longer-
lived SRSs. ATP binds to purine type 2X7 receptors (P2X7R) on nearby cells to
trigger the processing and release of IL1 and TNF from glia. Intracerebroventricular
administration of an ATP agonist or ATP antagonists increases or decrease sleep,
respectively. P2X7R knockout animals have reduced time spent in NREMS and EEG
slow wave activity after sleep deprivation. There are diurnal variations in cortical
P2X7R mRNA levels. ATP is also catabolized by ecto- enzymes into adenosine, a
well characterized SRS

The main sleep-wake homeostatic mechanism appears to refer specifically

to non-REM sleep, and particularly deep or slow-wave sleep. Thus, in general, the
pressure to sleep is a pressure to enter into deep non-REM sleep, a pressure that is
only relieved by a period of actual deep non-REM sleep. It is still not entirely clear
whether there is a similar mechanism for REM sleep, although the indications are that
there may well be. So, the loss of REM sleep also leads to an increase in the tendency
to enter REM sleep (also known as rebound sleep), but, unlike the case with slow-
wave sleep, this loss appears to be compensated up to a certain extent only, and with
certain differences between different animals.
On awakening in the morning, a phenonemenon known as sleep
inertia sometimes takes hold, which manifests itself as a general feeling of grogginess
and impaired motor activity which may last for up to half-an-hour after waking, and
which may be accompanied by a distinct feeling of wanting to return to sleep, even at
times appropriate for normal waking. This is most likely to occur when waking from
deep slow-wave sleep rather than from light sleep or REM sleep, and may be more
severe (and last longer) after waking from a sleep period or nap following a prolonged
period of wakefulness or accumulated sleep debt. It may be caused by an excessive
build-up of adenosine (through the normal sleep-wake homeostasis process) that has
not fully dissipated by the time of awakening.