RAS And Other neural substrates in maintaining sleep/wakefulness

Sleep is a natural, periodically recurring state of inactivity, characterized by the loss

of consciousness and reduced responsiveness to external stimuli. In contrast, wakefulness is the
absence of sleep and is marked by consciousness, awareness and activity. Observation and
speculation about the state of sleep was possible for thousands of years. However, it was only in
1929 that the German psychiatrist, Hans Berger discovered that the electrical activity of the brain
could be recorded as brain waves and that these waves changed as wakefulness gave way to
sleep. This discovery of the electroencephalogram (EEG), as it is now known, made the
objective study of sleep possible. By detecting synchronous activity of cortical neurons and
recording voltage fluctuations in terms of the amplitude of the resulting waves and their
frequency, the EEG is thus used to differentiate changes in alertness and sleep stages. For the
analysis of the states of sleep and wakefulness, EEG frequencies are conveniently grouped into
bands:
● Delta, 0.5 to 4 Hz
● Theta, 4 to 8 Hz
● Alpha, 8 to 12 Hz
● Sigma, 12 to 14 Hz
● Beta, 14 to 30 Hz
● Gamma, 30 to 50 Hz
EEG data are combined with those from concurrent recording of eye movements from the
electrooculogram (EOG), and muscle tone from the electromyogram (EMG) to define the states
of sleep and wakefulness. Other signals, such as breathing rate, are also recorded during
polysomnographic evaluation of human sleep in the clinic but they are not required for state
definition. In animals, EEG and EMG signals can be sufficient to define the states of sleep and
wakefulness.
Wakefulness, as shown in Figure, is defined by a low voltage fast frequency EEG pattern, called
desynchronized or activated EEG, that consists primarily of frequencies in the beta and gamma
ranges. The EOG and EMG recordings also show high activity during wakefulness.

In contrast to wakefulness, sleep is characterized by higher voltages and slower waves, a pattern
called synchronized EEG. Non-rapid eye movement (NREM) sleep is the usual term for this
state. In humans, NREM sleep is further subdivided into four Stages, 1 through 4, which depend
on the extent of EEG slowing, especially in the delta frequency range. Stage 2 is notable for the
presence of spindles, which are waxing and waning bursts of frequencies in the sigma band.
Stages 3 and 4, with delta waves comprising more than 50% of the signal, are further grouped
under the term slow wave sleep (SWS). During this state, the EOG can show gradual rolling eye
movements and there is low or minimal muscle activity. In animal studies, the terms SWS and
NREM sleep are often used synonymously as the state is rarely subdivided in terms of EEG
slowing.

Slow sleep is interrupted by periods of rapid eye movement (REM, i.e., active or paradoxical)
sleep, when, despite all the overt signs of continuing sleep, the activity of the brain is remarkably
different. In fact, the EEG in humans during REM sleep is essentially identical to that recorded
during wakefulness, but the EOG reveals rapid bursts of eye movements, hence the name of the
state. In rodents, the EEG is typically dominated by frequencies in the theta band during REM
sleep and phasic activity appears as twitching of the vibrissae as well as eye movements.
Importantly, and in all species, the EMG shows the complete loss of muscle tone (i.e., atonia)
that is a characteristic of REM sleep.

Reticular Activating System

● The reticular activating system (RAS) is a network of neurons located in the brain stem
that project anteriorly to the hypothalamus to mediate behavior, as well as both
posteriorly to the thalamus and directly to the cortex for activation of awake,
desynchronized cortical EEG patterns.
● The RAS receives input from visceral, somatic, and sensory systems.
● The neurotransmitters employed in this system include acetylcholine,
serotonin, noradrenalin, dopamine, histamine, and hypocretin (orexin).

The RAS is a component of the reticular formation, found in the anterior-most segment of the
brainstem. The reticular formation receives input from the spinal cord, sensory pathways,
thalamus, and cortex and has efferent connections throughout the nervous system.
The RAS itself is primarily composed of four main components that each contain groupings of
nuclei. These are the locus coeruleus, raphe nuclei, posterior tuberomammillary
hypothalamus, and pedunculopontine tegmentum. Each is unique in the neuropeptides they
release. In large part, these centers are activated by the lateral hypothalamus (LH), which
releases the neuropeptide orexin in response to the light hitting the eyes, which then stimulates
arousal and the transition from sleep to waking.

1. The locus coeruleus is located within the upper dorsolateral pons of the brainstem. The
LC is the main source of noradrenergic transmission to the cortex. LC neurons fire
tonically at 1–3 Hz during wakefulness, reduce their activity during NREM sleep and are
nearly silent during REM sleep. It is activated directly by orexin from the lateral
hypothalamus, and in response, releases norepinephrine. Its excitatory functions are
widely distributed within the brain, acting on both the alpha and beta receptors of neurons
and glial cells distributed throughout the cortex. It functions primarily upon waking and
in arousal.

2. The raphe nuclei are located midline throughout the brainstem within the pons, midbrain,
and medulla. The majority of neurons located in the raphe nuclei are serotonergic. The
more rostral raphe nuclei appear to be important in various bodily functions, including
pain sensation and mood regulation. In the context of the RAS, these nuclei communicate
with the suprachiasmatic nucleus, playing a role in circadian rhythms, and contributing to
arousal and attention.

3. The tuberomammillary nucleus is located within the posterior aspect of the

hypothalamus. The neurons that make up these nuclei are primarily histaminergic and
serve as the primary source of histamine projections in the brain. They are important in
both wakefulness and cognition, projecting in large part to the forebrain where they play
an important role in arousal.

4. The lateral and dorsal pedunculopontine tegmentum contains primarily cholinergic

neurons in neighboring groups within the midbrain and pons. Cholinergic neurons project
to the thalamus and cortex, promoting desynchronization of the brain, allowing the body
to switch from slow sleep rhythms to high frequency, low amplitude wake rhythms.
Lateral Hypthalamus and Posterior Hypothalamus
5. Neurons in the lateral and posterior hypothalamus are the sole source of the awake-
promoting neuropeptides hypocretin 1 (Hcrt1) and hypocretin 2 (Hcrt2), also known
as Orexin A and Orexin B, respectively.
6. Clinical injury to the LH and PH often causes severe sleepiness; these individuals can
sleep 15–20 hours each day, and when woken, they seem apathetic and quickly lapse
back to sleep. Loss of the orexin neurons causes sleepiness, but it does not increase the
total amount of sleep so researchers have been searching for other wake-promoting
systems in these regions.

The other neural substrates in maintaining sleep/wakefulness

Hypocretin Neurons

- Two decades ago, hypocretin/orexin (Hcrt) neurons of the lateral hypothalamus (LH)
emerged as essential orchestrators of sleep and wakefulness. The hypocretins are two
alternatively spliced neuropeptides, Hcrt-1 and Hcrt-2, produced from a single-
precursor gene.

- Orexin/hypocretin neurons have a crucial role in the regulation of sleep and

wakefulness. Recent optogenetic studies revealed that the activation or inhibition of
orexin neuronal activity affects the probability of sleep/wakefulness transition in the
acute phase.

- Narcoleptic patients show symptoms of fragmented sleep and wakefulness, sleep-

onset rapid eye movement (REM), and cataplexy, a sudden bilateral postural muscle
weakness usually precipitated by emotional stimuli.

- In addition, the concentration of orexin-A (hypocretin-1) peptide in the cerebrospinal

fluid (CSF) of narcoleptic patients is greatly reduced, and a specific degeneration of
orexin neurons has also been reported. Indeed, central administration of orexin
 induces wakefulness and decreases slow-wave sleep (SWS) and REM
sleep durations.

- Orexin neurons are thought to be phasically active during wakefulness and silent
during SWS.

- These findings indicate that the orexin system plays a crucial role in the regulation of
sleep/wakefulness, especially in the maintenance of arousal.

- Thus, Hcrt neurons show high discharge rates during active waking and goal-oriented
behaviors, decrease their activity during quiet wakefulness, and are silent during
sleep.

The Basal Forebrain

- The BF contains three major cell groups: cholinergic, GABAergic, and glutamatergic
neurons. This brain structure has been traditionally regarded as a wake-promoting
region, as its activation induce wakefulness and its lesion results in slow oscillations
in the EEG and a coma-like state.

- Cholinergic neurons of the BF are strongly activated during wakefulness and REM
sleep and their optogenetic stimulation induces cortical activation and transitions
from NREM sleep to wakefulness.

- Nonetheless, chemogenetic activation of BF cholinergic neurons mainly reduces delta

activity during NREM sleep, rather than substantially increasing wakefulness.

The Ventral Tegmental Area

- The VTA is a key structure in the regulation of motivational processes, and contains
dopaminergic, GABAergic, and glutamatergic neurons.
 - During wakefulness, VTA dopaminergic neurons are strongly activated by various
salient stimuli, including sensory cues, social cues, and rewards.

- Chemogenetic inhibition of VTA dopaminergic neurons during the dark/active phase

promotes sleep—strongly suggesting that activity in these neurons is necessary for
wakefulness.

- Glutamate levels in the posterior hypothalamus and cortex rise progressively during
wakefulness, decline during NREM sleep and increase rapidly at the onset of REM
sleep

- GABA has been traditionally viewed as a sleep-promoting neurotransmitter. Most

sleep-promoting populations are GABAergic, including those in the preoptic area and
parafacial zone. Sleep-promoting medications typically target and enhance
GABAergic signaling.

- Nonetheless, recent studies have identified several GABAergic populations in the LH

(lateral hypothalamus) and BF (basal forebrain) that promote wakefulness and
cortical activation. 

Circadian Rhythms
The timing and rhythmicity of the sleep‐wake cycle is matched to the solar day‐night cycle in
humans. This rhythmical pattern is generated internally but is modified by environmental factors,
particularly the light‐dark cycle. The endogenous nature of the circadian rhythm is verified by
the persistence of these rhythms when environmental conditions are held constant. For example,
a human kept in isolation without access to a clock or a periodic light‐dark cycle will maintain a
regular sleep‐wake cycle. According to Czeisler and colleagues, although the rhythm is
maintained, the periodicity of the sleep‐wake cycle under this free‐running condition is
approximately 24.2 hours. However, under the influence of the environmental light‐dark cycle,
this rhythm is entrained to the 24‐hour solar day. The environmental cues that are able to entrain
the internal clock mechanism are called zeitgebers. The most potent zeitgeber for sleep‐wake
rhythms in most organisms is light.

The Suprachiasmatic Nucleus

The anatomical structure serving as the internal circadian rhythm generator is
the suprachiasmatic nucleus (SCN) of the anterior hypothalamus. Lesions of the SCN in rodents
abolish circadian rhythmicity, and disconnection of the SCN from the rest of the brain also
results in a loss of circadian rhythms in the brain despite continued fluctuations within the SCN.
Furthermore, in animals with ablations of the SCN, transplantation of fetal SCN tissue restores
circadian rhythm.
Entrainment of these neurons occurs via the visual pathways linking photoreceptors of the retina
to the SCN.
There are two pathways:
(1) a direct pathway called the retinohypothalamic tract (RHT) and
(2) an indirect pathway called the geniculohypothalamic tract (GHT).
Photoreceptors in the retina transduce light into nerve impulses and transmit information
to ganglion cells, distributed over the entire retina. The ganglion cells contribute to the RHT,
which travels through the optic nerve and optic chiasm. In the chiasm, two‐thirds of the axons
cross and one‐third remain uncrossed. The RHT projects directly to the SCN. Collateral
processes from the RHT continue in the optic tract to the lateral geniculate complex. From the
lateral geniculate, the GHT projects to the SCN as the indirect pathway.
Efferent fibers from the SCN project to intrahypothalamic areas—encompassing the preoptic
area, paraventricular nucleus, retrochiasmatic area, dorsomedial area, and extrahypothalamic
sites, including the thalamus, basal forebrain, and periaqueductal gray. From these areas
information is further relayed to the effector organs for particular biological rhythms.
In addition to controlling the circadian variability of the sleep‐wake cycle, the SCN drives a
similar circadian variability in locomotor activity, food intake, water intake, sexual behavior,
core body temperature, and hormonal levels. Thus, cortisol is highest in the early morning hours
between 4:00 am and 8:00 am, and thyroid‐stimulating hormone increases just before sleep.
Hormones both influence and are influenced by the circadian clock.
The Pineal Gland and Melatonin
The pineal gland is a neuroendocrine gland that synthesizes and secretes melatonin (N‐acetyl‐5‐
methoxytryptamine). The afferent input to the pineal gland is transmitted from the retinal
photoreceptors through the SCN and sympathetic nervous system. The circadian rhythm of
melatonin is controlled by the SCN but is strongly entrained by light. The two effects of light are
to regulate melatonin secretion in accordance with diurnal light‐dark cycles and to suppress
melatonin if given in brief intense pulses. Melatonin secretion increases rather abruptly in the
evening, approximately 2 hours before typical bedtime (dim light melatonin onset), and then
continues elevated during the night, reaching a peak level between 2:00 am and 4:00 am, then
gradually falls during the latter part of the night and is present at very low levels during the day.
Exogenous melatonin has been used with some success to avoid jet lag and may be useful for
treatment of phase‐shifted sleep and sleep disturbance due to shift work. Melatonin is available
through health food stores and has received strong public attention. However, there are no
proven indications for melatonin.
NREM Sleep
Sleep spindles usually arise from the gamma‐aminobutyric acid (GABA)‐ergic neurons in
the reticular thalamic nucleus. These neurons have intrinsic oscillations with spontaneous
slow depolarization on which rhythmical spikes are superimposed and serve as drivers for
thalamocortical projection neurons. Dissection of the reticular thalamic region from the
thalamocortical region or specific kainic acid lesions of reticular thalamic nuclei eliminates
spindles. On scalp recordings, spindles occur maximally over the frontal and vertex areas. Depth
electrode recordings in humans show that thalamic spindles are earlier and more frequent than
those recorded on the scalp. Spindles occurring in the frontal leads may also originate in the
supplementary cortex.
The defining feature of Stages 3 and 4 sleep is the delta or slow wave. Thalamocortical cells are
capable of generating delta waves, but other areas are involved as well, as shown by lesions of
the anterior hypothalamus, preoptic region, and basal forebrain, all of which can abolish delta
waves.

REM Sleep
The anatomical substrates for the different components of REM sleep are as follows:

An important substrate is cortical desynchronization. The origin of the mixed frequency activity
is the mesencephalic reticular formation. The reticular cells begin firing approximately 15
seconds before activation is manifest in the cortex, and their projections extend to the
intralaminar nuclei of the thalamus with widespread projections to cortex.

Hippocampal theta activity is highly synchronous activity with a frequency of 5 to 10 Hz, which

is generated in the dentate gyrus and medial entorhinal cortex. It involves the rostral pontine
reticular formation in the area of the nucleus pontis oralis.
Muscle atonia, except for respiratory and ocular muscles, is a tonic event of REM sleep.  Muscle
paralysis arises at the spinal cord level, from a centrally mediated hyperpolarization of the alpha
motor neurons through the action of the inhibitory neurotransmitter glycine.
Muscle twitches are superimposed on the tonic muscle paralysis. The twitches arise from
descending excitatory impulses, which transiently overcome motor neuron inhibition.
Rapid eye movements are another phasic event of REM sleep. Horizontal eye movements arise
from burst neurons in the parabducens reticular formation in the pons, and vertical eye
movements are associated with activation of the midbrain reticular formation. Positron emission
tomography has shown that REM‐related eye movements involve cortical areas similar to those
used during wakefulness.

PGO activity is a phasic feature of REM sleep, generated in the pons and projected through the
lateral geniculate body and other thalamic nuclei to the occipital cortex. PGO activity is of two
types: Type 1 occurs independent of eye movements, and type 2 occurs simultaneously with eye
movements. PGO spike activity has been associated with fragmentary images or dreams.
Autonomic nervous system lability, with profound sympathetic activation and fluctuations in
respirations, heart rate, and blood pressure, involves the parabrachial nuclei of pons. Other
features of REM sleep include penile erections not associated with sexual stimulation or dream
content and thermoregulatory suspension leading to a pseudopoikilothermic state. Additionally,
there is an increase in cerebral metabolism and blood flow compared with NREM sleep,
particularly in the pons, thalamus, and cingulate cortex.
The regulation of REM sleep is primarily at the level of the brain stem, with REM ‐on and REM ‐
off nuclei. Although the putative trigger zone initiating REM sleep is not identified, the activity
of brain stem areas during REM sleep has been studied, both electrically and pharmacologically.
Brain stem nuclei with activity immediately preceding and persisting during REM sleep are the
cholinergic cells in the dorsolateral tegmentum: the lateral dorsal tegmental (LDT) and the
pedunculopontine tegmental (PPN) nuclei.
These two nuclei comprise the main concentration of brain stem cholinergic neurons. The
projection areas of these nuclei include the basal ganglia; the limbic areas, including the preoptic
area; the thalamic nuclei, including the lateral geniculate nuclei; and the cortex. The PPN plays a
role in numerous feedback loops, involving locomotion and rhythmical functions, specifically
control of sleep‐wake cycles and generation of REM sleep. The cholinoceptive REM triggering
zone located in the paramedian reticular formation receives input from LDT and PPN. Inhibition
of these REM‐on nuclei appears to arise from nearby REM‐off cells, primarily the serotonergic
neurons of the dorsal raphe and adrenergic neurons of the locus coeruleus.
The reciprocal‐interaction model proposed by Hobson posits that control of REM sleep arises
from anatomically distributed and neurochemically integrated populations of cells.
This model is summarized by McCarley as involving four steps:
(1) positive feedback of REM‐on neurons through excitatory interconnections with reticular
neurons;
(2) excitation of REM‐off neurons by REM‐on neurons mediated through cholinergic pathways,
although the reticular formation may actually be the origin of this process;
(3) inhibition of REM‐on neurons by REM‐off neurons located in the dorsal raphe and locus
coeruleus; and
(4) inhibitory feedback of REM‐off neurons through recurrent collateral or some other method of
serotonin and norepinephrine feedback.
The neuroanatomical areas involved in the generation of REM sleep have largely been identified
through transections at different levels in the neuraxis. In transections separating the forebrain
from the brain stem, REM sleep features are recorded caudal to the cut. These features include
atonia, rapid eye movements, PGO spike bursts, and REM‐like activation of the reticular
formation. However, in this transection, thermoregulatory control is lost with an inverse
relationship between temperature and amount of REM sleep. Transections between the locus
coeruleus and the red nucleus, separating the pons from the midbrain, result in atonia, PGO spike
bursts, rapid eye movements, and activation of the reticular formation in a rhythmical pattern
caudal to the transection.
Transections between the medulla and the pons result in a regular cycle of REM above the
transection, with the exception of the generation of muscle atonia. Taken together, these
experiments provide evidence that REM sleep is generated primarily in the pons.
REM sleep is the form of sleep in which many dreams occur. When awakened during an episode
of REM, the sleeper will report the contents of the dream approximately 85% of the time. The
function of dreaming has remained elusive. Both physiological (related to memory and learning)
and psychological roles have been proposed.