Neurophysiology and neurochemistry of sleep

Sergey Skudaev

It is known that sleep is very important for health and essential for surviving. Sleep deprivation leads to death within three weeks. However the purpose of sleep still is not clearly understood. Two fundamental processes carry out regulation of sleep: circadian rhythm and homeostasis. Circadian rhythm determines temporal organization of daily activity of the hormonal and nervous system. For example, rats are awake at night and sleep during the day. Homeostatic mechanisms restore deviations in body conditions caused by environmental stimuli or endogenous processes. For example, prolonged wakefulness increases the length of sleep. Krueger J.M et al 1998 At the beginning of 20th century, scientists believed that sleep is a rest state of our brain and body. In 1950s it was discovered that brain activity, brain blood flow; blood pressure and heart rate in some sleep stages might be the same as in wakefulness. No longer is sleep considered as a passive condition or as an absent of wakefulness. Brain cell activity is not inhibited, but reorganized in sleep. Cells of the cerebral cortex in sleep are united in synchronized activity (chorus), while in wake state some cells are inhibited, and some are active. It has been shown that sleep is essential for adaptation to stress and to learning. Consolidation of short-term memory in long term takes place in sleep. In sleep our brain is planning our future actions and is testing their outcomes.

Sleep states and stages
Sleep consists of two different states: Non-REM sleep and REM sleep. REM stands for Rapid Eye Movement. Before we can discuss what it means, we have to learn some basic facts about Electroencephalogram (EEG). EEG is a recording of electrical brain activity. To register EEG, electrical wires with silver contacts are applied to patient scalp or brain surface and connected to an amplifier and a recording device. The procedure of recording EEG is similar to that of EKG.

The first human EEG was recorded by Hans Berger in 1924. He proved that EEG waves are generated by the brain. His work was published in 1929[1]. The EEG waves are formed as a result of summation of individual neuron activity. The more synchronized neuronal activity is, the higher voltage waves are registered in EEG. The less synchronized neuronal activity is, the lower the EEG waves are. A normal EEG of relaxed person who closed his/her eyes usually displays wellsynchronized waves with frequency around 10 per second and amplitude about 50 micro volts. These waves are called alpha waves or alpha rhythms. A light or sound stimuli cause desynchronization of EEG; alpha waves are replaced with low voltage and high frequency beta waves. Beta waves may appear without external stimuli, when person performs a calculation or is involved in some other activity. Grey Walter [] 1952. During sleep EEG waves are changing from high frequency and low voltage to low frequency and high voltage (synchronization of neuronal activity). About every 70-80 minutes of sleep the EEG becomes similar to that in wake state; blood flow through the brain increases, tonus of skeletal muscles dramatically drops, and rapid eye movement (REM) is observed. This state of sleep is called REM sleep. Persons who wake up in REM sleep describe vivid colorful visual dreams. REM sleep is also called Fast-Waves Sleep, while Non-REM sleep is called Slow Waves Sleep. Two graduate students: Eugene Aserinsky and Nathaniel Kleitman from the laboratory of William C. Dement [] at the University of Chicago discovered and described REM sleep in the early 50s. At birth, REM sleep composes 90% of the total amount of sleep. Sleep in adult human comprises of 90 minutes cycles of Non-REM and REM sleep. REM sleep constitutes about 20% of total sleep time. First REM sleep episode lasts about 5-10 minutes. The length of the REM sleep phase increases toward the morning. Deprivation of REM sleep during one night leads to increasing the total REM sleep the next night. Long deprivation of REM sleep may cause hallucination and psychotic disorders. The muscle atonia in REM sleep increases resistance of the upper airway and as a result obstructive sleep apnea (OSA) may occur in some individuals. OSA is characterized by the frequent temporary interruption of breathing during sleep. Inhibition of hypoglossal motoneurons, which control tongue movement, may contribute to OSA during REM sleep. Bellingham M. C, Funk G.D [] (2000). It is believed that Sudden Infant Death Syndrome (SIDS) also occurs in REM sleep. Deprivation of REM sleeps in rats for as long as 20 days causes loss of weight in spite of increased food consumption. M. Koban and K. L. Swinson [] 2005. REM sleep deprivation in rats usually is achieved with a very simple method. A flowerpot is placed upside down in bath with water. A rat is sitting on the top of the flowerpot. In REM sleep stage rat muscles are loosing tonus and as a result, the rat falls in the water and wakes up. Then it climbs back on the top of the flowerpot and may sleep in Non-REM stage until it

falls in the water in the next REM stage. REM sleep deprived rat metabolism rose to 166% of baseline. Non-REM sleep consists of four stages. A. Rechtschaffen, J. Siegel [] 2000. The first stage of sleep is characterized by transition from wakefulness to sleep. It lasts few minutes. EEG of the first stage shows low-amplitude mix frequency waves. EEG in the second stage shows periods of well-synchronized sinusoidal waves 12-14 per second. These groups of waves are spindle shape and are called sleep spindles. In the third sleep stage slow, high-amplitude waves appear in the EEG every 0.5-2 seconds. The fourth stage is characterized by dominating slow high-amplitude waves. In 3 and 4 stages of sleep neuronal activity, blood pressure and heart rate is low. Global decrease of cerebral blood flow (CBF), oxygen and glucose metabolism are observed in slow wave sleep (SWS). No difference in CBF, oxygen and glucose metabolism were observed between REM sleep and wakefulness. Regional cerebral blood flow (rCBF ) was significantly decreased in the pons, midbrain, thalamus and basal forebrain during SWS. There was no significant decrease of CBF in primary or secondary sensory cortex, which implies that sensory systems remain functional during the sleep. Kajimura N. et al. 1999. Hofle N et al. 1997.

Neuroanatomy and physiology of sleep and wakefulness
It was thought that wakefulness is maintained by external and internal sensory stimulation of cerebral cortex and inhibition of sensory stimulation leads to sleep. In 1949 Moruzzi and Magoun [] dramatically changed existing concept of wakefulness and sleep. They discovered that stimulation of reticular formation maintains wakefulness even if sensory pathways are destroyed. The reticular formation is located in the brain stem and receives inputs from the ascending tracts, which are originated from spinal cord and brain stem. These ascending tracts give branches to the reticular formation and then pass to the thalamus. After relay in the thalamus, they reach the cerebral cortex. Lesion of the reticular formation causes a comatose state while ascending sensory pathways are intact. Later it was discovered that stimulation of the posterior hypothalamus produces EEG activation, while stimulation of the anterior hypothalamus promotes sleep. The pons, midbrain tegmentum, and the thalamus comprise the ascending reticular activating system. Steriade and McCarley 1990. It is believed that the thalamic inhibitory mechanism is responsible for transition from wakefulness to sleep. The sleep spindles are generated in the thalamus from onset of sleep. It is thought that spindle waves block activating input from the stem reticular

system to the cerebral cortex at the thalamic level and as a result initiate and maintain sleep. B.M.Evans [] 2002. The medullar reticular system also inhibits the midbrain activating reticular formation promoting sleep. Moruzzi, Magoun [] 1949.

Neurochemistry of sleep
Neural cell and synapses
Neural cell or neuron has many dendrites, which look like trees and serve for receiving information. One long processor - axon serves for output information. The body of neural cell and its dendrites are covered with thousand of synapses. Through these synapses, the dendrites receive messages from the other neurons. These messages can inhibit or increase neuron activity. In the latter case, neuron may generate action potential, which spreads along the axon. An axon terminal forms a synapse, a structure comprised of axon terminal, presynaptic membrane, synaptic cleft and postsynaptic membrane. Please see figure 1.

Figure 1. Synaptic Transmission The synapse contains vesicles with neurotransmitter. When action potential reaches axon terminal, its membrane becomes permeable to Ca++ ions. Ca++ ions flow inside the terminal and cause vesicles to move to presynaptic membrane and release transmitter in synaptic cleft. The transmitter molecules passively spread in the synaptic cleft and reach postsynaptic membrane receptors. Transmitter binds to receptors and change potential of postsynaptic membrane. Message is transferred to the next neuron. There are many different neurotransmitters or neuromediators: norepinephrine, dopamine, serotonin, acetylcholine, gamma-aminobutiric acid (GABA), glutamine, etc. Some transmitters are inhibitory some are excitatory; some are either, depending on receptor type.

Noradrenergic system (the locus coeruleus)
The locus coeruleus ("Blue spot") was discovered in 1786 by Félix Vicq-d'Azyr. . It is located in the dorsolateral pons. In 1960s it was discovered that the locus coeruleus is the major source of norepinephrine (noradrenalin) in the brain. Its neurons produce

norepinephrine and send projections to spinal cord, brainstem, midbrain, cerebellum, hippocampus, thalamus, and cerebral cortex. The locus coeruleus (LC) receive projections from many different brain regions, which release such wide specter of neurotransmitters as opiates, glutamate, GABA, serotonin, epinephrine. Aston-Jones G et al. 1991. The suprachiasmatic nucleus, known as circadian pacemaker, activates LC through dorsomedial hypothalamus executing circadian regulation of arousal. Spontaneous activity of LC neurons depends on sleep-wake cycle. The highest neuronal activity, which is observed during wakefulness, is decreasing during Non-REM sleep and absent during REM sleep. It is thought that LC is a part of activating system, which promotes cortical EEG arousal and hippocampal theta rhythm. Corticotropin-releasing factor (CRF), which is produced by neuroendocrine cells in the paraventricular nucleus of the hypothalamus and initiates adrenocorticotropin release from the anterior pituitary during stress, stimulates locus coeruleus neurons. LC plays major role not only in regulation of sleep-wakefulness, but also in stress related behavior.

Hypocretin (Orexin)
Recently, a neuropeptide called hypocretin/orexin, which has an important role in the sleep regulation, was identified independently by two groups of scientists. De Lecca et al. [] 1998. Sakurai T et al. [] 1998. The cells producing hypocretin/orexin are located in the dorso-lateral hypothalamus. These cells send their excitatory projections to cholinergic pontine reticular formation, spinal cord, locus coeruleus, dorsal raphe nuclei, amygdale, and basal forebrain. The hypocretins are thought to play a primary role in the control of sleep and wakefulness as well as attention, learning, memory, feeding-energy regulation and modulation of pain at all levels of spinal cord. Ebrahim I.O. et al. []2002 Human narcolepsy, a disorder, which is characterized by excessive daytime sleepiness, with irresistible sleep attack or cataplexy (a sudden loss of muscle tone) , links to an 8595% decrease of hypocretin neurons. Hypocretin neurons stimulate noradrenergic, serotonergic and histaminergic neurons promoting wakefulness and their activity decreases during Non-REM sleep. The other authors suggest that hypocretin neurons increase activity of acetylcholinergic neurons during active waking and REM sleep. Kiyashchenko L. et al. [] 2002.

Serotonergic System (The Raphe nuclei)
The raphe nuclei are located in the brain stem and send projection to almost every area of the brain." In order from the caudal to the rostral, the raphe nuclei are known as the nucleus raphe obscurus, the raphe magnus, the raphe pontis, the raphe pallidus, the

nucleus centralis superior, nucleus raphe dorsalis, nuclei linearis intermedius and linearis rostralis " The dorsal raphe nucleus (DRN) contains the largest pool of serotonergic neurons in the brain. The raphe sends predominantly inhibitory serotonergic projections to the dentate gyrus and moderate projections to Ammon's horn regions of the hippocampus. These projections end on the hippocampal interneurons. Microinjections of the 5-HT1a receptor agonist 8-hydroxy-2-(di-n-propylamino)-tetralin (8-OH-DPAT) in median raphe increases anxiety. This compound inhibits serotonergic neurons in the median and dorsal raphe. The microinjection of the 8-OH-DPAT in median raphe increases the hippocampal theta rhythm amplitude and the movement velocity of the freely behaving rats. Nitz D.A., McNaughton B.L. 1999. The dorsal raphe sent major projections to the dorsolateral pons which promotes REM sleep. The microinjections of the 8-OH-DPAT in the dorsal raphe increases REM sleep. The limbic cortical and diencephalic structures control the activity of the median and dorsal raphe serotonergic neurons by modulation of GABAergic neurons in the raphe nuclei. Varga et al. 2001, 2003. The activity of serotonergic neurons of the dorsal raphe nuclei decreases from waking through slow wave sleep to REM sleep, while the activity of the GABAergic neurons increases. The GABAergic neurons in the raphe nuclei may decrease the ascending serotonergic output by direct inhibition of serotonergic neurons or increase it by inhibition of GABA interneurons. The serotonergic ascending pathway inhibits hippocampal theta rhythm and causes desynchronization of EEG. The GABA interneurons also inhibit glutamatergic projections from median raphe to the limbic theta rhythm generators. The blockade of the GABA receptors activates glutamatergic pathway and promote theta rhythm. Li et al. 2005. Lesions in centralis superior raphe (CeSR) and reticularis pontis nuclei (RPN) in cats decrease slow wave sleep (SWS) and parodoxal sleep (PS) and increase wakefulness. Drowsiness was increase during the light "day time" phase but not during the dark phase. It is known that circadian rhythm may be controlled by changes in serotonin concentration by an endogenous pacemaker (the suprachiasmatic nucleus) and by the light. The present study supports the concept of possible control of sleep - wakefulness cycle by the raphe nuclei. Arpa J et al. 1998. It is known that morphine increases release of serotonin in DRN by inhibiting GABAergic projections to DRN, which imply participation of DRN in morphine addiction. The nucleus raphe magnus (NRM) and dorsal raphe nucleus (DRN) are involved in central modulation of pain by descending inhibitory pathways to spinal cord. Cucchiaro G. et al.[] 2005

The Role Acetylcholine and Histamine in Sleep

The nucleus basalis magnocellularis, which is located in the forebrain, is a major source of cholinergic projections to neocortex, amygdala and medium septum-banda diagonalis complex. Cholinergic neurons play a critical role in the cognitive function and behavior arousal and EEG activation. AC release is greater during REM sleep than during the wakefulness and low during the Non-REM sleep. Vazquez J et al 2001. The extensive loss of cholinergic neurons in Alzheimer's disease is responsible for decline of the cognitive functions. Anticholinergic drugs negatively affect memory and learning processes. Recent study indicated that histaminergic neurons also play an important role in memory and learning by direct influence on memory or by modulating release of acetylcholine. (AC). Blandina P. et al 2004. Histominergic neurons are located in the tuberomammillary nucleus (TMN) of the hypothalamus. TMN send projections to different brain areas and modulates activity of cholinergic neurons. Histaminergic neurons promote wakefulness and emotional memory. Their activity is high during wakefulness and attention and low during sleep. Histaminergic neurons directly stimulate the neocortex through the hypothalamo-cortical projections and indirectly by activation of the raphe nuclei. Histominergic descending projections activate cholinergic neurons in the mesopontine tegmentum, which activate neocortex through thalamo- and hypothalamo-cortical projections. It is known that the baselateral amygdala (BLA) activity related to emotional memory. Histominergic neurons regulate acetylcholine release in amygdala. Microinjections of histamine in BLA impaired learning in animals conditioned to escape from the punishment box. In the neocortex, microinjections of histamine decreases cholinergic tone through H3 receptors. The systemic administration of H3 receptor agonists impairs rat performance in learned tasks. The recent study shows that nitric oxide synapses exist on the cholinergic neurons of the laterodorsal and pedunculopontine tegmental nuclei, which send projections to the medial pontine reticular formation (mPRF). Stimulation of mPRF evokes REM sleep-like state and causes hypotonia of the upper airway muscles. mPRF microinjection of Ng-nitro-Larginine (NLA), which inhibits nitric oxide synapses in mPRF, significantly decrease the duration of REM sleep. It is thought that nitric oxide increases the release of AC in mPRF and promotes REM sleep. Leonard T.O. et al.1997.

The substantia nigra is the major source of dopamine in the brain. Noradrenergic and serotonergic neurons become silent during REM sleep whereas dopaminergic neurons remain active which produce "psychotic-like mental activity of dreaming" Gottesmann C. (2002)

Reduction in the number of dopaminergic neurons in the substantia nigra causes REM sleep behavior disorder (RBD), which is characterized by a loss of skeletal muscle atonia during REM sleep. Patients with RBD have aggressive dreams in which they can injure their bed partners. The dopamine neuron loss increases activity of the globus pallidum, which inhibits midbrain structures. These structures inhibit the spinal motoneurons and their inhibition prevents development of skeletal muscle atonia during REM sleep. Eisensehr et al 2000.

Neuropeptid S
One more group of neurons was discovered recently between locus coeruleus and Barrington nucleus. These neurons release neuropeptid S (NPS) that elevates arousal and at the same time produce anxiolytic-like effect. NPS receptors were found in neocortex, thalamus, hypothalamus and amygdale. It is well known that the amygdale participate in modulation of fear and anxiety. Probably, a unique anxiolytic effect of NPS is related to modulation of the amygdale activity by NPS. Reinscheid R.K. and Xu Y.2005

Sleep and memory
Since important role of acetylcholine in memory consolidation is well proven, it was proposed that REM sleep during which high level of acetylcholine is observed must be involved in memory consolidation. The memory study surprisingly proved that slowwave sleep deprivation impairs later memory consolidation after learning declarative and procedural tasks. How slow-wave sleep with low level of acetylcholine influents memory consolidation? It is proposed that there are two stage of memory consolidation. In the first stage during the wakefulness and REM sleep neocortex sends newly acquired information to the hippocampus, while acetylcholinergic projections release acetylcholine and suppress hippocampal feedback to neocortex. During slow-wave sleep, when acetylcholine level is low and hippocampal feedback to the neocortex is released, memory traces temporary stored in hippocampal circuitry are transmitt back to the neocortex. It is thought that the release of acetylcholine in the neocortex controls the flow of information from hippocampus to the neocortex. The improvement of the declarative tasks performance after slow-wave sleep can be blocked by anticholinesterase drug. In the same experiments, the performance of procedural tasks was not impaired, which suggest that procedural memory consolidation may depend more on REM sleep. It is proposed that REM sleep with high level of acetylcholine enhance synaptic plasticity and memory consolidation. Guis, S & Born 2004 in Power A.E. 2004.


In spite of the great progress in sleep research, it is still not possible to draw the whole picture of sleep mechanisms and understand the sleep purpose. Many neuroanatomic structures with different neurotransmitters at all levels of brain from the brainstem medulla to the thalamus and cerebral cortex participate in regulation of the sleep-wake cycle. Probably, understanding sleep is almost the same as understanding the brain. It does not mean that we cannot treat sleep disorders. The recent progress in neurochemistry and pharmacology of sleep gives us some hope.

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