This review summarizes the brain mechanisms controlling sleep and wakefulness. Wakefulness promoting systems cause low-voltage, fast activity in the electroencephalogram (EEG). Multiple interacting neurotransmitter systems in the brain stem, hypothalamus, and basal forebrain converge onto common effector systems in the thalamus and cortex. Sleep results from the inhibition of wake-promoting systems by homeostatic sleep factors such as adenosine and nitric oxide and GABAergic neurons in the preoptic area of the hypothalamus, resulting in large-amplitude, slow EEG oscillations. Local, activity-dependent factors modulate the amplitude and frequency of cortical slow oscillations. Non-rapid-eye-movement (NREM) sleep results in conservation of brain energy and facilitates memory consolidation through the modulation of synaptic weights. Rapid-eye-movement (REM) sleep results from the interaction of brain stem cholinergic, aminergic, and GABAergic neurons which control the activity of glutamatergic reticular formation neurons leading to REM sleep phenomena such as muscle atonia, REMs, dreaming, and cortical activation. Strong activation of limbic regions during REM sleep suggests a role in regulation of emotion. Genetic studies suggest that brain mechanisms controlling waking and NREM sleep are strongly conserved throughout evolution, underscoring their enormous importance for brain function. Sleep disruption interferes with the normal restorative functions of NREM and REM sleep, resulting in disruptions of breathing and cardiovascular function, changes in emotional reactivity, and cognitive impairments in attention, memory, and decision making.
The purpose of sleep is one of the great unsolved mysteries of biology and has fascinated people for millennia. Although the function or functions of sleep are still unresolved, great progress has been made in understanding the brain mechanisms that control sleep and wakefulness. An understanding of these mechanisms is of paramount importance to our society. Sleeping tablets are among the most widely prescribed medicines, and disturbances in sleep are associated with a wide range of medical and psychiatric conditions. Conversely, an increase in sleep is one important mechanism that the body uses to combat infection and maintain optimal health. Adequate sleep is also essential for optimal cognitive function; lack of sleep has been implicated in major industrial disasters as well as car and workplace accidents. In this unusually comprehensive review we summarize current knowledge regarding the brain mechanisms which control wakefulness, non-rapid-eye-movement (NREM) sleep, and rapid-eye-movement (REM) sleep.
A. Characteristics of Sleep-Wake States
Sleep is defined in the sleep laboratory, in both humans and animals, by recording the electrical field activity of large groups of cortical neurons and muscle cells. Thus scalp electrodes record the electroencephalogram (EEG), electrodes placed on or in skeletal muscles record the electromyogram (EMG), whereas electrodes placed over or near the muscles responsible for horizontal eye movement record the electro-oculogram (EOG). Deep brain electrodes are used to record the activity of individual brain areas or individual neurons. These so-called polysomnographic recordings are used to define the states of wakefulness and sleep as follows (FIGURE 1): wakefulness is defined by low-voltage fast EEG activity (LVFA) and high muscle tone, NREM sleep is characterized by high-amplitude low-frequency EEG and decreased muscle tone, whereas REM sleep has LVFA coupled with a complete loss of muscle tone (REM muscle atonia) and characteristic rapid eye movements which contrast with the slow rolling eye movements observed during NREM. Further characteristics of these three states and the brain circuitry which generates them are discussed in sections II–IV. A summary of studies involving inactivation of different parts of the brain controlling sleep and wake is provided in TABLE 1. The location of these brain regions is shown in FIGURE 2.
B. Control of Sleep Timing and Intensity
The timing, depth, and duration of sleep are controlled by the interaction of time of day (circadian control, process C) and by the duration of prior wakefulness (homeostatic control, process S) as proposed in the two-process model of Borbely (122). The cellular mechanisms in the suprachiasmatic nucleus (SCN) which generate circadian rhythms are not covered herein, since they have been reviewed extensively elsewhere (1263). The output pathways from the SCN that control the circadian timing of NREM and REM sleep are covered in sections III and IV. Homeostatic control of sleep is also covered in these sections.
C. Effects of Sleep Loss on Cognition
An important function ascribed to sleep is offline processing of information encountered during the day and consolidation of memory. Conversely, loss of sleep, either voluntarily or due to an underlying medical disorder, is associated with substantial impairments in cognitive function. The mechanisms underlying these impairments are discussed in Section V.
D. Sleep Ontogeny
Sleep is the predominant behavioral state in developing animals (645, 1070), and REM sleep is proportionally more abundant in young mammals (1070). As such, sleep, in particular REM sleep, has been suggested to play an important role in the elaboration of neuronal circuitry during development (1070). The circuitry controlling sleep and wakefulness appears to mature early in development (606), although cycling between states is more frequent in younger animals (111, 645). EEG signs of sleep and wakefulness do not become “adultlike” until the later full development of the cortex (110, 377, 585, 1144). In fact, in humans, the development of fast EEG synchrony typical of wakefulness continues through adolescence, reflecting the prolonged maturation of the cortex in higher primates (1318).
E. Sleep Phylogeny
A form of NREM sleep appears to be present in most animals investigated to date (185, 1478), which is one of the arguments in favor of sleep performing a vital function (1174). A distinct REM sleep state only appears in mammals, although a primitive form is evident in reptiles and birds (1175). Sleep physiology is adapted to the particular features of different animals. For example, dolphins and other cetaceans exhibit unihemispheric sleep (1174). The distribution of the durations of sleep bouts in mammals is exponential with time scales that vary across species from mice to humans that are proportional to body mass and metabolic rate, indicating a connection with energy metabolism (742, 1174; discussed more fully in sect. III). Whereas most early animal studies of sleep used cats, dogs, and rats as experimental subjects, more recently there has been an explosion of interest in using more genetically tractable organisms to identify and study the genes and proteins involved in controlling sleep (1478). This work is reviewed in section VI. Interestingly, this work suggests that even organisms such as the fly Drosophila melanogaster (492, 1159) and the worm Caenorhabditis elegans (1042) have a “rest state” with similarities to mammalian sleep. Furthermore, several homologs of genes controlling rest in these species play a role in the control of mammalian sleep (230).
F. Sleep Disorders
Polysomnographic recordings are used not only in experimental studies but also in clinical sleep laboratories to identify sleep disorders such as sleep apnea and narcolepsy which involve a dissociation and fragmentation of waking, NREM, and REM (780). Disorders of sleep and the brain mechanisms that underlie them are discussed in section VII.
A. Electrographic Signs of Wakefulness
Synchronized electrical activity in large numbers of cortical neurons provides the basis for observable extracellular field potential changes in the EEG. Summed synaptic currents from the apical dendrites of pyramidal neurons are the main contributors to these EEG waves, although intrinsic membrane properties and neuronal firing also contribute (178). Faster frequency EEG rhythms (LVFA) typical of wakefulness and REM sleep are of low amplitude and involve synchronized activity in small, functionally interrelated areas. Lower frequency rhythms such as the theta rhythm occur over more widespread areas and synchronize faster, locally generated fast rhythms (beta/gamma). These EEG rhythms are thought to provide a temporal framework for higher-order brain functions such as attention, memory formation, and conscious awareness by binding together the firing of neurons within cortical areas and by synchronizing cortical and subcortical sites (178, 1238). During quiet or drowsy wakefulness, the slower EEG frequencies become more prevalent. Alpha rhythms appear in posterior cortical recordings whilst theta rhythms increase in frontal cortical regions.
1. Gamma/beta rhythms (15–120 Hz)
Low-amplitude gamma (30–120 Hz) and beta (15–30 Hz) frequency rhythms are a prominent feature of the EEG during quiet waking (baseline or spontaneous gamma; FIGURE 1) and are enhanced in particular cortical areas following presentation of sensory stimuli (evoked or steady-state gamma). Gamma rhythms often occur concurrently with theta rhythms during active waking and during REM sleep (187, 739, 880), particularly following phasic REM periods with PGO wave activity (26). Gamma rhythms also occur during the brief upstate of the slow oscillation during NREM sleep (see sect. III) (1219). In some studies, gamma rhythms have been subdivided into two frequency bands: low gamma (30–70 Hz) and high gamma (70–120 Hz), which arise in different cortical layers and have different pharmacological modulation properties (35, 950). We here primarily discuss low gamma. Gamma rhythms are generated by cortical networks of fast-spiking [especially parvalbumin (PV)-Pos] interneurons targeting the cell bodies of glutamatergic neurons (FIGURE 3). Rhythmic inhibition and disinhibition of the pyramidal neurons are responsible for the observed field potentials with rate being set by the decay time of the inhibitory synaptic currents. In turn, the interneurons are driven by excitatory input from the pyramidal neurons. Synchrony is enhanced by electrical synapses mediated by gap junctions between interneuronal networks and between the axons of pyramidal neurons as well as by interneuron-interneuron chemical synapses (1405).
Gamma rhythms are generated locally in the neocortex but are modulated by subcortical inputs. The ability to elicit gamma rhythms in isolated brain slices in vitro (163, 366, 1407), together with current-source density and cross-correlational analysis in vivo (26, 1218), suggests that gamma rhythms are generated locally in the cortex. However, their dependence on behavioral state and stimulus presentation indicates that their occurrence is also dependent on subcortical inputs. In fact, gamma rhythms are enhanced by stimulation of the mesencephalic reticular formation, the origin of the ascending reticular activating system (502, 903). Further information on the subcortical control of gamma rhythms is provided in section IIC.
Fast-spiking interneurons containing PV generate gamma rhythms. Evidence supports the conclusion that beta and gamma rhythms are generated by GABAergic interneurons, in particular fast-spiking, PV GABAergic interneurons which synapse on the cell bodies and axon initial segments of pyramidal neurons. 1) In vivo, fast spiking interneurons discharge at gamma frequency and their firing is phase-locked to the extracellularly recorded oscillation (133, 1026, 1311). 2) In vitro, gamma and beta rhythms are completely blocked by GABAA receptor antagonists (366, 1407), and gamma frequency is inversely correlated with the decay time constant of inhibitory synaptic currents (1406). 3) Optogenetic stimulation of PV neocortical interneurons in vivo (via genetic introduction of bacterial light-activated ion channels) can elicit gamma rhythms, whereas optogenetic inhibition reduces gamma (193, 1194). 4) Wavelet analysis of local field potentials in the CA3 region of the hippocampus combined with simultaneous intracellular recordings from pyramidal neurons during cholinergically induced gamma rhythms revealed that perisomatic inhibitory currents generated the majority of the field potential (955). 5) In human visual cortex, GABA concentration measured by magnetic resonance spectroscopy predicts peak gamma frequency and orientation discrimination performance (331, 906). 6) Gene linkage analysis indicates significant linkage between the beta frequencies of the human EEG and GABAA receptor genes (1015).
PV knockout mice have enhanced gamma oscillations (1379), suggesting PV itself may not be required, although developmental compensation may have taken place. Alterations in PV neurons may be responsible for dysfunctional gamma rhythms in schizophrenia and other disorders that are associated with cognitive abnormalities (1319, 1428).
A) BETA OSCILLATIONS.
Beta frequency EEG oscillations (15–30 Hz) are thought to represent one or more of the following: 1) a slow gamma oscillation, 2) a subharmonic of ongoing gamma whereby inhibitory neurons fire at gamma frequencies but some excitatory neurons remain refractory for longer periods so that they only fire on a proportion of the gamma cycles, or 3) a rhythm with its own distinct underlying properties (653). Computational modeling suggests that beta rhythms are more effective than gamma rhythms in synchronizing activity between spatially distant brain loci (653).
2. Alpha rhythms (8–14 Hz)
The two most well-known alpha rhythms in humans are the occipital alpha rhythm which dominates the EEG during relaxed wakefulness (FIGURE 1) and the Rolandic mu rhythm observed over somatosensory cortex in the absence of movement (536). Occipital α rhythms were one of the first described EEG rhythms (7, 92). They are commonly observed during relaxed wakefulness in parietal and occipital cortex areas including primary visual cortex and are suppressed by eye opening and visual stimuli (961). Alpha rhythms may play an important role in internally directed thought processes since they are strengthened during tasks requiring mental arithmetic and visual imagery (1052).
A) THALAMOCORTICAL MECHANISMS GENERATING ALPHA RHYTHMS.
The mechanisms underlying the generation of alpha rhythms were little understood until recently (536). Alpha rhythms result from an interaction of thalamic and neocortical circuitry, together with a moderate level of brain stem cholinergic input. At the level of the visual cortex, alpha waves are due to a dipole located at the level of the cell bodies of pyramidal neurons in layer V and basal dendrites of pyramidal neurons in layers IV where thalamic input terminates (747, 748). At the level of the thalamus, the firing of two groups of thalamocortical relay neurons in the lateral geniculate nucleus are suppressed at either the positive or negative peak of the alpha rhythm through phasic inhibition. For the occipital alpha rhythm, local lateral geniculate GABAergic interneurons excited by high-threshold bursting thalamocortical neurons are important in periodically silencing thalamocortical neurons, whereas for the mu rhythm the GABAergic reticular nucleus may fulfill this role.
In vitro work in the cat thalamus suggests that alpha rhythms require stimulation of muscarinic cholinergic receptors (mimicking brain stem input) or stimulation of metabotropic glutamate receptor stimulation (mimicking cortical input). Stimulation of these receptors leads to depolarization and the generation of an afterdepolarizing potential (ADP) in the gap junction-coupled network of high-threshold bursting thalamocortical neurons (537, 751, 752), leading to synchronized firing. Concerning these two mechanisms, in vivo microdialysis experiments suggest that the brain stem muscarinic input is more important (752). Interestingly, the number of spikes in a burst and the interburst frequency (2–14 Hz) are dependent on the level of muscarinic receptor activation so that the transition from alpha to the slower theta frequency waves in early (light) sleep or drowsy wakefulness may reflect a gradual withdrawal of brain stem cholinergic input (536, 537).
3. Theta rhythms (4–8 Hz)
Theta-rhythms occur prominently during waking associated with movement in rodents, during tasks requiring attention/memory in humans, and during REM sleep in all mammals (FIGURE 1). They provide a temporal code for pyramidal/granule cell firing important for spatial navigation and episodic memory formation and facilitate synaptic plasticity (178, 587, 1362). In rodents and other lower mammals, very regular theta rhythms, also called rhythmic slow activity, have been studied most closely in the hippocampus and related temporal lobe structures, which in these species are located close to the dorsal surface of the brain (FIGURE 2) and strongly influence the EEG signal during movement and REM sleep. In humans, where the temporal lobe is located ventrally, theta rhythms are recorded and studied mainly in frontal and midline cortices that are part of the default network. Interestingly, in both animals and humans, theta-band activity increases strongly in frontal-midline areas during the course of sleep deprivation and is correlated with sleep drive (365, 1386). However, this theta activity is less regular than that generated by the hippocampus and may result from different mechanisms. Here, the mechanisms underlying hippocampal theta are discussed first followed by mechanisms that may be involved in human frontal-midline theta.
The medial septum drives hippocampal theta. A major afferent input to the hippocampus arises in the rostral basal forebrain (medial septum, vertical limb of the diagonal band; MS/vDB, FIGURE 2) via the fimbria-fornix. Withdrawal of this input by lesions, pharmacological inactivation or transection completely abolishes hippocampal theta rhythm (30, 106, 445, 1090). MS/vDB neurons fire rhythmically in phase with theta rhythm (997, 998). Thus the MS/vDB is thought to be a pacemaker for hippocampal theta (FIGURE 4). Selective lesion of MS/vDB cholinergic neurons reduce the amplitude but do not change the frequency of hippocampal theta (697). In contrast, kainic acid lesion of the MS/vDB, which largely spares cholinergic neurons but kills PV GABAergic projection neurons, and likely other noncholinergic neurons, eliminated hippocampal theta (1463). In vivo, single-unit recordings from identified PV neurons reported bursts of action potentials at theta frequency which are synchronized with the ongoing hippocampal theta activity (125, 1182). Within the burst, action potential firing rates are at gamma frequencies, providing an explanation of the phase-locking of gamma rhythms to theta rhythms. Two populations of PV MS/vDB neurons fired anti-phasically, i.e., one population fired at the peak of hippocampal theta, whereas the other fired at the trough (125). In contrast to the GABAergic neurons, slow-firing cholinergic neurons fired only single action potentials in synchrony with theta rhythm (1182). Together these data suggest that PV GABAergic MS neurons are crucial pacemakers for hippocampal theta, whereas cholinergic neurons modulate the amplitude. MS/vDB PV projection neurons selectively innervate the PV hippocampal interneurons (basket and chandelier neurons) responsible for controlling firing of principal neurons (385). Thus hippocampal theta rhythm is (at least partially) generated by rhythmic inhibition and disinhibition of hippocampal pyramidal and dentate granule cells (176). In addition, rhythmic input from the entorhinal cortex plays a large role in the observed variation in extracellular potential (176).
A) BRAIN STEM CONTROL OF THETA RHYTHM.
The ascending pathways from the brain stem which generate theta rhythm are still an active area of investigation (1365). Precise mapping studies by Vertes and colleagues (1356, 1365) revealed that the most effective brain stem stimulation sites for theta generation are located within the nucleus pontis oralis (PnO) region of the brain stem reticular formation (FIGURE 2). Extracellular single-unit recordings in the PnO of freely moving rats identified cells that fire in association with states when theta rhythm is present. However, these cells did not fire rhythmically, but fired tonically at high rates (60–100 Hz) (935, 1355). Thus these neurons are unlikely to be involved in coding the frequency of hippocampal theta rhythm.
Tonic brain stem input is converted into rhythmic firing in the supramammillary nucleus (FIGURE 4). Although it was originally assumed that tonic firing in the reticular formation is translated into rhythmic firing in the MS/vDB, anatomical tracing studies revealed that few neurons in the reticular formation project directly to the MS/vDB (1357, 1359). Thus at least one additional nucleus is likely interposed between these two areas. Anatomical tracing and physiological mapping studies using the local anesthetic procaine suggested that MS/vDB projecting, glutamatergic neurons containing the calcium-binding protein calretinin in the supramammillary nucleus (SuM; FIGURE 2) may fulfill this role (638, 710, 1360, 1366) (FIGURE 4). Single-unit SuM recordings in urethane-anesthetized animals reported single spike or rhythmic burst firing phase locked with hippocampal theta (105, 637, 639, 649). Rhythmic SuM firing is not due to descending inputs from the septum or hippocampus since it was not altered by inactivation of the MS/vDB with the local anesthetic procaine (639). However, SuM procaine injection blocked the ability of PnO stimulation to elicit theta (638). Thus it was proposed that the SuM translates tonic firing of the reticular input into phasic bursting at the frequency of hippocampal theta (636). However, in contrast to experiments in urethane-anesthetized animals, in freely moving animals procaine causes only a small reduction in the frequency of hippocampal theta (845, 1286), suggesting that additional pathways are involved. The precoeruleus region of the pons (758), located just rostral to the locus coeruleus, provides the major brain stem glutamatergic input to the MS/vDB. In addition, the nucleus incertus of the medulla (933) projects to the MS/vDB and SuM regions. Although these areas have been implicated in brain stem theta generation, rhythmically firing neurons have not been recorded in these regions. Thus they may relay the activity of the reticular formation (especially PnO) to the MS/vDB and SuM. In contrast, the GABAergic ventral tegmental nucleus of Gudden, located just ventral to the dorsal raphe, contains intrinsically bursting neurons (155) which fire rhythmically at theta frequencies during waking and REM sleep and may generate theta rhythmicity in the limbic Papez circuit through their interconnections with the medial mammillary body (81, 648).
B) THETA RHYTHMS IN HUMANS.
In humans, where the hippocampal formation and temporal formation are located ventrally, theta oscillations are most commonly recorded just anterior to the Fz electrode site over frontal and midline cortices (prefrontal and anterior cingulate) (862). Mechanisms that may be responsible for generating this frontal-midline theta (FM-theta) are as follows: 1) FM-theta may be generated through direct or indirect projections from the hippocampal formation, synchronizing information flow between hippocampus and neocortex (1184). However, FM-theta is not always coherent with hippocampal theta (862). 2) FM-theta may represent a slow alpha-rhythm generated by thalamocortical loops during drowsiness (see preceding section). 3) FM-theta may be generated by pacemaker GABAergic (PV-Pos), cholinergic, and glutamatergic projections from the caudal basal forebrain (BF). GABAergic PV-Pos and cholinergic neurons in the caudal BF show similar firing patterns (321, 481, 698) and projections to interneurons and pyramidal neurons in the neocortex (386) as their counterparts in the rostral BF which project to the hippocampus. Furthermore, the firing of ensembles of noncholinergic BF neurons are correlated with prefrontal cortex field potentials (729). Further research is required to determine the contribution of these three mechanisms to FM-theta. They are not mutually exclusive, and any of them may contribute under different conditions.
B. Brain Stem Reticular and Basal Forebrain Activating Systems
The work of a handful of researchers in the first half of the 20th century allowed the development of current ideas of how LVFA typical of wakefulness and REM sleep is generated. Frederic Bremer (136) found that transection of the brain of cats at the midcollicular level (“cerveau isole” preparation) led to “sleeplike” behavior and slow waves in the cortex. In contrast, transection at the junction of the brain stem and spinal cord (“encephale isole” preparation) did not alter the normal cyclic alternation of sleep-wake states and demonstrated that sensory input from the spinal cord was not necessary for wakefulness to occur. Later work by Giuseppe Moruzzi and Horace Magoun showed that electrical stimulation of the midbrain reticular formation in anesthetized cats caused the appearance of an “activated” EEG similar to that seen during waking (898). Together these findings led to the important concept of the “ascending reticular activating system (ARAS),” a network (reticulum) of nerve fibers ascending from the brain stem, which through multiple intermediary sites causes activation of the forebrain during waking and REM sleep (FIGURE 5). The activity of brain stem reticular neurons, the origin of both of these pathways, reliably predicts the onset of changes in behavioral state (818, 1228, 1232, 1236).
The ARAS consists of dorsal and ventral pathways (FIGURE 5). Axonal tracing studies coupled with histochemical or immunohistochemical visualization of particular neurotransmitter systems revealed the anatomical pathways transferring brain stem activity to the cerebral cortex (573, 1228). Single-unit recordings and indirect measures of neuronal activity (using immunohistochemical detection of the immediate early gene product Fos) defined the neurons in these areas whose activity is correlated with wakefulness or sleep (575, 1228). Two main pathways have been identified (FIGURE 5).
1. The dorsal pathway of the ARAS (Figure 5)
This comprises midbrain, pontine, and medullary reticular formation glutamate neurons (261, 916, 1227, 1235) and cholinergic neurons in the pedunculopontine and laterodorsal tegmental nuclei (PPT/LDT) (473, 1235) which innervate the midline and intralaminar (nonspecific) thalamocortical projection system (paraventricular, parataenial, intermediodorsal, centrolateral, paracentral, centromedial, rhomboid, reuniens, centromedian and parafascicular thalamic nuclei). These thalamic nuclei project to widespread and overlapping neocortical areas (559, 578, 750, 970, 1203), although each nucleus has some selectivity in their density of projections to their neocortical targets (1327). Stimulation of the nonspecific nuclei yields widespread cortical responses and elicits fast cortical rhythms (478, 559, 891), while electrical stimulation in sensory relay nuclei elicits short responses in local areas of sensory cortex. In addition to thalamic projections, the brain stem cholinergic (LDT/PPT) neurons also innervate the dopaminergic and GABAergic neurons of the midbrain ventral tegmental area of Tsai (260), which are involved in reward processes and project prominently to the nucleus accumbens and prefrontal cortex. Surprisingly, experiments in rodents (177, 397) and cats (1370) showed that very large lesions of the thalamus appear to have very little effect on cortical activation and the sleep-wake cycle in general, aside from a loss of sleep spindles, suggesting that the dorsal pathway is not absolutely necessary. However, a complete and selective ablation of thalamus is hard to achieve with lesion techniques, and it remains possible that a small thalamic projection remained after these lesions which was sufficient to maintain function. These findings in animals are also seemingly at variance with human studies of coma patients (see sect. VII) and imaging studies of sleep-wake and anesthesia which suggest that changes in reticular and thalamic function precede changes in the cortical EEG (66, 152, 928). At the very least, one can conclude that under normal conditions, the dorsal pathway is involved in and shapes cortical activation.
2. The ventral pathway of the ARAS (Figure 5)
This comprises fibers of the medial forebrain bundle which pass through and make contact with neurons in the midbrain, posterior/lateral hypothalamus, and basal forebrain (BF) on the way to the cortex. The ascending fibers from the brain stem include glutamatergic (parabrachial), noradrenergic (locus coeruleus, LC), serotonergic (dorsal and median raphe), and dopaminergic (periaqueductal gray) neurons. These systems synapse onto glutamatergic, histaminergic, and orexinergic/hypocretin neurons in the posterior/lateral hypothalamus (575). All of these systems converge onto caudal BF cholinergic, GABAergic, and glutamatergic neurons which project to and activate the neocortex (305, 316, 573, 1148, 1228). A branch of this system innervates the rostral BF theta rhythm generator.
In contrast to the thalamic lesions discussed above, a recent study showed that large lesions of the BF, or of the brain stem parabrachial nucleus (PB), which provides the major brain stem glutamatergic input to the BF, led to a comatose state in rats (397), whereas, as discussed above, thalamic lesions had little effect. However, it is important to note that in this study, orexin-saporin was used to lesion the BF and PB, whereas ibotenate was used to lesion the thalamus; thus the two experiments are not directly comparable. Orexin-saporin is a relatively new lesioning tool that requires further study. In particular, it is important to determine if very large lesions, resulting in widespread neuronal death, also affect fibers of passage.
Direct projections to the cortex and the nonspecific thalamic nuclei also arise from brain stem noradrenergic and serotonergic neurons as well as the hypothalamic histaminergic and orexinergic/hypocretin neurons. In section IIC, we discuss the role of the different components of the ARAS, subdivided according to neurotransmitter phenotype.
3. Default network
One novel finding from human imaging studies is the existence of a so-called “default network” of functionally interconnected cortical regions that are active when individuals are left to think to themselves and are not involved in responding to the external environment (1040). Anatomically, the default network consists of regions along the anterior and posterior midline, the lateral parietal cortex, prefrontal cortex, and temporal lobe (33, 1040). Upon presentation of external stimuli requiring a response, the default network regions show a decrease in activity, in contrast to other cortical areas that show increases or no change. Thus, while animal studies have often considered cortical activation as being fairly uniform throughout cortical regions, human imaging studies show that this is not the case. Future studies should distinguish how the ascending systems controlling the default network differ from those affecting other cortical areas.
C. Neurotransmitter Systems Promoting Wakefulness
Multiple neurotransmitter systems contribute to the promotion of wakefulness. However, none of them appears to be absolutely essential. In this section we describe the effects of inactivation or stimulation of these systems, the mechanisms by which they act, and their possible function during wakefulness.
The cholinergic system promotes high-frequency oscillatory activity typical of wakefulness and REM sleep. The BF cholinergic system has an additional role in the homeostatic sleep response to prolonged waking (discussed more fully in sect. III). The important role of brain stem cholinergic neurons in REM sleep control is discussed in section IV.
Neurons involved in sleep-wake control that release acetylcholine are located in the BF and in the mesopontine tegmentum (LDT/PPT) of the brain stem (44, 849) (Figs. 2 and 6). Identified, cortically-projecting cholinergic neurons in the caudal BF (substantia innominata, horizontal limb of the diagonal band, magnocellular preoptic area, nucleus basalis) fire fastest during both wakefulness and REM sleep (481, 698), and their firing is correlated with cortical activation (321, 698, 789). In particular, caudal BF cholinergic neurons fire bursts of spikes in association with neocortical theta rhythms (698). Rostral BF (MS/vDB) cholinergic neurons projecting to the hippocampus also fire in association with hippocampal theta rhythm but fire only single spikes per cycle (1182). Wake/REM-on neurons have also been recorded in cholinergic brain stem areas although, to date, the firing of identified brain stem cholinergic (LDT/PPT) neurons projecting to the thalamus has not been recorded across the sleep-wake cycle. In urethane-anesthetized animals, identified brain stem cholinergic neurons fire in association with cortical activation produced by tail pinch (126). Consistent with the firing patterns of cortical and thalamic-projecting cholinergic neurons, acetylcholine levels are highest in these areas during wakefulness and REM sleep (200, 560, 1412). Thus increased activity of both brain stem and BF cholinergic systems is associated with states when cortical activation and conscious awareness occur (572, 993, 1429).
BF cholinergic neurons projecting to the neocortex promote LVFA. Caudal BF neurons affect electrographic activity via a direct projection to the cortex (305, 450, 500, 1115, 1148, 1431). Intracellular recordings from cortical neurons in vivo and in vitro have revealed a plethora of cholinergic effects that lead to increased excitability and a facilitation of fast EEG rhythms at the expense of slow oscillations typical of NREM sleep (827, 1216). Prominent muscarinic effects include the following: 1) a depolarization of pyramidal neurons via block of a leak potassium conductance (M-current) and activation of mixed cation channels; 2) facilitation of subthreshold oscillations in the beta/gamma range (20–40 Hz), and 3) blockade of slow afterhyperpolarizations. Nicotinic actions include presynaptic facilitation of glutamate release (443) and depolarization of interneurons (20, 579). In vivo, application of agents which depolarize cholinergic neurons in vitro (22, 334, 375, 619) increases theta and gamma cortical activity, together with waking and REM sleep. In particular, the action of neurotensin is noteworthy, since it appears to be selective for cholinergic neurons (191). Conversely, application of serotonin, which hyperpolarizes BF cholinergic neurons (618), reduces gamma activity (189).
A) CHOLINERGIC ELICITATION OF FAST EEG RHYTHMS IN VITRO AND IN VIVO.
In vitro, application of cholinergic agonists causes theta and gamma/beta rhythms in isolated hippocampal (366, 534, 1165) or neocortical areas (107, 163, 950) and promote alpha or theta oscillations in thalamic relay nuclei such as the lateral geniculate nucleus (751, 752). The cholinergic neuromodulatory system is unique in this regard since only cholinergic or glutamatergic agonists have been shown to induce oscillatory activity in vitro. Early, in vivo studies in urethane or ether anaesthetized rats and rabbits established that one form of theta activity (type I theta, 4–7 Hz) was abolished by systemic administration of the muscarinic antagonist atropine sulfate (667). Both brain stem (LDT/PPT) cholinergic neurons projecting to the diencephalon and MS/vDB cholinergic neurons projecting to the hippocampus and neocortex promote theta activity. Infusion of the cholinergic agonist carbachol into the brain stem (PnO) or SuM/posterior hypothalamus increases hippocampal theta (635, 944, 1363) whilst selective lesion of MS/vDB cholinergic neurons reduces the amplitude of hippocampal theta (697). Muscarinic receptor blockade weakens the coupling between gamma and theta rhythms (501), suggesting that the enhanced acetylcholine release that occurs during waking and REM sleep promotes this coupling. Thus acetylcholine promotes the cortical rhythms typical of wakefulness and REM sleep and the coupling of gamma to theta rhythms.
B) BRAIN STEM CHOLINERGIC PROJECTIONS TO THE THALAMUS.
While BF cholinergic neurons promote cortical activation via a direct projection to the cortex, brain stem cholinergic neurons do so via their projections to the thalamus, comprising a major component of the dorsal ARAS pathway (FIGURE 5). Anterograde and retrograde tracing studies coupled with choline acetyltransferase immunohistochemistry revealed a massive cholinergic projection to the thalamus (291, 473, 474, 940, 974, 1193, 1235, 1430) which, depending on the thalamic region studied, make up 25–85% of the projection from all neurons in the pontine tegmentum. A minor cholinergic projection to the thalamus, especially the reticular nucleus and anterior nuclei, arises from BF (976).
Similar to BF cholinergic neurons, the firing of brain stem cholinergic neurons correlates with, and anticipates, cortical activation and deactivation (126, 336, 615, 1221). In vivo, electrical stimulation of brain stem areas containing cholinergic neurons enhances beta/gamma frequency firing in thalamocortical neurons and in the EEG (1226). In vitro, cholinergic agonists depolarize relay neurons via a muscarinic (M1/M3, Gαq G protein) receptor-mediated block of leak potassium conductance. This depolarization facilitates single-spike firing at the expense of the rhythmic bursting observed during NREM sleep (143, 826, 832). Acetylcholine directly depolarizes ventral tegmental area dopaminergic neurons via nicotinic receptors containing α4-α7 and β2 subunits (646, 1002), and by activation of muscarinic M1-like (probably M5) receptors (680, 1461), which increases burst firing (743) and facilitates dopamine release in target regions such as the nucleus accumbens (373).
In addition to acetylcholine, brain stem cholinergic neurons also release the gaseous neurotransmitter NO. In vitro, electrical stimulation of LDT produced NO (707), whereas in vivo studies showed that NO is released in the thalamus (1414) and medial pontine reticular formation (709) in relation to behavioral state. Administration of NO donors enhances neuronal activity in the thalamus and neocortex (265), while NOS inhibitors cause inhibition of thalamic cell activity. NO dampens the oscillatory activity of thalamocortical relay neurons by altering the voltage dependence of the hyperpolarization activated cation current, Ih (967).
C) EFFECTS OF CHOLINERGIC LESIONS.
While electrical or pharmacological stimulation of cholinergic neurons is highly effective in stimulating LVFA, lesioning of brain stem or BF cholinergic neurons does not lead to pronounced changes in 24-h amounts of wakefulness. Selective lesioning of BF cholinergic neurons using the toxin 192IgG-saporin led to relatively minor changes in wakefulness (94, 611). However, high-frequency EEG power, especially gamma-activity, was strongly reduced with extensive lesions of caudal cholinergic BF neurons (94, 600) but was unchanged with less complete lesions (611, 1398, 1399). More consistently, IgG192-saporin lesions of MS/vDB cholinergic neurons reduced the amplitude of hippocampal theta rhythm (80, 415, 697, 1463). Lesioning of the cholinergic neurons reduced the homeostatic response to sleep deprivation, but again this required an extensive destruction of cholinergic neurons (102, 592, 611). Thus it appears that there is considerable redundancy in the cholinergic system, and effects are only seen with extensive lesions.
Overall, the evidence suggests that serotonin promotes a quiet waking state with reduced cortical activation. Serotonin also plays an important role in suppression of REM sleep (sect. IV) and in the response to stress, which may account for some aspects of stress-related sleep disorders (sect. VII).
Serotonin neurons are clustered in several nuclei along the midline of the brain stem in the raphe nuclei (FIGURE 2) (553). Early experiments where serotonin levels were depleted erroneously suggested that serotonin promotes sleep (581, 899). Recent experiments examining mice in which serotonin neurons are genetically deleted suggest that insomnia resulting from disruption of serotonin signaling was due to a disruption of thermoregulation, leading to an increase in motor activity to generate heat (162). In contrast to the early depletion experiments, recording of the electrical discharge of serotonin neurons (839, 1308) and measurements of serotonin release (1020, 1411) revealed that serotonin neurons are wake-active, suggesting that serotonin is wake-promoting. Neuronal firing decreased during NREM sleep and ceased during REM sleep. Accordingly, systemic application of serotonergic receptor agonists increases waking and reduces NREM and REM sleep (884). Serotonergic suppression of NREM sleep is likely due to a 5-HT1A receptor-mediated postsynaptic inhibition of sleep-active VLPO neurons (403), whereas the inhibition of REM sleep involves a postsynaptic inhibition of REM-on brain stem cholinergic neurons (130, 522).
A) MECHANISMS BY WHICH SEROTONIN PROMOTES WAKEFULNESS.
Serotonin promotes waking via depolarization of histaminergic tuberomammillary neurons (344) and BF GABA neurons projecting to the hippocampus (23) and neocortex (154). Serotonin has complex effects on the thalamus. A direct depolarization of lateral geniculate neurons and other first-order thalamic relay neurons via a 5-HT7 receptor-mediated modulation of hyperpolarization-activated cation conductance was initially reported (205, 206, 829, 1342), an action which blocks spindle oscillations (696). However, most higher-order relay and nonspecific nuclei are inhibited by serotonin (877) via a combination of a direct 5-HT1A-mediated postsynaptic hyperpolarization and an indirect increase in inhibitory input due to depolarization of GABAergic thalamic reticular nucleus neurons (833). Sensory relay neurons may also be inhibited by serotonin (1071) through a depolarization of local interneurons (877, 969, 1109). However, serotonin also facilitates glutamate release from thalamocortical terminals via 5-HT2A receptors (10, 11), the main target of hallucinogenic drugs such as lysergic acid diethylamine (LSD) which act as partial agonists of this receptor (794). Serotonin blocks the slow afterhyperpolarizations of intralaminar thalamic (430), hippocampal (1302), and neocortical pyramidal neurons (40, 1475) via activation of receptors coupled to stimulation of adenylyl cyclase (5-HT4/5-HT7), allowing the faster firing typical of wakefulness.
B) STATE-DEPENDENT FIRING OF SEROTONIN NEURONS.
Most serotonin neurons fire in a slow, tonic fashion across the sleep-wake cycle (839, 1308). However, a subpopulation also fires in bursts (471). In contrast to norepinephrine and histamine neurons, most serotonin neurons recorded in vitro do not fire action potentials spontaneously (1335). Thus afferent input from other wake-active systems is required to maintain their firing (158, 713, 1095, 1335). Serotonin neurons are depolarized by norepinephrine, histamine, and orexins via activation of a long-lasting inward current due to the opening of mixed cation channels (158, 735, 1335), likely of the transient receptor potential family (1151). Unlike the other wake-active neuromodulatory systems discussed here, serotonin neurons promote a state of quiet or relaxed waking; single-unit recordings report highest activity during feeding and decreased firing during active waking (554). Serotonin neurons are also activated by stress (476), and 5-HT1A knockout mice lack the rebound of REM sleep observed following the stress of immobilization (130).
C) SEROTONIN INHIBITS THETA AND GAMMA RHYTHMS.
Serotonin acts in opposition to the cholinergic system (FIGURE 6), inhibiting both BF (618) and brain stem cholinergic neurons (763, 1280), resulting in a blockade of fast rhythms (especially theta and gamma) promoted by activation of the cholinergic system. In particular, median raphe (MR) serotonergic neurons inhibit hippocampal theta rhythm (1365). Electrical or pharmacological stimulation of the MR abolishes theta rhythm in both anesthetized and unanesthetized rats (51, 629, 1356, 1451), whereas lesions or pharmacological inactivation of MR result in continuous theta (630, 1451). An involvement of serotonin in these effects was suggested by the following findings: 1) treating rats with p-chlorophenylalanine, resulting in a 60–80% depletion of forebrain serotonin, blocked the effects of MR electrical stimulation (51); 2) continuous theta in raphe lesioned animals could be interrupted by administration of the serotonin precursor l-5-hydroxytryptophan (1451); and 3) inhibition of MR serotonin neurons with 5-HT1A agonists generates theta rhythm in urethane-anesthetized rats (631, 1364). Similarly, serotonin inhibits caudal BF cholinergic neurons (618) and reduces EEG gamma activity (189).
Norepinephrine neurons are generally thought of as part of the central flight-or-fight response, being particularly important in waking associated with stressful situations. Norepinephrine also plays an important role in the maintenance of muscle tone during waking and suppression of REM sleep (see sects. IV and VII).
Norepinephrine neurons are located in small clusters throughout the brain stem (194). The most prominent noradrenergic innervation of the forebrain arises from the LC (FIGURE 2). It is this nucleus that has been studied most closely with respect to the sleep-wake cycle. LC neurons fire most rapidly during wakefulness and are activated further by stressful stimuli (1050), but their firing slows during NREM sleep and ceases prior to and during REM sleep (511). Norepinephrine strongly excites many neurons of the ARAS (FIGURE 6), mainly via α1 receptors, including thalamic relay neurons (829), serotonin dorsal raphe (DRN) neurons (9, 68, 158, 962, 1335), BF cortically-projecting cholinergic (375) and GABAergic neurons (154). Norepinephrine inhibits neurons in the sleep-active ventrolateral (403) and median preoptic nuclei (63), as well as REM-on brain stem cholinergic neurons (706, 1280), by acting on postsynaptic α2 receptors and activating an inwardly rectifying potassium conductance. β-Receptors inhibit slow calcium-dependent afterhyperpolarizations of cortical pyramidal neurons, allowing the faster firing typical of wakefulness (463) and blocking the slow oscillations typical of NREM sleep (1220).
Studies utilizing neurotoxic or electrolytic lesions of the LC or norepinephrine system reported minor changes in the amount of wakefulness (100, 244, 576, 758, 881). However, depletion of norepinephrine using peripheral administration of the toxin DSP-4 reduced the expression of ∼20% of waking-related gene transcripts, particularly those involved in synaptic plasticity and cellular stress responses (238, 241, 244). Studies of long-term potentiation implicate noradrenergic β-receptors in promotion of synaptic plasticity (519, 1202). Thus one important function of norepinephrine released during waking appears to be the promotion of synaptic plasticity required for memory formation, in particular emotional memory (1312).
Histamine neurons were first implicated in wake promotion due to the sedative side effects of first-generation antihistamines (H1 receptor antagonists) that cross the blood-brain barrier and affect central histaminergic systems (159, 1402). More recent studies have clearly shown that histamine neurons in the tuberomammillary nucleus (TMN; FIGURE 2) are slow firing (<10 Hz) and have a wake-on, NREM-slow and REM-off firing pattern (564, 1264, 1338). In vitro, histamine neurons are spontaneously active (464, 1242) due to the activity of a persistent tetrodotoxin-sensitive sodium current (1257). They are excited directly by orexins (342) and serotonin [via 5-HT2C receptors (344)] and indirectly by norepinephrine [through inhibition of GABAergic inputs (1243)]. Histamine has excitatory effects on most nuclei of the ARAS (FIGURE 6; Refs. 159, 465) and, accordingly, injection of histamine into many nuclei of the ARAS promotes wakefulness (722). Conversely, histamine inhibits sleep-active projection neurons of the VLPO via excitation of local inhibitory interneurons, leading to a promotion of wakefulness (736).
Modest decreases/increases in waking have been observed following pharmacological suppression or activation, respectively, of the histamine system (159, 722). However, inactivation of the histamine system via lesions (302, 413), knockout of the histamine H1 receptor (528), administration of an irreversible inhibitor of the histamine synthesizing enzyme histidine decarboxylase (HDC; Refs. 551, 642, 1152) or knockout of HDC (29, 978) have relatively minor effects on 24-h amounts of waking or cortical activation suggesting that, similar to the other aminergic systems, the histamine system is not absolutely essential for wakefulness. Histamine neurons maintain their level of firing during cataplectic attacks in narcoleptic animals (in contrast to norepinephrine and serotonin neurons) implicating them in the preservation of consciousness which accompanies the cataplectic state (564). In addition, increased activation of histamine neurons as measured by Fos activity has been observed during feeding anticipatory behavior (851, 1323). More fine-grained analysis of sleep and wakefulness in HDC knockout animals revealed a deficit in wakefulness when placed in a novel, potentially dangerous environment (29, 978). This is consistent with a role for histamine in stress- or danger-induced arousal (159).
Orexins/hypocretins were discovered relatively recently by two groups who gave them their two names (290, 1101). We will use the term orexins for these peptide neurotransmitters in this review. Orexins consolidate wakefulness (increase the duration of long waking bouts), suppress REM sleep (sect. IV), and enhance wakefulness in periods of starvation (1452). Considerable evidence links them to the sleep disorder narcolepsy (see sect. VII).
A) OREXINS PROMOTE WAKEFULNESS.
Early work showed that intracerebroventricular application of orexin A dose-dependently increases wakefulness in rats (1005). More recent work using light-activation of orexin neurons via viral vector-mediated introduction of channelrhodopsins (6) found that excitation of orexin neurons in the lateral hypothalamus at frequencies above 5 Hz increased the probability of a transition from sleep to wakefulness. Conversely, administration of recently developed orexin receptor antagonists increased both NREM and REM sleep in animals and humans at the expense of wakefulness (140).
B) OREXIN NEURONS INCREASE WAKING IN RESPONSE TO LOW FOOD AVAILABILITY.
One function of the orexin system may be to integrate nutritional state with arousal (4, 1416, 1452). Orexin neurons respond to a wide variety of peripheral and central signals indicating nutritional state (164, 268, 393, 1048, 1452). Several metabolic signals which increase with feeding, such as glucose, leptin, and neuropeptide Y, inhibit orexin neurons in vitro (164, 393, 1452). In contrast, orexin neurons are activated by fasting in non-human primates (308), and given their wake-promoting effects, they are likely to be primary mediators of the increase in waking and suppression of sleep caused by limited availability of food. In fact, orexin knockout mice fail to respond to fasting with an increase in waking and activity (1452).
C) OREXIN NEURONS ARE WAKE-ACTIVE.
Orexin neurons are most active during waking as assessed by Fos immunohistochemistry (351, 872) and measurements of peptide release (641). In the squirrel monkey, which has a sleep-wake cycle similar to that of humans, orexin levels peaked in the latter third of the day and remained elevated during 4 h of extended wakefulness, consistent with a role for orexins in consolidating wakefulness in opposition to accumulating sleep drive (1472). Single-unit recordings in the rat from the area where orexin neurons are located revealed one group of slow-firing neurons that were wake-active and REM-off (13, 666). Later recordings in freely moving rats confirmed that this population corresponds to orexin neurons, determined by electrophysiological criteria (856) or post hoc immunohistochemical staining (699, 1265). Orexin neurons fire fastest during active waking, decrease firing during quiet waking, and cease firing during sleep, except during microarousals or immediately preceding the arousal from sleep.
In vitro, intracellular recordings from identified orexin neurons revealed that they have a depolarized resting membrane potential (333, 715), leading to spontaneous firing in the absence of injected current or application of neurotransmitter agonists. In addition, they are excited by a positive feedback loop involving local orexin release, activation of orexin type 2 receptors (1455), and excitation of local glutamatergic inputs (715). This positive feedback loop may help to synchronize the firing of the whole orexin neuron population. Furthermore, glutamatergic inputs to orexin neurons are potentiated via a cAMP-dependent mechanism during prolonged waking (1047), which is a mechanism suggested to be important in the maintenance of wakefulness in the face of increased sleep pressure (1299). However, recent optogenetic stimulation experiments found that sleep deprivation blocks the ability of orexin to activate its downstream targets and enhance waking (195).
D) CONTROL OF OREXIN NEURONS BY AFFERENT INPUTS.
Orexin neurons receive afferent inputs from other nodes of the sleep-wake circuitry (FIGURE 6) as well as from areas involved in emotional regulation such as the amygdala and lateral septum (1102, 1466). They are excited by acetylcholine via M3 muscarinic receptors (84, 947, 1454) but inhibited by serotonin via a postsynaptic activation of 5-HT1A receptors (715, 905). This inhibitory action is also observed in vivo since intracerebroventricular application of an 5-HT1A antagonist, WAY100635, increased locomotor activity during the dark (active) phase in wild-type mice, but not in orexin/ataxin-3 mice in which orexin neurons are ablated (905). Both inhibitory (715, 716, 1453) and excitatory (84, 1453, 1454) effects of norepinephrine on orexin neurons have been reported in recordings from mouse and rat brain slices. The inhibitory response is mediated by α2 receptors activating inwardly rectifying potassium conductance (716, 1453), whereas the excitatory action is due to activation of α1 receptors and activation of a nonselective cationic current (1453). In the rat, it has been suggested that the response to norepinephrine shifts from an excitation to an inhibition during a short period (2 h) of sleep deprivation (452). In addition, norepinephrine increases the frequency of inhibitory postsynaptic currents (IPSCs) via an effect on presynaptic GABAergic terminals (716, 1453). In vitro, dopamine inhibits orexin neurons via D2 receptors (716), whereas in vivo, systemic dopaminergic agonists increase their activity as assessed by Fos immunohistochemistry, likely by an indirect action (161). Orexin neurons are unaffected by histamine, which is somewhat surprising, considering the close proximity of histamine and orexin neurons in the hypothalamus (342).
E) INHIBITION OF OREXIN NEURONS DURING SLEEP.
The spontaneous activity of orexin neurons in vitro suggests that they must be actively inhibited during NREM and REM sleep when their activity level slows markedly. This inhibition likely arises from GABAergic neurons in the preoptic area and BF. Orexin neurons are postsynaptically inhibited by both GABAA and GABAB receptors (15, 715, 1150, 1445), and GABAB receptors also mediate a presynaptic inhibition of glutamatergic and GABAergic inputs (1445). In vivo, antagonism of GABAA receptors increases the firing rate of wake-on (presumed orexinergic) neurons in the perifornical hypothalamus during NREM sleep, indicating an inhibitory GABA receptor-mediated tone during this state (15). Mutant mice with a constitutive loss of GABAB receptors in orexin neurons (via knockout of the GABA-B1 gene) have fragmented sleep-wake cycles, due to an upregulation of inhibitory tone which shunts (short-circuits) excitatory and inhibitory inputs (811). Feedback control of orexin neurons may occur through the release of coexpressed dynorphin peptides (223), which cause a hyperpolarization, inhibition of calcium channels, and reduction of excitatory synaptic inputs (717), although direct evidence for feedback control by this mechanism is lacking at present. In addition, orexin neurons are inhibited by the sleep homeostatic factor adenosine via A1 receptors (14, 1277) (see sect. III).
F) DOWNSTREAM EFFECTORS OF OREXIN PROMOTION OF WAKEFULNESS.
How do the orexins consolidate wakefulness? Anatomical studies demonstrated a strong innervation of sleep-wake circuitry by the orexin neurons, particularly the aminergic nuclei (273, 524, 1000). The strongest projection was found to the LC which expresses exclusively the type I receptor, whereas most other sleep-wake nuclei express the type II receptor or both type I and II (249, 506, 793, 853, 1307). In vivo, injections of orexin A into the LC enhanced wakefulness at the expense of REM sleep (128, 467), whereas in vitro recordings revealed a postsynaptic excitation mediated by activation of nonselective cation channels and blockade of leak potassium channels (524, 552, 904, 1326). Similarly, in vitro studies showed that orexins had excitatory effects on serotonergic DRN neurons (157, 158, 651, 735), histaminergic tuberomammillary neurons (85, 342, 1456), BF (47, 334), and brain stem cholinergic neurons (169, 651) and ventral tegmental area dopamine and GABA neurons (660). Furthermore, orexins target neurons in the dorsal ARAS pathway, exciting neurons in the reticular formation (160), nonspecific thalamic nuclei (83, 312, 436, 527), thalamocortical terminals (685), and deep layer VI cortical neurons (86). In addition to the LC, in vivo studies showed wake-promoting effects of orexins in the BF (348, 1279), tuberomammillary nucleus (530), laterodorsal tegmentum (1442), and reticular formation (1392). Orexins also directly increase muscle tone via excitation of spinal cord motoneurons (1457).
It was proposed that orexins exert their wake-promoting action through stimulation of the histamine system since orexins excite histamine neurons in vitro (342), and the wake-promoting effect of intracerebroventricular orexin A is reduced/lost in HDC knockouts and histamine H1 receptor knockouts (530) or with application of a histamine H1 receptor antagonist (1163). Furthermore, low histamine levels have been reported in the brains of narcoleptic dogs (922) and in the cerebrospinal fluid (CSF) of human narcoleptics, particularly in unmedicated patients (597, 925). However, the dependence of the intracerebroventricular effect of orexin A application on the histamine system may simply reflect the close proximity of histamine neurons to the ventricular system, compared with other postsynaptic targets. In contrast to orexin knockout animals, HDC or histamine H1 receptor knockout animals do not have reduced duration of sleep-wake states (29), and optogenetic stimulation of orexin neurons is still able to increase the probability of awakening in HDC knockout animals. However, expression of the orexin type II receptor in histamine neurons and other areas surrounding the TMN in mice lacking type II receptors was sufficient to consolidate wakefulness, although sleep was still fragmented (869). Orexins actions at other sites are likely to be similarly important. For instance, optogenetic inhibition of LC norepinephrine neurons inhibited the wake-promoting effect resulting from optogenetic excitation of orexin neurons (196).
6. Neuropeptide S
Like the orexins, neuropeptide S (NPS) is a recently discovered peptide activating a previously “orphan” G protein-coupled receptor activating phospholipase C (1449). NPS is coexpressed in glutamate-producing neurons located just rostral to the LC (precoeruleus region) which project to widespread areas of the brain, including sleep-wake regulatory regions such as the midline thalamic nuclei, lateral hypothalamus, and preoptic area (1448, 1449). Intracerebroventricular application of NPS increased locomotor activity and decreased sleep in rats (1449), whereas NPS receptor knockout mice had reduced exploratory activity in a novel environment (317). In addition to its role in promoting wakefulness, recent experiments suggest a role for the peptide in controlling fear and anxiety (586, 1449).
Pharmacological agents increasing dopaminergic tone such as amphetamines and modafinil (Provigil) are the most potent wake-promoting substances currently known. As such, they are commonly prescribed to treat sleep disorders involving excessive daytime sleepiness (see sect. VII). Although these substances can enhance the release of other neuromodulators such as serotonin and norepinephrine, their effects are abolished in dopamine transporter (DAT) knockout animals (1425), confirming that their main effect is on dopaminergic systems (129). Additional evidence supporting a role for dopaminergic systems in promotion of wakefulness comes from analysis of D2 receptor knockout mice that exhibit a significant decrease in waking amounts due to a shorter wake bout duration and a concomitant increase in sleep (1032). One possible mechanism explaining this effect is a disinhibition of intralaminar thalamic neurons via indirect basal ganglia-thalamic pathways (1135). In Parkinson's disease, where dopamine neurons in the substantia nigra degenerate, waking is interrupted by sleep episodes (1087). However, dopamine neurons are not the only neurons to be affected by this disease.
A) VENTRAL TEGMENTAL AREA DOPAMINE NEURONS.
While the average firing rate of dopamine neurons in the ventral tegmental area (VTA) and substantia nigra does not vary across sleep-wake states (858), VTA dopamine neurons fire more bursts during waking and REM sleep, resulting in increased release of dopamine in target areas such as the nucleus accumbens and prefrontal cortex (271). In particular, increased bursting is observed in the presence of rewarding or aversive stimuli requiring an alerting response (1209). VTA neurons are excited in vitro by several neuromodulators that promote arousal such as orexins, substance P, and corticotrophin releasing hormone (658, 660).
B) VENTRAL PERIAQUEDUCTAL GRAY DOPAMINE NEURONS.
Dopaminergic neurons in the ventral periaqueductal gray (vPAG)/DRN (FIGURE 2) show Fos activity during waking but not during sleep (757). Selective lesioning of these neurons by injections of 6-hydroxydopamine (63% loss) or nonselective lesions with ibotenic acid (80% loss) resulted in a marked (>20%) reduction in 24-h amounts of wakefulness, one of the most pronounced effects of lesions on wakefulness reported to date (757). In contrast, lesions of the serotonergic neurons in this area were without effect on 24-h amounts of sleep and waking. Retrograde tracing studies showed that dopaminergic vPAG neurons project to other parts of the ARAS such as the BF and midline thalamus, and receive input from sleep-active VLPO neurons (757). These data all support a role for these neurons in control of wakefulness, but electrophysiological recordings from these neurons across behavioral state are lacking at present.
GABAergic neurons and glutamatergic neurons (reviewed below) are very abundant and widely distributed in the brain. Hence, it is not surprising that some populations of the neurons utilizing these two neurotransmitters are involved in promoting wakefulness, whereas others are associated with sleep. Thus, although pharmacological agents potentiating the activity of GABAergic systems have been most closely linked with sleep (see sects. III and IV), select GABAergic subpopulations in the cortex (especially PV interneurons, sect. IIA) and in subcortical sites, are thought to be critical in the production of cortical LVFA. Cortically-projecting GABA neurons are located in the BF (386, 450, 500), hypothalamus [colocalized in histamine (1371), and melanin-concentrating hormone neurons (60)] and in the VTA (1206). Hypothalamic melanin-concentrating hormone neurons fire predominantly during sleep (482) and so are unlikely to contribute to wakefulness. While the activity of histamine neurons is correlated with wakefulness, the function of GABA in histamine neurons is unclear, especially since it would be expected to counteract excitatory actions of histamine on target neurons. GABAergic neurons in the thalamic reticular nucleus play a crucial role in thalamocortical rhythms during sleep and wakefulness (see sect. III).
A) BF AND VTA GABA NEURONS.
GABAergic neurons in the BF and VTA (FIGURE 2) in particular appear to be important for cortical LVFA since a fast-firing subpopulation of these neurons increases their activity during waking and REM sleep (481, 700). Many GABAergic BF neurons projecting to the cortex contain PV (451). Preliminary studies (154) showed that identified cortically-projecting BF GABA neurons are excited by neurotransmitters promoting cortical activation (acetylcholine, norepinephrine, histamine, orexins), likely accounting for their faster firing rate during waking and REM sleep (481). Rostral and caudal PV GABAergic projection neurons synapse onto hippocampal (385) and neocortical PV-positive neurons (386) which control hippocampal and cortical gamma rhythms, respectively (see sect. IIA). Other subpopulations of BF GABA neurons that are likely sleep related project to the thalamic reticular nucleus (49) and lateral hypothalamus (449). The firing of cortically projecting GABA neurons in the BF (481) but not VTA (700) was correlated with gamma activity in the EEG. However, VTA GABA neurons increased their firing prior to intracranial self-stimulation of the medial forebrain bundle, indicating that they may be involved in the attentive processes related to brain reward (1205). VTA GABA neurons are excited by the wake-promoting orexins (660) and by histamine (659). Ibotenic acid lesions of the rostral BF (MS/vDB), which preferentially affect noncholinergic neurons, abolish theta and gamma rhythms in the hippocampus (see sect. IIA). Similarly, chemical lesions of the caudal BF have dramatic effects on cortical LVFA and attention that are correlated with the loss of PV-positive GABA neurons (166, 397, 611).
B) STRIATAL MEDIUM SPINY NEURONS.
GABAergic medium spiny neurons in the striatum receive a massive glutamatergic cortical input and control the activity of thalamocortical neurons. Transitions from NREM sleep to wakefulness convert the firing of striatal neurons from fast cyclic firing, synchronized with cortical field potentials, to an irregular pattern of action potentials triggered by disorganized depolarizing synaptic events of variable amplitude (777). Cell body specific lesions of the rostral striatum reduce waking by ∼15% and produce cortical slowing of the EEG (1029). Conversely, lesions of the globus pallidus, the main recipient of inhibitory striatal projections, increase waking by 46%. Improved function in minimally conscious patients produced by stimulation of the nonspecific thalamic nuclei (1136) may be mediated by increased cortico-striatal-thalamic interplay (1135).
The vast majority (>90%) of glutamatergic projections to the cortex arise from the thalamic relay nuclei innervating cortical layers III and IV, and from nonspecific thalamic nuclei innervating layers I and VI (540, 750). In addition, the BF (500), claustrum, amygdala, VTA, laterodorsal tegmentum, and hypothalamus (540) provide minor glutamatergic projections to the cortex. Vesicular glutamate transporters are expressed in cortically projecting orexin neurons in the perifornical hypothalamus (1077) and serotonergic DRN neurons (439), suggesting that glutamate is a cotransmitter in these neurons. Furthermore, glutamate is the major neurotransmitter released from rostral midbrain brain stem reticular formation neurons projecting to the thalamus. Dissociative anesthetic agents such as ketamine inhibit glutamatergic NMDA receptors, whereas pharmacological agents that prolong the decay of AMPA receptor currents (AMPAkines) are proposed to enhance attention and cognition.
A) THALAMIC INTRALAMINAR AND RELAY NEURONS.
The thalamus is an important component of the dorsal branch of the ARAS involving the nonspecific thalamic nuclei (FIGURE 5), as well as the specific relay nuclei which convey external sensory information to the cortex. EEG rhythms typical of wakefulness are sculpted through interactions between the thalamocortical relay neurons, corticothalamic pyramidal neurons, and GABAergic neurons in the thalamic reticular nucleus. At the onset of conscious states (i.e., wakefulness and REM sleep), thalamic relay neurons are excited by the action of acetylcholine, norepinephrine, and histamine, leading to a switch in firing pattern from synchronized burst firing (typical of NREM sleep) to tonic firing able to faithfully transmit sensory information to the cortex (826, 828, 1228). This switch in firing pattern is due to a depolarization mediated by a block of leak potassium conductance by Gq-coupled receptors (muscarinic M1/3, norepinephrine α1, histamine H1) and a block of the pacemaker current Ih by Gs/adenylyl cyclase-coupled receptors (muscarinic M2/M4, norepinephrine β, serotonin 5-HT4,6, histamine H2). In a thalamocortical slice preparation, coincident stimulation of nonspecific thalamic nuclei (centrolateral intralaminar nucleus) or direct stimulation of layer I together with relay nucleus stimulation induced supralinear summation of the two inputs in cortical output layer V, providing a possible mechanism by which the nonspecific nuclei promote arousal (741).
10. Effector systems of neurotransmitters promoting wakefulness
The effector systems used by the neurotransmitter systems involved in generation of wakefulness have been studied by in vitro electrophysiology, pharmacology, and genetic methods (see sect. VI). The majority of the receptors implicated in cortical LVFA and wakefulness are either ionotropic (glutamatergic AMPA, kainate and NMDA receptors, GABAA receptors, nicotinic acetylcholine and serotonin 5-HT3 receptors) or metabotropic receptors coupled to Gq G proteins and the beta form of the enzyme phospholipase C (glutamatergic mGluR1 and mGluR5, cholinergic muscarinic M1, M3, M5, norepinephrine α1, histamine H1, serotonin 5-HT2, orexin type I and type II receptors). Phospholipase C (PLC)-β occurs in four isoforms. Mice lacking the β1 or β4 subunits of PLC have disrupted theta rhythms and other EEG abnormalities (595, 1166). Activation of these metabotropic receptors causes a depolarization in target neurons mediated by one or a combination of three mechanisms: 1) blockade of leak potassium conductances (two-pore potassium channels) (1348); similar to deletion of PLC-β isoforms, mice lacking the TASK3 two-pore potassium channel have deficient theta oscillations and altered sleep behavior (965); 2) activation of mixed cation channels [likely of the transient receptor potential (TRP) family] (1151); and 3) activation of electrogenic sodium-calcium exchangers (343, 1434). In addition, effects on other intrinsic membrane currents contribute to the activation of thalamocortical and limbic neurons (828, 918). For the most part, the role of individual subunits of these channels/transporters in the control of sleep-wake behavior remains to be determined.
Studies involving stimulation of the brain areas and neurotransmitter systems comprising the ARAS consistently report EEG activation and wakefulness as a result. These studies include both older techniques of electrical stimulation or infusion of pharmacological agents as well as state-of-the-art optogenetic techniques where light-activated ion channels are introduced into the desired neuronal population by genetic engineering techniques (6, 1473). In contrast to the stimulation experiments, studies where localized inactivation of individual neurotransmitter systems or nuclei of the ARAS have been performed (summarized in TABLE 1) generally produce relatively minor changes in cortical EEG or the amount of wakefulness in a 24-h period (see sect. IIC), with the possible exception of the parabrachial nucleus (see sect. IIB). There are several possible explanations for this dichotomy between stimulation and inactivation experiments. First, the ARAS systems are strongly interconnected, mutually excitatory to each other (FIGURE 6) and converge onto common effector systems at the level of thalamic and cortical neurons (826, 918). Thus there is considerable redundancy in the system, and inactivation of any individual component of the system is compensated for by the other systems. This is perhaps not surprising considering the enormous adaptive advantage of wakefulness! A second possibility for the mild effects of loss-of-function experiments is that the systems so far targeted are not absolutely required for wakefulness. The majority of studies have focused on neuromodulatory systems, whereas selective inactivation of glutamatergic and GABAergic systems projecting to the neocortex have not been tested due to technical difficulties in targeting these systems. The neuromodulatory systems are clearly able to generate cortical activation when stimulated but may only be required for specific aspects of wakefulness. Specific roles for these systems could be 1) facilitation of LVFA (acetylcholine); 2) inhibition of sleep-active neurons (norepinephrine, serotonin, acetylcholine; see sect. III); 3) maintenance of high muscle tone (norepinephrine) during waking (see sect. IVA); 4) consolidation of wake periods (orexins); 5) maintenance of waking in a novel environment (histamine); 6) enhanced arousal in the presence of rewarding stimuli (dopamine, acetylcholine); 7) enhanced arousal in the presence of aversive stimuli (norepinephrine, serotonin, histamine); and 8) consolidation of memories through enhancement of synaptic plasticity (acetylcholine, norepinephrine, serotonin, histamine, dopamine, orexins). Methods to selectively stimulate these systems (e.g., using optogenetic techniques) together with whole brain imaging will be helpful in further delineating their function.
III. NREM SLEEP
Subjectively experienced as a loss of conscious awareness, the onset of sleep is heralded in the EEG by the replacement of LVFA by large-amplitude, slow (<4 Hz) waves and the appearance of thalamocortical spindles (FIGURE 1). These EEG changes are due to the progressive reduction in firing of neurons in the ARAS. This section describes the mechanisms underlying the EEG signs of NREM sleep (also called slow-wave sleep) and the mechanisms that cause the circadian and homeostatic inhibition of wake-promoting ARAS neurons.
A. Electrographic Signs of NREM Sleep
In humans, the different stages of NREM sleep are classified according to the criteria established by Rechtschaffen and Kales (1053) (FIGURE 1). Stage 1 NREM sleep exhibits theta activity at frontal sites and alpha activity posteriorally, similar to drowsy waking (sect. II). Stage 2 NREM sleep is characterized by the appearance of sleep spindles (7–15 Hz) and K-complexes in the EEG. Stages 3 and 4 of NREM sleep (deep sleep) exhibit prominent, high-amplitude slow, delta waves (1–4 Hz). The cortical slow oscillation (0.5–1 Hz) discovered by Steriade and colleagues synchronizes the activity of cortical and thalamic neurons that generate spindle and delta waves throughout NREM sleep (1217). In animals, NREM sleep is not usually subdivided into these four stages, but deep (delta) sleep may be distinguished from light NREM sleep. NREM sleep is also characterized by low skeletal muscle tone and slow, rolling eye movements. Here we first describe phasic events occurring during NREM sleep in the thalamocortical system (spindles) and hippocampal formation (sharp wave-ripple complexes) and then discuss the delta and slow oscillations typical of deep NREM sleep.
1. Thalamocortical spindles
Spindles and K-complexes are the defining features of stage 2 NREM sleep in humans (FIGURE 1). K-complexes represent a combination of one cycle of the neocortical slow oscillation followed by a spindle in thalamocortical neurons (27, 28, 197). Several lines of evidence support the contention that spindles are generated in the thalamic GABAergic reticular and perigeniculate nuclei (394, 1228): 1) spindles occur even in the absence of the cerebral cortex (1215); 2) large thalamic lesions (177, 397, 1370) or specific lesions/deafferentation of the thalamic reticular nucleus abolish spindles in thalamocortical neurons (1223); 3) spindle activity can be recorded in the deafferented reticular nucleus (1224); 4) spindles are absent in the anterior part of the thalamus, which does not receive afferents from the thalamic reticular nucleus (i.e., the anterodorsal, anteromedial, and anteroventral nuclei; Ref. 975), and in their projection areas (cingulate cortex, habenular nucleus). Although the thalamic reticular nucleus is the generator of spindles, in intact animals spindles are initiated and terminated in concert with delta and slow oscillations in corticothalamic and thalamocortical neurons due to the extensive interconnections of these cells (828).
A) CELLULAR MECHANISMS UNDERLYING SPINDLES.
As aminergic inputs are slowly withdrawn during early NREM sleep, long-lasting (50 ms) bursts of action potentials are generated in reticular nucleus neurons due to activation of low-threshold (T-type) calcium channels. These channels are of the Cav3.2 and CaV3.3 subtype (1269), which allow bursting at the resting membrane potential. Bursts at spindle frequencies lead to large and long-lasting inhibitory synaptic potentials (IPSPs) in thalamocortical neurons which remove the inactivation of T-type (Cav3.1) (1269) calcium channels. Thus, at the offset of the IPSPs, when the cell becomes more depolarized, the low-threshold calcium channels are activated, calcium enters the cell, resulting in a low-threshold calcium spike crowned by a short (5–15 ms) burst of sodium-dependent action potentials in the thalamocortical neurons. This burst in thalamocortical neurons leads to EPSPs in cortical neurons and to action potentials which together make up the spindle recorded in the EEG. Synchronization of spindles is achieved via recurrent inhibitory and electrical synaptic connections between thalamic reticular neurons (828). Spindles can also be recorded in cortical projection sites such as the basal ganglia (298), possibly providing a substrate for procedural learning during sleep. Spindles decline during deep sleep due to the increased hyperpolarization of thalamocortical relay neurons but may reappear just prior to the transition to REM sleep when thalamocortical relay neurons become more depolarized again (due to increased ascending brain stem excitation).
B) INHIBITION OF SPINDLES DURING WAKEFULNESS AND REM.
In vivo, extracellular and intracellular recording studies revealed that thalamic reticular neurons fire tonically during waking and switch to burst firing during NREM sleep, similar to thalamocortical relay neurons (802, 1222). Tonic firing and inhibition of bursts/spindles during waking is likely due to excitation of these thalamic reticular neurons by norepinephrine and serotonin (833) released from ascending projections arising in the LC and DRN. Norepinephrine, acting via α1 receptors and serotonin, acting via 5-HT2 receptors, causes a depolarization by block of a leak potassium conductance leading to an inactivation of low-threshold calcium channels responsible for bursting (833). Spindles can also be inhibited by input from other ARAS systems, in particular brain stem cholinergic (974) and BF cholinergic and GABAergic inputs (49, 976). The mechanism underlying inhibition of spindle activity during REM sleep is less clear but has been proposed to be due to input from REM-on cholinergic neurons which hyperpolarize thalamic reticular neurons via a muscarinic M2 receptor mediated inhibition of leak potassium conductance (192, 831).
2. Hippocampal sharp waves and high-frequency ripples
High-frequency (100–400 Hz) field potentials termed sharp wave-ripple complexes can be recorded in the hippocampus and associated areas during quiet wakefulness and NREM sleep in rodents (174, 180, 181) and in humans (132). Sharp-waves occur in the CA3 region of the hippocampus when the highly interconnected CA3 pyramidal network is released from the control exerted by subcortical inputs, especially the MS/vDB. Sharp-wave like epileptiform waves occur in the hippocampus following transection of the fimbria-fornix, the main fiber bundle carrying ascending fibers from the MS/vDB and other ARAS systems (182). Ripple occurrence is also increased by pharmacological blockade of histamine H1 receptors, which mediate histaminergic excitation of MS/vDB neurons (647, 1012) or blockade of serotonin 5-HT3 receptors (1012), which excite inhibitory hippocampal/dentate interneurons. When released from inhibition, the synchronized firing of CA3 pyramidal neurons leads to a concerted activation of Schaffer-collateral synapses in the CA1 region and subsequently of subicular and downstream retrohippocampal cortical structures (227). Feed-forward and feedback activation of hippocampal GABAergic interneurons leads to a high-frequency oscillation in the membrane potential of pyramidal neurons due to IPSPs, reflected as a high-frequency ripple in the extracellular potential (1462). Phase-locked interneurons fire at high frequencies on every cycle of the extracellularly recorded oscillation and entrain the firing of pyramidal neurons, which fire at lower frequencies (643, 1462). Accordingly, ripple frequency is reduced by pharmacological prolongation of GABAA receptor-mediated currents (1013). Surprisingly, ripple amplitude and entrainment of pyramidal neurons were increased in mice lacking the GluR1 subunit of AMPA-type glutamatergic receptors specifically on PV-positive interneurons, possibly as a result of developmental compensation (1035). Although many pyramidal neurons contribute to each sharp wave/ripple, each wave of a ripple reflects the firing of a discrete subpopulation of these neurons (228, 1462). Modeling studies suggest that electrical coupling between the axons of pyramidal neurons is required to synchronize their activity (1306). In support of this idea, the occurrence of ripples in vitro was reduced in mice lacking one type of gap junction protein (connexin 36), and the intraripple frequency was reduced (781). However, in another report, the occurrence of in vitro kainate-induced sharp waves was actually increased in these mice (957).
3. Delta (1–4 Hz) and slow (<1 Hz) oscillations
Delta and slow oscillations in the cortex and thalamus are typical of the deeper stages of NREM sleep (FIGURE 1). They result from increased withdrawal of excitatory neuromodulatory inputs (primarily cholinergic and aminergic), resulting in a more hyperpolarized membrane potential of the pyramidal/thalamic relay neurons. Delta oscillations are best understood at the thalamic level. Recordings in vivo from thalamocortical neurons revealed that stereotyped high-frequency bursts of action potentials occur at delta frequencies interspersed with silent periods (25, 314, 817, 932, 1225), a pattern which can be abolished by brain stem cholinergic stimulation or by increases in ambient light (25, 932, 1225). The ability of thalamocortical neurons to generate burst firing in the delta frequency range is due to their intrinsic membrane properties (556, 557, 711, 830, 1199). Hyperpolarization resulting from the activation of calcium-dependent potassium conductances after a burst of action potentials or from inhibitory synaptic inputs leads to the opening of hyperpolarization-activated, cAMP-modulated cation (HCN) channels causing the so-called H-current (Ih). This slowly activating current provides a depolarizing drive towards the threshold for action potentials and is a major contributor to the duration of the interburst interval (830, 968). Ih is modulated during waking by activation of neurotransmitter receptors coupled to stimulation of cAMP (e.g., norepinephrine β, histamine H2) and by release of NO from cholinergic projections (967, 968) resulting in a shift in the activation curve to more positive membrane potentials and reducing the ability of the cells to generate intrinsic oscillations (828, 829). As well as activating Ih, hyperpolarizations result in deinactivation of low-threshold calcium channels, allowing their subsequent activation once the membrane potential reaches less negative potentials (828). Opening of these calcium channels leads to a low-threshold spike (LTS) and a burst of action potentials (556–558). Bursts of action potentials in thalamocortical neurons lead to a prominent burst in large numbers of cortical pyramidal neurons. Bursting of corticothalamic neurons potentiates intrinsic rhythms in thalamocortical neurons and entrains their firing through excitation of thalamic reticular neurons leading to rhythmic hyperpolarizations in thalamocortical neurons, creating increased network synchronization (25, 1225). Calcium influx through the low-threshold channels allows activation of calcium-dependent potassium conductances, restarting the cycle. Ascending influences during waking or REM sleep block this cycle by acting on PLC-coupled receptors that block a leak potassium conductance causing inactivation of the low-threshold calcium channels and bringing the membrane potential out of the range of the H-current (826, 828).
A) ROLE OF LOW-THRESHOLD CALCIUM CHANNELS IN DELTA WAVES.
Low-threshold bursts in thalamocortical neurons were abolished in mice constitutively lacking the Cav3.1 calcium-channel gene (695) or in mice with a targeted deletion of Cav3.1 in rostral-midline thalamus (32). Delta waves were abolished in these mice with knockouts in the whole brain or thalamus, whereas deletions of Cav3.1 channels in cortical neurons did not affect delta waves (32). Loss of delta waves was associated with fragmented sleep with a higher incidence of brief arousals. Similar to thalamocortical neurons, bursting in thalamic reticular neurons is regulated by calcium dynamics involving low-threshold calcium channels, endoplasmic reticulum calcium ATPases which sequester intracellular calcium, and small-conductance calcium-dependent potassium (SK) channels (266). Like Cav3.1 knockouts, mice lacking the SK2 channels responsible for slow afterhyperpolarizations in thalamic reticular nucleus neurons had disrupted sleep and a threefold reduction of low-frequency rhythms during NREM sleep (266).
B) THE NEOCORTICAL SLOW OSCILLATION (0.5–1 HZ).
This phenomenon, discovered by Steriade in anesthetized cats (1230), was subsequently observed in naturally sleeping animals (28, 1237, 1289) and in humans (27, 272). Somewhat confusingly, despite its name, the so-called slow oscillation does not necessarily imply rhythmicity. High-density EEG recordings in humans revealed that each cycle of the slow oscillation represents a traveling wave originating most frequently in prefrontal-orbitofrontal regions and propagating towards more posterior cortical areas (809). The slow oscillation occurs throughout all NREM sleep stages and serves to bind together the other EEG phenomena of NREM sleep such as spindles and delta waves (1217, 1228, 1229). Slow-wave activity (SWA; 0.5–4 Hz), a combination of EEG power in the frequency bands reflecting the slow oscillation and delta oscillations, is widely considered to be a measure of sleep need and/or intensity (3, 1292). Periods of sleep deprivation cause increases of SWA in the subsequent sleep period in both animals and in humans. SWA is highest at the beginning of the sleep period and progressively decreases during sleep. Naps during the day also reduce SWA in the subsequent night (1292). The hypothesized relationship between SWA and synaptic strength is discussed in section IIID. The slow oscillation is generated within the cortex since it is abolished in thalamic neurons following removal of the cortex (1290), and it persists following disconnection of subcortical inputs (1231) and can occur in vitro in cortical slices, following manipulation of the ionic milieu bathing the slices (269, 1110). However, in intact animals, the slow oscillation strongly influences the activity of the thalamus through corticothalamic projections and conversely the thalamus influences the cortex through thalamocortical projections (264, 535, 1229, 1231).
C) CELLULAR MECHANISM CAUSING UP AND DOWN STATES.
Intracellular recordings from cortical neurons in vivo (259, 1374) and in vitro (1110) revealed that the slow oscillation consists of prolonged depolarizations associated with extracellular gamma frequency activity (UP states) separated by prolonged hyperpolarizations (DOWN states) when most cortical neurons are silent (255, 256). These states are well-synchronized over widespread areas of cortex (1374). The UP states are due to a barrage of excitatory synaptic inputs mediated by glutamatergic AMPA/kainate and NMDA receptors and activation of a persistent sodium current and usually begin in deeper cortical layers, possibly due to an increased frequency of excitatory spontaneous synaptic potentials (211). Consistent with this idea, the frequency and amplitude of miniature excitatory postsynaptic currents in pyramidal neurons of frontal cortex was enhanced following waking and decreased following sleep (737). Fast inhibitory GABAA receptor-mediated potentials also occur during the UP state due to input from GABAergic interneurons activated by the firing of principal glutamatergic neurons. The hyperpolarizing phase is due to withdrawal of excitatory input.
B. Generation and Maintenance of NREM Sleep
An involvement of the preoptic area (PO)/BF (FIGURE 2) in the control of sleep has been inferred since the observations by Constantin von Economo of damage to this area in the brains of patients with persistent insomnia following the influenza pandemic in the early years of the 20th century (1377). Extensive lesions of the PO/BF led to long-lasting insomnia in the cat (840, 1105), whereas warming caused increases in sleep (1068). Whereas most brain neurons exhibit a wake-On, a wake/REM-on or state-indifferent firing patterns across the sleep-wake cycle, the PO/BF is unusual in that it contains a large number of neurons utilizing the neurotransmitter GABA which have sleep-on, wake-off firing patterns. Many of the sleep-active neurons in the medial and lateral preoptic area are also temperature sensitive, likely explaining the coupling of body temperature and sleep (16). In the BF, caudally projecting, possibly sleep-active, GABA neurons (449) are intermingled with cortically projecting cholinergic, GABAergic, and glutamatergic neurons (451) which increase firing in association with cortical activation (481).
1. Ventrolateral preoptic nucleus
With the use of Fos immunohistochemistry to identify neurons that had been recently active, a cluster of sleep-active neurons was identified in the ventrolateral preoptic nucleus (VLPO) (FIGURE 2) of the rat (1162). These neurons contained the inhibitory neurotransmitters GABA and galanin and projected heavily to nuclei of the ARAS, especially the histaminergic tuberomammillary nucleus (406, 1161, 1162, 1210). Single-unit recordings targeting this area confirmed that it contains sleep-active neurons (1254). Extensive neurotoxic lesions of the central cluster of the VLPO in the rat led to a large decrease in delta power and NREM sleep time and a fragmentation of the sleep-wake cycle (756), effects which persisted for at least 3 wk postlesion. Furthermore, the number of remaining Fos-immunoreactive neurons was linearly related to NREM sleep time and EEG delta power.
In vitro recordings in the rat determined that many VLPO neurons are multipolar, triangular-shaped neurons, which exhibit a LTS. All of these were inhibited by norepinephrine and acetylcholine, and the majority were also inhibited by serotonin (5-HT1A) (403). Activation of non-α7 containing nicotinic receptors enhances the release of norepinephrine onto VLPO neurons, indicating a synergistic inhibitory action of acetylcholine and norepinephrine (1091). Other VLPO neurons, possibly local interneurons, had a fusiform/bipolar shape, lacked an LTS, and were excited by serotonin and an adenosine A2a receptor agonist (403, 404). Initial experiments suggested that histamine and orexin did not affect the firing rate of VLPO neurons, but more recent experiments have revealed an indirect histaminergic inhibition due to excitation of local inhibitory interneurons (736). Retrograde tracing revealed surprisingly few cholinergic projections to VLPO but prominent projections from the histaminergic tuberomammillary nucleus, norepinephrine neurons in the LC and ventrolateral medulla, and serotonergic neurons in the dorsal median and central linear raphe nuclei (222). However, somatodendritic release of acetylcholine from nearby cholinergic neurons in the HDB and MCPO is a possibility. Interestingly, VLPO neurons also receive direct inputs from the retina (759) and indirect projections from the suprachiasmatic nucleus via the dorsomedial hypothalamus (222, 306, 1250), one pathway by which light exposure could affect sleep. In vitro studies also revealed that VLPO neurons are excited by adenosine through an indirect mechanism: A1 receptor-mediated presynaptic inhibition of inhibitory synaptic inputs (203, 888). In addition, activation of adenosine A2a receptors by infusion of an A2a agonist in the subarachnoid space underlying the VLPO area increases the activity of VLPO neurons in vivo (1128).
2. Median preoptic area
Single-unit recordings and Fos studies have defined another preoptic nucleus containing a large population of GABAergic sleep-active neurons, the median preoptic nucleus (MnPO), located just dorsal to the third ventricle (FIGURE 2) (432, 1094, 1252). Like VLPO neurons, MnPO neurons project to and inhibit wake-promoting neurons of the ARAS in the perifornical lateral hypothalamus, DRN, and LC (1251, 1321). Inactivation of the MnPO by infusion of the GABAA receptor agonist muscimol led to a prolonged waking state in rats (1251). On the other hand, high-frequency electrical stimulation or perfusion with glutamate or the GABAA receptor antagonist bicuculline enhanced NREM sleep and inhibited the activity of wake-active neurons in the perifornical hypothalamus (1251). MnPO neurons recorded in vitro were dose-dependently inhibited by norepinephrine via α2 receptor-mediated activation of inwardly rectifying potassium channels (63), possibly explaining their silence during waking. Experiments comparing the extent of Fos during spontaneous sleep, sleep deprivation, and recovery from sleep deprivation suggest that MnPO neurons are active during sleep deprivation, whereas VLPO neurons are mainly active during sleep (462, 871, 994). Thus MnPO neurons increase their activity in response to increased homeostatic sleep pressure, whereas VLPO neurons may function to consolidate and maintain sleep and regulate sleep depth (462).
3. Wake-NREM transitions and the flip-flop model
Von Economo (1378) was the first to propose the existence of an anterior hypothalamic sleep-promoting area and a posterior hypothalamic waking center. More recent anatomical tracing experiments revealed that neurons in the core of the VLPO project heavily to wake-promoting histamine neurons in the tuberomammillary nucleus (TMN) of the posterior hypothalamus and also to wake-promoting serotonin DRN neurons and norepinephrine LC neurons in the brain stem (1161). Around the same time, pharmacological and electrophysiological experiments showed that GABA and galanin inhibit TMN, DR, and LC neurons (420, 421, 726, 1003, 1141), whereas serotonin and norepinephrine inhibit most VLPO neurons (403). Similarly, histamine excites a subpopulation of inhibitory interneurons in the VLPO via H1 and H2 receptors and thereby causes an indirect inhibition of VLPO projection neurons (736). In addition, histamine neurons appear to utilize GABA as a cotransmitter (677); thus TMN neurons can potentially also inhibit VLPO neurons through release of GABA. These mutually inhibitory interactions between VLPO neurons and TMN/DRN/LC neurons were conceptualized in the form of a flip-flop switch (838, 1117) such that activation of VLPO leads to inactivity of TMN/DRN/LC neurons and promotes sleep, whereas activation of TMN/DRN/LC leads to inactivity of VLPO neurons and promotes wakefulness (FIGURE 7). A crucial aspect of this model is that the two halves of the switch, by strongly inhibiting each other, create a feedback loop that is stable in only two states such that intermediate states of sleep and wakefulness are very brief. A further component to the model was the proposal that orexins stabilize behavioral state via their strong excitatory actions on wake-promoting neurons. Analysis of orexin knockout mice revealed that they have many more transitions between wake, NREM, and REM states than do wild-type mice, supporting this model (870). Although intuitively appealing, this model has a few weaknesses. First, the model does not well represent the changes in firing of all the neuronal subpopulations involved. While sleep-active preoptic neurons have fast transitions around state-transitions (1266), the firing rate of wake-active BF neurons changes more slowly (1266) and thus more closely resembles a latch than a switch. Furthermore, recordings of histamine neurons showed that they begin firing after the onset of EEG activation during NREM→Wake transitions (1264), which would seem inconsistent with them being involved in causing the state change. Second, the mechanism responsible for turning the switch on and off is unclear since a switch remains in one state unless a third mechanism causes a transition. Possible candidates for facilitators of the wake-sleep transition are sleep homeostatic factors that slowly build up during wakefulness and are discussed in the following section.
C. NREM Sleep Homeostasis
Homeostatic control of sleep refers to the increased propensity for sleep during prolonged waking and the prolonged sleep time and depth of sleep (reflected as increased EEG slow wave activity) following a period of sleep deprivation (123, 311). Sleep homeostasis is considered to reflect the accumulation of sleep homeostatic factors during waking, particularly in the BF and cortex, in a manner related to brain energy usage (see sect. IIID). Sleep homeostatic factors inhibit the activity of ARAS neurons as well as cortical neurons and thereby facilitate the slow oscillations typical of NREM sleep.
1. Sleep factors
The search for sleep-promoting factors dates back 100 years when Ishimori (548) and Legendre and Pieron (701) reported that injection of CSF from a sleep-deprived dog into the cisterna magna of a normal animal induced sleep. Later in 1967, Pappenheimer et al. (972) performed similar experiments where CSF from the cisterna magna of goats deprived of sleep for 72 h induced sleep in cats and rats following intraventricular application. These pioneering studies suggested that endogenous humoral factors are induced and accumulate during waking and generate a homeostatic sleep response. This led to a series of investigations in search of humoral sleep-promoting substances (545), leading to the identification of several substances including 1) delta sleep inducing peptide (438); 2) uridine (517); 3) oxidized glutathione, originally designated as SPS-B (516); 4) muramyl dipeptide (N-acetylmuramyl-l-alanyl-d-isoglutamine), originally described as Factor S (971); and 5) prostaglandin D2 (1317). In the following years, a variety of additional endogenous sleep-inducing substances were identified including peptides, growth factors, and cytokines as well as neuromodulators such as adenosine and NO. Homeostatic sleep factors should fulfill the following criteria: 1) administration of the substance induces sleep; 2) the levels of the substance in the brain should increase with increasing sleep propensity; and 3) the substance should act on brain regions and neurons involved in the regulation of sleep or wakefulness. Recent studies have focused extensively on the role of adenosine, nitric oxide, prostaglandin D2, and cytokines in sleep regulation and the following sections will review the latest research on these factors.
The neuromodulator adenosine links energy metabolism, neuronal activity, and sleep (79, 91, 319, 446). The hypnogenic effects of adenosine were first described in cats by Feldberg and Sherwood (359) and later in dogs by Haulica et al. (483). Systemic and central administrations of adenosine or adenosine A1 receptor agonists induced sleepiness and impaired vigilance (91, 226, 320, 1022, 1037, 1038, 1373) by inhibition of wake-active neurons. Adenosine A2A receptors are also implicated in mediating the somnogenic effects of adenosine by excitation of sleep active neurons (485, 531, 1123). Stimulants such as caffeine and theophylline counteract the sleep-inducing effects of adenosine by serving as antagonists at both A1 and A2A adenosine receptors (384, 1192).
Adenosine levels correlate with time spent awake. Endogenous, extracellular adenosine levels in the BF (102, 591, 843, 1017, 1018) and cortex (591, 1017) increase in proportion with time spent awake (FIGURE 8). Thus adenosine induces sleep and adenosine levels track sleep need, fulfilling the criteria for adenosine being a homeostatic sleep factor. Measurements of extracellular adenosine levels across the sleep-wake cycle and in response to sleep deprivation revealed that adenosine levels rise only in select regions of the brain (1017, 1247). In particular, adenosine levels correlate with time awake in the region of the caudal BF containing cortically projecting wake-active neurons, and in the cortex itself. In contrast, adenosine levels did not follow this pattern in other brain areas such as the preoptic area of the hypothalamus, ventral thalamus, DRN, or pedunculopontine tegmentum. BF adenosine levels also rise when rats are exposed to a sleep fragmentation protocol (844), possibly explaining excessive daytime sleepiness in sleep disorders where sleep is fragmented (see sect. VII).
I) Mechanisms underlying adenosine increases during wakefulness. Increased levels of extracellular adenosine during prolonged wakefulness are caused by interactions between neuronal and glial mechanisms. Glutamatergic stimulation of the BF elevates extracellular adenosine and increases sleep (1409). Selective activation of glutamatergic NMDA receptors on hippocampal pyramidal (790) or on brain stem cholinergic neurons (134) also leads to slow adenosine release and inhibition of neuronal activity. In the BF, cell-specific lesion of cholinergic neurons attenuates the sleep deprivation-induced increase of adenosine (102, 592), suggesting either that increases in extracellular adenosine are derived from these neurons or that they release an essential signal for extracellular adenosine accumulation. Such a signal may be NO (see next section). There is also strong recent evidence in support of an astrocytic origin of adenosine via so-called “gliotransmission.” Neurotransmitter release, especially glutamate release, triggers a rise in astrocytic calcium which triggers gliotransmision of ATP along with other neurotransmitters such as glutamate and d-serine (360, 979). In turn, breakdown of the extracellular ATP released by glia yields adenosine, which depresses neuronal activity (981, 1474). Blockade of vesicular release via transgenic expression of a dominant-negative SNARE domain specifically in astrocytes (dn-SNARE mice) blocked the accumulation of homeostatic sleep pressure following sleep deprivation as reflected by slow-wave activity and prevented the sleep-suppressing effects of an adenosine A1 receptor antagonist (360, 472), suggesting that blocking gliotransmission affected sleep by reducing the accumulation of extracellular adenosine.
II) Adenosine mediates its sleep-promoting effects through activation of both A1 and A2A receptors. Electrophysiological, behavioral, and molecular evidence suggest that in wake-active areas, the effects of adenosine are primarily mediated via A1 receptors. In vitro studies have demonstrated inhibitory postsynaptic effects mediated by activation of inwardly rectifying potassium channels on brain stem (48, 1041) and BF cholinergic neurons (45), orexin neurons (738), and hippocampal/neocortical pyramidal neurons (417, 834). A weaker postsynaptic inhibitory effect mediated via an A1 receptor-mediated shift of the activation threshold of the hyperpolarization-activated current Ih is observed in thalamic relay neurons (826) and BF noncholinergic neurons (45). Adenosine further dampens neuronal activity and promotes sleep via presynaptic inhibitory effects on excitatory glutamatergic inputs to cortical glutamatergic neurons (446) and wake-active cholinergic (48, 134, 897, 1332) and orexin (1444) neurons, as well as on inhibitory GABAergic inputs to sleep-active VLPO neurons (203, 888). Infusion of A1 receptor agonists in the BF, laterodorsal tegmentum, lateral hypothalamus, and prefrontal cortex increases sleep, whereas infusion of A1 receptor antagonists in the same areas increases waking (14, 1022, 1039, 1247, 1277, 1332). Although adenosine A1 receptors have effects in multiple regions of the brain controlling sleep-wakefulness, as mentioned above, to date adenosine levels have only been shown to increase with prolonged wakefulness in the BF and neocortex. Consistent with the BF being a crucial site mediating adenosine effects, local perfusion of an A1 receptor antagonist in this region activated wake-active neurons (17, 1276), and localized suppression of A1 receptor expression using antisense oligonucleotides significantly reduced spontaneous sleep time as well as the homeostatic sleep response (1282). In contrast, adenosine A1 receptor blockade in the lateral hypothalamus did not block the homeostatic sleep response (1277). While sleep homeostasis was intact in constitutive A1 receptor knockout mice (1214), conditional deletion of A1 receptor in forebrain and brain stem after 6–8 wk of age, circumventing developmental compensation, not only resulted in a decreased homeostatic sleep response after sleep restriction but also led to a failure in working memory consolidation (sect. V) (99).
Prolonged sleep deprivation upregulates A1 receptor mRNA and protein in BF and cortex in both rats and humans (74, 76, 340, 341). Upregulation of adenosine receptors provides an additional level of homeostatic control beyond rises in extracellular adenosine levels. The intracellular signaling pathway leading to this positive feedback regulation was revealed in experiments in rat BF (79) (FIGURE 8). The A1 adenosine receptor, coupled to the inhibitory Gi-3 G protein, is capable of “dual signaling,” i.e., inhibition of adenylyl cyclase and stimulation of PLC (422). Increased stimulation of the A1 receptor during sleep deprivation activates the PLC pathway, mobilizing intracellular calcium which in turn activates the transcription factor NF-kB and upregulates A1 receptor expression (73, 74).
What is the involvement of the adenosine A2A receptor in the homeostatic sleep response of adenosine? A2A receptors are coupled to the stimulatory Gs subunit and activate adenylyl cyclase. In contrast to the A1 receptor with its wide distribution in brain, the distribution of A2A receptor is more restricted to basal ganglia structures such as striatum, nucleus accumbens, and olfactory tubercle with much lower abundance in other areas such as the hippocampus, neocortex, BF, and other sleep-wake regulatory structures (1076). In rats, selective A2A receptor agonists such as CGS21680 administered to the subarachnoid space adjacent to BF and ventrolateral preoptic area (VLPO) induce NREM sleep (485, 1123, 1124, 1320). The local application of the A2A receptor selective agonist CGS21680 increases Fos expression in the VLPO (1128). Consistent with this are the observations that adenosine excites one subpopulation of sleep-promoting VLPO neurons via A2A receptors (404), and administration of CGS21680 into the lateral preoptic area close to the VLPO induces sleep (850). In A2A receptor knockout mice, the homeostatic sleep response following sleep deprivation (1320) and the wake-promoting effects of caffeine were blocked (529), although given the strong expression of this receptor in the basal ganglia, effects on motivation or motor behavior may confound these results. Accordingly, the locomotor stimulatory effects of caffeine mediated via A2A receptor blockade were shown to require the presence of A2A receptors in the nucleus accumbens (694).
The importance of adenosine as a sleep factor is supported by studies of enzymes involved in adenosine metabolism. Adenosine deaminase is involved in clearance of adenosine from the extracellular space. Blocking its activity using coformycin increased extracellular adenosine and sleep (949). The enzyme adenosine kinase phosphorylates adenosine to adenosine monophosphate, and blocking adenosine kinase activity with ABT-702 increased sleep in rat (1036). These data from rats are consistent with findings in mice that a genomic region encoding adenosine deaminase influences the rate at which NREM sleep need accumulates during wakefulness (sect. VI) (378). In humans, a genetic variation of the adenosine deaminase gene resulting in an amino acid substitution (asparagine for aspartic acid) results in decreased enzyme activity and is associated with increased sleep time and sleep intensity (687, 1059).
NO is a small gaseous molecule with multiple roles in the control of sleep and wakefulness (407) (see also sects. II and IV). In the brain, it is predominantly synthesized under basal conditions by neuronal NO synthase (nNOS) and endothelial NO synthase (eNOS) in blood vessels. nNOS is highly expressed in brain stem cholinergic neurons and participates in their control of cortical activation and REM sleep. In this section we focus on the role of NO in the homeostatic regulation of NREM sleep.
I) NO promotes NREM sleep. The enzymatic activity of NOS is highest in the rat brain when animals are awake (57), and NO itself is detected in higher quantities in the cortex during waking (167). Systemic or intracerebroventricular (icv) administration of NOS inhibitors at light onset reduced NREM sleep within the first few hours in rats (199, 324, 325, 599, 882, 883, 1062) and in rabbits (601). Thus, although some studies (1347) provided contradictory evidence, most evidence suggests that NO promotes NREM sleep. Recent studies suggest that NO produced by the inducible isoform of NOS (iNOS), is responsible for this effect. Microdialysis infusion of an iNOS selective inhibitor, during sleep deprivation prevented NREM sleep rebound (593), while an inhibitor of nNOS decreased REM recovery but did not affect NREM recovery (593). Consistent with these findings, iNOS knockout mice had less NREM sleep during the dark period (218). The induction of sleep deprivation-induced iNOS occurs in neurons within the BF (590). The iNOS expressing neurons also express Fos during sleep deprivation, suggesting they are wake active and the number of iNOS/Fos double-labeled neurons positively correlates with the sleep pressure following prolonged sleep deprivation (590). Furthermore, NO itself also rises during the early stages of sleep deprivation as assessed by the dye 4,5-diaminofluorescein diacetate (DAF-2/DA), which fluoresces upon binding NO. Currently, the cellular signaling pathway by which sleep deprivation leads to the induction of iNOS is unknown.
II) How does NO produced by iNOS cause sleep in the BF? NO has multiple cellular effectors, but one which may be particularly important in the context of the homeostatic sleep drive is release of adenosine (FIGURE 8). In vitro studies in culture and brain slices have shown that NO donors cause release of adenosine, which in turn inhibits neuronal activity (149, 352, 1075). Multiple pathways for NO-stimulated adenosine release may exist including inhibition of adenosine kinase (1075), inhibition of glycolysis and the mitochondrial electron-transport chain with a subsequent decrease in ATP/ADP ratio (150), and stimulation of neurotransmitter release leading to corelease of ATP which is subsequently degraded to adenosine (142). In vivo, NO production and adenosine increased concurrently in the BF during sleep deprivation (589, 593). Furthermore, under nondeprived conditions, microdialysis perfusion of an NO donor, DETA/NO, mimicked the ability of sleep deprivation to increase adenosine and NREM sleep (589). Recent in vitro electrophysiological evidence suggests that NO causes an initial excitation of cholinergic and cortically projecting GABAergic BF neurons which is followed by a long-lasting inhibition that can be reversed with an adenosine A1 receptor antagonist, suggesting mediation by adenosine (154). Similarly, in vivo, the effects of NO donors on BF neurons were found to be dose-dependent, with lower doses favoring excitation and higher doses leading to more inhibition (662, 663). Similar mechanisms may also be active in the perifornical lateral hypothalamus (661).
III) Sleep-active cortical interneurons contain nNOS. The presence of nNOS has been recently described in a small subset of cortical interneurons which are sleep active as determined by Fos immunohistochemistry (416, 620). In rats, mice, or hamsters, a majority of cortical GABAergic interneurons that express nNOS also express Fos during the recovery sleep that follows sleep deprivation. Fos expression in these sleep-active, nNOS-immunoreactive neurons parallels changes in the intensity of slow-wave activity in the EEG, and thus these neurons are suggested to be part of the neurobiological substrate that underlies homeostatic sleep regulation (416, 620, 984).
C) PROSTAGLANDIN D2.
Prostaglandins are lipid signaling molecules produced from arachidonic acid through the cyclooxygenase pathway. The most abundant prostanoid in the brain, prostaglandin D2 (PGD2), meets all the criteria of a potent sleep-factor (485, 531, 943). Infusion of PGD2 into the third ventricle or preoptic area of rats (546, 1316, 1317) or the third ventricle of monkeys (953) increased sleep in a dose-dependent manner. Further investigations in rats showed that the levels of PGD2 in CSF increase with increasing time awake and propensity to sleep (963, 1043). The effects of PDG2 are mediated via the prostaglandin receptor 1 (DP1 R) as shown by the absence of sleep-inducing effects of PGD2 infusions in DP1R knockout mice (867). Blocking the PGD2 receptor with a selective antagonist, ONO-412, reduces sleep time (485). Lipocalin-type prostaglandin synthase (L-PGDS) is expressed in the brain and has been associated with sleep-wake regulation (1031). Animals that overexpress human L-PGDS show a significant increase in NREM sleep that is positively correlated with the increases in PGD2 produced in the brain (1004). Inhibitors of L-PGDS, such as selenium-based compounds, inhibit sleep, an effect that is reversed by subsequent administration of PGD2 (812). In L-PGDS knockout mice, the PDG2 levels do not increase following sleep deprivation (485).
The somnogenic effects of PGD2 are predominantly mediated via the membranes surrounding the brain in the leptomeninges/arachnoid space (485). The expression of both L-PGDS and DP1R is mainly observed in the rostroventral area of the subarachnoid space near the BF which is the most potent site for the sleep promoting effects of PGD2; in this area, L-PGDS and DP1R expression is colocalized in arachnoid trabecular cells (95, 867) along with the synthesizing and degradation enzymes for adenosine (949). PGD2 infusion induces Fos expression in the sleep active neurons of the VLPO area (1126). Furthermore, the sleep-promoting effects of PGD2 have been shown to be mediated by A2A receptors in the VLPO. Subarachnoid administration of an A2A agonist induces Fos expression in the VLPO and increases NREM sleep (1128). The activation of DP1R by PGD2 in the meninges is followed by an increase in adenosine that acts on the A2A receptor in the sleep promoting preoptic area (867). Blocking the A2A receptor with the selective antagonist KF17837 also blocks the sleep-inducing effects of PGD2. The role of adenosine in the leptomeninges has been demonstrated by infusion of A2A selective agonist CGS 21680, leading to increased Fos expression in VLPO and increased sleep (518, 1128). Like adenosine, PGD2 is implicated in the homeostatic sleep response as animals that lack L-PDGS or PGD2 receptors fail to exhibit a sleep rebound in response to sleep deprivation (484, 485) and infusion of PGD2 mimics the effects of prolonged wakefulness in promoting sleep (1125).
Clinical data suggest that the PGD2-A2A sleep inducing system may be particularly important in mediating enhanced sleep in pathological states. For instance, synthesis of PGD2 was massively upregulated in the CSF of patients with African sleeping sickness (991). L-PGDS was also upregulated in sleep apnea patients exhibiting excessive daytime sleepiness (EDS) compared with controls or patients without EDS (70).
Cytokines are best known for their role in the immune system response to infection which includes enhanced sleep (543). Several cytokines and their receptors are present in the brain, and even in the absence of immune challenge, they are involved in sleep regulation (543, 669). Among the different cytokines, the most convincing evidence for a sleep-regulatory role is available for interleukin-1 (IL-1) and tumor necrosis factor-α (TNF-α). Administration of either of these cytokines increases NREM sleep in mice, rats, rabbits, cats, and sheep (603, 669, 670, 943). In humans, IL-1 administration results in fatigue and sleepiness (603, 669, 670, 943). Consistent with their somnogenic role, the endogenous brain and plasma levels of IL-1 and TNF-α increase with increased propensity to sleep. For example, in rat, the mRNA and protein expression of IL-1 and TNF-α in brain shows diurnal variation with changes paralleling the amount of NREM sleep (135, 368, 1262). In cat cerebrospinal fluid, the changes in IL-1-like activity correspond to sleep-wake behavior (762). In humans, plasma concentrations of IL-1 peak at sleep onset (874). The mRNA levels of these cytokines increase during sleep deprivation (1262).
The somnogenic effects of IL-1 and TNF-α are mediated through the IL-1 type 1 receptor and the TNF 55-kDa receptor. Mice lacking these receptors showed reduced sleep and failed to respond to IL-1 and TNF-α (353, 354). Antagonists against IL-1 and TNF-α receptors reduce sleep (954, 1267, 1368). Reduction of sleep in IL-1 receptor knockout mice was predominantly observed during the dark period, whereas TNF receptor knockout mice showed decreased sleep during the transition from the dark to the light period, suggesting involvement in the circadian component of sleep regulation. Accordingly, TNF-α has been shown to inhibit the expression of clock genes by interfering with the interaction of CLOCK-BMAL1 with the E-box regulatory element (198).
I) Mechanisms underlying cytokine-induced sleep. It has been suggested that the release of extracellular ATP associated with neurotransmitter release during waking promotes astrocytic production of IL-1 and TNF-α via activation of P2 purinergic receptors (97, 1253). In addition to effects on cortical synapses (see next section), cytokines may act to induce sleep by affecting monoaminergic neurons that express the IL-1 receptor. Administration of IL-1 into the DRN or LC (293) induces sleep (409, 788), and blocking the 5-HT2 receptor attenuates the sleep-inducing effects of IL-1 (542).
2. Local regulation of sleep homeostasis
Sleep is normally a global, coordinated phenomenon affecting the whole organism and nervous system. However, recent evidence suggests that slow waves and spindles can be induced or modulated locally in cortical areas (532, 671, 921, 1383). The most striking example of local sleep is the unihemispheric sleep exhibited by cetaceans (766). In rodents, multiunit recordings showed that groups of cortical neurons display coordinated off-states, associated with local slow waves as the duration of wakefulness increases, even while the scalp EEG shows a LVFA pattern typical of wakefulness (1383). Dissociation of different sleep/wake phenomena is also a feature of several sleep disorders (778). A number of cortically produced neuromodulators could be involved in local modulation of sleep intensity. These include 1) adenosine and NO (discussed above); 2) TNF-α, which is a regulator of sleep and sleep intensity and also regulates synaptic homeostasis (1213); 3) brain-derived neurotrophic factor (BDNF), which is involved in the establishment of neuronal circuitry during development and promotes synaptic plasticity in the adult. Cortical infusion of BDNF locally enhances NREM slow-wave activity, whereas infusion of a BDNF antibody or an inhibitor of trkB receptors causes the reverse effect (355). 4) Cortistatin is a recently discovered peptide neuromodulator structurally related to somatostatin which is expressed in cortical and hippocampal interneurons and hyperpolarizes hippocampal pyramidal neurons (289). Intraventricular administration of cortistatin increased cortical slow waves (127, 289). Furthermore, the cortistatin transcript was upregulated following sleep deprivation (127, 232). 5) Growth hormone releasing hormone (GHRH): local administration of a GHRH antagonist or siRNA targeting GHRH to the somatosensory cortex increased EEG delta power during NREM sleep but not during waking (718). Use-dependent alterations in the release of these neuromodulators may account for local cortical changes in synaptic weights and/or cortical region-dependent alterations in NREM delta activity (532, 609, 865, 1381).
D. Functional Aspects of NREM Sleep
1. Brain energy metabolism
The brain constitutes only 2% of the body mass. However, brain oxygen and glucose utilization account for ∼20% of those of the whole organism (775), consistent with the high energetic costs incurred by neural tissue, both whilst processing information and at rest (54). Compared with wakefulness, sleep reduces brain energy demands, as suggested by the 44% reduction in the cerebral metabolic rate (CMR) of glucose (791) and a 25% reduction in the CMR of O2 (774) during sleep.
A) LOCAL CHANGES IN ATP USAGE DURING THE SLEEP-WAKE CYCLE.
Under normal physiological conditions, use-dependent variations in the CMR of glucose and O2 maintain a balance in brain energetics at all times to maintain a stable level of the high-energy molecule ATP. At the molecular level, this is achieved due to tightly coupled ATP biosynthesis/usage and an efficient buffering of ATP energy via paired creatine kinase reactions to conserve ATP energy in the form of phosphocreatine and its release, as needed, in the cell (1477). However, recent studies indicate that transient changes in brain ATP levels can occur in a brain region specific- and treatment-dependent manner. For example, altered ATP levels have been observed in response to localized electrical stimulation, glucose deprivation, or manipulations of Na+-K+-ATPase activity (56, 225, 764, 773). In a recent rat study, ATP tissue levels showed a surge in the initial hours of spontaneous sleep in wake-active but not in sleep-active brain regions of rat (323). The surge was dependent on sleep but not on time of day, since preventing sleep by gentle handling of rats for 3 or 6 h delayed the surge in ATP. A significant positive correlation was observed between the surge in ATP and EEG NREM delta activity (0.5–4.5 Hz) during spontaneous sleep. Inducing sleep and delta activity by adenosine infusion into the BF during the normally active dark period also increased tissue levels of ATP. Taken together, these observations suggest that a surge in ATP occurs when the neuronal activity is reduced, as occurs during sleep. The levels of the cellular energy sensor, phosphorylated AMP-activated protein kinase (P-AMPK), show reciprocal changes to ATP levels. Thus P-AMPK levels are lower during the sleep-induced ATP surge than during periods of wakefulness or sleep deprivation. Together, these results suggest that the sleep-induced surge in ATP and the decrease in P-AMPK levels set the stage for increased anabolic processes during sleep and provide insight into the molecular events leading to the restorative biosynthetic processes occurring during sleep (323) (FIGURE 9).
B) ADENOSINE AS A REGULATOR OF BRAIN ENERGY.
One of the proposed functional theories for adenosine's role in sleep-wake behavior suggests that adenosine, a byproduct of energy metabolism, may serve as a homeostatic regulator of brain energy during sleep (91, 1130). Extracellular adenosine concentrations rise with increased metabolism and neural activity (1027). Wakefulness has a greater metabolic rate than NREM sleep (774, 791), and accordingly, extracellular adenosine levels in neostriatum and hippocampus were higher during the circadian active period and lower during the circadian inactive period in rats (541). Differential pulse voltammetry using a glucose sensor in cortex reveal that extracellular glucose levels are higher during NREM sleep compared with waking, an observation consistent with the idea that energy metabolism and glucose utilization/breakdown decrease during NREM sleep compared with waking (915). Thus changes in adenosine levels during spontaneous sleep-wake and sleep deprivation conditions may be direct reflections of ATP breakdown.
C) HYPOTHALAMIC NEUROMODULATORS LINKING METABOLISM AND THE SLEEP-WAKE CYCLE.
Several hypothalamic neurotransmitter systems link energy usage and arousal (658). The role of the orexins was discussed in section II. Several recent studies have suggested that the GABAergic/MCH neurons may be involved in connecting metabolism and sleep (1353). Mice lacking MCH or the MCH 1 receptor eat less, are lean, and have a higher metabolic rate (804, 1164). Recordings from identified MCH neurons across the sleep-wake cycle revealed that they are silent during wake and fire occasionally during NREM sleep but maximally during REM sleep (482). This silence during waking is likely due to direct inhibition by norepinephrine, serotonin, and acetylcholine (1325). Intracerebroventricular injections of MCH increased REM sleep via an increase in the number of REM episodes and increased NREM sleep to a lesser extent (1353), while an MCH1 receptor antagonist decreased NREM and REM sleep (12). The mechanisms connecting the energy conservation and sleep promoting functions of this peptide remain to be established.
2. Synaptic homeostasis
Together, the slow oscillation and cortical delta waves are termed SWA. Recent evidence suggests that sleep intensity, as measured by SWA, can be modulated locally in the cortex in a use-dependent fashion (532, 671, 1298). What might be the function of such a local regulation? Learning and synaptic plasticity studies, together with the results of experiments investigating genomic and proteomic changes during sleep (see sect. V), led Cirelli and Tononi to propose the synaptic homeostasis theory of sleep (1298). This theory proposes the following:
1) Wakefulness is associated with net synaptic potentiation. Gene and protein studies showed that animals killed following prolonged periods of wakefulness have upregulated levels of genes/proteins implicated in long-term potentiation, whereas genes/proteins implicated in long-term depression were increased following periods of sleep (241, 242). A recent ex vivo slice study showed that the frequency and amplitude of miniature excitatory postsynaptic currents (EPSCs) recorded from pyramidal neurons in the prefrontal cortex were higher following a prolonged period of waking and decreased after sleep, independent of time of day (737). Although consistent with the hypothesis, it should be noted that changes in miniature EPSCs may not accurately reflect action potential-dependent changes in synaptic strength. Several other theories of sleep and memory are based on the premise that long-term potentiation is induced during sleep (53, 175, 1417), especially during the sharp-wave/ripple complexes and spindles of NREM sleep (175, 179) or in association with the theta rhythm during REM sleep (534) (sect. V). More direct evidence of synaptic potentiation was found in Drosophila (172), where synapse size and number increased with waking and decreased during sleep.
2) Synaptic potentiation is coupled with the homeostatic regulation of cortical slow-wave activity. This idea is supported by large-scale computational models of the thalamocortical system (350, 507). It is further bolstered by experiments in which application of neuromodulators previously shown to enhance LTP, locally enhanced SWA in subsequent sleep periods (see sect. IIIC above) and learning experiments showing local increases in SWA in cortical areas following learning of a particular task (532). For instance, in an experiment pairing transcranial magnetic stimulation of the motor cortex with median nerve stimulation, subjects showing a potentiation of local field potentials also had a local increase in slow-wave activity during subsequent sleep (533).
3) SWA leads to synaptic downscaling, allowing improved cognitive performance following NREM sleep. Local neocortical field potential recordings in rats (1385) and high-density EEG recordings in humans (1063) showed that the slope and amplitude of slow waves were lower at the end of the sleep phase compared with those during sleep at the beginning of the sleep phase, findings which are consistent with synaptic downscaling if, as suggested by modeling studies, slow wave slope and amplitude reflect synaptic strength. Furthermore, single and multiunit recordings showed that the firing rate of neocortical neurons was higher following a period of sustained wakefulness, whereas firing rates and synchrony decreased following a period of sustained sleep (1384). These findings are also consistent with the increased energetic cost of wakefulness discussed in the previous section.
Although the synaptic homeostasis theory is an attractive hypothesis supported by a large amount of evidence, several questions remain. 1) Many different forms of NMDA receptor-dependent and NMDA receptor-independent synaptic plasticity have been described. Which of these are involved in sleep-dependent changes? 2) Are homeostatic changes restricted to synapses between glutamatergic neurons? So far experiments have targeted cortical regions and not investigated other brain areas such as the striatum and cerebellum where GABA neurons predominate and homosynaptic long-term depression of glutamatergic synapses is the predominant form of synaptic modification associated with learning. Furthermore, how inhibitory synapses change across the sleep-wake cycle remains unclear. Experiments in zebrafish revealed circadian and homeostatic changes in synapses from orexin neurons (36), suggesting that neuromodulatory synapses also change according to the sleep-wake cycle. 3) What happens to synapses during REM sleep?
The induction of sleep is mediated by the build-up of homeostatic sleep factors such as adenosine and NO in the BF and cortex and by increased activity of median preoptic nucleus GABA neurons that inhibit the wake-promoting neurons of the ARAS. Circadian influences are mediated by retinal and indirect SCN projections to GABAergic sleep-promoting neurons in the VLPO and other regions of the preoptic area and BF. Once initiated, sleep and the silence of cortically projecting wake-active neurons is maintained by increased firing of VLPO and other preoptic/BF GABA neurons and subsequent postsynaptic inhibition of ARAS neurons via activation of GABA and galanin receptors. As ascending excitatory influences are progressively withdrawn, cortical and thalamic neurons become increasingly hyperpolarized entering into the range of membrane potentials conducive to rhythmic bursting due to activation of Ih and It currents and leading to the characteristic EEG phenomena of NREM sleep. During early NREM sleep, effective cortical connectivity (assessed by transcranial magnetic stimulation) begins to break down (808), and in deep sleep, the activity of the brain regions comprising the default network becomes decoupled, in particular the frontal cortex (523). Local cortical differences in delta power during NREM sleep reflect the extent to which that cortical area was active during the prior waking period and the duration of prior waking, possibly reflective of increased synaptic potentiation during waking with respect to sleep. Energy usage is high during waking and during sleep deprivation, due to increased neuronal firing, synaptic activity, and synaptic potentiation. This increased energy usage of waking is reflected in increased release of sleep homeostatic factors such as adenosine during waking and a surge of ATP production which occurs during the early NREM sleep that follows waking. Synaptic activity and plasticity are major components of brain energy usage (775), potentially tying together the proposed energetic and synaptic plasticity functions of NREM sleep. Under normal conditions, wakefulness transitions first into NREM sleep and then later transitions into REM sleep. The mechanisms underlying REM sleep and the cyclic oscillation of NREM and REM sleep during the night are described next.
IV. REM SLEEP
Dreaming, which occurs most frequently in REM sleep, has inspired and fascinated artists, writers, and scientists for centuries. However, REM sleep was only defined as a separate brain state relatively recently. Experiments by Aserinsky, Dement, and Kleitmann in humans (50, 299) and Jouvet in animals (580) showed that REM sleep is defined by a distinct constellation of tonic and phasic features of the EEG and EMG. The association of an activated cortical EEG reminiscent of waking, together with paralysis of antigravity muscles (FIGURE 1), led Jouvet to term this state Le sommeil paradoxal (paradoxical sleep). In young animals, where REM is the predominant sleep state (585, 1070), it is also known as active sleep (NREM sleep is called quiet sleep in this terminology). Awakening human subjects during REM sleep commonly led to reports of dreaming (300); thus REM sleep has also been called dream sleep, although some dreaming also occurs in NREM sleep (509). Since the discovery of REM sleep, research on REM sleep can be subdivided into three main areas: 1) delineation of the neurons, circuits, and neurotransmitter effector systems responsible for the individual signs of REM sleep; 2) control of when and for how long REM sleep is expressed (REM sleep master control mechanisms); and 3) investigation of the relation of REM sleep to learning and memory. In this section we focus on the first two of these areas and the relation of REM sleep to dreaming; studies of REM sleep and learning and memory are covered in section V.
The terminology used to describe the reticular formation regions involved in REM-sleep control (FIGURE 2) differs between brain atlases, between species, and between different investigators. In the cat, the REM control area was defined as a region immediately ventral to the main cluster of noradrenergic neurons in the LC and dorsal to the gigantocellular tegmental field (FTG). This region was termed subcoeruleus (SubC) which is the term used in this review. A subset of this area, containing caudally projecting neurons involved in REM muscle atonia, and with tonic firing patterns very specific to REM sleep (1093) was termed the peri-LC alpha. Slightly lateral to this area, neurons with burst firing patterns correlated with PGO waves were found in the parabrachial area (276, 824, 1092), which at its most rostral extent also includes the cholinergic PPT area. In the rat and mouse brain atlases of Paxinos and colleagues (988, 989) the REM control region is also termed SubC (dorsal or alpha parts); however, in these species the region immediately ventral to the LC is cell-poor and the REM-control neurons (particularly those involved in muscle atonia) appear to be located slightly more rostrally, ventral to the cholinergic neurons of the laterodorsal tegmental nucleus (LDT). Hence, the functionally equivalent region in the rat or mouse has been termed the sublaterodorsal nucleus (SLD). This area corresponds to the rostral part of the subcoeruleus area as defined by Paxinos and Watson in the rat (160). REM-on neurons in this area are primarily glutamatergic, as indicated by vGLUT2 in situ hybridization and Fos immunohistochemistry (248). Other nearby reticular formation areas containing glutamatergic neurons such as the nucleus pontis oralis (PnO) and nucleus pontis caudalis (PnC) play a role in particular aspects of REM sleep such as theta rhythm generation or rapid eye movements.
I) Spike discharge characteristics of neurons in regions of the brain stem involved in REM control (see also sect. IVB). Intracellular recordings from medullary and pontine reticular formation neurons in naturally sleeping cats revealed that the excitability of these neurons was greater during REM than during NREM sleep or waking, i.e., there is a tonic depolarization during REM (209, 549) (FIGURE 10). Phasic depolarizations associated with action potential firing were superimposed upon this tonic depolarization (549). In contrast, the membrane potential was more hyperpolarized during waking and NREM sleep with phasic depolarizations occurring with motor activity during waking. When recorded in vitro, SubC “reticular” neurons fire tonically at high rates with little adaptation when depolarized (160), as in other reticular areas (418, 934). Identified GABAergic neurons in this area show similar properties (156), making it difficult to distinguish GABAergic from glutamatergic neurons solely on the basis of intrinsic membrane properties. A subset of the reticular formation neurons fire stereotyped high-frequency bursts of action potentials due to the presence of low-threshold calcium channels that are deinactivated by hyperpolarization (160, 448). Cholinergic neurons also exhibit this property (whereas most aminergic brain stem neurons do not). These neurons may be involved in phasic phenomena of REM sleep such as PGO waves. Interestingly, many presumed REM-on neurons in the SubC exhibit “spikelets” (small alterations in membrane potential resembling low-pass filtered action potentials), indicating that they are likely to be electrically coupled (489) and suggesting a possible mechanism by which their firing may be synchronized.
A. REM Sleep Signs
1. Electrographic signs of REM sleep
Ascending pathways from the brain stem areas that control activation of the neocortex during waking and REM sleep were covered in section II. We focus here on REM specific aspects of hippocampal theta rhythms and PGO waves, which appear prior to the REM state and define the transitional REM period (t-REM) in between NREM and REM sleep.
A) THETA ACTIVITY.
Theta rhythmic activity is a prominent feature of the EEG during REM sleep in rodents (FIGURE 1) and other lower mammals (1069). Early studies in rats and rabbits established that there are two forms of theta activity. Type I (4–7 Hz) was observed when the animals were under urethane or ether anesthesia and during behavioral immobility and was abolished by systemic administration of the muscarinic antagonist atropine sulfate (667). A higher frequency form of theta (type II theta, 7–12 Hz) was observed during waking associated with movement and was abolished by urethane anesthesia. Theta occurring during REM sleep appears to represent a combination of type I and type II, since atropine sulfate abolished continuous lower-frequency theta during tonic REM periods whilst leaving intact intermittent, higher frequency hippocampal theta during periods with muscle twitches (1069, 1166). Similarly, mice with a knockout of phospholipase β1 (an effector for M1-type muscarinic receptors) lacked type I theta activity and had only intermittent theta activity during REM sleep (1166). A recent genetic study suggests that theta generation during REM is different from theta during waking. A deficiency in short-chain fatty acid β-oxidation (affecting the enzyme short-chain acylcoenzyme A dehydrogenase, Acads) caused a slowing (by ∼1 Hz) of peak theta frequency during REM sleep in mice but did not affect waking theta (1260).
I) Theta rhythm during REM sleep in humans. Low-frequency (4–7 Hz) theta activity has also been observed in the human hippocampus (of epileptic patients) during sleep but, in contrast to rodents, theta rhythm was not observed continuously but rather was limited to short (1 s) epochs (188). The occurrence of these short theta epochs in humans was not correlated with the occurrence of rapid eye movements. A further contrast to rodents was the finding that theta activity was not observed in the basal temporal lobe or frontal cortex during REM (188). Another study has demonstrated a type of rhythmic slow activity in the hippocampus during REM sleep with a slightly lower peak frequency (1.5–3 Hz; delta), leaving open the possibility that these EEG signals in humans are equivalent to the type I and type II theta but that the peak frequencies for the two types are shifted to lower values (113).
II) Brain stem generation of theta rhythm during REM sleep. The ascending pathways controlling forebrain theta rhythm are discussed in section IIA. One recent study (758) suggested that a region just dorsal to the LC termed precoeruleus is necessary for theta activity during REM sleep. This region was found to provide the major glutamatergic input to the MS/vDB and contained cells that were active during REM sleep (contained Fos immunoreactivity). Ibotenic acid lesions of this area abolished theta rhythm during REM sleep (758). However, this part of the study was based on a small number of animals, and no quantification of damage to surrounding regions containing cholinergic neurons (known to be important for control of theta rhythm) was reported. Therefore, the precise role of the precoeruleus in theta rhythm generation during REM sleep awaits further confirmation with more targeted lesions (i.e., lesions affecting only glutamatergic neurons).
B) PGO WAVES.
Synchronized electrical field potentials in the pons, lateral geniculate nucleus, and occipital cortex (PGO waves) occur singly at high amplitude in the period immediately preceding the onset of REM sleep (transitional REM period; 30–90 s) and in bursts of lower amplitude during REM sleep itself (98, 146, 274, 580, 584, 1228). They are considered the source of dreaming episodes and visual imagery during REM sleep (1228) since they occur simultaneously with rapid eye movements associated with gaze direction in dream imagery (300, 825) and are prominent in visual thalamocortical circuits (144, 145, 901). PGO waves have been studied most intensively in cats, where the largest potentials can be recorded in the LGN and occipital cortex. However, more recent studies in both animals and humans describe a widespread activation of limbic, parahippocampal and many thalamocortical systems during these phasic REM events (26, 274, 1395). The pontine component of PGO waves has been