REVIEW OF SLEEP NEUROBIOLOGY FROM A CLINICAL PERSPECTIVE

DOI: 10.5665/SLEEP.1112

Sleep Neurobiology from a Clinical Perspective

Rodrigo A. España, PhD1; Thomas E. Scammell, MD2
Department of Physiology and Pharmacology, Wake Forest University Health Sciences, Winston Salem, NC; 2Department of Neurology, Beth Israel
1

Deaconess Medical Center, Boston, MA

Many neurochemical systems interact to generate wakefulness and sleep. Wakefulness is promoted by neurons in the pons, midbrain, and pos-
terior hypothalamus that produce acetylcholine, norepinephrine, dopamine, serotonin, histamine, and orexin/hypocretin. Most of these ascending
arousal systems diffusely activate the cortex and other forebrain targets. NREM sleep is mainly driven by neurons in the preoptic area that inhibit
the ascending arousal systems, while REM sleep is regulated primarily by neurons in the pons, with additional influence arising in the hypothala-
mus. Mutual inhibition between these wake- and sleep-regulating regions likely helps generate full wakefulness and sleep with rapid transitions
between states. This up-to-date review of these systems should allow clinicians and researchers to better understand the effects of drugs, lesions,
and neurologic disease on sleep and wakefulness.
Keywords: Waking, arousal, locus coeruleus, tuberomammillary nucleus, dorsal raphe nucleus, thalamus, ventrolateral preoptic area
Citation: España RA; Scammell TE. Sleep neurobiology from a clinical perspective. SLEEP 2011;34(7):845-858.

INTRODUCTION of promoting arousal.3 Soon after the discovery of REM sleep

Sleep medicine physicians often encounter questions that re-
quire an understanding of the neurobiology of sleep: How do
certain brain injuries produce coma or hypersomnolence? Why
do antidepressants often reduce REM sleep? Why do people with
narcolepsy have trouble staying awake? How do amphetamines
improve alertness and wakefulness? To help with these and simi-
lar questions, this paper provides an overview of the basic circuits
that control sleep and wakefulness. This paper has evolved from
one we wrote several years ago1 and has been updated to include
many of the latest discoveries on the circuits and neurochemistry
of sleep, more information on drugs that are used in clinical prac-
tice, and some thoughts on medications that are now in clinical tri-
als. We hope this will provide the reader with useful perspectives
on sleep disorders, how drugs influence sleep and wakefulness,
and how injuries in different brain regions may affect sleep.
Almost 100 years ago, clinicians and pioneer neuroscientists
began to identify the general brain regions that regulate sleep
and wakefulness. After an epidemic of encephalitis lethargica
around 1915-1920, Baron Constantin von Economo found that
patients with encephalitis of the posterior hypothalamus and
rostral midbrain often had crushing sleepiness, whereas those
with injury to the preoptic area usually had severe insomnia.2
He thus hypothesized that the preoptic area and adjacent an-
terior hypothalamus contained neurons that promoted sleep,
whereas neurons in the posterior hypothalamus and rostral
midbrain promoted wakefulness. In the 1940s, Moruzzi and
Magoun found that stimulation of the rostral reticular forma-
tion caused the EEG of an anesthetized animal to switch from
slow waves to the low-voltage desynchronized pattern typical
of wakefulness, suggesting that this general region is capable
Submitted for publication February, 2011
Submitted in final revised form March, 2011
Accepted for publication March, 2011
Address correspondence to: T. E. Scammell: Department of Neurology,
Beth Israel Deaconess Medical Center, 330 Brookline Ave., Boston, MA
02215; Tel: (617)-735-3260; Fax: (617)-735-3252; E-mail: tscammel@
bidmc.harvard.edu
SLEEP, Vol. 34, No. 7, 2011
in the mid-1950s, Jouvet and others established that this state
is driven by circuitry in the pons.4-6 Over the last few decades,
the latest generations of researchers and clinicians have built on
these ideas and identified many distinct systems, each of which
contributes to specific aspects of sleep-wake behavior.
The Reticular Formation
The reticular formation is a heterogeneous region that runs
through the core of the brainstem from the medulla up to the
midbrain and into the posterior hypothalamus. Soon after Moru-
zzi and Magoun showed that the rostral reticular formation can
activate the cortex, experimental lesions in animals and clini-
cal observations in patients with strokes or tumors confirmed
that the rostral reticular formation is necessary for generating
wakefulness, as these injuries often produce hypersomnolence
or coma.7,8 Thus, many researchers hypothesized that the re-
ticular formation received inputs from a number of sensory
systems and promoted wakefulness via excitatory projections
to the thalamus, hypothalamus, and basal forebrain. More re-
cently, researchers have reconsidered the idea of a monolithic
reticular formation and instead attribute its functions to the
activity of specific systems that promote arousal using ace-
tylcholine, glutamate, or monoamine neurotransmitters (e.g.,
norepinephrine, histamine, serotonin, and dopamine). These
neurotransmitters are generally considered to produce arousal
through widespread, often excitatory effects on target neurons.
In addition, they can act as neuromodulators to enhance other
excitatory or inhibitory inputs to these cells. This modulation
can thus amplify neuronal signals over much of the brain to
recruit the many systems necessary for waking behaviors. Thus,
the term reticular formation is helpful anatomically, but more
insight can be gained from understanding the specific cells and
pathways contained within this general region.
Wake-Promoting Neurochemical Systems
Acetylcholine (ACh)
The basal forebrain (BF) and brainstem contain large groups
of cholinergic neurons that promote wakefulness and REM
845 Sleep Neurobiology for the Clinician—España and Scammell
 cholinergic neurons in the LDT/PPT are mainly active during
wakefulness and REM sleep, and promote cortical activation by
releasing ACh into the thalamus.17,18
Pharmacological studies that manipulate ACh neurotrans-
mission offer further evidence for its importance in the control
of sleep and wakefulness. ACh, nicotine, and muscarinic recep-
tor agonists such as pilocarpine produce desynchronized corti-
BF Thalamus cal activity and increases wakefulness.19,20 Similar effects occur
(ACh/GABA)
with physostigmine which blocks enzymatic degradation of
LDT ACh. In contrast, agents that reduce ACh signaling, including
PPT
(ACh) the muscarinic antagonists scopolamine and atropine, produce
Raphe immobility and EEG slow waves.21,22
TMN (5-HT) LC
(HA) (NE)
SN/VTA
vPAG Norepinephrine (NE)
(DA) NE is produced by several brainstem nuclei and may help
generate arousal during conditions that require high attention
or activation of the sympathetic nervous system. The major
Figure 1—A variety of neurochemical systems promote arousal via source of NE to the forebrain is the locus coeruleus (LC), an
projections to the forebrain. Cortical and subcortical regions are excited elongated nucleus just beneath the floor of the fourth ventricle.
by monoaminergic neurotransmitters including norepinephrine (NE) from LC neurons fire most rapidly during wakefulness, are much less
the locus coeruleus (LC), serotonin (5-HT) from the dorsal and median active during NREM sleep, and are nearly silent during REM
raphe nuclei, histamine (HA) from the tuberomammillary nucleus (TMN); sleep.23,24 Extracellular levels of NE are linearly related to LC
and dopamine (DA) from the substantia nigra, ventral tegmental area,
neuronal activity, with the highest rates of release observed dur-
and ventral periaqueductal gray (SN/VTA/vPAG). Neurons of the basal
forebrain (BF) promote cortical activation using acetylcholine (ACh) ing wakefulness.25 NE is also made by neurons in the ventral
and γ-aminobutyric acid (GABA). Neurons in the laterodorsal and medulla that mediate autonomic responses, and though much
pedunculopontine tegmental nuclei (LDT/PPT) release ACh to excite less studied, these cells may also promote arousal.26
neurons in the thalamus, hypothalamus, and brainstem. Pharmacological studies provide some of the strongest evi-
dence that NE regulates wakefulness and sleep. For example,
infusion of NE or the noradrenergic agonists isoproterenol and
phenylephrine into the lateral ventricle or BF increases behav-
Table 1—Activity profiles of neurotransmitter systems across sleep/
wakefulness
ioral and EEG indices of wakefulness.27 LC neurons are nor-
mally inhibited by NE via α2 receptors, and blockade of this
Neurotransmitter Wakefulness NREM sleep REM sleep negative feedback with yohimbine increases LC activity and
Acetylcholine ↑↑ — ↑↑ also increases wakefulness.28,29 Conversely, bilateral inactiva-
Monoamines ↑↑ ↑ — tion of the LC with the α2-agonist clonidine, or co-administra-
Orexin/Hypocretin ↑↑ — — tion of both α1 and β noradrenergic antagonists (prazosin and
MCH — — ↑↑ timolol) increases NREM sleep.30,31
VLPO/MNPO — ↑↑ ↑↑ The NE system may be especially important in promoting
arousal under conditions that require responding to a behav-
Neuronal activity: ↑↑, rapid firing rate; ↑, slower firing rate; —, little or iorally important stimulus, a cognitive challenge, or stress. In
no firing. broad terms, an animal may be drowsy and inattentive if LC
activity is too low, distractible and anxious if LC activity is
too high, but optimally attentive and aroused with intermedi-
sleep and also participate in learning, memory, and cognition. ate levels of activity. NE tone is clearly linked to cognition as
The BF is a region surrounding the front of the hypothalamus LC neurons in monkeys fire phasically in response to a salient
that includes the medial septum, magnocellular preoptic nucle- stimulus that signals a reward such as food, but these cells do
us, diagonal band of Broca, and substantia innominata (Figure not respond to a distracting stimulus.32 Integrating these ideas,
1). Most BF cholinergic neurons are active during wakefulness Aston-Jones and colleagues have proposed that activity in the
and REM sleep, and they directly promote fast EEG rhythms LC may promote arousal in a way that optimizes attention and
via projections to the cortex and hippocampus (Table 1).9-12 The task performance.33 In addition, the LC and NE neurons in the
BF also contains a large population of neurons that produce the ventral medulla are active during stress.23,34 The necessity of
inhibitory neurotransmitter γ-aminobutyric acid (GABA), and this system is clear in mice lacking NE because after exposure
these likely activate the cortex by reducing activity in inhibitory to a mild stressor, they fall asleep more rapidly than control
cortical interneurons.13,14 mice.35 Similarly, rats with lesions of the LC show considerably
A second major group of cholinergic neurons is found in the less behavioral arousal and cortical activation when confronted
pons within the laterodorsal and pedunculopontine tegmental with novel stimuli.36 On the other hand, excessive NE tone with
nuclei (LDT/PPT). In contrast to the BF, LDT/PPT neurons anxiety might contribute to insomnia, and the α1 antagonist pra-
primarily project to subcortical regions including the thala- zosin can reduce the vivid nightmares and nighttime arousal of
mus, lateral hypothalamus, and BF.15,16 Like most BF neurons, PTSD.37
SLEEP, Vol. 34, No. 7, 2011 846 Sleep Neurobiology for the Clinician—España and Scammell
 Table 2—Effects of commonly used drugs on sleep and waking
Drug Type Examples Pharmacologic Effect Neurobiologic Mechanism Clinical Effects

Selective serotonin Fluoxetine Increase extracellular levels 5-HT inhibits REM sleep- Decreased REM sleep
reuptake inhibitors Fluvoxamine of 5-HT producing cells
(SSRIs) Citalopram

Tricyclic Amitriptyline Increase extracellular levels 5-HT and NE inhibit REM Decreased REM sleep
antidepressants Nortriptyline of 5-HT and NE sleep-producing cells
Clomipramine
Desipramine

Traditional, Amphetamine Increase extracellular levels Increased DA and NE Increased wakefulness

amphetamine-like Dextroamphetamine of DA and NE signaling
stimulants Methylphenidate

Wake-promoting, non- Modafinil Increase extracellular levels Increased DA signaling Increased wakefulness
traditional stimulants Armodafinil of DA

Benzodiazepines Diazepam Enhance GABA signaling via GABA inhibits the arousal Increased sleep
Clonazepam GABAA receptors systems
Lorazepam
Triazolam

Non-benzodiazepine Zolpidem Enhance GABA signaling via GABA inhibits the arousal Increased sleep
sedative hypnotics Zaleplon GABAA receptors systems
Zopiclone

Classic antihistamines Diphenhydramine Block HA H1 receptors Reduced HA signaling Increased sleep

Triprolidine

Typical antipsychotics Haloperidol Block DA receptors Reduced DA signaling Increased sleep

Chlorpromazine

Histamine (HA) daytime sleepiness observed with narcolepsy.50-54 This wake-

HA plays an essential role in promoting wakefulness, yet
little is known about which aspects of arousal it governs.38 The
tuberomammillary nucleus (TMN) is a small cluster of cells
adjacent to the mammillary body at the base of the posterior
hypothalamus. Though few in number, these cells innervate
much of the forebrain and brainstem and are the sole source of
HA in the brain. Similar to the pattern seen in the LC and other
monoaminergic nuclei, TMN firing rates and HA release are
highest during wakefulness, lower during NREM sleep and
lowest during REM sleep.39,40 Administration of HA or an H1-
receptor agonist increases cortical activation and wakefulness
while reducing NREM and REM sleep.41,42 In contrast, drugs
that reduce HA signaling, including classical antihistamines,
such as the H1 receptor antagonists diphenhydramine, pyril-
amine, and low dose doxepin increase NREM and REM sleep
(Table 2).41-46
Over the last several years, a new class of wake-promoting
drugs has been developed to target the autoinhibitory histamine
H3 receptors.47,48 The clinical rationale for these agents appears
strong, as many people with narcolepsy or idiopathic hyper-
somnia have reduced HA levels,49 and blockade of H3 recep-
tors should increase HA signaling. For example, H3 antagonists
(or reverse agonists) such as ciproxifan or tiprolisant promote
wakefulness and EEG desynchrony and improve the excessive
SLEEP, Vol. 34, No. 7, 2011
promoting effect is likely mediated by increased HA tone as
the response to an H3 antagonist is absent in mice lacking H1
receptors.55
Which aspects of arousal are mediated by the HA system
remains unclear. HA improves attention and psychomotor per-
formance,56 and it may promote motivated behaviors such as
food seeking.57 In addition, mice lacking HA have less wake-
fulness at the beginning of their active period,58 suggesting that
HA may be especially important for initiating arousal. As sleep
inertia upon awakening is common in many patients with id-
iopathic hypersomnia, it is possible that low HA signaling is a
contributing factor.
Serotonin (5-HT)
Understanding how 5-HT promotes arousal is challenging
because: there are many sources of 5-HT; 5-HT binds to at least
15 different receptors with varied effects, and 5-HT has been
shown to influence many other aspects of behavior including
mood, anxiety, aggression, and appetite. 5-HT is produced by
neurons in the dorsal raphe nucleus and other raphe nuclei scat-
tered along the midline of the brainstem, and together these
neurons innervate many brain regions that can influence sleep/
wake behavior, including the preoptic area, basal forebrain, hy-
pothalamus, and thalamus. Early studies suggested that 5-HT
847 Sleep Neurobiology for the Clinician—España and Scammell

DA DA terminal
DAT
DA receptor
VMAT
Post-synaptic
neuron
Figure 2—A prototypical dopamine synapse. Under normal conditions,
action potentials in a DA nerve terminal cause DA-filled vesicles to fuse
with the presynaptic membrane, and DA is released into the synaptic cleft
where it can bind to postsynaptic DA receptors. DA is removed from the
synaptic cleft primarily by the DA transporter (DAT). Once DA is back
inside the presynaptic terminal, it is repackaged into synaptic vesicles
for future release via the vesicular monoamine transporter (VMAT).
Amphetamines increase the synaptic concentration of DA through two
main mechanisms: Amphetamines interfere with the reuptake of DA
through the DAT, and they disrupt vesicular packaging of DA which
increases cytosolic levels of DA which can then leak out through the DAT
via reverse transport.
might help produce NREM and possibly REM sleep, but more
recent work indicates 5-HT generally promotes wakefulness
and suppresses REM sleep. The firing rates of dorsal raphe
neurons and extracellular 5-HT levels are highest during wake-
fulness, much lower during NREM sleep, and lowest during
REM sleep—a pattern very similar to that of the NE and HA
systems.59,60 In support of this wake-promoting role, agonists
of the 5-HT1A, 5-HT1B, 5-HT2, or 5-HT3 receptors increase
wakefulness.61-65 Of clinical relevance, similar effects occur
with selective serotonin reuptake inhibitors (SSRIs) such as
fluoxetine and citalopram that increase wakefulness and reduce
REM sleep in both people and rodents.66-70 In addition, drugs
that block 5-HT2 receptors such as ritanserin or agomelatine are
thought to promote NREM sleep and thus are under develop-
ment as treatments for insomnia.62,64,71-73
Dopamine (DA)
DA has been implicated in the regulation of a variety of be-
havioral and physiological processes including motor function,
motivation, reward, and learning. Additionally, DA exerts potent
wake-promoting effects that are of great clinical relevance. For
example, sleepiness is common with DA antagonists such as hal-
SLEEP, Vol. 34, No. 7, 2011
operidol or chlorpromazine or in patients with Parkinson’s dis-
ease who have a loss of DA-producing neurons.74-76 Additionally,
D2 agonists like ropinirole can produce sleepiness via activation
of autoinhibitory D2 receptors that reduce DA signaling.77,78
However, it is unclear which DA neurons actually promote
arousal. DA-producing neurons are most abundant in the sub-
stantia nigra and ventral tegmental area, yet cells in these re-
gions fire in relation to movement or reward but, in general,
have not been found to alter their rates of firing across sleep and
wakefulness.79-82 Nevertheless, extracellular levels of DA are
high during periods of wakefulness and lower during NREM
sleep, suggesting that some DA neurons must be wake-active.81
One candidate population sits in the ventral periaqueductal gray
of the pons, and lesions of these wake-active DA neurons pro-
duce moderate reductions in wakefulness.83 The conditions un-
der which these or other DA wake-promoting neurons fire are
unknown, but in general, DA may naturally promote arousal
when an individual is highly motivated or physically active.
Drugs that increase DA signaling are used frequently to im-
prove excessive daytime sleepiness. Classical stimulants such
as methylphenidate and amphetamine increase extracellular
levels of DA by disrupting the function of the DA transporter
(DAT), thereby increasing extracellular levels of DA (Figure
2).84 These drugs are usually very effective, but because they
enhance DA signaling in reward and motor pathways, they
have high abuse potential and can elicit tics or other movement
disorders. At higher doses, these stimulants can also block the
reuptake of NE and 5-HT which can result in tachycardia, ar-
rhythmias, mania, and psychosis.
Modafinil is frequently prescribed for treating the sleepiness
of narcolepsy and some other disorders. Clinically, it promotes
wakefulness effectively, usually with fewer side effects than
encountered with classical stimulants. Like amphetamines,
modafinil disrupts DAT function in humans and rodents,85,86
and this is a necessary part of its wake-promoting mechanism,
as mice lacking the DAT show no increase in wakefulness
with modafinil,87 and D1 and D2 receptor antagonists can block
modafinil-induced wakefulness.88 Still, if modafinil acts via the
DAT, it seems surprising that it has less abuse potential than
amphetamines. One possible explanation is that amphetamines
produce a dramatic efflux of DA into the synapse via reverse
transport through the DAT, and this may be very reinforcing. In
contrast, modafinil may simply block reuptake of DA through
the DAT, leading to more modest rises in DA that are not as
reinforcing. A better understanding of these mechanisms could
drive the discovery of even better wake-promoting medications.
Orexin/Hypocretin
The excitatory neuropeptides orexin-A and -B (also known
as hypocretin-1 and -2) are synthesized by neurons in the lateral
and posterior hypothalamus and play essential roles in the regu-
lation of wakefulness and sleep.89,90 The orexin neurons project
widely and heavily innervate all the arousal regions described
above, with particularly dense innervation of the LC and TMN
(Figure 3).89,91 Orexins excite target neurons through the OX1
and OX2 receptors. Like most other wake-promoting neurons,
orexin neurons fire mainly during wakefulness, especially dur-
ing active exploration, and are silent during NREM and REM
sleep.92,93 Orexin levels are highest during wakefulness,94 and
848 Sleep Neurobiology for the Clinician—España and Scammell
when injected into the brain, orexins increase arousal and be-
havioral activity while suppressing NREM and REM sleep.95-97
Consistent with this, selective optogenetic activation of orexin
neurons can trigger brief awakenings from sleep.98,99 Addition-
ally, orexin receptor antagonists such as almorexant reduce sleep
latency and increase the amounts of REM and NREM sleep.100-102
The most compelling evidence that orexins are necessary for
the regulation of wakefulness and sleep was the discovery that
narcolepsy with cataplexy is associated with a loss of orexin
signaling.103-107 Dogs with a mutation of the OX2 receptor gene
display many of the classic symptoms of narcolepsy, including
cataplexy when presented with palatable foods.104 Further, mice
lacking the orexin peptides or the orexin-producing neurons
have severe sleepiness and cataplexy.103,108-111 Most importantly,
people with narcolepsy with cataplexy have a severe (85% to
95%) loss of the orexin neurons and very low CSF levels of
orexin-A.106,107,112 Less severe loss (20% to 60%) of the orexin
neurons also occurs in other disorders that can cause sleepiness
such as Parkinson disease, multiple system atrophy, and trau-
matic brain injury.113-117
In just the last 10 years, much has been learned about the
ways in which orexins promote arousal. In general, it may be
best to think of this as a system for sustaining wakefulness as
people and mice with narcolepsy have approximately normal
amounts of wakefulness, but have great difficulty maintain-
ing long periods of wakefulness.110 Orexins may also stabilize
sleep as people with narcolepsy often have fragmented sleep,
and orexins certainly regulate REM sleep as discussed below.
In addition, orexins promote arousal responses to homeostatic
challenges and drive motivated behaviors such as seeking food.
Orexins directly excite neurons of the mesolimbic reward path-
ways, and orexin antagonists can reduce the motivation to seek
drugs of abuse.118-121 The orexin neurons are also activated by
humoral indicators of hunger such as low glucose or high levels
of ghrelin,122,123 and while normal mice have a clear increase in
arousal when deprived of food, mice lacking the orexin neurons
show little response.124 Thus, one can view the orexin system
as helping sustain wakefulness across much of the day, and in-
creasing arousal in motivating conditions.
Cortical and Thalamic Activity across Sleep and Wakefulness
All the arousal systems we have discussed thus far are lo-
cated in the BF, hypothalamus, or brainstem and exert diffuse
effects on the cortex and many other target regions. These sub-
cortical systems are essential for the generation of sleep/wake
states and for the regulation of the transitions between these
states. However, patterns of EEG activity and consciousness it-
self arise from interactions between these subcortical systems,
the thalamus, and the cortex.
Thalamic neurons relay information to and from the cortex
and have intrinsic electrical characteristics that help generate
some of the cortical rhythms seen in NREM sleep.125,126 The
thalamus contains two major types of neurons, glutamatergic
thalamocortical projection neurons that relay sensory, motor,
and limbic information to the cortex, and GABAergic neurons
in the reticular nucleus of the thalamus that are innervated by
the projection neurons and cortex and in turn inhibit the projec-
tion neurons. These reciprocal connections are thought to drive
some cortical rhythms, including sleep spindles.127 Thalamic
SLEEP, Vol. 34, No. 7, 2011
BF Orexin
(ACh/GABA)
LDT
PPT
(ACh)
Raphe
TMN (5-HT) LC
(HA) (NE)
SN/VTA
vPAG
(DA)
Figure 3—Orexin/hypocretin-producing neurons in the lateral
hypothalamus innervate all of the ascending arousal systems, as well as
the cerebral cortex.
neurons are hyperpolarized during NREM sleep, promoting a
pattern of burst firing and reducing their responsiveness to in-
coming sensory stimuli.128 During wakefulness and REM sleep,
ACh depolarizes thalamic neurons to suppress spindles and
slow waves and promote the transmission of single spikes that
efficiently transmit information to the cortex and drive desyn-
chronized cortical activity.129 During wakefulness, monoamines
bolster this effect.119 Extensive damage to the thalamus severely
impairs consciousness and the ability to interact with the envi-
ronment, but the general patterns of wakefulness, NREM, and
REM sleep persist, suggesting that the thalamus is not required
for the basic generation of sleep states.130-133
The cortex contains a wide variety of neurons, and much less
is known about their activity in relation to sleep/wake states.
The EEG reflects broad patterns of excitatory and inhibitory
post-synaptic potentials, mainly arising from the dendrites of
pyramidal neurons. During wakefulness and REM sleep, these
potentials are desynchronized, resulting in low-amplitude fast
activity, but during NREM sleep these signals are synchronized,
resulting in high-amplitude slow activity. Release of ACh and
monoamines during wakefulness generally excites cortical neu-
rons and increases their responsiveness to incoming sensory
stimuli. Delta waves likely arise from interactions amongst cor-
tical neurons and may also be influenced by the BF and other
subcortical sites. Recent work has identified a population of
widely projecting GABAergic neurons within the cortex that are
uniquely active during NREM sleep, suggesting that these cells
may broadly inhibit other cortical neurons, helping generate
slow waves during NREM sleep.134 In addition, the intensity of
cortical slow waves may reflect prior local activity and changes
in synaptic strength, as slow waves during NREM sleep are in-
creased over supplementary motor cortex after learning a motor
task but decreased with arm immobilization.135-137
The Arousal Network: Interactions among Wake-Promoting
Neurotransmitter Systems
Each of the arousal systems presented above is independent-
ly capable of promoting wakefulness, yet these systems work
849 Sleep Neurobiology for the Clinician—España and Scammell

Orexin
VLPO
LDT
PPT
Raphe
TMN LC
SN/VTA
vPAG
Figure 4—NREM sleep pathways. Ventrolateral preoptic area (VLPO)
neurons are active during NREM sleep and reduce activity in the
ascending arousal systems using GABA and galanin. A subset of VLPO
neurons is also active during REM sleep.
together to generate behavioral arousal. Anatomically, there are
many interconnections between the systems. For instance, ACh
and 5-HT fibers innervate and excite LC neurons, and nearly
all wake-promoting neurons respond to HA, NE, and orexin. In
addition, these neurotransmitters often produce similar effects
on their targets. For example, all the arousal systems excite tha-
lamic and cortical neurons. These interconnections and parallel
effects may explain why injury to any one of the arousal sys-
tems often produces little lasting effect on wakefulness. Func-
tionally, this is adaptive, as it helps ensure that wakefulness will
still occur after injury to any one of the arousal systems. In fact,
there are only a few brain regions in which lesions produce last-
ing reductions in arousal. One is the rostral reticular formation
in the midbrain and posterior hypothalamus in which lesions
from strokes or tumors can produce severe hypersomnolence
or even coma, probably from damage to many of the ascending
monoaminergic and cholinergic pathways.
Wakefulness is a complex and dynamic state, arising from
networks of neurons driven by homeostatic, affective, cogni-
tive, and motivational processes. Thus, it is likely that each
arousal system helps promote specific aspects of behavioral
arousal so that individuals can detect sensory and internal stim-
uli and generate appropriate motor and affective responses.138
For example, NE, HA, and ACh may be particularly important
for enhancing attention and responding to novel, stressful, or
salient stimuli. Similarly, through its limbic and striatal projec-
tions, DA may promote arousal especially when an individual is
motivated or physically active. The orexin peptides help sustain
wakefulness and also may help drive goal-oriented behaviors
and locomotion. So, while lesions of some arousal systems ap-
pear to have little effect on the amounts of wakefulness, deficits
in arousal may be best revealed by carefully examining the re-
sponse to specific circumstances and challenges.
SLEEP, Vol. 34, No. 7, 2011
NREM Sleep-Promoting Systems
Preoptic area
In the early 20th century, most researchers thought that sleep
was a passive consequence of inactivity in the arousal systems,
but many experiments have now shown that specific neurons
actively promote sleep. Baron von Economo first observed that
insomnia was common in patients with encephalitis injuring the
preoptic area (the rostral end of the hypothalamus, just above the
optic chiasm) and the adjacent BF.2 This observation suggested
that this region might contain neurons that promote sleep, and
subsequent research in animals identified sleep-active neurons in
the ventrolateral preoptic area (VLPO) and median preoptic area
(MNPO).139,140 Many neurons in these nuclei fire most frequently
during NREM sleep and to some degree during REM sleep but
are virtually silent during wakefulness.141-143 Interestingly, these
sleep active VLPO neurons show particularly high rates of firing
during deep NREM sleep, and MNPO neurons often begin firing
just before NREM sleep. Lesions of the preoptic area and specifi-
cally of the VLPO markedly reduce sleep, and the sleep that does
occur is light and fragmented.144,145 Collectively, these observa-
tions suggest that MNPO neurons may help initiate sleep, whereas
VLPO neurons may be necessary for the maintenance of sleep.
Anatomically, the VLPO and MNPO are well positioned to
promote sleep. The neurons in these nuclei contain the inhibi-
tory neurotransmitter GABA and the inhibitory neuropeptide
galanin,146,147 and they innervate all the arousal-promoting re-
gions, including the LDT/PPT, LC, DR, TMN, and also the
orexin neurons (Figure 4). Thus, the VLPO and MNPO are hy-
pothesized to promote sleep by coordinating the inhibition of
arousal regions during NREM and REM sleep.146,148,149
Other brain regions contain neurons active in NREM sleep, but
these populations are less well understood. For example, parts of
the BF and lateral hypothalamus contain scattered GABAergic
neurons that are active during NREM sleep.150-152 Some of these
cells may directly innervate the cortex, and it is possible that they
modulate cortical networks to promote slow wave activity.
Many of the medications now used to treat insomnia do so
by promoting GABA signaling. Benzodiazepines, (e.g., diaz-
epam), barbiturates (e.g., pentobarbital), and the newer non-
benzodiazepine agents (e.g., zolpidem) all bind to GABA-A
receptors to enhance the effects of GABA.153,154 Gamma hy-
droxybutyrate (sodium oxybate) promotes very deep sleep,
most likely by binding to GABA-B receptors.155 These drugs
may promote sleep by boosting signaling by the VLPO and
other NREM sleep-active populations, but GABA can inhibit
neurons throughout the brain, and the precise pathways through
which these drugs work remain unclear.
REM Sleep-Promoting Systems
Soon after the discovery of REM sleep in the mid-1950s,4,5
researchers learned that the pons plays an essential role in the
generation of REM sleep.6 After several decades of work, much
has been learned, but the specific pathways that generate this
state are still debated (Figure 5).
Acetylcholine
Many researchers have hypothesized that REM sleep is con-
trolled by cholinergic neurons located in the LDT/PPT. These are
850 Sleep Neurobiology for the Clinician—España and Scammell
 REM-on
REM atonia Motor
REM-off neurons

A B
thalamus thalamus

MCH

TMN
LDT LDT
PPT PPT
vlPAG LC
LC LPT DR
DR
SLD

Spinal Spinal
interneurons interneurons

Ventromedial Ventromedial
Medulla Medulla

REM-on REM-on
REM atonia Motor REM atonia Motor
REM-off neurons
REM-off neurons

Figure 5—Pathways that control REM sleep. (A) A classic perspective on REM sleep control involves interactions between the cholinergic and aminergic
systems. REM sleep-active cholinergic neurons in the LDT/PPT activate thalamo-cortical signaling and drive atonia by exciting neurons in the ventromedial
B
medulla that inhibit motor neurons. During REM sleep, monoaminergic neurons including the LC, DR, and TMN become silent, which disinhibits the LDT/PPT
of motor neurons by NE and 5-HT. (B) Recent observations have expanded on the classic view of REM sleep control. In this model,
and lessens the excitation thalamus
mutual inhibition between REM sleep-on neurons of the sublaterodorsal nucleus (SLD) and REM sleep-off neurons of the ventrolateral periaqueductal gray
and lateral pontine tegmentum (vlPAG/LPT) is thought to regulate transitions into and out of REM sleep. During REM sleep, SLD neurons activate GABA/
MCH
glycine neurons in the ventromedial medulla and spinal cord that inhibit motor neurons. At most times, the vlPAG/LPT inhibits the SLD, but during REM sleep,
the vlPAG/LPT may be inhibited by neurons making melanin concentrating hormone (MCH) and other neurotransmitters. Solid lines depict pathways active
during REM sleep, while dashed lines are pathways inactive during REM sleep.
LDT
PPT
vlPAG LC
the same nuclei that contain wake-promoting
LPT DRcells, but a subpopu- clearly important for atonia as drugs that block glycine signal-
lation of these cholinergic neurons
SLD
are active in both wakefulness ing such as strychnine can markedly increase muscle tone in
and REM sleep or are selectively active in REM sleep.9,156-158 When REM sleep and wakefulness.168,169
injected into the lateral pontine tegmentum (LPT; a heterogeneous
region extending rostrally from the PPT that is lateral to the peri- Monoamines
aqueductal gray), drugs that enhance ACh signaling such as the Monoamines such as NE and 5-HT increase muscle tone
cholinergic agonist carbachol or the acetylcholinesteraseSpinalblocker, by directly exciting motor neurons.170-173 In the genioglossus
interneurons
neostigmine, elicit intense and long-lasting REM sleep.159-161 Con- muscle, withdrawal of this excitatory tone contributes more to
versely, cholinergic antagonists reduce the duration of REM sleep atonia than the inhibitory effects of GABA and glycine.174,175
Ventromedial
bouts.162,163 Furthermore, large lesions that include the LDT/PPT Whether this applies to most muscles is unknown, but it is clear
Medulla
produce significant reductions in REM sleep,164,165 suggesting that that atonia during REM sleep is probably due to a combination
the LDT/PPT is necessary for REM sleep. of inhibition (GABA and glycine) and a loss of excitation (NE
Neurons REM-on
in the LDT/PPT may help generate the cortical ac- and 5-HT).
Motor
atonia of REM REM
REM-off
tivation and atonia
sleep. The LDT/PPT is the main
neurons Monoamines also inhibit REM sleep itself. During wakeful-
source of ACh to the thalamus, and ACh depolarizes thalamic ness, and to some degree during NREM sleep, the REM-active
neurons to promote the transmission of information through cholinergic neurons are inhibited by 5-HT, NE, and HA.176 This
the thalamus, driving the cortical activation that is probably re- interaction between cholinergic and monoaminergic populations
quired for the complex dreams of REM sleep. The LDT/PPT forms the foundation of the classic model explaining the alterna-
neurons may also activate atonia-promoting neurons in the ven- tion of NREM and REM sleep across the night (Figure 5A).177
tromedial medulla.158,166 These medullary cells release GABA These monoaminergic effects on motor tone and REM sleep
and another inhibitory neurotransmitter glycine onto spinal and may account for many phenomena commonly seen by sleep cli-
brainstem motor neurons during REM sleep, producing hy- nicians. NE and 5-HT reuptake inhibitors often increase muscle
perpolarization and inhibition.167 This descending inhibition is tone during sleep and can unmask REM sleep behavior disor-
SLEEP, Vol. 34, No. 7, 2011 851 Sleep Neurobiology for the Clinician—España and Scammell
der (RBD) and worsen periodic limb movements of sleep.178
These drugs and other antidepressants also strongly suppress
REM sleep, and thus can markedly reduce REM sleep during
overnight polysomnograms or during the MSLT.179
GABA
Over the last few years, new observations have expanded on
the classic model of REM sleep control (Figure 5B). One region
that has received significant attention is the sublaterodorsal nu-
cleus (SLD; also termed the subcoeruleus, or LCα), which is a
small cluster of cells ventral to the LC that produce GABA or
glutamate.180 Many neurons in the SLD are active during REM
sleep,158,181-183 and they project to the ventromedial medulla and
ventral horn of the spinal cord, providing pathways through
which they may inhibit motor neurons. Activation of the SLD
region elicits atonia and REM sleep-like EEG activity,158 while
inhibition of the SLD promotes wakefulness and reduces REM
sleep. Most importantly, lesions of the SLD region disrupt
REM sleep atonia and reduce REM sleep.165,180,182,184,185 Neuro-
nal loss near the SLD has been reported in some patients with
RBD,186 suggesting that injury to the SLD may contribute to the
inadequate atonia of RBD.
Another new perspective on the classic view of REM sleep is
that the SLD neurons may be strongly inhibited by REM sleep-
suppressing neurons in the mid-pons.149,182,187 These GABAergic
cells are scattered from the ventral part of the periaqueductal
gray out into the lateral pontine tegmentum (vlPAG/LPT) and
lesions of this region substantially increase REM sleep.188 The
vlPAG/LPT inhibits the SLD, and the SLD may in turn inhibit
the vlPAG/LPT, giving rise to a mutually inhibitory circuit that
may regulate transitions between NREM and REM sleep.149,182
Melanin-concentrating hormone (MCH)
Mixed in with the orexin neurons of the lateral hypothalamus
are a large number of REM sleep-active neurons that produce
both MCH and GABA.189-191 These cells innervate nearly all
the same target regions as the orexin neurons including the DR
and LC,192,193 yet in contrast to the excitatory effects of orex-
ins, both MCH and GABA are inhibitory. Electrophysiological
recordings demonstrate that MCH neurons fire at a high rate
during REM sleep, with much less firing during NREM sleep
and complete inactivity during wakefulness.152 The amount of
REM sleep is increased by infusions of MCH into the lateral
ventricles and decreased by a MCH antagonist.191,194 Consistent
with these observations, mice lacking MCH spend less time in
NREM and REM sleep.195 Thus, it seems likely that the MCH
neurons promote REM sleep by inhibiting the arousal regions.
This pattern is strikingly opposite to that of the orexin neurons
and much remains to be learned about how the activity of these
intertwined systems is organized.
Mechanisms that Regulate the Transitions between Sleep and
Wakefulness
The systems that promote wakefulness, NREM, and REM
sleep dynamically interact in a variety of ways to ensure rapid
and complete transitions between sleep/wake states.148,149 The
VLPO and other sleep-promoting preoptic neurons inhibit
monoaminergic and cholinergic wake-promoting neurons, and
the preoptic neurons themselves are inhibited by NE, 5-HT, and
SLEEP, Vol. 34, No. 7, 2011
ACh.196,197 During wakefulness, high monoaminergic and cho-
linergic tone should thoroughly silence the VLPO, thus disin-
hibiting the arousal regions and helping ensure the production
of complete wakefulness. Conversely, during sleep, preoptic
neurons become active and inhibit the arousal regions, thus
disinhibiting their own firing. This mutual inhibition should
produce stable wakefulness and sleep while facilitating rapid
transitions between sleep and wakefulness and minimizing time
in drowsy, intermediate states. Similar mutually inhibitory cir-
cuits may regulate REM sleep as REM sleep-active neurons in
the SLD inhibit and are inhibited by neurons in the vlPAG/LPT
that are inactive in REM sleep.182
The orexin neuropeptides probably reinforce these mutually
inhibitory systems. Orexins may stabilize wakefulness by en-
hancing activity in the arousal systems, ensuring full alertness
and long periods of wakefulness despite rising homeostatic
pressure across the day.94 During wakefulness and perhaps to
a lesser degree in NREM sleep, orexins may excite a variety of
neurons that inhibit REM sleep, including monoaminergic neu-
rons, the vlPAG/LPT, and GABAergic inputs to the SLD.96,198-
201
However, loss of the orexin neurons in narcolepsy with
cataplexy results in persistent sleepiness, frequent transitions
between states, and odd states such as cataplexy and hypnago-
gic hallucinations in which it seems that elements of REM sleep
mix into wakefulness. Collectively, these symptoms may be
best thought of as “behavioral state instability,” a phenomena
that is likely caused by loss of the stabilizing effects of orexins
on the mutually inhibitory circuits that regulate wakefulness,
NREM, and REM sleep.110,202
Somnogens
Most of the preceding text describes neural pathways that reg-
ulate sleep/wake states, but these states can also be influenced by
diffusible or circulating factors that act upon many brain regions
to promote sleep. In fact, more than 100 years ago, researchers
found that the CSF of sleep deprived dogs contained somnogens,
substances that promote sleep.203,204 Much evidence now suggests
that adenosine, cytokines, prostaglandins, and probably addition-
al substances serve as natural sleep-generating signals.205
Adenosine
During wakefulness, brain metabolic activity is high, and
adenosine may promote sleep in response to this metabolic
challenge.206-208 When cells have ample energy, nearly all ad-
enosine is phosphorylated to ATP and adenosine levels are low.
However, when cells are fatigued, ATP production is lower,
adenosine levels rise, and then adenosine acts as an inhibitory
neuromodulator. For example, adenosine reduces the activ-
ity of most wake-promoting neurons, but disinhibits VLPO
neurons. With prolonged wakefulness, adenosine levels rise
in the basal forebrain and other regions, and levels then fall
during recovery sleep.209 Most likely, the extracellular levels
of adenosine are governed by the activity of astrocytes, the
support cells of the brain, because manipulations of astrocytes
reduce the usual increases in sleep and delta power after sleep
deprivation.207,209,210 Furthermore, adenosine receptor agonists
increase sleep and NREM delta power, while caffeine and
other drugs that block adenosine receptors promote wakeful-
ness.207,211-213 As caffeine promotes arousal after sleep depriva-
852 Sleep Neurobiology for the Clinician—España and Scammell
tion,214 it seems likely that adenosine is an important mediator
of everyday sleepiness.
Cytokines
Cytokines are intercellular signaling peptides released by
immune cells, neurons, and astrocytes, and several cytokines,
including interleukin-1β (IL-1β) and tumor necrosis factor-α
(TNF-α), promote sleep.215 Administration of IL-1β into the
preoptic area of rats reduces firing rates of wake-active neurons
and promotes NREM sleep.216 Similarly, TNF-α infusions into
the preoptic area also promote NREM sleep.217 During infec-
tions, bacterial cell wall products such as lipopolysaccharide
and muramyl dipeptide may trigger production of these cyto-
kines that then increase NREM sleep and reduce REM sleep.218
In addition, these cytokines may promote spontaneous, physio-
logical sleep as IL-1β and TNF-α mRNA and protein levels are
highest around sleep onset, and blockade of IL-1β and TNF-α
signaling with antagonists, antibodies, or deletion of their re-
ceptors reduces spontaneous NREM sleep.219-221
Prostaglandin D2
Prostaglandin D2 (PGD2) is a lipid derived from fatty ac-
ids that potently promotes NREM sleep.211 Unlike adenosine
or cytokines which are made in the brain parenchyma, PGD2
is likely synthesized in the basal meninges just below the hy-
pothalamus.222 PGD2 levels in cerebrospinal fluid are highest
during the sleep period,223 and PDG2 levels increase with sleep
deprivation.224 Infusions of PGD2 just below or within the pre-
optic area activate neurons in the VLPO and increase NREM
and REM sleep, perhaps by increasing local concentrations of
adenosine.225-229 Like cytokines, PGD2 may contribute to the
sleepiness seen with inflammation as patients with African
sleeping sickness display increased CSF levels of PGD2.230
Process C and Process S
The two-process model provides a useful macroscopic per-
spective on the dynamic control of sleep and wakefulness. It is
likely that a homeostatic factor (process S) accumulates dur-
ing wakefulness and declines during sleep, and this factor in-
teracts with a circadian process (process C) that helps regulate
the timing of wakefulness and REM sleep.231-233 After a period
of wakefulness, delta power in NREM sleep is thought to be a
good indicator of Process S,234,235 and somnogens such as ad-
enosine may be the neurobiologic equivalent of Process S as
disruption of adenosine signaling can blunt the usual increase in
NREM sleep and the intense EEG delta power seen after sleep
deprivation.210
Process C is driven by the suprachiasmatic nucleus (SCN),
the master pacemaker that regulates the circadian rhythms of
sleep, wakefulness, and most other physiologic rhythms.236 The
activity of individual SCN neurons is strikingly rhythmic, es-
pecially when coupled with other SCN neurons.237 This rhyth-
micity arises from positive and negative feedback loops in the
transcription and translation of several genes.238 To synchronize
its activity with the environmental light-dark cycle, the SCN
uses luminance information from photosensitive retinal gan-
glion cells that contain the photopigment melanopsin.239 SCN
neurons then relay these timing signals to the adjacent subpara-
ventricular zone, using neuropeptides such as prokineticin 2 or
SLEEP, Vol. 34, No. 7, 2011
transforming growth factor-α.240,241 This signal is then passed
through the dorsomedial nucleus of the hypothalamus and on
to brain regions that regulate sleep and wakefulness such as the
LC, VLPO, and lateral hypothalamus.242 The SCN also regu-
lates the daily rhythm of body temperature, and through these
cycles in temperature, the SCN can entrain circadian activity
in cells throughout the body.243 Circadian rhythms are closely
linked to metabolism, and a breakdown in this coordination of
central and peripheral rhythms may contribute to the obesity
and glucose intolerance that is common in people with shift
work sleep disorder or insufficient sleep.244
CONCLUSIONS
Since the days of von Economo and then Moruzzi and Ma-
goun, much has been learned about the neurobiology of sleep
and wakefulness. We now know that neurons producing ACh and
monoamines such as NE, 5-HT, DA, and HA promote various as-
pects of wakefulness. In addition, orexins/hypocretins help sus-
tain long periods of wakefulness while suppressing REM sleep.
NREM sleep is mainly regulated by neural pathways originating
in the VLPO and other preoptic regions, yet it is also influenced
by diffusible somnogens such as adenosine. REM sleep is driven
by neurons in the pons that make ACh and GABA. These dis-
coveries provide a useful framework to better understand sleep
disorders and the effects of medications on sleep.
Nevertheless, despite these advances, many questions of
clinical importance remain unanswered. What goes wrong in
these circuits to cause parasomnias such as sleepwalking and
periodic limb movements of sleep? Under what conditions are
specific wake- and sleep-promoting systems especially nec-
essary? How is sleep restorative? What are the functions of
NREM and REM sleep? Undoubtedly, future sleep research
will provide helpful insights into the underlying causes of sleep
disorders and lead to new and more powerful therapeutics to
treat them.
ACKNOWLEDGMENTS
The authors thank Dr. P. Valko, D. Kroeger, and C. Burgess
for their thoughtful comments on this manuscript. Writing of
this article was partially supported by research grants from the
NIH (NS055367, HL095491).
DISCLOSURE STATEMENT
This was not an industry supported study. Dr. Scammell has
consulted for Merck and Cephalon. Dr. España has indicated no
financial conflicts of interest.
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858 Sleep Neurobiology for the Clinician—España and Scammell
 Review TRENDS in Pharmacological Sciences
From waking to sleeping: neuronal and
chemical substrates
Barbara E. Jones
Department of Neurology and Neurosurgery, McGill University, Montreal Neurological Institute, Montreal,
Quebec H3A 2B4, Canada
Multiple arousal systems maintain waking through the
actions of chemical neurotransmitters that are released
from broadly distributed nerve terminals when the
neurons fire. Among these, noradrenaline-, histamine-
and orexin-containing neurons fire during waking with
behavioral arousal, decrease firing during slow-wave
sleep (SWS) and cease firing during paradoxical sleep
(PS), which is also known as rapid-eye-movement sleep.
By contrast, acetylcholine (ACh)-containing neurons
discharge during waking, decrease firing during SWS
and fire at high rates during PS in association with
fast cortical activity. Neurons that do not contain ACh,
including GABA-containing neurons in the basal fore-
brain and preoptic area, are active in a reciprocal manner
to the neurons of the arousal systems: one group
discharges with slow cortical activity during SWS, and
another discharges with behavioral quiescence and loss
of postural muscle tone during SWS and PS. The reci-
procal activities and interactions of these wake-active
and sleep-active cell groups determine the alternation
between waking and sleeping. Selective enhancement
and attenuation of their discharge, transmitter release
and postsynaptic actions comprise the substrates for
the major stimulant and hypnotic drugs.
The neuronal basis of the sleep–wake cycle
From early pharmacological and lesion studies, and more-
recent gene-knockout studies, we know that wakefulness
is maintained by multiple neuronal systems that use
different chemical neurotransmitters (reviewed in [1])
(Figure 1). These multiple systems are partially redun-
dant because no one system appears to be absolutely
necessary for wakefulness, although each contributes
in a unique way to its generation and maintenance [1].
They include glutamate-, noradrenaline (NA)-, dopamine
(DA)-, 5-hydroxytryptamine (5-HT)-, histamine-, orexin
[Orx (also known as hypocretin)]- and acetylcholine
(ACh)-containing neurons. These systems share some
features including either widespread or diffuse projections
to the cerebral cortex, subcortical relays and brainstem or
spinal cord, and mutually excitatory influences on each
other that evoke concerted effects under many conditions.
Single-unit-recording studies show that many neurons
in these subcortical arousal systems discharge at their
highest rates during waking, diminish their activity
Corresponding author: Jones, B.E. (barbara.jones@mcgill.ca).
Available online 23 September 2005
www.sciencedirect.com 0165-6147/$ - see front matter Q 2005 Elsevier Ltd. All rights reserved. doi:10.1016/j.tips.2005.09.009
Vol.26 No.11 November 2005
during slow-wave sleep (SWS) and become silent during
paradoxical sleep (PS), which is also known as rapid-eye-
movement sleep (REMS). It has been suggested that they
are silenced by other neurons that become active during
sleep. Precise knowledge of the activity profiles of
chemically identified neurons during the sleep–wake
cycle has thus been sought to understand how the
activities of specific groups of neurons determine the
alternation from waking to sleeping. Such knowledge also
helps understanding of how the major stimulant and
hypnotic pharmacological agents work.
Waking is characterized by cortical activation, which is
evident on electroencephalograms (EEGs) recorded from
the surface of the cortex by low-amplitude fast activity
(particularly in a gamma range of 30–60 Hz) [2], and
behavioral arousal, which is evident on the electromyo-
gram (EMG) by high-amplitude activity in the postural
muscles, particularly in the neck (Figure 1). Sleep is
composed of two states: SWS is characterized by high-
amplitude slow EEG activity in a delta range (1–4 Hz) and
behavioral quiescence, which is evident as reduced EMG
activity in the postural muscles; by contrast, PS is charac-
terized by the paradoxical association of fast EEG activity
with complete EMG atonia in the postural muscles. Here,
I review how increasingly refined techniques have been
used to measure the activity profiles of chemically
identified neurons in relation to the sleep–wake states
and their respective EEG and EMG correlates to uncover
the manner in which specific neurons and their neuro-
transmitters influence waking and sleeping.
Arousal systems
Ascending reticular activating system
As established by the early work of Moruzzi and Magoun
in the 1940s and 1950s, the brainstem reticular formation
(RF) is crucial for maintaining cortical activation and
behavioral arousal of waking (reviewed in [1]). Projections
from neurons concentrated in the oral pontine and mesen-
cephalic RF ascend into the forebrain where they stimu-
late cortical activation via a dorsal relay in the thalamus
and a ventral relay through the hypothalamus and basal
forebrain (Figure 1). Neurons concentrated in the caudal
pontine and medullary RF facilitate postural muscle tone
through descending projections onto motor neurons and
other neurons in the spinal cord. The RF projection
neurons are likely to use glutamate as a neurotransmitter
[3]. Neurons of the diffuse thalamocortical projection
 Review TRENDS in Pharmacological Sciences Vol.26 No.11 November 2005 579

Fast EEG (W, PS):

Slow EEG (SWS):

Cx

Hi

Th
CPu ic
Rt Mes RF
LDTg
GP scp LC
ac PPTg
PnO 7g Sol s
SI VTA
POA PH SN PnC
Gi
opt TM
Fast EEG active (gamma+, delta–; W-PS) GiA
GiV W EMG:
ACh
Glu Slow EEG active (gamma–,delta+; SWS) SWS EMG:
GABA GABA (α2-adrenoceptor) PS EMG:
Behavioral wake active (EMG+; W)
NA
H Behavioral sleep active (EMG–; SWS-PS)
Orx GABA (α2-adrenoceptor)
Glu

Figure 1. The rat brain depicting neurons and their chemical neurotransmitters, and pathways that influence either cortical activity or behavior across the sleep–wake cycle.
Wake (W) is characterized by fast gamma activity on the cortical EEG (upper left) and high postural-muscle tone on the neck EMG (lower right); SWS is characterized by a slow
delta EEG (upper left) and low tone on the EMG (lower right); and PS is characterized by a fast gamma EEG (upper left) and atonia on the EMG (lower right). Neurons that are
active during waking (pale red symbols; see Figures 2 and 3) include those with ascending projections to the cortex, which stimulate fast cortical activity, and others with
descending projections to the spinal cord, which stimulate postural-muscle tone and behavioral waking. Neurons with predominantly ascending projections discharge in
association with fast EEG activity (gammaC), cease firing with delta activity (deltaK), and are active during W and PS (filled pale red symbols). These neurons include
ACh-containing (ACh), glutamate-containing (Glu) and some GABA-containing (GABA) neurons. Neurons that have either more diffuse or descending projections discharge
in association with behavioral arousal and EMG activity (EMGC), cease firing with muscle atonia, are active during W and are silent during PS (open pale red symbols). These
neurons include NA-containing (NA), histamine-containing (H), Orx-containing (Orx) and some putative glutamate-containing (Glu) neurons. Neurons that are active during
sleep (blue and green symbols) include cells whose projections ascend towards the cortex, which dampen fast cortical activity, and those with descending projections to the
brainstem and spinal cord, which diminish behavioral arousal and muscle tone. Neurons with projections to the cortex discharge in association with slow EEG activity
(gammaK/deltaC) during SWS (blue triangles). These include some GABA-containing neurons in the basal forebrain and preoptic area that bear a2-adrenoceptors and are
inhibited by NA. Also shown are GABA-containing neurons of the nucleus reticularis in the thalamus. These discharge in bursts with sleep spindles and slow waves to inhibit
and pace thalamocortical relay neurons. In the basal forebrain and preoptic area, presumed GABA-containing neurons with descending projections and a2-adrenoceptors
increase firing as muscle tone decreases (EMGK) during SWS and PS (green symbols). Also shown are GABA-containing (and/or glycine-containing) neurons in the ventral
medulla that project directly to the spinal cord where they might inhibit motor neurons in the neck and other regions during sleep. Abbreviations: 7g, genu 7th nerve; ac,
anterior commissure; CPu, caudate putamen; Cx, cortex; EEG, electroencephalogram; EMG, electromyogram; Gi, gigantocellular RF; GiA, gigantocellular, a part RF; GiV,
gigantocellular, ventral part RF; GP, globus pallidus; Hi, hippocampus; ic, internal capsule; LDTg, laterodorsal tegmental nucleus; Mes RF, mesencephalic RF; opt, optic tract;
PH, posterior hypothalamus; PnC, pontine, caudal part RF; PnO, pontine, oral part RF; POA, preoptic area; PPTg, pedunculopontine tegmental nucleus; Rt, reticularis nucleus
of the thalamus; s, solitary tract; scp, superior cerebellar peduncle; SI, substantia innominata; SN, substantia nigra; Sol, solitary tract nucleus; Th, thalamus; TM,
tuberomammillary nuclei; VTA, ventral tegmental area. Modified from [71].

relay, which project widely to the cerebral cortex to
stimulate cortical activation, and some cortically project-
ing neurons of the basal forebrain also use glutamate
[4–6]. Thus, glutamate-containing neurons represent the
backbone of the cortical-activating and behavioral-arousal
systems in the brain (Figure 1). Given the importance of
glutamate, it is not surprising that many anesthetics
(including inhalant anesthetics and ketamine) attenuate
glutamate-mediated neurotransmission [7]. Glutamate-
containing neurons of the arousal systems are regulated
by neuromodulatory transmitters that are released by
other arousal systems.
NA-containing locus coeruleus neurons
NA-containing locus coeruleus (LC) neurons stimulate
cortical activation and behavioral arousal by diffuse
projections through the forebrain, brainstem and spinal
cord (Figure 1) [3]. LC neurons discharge during waking,
particularly during active waking, decrease firing during
SWS and cease firing during PS [8,9]. NA either excites or
www.sciencedirect.com
inhibits target neurons in the brain and spinal cord,
depending on the adrenoceptors on each neuron; in
general, a1-adrenoceptors are associated with depolariz-
ation through closing KC channels, and a2-adrenoceptors
are associated with hyperpolarization by opening
KC channels. Through its receptors, NA selectively excites
other systems that are involved in waking and inhibits
those involved in sleep. Stimulation of a1-adrenoceptors
by NA excites many neurons through the forebrain
[including ACh-containing neurons (see later)], brainstem
and spinal cord (including motor neurons). Some neurons
in the brain [including putative sleep-promoting neurons
in the basal forebrain and preoptic area (see later)] are
inhibited by NA acting through a2-adrenoceptors.
NA-containing neurons and terminals also bear
a2-adrenoceptors that act through KC and Ca2C channels
to diminish activity and NA release. Drugs that antagon-
ize a1-adrenoceptors, such as prazosin, facilitate sleep
onset, probably by blocking the postsynaptic action of
NA on many of its target neurons [10]. By contrast,
580 Review TRENDS in Pharmacological Sciences
a2-adrenoceptor antagonists such as yohimbine delay
sleep, the main cause of which is likely to involve blocking
inhibitory a2-adrenoceptor autoreceptors on NA-containing
neurons and, thus, enhancing NA release and stimulation
of a1-adrenoceptors. Conversely, a2-adrenoceptor agonists
such as clonidine, which inhibit NA release, diminish
arousal and are hypnotic and anesthetic agents [11].
Drugs that either stimulate the release, or block the
reuptake, of NA, the most important of which include the
amphetamine compounds, either enhance or prolong
wakefulness. These drugs are used to treat the sleep-
iness (hypersomnolence) and loss of muscle tonus
(cataplexy) that occur with narcolepsy, which is the
sudden onset of SWS and/or PS from waking [10]. The
more recently used drug modafinil, which is used to treat
hypersomnolence, also appears to enhance NA-mediated
neurotransmission [10,12,13].
Mesencephalic DA-containing neurons
In the mesencephalic tegmentum, DA-containing neurons
of the substantia nigra and ventral tegmental area are
also important for arousal (reviewed in [1]). These neurons
project to the striatum, basal forebrain and cortex.
Presumed DA-containing neurons discharge in bursts of
spikes in association with aroused and often positively
rewarding states [14]. Such enhanced activity can occur
during both waking and PS [15], which indicates that
DA normally stimulates central arousal with positive
emotion, but not necessarily behavioral arousal and
increased postural muscle tone. Drugs that either block
the reuptake of DA and NA, such as cocaine, or stimulate
their release, such as amphetamine, commonly have both
arousing and positively rewarding effects [16]. It is pos-
sible that the enhanced release of DA and NA caused by
these drugs promotes both cortical activation and behav-
ioral arousal of waking and, thus, they are useful in
treating hypersomnolence and cataplexy [10].
5-HT-containing raphe neurons
5-HT-containing neurons in the brainstem raphe (which
means midline) nuclei discharge, like the NA-containing
neurons, maximally during waking, decrease discharge
during SWS and cease firing during REMS or PS [17],
which indicates that they promote wakefulness. However,
unlike ACh-containing neurons, 5-HT-containing neurons
appear to be active in association with less-aroused
waking states, such as during grooming and rhythmic
movement in animals. Furthermore, they attenuate
cortical activation through inhibitory influences on other
neurons of the activating systems, the most important
of which are ACh-containing neurons (reviewed in [1]).
Tricyclic antidepressant drugs that act relatively selec-
tively on the reuptake of 5-HT (such as fluoxetine) have
sedative effects during the day and variable effects on
sleep at night, often including increased movements and
muscle tone (reviewed in [18]).
ACh-containing pontomesencephalic neurons
ACh-containing neurons of the pontomesencephalic teg-
mentum project, in parallel with neurons of the RF, to the
non-specific thalamocortical projection system where they
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Vol.26 No.11 November 2005
stimulate cortical activation (Figure 1) [3,19,20]. To a
lesser degree, they also project through the ventral
extrathalamic pathway to the posterior hypothalamus
and basal forebrain, and to the brainstem RF. Although
neurons have not been identified by their neurotrans-
mitter in recording studies in the brainstem, cells in the
region of ACh-containing pontomesencephalic neurons
in the laterodorsal and pedunculopontine tegmental
nucleus (Figure 1) discharge during waking, decrease
firing during SWS and increase firing during PS [19,21].
Expression of FOS, which reflects neural activity, also
occurs in immunohistochemically identified ACh-contain-
ing neurons following PS rebound after deprivation [22].
Thus, the discharge of ACh-containing neurons occurs
in association with cortical activation but is not linked
to behavioral arousal. Indeed, it is well accepted that
injection of ACh agonists such as carbachol into the
pontomesencephalic tegmentum causes cortical activation
accompanied by muscle atonia, a state that resembles PS
(reviewed in [23]). Accordingly, ACh can act on different
target-cell populations to promote cortical activation and
to inhibit muscle tone. In the thalamus, ACh acts on
nicotinic ACh receptors (nAChRs) and muscarinic ACh
receptors to facilitate cortical activation [20,24]. It excites
thalamocortical relay neurons through direct excitatory
actions of nAChRs and M1 ACh receptors on relay neurons
and through indirect facilitation by inhibitory actions of
M2 ACh receptors on GABA-containing thalamic reticu-
laris neurons. Similarly, in the brainstem RF ACh might
act at different receptors to excite some neurons that are
involved in either cortical activation or motor inhibition,
and to inhibit other neurons, such as reticulospinal
neurons, that excited motor neurons (reviewed in [23]).
Normally, there is a balance between ACh-mediated and
NA-mediated neurotransmission, such that activation of
both types of neurons maintains a waking state with both
muscle tone and cortical activation. PS and loss of muscle
tonus can occur when NA-containing neurons are inactive
and ACh-containing neurons are active. This association
first became evident in the 1970s in an animal model
in which acetylcholinesterase inhibitors were used to
enhance ACh activity: when administered alone, the
inhibitors stimulated waking, but they stimulated PS
when administered following depletion of the catechol-
amines by previous administration of reserpine (reviewed
in [23]). In humans, acetylcholinesterase inhibitors stimu-
late cortical activation with prolonged waking when
administered during waking, but precipitate REMS
when administered during sleep [25] when NA-mediated
and other arousal systems are inactive.
Histamine-containing tuberomammillary neurons
Histamine-containing neurons located in the tuberomam-
millary nuclei (TM) of the posterior hypothalamus
stimulate cortical activation through diffuse projections
[26,27] (Figure 1). Presumed, although unidentified,
histamine-containing neurons discharge during waking,
decrease firing during SWS and cease firing during PS
[28]. Through H1 and H2 receptors, histamine depolarizes
and excites multiple neurons of the arousal systems in
addition to cortical neurons. Interestingly, histamine does
 Review TRENDS in Pharmacological Sciences Vol.26 No.11 November 2005 581

not inhibit putative sleep-promoting neurons of the pre- ACh-containing basal forebrain neurons
optic region (see later) [29]. Acting largely on H1 receptors, ACh-containing neurons of the basal forebrain have long
antihistamine drugs prescribed for alleviating allergic been known to have an important role in cortical acti-
responses often have side-effects of somnolence and thus vation (reviewed in [38]). They receive input from other
have also been prescribed as hypnotic agents [18,30]. brainstem and hypothalamic arousal systems and, in
turn, have widespread projections to the cortical mantle
(Figure 1). Their stimulation by local delivery of agonists
Orx-containing hypothalamic neurons of the arousal systems, such as AMPA and NA, evokes fast
Orx is a recently discovered peptide in the brain. Like cortical activity, particularly high-frequency gamma
its receptor, Orx is necessary for the maintenance of activity. Conversely, inactivation by lidocaine produces a
waking because narcolepsy occurs in transgenic mice and loss of fast cortical activity and predominance of slow-
dogs with spontaneous mutations that lack Orx or Orx wave activity. In FOS expression studies, ACh-containing
receptors [30–33]. Orx-containing neurons are located in neurons were active during continuous waking
the posterior hypothalamus which has long been known to enforced by sleep deprivation (Figure 2a–c) [39]. In recent
be important in maintaining waking and engaging the electrophysiological studies, ACh-containing basal
sympathetic nervous system (Figure 1). It is likely that forebrain neurons were recorded and identified in
these neurons receive inputs from the ascending fibers naturally waking/sleeping rats [40]. In contrast to
that pass through the lateral hypothalamus from the presumed NA-containing neurons in the LC, presumed
brainstem-activating systems, including the LC, and they histamine-containing neurons in the TM and identified
are excited by NA [34]. Orx stimulates cortical activation, Orx-containing hypothalamic neurons, but like presumed
behavioral arousal and autonomic changes by diffuse pro- ACh-containing neurons in the brainstem, ACh-contain-
jections and excitatory influences on the cerebral cortex, ing neurons in the basal forebrain are active during both
nonspecific thalamocortical projection system, basal fore- waking and PS. Their discharge rate correlates positively
brain ACh-containing neurons, histamine-containing with the power of fast gamma (30–60 Hz) and with theta
TM neurons, NA-containing LC neurons, and spinal cord (4–8 Hz) activity on the EEG across sleep–wake states,
motor and sympathetic neurons (reviewed in [35]). Recently, correlates negatively with slow delta activity (1–4 Hz),
Orx-containing neurons have been recorded in vivo and and does not correlate with the amplitude of the EMG
found to discharge during waking, decrease firing during (Figure 3a–d). Thus, ACh-containing neurons in the basal
SWS and cease firing during PS [36,37]. They are, thus, forebrain stimulate high frequency gamma and theta
assumed to stimulate arousal, and antagonize cortical activity during both waking and PS. This action in the
deactivation and loss of muscle tonus, which occurs in cortex might be mediated by activation of either nAChRs
their absence in cases of narcolepsy. Given their inner- or muscarinic ACh receptors on different cortical inter-
vation and excitation of all other arousal systems, Orx- neurons and/or pyramidal cells [20,38]. As one of the most
containing neurons appear to have a central role in common stimulants, nicotine elicits cortical activation,
stimulating and maintaining waking. However, Orx does and blocking nAChRs diminishes this cortical activation.
not inhibit sleep-promoting neurons in the preoptic region Blocking muscarinic ACh receptors with either scopol-
(see later) [35]. amine or atropine prevents fast cortical activity and

Distribution of ACh and GABA cells FOS expression: sleep deprivation (waking) FOS expression: sleep recovery (sleeping)

MnPO MnPO
MnPO
MPO
MPO MPO
SIa LPO
LPO LPO SIa
LPO LPO SIa
LPO
MPO MPO MPO
SIa SIa VLPO VLPO
VLPO SIa
DBB DBB
MCPO MCPO DBB MCPO
MCPO MCPO MCPO

c-FOS+
(a) ChAT+ (b) (c) c-FOS+, ChAT+

MnPO MnPO MnPO

LPO MPO
MPO
SIa LPO MPO SIa
SIa LPO
LPO MPO
LPO MPO e LPO

SIa VLPO
SIa VLPO SIa
DBB VLPO
DBB
MCPO DBB
MCPO MCPO MCPO
MCPO MCPO

c-Fos+
(d) A 9.0 A 8.6 GAD+ (e) A 9.0 A 8.6
1 mm (f) A 9.0 A 8.6 c-FOS+, GAD+

Figure 2. ACh-containing and GABA-containing neurons in levels wA9.0 and A8.6 of the basal forebrain and preoptic area of the rat are active during the wake–sleep cycle.
(a) ACh-containing neurons are immunostained for choline acetyltransferase (ChAT) (open red circles). (d) GABA-containing neurons are immunostained for glutamic acid
decarboxylase (GAD) (open blue triangles). FOS expression, which indicates activity, was examined following 3 h of sleep deprivation with continuous waking (b,e), and
during sleep recovery with almost continuous sleeping (O90% of the time) (c,f). Most of the neurons that express FOS (black dots) also release ACh (filled red circles)
following waking (b) but not following sleeping (c). Neurons that release GABA express FOS (filled blue triangles) following sleep deprivation (e) and following sleep recovery
(f). Thus, the proportion of cells that express FOS and release GABA increases significantly with sleep recovery. Most GABA-containing neurons that express FOS during
sleep immunostain for a2-adrenoceptors. Abbreviations: DBB, diagonal band of Broca nuclei; LPO, lateral preoptic area; MCPO, magnocellular preoptic area; MPO, medial
preoptic area; MnPO, median preoptic nucleus; SIa, substantia innominata, anterior part; VLPO, ventrolateral preoptic area. Modified from [39].

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582 Review TRENDS in Pharmacological Sciences Vol.26 No.11 November 2005

(a) Fast EEG: gamma (b) Slow EEG: delta (c) EMG

1.0 1.0 1.0

Normalized gamma power

Normalized EMG amplitude

0.8

Normalized delta power

0.8 0.8

0.6 0.6 0.6

0.4 0.4 0.4

0.2 0.2 0.2

0.0 0.0 0.0

aW qW tSWS SWS tPS PS aW qW tSWS SWS tPS PS aW qW tSWS SWS tPS PS

(d) Gamma+/delta–: W-PS active units (e) EMG+: W active units

1.0 1.0
0.9 0.9
Normalized average spike rate

Normalized average spike rate

0.8 0.8
0.7 0.7
0.6 ACh 0.6
0.5 Glu Glu 0.5
0.4 GABA 0.4
0.3 0.3
0.2 0.2
0.1 0.1
0.0 0.0
aW qW tSWS SWS tPS PS aW qW tSWS SWS tPS PS

(f) Gamma–/delta+: SWS active units (g) EMG–: SWS-PS active units

1.0 1.0
0.9 0.9
Normalized average spike rate
Normalized average spike rate

0.8 0.8
0.7 0.7
0.6 0.6
0.5 0.5
GABA (α2– adrenoceptor)
0.4 0.4
0.3 0.3
0.2 0.2
0.1 0.1
0.0 0.0
aW qW tSWS SWS tPS PS aW qW tSWS SWS tPS PS
TRENDS in Pharmacological Sciences

Figure 3. Sleep–wake-related EEG, EMG and unit activity. (a) Normalized gamma power, (b) delta power and (c) EMG amplitude across all sleep–wake stages, and
(d–g) normalized, average unit-spike rate for groups of cells in the basal forebrain are shown. (d) Neurons whose discharge correlates positively with gamma EEG activity,
correlates negatively with delta EEG activity (gammaC/deltaK), and which fire maximally during W and PS include the putative ACh-containing, glutamate-containing and
GABA-containing cells (represented in Figure 1). (e) Neurons whose discharge correlates positively with EMG amplitude (EMGC) and which fire maximally during W include
putative glutamate-containing cells (represented in Figure 1). (f) Neurons whose discharge correlates negatively with gamma and correlates positively with delta EEG activity
(gammaK/deltaC) and which fire maximally during SWS include putative GABA-containing neurons that bear a2-adrenoceptors (represented in Figure 1). (g) Neurons
whose discharge correlates negatively with EMG amplitude (EMGK) and which fire at higher rates during SWS and PS include putative GABA-containing neurons that bear
a2-adrenoceptors (represented in Figure 1). Abbreviations: aW, active wake; qW, quiet wake; tSWS, transition to slow-wave sleep; SWS, slow-wave sleep; tPS, transition to
paradoxical sleep; PS, paradoxical sleep. Based on data from [53].

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 Review TRENDS in Pharmacological Sciences
results in a predominance of slow EEG activity even
while animals appear to be awake and moving with full
postural-muscle tone (reviewed in [1]). Thus, basal
forebrain ACh-containing neurons have a crucial role in
stimulating and maintaining cortical activation during
waking and PS.
In addition to ACh-containing neurons, neurons in
the basal forebrain that do not contain ACh also increase
their discharge in association with cortical activation
(Figure 3d). As established in urethane-anesthetized
animals, these ‘cortical activation-on’ cells include puta-
tive glutamate-containing and a smaller number of
GABA-containing neurons that project to the cerebral
cortex (Figure 1) [6,41,42]. In addition, other neurons in
the basal forebrain that do not project to the cortex are
more active with arousal. These neurons probably contain
glutamate and they might correspond to unidentified
neurons that have been recorded in the basal forebrain in
naturally waking/sleeping rats that discharge in associ-
ation with EMG activity and are presumed to project
caudally to increase behavioral arousal and postural-
muscle tone (Figure 3e) [43,44].
Sleep-promoting neurons
GABA-containing neurons in the basal forebrain and
preoptic area
Since early studies in the 20th century it has been known
that neurons in the basal forebrain and preoptic area have
an important role in promoting sleep because lesions in
these areas result in insomnia (reviewed in [1,45]).
Neurons were also recorded in these areas that discharge
at higher rates during sleep than during waking [46–49].
In the basal forebrain, a sleep-promoting role is, pre-
sumably, fulfilled by neurons that co-distribute with
ACh-containing neurons that promote cortical activation.
In contrast to ACh-containing and presumed glutamate-
containing neurons in the basal forebrain, it is evident
from FOS expression that a significant proportion of
GABA-containing neurons in this area are active during
sleep-recovery following sleep-deprivation (Figure 2d–f )
[39]. As has also been shown in similar paradigms
[27,50,51], GABA-containing neurons in the adjacent
preoptic area, including the midline, medial, lateral and
ventrolateral areas, are also active during sleep recovery.
Across regions, the proportion of FOS-positive neurons
that release GABA increases significantly during sleep
[39]. The results with FOS parallel results from recording
studies in anesthetized rats, which show that, whereas
some GABA-containing neurons in the basal forebrain
discharge at their highest rates in association with cortical
activation (i.e. cortical activation-on), the majority
(w60%) discharge at their highest rates in association
with cortical de-activation and slow-wave activity
(‘cortical activation-off ’) cells [52]. Some of the cortical
activation-off cells are activated antidromically from the
cerebral cortex, whereas others are not and, thus, are
presumed to project either locally or caudally to either the
hypothalamus or the brainstem. The cortically projecting,
cortical activation-off GABA-containing cells might corre-
spond in part to chemically unidentified sleep-active
neurons that have been recorded in head-fixed naturally
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Vol.26 No.11 November 2005 583
waking/sleeping animals. The discharge of these cells
correlates negatively with gamma EEG activity and
correlates positively with delta EEG activity (Figure 3f )
[53]. GABA-containing neurons in the basal forebrain
project with ACh-containing neurons to the cerebral
cortex, where they might influence directly interneurons
and pyramidal neurons [6,42]. The other cortical acti-
vation-off GABA-containing cells in the basal forebrain
might correspond to neurons whose discharge correlates
negatively with EMG amplitude and which might, thus,
dampen behavioral arousal and muscle tone through
descending projections to the posterior hypothalamus
and/or brainstem (Figure 3g). GABA-containing neurons
of the basal forebrain and the preoptic area project
caudally into the posterior lateral hypothalamus where
they appear to innervate many neurons including
Orx-containing neurons [43,44,54]. Neurons of the basal
forebrain and preoptic area might also promote sleep by
descending projections to either histamine-containing
neurons or NA-containing neurons in the LC [27,55].
GABA-containing, sleep-active neurons must be dis-
tinguished from other GABA-containing neurons by the
way in which they are modulated by the major chemical
neurotransmitters of the arousal systems. In vitro
pharmacological studies show that NA depolarizes and
excites ACh-containing neurons in the basal forebrain, but
hyperpolarizes and inhibits some of the neurons in this
area that do not release ACh [56]. Similarly, most neurons
in the ventrolateral preoptic area are hyperpolarized by
NA, and these putative sleep-promoting neurons syn-
thesize GABA [29]. In the in vivo recording experiments,
dual immunostaining shows that the cortical activation-
off, GABA-containing cells bear a2-adrenoceptors [57].
These cells are heterogeneous in their firing properties;
they include neurons that burst with cortical slow waves
and can be activated antidromically from the cortex, and
others that discharge tonically and slowly and are not
activated antidromically from the cortex [52]. Thus, they
comprise both types of sleep-active neurons. In addition,
the GABA-containing neurons that express FOS during
sleep recovery also bear a2-adrenoceptors (Figure 2) [39].
Sleep-active neurons in the basal forebrain and preoptic
area are, thus, inhibited by NA-mediated stimulation of
a2-adrenoceptors. Accordingly, these neurons are inhi-
bited by NA during waking and disinhibited when
NA-containing LC neurons decrease their discharge with
drowsiness, to become active and promote SWS.
GABA-containing neurons in brainstem and thalamus
GABA-containing neurons in other areas also appear to be
active selectively during sleep to inhibit wake-active
neurons in many areas, including the brainstem RF and
LC where local GABA-containing neurons are distributed
and express FOS with sleep [3,22,58]. Together with
glycine-containing neurons [59], GABA-containing neu-
rons in the caudal medullary reticular formation that
project to the spinal cord are active during PS and might
inhibit spinal motor neurons directly (Figure 1).
In the thalamus, GABA-containing neurons in
the thalamic reticularis inhibit thalamocortical relay
neurons, including those of the nonspecific
584 Review TRENDS in Pharmacological Sciences
thalamocortical-projection system, to dampen cortical
activation [60]. Moreover, their burst pattern of discharge
during SWS triggers spindle activity (12–14 Hz) and
induces delta oscillations through the hyperpolarizing
actions of GABA on the relay neurons.
GABA receptors and hypnotic drugs
GABA, the main inhibitory neurotransmitter in the brain,
is not released in a state-selective manner throughout the
brain. However, specific GABA-containing neurons,
including those that bear a2-adrenoceptors in the basal
forebrain and preoptic area, and GABA-containing cells
in the brainstem and the thalamic reticularis nucleus,
release GABA in a state-selective manner according to
their discharge profile, and inhibit target neurons of the
arousal systems. It is, thus, not surprising that common
hypnotic drugs and anesthetics enhance GABA-mediated
neurotransmission [7,30,61–63]. Many of these agents act
at GABAA receptors, which are pentameric structures
comprising different subunits and linked to a ClK ion
channel. Many hypnotic agents, including fluorazepam
and zolpidem, modulate agonist activity by binding to a
benzodiazepine (BZD)-recognition site located between
the a- and g-subunits. Enhancing the postsynaptic action
of GABA on the ClK ionophore augments the inhibitory
influence of sleep-promoting neurons on target neurons
that bear the BZD-recognition site. BZD agonists enhance
spindling activity, which is driven by the effects of
GABA-containing reticularis thalamic neurons on the
thalamocortical relay neurons [60,64] that bear
BZD-sensitive (abg) GABA A -receptor subunits [65].
Other agents, such as muscimol and gaboxadol, which
are direct agonists at most GABAA receptor subtypes
(including those that contain ab-subunits but might lack
g-subunits and instead contain d-subunits), promote
cortical slow-wave activity and SWS [66,67]. Independent
of the release of GABA and, thus, the activity of
GABA-containing sleep-active neurons, GABAA receptor
agonists can promote sleep by tonic inhibition of neurons
of the arousal systems, including ACh-containing and
NA-containing systems. Agonists of GABAB receptors,
which are G-protein-coupled receptors linked to
KC channels, such as baclofen and gamma-hydroxybuty-
rate, also enhance cortical slow-wave activity and sleep,
and simultaneously diminish peripheral muscle tone
[18,68,69]. Opening KC channels through GABAB
receptor activation would hyperpolarize neurons that
bear these receptors throughout the neuraxis, including
neurons of the arousal systems such as NA-containing
neurons in the LC [70]. GABAB receptor agonists can thus
directly or indirectly promote sleep with slow-wave
oscillations in the forebrain and hypotonus or atonia
in the periphery.
Concluding remarks
Waking is maintained by multiple, parallel, partially
redundant arousal systems that use discrete neurotrans-
mitters. Several of these, including NA-containing neu-
rons in the LC, histamine-containing neurons in the TM
and Orx-containing neurons in the hypothalamus, dis-
charge during behavioral arousal and waking, and cease
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Vol.26 No.11 November 2005
discharge during SWS and PS. By diffuse projections and
excitatory actions, these neurons simultaneously stimu-
late cortical activation, behavioral arousal and postural-
muscle tone. NA-containing LC neurons also uniquely
inhibit sleep-promoting neurons through a2-adrenocep-
tors. ACh-containing neurons in the brainstem and basal
forebrain are active in association with the cortical acti-
vation that occurs during both waking and PS. Further-
more, they induce muscle atonia when not counterbalanced
by other arousal systems, most notably NA-containing
and Orx-containing neurons. GABA-containing neurons
that bear a2-adrenoceptors and discharge during sleep
co-distribute with ACh-containing neurons in the basal
forebrain and with other neurons across the preoptic area.
Some of these neurons are associated with cortical slow-
wave activity during SWS, whereas others are associated
with diminishing muscle tone through SWS into PS. It is
likely that these sleep-active neurons promote sleep when
they are relieved from inhibition by noradrenergic affe-
rents. Furthermore, they are likely to act via cortical
projections that inhibit cortical neurons, local projections
that inhibit either local ACh-containing neurons or
other neurons, and descending projections that inhibit
Orx-containing and NA-containing neurons. These neu-
ronal and chemical substrates, which determine the
passage from waking to sleep, are the targets of common
stimulant and hypnotic drugs.
Acknowledgements
Most of the recent research presented was funded by grants from the
Canadian Institutes of Health Research (CIHR) and U.S. National
Institutes of Health (NIH) and performed at the Montreal Neurological
Institute (MNI) by Maan Gee Lee, Ian Manns, Oum Hassani, Mandana
Modirrousta, Pablo Henny and Lynda Mainville to whom I am most
grateful. I am also thankful to my collaborators, Angel Alonso at the
MNI and Michel Muhlethaler and colleagues at the Centre Medicale
Universitaire (CMU) in Geneva, whose work is also presented.
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COMMENTARY

GABA MECHANISMS AND SLEEP

CLAUDE GOTTESMANN
Laboratoire de Psychophysiologie, Faculte¤ des Sciences, Universite¤ de Nice-Sophia Antipolis, 06108 Nice Cedex 2, France

AbstractöGABA is the main inhibitory neurotransmitter of the CNS. It is well established that activation of GABAA
receptors favors sleep. Three generations of hypnotics are based on these GABAA receptor-mediated inhibitory processes.
The ¢rst and second generation of hypnotics (barbiturates and benzodiazepines respectively) decrease waking, increase
slow-wave sleep and enhance the intermediate stage situated between slow-wave sleep and paradoxical sleep, at the
expense of this last sleep stage. The third generation of hypnotics (imidazopyridines and cyclopyrrolones) act similarly
on waking and slow-wave sleep but the slight decrease of paradoxical sleep during the ¢rst hours does not result from an
increase of the intermediate stage. It has been shown that GABAB receptor antagonists increase brain-activated behav-
ioral states (waking and paradoxical sleep: dreaming stage). Recently, a speci¢c GABAC receptor antagonist was syn-
thesized and found by i.c.v. infusion to increase waking at the expense of slow-wave sleep and paradoxical sleep.
Since the sensitivity of GABAC receptors for GABA is higher than that of GABAA and GABAB receptors, GABAC
receptor agonists and antagonists, when available for clinical practice, could open up a new era for therapy of troubles
such as insomnia, epilepsy and narcolepsy. They could possibly act at lower doses, with fewer side e¡ects than currently
used drugs. This paper reviews the in£uence of di¡erent kinds of molecules that a¡ect sleep and waking by acting on
GABA receptors. ß 2002 IBRO. Published by Elsevier Science Ltd. All rights reserved.

Key words: GABAA , GABAB , GABAC , biological rhythm.

CONTENTS

GABAA RECEPTOR COMPLEX 232

GABAA binding site 232
Barbiturate binding site 234
Benzodiazepine binding site 234
New-generation hypnotics 234
Steroid binding site 235
GABAB RECEPTORS 235
GABAC RECEPTORS 235
CONCLUSION 235
NOTE ADDED IN PROOF 236
ACKNOWLEDGEMENTS 236
REFERENCES 236

Insomnia is one of the most frequent complaints
encountered in a doctor’s o⁄ce. Although it is most
often related to anxiety, it commonly re£ects a lack of
central inhibitory processes. Bubno¡ and Heidenhain
(1881) seem to have been the ¢rst to describe such central
inhibitory processes. They showed that low-intensity
peripheral and cortical stimulations were able to inhibit
cortical stimulation-induced motor activities. For sleep^
waking behavior, it was Hess (1931) who ¢rst claimed
*Tel. : +33-4-92-07-61-66; fax: +33-4-92-07-61-62.
E-mail address: gottesma@unice.fr (C. Gottesmann).
Abbreviations : CACA, cis-4-amino crotonic acid; EEG, electroen-
cephalogram; TPMPA, (1,2,5,6-tetrahydropyridine)-methylphos-
phonic acid.
231
that sleep involved inhibitory processes: ‘the essential
mechanism of sleep cannot be explained di¡erently as
by active inhibition of some functions of the organism’.
Although Creutzfeldt et al. (1956) observed true inhibi-
tory phenomena at the cortical cellular level, it was
Evarts et al. (1960) who studied the recovery cycle of
cortical evoked potentials induced by radiation stimula-
tion and demonstrated inhibitory mechanisms during
sleep^waking behavior. From the neurochemical stand-
point, since the pioneering work of Krnjevic et al. (1966),
it has been acknowledged that GABA is the principal
inhibitory transmitter and nowadays it is estimated that
20% at least of brain neurons are GABAergic (Parades
and Agmo, 1992).
The pentameric GABAA ionotropic receptor (Fig. 1,
top) was the ¢rst to be identi¢ed, with several classes of

NSC 5488 19-4-02 Cyaan Magenta Geel Zwart

232
subunits K, L, Q, b, N, Z, O and a: K comprising six
subunits in rats, L four subunits, Q three subunits, and
b three subunits (Mo«hler et al., 1990; Duncan et al.,
1995; Schmid et al., 1995; Barnard et al., 1998; Davies
et al., 2000); the Q2 binding site has been shown to com-
prise two variants, Q2L and Q2S, which di¡er by eight
amino acids (Duncan et al., 1995). The GABAA receptor
complex consists of a Cl3 ionophore principally coupled
to GABA, barbiturate, benzodiazepine, steroid, and pic-
rotoxin binding sites (McDonald and Olsen, 1994).
GABA’s in£uence on its own binding site has been
most often studied by the antagonist bicuculline and
the agonist muscimol (Sieghart, 1995). However, many
compounds binding to other sites have given rise to
numerous studies on sleep. The second receptor to be
identi¢ed was the GABAB receptor (Hill and Bowery,
1981) which is coupled to Ca2þ and Kþ ion channels
and functions by a metabotropic pathway with a second
messenger. The a⁄nity of GABA to GABAB receptors is
lower than for GABAA receptors (Chu et al., 1990). This
receptor was ¢rst blocked by phaclofen and the agonist
most often used is baclofen. The modulation of this
receptor has given rise to only a few studies on sleep.
The third receptor identi¢ed is that for GABAC (Fig. 1,
bottom). Indeed, as early as 1975 (Johnston et al., 1975)
and again later (Parades and Agmo, 1992), it appeared
that some GABA analogues acted on bicuculline- and
baclofen-insensitive receptors. These GABAC receptors
(Drew et al., 1984) were ¢rst observed at the retinal
level (Feigenspan et al., 1993; Quian and Dowling,
1994), then at the central brain level (Bormann and
Feigenspan, 1995; Boue-Grabot et al., 2000). This recep-
tor comprises a Cl3 ionophore and several subunits (b1,
b2, b3) (Ogurusu et al., 1997). It is also more sensitive to
GABA than the GABAA receptor and its desensitization
is also di¡erent (Quian and Dowling, 1994): ‘while the
amplitude of GABAA receptor-mediated currents
decreases notably in the presence of agonists, the time-
course of GABAC receptor responses is more sustained’
(Bormann and Feigenspan, 1995).
Up to now, insomnia has been principally treated by
compounds acting at the GABAA receptor level. I pro-
pose to analyze the function of the di¡erent kind of
GABA receptors and their clinical potentialities.
GABAA RECEPTOR COMPLEX
GABAA binding site
The function of the GABA receptor binding site is to
open a chloride channel. Some time ago it was shown
that the global central increase of GABA by i.c.v. infu-
sion of GABA or inhibition of GABA transaminase
increased slow-wave sleep and induced a 64% drop of
paradoxical sleep without rebound e¡ect in cats
(Karadzic, 1966; Holmes and Sugden, 1975), and did
not in£uence the recovery of paradoxical sleep after
selective deprivation in rats (Juan de Mendoza et al.,
1973). In humans it seemed to have no signi¢cant in£u-
ence on paradoxical sleep but decreased sleep latency
NSC 5488 19-4-02
C. Gottesmann
and the amount of waking (Schneider et al., 1977).
More recent data have established that inhibition of
uptake (Fink-Jensen et al., 1992) has little or no e¡ect
on the duration of sleep stages (Lancel et al., 1998). The
more speci¢c in£uence of GABA on sleep mechanisms
was principally studied through the action of its agonist
muscimol. Mendelson and Martin (1990) found no in£u-
ence on slow-wave sleep and paradoxical sleep in rats.
Lancel et al. (1996a, 1997a) found an increase in both
and the low frequencies of electroencephalogram (EEG)
(2^6 c/s) were increased during slow-wave sleep. A sim-
ilar enhancement of slow-wave activity and the corre-
sponding sleep stage was observed by Lancel (1997) in
rats with another agonist (gaboxadol), the same e¡ect
occurring in humans (Faulhaber et al., 1997). Muscimol
was also used by infusion to identify local structure func-
tions. Injection in the anterior hypothalamus which is
involved in sleep-generating processes (Von Economo,
1928; Nauta, 1946; Sterman and Clemente, 1962;
Bremer, 1973; Szymusiak and McGinty, 1986; Ogawa
and Kawamura, 1988; Sallanon et al., 1989; Cirelli et
al., 1995) produced transient insomnia (Lin et al.,
1989), and recent results have con¢rmed that anterior
hypothalamic neurons (of the ventrolateral preoptic
nucleus) favoring sleep are GABAergic (Gallopin et al.,
2000). Moreover, it is known that anterior hypothalamic
GABAergic neurons project to the posterior hypothala-
mus (Gritti et al., 1994) and muscimol injection to pos-
terior hypothalamus target neurons which are involved
in the generation of wakefulness (Von Economo, 1928;
Ranson, 1939; Nauta, 1946; McGinty, 1969; Lin et al.,
1999) promoted slow-wave sleep and inhibited paradox-
ical sleep (Lin et al., 1989) even in cats with anterior
hypothalamic lesions (Sallanon et al., 1989). Further-
more, there is an increased release of GABA during
slow-wave sleep in this posterior hypothalamic area
(Nitz and Siegel, 1996). At the brainstem level, muscimol
infusion in the rat pontine reticular formation increases
waking and decreases slow-wave sleep, and the latency of
paradoxical sleep occurrence is prolonged (Camacho-
Arroyo et al., 1991) in such a way that it seems to cor-
respond to a suppression followed by a rebound of this
sleep stage. Similar results were obtained recently in cats
(Xi et al., 1999a), where antisense nucleotide against glu-
tamic acid decarboxylase (when injected in the pons) in-
duces an increase of paradoxical sleep (Xi et al., 1999b).
Other data suggest that brainstem ‘GABAergic neurons
could be responsible for inhibiting ‘paradoxical sleep-on’
neurons during slow-wave sleep and waking and disinhi-
biting them during paradoxical sleep’ (Mallick et al.,
1999; Maloney et al., 2000). All conclusions based on
results with muscimol have to be approached with cau-
tion since this compound is also a GABAC receptor ago-
nist (Bormann and Feigenspan, 1995) and, as already
mentioned, the sensitivity of this receptor for GABA is
higher than for the GABAA receptor. Several studies
related to GABA were also undertaken at midbrain
and pontine monoaminergic levels. Nitz and Siegel
(1997a) showed the highest amount of GABA release
during paradoxical sleep in the dorsal raphe nucleus
which contains glutamic acid decarboxylase-labeled neu-
Cyaan Magenta Geel Zwart
 GABA and sleep 233

Fig. 1. Schematic illustration of the GABAA and GABAC receptors. (a) The GABAA receptor, which is a Cl3 pore, has
binding sites for barbiturates, benzodiazepines and neurosteroids. The GABA responses are blocked competitively by bicucul-
line and non-competitively by picrotoxinin. The GABAB receptor agonist baclofen is without e¡ect, like the GABAC receptor
agonist, CACA, and antagonist, TPMPA. The vertebrate GABAA receptor is built from several subunits, some examples of
which are shown on the right-hand side. (b) Each subunit comprises four transmembrane domains. (c) The GABAC receptor
is activated by CACA and antagonized by TPMPA. It is blocked by picrotoxinin. It comprises three subunits (see on the
right). Both receptors are modulated intracellularly by protein kinases. Reprinted from Bormann (2000) with permission of
Elsevier.

rons (Ford et al., 1995) and receives a¡erents from di¡er-
ent brain levels (Gervasoni et al., 2000). Moreover, the
infusion of muscimol increases the amount of paradox-
ical sleep (Sastre et al., 1996, 1999; Nitz and Siegel,
1997a). The locus coeruleus, which also contains some
GABAergic interneurons (Ijima and Ohtomo, 1988),
receives GABAergic terminals from the medullary pre-
positus hypoglossi nucleus (Ennis and Aston-Jones,
1989). Recent ¢ndings show that the highest amount of
GABA occurs in the locus coeruleus during paradoxical
sleep (Nitz and Siegel, 1997b) and that the local infusion
of the GABA receptor antagonist picrotoxin reduces the
duration of paradoxical sleep phases (Kaur et al., 1997).
It is noteworthy that the locus coeruleus and the dorsal
raphe nucleus have been shown to be permissive struc-
tures for paradoxical sleep appearance (Hobson et al.,
1975). However, partial results involving only few cells
show that neurons of the locus coeruleus are able to ¢re
without suppressing paradoxical sleep (Gervasoni et al.,
1998) as can dorsal raphe nucleus neurons in the normal
animal (Gervasoni et al., 2000) or in the cat with aboli-
tion of paradoxical sleep atonia (by pontine lesions)
(Trulson et al., 1981). These data strongly suggest that
the GABAergic inhibition of one of these neuron areas is
su⁄cient for the appearance of this sleep stage. It should
be noted that, for the raphe nucleus, the inhibition of
serotonergic neuron ¢ring during paradoxical sleep could
be partly consecutive to disfacilitation of serotonergic
neurons (Levine and Jacobs, 1992; Sakai and Crochet,
2000). At the level of the medulla itself, which plays an
important role in processes inducing slow-wave sleep
(Batini et al., 1958; Magnes et al., 1961; Bonvallet and

NSC 5488 19-4-02 Cyaan Magenta Geel Zwart

234 C. Gottesmann
Allen, 1963) and paradoxical sleep (Webster et al., 1986;
Vanni-Mercier et al., 1991; Gottesmann et al., 1995;
Jouvet et al., 1995), there are GABAergic interneurons.
These ‘could T be responsible for the inhibition of para-
doxical sleep-o¡ cells presumed to be in part serotonergic
or (indirectly for) the disinhibition of paradoxical sleep-
on cells, presumed to be in part cholinergic’ (Holmes et
al., 1994; Holmes and Jones, 1994).
Barbiturate binding site
Barbiturates stimulate K1 and L1 sites and favor the
binding of GABA. ‘At higher doses, barbiturate mole-
cules can activate the chloride channel directly, in the
absence of GABA’ (Mo«hler, 1992). Barbiturates (synthe-
sized in 1862) have long been known to induce sleep and
they have had clinical applications since 1903. They pro-
mote slow-wave sleep and inhibit paradoxical sleep (with
a rebound e¡ect) (Oswald, 1968). However, in rats
(Gottesmann, 1964, 1996) and cats (Gottesmann et al.,
1995; Gottesmann, 1996) these ¢rst-generation hyp-
notics, at low and medium doses, massively extend the
‘intermediate stage’ (Gottesmann, 1967, 1972), the turn-
ing point at the junction of slow-wave sleep and para-
doxical sleep. This stage, also identi¢ed in mice (Glin et
al., 1991), is characterized by high-amplitude cortical
spindles and a low-frequency hippocampal theta rhythm.
Several arguments suggest that it corresponds to a tran-
sient disconnection of the forebrain from the brainstem,
thus constituting a physiological cerveau isole¤ stage
(Bremer, 1935). Indeed, the thalamic responsiveness
which is controlled by midbrain facilitating in£uences
(Dumont and Dell, 1958; Steriade, 1970) is the lowest
of all sleep^waking stages (Gandolfo et al., 1980) and
intercollicular transections induce a continuous inter-
mediate stage in rats (Gottesmann et al., 1980) and
cats (Gottesmann et al., 1984). Finally, barbiturates sup-
press pontine activation responsible for the central and
peripheral characteristics of paradoxical sleep
(Gottesmann, 1967, 1969, 1996). Consequently, stimula-
tion of the barbiturate binding site inhibits the midbrain
as well as the pontine and mesopontine reticular core
responsible for waking (Moruzzi and Magoun, 1949;
Steriade, 1996) and paradoxical sleep (Jouvet and
Mounier, 1960; Sakai, 1988; Datta and Siwek, 1997)
respectively.
Benzodiazepine binding site
In 1971 the era of benzodiazepines came fully into
being with £urazepam (Mitler, 2000). The binding site
of benzodiazepines on the GABAA receptor complex ‘is
distinct, but is nonetheless functionally coupled with the
GABAA binding site and they regulate each other in an
allosteric manner. Benzodiazepine agonists enhance the
a⁄nity of GABA for its receptor and hence its inhibitory
function. Similarly, GABA (or GABAA receptor ago-
nists, like muscimol) increases the a⁄nity of benzodiaze-
pine agonists for their receptor’ (Sadzot and Frost,
1990). However, it is worth mentioning two studies
NSC 5488 19-4-02 Cyaan Magenta Geel Zwart
which show that there is no potentiation of sleep by
stimulation of the GABAA and benzodiazepine binding
sites (Mendelson and Martin, 1990; Lancel et al., 1997a).
The stimulation of the benzodiazepine binding site pro-
motes slow-wave sleep in humans, particularly stage II
(with spindle enhancement) at the expense of stages III
and IV, and inhibits paradoxical sleep (and its eye move-
ments) (Gaillard et al., 1973; Monti and Altier, 1973;
Borbely et al., 1985; Mendelson and Martin, 1990;
Lancel et al., 1996a). In animals most benzodiazepines,
like barbiturates, increase the intermediate stage and
decrease or suppress paradoxical sleep (Gandolfo et al.,
1994), except for midazolam which increases both the
intermediate stage and paradoxical sleep in rats at 1
and 3 mg/kg injected i.p. (Gandolfo et al., 1994) and
increases paradoxical sleep in rabbits at 1 mg/kg injected
i.v. (Scherschlicht and Marias, 1983), while Lancel et al.
(1996a) found a decrease of paradoxical sleep at 3 mg/kg
i.p. in rats. At the central level, benzodiazepines injected
in the dorsal raphe nucleus increase waking (Mendelson
et al., 1987); this result recalls the insomnia induced by
raphe nucleus lesion in cats (Jouvet and Renault, 1966).
New-generation hypnotics
The third generation of sedative-hypnotic compounds
comprises principally the imidazopyridines (zolpidem)
and the cyclopyrrolones (zopiclone) which also bind to
the GABAA receptor complex. Zolpidem (Depoortere et
al., 1986), which binds to K1, L2, Q2 subunits with a
preference for the Q2L variant (Duncan et al., 1995), in-
duces sleep at much lower doses than benzodiazepines
(Depoortere et al., 1986). It reduces sleep latency in
humans (Lund et al., 1988; Declerck et al., 1992), but
does not induce signi¢cant changes in night sleep distri-
bution although there is a decrease of EEG low fre-
quency bands (910 Hz), an enhancement of the
frequency range of spindles (Lancel and Steiger, 1999)
and a decrease of paradoxical sleep (Brunner et al.,
1991; Declerck et al., 1992). In rats, zolpidem increases
slow-wave sleep (Depoortere et al., 1995), and decreases
paradoxical sleep during the ¢rst 2 h (Gottesmann et al.,
1994) but not on a 6-h recording (Gottesmann et al.,
1994; Depoortere et al., 1995). Zopiclone (Stutzmann
et al., 1993) acts as a partial agonist at the benzodiaze-
pine receptor level (Concas et al., 1994). ‘The di¡erences
between zopiclone and the classical benzodiazepines that
have been reported previously must be due to distinct
interactions of these ligands with recognition site
domains other than that represented by histidine 101’
(Davies et al., 2000). It reduces sleep latency in humans,
decreases stage I, increases stage II, has almost no e¡ect
on stages III and IV and reduces paradoxical sleep
(Stutzmann et al., 1993; Lancel and Steiger, 1999). In
rats, it also decreases sleep latency and increases the
latency of the intermediate stage and paradoxical sleep.
This last stage is decreased by up to 6 h, but the inter-
mediate stage is never increased as is observed under
barbiturates and most benzodiazepines (Gauthier et al.,
1997a,b; Gottesmann et al., 1998).

Steroid binding site
Only a few studies have been speci¢cally devoted to
the steroid binding site for sleep mechanisms. It is well
known that pregnancy is often associated with sleepiness
and a tendency to increased daily sleeping time. Lancel et
al. (1997b) studied allopregnanolone, which is a neuro-
active steroid, and Edgar et al. (1997) studied pregnano-
lone, another neuroactive steroid, and an active analogue
of it. They induced an increase in slow-wave sleep, allo-
pregnanolone decreasing the EEG slow frequencies (97
c/s) and increasing the spindle frequencies (v13 c/s). A
steroid precursor, pregnenolone, did not change the
sleep^waking cycle distribution but promoted the cortical
slow waves (0.5^4 c/s) within slow-wave sleep (Lancel et
al., 1994). However, studying the in£uence of progester-
one and allopregnanolone with more detailed sleep
stages, Lancel et al. (1996b, 1997b) were able to observe
a pronounced increase in the intermediate stage and a
decrease of paradoxical sleep with progesterone. This
e¡ect could be related to the bioconversion (of proges-
terone) into neuroactive metabolites (Lancel, 1999) like
allopregnanolone and, to a lesser extent, pregnanolone.
Consequently, all agonists of GABAA receptor bind-
ing sites (except the picrotoxin binding site) favor sleep,
although physiological sleep is better respected only with
third generation hypnotics.
GABAB RECEPTORS
These receptors are involved in sleep^waking regula-
tion as shown by lethargic (lh/lh) mice which have
increased numbers of GABAB receptors in the cortex
and thalamus (Lin et al., 1993, 1995). Moreover,
GABAB receptor antagonists infused in the thalamus
decrease EEG slow waves and deep slow-wave sleep
while light slow-wave sleep is increased (Juhasz et al.,
1994). These compounds are used to alleviate absence-
epilepsy in humans (Marescaux et al., 1992; Bittinger et
al., 1993) partly because of their thalamic impact on
relay and reticular nuclei (Lin et al., 1992), this brain
level having the highest number of GABAB receptors
(Crunelli and Leresche, 1991). At this level, the reduction
of GABAA receptor-mediated inhibition markedly
enhances GABAB receptor inhibitory postsynaptic
potentials in relay cells (Krosigk et al., 1993). Sleep stud-
ies were also undertaken in humans. Guilleminault and
Flagg (1984), in a double-blind study, found a dose-
related increase in slow-wave sleep and a reduction of
paradoxical sleep with the agonist baclofen, while
Finnimore et al. (1995) observed an increase in both
slow-wave sleep and paradoxical sleep. In rats the recep-
tor antagonist (CGP 35348) increased slow-wave sleep
and paradoxical sleep in one study (Puigcerver et al.,
1996) and increased waking and paradoxical sleep in
another (Gauthier et al., 1997a,b). The ¢ndings of the
last study are more in accordance with expected physio-
logical results since these two behavioral stages are char-
acterized by activation of the midbrain (Moruzzi and
Magoun, 1949; Steriade, 1996) and pons (Gottesmann,
NSC 5488 19-4-02
GABA and sleep 235
1967, 1969; McCarley and Hobson, 1971; McGinty et
al., 1974; Moroz et al., 1977; Vertes, 1977). In the same
way, phaclofen, another antagonist, increased paradoxi-
cal sleep when infused in the pontine reticular oralis
nucleus of the cat, while waking and slow-wave sleep
were not a¡ected (Xi et al., 2001).
The study of these agonists and antagonists of the
GABAB receptor con¢rms the sleep-inducing properties
of this receptor.
GABAC RECEPTORS
Hitherto, it has been di⁄cult to study the in£uence of
the GABAC receptor on behavior since the agonist trans-
4-aminocrotonic acid (TACA) and its cis-enantiomer
CACA, the ¢rst compounds acting on this receptor to
be identi¢ed, were not speci¢c, like the ¢rst antagonists
(Bormann and Feigenspan, 1995; Quian and Dowling,
1996). However, in 1996 the ¢rst selective antagonist was
uncovered: (1,2,5,6-tetrahydropyridine)-methylphos-
phonic acid (TPMPA) (Murata et al., 1996; Ragozzino
et al., 1996).
The ¢rst results show that GABA acting at these
receptors is also involved in sleep^waking regulation.
Indeed, TPMPA increases both quiet and active waking
(with hippocampal theta rhythm), decreases total slow-
wave sleep, essentially by decreasing the slow-wave stage
(spindles and the intermediate stage are not signi¢cantly
modi¢ed), and also decreases paradoxical sleep (Arnaud
et al., 2001).
Regarding the decrease in paradoxical sleep, there are
con£icting results with compounds acting on GABAA
and GABAB receptors. It can be hypothesized that the
decrease in this sleep stage induced by the GABAC
receptor antagonist is related to corresponding receptors
located at the neuron level in the dorsal raphe and locus
coeruleus nuclei (Nitz and Siegel, 1997a,b; Gervasoni et
al., 1998, 2000). Their blockade may induce a disinhibi-
tion of monoaminergic in£uences antagonistic to this
sleep stage (and otherwise favoring waking; Berlucchi,
1997). The contradictory result obtained with GABAA
and GABAB receptor acting compounds seems to be
linked to an inhibition of paradoxical executive processes
(Gottesmann, 1967, 1969, 1996; McCarley and Hobson,
1971; Sakai, 1988; Xi et al., 1999b, 2001).
CONCLUSION
The three currently identi¢ed GABA receptors have
similar hypnotic e¡ects when stimulated. Some di¡er-
ences appear when the quality of sleep is examined.
Slow-wave sleep is enhanced in all cases. It can be
strongly hypothesized that when antagonists promote
waking, agonists, where available, will increase sleep.
The intermediate stage is extended at the expense of
paradoxical sleep by barbiturates, most benzodiazepines
and steroids but not by new-generation hypnotics.
GABAB and GABAC receptor antagonists used so far
are not appropriate compounds for the study of this
Cyaan Magenta Geel Zwart
236 C. Gottesmann

e¡ect on the intermediate stage, because of its natural
shortness; speci¢c agonists would be more appropriate.
Finally, paradoxical sleep is decreased by agonistic
modulators of GABAA receptors, and increased by
GABAB receptor antagonists ^ parallel results ^
whereas the ¢rst available GABAC receptor antagonist
decreases this sleep stage. Altogether, these results show
the complexity and richness of brain regulation process-
es and could explain why a global in vivo increase in
extracellular GABA by various methods leads to di¡er-
ent results.
It seems probable that future research will be directed
towards GABAC receptors which could have an impor-
tant function in global behavior organization. Indeed,
they are more sensitive to GABA than GABAA and
GABAB receptors and their desensitization is much
slower (Chebib and Johnston, 1999; Bormann, 2000).
Thus, low concentrations of GABA should act preferen-
tially on GABAC receptors. To speculate somewhat, spe-
ci¢c agonists, when identi¢ed, could be useful
compounds in clinical use as hypnotics and perhaps
anti-epileptics, with fewer side e¡ects than current med-
ications. It is also not excluded that antagonists could be
e¡ective on narcoleptic attacks since there is GABAergic
participation in recent molecules which relieve this dis-
ease (Lin et al., 2000).
NOTE ADDED IN PROOF
Kauer et al. (2001) con¢rmed that the medulla prepo-
situs hypoglossi nucleus regulates paradoxical sleep by
GABAergic neurons which inhibit locus coeruleus neu-
rons.
AcknowledgementsöI thank Professor G. Morgan for correction
of the English.

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(Accepted 21 January 2002)

NSC 5488 19-4-02 Cyaan Magenta Geel Zwart