Hypocretin/Orexin Neuropeptides: Participation in the Control of Sleep-
Wakefulness Cycle and Energy Homeostasis
A. Nuñez*, M.L. Rodrigo-Angulo, I. De Andrés and M. Garzón
Current Neuropharmacology, 2009, 7, 50-59
1570-159X/09 $55.00+.00 ©2009 Bentham Science Publishers Ltd.
Departamento de Anatomía, Histología y Neurociencia, Facultad de Medicina, Universidad Autónoma de Madrid, Ma-
Abstract: Hypocretins or orexins (Hcrt/Orx) are hypothalamic neuropeptides that are synthesized by neurons located
mainly in the perifornical area of the posterolateral hypothalamus. These hypothalamic neurons are the origin of an exten-
sive and divergent projection system innervating numerous structures of the central nervous system. In recent years it has
become clear that these neuropeptides are involved in the regulation of many organic functions, such as feeding, ther-
moregulation and neuroendocrine and cardiovascular control, as well as in the control of the sleep-wakefulness cycle. In
this respect, Hcrt/Orx activate two subtypes of G protein-coupled receptors (Hcrt/Orx1R and Hcrt/Orx2R) that show a
partly segregated and prominent distribution in neural structures involved in sleep-wakefulness regulation. Wakefulness-
enhancing and/or sleep-suppressing actions of Hcrt/Orx have been reported in specific areas of the brainstem. Moreover,
presently there are animal models of human narcolepsy consisting in modifications of Hcrt/Orx receptors or absence of
these peptides. This strongly suggests that narcolepsy is the direct consequence of a hypofunction of the Hcrt/Orx system,
which is most likely due to Hcrt/Orx neurons degeneration.
The main focus of this review is to update and illustrate the available data on the actions of Hcrt/Orx neuropeptides with
special interest in their participation in the control of the sleep-wakefulness cycle and the regulation of energy homeosta-
sis. Current pharmacological treatment of narcolepsy is also discussed.
Key Words: Posterior lateral hypothalamic area, hypocretin neurons, orexin neurons, perifornical area, sleep-wakefulness,
Hypocretins/orexins (Hcrt/Orx) are hypothalamic neu-
ropeptides that are synthesized by neurons located mainly in
the perifornical area of the posterolateral hypothalamus.
These hypothalamic neurons are the origin of an extensive
and divergent projection system innervating numerous struc-
tures of the central nervous system (CNS). Hcrt/Orx neu-
ropeptides are involved in the regulation of many organic
functions, such as feeding, thermoregulation and neuroendo-
crine and cardiovascular control, as well as in the control of
the sleep-wakefulness cycle and expression of narcolepsy.
Since the discovery of the Hcrt/Orx neuropeptides in 1998
much information has been gathered about their actions in
enhancing wakefulness and EEG activation. As well as in-
creasing wakefulness and food intake, administration of
Hcrt/Orx neuropeptides also affects blood pressure, hormone
secretion and locomotor activity (see for recent review ).
Two independent research groups (the De Lecea and Sa-
kurai groups) simultaneously described the existence of two
peptides synthesized by hypothalamic neurons [18,75]. De
Lecea and collaborators observed that these peptides are ex-
pressed by neurons in the posterolateral hypothalamus that
are very similar to the secretin-related peptides, so they
*Address correspondence to this author at the Dept. Anatomía, Histología y
Neurociencia. Fac. Medicina, Universidad Autónoma de Madrid, c/ Arzo-
bispo Morcillo 4, 28029 Madrid, Spain; Tel: 34-91 497 3755; Fax: 34-91
4975338; E-mail: email@example.com
named them hypocretin-1 and hypocretin-2 (Hcrt-1 and Hcrt-
2; ). At the same time, Sakurai et al. [74,75] reported
that central administration of these peptides increased feed-
ing behavior and called them orexin A (OrxA) and orexin B
(OrxB). Hcrt/Orx neuropeptides act on two types of recep-
tors (ORX1R and ORX2R; also known as Hcrtr1R and
Hcrtr2R; ), which are expressed throughout the CNS
Mammalian Hcrt/Orx1 is a 33 amino acid peptide with a
molecular mass of roughly 3.5 kDa; it possesses an N-
terminal pyroglutamyl residue, a C-terminal amidation, and
two intramolecular disulfide bridges, Cys6–Cys12 and Cys7-
Cys14. The amino acid sequence of Hcrt/Orx1 is remarkably
well preserved in humans, cattle, rats, mice , and pigs
Mammalian Hcrt/Orx-2 is a 28 amino acid peptide with a
molecular mass of about 2.9 kDa and a C-terminal amida-
tion. The structure of Hcrt/Orx-2 in solution has been deter-
mined by magnetic resonance imaging , and consists of
two stable alpha-helices connected by a short linker. It shows
46% (13/28) amino acid identity to Hcrt/Orx1. Rat and
mouse Hcrt/Orx-2 are identical, and only one and two amino
acid residues are changed in the porcine and human counter-
parts, respectively. Hcrt/Orx neuropeptides that have also
been described in the frog Xenopus laevis has a high similar-
ity to the mammalian peptides . The structure of
Hcrt/Orx belongs to the incretin family of neuropeptides and
has been strongly conserved during the evolution,
The Hcrt/Orx gene is located in chromosome 17q21-q24
. In humans this gene consists of two exons and one in-
Hypocretin/Orexin Neuropeptides Current Neuropharmacology, 2009, Vol. 7, No. 1 51
tron, and encodes a 131 amino acid precursor peptide, pre-
pro-Hcrt/Orx. This precursor possesses an N-terminal 33
residue secretory signal peptide, and is cleaved at sites of
basic amino acid residue pairs by prohormone convertases to
yield Hcrt/Orx1 and Hcrt/Orx2 . The amino acid identity
between human and rat prepro-Hcrt/Orx is 83%, with most
substitutions occurring near the C-terminus. Given this struc-
ture, the existence of a third functional peptide derived from
the C-terminal part of the precursor is unlikely.
The Hcrt/Orx neurons in the rat are restricted to the tu-
beral region of the hypothalamus, particularly the periforni-
cal region (PeF) and the lateral hypothalamic area (LHA)
[18,75]. In the cat, Hcrt/Orx neurons are also concentrated in
the same tuberal region, but extend widely to other hypotha-
lamic areas [89,106] (Fig. 2). Hcrt/Orxergic neurons are
variable in size (diameter of cell body of 15–40 ?m) and
shape (spherical, fusiform, multipolar) [16,17,63], and they
have been assumed to number from 1,100 to 3,400 in the
whole rat brain [34,69]. The human LHA has been estimated
to hold about 50,000-80,000 Hcrt/Orx neurons . Hcrt/
Orx axons are very heterogeneous in morphology; they can
be either thick and very varicose or thin and slightly varicose
. Although Hcrt/Orx neurons are scarce, they have a pro-
fuse projection system to numerous brain regions involved in
arousal and cortical activation and in sleep-wakefulness cy-
cle regulation. Among the main structures innervated by
Hcrt/Orx neurons are the hypothalamus itself, the locus co-
eruleus (LC), the dorsal raphe nucleus (DR), and the cerebral
cortex [50,53]. Hcrt/Orx neurons also innervate the brain-
stem reticular formation, including the REM sleep inducing
region located in the ventral portion of the oral pontine re-
ticular nucleus (vRPO)  (Fig. 3).
Two Hcrt/Orx receptors (Hcrt/Orx1R and Hcrt/Orx2R)
have been described. They show 64% amino acid identity
and their structure is similar to most other peptidergic recep-
tors, to which they show an approximately 25–35% amino
acid identity [75,76]. The amino acid homology between
human and rat Hcrt/Orx receptors is 94% for Hcrt/Orx1R
and 95% for Hcrt/Orx2R. The respective affinities (ex-
pressed as EC50, the concentration of ligand needed to elicit
half-maximum receptor response) of Hcrt/Orx1 and 2 for
Hcrt/Orx1R are 30 nM and 2500 nM. However, Hcrt/Orx1
and 2 have affinities of 34 nM and 60 nM, respectively, for
Hcrt/Orx2R . This indicates that Hcrt/Orx2R is a nonse-
lective, high-affinity receptor for both Hcrt/Orx neuropep-
tides, whereas Hcrt/Orx1R is selective for Hcrt/Orx1 alone.
Hcrt/Orx receptors are highly specific for Hcrt/Orx neurop-
eptides; neuropeptide Y, secretin, ?-melanocortin, and other
neuropeptides do not activate Hcrt/Orx receptors [37,83]
Hcrt/Orx1R couple exclusively to the Gq/11 subclass of
heterotrimeric G proteins, whereas Hcrt/Orx2R can couple to
Fig. (1). Schematic depiction of hypocretin/orexin system. Hypocretin-1/Orexin A (Hcrt-1/OrxA) and hypocretin-2/Orexin B (Hcrt-2/OrxB)
are derived from a common precursor peptide, pre-pro-hypocretin. After removal of the N-terminal secretory signal sequence, pre-pro-
hypocretin is cleaved at specific sites having basic amino acid residues to yield the two mature peptides. Hcrt-1/OrxA possesses two disulfide
bridges while Hcrt-2/OxB is linear. The actions of hypocretins are mediated through interaction with two heterotrimeric G protein-coupled
receptors (Hcrt/Orx1R and Hcrt/Orx2R), whose distribution in the central nervous system is regionally specific. Hcrt/Orx1R is more selective
for Hcrt-1/OrxA, while Hcrt/Orx2R is equally specific for both peptides. Hcrt-1/OrxA is linked exclusively to excitatory G proteins of the Gq
subclass, whereas Hcrt-2/OxB couples in vitro to excitatory Gq and/or inhibitory Gi/o. Signaling through Gq pathway results in increase of
intracellular Ca2+, most probably via activation of phospholipase C-b with subsequent triggering of the phosphatidylinositol cascade and
activation of protein kinase C. The Ca2+ influx likely induces depolarization. Signaling via inhibitory Gi/o pathway may occur through hy-
perpolarization due to K+ efflux (GIRK channel-mediated). Figure modified from .
52 Current Neuropharmacology, 2009, Vol. 7, No. 1Nuñez et al.
Gq/11 or Gi/o proteins. Signaling through the Gq pathway
results in nonselective cation channel activation leading to
cellular depolarization, while Gi/o signaling activates in-
wardly-rectifying K+ channels and leads to cellular hyperpo-
larization. Thus it is thought that Hcrt/Orx1R-mediated sig-
naling is excitatory through the Gq/11-mediated stimulation
of phospholipase C, while Hcrt/Orx2R-mediated signaling
can be either excitatory (when coupled to Gq/11) or inhibi-
tory through adenylate cyclase inhibition (when coupled to
Gi/o), depending on the postsynaptic neurons .
The receptors are distinct gene products (hcrt-r1 and
hcrt-r2) that show an apparently segregated form of mRNA
expression in the rat. For example, hcrt-r1 mRNA is present
in the LC, whereas hcrt-r2 mRNA is barely detectable .
Rat Hcrt/Orx1R and Hcrt/Orx2R mRNAs are detected on
postnatal day 1 and embryonic day 18, respectively, suggest-
ing the presence of Hcrt/Orx receptors at an early stage in
hypothalamic development .
The mRNA distribution of Hcrt/Orx1R and of Hcrt/
Orx2R have been mapped in the complete adult rat brain.
Hcrt/Orx1R mRNA was located in the prefrontal and infra-
limbic cortex, hippocampus, paraventricular thalamic nu-
cleus, ventromedial hypothalamic nucleus, DR, and LC.
Hcrt/Orx2R mRNA was detected in cerebral cortex, basal
forebrain (BF) cholinergic nuclei, hippocampus, midline and
intralaminar thalamus, raphe nuclei, and hypothalamic nuclei
such as the tuberomammillary nucleus (TMN), dorsomedial,
paraventricular, and ventral premammillary nuclei .
The distribution of Hcrt/Orx receptors is on the whole
consistent with the location of the Hcrt/Orx axons and Hcrt/
OrxR mRNA-expressing neurons. Thus, the distribution pat-
terns of Hcrt/Orx1R and Hcrt/Orx2R coincide in some re-
gions but are distinct and complementary in some others.
This suggests different physiological roles for each receptor
subtype. Most of the noradrenergic LC neurons and cho-
linergic neurons in the pedunculopontine (PPT) and latero-
dorsal tegmental (LDT) nuclei express Hcrt-r1 mRNA and
Hcrt/Orx1R. In contrast, serotonergic DR neurons and do-
paminergic ventral tegmental area (VTA) neurons express
Hcrt-r1 mRNA and Hcrt/Orx1R and Hcrt-r2 mRNA and
Hcrt/Orx2R in a more balanced manner. In the forebrain, the
histaminergic TMN exclusively expresses Hcrt-r2 mRNA
Fig. (2). Distribution of Hcrt/Orx neurons in the cat hypothalamus.
A: Microphotograph of a coronal section of cat hypothalamus
showing the distribution of orexinergic neurons as result of the
immunoreaction for anti-Orexin A antiserum. No counterstaining.
B: High magnification of area squared in A. DHA. dorsal hypotha-
lamic area, LHA: lateral hypothalamic area, PeF: perifornical re-
gion, 3V: third ventricle. Calibration bars: A, 500 ?m, B, 100 ?m.
Fig. (3). Sagittal scheme of the rat brain illustrating hypocretinergic influences on the cerebral cortex and wakefulness-promoting structures.
Hypocretin/orexin (Hcrt/Orx) hypothalamic neurons send axons to both the cerebral cortex and neurochemically-specific neuronal groups
projecting to the cortex, which are most involved in wakeulness maintenance and cortical activation. These groups are the noradrenergic
locus coeruleus (LC), serotonergic dorsal raphe nucleus (RDo), cholinergic laterodorsal tegmental (LDT) and peduculopontine tegmental
(PPT) nuclei, dopaminergic ventral tegmental area (VTA), histaminergic tuberomammilary nucleus (TMN) and cholinergic basal forebrain
(BF) In the pontine tegmentum, Hcrt/Orx axons reach DOPT, where Hcrt/Orx enhance wakeulness, and also vRPO, where Hcrt/Orx suppress
REM. Figure modified from .
Hypocretin/Orexin Neuropeptides Current Neuropharmacology, 2009, Vol. 7, No. 1 53
than CNS, such as the human adrenal zona fasciculata-
reticularis and adrenal medulla, which show very low levels
of OX2R mRNA . However, Jöhren and coworkers 
demonstrated that the amount of OX1R mRNA in the pitui-
tary gland and of OX2R mRNAs in adrenal glands is higher
in male than in female rats. These results suggest a sexually
dimorphic role for Hcrt/Orx neuropeptides in peripheral or-
gans that is still poorly defined.
Hcrt/Orx peptides have been shown to exert excitatory
actions on noradrenergic LC neurons, histaminergic TMN
neurons, and cholinergic mesopontine and BF neurons [11,
Whole-cell patch clamp recordings in slices from neurons
of the rat LHA, superficial dorsal horn or laterodorsal teg-
mentum demonstrated an increase in the frequency of spon-
taneous and evoked excitatory or inhibitory postsynaptic
potentials (EPSPs and IPSPs, respectively) when Hcrt/Orx
was administered [13,31,92,93]. Also, cortical neurons in
layer VI are activated by Hcrt/Orx through the closure of a
potassium conductance .
Moreover, Hcrt/Orx, acting on Hcrt/OrxR2 receptors, has
been reported to depolarize neurons and increase their excit-
ability either by activating an inward current [22,98] or by
inhibiting an outward current . The former occurs in TMN
 and hippocampal  neurons and involves the activa-
tion of a Na+/Ca2+ exchange current. Moreover, activation of
postsynaptic Hcrt/OrxR2 receptors also stimulates a Na+/
Ca2+ exchange current in arcuate Type-C GABAergic neu-
rons, thereby producing membrane depolarization and an
increased firing rate. This effect is dependent on an increase
in cytosolic Ca2+ concentration, which is probably derived
from intracellular stores .
Van den Pol and colleagues . have studied the second
messenger system involved in Hcrt/Orx signaling. Both
types of Hcrt/Orx increase Ca2+ influx in medial and lateral
hypothalamic neurons, as measured by fura-2 imaging, in
about one third of hypothalamic neurons, probably by open-
ing a plasmatic membrane Ca2+ channel. Hcrt/Orx responses
are completely blocked by the PKC-specific inhibitor bisin-
dolylmaleide, suggesting that Hcrt/Orx may work via Gq-
activated PKC, resulting in Ca2+ channel phosphorylation
that has been reported to increase Ca2+ conductance .
More recent studies have shown that Hcrt/Orx may be linked
to the adenyl cyclase pathway , probably via an interac-
tion between Hcrt/Orx-2 neuropeptides and Gi proteins
HYPOCRETINS/OREXINS AND ENERGY HOMEO-
The hypothalamus has long been implicated in the regu-
lation of food intake, body mass, body temperature and en-
ergy balance. The LHA would be responsible for the initia-
tion of food intake, while the basomedial hypothalamic nu-
clei are associated with the cessation of food intake [7,8].
Moreover, Hcrt/Orx1 also increases food intake in satiated
rats when infused intracerebroventricularly [102,103]. Fur-
OX1R mRNA has also been detected in structures other
thermore, intraperitoneal injection of the selective Hcrt/
Orx1R antagonist (SB-334867-A) significantly reduced food
intake and increased resting behavior in rats [35,73].
The molecular bases of food intake control are the appe-
tite-stimulating (orexigenic) neuropeptides, such as melanin-
concentrating hormone (MCH) , galanin , and dynor-
phin , which have been reported in the LHA neurons. In
addition to food intake, Hcrt/Orx neuropeptides have also
been implicated in the regulation of drinking behavior .
The Hcrt/Orx system is activated in situations in which
little food is available, since 48-h fasting increases prepro-
Hcrt/Orx mRNA levels in rats . Insulin-induced hypogly-
cemia activates of Hcrt/Orx neurons, as determined by im-
munohistochemical staining against Fos protein . Fasting
in humans (ten nonobese females) results in an increase in
plasma Hcrt/Orx1 paralleled by a reduction in plasma leptin
Consequently, data indicate that Hcrt/Orx neurons are
involved in an appetite regulatory circuit that includes the
circulating hormone leptin, which is secreted by adipocytes
according to total body adipose mass. The actions of leptin
are partly mediated by the LHA, where it decreases the firing
rate of both glucose-sensitive and glucose-insensitive neu-
rons. In contrast, Hcrt/Orx1 increases the activity of glucose-
sensitive neurons . Patch-clamp measurements in iso-
lated Hcrt/Orx neurons indicate that leptin, as well as high
extracellular glucose levels, can directly decrease the neu-
ronal firing rate and intracellular Ca2+ concentrations .
Exogenously administered Hcrt/Orx neuropeptides them-
selves also reduce the firing rate of these neurons. It is there-
fore likely that some of the leptin-sensitive and glucose-
sensitive neurons in the LHA described by Shiraishi and
coworkers  are in fact Hcrt/Orx neurons, and that these
cells express inhibitory Hcrt/Orx autoreceptors.
It has also been pointed out that Hcrt/Orx can play a role
in the control of body temperature. Anatomical evidences
have demonstrated polysynaptic connections to thermogenic
sites, such as the brown adipose tissue, from Hcrt/Orx neu-
rons in the lateral hypothalamus suggesting the possibility
that these neurons represent the anatomical substrate for two
independent components for energy homeostasis, feeding
and thermogenesis [67,68]. On the other hand, intracere-
broventricular injections of Hcrt1/OrxA in mice neither in-
creased the metabolic rate nor modified the body tempera-
ture, while the receptor antagonist SB-334867-A injected
intraperitoneally acts as a thermogenic agent producing a
significant increase in energy expenditure [36,51]. These two
different effects can be due to that the antagonist has a direct
effect on peripheral thermogenic sites although orexin re-
lease at these sites has not been demonstrated . Since a
close relationship between body temperature cycle and sleep-
wakefulness cycle has been widely demonstrated (se for re-
view ), it could be possible that Hcrt/Orx participate in
the mediation of this relationship.
HYPOCRETINS/OREXINS AND SLEEP-WAKEFUL-
The stages that characterize the sleep-wakefulness cycle
are distinguished by different electrophysiological patterns in
54 Current Neuropharmacology, 2009, Vol. 7, No. 1Nuñez et al.
the electroencephalogram (EEG) and in other bioelectrical
signals. Wakefulness is characterized by low-amplitude and
fast EEG, while slow wave sleep (non-REM sleep) by high
amplitude and slow EEG waves. This pattern develops fur-
ther into high-frequency EEG waves that define the stage of
REM sleep. Switching among these states is controlled in
part by the activities of hypothalamic neurons and several
areas located in the brainstem.
The Hcrt/Orx neuropeptides have been implicated in the
control of the sleep-wakefulness cycle. Since the Hcrt/Orx
neuropeptides were discovered, much data has been col-
lected about their ability to enhance wakefulness and cortical
EEGactivation.Intracerebroventricularinfusion of Hcrt/Orx1
produces an increase in wakefulness at the expenses of non-
REM sleep and a remarkable decrease in REM sleep .
Moreover, most of the neurons within the PeF area, includ-
ing the Hcrt/Orx neurons, increase their firing rate during
alert wakefulness and decrease their activity during slow
wave sleep and REM sleep in absence of twitches [2,44,
48,58]. However, Torterolo and coworkers  reported that
significant c-fos expression in Hcrt/Orx-containing cells was
detected during both active wakefulness and the carbachol
induced REM sleep-like state. They found that 79% of the
total number of hypocretinergic neurons detected were active
during active wakefulness, approximately 34% of them were
active during carbachol induced REM sleep, and only 2%
were active during quiet wakefulness. Moreover, Kiyash-
chenko and coworkers  described maximal Hcrt-1 re-
lease in the hypothalamus and basal forebrain during both
REM sleep and active wakefulness and minimal release dur-
ing slow wave sleep. Thus, it is possible that the level of
Hcrt/Orx1 may dependent on the intensity of motor system
activation (see below) since central motor systems reach
discharge levels equal to or greater than those of active wak-
ing during REM sleep and have minimal discharge during
slow wave sleep [80,81].
The implication of Hcrt/Orx in sleep-wakefulness control
is certainly the consequence of the existence of strong ana-
tomical connections from Hcrt/Ox neurons to the major areas
responsible for the generation of the different sleep-wake-
fulness states [24,25,63,69,107] (Fig. 3). Hcrt/Orx neuropep-
tides excite DR, LC, TMN, LDT and PPT nuclei, as well as
BF cholinergic neurons, by activating postsynaptic receptors
in these neurons [11,21,23,32,39]. These “wake-active” nu-
clei are implicated in maintaining wakefulness. Accordingly,
Hcrt/Ox neuropeptides promote wakefulness when adminis-
tered in these regions [11,24,85,99]. Monoaminergic neurons
in these nuclei are most active during wakefulness, slow
down during non-REM sleep, and nearly cease firing during
REM sleep, probably due to a decrease of the excitatory
In relation with the control of REM sleep generation,
Hcrt/Orx projections and receptors have been identified in
cholinoceptive areas of the pontine reticular formation in-
volved in REM generation and control of REM-polygraphic
signs [30,53,97,107]. Furthermore, Hcrt/Orx enhances ace-
tylcholine and GABA release in these areas [6,96]. However,
altering Hcrt/Orx neurotransmission in the pontine tegmen-
tum has led to conflicting results in behaving animals. Some
studies have reported a facilitation of REM sleep after
Hcrt/Orx increase in the pontine tegmentum [99-101] but
others groups have reported a Hcrt/Orx inhibitory action on
REM sleep [10,84].
These discrepancies may be the result of the different
cellular actions produced by Hcrt/Orx at the level of the dor-
sal oral pontine tegmentum (DOPT) and in the ventral part of
the oral pontine reticular nucleus (vRPO), which is impli-
cated in REM sleep generation [27,28,60,72], Hcrt/Orx in
DOPT was recently found to produce excitatory electro-
physiological responses in both cholinergic and noradrener-
gic cells . In contrast, we have demonstrated that ionto-
phoretic application of Hcrt/Orx through a barrel micropi-
pette in the vRPO induces inhibition by activation of
GABAA receptors because is blocked by application of the
GABAA antagonist bicuculline . There is a specific
Hcrt/Orx projection from the PeF area to the vRPO .
Therefore, the PeF area might control REM generation
through a hypocretinergic projection that would activate
Recent experiments in our laboratory have shown that
Hcrt/Orx neuropeptides have a wake-promoting and sleep-
suppressing actions when acting in the DOPT and a direct
and exclusive inhibition of REM sleep when acting in the
vRPO  (Fig. 4). Also a defacilitating action on REM
sleep could be secondarily produced by the wake-promoting
and sleep-suppressing actions of Hcrt/Orx in other pontine
areas such as the principal LC and LDT nuclei [11,99]. The
loss of Hcrt/Orx signaling in narcolepsy disease would im-
pair these actions and could remove the defacilitat-
ing/inhibiting actions on REM generation of the Hcrt/Orx
signal in these pontine regions during wakefulness; conse-
quently, patients would fall directly into REM while still in a
wakefulness period (see below).
Hcrt/Orx neurons may be also involved in motor activity.
Hcrt/Orx cells discharge during active waking, when pos-
tural muscle tone is high in association with movements,
decrease discharge during quiet waking in the absence of
movements, and virtually cease firing during sleep, when
postural muscle tone is low or absent [2, 48, 58]. However,
Hcrt/Orx-containing neurons are also activated during car-
bachol-induced REM sleep with muscular twitches . The
relationship between hypocretinergic system activation and
motor activation is reinforced by decrease in Hcr/Orx1 levels
in CSF of rats after long-term immobilization and its in-
creased levels after short-term forced swimming [54,86]. The
peptide concentration in dialysates from the hypothalamus
was significantly higher during active waking than during
slow-wave sleep . Moreover, systemic, intracerebroven-
tricular, and intraparenchymal injection of Hcrt/Orx in-
creases motor activity [42,86].
In agreement with a putative role for the hypocretinergic
system in motor functions, Hcrt/Orx terminals have been
found in the ventral horn where motoneuron cell bodies are
located . In addition, application of hypocretin depolar-
izes lumbar motoneurons by means of presynaptic and post-
synaptic mechanisms that result in the facilitation of their
discharges . These authors propose that this action of
Hcrt on motor output is important in the physiological regu-
Hypocretin/Orexin Neuropeptides Current Neuropharmacology, 2009, Vol. 7, No. 1 55
lation of motor activity in situations that involve certain hy-
Another question of interest is the mechanism for cir-
cadian regulation of Hcrt/Orx neurons. As mentioned above,
Hcrt/Orx neuron activity follows a circadian rhythm as dem-
onstrated by both Fos-immunostaining  and Hcrt/Orx
peptide levels measured in the rat cisterna magna .
Hcrt/Orx neurons in rats and humans were recently shown to
be directly innervated by neurons of the suprachiasmatic
nucleus, a structure that is responsible for regulation of cir-
cadian processes . Hcrt/Orx neurons may therefore be a
relay station for circadian sleep/wake control by the su-
HYPOCRETINS/OREXINS AND NARCOLEPSY
Idiopathic narcolepsy is more frequent than commonly
thought, having approximate prevalences 1 in 1,000–2,000 in
the United States  and 1 in 600 in Japan . This neuro-
logical disorder is characterized by a primary disturbance in
sleep-wakefulness organization. The onset of narcolepsy
most often occurs during adolescence and the symptoms
gradually reach a certain severity within several years, after
which patient condition neither worsens nor improves.
Narcoleptic patients suffer from severe daytime hyper-
somnolence, combined with night time insomnia and sleep
fragmentation, which produces a constant feeling of tired-
ness in these subjects. In healthy human subjects the latency
Fig. (4). Mean time spent ± SEM of the sleep-wakefulness cycle states by animals with Hcrt-1 microinjections in either the dorsal oral pon-
tine tegmentum (DOPT) or the ventral oral pontine tegmentum (vRPO) in each of the first 3 h of polygraphic recordings in baseline and after
Hcrt-1 1000 mM dose experiments. *Statistically significant difference in comparison with baseline. Post hoc analyses (Fisher's test, P <
56 Current Neuropharmacology, 2009, Vol. 7, No. 1Nuñez et al.
for REM sleep after the onset of non-REM sleep is around
90–100 min. In contrast, in narcoleptic patients, REM sleep
latency is frequently shortened to less than 15 min, some-
times being so short that even direct transitions from wake-
fulness to REM sleep occur, something which can under-
standably cause embarrassing and even dangerous situations.
This “sleep-onset REM period” is regarded as the diagnostic
indication for narcolepsy.
However, the most striking feature of the disease is cata-
plexy, a sudden bilateral loss of skeletal muscle tone during
wakefulness; it is most often triggered by a strong positive
swing of emotion such as laughter (a trigger in 80% of cases)
. Cataplectic attacks normally last from a few seconds to a
few minutes and range in severity from slurred speech, head
dropping, and knee jerking to complete collapse to the floor
despite maintained consciousness . All these clinical
symptoms suggest that narcolepsy is a dysfunction of vigi-
lance state boundary control, in which the fundamental
pathophysiology involves an abnormal and premature intru-
sion of REM sleep into the state of wakefulness.
Current pharmacological treatment of narcolepsy is based
on two approaches, although a host of different therapies are
in use . Excessive daytime sleepiness is currently treated
with either amphetamine-like stimulants or the stimulant
modafinil, both of which increase the catecholaminergic
tone. Amphetamines increase catecholamine release and also
reduce catecholamine uptake by inhibiting monoamine
transporters, however they have considerable sympathetic
side effects. Modafinil is structurally unrelated to ampheta-
mines and presently constitutes a better first-line treatment
for excessive daytime sleepiness and sleep attacks. Although
the mechanism of action of modafinil is not yet fully under-
stood, it is thought to consist mainly in inhibition of the do-
pamine transporter. Interestingly, administration of modafinil
or amphetamine-like stimulants to mice increases Fos-
expression in Hcrt/Orx neurons of the hypothalamus  or
of the TMN . Since both amphetamines and modafinil
also enhance wakefulness in Hcrt/Orx-deficient narcoleptic
subjects, it appears that their sites of action are largely inde-
pendent of the Hcrt/Orx system, and their advantageous ac-
tions in narcolepsy would be purely symptomatic.
Despite promotion of wakefulness, these stimulants do
not improve other REM sleep-related narcolepsy symptoms.
For the treatment of cataplexy, tricyclic antidepressants such
as imipramine, protryptiline, and clomipramine have been
commonly used and are still widely prescribed. These drugs
act by blocking reuptake of noradrenaline and serotonin, and
they have considerable anticholinergic side effects . The
newer antidepressants, such as fluoxetine, are clinically less
effective, although they have significantly less side effects.
Sodium oxybate is, at present, the first-line treatment for
cataplexy. It is the sodium salt of the natural neurotransmit-
ter gamma-hydroxybutyric acid, and it binds to its own re-
ceptors at physiologic concentrations; however, when used at
higher pharmacological concentrations, sodium oxybate acts
mainly through GABAB receptors.
Animal models of human narcolepsy consist in modifica-
tions of Hcrt/Orx receptors  or absence of these peptides
. Hcrt/Orx knockout mice display a severe narcolepsy-
like phenotype . This is also evident in double receptor
knockout (Hcrt/Orx1R- and Hcrt/Orx2R-null) mice. In con-
trast, knockout mice for either Hcrt/Orx1R or Hcrt/Orx2R
show phenotypes that is somewhat different. Hcrt/Orx1R
deficient mice only exhibit slightly increased sleep fragmen-
tation and lack evident behavioral abnormalities. Hcrt/Orx2R
knockout mice also show a mild narcoleptic phenotype, in
which fragmentation of sleep is present but abnormalities of
REM sleep, such as direct transitions from wakefulness to
REM sleep, are either absent or much less frequent than in
double-null animals. These data suggest that Hcrt/Orx2R is
critical for normal regulation of wakefulness/non-REM tran-
sitions, whereas the intense deregulation of REM sleep con-
trol present in the narcoleptic syndrome relies on signaling
disruption through both Hcrt/Orx1R and Hcrt/Orx2R.
Nowadays it is assumed that narcolepsy is the direct con-
sequence of Hcrt/Orx neuron degeneration, and therefore
indicates widespread Hcrt/Orx hypofunction. There are dif-
ferent reasons to link Hcrt/Orx and human narcolepsy. Nar-
coleptic patients have fewer Hcrt/Orx neurons in the postero-
lateral hypothalamus than control subjects [70,86], and their
cerebrospinal fluid shows lower or untraceable Hcrt/Orx
levels . Moreover, gliosis has been reported in the peri-
fornical area in some narcoleptic patients [70,86]. All these
observations, together with the well known association be-
tween narcolepsy and specific antigens of the major histo-
compatibility system (HLA), suggest that an autoimmune
process might be the triggering factor initiating hypothalamic
Hcrt/Orx neuron degeneration in narcolepsy. The astrocytic
marker GFAP (glial fibrillary acidic protein) for gliosis
seems to be present in a few narcoleptic patients, and might
be found in more since the analyzed tissue had been stored
for a long time and could have lost immunoreactivity .
Although Hcrt/Orx neuronal degeneration is the most ac-
cepted hypothesis for human narcolepsy, other possible
causes, including defects in the synthesis of Hcrt/Orx or their
receptors cannot be rejected. Hereditary canine narcolepsy
caused by a mutation in hcrt2R/ox2R  or rodent models
of narcolepsy due to deletion of the Hcrt/Orx gene  have
been well documented.
At the present time, Hcrt/Orx neuropeptides are consid-
ered to be neuromodulators that enhance the waking state
through increasing the activity of several neuronal popula-
tions; they also inhibit REM sleep by acting on the vRPO
(see above). Impairment of the Hcrt/Orx neuron projection
system or actions would provoke, on one hand, hypoactivity
of the ascending activating systems, and, on the other hand,
disinhibition of the vRPO and REM sleep triggering. This
hypothesis could explain the great number of transitions be-
tween wakefulness and sleep, REM sleep fragmentation and
hypersomnia present in narcoleptic patients.
The Hcrt/Orx neuropeptide system has proven to be a
novel mechanism by which the brain regulates arousal and
sleep/wake states. Also, these neuropeptides contribute to
regulation of energy homeostasis. The link between narco-
lepsy and Hcrt/Orx deficiency in animals and humans has
provided a better understanding of sleep-wakefulness regula-
tion and the cause of narcolepsy. Different studies clearly
Hypocretin/Orexin Neuropeptides Current Neuropharmacology, 2009, Vol. 7, No. 1 57
demonstrate that Hcrt/Orx neuropeptides favored the activity
of neurons implicated in wakefulness generation while at the
same time, they inhibit neurons involved in REM sleep gen-
Discovery of the pathogenic mechanisms that underlie
the loss of Hcrt/Orx neurons in humans will constitute a cru-
cial boost for narcolepsy research in the future. That infor-
mation is essential for the prevention and treatment of this
This work was supported by Grants BFI 2003-00809 and
BFU2006-07430 from the Spanish Ministry of Education
and Science. We thank Ms Marta Callejo for assistance with
the experiments and Ms Carol F. Warren for revision of Eng-
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Received: July 24, 2008 Revised: August 19, 2008 Accepted: September 17, 2008