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The Journal of Neuroscience, October 15, 2000, 20(20):7760-7765
Hypocretin-1 Modulates Rapid Eye Movement Sleep through
Activation of Locus Coeruleus Neurons
Patrice
Bourgin1,
Salvador
Huitrón-Reséndiz2,
Avron
D.
Spier1,
Véronique
Fabre2,
Beatriz
Morte1,
José R.
Criado2,
J. Gregor
Sutcliffe1,
Steven J.
Henriksen2, and
Luis
de
Lecea1, 2
Departments of 1 Molecular Biology and
2 Neuropharmacology, The Scripps Research Institute, La
Jolla, California 92037
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ABSTRACT |
The hypocretins (hcrts), also known as orexins, are two recently
identified excitatory neuropeptides that in rat are produced by ~1200
neurons whose cell bodies are located in the lateral hypothalamus. The
hypocretins/orexins have been implicated in the regulation of rapid eye
movement (REM) sleep and the pathophysiology of narcolepsy. In the
present study, we investigated whether the locus coeruleus (LC), a
structure receiving dense hcrtergic innervation, which is quiescent
during REM sleep, might be a target for hcrt to regulate REM sleep.
Local administration of hcrt1 but not hcrt2 in the LC suppressed REM
sleep in a dose-dependent manner and increased wakefulness at the
expense of deep, slow-wave sleep. These effects were blocked with an
antibody that neutralizes hcrt binding to hcrt receptor 1. In
situ hybridization and immunocytochemistry showed the presence
of hcrt receptor 1 but not the presence of hcrt receptor 2 in the LC.
Iontophoretic application of hcrt1 enhanced the firing rate of LC
neurons in vivo, and local injection of hcrt1 into the
LC induced the expression of c-fos in the LC area. We
propose that hcrt receptor 1 in the LC is a key target for REM sleep
regulation and might be involved in the pathophysiological mechanisms
of narcolepsy.
Key words:
norepinephrine; orexin; orexin receptors; c-fos; arousal; microinjection; immunocytochemistry
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INTRODUCTION |
The hypocretins (hcrt1 and hcrt2),
also called orexins, are two neuropeptides derived from the same
precursor, which are expressed in a small set of neurons in the
perifornical area of the hypothalamus (de Lecea et al., 1998 ; Sakurai
et al., 1998). The hypocretins are neuroexcitatory (de Lecea et al.,
1998; van den Pol et al., 1998) and bind to two different
G-protein-coupled receptors, hcrt receptors 1 and 2 (hcrtr1 and hcrtr2,
also known as OX1 and OX2 receptors) with different affinities (Sakurai
et al., 1998). Recently, evidence has emerged that confirms a role for
the hypocretins in arousal states. Lin et al. (1999) mapped the canine
narcolepsy mutation (canarc-1) to hcrtr2. Knock-out experiments in
mice demonstrated that the absence of hypocretin causes alterations in
sleep architecture, particularly on the amount of rapid eye movement
(REM) sleep during the dark period (Chemelli et al., 1999). In
addition, hcrt-deficient mice display electroencephalographic patterns
and behaviors that resemble those of narcoleptic attacks.
Intracerebroventricular infusion of nanomolar amounts of hypocretin has
recently been shown to increase arousal, reduce REM sleep, and affect
neuroendocrine balance (Hagan et al., 1999). Nishino and colleagues
(2000) found that seven of nine patients with narcolepsy had
undetectable hcrt1 in CSF. These independent studies indicate
that the hypocretins have a major role in the regulation of sleep, but
the role of different brain structures and the contribution of each of
the hcrt receptors remain unknown.
The projections of hcrt-containing neurons extend widely throughout the
brain (Peyron et al., 1998; Date et al., 1999). Four main hcrtergic
afferent regions can be recognized from anatomical studies (Peyron et
al., 1998): an intrahypothalamic field; an ascending pathway through
the basal ganglia, septum, and cerebral cortex; a medial pathway that
connects a variety of thalamic nuclei; and a descending pathway that
reaches the locus coeruleus (LC), dorsal raphe, and spinal cord. These
pathways are consistent with the expression patterns of hypocretin
receptors 1 and 2 (Trivedi et al., 1998) and strongly suggest that the
hypocretinergic system is involved in many different physiological
functions, including feeding, blood pressure, hormone release, and arousal.
Neuroanatomical distribution of hcrt fibers suggest several brain
regions relevant to the role of the hcrts in the regulation of the
sleep-wakefulness cycle. Noradrenergic neurons in the LC are active
during wakefulness, decrease their activity during non-REM sleep, and
are virtually silent during REM sleep (Hobson et al., 1975; Aston-Jones
and Bloom, 1981). Because the LC receives the densest hcrtergic
innervation, the purpose of the present study was to determine whether
the LC is a physiological target for hcrt to regulate the
sleep-wakefulness cycle and to assess the respective roles of the
hcrtrs in arousal and sleep. We show that administration of hcrt1 to
the LC dramatically decreases the amount of REM sleep. Moreover,
injection of hcrt1 induced the expression of c-fos in the
LC, and iontophoretic application enhanced the firing rate of LC
neurons in vivo. Our results directly implicate hcrt1 and
hcrtr1 in the modulation of REM sleep.
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MATERIALS AND METHODS |
Anatomical studies
Adult male Sprague Dawley rats (250-300 gm; n = 3) were anesthetized with CO2, perfused
intracardially with 4% paraformaldehyde in PBS, pH 7.4, and used for
in situ hybridization and immunohistochemistry as previously
described (de Lecea et al., 1997). For immunocytochemistry the
following primary rabbit polyclonal antisera were used: 1:500 hcrt1
(Chemicon, Temecula, CA), 1:200 hcrtr2 (Santa Cruz Biotechnology, Santa
Cruz, CA); 1:10,000 c-fos (Ab-5; Calbiochem, La Jolla, CA); and 1:400 tyrosine hydroxylase (TH) monoclonal (Chemicon). For double
immunofluorescence, sections were incubated with Cy2-labeled anti-mouse
IgG and rhodamine anti-rabbit IgG, washed, and visualized under a Zeiss
(Thornwood, NY) microscope using a 25× multi-immersion objective.
Images were acquired with a charge-coupled device 1300Y camera (Roper
Scientific) using IPLabs software and processed with Adobe
(Mountain View, CA) Photoshop. For the c-fos
immunocytochemistry, rats were injected with hcrt1 (25 pmol) at noon
and killed 60 min afterward. c-fos-positive cells
were counted in five sections per animal (25 µm thick) from three
different animals.
In vivo experiments
Studies in freely moving animals. Experiments were
performed on a total of 14 adult male rats (Sprague Dawley, 250-300
gm), kept under controlled environmental conditions (12 hr light/dark cycle, 23 ± 1°C, and food and water ad libitum) and
handled in agreement with the ethical rules for experimentation on
laboratory animals (US Department of Health and Human Services
publication 80-23; Office of Science and Health Reports, Division of
Research Resources, National Institutes of Health, Bethesda, MD, 1980).
Rats (n = 14) were anesthetized under halothane
(1-2%) and implanted with electrodes for recording the
electroencephalogram (EEG) and electromyogram. In addition, two
stainless steel guide cannulas and a dummy stylus were stereotaxically
implanted and positioned 3 mm above the right and left LC [Paxinos and
Watson, 1986; from the ear bar zero: posterior (P),  0.8 mm; lateral
(L), +1.3 mm; and H, +2.3 mm, using a posterior angle of
20° from vertical]. After surgery, rats were allowed 7-10 d for
recovery. All compounds were dissolved in saline. For intracerebral
injections, a total volume of 0.2 µl was injected through a smaller
cannula 3 mm longer than the guide tube at the rate of 0.1 µl/min,
after which the cannula was left in place for another 2 min. Rats were
injected bilaterally, except for one animal that was injected
unilaterally. Infusion sites were verified histologically (frontal
brain sections of 25 µm, cresyl violet and methylene blue staining).
Polygraphic recordings and behavioral observation began immediately
after the infusion for 5 hr (12 P.M. to 5 P.M.). Recordings were
performed after microinjection of saline for baseline, hcrt1 (2.5 and
25 pmol), hcrt2 (25 pmol), affinity-purified antisera (1.4 mg/ml), and
hcrt1 (25 pmol) preincubated with antiserum for 1 hr. Recordings were
also performed on the day after a drug treatment after a sham
microinjection to verify that the sleep and wakefulness amounts had
returned to control values. Infusions of saline, hcrt1, hcrt2, and/or
antiserum were given in random order. The hcrt peptides were obtained
from Peninsula Laboratories (Belmont, CA) and the peptide synthesis
core facility at The Scripps Research Institute.
Studies in halothane-anesthetized rats. Male Sprague Dawley
rats (310-330 gm) were anesthetized with halothane (3.0-4.0%) and
placed into a stereotaxic apparatus. Body temperature was monitored and
maintained at 37.0 ± 0.1°C by a feedback-regulated heating pad.
Halothane anesthesia was maintained at 0.75% after surgery.
Extracellular potentials were recorded by a single 3.0 M
NaCl-filled micropipette (5-10 M , 1-2 µm
inner diameter) cemented 20-40 µm distal to a four-barrel
micropipette (30-80 M barrels), and amplified with an
AXOPROBE-1A amplifier. Microelectrode assemblies were
stereotaxically oriented into the LC [coordinates (from bregma): P,
12.35; L, 1.1; and ventral, 6.8-7.5, using a posterior angle of 20°
from vertical]. Single-unit activity was filtered at 1-3 kHz (3
dB). Only spikes that had a >3:1 signal-to-noise ratio were evaluated.
Acquisition, analysis, and processing of data were performed by
customized LabView software. Extracellularly recorded single-unit
action potentials were discriminated temporospatially by a peak
detector digital-processing algorithm.
Statistical analysis
Polygraphic recordings were analyzed visually every 15 sec epoch
into wakefulness (W), slow-wave sleep 1 (SWS1), SWS2, and REM sleep,
according to standard criteria. Data were analyzed for 2 and 4 hr
periods and expressed as mean ± SEM (total minutes per state per
recording time). The following variables were examined: time spent in
W, SWS1, SWS2, or REM sleep; duration and number of REM sleep episodes;
and latency of SWS2 and REM sleep onset (interval between the time of
injection and the first SWS2 or REM sleep episode of at least 15 sec
duration). The effects of hcrt1, hcrt2, antiserum, and antiserum and
hcrt1 were analyzed by comparing sleep-wakefulness amounts after
bilateral intra-LC administration of these compounds with those after
administration of saline. A decrease in REM sleep duration of at least
75% of baseline (saline injection in the same rat) was considered a
significant response. The  2 test with
the Yates correction for continuity was used to test the relationship
between the injection site and the effect of hcrt1 on the sleep-wake
cycle. Fisher's exact test (used because of low expected frequencies)
and ANOVA were used to compare the number of responders to hcrt1 with
the number of responders to hcrt2. Data from responder rats had the
same profile and were pooled for statistical analysis. Grouped data
were statistically compared by an ANOVA for comparison across collapsed
groups of data, with p < 0.05 as the critical limit.
Comparisons among individual means were made by Fisher's least
significant difference (LSD) post hoc test after ANOVA. The
effect of hcrt1 on the firing rate of LC neurons was tested using the
Wilcoxon matched pair test.
Antibody blockade
COS-7 cells, transiently transfected with human hcrtr1 cDNA
(Receptor Biology), were diluted with growth medium (high-glucose DMEM
and 10% fetal bovine serum) at a density of 50,000 cells per well.
Cell culture medium was removed, and cells washed briefly in binding
buffer [10 mM HEPES, pH 7.4, 5 mM
MgCl2, 1% BSA, and protease inhibitors (50 µg/ml bacitracin, 50 µg/ml leupeptin, and 0.1 mM
phenylmethylsulfonyl fluoride)]. Cells were incubated with a final
concentration of 0.2 nM
125I-hcrt1 (2200 Ci/mmol; New England
Nuclear, Boston, MA) diluted in binding buffer for 45 min at room
temperature. Nonspecific binding was defined by addition of 10 µM unlabeled hcrt1. The anti-hcrt antiserum 2123 (Peyron
et al., 1998) and a control antibody directed against preprohypocretin
C-terminal peptide (de Lecea et al., 1998) were included in the binding
reactions at various concentrations. The reactions were stopped by
aspiration of the binding reaction mixture and two rapid washes with
ice-cold wash buffer (HEPES, pH 7.4, and 1% BSA). Cells were suspended
in 150 ml of 0.2 M HCl, and radioactivity was counted in a
gamma counter.
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RESULTS |
hcrtr1 immunoreactivity in the locus coeruleus
The hypocretins/orexins exert their actions via binding to
two G-protein-coupled receptors, hcrtr1 and hcrtr2. To determine whether hcrtrs were present in areas related to sleep modulation, we
performed in situ hybridization and immunohistochemical
staining of rat brain sections using antibodies specific to hcrtr1 and hcrtr2. Hcrtr1 mRNA is highly expressed in the LC (Fig.
1A), whereas hcrtr2
mRNA signals were not detected in this region (data not shown).
Consistent with this result, hcrtr1 immunoreactivity was found in most
if not all neurons in the LC (Fig. 1). hcrtr1-immunoreactive cells
co-localized with tyrosine hydroxylase-labeled neurons in the LC,
indicating that hcrtr1 is expressed in the noradrenergic population of
this nucleus (Fig. 1C). Adjacent nuclei, including the
laterodorsal tegmental nucleus (LDT), and brainstem motor nuclei also
contained immunoreactive cells positive for hcrtr1. The paraventricular
nucleus of the hypothalamus, the medial thalamus, and layers II and III
of the cerebral cortex also showed prominent immunoreactivity for
hcrtr1. Staining was suppressed by preincubation with the peptide
immunogen.
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Figure 1.
Localization of hcrtr1 in the LC.
A, Dark-field photomicrograph of the LC hybridized with
an hcrtr1 riboprobe. B, Immunocytochemical detection of
hcrtr1. Note that most cells in the LC are labeled with the antibody.
Scale bar, 50 µm. C-E, Immunofluorescence micrographs
showing overlap (yellow, arrows)
between hcrtr1-positive (red) and TH-immunoreactive
(green) neurons in the LC region. Scale bar, 25 µm.
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hcrt1 administration in the LC affects sleep-wake cycle
We tested the effects on the sleep-wake cycle of local bilateral
application of hcrt1 and hcrt2 to the LC area. To test the specificity
of the site of injection, we mapped the injection sites histologically
according to the atlas of Swanson (1992) (Fig.
2). A decrease in REM sleep duration with
25 pmol of hcrt1 superior to 75% of baseline (saline injection in the
same rat) was considered a significant response. This criterion was
chosen on the basis of our original hypothesis of a possible effect of hcrt1 on REM sleep. The number of responder rats in the group of
animals injected in the LC (unilaterally or bilaterally) was statistically different from the number of responders in the group that
received injections located outside the LC
(2 = 10.29; p < 0.01).
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Figure 2.
Schematic representation of hcrt injection sites.
Drawings show coronal sections at five different levels of the
brainstem (interaural, 1.3 to +0.7; Swanson, 1992). The black
circles represent sites for which no changes in the
sleep-wakefulness cycle were observed after injection. The
triangles and diamonds correspond to the
positive sites (at least one site is located in the LC) for which
significant changes in W, SWS2, and REM sleep were observed after
bilateral infusion of the 25 pmol of hcrt1. For each rat the bilateral
sites are represented by a triangle or a
diamond and one color. One rat received
unilateral injections (red triangle).
Mo5, Motor trigeminal nucleus; SLC,
subcoeruleus nucleus; PRF, pontine reticular formation;
PB, parabrachial nucleus; DR, dorsal
raphe; PPT, pedunculopontine tegmentum;
PAG, periacqueductal central gray.
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From the responding animals, two rats received the injections in both
LC regions; one animal received the injection in the LC area and
subcoeruleus nucleus; another rat had the injection site in the LC and
another site adjacent to LC; and two rats received the injection in
only one LC side, and the other injection site was located outside the
LC region. In one of these animals the LC injection site was located in
an area that overlaps both with the LC and the fourth ventricle.
Another rat was injected in only one site corresponding to the right LC
area (Fig. 2). In rats receiving hcrt1 in only one side, the effect
profile on the sleep-wakefulness cycle was indistinguishable from that
observed in rats injected bilaterally. In contrast, in nonresponding
rats, injections were performed in the LDT or pontine reticular
formation, outside the LC region (n = 7). In the same
manner, injections located in the fourth ventricle (one site;
n = 2) were also negative for REM sleep blockade and
wakefulness promotion.
Local administration of hcrt1 into the LC area produced significant
changes in the amounts of REM sleep
(F(4,24) = 21.8; p < 0.001), SWS2 (F(4,24) = 8.4;
p < 0.001), and wakefulness
(F(4,24) = 16.9; p < 0.001). In contrast, our data showed that hcrt1 had no effect on SWS1
(F(4,24) = 1.2; p > 0.05). As shown in Figure 3, hcrt1 (25 pmol) induced significant changes characterized by a decrease in REM
sleep amounts (95% of control levels; p < 0.05, Fisher's LSD post hoc comparisons) and SWS2 amounts
(33%; p < 0.05, Fisher's LSD) associated with an
increase in wakefulness (+62%; p < 0.05, Fisher's
LSD) during the 0-4 hr period after injection compared with baseline.
A lower dose of hcrt1 (2.5 pmol) also induced a significant decrease in
REM sleep compared with baseline (p < 0.05, Fisher's LSD) but at a lower magnitude (31%, Fisher's LSD)
compared with the higher dose. However, the lower dose induced no
significant changes in the amounts of SWS and wakefulness (Fig.
3B). All these effects were observed during 4 hr of
recording time and tended to recover by the fifth hour (Fig.
3A). No behavioral abnormalities were noticed in rats
injected with hcrt1.
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Figure 3.
Effects of hcrt1 and hcrt2 on the amounts of W,
SWS1, SWS2, and REM sleep after microinjection into the LC.
A, Bars represent the amounts of the
states of vigilance (mean ± SEM) expressed in minutes (saline,
n = 7; hcrt1, 25 pmol, n = 7;
hcrt1, 2.5 pmol, n = 5; hcrt2, 25 pmol,
n = 4; hcrt1, 25 pmol, + antiserum,
n = 4). *Significant difference
(p < 0.05, Fisher's exact test)
from baseline (rats treated with saline; open bars).
B, Representative hypnographs obtained from one rat
during 5 hr after the infusion of the vehicle (bottom)
and 25 pmol of hcrt1 (top) into the right LC.
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We then explored whether changes in the sleep-wake cycle produced by
local application of hcrt1 into the LC area were equally distributed
throughout the 4 hr recording session (Table
1). Injection of 25 pmol of hcrt1
decreased the duration of SWS2 episodes (F(1,14) = 5.9; p < 0.05), increased the duration of W epochs during the first 2 hr
(F(1,14) = 4.9; p < 0.05), and increased the number of W episodes during the 2-4 hr
interval (F(1,14) = 4.6;
p = 0.05). Changes in REM sleep were explained by a
decrease in the number (hcrt1: 25 pmol, 14%; 1-2 hr,
F(1,14) = 62.3; p < 0.001; 2-4 hr, F(1,14) = 21.4;
p < 0.001) and the duration (hcrt1: 25 pmol, 19%;
1-2 hr, F(1,14) = 86.1;
p < 0.001; 2-4 hr,
F(1,14) = 29.2; p < 0.001) of the REM sleep episodes. Latencies to SWS2 and REM sleep were
also significantly increased after injection of 25 pmol of hcrt1 (SWS2,
F(1,16) = 18.9; p < 0.001; REM sleep, F(1,16) = 31.7;
p < 0.001) compared with animals injected with saline
(Table 1). Recordings after 4 hr did not show significant changes
compared with EEGs of control rats or with baseline EEGs. No
significant changes in the duration of the vigilance states were
observed after administration of 25 pmol of hcrt2 compared with saline
(SWS, Fisher's LSD, p > 0.05; REM sleep, Fisher's LSD, p > 0.05; W, Fisher's LSD, p > 0.05; n = 4), and the number of positive responses was
significantly different from that of hcrt1 injection (Fisher's exact
test, p = 0.014; n = 4).
Antibody blockade
In the search for reagents that would block hypocretin activity,
we tested the ability of different antibodies to neutralize hcrt
binding to cells transfected with hcrtr1. A concentration-dependent inhibition of 125I-hcrt1 binding to hcrtr1
expressed in COS7 cells by antibody 2123 was observed (data not shown).
The control antibody did not appreciably decrease
125I-hcrt1 binding to hcrtr1. Antiserum
alone did not modify the amounts of sleep and wakefulness when infused
into the same site as hcrt1 (n = 2; data not shown).
However, preincubation of 25 pmol of hcrt1 with the antiserum
completely prevented the promoting effect on W of hcrt1 as well as the
decrease in SWS2 and REM sleep induced by hcrt1 infusion
(n = 4; data not shown).
Hypocretins are excitatory in the LC in vivo
To determine whether the observed effect of hcrt1 was direct or,
alternatively, through activation of neurons adjacent to the locus
coeruleus, we applied the hcrt1 by iontophoresis in the LC and
performed single-unit recordings in this region. Identification of LC
neurons in vivo was done as previously described
(Aston-Jones and Bloom, 1981; Harley and Sara, 1992). The mean
frequency of the firing rate of LC neurons was significantly increased
by microiontophoretic application of hcrt1 (30 to 50 nA) from
2.95 ± 0.22 Hz during baseline recordings to 4.20 ± 0.96 Hz
(Wilcoxon matched pair test, z = 2.20;
p = 0.03; n = 6; Fig.
4).
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Figure 4.
Activation of hypocretin 1 receptors increases the
firing rate of locus coeruleus neurons. Top tracing,
Filtered recorded signal demonstrating the waveforms of the action
potential. Calibration: 100 µV, 5 msec. Bottom, Rate
meter record demonstrating that in situ
microelectrophoretic application of hypocretin 1 (1 mg/ml) produced a
marked activation of a spontaneously active LC neuron. Calibration: 5 Hz, 5 sec.
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Hypocretins stimulate c-fos in the LC
To determine the extent of neuronal activation on infusion of
hcrt1 in the LC area, we used the immediate early gene product c-fos as an immunocytochemical marker. Rats were infused
with saline (n = 5) and with hcrt1 (n = 6) and killed 60 min after the injection. c-fos-like
immunoreactivity was observed in LC cells in animals treated with
hcrt1, whereas little or no immunoreactivity in this area was observed
after injection of saline (Fig. 5). The
number of c-fos-positive cells was quantified in LC sections from three animals for each condition: 164 ± 11 c-fos-positive cells were found in hcrt1-injected versus
31 ± 13 c-fos-positive cells in saline-treated animals
(Student's t test, p < 0.01;
n = 3). Only a few positive immunoreactive cells were
observed in the cerebral cortex and hypothalamus.
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Figure 5.
Injection of hcrt1 stimulates c-fos
immunoreactivity in the LC. Photomicrographs show c-fos
immunoreactivity in the LC area after injection of saline
(A) or iontophoretic administration of hcrt1
(B). Scale bar, 100 µm.
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DISCUSSION |
The hypocretins have been implicated in a variety of physiological
functions, including feeding (Sakurai et al., 1998), neuroendocrine regulation, and arousal (Chemelli et al., 1999; Hagan et al., 1999; Lin
et al., 1999), as well as in the pathophysiology of narcolepsy. Here we
have shown that administration of hcrt1 but not hcrt2 to the LC has
dramatic effects on sleep architecture. We have detected mRNA
expression and immunoreactivity to hcrtr1, whereas neither mRNA nor
protein expression for hcrtr2 was observed in the LC area. Finally,
administration of hcrt1 increased the firing rate of LC neurons and
induced c-fos in the LC area. These data strongly suggest
that the alterations in sleep architecture induced by hcrt1 are the
consequence of a neuroexcitatory effect on LC neurons through the hcrtr1.
hcrt1 application suppresses REM sleep and leads to a 70% increase in
wakefulness and a 48% decrease in SWS2 after either unilateral or
bilateral injections into the LC area. Bilateral injections in nearby
structures had no effects on the sleep-wakefulness cycle, especially
microinjections into the LDT, a cholinergic nucleus considered a
"REM-on" structure that receives a relatively dense hcrtergic
innervation. Injections into the pontine reticular formation, a nucleus
that does not contain hcrt-immunopositive fibers (Peyron et al., 1998)
and that is in close proximity to the "REM sleep induction zone"
(Ahnaou et al., 1999; Kohlmeier and Reiner, 1999), did not show any
effect. In contrast to published results (Hagan et al., 1999; Piper et
al., 2000), local injections of the hcrts into the fourth ventricle
(without any associated injection site located into the LC area) did
not cause any significant effect on sleep-wakefulness cycle,
presumably because the dose of the locally applied peptide was 100 times lower than the dose used for intracerebroventricular infusion. In
addition, the observed effects were dose-dependent, indicating that the
conditions used in the present study were not saturating. This,
together with blockade of the effects on sleep and wakefulness by
previous incubation with an anti-hcrt-specific antiserum, supports the
specificity of hcrt-induced effects. It is noteworthy that injection of
the lower dose of hcrt1 caused a selective reduction of REM sleep without significantly affecting other states of vigilance, which suggests that the physiological role of hcrt1 is mainly related to REM
sleep, and that the alterations of the sleep-wake cycle seen at the
higher dose are the result of overstimulation of LC noradrenergic cells.
LC neuronal activity is highly related to the status of wakefulness and
REM sleep. It has been known for >20 years that monoaminergic neurons
in the LC area are active during W, decrease their firing rate during
SWS, and are virtually quiescent during REM sleep (Hobson et al., 1975;
Aston-Jones and Bloom, 1981). Unilateral lesion of the LC in cats
enhances REM sleep (Caballero and De Andres, 1986), and stimulation of
cholinergic afferents promotes increased cortical desynchronization
(Berridge and Foote, 1991), whereas 6-hydroxydopamine lesions do not
cause changes in the behavioral state but affect immediate early gene
expression in the cortex (Cirelli et al., 1996). Although
interpretation of these data is difficult because of the complexity of
the neuroanatomical distribution of neurons in this area, the LC is
currently regarded, in part, as a "REM-off" structure in the
proposed model of reciprocal interaction of REM sleep regulation
(Hobson et al., 1975). Thus, our results directly implicate the
hcrtergic system on the regulation of the wakefulness cycle by the LC.
Considering that the raphe nucleus, another REM-off area, also
receives hcrt innervation and expresses hcrt receptors (Trivedi et al.,
1998), further experiments are needed to determine the role of the
hcrts in REM sleep regulation in other brainstem nuclei.
The hypocretins are excitatory in the hypothalamus and spinal cord (de
Lecea et al., 1998; van den Pol, 1999). Here we have shown that
in vivo administration of hcrt1 increases activity of LC
neurons. This is consistent with recent data showing an effect of hcrt1
(Hagan et al., 1999; Horvath et al., 1999) on LC slices in
vitro and with data demonstrating that mild electrical stimulation
of the LC suppresses REM sleep (Singh and Mallick, 1996). However, in
contrast to a previous study describing stimulation of LC neurons
in vitro by hcrt2 (Horvath et al., 1999), we did not observe
physiological responses to LC administration of hcrt2. Because the
in vitro assay was done in saturating concentrations of the
peptide, the excitatory effect of hcrt2 on LC slices may be mediated
through hcrtr1.
The excitatory effect of hcrt1 raises the question of which cell type
is activated in the LC area. Increased cell firing reported in this
study may be assigned to noradrenergic cells because of their
predominant distribution within this area and their
electrophysiological firing pattern. Also, TH mRNA and norepinephrine
concentration are increased in the LC after 72 hr of REM sleep
deprivation (Porkka-Heiskanen et al., 1995), and the percentage of REM
sleep negatively correlates with noradrenergic (NA)
c-fos-positive cells (Maloney et al., 1999). Moreover,
studies in cats have recently shown that norepinephrine application on
peri-LC , an area next to the LC, suppresses REM sleep with an
increase in W and a decrease in SWS (Crochet and Sakai, 1999). Despite
the large amount of data implicating NA cells in the modulation of REM
sleep, it is possible that hcrt1 stimulates cholinergic cells in the
area, which are also known to promote wakefulness. Indeed, cholinergic
and noradrenergic cells are in close proximity in the LC area and are
critical for wakefulness promotion. However, generation of the state of
REM sleep may depend on the cessation of NA LC neurons and simultaneous activation of cholinergic neighboring neurons (Jones, 1991). Thus, it
is likely that the REM suppressor effect of hcrt1 reported here is the
consequence of a stimulation of NA cells in the LC area and not of a
stimulation of cholinergic cells. Moreover, our data showing TH
staining of hcrtr1-positive cells support the idea that the observed
effects are attributable to activation of NA neurons.
Interestingly, the effects of hcrt1 reported here are reciprocal to
those observed in hcrt/orexin knock-out mice (Chemelli et al., 1999),
in which REM and SW sleep are increased during the dark period, whereas
wakefulness is decreased. The observed phenotype of hcrt deficiency
could be explained by lack of hcrtergic activation of LC neurons and
suggests that the LC area is a key target for the hcrts to regulate the
sleep-wakefulness cycle. LC neurons cease to fire during cataplexy (Wu
et al., 1999), suggesting that the signaling mechanisms in LC neurons
are impaired in narcolepsy. Narcoleptic patients show a dramatic
increase in sleep fragmentation, although sleep homoeostasis is
relatively well preserved during the dark period (Nishino and Mignot,
1997). Thus, hcrts might be required to modulate the firing of REM-off
neurons in the brainstem and to stabilize sleep transitions. Absence of
hcrt signaling in narcolepsy may thus result in disregulation of the
mechanism that establishes boundaries between sleep states.
The lack of effect of hcrt2 administration in the LC area and the
absence of hcrtr2 immunoreactivity or hybridization signals strongly
suggest that hcrt1 regulates REM sleep through interaction with hcrtr1.
The role of hcrtr2, which has been shown to be responsible for canine
narcolepsy and is not expressed in the LC in rats, remains to be
determined. Also, this raises the question of the possible
relationship between hcrtr1 and human narcolepsy. Further studies will
be required to assess the roles of the hcrt receptors and the brain
regions involved in different components of the disease (i.e.,
cataplexy, sleep fragmentation, and REM sleep attacks).
Together, our data indicate that hcrt1 in the LC area modulates REM
sleep by acting on the firing rate of noradrenergic neurons through
hcrtr1 receptors and suggest that the LC is a key target of the
hcrtergic system for sleep-wakefulness cycle regulation. However, the
possible implication of hcrtergic transmission within the LC area in
the pathophysiological mechanisms of human narcolepsy remains to be explored.
| |
FOOTNOTES |
Received Feb. 4, 2000; revised July 28, 2000; accepted July 28, 2000.
This work was supported by National Institutes of Health Grants MH58543
(L.d.L.), GM32355 (J.G.S.), DA12669 (J.R.C.), and DA08301 (S.J.H.).
P.B. was supported by Fondation Simone et Cino del Duca and Fondation
Phillippe. B.M. was supported by a fellowship from Comunidad de Madrid.
We thank Angels Almenar for help with immunofluorescence and Juliette
Hinsche for technical assistance.
Correspondence should be addressed to Dr. Luis de Lecea, Department of
Molecular Biology, MB-10, The Scripps Research Institute, 10550 North
Torrey Pines Road, La Jolla, CA 92037. E-mail: llecea{at}scripps.edu.
| |
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Am J Physiol Regulatory Integrative Comp Physiol,
November 1, 2002;
283(5):
R1079 - R1086.
[Abstract]
[Full Text]
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L. Bayer, E. Eggermann, B. Saint-Mleux, D. Machard, B. E. Jones, M. Muhlethaler, and M. Serafin
Selective Action of Orexin (Hypocretin) on Nonspecific Thalamocortical Projection Neurons
J. Neurosci.,
September 15, 2002;
22(18):
7835 - 7839.
[Abstract]
[Full Text]
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M. Wu, Z. Zhang, C. Leranth, C. Xu, A. N. van den Pol, and M. Alreja
Hypocretin Increases Impulse Flow in the Septohippocampal GABAergic Pathway: Implications for Arousal via a Mechanism of Hippocampal Disinhibition
J. Neurosci.,
September 1, 2002;
22(17):
7754 - 7765.
[Abstract]
[Full Text]
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B. Yang and A. V. Ferguson
Orexin-A Depolarizes Dissociated Rat Area Postrema Neurons through Activation of a Nonselective Cationic Conductance
J. Neurosci.,
August 1, 2002;
22(15):
6303 - 6308.
[Abstract]
[Full Text]
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L. I. Kiyashchenko, B. Y. Mileykovskiy, N. Maidment, H. A. Lam, M.-F. Wu, J. John, J. Peever, and J. M. Siegel
Release of Hypocretin (Orexin) during Waking and Sleep States
J. Neurosci.,
July 1, 2002;
22(13):
5282 - 5286.
[Abstract]
[Full Text]
[PDF]
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M.-C. Xi, S. J. Fung, J. Yamuy, F. R. Morales, and M. H. Chase
Induction of Active (REM) Sleep and Motor Inhibition by Hypocretin in the Nucleus Pontis Oralis of the Cat
J Neurophysiol,
June 1, 2002;
87(6):
2880 - 2888.
[Abstract]
[Full Text]
[PDF]
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A. N van den Pol, P. K Ghosh, R.-j. Liu, Y. Li, G. K Aghajanian, and X.-B. Gao
Hypocretin (orexin) enhances neuron activity and cell synchrony in developing mouse GFP-expressing locus coeruleus
J. Physiol.,
May 15, 2002;
541(1):
169 - 185.
[Abstract]
[Full Text]
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B. Y. Mileykovskiy, L. I. Kiyashchenko, and J. M. Siegel
Muscle Tone Facilitation and Inhibition After Orexin-A (Hypocretin-1) Microinjections Into the Medial Medulla
J Neurophysiol,
May 1, 2002;
87(5):
2480 - 2489.
[Abstract]
[Full Text]
[PDF]
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S. Burlet, C. J. Tyler, and C. S. Leonard
Direct and Indirect Excitation of Laterodorsal Tegmental Neurons by Hypocretin/Orexin Peptides: Implications for Wakefulness and Narcolepsy
J. Neurosci.,
April 1, 2002;
22(7):
2862 - 2872.
[Abstract]
[Full Text]
[PDF]
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M. Tafti and P. Franken
Functional Genomics of Sleep and Circadian Rhythm: Invited Review: Genetic dissection of sleep
J Appl Physiol,
March 1, 2002;
92(3):
1339 - 1347.
[Abstract]
[Full Text]
[PDF]
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M. N. Alam, H. Gong, T. Alam, R. Jaganath, D. McGinty, and R. Szymusiak
Sleep-waking discharge patterns of neurons recorded in the rat perifornical lateral hypothalamic area
J. Physiol.,
January 15, 2002;
538(2):
619 - 631.
[Abstract]
[Full Text]
[PDF]
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L. I. Kiyashchenko, B. Y. Mileykovskiy, Y.-Y. Lai, and J. M. Siegel
Increased and Decreased Muscle Tone With Orexin (Hypocretin) Microinjections in the Locus Coeruleus and Pontine Inhibitory Area
J Neurophysiol,
May 1, 2001;
85(5):
2008 - 2016.
[Abstract]
[Full Text]
[PDF]
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T. C. Chou, C. E. Lee, J. Lu, J. K. Elmquist, J. Hara, J. T. Willie, C. T. Beuckmann, R. M. Chemelli, T. Sakurai, M. Yanagisawa, et al.
Orexin (Hypocretin) Neurons Contain Dynorphin
J. Neurosci.,
October 1, 2001;
21(19):
RC168 - RC168.
[Abstract]
[Full Text]
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M. N. Alam, H. Gong, T. Alam, R. Jaganath, D. McGinty, and R. Szymusiak
Sleep-waking discharge patterns of neurons recorded in the rat perifornical lateral hypothalamic area
J. Physiol.,
January 15, 2002;
538(2):
619 - 631.
[Abstract]
[Full Text]
[PDF]
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TOP
ABSTRACT
INTRODUCTION
