Selective Activation of the Extended Ventrolateral Preoptic Nucleus
during Rapid Eye Movement Sleep
Jun Lu,1Alvhild A. Bjorkum,1,2Man Xu,1Stephanie E. Gaus,1Priyattam J. Shiromani,3and Clifford B. Saper1
1Department of Neurology, Beth Israel Deaconess Medical Center, Boston, Massachusetts 02215,2Department of
Physiology, University of Bergen, Bergen N-5009, Norway, and3Department of Neurology, Veterans Affairs Medical
Center, West Roxbury Harvard Medical School, Boston, Massachusetts 02132
We found previously that damage to a cluster of sleep-active
neurons (Fos-positive during sleep) in the ventrolateral preoptic
nucleus (VLPO) decreases non-rapid eye movement (NREM)
sleep in rats, whereas injury to the sleep-active cells extending
dorsally and medially from the VLPO cluster (the extended
VLPO) diminishes REM sleep. These results led us to examine
whether neurons in the extended VLPO are activated during
REM sleep and the connectivity of these neurons with pontine
sites implicated in producing REM sleep: the laterodorsal teg-
mental nucleus (LDT), dorsal raphe nucleus (DRN), and locus
ceruleus (LC). After periods of dark exposure that triggered
enrichment of REM sleep, the number of Fos-positive cells in
the extended VLPO was highly correlated with REM but not
NREM sleep. In contrast, the number of Fos-positive cells in the
VLPO cluster was correlated with NREM but not REM sleep.
Sixty percent of sleep-active cells in the extended VLPO and
90% of sleep-active cells in the VLPO cluster in dark-treated
animals contained galanin mRNA. Retrograde tracing from
the LDT, DRN, and LC demonstrated more labeled cells in the
extended VLPO than the VLPO cluster, and 50% of these in
the extended VLPO were sleep-active. Anterograde tracing
showed that projections from the extended VLPO and VLPO
cluster targeted the cell bodies and dendrites of DRN sero-
toninergic neurons and LC noradrenergic neurons but were not
apposed to cholinergic neurons in the LDT. The connections
and physiological activity of the extended VLPO suggest a
specialized role in the regulation of REM sleep.
Key words: laterodorsal tegmental nucleus; dorsal raphe nu-
cleus; locus ceruleus; galanin; GABA; c-Fos
Recent studies show that the ventrolateral preoptic nucleus
(VLPO) contains a cluster of sleep-active neurons (Sherin et al.,
1996, 1998; Szymusiak et al., 1998). These sleep-active cells
contain galanin and GABA (Sherin et al., 1998; Gaus et al., 2002)
and project to many components of the arousal system including
the histaminergic tuberomammillary nucleus (TMN), the sero-
toninergic dorsal raphe nucleus (DRN), and the noradrenergic
locus ceruleus (LC) (Sherin et al., 1998; Steininger et al., 2001),
suggesting that they may inhibit the ascending monoaminergic
arousal system during sleep. After preoptic lesions, loss of neu-
rons in the VLPO cluster correlates closely with the loss of
non-rapid eye movement (NREM) sleep (Lu et al., 2000). Al-
though REM sleep is also diminished after lesions of the VLPO
region, this is not correlated with the loss of cells in the VLPO
cluster, suggesting that cells nearby but outside the VLPO cluster
regulate REM sleep. Numerous galaninergic cells are located
dorsally and medially to the VLPO cluster (the extended
VLPO), and many of them are sleep-active (Fos-positive) during
sleep and are galaninergic (Gaus et al., 2002). We found that loss
of sleep-active cells in the extended VLPO is correlated with a
decrease in REM sleep but not NREM sleep (Lu et al., 2000).
This hypothetical role of the extended VLPO cells in REM
sleep control is consistent with the observations that some cells in
the VLPO region fire faster during REM sleep compared with
during NREM sleep or wakefulness (Koyama and Hayaishi,
1994; Osaka and Matsumura, 1994; Szymusiak et al., 1998). How-
ever, these single-unit recording studies could not determine
whether the REM-active neurons were galaninergic. In addition,
very little is known about the projections of the extended VLPO
neurons compared with the VLPO cluster.
To address these questions, we developed a model for iden-
tifying anatomically the REM active neurons in the VLPO
area. Previous studies had found that exposing rats to darkness
during the daily light cycle when they are usually asleep trig-
gers REM sleep (Alfoldi et al., 1991; Benca et al., 1991, 1998;
Miller et al., 1998). We therefore correlated the number of
sleep-active cells in the extended VLPO with REM sleep
during dark exposure. We also determined by the combination
of anterograde and retrograde tracing with immunocytochem-
istry and in situ hybridization whether the sleep-active cells in
the extended VLPO during periods of enhanced REM sleep
contain galanin and project to pontine sites believed to control
production of REM sleep [i.e., the laterodorsal tegmental
nucleus (LDT), DRN, and LC].
MATERIALS AND METHODS
Pathogen-free adult male Sprague Dawley rats (275–300 gm; n ? 82)
purchased from Harlan Sprague Dawley (Indianapolis, IN) were used.
The rats were individually housed and had access to food and water ad
libitum. All animals were housed under controlled conditions (12 hr of
light starting at 7:00 A.M.; 200 lux) in an isolated ventilated chamber
Received Aug. 20, 2001; revised March 12, 2002; accepted March 13, 2002.
This work was supported by United States Public Health Service Grants NS3397,
HL60292, AG47755, and MH12058. We thank Quan Ha, Alex Adler, and Minh Ha
for technical assistance.
Correspondence should be addressed to Dr. Clifford B. Saper, Department of
Neurology, Beth Israel Deaconess Medical Center, 330 Brookline Avenue, Boston,
MA 02215. E-mail: firstname.lastname@example.org.
Copyright © 2002 Society for Neuroscience 0270-6474/02/224568-09$15.00/0
The Journal of Neuroscience, June 1, 2002, 22(11):4568–4576
maintained at 20–22°C. All protocols were approved by the Institutional
Animal Care and Use Committees of Beth Israel Deaconess Medical
Center and Harvard Medical School.
Implantation for EEG/EMG. After animals were anesthetized with chlo-
ral hydrate, the skull was exposed. Four screw electrodes were implanted
into the skull, in the frontal (two electrodes) and parietal (two elec-
trodes) bones of each side, and two flexible wire electrodes were placed
in the nuchal muscles. The incisions were closed by wound clips. The
electrodes were soldered to sockets that were connected via flexible
recording cables and a commutator to a Grass polygraph (Grass Instru-
ments, West Warwick, RI) and a computer.
Dark treatment and EEG recording. After 1 week on a 12 hr light/dark
schedule (lights on at 7:00 A.M.), including 2 d of adaptation to the
EEG/EMG cables, animals were exposed to extended darkness or nor-
mal light from 7:00 A.M. to 10:00 A.M. EEG/EMG was continuously
recorded during this period. After the dark or light exposure, animals
were killed by vascular perfusion (see below).
Sleep analyses. The EEG/EMG signals were amplified by a poly-
graph (Grass Instruments) and digitized by an Apple Macintosh (Cu-
pertino, CA) power computer running Icelus (G Systems Inc., Plano,
TX). Wake–sleep states were manually scored in 12 sec epochs on the
digitized EEG/EMG. Wakefulness was identified by the presence of a
desynchronized EEG and phasic EMG activity. NREM sleep con-
sisted of a high-amplitude slow-wave EEG together with a low-EMG
tone relative to waking. REM sleep was identified by the presence of
regular theta activity coupled with low-EMG tone relative to NREM sleep.
The amount of time spent in a wake state, NREM sleep, and REM sleep
was determined for each hour.
Histology and tracing
Tracer injections. Under chloral hydrate anesthesia (7% in saline; 350
mg/kg), a fine glass pipette containing a 2.5% solution of the anterograde
tracer Phaseolus vulgaris leucoagglutinin (PHA-L) in saline was lowered
into the extended VLPO and VLPO cluster stereotaxically [anteropos-
terior (AP), ?0.6 mm; dorsoventral (DV), ?8.5 mm; mediolateral (ML),
?1.0 mm; with the bitebar at ?3.3 mm (Paxinos and Watson, 1986)].
PHA-L was injected by iontophoresis with a current of 5 ?A for 15 min
(7 sec on and 7 sec off) to give a discrete injection site (?200–300 ?m in
diameter). After 2 additional min, the pipette was slowly withdrawn.
Cholera toxin subunit B (CTB; List Biological, Campbell, CA) was
injected before implantation for EEG/EMG using an air-pressure deliv-
ery system to inject 6 nl of CTB solution (1% in saline). CTB targets
included the central DRN (AP, ?7.80 mm; ML, 0 mm; DV, ?5.6 mm),
the lateral wing of the DRN (AP, ?7.80 mm; ML, 0.4 mm; DV, 5.6 mm),
the LDT (AP, ?8.0 mm; ML, 0.8 mm; DV, 5.8 mm), and the LC (AP,
?9.30 mm; ML, 1.0 mm; DV, 6.0 mm). Incisions were closed with wound
clips and animals survived for 7 d.
Perfusion and histology. Animals were deeply anesthetized by chloral
hydrate (350 mg/kg) and then perfused through the heart with 50 ml of
saline followed by 500 ml of 10% formalin. The brains were removed and
postfixed in 10% formalin for 4 hr and then equilibrated in 20% sucrose
in PBS at 4°C.
Immunohistochemistry. The brains were sectioned on a freezing mic-
rotome into four series (40 ?m thickness) for immunostaining only or
five (30 ?m thickness) series of sections for immunostaining plus in situ
hybridization. Sections were washed in PBS (three changes) and incu-
bated in PBS containing 0.3% Triton X-100 (PBT) and 0.2% sodium
azide for 1 hr at room temperature, followed by the primary antiserum:
Fos, Ab-5, 1:150,000, rabbit (Oncogene Sciences, Uniondale, NY); CTB,
1:100,000, goat (List Biological); PHA-L, 1:10,000, goat (Vector Labo-
ratories, Burlingame, CA); choline acetyltransferase (ChAT), 1:20,000,
rabbit UO93 (gift from Dr. Louis Hersh, University of Kentucky);
tyrosine hydroxylase (TH), 1:200, TE-101, rabbit (Eugene Tech, Eugene,
OR); 5-HT, 1:10,000, goat (Chemicon, Temecula, CA) for 1 d at room
temperature. Sections were then washed in PBS and incubated in biotin-
ylated secondary antiserum (against appropriate species IgG; 1:1000 in
PBT) for 1 hr, washed again in PBS, and incubated in avidin–biotin
complex reagents for 1 hr. Sections were then washed again and incu-
bated in a 0.06% solution of 3,3-diaminobenzidine tetrahydrochloride
(DAB; Sigma, St. Louis, MO) plus 0.02% H2O2.The sections were
stained brown with DAB only or black by adding 0.05% cobalt chloride
and 0.01% nickel ammonium sulfate to the DAB solutions. Fos and
PHA-L staining was done using the black reaction, and CTB, ChAT,
5-HT, and TH staining was done using the brown reaction, as described
previously (Chou et al., 2002). For double labeling of Fos and galanin
mRNA, sections were stained for Fos with the brown reaction and then
used for in situ hybridization.
In situ hybridization. The sections (30 ?m thickness) with Fos staining
were acetylated and hybridized overnight (55°C) with a
cRNA probe synthesized from a plasmid containing the complete coding
sequence of rat galanin (Vrontakis et al., 1987). After washing, the tissue
was treated with RNase-A (Boehringer Mannheim, Indianapolis, IN)
followed by a succession of washes of increasing stringency (1 hr each in
2? SSC/1 mM DTT, 50°C; 0.2? SSC/1 mM DTT, 55°C; 0.2? SSC/1 mM
DTT, 60°C), and then dehydrated in alcohols and air-dried. The sections
were exposed to x-ray film (Eastman-Kodak, Rochester, NY) for 2–3 d,
and then the slides were dipped in Kodak NTB-2 emulsion and exposed
for 1 month. Slides were developed in Kodak D-19, fixed, and then
dehydrated and coverslipped.
Cell counting. Fos-positive cells were counted in brightfield by an
observer who was blinded to physiological treatment, in three consec-
utive sections (AP level: from ?0.3 mm to ?0.7 mm) through the
midpart of the VLPO and extended VLPO. The spacing between the
sections was 160 ?m. For experiments in which there was no galanin-
staining present to define the borders of the VLPO, we constructed a
set of counting boxes based on the distribution of galaninergic neurons:
the VLPO cluster box was 300 ?m wide by 300 ?m high, placed along
the base of the brain just medial to the diagonal band of Broca, as
shown in Figure 1B. The medial extended VLPO box was medial to
the VLPO cluster and 400 ?m wide by 300 ?m high (Fig. 1A); the
dorsal extended VLPO box was 200 ?m wide by 300 ?m high, posi-
tioned above the VLPO cluster and medial extended VLPO boxes,
and centered over their border. Because the mean diameter of Fos-
labeled nuclei was not different between experimental groups and
controls, and we were interested only in relative cell numbers; we did
not apply a correction factor for double counting (Guillery and Her-
We used Student’s two-tailed t test to compare the differences in the
amounts of NREM and REM sleep time and in the numbers of Fos-
positive and double-labeled cells in the extended VLPO and VLPO
cluster, under control conditions and after dark treatment.
For correlational analysis, the mean number of Fos-immunoreactive
(Fos-ir) cells (per section per side) in the extended VLPO or the VLPO
cluster was plotted against amounts of NREM sleep or REM sleep
during the hour before perfusion. Pearson correlation coefficients and p
values were calculated.
The relationship of Fos-positive neurons in the
extended VLPO and sleep
To determine the relationship of the Fos-ir cells in the extended
VLPO and REM sleep, on the day of the experiment we exposed
animals (n ? 19) to dark from 7:00 A.M. to 10:00 A.M. (dark
treatment) after 1 week on a 12 hr light/dark cycle. Control
animals (n ? 10) were exposed to light from 7:00 A.M. to 10:00
A.M. as usual. Dark treatment increased REM sleep consistently
across the 3 hr period. NREM sleep occupied 65.2 ? 4.2%
(mean ? SEM) and REM sleep occupied 13.1 ? 4.9% of the first
hour; NREM was 60.2 ? 4.5% and REM was 16.1 ? 2.1% in the
second hour; and NREM was 56.1 ? 4.5% and REM was 17.1 ?
3.4% of the third hour. Over the third hour, dark treatment
increased REM sleep by twofold over controls (dark, 17.1 ?
3.4%; light, 8.5 ? 3.1%; p ? 0.001),but had no significant effect on
NREM sleep (dark, 56.1 ? 4.5%; light, 62.2 ? 8.4%; p ? 0.05).
Dark-treated animals had a significant increase in the number
of Fos-ir cells compared with controls in the medial extended
VLPO (22.9 ? 1.3 vs 12.8 ? 1.0 cells ? section?1? side?1; p ?
0.01) and dorsal extended VLPO (13 ? 1.3 vs 6.6 ? 1.7
cells ? section?1? side?1; p ? 0.01). However, the number of
Fos-ir cells in the VLPO cluster did not differ (dark treatment,
Lu et al. • Extended VLPO and REM SleepJ. Neurosci., June 1, 2002, 22(11):4568–4576 4569
20.4 ? 5.3 cells ? section?1? side?1vs controls, 18.0 ? 5.5
cells ? section?1? side?1; p ? 0.05).
Putting together the light- and dark-treated animals, the num-
ber of Fos-ir cells in the medial extended VLPO (r ? 0.76; p ?
0.01) or dorsal extended VLPO (r ? 0.71; p ? 0.01) was signif-
icantly correlated with REM sleep during the previous hour but
was not significantly correlated with NREM sleep (r ? 0.12, 0.34;
p ? 0.05) (Fig. 2). In contrast, the number of VLPO cluster cells
(12% REM sleep, A) and in an animal exposed to dark treatment (30% REM sleep, B) during the early part of the sleep cycle. The counting boxes used
for the VLPO cluster and the dorsal and medial extended VLPO are shown in B. These sections are approximately at the level of AP ?0.5 in Paxinos
and Watson (1986). OC, Optic chiasm.
A pair of photomicrographs showing the distributions of Fos-ir cells in the extended VLPO and VLPO cluster in an animal exposed to light
with the amounts of REM sleep or NREM sleep that the animals experienced during the hour before perfusion.
Correlation (illustrated by solid line) of the number of Fos-ir cells in the extended VLPO and VLPO cluster in light- and dark-treated animals
4570 J. Neurosci., June 1, 2002, 22(11):4568–4576Lu et al. • Extended VLPO and REM Sleep
was not significantly correlated with REM sleep (r ? 0.07; p ?
0.05) (Fig. 2); however, it was significantly correlated with NREM
sleep (r ? 0.76; p ? 0.01) (Fig. 2).
Sleep-active cells in the extended VLPO and VLPO
cluster of dark-treated animals contain galanin
The relationship of Fos-ir cells and neurons containing galanin
mRNA in the extended VLPO was determined in rats that
received dark treatment (n ? 10) versus controls (n ? 7) (Fig. 3).
In the dark-treated animals, 90.2 ? 8.2% of the Fos-positive cells
in the VLPO cluster and 60.5 ? 6.1% of Fos cells in the extended
VLPO contained galanin mRNA. In control rats, a high percent-
age of Fos-ir cells also contained galaninergic mRNA in the
VLPO cluster (93.2 ? 9.5%) and the extended VLPO (80.7 ?
Relationship of Fos-positive and retrogradely labeled
cells after injections of CTB into the LDT, LC, and DRN
after dark treatment
To determine whether the cells in the extended VLPO and
VLPO cluster that project to the LDT, DRN, and LC expressed
Fos during REM sleep, we injected the retrograde tracer CTB
into the region containing the LDT (n ? 10), LC (n ? 12), or
DRN (central area, n ? 10; lateral wing, n ? 9). The animals were
then perfused as described above after dark treatment, and the
brains were stained for CTB (brown) and Fos (black). All cell
counts were done on the ipsilateral side of the brain, which
contained the heaviest retrograde labeling. Animals in this series
were analyzed if they spent ?50% of the hour before death in
NREM sleep and ?10% in REM sleep.
In seven rats that had injections into the LDT (Fig. 4), many
more CTB-labeled cells were found in the extended VLPO
(5.8 ? 2.5 cells/ipsilateral section; p ? 0.05) than in the VLPO
cluster (2.8 ? 0.9 cells/ipsilateral section). Of CTB-labeled cells,
62.2 ? 19.5% in the extended VLPO and 95.0 ? 5.0% in the
VLPO cluster were Fos-positive (Fig. 5). Three control rats in
which the CTB injections were dorsal to the LDT showed no
retrogradely labeled cells in either the extended VLPO or the
Five of 12 injections aimed at the LC largely filled the nucleus;
in the rest of the animals, the injections were medial, lateral, or
ventral to the LC, and these rats were used as anatomical controls.
In the five sleeping rats with injections filling the LC, more
CTB-labeled cells were found in the extended VLPO than in the
VLPO cluster (6.5 ? 1.5 vs 3.0 ? 1.4 cells/ipsilateral section; p ?
0.05). In these dark-treated animals, 46.6 ? 7.4% of the CTB-
labeled cells in the extended VLPO and 56.0 ? 3.1% of CTB-
labeled cells in the VLPO cluster were Fos-positive (Fig. 5). We
hybridization) in the extended VLPO and VLPO cluster. Arrowheads indicate double-labeled cells. Many Fos-ir cells in the extended VLPO and
particularly in the VLPO cluster contain galanin mRNA. A shows the VLPO complex with boxes that are shown at higher magnification in B–D. Note
that these fields are not equivalent to the counting boxes shown in Figure 1. OC, Optic chiasm.
Photomicrographs showing dual labeling of Fos (brown immunostaining) and galanin mRNA (black silver grains representing in situ
Lu et al. • Extended VLPO and REM SleepJ. Neurosci., June 1, 2002, 22(11):4568–4576 4571
also noticed CTB-labeled cells scattered in the medial preoptic
area and dorsolateral preoptic area, but these cells were not
Fos-positive during sleep (Fig. 5). The injections in the areas
immediately medial (R2103), lateral (R2091 and R2099), or ven-
tral (R2090 and R2098) to the LC did not retrogradely label cells
in the extended VLPO or the VLPO cluster (Fig. 4).
In 10 rats with injections of CTB into the central part of the
DRN, more retrogradely labeled cells were found in the extended
VLPO than in the VLPO cluster (5.5 ? 2.1 vs 2.4 ? 1.1 cells/
ipsilateral section; p ? 0.05). Only 15.0 ? 8.2% of CTB-labeled
cells in the extended VLPO and 12.5 ? 4.5% of CTB-labeled
cells in the VLPO cluster were Fos-positive (Fig. 5). We found
numerous CTB-labeled cells in the diagonal band nucleus, the
median preoptic nucleus, and the dorsolateral preoptic area, but
very few cells in these regions were Fos-positive during sleep.
Four rats that had CTB injections into the lateral wing of the
dorsal raphe nucleus (also called the paradorsal raphe nucleus;
see injection 2203 in Fig. 4A) and were asleep for most of the
hour before perfusion (NREM, 53.5 ? 5.6%; REM, 18.2 ? 4.3%)
showed retrogradely labeled cells in the extended VLPO (3.0 ?
0.8 cells/ipsilateral section) and VLPO cluster (3.0 ? 0.81 cells/
ipsilateral section). In these animals, a large percentage of retro-
gradely labeled neurons was also Fos-positive in the VLPO
cluster (64.2 ? 27.4%) and extended VLPO (56.5 ? 7.0%). Two
rats that had injections ventral to the lateral wing of the DRN
(experiments R2222 and R2223) (Fig. 4A) showed no double-
labeled cells in the VLPO cluster and very few in the extended
VLPO. Two other rats that had injections into the lateral wing of
the DRN and ?70% wakefulness during the hour before perfu-
sion also showed no double-labeled cells in the extended VLPO
and VLPO cluster.
Anterograde tracing of inputs from the extended VLPO
and VLPO cluster to the LDT, DRN, and LC
To determine the relationship of efferents from the extended
VLPO and VLPO cluster to chemically identified neurons in the
LDT, DRN, and LC, PHA-L was injected into the extended
VLPO and the VLPO cluster in five rats, and sections through
the brainstem were immunostained for PHA-L (black) axons and
with antisera against 5-HT, ChAT, or TH (brown).
The VLPO complex was successfully labeled in four cases: two
involving the cluster plus part of the extended VLPO and two
involving primarily the extended VLPO (Fig. 4D). All of them
gave rise to a similar pattern of efferent projection. In case R2133,
the PHA-L injection filled the VLPO cluster and dorsal extended
VLPO and partially filled the medial extended VLPO. Labeled
efferents terminated extensively in the LDT, LC, and DRN, as
well as in the median raphe nucleus.
In the LDT, labeled axons primarily terminated in the dorsal
LDT region. The efferent terminal field only partially overlapped
with the region occupied by cholinergic cells. Double staining
showed that labeled terminals did not form appositions with the
ChAT-ir cell bodies or proximal dendrites in the LDT, but rather
appeared to terminate in the region between the cholinergic cell
bodies (Fig. 6). Using Nissl staining, we found that some termi-
nals apposed small-sized cells in the LDT region. Compared with
the LDT, relatively few labeled axons terminated in the cholin-
ergic pedunculopontine tegmental nucleus (PPT) and the efferent
terminals did not appose cholinergic cells.
In the LC, terminal boutons were located predominantly in the
ventral LC, especially in the region containing a dense bundle of
LC (C) as well as four injection sites of PHA-L in the VLPO region (D). Gray shading shows borders of key nuclei; gray lines show other brain structures.
Br, Barrington’s nucleus; HDB, horizontal limb of the nucleus of the diagonal band of Broca; mlf, medial longitudinal fasciculus; MnRn, median raphe
nucleus; OC, optic chiasm; scp, superior cerebellar peduncle; VTg, ventral tegmental nucleus.
Camera lucida drawings showing selected injection sites (black solid and dashed lines) of CTB in the region of the DRN (A), LDT (B), and
4572 J. Neurosci., June 1, 2002, 22(11):4568–4576Lu et al. • Extended VLPO and REM Sleep
noradrenergic dendrites, where many boutons apposed TH-ir
dendrites. In addition, labeled terminals also were apposed to the
noradrenergic cell bodies in the core of the LC (Fig. 6).
In the DRN, efferent terminals were heavily distributed through-
out the nucleus and especially in its lateral wing. Double staining
demonstrated that many labeled terminals apposed serotoninergic
cell bodies and dendrites in the DRN (Fig. 6), although many
terminated on unlabeled (presumably nonserotoninergic) cells. La-
beled axons also terminated heavily in the ventrolateral periaque-
ductal gray matter (PAG), dorsolateral to the lateral wing of the
Cases in which the injections involved the extended VLPO but
avoided the VLPO cluster (e.g., experiments R2132 and R2134)
(Fig. 4D) showed very similar distributions of efferent terminals
with respect to appositions onto the monoaminergic and cholin-
ergic neurons in the midbrain and pons. The only major exception
was a relatively smaller number of terminals onto serotoninergic
neurons in the central DRN in these cases.
Our principal findings were that in dark-treated rats with in-
creased amounts of REM sleep, the number of Fos-positive cells
in the extended VLPO was positively correlated with REM sleep
time but was not correlated with NREM sleep time. In contrast,
the number of Fos-positive cells in the VLPO cluster was posi-
tively correlated with NREM sleep time but was not correlated
with REM sleep time. A high percentage of Fos-positive cells in
the VLPO cluster (?90%) and the extended VLPO (?60%)
contained galanin mRNA. By injecting retrograde tracer into the
LDT and LC, we found that the extended VLPO of dark-treated
rats contained more retrogradely labeled cells than the VLPO
cluster, and that ?50% of the labeled cells were Fos-positive in
dark-treated sleeping animals. Although we found that numerous
retrogradely labeled cells in the extended VLPO project to the
central part of the DRN, a relatively small number of them
(12–15%) were Fos-positive during sleep in dark-treated animals.
However, injections of CTB into the lateral wing of the DRN
yielded more retrogradely labeled and double-labeled cells (CTB
plus Fos) in the VLPO cluster (64%). Anterograde tracing
showed that the efferents of the extended VLPO and VLPO
cluster apposed the serotoninergic cell bodies and proximal den-
drites in the DRN especially in the lateral wing region and the
noradrenergic cell bodies in the LC, but there were few apposi-
tions with the cholinergic cells in the LDT or PPT. We hypoth-
H) after the injections of CTB into the central DRN (A, experiment R2049), LDT (D, experiment R1969), and LC (G, experiment R2219), respectively,
in the rats exposed to 3 hr of darkness. In B, E, and H, the VLPO cluster is identified by a large arrow and the extended VLPO by a large double-headed
arrow. The rectangular boxes identify the fields that are magnified in C, F, and I, respectively. Double-labeled cells in the extended VLPO and VLPO
cluster are indicated by small arrows and single CTB labeled cells are indicated by small arrowheads.
Dual labeling of CTB retrograde transport (brown) and Fos expression (black) in cells within the extended VLPO and VLPO cluster (B, E,
Lu et al. • Extended VLPO and REM SleepJ. Neurosci., June 1, 2002, 22(11):4568–4576 4573
esize that the extended VLPO may enhance REM sleep by its
influence on the LDT, DRN, and LC.
Four methods have been used previously to study brain activity
during enhanced REM sleep in rats and cats: auditory stimula-
tion, recovery from REM sleep deprivation (platform method),
injection of carbachol into the brainstem, and dark treatment.
Using the first two methods, Merchant-Nancy et al. (1995) found
significant Fos induction in the brainstem (nucleus of the solitary
tract, LDT, PPT, and parabrachial nucleus) and diencephalon
(suprachiasmatic nucleus, lateral hypothalamus), and amygdaloid
nucleus. Injection of carbachol into a region of the pontine
reticular formation that receives the LDT/PPT cholinergic effer-
ents (Semba et al., 1990) causes prolonged and persistent REM
sleep in cats and rats (Shiromani et al., 1992, 1995; Marks and
Birabil, 1998; Horner and Kubin, 1999) and produces Fos in both
cholinergic and noncholinergic cells such as GABAergic ones in
the LDT and PPT (Shiromani et al., 1992, 1995; Yamuy et al.,
1998; Xi et al., 1999; Torterolo et al., 2001). Similar Fos expres-
sion patterns in the LDT/PPT have also been observed during
recovery from REM sleep deprivation (Maloney et al., 1999). In
contrast, Fos immunoreactivity was not expressed in the sero-
toninergic DRN cells and noradrenergic LC cells during REM
sleep (Maloney et al., 1999). These results are consistent with the
hypothesis that REM sleep is triggered by activity of cholinergic
LDT/PPT neurons, occurring in association with inactivity of the
monoaminergic cells in the DRN and LC (Maloney et al., 1999,
2000; see below). However, these previous studies did not exam-
ine the hypothalamus in detail.
We chose to use dark treatment to induce REM sleep because
it does not involve inherently stressful or invasive procedures.
The mechanism by which dark treatment triggers REM sleep is
not clear, although this phenomenon appears to occur in albino
but not pigmented rats (Benca et al., 1991, 1998). Direct retinal
projections have been found to the sleep-active, galaninergic cells
in the extended VLPO and VLPO cluster (Lu et al., 1999) and
to the DRN (Shen and Semba, 1994; Fite et al., 1999). This
pathway could potentially mediate the effect of dark treatment on
REM sleep. However, Miller et al. (1998) reported that lesions of
the pretectum and superior colliculus could diminish the dark-
mediated increase in REM sleep.
We realize that demonstration of synaptic contacts from the
extended VLPO and VLPO cluster with neurons in the brain-
stem will require electron microscopy. However, the spatial rela-
tionship of the terminal boutons with cell bodies in our materials
suggests the likelihood of synaptic contacts.
Brainstem regulation of REM sleep
It is well established that the cholinergic cells in the LDT/PPT,
which show increased firing rates during REM sleep and are
almost inactive during NREM sleep, play a central role in gen-
erating REM sleep (Kayama et al., 1992; Sakai and Koyama,
1996). Lesions of the LDT abolish REM sleep in cats (Webster
and Jones, 1988), and electrical stimulation of the LDT or gluta-
mate injection into the PPT increases REM sleep (Thakkar et al.,
1996; Datta and Siwek, 1997). Injection of the cholinergic agonist
carbachol into the medial pontine reticular formation, a target of
the PPT (Rye et al., 1987), induces prolonged and persistent
REM sleep in cats and rats (Shiromani et al., 1992, 1995; Marks
and Birabil, 1998).
Serotoninergic cells in the DRN and noradrenergic cells in the
LC are active during wakefulness, less active during NREM
sleep, and inactive during REM sleep (Heym et al., 1982; Fornal
et al., 1985; Sakai, 1986; Reiner and McGeer, 1987; Yamuy et al.,
1995, 1998; Thakkar et al., 1998; Gervasoni et al., 2000). Both cell
neurons in the DRN, LDT, and LC that are stained (brown) for serotonin, choline acetyltransferase, or tyrosine hydroxylase, respectively. In A, the
efferent terminals concentrate dorsal to the cluster of the cholinergic cells in the LDT. Occasionally we found that terminal boutons were near choline
acetyltransferase-immunoreactive neurons such as in B; however careful observation indicated that such boutons were not on the same plane as the
cholinergic cell body. In C and D, efferent terminal boutons clearly appose serotonin-immunoreactive neurons in the DRN. E and F show labeled efferent
axons apposing tyrosine hydroxylase-immunoreactive neurons in the LC.
Photomicrographs showing the relationships of anterogradely labeled efferent axons (black) from the extended VLPO and VLPO cluster with
4574 J. Neurosci., June 1, 2002, 22(11):4568–4576Lu et al. • Extended VLPO and REM Sleep
groups project to the LDT/PPT (Semba and Fibiger, 1992; Honda
and Semba, 1994; Leonard et al., 1995; Steininger et al., 1997)
where they are thought to inhibit the cholinergic neurons. The
inhibition of serotoninergic and noradrenergic neurons by the
extended VLPO would thereby promote REM sleep (Jones,
1991; Hobson et al., 1998; Crochet and Sakai, 1999).
The quiescence of sertoninergic cells during REM sleep is
caused by GABAergic inputs (Levine and Jacobs, 1992; Wang et
al., 1992; Nitz and Siegel, 1997a; Gervasoni et al., 2000). Because
virtually all VLPO and extended VLPO neurons that contain
galanin also contain GABA, the extended VLPO may be a
critical source of inhibition of monoaminergic regions during
REM sleep (Sherin et al., 1998; Gervasoni et al., 2000; J. E.
Sherin and C. B. Saper, unpublished observations). Stimulation of
the preoptic area by warming, which increases the firing of many
sleep-active neurons, inhibits firing activity in the serotoninergic
cells in the DRN (Guzman-Marin et al., 2000). Similarly
GABAergic control of the LC is also thought to derive from the
preoptic area (Luppi et al., 1995; Nitz and Siegel, 1997b; Sherin
et al., 1998; Luppi et al., 1999). Sherin et al. (1998) attributed this
projection primarily to the VLPO, but their injections of antero-
grade tracer included the medial and dorsal extended VLPO as
well as the VLPO cluster. Our results indicate that much of the
input to the DRN and LC attributed to the VLPO actually
originates from the extended VLPO.
We also identified an intense projection from the extended
VLPO and VLPO cluster to the ventrolateral PAG. The ventro-
lateral PAG contains GABAergic cells (Gervasoni et al., 2000),
and injection of a GABAergic agonist into the ventrolateral
periaqueductal gray matter increases REM sleep (Sastre et al.,
1996). The mechanism by which GABAergic cells in the periaq-
ueductal gray matter may contribute to REM sleep remains to be
determined, but our finding suggests another potential pathway
by which the extended VLPO and VLPO cluster may influence
Preoptic regulation of REM and NREM sleep
The sleep-active cells in the VLPO cluster were originally de-
fined by their expression of Fos protein during sleep, their ex-
pression of the neurotransmitters galanin and GABA, and their
projection to the histaminergic cells in the TMN (Sherin et al.,
1996, 1998). Our previous studies (Sherin et al., 1998; Lu et al.,
2000; Gaus et al., 2002) also found that many cells extending
beyond the VLPO cluster in a dorsal and medial direction possess
the same qualities, and we speculated that they might project
topographically to different targets. We found that lesions of the
VLPO cluster primarily were correlated with loss of NREM
sleep, whereas lesions of the extended VLPO correlated with loss
of REM sleep (Lu et al., 2000). Here we show in dark-treated rats
that the sleep-active cells in the extended VLPO are galaninergic
cells, that their activity is highly correlated with REM sleep, and
that they do indeed have slightly different projections from neu-
rons in the VLPO cluster, which are consistent with a role in
regulating REM sleep.
During NREM sleep, the sleep-active neurons in the VLPO
cluster would inhibit the activity of the cells in the TMN, DRN,
and LC by releasing galanin and GABA, thus maintaining slow-
wave sleep. During the transitions from NREM to REM sleep,
the firing of DRN and LC is further decreased (Heym et al., 1982;
Fornal et al., 1985; Sakai, 1986; Reiner and McGeer, 1987). We
propose that this transition may be attributable at least in part to
the recruitment of inhibitory neurons in the extended VLPO that
further decrease LC and DRN firing, thus disinhibiting the LDT
and PPT cholinergic cells. In addition, if extended VLPO effer-
ents end on inhibitory interneurons in the LDT/PPT, they could
further promote their firing during the transition to REM sleep.
The connections of the extended VLPO neurons and their REM-
active pattern would make them prime candidates to fulfill this
Alfoldi P, Franken P, Tobler I, Borbely AA (1991) Short light-dark
cycles influence sleep stages and EEG power spectra in the rat. Behav
Brain Res 43:125–131.
Benca RM, Bergmann BM, Leung C, Nummy D, Rechtschaffen A (1991)
Rat strain differences in response to dark pulse triggering of paradox-
ical sleep. Physiol Behav 49:83–87.
Benca RM, Gilliland MA, Obermeyer WH (1998) Effects of lighting
conditions on sleep and wakefulness in albino Lewis and pigmented
Brown Norway rats. Sleep 21:451–460.
Chou TC, Bjorkum AA, Gaus SE, Lu J, Scammell TE, Saper CB (2002)
Afferents to the ventrolateral preoptic nucleus. J Neurosci 22:977–990.
Crochet S, Sakai K (1999) Effects of microdialysis application of mono-
amines on the EEG and behavioural states in the cat mesopontine
tegmentum. Eur J Neurosci 11:3738–3752.
Datta S, Siwek DF (1997) Excitation of the brain stem pedunculopon-
tine tegmentum cholinergic cells induces wakefulness and REM sleep.
J Neurophysiol 77:2975–2988.
Fite KV, Janusonis S, Foote W, Bengston L (1999) Retinal afferents to
the dorsal raphe nucleus in rats and Mongolian gerbils. J Comp Neurol
Fornal C, Auerbach S, Jacobs BL (1985) Activity of serotonin-
containing neurons in nucleus raphe magnus in freely moving cats. Exp
Gaus SE, Strecker RE, Tate BA, Parker RA, Saper CB (2002) Ventro-
lateral preoptic nucleus contains sleep-active, galaninergic neurons
across the mammalian spectrum. Neuroscience, in press.
Gervasoni D, Peyron C, Rampon C, Barbagli B, Chouvet G, Urbain N,
Fort P, Luppi PH (2000) Role and origin of the GABAergic innerva-
tion of dorsal raphe serotonergic neurons. J Neurosci 20:4217–4225.
Guillery RW, Herrup K (1997) Quantification without pontification:
choosing a method for counting objects in sectioned tissues. J Comp
Guzman-Marin R, Alam MN, Szymusiak R, Drucker-Colin R, Gong H,
McGinty D (2000) Discharge modulation of rat dorsal raphe neurons
during sleep and waking: effects of preoptic/basal forebrain warming.
Brain Res 875:23–34.
Heym J, Steinfels GF, Jacobs BL (1982) Activity of serotonin-containing
neurons in the nucleus raphe pallidus of freely moving cats. Brain Res
Hobson JA, Stickgold R, Pace-Schott EF (1998) The neuropsychology of
REM sleep dreaming. NeuroReport 9:R1–R14.
Honda T, Semba K (1994) Serotonergic synaptic input to cholinergic
neurons in the rat mesopontine tegmentum. Brain Res 647:299–306.
Horner RL, Kubin L (1999) Pontine carbachol elicits multiple rapid eye
movement sleep-like neural events in urethane-anaesthetized rats. Neu-
Jones BE (1991) The role of noradrenergic locus coeruleus neurons and
neighboring cholinergic neurons of the pontomesencephalic tegmen-
tum in sleep-wake states. Prog Brain Res 88:533–543.
Kayama Y, Ohta M, Jodo E (1992) Firing of “possibly” cholinergic
neurons in the rat laterodorsal tegmental nucleus during sleep and
wakefulness. Brain Res 569:210–220.
Koyama Y, Hayaishi O (1994) Firing of neurons in the preoptic/anterior
hypothalamic areas in rat: its possible involvement in slow wave sleep
and paradoxical sleep. Neurosci Res 19:31–38.
Leonard CS, Kerman I, Blaha G, Taveras E, Taylor B (1995) Interdigi-
tation of nitric oxide synthase-, tyrosine hydroxylase-, and serotonin-
containing neurons in and around the laterodorsal and pedunculopon-
tine tegmental nuclei of the guinea pig. J Comp Neurol 362:411–432.
Levine ES, Jacobs BL (1992) Neurochemical afferents controlling the
activity of serotonergic neurons in the dorsal raphe nucleus: microion-
tophoretic studies in the awake cat. J Neurosci 12:4037–4044.
Lu J, Shiromani P, Saper CB (1999) Retinal input to the sleep-active
ventrolateral preoptic nucleus in the rat. Neuroscience 93:209–214.
Lu J, Greco M, Shiromani P, Saper CB (2000) Effects of the lesions of
the ventrolateral preoptic nucleus on NREM and REM sleep. J Neu-
Luppi PH, Aston-Jones G, Akaoka H, Chouvet G, Jouvet M (1995)
Afferent projections to the rat locus coeruleus demonstrated by retro-
grade and anterograde tracing with cholera-toxin B subunit and Phaseo-
lus vulgaris leucoagglutinin. Neuroscience 65:119–160.
Luppi PH, Gervasoni D, Peyron C, Rampon C, Barbagli B, Boissard R,
Lu et al. • Extended VLPO and REM SleepJ. Neurosci., June 1, 2002, 22(11):4568–4576 4575
Fort P (1999) Norepinephrine and REM sleep. In: Rapid eye move- Download full-text
ment sleep (Mallick BN, Inoue S, eds), pp 107–122. New Delhi, India:
Maloney KJ, Mainville L, Jones BE (1999) Differential c-Fos expression
in cholinergic, monoaminergic, and GABAergic cell groups of the
pontomesencephalic tegmentum after paradoxical sleep deprivation
and recovery. J Neurosci 19:3057–3072.
Maloney KJ, Mainville L, Jones BE (2000) c-Fos expression in
GABAergic, serotonergic, and other neurons of the pontomedullary
reticular formation and raphe after paradoxical sleep deprivation and
recovery. J Neurosci 20:4669–4679.
Marks GA, Birabil CG (1998) Enhancement of rapid eye movement
sleep in the rat by cholinergic and adenosinergic agonists infused into
the pontine reticular formation. Neuroscience 86:29–37.
Merchant-Nancy H, Vazquez J, Garcia F, Drucker-Colin R (1995) Brain
distribution of c-fos expression as a result of prolonged rapid eye
movement (REM) sleep period duration. Brain Res 681:15–22.
Miller AM, Obermeyer WH, Behan M, Benca RM (1998) The superior
colliculus-pretectum mediates the direct effects of light on sleep. Proc
Natl Acad Sci USA 95:8957–8962.
Nitz D, Siegel J (1997a) GABA release in the dorsal raphe nucleus: role
in the control of REM sleep. Am J Physiol 273:R451–R455.
Nitz D, Siegel JM (1997b) GABA release in the locus coeruleus as a
function of sleep/wake state. Neuroscience 78:795–801.
Osaka T, Matsumura H (1994) Noradrenergic inputs to sleep-related
neurons in the preoptic area from the locus coeruleus and the ventro-
lateral medulla in the rat. Neurosci Res 19:39–50.
Paxinos G, Watson C (1986) The rat brain in stereotaxic coordinates.
San Diego: Academic.
Reiner PB, McGeer EG (1987) Electrophysiological properties of corti-
cally projecting histamine neurons of the rat hypothalamus. Neurosci
Rye DB, Saper CB, Lee HJ, Wainer BH (1987) Pedunculopontine teg-
mental nucleus of the rat: cytoarchitecture, cytochemistry, and some
extrapyramidal connections of the mesopontine tegmentum. J Comp
Sakai K (1986) Central mechanisms of paradoxical sleep. Brain Dev
Sakai K, Koyama Y (1996) Are there cholinergic and non-cholinergic
paradoxical sleep-on neurones in the pons? NeuroReport 7:2449–2453.
Sastre JP, Buda C, Kitahama K, Jouvet M (1996) Importance of the
ventrolateral region of the periaqueductal gray and adjacent tegmen-
tum in the control of paradoxical sleep as studied by muscimol micro-
injections in the cat. Neuroscience 74:415–426.
Semba K, Fibiger HC (1992) Afferent connections of the laterodorsal
and the pedunculopontine tegmental nuclei in the rat: a retro- and
antero-grade transport and immunohistochemical study. J Comp Neu-
Semba K, Reiner PB, Fibiger HC (1990) Single cholinergic mesopontine
tegmental neurons project to both the pontine reticular formation and
the thalamus in the rat. Neuroscience 38:643–654.
Shen H, Semba K (1994) A direct retinal projection to the dorsal raphe
nucleus in the rat. Brain Res 635:159–168.
Sherin JE, Shiromani PJ, McCarley RW, Saper CB (1996) Activation of
ventrolateral preoptic neurons during sleep. Science 271:216–219.
Sherin JE, Elmquist JK, Torrealba F, Saper CB (1998) Innervation of
histaminergic tuberomammillary neurons by GABAergic and galanin-
ergic neurons in the ventrolateral preoptic nucleus of the rat. J Neurosci
Shiromani PJ, Kilduff TS, Bloom FE, McCarley RW (1992) Cholinergi-
cally induced REM sleep triggers Fos-like immunoreactivity in dorso-
lateral pontine regions associated with REM sleep. Brain Res
Shiromani PJ, Malik M, Winston S, McCarley RW (1995) Time course
of Fos-like immunoreactivity associated with cholinergically induced
REM sleep. J Neurosci 15:3500–3508.
Steininger TL, Wainer BH, Blakely RD, Rye DB (1997) Serotonergic
dorsal raphe nucleus projections to the cholinergic and noncholinergic
neurons of the pedunculopontine tegmental region: a light and electron
microscopic anterograde tracing and immunohistochemical study.
J Comp Neurol 382:302–322.
Steininger TL, Gong H, McGinty D, Szymusiak R (2001) Subregional
organization of preoptic area /anterior hypothalamic projections to
Szymusiak R, Alam N, Steininger TL, McGinty D (1998) Sleep-waking
discharge patterns of ventrolateral preoptic/anterior hypothalamic neu-
rons in rats. Brain Res 803:178–188.
Thakkar MM, Portas C, McCarley RW (1996) Chronic low-amplitude
electrical stimulation of the laterodorsal tegmental nucleus of freely
moving cats increases REM sleep. Brain Res 723:223–227.
Thakkar MM, Strecker RE, McCarley RW (1998) Behavioral state con-
trol through differential serotonergic inhibition in the mesopontine
cholinergic nuclei: a simultaneous unit recording and microdialysis
study. J Neurosci 18:5490–5497.
Torterolo P, Yamuy J, Sampogna S, Morales FR, Chase MH (2001)
GABAergic neurons of the laterodorsal and pedunculopontine tegmen-
tal nuclei of the cat express c-fos during carbachol-induced active sleep.
Brain Res 892:309–319.
Vrontakis ME, Peden LM, Duckworth ML, Friesen HG (1987) Isola-
tion and characterization of a complementary DNA (galanin) clone
from estrogen-induced pituitary tumor messenger RNA. J Biol Chem
Wang QP, Ochiai H, Nakai Y (1992) GABAergic innervation of seroto-
nergic neurons in the dorsal raphe nucleus of the rat studied by electron
microscopy double immunostaining. Brain Res Bull 29:943–948.
Webster HH, Jones B (1988) Neurotoxic lesions of the dorsolateral
pontomesencephalic tegmentum-cholinergic cell area in the cat. II.
Effects upon sleep-waking states. Brain Res 458:285–302.
Xi M-C, Morales FR, Chase MH (1999) A GABAergic pontine reticular
system is involved in the control of wakefulness and sleep. Sleep Res
Yamuy J, Sampogna S, Lopez-Rodriguez F, Luppi PH, Morales FR,
Chase MH (1995) Fos and serotonin immunoreactivity in the raphe
nuclei of the cat during carbachol-induced active sleep: a double-
labeling study. Neuroscience 67:211–223.
Yamuy J, Sampogna S, Morales FR, Chase MH (1998) c-fos expression
in mesopontine noradrenergic and cholinergic neurons of the cat during
carbachol-induced active sleep: double-labeling study. Sleep Res On-
cell groups.J Comp Neurol
4576 J. Neurosci., June 1, 2002, 22(11):4568–4576Lu et al. • Extended VLPO and REM Sleep