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The Journal of Neuroscience, March 1, 2001, 21(5):1656-1662
Fos Expression in Orexin Neurons Varies with Behavioral State
Ivy V.
Estabrooke1,
Marie T.
McCarthy1,
Emily
Ko2,
Thomas C.
Chou3,
Richard M.
Chemelli4,
Masashi
Yanagisawa4,
Clifford B.
Saper1, 3, and
Thomas E.
Scammell1
1 Department of Neurology, Beth Israel Deaconess
Medical Center, Boston, Massachusetts 02115, 2 Department of Psychology, Harvard University,
Cambridge, Massachusetts 02138, 3 Program in Neuroscience,
Harvard Medical School, Boston, Massachusetts 02115, and
4 Department of Molecular Genetics, University of Texas
Southwestern Medical Center, Dallas, Texas 75235
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ABSTRACT |
The neuropeptide orexin (also known as hypocretin) is hypothesized
to play a critical role in the regulation of sleep-wake behavior. Lack
of orexin produces narcolepsy, which is characterized by poor
maintenance of wakefulness and intrusions of rapid eye movement
(REM) sleep or REM sleep-like phenomena into wakefulness. Orexin
neurons heavily innervate many aminergic nuclei that promote wakefulness and inhibit REM sleep. We hypothesized that orexin neurons
should be relatively active during wakefulness and inactive during
sleep. To determine the pattern of activity of orexin neurons, we
recorded sleep-wake behavior, body temperature, and locomotor activity
under various conditions and used double-label immunohistochemistry to
measure the expression of Fos in orexin neurons of the perifornical region. In rats maintained on a 12 hr light/dark cycle, more orexin neurons had Fos immunoreactive nuclei during the night period; in
animals housed in constant darkness, this activation still occurred
during the subjective night. Sleep deprivation or treatment with
methamphetamine also increased Fos expression in orexin neurons. In
each of these experiments, Fos expression in orexin neurons correlated
positively with the amount of wakefulness and correlated negatively
with the amounts of non-REM and REM sleep during the preceding 2 hr. In
combination with previous work, these results suggest that activation
of orexin neurons may contribute to the promotion or maintenance of
wakefulness. Conversely, relative inactivity of orexin neurons may
allow the expression of sleep.
Key words:
orexin; hypocretin; Fos; wake; wakefulness; sleep; REM; lateral hypothalamus; perifornical region; hypothalamus; thermoregulation; rat
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INTRODUCTION |
The neuropeptide orexin (also known
as hypocretin) is hypothesized to play a critical role in the
regulation of wakefulness and sleep. Orexin is synthesized in neurons
of the perifornical region and lateral hypothalamic area, and orexin
fibers innervate brain regions known to regulate behavioral state such
as the raphe nuclei, the locus coeruleus, the tuberomammillary nucleus,
and the cholinergic neurons of the basal forebrain and pons (Peyron et
al., 1998 ; Chemelli et al., 1999; Date et al., 1999). Orexin increases
the firing rate of postsynaptic neurons via OX1 and OX2 receptors
(Sakurai et al., 1998; Hagan et al., 1999). Recent observations
demonstrate that impaired orexin transmission may result in narcolepsy,
which is characterized by poor maintenance of wakefulness and
intrusions of rapid eye movement (REM) sleep or REM sleep-like
phenomena into wakefulness (for review, see Siegel, 1999). The brains
of narcoleptics exhibit a nearly complete loss of neurons expressing
orexin mRNA or peptide (Peyron et al., 2000; Thannickal et al., 2000),
and individuals with narcolepsy often have unmeasurably low orexin
levels in CSF (Nishino et al., 2000). Canine narcolepsy is caused by
mutations of the OX2 receptor (Lin et al., 1999), and orexin
knock-out mice have a phenotype very similar to canine and human
narcolepsy (Chemelli et al., 1999). Additionally, the wakefulness
produced by modafinil, a drug used to treat the excessive sleepiness of
narcolepsy in humans, is accompanied by increased orexin neuron
activity in rodents as indicated by increased expression of the
transcription factor Fos (Chemelli et al., 1999; Scammell et al.,
2000). These observations suggest that orexin may be necessary for the
maintenance of wakefulness and the suppression of REM sleep, but the
relationship of orexin neuron activity to behavioral state is unknown.
We hypothesized that orexin neurons should be most active during
wakefulness and least active during sleep. Extracellular recordings of
the perifornical area and lateral hypothalamus would be difficult to
interpret because the orexin neurons are intermingled with many other
populations. Therefore, we used double immunohistochemical staining for
Fos and orexin in combination with physiological recordings of
sleep-wake behavior to investigate the relationship between orexin
neuron activity and behavioral state in rats.
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MATERIALS AND METHODS |
Experimental design
We performed three experiments to determine whether orexin
neuron activity varies with behavioral state. In each experiment, rats
were instrumented for electroencephalogram (EEG) and electromyogram (EMG) recordings, as well as the telemetric measurement of locomotor activity (LMA) and body temperature
(Tb). After the experiments, the rats were perfused, and the brain sections were stained for Fos and
orexin using double immunohistochemistry. Each experimental group
contained six to eight animals.
Experiment 1. To determine whether the activity of orexin
neurons changes with circadian variations in sleep-wake behavior, animals were maintained on a 12 hr light/dark (LD) cycle and
killed at zeitgeber times (ZTs, in hours) 3, 9, 15, or 21; ZT 0 is morning light onset. To eliminate photic influences, two additional
groups of rats were kept in constant darkness (DD) for 4 d and
then killed at circadian times (CTs) 3 or 15; CT 0 is the circadian
phase at which lights would normally turn on.
Experiment 2. To determine whether orexin neurons are more
active during the wakefulness induced by stimulants, we treated rats
with methamphetamine or vehicle at ZT 17 and killed the rats 2 hr later.
Experiment 3. To determine whether the wake-related activity
of orexin neurons is influenced by the time of day, we deprived rats of
sleep for 2 hr, from ZT 5 to ZT 7 or ZT 17 to ZT 19, and then killed
them. Very dim light (0.35 lux) was maintained during the nighttime
sleep-deprivation experiments to allow observation of the animals.
Sleep deprivation was performed by tapping lightly on the rats'
cages. When light tapping did not awaken the animals, crumpled
paper was inserted into the cages to stimulate the rats. If needed,
louder tapping of the cage was repeated. The number of stimuli applied
during the nighttime sleep deprivation was equivalent to that applied
during the day. Separate groups of unhandled control rats were killed
at ZT 7 and ZT 19.
Animals and surgery
Seventy-eight male, Sprague Dawley rats (Harlan) weighing
270-330 gm were housed individually in a pathogen-free barrier
facility maintained at 21.5-22.5°C with lights on at 7:00 A.M. and
off at 7:00 P.M. Rats had food and water available ad
libitum. Under chloral hydrate anesthesia (350 mg/kg, i.p.), each
animal was surgically implanted with four EEG screws (anteroposterior:
+3,  4; lateral: +2, 2, from bregma) lightly contacting the
dura, and EMG wires (Plastics One, Roanoke, VA) were placed in the
nuchal muscles. All leads were connected to a six-channel connector
(Plastics One) and affixed to the skull with dental acrylic. A
telemetric temperature-activity transmitter (TA10TA-F40; Data Sciences
International, St. Paul, MN) was placed in the peritoneal cavity. In
experiment 2, we administered drugs via an 80 cm SILASTIC catheter (1 mm, inner diameter; Baxter Scientific, Boston, MA) to avoid
stress caused by handling. The catheter was implanted in the peritoneal cavity, tunneled subcutaneously to the scalp, cemented in place with
dental acrylic, and protected externally by a spring. This intraperitoneal catheter was filled with sterile heparinized saline and
flushed weekly and 3 d before the experiment. All animals recovered for at least 14 d and were allowed to acclimate to
commutators for at least 3 d before the start of physiological
recordings. The Institutional Animal Care and Use Committees of Beth
Israel Deaconess Medical Center and Harvard Medical School approved all procedures.
For experiment 2, methamphetamine HCl (U.S. Pharmacopeia, Rockville,
MD) was suspended in a solution of 0.25% methylcellulose (Dow
Chemical, Midland, MI), pH 7.4, in 0.9% pyrogen-free saline (Sigma,
St. Louis, MO). Methamphetamine (0.5 mg/kg) or methylcellulose control
vehicle was administered in a volume of 0.7 ml via the chronic
intraperitoneal catheter. Catheters were then flushed with 1 ml of
0.9% saline to guarantee that all of the drug was flushed into the
peritoneal cavity, and animals were killed 2 hr later.
Physiological recordings
EEG and EMG signals were recorded from all animals. These
signals were amplified using Grass model 12 amplifiers and digitally acquired using ICELUS software (Mark Opp, University of Texas at
Galveston, Galveston, TX). EEG signals were amplified 5000×, bandpass
filtered at 0.3-30 Hz, and sampled at 128 Hz. EMG signals were
amplified 5000×, bandpass-filtered at 10-100 Hz, sampled at 128 Hz,
and integrated into 1 sec values. Behavioral states were scored as
wake, non-REM (NREM), or REM in 12 sec epochs by a single examiner who
was blinded to the treatment condition. Tb and LMA were monitored every 5 min
using Dataquest software (Data Sciences International) and integrated
into 30 min intervals. The animals of experiment 1 that were housed in
constant darkness for 4 d had a free-running period of 24 hr in
their Tb rhythm, and their
Tb minima occurred within 10 min of
the minima of rats housed in LD conditions as determined by cosinor
analysis. Telemetric measures of LMA varied considerably between
animals, and thus LMA was normalized for each animal by dividing by the
mean LMA over the 24 hr before the experiment. Because orexin neurons
have been hypothesized to play a role in thermoregulation, we also calculated the change in Tb over the 2 hr before death by subtracting the mean
Tb in the second hour before death
from the mean temperature over the hour before death.
Tb and LMA data from one CT 3 rat of
experiment 1 were excluded from analysis because of a malfunctioning transmitter.
Histology and immunohistochemistry
Animals were anesthetized deeply with chloral hydrate (700 mg/kg, i.p.) and perfused transcardially with 100 ml of 0.9% saline and 500 ml of phosphate-buffered 10% formalin, pH 7.0 (Sigma). Brains
were removed, post-fixed for 4 hr in formalin, and then allowed to
equilibrate in 20% sucrose in 0.1 M PBS with 0.02% sodium azide (Sigma). Brains were then sectioned (1:5 series, 30 µm)
on a freezing microtome and stored in PBS-azide at 4°C. Hypothalamic
sections were selected from one series of each brain and stained
immunohistochemically for Fos using previously described methods
(Elmquist et al., 1996). Briefly, tissue was incubated in rabbit
anti-Fos antiserum (Ab-5, 1:100,000) (Oncogene Research Products,
Cambridge, MA) for 48 hr with 3% normal donkey serum (Jackson
ImmunoResearch, West Grove, PA). A biotinylated donkey anti-rabbit
secondary antiserum (Jackson ImmunoResearch) was used at a dilution of
1:1000. Tissue was then reacted with avidin-biotin complex (Vectastain
ABC Elite kit; Vector Laboratories, Burlingame, CA) for 1 hr, and
Fos-immunoreactive (IR) nuclei were visualized by reaction with
3,3'-diaminobenzidine (DAB; Sigma), 3%
H2O2, 0.01%
NiSO4, and 0.01%CoCl2.
After Fos staining, sections were rinsed in PBS-azide and incubated
overnight in rabbit anti-orexin-A primary antiserum (1:5000) (M. Yanagisawa, University of Texas Southwestern Medical Center) and
3% normal donkey serum (Jackson ImmunoResearch). Sections were then
incubated for 2 hr in donkey anti-rabbit secondary antiserum (1:500)
(Jackson ImmunoResearch) and 3% normal donkey serum. The staining was
completed as described above with the exclusion of the
NiSO4/CoCl2 in the DAB step
to yield a brown cytoplasmic product in orexin-IR neurons. The orexin antiserum used in experiment 2 was a different lot than that used in
experiments 1 and 3. The orexin immunostaining was blocked by
preadsorption with 50 µg/ml of orexin-A, and omission of the primary
antiserum resulted in no specific staining. The Fos antiserum was
previously characterized in our laboratory (Elmquist et al., 1996).
Cell counts
A single examiner, who was blinded to treatment conditions,
performed all counts using a Leitz Laborlux microscope. Perifornical Fos-IR nuclei, orexin-IR neurons, and double-labeled neurons were counted in sections beginning 300 µm behind the paraventricular nucleus of the hypothalamus using a horizontally oriented 1 × 0.4 mm box centered just dorsal to the fornix (Fig.
1). Cells were counted on both sides of
the brain in three consecutive sections 150 µm apart. The six counts
per animal were then averaged. Although the orexin neurons are
concentrated in the perifornical region, the number of orexin neurons
within the counting box varied by as much as a factor of two between
animals. To correct for this variation in sampling of orexin neurons,
we calculated the percentage of double-labeled cells for each animal
(double-labeled neurons/orexin-IR neurons) as a measure of orexin
neuron activity. Fos expression was not limited to the orexin-IR
neurons, and we defined these as "Fos-IR, non-orexin-IR" neurons,
although some non-orexin-IR neurons may simply have orexin
concentrations below our detection limit. In experiment 1, cells also
were counted in the region medial to the perifornical counting box
using a vertically oriented 0.4 × 0.6 mm box and in the lateral
hypothalamic area using a vertically oriented 0.7 × 1.0 mm box;
these boxes abutted the edges of the perifornical counting box as shown
in Figure 1. Within these medial and lateral areas, orexin neurons are
more numerous caudally, so counts in these regions began with the third
section on which perifornical counts were performed.
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Figure 1.
Camera lucida drawing illustrating the cell
counting box in the perifornical region. In experiment 1, cells also
were counted within boxes that sampled the lateral hypothalamic area
and the area medial to the perifornical region. VMH,
Ventromedial hypothalamic nucleus; Arc, arcuate
hypothalamic nucleus; f, fornix; opt,
optic tract; 3V, third ventricle. Each
dot represents an orexin-IR neuron.
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Statistical analysis
Mann-Whitney or Kruskal-Wallis rank sum tests were used to
compare cell counts between groups, and these same tests were used for
analysis of physiological variables. In experiments with multiple groups, a post hoc Scheffe test was used to identify
significant pairwise differences. Cell counts were not corrected for
double-counting errors (Guillery and Herrup, 1997) because there was no
change in the size of labeled cells or nuclei across groups, and only relative, not absolute, values were sought. Cell counts were correlated with physiological variables using the Pearson correlation coefficient. In our previous work, we have correlated Fos expression with
sleep-wake behavior in the hour before death. In preliminary
experiments, we found that orexin neuron activation correlated slightly
better with behavior over the 2 hr before death, and this longer
interval was used throughout our analyses.
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RESULTS |
Experiment 1: circadian variations in orexin neuron activity
Under LD conditions, the number of double-labeled neurons
(orexin-IR neurons with Fos-IR nuclei) varied across the four time points, with higher levels of double-labeling in the two nighttime groups (Table 1, Fig.
2). The percentage of double-labeled
neurons also varied across the four groups (p < 0.0001) (Fig. 3), with either of the
daytime groups differing from either of the nighttime groups on
pairwise comparisons (p < 0.02). In addition,
the total number of Fos-IR neurons and the number of Fos-IR,
non-orexin-IR neurons in the perifornical region differed across the
four time points, with more Fos-IR neurons in the two nighttime groups. Pairwise differences in the total number of Fos-IR neurons or the
number of Fos-IR, non-orexin-IR neurons were evident between either of
the daytime groups and either of the nighttime groups (p < 0.01), except that the ZT 9 group did not
differ from the ZT 21 group.
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Table 1.
Perifornical region cell counts, sleep-wake behavior,
Tb, and LMA of rats killed at different times
under LD or DD conditions
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Figure 2.
A, Fos-IR nuclei are uncommon in
perifornical orexin-IR neurons from a rat killed at ZT 3. B, A rat killed at ZT 15 has many Fos-IR nuclei in
orexin-IR neurons. Arrow, Orexin-IR neuron;
arrowhead, Fos-IR nucleus; double arrow,
Fos-IR/orexin-IR neuron. Scale bar, 50 µm.
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Figure 3.
Fos expression in perifornical orexin neurons is
higher during the night in rats maintained on a 12 hr LD cycle
(ZT 3 through ZT 21 groups). In rats
maintained in constant darkness (CT 3 and CT
15 groups), the percentage of double-labeling also is higher
during the subjective night.
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Animals maintained in constant darkness showed a similar increase in
the percentage of double-labeled neurons during the subjective night
(p = 0.007). The total number of Fos-IR neurons
and the number of Fos-IR, non-orexin-IR neurons also were higher during the subjective night. Comparison of the LD and DD groups revealed no
differences in the number of Fos-IR neurons, orexin-IR neurons, or Fos-IR, non-orexin-IR neurons at CT 3 versus ZT 3, although the
percentage of double-labeled neurons was slightly decreased (p = 0.02). The CT 15 and ZT 15 groups did not
differ in any of these measures.
During the 2 hr before death, the amounts of wakefulness were higher in
the two LD nighttime groups compared with the daytime groups, and this
pattern persisted in the DD groups. Higher
Tb and greater LMA accompanied this
increased wakefulness during the (subjective) night. The change in
Tb over the final 2 hr did not differ
between groups. Under LD conditions, the percentage of
double-labeled cells correlated positively with the amount of
wakefulness and negatively with the amounts of NREM and REM sleep
(Table 2). The percentage of
double-labeled cells also correlated with
Tb and LMA but not with the change in
Tb over the 2 hr before death. These
relationships persisted in constant darkness.
To determine whether this pattern of wake-related Fos was evident in
all orexin neurons, we counted cells in the perifornical, medial, and
lateral parts of the orexin field. Compared with rats killed at ZT 3, rats killed at ZT 15 had a greater percentage of Fos-IR, orexin-IR
neurons within the medial and perifornical regions, but no diurnal
variation was evident in the lateral region (Fig.
4).
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Figure 4.
Orexin neurons in the perifornical region have the
greatest diurnal variation in Fos expression. Orexin neurons medial to
the perifornical region have a less striking diurnal variation, but Fos
expression does not vary in orexin neurons of the lateral hypothalamic
area. *p = 0.05; **p = 0.01.
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Experiment 2: orexin neuron activity during treatment
with methamphetamine
Rats treated with methamphetamine had a greater percentage of
double-labeled neurons compared with controls (p = 0.003) (Fig. 5). Fos expression in
perifornical non-orexin-IR neurons was also increased, although the
number of orexin-IR neurons did not differ (Table
3).
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Table 3.
Perifornical cell counts, sleep-wake behavior,
Tb, and LMA of animals treated with vehicle or
methamphetamine (0.5 mg/kg) at ZT 17 and killed 2 hr later
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Methamphetamine-treated animals were significantly more awake in the 2 hr between injection and death (ZT 17 to ZT 19) than control animals.
This increased wakefulness was accompanied by a small increase in LMA,
but there was no significant difference in
Tb or the change in
Tb after drug administration. The
percentage of double-labeled neurons correlated positively with the
amount of wakefulness and negatively with the amounts of NREM and REM sleep. The percentage of double-labeled neurons did not correlate with
LMA, Tb, or the change in
Tb.
Experiment 3: orexin neuron activity during sleep deprivation
Animals that were sleep deprived from ZT 5 to ZT 7 had a greater
percentage of double-labeled neurons than did the unhandled control
rats (p = 0.001) (Fig.
6). The number of Fos-IR, non-orexin-IR neurons in the perifornical region also increased with sleep
deprivation, although the number of orexin-IR neurons did not change
(Table 4). Orexin neuron Fos expression
correlated positively with the amounts of wakefulness, LMA, and
Tb and negatively with the amounts of
NREM and REM sleep.
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Figure 6.
Sleep deprivation from ZT 5 to ZT 7 increases Fos
expression in perifornical orexin-IR neurons. Sleep deprivation from ZT
17 to ZT 19 marginally increases Fos immunoreactivity in orexin-IR
neurons. *p = 0.04; **p < 0.0001.
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Table 4.
Perifornical cell counts, sleep-wake behavior,
Tb, and LMA for animals sleep deprived from ZT
5 to ZT 7 and animals sleep deprived from ZT 17 to ZT 19
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Sleep deprivation from ZT 17 to ZT 19 produced a small increase in the
percentage of double-labeled neurons (p = 0.04),
but the absolute number of double-labeled neurons was unchanged. The total number of Fos-IR neurons was increased, although there was no
difference in the number of orexin-IR neurons. Sleep deprivation modestly increased the amount of wakefulness, but there were no significant differences in Tb, change
in Tb, or LMA between the two groups,
and Fos expression in orexin-IR neurons did not correlate with any of
these variables.
Across all three experiments, the percentage of double-labeled neurons
correlated positively with the amount of wakefulness over the previous
2 hr (r = 0.78; p < 0.0001) and
negatively with the amounts of NREM (r = 0.78;
p < 0.0001) and REM sleep (r = 0.63;
p < 0.0001) (Fig. 7).
This relation with behavioral state was most evident at the extremes of
behavior; rats with >80% wakefulness had moderate to large numbers of
double-labeled neurons, whereas those with substantial amounts of NREM
or REM sleep had very few double-labeled cells. To determine the
relative contributions of NREM and REM sleep, we performed a stepwise
regression analysis which revealed that the percentage of
double-labeled neurons was strongly related to the amount of NREM sleep
(r = 0.78; p < 0.0001) with little
relation to the amount of REM sleep (r = 0.09) once the effects of NREM were removed. The percentage of double-labeled neurons also correlated well with LMA (r = 0.68;
p < 0.0001) and Tb
(r = 0.59; p < 0.0001). Although these
animals differed in handling, drug administration, ambient light
exposure, and time of death, these correlations were evident under all
conditions.
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Figure 7.
Across all three experiments, Fos expression in
perifornical orexin-IR neurons correlates positively with the amount of
wakefulness (r = 0.78; p < 0.0001) and negatively with the amounts of NREM (r = 0.78; p < 0.0001) and REM sleep
(r = 0.63, p < 0.0001).  ,
Experiment 1;  , experiment 2;  , experiment 3.
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DISCUSSION |
We used Fos immunohistochemistry to investigate the relation
between orexin neuron activity, behavioral state,
Tb, and LMA. We found that Fos
expression in perifornical orexin neurons was common after periods of
wakefulness but uncommon after periods of NREM and REM sleep. Orexin
neuron Fos expression exhibited a circadian pattern under both LD and
DD conditions; in experiments 1 and 3, this Fos expression also
correlated with Tb and LMA.
Several methodological issues warrant discussion. First, Fos is a
useful marker of activity in neurochemically defined neurons, often
indicating an increase in synaptic activity or a rise in the
intracellular concentrations of cAMP or calcium (for review, see
Cirelli and Tononi, 2000). Still, Fos immunohistochemistry has some
limitations. Fos protein may persist in neurons for several hours after
a stimulus (Schwartz et al., 1994), and its presence may reflect the
summation of neuronal activity over time. Thus, the correlation of
orexin neuron activity with sleep-wake behavior must be interpreted
cautiously; for example, although rats with >15% REM sleep had
especially low levels of Fos expression, these animals often had
increases in their total amount of sleep, and we cannot determine
whether orexin neurons are inactive during REM sleep in particular.
Identifying whether orexin neurons are active during specific bouts of
wakefulness or sleep must await techniques with better temporal
resolution. In addition, we cannot be certain whether an increase in
Fos corresponds to changes in firing rates or the release of
transmitters from orexin nerve terminals.
Second, we used a different lot of orexin antiserum in experiment 2 than we used in experiments 1 and 3, and this antiserum consistently
labeled fewer orexin neurons per brain section. Although this
influenced the absolute counts of orexin-IR neurons, it is unlikely to
have substantially biased the results because the percentage of
double-labeled neurons after 2 hr of methamphetamine-induced wakefulness was comparable with that seen after 2 hr of sleep deprivation.
Third, many researchers have found that methamphetamine increases
Tb, especially at high doses, and even
0.5 mg/kg can produce a rise of 0.3°C (Edgar and Seidel, 1997).
Although we found that this dose produced increases in LMA and
wakefulness similar to those reported by Edgar and Seidel (1997), we
did not observe an increase in Tb.
Most previous studies administered methamphetamine during the day,
whereas we gave it at ZT 17, a time when
Tb is relatively elevated. Perhaps the
hyperthermic effects of methamphetamine were masked by this elevated
nighttime Tb.
Finally, sleep deprivation can induce stress, particularly when
performed during the animal's normal sleep period. Orexin neurons may
influence autonomic functions associated with stress, such as increases
in pulse, blood pressure, and Tb
(Hagan et al., 1999; Samson et al., 1999); therefore it is difficult to
ascertain whether the increase in Fos produced by sleep deprivation
during the day is related to increased wakefulness or stress. However, stress is unlikely to be a major factor because sleep deprivation during the night did not significantly increase orexin neuron Fos
expression compared with unhandled controls, and the amount of handling
was comparable with that used during the daytime experiments. Additionally, the animals of experiments 1 and 2 were not handled. Thus, across all experiments, increased wakefulness was the factor most
consistently associated with orexin neuron activation.
All three experiments demonstrated a positive correlation between Fos
expression in perifornical orexin neurons and the percentage of
wakefulness in the 2 hr preceding death. These relationships persisted
in constant darkness, indicating that the activity of orexin neurons
does not depend on photic stimuli. Orexin neurons also were active in
association with the wakefulness produced by methamphetamine, as we
have previously shown with the wake-promoting drug modafinil (Chemelli
et al., 1999; Scammell et al., 2000). Fos expression in orexin-IR
neurons correlated with the amount of wakefulness after daytime sleep
deprivation but not after sleep deprivation at night, possibly because
the abundant wakefulness in the nighttime control group diminished the
contrast between groups. Our analysis of Fos expression in different
parts of the orexin field demonstrates that orexin neurons in the
perifornical region exhibit more diurnal variation in activity than
orexin neurons in the lateral hypothalamic area. Although it is
possible that this finding simply reflects a tendency for some orexin
neurons to express Fos more readily, it also may indicate a functional heterogeneity across the orexin field. Abrahamson and Moore (1999) have
demonstrated that orexin neurons within different parts of the orexin
field may project to separate targets, and our results suggest that the
perifornical orexin neurons may be involved more closely with the
control of sleep-wake behavior. Our experimental design does not allow
us to determine whether this orexin neuron activity promotes
wakefulness or is a consequence of wakefulness, but these associations
demonstrate a consistent relationship between the activity of
perifornical orexin neurons and behavioral state. Taheri and colleagues
(2000) have demonstrated diurnal variations in prepro-orexin mRNA as
well as in the concentration of orexin-A within the preoptic area and
pons. This evidence, in conjunction with the finding that
intracerebroventricular injection of orexin increases wakefulness
(Hagan et al., 1999), suggests that increased orexin neuron activity
may contribute to the promotion or maintenance of wakefulness.
Across all of these experiments, perifornical orexin neuron activity
was negatively correlated with the amounts of NREM and REM sleep, and
decreased orexin signaling may allow the expression of sleep.
Narcoleptic humans with a loss of orexin neurons (Peyron et al., 2000;
Thannickal et al., 2000) and dogs with exon-skipping mutations of the
OX2 receptor gene (Lin et al., 1999) have intrusions of REM sleep and
REM-like behaviors into the waking period. Orexin knock-out mice have a
similar dysregulation of REM sleep and also have increased amounts of
REM and NREM sleep during the dark period (Chemelli et al., 1999).
Thus, impaired orexin signaling may allow inappropriate expression of
REM and NREM sleep. In normal individuals, physiologically decreased
orexin signaling may allow the emergence of REM and possibly NREM sleep.
In experiments 1 and 3, orexin neuron activity correlated positively
with body temperature and LMA over the 2 hr preceding death. These
associations are not surprising because wakefulness is associated with
increases in Tb and LMA. Still, it
remains possible that orexin neurons may play a role in the control of Tb and LMA, because Hagan and
colleagues (1999) have demonstrated that intracerebroventricular
injections of orexin-A increase LMA. Also, we cannot rule out
the possibility that orexin neurons may influence other wake-associated
behaviors such as eating and glucose regulation (Ida et al., 1999;
Moriguchi et al., 1999; Sweet et al., 1999).
Fos expression in perifornical non-orexin-IR neurons also correlated
with wakefulness, Tb, and LMA and
maintained a circadian pattern. Although some of these "non-orexin"
neurons may simply have concentrations of orexin below our detection
limit, most probably contain other neurotransmitters. Some of these
neurons produce melanin-concentrating hormone (MCH), a neuropeptide
implicated in feeding and autonomic control (Elmquist et al., 1999).
Like the orexin neurons, these MCH neurons innervate areas implicated in state control such as the tuberomammillary nucleus, suggesting a
role in the control of behavioral state. However, we found no substantial diurnal variation in MCH neuron Fos expression (our unpublished data). The circadian variation in non-orexin neuron Fos
probably occurs in other perifornical neurons such as those producing
cocaine and amphetamine-regulated transcript, neurotensin, GABA, and
glutamate, but the function of these cells is largely unknown. Some of
these signaling molecules may influence behavioral state, but as yet,
only orexin is clearly necessary for normal state control.
Our observation that orexin neurons are active during wakefulness
fits well with several recent findings. Orexin fibers heavily innervate
several wake-promoting nuclei such as the locus coeruleus, the dorsal
raphe, and the tuberomammillary nucleus, and orexin increases the
firing rate of locus coeruleus neurons. The sleep-wake behavior of
orexin-deficient narcoleptic humans, orexin knock-out mice, and OX2
receptor-deficient dogs demonstrates that the orexin system is
necessary to maintain wakefulness and suppress REM sleep. We have shown
that orexin neurons are active during wakefulness, and these neurons
may maintain or promote wakefulness by stabilizing or enhancing the
activity of aminergic arousal regions. Conversely, decreased orexin
neuron activity may be necessary for the production of sleep.
Further studies characterizing the factors that influence the activity
of orexin neurons and defining how orexin is necessary for state
regulation should greatly increase our understanding of the mechanisms
underlying the control of sleep and wakefulness.
| |
FOOTNOTES |
Received Sept. 1, 2000; revised Nov. 27, 2000; accepted Dec. 11, 2000.
This study was supported by United States Public Health Service Grants
MH01507 and HL60292. We are grateful to Janet Mullington for her advice
on statistics, Stephanie Gaus for help with preliminary experiments,
and Courtney Sears for excellent technical assistance.
Correspondence should be addressed to Dr. Thomas E. Scammell,
Department of Neurology, Beth Israel Deaconess Medical Center, 77 Avenue Louis Pasteur, Boston, MA 02115. E-mail:
tscammel{at}caregroup.harvard.edu.
| |
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ABSTRACT
INTRODUCTION
