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The Journal of Neuroscience, November 15, 2000, 20(22):8620-8628
Hypothalamic Arousal Regions Are Activated during
Modafinil-Induced Wakefulness
Thomas E.
Scammell1,
Ivy V.
Estabrooke1,
Marie
T.
McCarthy1,
Richard M.
Chemelli2,
Masashi
Yanagisawa2,
Matthew S.
Miller3, and
Clifford B.
Saper1, 4
1 Department of Neurology, Beth Israel Deaconess
Medical Center, Boston, Massachusetts, 2 Department of
Molecular Genetics, University of Texas Southwestern Medical Center at
Dallas, Dallas, Texas, 3 Cephalon, Inc., West Chester,
Pennsylvania, and 4 Program in Neuroscience, Harvard
Medical School, Boston, Massachusetts
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ABSTRACT |
Modafinil is an increasingly popular wake-promoting drug used for
the treatment of narcolepsy, but its precise mechanism of action is
unknown. To determine potential pathways via which modafinil acts, we
administered a range of doses of modafinil to rats, recorded sleep/wake
activity, and studied the pattern of neuronal activation using Fos
immunohistochemistry. To contrast modafinil-induced wakefulness with
spontaneous wakefulness, we administered modafinil at midnight, during
the normal waking period of rats. To determine the influence of
circadian phase or ambient light, we also injected modafinil at noon on
a normal light/dark cycle or in constant darkness. We found that 75 mg/kg modafinil increased Fos immunoreactivity in the tuberomammillary
nucleus (TMN) and in orexin (hypocretin) neurons of the perifornical
area, two cell groups implicated in the regulation of wakefulness. This
low dose of modafinil also increased the number of Fos-immunoreactive
(Fos-IR) neurons in the lateral subdivision of the central nucleus of
the amygdala. Higher doses increased the number of Fos-IR neurons in
the striatum and cingulate cortex. In contrast to previous studies,
modafinil did not produce statistically significant increases in Fos
expression in either the suprachiasmatic nucleus or the anterior
hypothalamic area. These observations suggest that modafinil may
promote waking via activation of TMN and orexin neurons, two regions
implicated in the promotion of normal wakefulness. Selective
pharmacological activation of these hypothalamic regions may represent
a novel approach to inducing wakefulness.
Key words:
modafinil; tuberomammillary nucleus; lateral hypothalamic
area; perifornical area; orexin; hypocretin; striatum; amygdala; suprachiasmatic nucleus; anterior hypothalamic area; Fos; dopamine; stimulant; narcolepsy
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INTRODUCTION |
Modafinil has been used for the
treatment of excessive sleepiness for over a decade, but its precise
mechanism of action is unknown. Traditional stimulants such as
amphetamine appear to promote wakefulness by facilitating neural
transmission by catecholamines, particularly dopamine (Nishino and
Mignot, 1997 ). Treatment of animals with inhibitors of catecholamine
synthesis such as  -methyl-p-tyrosine (MPT)
substantially blocks the effects of amphetamine (Lin et al., 1992). In
contrast, the wake-promoting effects of modafinil are not blocked by
MPT (Lin et al., 1992), suggesting that modafinil may promote
wakefulness via novel mechanisms.
To determine potential neuronal targets for modafinil, Lin et al.
(1996) treated cats with modafinil or amphetamine and studied the
pattern of neuronal activation as indicated by the expression of Fos.
The transcription factor Fos is expressed in physiologically activated
brain regions after a variety of stimuli, thus serving as an indicator
of neuronal activation (Sagar et al., 1988). In contrast to the Fos
pattern induced by amphetamine, Lin found that a comparable
wake-promoting dose of modafinil induced very little Fos in
dopamine-responsive areas such as the striatum. However, modafinil
strongly induced the expression of Fos in the anterior hypothalamic
area (AHA) and the dorsolateral portion of the suprachiasmatic nuclei
(SCN). Lin hypothesized that these regions play an important role in
coordinating the activity of hypothalamic and basal forebrain regions
that promote wakefulness. Subsequent work in rats treated with
modafinil also demonstrated increased Fos immunoreactivity in the SCN
and AHA (Engber et al., 1998a).
Although these previous studies provide many important observations,
they are limited in several ways. Neither of these studies combined
anatomic observations with sleep/wake recordings, thereby hindering
correlations between patterns of Fos expression and behavior.
Additionally, the researchers administered modafinil via gavage or
acute intraperitoneal injection, techniques that could produce stress
and confound the results. Finally, animals in these studies were
treated with only a single dose of modafinil at approximately noon, but
because sleep/wake behavior varies considerably over the day, the
neural and behavioral responses to modafinil may also vary.
The purpose of our experiments was to define the pattern of neuronal
activation induced by modafinil in rats. We administered modafinil via
chronic intraperitoneal catheters at different doses and at different
times of day in combination with physiological recordings of sleep/wake
behavior. We then used immunohistochemistry to identify
Fos-immunoreactive (Fos-IR) neurons. We also used double
immunohistochemistry for Fos and orexin (also known as hypocretin),
because we have found previously that modafinil increases Fos
expression in orexin neurons of mice (Chemelli et al., 1999). We found
that modafinil strongly induced Fos in neurons of the tuberomammillary
nucleus (TMN) and in orexin neurons of the perifornical area (PFx), two
cell groups implicated in the control of wakefulness.
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MATERIALS AND METHODS |
Experimental design
To gain insight into modafinil's wake-promoting mechanism, we
performed three experiments. In all experiments, rats were killed 2 hr
after treatment, and the brains were analyzed by the use of
immunohistochemistry for Fos. In experiments 1 and 2, rats were
instrumented for electroencephalogram (EEG) and electromyogram (EMG)
recordings as well as telemetric measurement of body
temperature. To avoid the stress of handling during
administration of modafinil or vehicle, chronic catheters were placed
in the peritoneal cavity for administration of modafinil or vehicle.
Experiment 1. To determine the pattern of neuronal
activation induced by modafinil, we administered modafinil (75 or 150 mg/kg) or vehicle at midnight to contrast modafinil-induced wakefulness with spontaneous wakefulness.
Experiment 2. In contrast to previous studies, experiment 1 did not demonstrate an increase in SCN or AHA Fos expression with modafinil. Because these differences may have been caused by
time-of-day effects, we administered modafinil (150 mg/kg) at noon as
done in the previous studies. These experiments still did not show any
changes in these regions, and we were concerned that morning light
might induce Fos in the SCN, masking any drug-induced changes. Thus,
additional groups of animals were maintained in constant darkness on
the day of the experiment, and a wider range of doses (75, 150, and 300 mg/kg) was studied.
Experiment 3. Because experiments 1 and 2 did not
demonstrate any modafinil-induced change in SCN or AHA Fos expression,
we replicated the protocol of Engber et al. (1998a).
Uninstrumented rats were handled daily and received direct
intraperitoneal injections of saline at noon for 4 d. On the fifth
day, they received modafinil (300 mg/kg) or vehicle at noon and were
killed 2 hr later.
Animals and recording environment
Sixty-five male Sprague Dawley rats (Harlan Sprague Dawley,
Indianapolis, IN) weighing 270-330 gm were housed individually in a
pathogen-free barrier facility in a room maintained at 21.5-22.5°C with lights on at 7 A.M. and off at 7 P.M. Rats had food and water available ad libitum. At least 3 d before each
experiment, rats were placed into a light-tight, sound-attenuated
recording chamber (Biocube; Hartford Systems) in an isolated room.
Light intensity was 100-150 lux inside each cage during the light
period and <0.5 lux during the dark period. The Institutional Animal
Care and Use Committees of Beth Israel Deaconess Medical Center and
Harvard Medical School approved all procedures.
Animal surgery
Under chloral hydrate anesthesia (350 mg/kg, i.p.), the rats of
experiments 1 and 2 were surgically implanted with four EEG screws
(anteroposterior, +3,  4; lateral, +2, 2 from bregma) lightly
contacting the dura and two EMG wires (Plastics One, Roanoke, VA) below
the nuchal muscles. The leads were connected to a six-channel connector
(Plastics One) that was affixed to the skull with dental acrylic. A
telemetric temperature transmitter (TA10TA-F40; Data Sciences
International, St. Paul, MN) was placed in the peritoneal cavity of all
but five rats. To administer drugs without handling the rats, an 80 cm
SILASTIC catheter (1 mm inner diameter; Baxter Scientific
Products) was inserted into the peritoneal cavity, subcutaneously
tunneled to the scalp, cemented in place with dental acrylic, and
protected externally by a spring. This intraperitoneal catheter was
filled with heparinized, pyrogen-free saline and flushed weekly and
3 d before the experiment. Animals recovered at least 14 d
and then acclimated to recording cables for 3 d before the start
of physiological recordings.
Drug administration
Modafinil (lot #PA 008; Cephalon, Inc., West Chester, PA) was
suspended in a solution of 0.25% methylcellulose, pH 7.4 (Dow Chemical, Inc., Midland, MI), in 0.9% pyrogen-free saline. The drug
was administered in a volume of 2.0 ml/kg at doses of 75, 150, or 300 mg/kg. Control animals received an equal volume of methylcellulose
vehicle. Catheters were then flushed with 1 ml of 0.9% saline to
ensure delivery of drug into the peritoneal cavity. A dim, red
flashlight (8 lux at 25 cm) was used during injections performed in the dark.
Histology and immunohistochemistry
Two hours after drug injections, animals were deeply
anesthetized with chloral hydrate (600 mg/kg, i.p.) and transcardially perfused with 100 ml of 0.9% saline followed by 500 ml of
phosphate-buffered 10% formalin, pH 7.0 (Sigma, St. Louis, MO). 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) at 4°C. Brains were sectioned (1:5 series; 30 µm) on a freezing microtome and stored in PBS-azide at 4°C. One
series from each brain was stained for Fos by the use of previously
described methods (Elmquist et al., 1996). Briefly, sections
were incubated for 48 hr at 4°C in anti-Fos rabbit polyclonal
antiserum (Ab-5; Oncogene Research Products; 1:100,000 dilution), 3%
donkey serum (Jackson ImmunoResearch, West Grove, PA), and PBS-azide
with 0.25% Triton X-100 (PBT-Az). Tissue was then rinsed in PBS,
incubated in biotinylated donkey anti-rabbit IgG (1:1000;
Jackson ImmunoResearch) for 1 hr at room temperature, and incubated
with peroxidase-conjugated avidin-biotin complex (ABC; Vector
Laboratories, Burlingame, CA) for 1 hr, followed by 0.05%
diaminobenzidine tetrahydrochloride (DAB) and 0.01%
H2O2 with 1%
NiSO4 and 0.5% CoCl2, to
produce a black reaction product in cell nuclei.
Double staining for Fos and orexin was performed on the tissue of
experiment 1 by the use of an antiserum directed against orexin-A
(M.Y., University of Texas Southwestern Medical Center at Dallas).
After the Fos staining, sections were rinsed in PBS-azide and incubated
in rabbit anti-orexin antiserum (1:5000) with 3% normal donkey serum
and PBT-Az overnight at room temperature. Sections were then incubated
in donkey anti-rabbit IgG (1:500; Jackson ImmunoResearch) for 1 hr at
room temperature, and the ABC and DAB steps were followed as above with
the omission of the NiSO4 and
CoCl2 in the DAB step to yield a brown
cytoplasmic product in orexin-IR neurons. All tissue was mounted on
gelatin-coated slides, dehydrated in ascending concentrations of
ethanol, dilipidated in xylenes, and coverslipped. The orexin
immunostaining was blocked by preadsorption with 50 µg/ml orexin-A,
and omission of the primary antisera resulted in no specific staining.
Physiological recordings
Intraperitoneal temperature and EEG and EMG signals were
recorded for at least 30 hr before and 2 hr after drug or vehicle injections. EEG and EMG signals were amplified using Grass model 12 amplifiers and digitally acquired using ICELUS software (Mark Opp;
University Texas at Galveston). 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 bins. Arousal states were scored in 12 sec epochs by a single examiner blinded to the treatment group of the
animals. Intraperitoneal temperature was monitored every 5 min using
Dataquest software (Data Sciences International) and integrated into 30 min bins.
Cell counts
The pattern of Fos immunoreactivity was examined throughout the
entire brain, and regions in which modafinil-treated rats appeared to
differ from controls were identified. To quantify these differences,
Fos-IR neurons were counted in regions with possible modafinil-induced
Fos by an examiner blinded to experimental conditions. For all nuclei,
bilateral counts were taken on three consecutive sections, 120 µm
apart, that contained the largest nuclear areas, and these six counts
were averaged. In experiment 1, Fos-IR nuclei were counted in regions
implicated in behavioral state control: the ventrolateral preoptic area
(VLPO), SCN, AHA, PFx, TMN, ventral tegmental area, dorsal raphe
nucleus, and locus coeruleus (LC). Traditional stimulants often
increase sympathetic activity, and therefore Fos-IR nuclei were also
counted in putative autonomic regulatory regions: the paraventricular
nucleus of the hypothalamus, laterodorsal subnucleus of the bed nucleus
of the stria terminalis (BSTLD), the lateral subdivision of the central nucleus of the amygdala (CeL), the central and external lateral subnuclei of the parabrachial nucleus, and the nucleus of the solitary
tract. Counts were also performed in other areas of interest: the
cingulate cortex and a region just posterior to the ventral tegmental
area referred to as the retro-ventral tegmental area. In most of these
regions, counts were performed within the region demarcated by the
borders of the nucleus as seen in dark field. Fos-IR nuclei were
counted in the VLPO and several other regions without clear borders by
the use of predetermined counting boxes as described previously
(Scammell et al., 1998). AHA nuclei were counted within a vertically
oriented 0.5 × 0.8 mm box with the medial edge along the
ventricular wall and the ventral edge bounded by the optic chiasm;
these counts began at the most caudal section that included the SCN,
but the SCN was excluded from AHA counts. In the cingulate cortex,
labeled nuclei were counted within a horizontally oriented 1 × 0.5 mm box with the ventral edge along the corpus callosum and the
medial edge at the pial surface of the cortex at the level of the
decussation of the anterior commissure. In the same sections, striatal
Fos-IR nuclei were counted within a 0.5 × 0.5 mm box with the
dorsomedial corner of the box 0.5 mm below and 0.5 mm lateral to the
dorsomedial corner of the striatum. PFx Fos-IR nuclei, orexin-IR
neurons, and double-labeled neurons were counted in sections beginning
360 µm caudal to the paraventricular nucleus of the hypothalamus
using a horizontally oriented 1 × 0.4 mm box centered just dorsal
to the fornix. Fewer regions were counted in experiments 2 and 3.
Statistical analysis
Mann-Whitney rank-sum tests with a Bonferroni correction were
used to compare physiological variables between the modafinil and
vehicle groups during the 2 hr after injection; Kruskal-Wallis tests
were used for comparisons of three or more groups. Data were analyzed
in 1 hr intervals, and p was considered significant if
<0.025 because two time points were studied. These same tests were
used for comparisons of Fos-IR cell counts; p was considered significant if <0.05. In experiments 1 and 2 in which multiple doses
of modafinil were tested, a post hoc Scheffe test was used to identify significant differences from vehicle with p < 0.05 considered significant. Counts of Fos-IR nuclei were not
corrected for double-counting errors (Guillery and Herrup, 1997)
because there was no change in sizes of labeled nuclei across groups
and only relative, not absolute, values were sought.
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RESULTS |
Experiment 1: injections of modafinil at midnight
In experiment 1, we administered modafinil (75 or 150 mg/kg)
or vehicle during the rat's normal waking period at midnight to
contrast spontaneous wakefulness with modafinil-induced wakefulness.
Behavioral effects of modafinil
During the baseline night, no differences in the amounts of wake,
nonrapid eye movement (NREM), or REM sleep were evident across the
three groups. After injection of vehicle on the night of the
experiment, control rats continued to have the same amount of each
behavioral stage that they had on the previous night (Fig. 1). However, rats treated with either 75 or 150 mg/kg modafinil spent >95% of the first postinjection hour
awake (p < 0.0001 for either dose). Low-dose
modafinil appeared to promote waking less effectively over time; during
the second hour after injection, 75 mg/kg modafinil increased waking to
80% (p = 0.02), whereas the 150 mg/kg dose
increased waking to 96% (p < 0.001). Neither dose of modafinil significantly affected body temperature.
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Figure 1.
Effects of modafinil on rats treated at midnight.
Modafinil produces clear increases in waking, whereas rats treated with
vehicle have no increase in waking. *p < 0.001.
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The pattern of neuronal activation induced by modafinil
Treatment with modafinil consistently increased Fos expression in
many brain regions, with 75 mg/kg modafinil inducing Fos in fewer
neurons compared with 150 mg/kg (Table
1).
Some of the most striking changes in Fos expression occurred within
specific hypothalamic areas implicated in the control of wakefulness
(Figs. 2,
3). After either dose of modafinil, the TMN had four times as many Fos-IR neurons as seen in the controls (Fig.
3). Neurons of the VLPO are active during sleep (Sherin et al., 1996;
Szymusiak et al., 1998), and because all animals were mainly awake, it
was not surprising that Fos-IR VLPO neurons were uncommon in all rats.
No changes in Fos expression were evident in the SCN or AHA (Fig.
3).
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Figure 2.
A series of line drawings illustrating the
distribution of Fos-IR neurons in individual rats after administration
of vehicle (A) or modafinil (75 mg/kg;
B) at midnight. Modafinil-treated rats have substantial
increases in Fos-IR neurons in hypothalamic arousal regions such as the
TMN and PFx, as well as in the
BSTLD and the CeL. Increased numbers of
Fos-IR neurons can also be seen in the cortex, islands of Calleja
(IC), and striatum, but not in the SCN or
AHA.
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Figure 3.
Effects of vehicle (A, C,
E) or modafinil (B, D, F) on Fos
expression in putative sleep/wake regulatory regions. Administration of
modafinil (150 mg/kg) at midnight substantially increases Fos
expression in the TMN, but little change is evident in
the SCN or AHA. Scale bars, 100 µm.
3V, Third ventricle; ox, optic
chiasm.
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In the PFx at the tuberal level of the hypothalamus, modafinil-treated
rats had twice as many Fos-IR neurons as controls. To determine whether
this Fos expression occurred in orexin neurons, we used double
immunohistochemistry for Fos and orexin. The number of PFx orexin-IR
neurons with Fos-IR nuclei was increased threefold by modafinil (Fig.
4). Modafinil slightly increased the
number of nonorexin-IR, Fos-IR neurons (total PFx Fos-IR neurons minus double-labeled neurons), but this change did not reach statistical significance. The number of orexin-IR neurons did not differ among groups.
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Figure 4.
Few orexin-IR neurons contain Fos in rats treated
with vehicle at midnight (A), but Fos-IR nuclei
are common in orexin-IR neurons of rats treated with modafinil (150 mg/kg; B). Single arrow, Orexin-IR
neuron; double arrows, Fos-IR and orexin-IR neuron.
Scale bar, 30 µm.
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In other hypothalamic areas, modafinil produced a moderate increase in
Fos expression within the paraventricular nucleus, but only small
increases were seen in the ventromedial preoptic nucleus, median
preoptic nucleus, supraoptic nucleus, and dorsomedial nucleus. No
consistent changes were evident in the arcuate nucleus, ventromedial
nucleus, or supramammillary region.
Modafinil-treated rats had more Fos immunoreactivity in the cortex than
did the controls. This Fos induction was evident across much of the
cortex but was often more pronounced in cingulate and pyriform cortex
with moderate amounts in frontal and parietal cortex; the spontaneously
awake control animals usually had a more diffuse and less intense
pattern of cortical Fos. Fos-IR neurons were always rare in the hippocampus.
Fos-IR neurons were uncommon in the basal ganglia of vehicle-treated
rats but common in modafinil-treated rats, especially in the caudate
and putamen (Fig. 5). Few Fos-IR neurons
were present in the ventral striatum, and none were seen in the globus
pallidus. Modafinil induced small but statistically insignificant
increases in Fos expression in the shell and core of the accumbens
nucleus. The olfactory tubercle of modafinil-treated rats had striking increases in the number of Fos-IR neurons in the islands of Calleja. No
change in Fos expression was evident in the basal forebrain.
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Figure 5.
Photomicrographs of several regions with notable
increases in Fos-IR neurons after treatment with modafinil (150 mg/kg)
at midnight. A, The IC have marked
increases in the number of Fos-IR cells, but Fos expression is
considerably less in other parts of the olfactory tubercle.
B, The striatum has large increases in Fos expression.
C, D, The BSTLD (C)
and CeL (D) also show intense Fos
expression that is not seen in other parts of the extended amygdala.
E, F, Modafinil also induces Fos immunoreactivity at the
mesopontine junction in the retro-ventral tegmental area
(retro-VTA; E) and in the
LC (F). Scale bars, 100 µm.
ac, Anterior commissure; cc, corpus
callosum; IPN, interpeduncular nucleus;
LV, lateral ventricle; opt, optic tract;
4V, fourth ventricle.
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In the thalamus, Fos-IR neurons were common in the paraventricular
nucleus of all animals with small increases in modafinil-treated rats.
Low-to-moderate numbers of Fos-IR cells were present in other midline
thalamic nuclei regardless of treatment. No consistent changes were
evident in the ventromedial or reticular nuclei. The lateral
habenular nuclei contained low levels of Fos expression in all rats,
with small increases in modafinil-treated animals.
Modafinil induced striking increases in the number of Fos-IR neurons in
the extended amygdala, particularly the BSTLD and the CeL. Modafinil
produced approximately a fourfold increase in Fos immunoreactivity in
the BSTLD and sixfold increases in the CeL. Subtle increases were
apparent in the ventral part of the bed nucleus of the stria terminalis
and in the medial subdivision of the central nucleus of the amygdala
with little change in other regions.
In the brainstem, the dorsal raphe nucleus and ventral tegmental area
contained only rare Fos-IR neurons with no significant differences
after treatment with modafinil. No qualitative differences were evident
in the pedunculopontine or laterodorsal tegmental nuclei. However,
modafinil consistently produced large increases in Fos expression in a
region that we refer to as the retro-ventral tegmental area (Fig. 5);
these Fos-IR neurons were clustered dorsolateral to the most caudal
portion of the interpeduncular nucleus, along the lateral margin of the
caudal linear and median raphe nuclei. The 150 mg/kg dose of modafinil
produced consistent increases in Fos expression in the LC (Fig. 5).
Fos-IR neurons were abundant among the transverse fibers of the pons in
all groups. Modafinil-treated rats had a small increase in Fos-IR
neurons within the caudal portion of the ventrolateral periaqueductal
gray. In the parabrachial nucleus, modafinil tended to increase Fos
expression in the external lateral subnucleus. After 150 mg/kg
modafinil, the nucleus of the solitary tract had a slight increase in
the number of Fos-IR neurons mainly in the medial subnuclei. No changes
were evident in the ventrolateral medulla.
Experiment 2: injections of modafinil at noon in light/dark or
constant dark conditions
In animals maintained on a normal light/dark (LD) cycle,
administration of modafinil (150 mg/kg) at noon markedly increased wakefulness over the next 2 hr (Fig. 6),
whereas vehicle had no effect compared with the baseline day. Baseline
recordings over the previous day revealed no differences between the
groups in the amounts of wake, NREM, or REM sleep. Neither modafinil
nor vehicle had any effect on body temperature.
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Figure 6.
Effects of modafinil on rats treated at noon on a
12:12 hr LD cycle. Modafinil markedly increases waking, whereas vehicle
has no effect. *p < 0.01.
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The pattern of Fos immunoreactivity was very similar to that seen in
experiment 1. In brief, modafinil-treated rats had clear increases in
the number of Fos-IR neurons in the TMN, with decreases in the VLPO
(Table 2). Brain sections were not double
stained for Fos and orexin, but the total number of Fos-IR PFx neurons had a small but statistically insignificant increase. Modafinil treatment increased expression of Fos in frontal and cingulate cortex,
the striatum, the CeL, and the BSTLD. The numbers of Fos-IR SCN and AHA
neurons were slightly increased but did not reach statistical
significance.
To examine the extent to which this pattern might be influenced
by morning light exposure, additional animals were kept in darkness on
the day of the experiment and treated at noon. These rats also were
treated with lower (75 mg/kg) and higher (300 mg/kg) doses of modafinil
to explore dose-response effects on Fos expression.
In comparison with the previous baseline day during which the
animals were on a regular LD cycle, rats housed in constant darkness
had no change in the hourly amounts of waking. However, this dark
exposure moderately increased REM sleep across the morning to 15% from
an average baseline of 8.5% (p < 0.01 for each
hour between 8 A.M. and noon) as has been described previously (Lisk and Sawyer, 1966; Benca et al., 1991). NREM sleep during this morning
period was mildly reduced to an average of 56.7% from 65.3% on the
baseline day with statistically significant reductions only during the
8-9 A.M. and 9-10 A.M. periods (p < 0.005).
Injections of modafinil produced dose-dependent increases in
wakefulness (Fig. 7). During the first
and second hours after injection, 150 or 300 mg/kg modafinil
significantly increased waking (p < 0.01), but
the wake-promoting effect of the 75 mg/kg dose was marginal during the
first hour (p = 0.06) and not evident during the
second hour (p = 0.55). None of the three doses
produced a significant change in body temperature.
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Figure 7.
Effects of modafinil on rats housed in constant
dark and treated at noon. Rats treated with vehicle have a small and
transient increase in waking, but those treated with modafinil have a
dose-dependent increase in waking. *p < 0.01.
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In most respects, the pattern of Fos immunoreactivity induced by
injections during the dark was similar to that described above. The
modafinil-treated groups had more Fos-IR neurons in the TMN and fewer
in the VLPO (Table 2). The PFx had a small but statistically
insignificant increase in Fos expression. Constant darkness reduced the
number of Fos-IR neurons in the SCN, but modafinil still did not
produce a statistically significant increase in the number of Fos-IR
neurons in either the SCN or AHA. The modafinil-treated rats had a
marked, dose-dependent increase in Fos-IR neurons in the striatum, CeL,
and BSTLD.
Experiment 3: injections of modafinil at noon in
uninstrumented animals
To ensure that the pattern of Fos immunoreactivity was not
influenced by the instrumentation or tethering of animals for
physiological recordings, we followed the experimental protocol used by
Engber et al. (1998a). As found in our other experiments,
modafinil substantially increased Fos expression in the TMN and
decreased Fos in the VLPO (Table 3). Fos
expression was increased in the striatum, CeL, and BSTLD. Modafinil
also produced a modest, statistically insignificant increase in PFx and
SCN Fos expression, but there was no change in the AHA.
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DISCUSSION |
Across a variety of conditions, we found that modafinil promoted
wakefulness and significantly increased Fos expression in the TMN and
in orexin neurons of the PFx, two areas implicated in the regulation of
wakefulness. We also found that higher doses of modafinil induced Fos
immunoreactivity in neurons of the striatum and several other regions.
In contrast to previous studies, we did not detect any statistically
significant changes in the number of Fos-IR neurons in the SCN or AHA.
Technical considerations
Our experiments demonstrate a pattern of Fos immunoreactivity that
differs from that described previously in cats (Lin et al., 1996) or
rats (Engber et al., 1998a), and some technical aspects of our
study deserve comment. Although modafinil sometimes produced small
increases in Fos within the SCN and AHA, this increase was variable and
never close to the 20-fold increase reported by Lin. We were initially
concerned that this might be caused by differences in the treatment of
our animals, but we were unable to detect any substantial changes in
these regions despite administration of modafinil at different times of
day, under light or darkness. Because exposure to dim light during
nighttime injections, instrumentation, or tethering might have
influenced our results, we replicated the methods of Engber and
colleagues in experiment 3, but we still saw no significant change in
the SCN or AHA. Engber described modafinil-induced Fos expression
within the SCN of rats, but their qualitative analysis of a small
number of rats may not be as reliable as our quantitative cell counts
in larger groups of rats. Direct comparison of our results with the cat
studies of Lin and colleagues is difficult because species differences
may exist and they did not describe their cell-counting technique. The
AHA lacks distinct borders, and our AHA counting box may not have been
homologous to the region described by Lin. Alternatively, if modafinil
induces Fos only in a subregion of the SCN or AHA, an increase could be obscured by dilution with counts from the entire nucleus. Still, we
found no increase in Fos in any preoptic or anterior hypothalamic regions. Finally, these previous studies used a different Fos antiserum
(Ab-2), and subtle differences in antibody specificity might contribute
to dissimilar results. These differences in staining and counting
techniques may explain some of the seeming discrepancies between studies.
Modafinil-induced neuronal activation
We found that even low doses of modafinil strongly increased Fos
expression in TMN and orexin neurons, and activation of these neurons
may be an essential component of modafinil's wake-promoting mechanism.
These cell groups are anatomically well positioned to facilitate
wakefulness with ascending projections to the cortex, basal forebrain,
and midline thalamus and descending projections to other
arousal-promoting regions such as the dorsal raphe nucleus, LC, and
pontine cholinergic regions (Inagaki et al., 1988; Peyron et al., 1998;
Chemelli et al., 1999). Both the TMN and PFx regions contain
wake-active neurons as demonstrated with extracellular recordings
(Vanni-Mercier et al., 1984; Steininger et al., 1996), and Fos
immunoreactivity is common in orexin and TMN neurons during spontaneous
wakefulness (Scammell et al., 1998) (I. Estabrooke, M. McCarthy, E. Ko,
T. Chou, R. Chemelli, M. Yanagisawa, C. Saper, and T. Scammell,
unpublished observations). The TMN is the sole neuronal source
of histamine in brain, and these cells are probably necessary for
promoting wakefulness because antihistamines or inhibitors of histamine
synthesis decrease wakefulness (Lin et al., 1988, 1994). Orexin may
play a related role in behavioral state control because orexin
knock-out mice or dogs with mutations in the orexin type-2 receptor
gene have a narcolepsy-like phenotype with poor maintenance of
wakefulness (Chemelli et al., 1999; Lin et al., 1999). Orexin increases
the firing rate of LC neurons (Hagan et al., 1999) and may have similar
excitatory effects on other arousal regions including the TMN (K. Eriksson, personal communication). Thus, the combined activity
of orexin and TMN neurons may play a key role in generating wakefulness
and the response to modafinil.
Modafinil generally did not induce Fos within brainstem arousal regions
such as the dorsal raphe nucleus or ventral tegmental area, although a
small increase was evident in the LC after 150 mg/kg modafinil.
Slightly lower doses of modafinil do not alter the firing rates of LC
neurons (Akaoka et al., 1991), and this LC activation may only play a
role with higher doses. Still, because the absence of Fos does not
exclude activation of specific neurons, we cannot determine whether
these brainstem arousal regions contribute to modafinil's
wake-promoting effects.
Increased activity of TMN neurons should inhibit preoptic sleep-active
neurons. The VLPO contains sleep-promoting neurons (Lu et al., 2000),
and microinjection of histamine into this region decreases sleep (Lin
et al., 1994). In agreement with this model, we found that during the
day, modafinil often decreased the activity of VLPO neurons. This
decrease was not evident among rats treated at midnight when rats from
all groups were mainly awake; most likely Fos immunohistochemistry is
unable to resolve differences in low VLPO activity in predominantly
awake rats.
These experiments demonstrate modafinil-induced activation of
wake-promoting regions and inhibition of a sleep-promoting region, but
Fos immunohistochemistry can only provide correlative observations; we
cannot determine whether this activation of TMN and orexin neurons
helps promote wakefulness or is simply a consequence of sustained
wakefulness. Further experiments to study the effects of modafinil in
orexin knock-out mice or TMN-lesioned animals are needed to test the
necessity of these regions.
Modafinil increases CeL metabolic activity as measured with the
2-deoxyglucose technique (Engber et al., 1998b), and we found that low doses of modafinil strongly increased Fos expression in the
CeL and BSTLD. These two regions have very similar cytoarchitecture, neurotransmitters, afferents, and efferents (Moga et al., 1989; Alheid
et al., 1995), so it is not surprising that they are jointly activated.
The CeL projects to the LHA, dorsal raphe nucleus, and distal dendrites
of the LC (Alheid et al., 1995) and may influence the activity of these
arousal regions. Although lesions of the central nucleus of the
amygdala do not alter the wake-promoting effects of modafinil
(Silvestri et al., 2000), functionally similar neurons in the BSTLD may
still promote wakefulness.
In contrast to amphetamines, modafinil has little potential to produce
addiction or increase blood pressure or heart rate (Broughton et al.,
1997; Mitler et al., 1998). Amphetamine increases Fos expression in the
accumbens nucleus (Johansson et al., 1994), but we found no increase
with modafinil. Modafinil (75 mg/kg) also induced little Fos
immunoreactivity in brain regions critical for autonomic control. At
higher doses, modafinil can slightly increase blood pressure, and 150 mg/kg modafinil produced small increases in Fos expression within
the paraventricular nucleus, external lateral subnucleus of the
parabrachial nucleus, and nucleus of the solitary tract. Although these
increases were not statistically significant, they may reflect
subtle autonomic activation.
Does modafinil act via dopaminergic or GABAergic mechanisms?
The pattern of Fos immunoreactivity induced by modafinil provides
some useful insights into neurochemical mechanisms via which modafinil
may activate neurons. The striatum and all other regions in which we
found increases in Fos-IR neurons receive moderate-to-substantial dopaminergic afferents. However, little evidence indicates that modafinil increases dopaminergic transmission. Modafinil does not
increase locomotor activity (Edgar and Seidel, 1997), bind to dopamine
receptors (Mignot et al., 1994), or alter the firing rates of
dopaminergic neurons in the ventral tegmental area (Akaoka et al.,
1991). Even high doses of modafinil fail to elicit ipsilateral rotation
in rats with unilateral lesions of the nigrostriatal pathway (Contreras
et al., 1997). Still, modafinil might increase dopaminergic
transmission by binding weakly to the dopamine transporter (Mignot et
al., 1994). All regions in which we found modafinil-induced Fos have
increased Fos after treatment with dopamine agonists (LaHoste et al.,
1993; Eaton et al., 1996; Wirtshafter, 1998) or drugs that promote
dopaminergic transmission such as amphetamine (Cole et al., 1992; Lin
et al., 1996).
All regions activated by modafinil in our study also receive at least
moderate GABAergic innervation. Modafinil reduces the outflow of GABA
in cortex, striatum, and posterior hypothalamus as measured by
microdialysis (Tanganelli et al., 1995; Ferraro et al., 1996, 1998).
GABA release in the posterior hypothalamus may promote sleep via
inhibition of the TMN (Yang and Hatton, 1997; Sherin et al., 1998) or
PFx (Lin et al., 1989), and a reduction in GABAergic activity could
disinhibit these hypothalamic regions resulting in increased
wakefulness. Currently, modafinil's neurochemical mechanisms remain
unclear, but increased dopaminergic or decreased GABAergic transmission
may play critical roles in promoting wakefulness.
Conclusions
Administration of modafinil to rats produced wakefulness in
association with activation of TMN and orexin neurons, two cell groups
implicated in the control of normal wakefulness. Activation of specific
arousal regions may underlie the wakefulness produced by modafinil in
people with narcolepsy or other forms of excessive sleepiness.
Selective pharmacological modulation of these regions may represent a
novel approach to producing wakefulness that is devoid of
amphetamine-like subjective effects.
| |
FOOTNOTES |
Received May 10, 2000; revised Aug. 31, 2000; accepted Aug. 31, 2000.
This study was supported by grants from Cephalon, Inc., and by United
States Public Health Service Grants MH 01507 and HL 60292. We are
grateful to Thomas C. Chou for his expert advice and to Courtney Sears
and Quan Hue Ha for excellent technical assistance.
Correspondence should be addressed to Dr. T. 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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TOP
ABSTRACT
INTRODUCTION
