OREXIN KNOCKOUT
Running Promotes Wakefulness and Increases Cataplexy in Orexin Knockout Mice
Rodrigo A. España, PhD1,2; Sarah L. McCormack, BS1; Takatoshi Mochizuki, PhD1; Thomas E. Scammell, MD1
Department of Neurology, Beth Israel Deaconess Medical Center, Boston, MA; 2Department of Physiology and Pharmacology, Wake Forest University
School of Medicine, Winston-Salem, NC
1
Study Objective: People with narcolepsy and mice lacking orexin/hypocretin have disrupted sleep/wake behavior and reduced physical activity. Our objective was to identify physiologic mechanisms through which
orexin deficiency reduces locomotor activity.
Design: We examined spontaneous wheel running activity and its relationship to sleep/wake behavior in wild type (WT) and orexin knockout
(KO) mice. Additionally, given that physical activity promotes alertness,
we also studied whether orexin deficiency reduces the wake-promoting
effects of exercise.
Measurements and Results: Orexin KO mice ran 42% less than WT
mice. Their ability to run appeared normal as they initiated running as
often as WT mice and ran at normal speeds. However, their running bouts
were considerably shorter, and they often had cataplexy or quick transitions into sleep after running. Wheel running increased the total amount
of wakefulness in WT and orexin KO mice similarly, however, KO mice
continued to have moderately fragmented sleep/wake behavior. Wheel
running also doubled the amount of cataplexy by increasing the probability of transitioning into cataplexy.
Conclusions: Orexin KO mice run significantly less than normal, likely
due to sleepiness, imminent cataplexy, or a reduced motivation to run.
Orexin is not required for the wake-promoting effects of wheel running
given that both WT and KO mice had similar increases in wakefulness
with running wheels. In addition, the clear increase in cataplexy with
wheel running suggests the possibility that positive emotions or reward
can trigger murine cataplexy, similar to that seen in people and dogs with
narcolepsy.
Keywords: Orexin, hypocretin, cataplexy, locomotor activity, running,
running wheels, obesity, motivation, positive affect
Citation: España RA; McCormack SL; Mochizuki T; Scammell TE. Running promotes wakefulness and increases cataplexy in orexin knockout
mice. SLEEP 2007;30(11):1417-1425.
INTRODUCTION
ness or cataplexy. Therefore, we examined spontaneous wheel running and its relationship to sleep-wake behavior.
Second, we examined whether orexin is necessary for the wakepromoting effects of exercise. In healthy people, exercise reduces
the likelihood of sleep and promotes alertness, whereas forced
bed rest has the opposite effects.14,15 In rodents, wheel running
increases wakefulness and markedly lengthens wake bouts.16-18
Although the arousing effects of exercise have long been recognized, the neural mechanisms through which physical activity promotes wakefulness remain largely unknown. The orexin
neurons are well-situated to mediate this effect because they receive inputs from brain regions that control locomotion (e.g., the
substantia nigra, periaqueductal gray, and midbrain locomotor
region),19 and they heavily innervate and excite numerous wakepromoting brain regions.20 Furthermore, the orexin neurons are
particularly active during locomotion as shown by extracellular
recordings and the expression of Fos.21-25 To test whether orexin
mediates the wake-promoting effects of exercise, we measured
wheel running in WT and orexin KO mice.
Last, we examined whether running increases cataplexy in
orexin KO mice. In people and dogs, cataplexy is often triggered
by positive emotions such as mirth or surprise, but it remains unknown whether positive emotions trigger cataplexy in narcoleptic
mice. Wheel running is rewarding for mice and probably elicits
positive emotions.26,27 Accordingly, wheel running provides an
excellent opportunity to better understand emotional triggering
of murine cataplexy.
NARCOLEPSY WITH CATAPLEXY IS ASSOCIATED WITH A
LOSS OF THE OREXIN (HYPOCRETIN)-CONTAINING NEURONS IN THE HYPOTHALAMUS.1-3 MICE LACKING OREXIN are an excellent model of narcolepsy as they have sleepiness
and episodes of sudden atonia resembling human cataplexy.4,5 In
addition, mice lacking the orexin neurons can become mildly obese
similar to that seen in human narcolepsy.6 In part, this obesity may
be a consequence of reduced physical activity because orexin-deficient mice have less spontaneous movement than WT mice.5,7,8 To
better understand the interaction of physical activity with sleepiness
and cataplexy, we examined the influence of wheel running on the
behavior of wild type (WT) and orexin knockout (KO) mice.
First, we examined whether sleepiness, cataplexy, or reduced motivation might contribute to the reduced locomotor activity (LMA)
observed in orexin KO mice. One possibility is that orexin KO mice
might be less motivated to run because they lack excitatory orexin
signaling to reward pathways between the ventral tegmental area
(VTA) and nucleus accumbens.9-13 Alternatively, given that orexin
promotes arousal and suppresses cataplexy, the reduced LMA in
orexin deficient animals could be a direct consequence of sleepiDisclosure Statement
This is not an industry supported study. Dr. Scammell has received research
support from Takeda and Jazz and has participated in speaking engagements for Cephalon, Jazz, Alexza, Lundbeck, and Takeda. The other authors
have reported no financial conflicts of interest.
METHODS
Submitted for publication January, 2007
Accepted for publication July, 2007
Address correspondence to: T.E. Scammell, Department of Neurology, Beth
Israel Deaconess Medical Center, 77 Avenue Louis Pasteur, Boston, MA
02115; Tel: (617) 667-0833; Fax: (617) 667-0810; E-mail: tscammel@bidmc.
harvard.edu
SLEEP, Vol. 30, No. 11, 2007
Animals
These experiments used 12-15 week old, male, homozygous
orexin KO mice (n = 11) and male WT littermates (n = 9). Mean
weights were 26 grams in each group. Mice had ad lib access to
1417
Running Wheels Increase Cataplexy—España et al
water and regular mouse chow (PicoLab Rodent Diet 20, Purina). Founder lines of mice were on a C57BL/6J-129/SvEV background4 and then backcrossed with C57BL/6J mice for seven to
eight generations. Mice were genotyped using PCR with a neo
primer, 5’-CCGCTATCAGGACATAGCGTTGGC, or a genomic
primer, 5’-GACGACGGCCTCAGACTTCTTGGG, and a genomic primer, 3’-TCACCCCCTTGGGATAGCCCTTCC, common to KO and WT mice. All experiments were approved by
the Institutional Animal Care and Use Committees of Beth Israel
Deaconess Medical Center and Harvard Medical School.
ately before atonia/theta correctly identified 95% of all cataplexy
episodes.30 C) The animal’s posture must be different than that
seen during sleep (i.e., the mouse cannot be curled in a sleeping
position). D) The animal’s location in the cage cannot be its usual
nest. Whenever behavior met criteria A, we examined integrated
infrared video recordings (Sleep Sign) in detail to determine if all
criteria were fulfilled.
Running Wheel Analyses
Running wheel activity was recorded using low torque, polycarbonate running wheels (Fast-Trac, Bio-Serv, Frenchtown, NJ)
modified with photodetectors to record wheel rotations. This style
of wheel was chosen because it does not interfere with the EEG
recording cable. Wheel rotations were recorded in 10-sec epochs
using a data acquisition system (Dataquest, Data Sciences International). Wheel running was analyzed using 2 approaches. First,
we examined running bouts that were defined as at least 2 wheel
rotations in the first 10-sec epoch of running and persistence of
running for at least one additional epoch. A running bout terminated if running ceased for at least 2 consecutive 10-sec epochs.
Next, we analyzed the average amount of wheel running as a
function of wakefulness duration by aligning the ends and beginnings of all wakefulness bouts from the dark period and then calculating the mean amount of running that occurred in each 10-sec
epoch of wakefulness. This second analysis included all wheel
activity, not just the rotations that occurred in running bouts. The
amount of running in each epoch appears lower with this approach because many wake bouts contain no running.
Surgery
Mice were anaesthetized with ketamine/xylazine (100 and 10
mg/kg, i.p.) and placed in a stereotaxic apparatus. Two stainlesssteel screws for recording the EEG were placed; one at 1 mm
rostral and 1.5 mm lateral to bregma and the other at 1 mm rostral and 1.5 mm lateral to lambda. EMG signals were acquired
with a pair of multistranded stainless steel wires (Cooner Wire,
Chatsworth, CA) inserted into the neck extensor muscles. All
leads were attached to a 2 x 2 pin head plug that was secured to
the skull using dental acrylic.
Experimental Protocol
One week after surgery, mice were transferred to recording cages
in a sound-attenuated chamber with a 12:12 hr light:dark (LD) cycle (30 lux; lights on at 07:00 and off at 19:00) and a constant temperature (22–24°C). Mice were connected to recording cables and
allowed to acclimate to running wheels for ≥10 d. On day 12, sleep/
wake behavior and running wheel rotations were recorded for 24
h in the “Wheel Run” condition. On day 13, running wheels were
locked and mice were allowed to adjust to this “Wheel Locked”
condition for 48 h. On day 16, sleep/wake behavior was recorded
for another 24 h with wheels locked. Prior reports and comparison with other recordings in our lab indicate that 2 days of locked
wheels is sufficient for behavior to stabilize.5,17,28 To ensure that running behavior had not changed over time, we then unlocked the
wheels on day 17. In all cases, mice readjusted to wheels within 48
h, running as much as they had on day 12 (F1,18 = 0.2, P=0.7).
Statistics and Analysis
We analyzed the daily amounts of running using a one-way
ANOVA with genotype (WT vs. KO) as the between-subjects
variable. For comparisons between genotypes and time (dark
phase vs. light phase), we used an omnibus 2-way, mixed design
ANOVA, with genotype as the between-subjects variable and
time as the repeated measure variable. We used the same technique for comparisons of genotype and wheel condition (Wheel
Run vs. Wheel Locked), with wheel condition as the repeated
measure variable. If the omnibus test showed significant main or
interaction effects, we used uncorrected one-way ANOVA’s to assess pair-wise comparisons between genotypes, time, and wheel
condition. To analyze the probability of cataplexy as a function
of wake bout length, we first calculated the absolute probability
of transitioning from wakefulness into cataplexy for each minute
of wakefulness and then used a two-way ANOVA with genotype
as the between subjects variable and wake bout length as the repeated measures variable. Results are shown as means ± SEM.
EEG/EMG recordings
Ipsilateral EEG and bilateral EMG signals were acquired using
Grass Model 12 amplifiers (Astro-Med, West Warwick, RI) and
digitized at 128 Hz using a sleep scoring system (Sleep Sign; Kissei Comtec, Matsumoto, Japan). The EEG/EMG signals were digitally filtered (EEG, 0.3–30 Hz; EMG, 2–100 Hz) and manually
scored in 10-sec epochs by a single observer blind to experimental conditions. Behavior was scored as wakefulness (low-voltage
EEG with frequent EMG activity), NREM sleep (high-voltage
EEG with slow waves, low voltage EMG), or REM sleep (lowvoltage, theta-dominant EEG with minimal EMG activity). Behavior was scored as cataplexy only when the following criteria
were met: A) Cataplexy is one or more epochs of EEG theta activity and muscle atonia immediately preceded and followed by
active wakefulness.4,5,29 B) At least 40 sec of wakefulness must
precede cataplexy to exclude any REM sleep that might follow a
brief awakening. This criterion was established from recent work
by the Nishino group showing that 40 sec of wakefulness immediSLEEP, Vol. 30, No. 11, 2007
RESULTS
Patterns of Wheel Running in WT and Orexin KO Mice
Over the first 12 days, WT (n=9) and orexin KO mice (n=11)
acclimated to running wheels, establishing steady, maximal
amounts of running (Figure 1). Running stabilized after 9 days
as indicated by similar amounts of running on days 10, 11, and
12 in each group (F1,18 = 0.17, P = 0.2). The amount of running
1418
Running Wheels Increase Cataplexy—España et al
35000
WT
KO
Total Running
(rotations/24hrs)
30000
25000
20000
Wheels Locked
15000
10000
5000
0
1
2
3
4
5
6
7
8
9
10
11
12 13
14
15
16
17
18
Days
Figure 1—Wild type (WT) and orexin knockout (KO) mice acclimate similarly to running wheels, however, KO mice consistently run less than
WT mice. Running wheels were locked on days 13 through 16. Sleep/wake EEG behavior was recorded on days 12 and 16. Wheels were unlocked
on day 17, and running returned to prior levels.
increased at a similar rate in both groups across the first 12 days
(increases of 850 ± 231 rotations/day in WT mice and 782 ± 175
rotations/day in orexin KO mice; F1,18 = 0.2, P = 0.6).
Although orexin KO mice showed a normal pattern of acclimation, they ran less than WT mice across all days (F1,18 = 9.37,
P<0.01). For example, on day 12, WT mice ran 29,423 ± 4774
rotations (~ 10.3 ± 1.7 km) whereas KO mice ran 16,985 ± 1514
rotations (~ 6.0 ± 0.5 km; F1,18 = 7.28, P<0.02; Figure 2). Additionally, orexin KO mice spent significantly less time running
than WT mice (4.1 ± 0.2 vs. 6.1 ± 0.5 hrs; F1,18 = 16.9, P < 0.001).
The timing of running in KO mice resembled that seen in WT
mice, with over 98% of running occurring during the dark period
in both groups (F1,18 = 87.5, P < 0.001; Figure 3). Locomotor intensity (wheel rotations per hour of wakefulness 31) was reduced
in orexin KO mice across the dark period (F(1,16) = 4.8, P < 0.05).
To investigate why orexin KO mice run less, we analyzed
additional aspects of running during the dark period on day 12
(Figure 4). Orexin KO mice initiated running as often as WT littermates (F1,18 = 0.09, P = 0.8) and ran at similar average (F1,18 = 1.14,
P = 0.3) and maximal speeds (top 10% of running epochs; F1,18 =
0.42, P = 0.5). However, the average length of a running bout was
significantly shorter in orexin KO mice (F1,18 = 9.4, P = 0.01).
These observations suggest that orexin KO mice have normal motivation to initiate running and they run at normal speeds, yet their
total running is reduced due to their shorter running bouts.
minute, and 68% of these transitions occurred within ten seconds
of the end of running. Occasionally, cataplexy occurred while
the animal was on the running wheel, however, more typically,
the mouse stumbled off of the wheel and had a cataplexy episode soon thereafter (Supplemental Movie). These observations
support the hypothesis that running bouts in orexin KO mice are
shorter because of imminent sleepiness or cataplexy.
To further explore these possibilities, we analyzed wheel running
around episodes of sleep and cataplexy. Prior to sleep, WT and KO
mice had comparable average amounts of running and gradually
decreased their running over the 5 min before sleep onset (Figure
5). After sleep, running gradually resumed, reaching steady levels
within 5 to 10 min. Perhaps sleepiness leads to the gradual reduction in running before sleep and sleep inertia hinders the expression
of running after sleep. If so, then KO mice show similar amounts of
sleepiness and sleep inertia around episodes of sleep.
On average, KO mice ran more in the minutes prior to cataplexy
than prior to sleep (F1,18 = 49.8, P < 0.001), and running persisted at
high levels until ceasing immediately before episodes of cataplexy.
After cataplexy, orexin KO mice rapidly resumed running, reaching steady, high amounts within 1 min. The greater amounts of running before cataplexy may reflect high emotional tone that helps
trigger cataplexy, and the abrupt onset and recovery from cataplexy
highlights its differences from sleep.
Together, these data indicate that orexin KO mice have short
sleep latencies and rapid transitions into cataplexy after running,
suggesting that sleepiness and imminent cataplexy may contribute to their short running bouts.
��������
Effects of Sleep/Wake Behavior on Wheel Running
Reduced running in orexin KO mice may be associated with
sleepiness or cataplexy. To explore these possibilities, we examined the pattern of wheel running in relation to sleep/wake behavior during the dark phase on day 12. On average, WT mice
were awake 5.5 ± 1.1 min before a running bout, whereas orexin
KO mice were awake only 2.6 ± 0.3 min before running (F1,18 =
8.0, P < 0.01). After a bout of running, WT mice remained awake
for 10.4 ± 2.8 min before transitioning into NREM sleep. In contrast, orexin KO mice remained awake for only 4.7 ± 0.8 min after
running (F1,18 = 4.7, P < 0.05). Additionally, in orexin KO mice,
35% of all running bouts were followed by cataplexy within one
SLEEP, Vol. 30, No. 11, 2007
Effects of Wheel Running on Sleep-Wake Behavior
To examine the impact of running wheels on sleep/wake behavior, we compared the Wheel Run and Wheel Locked conditions.
Consistent with prior observations 16-18, wheel running increased
and consolidated wakefulness during the dark period but had no
significant effect on sleep/wake behavior during the light period
(Figure 6 and Supplemental Table). During the dark period, wheel
running increased the amount of time spent awake by approximately 20% in WT and orexin KO mice (F1,18 = 68.5, P < 0.001).
1419
Running Wheels Increase Cataplexy—España et al
A
5000
40000
WT
KO
4000
30000
Running
(rotations/hr)
Total Running
(rotations/24hrs)
A
*
20000
3000
** *
2000
**
*
1000
10000
0
0
B
WT
7pm
KO
Wheel Lock
8
Wheel Run
7
6
6
5
Cataplexy
(bouts/hr)
Time Spent Running
(hours/24hrs)
7pm
B
7
**
4
3
5
**
*
4
3
*
2
2
1
1
0
7am
0
WT
7pm
KO
7am
7pm
Figure 3—A) Orexin KO mice run less than WT mice, however,
the temporal pattern of running is preserved. In both groups, 98%
of running occurs during the dark period. B) In orexin KO mice,
cataplexy is more frequent with running wheels unlocked, but the
distribution of cataplexy across the dark period is similar to that seen
with locked wheels. *P < 0.05; **P < 0.01.
Figure 2—Orexin KO mice run less than WT mice. A) Over 24
hours, KO mice run 42% less than WT mice. B) Orexin KO mice
spend less time running than WT mice. Data shown here and on subsequent figures are from day 12, when mice were fully acclimated to
wheels. *P < 0.05; **P < 0.01.
Thus, the increase in total wakefulness with wheel running occurs
even in the absence of orexin.
Wheel running also consolidated wakefulness. The number of
wake bouts was reduced in both groups (F1,18 = 21.8, P < 0.001),
and the average duration of wake bouts increased by 75% in orexin
KO mice and by 290% in WT mice (F1,18 = 30.3, P < 0.001). Wheel
running shifted the distribution of wakefulness into longer wake
bouts, with approximately half of all wakefulness occurring in very
long bouts (longer than 42.5 min) in WT mice (Supplemental Figure). Orexin deficiency disrupts the production of long wake bouts,5
and this shift to longer bouts was less prominent in KO mice.
In both groups, wheel running reduced the amount of dark period NREM sleep by 40%-50% (F1,18 = 60.2, P < 0.001) and decreased the amount of REM sleep by 60%-70% (F1,18 = 82.2, P <
0.001) mainly through a reduction in the number of NREM and
REM bouts. This reduction in sleep had little effect on measures
of sleep homeostasis. EEG delta power during either dark or light
period NREM sleep was not increased (F1,18 = 3.0, P = 0.1) and
there were no significant increases in the amounts of NREM (F1,18
= 3.4, P = 0.08) or REM (F1,18 = 2.8, P = 0.11) sleep during the
light period.
Compared to WT mice, orexin KO mice still had fragmented
wakefulness and sleep, despite the wake-promoting effects of
wheel running (Figure 6). During the dark period, KO mice had
slightly less wakefulness (F1,18 = 14.2, P < 0.001) and much shorter
wake bouts than WT mice (F1,18 = 40.6, P < 0.001). Additionally,
the durations of NREM and REM bouts were shorter in KO mice
(NREM, F1,18 = 28.4, P < 0.001; REM, F1,18 = 16.3, P < 0.001),
though the total amounts of each sleep state were similar in both
groups. Overall, wheel running increased the total amount of
wakefulness but only partially improved the fragmented sleep/
wake behavior of orexin KO mice.
Figure 3
Effects of Running Wheels on Cataplexy
Wheel running substantially increased cataplexy in orexin
KO mice. Compared to the Wheel Locked condition, orexin KO
mice with unlocked wheels spent nearly twice as much time in
cataplexy (F1,18 = 11.6, P < 0.01). This increase was caused by a
doubling in the number of cataplexy bouts during the dark period
(F1,18 = 22.2, P < 0.001) with only a slight decrease in cataplexy
duration. Most cataplexy bouts (78%) were preceded by some
wheel running in the prior minute. During the light period, cataplexy remained rare and unaffected by the presence of a functional running wheel despite less dark period REM sleep.
To examine whether the increase in cataplexy with wheel running is a consequence of prolonged wakefulness, we analyzed
cataplexy as a function of wake bout length. With running wheels
locked, nearly all episodes of cataplexy occurred during the first
15 min of wakefulness, and on average, orexin KO mice had about
18 episodes of cataplexy during the dark period (Figure 7). With
running wheels unlocked, most cataplexy still occurred during
the first 15 min of wakefulness, but it occurred more frequently,
resulting in an average of 40 episodes of cataplexy during the
dark period. With locked wheels, the absolute probability of transitioning from wakefulness into cataplexy in any minute gradually rose over the first 10 min of wakefulness and then remained
roughly constant. However, when running wheels were unlocked,
��������
SLEEP, Vol. 30, No. 11, 2007
1420
Running Wheels Increase Cataplexy—España et al
the probability of entering cataplexy was generally higher during
the first 10 min of wakefulness (F1,18 = 5.65, P<0.05). In addition, for both the Wheel Run and the Wheel Locked conditions,
the longer a KO mouse was awake, the more likely it was to experience a cataplexy episode (F15,119 = 60.95, P<0.001). Thus, at
least 2 factors contribute to the increase in cataplexy with wheel
running: a time-dependent increase in cataplexy probability (cataplexy is more likely with prolonged wakefulness); and a timeindependent increase in the probability of cataplexy (cataplexy is
generally more likely with wheel running).
A
Number of
Running Bouts
125
100
75
50
25
DISCUSSION
0
The current studies demonstrate that running is reduced in orexin KO mice because their running bouts are considerably shorter
than normal. Transitions into sleep or cataplexy occur shortly after running in KO mice, and running bouts may be shortened by
sensations of sleepiness or imminent cataplexy. In addition, wheel
running doubles the amount of cataplexy, suggesting that cataplexy is triggered by high levels of activity or possibly heightened
emotional tone associated with running.
Average Running Speed
(rotations/min)
B
Possible Causes of Reduced Wheel Running in Orexin KO Mice
Orexin KO mice run much less than WT littermates, largely
due to shortened running bouts. Sleepiness and cataplexy are two
obvious explanations. After a bout of running, orexin KO mice
fall asleep more quickly than WT mice, and 35% of all running
bouts are followed by cataplexy within 1 min. These associations
do not prove that sleepiness or cataplexy directly truncate running
bouts, but these hypotheses could be tested by examining wheel
running in orexin KO mice treated with cataplexy-suppressing or
wake-promoting drugs that have little effect on locomotor activity
(e.g. modafinil31,32).
Alternatively, orexin KO mice may run less because they are
less motivated to continue running or they find running less rewarding. Orexin KO mice appear motivated to initiate running
as often as WT mice (normal number of running bouts), however, their failure to sustain running could reflect an inability to
remain motivated. Wheel running can be viewed as a naturally
rewarding and potentially addictive behavior that is dependent
on the release of dopamine from mesolimbic projections to the
nucleus accumbens.26,27 Orexin neurons are activated in anticipation of a food or drug reward,10 and they innervate and excite
neurons in the VTA and nucleus accumbens.20,33 Direct injection
of orexin into the nucleus accumbens increases locomotor activity,12 and the locomotor-enhancing effects of orexin are blocked
by dopamine antagonists.9 Considered together, these studies
demonstrate that orexin strengthens reward signals by enhancing signaling in the VTA and nucleus accumbens, and suggest
that orexin KO mice may run less because they find running less
rewarding.
Orexin has also been implicated in other systems that may directly influence running. The orexin neurons innervate areas that intimately control motor behavior such as the substantia nigra, midbrain
locomotor region, and motor neurons.20 Injection of orexin into the
midbrain locomotor region of decerebrate cats reduces the amount
of electrical stimulation needed to trigger locomotion,34 and when
applied near motor neurons, orexin increases muscle tone.35,36 However, direct effects of orexin on motor systems seem an unlikely exSLEEP, Vol. 30, No. 11, 2007
150
Maximal Running Speed
(rotations/min)
Duration of Running
Bouts (mins)
WT
KO
WT
KO
80
60
40
20
150
120
90
60
30
0
D
KO
100
0
C
WT
5
4
3
**
2
1
0
WT
KO
Figure 4—Orexin KO mice initiate but do not sustain bouts of running. A) Orexin KO mice begin running as frequently as WT mice.
B and C) The average running speed and maximal speed (top 10%
of running epochs) in KO mice are similar to WT littermates. D) The
mean duration of running bouts is much shorter in orexin KO mice.
**P < 0.01.
1421
Running Wheels Increase Cataplexy—España et al
���
KO cataplexy
KO sleep
WT sleep
8
Average Running
(rotations/10 sec)
7
6
5
4
3
2
1
0
-10 -9 -8
-7 -6
-5
-4
-3
-2
-1
0 0
1
2
3
4
5
6
7
8
9
10
Minutes Before or After Sleep/Cataplexy Episode
Figure 5—The patterns of locomotor activity around sleep and cataplexy. Both WT and orexin KO mice have gradual reductions in wheel running prior to sleep, and then wheel running gradually increases after sleep. In contrast, prior to cataplexy, orexin KO mice have larger amounts
of running that abruptly stop 10 to 20 seconds before cataplexy, and then they resume running within a minute after cataplexy. Graphs show the
mean amount of wheel running in each 10-sec wake epoch, aligned to the end or beginning of a bout of wakefulness. (Many wake bouts contain
no running, so this does not represent running speed.)
planation for reduced running because orexin KO mice have normal
running speeds and initiate running as frequently as WT mice.
Overall, our experiments suggest that orexin KO mice run
less due to sleepiness and cataplexy. Future studies could examine whether reduced motivation or decreased activation of motor
pathways also contribute to this deficit.
cataplexy. These results suggest that cataplexy can be triggered
by running or the internal states that accompany running. Vigorous physical activity itself is unlikely to be the trigger because
cataplexy is uncommon during unexciting exercise in people with
narcolepsy, though this should be tested in animal experiments
using forced exercise. Laughter, mirth, and surprise frequently
trigger cataplexy in people with narcolepsy,38 and play and highly
palatable food often trigger cataplexy in narcoleptic dogs.39 Thus
far, there has been little evidence that positive emotions trigger
cataplexy in mice, although cataplexy is sometimes preceded by
behaviors that could be associated with positive emotions such as
climbing and burrowing.4 Wheel running is rewarding for mice as
they will work to access wheels,40 and though it is speculative, it
is possible that wheel running induces strong, positive emotions
that trigger cataplexy.
Alternatively, the increase in cataplexy could be caused by increased wakefulness with reduced sleep and increased REM sleep
pressure. First, cataplexy occurs during wakefulness, and with
wheel running, mice are simply awake more, with more time at
risk for a transition into cataplexy. However, this explanation appears insufficient as wheel running doubles the amount of cataplexy, yet the total amount of wakefulness increases only 20%. A
second explanation is that cataplexy increases with wheel running
because wheel running lengthens wake bouts and the probability
of cataplexy increases with the duration of wakefulness; thus, with
wheel running, mice spend more time at risk in long wake bouts.
However, the lengthening of wake bouts with wheel running is
relatively small in orexin KO mice. A third perspective is that the
reduction in dark period sleep with wheel running could increase
the pressure for cataplexy because cataplexy is more frequent in
dogs with less REM sleep41 and clinical observations suggest that
sleep deprivation worsens cataplexy perhaps by increasing REM
sleep pressure.42 Still, the reduction in dark period sleep with wheel
running appears not to generate much sleep pressure as there is
no significant increase in light period sleep, delta power in NREM
The Effects of Running Wheels on Wakefulness
The behavioral response of WT and orexin KO mice to wheel
running confirms that physical activity promotes arousal. In
healthy people, exercise promotes wakefulness, whereas forced
bed rest often results in more sleep and shorter bouts of wakefulness.14,15 Consistent with prior studies,16-18 we found that wheel
running increases the total amount of wakefulness during the dark
period by about 20% and lengthens the duration of wake bouts in
both WT and orexin KO mice. The orexin neurons are innervated
by brain regions that influence locomotor activity including the
substantia nigra,19 and the orexin neurons are especially active
during periods of robust locomotor activity.21-25 These observations suggested that orexin might be required for the wake-promoting effects of wheel running, but this mechanism now appears
unlikely as both WT and orexin KO mice showed proportionately
similar increases in total wakefulness in the presence of running
wheels. Although orexin is necessary for producing long periods
of wakefulness,5 the arousing effects of wheel running may be
mediated by other signaling molecules such as norepinephrine.37
The Effects of Running Wheels on Cataplexy
Wheel running doubles the amount of cataplexy in orexin KO
mice, in part, due to an increase in the amount of wakefulness
and longer bouts of wakefulness. However, even after correcting for these changes, wheel running increases the probability of
cataplexy per minute of wakefulness and running often precedes
SLEEP, Vol. 30, No. 11, 2007
Figure 5
1422
Running Wheels Increase Cataplexy—España et al
WT
KO
100
**
Percentage of
Time
80
Percentage of
Time
100
60
40
20
60
40
**
WAKE
NREM
150
150
Number of Bouts
180
60
**
30
**
120
WAKE
NREM
REM
**
Cataplexy
**
90
**
60
**
**
0
WAKE
REM
50
NREM
REM
Cataplexy
25
40
**
Bout Duration
(mins)
Bout Duration
(mins)
NREM
30
**
0
**
WAKE
180
90
**
0
REM
120
Wheel Run
**
20
**
0
Number of Bouts
80
Wheel Lock
30
20
10
20
15
10
*
0
WAKE
NREM
5
0
REM
**
*
WAKE
**
NREM
REM
*
Cataplexy
Figure 6—Wheel running consolidates sleep/wake behavior during the dark period, especially in WT mice. Wheel running in WT mice produces
more wakefulness during the dark period, and wakefulness occurs in considerably longer bouts. A similar pattern occurs in orexin KO mice, though
the increase in wake bout duration is smaller. Additionally, wheel running doubles the number of cataplexy episodes in orexin KO mice. *P < 0.05;
**P < 0.01.
sleep during the dark and light periods is not higher, and the distribution of cataplexy across the dark period is unaltered (no increase
in cataplexy towards the end of the dark period as one might expect
with rising sleep pressure). The increased risk of cataplexy per minute of wakefulness suggests that positive emotions could trigger
cataplexy with wheel running, although long bouts of wakefulness
and less sleep may also contribute.
reduced motivation likely underlie this inability to sustain running, and future studies could test the importance of these factors
by treating KO mice with drugs that improve alertness and suppress cataplexy and by studying motivation and reward in formal
operant conditions. An improved understanding of these factors
should provide new perspectives on the mild obesity that is common in people with narcolepsy.
Wheel running doubles the amount of cataplexy in orexin KO
mice, and thus on a practical level, wheel running provides a
simple method for increasing cataplexy that should be useful in
future cataplexy experiments. Whether this increase in cataplexy
is caused by physical activity or positive emotions remains un-
��������
Implications and Future Directions
These experiments demonstrate that orexin KO mice run less
because their running bouts are shorter. Sleepiness, cataplexy, or
SLEEP, Vol. 30, No. 11, 2007
1423
Running Wheels Increase Cataplexy—España et al
A
50
Wheel Lock
Cumulative Wake to
Cataplexy Transitions
45
Wheel Run
40
35
30
25
20
15
10
5
0
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
>15
Wake Bout Length (min)
Probability of Cataplexy
per Minute of Wakefulness (%)
B
20
Wheel Lock
Wheel Run
15
**
10
**
5
*
**
**
**
0
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
Wake Bout Length (min)
Figure 7—Wheel running increase the probability of cataplexy. A) With running wheels locked, the cumulative number of transitions into cataplexy gradually increases over the first 15 min of wakefulness. With wheels unlocked, the number of transitions accumulates more rapidly. In both
conditions, a few additional episodes of cataplexy occur after 15 min of wakefulness. B) The probability of transitioning into cataplexy in any
minute of wakefulness is higher with running wheels unlocked. This difference is most apparent in the first 10 min of wakefulness and disappears
by 15 min, probably because of a time-dependent increase in cataplexy.
certain, but ongoing studies suggest that murine cataplexy can be
triggered by other rewards such as anticipation of food, especially
highly palatable food 43. If so, this should provide new opportunities to understand the neurobiology of positive emotions and how
emotions trigger cataplexy in people with narcolepsy.
ACKNOWLEDGMENTS
KO mice were a kind gift from Takeshi Sakurai of the University
of Tsukuba. Cecilia Diniz Behn provided helpful advice on calculating state transition probabilities.
Work performed at: Beth Israel Deaconess Medical Center
Disclosures: This research was supported by grants from the
NIH and Sleep Research Society
Figure 7
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1425
Running Wheels Increase Cataplexy—España et al
WT
Wheel Lock
Wheel Run
60
45
30
15
55
0
12
>2
50
-2
5
70
64
32
012
0
0
031
16
*
**
80
**
0
-1
5
80
10
40
-3
0
**
063
**
0
-7
0
Percentage of Total
Wakefulness
75
Bout Duration (sec)
KO
60
45
30
15
**
55
0
>2
50
-2
5
80
70
64
12
Bout Duration (sec)
012
0
063
32
16
031
0
0
-1
5
-7
0
**
80
10
-3
0
0
40
Percentage of Total
Wakefulness
75
Supplemental Figure 1—Running wheels consolidate wakefulness
during the dark period. These time-weighted frequency histograms
show that with running wheels unlocked, less wakefulness occurs in
very short bouts and more wakefulness occurs in long to very long
bouts. Note that even with unlocked running wheels, the wakefulness of orexin KO mice mainly occurs in mid-length bouts. *, p <
0.05; **, p < 0.01.
�������������������
SLEEP, Vol. 30, No. 11, 2007
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Running Wheels Increase Cataplexy—España et al
Supplemental Table 1—Effects of Running Wheels on Sleep/Wake Behavior.
Wake
WT
KO
NREM
WT
KO
REM
WT
KO
Cataplexy
KO
Wake
WT
KO
NREM
WT
KO
REM
WT
KO
Cataplexy
KO
WR
% of hour
WL
DARK PHASE
Mean bout duration (sec)
WR
WL
Number of bouts
WR
WL
86.0 ± 1.5**
78.8 ± 1.0**
71.5 ± 1.9
66.3 ± 1.3
1697 ± 267 **
366 ± 26 **
435 ± 26
210 ± 11
26 ± 3**
97 ± 6 **
76 ± 4
139 ± 6
12.7 ± 1.3**
15.2 ± 1.0**
25.4 ± 1.8
26.6 ± 1.3
228 ± 21 *
119 ± 7*
162 ± 17
99 ± 5
25 ± 3**
57 ± 6**
75.2 ± 5
121.2 ± 7
1.2 ± 0.2**
1.3 ± 0.2**
3.1 ± 0.3
4.1 ± 0.4
59 ± 5
44 ± 4 **
59 ± 2
55 ± 3
9 ± 1**
13 ± 1 **
24.4 ± 3
31.5 ± 2.5
4.7 ± 0.5**
2.9 ± 0.4
52 ± 4*
63 ± 6
40 ± 4**
18 ± 2
WR
% of hour
WL
LIGHT PHASE
Mean bout duration (sec)
WR
WL
Number of bouts
WR
WL
34.9 ± 1.5
35.3 ± 1.0
37.9 ± 1.4
36.6 ± 0.9
123 ± 27
90 ± 11
106 ± 12
97 ± 10
128 ± 10
170 ± 10
161 ± 15
166 ± 6
57.0 ± 1.3
56.3 ± 1.0
54.6 ± 0.9
55.3 ± 0.8
196 ± 38
145 ± 26
151 ± 30
145 ± 18
130 ± 10
173 ± 9
161 ± 15
166 ± 7
8.0 ± 0.3
8.2 ± 0.4
7.4 ± 0.8
7.6 ± 0.4
64 ± 5
57 ± 57
72 ± 19
69 ± 30
54 ± 2
74 ± 4
47 ± 4
64 ± 4
0.3 ± 0.1
0.4 ± 0.1
56 ± 23
92 ± 60
2±1
2±1
Running wheels increased and consolidated wakefulness during the dark period but had no effects on behavior during the light period. The total
amount of wakefulness increased in both WT and orexin KO mice, but KO mice continued to have relatively short bouts of wakefulness even with
running wheels. In orexin KO mice, running wheels doubled the number of bouts of cataplexy. *, p < 0.05; **, p < 0.01.
SLEEP, Vol. 30, No. 11, 2007
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Running Wheels Increase Cataplexy—España et al
Supplemental Movie 1—A short episode of cataplexy that occurs during wheel running. An orexin KO mouse runs quickly, climbs on top of
the running wheel, and then slides off with hind legs still on the wheel. This cataplexy bout lasts only 20-30 sec, and the mouse then resumes
running. During cataplexy, the EEG contains theta activity (top trace), and the EMG shows very low tone with some EKG artifact (lower trace).
Scoring of behavior: W, wakefulness; C, cataplexy; C*, cataplexy with artifact (excluded from EEG spectral analysis). (Movie file available on
journalsleep.org)
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Running Wheels Increase Cataplexy—España et al