that Hcrt overexpression promotes and consolidates wakefulness and inhibits rest. Similar to humans with insomnia, Hcrt-
sleep and wakefulness are regulated, and the function of sleep
remains controversial (Hendricks et al., 2000a; Greenspan et al.,
2001; Hobson, 2005; Saper et al., 2005; Siegel, 2005). The genetic
mechanisms that regulate sleep are of great medical interest, be-
cause ?10% of Americans suffer chronic sleep disorders (Colten
and Altevog, 2006). Insomnia, defined as an impaired ability to
2001), accounts for ?50% of sleep-related complaints (Colten
and Altevog, 2006).
The identification of defective hypocretin/orexin (Hcrt) sig-
naling as a cause of mammalian sleep disorders highlighted the
power of genetic approaches to sleep research. Dogs and mice
that lack either the neuropeptide Hcrt or its G-protein-coupled
receptors have symptoms of narcolepsy, a syndrome character-
ized by excessive daytime sleepiness, fragmented sleep/wake
states, and sudden loss of muscle tone during waking (cataplexy)
(Chemelli et al., 1999; Lin et al., 1999; Hara et al., 2001; Willie et
al., 2003; Mochizuki et al., 2004; Siegel, 2004). Conversely, direct
injection of Hcrt protein into the brain increases locomotor ac-
tivity and decreases sleep for a few hours (Sakurai et al., 1998;
Hagan et al., 1999; Ida et al., 1999; Yamanaka et al., 1999, 2002;
Bourgin et al., 2000; John et al., 2000; Nakamura et al., 2000;
Piper et al., 2000; Huang et al., 2001; Jones et al., 2001; Espana et
al., 2002; Fujiki et al., 2003; Mieda et al., 2004; Nakamachi et al.,
2006). These studies led to the hypothesis that Hcrt signaling
maintains wakefulness. However, the long-term effects of in-
creased Hcrt signaling on behavior remain unknown but will
greatly impact the therapeutic potential of drugs that stimulate
Hcrt signaling (Zeitzer et al., 2006).
Here we introduce the zebrafish as a model system to analyze
the ancestral roles of Hcrt signaling and to determine the effects
of long-term Hcrt overexpression on behavior. Previous studies
al., 2000; Zhdanova et al., 2001; Saper et al., 2005; Siegel, 2005).
Assays for locomotor activity revealed that zebrafish larvae are
less active and exhibit increased arousal thresholds to a mechan-
ical stimulus at night (Zhdanova et al., 2001). It has also been
shown that the single zebrafish Hcrt ortholog is expressed in
neurons in the posterior hypothalamus (Kaslin et al., 2004;
Faraco et al., 2006) that project to monoaminergic and cholin-
al., 2000). These studies raised the questions of whether Hcrt
regulates sleep/wake states in zebrafish and whether the Hcrt cir-
Neuroscience (A.F.S.). D.A.P. was supported by a fellowship from the Helen Hay Whitney Foundation. J.R. is a
Correspondence should be addressed to David A. Prober or Alexander F. Schier, Department of Molecular and
13400 • TheJournalofNeuroscience,December20,2006 • 26(51):13400–13410
we generated an inducible, long-lasting genetic model of Hcrt
overexpression in zebrafish larvae and developed high-
throughput locomotor activity and arousal assays to study sleep/
Isolation of zebrafish hcrt receptor
Full-length hcrt receptor (HcrtR) cDNA was amplified by performing
reverse transcription-PCR with Superscript II (Invitrogen, Carlsbad,
CA) using primers based on sequence from 5? rapid amplification of
Ensembl exon prediction. The hcrt receptor cDNA sequence has been
deposited in GenBank under accession number EF122429.
In situ hybridization
ing standard protocols and developed using ni-
indolyl-phosphate (Roche, Indianapolis, IN).
Double-fluorescent in situ hybridizations were
performed using DIG- and 2,4-dinitrophenol
(DNP)-labeled riboprobes and the TSA Plus
DNP System (PerkinElmer, Wellesley, MA).
Samples were fixed in 4% paraformaldehyde/
PBS overnight at 4°C and then washed with
0.25% Triton X-100/PBS (PBTx). Brains were
manually dissected and blocked for at least 1 h
incubations were performed in block solution
overnight at 4°C. Primary antibodies were rab-
bit anti-orexin A (AB3704, 1:500; Chemicon,
Temecula, CA), rabbit anti-dopamine ? hy-
droxylase (AB1585, 1:100; Chemicon), mouse
anti-tyrosine hydroxylase (MAB318, 1:100;
Chemicon), rabbit anti-histamine (AB5885,
1:1000; Chemicon), and rabbit anti-serotonin
that the rabbit anti-orexin A antibody specifi-
cally labels hypothalamic Hcrt neurons in adult
zebrafish (Kaslin et al., 2004). Alexa 594-
conjugated secondary antibodies (1:500; Invitro-
gen) were used. Samples were mounted in 100%
Generation of transgenic fish
hcrt–enhanced green fluorescent protein. A 2.4
kb fragment of Fugu rubripes genomic DNA
containing 2 kb of upstream sequence, the pu-
tative hcrt first exon, intron, and the beginning
of the second exon (Ensembl Fugu Assembly
Fidelity kit (Roche). This sequence was sub-
protein (EGFP) and flanked by adeno-
associated viral inverted terminal repeat ele-
ments (Hsiao et al., 2001) and sites recognized
al., 2002). For transient expression experi-
ments, ?20 pg of plasmid DNA was injected
into embryos at the one-cell stage. Live hcrt–
EGFP-expressing larvae were mounted in 0.8%
low-melting agarose and imaged on a Zeiss
LSM510 confocal microscope.
Heat-shock promoter driven Hcrt. Full-length
RACE (FirstChoice RLM-RACE; Ambion) and cloned downstream of
Locomotor activity analysis. Larvae were raised on a 14/10 h light/dark
placed in each of 80 wells of a 96-well plate (7701-1651; Whatman,
Clifton, NJ), which allowed simultaneous tracking of each larva and
prevented the larvae from interfering with the activity of each other.
Locomotor activity was monitored for several days using an automated
video-tracking system (Videotrack; ViewPoint Life Sciences, Montreal,
Proberetal.•HypocretinCausesInsomniaPhenotypeinZebrafishJ.Neurosci.,December20,2006 • 26(51):13400–13410 • 13401
Quebec, Canada) with a Dinion one-third inch Monochrome camera
(model LTC0385; Bosch, Fairport, NY) fitted with a fixed-angle
megapixel lens (M5018-MP; Computar) and infrared filter, and the
movement of each larva was recorded using Videotrack quantization
mode. The 96-well plate and camera were housed inside a custom-
modified Zebrabox (ViewPoint Life Sciences) that was continuously il-
luminated with infrared lights and was illuminated with white lights
filled with circulating water to maintain a constant temperature of
28.5°C. The Videotrack threshold parameters for detection were
matched to visual observation of the locomotion of single larva. The
Videotrack quantization parameters were set as follows: detection
threshold, 40; burst (threshold for very large movement), 25; freeze
(threshold for no movement), 4; bin size, 60 s. The data were further
analyzed using custom PERL software and Visual Basic Macros for Mi-
crosoft (Seattle, WA) Excel. Any 1 min bin with zero detectable move-
a continuous string of rest minutes. Sleep latency was defined as the
length of time from lights out to the start of the first rest bout. An active
minute was defined as a 1 min bin with any detectable activity. An active
shock promoter-driven Hcrt (HS-Hcrt) and 219 wild-type (WT) larvae
leakiness of the heat-shock promoter (Thummel et al., 2005). This was
more often observed in experiments performed under constant lighting
conditions, in which circadian oscillations do not dampen the effect of
overexpressed Hcrt (see Fig. 7A) (supplemental Fig. 9A, available at ww-
w.jneurosci.org as supplemental material).
Behavioral state transition analysis. To calculate transition frequencies
between inactive, low-active, and high-active states, the total number of
transitions from one state to another was divided by the total number of
minutes spent in that state. A low-active state was defined as any 1 min
period with activity that lasted 1 s or less, whereas a high-active state was
WT larvae in a normal LD cycle spend ?30% of their time in each state.
Arousal threshold analysis. White lights were manipulated during the
first and second nights after heat shock with an automated timer to
precisely regulate each transition. Transitions were from full lights on to
except that the recorded bin size was shortened to 1 s to ensure that
transition and the first 1 s bin of activity. A total of 780 HS-Hcrt and 800
WT responses were recorded in three independent experiments. The
Institutional and Animal Care and Use Committee of Harvard Univer-
sity approved all animal experiments.
To study the Hcrt circuit in zebrafish, we cloned the zebrafish
Hcrt and HcrtR orthologs. We found that the zebrafish genome
encodes a single HcrtR ortholog that is more related to mamma-
lian HcrtR2 (70%) than HcrtR1 (60%) (supplemental Fig. 1C,D,
available at www.jneurosci.org as supplemental material). We
also confirmed that the single zebrafish hcrt gene encodes Hcrt1
jneurosci.org as supplemental material) (Faraco et al., 2006).
Using in situ hybridization, we found that the hcrt receptor is
neurons along the spinal cord (Fig. 1D). Using in situ hybridiza-
tion (Fig. 1A,B), an Hcrt1-specific antibody (Fig. 1E,F), and
transgenic fish in which EGFP expression is regulated by the hcrt
promoter (Fig. 1F), we confirmed that hcrt is expressed in 7–10
neurons in the posterior hypothalamus on the fifth day of devel-
opment (Faraco et al., 2006).
Mammalian Hcrt neurons project to widespread regions of
arousal (Peyron et al., 1998; Chemelli et al., 1999; Date et al.,
Nakamura et al., 2000). Using our hcrt–EGFP transgenic line, we
observed that, by the fifth day of development, the axons of Hcrt
neurons project to the four to five noradrenergic neurons of the
locus ceruleus (Fig. 1G,H). We also observed extensive colocal-
of tyrosine hydroxylase-expressing dopaminergic neurons (sup-
plemental Fig. 2, available at www.jneurosci.org as supplemental
material). Double-fluorescent in situ hybridization revealed that
the hcrt receptor is expressed in all of the diencephalic dopami-
nergic neurons (Fig. 1J,K) and the noradrenergic neurons of the
locus ceruleus (Fig. 1L,M), suggesting that Hcrt may directly
modulate the activity of these neurons. Thus, the projection pat-
tern of Hcrt neurons during early zebrafish larval development
2004) and mammals (Peyron et al., 1998; Chemelli et al., 1999;
Date et al., 1999; Hagan et al., 1999; Horvath et al., 1999; Nambu
et al., 1999; Nakamura et al., 2000). However, unlike mammals
EGFP transgene labels a single Hcrt neuron, which was repeatedly imaged up to day 11 of
13402 • J.Neurosci.,December20,2006 • 26(51):13400–13410Proberetal.•HypocretinCausesInsomniaPhenotypeinZebrafish
the raphe serotonergic or tuberomammillary histaminergic neu-
(see below), Hcrt neurons do not directly project to serotonergic
or histaminergic neurons.
A recent report used an hcrt–EGFP transgene to label Hcrt
neurons (Faraco et al., 2006). Using a similar reagent, we ex-
tended these studies by performing time-lapse imaging of indi-
vidual Hcrt neurons and made two novel observations. First, we
found that all Hcrt neurons extend their axons toward the spinal
2) (supplemental Figs. 3–6, available at www.jneurosci.org as
supplemental material) (data not shown). This contrasts with
mammals, in which only a subset of Hcrt neurons project axons
down the spinal cord (van den Pol, 1999), and indicates that the
small number of zebrafish Hcrt neurons efficiently innervate the
trunk. Second, we observed the development of extensive den-
dritic and axonal arbors as early as 30 h postfertilization (hpf)
org as supplemental material), many of which undergo dynamic
growth (Fig. 2D,F) (supplemental Figs. 5, 6, available at www.
jneurosci.org as supplemental material) and retraction (supple-
mental Fig. 6, available at www.jneurosci.org as supplemental
material) over the first 2 weeks of development. Together, these
but also in zebrafish larvae, Hcrt is expressed in neurons in the
posterior hypothalamus that project to aminergic neurons that
express the Hcrt receptor. In contrast to mammals, however,
zebrafish larvae have ?100-fold fewer Hcrt neurons (de Lecea et
al., 1998; Lin et al., 1999; Peyron et al., 2000), whose growth can
be monitored during development. These results establish the
zebrafish as a simple yet powerful system to study the develop-
ment and circuitry of Hcrt neurons.
The effect of ectopic Hcrt has been investigated in a variety of
animals by injecting Hcrt peptide into the brain (Sakurai et al.,
7 d postfertilization brains labeled with an Hcrt1-specific antibody from HS-Hcrt transgenic
larva that either were not (top) or were (bottom) heat shocked 48 h earlier. The non-heat-
shocked brain shows endogenous Hcrt protein, whereas the heat-shocked brain shows high
spike in activity during the afternoon on days 6 and 7 resulted from the addition of water to
1 h preceding and after lights out. Black squares represent 1 min periods during which any
least 1 min with no activity. D–H, Combined results from 10 independent experiments are
shown. D–G, Each bar represents the mean ? SEM of 302 HS-Hcrt or 219 WT larvae. Hcrt
overexpression increases active bout length (D), decreases rest bout length at night (E), de-
creases total time at rest (F), and decreases the number of rest bouts (G) (**p ? 0.01 by
significantly different between HS-Hcrt and WT larvae ( p ? 0.05 by two-tailed Student’s t
with 38% for WT, and the probability that WT larvae will transition from the inactive to the
Proberetal.•HypocretinCausesInsomniaPhenotypeinZebrafishJ.Neurosci.,December20,2006 • 26(51):13400–13410 • 13403
1998; Hagan et al., 1999; Ida et al., 1999;
John et al., 2000; Nakamura et al., 2000;
et al., 2001; Espana et al., 2002; Yamanaka
et al., 2002; Fujiki et al., 2003; Mieda et al.,
technique is invasive, labor intensive, and
only produces transient effects, we gener-
ated a genetic model of Hcrt overexpres-
sion that is non-invasive, easy to induce,
a heat-shock promoter to drive hcrt ex-
pression (HS-Hcrt) (Fig. 3A). After heat
shock, these larvae express hcrt mRNA in
all cells (supplemental Fig. 7B,F, available
terial). Interestingly, overexpressed Hcrt1
peptide is only found in a subset of neu-
rons in the CNS (Fig. 3A) (supplemental
Fig. 7D,H, available at www.jneurosci.org
as supplemental material), likely attribut-
able to the restricted expression of a pro-
ture Hcrt1 peptide levels begin to increase
3 h after heat shock (supplemental Fig. 7,
available at www.jneurosci.org as supple-
mental material) and return to normal 3–4 d later (data not
To determine whether Hcrt regulates sleep/wake states in ze-
brafish, we characterized these behaviors in WT and HS-Hcrt
larvae. The existence of sleep-like states has been established in
organisms ranging from flies and zebrafish to mice and humans
(Campbell and Tobler, 1984; Hendricks et al., 2000a,b; Shaw et
al., 2000; Zhdanova et al., 2001; Hobson, 2005; Pack et al., 2006).
cordings to define sleep and wake states based on patterns of
brain activity. This technique provides precise information re-
for high-throughput experiments (Pack et al., 2006). As an alter-
native means of monitoring sleep/wake states, behavioral studies
have shown that sleep-like states can be identified as periods of
and Tobler, 1984; Hendricks et al., 2000a; Shaw et al., 2000;
Greenspan et al., 2001; Shaw and Franken, 2003; Huber et al.,
2004). Studies in Drosophila assay locomotor activity by record-
levels are measured using locomotor responses to a variety of
mechanical, auditory, and thermal stimuli (Hendricks et al.,
2000b; Shaw et al., 2000; Huber et al., 2004; Cirelli et al., 2005).
Similarly, a study monitoring the locomotor activity of 7- to 14-
d-old zebrafish larvae demonstrated sleep/wake states and in-
creased arousal thresholds in response to a mechanical stimulus
at night (Zhdanova et al., 2001). To determine the earliest point
in zebrafish development in which a sleep-like state could be
studied, we developed an automated video-tracking system to
monitor the locomotor activity of large numbers of larvae in a
high-throughput manner (Fig. 4). Individual larvae were placed
in each of 80 wells of a 96-well plate on the fourth day of devel-
opment, and the locomotor activity of each larva was monitored
for several days using an infrared camera. We defined an active
3C). A rest bout was defined as a 1 min period with zero activity,
because 1 min of inactivity is associated with significant changes
in arousal threshold, as is observed in sleep (see below). We ob-
served that WT larvae exhibit robust locomotor activity begin-
ning on the fifth day of development and are much more active
Hcrt overexpression significantly increases locomotor activity
(Fig. 3B) and the length of active bouts (Fig. 3C,D) during both
the day and night (for movies, see http://www.mcb.harvard.edu/
schier). For example, the average active bout length during the
14 min in WT. These results suggest that Hcrt overexpression
stimulates locomotor activity and consolidates active states.
Hcrt-overexpressing larvae also spend less time in the resting
state (Fig. 3F,H) and have significantly fewer (Fig. 3C,G) and
inent at night. For example, on the night after heat shock, Hcrt-
overexpressing larvae have an average of 26 rest bouts compared
with 64 for WT, and Hcrt-overexpressing larvae have a median
sleep latency of 77 min compared with 13 min for WT. Thus,
Hcrt-overexpressing larvae are severely impaired in their ability
the hallmark symptoms of insomnia (American Academy of
Sleep Medicine, 2001; Mahowald and Schenck, 2005).
We next developed and applied an assay to test whether Hcrt
modulates arousal thresholds, a key criterion for sleep/wake reg-
ulators. We found that most larvae become active for several
minutes after exposure to sudden darkness, perhaps in response
(for movie, see http://www.mcb.harvard.edu/schier). Almost all
larvae become active within 15 s of dark onset if they display any
activity during the previous minute (Fig. 6A). In contrast, 1 min
or more of rest immediately before dark onset reduces the num-
ber of responding WT larvae (Fig. 6A) and increases the average
response latency (Fig. 6B). These results indicate that rest before
is continuously illuminated by infrared lights and is illuminated by white lights from 9:00 A.M. to 11:00 P.M. The larvae are
Zebrafish larval locomotor activity assay. WT or HS-Hcrt transgenic fish are mated, embryos are collected, and
13404 • J.Neurosci.,December20,2006 • 26(51):13400–13410 Proberetal.•HypocretinCausesInsomniaPhenotypeinZebrafish
dark onset increases arousal thresholds in WT larvae. Responses
those after 1 min of rest (Fig. 6A,B), indicating that as little as 1
min of rest can be considered a sleep-like state. Strikingly, Hcrt-
overexpressing larvae have lower arousal thresholds than WT
larvae. A larger proportion of Hcrt-overexpressing larvae re-
spond to darkness after rest (Fig. 6A), and response latency is
shorter for Hcrt-overexpressing larvae compared with WT (Fig.
6B). Thus, Hcrt-overexpressing larvae have reduced arousal
thresholds and, similar to humans with chronic insomnia, are
hypersensitive to arousing stimuli (Bonnet and Arand, 2000;
American Academy of Sleep Medicine, 2001; Nofzinger et al.,
2004; Mahowald and Schenck, 2005).
As an alternative means of assaying arousal levels in Hcrt-
overexpressing larvae, we investigated the nature of transitions
between active and resting states. We subdivided the active state
into a high-active state (active for ?1 s per minute) and a low-
active state (active for ?1 s per minute) and determined the
when WT larvae are in the inactive state at night, they transition
into the low-active state with a frequency of 36% and into the
high-active state with a frequency of 13% (Fig. 3I). Thus, when
to first enter a low-active state before becoming more highly
active. Hcrt-overexpressing larvae at rest have similar transition
rates into the low-active state (frequency of 33% for HS-Hcrt vs
twice as much time in the high-active state and a third as much
time in the inactive state as WT. Thus, Hcrt overexpression de-
stabilizes the inactive and low-active states in favor of the high-
active state. These results are consistent with our finding that
Hcrt-overexpressing larvae are hyperaroused (Fig. 6).
day, revealing that Hcrt signaling modulates but does not extin-
guish circadian regulation of locomotor activity (Fig. 3B). Con-
versely, we asked whether normal circadian oscillations are re-
have not been performed in other systems because inducible,
long-term Hcrt expression systems have not been established.
WT larvae raised in either constant dark (DD) or constant light
(LL) conditions exhibit little or no oscillation in their locomotor
activity (supplemental Fig. 8, available at www.jneurosci.org as
supplemental material) (Hurd and Cahill, 2002). Remarkably,
Hcrt overexpression increases locomotor activity and decreases
effect is particularly dramatic in DD conditions, in which WT
larvae exhibit very low activity levels. For example, in Hcrt-
overexpressing larvae, the average active bout length increases
23-fold and the number of sleep bouts decreases sevenfold com-
pared with WT from 12–24 h after heat shock. Furthermore, the
in larvae maintained in 14/10 h LD conditions. For example,
Proberetal.•HypocretinCausesInsomniaPhenotypeinZebrafishJ.Neurosci.,December20,2006 • 26(51):13400–13410 • 13405
during the night after heat shock, the average active bout length
of Hcrt-overexpressing larvae is ?2.5-fold longer in DD or LL
than in LD (compare Figs. 3D, 7B) (supplemental Fig. 9B, avail-
able at www.jneurosci.org as supplemental material), and Hcrt-
overexpressing larvae in DD or LL spend less than half as much
Fig. 9D, available at www.jneurosci.org as supplemental
Dramatic differences in state transitions were also observed
under constant lighting conditions. For example, in DD condi-
tions, Hcrt-overexpressing larvae are far more likely than WT to
53% for HS-Hcrt vs 15% for WT) (Fig. 7G). HS-Hcrt larvae are
also far more likely than WT larvae to transition from the low-
active to the high-active state (frequency of 71% for HS-Hcrt vs
from either active state (HS-Hcrt low-active to inactive fre-
quency, 8 vs 21% for WT; HS-Hcrt high-active to inactive fre-
quency, 2 vs 10% for WT) (Fig. 7G). The effect of overexpressed
Hcrt in DD or LL is also greater than in LD. For example, Hcrt-
overexpressing larvae spend 64% of the time in the high-active
state in LD (Fig. 3I) but 86 or 90% of the time in DD or LL
conditions, respectively (Fig. 7G) (supplemental Fig. 9G, avail-
able at www.jneurosci.org as supplemental material). Further-
of the time in DD or LL conditions, respectively. Because the
effect of overexpressed Hcrt in DD or LL conditions is signifi-
cantly greater than in LD, we conclude that normal circadian
oscillations dampen the effects of Hcrt signaling. These results
circadian oscillations in locomotor activity.
Our results reveal that the neural circuitry and function of the
Hcrt system is conserved from mammals to zebrafish. We find
that 5-d-old wild-type larvae exhibit robust locomotive sleep/
wake behaviors and have higher arousal thresholds during peri-
ods of rest, indicative of a sleep-like state (Campbell and Tobler,
1984; Hendricks et al., 2000a; Shaw et al., 2000; Greenspan et al.,
2001; Zhdanova et al., 2001; Shaw and Franken, 2003). Our in-
ducible Hcrt transgenic expression system reveals that Hcrt-
overexpressing larvae are hyperaroused and have dramatically
to humans with insomnia (Bonnet and Arand, 2000; American
Academy of Sleep Medicine, 2001; Nofzinger et al., 2004; Ma-
howald and Schenck, 2005). Strikingly, Hcrt can increase loco-
motor activity independent of normal circadian oscillations in
locomotor activity, and these oscillations dampen the effects of
Hcrt overexpression. Because our experiments were performed
feeding, thermoregulation, social interactions, or other complex
homeostatic processes or behaviors in which Hcrt has been im-
plicated in mammals (Sakurai et al., 1998; Szekely et al., 2002;
Yamanaka et al., 2003; Harris et al., 2005; Borgland et al., 2006;
D’Anna and Gammie, 2006). Our experiments therefore clarify
the primary function of Hcrt and suggest that the most basal and
ancestral role of Hcrt signaling is the promotion of wakefulness.
Our experiments also establish zebrafish as a system to study the
genetics and neural circuitry of sleep.
Our results indicate that the Hcrt neural circuit in zebrafish
larvae has strong similarity to its counterparts in adult zebrafish
and mammals. Hcrt is exclusively expressed in the zebrafish pos-
terior hypothalamus (Fig. 1) (Kaslin et al., 2004; Faraco et al.,
2006) as in mammals (de Lecea et al., 1998; Sakurai et al., 1998),
1998; Sakurai et al., 1998; Lin et al., 1999; Peyron et al., 2000).
Although this highly specific expression is regulated at the tran-
peptide (supplemental Fig. 7, available at www.jneurosci.org as
diencephalic dopaminergic neurons and the noradrenergic neu-
rons of the locus ceruleus, as they do in mammals (Peyron et al.,
1998; Chemelli et al., 1999; Date et al., 1999; Hagan et al., 1999;
Horvath et al., 1999; Nambu et al., 1999; Nakamura et al., 2000)
and adult zebrafish (Kaslin et al., 2004). These neurons express
the single zebrafish Hcrt receptor ortholog, indicating that they
are likely directly activated by Hcrt. In contrast to mammals
(Peyron et al., 1998; Date et al., 1999; Nambu et al., 1999) and
30% for WT larvae but only by 10% for Hcrt-overexpressing larvae. For each time point ana-
lyzed, significantly more Hcrt-overexpressing larvae respond than WT larvae (*p ? 0.05;
of time. Hcrt-overexpressing larvae respond significantly faster than WT larvae (*p ? 0.05;
**p ? 0.01 by two-tailed Student’s t test). One minute of prestimulus rest increases the re-
prestimulus rest are all significantly different from those after 0 min of rest ( p ? 0.001).
after 1 min of rest ( p ? 0.05). A total of 780 HS-Hcrt and 800 WT responses from three
13406 • J.Neurosci.,December20,2006 • 26(51):13400–13410Proberetal.•HypocretinCausesInsomniaPhenotypeinZebrafish
adult zebrafish (Kaslin et al., 2004), we do not observe Hcrt pro-
jections to the raphe serotonergic or tuberomammillary hista-
modulation of serotonergic and histaminergic neurons may not
be necessary to mediate Hcrt function and may therefore reveal
the basic neural circuitry required for Hcrt function. Moreover,
brain structures are sufficient to mediate Hcrt function. The ac-
cessibility of zebrafish for in vivo imaging (Higashijima et al.,
2003; Gahtan and Baier, 2004) and the small number of Hcrt
neurons should make zebrafish a powerful system to study the
development and function of Hcrt neural circuits.
To generate a non-invasive, inducible, and robust model of
Hcrt overexpression, we created transgenic zebrafish that ex-
press Hcrt under the control of a heat-shock promoter. Most
previous Hcrt overexpression studies required the invasive and
labor-intensive injection of Hcrt peptide into the brain, which
thus required that behavior be assayed immediately after han-
1999; Bourgin et al., 2000; John et al., 2000; Nakamura et al.,
2000; Piper et al., 2000; Huang et al., 2001; Jones et al., 2001;
Espana et al., 2002; Yamanaka et al., 2002; Fujiki et al., 2003;
Nakamachi et al., 2006). One study used transgenic mice that
constitutively overexpressed Hcrt in the brain to rescue Hcrt de-
ficiency, but the effects of Hcrt-overexpression on wild-type be-
havior were not described (Mieda et al., 2004). In contrast to
least 3 d, allowing us to assay behavior long after the heat shock.
We find that Hcrt overexpression promotes and consolidates ac-
tive states, decreases the frequency and length of rest bouts, in-
old. Hcrt-overexpressing larvae also spend more time in highly
sition from states of no or low activity into a high-activity state.
Our findings indicate that long-term overexpression of Hcrt in-
creases wakefulness and arousal, whereas long-term absence of
ever, both loss- and gain-of-Hcrt function result in altered sleep/
wake states and abnormal transitions between activity states
(Chemelli et al., 1999; Hara et al., 2001; Willie et al., 2003; Mo-
allow optimal control of sleep/wake states, providing a potential
challenge to the development of therapeutics that target the Hcrt
We found that Hcrt-overexpressing larvae are hyperaroused
and have impaired abilities to initiate and maintain rest at night,
insomnia (American Academy of Sleep Medicine, 2001). Al-
though there is no direct evidence implicating Hcrt in insomnia,
it has been proposed that chronic insomnia is a general disorder
are slightly more active than WT larvae before heat shock, presumably attributable to leaky
sure to low levels of red light immediately before and after heat shock, to handling of the
Hcrt overexpression dramatically consolidates active states and reduces rest in
HS-Hcrt larvae in the inactive and low-active states are more likely to transition to the high-
Proberetal.•HypocretinCausesInsomniaPhenotypeinZebrafishJ.Neurosci.,December20,2006 • 26(51):13400–13410 • 13407
of hyperarousal that may involve dysfunction of the Hcrt system
are regulated by minimal inhibitory input, making them suscep-
tible to abnormal excitation (Horvath and Gao, 2005). Although
one study found normal Hcrt levels in human subjects with in-
somnia (Mignot et al., 2002), only 12 subjects were studied. Fur-
thermore, this study would not detect other Hcrt-dependent ef-
fects, such as hyperactivity of Hcrt-expressing neurons or
activation of Hcrt signaling downstream of the Hcrt ligand. Al-
though models with reduced sleep have been generated via brain
lesions in mammals (Lu et al., 2000) or through genetic loss-of-
function in Drosophila (Cirelli et al., 2005), our novel gain-of-
function model is uniquely suited for the large-scale testing of
environmental signals and drugs that suppress insomnia in ver-
tebrates. Such models are urgently needed, because insomnia ac-
counts for ?50% of sleep-related complaints (Colten and Al-
Our experiments reveal novel interactions between Hcrt sig-
naling and the circadian system that were not identified previ-
ously because a long-term Hcrt overexpression system was not
than during the day. This indicates that Hcrt-overexpression
on locomotor activity. Second, Hcrt overexpression more dra-
matically increases locomotor activity under constant lighting
motor activity. Previous studies showed that the mammalian su-
prachiasmatic nucleus (SCN), which acts as the master pace-
Hcrt neurons (Abrahamson et al., 2001; Aston-Jones et al., 2001;
Chou et al., 2003; Deurveilher and Semba, 2005; Yoshida et al.,
2006) and that Hcrt levels fluctuate with a circadian rhythm that
is abolished after SCN ablation (Deboer et al., 2004; Zhang et al.,
2004). These findings suggest that the circadian system regulates
Hcrt levels, perhaps by regulating the activity of Hcrt neurons.
rhythms (Mochizuki et al., 2004), indicating that the circadian
system can function independently of Hcrt signaling. Interest-
ingly, SCN neurons indirectly project to and regulate the activity
of locus ceruleus neurons (Aston-Jones et al., 2001), suggesting
that both the circadian and Hcrt systems likely regulate sleep/
wake states, at least in part, by modulating the activity of locus
ceruleus neurons. Together, the mammalian loss-of-function
and our gain-of-function studies suggest that the Hcrt and circa-
dian systems can function independently of each other but also
appear to cross regulate. Additional study is required to clearly
common mechanisms to regulate sleep/wake states.
More generally, our study establishes zebrafish as a model
system for the genetic analysis of sleep. Zebrafish will comple-
ment mammals and Drosophila because it combines the experi-
al., 2004; Gross et al., 2005; Zon and Peterson, 2005) with neural
assay locomotor activity and arousal in zebrafish allows us to
simultaneously analyze hundreds of larvae. A similar technology
has provided the basis for large-scale genetic screens in Drosoph-
ila (Cirelli et al., 2005). Our system should therefore allow high-
throughput zebrafish screens for novel genes and drugs that reg-
Abrahamson EE, Leak RK, Moore RY (2001) The suprachiasmatic nucleus
projects to posterior hypothalamic arousal systems. NeuroReport
AmericanAcademyofSleepMedicine (2001) ICSD—Internationalclassifi-
American Academy of Sleep Medicine.
Aston-Jones G, Chen S, Zhu Y, Oshinsky ML (2001) A neural circuit for
circadian regulation of arousal. Nat Neurosci 4:732–738.
Bonnet MH, Arand DL (2000) Activity, arousal, and the MSLT in patients
with insomnia. Sleep 23:205–212.
Borgland SL, Taha SA, Sarti F, Fields HL, Bonci A (2006) Orexin A in the
VTA is critical for the induction of synaptic plasticity and behavioral
sensitization to cocaine. Neuron 49:589–601.
Bourgin P, Huitron-Resendiz S, Spier AD, Fabre V, Morte B, Criado JR,
Sutcliffe JG, Henriksen SJ, de Lecea L (2000) Hypocretin-1 modulates
rapid eye movement sleep through activation of locus ceruleus neurons.
J Neurosci 20:7760–7765.
CampbellSS,ToblerI (1984) Animalsleep:areviewofsleepdurationacross
phylogeny. Neurosci Biobehav Rev 8:269–300.
Chemelli RM, Willie JT, Sinton CM, Elmquist JK, Scammell T, Lee C, Rich-
ardson JA, Williams SC, Xiong Y, Kisanuki Y, Fitch TE, Nakazato M,
Hammer RE, Saper CB, Yanagisawa M (1999) Narcolepsy in orexin
knockout mice: molecular genetics of sleep regulation. Cell 98:437–451.
Chou TC, Scammell TE, Gooley JJ, Gaus SE, Saper CB, Lu J (2003) Critical
role of dorsomedial hypothalamic nucleus in a wide range of behavioral
circadian rhythms. J Neurosci 23:10691–10702.
Reduced sleep in Drosophila Shaker mutants. Nature 434:1087–1092.
Colten HR, Altevog BM (2006) Sleep disorders and sleep deprivation: an
unmet public health problem. Washington, DC: National Academy of
D’AnnaKL,GammieSC (2006) Hypocretin-1dose-dependentlymodulates
maternal behaviour in mice. J Neuroendocrinol 18:553–566.
rai T, Yanagisawa M, Nakazato M (1999) Orexins, orexigenic hypotha-
latory systems. Proc Natl Acad Sci USA 96:748–753.
Deboer T, Overeem S, Visser NA, Duindam H, Frolich M, Lammers GJ,
Meijer JH (2004) Convergence of circadian and sleep regulatory mech-
anisms on hypocretin-1. Neuroscience 129:727–732.
Battenberg EL, Gautvik VT, Bartlett II FS, Frankel WN, van den Pol AN,
hypothalamus-specific peptides with neuroexcitatory activity. Proc Natl
Acad Sci USA 95:322–327.
Deurveilher S, Semba K (2005) Indirect projections from the suprachias-
Stainier DY, Zwartkruis F, Abdelilah S, Rangini Z, Belak J, Boggs C
(1996) A genetic screen for mutations affecting embryogenesis in ze-
brafish. Development 123:37–46.
Espana RA, Plahn S, Berridge CW (2002) Circadian-dependent and
circadian-independent behavioral actions of hypocretin/orexin. Brain
Faraco JH, Appelbaum L, Marin W, Gaus SE, Mourrain P, Mignot E (2006)
Regulation of hypocretin (OREXIN) expression in embryonic zebrafish.
J Biol Chem 281:29753–29761.
Fujiki N, Yoshida Y, Ripley B, Mignot E, Nishino S (2003) Effects of IV and
coleptic dogs and IV hypocretin-1 replacement therapy in a hypocretin-
ligand-deficient narcoleptic dog. Sleep 26:953–959.
Gahtan E, Baier H (2004) Of lasers, mutants, and see-through brains: func-
tional neuroanatomy in zebrafish. J Neurobiol 59:147–161.
Granato M, van Eeden FJ, Schach U, Trowe T, Brand M, Furutani-Seiki M,
Haffter P, Hammerschmidt M, Heisenberg CP, Jiang YJ, Kane DA, Kelsh
RN, Mullins MC, Odenthal J, Nusslein-Volhard C (1996) Genes con-
trolling and mediating locomotion behavior of the zebrafish embryo and
larva. Development 123:399–413.
Greenspan RJ, Tononi G, Cirelli C, Shaw PJ (2001) Sleep and the fruit fly.
Trends Neurosci 24:142–145.
JG (1998) The hypocretins:
13408 • J.Neurosci.,December20,2006 • 26(51):13400–13410Proberetal.•HypocretinCausesInsomniaPhenotypeinZebrafish
S, Hopkins N, Dowling JE (2005) Identification of zebrafish insertional
Hagan JJ, Leslie RA, Patel S, Evans ML, Wattam TA, Holmes S, Benham CD,
Taylor SG, Routledge C, Hemmati P, Munton RP, Ashmeade TE, Shah
AS, Hatcher JP, Hatcher PD, Jones DN, Smith MI, Piper DC, Hunter
AJ, Porter RA, Upton N (1999) Orexin A activates locus coeruleus
cell firing and increases arousal in the rat. Proc Natl Acad Sci USA
Shoji W (2000) Laser-induced gene expression in specific cells of trans-
genic zebrafish. Development 127:1953–1960.
Hara J, Beuckmann CT, Nambu T, Willie JT, Chemelli RM, Sinton CM,
Sugiyama F, Yagami K, Goto K, Yanagisawa M, Sakurai T (2001) Ge-
netic ablation of orexin neurons in mice results in narcolepsy, hypopha-
gia, and obesity. Neuron 30:345–354.
Harris GC, Wimmer M, Aston-Jones G (2005) A role for lateral hypotha-
lamic orexin neurons in reward seeking. Nature 437:556–559.
Hendricks JC, Sehgal A, Pack AI (2000a) The need for a simple animal
model to understand sleep. Prog Neurobiol 61:339–351.
AI (2000b) Rest in Drosophila is a sleep-like state. Neuron 25:129–138.
Higashijima S, Masino MA, Mandel G, Fetcho JR (2003) Imaging neuronal
activity during zebrafish behavior with a genetically encoded calcium
indicator. J Neurophysiol 90:3986–3997.
Hobson JA (2005) Sleep is of the brain, by the brain and for the brain.
HorvathTL,GaoXB (2005) Inputorganizationandplasticityofhypocretin
Horvath TL, Peyron C, Diano S, Ivanov A, Aston-Jones G, Kilduff TS, van
Den Pol AN (1999) Hypocretin (orexin) activation and synaptic inner-
vation of the locus coeruleus noradrenergic system. J Comp Neurol
Hsiao CD, Hsieh FJ, Tsai HJ (2001) Enhanced expression and stable trans-
mission of transgenes flanked by inverted terminal repeats from adeno-
associated virus in zebrafish. Dev Dyn 220:323–336.
Huang ZL, Qu WM, Li WD, Mochizuki T, Eguchi N, Watanabe T, Urade Y,
Hayaishi O (2001) Arousal effect of orexin A depends on activation of
the histaminergic system. Proc Natl Acad Sci USA 98:9965–9970.
Huber R, Hill SL, Holladay C, Biesiadecki M, Tononi G, Cirelli C (2004)
Sleep homeostasis in Drosophila melanogaster. Sleep 27:628–639.
Hurd MW, Cahill GM (2002) Entraining signals initiate behavioral circa-
dian rhythmicity in larval zebrafish. J Biol Rhythms 17:307–314.
IdaT,NakaharaK,KatayamaT,MurakamiN,NakazatoM (1999) Effectof
tide, orexin and neuropeptide Y, on the various behavioral activities of
rats. Brain Res 821:526–529.
John J, Wu MF, Siegel JM (2000) Systemic administration of hypocretin-1
reduces cataplexy and normalizes sleep and waking durations in narco-
leptic dogs. Sleep Res Online 3:23–28.
Jones DN, Gartlon J, Parker F, Taylor SG, Routledge C, Hemmati P, Munton
RP, Ashmeade TE, Hatcher JP, Johns A, Porter RA, Hagan JJ, Hunter AJ,
Upton N (2001) Effects of centrally administered orexin-B and
Psychopharmacology (Berl) 153:210–218.
Kaneko M, Cahill GM (2005) Light-dependent development of circadian
gene expression in transgenic zebrafish. PLoS Biol 3:e34.
KaslinJ,NystedtJM,OstergardM,PeitsaroN,PanulaP (2004) Theorexin/
hypocretin system in zebrafish is connected to the aminergic and cholin-
ergic systems. J Neurosci 24:2678–2689.
S, Mignot E (1999) The sleep disorder canine narcolepsy is caused by a
mutation in the hypocretin (orexin) receptor 2 gene. Cell 98:365–376.
Lu J, Greco MA, Shiromani P, Saper CB (2000) Effect of lesions of the ven-
trolateral preoptic nucleus on NREM and REM sleep. J Neurosci
Mahowald MW, Schenck CH (2005) Insights from studying human sleep
disorders. Nature 437:1279–1285.
Mieda M, Willie JT, Hara J, Sinton CM, Sakurai T, Yanagisawa M (2004)
Orexin peptides prevent cataplexy and improve wakefulness in an orexin
neuron-ablated model of narcolepsy in mice. Proc Natl Acad Sci USA
Mignot E, Lammers GJ, Ripley B, Okun M, Nevsimalova S, Overeem S,
Vankova J, Black J, Harsh J, Bassetti C, Schrader H, Nishino S (2002)
The role of cerebrospinal fluid hypocretin measurement in the diagnosis
of narcolepsy and other hypersomnias. Arch Neurol 59:1553–1562.
TE (2004) Behavioral state instability in orexin knock-out mice. J Neu-
MuellerT,WullimannMF (2005) Atlasofearlyzebrafishdevelopment.Ox-
Nakamachi T, Matsuda K, Maruyama K, Miura T, Uchiyama M, Fu-
nahashi H, Sakurai T, Shioda S (2006) Regulation by orexin of feed-
ing behaviour and locomotor activity in the goldfish. J Neuroendocri-
T (2000) Orexin-induced hyperlocomotion and stereotypy are medi-
ated by the dopaminergic system. Brain Res 873:181–187.
Distribution of orexin neurons in the adult rat brain. Brain Res
Nofzinger EA, Buysse DJ, Germain A, Price JC, Miewald JM, Kupfer DJ
Am J Psychiatry 161:2126–2128.
Orger MB, Gahtan E, Muto A, Page-McCaw P, Smear MC, Baier H (2004)
Behavioral screening assays in zebrafish. Methods Cell Biol 77:53–68.
R, Kay T, Lian J, Svenson K, Peters LL (2006) A novel method for high
throughput phenotyping of sleep in mice. Physiol Genomics, in press.
Peyron C, Tighe DK, van den Pol AN, de Lecea L, Heller HC, Sutcliffe JG,
Kilduff TS (1998) Neurons containing hypocretin (orexin) project to
multiple neuronal systems. J Neurosci 18:9996–10015.
S, Aldrich M, Reynolds D, Albin R, Li R, Hungs M, Pedrazzoli M, Padi-
garu M, Kucherlapati M, Fan J, Maki R, Lammers GJ, Bouras C, Kucher-
lapati R, Nishino S, Mignot E (2000) A mutation in a case of early onset
narcolepsy and a generalized absence of hypocretin peptides in human
narcoleptic brains. Nat Med 6:991–997.
Piper DC, Upton N, Smith MI, Hunter AJ (2000) The novel brain neu-
ReichertH,WullimannMF,RuppB (1996) Neuroanatomyofthezebrafish
brain: a topological atlas. Basel: Birkhauser.
Sakurai T, Amemiya A, Ishii M, Matsuzaki I, Chemelli RM, Tanaka H,
Williams SC, Richardson JA, Kozlowski GP, Wilson S, Arch JR, Buck-
ingham RE, Haynes AC, Carr SA, Annan RS, McNulty DE, Liu WS,
Terrett JA, Elshourbagy NA, Bergsma DJ, Yanagisawa M (1998)
Orexins and orexin receptors: a family of hypothalamic neuropeptides
and G protein-coupled receptors that regulate feeding behavior. Cell
Saper CB, Cano G, Scammell TE (2005) Homeostatic, circadian, and emo-
tional regulation of sleep. J Comp Neurol 493:92–98.
ShawPJ,FrankenP (2003) Perchancetodream:solvingthemysteryofsleep
through genetic analysis. J Neurobiol 54:179–202.
Shaw PJ, Cirelli C, Greenspan RJ, Tononi G (2000) Correlates of sleep and
waking in Drosophila melanogaster. Science 287:1834–1837.
Siegel JM (2004) Hypocretin (orexin): role in normal behavior and neuro-
pathology. Annu Rev Psychol 55:125–148.
Siegel JM (2005) Clues to the functions of mammalian sleep. Nature
Szekely M, Petervari E, Balasko M, Hernadi I, Uzsoki B (2002) Effects of
orexins on energy balance and thermoregulation. Regul Pept 104:47–53.
JS (2002) I-SceI meganuclease mediates highly efficient transgenesis in
fish. Mech Dev 118:91–98.
Thummel R, Burket CT, Brewer JL, Sarras Jr MP, Li L, Perry M, McDermott
JP, Sauer B, Hyde DR, Godwin AR (2005) Cre-mediated site-specific
recombination in zebrafish embryos. Dev Dyn 233:1366–1377.
van den Pol AN (1999) Hypothalamic hypocretin (orexin): robust innerva-
tion of the spinal cord. J Neurosci 19:3171–3182.
Proberetal.•HypocretinCausesInsomniaPhenotypeinZebrafishJ.Neurosci.,December20,2006 • 26(51):13400–13410 • 13409
Willie JT, Chemelli RM, Sinton CM, Tokita S, Williams SC, Kisanuki YY, Download full-text
Marcus JN, Lee C, Elmquist JK, Kohlmeier KA, Leonard CS, Richardson
JA, Hammer RE, Yanagisawa M (2003) Distinct narcolepsy syndromes
in Orexin receptor-2 and Orexin null mice: molecular genetic dissection
of non-REM and REM sleep regulatory processes. Neuron 38:715–730.
Yamanaka A, Sakurai T, Katsumoto T, Yanagisawa M, Goto K (1999)
Chronic intracerebroventricular administration of orexin-A to rats in-
Yamanaka A, Tsujino N, Funahashi H, Honda K, Guan JL, Wang QP, Tomi-
naga M, Goto K, Shioda S, Sakurai T (2002) Orexins activate histamin-
ergic neurons via the orexin 2 receptor. Biochem Biophys Res Commun
Yamanaka A, Beuckmann CT, Willie JT, Hara J, Tsujino N, Mieda M, Tomi-
Hypothalamic orexin neurons regulate arousal according to energy bal-
ance in mice. Neuron 38:701–713.
Yoshida K, McCormack S, Espana RA, Crocker A, Scammell TE (2006) Af-
ferents to the orexin neurons of the rat brain. J Comp Neurol
Zeitzer JM, Nishino S, Mignot E (2006) The neurobiology of hypocretins
macol Sci 27:368–374.
Zhang S, Zeitzer JM, Yoshida Y, Wisor JP, Nishino S, Edgar DM, Mignot E
(2004) Lesions of the suprachiasmatic nucleus eliminate the daily
rhythm of hypocretin-1 release. Sleep 27:619–627.
Zhdanova IV, Wang SY, Leclair OU, Danilova NP (2001) Melatonin pro-
motes sleep-like state in zebrafish. Brain Res 903:263–268.
ZonLI,PetersonRT (2005) Invivodrugdiscoveryinthezebrafish.NatRev
Drug Discov 4:35–44.
13410 • J.Neurosci.,December20,2006 • 26(51):13400–13410Proberetal.•HypocretinCausesInsomniaPhenotypeinZebrafish