Sleep–wake regulation and hypocretin–melatonin
interaction in zebrafish
Lior Appelbauma,b, Gordon X. Wangc, Geraldine S. Marod, Rotem Morie, Adi Tovine, Wilfredo Marina,b,
Tohei Yokogawaa,b, Koichi Kawakamif, Stephen J. Smithc, Yoav Gothilfe, Emmanuel Mignota,b,
and Philippe Mourraina,g,1
aCenter for Narcolepsy, Department of Psychiatry and Behavioral Sciences,bHoward Hughes Medical Institute,cDepartment of Molecular and Cellular
Physiology, Beckman Center, anddDepartment of Biology and Pathology Stanford University, Palo Alto, CA 94305;eDepartment of Neurobiology, George S.
Wise Faculty of Life Sciences, Tel Aviv University, Tel Aviv 69978, Israel;fDivision of Molecular and Developmental Biology, National Institute of Genetics,
Mishima, Shizuoka 411-8540, Japan; andgInstitut National de la Sante ´ et de la Recherche Me ´dicale, Inserm U784, Ecole Normale Supe ´rieure,
75005 Paris, France
Edited by Joseph S. Takahashi, University of Texas Southwestern Medical Center, Dallas, TX, and approved October 20, 2009 (received for review
June 15, 2009)
In mammals, hypocretin/orexin (HCRT) neuropeptides are important
sleep–wake regulators and HCRT deficiency causes narcolepsy. In
addition to fragmented wakefulness, narcoleptic mammals also dis-
zebrafish to study the potential mediators of HCRT-mediated sleep
consolidation. Similar to mammals, zebrafish HCRT neurons express
vesicular glutamate transporters indicating conservation of the exci-
stably expressing hcrt:EGFP revealed parallels with established mam-
malian HCRT neuroanatomy, including projections to the pineal
is a major sleep-inducing hormone in zebrafish, we further studied
how the HCRT and melatonin systems interact functionally. mRNA
level of arylalkylamine-N-acetyltransferase (AANAT2), a key enzyme
night. Moreover, HCRT perfusion of cultured zebrafish pineal glands
induces melatonin release. Together these data indicate that HCRT
can modulate melatonin production at night. Furthermore, hcrtr?/?
fish are hypersensitive to melatonin, but not other hypnotic com-
pounds. Subthreshold doses of melatonin increased the amount of
neurons-pineal gland circuit able to modulate melatonin production
and sleep consolidation.
pineal gland ? sleep consolidation
preproprotein is exclusively expressed in neurons restricted to
the lateral hypothalamus (LH) organized as a single compact
cluster in each hemi-brain (1–3). HCRT neuron number may
vary from a few thousand in a rodent LH to 50,000–80,000 in the
human LH. This cluster organization is conserved in all mam-
mals investigated (4). Despite its restricted expression, HCRT is
a critical regulator of the sleep–wake cycle and is further
implicated in food intake regulation, energy homeostasis,
arousal, drug addiction, stress, and cardiovascular function.
Interestingly, the complexity of HCRT physiological function is
reflected in the diversity of HCRT anatomic projections and
HCRT receptor expression sites in the central nervous system.
From their discrete location in the LH, HCRT neurons send
widespread projections throughout the brain and the spinal cord
(3, 5). This broad fiber distribution is consistent with the diffuse
expression patterns of the two HCRT G protein-coupled recep-
tors (HCRTR1/OX1R and HCRTR2/OX2R) (2, 6).
HCRT deficiencies produce narcolepsy, a disorder character-
ized in mammals by excessive sleepiness during the normal wake
periods, direct transitions from wake to REM sleep, and sudden
ypocretin 1 and 2 (HCRT 1 and 2, also known as orexin A
and B) are two neuropeptides originally isolated in rats, that
loss of muscle tonus while still being awake (cataplexy) (4, 7).
Moreover, HCRT is crucial to maintain wakefulness and has
been extensively shown to have a wake-promoting role. HCRT
neurons are active during wakefulness (8) and intracerebroven-
tricular administration of HCRT induces a dose-dependent
increase of time spent awake (9). Furthermore, HCRT also
appears to play an important role in sleep regulation and
consolidation. Individuals with narcolepsy and similarly affected
dogs and mice also suffer sleep fragmentation (10, 11), even
when pharmacologically kept awake during the daytime to
increase homeostatic pressure for sleep (12).
We, and others, have previously characterized the HCRT system
in zebrafish (13–16), a simpler model to understand the anatomy
and function of the HCRT neuronal network and its evolution
across vertebrates. Unlike mammals, zebrafish have only one
receptor with the mammalian HCRTR2, zebrafish hcrtr?/? null
mutants do not have a fragmented wake during the daytime or
cataplexy-like behavior but only possess the sleep fragmentation
phenotype (16). Moreover, in contrast to the mammalian HCRT
system involving thousands of neurons in a complex neuronal
circuit, the larval and adult zebrafish hypothalamus contain ap-
proximately 20 and 60 HCRT-positive neurons, respectively (13–
16). In this study, we show a neuroanatomical and functional
connection between the HCRT and melatonin systems, supporting
a role for a HCRT/melatonin pathway in sleep consolidation.
Zebrafish HCRT Neurons Are Mostly Glutamatergic. In mammals, the
majority of HCRT neurons are glutamatergic, a phenotype con-
sistent with the wake-promoting influence and neuroexcitatory
nature of HCRT neuropeptides (17). However, HCRT signaling
us to examine the excitatory or inhibitory nature of the zebrafish
HCRT neurons. We studied their fast neurotransmitter phenotype
using double in situ hybridization (ISH) of hcrt with vesicular
glutamate transporter genes vglut1, vglut2a, vglut2b (markers of
excitatory glutamate), and glutamate decarboxylase gene gad67
(marker of inhibitory GABA). Colocalization with hcrt was deter-
mined by confocal microscopy. We found that no HCRT neurons
Author contributions: L.A., G.X.W., G.S.M., Y.G., E.M., and P.M. designed research; L.A.,
G.X.W., G.S.M., R.M., A.T., W.M., T.Y., and P.M. performed research; K.K., S.J.S., and Y.G.
contributed new reagents/analytic tools; L.A., G.X.W., G.S.M., W.M., T.Y., Y.G., E.M., and
P.M. analyzed data; and L.A., E.M., and P.M. wrote the paper.
The authors declare no conflict of interest.
This article is a PNAS Direct Submission.
1To whom correspondence should be addressed. E-mail: firstname.lastname@example.org.
This article contains supporting information online at www.pnas.org/cgi/content/full/
December 22, 2009 ?
vol. 106 ?
are not GABAergic. Similarly, HCRT neurons in majority do not
express vglut1 (Fig. 1 B and B1), however occasional coexpression
in a few ventralmost HCRT cells was observed. In contrast, a large
majority of the HCRT neurons express one of the vglut2 genes
suggesting a glutamatergic phenotype (Fig. 1 C and D, and asso-
ciated close-up panels C1–C4 and D1–D4). Of note, vglut2a- or
1 C2–C4, arrow). These results are reminiscent of the rat, where
none of the HCRT neurons express gad67, a minority expresses
vglut1 and a majority expresses vglut2 (17), suggesting a conserva-
tion of the developmental program underlying the HCRT cell
identity across vertebrates. Functionally, the glutamatergic pheno-
type of HCRT neurons in zebrafish suggests a conservation of the
excitatory nature of these neurons between fish and mammals.
Architecture of the Larval and Adult Zebrafish HCRT System. We
isolated the zebrafish hcrt gene promoter (14) and generated a
specific stable hcrt:EGFP transgenic line (Fig. S1) to perform high
resolution imaging of the zebrafish HCRT circuit, and its potential
connectivity with sleep/wake-regulating brain regions. Two-photon
microscopy analysis of living hcrt:EGFP larvae revealed the ipsi-
lateral track projecting to the spinal cord (14, 15) and bilateral
projections proceeding anteriorly through the telencephalon to-
ward both olfactory bulbs (OB, Fig. 2A and close up in B),
reminiscent of the rat dorsal descending and ventral ascending
pathways respectively. In addition, contralateral projections were
observed ventrally to the HCRT cell bodies (Fig. 2 C and D, arrow)
and in the commissura anterior to the OB (Fig. 2B, arrow).
In adult hcrt:EGFP zebrafish (Fig. 3), HCRT fibers are found in
subpallium midline (Fig. 3A), similar to the rat dorsal and ventral
ascending pathways respectively (3). As in mammals, the highest
density of axonal projections is detected in the hypothalamic
periventricular region (Fig. 3 B–D) and HCRT cell bodies are
organized as two compact clusters facing each other without direct
contralateral projection to their counterpart (Fig. 3 D and D1).
the habenular commisure (Fig. 3B1), the commissura tecti, the
commissura posterior, and through the most ventral enclosure of
the periventricular hypothalamus (Fig. 3D). Fibers were also ob-
served around the tectal ventricule in the periventricular gray zone
of the optic tectum (Fig. 3 C and D), a structure homologous to the
fibers have also been described in rats (3). Finally, posterior
projections are sent dorsally and ventrally in the subventricular
region of the rhombencephalon (Fig. 3E) similar to that observed
in rat (3, 5).
to confirm the projection architecture observed in our hcrt:EGFP
transgenics. Consistently, the fiber distribution matched the HCRT
receptor mRNA expression profile (Fig. 3, compare panels A vs. F,
B vs. G, C vs. H, D vs. I, and E vs. J), except in the hypothalamus.
Indeed, abundant projections around the HCRT cell bodies do not
have corresponding receptor expression (see Fig. 3 D vs. I) sug-
gesting those processes are likely dendritic. Overall, our stable
(A1–D1) Double fluorescent ISH between hcrt mRNA and fast neurotransmit-
ter phenotype markers as visualized using confocal microscopy on adult brain
sections (reconstructed stacks of 0.5- or 1-?m sections). (C2–C4) and (D2–D4):
single-plane, high-magnification pictures of hcrt cells (green, C2, D2), vglut2a
or -b cells (red, C3, D3), and merged views (C4, D4). Arrowheads indicate cells
coexpressing hcrt and vglut2a or vglut2b. Note the frequent colocalization.
Absence of coexpression is occasionally observed (arrow). [Scale bar, 100 ?m
(A–D), 20 ?m (A1–D1, C2–C4, and D2–D4).]
Zebrafish HCRT neurons are glutamatergic. (A–D) and close-ups
entire HCRT circuit of a living larva. (A–C) Two-photon
imaging of a 7dpf stable transgenic larva with head to
the left, dorsal views. (A and close-up B) Composite
picture showing the HCRT cell bodies in the dienceph-
alon (Di) and their processes in the hindbrain (HB) and
toward the olfactory bulbs (OB). In A and B, white
areas on both sides of the larva correspond to skin
autofluorescence. Commissural projections are ob-
served ventrally to the HCRT cell body clusters (C,
arrow) and in the anterior telencephalon (B, arrow,
commissura anterior). (D) Mosaic expression of a non-
integrated hcrt:EGFP transgene allowing the observa-
tion of a single HCRT neuron harboring both commis-
sural (arrow) and ipsilateral processes.
A stable hcrt:EGFP transgenic line reveals the
Appelbaum et al. PNAS ?
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hcrt:EGFP transgenic line documents considerable conservation of
the HCRT circuit architecture across vertebrates and is therefore a
HCRT Neurons Project to the Pineal Gland Where hcrtr Is Expressed.
Through 2-photon imaging of hcrt:EGFP larvae, we identified
axons emerging from both HCRT cell clusters and forming an arch
reaching the most dorsal region of the midbrain-telencephalon
boundary (Fig. 4A, dorsal view and B, frontal view). These fibers
were located superficially, just underneath the skull, suggesting an
innervation of the pineal gland. In all vertebrates, the pineal gland
influences daily and annual physiological changes mediated by the
secretion of melatonin at night (18, 19). In zebrafish, melatonin
mediates the circadian clock output and also promotes sleep (20,
21). To demonstrate that HCRT neurons project into the pineal
aanat2:EGFP fish expresses EGFP specifically in melatonin pro-
ducing photoreceptors of the pineal gland (22). In the resulting
double transgenic fish, HCRT axons were indeed found to project
into the pineal gland (Fig. 4C). This observation was confirmed in
adult brains, where, in addition to the commissural innervation of
the habenula (Ha, Fig. 3B1), HCRT neurons innervate the pineal
gland stalk (PS, Fig. 4 D and E). This HCRT-pineal connection is
an exceptional case of a direct neuropeptidergic innervation of the
fish pineal gland. The importance of this projection was substan-
tiated by the detection of hcrtr mRNA in the pineal gland of both
larvae (Fig. 4 F–H) and adults (Fig. 4 I–K), strongly suggesting that
pineal gland activity could be modulated by HCRT circuitry.
HCRT Increases Melatonin Production in Cultured Pineal Glands. To
test whether HCRT peptides affect melatonin production in the
pineal gland, we used a well-established perfusion assay of adult
zebrafish pineal glands (SI Materials and Methods and Fig. S2).
system for 3 days. As expected, isolated pineal glands of fish
and dark–dark (DD) cycles (Fig. 5A and B). Custom-made mature
zebrafish HCRT-1 peptide (10?6M) (16) was applied to individual
21–23, when melatonin level is declining. HCRT-1 application led
to a significant increase in melatonin production (up to 70%, P ?
0.05, n ? 8), which lasted for the duration of HCRT application
(Fig. 5B), indicating a stimulating effect of HCRT-1 on melatonin
production. In contrast, minimum essential medium (MEM) ap-
plication did not affect melatonin production in control pineal
glands (n ? 12, Fig. 5A). These results demonstrate that the
zebrafish pineal gland can respond to HCRT excitatory input and
suggest the existence of a functional HCRT/pineal gland neuronal
circuit in zebrafish.
aanat2 Expression Is Down-Regulated in hcrtr?/? Pineal Gland. We
next tested whether this putative circuit is functional in vivo and
analyzed whether the absence of HCRT input in hcrtr?/? back-
ground could affect pineal gland melatonin production. To do so,
stacks of 0.5- or 1-?m sections). Note the compact organization of the HCRT cell bodies in the periventricular hypothalamus (D and dashed-box close up in D1).
(A vs. F), telencephalon-midbrain boundary (B and B1 vs. G), anterior diencephalon and mesencephalon (C vs. H), mid diencephalon and mesencephalon (D vs.
I), rhombencephalon (E vs. J). Chab, commissura habenularum; Ctec, commissura tecti; Cpost, commissura posterior; CC, crista cerebellaris; CP, central posterior
thalamic nucleus; D, dorsal telencephalic area; Dm, medial zone of D; Dc, central zone of D; Dl, lateral zone of D; DIL, diffuse nucleus of the inferior lobe; Ha,
habenula; Hv, ventral zone of the periventricular hypothalamus; Hd, dorsal zone of the periventricular hypothalamus; IMRF, intermediate reticular formation;
octaval population; TBS, tractus bulbospinalis; TeO, optic tectum; TPp, periventricular nucleus of posterior tuberculum; V, ventral telencephalic area; Vv, ventral
nucleus of V; Vd, dorsal nucleus of V.
Adult zebrafish HCRT circuit. (A–E) Confocal imaging of 100 ?m transversal brain sections from a stable hcrt:EGFP transgenic adult fish (reconstructed
www.pnas.org?cgi?doi?10.1073?pnas.906637106 Appelbaum et al.
we studied the level of expression of AANAT the key enzyme of
melatonin production (19). In zebrafish pineal glands, aanat2
mRNA level is rhythmic, and is a robust indicator of melatonin
six hcrtr?/? adult pineal glands collected at 1:00 AM (ZT16) were
measured using quantitative PCR. Strikingly, we observed a sig-
nificant reduction of 31% of aanat2 expression level in hcrtr?/?
pineal glands (P ? 0.05, Fig. 5C), indicating that melatonin pro-
duction is decreased in absence of HCRT signal. This result
demonstrates the existence of a functional HCRT-pineal gland
circuit and may explain the fragmented sleep phenotype of
hcrtr?/? fish in the dark.
hcrtr?/? Larval and Adult Mutants Are Hypersensitive to the Sleep
Inducing Effects of Melatonin. To further investigate whether mel-
atonin signaling down-regulation is responsible for the hcrtr?/?
melatonin and other hypnotic drugs on hcrtr?/? fish and wild-type
siblings. We treated larvae with melatonin and compounds from
four different classes of hypnotics (barbiturate, benzodiazepine,
anti-histaminergic, and ?2 adrenergic agonist) and monitored their
activity over 24 h under constant dim light conditions (Fig. 6 and
Fig. S3). No significant differences were found under drug-free
conditions between genotypes (Fig. 6A). However, with melatonin
exposure, hcrtr?/? larvae were more sedated than the wild-type
Dorsal and frontal views of the brain of a 7 dpf hcr-
copy. HCRT axons (arrows) projecting toward the pi-
neal gland are observed. (C) A dorsal image of 6 dpf
transgenic larva carrying two transgenes; an EGFP re-
porter driven by hcrt (hcrt:EGFP) and the pineal-
specific aanat2 (aanat2:EGFP) promoters, demon-
strate direct axon projection (arrow) to the pineal
gland. (D and E) Close-ups of two adjacent transversal
hcrt:EGFP adult brain sections showing HCRT projec-
tions to the habenula and the pineal gland stalk. (F)
Lateral and (G and H) dorsal views of whole-mount in
situ hybridization of 2-dpf embryos. (F) hcrtr mRNA is
the pineal gland (arrow). Double ISH experiment with
aanat2 demonstrates that hcrtr is expressed in the
pineal gland during the day (G) and the night (H).
Similarly, in adult animals, hcrtr is expressed in the
pineal gland (I). aanat2 (J) and egfp (K) probes were
used as positive and negative controls, respectively.
Adult pineal glands (I–K) were removed with the up-
per skull and skin hence presence of brown melano-
phores cells in the preparations.
The HCRT-pineal gland circuit. (A and B)
circadian rhythm of melatonin release of melatonin from zebrafish pineal glands cultured in constant darkness. MEM medium application does not affect
melatonin production in 12 control pineal glands. (B) zebrafish HCRT-1 application (10?6M) stimulates melatonin production (ZT 21–23) (n ? 8, P ? 0.05). The
production lasts for the duration of HCRT application. (C) Quantitative PCR analysis of aanat2 mRNA level in six wild-type sibling and 6 hcrtr?/? pineal glands
collected during the night (ZT 16). Note the significant decrease (31%, P ? 0.05) of aanat2 expression in pineal glands devoid of functional HCRT input.
HCRT modulates pineal gland melatonin production. (A and B) HCRT induces melatonin release from cultured zebrafish pineal glands. (A) Normal
Appelbaum et al.PNAS ?
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siblings (P ? 0.0001, Fig. 6A). In contrast, hcrtr?/? and wild-type
suggesting, in this mutant, an up-regulation of the actors down-
melatonin signaling (24).
We next analyzed the impact of melatonin application on the
sleep dynamics of adult hcrtr?/? and wild-type sibling fish. Based
small concentration of melatonin could abrogate the sleep frag-
mentation phenotype, we used a subthreshold dose of melatonin:
the highest dose that could be administered to wild-type adults
without inducing hypnotic effects; that is, 1 ?M. Sleep time, sleep
cycle in wild-type and mutant adult fish using our previously
described Adult Fish Sleep Recording System (AFSRS) (16). For
each individual fish, sleep patterns in the presence of melatonin
were compared to records obtained during the previous night
were not affected by this low dose of melatonin, we found that
hcrtr?/? fish were strongly sedated. They displayed an approxi-
mate 30% increase in sleep (P ? 0.01) (Fig. 6C), and a strong
increase of sleep consolidation indicated by a 40% decrease in the
number of sleep–wake transitions (P ? 0.005) (Fig. 6D), and a
100–150% increase in mean sleep bout length (P ? 0.005) during
the night (Fig. 6 B and E). Together, these data indicate that
hypersensitivity to melatonin is a strong and specific feature of
hcrtr?/? fish. Further, it demonstrates a behavioral-level of inter-
the absence of functional HCRT pathway affects sleep-promoting
melatonin signaling at a level yet to be discovered.
In mammals, HCRT has a wake-promoting role (4). In addition to
this function, evidence supports a major role of HCRT in the
consolidation not only of wake state but also of sleep state. In
human, dog, and mouse, HCRT deficiency is associated with both
wake and sleep fragmentation (10, 11). Moreover, sleep fragmen-
tation persists in narcoleptic patients even when pharmacologically
kept awake during the daytime (12), excluding a simple wake
rebound hypothesis. Furthermore, mice with HCRT neurons de-
void of GABA-B receptors also display both fragmented sleep and
wake without reduction of total sleep amount (25). Finally, al-
though studies have shown that HCRT neurons decrease firing
(8). This is also reflected at the extracellular levels, where HCRT
levels stay high during sleep (26) and could regulate it (27). We
therefore propose that HCRT has a dual function on promoting
is very likely conserved. Ubiquitous HCRT overexpression in
transgenic fish increases wakefulness (15), while hcrtr?/? mutants
existence of compensatory system(s) or wiring differences. Never-
theless commonalities observed in these experiments (15, 16)
suggest that HCRT function is conserved in zebrafish and can be
implicated in both wake promotion and sleep consolidation.
Here, we demonstrate conservation of glutamatergic phenotype
of HCRT neurons across vertebrates, and we further show fish
HCRT circuit homology with the mammalian system (3, 5), includ-
ing the direct HCRT innervation of the pineal gland. In sheep (28),
rat (29), and pig (30), HCRT fibers and/or HCRT receptors were
indeed also found in the pineal gland, demonstrating conservation
across diurnal and nocturnal vertebrates. In all vertebrates, diurnal
or nocturnal, melatonin is secreted in the dark by the pineal gland
and controls daily and seasonal changes in physiology (18, 19).
Melatonin is also a well-established sleep inducer in zebrafish (21),
albeit a weaker one in humans. In mammals, light acts via retino-
hypothalamic projections to entrain the central oscillator located in
the suprachiasmatic nucleus (SCN). In turn, the SCN synchronizes
the pineal gland. In contrast, in teleosts, phototransduction, oscil-
lator, and melatonin production are located in the photoreceptor
cells of the pineal gland. Thus, the fish pineal gland is considered
as an autonomous organ able to rhythmically produce melatonin
(19, 20). There are only a few described examples of pineal
innervations in teleosts (20, 31). In this study, we show HCRT
innervation of the pineal gland and that HCRT signal modulates
pineal melatonin production. This is an example of peptidergic
innervation and functional control of the fish pineal gland. Thus,
the dogma that pineal melatonin production in teleost is totally
independent of CNS regulation may need to be revisited.
tonin sleep-promoting effect. (A) Larvae were kept
monitoring system (white bars represent the subjec-
tive day period). hcrtr?/? and wild-type sibling 5 dpf
larvae demonstrate similar rhythmic activity that peak
during the day. hcrtr?/? larvae were significantly
more sensitive to melatonin’s (1 ?M) hypnotic effect.
by white and black bars) in AFSRS [SI Materials and
Methods and (16)] and fine sleep architecture was
analyzed. (B) Representative sleep bout pattern of an
adult hcrtr?/? mutant. Subthreshold dose of melato-
nin (1 ?M) was added at the beginning of the second
first night. hcrtr?/? adults were more sensitive to
melatonin as, after its administration, total sleep time
increased (C), the number of sleep/wake transition
decreased (D) and sleep bout length increased (E), as
compared to wild-type siblings.
hcrtr?/? fish are hypersensitive to the mela-
www.pnas.org?cgi?doi?10.1073?pnas.906637106 Appelbaum et al.
This study not only describes a neuroanatomical connection Download full-text
between two major sleep/circadian systems, it also demonstrates
and behavioral levels. Application of zebrafish HCRT on wild-type
pineal glands consistently stimulates the release of melatonin. This
promoting effect was confirmed in vivo by the down-regulation of
aanat2 in hcrtr?/? fish pineal glands, suggesting that the HCRT
signaling pathway likely modulates melatonin production by acting
upstream of the transcriptional cascade controlling aanat2 expres-
sion (32). Furthermore, hcrtr?/? fish are strikingly hypersensitive
to the hypnotic effects of melatonin. This effect is specific, as four
other classes of hypnotics (Fig. S3) show no differences between
hcrtr?/? fish and wild-type siblings. This hypersensitivity may be
interpreted in different ways. A first possibility could be that
hcrtr?/? fish are sleepier, and thus more sensitive to sleep induc-
tion by melatonin. In this case, however, we would have expected
similar results with other hypnotics. An attractive alternative hy-
pothesis supported by our results, could be that HCRT promotes,
at least partially, sleep consolidation in the dark through the
stimulation of the melatonin sleep-promoting system. This would
explain sleep disruption in the hcrtr?/? fish when in the dark, but
not the light that is a suppressor of melatonin release of its own.
Furthermore, in the absence of HCRT signaling, the downstream
melatonin pathway and/or circuit could be up-regulated to com-
pensate for the decrease of melatonin production, thus explaining
loop has been previously reported in mammals and birds where
melatonin receptor numbers increased after pinealectomy (24).
(Fig. 3) strongly resemble melatonin receptors’ expression pattern
(Fig. S4) especially in the periventricular gray zone of the optic
tectum, and the periventricular thalamus and hypothalamus. Thus,
HCRT and melatonin pathways could also interact at downstream
levels in brain regions where both HCRT and melatonin receptors
are coexpressed. These potential downstream interconnections
might also be responsible for the hcrtr?/? melatonin hypersensi-
tivity phenotype. Overall, future studies will be required to inves-
tigate the full extent of melatonin and HCRT pathway collabora-
tion (synergy or compensation) and to further test the wake-
promoting and sleep-consolidating dual function of HCRT.
is relevant to humans is unknown at this stage. Studies in narco-
leptics have not shown dramatic differences in melatonin release,
although light regime was not controlled (33). In addition, humans
display variable sensitivity to exogenous melatonin’s hypnotic ef-
fects. Melatonin acts as a mild hypnotic when administered during
although notably in Smith Magenis syndrome strong beneficial
effects on disturbed nocturnal sleep have been documented (35).
The use of melatonin for the treatment of insomnia in narcolepsy
has only been reported in a single case, with a dramatic REM sleep
promoting effect (36). As this is not in line with clinical impression,
a more systematic evaluation is needed.
In summary, because HCRT is not present in nonvertebrate
lineages, zebrafish provide an excellent simplified system in which
to dissect how HCRT neurons and their projections may affect the
sleep/wake cycle and other behaviors. This work reveals a sleep
mediators of the HCRT system in regulating sleep.
Materials and Methods
The methods are described in detail in SI Materials and Methods. Imaging was
performed using a custom-made two-photon laser-scanning microscope and
laboratory. Behavior was monitored with either Adult Fish Sleep Recording
System or ViewPoint system.
ACKNOWLEDGMENTS. The authors thank Dr. David C. Klein for fruitful discus-
sion, Dr. Jamie M. Zeiter for his advices in statistical analysis, Drs. Matthew P.
Klassen and Shawn M. Burgess for critical reading of the manuscript, Laura
Alexander, Gemini Skariah, and Drs. Sarit Lampart and Oren Levy for their
technical assistance. This work was supported by Howard Hughes Medical Insti-
tute and National Institutes of Health Grant R01 NS062798 (to E.M.).
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