Neuropeptide Y regulates sleep by modulating noradrenergic signaling

Neuropeptide Y regulates sleep by modulating noradrenergic

signaling

Chanpreet Singh

Jason Rihel

, and

David A. Prober

1,*

Division of Biology and Biological Engineering, California Institute of Technology, Pasadena, CA,

91125, USA

Department of Cell and Developmental Biology, University College London, London, WC1E 6BT,

SUMMARY

Sleep is an essential and evolutionarily conserved behavioral state whose regulation remains

poorly understood. To identify genes that regulate vertebrate sleep, we recently performed a

genetic screen in zebrafish, and here we report the identification of neuropeptide Y (NPY) as both

necessary for normal daytime sleep duration and sufficient to promote sleep. We show that

overexpression of NPY increases sleep, whereas mutation of

npy

or ablation of

npy

-expressing

neurons decreases sleep. By analyzing sleep architecture, we show that NPY regulates sleep

primarily by modulating the length of wake bouts. To determine how NPY regulates sleep, we

tested for interactions with several systems known to regulate sleep, and provide anatomical,

molecular, genetic and pharmacological evidence that NPY promotes sleep by inhibiting

noradrenergic signaling. These data establish NPY as an important vertebrate sleep/wake regulator

and link NPY signaling to an established arousal-promoting system.

eTOC Blurb

Based on a genetic screen, Singh et al identify NPY signaling and

npy

-expressing neurons as

regulators of zebrafish sleep. They show that NPY promotes sleep by inhibiting noradrenergic

signaling, thus linking NPY signaling to an established arousal-promoting system.

Keywords

Sleep; neuropeptide Y; hypothalamus; locus coeruleus; noradrenaline; locomotor activity; arousal;

genetics

Corresponding author: dprober@caltech.edu.

Lead Contact:

David A. Prober

AUTHOR CONTRIBUTIONS

DAP and JR performed the genetic screen. CS and DAP conceptualized and designed the experiments, and generated reagents. CS

performed the experiments and analyzed the data. CS and DAP wrote the paper with assistance from JR. DAP supervised the project.

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Author manuscript

Curr Biol

. Author manuscript; available in PMC 2018 December 18.

Published in final edited form as:

Curr Biol

. 2017 December 18; 27(24): 3796–3811.e5. doi:10.1016/j.cub.2017.11.018.

Author Manuscript

INTRODUCTION

Sleep is among most basic needs of living organisms, yet mechanisms that regulate sleep

remain poorly understood. Several neuropeptides have been implicated in regulating

mammalian sleep [

], including hypocretin [

–

], which promotes wakefulness, and galanin

[

–

] and melanin concentrating hormone [

–

], which promote sleep, suggesting that

examining additional neuropeptides may identify novel mechanisms that regulate sleep.

Identifying these mechanisms using mammalian models has been challenging due to their

poor amenability for large-scale screens, although such screens are possible [

]. As an

alternative approach, several groups have used behavioral criteria to study sleep-like states in

simpler model organisms that are amenable to screens, including

Drosophila

[

–

elegans

[

–

], and zebrafish [

–

]. In particular, several groups have demonstrated

behavioral, anatomical, genetic and pharmacological conservation of sleep between

zebrafish and mammals, establishing zebrafish as a vertebrate sleep model [

–

We previously described a screen for genes whose overexpression affects zebrafish sleep,

and reported that the neuropeptide neuromedin U is necessary and sufficient for normal

levels of arousal [

]. Here we demonstrate that another neuropeptide identified in the

screen, neuropeptide Y (NPY), is necessary for daytime sleep and sufficient to promote

sleep.

NPY is widely expressed in the brain and has been implicated in regulating endocrine,

behavioral and circadian processes [

], and is perhaps best known for its role in promoting

feeding [

–

]. NPY has also been shown to affect sleep, but its role in this behavioral state

remains unclear. Several studies showed that injection of

in vitro

synthesized NPY into the

rodent brain [

–

] or intravenously in young healthy [

] or depressed [

] humans can

induce sleep or reduce locomotor activity. However, other rodent studies reported the

opposite effect [

–

]. The basis for these disparate reports is unclear, but may be due to

different sites and doses of NPY injection, or the use of

in vitro

synthesized peptide that may

vary in different preparations and from endogenous NPY. Understanding the role of NPY in

mammalian sleep is also confounded by links between mechanisms that regulate feeding and

sleep [

–

]. Indeed, reports of wake-promotion by injected NPY also observed increased

feeding [

–

], suggesting that the increased wakefulness may result from increased

feeding.

npy

mutant mice exhibit several phenotypes, including increased anxiety,

depression-like behavior, and cognitive deficits [

], and are less susceptible to diet-

induced obesity [

]. However, an analysis of sleep in these animals and a role for

npy

expressing neurons in sleep has not been described. As a result, the role of NPY in

vertebrate sleep remains unclear.

Here we show that NPY is sufficient to promote sleep in zebrafish, whereas loss of

npy

-expressing neurons results in less daytime sleep. We also show that NPY promotes sleep

by inhibiting the wake-promoting noradrenergic system, providing a mechanistic basis for

sleep regulation by NPY. Together with the requirement of noradrenergic signaling for the

wake-promoting function of hypocretin [

], these results suggest that the noradrenergic

system integrates neuropeptidergic signals that regulate sleep/wake states.

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RESULTS

Overexpression of human NPY reduces locomotor activity and increases sleep in

zebrafish

We previously performed a screen to identify genes that affect larval zebrafish sleep [

We injected >1200 unique plasmids in which a heat shock-inducible promoter (

hsp

)

regulates the expression of genes that encode for secreted proteins into wild-type (WT)

zebrafish embryos at the one-cell stage. We used human open reading frames (ORFs)

encoding secreted proteins from the hORFeome 3.1 library [

] because there was no

resource of zebrafish ORFs. Co-injection of each plasmid with

tol2 transposase

mRNA

resulted in incorporation of the

hsp

-regulated transgene into the genome in many cells of

each animal and enabled heat shock-induced overexpression [

]. We then compared sleep/

wake behaviors in injected animals before and after heat shock and to negative control

animals injected with a

hsp:egfp

plasmid. One gene whose overexpression increased sleep at

night (Z-score=1.8) encoded human NPY (Figure S1A). Even though zebrafish exhibit high

levels of sleep at night, NPY-overexpressing animals were 28% less active and slept 34%

more than control animals during the night after heat shock (

<0.05 and

<0.01, two-tailed

Student’s

test) (Figures S1B–S1G). We observed a similar phenotype during the day before

heat shock that did not reach statistical significance, consistent with leaky expression from

the

hsp

promoter that often is observed using this transient injection assay, but is not

observed using stable transgenic lines [

Overexpression of zebrafish NPY reduces locomotor activity and increases sleep in

zebrafish

Using reciprocal BLAST searches, we identified a single zebrafish

npy

ortholog, which

encodes for a preproprotein that generates a predicted 36 amino-acid mature peptide that is

89% identical to the human and mouse orthologs (Figure S1H).

npy

is widely expressed in

the mammalian brain, particularly in the hypothalamus, amygdala, locus coeruleus (LC) and

cerebral cortex [

]. Using

in situ

hybridization (ISH) with an

npy

-specific probe,

immunostaining for total extracellular signal-regulated kinase (t-ERK), and image

registration to the Z-brain atlas [

], we found that

npy

is similarly expressed in several

discrete nuclei within the larval zebrafish brain (Figures S1I–S1N and Movie S1). We also

observed

npy

expression in the retina (data not shown) but not in other tissues.

To test whether overexpression of zebrafish NPY affects sleep, we generated

Tg(hsp:npy)

zebrafish.

(

hsp:npy

) animals and their WT siblings had similar amounts of locomotor

activity and sleep before heat-shock (Figures 1A–1D). However, following a heat shock at 3

p.m.,

Tg(hsp:npy)

animals were 50% less active (Figures 1A and 1B) and slept 111% more

(Figures 1C and 1D) than their WT siblings for the rest of the day (

<0.0001, two-tailed

Student’s

test). The phenotype resulted from a 230% increase in the number of sleep bouts

(Figure 1E) and an 85% decrease in the length of wake bouts (Figure 1G) (

<0.0001, two-

tailed Student’s

test), with a smaller decrease in the length of sleep bouts (Figure 1F), and

thus is primarily due to fragmentation of the wake state.

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The increase in sleep after the heat shock-induced pulse of NPY overexpression dampened

by nighttime. A previous study showed that the circadian system inhibits sleep in the

evening, when homeostatic sleep drive is high [

], suggesting the circadian system might

limit NPY overexpression-induced sleep to the day. To test whether NPY overexpression can

also increase sleep at night, we heat shocked animals during the last hour of the day. We

found that

Tg(hsp:npy)

animals were 46% less active (Figures S2A–S2C) and slept 54%

more (Figures S2D–S2F) than their WT siblings during the night (

<0.0001, two-tailed

Student’s

test), similar to the daytime phenotype when NPY overexpression was induced in

the afternoon. This phenotype was due to longer sleep bouts (Figure S2H) and shorter wake

bouts (Figure S2J), with no change in the number of sleep bouts (Figure S2G). These results

suggest that dampening of NPY-induced sleep at night following heat shock in the afternoon

is due to declining levels of NPY rather than effects of the circadian clock.

Light affects locomotor activity and sleep in zebrafish [

], as it does in mammals [

To determine whether light affects NPY overexpression-induced sleep, we entrained larvae

by raising them in 14:10 hour light:dark (LD) conditions for four days, and then transferred

them to constant dark before inducing NPY overexpression. NPY-overexpressing animals

were 54% less active and slept 80% more than WT siblings during the rest of the subjective

day (Figures S2K–S2N) (

<0.0001, two-tailed Student’s

test). This phenotype was due to

more sleep bouts and shorter wake bouts, with no change in the length of sleep bouts

(Figures S2O–S2Q). Hence, NPY overexpression promotes sleep independent of lighting

condition and circadian phase.

Overexpression of NPY increases arousal threshold

Sleep is distinguished from quiet wakefulness by reduced sensory responsiveness [

Because NPY overexpression increases sleep, we asked whether it also alters arousal

threshold by monitoring responses to mechano-acoustic stimuli. We found that the stimulus

intensity at which we observed the half-maximal response (effective tap power 50, ETP

)

was 290% higher for

Tg(hsp:npy)

animals than their WT siblings (Figure 1H) (

<0.05 by

extra sum-of-squares F test). Thus, NPY overexpression increases arousal threshold,

consistent with increased sleep. We next asked if NPY overexpression affects arousal in

awake and/or sleeping animals by allowing 5 minutes between trials. According to the

behavioral definition of sleep, we scored animals as awake if they moved during the minute

before a stimulus was delivered. We used stimulus intensities of 2.3, 3.0 and 4.0 arbitrary

units, which were lower than the ETP

values of both

Tg(hsp:npy)

and WT animals. NPY-

overexpressing animals were less responsive to these stimuli than WT siblings during both

awake (Figure 1I) and sleep (Figure 1J) states. These data suggest that NPY overexpression

decreases arousal in awake animals and increases sleep depth in sleeping animals.

npy

mutant zebrafish are more active and sleep less during the day

We next asked whether endogenous

npy

is required for normal sleep/wake behaviors by

using the zinc finger nuclease method to generate zebrafish containing a predicted null

mutation in the

npy

open reading frame [

]. We isolated zebrafish containing a 17-

nucleotide deletion in the second exon of the

npy

gene [

], which results in a translational

frame shift at the beginning of the mature peptide domain (Figure 2A), generating a protein

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that lacks the mature peptide domain and thus is likely nonfunctional. Homozygous mutant

animals are viable and fertile, and lack obvious developmental defects.

Consistent with the NPY overexpression phenotype,

npy

−/− larvae were 23% more active

and slept 36% less during the day than their

npy

+/+ siblings (

<0.0001 and

<0.01, one-

way ANOVA, Holm-Sidak test) (Figures 2B, 2C, 2E and 2F). These effects were due to

fewer sleep bouts (Figure 2H) and longer wake bouts (Figure 2L), with no effect on the

length of sleep bouts (Figures 2J). Thus, reduced daytime sleep in

npy

−/− animals is due to

consolidation of the wake state. We did not observe

npy

−/− phenotypes at night. These data

indicate that endogenous

npy

is required for normal daytime sleep amounts.

Microinjection of NPY into the hamster suprachiasmatic nucleus phase shifts the locomotor

activity circadian rhythm in constant light (LL) [

], suggesting that NPY may regulate

entrainment or expression of circadian rhythms. To test whether endogenous

npy

is required

for circadian regulation of locomotor activity and sleep, we tested larvae that were entrained

for 4 days in LD, then monitored for 24 hours in LD and then for 48 hours in LL. Absence

npy

had no obvious effect on the locomotor activity or sleep circadian period length or

phase (Figures 3A and 3D). As expected, in LD

npy

−/− animals were more active (Figures

3A and S3A) and slept less (Figures 3D and S3C) than their

npy

+/+ and

npy

+/− siblings

during the day, with no phenotype at night. The daytime phenotype was due to fewer sleep

bouts and longer wake bouts (Figures S3E and S3I). Following the shift to LL,

npy

−/−

animals were more active by 30% and 26% during the subjective day and night, respectively,

compared to their

npy

+/+ siblings (

<0.001 and

<0.01, one-way ANOVA, Holm-Sidak

test) (Figures 3A–3C).

npy

−/− larvae also slept ~40% less during the subjective day and

night (

<0.0001 and

<0.001, one-way ANOVA, Holm-Sidak test) (Figures 3D–3F). These

phenotypes were primarily due to longer wake bouts (Figures 3K and 3L), although there

were also fewer (Figures 3G and 3H) and shorter (Figures 3I and 3J) sleep bouts. These

results indicate that

npy

is not required for circadian regulation of locomotor activity or

sleep in zebrafish larvae, but rather regulates sleep in a light-dependent manner.

Ablation of

npy

-expressing neurons increases locomotor activity and decreases sleep

As an alternative approach to test the hypothesis that NPY is necessary for normal sleep

duration, we ablated

npy

-expressing neurons. To this end, we generated

Tg(npy:kalta4)

zebrafish, in which NPY neurons express an optimized version of the transcriptional

activator Gal4 (KalTA4). To verify the specificity of this transgene, we performed double

fluorescent ISH (FISH) using probes specific for

npy

and

kalta4

. We observed that

kalta4

expressed in >80% of

npy

-expressing neurons (>95% for some brain regions), and that

>92% of

kalta4

-expressing neurons express

npy

(Figure S5A and Table S1). We mated these

fish to

Tg(uas:nfsb-mcherry)

animals [

], resulting in the expression of nitroreductase

(nfsb) in

npy

-expressing neurons (Figure 4A). Nitroreductase is a bacterial protein that

converts the inert prodrug metronidazole (MTZ) into a cytotoxic DNA crosslinking agent,

thus enabling drug-inducible ablation of the targeted cell type [

]. We treated

Tg(npy:kalta4);Tg(uas:nfsb-mcherry)

and

Tg(npy:kalta4)

sibling control animals with MTZ

or DMSO vehicle control for 48 hours (from 3 days post-fertilization (dpf) to 5 dpf). MTZ

treatment almost completely eliminated mCherry-labeled cells in double transgenic animals

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(Figures 4A–4C), indicating loss of most

npy

-expressing neurons. Consistent with these

observations, we detected TUNEL labeling in

npy

-expressing neurons in

Tg(npy:kalta4);Tg(uas:nfsb-mcherry)

animals treated with MTZ, but not in those treated

with DMSO (Figures S5B–S5D), indicating that MTZ treatment induces apoptosis of

npy

expressing neurons. Consistent with the

npy

−/− phenotype,

npy

-ablated animals were 23%

more active (Figures 4C and 4D) and slept 28% less (Figures 4F and 4G) (

<0.01 and

<0.05, two-tailed Student’s

test) compared to sibling controls during the day. This

phenotype was due to fewer sleep bouts (Figure 4I) and longer wake bouts (Figure 4M),

indicating consolidation of the wake state, similar to

npy

−/− animals. To confirm that the

Tg(uas:nfsb-mcherry)

transgene alone does not cause a behavioral phenotype, we crossed

Tg(npy:kalta4)/+;Tg(uas:nfsb-mcherry)/+

to WT fish, excluded animals that were positive

for mCherry, and treated the remaining animals with MTZ. We observed no difference in

locomotor activity or sleep among animals of these three genotypes (Figure S4). The cell

ablation phenotype was slightly weaker than that of the

npy

mutant, likely because the

npy:kalta4

transgene is not expressed in all

npy

-expressing neurons. Because a small number

of neurons express

kalta4

but not

npy

in some brain regions (8% in the subpallium, <5% in

other brain regions; Figure S5A and Table S1), it is possible that ablation of these NPY-

negative cells is responsible for the behavioral phenotype. However, this is unlikely to be the

case due to the small number of cells involved and because the NPY neuron ablation

phenotype is consistent with the

npy

mutant phenotype, suggesting that both NPY and

npy

expressing neurons are necessary for normal daytime sleep amount.

The NPY overexpression phenotype is not blocked by manipulation of several pathways

known to regulate sleep

To identify genetic mechanisms through which NPY affects sleep, we tested whether the

NPY overexpression phenotype is suppressed in zebrafish containing mutations in other

genes implicated in regulating sleep (Table S2). We found that the NPY overexpression

phenotype persisted in larvae containing null mutations in

histidine decarboxylase

(

hdc

)

[

hypocretin receptor

(

hcrtr

) [

corticotropin releasing hormone a

(

crha)

(Singh et al.,

unpublished)

, crhb

(Singh et al., unpublished) or arylalkylamine

N-acetyltransferase 2

(

aanat2

) [

] (data not shown). These data suggest that NPY promotes sleep via other

mechanisms.

NPY promotes sleep by inhibiting noradrenergic signaling

Pharmacological and genetic studies in mammals and zebrafish have shown that

norepinephrine (NE) plays an important role in promoting arousal [

], and the LC is the

primary source of NE in the brain [

]. We obtained several lines of evidence suggesting

that NPY promotes sleep by inhibiting NE signaling. First, a nucleus of 3–5

npy

-expressing

neurons is located adjacent to, and sends projections that form close contacts with, LC

neurons (Figures 5A–5H and Movie S2). While this does not prove a direct interaction

between the two neuronal populations, it is consistent with our functional evidence that NPY

promotes sleep by inhibiting NE signaling (see below). The zebrafish genome contains

seven annotated

npy receptor

genes [

]. Using FISH, we did not detect

npy receptor

expression in LC neurons, although we observed expression of

npy receptor y1

(

npy1r

)

(Figure 5I) and

npy receptor y2 like

(

npy2rl

) (Figure 5J) near the LC. The other

npy

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receptors

showed expression in other brain regions (

npy8ar

and

npy8br

) or no detectable

pattern of expression (

npy2r

npy4r

and

npy7r

) (data not shown). These results suggest that

NPY indirectly affects NE signaling, although a

npy receptor

might be expressed in LC

neurons at levels too low to be detected using FISH, a common problem for G-protein

coupled receptors (GPCRs).

Second, we found that the sedating effects of NPY overexpression and loss of NE signaling

are not additive. We made this observation by overexpressing NPY in larvae that lack NE

synthesis due to mutation of

dopamine beta hydroxylase

(

dbh

) [

], or that lack NE

signaling due to treatment with the

-1-adrenergic receptor antagonist prazosin. Both

genetic and pharmacological inhibition of NE signaling increase sleep in zebrafish [

]. If

NPY promotes sleep by inhibiting NE signaling, then overexpression of NPY should not

further increase sleep in

dbh

−/− larvae or in WT larvae treated with prazosin. Alternatively,

if NPY promotes sleep via a NE-independent mechanism, then the combined effects of NPY

overexpression and loss of NE signaling on sleep should be additive. Because the behavior

dbh

+/− animals is indistinguishable from that of their

dbh

+/+ siblings [

], we compared

dbh

+/− and

dbh

−/− siblings to reduce the number of comparisons in each experiment, and

thus increase the number of animals per condition. Prior to heat shock-induced NPY

overexpression,

dbh

−/− larvae were 40% less active and slept >100% more than their

dbh

− siblings for both

Tg(hsp:npy)

animals and their non-transgenic siblings (Figures 6A–6D)

(

<0.01, two-way ANOVA, Holm-Sidak test). NPY overexpression decreased locomotor

activity by 54% and increased sleep by 60% in

Tg(hsp:npy);dbh+/

− animals compared to

dbh+/

− siblings (Figures 6A–6D) (

<0.0001 and

<0.05, Two-way ANOVA, Holm-Sidak

test). However, overexpression of NPY did not further affect the sleep/wake behavior of

dbh

−

− animals, as activity and sleep amounts were indistinguishable for

Tg(hsp:npy);dbh

−/−

and

dbh

−/− animals (Figures 6A–6D). We obtained similar results for NPY overexpression

in prazosin-treated animals compared to DMSO vehicle-treated controls (Figures S6A–

S6D). To confirm that the failure of NPY overexpression to enhance sleep in

dbh

−/− or

prazosin-treated animals is not due to a ceiling effect for sleep, we found that treatment with

melatonin, an alternative sedative, enhanced sleep induced by overexpression of NPY

(Figures S7A–S7D) or prazosin (Figures S7E–S7H).

Third, we found that the increased locomotor activity and reduced sleep observed in

npy

−/−

animals compared to their

npy

+/+ siblings was abolished by treatment with prazosin. We

made this observation by treating

npy

+/+,

npy

+/− and

npy

−/− larvae with either DMSO or

prazosin. If NPY promotes sleep by inhibiting NE signaling, then loss of NPY should not

affect prasozin-induced sleep. Alternatively, if NPY promotes sleep via a NE-independent

mechanism, then loss of NPY should affect sleep amount in prazosin-treated animals.

Consistent with the former possibility, we found that prazosin decreased activity and

increased sleep, and this phenotype was indistinguishable for

npy

+/+,

npy

+/− and

npy

−/−

siblings (Figures 6E–6J).

Fourth, we found that NPY regulates

dbh

expression in the LC. NPY overexpression

decreased

dbh

mRNA in the LC by 38% at 3 hours post-heat shock in

Tg(hsp:npy)

animals

compared to WT siblings (

<0.05, two-tailed Student’s

test) (Figures 7A and 7D). This

time point coincides with the maximal effect of NPY overexpression on locomotor activity

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and sleep (Figures 1A and 1C), suggesting that NPY overexpression-induced sleep may

result from reduced

dbh

expression, and thus reduced NE levels. However, effects of NPY

overexpression on behavior begin within the first hour after heat shock, and we only

observed a trend of decreased

dbh

mRNA at 1 and 2 hours post-heat shock that was not

statistically significant (Figure 7D). These observations suggest that reduced

dbh

expression

may not be the primary cause of NPY overexpression-induced sleep, but may rather be a

secondary effect that maintains NPY-induced sleep, perhaps resulting from decreased LC

neuron activity. We also tested whether NPY overexpression affects the level of

tyrosine

hydroxylase

(

), which acts upstream of

dbh

in the NE synthesis pathway. We found that

NPY overexpression did not significantly change

mRNA expression in the LC at 1, 2 or 3

hours post-heat shock (data not shown). Reduced

dbh

expression was not simply a

consequence of increased sleep, as

dbh

mRNA level was unaffected following

overexpression of the sleep-promoting neuropeptide prokineticin 2 (Prok2) [

] (Figure 7E)

or treatment with the sedative melatonin (Figure 7E). The interaction between NPY and

dbh

appears to be specific, as NPY overexpression did not affect expression of other genes

involved in promoting arousal, including the neuropeptides

hypocretin

(

hcrt

) [

] or

adenylate cyclase activating polypeptide 1a

(

adcyap1a

) (Singh and Prober, unpublished)

(Figures 7B, 7C and 7E). These results indicate that overexpression of NPY selectively

decreases the level of

dbh

mRNA in the LC, presumably resulting in decreased NE levels

and thus increased sleep. In support of this finding, we observed that

dbh

mRNA level was

33% higher in the LC of

npy

−/− animals compared to their

npy

+/− and

npy

+/+ siblings

during the day (Figures 7F and 7G) (

<0.05, one-way ANOVA, Holm-Sidak test).

Moreover,

dbh

mRNA level in the LC of WT animals was 25% lower at night compared to

the day (

<0.05, two-tailed Student’s

test) (Figures 7H). This result demonstrates a

correlation between the wake circadian phase of this diurnal species and the level of

dbh

mRNA in the LC, and suggests that changes in NE levels contribute to the regulation of

normal sleep/wake states. Taken together, these results are consistent with a model in which

NPY promotes sleep by inhibiting NE signaling.

DISCUSSION

NPY has been shown to affect sleep in mammals, but its role in sleep has been unclear.

Infusion of NPY in rodents has been reported to increase [

–

] or decrease [

–

] sleep.

These opposite effects may be due to different sites of injection or dosage, or the use of

vitro

synthesized NPY that may lack modifications present on endogenously produced

peptide. These studies are also confounded by other functions of NPY. For example,

experiments in rats found the wake-promoting effects of NPY to be associated with feeding

behaviors [

–

], and NPY can induce hypothermia [

] and increase social interactions

[

], which may affect sleep. In agreement with some rodent studies, intravenous NPY

injection promoted sleep in both healthy [

] and depressed [

] humans. Reduced NPY

was observed in humans with major depression who report sleep disturbances [

] and in

humans with primary insomnia [

], consistent with a sleep-promoting role for NPY.

Reduced NPY was also found in individuals with post-traumatic stress disorder (PTSD) [

]

and could contribute to the insomnia and fragmented sleep experienced by these patients.

npy

-expressing neurons are also implicated in mammalian sleep. For example, GABAergic

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cortical interneurons co-expressing

neuronal nitric oxide synthase

(

nnos

) and

npy

express

fos

, a marker of neuronal activity, during sleep in rodents [

]. Furthermore, extracellular

single-unit activity in the basal forebrain of anaesthetized rats showed increased firing of

npy

-expressing neurons during slow wave sleep [

To address the role of endogenous NPY in sleep, we performed genetic gain- and loss-of-

function studies using zebrafish larvae. These studies are performed before the onset of

feeding, when larvae receive nutrients from the yolk sac [

], and before the onset of social

interactions [

]. Furthermore, because zebrafish are poikilothermic, thermoregulation is

unlikely to be a factor in studies of zebrafish sleep. Thus, zebrafish larvae allow the role of

NPY in sleep to be addressed without complications of mammalian models. We found that

overexpression of NPY suppresses locomotor activity and increases sleep during the day and

night, whereas

npy

mutant zebrafish are more active and sleep less during the day. Analysis

of sleep architecture revealed that NPY overexpression results in shorter wake bouts,

whereas

npy

mutants have longer wake bouts, suggesting that NPY regulates consolidation

of the wake state. Consistent with this phenotype, ablation of

npy

-expressing neurons

resulted in decreased sleep during the day, again due to longer wake bouts. The daytime

specificity of the loss-of-function phenotype could be explained by the presence of

redundant sleep-promoting systems at night, the primary sleep phase of zebrafish. Consistent

with our observations, overexpression in

Drosophila

of neuropeptide F (NPF), a

Drosophila

homolog of NPY, or its receptor NPFR1, promotes sleep [

], although stimulation of NPF

neurons was recently shown to promote wakefulness and feeding [

]. This discrepancy

could arise from differences in nutritional status [

]. The

Drosophila

short neuropeptide F

(sNPF) is also thought to promote sleep [

] and has been referred to as an NPY ortholog,

but is more likely an ortholog of vertebrate RFamide peptides [

]. In

C. elegans

, locomotor

quiescence during lethargus is abolished in mutants lacking the receptor

npr-1

and reduced

in mutants lacking the

npr-1

ligands

flp-18

and

flp-21

[

npr-1

mutants are also more

responsive to oxygen and pheromones, resulting in altered foraging and accelerated

locomotion [

–

]. While NPR-1 is related to NPY receptors [

], FLP-18 and FLP-21 are

more similar to RFamide peptides [

]. Combined with our results, these studies

establish NPY as a conserved sleep promoting neuropeptide, and the human studies

described above suggest this function is conserved in humans.

npy

is widely expressed in the mammalian brain, particularly in the hypothalamus,

amygdala, LC and cerebral cortex [

]. Similar to mammals, NPY is expressed in

several discrete brain regions in zebrafish larvae. Because of this broad expression pattern,

NPY could act via several known sleep/wake regulators. First,

npy

-expressing neurons

innervate

hcrt

-expressing neurons, and NPY inhibits

hcrt

neurons in mouse brain slices [

Second, a hypothalamic population of

npy

-expressing neurons project to the histaminergic

tuberomammillary nucleus in rodents [

]. Third, corticotropin releasing hormone (CRH)

impairs sleep and enhances vigilance [

], and NPY enhances inhibitory synaptic

transmission in

crh

-expressing neurons in amygdala brain slices [

]. Fourth, melatonin

promotes sleep in diurnal vertebrates, including humans [

], and application of NPY to rat

pineal explants increases melatonin production [

]. To determine whether any of these

pathways underlie the sleep-promoting effects of NPY, we tested whether the NPY

overexpression phenotype is blocked in zebrafish mutants in which these pathways are

Singh et al.

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affected, but found this not to be the case. We also found that NPY overexpression increased

sleep in WT and melatonin-treated animals to a similar extent. These observations suggest

that NPY does not affect sleep by modulating these pathways.

In contrast to these negative results, we made several observations suggesting that NPY

promotes sleep by inhibiting NE signaling. Pharmacological and genetic studies in mammals

and zebrafish have shown that NE promotes arousal, and that inhibition of NE signaling

increases sleep [

]. We found that overexpression of NPY did not enhance the

increased sleep observed in

dbh

−/− animals and prazosin-treated WT animals, suggesting

that NPY overexpression promotes sleep by inhibiting NE signaling. Consistent with this

possibility, we found that prazosin treatment abolished the decreased sleep observed in

npy

mutants, suggesting that elevated NE signaling underlies the

npy

mutant phenotype. In

support of these functional interactions, we found that NPY overexpression decreases the

level of

dbh

mRNA in the LC, the primary source of NE in the brain [

], and thus likely

reduces NE levels. We observed a trend of reduced

dbh

mRNA levels at 1 and 2 hours after

induction of NPY overexpression, and a significant reduction at 3 hours post-heat shock.

These observations suggest that reduced

dbh

expression may not be the primary cause of

NPY overexpression-induced sleep, but rather may be a secondary effect that maintains

NPY-induced sleep, perhaps resulting from decreased LC neuron activity. Consistent with

this possibility, NPY can inhibit LC neurons in rodent brain slices [

100

]. However, the

maximal effect of NPY overexpression on behavior occurred at ~3 hours post-heat shock,

coinciding with a significant reduction in

dbh

expression in the LC, consistent with NPY

directly promoting sleep by decreasing

dbh

expression, and thus NE production, in the LC.

Moreover, we found that

npy

mutants have elevated

dbh

expression in the LC, presumably

resulting in increased NE levels and increased arousal. It was recently shown that

dbh

expression undergoes a circadian oscillation in whole zebrafish larvae [

101

]. Consistent with

this observation, we found that the level of

dbh

mRNA in the LC is lower at night compared

to the day, suggesting that NE levels contribute to the diurnal sleep/wake cycle.

Consistent with an interaction between NPY and the LC, we identified a small population of

npy

-expressing neurons that is adjacent to, and appears to innervate, the LC. This

observation contrasts with mammals, where

npy

and

dbh

are co-expressed in LC neurons

[

102

103

]. We were unable to detect expression of NPY receptors in LC neurons,

suggesting that NPY may indirectly affect NE signaling. However, expression of GPCRs,

the protein class of NPY receptors, is notoriously difficult to detect, and we thus cannot rule

out the possibility that a NPY receptor is expressed in LC neurons. We did observe

expression of

npy1r

and

npy2rl

in cells near the LC, suggesting the possibility of local

indirect interactions between NPY neurons and the LC. Thus, while the anatomic interaction

between the NPY and NE systems appears to differ in zebrafish and mammals, the

functional relationship between the systems may be conserved. Taken together, these

observations suggest that NPY could regulate sleep by directly affecting the firing of LC

neurons and/or the level of NE. Alternately, the site of interaction between NPY and NE in

sleep may lie in a network of neurons near the LC or elsewhere in the brain.

In both mammals and zebrafish, NE is necessary for the wake-promoting functions of Hcrt

signaling and

hcrt

-expressing neurons [

]. Here we provide evidence that NE signaling

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mediates the sedating effect of NPY, suggesting a central role for the NE system in

neuropeptidergic regulation of sleep/wake states. While Hcrt and NPY have opposite effects

on sleep via NE signaling, both neuropeptides promote feeding via neurons in the

hypothalamus [

104

], suggesting a segregation of neuronal circuits through which these

neuropeptides regulate sleep and feeding. While an interaction between NPY and the LC has

been shown to control stress responses in rodents [

105

], to our knowledge this is the first

demonstration of an interaction between NPY and the NE system in the context of sleep.

Finally, we note that cerebrospinal fluid levels of NPY are reduced in individuals suffering

from PTSD who have sleep disturbances [

], and treatment with prazosin reduces

nightmares and improves sleep in these individuals [

106

]. Since we found that

npy

mutant

zebrafish have elevated

dbh

expression, and presumably more NE, the reduced NPY

observed in in PTSD might cause increased NE levels, thereby disrupting sleep. These

observations suggest that NPY might be therapeutic for at least some aspects of PTSD.

In summary, our results identify NPY as a regulator of sleep/wake behaviors in zebrafish and

suggest that NPY promotes sleep by inhibiting NE signaling. These results highlight a

central role for NE signaling in regulating sleep, and suggest that modulation of NPY

signaling may be a useful therapeutic approach for sleep disorders.

STAR METHODS

CONTACT FOR REAGENT AND RESOURCE SHARING

Further information and requests for reagents may be directed to, and will be fulfilled by, the

Lead Contact David A. Prober (dprober@caltech.edu).

EXPERIMENTAL MODEL AND SUBJECT DETAILS

Zebrafish experiments and husbandry followed standard protocols [

107

] in accordance with

Caltech Institutional Animal Care and Use Committee guidelines. Larval zebrafish were

studied before the onset of sexual differentiation and all behavioral experiments were

performed using siblings with the same genetic background, differing only in the presence of

a transgene, mutation of a specific gene, or treatment with drugs and appropriate vehicle

controls. The age of animals used in each experiment is described in the manuscript, in each

figure legend, and/or in the STAR Methods.

Transgenic and mutant zebrafish

Tg(hsp:npy)

ct853Tg:

Full-length zebrafish

npy

cDNA was isolated using 5

′

and 3

′

RACE

(FirstChoice RLM-RACE, AM1700, Thermo Fisher Scientific) and the open reading frame

was cloned downstream of the zebrafish

hsp70c

promoter [

] in a vector containing

flanking I-SceI endonuclease recognition sites. The same zebrafish

npy

gene was cloned in a

previous study [

108

], but the gene isolated in our study contains an arginine residue located

C-terminal to the mature peptide domain that was reported as an alanine residue in the

previous study [

108

]. The sequence reported in our study is the same as that reported by the

zebrafish genome sequencing project (

www.ensembl.org/Danio_rerio

). The alanine residue

described in the previous report [

108

] is therefore likely either a sequencing error or a

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polymorphism in the fish strain used. Stable transgenic lines were generated by injecting

plasmids with I-SceI (R0694, New England Biolabs Inc.) into zebrafish embryos at the one-

cell stage. Transgenic founders were identified by outcrossing potential founders, heat

shocking progeny at 5 dpf, fixing animals 30 minutes after heat shock and performing ISH

using an

npy

-specific probe.

Tg(hsp:npy)

fish were genotyped using the primers 5

′

CCGCCACCATGAATCCA-3

′

and 5

′

-GGTTTGTCCAAACTCATCAATGT-3

′

, which

generate a 370 bp band. We generated two independent

Tg(hsp:npy)

stable transgenic lines

that produced similar phenotypes, but all data shown in the paper are from the line that

produced stronger phenotypes.

npy

mutant ct811:

npy

mutant zebrafish were generated using the zinc finger nuclease

method [

]. The mutant contains a 17 bp deletion (AGCCCGACAACCCGGGA) after

nucleotide 94 of the open reading frame, resulting in a translational frame shift beginning at

the fourth amino acid of the mature peptide domain. Mutant animals were genotyped using

the primers 5

′

-ATAAATTGCGCATCAGCACA-3

′

and 5

′

TGAGGAAGAATTTGAGACTACGC-3

′

, which produce a 281 or 264 bp band for the WT

or mutant allele, respectively.

npy

heterozygous mutants were outcrossed to the parental

TLAB strain for four generations before use in behavioral experiments. Homozygous

npy

mutants are viable, fertile, lack obvious developmental defects and are morphologically

indistinguishable from WT animals.

Tg(npy:kalta4)

ct852Tg:

We used bacterial artificial chromosome (BAC) recombineering

[

109

] to insert an optimized version of the transcriptional activator Gal4 (KalTA4) [

109

] at

the

npy

start codon of a BAC (zK50N10SP6; HUKGB735N1050Q, Source BioScience))

containing 288 kb of genomic sequence, including 145 kb upstream and 143 kb downstream

of the

npy

gene. Primers of 70 nucleotides (pIndigoBAC_HA1_iTol2_F and

pIndigoBAC_HA1_iTol2_R, Table S3) were used to amplify the long terminal repeats of the

medaka Tol2 transposon to enable single-copy integration of the BAC into the zebrafish

genome, using the plasmid

pIndigoBAC-536

[

109

] as template.

npy

-specific primers were

designed that contain 50 nucleotide homology arms around the

npy

start codon (positions

−53 to −4 and +4 to +53) with ~20 nucleotide ends (Homology arm F and Homology arm R,

Table S3 to amplify a KalTA4_kanamycin cassette from the plasmid

pCS2+_kalta4_kanR

[

109

]. These plasmids were a kind gift from Dr. Stefan Schulte-Merker. The modified BAC

was purified using the Nucleobond BAC 100 kit (740579, Macherey-Nagel) and injected

into zebrafish embryos at the one- or two-cell stage at a concentration of 50 ng/μL, along

with

tol2 transposase

mRNA at a concentration of 50 ng/μL. Transgenic lines were identified

by mating potential founders to WT TLAB fish, and progeny were genotyped using the

primers 5

′

-CGCTATCATTTATAGATTTTTGCAC-3

′

and 5

′

AGTAGCGACACTCCCAGTTG-3

′

, which produce a 220 bp band in transgenic animals.

Transgenic founders were crossed to the

Tg(uas:nfsb-mcherry)

line [

] and the strongest

line was identified by fluorescence microscopy.

Other transgenic and mutant lines:

The

Tg(dbh:EGFP)

transgenic line [

110

dbh

mutant

[

hcrtr

mutant [

hdc

mutant [

], and

aanat2

mutant [

] have been previously

described. The

crha

and

crhb

mutants are unpublished (Singh and Prober unpublished).

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METHOD DETAILS

Locomotor activity assay—

At 4 dpf, individual larvae were placed into each well of a

96-well plate (7701–1651, GE Healthcare Life Sciences) containing 650 μL of E3 embryo

medium (5 mM NaCl, 0.17 mM KCl, 0.33 mM CaCl

, 0.33 mM MgSO

, pH 7.4). Plates

were sealed with an optical adhesive film (4311971, Applied Biosystems) to prevent

evaporation, except in experiments where drugs were added. The sealing process introduces

air bubbles in some wells, which are excluded from analysis. In experiments using

transgenic animals, larvae were blindly assigned a position in the plate, and were genotyped

after the behavioral experiment was completed. Locomotor activity was monitored using an

automated videotracking system (Viewpoint Life Sciences) with a Dinion one-third inch

monochrome camera (Dragonfly 2, Point Grey) fitted with a fixed-angle megapixel lens

(M5018-MP, Computar) and infrared filter. For heat shock-induced overexpression

experiments, larvae were heat shocked at 37°C for 1 hour starting at either 3 p.m. or 10 p.m.

at 5 dpf. The movement of each larva was captured at 15 Hz and recorded using the

quantization mode in 1-minute time bins. The 96-well plate and camera were housed inside

a custom-modified Zebrabox (Viewpoint Life Sciences) that was continuously illuminated

with infrared LEDs, and illuminated with white LEDs from 9 a.m. to 11 p.m., except as

noted in constant light or constant dark experiments. The 96-well plate was housed in a

chamber filled with recirculating water to maintain a constant temperature of 28.5°C. The

parameters used for detection were: detection threshold, 15; burst, 29; freeze, 3, which were

determined empirically. Data were processed using custom PERL and Matlab (The

Mathworks, Inc.) scripts, and statistical tests were performed using Prism 6 (GraphPad).

A movement was defined as a pixel displacement between adjacent video frames preceded

and followed by a period of inactivity of at least 67 ms (the limit of temporal resolution).

Any one-minute period with no movement was defined as one minute of sleep based on

arousal threshold changes [

]. A sleep bout was defined as a continuous string of sleep

minutes. Average activity was defined as the average amount of activity in seconds/hour,

including sleep bouts.

Arousal threshold assay—

The arousal threshold assay was performed as described [

Animals were heat shocked at 5 dpf from 12 p.m. to 1 p.m, and taps of 14 different

intensities were applied in a random order from 3 p.m. to 10 p.m. Thirty trials were

performed at each stimulus intensity, with a 1-minute inter-trial interval. The background

probability of movement was calculated by identifying for each genotype the fraction of

larvae that moved 5 seconds prior to all stimuli delivered. This value was subtracted from the

average response fraction value for each tap event. A response is defined as any movement

that occurred within 1 second after a tap was delivered. Data was analyzed using Matlab

(Mathworks, Inc.) and dose-response curves were constructed using the Variable Slope

log(dose) response curve fitting module of Prism (Graphpad) and fitted using ordinary least

squares. The effective tap power 50 (ETP

) was defined as the tapping intensity at which

50% of the maximum number of responding larvae occurs, based on the fitted curve.

Tapping experiments with a 5-minute inter-trial interval were performed using three tap

intensities of 2.3, 3.0 and 4.0 arbitrary units to assess the response of awake and sleeping

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larvae to the stimuli. These stimulus intensities were chosen because they were lower than

the ETP

of animals of both genotypes. Animals were heat shocked at 5 dpf from 12 p.m.

to 1 p.m., and thirty-three trials were performed at each stimulus intensity in a random order

from 3:00 p.m. to 10:30 p.m. Behavioral responses were analyzed as described above. Three

independent experiments for were performed for both 1-minute and 5-minute tapping assays,

and one representative experiment for each is shown.

In situ

hybridization (ISH)—

Animals were fixed in 4% paraformaldehyde (PFA) in

phosphate buffered saline (PBS) for 16 hours at room temperature. ISH was performed using

digoxygenin (DIG) labeled antisense riboprobes (DIG RNA Labeling Kit, 11175025910,

Sigma-Aldrich), followed by incubation with a sheep anti-digoxigenin-POD antibody

(1:400; 11207733910, Sigma-Aldrich), and developed using the TSA Plus Fluorescein and

Cyanine 3 System (NEL753001KT, PerkinElmer). Double-fluorescent ISH was performed

using DIG- and fluorescein-labeled riboprobes (Fluorescein RNA Labeling kit,

11685619910, Sigma-Aldrich), and the TSA Plus Fluorescein and Cyanine 3 System

(NEL753001KT, PerkinElmer) using a previously described protocol [

]. Probes specific

for

npy

dbh

adcyap1a

kalta4

npy1r

npy2r

npy2rl

npy4r

npy7r

npy8ar

and

npy8br

were

synthesized using standard protocols [

111

]. The

npy

probe was transcribed using a PCR

product amplified from a zebrafish cDNA library using the primers Forward: 5

′

CCACAGAGCAAGAATTCCAA-3

′

and Reverse: 5

′

CAGTCATTATTGTTCTCCTTTGC-3

′

, and then serially amplified with the same Forward

primer and the Reverse Primer with a T7 promoter sequence added: 5

′

TAATACGACTCACTATAGGGCAGTCATTATTGTTCTCCTTTGC-3

′

. The

kalta4

probe

was transcribed using the plasmid

pCS2+_kalta4_kanR

[

109

] as a template after

linearization with BamH1 and using T7 RNA polymerase (10881767001, Sigma-Aldrich). A

probe specific for

dbh

has been previously described [

112

]. Probes specific for

adcyap1a

npy1r

npy2r

npy2rl

npy4r

npy7r

npy8ar

and

npy8br

were generated as described for the

npy

-specific probe using the primers listed in Table S3.

Immunohistochemistry (IHC)—

Samples were fixed in 4% PFA in 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 hour in 2% goat serum/2% dimethyl sulfoxide (DMSO)/PBTx at

room temperature or overnight at 4°C. Primary antibody incubations were performed in

blocking solution overnight at 4°C using chicken anti-GFP (1:400, GFP-1020, Aves L abs,

Inc.) and rabbit anti-DsRed (1:100, 632496, Clontech Laboratories, Inc.). Secondary

antibody incubations were performed in blocking solution overnight at 4°C using Alexa Fluo

r 488 goat anti-chicken (1:500, A-11039, Thermo Fisher Scientific) and Alexa Fluor 568

goat anti-rabbit (1:500, A-11011, Thermo Fisher Scientific) antibodies. Samples were

mounted in 50% glycerol/PBS and imaged using a Zeiss LSM 780 confocal microscope with

a 25× 0.8 NA water immersion objective (LD LCI Plan-Apochromat 25x/0.8 1mm Corr DIC

M27). Images were processed using Fiji [

113

Z-brain registration—

WT larvae were fixed at 6 dpf and ISH was performed using an

npy

-specific probe on dissected brains as described above, followed by IHC using mouse

anti-t-ERK primary antibody (1:500, 4696, Cell Signaling Technology) and Alexa Fluor 488

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goat anti-mouse secondary antibody (1:500, A32723, Thermo Fisher Scientific). Imaging

was performed using a Zeiss 780 confocal microscope, using a 20× 1.0 NA water dipping

objective (W Plan-Apochromat 20x/1.0 DIC CG=0.17 M27 75mm) and imaged at

~0.8/0.8/2 μm voxel size (x/y/z) using the Zeiss tiling function and the pairwise stitching

function of Fiji [

113

]. Non-rigid image registration was performed using the Computational

Morphometry Toolkit (CMTK,

http://www.nitrc.org/projects/cmtk/

) as previously described

[

]. t-ERK staining was used to register to the t-ERK reference brain [

], which was then

used to align

npy

ISH labeling. Registered brains were analyzed using the Z-Brain browser

(MATLAB) [

] to identify anatomical regions expressing

npy

. Using Fiji, the registered

brain showing

npy

expression was merged to the database ‘Anti-

tERK_6dpf_MeanImageOf193Fish’ from ‘AnatomyLabel DatabaseDownsampled’ from the

Z-Brain Downloads [

] to show the expression of

npy

relative to t-ERK in the reference 6

dpf zebrafish larva. The combined stack was converted into a movie and processed in

Windows Movie Maker to add anatomical labels.

Image processing in Imaris and Fiji—

Surface rendering to reconstruct projections of

npy

- and

dbh

-expressing neurons was performed using Imaris 9 (Bitplane). To perform

surface rendering, we used the Volume function followed by the Normal Shading mode to

add a depth effect to the 2-dimensional z-stack imaged using a 63× 1.4 NA oil immersion

objective (Plan-Apochromat 63x/1.4 oil DIC M27), and then displayed the image in the 3-

dimensional isometric view. We then used the Interactive Software Histogram to select a

threshold that included as much of the neuronal projections as possible while excluding any

background. Areas of overlap between projections from

npy

- and

dbh

-expressing neurons

were magnified 4-fold and saved as TIFF images.

To identify the sources of overlapping projections, a 63x z-stack of

npy

-expressing and

dbh

expressing neurons was converted to an 8-bit stack. Projections from a single

npy

-expressing

neuron and a single

dbh

-expressing neuron were manually traced using the Simple Neurite

Tracer plugin in Fiji. Tracings were then filled-in using the same plugin, with an exemplar

npy

-expressing neuron labeled magenta and an exemplar

dbh

-expressing neuron labeled

green, and saved as individual z-stacks. These z-stacks were then merged with the original z-

stack to so that the traced

npy

-expressing and

dbh

-expressing neurons were overlaid on the

original images. As a result, the traced

npy

-expressing neuron appears magenta and the

traced

dbh

-expressing neuron appears yellow. This merged image stack is shown in Movie

S2.

TUNEL staining—

Tg(npy:kalta4);Tg(uas:nfsb-mcherry

) larvae were treated with DMSO

or 10 mM MTZ for 18 hours starting at 3 dpf, and then were fixed in 4% PFA in PBS for 16

hours at 4°C, and subjected to a TUNEL Assay (

In Situ

Cell Death Detection Kit,

11684795910, Sigma-Aldrich) according to the manufacturer’s instructions.

Analysis and quantification of

dbh

expression using ISH—

dbh

ISH was

performed by incubating fixed 5 dpf brains with a DIG-labeled

dbh

antisense riboprobe,

followed by a sheep anti-digoxigenin-POD antibody (1:400; 11207733910, Sigma-Aldrich),

and developed using the TSA Plus Cyanine 3 System (NEL753001KT, PerkinElmer).

Samples were developed using the cyanine 3 substrate at 1:300 for 5 minutes to avoid

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saturation. Brains were imaged using a Zeiss LSM 780 confocal microscope using a 561 nm

laser and a 25× 0.8 NA water immersion objective (LD LCI Plan-Apochromat 25×/0.8 1mm

Corr DIC M27). To quantify

dbh

expression in

Tg(hsp:npy)

animals, larvae were heat

shocked from 3 p.m. to 4 p.m. and samples were collected at the indicated times after heat

shock. To quantify

dbh

expression in

npy

mutants, samples were collected at 4 p.m. Both

experiments used siblings whose brains were processed for ISH in the same tube, imaged,

quantified and then genotyped by PCR. To compare

dbh

expression levels during the day

and night, day samples were collected at 4 p.m. and night samples were collected at 2 a.m.

After fixation, a small nick was made in the forebrain of night samples to enable their

identification at the end of the experiment. Day and night samples were then placed together

in the same tube, processed for ISH, imaged and then quantified. Three independent

experiments were performed and images of representative samples are shown. For

quantification of

dbh

mRNA level, confocal z-stacks were obtained as described above.

Using Fiji [

113

], each z-stack was converted into a maximum intensity projection, converted

into 8-bit grayscale, and thresholded to select only the fluorescent ISH signal. This function

was applied to all images in an experiment to determine a threshold level that was optimal

for most images, and this threshold was then used for all images in an experiment. The

Analyze-Set Measurements function was used to select Integrated Density as the

measurement parameter and Limit to Threshold was selected to measure only the

thresholded region. Fluorescent intensity was then measured by the Analyze-Measure

function.

QUANTIFICATION AND STATISTICAL ANALYSIS

All line graphs show a 1 hour forward moving average plotted in 10 minute bins, except

Figures S1B and S1E, which show data plotted in 10 minute bins. Line and bar graphs show

mean ± standard error of the mean (SEM). In all statistical tests, the significance threshold

was set to

<0.05. Parametric statistical tests were used because the data followed an

approximately normal distribution. For behavioral experiments that compared two

genotypes, statistical significance was assessed using a two-tailed Student’s

test. For

npy

mutant experiments, which compared animals of three different genotypes, one-way

ANOVA followed by the Holm-Sidak correction for multiple comparisons was performed to

test for significant pair-wise comparisons among all genotypes. The Holm-Sidak test was

used to focus on significance but not confidence intervals. For experiments in which NPY

was overexpressed in various mutant backgrounds or in which NPY overexpression was

combined with drug treatments, statistical significance was assessed using two-way ANOVA

followed by the Holm-Sidak correction for multiple comparisons. For experiments in which

npy

mutants were treated with drugs, statistical significance was assessed using two-way

ANOVA followed by Holm-Sidak correction for multiple comparisons. For quantification of

ISH data, statistical significance was assessed using a two-tailed Student’s

test for

experiments that compared two samples, and one-way ANOVA followed by the Holm-Sidak

correction for multiple comparisons for experiments that compared three or more samples.

Behavioral data was processed using Matlab (MathWorks), graphs were generated using

Excel (Microsoft), and statistical analyses were performed using Prism 6 (Graphpad). The

number of animals and statistical test used are stated in each figure or figure legend.

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