Current Biology, Volume
27
Supplemental Information
Neuropeptide Y Regulates Sleep
by Modulating Noradrenergic Signaling
Chanpreet Singh, Jason Rihel, and David A. Prober
0
1
2
3
0
1
2
3
4
5
6
7
0
4
8
12
0
20
40
60
Night 4 Night 5
Day 5 Pre Day 5 Post
Night 4 Night 5
Day 5 Pre Day 5 Post
hsp:npy_Hs
Z=1.8
Z
-
Score
0
20
40
60
80
0
1
2
3
4
5
6
7
h
sp:EGFP
(n=16)
hsp:npy
(n=36)
Day 5
Night 5
Activity (s/10 min)
Sleep (min/10 min)
*
**
Night 4
Day 5
Night 5
Night 4
B
C
D
E
F
G
A
Number of genes
Sleep during night post HS
M
a
p
N
d
v
a
p
NPY_Hs
NPY_Mm
NPY_Dr
H
I
J
K
L
Activity (s/10 min)
Sleep (min/10 min)
Dorsal
Ventral
Figure
S
1
.
A
genetic
screen
identifies
a
sleep
-
promoting
role
for
NPY
and
zebrafish
npy
is
widely
expressed
in
the
brain
.
Related
to
Figure
1
.
(
A
)
Histogram
depicting
the
total
amount
of
sleep
during
the
night
after
heat
shock
for
~
1200
human
genes
tested
in
the
larval
zebrafish
genetic
screen
.
Larvae
overexpressing
human
NPY
had
a
Z
-
score
of
1
.
8
.
Overexpression
of
human
NPY
decreased
locomotor
activity
(
B
-
D
)
and
increased
sleep
(
E
-
G
)
compared
to
EGFP
-
overexpressing
controls
during
the
night
following
heat
shock
(indicated
by
yellow
bar)
.
(
H
)
Amino
acid
sequence
alignment
of
human
(Hs),
mouse
(Mm)
and
zebrafish
(
Dr
)
NPY
mature
peptide
sequences
.
Colors
indicate
residues
with
similar
properties
.
(
I
-
L
)
ISH
using
an
npy
-
specific
probe
reveals
discrete
yet
widespread
nuclei
of
npy
expression
in
a
6
-
dpf
zebrafish
.
Images
show
4
different
focal
planes,
with
the
most
dorsal
image
at
left
.
The
full
image
stack
of
npy
expression
throughout
the
brain
mapped
onto
the
Z
-
brain
atlas
is
shown
in
Movie
S
1
.
(
M,N
)
Schematic
drawings
illustrate
relative
positions
of
different
npy
-
expressing
populations
in
ventral
(
M
)
and
lateral
(
N
)
views
.
npy
is
expressed
in
the
olfactory
bulb
(a),
telencephalon
(b),
preoptic
area
(c),
posterior
tuberculum
(d),
Intermediate
lateral
hypothalamus
(e),
caudal
medial
hypothalamus
(f),
pretectum
(g),
torus
semicircularis
(h),
tectum
(
i
),
locus
coeruleus
(j),
medial
rhombomere
(k)
and
subpallium
(l)
.
a,
anterior
;
p,
posterior
;
d,
dorsal
;
v,
ventral
.
Scale
bar
:
100
μm
.
Mean
(
B,E
)
and
mean
±
SEM
(
C,D,F,G
)
are
shown
.
n=number
of
animals
.
*
P
<
0
.
05
,
**
P
<
0
.
01
by
two
-
tailed
Student’s
t
test
.
0
50
100
150
0
10
20
30
40
50
60
0
50
100
150
200
250
300
350
Day 5
Night 5
0
10
20
30
40
50
60
0
2
4
6
8
0
2
4
6
8
WT (n=121)
hsp:npy
(n=131)
Day 5
Night 5
Pre
-
HS
Post
-
HS
Pre
-
HS
Post
-
HS
A
D
H
G
****
WT (n=74)
hsp:npy
(n=79)
Day 5
Night 5
Day 6
Night 6
K
M
0
10
20
30
40
50
Pre
-
HS
Post
-
HS
****
0
20
40
60
80
100
120
Pre
-
HS
Post
-
HS
****
L
N
O
P
Q
0
2
4
6
8
10
12
14
****
Pre
-
HS
Post
-
HS
0
1
2
3
4
5
Pre
-
HS
Post
-
HS
0
5
10
15
20
****
Pre
-
HS
Post
-
HS
Activity (s/h)
Sleep (min/h)
Sleep bouts/h
Sleep bout length (min)
0
20
40
60
80
100
120
140
0
1
2
3
4
5
6
7
Pre
-
HS
Post
-
HS
I
J
****
0
10
20
30
40
50
60
0
2
4
6
8
0
10
20
30
40
50
0
50
100
150
200
250
300
Pre
-
HS
Post
-
HS
Pre
-
HS
Post
-
HS
B
C
E
F
****
****
Wake bout length (min)
Activity (s/h)
Sleep (min/h)
Sleep bouts/h
Sleep bout length (min)
Wake
bout length (min)
Activity (s/h)
Sleep (min/h)
Activity (s/h)
Sleep (min/h)
Day 5
Night 5
Day 6
Night 6
Figure
S
2
.
NPY
overexpression
increases
sleep
at
night
and
during
subjective
day
in
constant
dark
.
Related
to
Figure
1
.
(
A
-
J
)
Heat
shock
-
induced
overexpression
of
zebrafish
NPY
during
the
last
hour
of
the
day
resulted
in
decreased
locomotor
activity
(
A
-
C
)
and
increased
sleep
(
D
-
F
)
during
the
following
night
.
NPY
overexpression
increased
the
length
of
sleep
bouts
(
H
)
and
decreased
the
length
of
wake
bouts
(
J
),
but
did
not
affect
the
number
of
sleep
bouts
(
G
)
.
Pre
-
HS
and
Post
-
HS
quantify
data
during
the
entire
day
before
and
entire
night
after
heat
shock
(indicated
by
yellow
bar),
respectively
.
(
K
-
Q
)
Larvae
were
entrained
in
14
:
10
hour
light
:
dark
cycles
for
4
days,
and
then
transferred
to
constant
dark
after
the
fourth
night
of
development
.
Heat
shock
-
induced
overexpression
of
zebrafish
NPY
during
the
subjective
day
resulted
in
decreased
locomotor
activity
(
K,L
)
and
increased
sleep
(
M,N
)
during
the
remainder
of
the
subjective
day
.
NPY
overexpression
increased
the
number
of
sleep
bouts
(
O
)
and
decreased
the
length
of
wake
bouts
(
Q
),
but
had
no
effect
on
the
length
of
sleep
bouts
(
P
)
.
Pre
-
HS
and
Post
-
HS
quantify
data
during
the
entire
subjective
day
before
and
after
heat
shock
(indicated
by
yellow
bar),
respectively
.
Mean
±
SEM
from
3
(
A
-
J
)
and
2
(
K
-
Q
)
experiments
are
shown
.
n=number
of
animals
.
****
P
<
0
.
0001
by
two
-
tailed
Student’s
t
test
.
0
2
4
6
8
10
12
0
10
20
30
40
50
*
**
C
D
0
50
100
150
200
250
300
350
0
10
20
30
40
Day 5
Night 5
+/+
+/
-
-
/
-
+/+
+/
-
-
/
-
A
B
*
0
1
2
3
4
*
**
0
2
4
6
8
10
E
F
0
1
2
3
4
0
1
2
3
4
5
6
G
H
0
20
40
60
80
100
120
*
0
1
2
3
4
I
J
Activity (s/h)
Sleep bouts/h
Sleep (min/h)
Sleep bout length (min)
Wake bout length (min)
+/+
+/
-
-
/
-
+/+
+/
-
-
/
-
+/+
+/
-
-
/
-
+/+
+/
-
-
/
-
+/+
+/
-
-
/
-
+/+
+/
-
-
/
-
+/+
+/
-
-
/
-
+/+
+/
-
-
/
-
Day 5
Night 5
npy
+/+ (
n=48)
npy
+/
-
(n=113)
npy
-
/
-
(n=44)
Figure
S
3
.
Loss
of
npy
reduces
daytime
sleep
in
animals
raised
in
LD
before
transition
to
LL
.
Related
to
Figure
3
.
Larvae
were
entrained
in
14
:
10
hour
LD
cycles
for
4
days
and
then
behaviorally
monitored
for
one
LD
cycle
before
transferring
to
LL
.
During
the
one
LD
cycle,
npy
-
/
-
larvae
were
more
active
(
A
),
and
slept
less
(
C
),
than
their
npy
+/+
and
npy
+/
-
siblings
during
the
day
.
These
phenotypes
were
due
to
fewer
sleep
bouts
(
E
),
and
longer
wake
bouts
(
I
)
.
Mean
±
SEM
from
3
experiments
is
shown
.
n=number
of
animals
.
*
P
<
0
.
05
,
**
P
<
0
.
01
by
one
-
way
ANOVA
with
Holm
-
Sidak
test
.
0
200
400
600
800
0
5
10
15
20
25
30
35
40
45
0
200
400
600
0
10
20
30
40
50
0
1
2
3
4
5
6
0
5
10
15
20
25
30
Day 6
Night 6
Day 7
Night 7
Day 6
Night 6
Day 7
Night 7
A
D
E
F
B
C
Day
Night
WT + MTZ (n=56)
Tg
(npy:kalta4)
+ MTZ (
n=44)
Tg
(
uas:nfsb
-
mcherry
)
+ MTZ (
n=32)
Activity (s/h)
Sleep (min/h)
Activity (s/h)
Sleep (min/h)
Figure
S
4
.
The
npy
:
kalta
4
and
uas
:
nfsb
-
mcherry
transgenes
do
not
affect
sleep/wake
behaviors
.
Related
to
Figure
4
.
Tg
(npy
:
kalta
4
)
,
Tg
(
uas
:
nfsb
-
mcherry
)
and
WT
sibling
larvae
were
treated
with
10
mM
MTZ
for
48
hrs
(from
3
dpf
to
5
dpf
),
and
their
behavior
was
monitored
from
the
morning
of
6
dpf
until
the
morning
of
8
dpf
.
Mean
±
SEM
from
3
experiments
is
shown
.
n=number
of
animals
.
No
significant
difference
was
observed
among
the
three
genotypes
in
their
activity
(
A
-
C
)
or
sleep
(
D
-
F
)
(
P
>
0
.
05
by
one
-
way
ANOVA
with
Holm
-
Sidak
test)
.
npy
merge
kalta4
npy
merge
kalta4
npy
merge
Telencephalon
Posterior
tuberculum
Torus
semicircularis
Tectum
kalta4
Olfactory bulb
Preoptic area
Intermediate
lateral
Hypothalamus
Pretectum
Locus
coeruleus
Medial
rhombomere
Subpallium
a
p
B
C
mCherry
TUNEL
merge
B
’
B
’’
B
’’’
mCherry
TUNEL
merge
C
’
C
’’
C
’’’
MTZ
MTZ
DMSO
mCherry
TUNEL
merge
D
’
D
’’
D
’’’
D
A
Caudal medial
Hypothalamus
Figure
S
5
.
Specific
expression
of
kalta
4
in
npy
-
expressing
neurons
and
TUNEL
labeling
of
apoptotic
cells
.
Related
to
Figure
4
.
(
A
)
Double
FISH
showing
kalta
4
and
npy
co
-
expression
using
probes
specific
for
kalta
4
and
npy
in
different
populations
of
npy
-
expressing
neurons
.
(
B
-
D
)
Tg
(npy
:
kalta
4
)
;
Tg
(
uas
:
nfsb
-
mcherry
)
animals
were
treated
with
DMSO
(
B
)
or
10
mM
MTZ
(
C,D
)
from
72
-
90
hpf
,
and
then
fixed
and
processed
for
TUNEL
.
TUNEL
labeling
was
observed
in
npy
-
expressing
neurons
of
animals
treated
with
MTZ
(
C,D
),
but
not
in
animals
treated
with
DMSO
(
B
)
.
Note
that
mCherry
fluorescence
is
weaker
in
MTZ
-
treated
animals
because
the
neurons
are
undergoing
apoptosis
.
Leftmost
panels
show
schematic
brain
diagrams
with
npy
expression
domains
colored
as
in
Figure
S
1
,
and
boxes
indicate
exemplar
regions
shown
in
the
fluorescent
images
.
a,
anterior
;
p,
posterior
.
Scale
bar
:
10
μm
.
0
10
20
30
40
50
60
n.s.
n.s.
****
0
100
200
300
400
0
100
200
300
400
0
10
20
30
40
50
60
Day 5
Night 5
Day 5
Night 5
A
B
C
D
Pre
-
HS Post
-
HS
Pre
-
HS Post
-
HS
*
****
****
****
****
DMSO (n=73)
hsp:npy
+ DMSO (n=63)
Prazosin
(
n=61)
hsp:npy
+
Prazosin
(
n=69)
Activity (s/h)
Sleep (min/h)
Activity (s/h)
Sleep (min/h)
Figure
S
6
.
The
sedating
effects
of
NPY
overexpression
and
inhibition
of
NE
signaling
are
not
additive
.
Related
to
Figure
6
.
Tg
(
hsp
:
npy
)
animals
and
their
WT
siblings
were
treated
with
100
μM
prazosin
or
DMSO
vehicle
control,
and
then
heat
shocked
(yellow
bars)
during
the
fifth
day
of
development
.
Prazosin
-
treated
animals
were
less
active
(
A,B
)
and
slept
more
(
C,D
)
during
the
day
before
and
after
heat
shock
.
DMSO
-
treated
Tg
(
hsp
:
npy
)
animals
were
less
active
and
slept
more
than
their
DMSO
-
treated
WT
siblings
during
the
day
after
heat
shock
.
NPY
overexpression
did
not
further
decrease
locomotor
activity
or
increase
sleep
in
prazosin
-
treated
Tg
(
hsp
:
npy
)
animals
.
Pre
-
HS
and
Post
-
HS
quantify
data
for
day
5
before
and
after
heat
shock,
respectively
.
Mean
±
SEM
from
3
experiments
is
shown
.
n=number
of
animals
.
n
.
s
.
=not
significant,
*
P
<
0
.
05
,
****
P
<
0
.
0001
by
two
-
way
ANOVA
with
Holm
-
Sidak
test
.
0
10
20
30
40
50
0
50
100
150
200
250
300
350
400
450
0
100
200
300
400
500
*
****
****
0
10
20
30
40
50
60
*
****
0
10
20
30
40
0
100
200
300
400
500
0
10
20
30
40
50
Day 5
Night 5
Day 5
Night 5
0
100
200
300
400
Pre
-
HS
Post
-
HS
Activity (s/h)
Sleep (min/h)
A
B
C
D
Pre melatonin
Post melatonin
****
Activity (s/h)
Sleep (min/h)
E
F
G
H
****
****
****
*
DMSO (n=95)
DMSO + Melatonin (n=92)
Prazosin
(
n=87)
Prazosin
+
Melatonin
(
n=92)
WT (n=54)
hsp:npy
(
n=42)
Melatonin
(
n=50)
h
sp:npy
+
Melatonin
(
n=46)
Day 5
Night 5
Day 5
Night 5
Pre
-
HS
Post
-
HS
Pre melatonin
Post melatonin
*
*
****
M
elatonin
M
elatonin
M
elatonin
M
elatonin
Activity (s/h)
Sleep (min/h)
Activity (s/h)
Sleep (min/h)
Figure
S
7
.
Melatonin
treatment
enhances
sleep
induced
by
overexpression
of
NPY
or
by
treatment
with
prazosin
.
Related
to
Figure
6
.
(
A
-
D
)
Tg
(
hsp
:
npy
)
animals
and
their
WT
siblings
were
heat
shocked
(yellow
bars)
during
the
fifth
day
of
development,
after
which
20
μM
melatonin
was
added
(arrows)
.
Tg
(
hsp
:
npy
)
animals
were
less
active
(
A,B
)
and
slept
more
(
C,D
)
than
their
WT
siblings
after
heat
shock
.
Both
Tg
(
hsp
:
npy
)
and
their
WT
siblings
showed
a
further
decrease
in
activity
(
A,B
)
and
increase
in
sleep
(
C,D
)
after
addition
of
melatonin
.
Pre
-
HS
and
Post
-
HS
quantify
data
for
day
5
before
and
after
heat
shock,
respectively
.
(
E
-
H
)
WT
animals
were
treated
with
either
100
μM
prazosin
or
DMSO
vehicle
control
starting
at
4
dpf
,
and
20
μM
melatonin
was
added
during
the
fifth
day
of
development
(arrows)
.
Prazosin
-
treated
animals
were
less
active
(
E,F
)
and
slept
more
(
G,H
)
than
DMSO
-
treated
siblings
.
Both
prazosin
-
and
DMSO
-
treated
animals
showed
a
further
decrease
in
activity
(
E,F
)
and
increase
in
sleep
(
G,H
)
following
addition
of
melatonin
.
Pre
melatonin
and
Post
melatonin
quantify
data
for
day
5
before
and
after
addition
of
melatonin,
respectively
.
Mean
±
SEM
for
2
(
A
-
D
)
and
3
(
E
-
H
)
experiments
are
shown
.
n=number
of
animals
.
*
P
<
0
.
05
,
****
P
<
0
.
0001
by
two
-
way
ANOVA
with
Holm
-
Sidak
test
.
Symbol
Brain region
%
k
alta4
-
expressing
neurons co
-
expressing
npy
%
npy
neurons co
-
expressing
kalta4
A
Olfactory
bulb
100.00
±
0.00
91.67
±
6.81
B
Telencephalon
98.92
±
1.07
95.10
±
1.97
C
Preoptic area
97.07
±
1.61
88.71
±
2.53
D
Posterior
tuberculum
98.72
±
1.28
89.31
±
3.31
E
Intermediate lateral
Hypothalamus
100.00
±
0.00
94.44
±
5.56
F
Caudal medial
Hypothalamus
100.00
±
0.00
88.89
±
11.12
G
Pretectum
98.04
±
1.96
83.06
±
1.17
H
Torus
semicircularis
95.77
±
2.41
88.34
±
4.52
I
Tectum
98.03
±
0.98
89.09
±
1.04
J
Locus
coeruleus
100.00
±
0.00
88.89
±
11.12
K
Medial
rhombomere
96.67
±
3.33
82.22
±
1.11
L
Subpallium
92.86
±
5.83
86.61
±
0.72
Table
S
1
.
Specificity
of
npy
:
KalTA
4
transgene
.
Related
to
Figure
4
.
The
specificity
of
kalta
4
expression
in
Tg
(npy
:
kalta
4
)
animals
at
5
dpf
was
assayed
by
double
FISH
using
probes
specific
for
kalta
4
and
npy
and
quantified
in
each
sub
-
population
of
npy
-
expressing
neurons
.
Mean
±
SEM
percentage
of
co
-
expression
in
4
animals
is
shown
.
Mutant
System Affected
histidine decarboxylase
no histamine
hypocretin
receptor
no
hypocretin
signaling
corticotropin
releasing hormone a
no
crha
corticotropin
releasing hormone b
no crhb
arylalkylamine
N
-
acetyltransferase
2
no melatonin
Table
S
2
.
Mutants
tested
for
effects
on
NPY
overexpression
-
induced
sleep
.
Related
to
Figure
6
.
The
effect
of
NPY
overexpression
on
locomotor
activity
and
sleep
was
compared
in
animals
that
were
homozygous
mutant
for
each
of
the
indicated
genes
to
their
WT
siblings
.
In
each
case
an
additive
phenotype
was
observed
.
Genotyping Primers
Primer Name
Primer Sequence
hsp:npy
genotype F
CCGCCACCATGAATCCA
hsp:npy
genotype R
GGTTTGTCCAAACTCATCAATGT
npy
mutant genotype F
ATAAATTGCGCATCAGCACA
npy
mutant genotype R
TGAGGAAGAATTTGAGACTACGC
npy:kalta4
genotype F
CGCTATCATTTATAGATTTTTGCAC
npy:kalta4
genotype R
AGTAGCGACACTCCCAGTTG
Primers for Riboprobe Synthesis
Primer Name
Primer Sequence
npy
riboprobe F
CCACAGAGCAAGAATTCCAA
npy
riboprobe R
CAGTCATTATTGTTCTCCTTTGC
adcyap1a
riboprobe F
ATGATTACGAGCAGCAAAACGACTC
adcyap1a
riboprobe R
TCACAAAGCCGGGAATTCAG
npy1r
riboprobe F
CTGACCGACAGCAGTGTGTT
npy1r
riboprobe R
CCGGTGGTGTAGGTGAGTTT
npy2r
riboprobe F
CGCAATTTACACACGGTGAC
npy2r
riboprobe R
TCCCTTACTGCCTCACTGCT
npy2rl
riboprobe F
GGCTTGTGTGGATGGATGTA
npy2rl
riboprobe R
TGTCGAGGTGGTAAACGATG
npy4r
riboprobe F
GTCCTAGGGGTGTGCATGTC
npy4r
riboprobe R
AATAGCAACAAGCTGGTGGTG
npy7r
riboprobe F
AAGAGACCAGCCTGGGAAAT
npy7r
riboprobe R
AAACTGCGAAGACCACGACT
npy8ar
riboprobe F
CCAGAAATCATGGGTGGAGT
npy8ar
riboprobe R
GCAAATGCAACCACAATCAC
npy8br
riboprobe F
CGAAGCGTTATGCAAAGTGA
npy8br
riboprobe R
TTGCTCAAGATGGAGCCTTT
Primers for BAC transgenesis
Primer Name
Primer Sequence
Homology arm F
AACAATAAATTGCGCATCAGCACAAACACGTTTGCTTTGTTTAATTGCAG
Homology arm R
AAGAGAAACGCGCACGCTGCCCAGCTCATCCACATCTTCATGTTTGGATT
pIndigoBAC_HA1_iTol2_
F
TTCTCTGTTTTTGTCCGTGGAATGAACAATGGAAGTCCGAGCTCATCGCTC
CCTGCTCGAGCCGGGCCCAAGTG
pIndigoBAC_HA1_iTol2_
R
AGCCCCGACACCCGCCAACACCCGCTGACGCGAACCCCTTGCGGCCGCA
TATTATGATCCTCTAGATCAGATC
Table S3. List of primers used in this
study.
Related to STAR Methods.