Neuron
Supplemental Information
Melatonin Is Required
for the Circadian Regulation of Sleep
Avni V.
Gandhi
,
Eric
Mosser
,
Grigorios
Oikonomou
,
and
David A.
Prober
G
!
Sleep Bout
Length (min)
!
1
!
2
!
3
!
4
!
5
!
0
!
L
!
**
!
**
!
**
!
**
!
0
!
2
!
4
!
6
!
8
!
10
!
Night 5
!
Night 6
!
K
!
Wake Bout
!
Length (min)
!
0
!
50
!
100
!
150
!
Day 5
!
Day 6
!
I
!
0
!
30
!
60
!
90
!
120
!
150
!
Sleep Latency
!
(min)
!
Day 5
!
Day 6
!
J
!
**
!
**
!
**
!
**
!
0
!
10
!
20
!
30
!
Night 5
!
Night 6
!
Figure S1
!
H
!
**
!
**
!
**
!
**
!
0
!
1
!
2
!
3
!
4
!
5
!
aanat2
-/-
!
aanat2
+/-
!
aanat2
+/+
!
n=64
!
n=112
!
n=63
!
1
Night
!
Day
!
1.5
!
1.2
!
0.9
!
0.6
!
0
!
***
!
0.3
!
Melatonin (pg/larva)
!
aanat2-/-
!
WT
!
n=3
!
n=3
!
B
!
D
0
!
100
!
300
!
200
!
400
!
aanat2-/-
!
aanat2+/-
!
5-HT Pixel Intensity
!
(x10
6
)/Pineal (a.u.)
!
Night
!
Day
!
*
!
*
!
***
!
Day
!
Night
!
aanat2
+/-
!
aanat2
-/-
!
5-HT
!
C
A
!
MS
T
Q
S
T
H
P
-
-
-
L
K
P
E
A
P
R
L
P
P
G
I
P
E
S
P
S
C
QR
R
H
T
L
P
A
S
E
F
R
C
L
T
P
E
D
A
V
S
A
F
E
I
E
R
E
A
F
I
S
V
L
G
V
C
P
L
Y
L
67
Aanat_Hs
M
-
-
L
N
I
N
S
-
-
-
L
K
P
E
A
L
H
L
P
L
G
T
S
E
F
L
G
C
QR
R
H
T
L
P
A
S
E
F
R
C
L
T
P
E
D
A
T
S
A
F
E
I
E
R
E
A
F
I
S
V
S
G
T
C
P
L
Y
L
65
Aanat_Mm
MS
T
P
S
V
H
C
-
-
-
L
K
P
S
P
L
H
L
P
S
G
I
P
G
S
P
G
R
QR
R
H
T
L
P
A
N
E
F
R
C
L
T
P
E
D
A
A
GV
F
E
I
E
R
E
A
F
I
S
V
S
G
N
C
P
L
N
L
67
Aanat_Oa
M
MA
P
QV
V
S
S
P
F
L
K
P
F
F
L
K
T
P
I
S
V
S
S
-
P
R
R
QR
R
H
T
L
P
A
S
E
F
R
N
L
T
P
Q
D
A
I
S
V
F
E
I
E
R
E
A
F
I
S
V
S
G
E
C
P
L
T
L
69
Aanat2_Dr
M
MA
P
QV
V
S
S
P
F
L
K
P
F
F
L
K
T
P
I
S
V
S
S
-
P
R
R
QR
R
H
T
L
P
A
S
E
F
R
N
L
T
P
Q
D
A
I
S
V
F
E
I
E
R
E
A
F
I
S
V
S
G
E
C
P
L
T
L
69
Aanat2_Dr d10
M
MA
P
QV
V
S
S
P
F
L
K
P
F
F
L
K
T
P
I
S
V
S
S
-
P
R
R
QR
R
H
T
L
P
A
S
E
F
R
N
L
T
P
Q
D
A
I
S
V
F
E
I
E
R
E
A
F
I
S
V
S
G
E
C
P
L
T
L
69
Aanat2_Dr d11
M
-
-
S
V
V
S
A
L
P
F
L
K
P
I
H
A
R
V
P
I
S
-
-
-
-
P
G
R
QR
R
H
T
L
P
A
S
E
F
R
S
L
N
A
E
D
A
I
S
V
F
E
I
E
R
E
A
F
I
S
V
S
G
E
C
P
L
H
L
64
Aanat1_Dr
M
-
-
S
V
V
S
A
L
P
F
L
K
P
I
H
A
R
V
P
I
S
-
-
-
-
P
G
R
S
T
P
R
T
QS
V
C
S
K
L
R
E
R
L
L
F
QS
L
E
N
V
L
F
T
W
M
K
S
G
T
F
S
L
S
V
L
S
F
64
Aanat1_Dr d40
D
E
I
R
H
F
L
T
L
C
P
E
L
S
L
G
W
F
E
E
GC
L
V
A
F
I
I
GS
L
W
D
K
E
R
L
MQE
S
L
T
L
H
R
S
GG
H
I
A
H
L
H
V
L
A
V
H
R
A
F
R
QQG
R
GP
137
Aanat_Hs
D
E
I
R
H
F
L
T
L
C
P
E
L
S
L
G
W
F
E
E
GC
L
V
A
F
I
I
GS
L
W
D
K
E
R
L
T
QE
S
L
T
L
H
R
P
GG
R
T
A
H
L
H
V
L
A
V
H
R
T
F
R
QQG
K
G
S
135
Aanat_Mm
D
E
V
Q
H
F
L
T
L
C
P
E
L
S
L
G
W
F
V
E
G
R
L
V
A
F
I
I
GS
L
W
D
E
E
R
L
T
QE
S
L
A
L
H
R
P
R
G
H
S
A
H
L
H
A
L
A
V
H
R
S
F
R
QQG
K
G
S
137
Aanat_Oa
D
E
V
L
V
F
L
GQ
C
P
E
L
S
M
G
W
F
E
E
G
Q
L
V
A
F
I
I
GS
G
W
D
K
E
K
L
E
QE
A
MS
T
H
V
P
D
S
P
T
V
H
I
H
V
L
S
V
H
R
H
C
R
QQG
K
G
S
139
Aanat2_Dr
D
E
V
L
V
F
L
GQ
C
P
E
L
S
M
G
W
F
E
E
G
Q
L
V
A
F
I
I
G
I
K
R
N
102
Aanat2_Dr d10
D
E
V
L
V
F
L
GQ
C
P
E
L
S
M
G
W
F
E
E
G
Q
L
V
A
F
I
I
G
98
Aanat2_Dr d11
D
E
V
R
H
F
L
T
L
C
P
E
L
S
L
G
W
F
E
Q
G
R
L
V
A
F
I
I
GS
L
W
D
Q
D
R
L
T
A
D
A
L
T
L
H
K
P
H
G
T
T
V
H
I
H
V
L
A
V
H
R
T
F
R
QQG
K
G
S
134
Aanat1_Dr
L
W
A
G
L
N
R
E
GL
S
L
L
S
78
Aanat1_Dr d40
I
L
L
WR
Y
L
H
H
L
GS
Q
P
A
V
R
R
A
A
L
M
C
E
D
A
L
V
P
F
Y
E
R
F
S
F
H
A
V
G
P
C
A
I
T
V
GS
L
T
F
M
E
L
H
C
S
L
R
GH
P
F
L
R
R
N
S
GC
207
Aanat_Hs
V
L
L
WR
Y
L
H
H
L
GS
Q
P
A
V
R
R
A
V
L
M
C
E
D
A
L
V
P
F
Y
E
K
F
G
F
Q
A
V
G
P
C
A
I
T
V
GS
L
T
F
T
E
L
Q
C
S
L
R
C
H
A
F
L
R
R
N
S
GC
205
Aanat_Mm
V
L
L
WR
Y
L
H
H
V
G
A
Q
P
A
V
R
R
A
V
L
M
C
E
D
A
L
V
P
F
Y
Q
R
F
G
F
H
P
A
G
P
C
A
I
V
V
GS
L
T
F
T
E
M
H
C
S
L
R
GH
A
A
L
R
R
N
S
D
R
207
Aanat_Oa
I
L
L
WR
Y
L
QY
L
R
C
L
P
GL
R
R
A
L
L
V
C
E
E
F
L
V
P
F
Y
QK
A
G
F
K
E
K
G
P
S
A
I
S
V
A
A
L
T
F
T
E
ME
Y
Q
L
G
G
L
A
Y
A
R
R
N
S
GC
209
Aanat2_Dr
102
Aanat2_Dr d10
98
Aanat2_Dr d11
I
L
L
WR
Y
L
QY
L
R
C
L
P
Y
V
R
R
A
V
L
M
C
E
D
F
L
V
P
F
Y
QK
S
G
F
K
GQ
G
P
S
E
I
I
V
GS
L
T
F
I
E
MY
Y
P
I
R
GH
A
F
V
R
R
N
S
GC
204
Aanat1_Dr
78
Aanat1_Dr d40
Aanat_Hs
!
Aanat_Mm
!
Aanat_Oa
!
Aanat2_Dr
!
Aanat2_Dr d10
!
Aanat2_Dr d11
!
Aanat1_Dr
!
Aanat1_Dr d40
!
Aanat_Hs
!
Aanat_Mm
!
Aanat_Oa
!
Aanat2_Dr
!
Aanat2_Dr d10
!
Aanat2_Dr d11
!
Aanat1_Dr
!
Aanat1_Dr d40
!
Aanat_Hs
!
Aanat_Mm
!
Aanat_Oa
!
Aanat2_Dr
!
!
!
Aanat1_Dr
!
0
!
0.1
!
0.2
!
0.3
!
0.4
!
aanat2
-/-
!
(n=37)
!
aanat2
+/-
!
(n=80)
!
aanat2
+/+
!
(n=38)
!
Response Probability
!
When Asleep
!
N
!
Stimulus Intensity
!
aanat2
-/-
!
aanat2
+/-
!
aanat2
+/+
!
n=26
!
n=41
!
n=24
!
Response Probability
!
M
!
0
!
0.1
!
0.3
!
0.2
!
0.4
!
1
!
10
!
100
!
aanat2
-/-
!
aanat2
+/-
!
aanat2
+/+
!
n=26
!
n=41
!
n=24
!
0
!
2
!
4
!
Sleep Bouts/hr
!
6
!
0
!
**
!
**
!
2
!
4
!
6
!
8
!
10
!
E
!
F
!
Figure S2
!
25
30
35
40
45
50
55
60
65
70
0
10
20
30
40
Zeitgeber Time (Hours)
Average Activity (seconds per 10 minutes
WT n=40
aanat2
−
CFP
−
NTR n=52
Activity (sec/10 min)
!
0
!
10
!
20
!
30
!
40
!
F
!
Sleep (min/10min)
!
0
!
2
!
4
!
6
!
8
!
Night 5
G
!
25
30
35
40
45
50
55
60
65
70
0
2
4
6
8
Zeitgeber Time (Hours)
Average Sleep (Bouts/10 minutes
WT n=40
aanat2
−
CFP
−
NTR n=52
NTR+ n=52
!
NTR–
!
n=40
!
Day 7
Night 6
Day 6
Night 7
Day 7
Night 6
Day 6
Night 7
DMSO
!
MTZ
!
Night 5
!
Night 6
!
Night 7
!
D
!
0
!
40
!
80
!
120
!
160
!
200
!
Night 5
!
Night 6
!
Night 7
!
MTZ n=3
!
DMSO n=3
!
**
!
**
!
E
!
CFP+ Cell Number
!
expose
larvae with
bright CFP to
15 mM MTZ
!
wash
out MTZ
!
add 15
mM MTZ
!
wash out MTZ,
test larvae with
weak CFP in
videotracker
!
Day 0
!
Day 1
!
Day 2
!
Day 3
!
Day 4
!
Day 5
!
Day 6
!
Day 7
!
Day 8
!
end
videotracker
experiment
!
C
!
aanat2
!
cfp-ntr
!
aanat2
!
cfp-ntr
!
A
!
A’
!
A”
!
B
!
B’
!
B”
!
aanat2
!
cfp-ntr
!
aanat2
!
cfp-ntr
!
Day 6
!
Day 7
!
3
!
2
!
1
!
0
!
Activity (sec/min)
!
H
!
Sleep (min/hr)
!
Night 6
!
Night 7
!
**
!
1.0
!
0.8
!
0.6
!
0.4
!
0.2
!
0
!
I
!
Night 6
!
Night 7
!
30
!
20
!
10
!
0
!
**
!
40
!
30
!
20
!
10
!
0
!
J
!
K
!
NTR+ n=159
!
NTR–
!
n=94
!
Day 6
Day 7
2
Figure S3
!
0
!
1
!
2
!
3
!
4
!
6 pm
!
12 am
!
6 am
!
12 pm
!
per1b
intensity
(x10
9
) (a.u.)
!
Q
!
DD
!
LD
!
DD
!
LD
!
12 am
!
6 pm
!
12 pm
!
6 am
!
per1b
!
DAPI
!
per1b
!
DAPI
!
I'
!
K'
!
M'
!
O'
!
I
!
M
!
O
!
I''
!
K''
!
M''
!
O''
!
J
!
J'
!
L
!
L'
!
N
!
N'
!
P
!
P'
!
J''
!
L''
!
N''
!
P''
!
I'''
!
K'''
!
M'''
!
O'''
!
J'''
!
L'''
!
N'''
!
P'''
!
aanat2
-/-
!
aanat2
+/-
!
aanat2
+/+
!
n=54
!
n=124
!
n=74
!
Sleep Bout
!
Length (min)
!
**
!
**
!
**
!
**
!
0
!
1
!
2
!
3
!
4
!
**
!
**
!
**
!
**
!
1
!
2
!
3
!
4
!
0
!
D
!
C
!
E
!
F
!
10
!
**
!
**
!
**
!
**
!
0
!
2
!
4
!
8
!
6
!
134-144
!
hpf
!
158-168
!
hpf
!
Wake Bout
!
Length (min)
!
**
!
**
!
**
!
**
!
0
!
4
!
8
!
12
!
120-134
!
hpf
!
144-158
!
hpf
!
H
!
6pm
!
12am
!
6am
!
12pm
!
6pm
!
arntl1a
LD WT
!
arntl1a
DD WT
!
0
!
1
!
2
!
3
!
4
!
5
!
G
!
Normalized
!
Expression (a.u.)
!
6pm
!
12am
!
6am
!
12pm
!
6pm
!
0
!
10
!
20
!
30
!
40
!
50
!
per1b
LD WT
!
per1b
DD WT
!
Normalized
!
Expression (a.u.)
!
**
!
**
!
*
!
A
!
0
!
4
!
8
!
12
!
Sleep Bouts/hr
!
**
!
**
!
*
!
**
!
B
!
0
!
4
!
8
!
12
!
3
K
!