Current Biology Supplemental Information · 2018. 4. 18. · Current Biology Supplemental...
Transcript of Current Biology Supplemental Information · 2018. 4. 18. · Current Biology Supplemental...
Current Biology Supplemental Information
Motor Behavior Mediated by Continuously Generated Dopaminergic Neurons in the Zebrafish Hypothalamus Recovers after Cell Ablation Adam D. McPherson, Joshua P. Barrios, Sasha J. Luks-Morgan, John P. Manfredi, Joshua L. Bonkowsky, Adam D. Douglass, and Richard I. Dorsky
th2:Gal4-VP16;UAS:NTR-mCherry
th2:NTR-EGFP
Swim bouts/sec % Time spent swimming
DMSOMTZ
nsns
DMSOMTZ
2
4
6
8
10
0.5
1.0
1.5
2.0
2.5
3.0
3.5
A
5 10 15 20 25Time (s)
20
40
60
80
100
LEDStimulus
30 35
5 10 15 20 25Time (s)
30 35
th2:Gal4-VP16;UAS:ChR2
20
40
60
Time (s)
5 10 15 20 25Time (s)
20
40
60
80
100
Tria
l #
WT controlsLED
Stimulus
30 35
5 10 15 20 25
Time (s)
30
20
40
60
Bou
ts/2
00 m
s
35
Tria
l #B
outs
/200
ms
B
D
0 100 200 300 400 500 600 700Time (s)
20
40
60
80
100
dMax
(AU
)
DMSO treated
0 100 200 300 400 500 600 700Time (s)
20
40
60
80
100
dMax
(AU
)
Mtz treated
C
Chr2-YFP+
Chr2-YFP+
Chr2-YFP-
Chr2-YFP-Stimulated Stimulated
Unstimulated Unstimulated
cfos
cfos
cfos
cfos
H
DMSO
MTZ
Et(Gal4-VP16)zc1066a;UAS;NTR-mCherry
Et(Gal4-VP16)zc1066a;UAS;NTR-mCherry
E
F Gth2:Gal4-VP16;UAS:ChR2-YFP
0.2
0.40.6
0.8
1.0
1.2
1.4
ChR2Control
1.6 p=0.048
Swim distance stim/prestimI
Control ChR2
J
3secprestim
3secstim
Control ChR2
time (s)
Velocity (mm/sec)
3secprestim
3secstim
Track Track
5mm 5mm
Velocity (mm/sec)
4
8
12
16
20
24
28
32
36
40
4
8
12
16
20
24
28
32
36
40
time (s)1.25 2.5 3.75 5 6.25 7.5 1.25 2.5 3.75 5 6.25 7.5
Fig. S1
0 100 200 300 400 500 600 700Time (s)
20
406080
100
dMax
(AG
U)
DMSO-->DMSO
120140
MTZ-->DMSO
0 100 200 300 400 500 600 700Time (s)
20
406080
100
dMax
(AG
U)
120140
MTZ-->MTZ
0 100 200 300 400 500 600 700Time (s)
20
406080
100
dMax
(AG
U)
120140
DMSO-->MTZ
0 100 200 300 400 500 600 700Time (s)
20
406080
100
dMax
(AG
U)
120140
800
A
B
C
D
Fig. S2
Figure S1 (Refers to Main Figure 3):
(A-B) Live 7 dpf larvae expressing th2:Gal4-VP16;UAS;NTR-mCherry (A) and
th2:NTR-EGFP (B). (C) Representative behavioral plots of single 8 dpf larvae from same
cohort analyzed in Figure 3. Each red arrowhead indicates a swim bout, defined as a
superthreshold spike in the running frame subtraction trace, which is plotted as a black
line. Y-axis indicates the maximum pixel displacement for each frame, in arbitrary units.
(D-E) Effects of ablation of hypothalamic radial glia on behavior in 8 dpf larvae. Larvae
expressing Et(Gal4-VP16)zc1066a; UAS;NTR-mCherry were treated with 0.5% DMSO or
5mM MTZ from 5-7 dpf as described for th2+ cell ablations. Error bars=SEM, n=6
larvae for each condition. (F) Live 7 dpf larva expressing th2:Gal4-VP16;UAS;ChR2-
YFP. (G) Expression of cfos in the posterior recess of 7 dpf larvae that were unstimulated,
or stimulated for 30 seconds with 470nm light once per minute for 10 minutes. The
experiment was performed twice on groups of 10 larvae for each condition, processed
simultaneously. While there was variability in expression between individual animals, in
blind scoring all the stimulated Chr2+ larvae showed higher cfos staining than larvae in
any other group. (H) Raster plots and cumulative counts of swim bouts before, during
(blue shading), and after light pulse summing all trials for one dish of 10 larvae
quantified in Figure 3K. Histogram bins are 200 ms wide. All fish exhibited a visually
mediated transient reduction in swimming at light offset. (I) Tracking analysis reveals
that fish expressing ChR2 exhibit an increased cumulative displacement during the 3s of
optogenetic stimulation versus the 3s baseline condition. Error bars=SEM, n=24 control
larvae, n=25 Chr2+ larvae. (J) Increased displacement reflects an increase in swim bout
frequency. In the left panels, swim trajectories are color-coded to indicate the baseline
(red) and stimulus (blue) periods. Right panels depict the instantaneous velocities vs. time
for each trial.
Figure S2 (Refers to Main Figure 4):
(A-D) Representative spike plots of a group of three juvenile fish from same cohort
analyzed in Figure 4. Each red arrowhead indicates a swim bout, defined as a
superthreshold spike in the running frame subtraction trace, which is plotted as a black
line.