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

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Chr2-YFP+

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Chr2-YFP-Stimulated Stimulated

Unstimulated Unstimulated

cfos

cfos

cfos

cfos

H

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MTZ

Et(Gal4-VP16)zc1066a;UAS;NTR-mCherry

Et(Gal4-VP16)zc1066a;UAS;NTR-mCherry

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F Gth2:Gal4-VP16;UAS:ChR2-YFP

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ChR2Control

1.6 p=0.048

Swim distance stim/prestimI

Control ChR2

J

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Control ChR2

time (s)

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Track Track

5mm 5mm

Velocity (mm/sec)

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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

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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.