Supplementary Table 1. Similar crawling speeds were measured using the DIAS and the MWT acquisition systems.

Acquisition System / AMPH (mM) a / Speed (cm/sec) ± SEM (n) b / Larva(e)/plate / % Increase in Speed c / MWW d
DIAS / 0 / 0.084 ± 0.002 (49) / 1 / 19% (***) / U = 613, P <0.001
60 / 0.100 ± 0.002 (54)
MWT / 0 / 0.090 ± 0.003 (24) / 2 to 4 / 22% (***) / U = 45, P <0.001
60 / 0.110 ± 0.002 (17)

a AMPH was dissolved in water (vehicle).

b (n) denotes the number of plates assayed.

c The increase in speed of larvae fed AMPH as compared to larvae fed vehicle.

d Significance was determined using the Mann-Whitney-Wilcoxon (MWW) test.

Supplementary Table 2. AMPH-induced hyperlocomotion of Drosophila larvae is dependent on TH but not on Tdc2.

Genotype a / AMPH (mM) b / Average Speed (cm/sec) ± SEM (n) c / % Increase in Speed d / MWW e
Tdc2R054 / 0 / 0.020 ± 0.0009 (31) / 65% / U = 112, P < 0.001
60 / 0.033 ± 0.002 (32)
w1118 / 0 / 0.080 ± 0.002 (51) / 25% / U = 345, P < 0.001
60 / 0.100 ± 0.002 (42)
TH-GAL4/
UAS-TH-RNAi / 0 / 0.073 ± 0.002 (33) / (NS) / U = 377, P > 0.05
60 / 0.069 ± 0.002 (31)

a TH-GAL4 flies also carried a UAS-Dicer2 transgene.

b AMPH was dissolved in water (vehicle).

c (n) denotes the number of plates assayed, with 2-3 larvae per plate.

d The increase in average speed of larvae fed AMPH as compared to larvae of the same genotype fed vehicle. NS = Non Significant.

e Significance was determined using the Mann-Whitney-Wilcoxon (MWW) test.

Supplementary Table 3. The mianserin-induced decrease in speed of locomotion of Drosophila larvae is dependent on Tdc2 but not on TH.

Genotype a / Mianserin (mM) b / Average Speed (cm/sec) ± SEM (n) c / % Decrease in Speed d / MWW e
Tdc2R054 / 0 / 0.020 ± 0.0009 (31) / (NS) / U = 418, P > 0.05
0.8 / 0.021 ± 0.001 (32)
w1118 / 0 / 0.062 ± 0.002 (44) / 21% / U = 453, P < 0.001
0.8 / 0.049 ± 0.001 (48)
TH-GAL4/
UAS-TH-RNAi / 0 / 0.071 ± 0.002 (37) / 20% / U = 237, P < 0.001
0.8 / 0.057 ± 0.002 (39)

a TH-GAL4 flies also carried a UAS-Dicer2 transgene.

b Mianserin was dissolved in water (vehicle).

c (n) denotes the number of plates assayed, with 2-3 larvae per plate.

d The decrease in average speed of larvae fed mianserin as compared to larvae of the same genotype fed vehicle. NS = Non Significant.

e Significance was determined using the Mann-Whitney-Wilcoxon (MWW) test.

Supplementary Table 4. AMPH-induced hyperlocomotion in the presence of mianserin is also dependent on TH.

Genotype / AMPH (mM) b / Average Speed (cm/sec) ± SEM (n) c / % Increase in Speed d / MWW e
TH-GAL4/+ a / 0 / 0.057 ± 0.001 (79) / 26% / U = 1174, P < 0.001
60 / 0.072 ± 0.001 (82)
TH-GAL4/
UAS-TH-RNAi a / 0 / 0.0575 ± 0.002 (39) / 8% / U = 598, P < 0.05
60 / 0.062 ± 0.002 (42)
UAS-TH-RNAi/+ / 0 / 0.053 ± 0.001 (41) / 36% / U = 139, P < 0.001
60 / 0.072 ± 0.002 (36)
DTHg ; ple / 0 / 0.043 ± 0.001 (25) / 28% / U = 81, P < 0.001
60 / 0.055 ± 0.001 (26)
DTHgFS±; ple / 0 / 0.052 ± 0.002 (24) / (NS) / U = 304, P > 0.05
60 / 0.052 ± 0.002 (29)

a TH-GAL4 flies also carried a UAS-Dicer2 transgene.

b AMPH was dissolved in 0.8 mM mianserin (vehicle).

c (n) denotes the number of plates assayed, with 2-3 larvae per plate.

d The increase in average speed of larvae fed AMPH as compared to larvae of the same genotype fed vehicle. NS = Non Significant.

e Significance was determined using the Mann-Whitney-Wilcoxon (MWW) test.

Supplementary Table 5. AMPH-induced hyperlocomotion in the presence of mianserin is also not dependent on Tdc2.

Genotype / AMPH (mM) a / Average Speed (cm/sec) ± SEM (n) b / % Increase in Speed c / MWW d
w1118 / 0 / 0.054 ± 0.002 (60) / 31% / U = 638, P < 0.001
60 / 0.071 ± 0.002 (59)
Tdc2R054 / 0 / 0.021 ± 0.001 (32) / 43% / U = 166.5, P < 0.001
60 / 0.030 ± 0.001 (33)

a AMPH was dissolved in 0.8 mM mianserin (vehicle).

b (n) denotes the number of plates assayed, with 2-3 larvae per plate.

c The increase in average speed of larvae fed AMPH as compared to larvae of the same genotype fed vehicle.

d Significance was determined using the Mann-Whitney-Wilcoxon (MWW) test.

Supplementary Table 6. RNAi-mediated depletion of dDAT in DA neurons blunts the response to AMPH.

Genotype a / AMPH (mM) b / Average Speed (cm/sec) ± SEM (n) c / % Increase in Speed d / MWW e
UAS-GFP-RNAi/
TH-GAL4 / 0 / 0.076 ± 0.002 (31) / 34% / U = 45.5, P < 0.001
60 / 0.102 ± 0.002 (33)
UAS-dDAT-RNAi/+ ;
TH-GAL4/+ / 0 / 0.072 ± 0.002 (42) / 10% / U = 572, P < 0.05
60 / 0.079 ± 0.002 (40)
UAS-dDAT-RNAi/+ / 0 / 0.071 ± 0.002 (31) / 23% / U = 101, P < 0.001
60 / 0.087 ± 0.002 (29)

a All TH-GAL4 flies also carried a UAS-Dicer2 transgene.

b AMPH was dissolved in 0.8 mM mianserin (vehicle).

c (n) denotes the number of plates assayed, with 2-3 larvae per plate.

d The increase in average speed of larvae fed AMPH as compared to larvae of the same genotype fed vehicle.

e Significance was determined using the Mann-Whitney-Wilcoxon (MWW) test.

Supplementary Table 7. AMPH-induced hyperlocomotion is dependent on DAT and its phosphorylation.

Genotype / AMPH (mM) a / Speed (cm/sec) ± SEM (n) b / % Increase in Speed c / MWW d
dDATfmn / 0 / 0.067 ± 0.002 (30) / (NS) / U = 365, P > 0.05
60 / 0.065 ± 0.002 (30)
dDATfmn;
UAS-hDAT/+ / 0 / 0.070 ± 0.002 (26) / (NS) / U = 240, P > 0.05
60 / 0.075 ± 0.002 (25)
dDATfmn;
TH-GAL4/+ / 0 / 0.070 ± 0.001 (27) / (NS) / U = 263, P > 0.05
60 / 0.073 ± 0.002 (24)
dDATfmn ;
UAS-hDAT/TH-GAL4 / 0 / 0.073 ± 0.002 (35) / 23% / U = 211, P < 0.001
60 / 0.090 ± 0.002 (38)
dDATfmn;
UAS-hDAT-S/A/TH-GAL4 / 0 / 0.068 ± 0.002 (36) / (NS) / U = 417, P > 0.05
60 / 0.064 ± 0.002 (31)
dDATfmn;
UAS-hDAT-S/D/TH-GAL4 / 0 / 0.054 ± 0.002 (34) / 24% / U = 231, P < 0.001
60 / 0.067 ± 0.002 (38)

a AMPH was dissolved in 0.8 mM mianserin (vehicle).

b (n) denotes the number of plates assayed, with 2-3 larvae per plate.

c The increase in average speed of larvae fed AMPH as compared to larvae of the same genotype fed vehicle. NS = Non Significant.

d Significance was determined using the Mann-Whitney-Wilcoxon (MWW) test.

Supplementary Table 8. RNAi-mediated depletion of dDAT in DA neurons blunts the response to MPH.

Genotype a / MPH (mM) b / Speed (cm/sec) ± SEM (n) c / % Increase in Speed d / MWW e
UAS-GFP-RNAi/
TH-GAL4 / 0 / 0.076 ± 0.002 (31) / 13% / U = 193.5, P < 0.001
3 / 0.086 ± 0.002 (31)
UAS-dDAT-RNAi/+ ;
TH-GAL4/+ / 0 / 0.074 ± 0.002 (32) / (NS) / U = 453, P > 0.05
3 / 0.074 ± 0.002 (30)
UAS-dDAT-RNAi/+ / 0 / 0.071 ± 0.003 (22) / 17% / U = 94, P = 0.001
3 / 0.083 ± 0.002 (22)

a All TH-GAL4 flies also carried a UAS-Dicer2 transgene.

b MPH was dissolved in 0.8 mM mianserin (vehicle).

c (n) denotes the number of plates assayed, with 2-3 larvae per plate.

d The increase in average speed of larvae fed MPH as compared to larvae of the same genotype fed vehicle. NS = Non Significant.

e Significance was determined using the Mann-Whitney-Wilcoxon (MWW) test.

Supplementary Table 9. MPH-induced hyperlocomotion is dependent on DAT but does not require its phosphorylation.

Genotype / MPH (mM) a / Speed (cm/sec) ± SEM (n) b / % Increase in Speed c / MWW d
dDATfmn / 0 / 0.068 ± 0.002 (20) / (NS) / U = 194.5, P > 0.05
3 / 0.068 ± 0.002 (20)
dDATfmn;
UAS-hDAT/+ / 0 / 0.056 ± 0.001 (41) / (NS) / U = 690, P > 0.05
3 / 0.058 ± 0.002 (39)
dDATfmn;
UAS-hDAT/TH-GAL4 / 0 / 0.052 ± 0.002 (35) / 25% / U = 255, P < 0.001
3 / 0.065 ± 0.001 (39)
dDATfmn;
UAS-hDAT-S/A/TH-GAL4 / 0 / 0.053 ± 0.001 (43) / 23% / U = 310, P < 0.001
3 / 0.065 ± 0.002 (43)
dDATfmn;
UAS-hDAT-S/D/TH-GAL4 / 0 / 0.054 ± 0.001 (39) / 15% / U = 407, P < 0.001
3 / 0.062 ± 0.001 (39)

a MPH was dissolved in 0.8 mM mianserin (vehicle).

b (n) denotes the number of plates assayed, with 2-3 larvae per plate.

c The increase in average speed of larvae fed AMPH as compared to larvae of the same genotype fed vehicle. NS = Non Significant.

d Significance was determined using the Mann-Whitney-Wilcoxon (MWW) test.

Supplementary Table 10. MPH-induced hyperlocomotion is not dependent on Flo.

Genotype a / MPH (mM) b / Speed (cm/sec) ± SEM (n) c / % Increase in Speed d / MWW e
w1118 / 0 / 0.052 ± 0.002 (33) / 23% / U = 208, P < 0.001
3 / 0.064 ± 0.002 (33)
Floe02554 / 0 / 0.066 ± 0.002 (35) / 17% / U = 361, P < 0.001
3 / 0.077 ± 0.002 (40)
UAS-Flo-RNAi/+ ;
TH-GAL4/+ / 0 / 0.061 ± 0.002 (30) / 18% / U = 173, P < 0.001
3 / 0.072 ± 0.001 (32)
UAS-Flo-RNAi/+ / 0 / 0.060 ± 0.002 (20) / 17% / U = 76.5, P < 0.001
3 / 0.070 ± 0.002 (21)

a All TH-GAL4 flies also carried a UAS-Dicer2 transgene.

b MPH was dissolved in 0.8 mM mianserin (vehicle).

c (n) denotes the number of plates assayed, with 2-3 larvae per plate.

d The increase in average speed of larvae fed MPH as compared to larvae of the same genotype fed vehicle.

e Significance was determined using the Mann-Whitney-Wilcoxon (MWW) test.

Supplementary Table 11. AMPH-induced hyperlocomotion is dependent on Flo.

Genotype / AMPH (mM) a / Average Speed (cm/sec) ± SEM (n) b / % Increase in Speed c / MWW d
UAS-hFlot1/+ ; Floe02554 ;
TH-GAL4/+ / 0 / 0.049 ± 0.002 (34) / 43% / U = 78, P < 0.001
60 / 0.070 ± 0.002 (37)
UAS-hFlot1/+ ; Floe02554 / 0 / 0.056 ± 0.002 (36) / (NS) / U = 532, P > 0.05
60 / 0.059 ± 0.002 (37)
Floe02554 / 0 / 0.062 ± 0.002 (34) / (NS) / U = 468, P > 0.05
60 / 0.058 ± 0.002 (34)

a AMPH was dissolved in 0.8 mM mianserin (vehicle).

b (n) denotes the number of plates assayed, with 2-3 larvae per plate.

c The increase in average speed of larvae fed AMPH as compared to larvae of the same genotype fed vehicle. NS = Non Significant.

d Significance was determined using the Mann-Whitney-Wilcoxon (MWW) test.


Supplementary Table 12. RNAi-mediated depletion of Flo in DA neurons blunts the response to AMPH.

Genotype a / AMPH (mM) b / Average Speed (cm/sec) ± SEM (n) c / % Increase in Speed d / MWW e
UAS-hFlot1/+;
UAS-Flo-RNAi/+ / 0 / 0.070 ± 0.003 (22) / 23% / U = 90, P < 0.001
60 / 0.086 ± 0.003 (25)
UAS-hFlot1/+;
UAS-Flo-RNAi/+ ; TH-GAL4/+ / 0 / 0.059 ± 0.001 (55) / 31% / U = 402, P < 0.001
60 / 0.077 ± 0.002 (53)
UAS-Flo-RNAi/+ ; TH-GAL4/+ / 0 / 0.060 ± 0.002 (40) / (NS) / U = 806, P > 0.05
60 / 0.064 ± 0.002 (47)
UAS-hFlot1C34A/+;
UAS-Flo-RNAi/+ ; TH-GAL4/+ / 0 / 0.065 ± 0.002 (27) / (NS) / U = 255, P > 0.05
60 / 0.061 ± 0.002 (26)

a All TH-GAL4 flies also carried a UAS-Dicer2 transgene.

b AMPH was dissolved in 0.8 mM mianserin (vehicle).

c (n) denotes the number of plates assayed, with 2-3 larvae per plate.

d The increase in average speed of larvae fed AMPH as compared to larvae of the same genotype fed vehicle. NS = Non Significant.

e Significance was determined using the Mann-Whitney-Wilcoxon (MWW) test.