Supplementary Figure Legends s5

Supplementary Figure Legends

Supplementary Figure 1. The Exelixis e02554 line contains a piggyBac RB transposable element insertion in the Flo gene. (a) Schematic drawing of the

Drosophila Flotillin1 (Flo) gene with piggyBac RB e02554 inserted in the second intron of the gene. The gene is 3136 bp long and the piggyBac is 6540 bp long. Arrowheads indicate the approximate positions of primers used to verify the insertion. (b) Gel electrophoresis image showing the PCR fragments run on a 1% w/v agarose gel and stained with ethidium bromide. Genomic DNA extracts from wild-type w1118 and from

Floe02554 were used as templates.

Predictions for PCR product size using these sets of primers:

FloS1 and FloAS: w1118 , 464 bp ; Floe02554, 7519 bp;

FloS1 and piggyBacAS: w1118 , 0 bp ; Floe02554, 284 bp

FloS2 and piggyBacAS: w1118 , 0 bp ; Floe02554, 799 bp rp49S and rp49AS: w1118 , 372 bp ; Floe02554, 372 bp

When both sense and antisense primers targeted sequences in the Flo gene (FloS1 and

FloAS), a band was observed only when DNA extracted from w1118 wild-type flies was used as template. No band was amplified when DNA extracted from Floe02554 flies was used due to the restriction set by the elongation time of our PCR protocol (1 min). When the antisense primer was targeted to sequences in the piggyBac (FloS1 and piggyBacAS or FloS2 and piggyBacAS), bands were observed only when DNA extracted from Floe02554 flies was used as template. Primers that amplify part of the rp49 gene were used to control for the quality of DNA.

1 Supplementary Figure 2. Schematic of the biosynthesis pathways of the biogenic amines dopamine, tyramine and octopamine. The amino acid tyrosine is the starting point for the synthesis of dopamine (DA), tyramine (TA) and octopamine (OA). Tyrosine is catalyzed either to L-dyhydroxy-phenylalanine (L-Dopa) by tyrosine hydroxylase (TH) or to tyramine (TA) by tyrosine decarboxylase (Tdc). Dopamine is synthesized from L-

Dopa by the enzyme Dopa decarboxylase (DDC) whereas octopamine (OA) is synthesized from TA by the enzyme tyramine -hydroxylase (TH).

Supplementary Figure 3. Drosophila larvae decrease their crawling speed in response to acute mianserin treatment. Mianserin led to a decrease in the speed of locomotion of wild-type (w1118) larvae in a dose-dependent manner. Mianserin at 0.33 mM, 0.66 mM and 1 mM led to a significant decrease by 19%, 26% and 26%, respectively. Asterisks indicate the significance of the decrease in crawling speed as compared to vehicle. Mann-Whitney-Wilcoxon (MWW) test, *** P < 0.001.

Supplementary Figure 4a. The locomotor response of larvae to mianserin is dependent on Tdc2 but not on TH, whereas the locomotor response to AMPH in the presence of mianserin is dependent on TH but not on Tdc2. Feeding larvae 0.8 mM mianserin had no effect on the speed of Tdc2R054 mutants whereas it led to a 20% decrease in the speed of locomotion of larvae expressing TH-RNAi in DA neurons.

Tdc2R054 mutants displayed a robust hyperlocomotion after feeding AMPH delivered in

0.8 mM mianserin compared to larvae of the same genotype fed only mianserin. These larvae increased their crawling velocity by 43%. In contrast, larvae expressing TH-RNAi in DA neurons exhibited a blunted response to AMPH delivered in 0.8 mM mianserin.

Also see Supplementary Tables 3, 4 and 5. b. TH is required for AMPH-induced hyperlocomotion in Drosophila larvae. Mutant larvae that lack TH selectively in the

2 nervous system (DTHgFS±; ple) did not display an increase in locomotion when fed

AMPH, while control larvae expressing genomic Drosophila TH driven by TH-GAL4

(expressed in most dopaminergic neurons) and Ddc-GAL4 (expressed in most dopaminergic and serotonergic neurons) show a significant response to AMPH (DTHg; ple, 28% increase). Asterisks indicate the significance of the increase in average crawling speed of larvae fed AMPH as compared to larvae of the same genotype fed vehicle (MWW test, NS P > 0.05, *P < 0.05, ***P < 0.001). Also see Supplementary

Table 4.