Serotonergic neurons regulate sucrose appetite and protein hunger.

A to C, Schematics of serotonergic neurons targeted by different Gal4 drivers; serotonergic neurons are marked in red. D to F, Expression of SerTDN under the control of the Sert-Gal4, R50H05-Gal4 or Tph-Gal4 drivers reduced sucrose intake in flies fed ad libitum but not starved flies. G to I, Hunger-induced protein intake is increased, when SerTDN is expressed in broad subsets of serotonergic neurons. K to L, Appetite-induced intake of a mixed diet consisting of 5% sucrose and 5% yeast is reduced only in flies in which SerTDN is expressed under the control of the Sert3-Gal4 driver. Data represents mean ± SEM (n = 16–37). For raw data see Supplementary Table S1. *P < 0.05; **P < 0.01; one-way ANOVA, with Tukey post-hoc test.

Activation of serotonergic neurons decreases appetite for sucrose and hunger for proteins

A, The CaFe-assays were combined with blue diodes with a light intensity of 1800 lx and a flicker frequency of 2 s 40 Hz, 16 s 8 Hz and 2 s of no light. B, Activation of serotonergic neurons decreased appetite-induced sucrose intake- C, Activation does not affect hunger-induced sucrose intake. D, Hunger-induced protein intake increases with activation of a broad subsets of serotonergic neurons. Data are mean ± SEM (n = 16-21). For raw data see Supplementary Table S1. Unpaired Student’s t-test for comparison between the groups: **P < 0.01, ***P < 0.001.

Influence of reduced sucrose appetite on appetitive and aversive STM

A, To assess appetitive short-term learning and memory, flies were first exposed to one odorant paired with a reward of 2 M sucrose followed by a second odorant during training. B, To asses aversive learning and memory, flies were exposed to the odor paired with an electric shock of 90V for 1 min followed by an exposure to a second odor (US) of 1 min. In the test situation 3 min after the training, they choose for 2 min between both odorants A, Expression of UAS-SerTDN under the control of Sert3-Gal4 significantly reduced the olfactory appetitive STM. B, Aversive STM is not affected in flies with expression UAS-SerTDN under the control of Sert3-Gal4. The letter “a” indicates a significant difference from random choice (one-sample t-test (P < 0.05)). N = 12. For raw data see Supplementary Table S1. Statistic differences between groups were determined by a One-way ANOVA followed by post-hoc Tukey HSD test; ** P < 0.01.

Sensory acuity tests in serotonergic neurons targeted by Sert3-Gal4

Mean ± s.e.m is shown. No significant differences between control and experimental groups were detected using ANOVA Turkey-Cramer post hoc analysis (P < 0.05). The letter a indicates significant differences from random choice as determined by One-sample sign test (P < 0.05). N = 12 - 18 for each test except sucrose sensitivity (N = 20).

The insulin-like receptor regulates appetite-induced sucrose intake.

A, Flies expressing InRCA under the control of the Ser3-Gal4 driver reduced their food intake when fed ad libitum with sucrose or sucrose and yeast, but not with yeast only. The food intake of hungry flies was not affected. B, Expression of InRDN under the control of the Ser3-Gal4 driver only reduced food intake in flies that were fed either sucrose or sucrose and yeast ad libitum. The data are mean food intake ± SEM (n = 12-36); for raw data see Supplementary Table S1. *P < 0.05, ** P < 0.01 based on ANOVA Tukey HSD test.

Insulin receptor function affects SerT expression.

A to A′′, Expression of SerT (in magenta), expression of serotonin (in green) and overlap (in white) in 10µm thick brain section showing the fans-shaped body (fb). B to B′′, The Tph-Gal4 driver targets the ExR3 neuron that innervates the fb and contributes to the layer 4 (l4) of the fb in the whole mount brain of a control fly (GFP expression in green and serotonin expression in magenta). C to E, The GFP signal (in green) of the UAS-SerT::GFP transgene under the control of the Tph-Gal4 driver changes the localization and intensity depending on D, InRCA, or E, InRDN , expression. To quantify the expression area of SerT::GFP, the width of layer 2 and layer 4 was measured. The nc82 antigen was used to label neurophils (in magenta) in the whole mount brain. l2: layer 2; l4: layer 4. N = 5 – 8 brains. The data were compared using ANOVA Tukey HSD test. For raw data see Supplementary Table S1. ** P < 0.01.

Insulin receptor function regulates neuron morphology and localization of SerT expression.

A, The Sert3-Gal4 driver target an SE1 serotonergic neuron in the SEZ (in green, GFP expression and in magenta, serotonin). B to D, SE1 neuron morphology was visualized using a membrane-bound red fluoresce protein (RFP). SerT::GFP expression (in green) changes upon co-expression of C, InRCA, or D, InRDN. The scale bar represents 10 µm. E to G, 5% sucrose intake was measured over 24 h. Expression of SerT::GFP under the control of Sert-Gal4 did not alter sucrose intake, but expression of F, InRCA, or G, InRDN. Combing expression with SerT::GFP restored reduced appetite when F, InRCA was co-expressed but not when G, InRDN was co-expressed. Data were compared using ANOVA Tukey HSD test. ** P < 0.01.

Potential pathways for regulation of sucrose appetite.

A, Expression of CG9911RNAi or CG10029RNAi under the control of the Sert-Gal4 driver does not alter sucrose appetite. B, Reduction of Sec24AB using an RNAi transgene on the third chromosome significantly reduced sucrose appetite. Sucrose appetite of flies carrying the RNAi transgene on the second chromosome under the control of the Sert-Gal4 driver is significantly reduced in compared to flies carrying one copy of the Gal4 driver. Data were compared using ANOVA Tukey HSD test. ** P < 0.01, *** P < 0.001. N = 25 - 30. For raw data see Supplementary Table S1. C, The taste neuron IR60b and D, the SE1 neuron are visualized using an UAS-mCD8::GFP transgene. The scale bar corresponds to 10 µm. E, Overlay of the IR60b neuron projection with the SE1 neuron projection pattern in the SEZ.