Sexual failure decreased sweet sensitivity.

(A) Schematic illustrating courtship conditioning strategy. (B) Percentage of males that performed feeding to 400 mM sucrose. (C) Fraction of Naïve, Failed and Satisfied male flies showing PER responses to different concentrations of sucrose. The PER experiment was performed immediately after conditioning unless otherwise indicated. Left: average PER responses of Naïve (blue), Failed (black), and Satisfied (red) flies (n=60-90). Right: MAT (mean acceptance threshold; the sucrose concentration where 50% of the flies show PER), plotted as a function of sexual state. (D) Fraction of male flies showing PER responses to different concentrations of sucrose. The PER experiment was performed immediately, 24 h, 48 h, and 72 h after conditioning (n=48-60). (E) Fraction of male flies showing PER responses to different concentrations of sucrose. Failed male flies were generated by two methods: one group was subjected to mated females (with cVA), and the other was subject to decapitated virgin females (without cVA) (n=48-60). (F) Fraction of male flies showing PER responses to different concentrations of sucrose. Failed-re-copulated males were generated by placing individual failed males with 25 virgin females in a food vial for 4.5 h. Data are shown as means ± SEM. Error bars represent SEM. n.s., p > 0.05; *p < 0.05; **p < 0.01; ***p < 0.001, ****p < 0.0001.

Dopamine signaling modulated the effect of sexual experience on sweet sensation.

(A) PER responses in male flies fed with 3IY, compared to male flies fed with vehicle (n=48-60). (B) PER responses in male flies with dopamine injection, compared to male flies injected with vehicle (n=48-60). (C-D) PER responses in male flies with indicated genotypes at 30°C (n=48-60). Data are shown as means ± SEM. Error bars represent SEM. n.s., p > 0.05; *p < 0.05; **p < 0.01; ***p < 0.001, ****p < 0.0001.

Dopamine neurons modulated the activity of Gr5a+ neurons upon sexual failure.

(A) Schematic diagram of in vivo calcium imaging paradigm. (B) Quantification of the in vivo calcium responses of Gr5a+ neurons to the indicated concentrations of sucrose (n=6-22). (C) Averaged traces of the calcium responses to 200 mM sucrose. The horizontal black bar represents sucrose application. The solid lines represent average trace, and shaded regions represent ±SEM. (D) Representative pseudocolor images of the calcium responses to 200mM sucrose. The dashed area was quantified to measure the response. (E) Top: Representative images from GRASP experiment between dopaminergic neurons and sweet GRNs. Bottom: Enlarged images of the SEZ region seen on the top. Scale bars, 20 μm. (F) Average fluorescence reported by TH>nsyb-spGFP1-10, Gr5a>CD4-spGFP11 (normalized to Naive) in the SEZ. Lines represent mean ± SEM, and dots indicate values for individual flies. (G) Top: Representative images of CaLexA-induced GFP reporter in dopaminergic neurons. Bottom: Enlarged images of the SEZ region shown on the top. Scale bars, 20 μm. (H-I) Average fluorescence reported by TH-GAL4/UAS-mLexA-NFAT, LexAop-CD2-GFP (normalized to Naive) in the SEZ (H), in the AL (I). Lines represent mean ± SEM, and dots indicate values for individual flies. Data are shown as means ± SEM. Error bars represent SEM. n.s., p > 0.05; *p < 0.05; **p < 0.01; ***p < 0.001, ****p < 0.0001.

The effect of sexual failure was modulated through two dopamine receptors, Dop1R1 and Dop2R.

(A-E) PER responses in male flies with indicated genotypes (n=48-75). (F) MAT calculation for the PER data from (A-E), plotted as a function of sexual state and type of flies. Data are shown as means ± SEM. Error bars represent SEM. n.s., p > 0.05; *p < 0.05; **p < 0.01; ***p < 0.001, ****p < 0.0001.

Dop1R1+ and Dop2R+ neurons regulated sexual experience-dependent sweet sensitivity.

(A-D) PER responses in male flies with indicated genotypes (n=48-60). Data are shown as means ± SEM. Error bars represent SEM. n.s., p > 0.05; *p < 0.05; **p < 0.01; ***p < 0.001, ****p < 0.0001.

Gr5a+ neurons received dopaminergic modulation through Dop1R1 and Dop2R.

(A-B) The expression of membrane-bound GFP (mCD8GFP, Green) in Gr5a+ neurons driven by Gr5a-LexA and membrane-bound RFP (mCD8RFP, Red) in dopamine receptors neurons driven by Dop1R1-Gal4 (A), and Dop2R-Gal4 (B). Scale bars, 20 μm. (C-D) PER responses in male flies with indicated genotypes (n=60-75). (E-F) Averaged traces of the calcium responses to 200mM sucrose in male flies with indicated genotypes (n=7-10). The horizontal black bars represent sucrose application. The solid lines represent average trace, and shaded regions represent ±SEM. Data are shown as means ± SEM. Error bars represent SEM. n.s., p > 0.05; *p < 0.05; **p < 0.01; ***p < 0.001, ****p < 0.0001.

Working model for the sexual failure-dependent modulation of sweet perception.

In the SEZ of the fly brain, dopaminergic neurons form synaptic connections with sweet-sensing Gr5a+ neurons, which express the dopamine receptors Dop1R1 and Dop2R. This connectivity enables Gr5a+ neurons to receive dopaminergic modulation, enhancing the flies’ sweet sensitivity. However, after mating failure, the activity of these SEZ dopaminergic neurons is selectively inhibited, and their synaptic connections with Gr5a+ neurons are weakened. This reduction in dopaminergic signaling ultimately decreases the flies’ sweet taste perception. This mechanism highlights a specific pathway by which unsuccessful mating experiences influence sensory processing through neural plasticity in dopamine-associated circuits.

Sexual failure decreased sweet sensitivity.

(A) Volume of 400mM sucrose consumed by males fed ad libitum (n = 35-60). (B) Fraction of Naïve, Failed and Satisfied male flies showing PER responses to different concentrations of D-Fructose (n=48-60). (C) Fraction of male flies showing PER responses to different concentrations of D-Glucose (n=48-60). Data are shown as means ± SEM. Error bars represent SEM. n.s., p > 0.05; *p < 0.05; **p < 0.01; ***p < 0.001, ****p < 0.0001.

Dopamine but not serotonin signaling modulated sexual experience-dependent sweet sensitivity.

(A) PER responses in Th mutant male flies (n=48-60). (B) PER responses in male flies injected with various doses of dopamine (n=48-60). (C) PER responses in male flies fed with DL-p-chlorophenylalanine (pCPA) (n=48-60). (D) PER responses in male flies fed with methysergide (n=48-60). (E-F) PER responses in male flies with indicated genotypes at 20°C(n=48-60). Data are shown as means ± SEM. Error bars represent SEM. n.s., p > 0.05; *p < 0.05; **p < 0.01; ***p < 0.001, ****p < 0.0001.

Dopaminergic neurons had functional connections with sweet GRNs.

(A) The expression of membrane-bound GFP (mCD8GFP, Green) in dopaminergic neurons driven by TH-Gal4 and membrane-bound RFP (rCD2RFP, Red) in Gr5a+ neurons driven by Gr5a-LexA. Scale bars, 20 μm. (B) Representative image of GRASP experiment to show connections between dopaminergic neurons and sweet GRNs in TH>nsyb-spGFP1-10, Gr5a>CD4-spGFP11 flies (left). Genetic controls of GRASP experiments were shown on the center and right. Scale bars, 20 μm. (C-D) Averaged traces of the calcium responses to 200mM sucrose in male flies with indicated genotypes (n=6-9). The horizontal black bars represent sucrose application. The solid lines represent average trace, and shaded regions represent ±SEM.

Dop1R1 and Dop2R were necessary for sexual failure-induced change in sweet sensitivity.

(A-D) PER responses in male flies with indicated genotypes (n=48-60). Data are shown as means ± SEM. Error bars represent SEM. n.s., p > 0.05; *p < 0.05; **p < 0.01; ***p < 0.001, ****p < 0.0001.

Dop1R2+ or DopEcR+ neurons did not affect sweet sensitivity following sexual failure.

(A-B) PER responses in male flies with indicated genotypes (n=48-60). (C) MAT, plotted as a function of sexual state and type of flies. Data are shown as means ± SEM. Error bars represent SEM. n.s., p > 0.05; *p < 0.05; **p < 0.01; ***p < 0.001, ****p < 0.0001.

Dopaminergic modulation of Gr5a+ neurons following sexual failure did not require DopEcR.

(A) The expression of membrane-bound GFP (mCD8GFP) in dopamine receptors neurons driven by Dop1R1-Gal4 (left), Dop2R-Gal4 (center), and DopEcR-Gal4 (right). Scale bars, 20 μm. (B) The expression of membrane-bound GFP (mCD8GFP, Green) in Gr5a+ neurons driven by Gr5a-LexA (left) and membrane-bound RFP (mCD8RFP, Red) in dopamine receptor neurons driven by DopEcR-Gal4. Scale bars, 20 μm. (C) PER responses in male flies with indicated genotypes (n=60-75). Data are shown as means ± SEM. Error bars represent SEM. n.s., p > 0.05; *p < 0.05; **p < 0.01; ***p < 0.001, ****p < 0.0001.