Dopamine signalling in mushroom bodies regulates temperature-preference behaviour in Drosophila

Abstract

The ability to respond to environmental temperature variation is essential for survival in animals. Flies show robust temperature-preference behaviour (TPB) to find optimal temperatures. Recently, we have shown that Drosophila mushroom body (MB) functions as a center controlling TPB. However, neuromodulators that control the TPB in MB remain unknown. To identify the functions of dopamine in TPB, we have conducted various genetic studies in Drosophila. Inhibition of dopamine biosynthesis by genetic mutations or treatment with chemical inhibitors caused flies to prefer temperatures colder than normal. We also found that dopaminergic neurons are involved in TPB regulation, as the targeted inactivation of dopaminergic neurons by expression of a potassium channel (Kir2.1) induced flies with the loss of cold avoidance. Consistently, the mutant flies for dopamine receptor gene (DopR) also showed a cold temperature preference, which was rescued by MB-specific expression of DopR. Based on these results, we concluded that dopamine in MB is a key component in the homeostatic temperature control of Drosophila. The current findings will provide important bases to understand the logic of thermosensation and temperature preference decision in Drosophila.

Figure 1. Dopamine biosynthetic pathway and calculation of the avoidance index.

(A) Dopamine biosynthetic pathway and enzymes (blue) with their inhibitors (red). TH converts tyrosine to L-DOPA, and then DDC converts L-DOPA to dopamine. AMPT is an inhibitor of TH, and HBH is an inhibitor of DDC. See text for the abbreviations. (B) Formulas for AI_Low and AI_High. AI values: 1, all flies resided in the intermediate temperature region; 0, all flies distributed evenly between the intermediate and the other regions; −1, all flies distributed in the low or high temperature region.

Figure 2. Dopamine biosynthesis is required for TPB.

(A–D) left, photographs of TPB tests; middle, distribution profiles of the tested flies; right, the calculated avoidance index. blue bars, AI_Low; red bars, AI_High.. All data are means ± SEMs. *, p<0.05; **, p<0.01. The flies did not go to the regions above 35°C in all TPB tests. The temperature regions below 35°C are shown in photographs. The photographs of TPB shown in each panel are representatives from repeated experiments. (A) TPB of pale⁴/+ flies and pale⁴/+ flies fed with 20 mM AMPT. (B) TPB of pale⁴/P{pale⁺⁸} flies. P{pale⁺⁸} is a genomic DNA of the pale locus. (C) TPB of Ddc^DE1/Ddc^DE1 flies and Ddc^DE1/Ddc^DE1 flies fed with 20 mM HBH. Genotypes of the tested flies and drugs treated are denoted in each panel.

Figure 3. Dopamine biosynthesis is critical for flies to avoid cold temperature.

(A) TPB photographs of TH-Gal4/+, TH-Gal4>TH, TH-Gal4>DDC, TH-Gal4>TH, DDC, and UAS-TH/+;UAS-DDC/+ flies. The photographs shown are representatives from repeated TPB experiments. (B) TPB of TH-Gal4>TH flies and TH-Gal4>DDC flies. (C) TPB of TH-Gal4>TH, DDC flies.

Figure 4. Dopaminergic neurons are essential for TPB.

The photographs of TPB shown in each panel are representatives from repeated experiments. (A) TPB of TH-Gal4>UAS-TNT flies. (B) TPB of DAT-Gal4 >UAS-TNT flies.

Figure 5. Temporal inactivation of dopaminergic neurons causes flies to lose normal TPB.

(A) TPB photographs of TH-Gal4>UAS-TNT:tub-Gal80^ts/+, TH-Gal4>UAS-Kir2.1:tub-Gal80^ts/+, DAT-Gal4>UAS-Kir2.1:tub-Gal80^ts/+, and their control fly lines. The flies were reared at 18°C all the time before the TPB assay. The photographs shown are representatives from repeated TPB experiments. (B) TPB photographs of TH-Gal4>UAS-TNT:tub-Gal80^ts/+, TH-Gal4>UAS-Kir2.1:tub-Gal80^ts/+, DAT-Gal4>UAS-Kir2.1:tub-Gal80^ts/+, and their control fly lines. The flies reared at 18°C and exposed to 32°C for 16 hr before the TPB assay. The photographs shown are representatives from repeated TPB experiments. (C) TPB of TH-Gal4>UAS-TNT:tub-Gal80^ts/+ flies reared at 18°C all the time before the TPB assay. (D) TPB of TH-Gal4>UAS-TNT:tub-Gal80^ts/+ flies reared at 18°C and exposed to 32°C for 16 hr before the TPB assay. UAS-TNT/+:tub-GAL80^ts/+ was used as a control (C, D). (E) TPB of TH-Gal4>UAS-Kir2.1:tub-Gal80^ts/+ flies reared at 18°C all the time before the TPB assay. (F) TPB of TH-Gal4>UAS-Kir2.1:tub-Gal80^ts/+ flies reared at 18°C and exposed to 32°C for 16 hr before the TPB assay. (G) TPB of DAT-Gal4>UAS-Kir2.1:tub-Gal80^ts/+ flies reared at 18°C all the time before the TPB assay. (H) TPB of DAT-Gal4>UAS-Kir2.1:tub-Gal80^ts/+ flies reared at 18°C and exposed to 32°C for 16 hr before the TPB assay. UAS-Kir2.1/+:tub-Gal80^ts/+ was used as a control (E–H). **, p<0.01 compared with TH-Gal4/+ or DAT-Gal4/+.

Figure 6. Dopamine receptor is required for TPB.

The photographs of TPB shown in each panel are representatives from repeated experiments. (A) TPB of DopR mutant dumb³/dumb³. (B) TPB of Df(3R)red-P52/+ flies, which is a heterozygous deficiency mutant of DopR. (C) TPB of trans-hetero allelic DopR mutant dumb³/Df(3R)red-P52.

Figure 7. Dopamine receptor in MB is important for normal TPB.

DopR expression patterns detected by DopR antisera in the adult brain (A–B). (A) DopR was expressed in MB and the central complex in w¹¹¹⁸ control flies as previously reported . Scale bar  =  100 µm. (B) DopR expression in dumb³/dumb³ flies. Scale bar  =  100 µm. The photographs of TPB shown in each panel are representatives from repeated experiments (C–D). (C) TPB of c309>DopR;dumb³/dumb³ flies. (D) TPB of MB247>DopR;dumb³/dumb³ flies.

Figure 8. DopR is required in MB for TPB.

The photographs of TPB shown in each panel are representatives from repeated experiments. (A) TPB of c309>DopR;Df(3R)red-P52/+ flies. (B) TPB of MB247>DopR;Df(3R)red-P52/+ flies.

Figure 9. DopR expression in the αβ lobes of MB is critical in TPB.

The photographs of TPB shown in each panel are representatives from repeated experiments. (A) TPB of c739>DopR;dumb³/dumb³ flies. (B) TPB of 1471>DopR;dumb³/dumb³ flies.

Figure 10. DopR is dispensable in the central complex for TPB.

The photographs of TPB shown in each panel are representatives from repeated experiments. (A) TPB of c161>DopR;dumb³/dumb³ flies. (B) TPB of OK348>DopR;dumb³/dumb³ flies.