Neural correlates of water reward in thirsty Drosophila

Abstract

Drinking water is innately rewarding to thirsty animals. In addition, the consumed value can be assigned to behavioral actions and predictive sensory cues by associative learning. Here we show that thirst converts water avoidance into water-seeking in naive Drosophila melanogaster. Thirst also permitted flies to learn olfactory cues paired with water reward. Water learning required water taste and <40 water-responsive dopaminergic neurons that innervate a restricted zone of the mushroom body γ lobe. These water learning neurons are different from those that are critical for conveying the reinforcing effects of sugar. Naive water-seeking behavior in thirsty flies did not require water taste but relied on another subset of water-responsive dopaminergic neurons that target the mushroom body β′ lobe. Furthermore, these naive water-approach neurons were not required for learned water-seeking. Our results therefore demonstrate that naive water-seeking, learned water-seeking and water learning use separable neural circuitry in the brain of thirsty flies.

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Figure 1: Thirsty flies approach water and can be trained with water reward.
Figure 2: Water-reinforced learning is independent of octopamine and is supported by dopaminergic signaling to mushroom body γ neurons.
Figure 3: Water learning requires reinforcing dopamine from specific neurons in the PAM cluster.
Figure 4: Naive thirst-dependent water-seeking requires different dopaminergic neurons than those required for water learning.

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Acknowledgements

We thank C.J. Burke for his extensive failings and teachings in the art of water learning. We also thank Y. Huang and R. Brain for technical support and the Bloomington stock center, T. Clandinin (Stanford University), D. Gohl (Stanford University), M. Silies (Stanford University), G. Rubin and the Janelia Farm Project, Y. Ben-Shahar (Washington University), K. Scott (University of California, Berkeley), T. Lee (Janelia Farm Research Campus) and J. Dubnau (Cold Spring Harbor) for fly lines. S.L. was supported by an EMBO Long-Term Fellowship. D.O. was supported by an EMBO Long-Term Fellowship and a Sir Henry Wellcome Postdoctoral Fellowship. V.C. was supported by a Andrew Mason Memorial Scholarship. S.W. is funded by a Wellcome Trust Senior Research Fellowship in the Basic Biomedical Sciences and by funds from the Gatsby Charitable Foundation and Oxford Martin School.

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Authors

Contributions

S.W. and S.L. conceived this project and designed all experiments. S.L. and V.C. designed and optimized the water-conditioning assay and performed all behavioral experiments. Live imaging was performed by D.O. using custom apparatus and software constructed and programmed by C.T. GAL4 lines were visually screened and selected by W.H. Anatomical data were produced by S.L. and W.H. The manuscript was written by S.W. and S.L.

Corresponding author

Correspondence to Scott Waddell.

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The authors declare no competing financial interests.

Integrated supplementary information

Supplementary Figure 1 Procedures to independently control fly hunger and thirst states.

(a) Protocol to produce exclusively hungry or thirsty flies for 6 h water memory retrieval. (b) Consumption assays confirm that flies housed on dry sugar for 6 h after training are thirsty but not hungry; on 1% agar for 6 h, hungry but not thirsty; and are fully satiated if kept on food. Flies kept on dry sugar for 6 h after training consume a significant amount of water in 2 min, whereas flies on 1% agar or fly food for 6 h do not drink (P>0.1 compared to zero, n=4; one sample t-test). Conversely, flies on 1% agar for 6 h after training eat a similar amount of 3M sucrose as flies starved on 1% agar for 21 h (P=0.07, n=4; ANOVA followed by post hoc Tukey HSD test), while the other two groups eat significantly less (P<0.0001, n=4; ANOVA followed by post hoc Tukey HSD test) and are not different from one another (P=0.33, n=4; ANOVA followed by post hoc Tukey HSD test). (c) Protocol to produce flies that are exclusively hungry or thirsty for 24 h sugar memory retrieval. (d) Consumption assays confirm that flies kept on 1% agar for 21 h are hungry but not thirsty; on drierite and dry sugar for 6 h, thirsty but not hungry; and are fully satiated if kept on food for 24 h. Flies on 1% agar for 21 h or fly food for 24 h do not drink (P>0.1 compared to zero, n=4; one sample t-test), whereas flies on drierite and dry sugar for 6 h consume an amount of water in 2 min that is indistinguishable from 16 h water deprived flies (P=0.14, n=4; ANOVA followed by post hoc Tukey HSD test). In contrast, flies kept on 1% agar for 21 h eat a significant amount of 3M sucrose while the other two groups eat significantly less (P<0.0001, n=4; ANOVA followed by post hoc Tukey HSD test) and are not different from one another (P=0.14, n=4; ANOVA followed by post hoc Tukey HSD test).

Supplementary Figure 2 Mutant ppk28 flies have normal olfactory acuity; control for Figure 1f.

Odor acuity of thirsty ppk28 flies is indistinguishable from that of wild-type flies, OCT (P=0.22, n=8; t-test) and MCH (P=0.5, n=8; t-test).

Supplementary Figure 3 Water consumption and olfactory acuity controls for DopR1 rescue experiment in Figure 2b.

(a) DopR1 mutant fly lines show normal levels of drinking (P>0.8, n=8; ANOVA followed by post hoc Tukey HSD test), except for c305a/UAS-DopR1; dumb2 flies that drink significantly more water in 2 min (P=0.0001, n=8; ANOVA followed by post hoc Tukey HSD test). d1 = dumb1; d2 = dumb2. (b) All thirsty DopR1 mutant flies show normal odor acuity to MCH (P=0.84, n=8; ANOVA) and most to OCT except UAS-DopR1; dumb2 flies that have reduced odor acuity to OCT (P<0.0001 compared to wild-type, n=8; ANOVA followed by post hoc Tukey HSD test). However, although the OCT acuity of all DopR1 mutant flies is generally lower than wild-type flies, there is no significant difference between the transgenic DopR1 mutant fly strains (P=0.42, n=8; ANOVA followed by post hoc Tukey HSD test).

Supplementary Figure 4 Permissive temperature, olfactory acuity and water consumption controls for Figure 2c.

(a) The 3 min memory performance of thirsty 0273; UAS- shits1 flies is significantly greater than that of UAS-shits1 (P=0.0016, n=8; ANOVA followed by post hoc Tukey HSD test), but indistinguishable from that of 0273-GAL4 control flies (P=0.43, n=8; ANOVA followed by post hoc Tukey HSD test) at permissive 23°C. Performance of thirsty R58E02; UAS-shits1 flies is not statistically different from that of either relevant control at permissive 23°C (P>0.5, n=8; ANOVA). (b) Thirsty 0273-GAL4; UAS-shits1 and R58E02-GAL4; UAS-shits1 flies show normal odor acuity to OCT (P=0.46, n=8; ANOVA) and MCH (P=0.67, n=8; ANOVA). (c) 0273-GAL4; UAS-shits1 flies drink significantly less water in 2 min (P<0.0001, n=8; ANOVA followed by post hoc Tukey HSD test), whereas R58B04-GAL4; UAS-shits1 drinking is not significantly different to the controls (P>0.45, n=8; ANOVA followed by post hoc Tukey HSD test).

Supplementary Figure 5 Additional experiments to accompany Figure 3, defining the role of the γ4 dopaminergic neurons in water learning.

(a) 3 min memory performance of thirsty R48B04; UAS-shits1 (JFRC100) flies is indistinguishable from that of controls at 23°C (P=0.34, n=8; ANOVA). (b) Drinking of R48B04-GAL4; UAS-shits1 (JFRC100) flies is not statistically impaired at 32°C (P=0.24, n=8; ANOVA). (c) Thirsty R48B04-GAL4; UAS-shits1 (JFRC100) flies show normal odor acuity to OCT (P=0.08, n=8; ANOVA) and MCH (P>0.46, n=8; ANOVA followed by post hoc Tukey HSD test), while thirsty R48B04-GAL4 flies display significantly different odor acuity to MCH (*P<0.04, n=8; ANOVA followed by post hoc Tukey HSD test). (d) R48B04 neurons are dopaminergic. Top panel shows the merged image of the below individual channels from a confocal projection through the PAM cluster in a R48B04-GAL4;UAS-CD8::GFP (green) brain costained with anti-TH antibody (magenta). Scale bar 40 μm. (e) A single confocal section through the mushroom body at the level of the γ4 and γ5 zones revealing the respective innervation by neurons labeled with 0104-GAL4 driven GFP (green) and R48B04-LexA driven RFP (magenta). (f) A single section from the same brain as shown in (e) at the level of the β´2 zone. Scale bar 20 μm. (g) Permissive temperature control for Fig. 3h. lexAop-shits1/R48B04-LexA;UAS-LexAi/0104-GAL4 flies show normal 3 min water memory performance at 23°C (P=0.89, n=8; ANOVA).(h) Water drinking control for Fig. 3h. Drinking of lexAop-shits1/R48B04-LexA; UAS-LexAi/0104-GAL4 flies is not significantly different from controls (P>0.08, n=8; ANOVA followed by post hoc Tukey HSD test). (i) Olfactory acuity control for Fig. 3h. Thirsty lexAop-shits1/R48B04-LexA; UAS-LexAi/0104-GAL4 flies have normal odor acuity to MCH (P=0.24, n=8; ANOVA).They displayed higher acuity to OCT than lexAop-shits1; UAS-LexAi controls (P=0.01, n=8; ANOVA followed by post hoc Tukey HSD test) but were indistinguishable from R48B04-LexA; 0104-GAL4 controls (P=0.35, n=8; ANOVA followed by post hoc Tukey HSD test). (j) Permissive temperature control for Fig. 3i and j. No memory was implanted without temperature shift during the second odor presentation (P=0.76, n=8; ANOVA). (k) Odor acuity control for Fig. 3i. Thirsty lexAop-TrpA1/R48B04-LexA; UAS-LexAi/0104-GAL4 flies show normal odor acuity to OCT (P=0.15, n=8; ANOVA) and MCH (P=0.34, n=8; ANOVA). (l) Permissive temperature control for Fig. 3l. R15A04-GAL80/R48B04-GAL4; UAS-shits1 flies exhibit normal 3min water memory performance at 23°C (P=0.998, n=8; ANOVA). (m) Water drinking control for Fig. 3l. R15A04-GAL80/R48B04-GAL4; UAS-shits1 drinking is indistinguishable from that of control flies (P=0.31, n=8; ANOVA). (n) Olfactory acuity controls for Fig. 3l. Odor acuity to OCT of thirsty R15A04-GAL80/R48B04-GAL4; UAS-shits1 flies was indistinguishable to that of controls (P=0.28, n=8; ANOVA). Acuity to MCH is also not significantly different from both controls (P=0.99, n=8 compared to UAS-shits1; P=0.06, n=8 compared to R15A04-GAL80; R48B04-GAL4; ANOVA followed by post hoc Tukey HSD test). However, the R15A04-GAL80; R48B04-GAL4 flies were statistically different from UAS-shits1 flies (P=0.04, n=8; ANOVA followed by post hoc Tukey HSD test).

Supplementary Figure 6 Additional experiments to accompany Figure 4, defining the role of the β′2 dopaminergic neurons in naive water-seeking.

(a) Permissive temperature control for Fig. 4b and d. Thirsty R48B04-GAL4; UAS-shits1 and 0104-GAL4; UAS-shits1 flies show normal water approach behavior at permissive 23°C (P=0.4, n=8; ANOVA). (b) Blocking R48B04 and 0104 neurons does not significantly alter water avoidance in sated flies (P=0.14, n≥8; ANOVA). (c) Permissive temperature control for Fig. 4c. Thirsty R48B04-GAL4; UAS-shits1 (JFRC100) flies show normal water approach behavior at 23°C (P=0.41, n=8; ANOVA). (d) Blocking R48B04 neurons with UAS-shits1 (JFRC100) does not alter water avoidance in sated flies (P=0.52, n=8; ANOVA). (e) Permissive temperature control for Fig. 4e. Thirsty R48B04-LexA/ LexAop-shits1; UAS-LexAi flies show normal water approach behavior at 23°C (P=0.36, n=8; ANOVA). (f) Sated R48B04-LexA/LexAop-shits1; UAS-LexAi flies show normal water avoidance behavior at the restricted temperature of 32°C (P=0.23, n=8; ANOVA). (g) Thirsty dumb1 mutant flies show normal naïve water-seeking behavior (P=0.9, n=8; ANOVA).

Supplementary Figure 7 Blocking R48B04 neurons enhances water memory expression in thirsty flies.

R48B04 neuron block immediately after training and during testing significantly enhances water memory expression (P<0.0001, n≥9; ANOVA).

Supplementary Figure 8 Blocking PAM dopaminergic neurons does not impair the proboscis extension response to water.

Blocking R48B04, 0273, or R58E02 neurons does not alter proboscis extension for water in thirsty flies (P=0.17, n≥9 for R48B04; P=0.35, n≥13 for 0273; P=0.36, n≥10 for R58E02; ANOVA).

Supplementary Figure 9 Water learning, wanting and liking can be mechanistically distinguished by manipulating subpopulations of R48B04 rewarding dopaminergic neurons.

Dopaminergic neurons innervating γ4 provide reinforcement for water learning and others to β′2 that are labeled by both R48B04 and 0104 are required for naïve water-seeking. Learned wanting and liking are apparently independent of the naïve wanting and learning neurons.

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Lin, S., Owald, D., Chandra, V. et al. Neural correlates of water reward in thirsty Drosophila. Nat Neurosci 17, 1536–1542 (2014). https://doi.org/10.1038/nn.3827

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