Supplementary Figure 1. Procedures to independently control fly hunger and thirst states.
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1 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).
2 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).
3 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; dumb 2 flies that drink significantly more water in 2 min (P=0.0001, n=8; ANOVA followed by post hoc Tukey HSD test). d 1 = dumb 1 ; d 2 = dumb 2. (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; dumb 2 flies that have reduced odor acuity to OCT (P< 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).
4 Supplementary Figure 4 Permissive temperature, olfactory acuity and water consumption controls for Figure 2c. (a) The 3 min memory performance of thirsty 0273; UAS-shi ts1 flies is significantly greater than that of UAS-shi ts1 (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-shi ts1 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-shi ts1 and R58E02-GAL4; UAS-shi ts1 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-shi ts1 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-shi ts1 drinking is not significantly different to the controls (P>0.45, n=8; ANOVA followed by post hoc Tukey HSD test).
5 Supplementary Figure 5 Additional experiments to accompany Figure 3, defining the role of the γ4 dopaminergic neurons in water learning. ts1 (JFRC100) (a) 3 min memory performance of thirsty R48B04; UAS-shi flies is indistinguishable from that of controls at 23 C (P=0.34, ts1 (JFRC100) n=8; ANOVA). (b) Drinking of R48B04-GAL4; UAS-shi flies is not statistically impaired at 32 C (P=0.24, n=8; ANOVA). (c)
6 Thirsty R48B04-GAL4; UAS-shi ts1 (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-shi ts1 /R48B04-LexA;UAS-LexAi/0104-GAL4 flies show normal 3min water memory performance at 23 C (P=0.89, n=8; ANOVA).(h) Water drinking control for Fig. 3h. Drinking of lexaop-shi ts1 /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-shi ts1 /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-shi ts1 ; 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-shi ts1 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-shi ts1 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-shi ts1 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-shi ts1 ; 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-shi ts1 flies (P=0.04, n=8; ANOVA followed by post hoc Tukey HSD test).
7 Supplementary Figure 6 Additional experiments to accompany Figure 4, defining the role of the βʹ 2 dopaminergic neurons in naive water-seeking. (a) Permissive temperature control for Fig. 4b and d. Thirsty R48B04-GAL4; UAS-shi ts1 and 0104-GAL4; UAS-shi ts1 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-shi ts1 (JFRC100) flies show normal water approach behavior at 23 C (P=0.41, n=8; ANOVA). (d) Blocking R48B04 neurons with UAS-shi ts1 (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-shi ts1 ; UAS-LexAi flies show normal water approach behavior at 23ºC (P=0.36, n=8; ANOVA). (f) Sated
8 R48B04-LexA/ LexAop-shi ts1 ; UAS-LexAi flies show normal water avoidance behavior at the restricted temperature of 32 C (P=0.23, n=8; ANOVA). (g) Thirsty dumb 1 mutant flies show normal naïve water-seeking behavior (P=0.9, n=8; ANOVA).
9 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).
10 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).
11 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.
12 Fly strains Description Figures Color Wild-type Canton-S 1f, 1g, 2a, 2b, S2, S3 ppk28 Loss-of-function mutant of 1f, 1g, S2 the osmosensitive ion channel required for water taste Tbh m18 Tyramine β hydroxylase 2a mutant that cannot synthesize octopamine. dumb 1 Loss-of-function allele of 2b, S3 dopamine receptor DopR1 UAS-DopR1; dumb 2 Loss-of-function allele in 2b, S3 dopamine receptor DopR1 UAS-DopR1/NP1131; Rescue DopR1 in MB γ and 2b, S3 dumb 2 subset of α β neurons in dumb 2 background UAS-DopR1/c739; dumb 2 Rescue DopR1 in MB αβ 2b, S3 neurons in dumb 2 background UAS-DopR1/c305a; dumb 2 Rescue DopR1 in MB α β 2b, S3 neurons in dumb 2 background UAS-DopR1/201Y; dumb 2 Rescue DopR1 in MB γ and αβ-core neurons in dumb 2 2b, S3 UAS-shi ts1 ts1 (JFRC100) UAS-shi A lexaop-shi ts1 ; UAS-LexAi Tdc2-GAL GAL4 R58E02-GAL4 TH-GAL GAL4 NP2583-GAL4 background A temperature-sensitive dominant-negative dynamin transgene under UAS control temperature-sensitive dominant-negative dynamin transgene under UAScontrol A temperature-sensitive dominant-negative dynamin transgene under LexAopcontrol and a LexA RNAi transgene under UAS control Labels most octopaminergic/tyraminergic neurons Labels entire PAM cluster of dopaminergic neurons and some MB output neurons Labels ~90 dopaminergic neurons in PAM cluster Labels all 12 dopaminergic neurons in PPL1 cluster and a few neurons in PAM cluster neurons innervating β1 and β2 zones neurons innervating β1 and α1 zones 2a, 2c-e,, 3d, 3k-m, 3o, 4b-e, 4f, S4, S5l-n, S6a-b, S7 3d, 4c, S5a, S5b, S5c, S6c, S6e 3k, 4e, S5g, S5h, S5i, S6d, S6f 2a 2c, 2e, S4a, S4b, S4c, S8 2c, 2e, S4a, S4c, S8 S4b 2d
13 R77E12-GAL4 R87D06-GAL4 R15A04-GAL4 R48B04-GAL GAL4 R48B04-LexA; 0104-GAL4 R15A04-GAL80; R48B04- GAL4 R48B04-LexA Tdc2-GAL4; UAS-shi ts GAL4; UAS-shi ts1 R58E02-GAL4; UAS-shi ts1 TH-GAL4; UAS-shi ts GAL4; UAS-shi ts1 NP2583-GAL4; UAS-shi ts1 R77E12-GAL4; UAS-shi ts1 R87D06-GAL4; UAS-shi ts1 R15A04-GAL4; UAS-shi ts1 neurons innervating γ5, β1 and β 2a zones neurons innervating α1 zones neurons innervating γ5, β2, β 1, α1 zones neurons innervating γ5, γ4, and β 2 zones neurons innervating γ5, γ4, β 2 and β2 zones LexA expressed in R48B04 neurons and GAL4 expressed in 0104 neurons neurons innervating γ4, and β 2 zones neurons innervating γ5, γ4, and β 2 zones Block Tdc2-GAL4 labeled neurons in a temperaturedependent Block 0273-GAL4 labeled neurons in a temperaturedependent Block R58E02-GAL4 labeled neurons in a Block TH-GAL4 labeled neurons in a temperaturedependent Block 0279-GAL4 labeled neurons in a temperaturedependent Block NP2583-GAL4 labeled neurons in a Block R77E12-GAL4 labeled neurons in a Block R87D06-GAL4 labeled neurons in a Block R15A04-GAL4 labeled neurons in a, 3d, 4b-c, S5a-c, S6a-d, S7, S8, 4d, 4f, S6a, S6b 3k, 3l, 3m, S5g, S5h, S5i, S5j, S5k 4e, S6e, S6f 3o, S5l, S5m, S5n 4e, S6e, S6f 2a 2c, 2e, S4a, S4b, S4c 2c, 2e, S4a, S4b, S4c 2d
14 R48B04-GAL4 UAS-shi ts1 R48B04-GAL4 UAS-shi ts1 (JFRC100) 0104-GAL4; UAS-shi ts1 lexaop-shi ts1 /R48B04-LexA; UAS-LexAi/0104-GAL4 lexaop-dtrpa1/r48b04- LexA; UAS-LexAi/0104- GAL4 UAS-shi ts1(jfrc100) ; R48B04- GAL4/R58E02-GAL80 R15A04-GAL80; R48B04- GAL4/UAS-shi ts1 lexaop-shi ts1 /R48B04-LexA; UAS-LexAi Supplementary table 1. Block R48B04-GAL4 labeled neurons in a Block R48B04-GAL4 labeled neurons in a Block 0104-GAL4 labeled neurons in a temperaturedependent Block PAM-γ4/5 dopaminergic neurons in a Activate PAM-γ4/5 dopaminergic neurons in a R58E02-GAL80 suppresses R48B04-GAL4 activity in PAM cluster to test whether phenotype results from R4804 blocking dopaminergic neurons Block PAM-γ4 and βʹ dopaminergic neurons in a Block R48B04-LexA labeled dopaminergic neurons in a, 4b, S6a, S6b, S7, S8 3d, 4c, S5a, S5b, S5c, S6c, S6d, 4d, 4f, S6a, S6b 3k, 4e, S5g, S5h, S5i, S6e, S6f 3l, 3m, S5j, S5k 3d, 3c, S5c, S6c, S6d 3o, S5l, S5m, S5n 4e, S6e, S6f Fly strains and their corresponding colors used in the bar-graphs.
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