P post hoc P post hoc (c) - Nature Research · amount of water in 2 min that is indistinguishable...
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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). Nature Neuroscience: doi:10.1038/nn.3827
Transcript of P post hoc P post hoc (c) - Nature Research · amount of water in 2 min that is indistinguishable...
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).
Nature Neuroscience: doi:10.1038/nn.3827
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).
Nature Neuroscience: doi:10.1038/nn.3827
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).
Nature Neuroscience: doi:10.1038/nn.3827
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).
Nature Neuroscience: doi:10.1038/nn.3827
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)
Nature Neuroscience: doi:10.1038/nn.3827
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 3min 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).
Nature Neuroscience: doi:10.1038/nn.3827
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-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 23ºC (P=0.36, n=8; ANOVA). (f) Sated
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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).
Nature Neuroscience: doi:10.1038/nn.3827
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).
Nature Neuroscience: doi:10.1038/nn.3827
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).
Nature Neuroscience: doi:10.1038/nn.3827
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.
Nature Neuroscience: doi:10.1038/nn.3827
Fly strains Description Figures Color Wild-type Canton-S 1f, 1g, 2a, 2b, S2, S3 ppk28 Loss-of-function mutant of
the osmosensitive ion channel required for water taste
1f, 1g, S2
Tbhm18 Tyramine β hydroxylase mutant that cannot synthesize octopamine.
2a
dumb1 Loss-of-function allele of dopamine receptor DopR1
2b, S3
UAS-DopR1; dumb2 Loss-of-function allele in dopamine receptor DopR1
2b, S3
UAS-DopR1/NP1131; dumb2
Rescue DopR1 in MB γ and subset of α′β′ neurons in dumb2 background
2b, S3
UAS-DopR1/c739; dumb2 Rescue DopR1 in MB αβ neurons in dumb2 background
2b, S3
UAS-DopR1/c305a; dumb2 Rescue DopR1 in MB α′β′ neurons in dumb2 background
2b, S3
UAS-DopR1/201Y; dumb2 Rescue DopR1 in MB γ and αβ-core neurons in dumb2 background
2b, S3
UAS-shits1 A temperature-sensitive dominant-negative dynamin transgene under UAS control
2a, 2c-e, 3a, 3d, 3k-m, 3o, 4b-e, 4f, S4, S5l-n, S6a-b, S7
UAS-shits1 (JFRC100) A temperature-sensitive dominant-negative dynamin transgene under UAS-control
3d, 4c, S5a, S5b, S5c, S6c, S6e
lexAop-shits1; UAS-LexAi A temperature-sensitive dominant-negative dynamin transgene under LexAop-control and a LexA RNAi transgene under UAS control
3k, 4e, S5g, S5h, S5i, S6d, S6f
Tdc2-GAL4 Labels most octopaminergic/tyraminergic neurons
2a
0273-GAL4 Labels entire PAM cluster of dopaminergic neurons and some MB output neurons
2c, 2e, S4a, S4b, S4c, S8
R58E02-GAL4 Labels ~90 dopaminergic neurons in PAM cluster
2c, 2e, S4a, S4c, S8 S4b
TH-GAL4 Labels all 12 dopaminergic neurons in PPL1 cluster and a few neurons in PAM cluster
2d
0279-GAL4 Labels PAM dopaminergic neurons innervating β1 and β2 zones
3a
NP2583-GAL4 Labels PAM dopaminergic neurons innervating β1 and α1 zones
3a
Nature Neuroscience: doi:10.1038/nn.3827
R77E12-GAL4 Labels PAM dopaminergic neurons innervating γ5, β1 and β′2a zones
3a
R87D06-GAL4 Labels PAM dopaminergic neurons innervating α1 zones
3a
R15A04-GAL4 Labels PAM dopaminergic neurons innervating γ5, β2, β′1, α1 zones
3a
R48B04-GAL4 Labels PAM dopaminergic neurons innervating γ5, γ4, and β′2 zones
3a, 3d, 4b-c, S5a-c, S6a-d, S7, S8
0104-GAL4 Labels PAM dopaminergic neurons innervating γ5, γ4, β′2 and β2 zones
3a, 4d, 4f, S6a, S6b
R48B04-LexA; 0104-GAL4 LexA expressed in R48B04 neurons and GAL4 expressed in 0104 neurons
3k, 3l, 3m, S5g, S5h, S5i, S5j, S5k
4e, S6e, S6f R15A04-GAL80; R48B04-GAL4
Labels PAM dopaminergic neurons innervating γ4, and β′2 zones
3o, S5l, S5m, S5n
R48B04-LexA Labels PAM dopaminergic neurons innervating γ5, γ4, and β′2 zones
4e, S6e, S6f
Tdc2-GAL4; UAS-shits1 Block Tdc2-GAL4 labeled neurons in a temperature-dependent manner
2a
0273-GAL4; UAS-shits1 Block 0273-GAL4 labeled neurons in a temperature-dependent manner
2c, 2e, S4a, S4b, S4c
R58E02-GAL4; UAS-shits1 Block R58E02-GAL4 labeled neurons in a temperature-dependent manner
2c, 2e, S4a, S4b, S4c
TH-GAL4; UAS-shits1 Block TH-GAL4 labeled neurons in a temperature-dependent manner
2d
0279-GAL4; UAS-shits1 Block 0279-GAL4 labeled neurons in a temperature-dependent manner
3a
NP2583-GAL4; UAS-shits1 Block NP2583-GAL4 labeled neurons in a temperature-dependent manner
3a
R77E12-GAL4; UAS-shits1 Block R77E12-GAL4 labeled neurons in a temperature-dependent manner
3a
R87D06-GAL4; UAS-shits1 Block R87D06-GAL4 labeled neurons in a temperature-dependent manner
3a
R15A04-GAL4; UAS-shits1 Block R15A04-GAL4 labeled neurons in a temperature-dependent manner
3a
Nature Neuroscience: doi:10.1038/nn.3827
R48B04-GAL4 UAS-shits1 Block R48B04-GAL4 labeled neurons in a temperature-dependent manner
3a, 4b, S6a, S6b, S7, S8
R48B04-GAL4 UAS-shits1
(JFRC100) Block R48B04-GAL4 labeled neurons in a temperature-dependent manner
3d, 4c, S5a, S5b, S5c, S6c, S6d
0104-GAL4; UAS-shits1 Block 0104-GAL4 labeled neurons in a temperature-dependent manner
3a, 4d, 4f, S6a, S6b
lexAop-shits1/R48B04-LexA; UAS-LexAi/0104-GAL4
Block PAM-γ4/5 dopaminergic neurons in a temperature-dependent manner
3k, 4e, S5g, S5h, S5i, S6e, S6f
lexAop-dTrpA1/R48B04-LexA; UAS-LexAi/0104-GAL4
Activate PAM-γ4/5 dopaminergic neurons in a temperature-dependent manner
3l, 3m, S5j, S5k
UAS-shits1(JFRC100); R48B04-GAL4/R58E02-GAL80
R58E02-GAL80 suppresses R48B04-GAL4 activity in PAM cluster to test whether phenotype results from R4804 blocking dopaminergic neurons
3d, 3c, S5c, S6c, S6d
R15A04-GAL80; R48B04-GAL4/UAS-shits1
Block PAM-γ4 and βʹ′ dopaminergic neurons in a temperature-dependent manner
3o, S5l, S5m, S5n
lexAop-shits1/R48B04-LexA; UAS-LexAi
Block R48B04-LexA labeled dopaminergic neurons in a temperature-dependent manner
4e, S6e, S6f
Supplementary table 1. Fly strains and their corresponding colors used in the bar-graphs.
Nature Neuroscience: doi:10.1038/nn.3827
