1. No category

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
2
c305a/UAS-DopR1; dumb flies that drink significantly more water in 2 min (P=0.0001, n=8; ANOVA followed by post hoc Tukey HSD
1
1
2
2
test). d = dumb ; d = dumb . (b) All thirsty DopR1 mutant flies show normal odor acuity to MCH (P=0.84, n=8; ANOVA) and most to
2
OCT except UAS-DopR1; dumb 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.
ts1
ts1
(a) The 3 min memory performance of thirsty 0273; UAS-shi flies is significantly greater than that of UAS-shi (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
ts1
followed by post hoc Tukey HSD test) at permissive 23˚C. Performance of thirsty R58E02; UAS-shi flies is not statistically different
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from that of either relevant control at permissive 23˚C (P>0.5, n=8; ANOVA). (b) Thirsty 0273-GAL4; UAS-shi and R58E02-GAL4;
ts1
ts1
UAS-shi 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
flies drink significantly less water in 2 min (P<0.0001, n=8; ANOVA followed by post hoc Tukey HSD test), whereas R58B04-GAL4;
ts1
UAS-shi 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.
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)
Nature Neuroscience: doi:10.1038/nn.3827
ts1 (JFRC100)
Thirsty R48B04-GAL4; UAS-shi
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 R48B04LexA 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)
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Permissive temperature control for Fig. 3h. lexAop-shi /R48B04-LexA;UAS-LexAi/0104-GAL4 flies show normal 3min water memory
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performance at 23˚C (P=0.89, n=8; ANOVA).(h) Water drinking control for Fig. 3h. Drinking of lexAop-shi /R48B04-LexA; UASLexAi/0104-GAL4 flies is not significantly different from controls (P>0.08, n=8; ANOVA followed by post hoc Tukey HSD test). (i)
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Olfactory acuity control for Fig. 3h. Thirsty lexAop-shi /R48B04-LexA; UAS-LexAi/0104-GAL4 flies have normal odor acuity to MCH
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(P=0.24, n=8; ANOVA).They displayed higher acuity to OCT than lexAop-shi ; 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; UASLexAi/ 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
ts1
temperature control for Fig. 3l. R15A04-GAL80/R48B04-GAL4; UAS-shi flies exhibit normal 3min water memory performance at 23˚C
ts1
(P=0.998, n=8; ANOVA). (m) Water drinking control for Fig. 3l. R15A04-GAL80/R48B04-GAL4; UAS-shi 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 R15A04ts1
GAL80/R48B04-GAL4; UAS-shi flies was indistinguishable to that of controls (P=0.28, n=8; ANOVA). Acuity to MCH is also not
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significantly different from both controls (P=0.99, n=8 compared to UAS-shi ; P=0.06, n=8 compared to R15A04-GAL80; R48B04GAL4; ANOVA followed by post hoc Tukey HSD test). However, the R15A04-GAL80; R48B04-GAL4 flies were statistically different
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from UAS-shi 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.
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(a) Permissive temperature control for Fig. 4b and d. Thirsty R48B04-GAL4; UAS-shi and 0104-GAL4; UAS-shi 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
ts1
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
(JFRC100) does not alter water avoidance in sated flies (P=0.52, n=8; ANOVA). (e) Permissive temperature control for Fig. 4e. Thirsty
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R48B04-LexA/ LexAop-shi ; UAS-LexAi flies show normal water approach behavior at 23ºC (P=0.36, n=8; ANOVA). (f) Sated
Nature Neuroscience: doi:10.1038/nn.3827
ts1
R48B04-LexA/ LexAop-shi ; UAS-LexAi flies show normal water avoidance behavior at the restricted temperature of 32˚C (P=0.23,
1
n=8; ANOVA). (g) Thirsty dumb 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
Wild-type
ppk28
Tbh
m18
dumb
1
UAS-DopR1; dumb
2
UAS-DopR1/NP1131;
2
dumb
UAS-DopR1/c739; dumb
2
UAS-DopR1/c305a; dumb
UAS-DopR1/201Y; dumb
UAS-shi
ts1
UAS-shi
ts1 (JFRC100)
ts1
lexAop-shi ; UAS-LexAi
Tdc2-GAL4
0273-GAL4
R58E02-GAL4
TH-GAL4
0279-GAL4
NP2583-GAL4
Nature Neuroscience: doi:10.1038/nn.3827
2
2
Description
Canton-S
Loss-of-function mutant of
the osmosensitive ion
channel required for water
taste
Tyramine β hydroxylase
mutant that cannot
synthesize octopamine.
Loss-of-function allele of
dopamine receptor DopR1
Loss-of-function allele in
dopamine receptor DopR1
Rescue DopR1 in MB γ and
subset of α′β′ neurons in
2
dumb background
Rescue DopR1 in MB αβ
2
neurons in dumb
background
Rescue DopR1 in MB α′β′
2
neurons in dumb
background
Rescue DopR1 in MB γ and
2
αβ-core neurons in dumb
background
A temperature-sensitive
dominant-negative dynamin
transgene under UAS
control
A 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
Labels PAM dopaminergic
neurons innervating β1 and
β2 zones
Labels PAM dopaminergic
neurons innervating β1 and
α1 zones
Figures
1f, 1g, 2a, 2b, S2, S3
1f, 1g, S2
2a
2b, S3
2b, S3
2b, S3
2b, S3
2b, S3
2b, S3
2a, 2c-e, 3a, 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
3a
3a
Color
R77E12-GAL4
R87D06-GAL4
R15A04-GAL4
R48B04-GAL4
0104-GAL4
R48B04-LexA; 0104-GAL4
R15A04-GAL80; R48B04GAL4
R48B04-LexA
Tdc2-GAL4; UAS-shi
ts1
0273-GAL4; UAS-shi
ts1
R58E02-GAL4; UAS-shi
TH-GAL4; UAS-shi
ts1
ts1
0279-GAL4; UAS-shi
ts1
NP2583-GAL4; UAS-shi
ts1
R77E12-GAL4; UAS-shi
ts1
R87D06-GAL4; UAS-shi
R15A04-GAL4; UAS-shi
ts1
ts1
Nature Neuroscience: doi:10.1038/nn.3827
Labels PAM dopaminergic
neurons innervating γ5, β1
and β′2a zones
Labels PAM dopaminergic
neurons innervating α1
zones
Labels PAM dopaminergic
neurons innervating γ5, β2,
β′1, α1 zones
Labels PAM dopaminergic
neurons innervating γ5, γ4,
and β′2 zones
Labels PAM dopaminergic
neurons innervating γ5, γ4,
β′2 and β2 zones
LexA expressed in R48B04
neurons and GAL4
expressed in 0104 neurons
Labels PAM dopaminergic
neurons innervating γ4, and
β′2 zones
Labels PAM dopaminergic
neurons innervating γ5, γ4,
and β′2 zones
Block Tdc2-GAL4 labeled
neurons in a temperaturedependent manner
Block 0273-GAL4 labeled
neurons in a temperaturedependent manner
Block R58E02-GAL4
labeled neurons in a
temperature-dependent
manner
Block TH-GAL4 labeled
neurons in a temperaturedependent manner
Block 0279-GAL4 labeled
neurons in a temperaturedependent manner
Block NP2583-GAL4
labeled neurons in a
temperature-dependent
manner
Block R77E12-GAL4
labeled neurons in a
temperature-dependent
manner
Block R87D06-GAL4
labeled neurons in a
temperature-dependent
manner
Block R15A04-GAL4
labeled neurons in a
temperature-dependent
manner
3a
3a
3a
3a, 3d, 4b-c, S5a-c,
S6a-d, S7, S8
3a, 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
3a
3a
3a
3a
3a
R48B04-GAL4 UAS-shi
ts1
R48B04-GAL4 UAS-shi
ts1
(JFRC100)
0104-GAL4; UAS-shi
ts1
ts1
lexAop-shi /R48B04-LexA;
UAS-LexAi/0104-GAL4
lexAop-dTrpA1/R48B04LexA; UAS-LexAi/0104GAL4
ts1(JFRC100)
UAS-shi
; R48B04GAL4/R58E02-GAL80
R15A04-GAL80; R48B04ts1
GAL4/UAS-shi
ts1
lexAop-shi /R48B04-LexA;
UAS-LexAi
Block R48B04-GAL4
labeled neurons in a
temperature-dependent
manner
Block R48B04-GAL4
labeled neurons in a
temperature-dependent
manner
Block 0104-GAL4 labeled
neurons in a temperaturedependent manner
Block PAM-γ4/5
dopaminergic neurons in a
temperature-dependent
manner
Activate PAM-γ4/5
dopaminergic neurons in a
temperature-dependent
manner
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
temperature-dependent
manner
Block R48B04-LexA labeled
dopaminergic neurons in a
temperature-dependent
manner
3a, 4b, S6a, S6b, S7,
S8
3d, 4c, S5a, S5b, S5c,
S6c, S6d
3a, 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
Supplementary table 1.
Fly strains and their corresponding colors used in the bar-graphs.
Nature Neuroscience: doi:10.1038/nn.3827