Neuron, Volume 64
Supplemental Data
Two Different Forms of Arousal in Drosophila Are Oppositely
Regulated by the Dopamine D1 Receptor Ortholog DopR via
Distinct Neural Circuits
Tim Lebestky
, Jung
-
Sook
C.
Chang,
Heiko Dankert, Lihi Zelnik,
Young
-
Cho Kim
,
Ky
ung
-
An Han, Fred W. Wolf, Pietro Perona,
and
Dav
id J. A
nderson
Supplemental Figure S1
Bout structure analy
sis of wild
-
type and DopR/DopR flies
2
-
puff trials were analyzed as in Figure 1D. Vertical bar indicates p
eriod
when puffs were delivered. (A) bout duration. (B) bout frequency. (C) bout
velocity. Error bars indicate S.E.M.
Supplemental Figure S2
Sensitization of stress
-
induced hy
peractiv
ity
(A) Response to single
-
puffs of naïve flies, or flies previously exposed to 6
successive puffs and allowed to settle for 10 minutes prior
to a single
-
puff
trial (“Sens”). (B
-
F) Parameters from experiment in (A). •••, p<.001.
Supplemental Figure S3
Mechanical startle facilitates sensitization of olfactory startle responses
(A) Olfactory startle curves for either naïve CS flies presented with MCH
for 5s (blue), or CS flies that had been sensitized by presentation of 6 air
puffs, followed by a 10 min. recovery period, prior to presentation of the
MCH stimulus (green). (B-F) Parameter plots from data in (A). •, p<.051,
••, p<.01. Note that baseline velocity is not statistically different, and
sensitized animals are not hyperactive prior to MCH presentation.
Sensitized animals display significant differences in peak velocity (B) and
distance travelled (F) in behavioral response to MCH presentation. MCH
odorant is made as a 50% dilution in paraffin oil, delivered through the
tubing of the Puff-o-Mat at 1 psi.
Supplemental Figure S4
Individual Behavioral Parameters for DopR mutants and excision lines
(A-C) Behavioral parameters calculated from the data in Figure 2B. (A)
Baseline velocity. (B) Peak velocity. (C) Peak velocity minus baseline
velocity. (D-F) Behavioral parameters from the data in Figure 2E. (D)
Baseline velocity. (E) Peak velocity. (F) Peak velocity minus baseline
velocity. •, p<.017; ••, p<.003; •••, p<.0003; Kruskal-Wallis ANOVA
followed by Mann-Whitney U-test with Bonferroni correction for multiple
comparisons.
Supplemental Figure S5
Df(3R)ED5634/DopR
PL00420
flies display exaggerated post-puff responses
after increased acclimation time in the puff-o-mat
(A) 1-puff trials of flies of the indicated genotypes. Puff intensity reduced to
10 psi to highlight the difference between mutant and wildtype genotypes.
Df(3R)ED5634
removes the entire DopR locus.
DopR
PL00420
is an
insertional piggyBac allele in the first intron. Q-RTPCR analysis indicated
that
DopR
PL00420
/Df(3R)ED5634
flies exhibit an 80% reduction in
DopR
transcript levels. (B-F) Parameter plots from the data in (A).
.
, p<.05 ;
..
,
p<.01 ;
...
, p<.001. After 40 minutes of acclimation in the puff-o-mat tubes,
the baseline velocities are statistically identical (B), but the post-puff
behavior of the
Df/DopR
PL00420
mutant flies is significantly different from
wildtype controls for all other parameters (C-F). n = 36 tubes per genotype.
DopR
PL00420
/+ showed no phenotypic difference from +/+ in any of the
parameters, while Df/+ showed a significant increase in peak-baseline
velocity and integrated velocity, and a trend to an increase in tau.
Supplemental Figure S6
DopR/DopR
flies still show exaggerated post-puff responses after
increased acclimatization time in the puff-o-mat
(A) Puff-o-mat response curves (2-puff trials) from wild-type and
DopR/DopR
flies allowed to acclimatize for 30 minutes in the puff-o-mat. (B-F) Parameter
plots from data in (A). •, p<.01, ••, p<.01.
Supplemental Figure S7
The
DopR
mutation increases sensitivity to puff number and puff intensity
(A-E) Analysis of behavioral parameters from the data in Figure 3A, B.
White bars, CS (
+/+
) flies; black bars,
DopR/DopR
flies. (F-J) Analysis of
behavioral parameters from the data in Figure 3C, D. (K-O) Analysis of
behavioral parameters from the data in Figure 3E, F. Note the suppression
of
τ
(
ν
) and distance traveled (O) by cocaine in CS flies (white bars), and
the lack of an effect of cocaine in
DopR/DopR
flies (black bars).Letters (a-
c or x-z) denote significant differences (p<.017) within genotypes, •’s
indicate significant differences between genotypes (black vs. white bars).
•••, p<.001, ••, p<.01, •, p<.05
Supplemental Figure S8
Rescue of
DopR/+
phenotype by Elav-Gal4
(A) 2-puff trials of flies of the indicated genotype. Elav-Gal4 was
backcrossed 6 generations into the same CS background as the
DopR
f02676
allele. (B-F) parameter plots from the data in (A). •, p<.05;
••, p<.01; •••, p<.001. Note that
Elav-Gal4; DopR/+
flies are statistically
indistinguishable from control (
Elav-Gal4; +/+
) flies.
Supplemental Figure S9
Neuroanatomical mapping of DopR function
(A1-E2) Additional Parametric analysis of the
data in (Figure 5A-E), respectively. “•••,”
p<.0003, “••,” p<.003, “•,” p<.017. (A1-C2)
Selective rescue of the ReSH phenotype by
restoration of DopR function in the Ellipsoid
Body by different Gal4 lines. 2-puff trials
applied to
DopR/+
heterozygotes (red,
“
DopR”
),
Gal4
/+ controls (blue), and
Gal4
/+;
DopR/+ double heterozygotes (green). (D1-
E2) Conditional rescue of
DopR
in adult
flies using
c547-Gal4
and
tubulin-Gal80
ts
.
“
DopR,”
DopR/DopR
mutants; “Rescue,”
c547-Gal4, DopR/DopR
; “
Gal80ts
,”
tub-
Gal80ts; c547-Gal4, DopR/DopR
. Flies
were raised at 18
o
C and tested at either 18
o
(D) or 30
o
(E). (A1, B1, C1, D1, E1)
Comparison of baseline velocity. (A2, B2,
C2, D2, E2) Comparison of peak velocity.
Supplemental Figure S10
Specificity of anti-DopR antibody staining in the Central Complex
Single confocal sections of whole mount brains double-labeled with
antibody to DopR (green) and FasII (magenta). MB, Mushroom Body;
FSB, fan-shaped body; NOD, noduli; EB, ellipsoid body. Panels A1 and
C1 were imaged at the same gain setting, as were panels B1 and D1.
(A3, B3, C3 and D3) represent merged images of the single channels
shown in (A1-2), (B1-2), (C1-2) and (D1-2), respectively. Scale bar in all
20X panels, 80
μ
m; in 40X panels, 40
μ
m.
Supplemental Figure S11
c547 rescue of
DopR/DopR
homozygous flies
The enhancer trap line c547 was recombined onto the
DopR
f02676
chromosome, to generate
c547,DopR/TM6
flies, from which the parental
stock the indicated genotypes were generated. (A) 2-puff response
curves of the indicated genotypes. (B-F) parameter plots from curves in
(A). p values are defined as in Supplemental Figure S5.