(A) Sample traces from mutant lines screened. Magenta curve represents the mean response of the piggyBac collection. Plots are ranked as outliers in principal component (PC) space. The plot encircled in red is the DopR mutation. (B) Upper, structure of the DopR^f02676 insertional allele; note the UAS in the first intron. Lower, levels of DopR mRNA determined by Q-RT-PCR in different genotypes. (C) Phenotype of DopR/DopR and DopR/+ flies in comparison to CS (+/+) controls (n=40 tubes per genotype). (D, E) Behavioral parameters in DopR mutants, calculated from the data in (C). (F) Phenotypic reversion of flies carrying a precise excision of the f02676 insertion (n=24 tubes per genotype). (G, H) Behavioral parameters in excision line, calculated from the data in (E). Insets in (C) and (F), comparison of genotypes after normalization to +/+ baseline. Additional behavioral parameters of (C) and (F) are available in Supplemental figure S5. In this and all following figures, ^•, p<.017; ^••, p<.003; ^•••, p<.0003; Kruskal-Wallis ANOVA followed by Mann-Whitney U-test with Bonferroni correction for multiple comparisons.

(A) Circadian activity of CS and DopR mutant flies (n=70 individuals per genotype). Note reduced activity of DopR/+ (green) and DopR/DopR (red) flies, relative to +/+ (blue), during the night phase. (B) DopR mutants still show an enhanced ReSH response during the night phase (n=25 tubes per genotype). 2-puff trials were performed at midnight (position of asterisk in 4A). Upper inset, comparison of curves after normalization to +/+ median baseline; Lower inset, τ values. (C-F) Total activity in the circadian monitor during different time periods. Letters on all bar graphs indicate significant differences (p<.017 after Bonferroni correction for multiple comparisons). (G-J) Sleep parameters in wild-type vs. DopR mutant flies during the night phase. (K-N) Cocaine stimulation of circadian activity is DopR-dependent. (K, L) activity plots of wild-type (K) or DopR/DopR (L) flies assayed without (blue) or with (red) 500 μg/ml cocaine. (M, N) Summary of data in (K, L). Note that the strongest effect of cocaine is at night (M) and is suppressed in DopR/DopR mutants (N). n=20 flies per genotype or drug treatment condition in (K-N). ^•••, p<.01.

(A-C) Confocal z-series of wholemount brains double-labeled with antibodies to nc82 (red) and GFP (green). Arrows indicate the ellipsoid body. (A) UAS-mCD8-GFP/+ ; c547/+. (B) 189y/UAS-mCD8-GFP. (C) c761/UAS-mCD8-GFP. (A1-A3), (B1-B3) and (C1-C3) illustrate double-labeling with antibodies to DopR (Green, panels A1, B1, C1) and to GFP (Magenta, panels A2, B2, C2). (D1-D3) illustrates relationship of DopR (D1) to TH⁺ dopaminergic fibers (D2) in the EB. All images represent single 1.7 μm optical sections of whole-mount stained brains. Panels (A1-A3) and (D1-D3) illustrate the same focal plane of the same specimen triply-labeled for DopR, GFP and TH, at low (A1-A3) and high (D1-D3) magnification. MB, mushroom body; EB, ellipsoid body; NOD, noduli. Open arrowheads in (A3) and (D3) indicate the ring of DopR expression in the R3 domain. Scale bars (A-C) = 80 μm, (A1-C3) = 40 μm, (D1-D3) = 20 μm.

(A) Summary of genetic rescue data indicating loci of DopR1 action in Drosophila. EB, ellipsoid body; LNv, lateral-ventral neurons (PDF⁺); MB, mushroom body. (B) Schematic illustrating how cocaine and the DopR mutation influence noctural arousal (sleep-wake transitions) and stress-induced arousal (ReSH behavior) in Drosophila.

(A) Schematic illustrating experimental set-up. (B) Mechanical stress induced by successive airpuffs (vertical arrows) causes persistent locomotor hyperactivity. Solid line represents mean velocity (n = 8 tubes, each containing 10 flies). Thin lines indicate traces from each tube, gray envelope S.E.M. (C) Initial acceleration computed during the interpuff-interval (5 sec following each airpuff). (D) Walking bout frequency prior to and following 6 airpuffs (pink line). (E) Exponential curve-fit to post-puff decay data. See Supplemental Methods for further details. (F) Puff “dose-response” curves. “1p” “2p” etc. indicates number of puffs (1p n=68 tubes, 2p n=64, 3p n=80, 4p n=72, 6p n=84). (G-J) Parameter values extracted from the data in (F) using the equation in (E). “Distance traveled” (J) is computed by integrating the area under the post-puff curve, after subtracting the pre-puff baseline. Lower-case letters indicate statistically significant differences (p<0.005; Kruskal-Wallis ANOVA followed by Mann-Whitney U-test, with Bonferroni correction for multiple comparisons).

(A, B) Puff-titration of CS (A) and DopR/DopR (B) flies (n=44 tubes for each genotype). DopR/DopR flies show heightened responses relative to CS at all puff conditions, and reach a peak response after 2 puffs (green). (C, D) Single-puff trials at the indicated puff intensities for wild-type CS (C, +/+) and DopR/DopR (D) flies (n=24 tubes for each condition). DopR/DopR mutants show increased responses at low puff intensities (D). (E, F) Cocaine suppresses stress-induced locomotor hyperactivity in a DopR-dependent manner (n=32 tubes for each condition). Flies fed with the indicated doses of cocaine were subjected to 2-puff trials. Note the suppression of post-puff activity by cocaine in CS (E) but not DopR/DopR (F) flies. Parametric analysis of data in (A-F) available in Supplemental Figure S6.

(A-C) 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). Superposition of the green and blue curves indicates phenotypic rescue. (A1-C2) Parametric analysis of the data in (A-C), respectively. “^•••,” p<.0003, “^••,” p<.003, “^•,” p<.017 (n=40 tubes per genotype in A, n=44 tubes per genotype in B,C). (D, E) 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°C and tested at either 18° (D) or 30° (E). The partial rescue at 18° likely reflects incomplete suppression of Gal4 by Gal80^ts. (D1-D2, E1-E2) Parameter plots of curves in (D) and (E), respectively (n=38 tubes per genotype in D, n = 32 tubes per genotype in E). (F-H) Double-dissociation of DopR function in learning vs. the ReSH assay. (F) MB247 Gal4 fails to rescue DopR (n=30 tubes per genotype). (G) MB247 rescues the DopR/DopR learning phenotype (green bar), while c547 does not (orange bar) (n=4 replicate experiments per genotype). (H) Expression of GFP in the mushroom body (MB247/+;UAS-mCD8-GFP/+).

(A-C) represent circadian activity of control (Gal4/+; blue curves), mutant (DopR/+; red curves) and rescue (Gal4/+; DopR/+, green curves), using Elav-Gal4, c547-Gal4 and pdf-Gal4 drivers, respectively (n=66 flies per genotype in A, n=46 flies per genotype in B, n=87 flies per genotype in C). Arrowhead in (C) indicates partial rescue of pre-dawn activity by pdf-Gal4. (D-F) Sleep (D, E) and nighttime activity (F) parameters calculated from the data in (A-C). The rescue percentages indicate potency of rescue calculated as [(P_Gal4;DopR - P_DopR)/(P_Gal4 - P_DopR)] × 100%, where P is the value of a given parameter for the genotype indicated by the subscript. “ns,” not significantly different (p>.017, using the Bonferroni correction for multiple comparisons).