The Neural Circuitry that Functions as a Switch for Courtship versus Aggression in Drosophila Males  Masayuki Koganezawa, Ken-ichi Kimura, Daisuke Yamamoto  Current Biology  Volume 26, Issue 11, Pages 1395-1403 (June 2016) DOI: 10.1016/j.cub.2016.04.017 Copyright © 2016 Elsevier Ltd Terms and Conditions

Figure 1 fru-Expressing LC1 Neurons Reciprocally Control Aggression and Courtship (A and B) Anatomical features of LC1 (somata are circled in A), revealed by immunostaining with anti-GFP (green; A, B, and B″) and anti-GABA antibodies (red; B′ and B″) in the brain that was counterstained with nc82 in a male of the genotype w; UAS>stop>mCD8::GFP/+; 9-10-GAL4/fruFLP. The number of LC1 neurons labeled was 12.8 ± 0.5 (mean ± SEM, n = 18). The optical sections containing LC1 cell bodies were stacked in (B). Yellow arrowheads indicate the location of cell bodies of LC1 neurons. Scale bar, 100 μm. (C–F) Quantification of male-to-male (C and E) and male-to-female (D and F) interactions observed in the pair of a test male and a target male fly with the same genotype as the test male or a CS female fly during the observation period of 3 min. To activate (C and D) and inactivate (E and F) LC1, dTrpA1 and TNT (or its inactive control, TNTin) were expressed, respectively, in addition to fruFLP and 9-10-GAL4. Total time spent for wing extension by the test male was measured to quantify courtship, whereas the total number of lunging, holding, tussling, and boxing by the test male was measured to quantify aggression. The total interaction time was taken as the sum of the durations of aggression and courtship. The genotypes and number of pairs are indicated below the graph. (G and H) Requirement of GABA in LC1 for the control of aggression and courtship. The genotypes of test flies were tubP>GAL80>; UAS-Gad1 RNAi (VALIUM20)/+; fruFLP, 9-10-GAL4/+ (G) or w; UAS>stop>dTrpA1/UAS-Gad1 RNAi (VALIUM20); fruFLP/9-10-GAL4 (H). (G) Total interactions, courtship, and aggression between the two males were enhanced when Gad1 knockdown was achieved in LC1 by a selective flipping out of tubP>GAL80 > with fruFLP. (H) Knockdown of Gad1 in LC1 mitigated aggression induced via dTrpA1. Data are shown as the mean ± SEM (C–H). Statistical significance was evaluated by Wilcoxon test (C and D), Mann-Whitney U test (E, F, and H), or Kruskal-Wallis test with Steel-Dwass multiple pairwise post hoc comparisons (G); ∗p < 0.05; ∗∗p < 0.01; ∗∗∗p < 0.001. See also Figures S1, S2, and S4 and Movie S1. Current Biology 2016 26, 1395-1403DOI: (10.1016/j.cub.2016.04.017) Copyright © 2016 Elsevier Ltd Terms and Conditions

Figure 2 fru-Expressing mAL Neurons Suppress Courtship and Aggression (A and B) Anatomical features of mAL (somata are circled in A), revealed by immunostaining with anti-GFP (green; A, B, and B″) and anti-GABA antibodies (red; B′ and B″) in the brain that was counterstained with nc82 in a male of the genotype w; UAS>stop>mCD8::GFP/+; 9-189-GAL4/fruFLP. The number of mAL neurons labeled was 16.0 ± 0.5 (mean ± SEM, n = 12). The optical sections containing mAL cell bodies were stacked in (B). Yellow arrowheads indicate the location of cell bodies of mAL neurons. Scale bar, 100 μm. (C–F) Quantification of male-to-male (C and E) and male-to-female (D and F) interactions observed in the pair of a test male and a target male fly with the same genotype as the test male or a CS female fly during the observation period of 3 min. To activate (C and D) and inactivate (E and F) mAL, dTrpA1 and TNT (or its inactive control, TNTin) were expressed, respectively, in addition to fruFLP and 9-189-GAL4. (G) Requirement of GABA in mAL for the control of aggression and courtship. Total interactions, courtship and aggression between the two males were enhanced when Gad1 knockdown was achieved in mAL by a selective flipping-out of tubP>GAL80 > with fruFLP. The genotype of test flies was tubP>GAL80>; UAS-Dcr2/UAS-mCD8::GFP; fruFLP, 9-189-GAL4/UAS-Gad1 RNAi (VDRC). Data are shown as the mean ± SEM (C–G). Statistical significance was evaluated by Wilcoxon test (C and D), Mann-Whitney U test (E and F), or Kruskal-Wallis test with Steel-Dwass multiple pairwise post hoc comparisons (G); ∗∗p < 0.01; ∗∗∗p < 0.001. See also Figure S2 and Movie S2. Current Biology 2016 26, 1395-1403DOI: (10.1016/j.cub.2016.04.017) Copyright © 2016 Elsevier Ltd Terms and Conditions

Figure 3 LC1 Releases the Aggression Program from the mAL-Mediated Inhibition (A) Aggression induced by the activation of LC1 was significantly reduced by the simultaneous activation of mAL. The genotype of test flies was w; UAS>stop>dTrpA1/+; fruFLP, 9-189-GAL4/9-10-GAL4. (B) Effects of LC1 activation, mAL synaptic block, and the combination of LC1 activation and mAL synaptic block on male-male interactions. The genotypes of test flies were (from left to right) +; UAS>stop>dTrpA1/+; fruFLP, 9-10-GAL4/+, +; R43D01-LexA/+; lexAop-shits/+ and +; R43D01-LexA/UAS>stop>dTrpA1; lexAop-shits/fruFLP, 9-10-GAL4. (C) R43D01-LexA preferentially drives expression in mAL neurons. The genotype of test flies was w; R43D01-LexA/lexAop-rCD2::GFP. (D) mAL neurons are Rdl-GAL4-positive. The genotype of test flies was w; UAS>stop>mCD8::GFP/Rdl-GAL4; fruFLP/+. Somata of mAL neurons are circled in (C) and (D). Scale bar, 100 μm. Data are shown as the mean ± SEM (A and B). Statistical significance was evaluated by Mann-Whitney U test (A) or Kruskal-Wallis test with Steel-Dwass multiple pairwise post hoc comparisons (B); ns, p > 0.05; ∗∗∗p < 0.001. Current Biology 2016 26, 1395-1403DOI: (10.1016/j.cub.2016.04.017) Copyright © 2016 Elsevier Ltd Terms and Conditions

Figure 4 Distinct Fractions of dsx-Expressing pC1 Neurons Each Induce Aggression and Courtship upon Relief from GABAergic Inhibition (A and B) Rdl knockdown restricted to brain dsx-expressing cells (A) promotes both courtship and aggression between males (B). The genotype of test males was tubP>GAL80>; Otd-FLP/UAS-mCD8::GFP; dsxGAL4/+ (A) or tubP>GAL80>; Otd-FLP/UAS-Rdl RNAi (VALIUM20); dsxGAL4/+ (B). Scale bar, 100 μm. Data are shown as the mean ± SEM. (C–F) The two divisions of pC1 each have distinct functions in the regulation of aggression and courtship. (C–E) The NP2631 enhancer trap defines all the composites of the pC1 neural cluster (dsx pC1; D), which can be decomposed into a fru-positive fraction (fru P1; C) and fru-negative fraction (dsx+/fru– pC1; E) by generating flies carrying a different combination of fruFLP, dsxFLP, and fru-GAL80 (fruLexA in combination with lexAop-GAL80) in addition to NP2631 and UAS>stop>mCD8::GFP or UAS-Rdl RNAi (VALIUM20). The numbers of fru P1, dsx pC1 and dsx+/fru– pC1 neurons labeled were 24.7 ± 0.7 (mean ± SEM, n = 12), 37.3 ± 0.6 (n = 12), and 21.9 ± 0.3 (n = 12), respectively. The detailed genotypes are given in the Supplemental Information. Scale bar, 100 μm (C, D, and E) or 50 μm (C′, D′, and E′). (F) Total male-to-male interactions, courtship, and aggression were compared among fru P1 (blue bar), dsx pC1 (green bar), and dsx+/dru– pC1 (red bar). Data are shown as the mean ± SEM. Statistical significance was evaluated by Kruskal-Wallis test with Steel-Dwass multiple pairwise post hoc comparisons; ns, p > 0.05; ∗∗∗p < 0.001. See also Movie S3. Current Biology 2016 26, 1395-1403DOI: (10.1016/j.cub.2016.04.017) Copyright © 2016 Elsevier Ltd Terms and Conditions

Figure 5 Functional Dichotomy of dsx-Expressing pC1 Neurons in the Regulation of Aggression and Courtship (A and B) dTrpA1-mediated activation of fru P1 (blue bar) primarily induces courtship but not aggression, whereas similar activation of dsx+/fru– pC1 (red bar) exclusively induces aggression in two-male assays (A) or single-male assays (B). The effects of activation of the entire pC1 cluster are also shown (green bar). (C) dTrpA1-induced courtship and aggression toward a decapitated male. Wing extension (i.e., courtship: left-hand-side graph) and attacks (i.e., aggression: right-hand-side graph) induced by activation of fru P1 (blue bar) or dsx+/fru– pC1 (red bar) were quantified. Data are shown as the mean ± SEM. Statistical significance was evaluated by Kruskal-Wallis test with Steel-Dwass multiple pairwise post hoc comparisons (A and B) or Wilcoxon test (C); ns, p > 0.05; ∗∗p < 0.01; ∗∗∗p < 0.001. See also Figures S1 and S3 and Movies S4 and S5. Current Biology 2016 26, 1395-1403DOI: (10.1016/j.cub.2016.04.017) Copyright © 2016 Elsevier Ltd Terms and Conditions

Figure 6 LC1 Neurons Inhibit the Courtship Center P1 and Disinhibit the Aggression Center pC1 (A) Effect of mAL coactivation on P1-dependent male-male interactions (courtship, left) and effect of mAL coactivation on pC1-dependent male-male interactions (aggression, right). (B) Pulse song generation in flies in which P1 was activated by dTrpA1 without or with coactivation of mAL or LC1. (C) A postulated relationship among LC1, mAL and two populations (dsx+/fru– pC1 and fru P1 (= dsx+/fru+ pC1)) of pC1 neurons in the control of aggression and courtship. Data are shown as the mean ± SEM (A and B). Statistical significance was evaluated by Mann-Whitney U test (A) or Kruskal-Wallis test with Steel-Dwass multiple pairwise post hoc comparisons (B); ∗p < 0.05; ∗∗p < 0.01; ∗∗∗p < 0.001. Current Biology 2016 26, 1395-1403DOI: (10.1016/j.cub.2016.04.017) Copyright © 2016 Elsevier Ltd Terms and Conditions