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. 2020 Nov 3;9:e58942. doi: 10.7554/eLife.58942

Figure 4. pC1d, but not pC1e, significantly increases aggressive social interactions in female flies.

(A) MIP (63x) image of the central brain of a female from the pC1dSS1 split-GAL4 line crossed with 20xUAS-CsChrimson::mVenus and stained with anti-GFP antibody. Images of the complete brain and ventral nerve cord of a female and male of the same genotype are shown in Figure 4—figure supplement 1A–C. (B) Percentage of flies engaging in aggressive behaviors over the course of a trial during which a 30 s 0.4 mW/mm2 continuous light stimulus (pink shading) was delivered, plotted as in Figure 1F. (C) Total time an individual spent performing aggressive behaviors during each of four 30 s periods: prior to, during, immediately following, and 30–60 s after the stimulus. Points represent individual flies. Note that we used 20xUAS-CsChrimson for these experiments to be consistent with the experiments done with aIPg split-GAL4 lines, but the levels of aggression observed with pC1dSS1 are actually higher when a weaker effector line (5xUAS-CsChrimson) is used (Figure 4—figure supplement 9A). Box-and-whisker plots show median and IQR; whiskers show range. Kruskal-Wallis and Dunn’s post hoc tests were used for statistical analysis. Asterisk indicates significance from 0: *p<0.05; **p<0.01; ****p<0.0001; n.s., not significant. (D) MIP (63x) image of the central brain of a female from the pC1eSS1 line crossed with 20xUAS-CsChrimson::mVenus and stained with anti-GFP antibody. Images of the complete brain and ventral nerve cord of a female and male of the same genotype are shown in Figure 4—figure supplement 2A–C. (E) Percentage of flies engaging in aggressive behaviors over the course of a trial during which a 30 s 0.4 mW/mm2 continuous light stimulus (pink shading) was delivered, plotted as in Figure 1F. No obvious differences between genotypes were observed. Data supporting the plots shown in the individual panels were as follows: B: 20xUAS-CsChrimson, n = 2 experiments; pC1dSS1, n = 3 experiments; EmptySS > 20xUAS-CsChrimson, n = 5 experiments; pC1dSS1 > 20xUAS-CsChrimson, n = 5 experiments. C: 20xUAS-CsChrimson, n = 29 flies; pC1dSS1, n = 48 flies; EmptySS > 20xUAS-CsChrimson, n = 46 flies; pC1dSS1 > 20xUAS-CsChrimson, n = 53 flies. E: 20xUAS-CsChrimson, n = 2 experiments; pC1eSS1, n = 2 experiments; EmptySS > 20xUAS-CsChrimson, n = 3 experiments; pC1eSS1 > 20xUAS-CsChrimson, n = 3 experiments. For all panels, data are representative of at least three independent biological repeats, one of which is shown here; see Supplementary file 3 for exact p-values.

Figure 4—source data 1. Source data for behavioural experiments in Figure 4.

Figure 4.

Figure 4—figure supplement 1. Expression patterns of pC1d split-GAL4 lines.

Figure 4—figure supplement 1.

(A–F) 20X MIP images of the brains and ventral nerve cords of the indicated split-GAL4 lines crossed to 20xUAS-CsChrimson::mVenus. The scale bar shown in A applies to panels A - F. (D'–F') Enlargements of the central brain of the images shown in D - F. (A–C) The SS56987 (pC1dSS1) expression pattern is shown; the neuropil reference channel shown in A (gray). Note that no expression in pC1d neurons is seen in the male nervous system which was imaged under identical conditions to the female nervous system shown in B. (D,D') Expression pattern of SS57598 (pC1dSS2). (E,E') Expression pattern of SS43274 (pC1dSS3). Note that this line expresses in both pC1d and pC1e. This line shows expression in males in a cell type clearly distinct from pC1d. Arrows indicate cell bodies. (F,F'). Expression pattern of SS59331 (pC1dSS4). Note that this line expresses in both pC1d and pC1e. Arrows indicate cell bodies.
Figure 4—figure supplement 2. Expression patterns of pC1e split-GAL4 lines.

Figure 4—figure supplement 2.

(A–E) 20X MIP images of the brains and ventral nerve cords of the indicated split-GAL4 lines crossed to 20xUAS-CsChrimson::mVenus. The scale bar shown in A applies to panels A-E. (D',E') Enlargements of the central brain of the images shown in D and E. (A–C) The SS59336 (pC1eSS1) expression pattern is shown; the neuropil reference channel is shown in A (gray). Note that no expression in aIPg neurons is seen in the male nervous system (C) which was imaged under identical conditions to the female nervous system shown in B. (D,D') Expression pattern of SS39313 (pC1eSS2). This line shows expression in males in a cell type clearly distinct from pC1e (not shown). (E,E') Expression pattern of SS59433 (pC1eSS3).
Figure 4—figure supplement 3. Morphologies of individual pC1d neurons.

Figure 4—figure supplement 3.

The images in all panels were generated by stochastic labeling of pC1d split-GAL4 lines using the MultiColor FlipOut method (Nern et al., 2015). Both brain hemispheres are shown. (A–D) Images from SS56987 (pC1dSS1). The arrow in (B) highlights a process that crosses the midline which is often seen in pC1d cells; see (D') and (F') in Figure 4—figure supplement 1 for additional examples. (A) The right and left hemisphere pC1d cells are shown. (E,F) Images from SS43274 (pC1dSS3).
Figure 4—figure supplement 4. Morphologies of individual pC1e neurons.

Figure 4—figure supplement 4.

The images in all panels were generated by stochastic labeling of pC1e split-GAL4 lines using the MultiColor FlipOut method (Nern et al., 2015). (A,B) Images from SS59336 (pC1eSS1). (A) The right and left hemisphere pC1e cells are shown. (C,D) Images from SS43274 (pC1dSS3). Note this line contains both pC1d and pC1e cells. See Figure 4—figure supplement 3 for pC1d cells.
Figure 4—figure supplement 5. Behavioral characterization of female flies after pC1d activation.

Figure 4—figure supplement 5.

Behaviors were measured over the course of a trial during which a 30 s 0.4 mW/mm2 continuous light stimulus (pink shading) was delivered. (A) Average percentage of flies per experiment engaging in aggressive behaviors over the 30 s period prior to or during stimulus delivery. (B,C,D) Percentage of female flies walking (B), touching (C), or chasing (D), plotted as in Figure 1F. (C', D') Average percentage of flies touching (C') or chasing (D') over the 30 s period prior to or during the stimulus delivery in C or D, respectively. Points represent separate experiments consisting of approximately 15 flies. EmptySS > 20xUAS-CsChrimson, n = 5 experiments; pC1dSS1 > 20xUAS-CsChrimson, n = 5 experiments. Data are representative of at least three independent biological repeats, one of which is shown here; see Supplementary file 3 for exact p-values. Box-and-whisker plots show median and IQR; whiskers show range. Mann-Whitney U-tests were used for statistical analysis. Asterisk indicates significance from 0: *p<0.05; **p<0.01; n.s., not significant.
Figure 4—figure supplement 6. Optogenetic activation of additional lines labeling pC1d split-GAL4 lines display similar behavioral results to pC1dSS1.

Figure 4—figure supplement 6.

(A) Percentage of flies engaging in aggressive behaviors over the course of a trial during which a 30 s 0.1 mW/mm2 continuous light stimulus (pink shading) was delivered, plotted as in Figure 1F. Note that two pC1d split-GAL4 lines label just pC1d and two others label both pC1d and pC1e, as indicated. Individual curves are shown for comparison. (B) Average percentage of flies engaging in aggressive behaviors over the 30 s period prior to or during the delivery of a 0.1 mW/mm2 stimulus. Note that the amount of aggression seen in lines expressing either in just pC1d or both pC1d and pC1e are not significantly different. Points represent separate experiments consisting of approximately 15 flies. Data supporting the plots shown in the individual panels were as follows: (A) EmptySS > Chrimson, n = 1 experiment; pC1dSS1 > Chrimson, n = 3 experiments; pC1dSS2 > Chrimson, n = 6 experiments; pC1dSS3 > Chrimson, n = 5 experiments; pC1dSS4 > Chrimson, n = 5 experiments. B: EmptySS > Chrimson, n = 1 experiment; pC1dSS1 > Chrimson, n = 3 experiments; pC1eSS1 > Chrimson, n = 3 experiments; pC1dSS3 > Chrimson, n = 5 experiments. Data was pooled from two independent biological replicates, which included separate parental crosses and were collected on different days. Data are representative of at least two independent biological repeats; see Supplementary file 3 for exact p-values. Box-and-whisker plots show median and IQR; whiskers show range. Kruskal-Wallis and Dunn’s post hoc tests were used for statistical analysis. Asterisk indicates significance from 0: n.s., not significant.
Figure 4—figure supplement 7. Optogenetic stimulation of pC1dSS1 > Chrimson males does not result in aggressive behavior.

Figure 4—figure supplement 7.

(A–B) Percentage of male flies touching (A) or performing aggressive behaviors (B) over the course of a trial during which a 30 s 0.4 mW/mm2 continuous light stimulus (pink shading) was delivered, plotted as in Figure 1F. 20xUAS-CsChrimson, n = 5 experiments; pC1dSS1, n = 4 experiments; EmptySS > 20xUAS-CsChrimson, n = 3 experiments; pC1dSS1 > 20xUAS-CsChrimson, n = 5 experiments. Data are representative of at least two independent biological repeats, one of which is shown here.
Figure 4—figure supplement 8. Optogenetic activation of aggression depends on feeding all trans-retinal.

Figure 4—figure supplement 8.

(A) Percentage of flies that engaged in aggressive behaviors during a trial in which a 30 s 0.1 mW/mm2 continuous light stimulus (pink shading) was delivered, plotted as in Figure 1F. Flies were raised with food supplemented with all trans-retinal (+ATR) or not (-ATR). (B) Average percentage of flies engaging in aggressive behaviors over the 30 s period prior to or during the stimulus delivery in A. Points represent separate experiments consisting of approximately 15 flies. +ATR: EmptySS > Chrimson, n = 8 experiments; pC1dSS1 > Chrimson, n = 9 experiments; -ATR: EmptySS > Chrimson, n = 7 experiments; pC1dSS1 > Chrimson, n = 8 experiments. Data was pooled from two independent biological replicates, which included separate parental crosses and were collected on different days. Data are representative of at least two independent biological repeats; see Supplementary file 3 for exact p-values. Box-and-whisker plots show median and IQR; whiskers show range. Kruskal-Wallis and Dunn’s post hoc tests were used for statistical analysis. Asterisk indicates significance from 0: **p<0.01; n.s., not significant.
Figure 4—figure supplement 9. Behavioral effects of stimulus delivery and effector strength.

Figure 4—figure supplement 9.

(A) Percentage of flies engaging in aggressive behaviors over the course of a trial during which a 30 s 0.1 mW/mm2 continuous light stimulus (pink shading) was delivered to flies carrying 5XUAS, 10xUAS, and 20xUAS effectors, data plotted as in Figure 1F. Individual curves are shown for comparison. (B) Percentage of flies engaging in aggressive behaviors over the course of a trial during which a 30 s 0.4, 0.1, or 0.04 mW/mm2 continuous light stimulus (pink shading) was delivered, plotted as in Figure 1F. Individual curves are shown for comparison. (C) Average percentage of flies engaging in aggressive behaviors over the 30 s period during stimulus delivery. Data supporting the plots shown in the individual panels were as follows: (A) EmptySS > 20xUAS-Chrimson, n = 2 experiments; pC1dSS1 > 20xUAS-Chrimson, n = 2 experiments; EmptySS > 10xUAS-Chrimson, n = 3 experiments; pC1dSS1 > 10xUAS-Chrimson, n = 2 experiments; EmptySS > 5xUAS-Chrimson, n = 3 experiments; pC1dSS1 > 5xUAS-Chrimson, n = 3 experiments. (B) 0.04 mW/mm2: EmptySS > Chrimson, n = 3 experiments; pC1dSS1 > Chrimson, n = 3 experiments; 0.1 mW/mm2: EmptySS > Chrimson, n = 4 experiments; pC1dSS1 > Chrimson, n = 4 experiments; 0.4 mW/mm2: EmptySS > Chrimson, n = 5 experiments; pC1dSS1 > Chrimson, n = 5 experiments. (C) 0.1 mW/mm2: EmptySS > 5xUAS-Chrimson, n = 6 experiments; pC1dSS1 > 5xUAS-Chrimson, n = 6 experiments; EmptySS > 20xUAS-Chrimson, n = 6 experiments; pC1dSS1 > 20xUAS-Chrimson, n = 7 experiments; 0.4 mW/mm2: EmptySS > 5xUAS-Chrimson, n = 6 experiments; pC1dSS1 > 5xUAS-Chrimson, n = 2 experiments; EmptySS > 20xUAS-Chrimson, n = 4 experiments; pC1dSS1 > 20xUAS-Chrimson, n = 6 experiments. Bars are mean ± S.E.M. Data was pooled from two independent biological replicates, which included separate parental crosses and were collected on different days. Data are representative of at least two independent biological repeats.
Figure 4—figure supplement 10. Behavioral effects of the frequency of optogenetic stimulation.

Figure 4—figure supplement 10.

(A,B) Blocks of 30 s photostimulation (gray bars) with increasing stimulation frequency separated by 30 (A) or 60 (B) second intervals were delivered sequentially to pC1dSS1females. Light was delivered at 0.1 mW/mm2 with at a 10 ms pulse width, but with increasing pulse frequency. The pulse period and pulse number during each period was as follows: 1000 ms, 30 (1 Hz); 200 ms, 150 (5 Hz); 100 ms, 300 (10 Hz); 50 ms, 600 (20 Hz); 33 ms, 909 (30 Hz); 20 ms, 1500 (50 Hz). pC1dSS1 > 20xUAS-Chrimson is shown in purple; EmptySS > 20xUAS-Chrimson is shown in gray. The mean is represented as a solid line and shaded error bars represent variation between experiments. (C) Average percentage of flies performing aggressive behaviors over the 30 s period during stimulus delivery in B. (D) Average percentage of flies performing aggressive behaviors over the following periods: the 30 s before the onset of the first stimulus (1 Hz), the 60 s periods between subsequent stimuli, and the 30 s after the last stimulus (50 Hz) in B. Points indicate separate experiments. Data supporting the plot shown in the panel A were as follows: EmptySS > 20xUAS-Chrimson, n = 8 experiments; pC1dSS1 > 20xUAS-Chrimson, n = 8 experiments; (B) EmptySS > 20xUAS-Chrimson, n = 3 experiments; pC1dSS1 > 20xUAS-Chrimson, n = 5 experiments. Data was pooled from two independent biological replicates, which included separate parental crosses and were collected on different days. Data are representative of at least two independent biological repeats; see Supplementary file 3 for exact p-values. Box-and-whisker plots show median and IQR; whiskers show range. Mann-Whitney U-tests were used for statistical analysis. Asterisk indicates significance from 0: *p<0.05; n.s., not significant.
Figure 4—figure supplement 11. pC1d activation also increases aggression against wild-type females and males.

Figure 4—figure supplement 11.

(A–C) Total time an individual spent performing aggressive behaviors over the 30 s period prior to or during stimulation, see Figure 2 legend for detail. (A'–C') Amount of time during a 30 s 0.1 mW/mm2 continuous stimulation period until first aggressive encounter. Points indicate individual flies. Dotted lines at 30 s indicate the end of the trial and error bars are mean ± S.E.M. Data supporting the plots shown in the individual panels were as follows: (A) EmptySS > 20xUAS-Chrimson, n = 22 flies; pC1dSS1 > 20xUAS-Chrimson, n = 22 flies; (B) EmptySS > 20xUAS-Chrimson, n = 8 flies; pC1dSS1 > 20xUAS-Chrimson, n = 8 flies; (C) EmptySS > 20xUAS-Chrimson, n = 7 flies; pC1dSS1 > 20xUAS-Chrimson, n = 8 flies. Data are representative of at least two independent biological repeats, one of which is shown here; see Supplementary file 3 for exact p-values. Box-and-whisker plots show median and IQR; whiskers show range. A Mann-Whitney U post hoc test was used for statistical analysis. Asterisk indicates significance from 0: *p<0.05; **p<0.01; ***p<0.001; ****p<0.0001.
Figure 4—figure supplement 12. pC1d inactivation did not significantly diminish aggressive behavior.

Figure 4—figure supplement 12.

Total time an individual spent performing aggressive behaviors over a 30 min trial; for more experimental detail see Figure 1—figure supplement 10E and Methods. Points indicate individual flies. pC1dSS1 > UAS-GFP, n = 42 flies; EmptySS > UAS-TNTe, n = 28 flies; pC1dSS1 > UAS-TNTe, n = 24 flies. Data are representative of at least two independent biological repeats, one of which is shown here; see Supplementary file 3 for exact p-values. Box-and-whisker plots show median and IQR; whiskers show range. Kruskal-Wallis and Dunn’s post hoc tests were used for statistical analysis. Asterisk indicates significance from 0: n.s., not significant.
Figure 4—figure supplement 13. Optogenetic activation of additional lines labeling pC1e.

Figure 4—figure supplement 13.

(A) Percentage of flies engaging in aggressive behaviors over the course of a trial during which a 30 s 0.1 mW/mm2 continuous light stimulus (pink shading) was delivered, plotted as in Figure 1F. (B) Average percentage of flies engaging in aggressive behaviors over the 30 s period prior to or during the delivery of a 0.1 mW/mm2 stimulus. Data supporting the plots shown in the individual panels were as follows: (A) EmptySS > Chrimson, n = 1 experiment; pC1eSS1 > Chrimson, n = 3 experiments; pC1eSS2 > Chrimson, n = 3 experiments; pC1eSS3 > Chrimson, n = 3 experiments. (B) EmptySS > 20xUAS-CsChrimson, n = 6 experiments; pC1eSS1 > 20xUAS-CsChrimson, n = 6 experiments. Data are representative of at least two independent biological repeats, one of which is shown here; see Supplementary file 3 for exact p-values. Box-and-whisker plots show median and IQR; whiskers show range. A Mann-Whitney U post hoc test was used for statistical analysis. Asterisk indicates significance from 0: n.s., not significant.
Figure 4—figure supplement 14. Behavioral effects of stimulus delivery and effector copy number.

Figure 4—figure supplement 14.

(A) Percentage of flies engaging in aggressive behaviors over the course of a trial during which a 30 s 0.1 mW/mm2 continuous light stimulus (pink shading) was delivered to flies carrying either 5xUAS, 10XUAS or 20xUAS effectors, time course plotted as in Figure 1F. (B) Percentage of flies engaging in aggressive behaviors over the course of a 2 min trial during which a 30 s 0.4, 0.1, or 0.04 mW/mm2 continuous light stimulus (pink shading) was delivered, plotted as in Figure 1F. (C,D) Blocks of 30 s photostimulation (gray bars) with increasing stimulation frequency separated by 30 (C) or 60 (D) second intervals were delivered sequentially to females. Light was delivered at 0.1 mW/mm2 with a 10 ms pulse width and increasing frequencies resulting in higher numbers of pulses given over the 30 s stimulus on period. The pulse period and pulse number during each period was as follows: 1000 ms, 30 (1 Hz); 200 ms, 150 (5 Hz); 100 ms, 300 (10 Hz); 50 ms, 600 (20 Hz); 33 ms, 909 (30 Hz); 20 ms, 1500 (50 Hz). Data supporting the plots shown in the individual panels were as follows: (A) EmptySS > 20xUAS-Chrimson, n = 2 experiments; pC1eSS1 > 20xUAS-Chrimson, n = 2 experiments; EmptySS > 10xUAS-Chrimson, n = 3 experiments; pC1eSS1 > 10xUAS-Chrimson, n = 2 experiments; EmptySS > 5xUAS-Chrimson, n = 3 experiments; pC1eSS1 > 5xUAS-Chrimson, n = 2 experiments. (B) 0.04 mW/mm2: EmptySS > Chrimson, n = 3 experiments; pC1eSS1 > Chrimson, n = 3 experiments; 0.1 mW/mm2: EmptySS > Chrimson, n = 4 experiments; pC1eSS1 > Chrimson, n = 3 experiments; 0.4 mW/mm2: EmptySS > Chrimson, n = 5 experiments; pC1eSS1 > Chrimson, n = 5 experiments. (C) EmptySS > 20xUAS-Chrimson, n = 8 experiments; pC1eSS1 > 20xUAS-Chrimson, n = 7 experiments; (D) EmptySS1 > 20xUAS-Chrimson, n = 1 experiment; pC1eSS1 > 20xUAS-Chrimson, n = 2 experiments. Data are representative of at least two independent biological repeats, one of which is shown here.
Figure 4—figure supplement 15. Comparison of activation phenotypes of pC1d, pC1e and pC1a-c.

Figure 4—figure supplement 15.

(A–C) Percentage of female flies engaged in aggressive behaviors (A) touching (B) or chasing (C) over the course of a trial during which a 30 s 0.1 mW/mm2 continuous light stimulus was delivered (pink shading), time courses plotted as in Figure 1F. EmptySS > 20xUAS-Chrimson, n = 4 experiments; pC1a-cSS > 20xUAS-Chrimson, n = 4 experiments; pC1dSS1 > 20xUAS-Chrimson, n = 5 experiments; pC1eSS1 > 20xUAS-Chrimson, n = 6 experiments. Data was pooled from two independent biological replicates, which included separate parental crosses and were collected on different days. Data are representative of at least two independent biological repeats. The pC1a-c line was provided by K. Wang and Barry Dickson.