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. 2020 Apr 21;9:e54229. doi: 10.7554/eLife.54229

Figure 4. DSK Functions Downstream of a subset of P1 Neurons to Modulate Fighting Behavior.

(A) Transsynaptic labeling by trans-Tango method identifies DSK neurons as postsynaptic partners of a subset of P1 neurons. Expression of the trans-Tango ligand in a subset of P1 neurons (red) (A1) induces postsynaptic signals (anti-HA, magenta) (A2) in the central brain. Green, anti-DSK antibody staining (A2). (A3–A5) and (A6–A8) are enlargements of dashed boxes in (A2). Genotypes of a subset of P1 neurons (P1a split-GAL4): +/y; R15A01.AD/R15A01.AD; R71G01-GAL4.DBD/R71G01-GAL4.DBD. (B) GRASP signals (B1) reveal synaptic connections between R71G01-LexA labeled P1 Neurons and DSK neurons. spGFP1–10 is expressed by DskGal4 drivers; spGFP11 is driven by the R71G01-LexA. Magenta, anti-DSK antibody. (B4–B6) are enlargements of the dashed box in (B3). White arrowheads point to areas in which GRASP signal co-localized with synaptic boutons revealed by anti-DSK antibody staining (B6). Scale bars represent 50 μm in (A1–A2, B1–B3). Scale bars represent 5 μm (A3–A8, B4–B6). (C) Dendrites of DSK neurons revealed by DskGAL4 driven UAS-Denmark expression (red). Axons of a subset of P1 neurons revealed by P1a split-GAL4 driven UAS-sytGFP expression (green). nc82 antibody (blue) was used to label neuropils. Scale bars represent 50 μm. (D–E) Enlargements of dashed boxes in (C). White arrowheads point to areas in which a subset of P1 axons and DSK dendrites overlap. Scale bars represent 5 μm. (F and G) Number of lunges per minute after initiation (F) and fighting latency (G) during thermogenetic activation of a subset of P1 neurons in the ΔDsk mutant background. The aggression-promoting effect of activating a subset of P1 neurons is suppressed by the ΔDsk mutants. ***p<0.001, n.s. indicates no significant difference (Kruskal-Wallis and post-hoc Mann-Whitney U tests).

Figure 4.

Figure 4—figure supplement 1. Controls for the trans-Tango and GRASP experiments.

Figure 4—figure supplement 1.

(A) Negative control demonstrates lack of postsynaptic signals (anti-HA, magenta) (A2) for the trans-Tango experiment. Red, anti-DSK antibody staining (A1). (B–C) Negative controls for the GRASP experiments. Magenta, anti-DSK antibody (B2, C2). Scale bars represent 50 μm.
Figure 4—figure supplement 2. Activation of a subset of P1 neurons could promote courtship in the ΔDsk mutant background.

Figure 4—figure supplement 2.

(A–B) Raster plots of unilateral wing extensions during 10 min. (C–D) The number (C) and total duration (D) of unilateral wing extensions in (A–B) during thermogenetic activation of a subset of P1 neurons in the ΔDsk mutant background (black: 21°C, red: 28°C). a subset of P1 neurons can promote courtship behavior in ΔDsk mutant background. **p<0.01, ***p<0.001 (Kruskal-Wallis and post-hoc Mann-Whitney U tests).
Figure 4—figure supplement 3. The design of aggression chamber.

Figure 4—figure supplement 3.

The aggression chamber consists of four acrylic plates. The bottom plate (Plate 4) contains 12 wells for food patches (diameter: 8 mm; depth: 3 mm). The lower (Plate 3) and upper plates (Plate 2) have 12 cylindrical arenas (diameter: 15 mm; height of each plates: 3 mm). A transparent film is sandwiched by Plate two and Plate three to separate the two flies and removal of the transparent film allows encounters of the two flies.