A small number of cholinergic neurons mediate hyperaggression in female Drosophila

Abstract

In the Drosophila model of aggression, males and females fight in same-sex pairings, but a wide disparity exists in the levels of aggression displayed by the 2 sexes. A screen of Drosophila Flylight Gal4 lines by driving expression of the gene coding for the temperature sensitive dTRPA1 channel, yielded a single line (GMR26E01-Gal4) displaying greatly enhanced aggression when thermoactivated. Targeted neurons were widely distributed throughout male and female nervous systems, but the enhanced aggression was seen only in females. No effects were seen on female mating behavior, general arousal, or male aggression. We quantified the enhancement by measuring fight patterns characteristic of female and male aggression and confirmed that the effect was female-specific. To reduce the numbers of neurons involved, we used an intersectional approach with our library of enhancer trap flp-recombinase lines. Several crosses reduced the populations of labeled neurons, but only 1 cross yielded a large reduction while maintaining the phenotype. Of particular interest was a small group (2 to 4 pairs) of neurons in the approximate position of the pC1 cluster important in governing male and female social behavior. Female brains have approximately 20 doublesex (dsx)-expressing neurons within pC1 clusters. Using dsx^FLP instead of 357^FLP for the intersectional studies, we found that the same 2 to 4 pairs of neurons likely were identified with both. These neurons were cholinergic and showed no immunostaining for other transmitter compounds. Blocking the activation of these neurons blocked the enhancement of aggression.

Keywords: Drosophila melanogaster; aggression; cholinergic; doublesex; female aggression.

Fig. 1.

Hyperaggression in female Drosophila. (A) Schematic of assay in which a R26E01 > TrpA1 female is paired with another female of the same genotype in a fighting chamber at 20 °C or 29 °C. (B) Shorter latencies to attack displayed during thermogenetic activation of R26E01 > TrpA1-positive neurons in same genotype group pairings of female flies (Kruskal–Wallis, H = 20.7, P = 0.0001; n = 10 to 15 flies) compared with control R26E01Gal4/+ (***P = 0.0002) and wild-type (**P = 0.0036) females. Dunn’s multiple-comparison post hoc test determined these values. (C) An increase in the total number of head butts during thermogenetic activation of R26E01 neurons (Kruskal–Wallis, H = 24.84, P < 0.0001; n = 13 to 17 pairs). R26E01 > TrpA1 females used more head butts compared with R26E01/+ (****P < 0.0001), TrpA1/+ (***P = 0.0006), and wild-type pairs (***P = 0.0007). Dunn’s multiple-comparison post hoc tests determined these values. (D) Differences were found in the number of times wings were elevated during fights (Kruskal–Wallis, H = 26.06, P < 0.0001; n = 14 to 19 pairs). R26E01 > TrpA1 activated females elevated their wings more often compared with control R26E01/+ (***P = 0.0003) and TrpA1/+ (****P < 0.0001) and wild-type (***P = 0.0008) females. Dunn’s multiple-comparison post hoc tests determined these values. (E) Schematic of the assay in which a R26E01 > TrpA1 female was paired with a wild-type female in a fighting chamber at 20 °C or 29 °C. (F) R26E01 > TrpA1 (orange bar) female flies initiated attacks 100% of the time against wild-type Canton-S (gray bars), compared with 45% for R26E01/+ (green bar) and 57% for TrpA1/+ (green bar) flies (χ² test, χ² = 76.05; ****P < 0.0001). (G) Differences in the number of head butts between groups observed at 29 °C (Kruskal–Wallis, H = 10, P = 0.0067; n = 7 to 11 flies). R26E01 > TrpA1 flies displayed more head butts against their wild-type opponents compared with R26E01/+ (**P = 0.0030) and TrpA1/+ (*P = 0.0193) flies. Dunn’s multiple-comparison post hoc tests determined these values. (H) Differences were also seen in the number of wings up and charging (Kruskal–Wallis, H = 25.0, P < 0.0001; n = 7 to 11 flies). R26E01 > TrpA1 flies displayed more wings up and charging compared with R26E01/+ (****P = 0.0010) and TrpA1/+ (****P = 0.0010) flies. Dunn’s multiple-comparison post hoc tests determined these values. (I) Canton-S female flies retreated more times from the food cup (Kruskal–Wallis, H = 18.39, P < 0.0001; n = 10 to 11 flies) when fighting with R26E01 > TrpA1 female flies than when fighting with control genotype R26E01/+ (***P = 0.0004) and TrpA1/+ (**P = 0.0015) flies. Dunn’s multiple-comparison post hoc tests determined these values. (J) Confocal image of female brain of UAS-mCD8::GFP/+;R26E01-Gal4/+ flies immunostained with anti-GFP antibody (green). Z-stack projection is shown. (Scale bars: 50 µm.) Data in B–D, F, and I: center line, median; boxes, first and third quartiles; whiskers, range. Circles represent individual values. NS, not significant at P > 0.05. Aggressive behaviors were measured over a 40-min period.

Fig. 2.

Female aggression is driven by a subset of neurons. (A) Intersectional strategy used to isolate neurons in the female brain. et-^FLP was used in combination with the R26E01-Gal4 driver and UAS > stop > TrpRPA1^myc. (B) Schematic of assay in which a female FLP^#∩R26E01 was paired with another female of the same genotype in a fighting chamber at 20 °C or 29 °C. (C) Fighting latencies (Kruskal–Wallis, H = 27.05, P = 0.0007; n = 15 to 20 flies) were significantly shorter for 210^FLP∩R26E01 (**P = 0.0042), 357^FLP∩R26E01 (**P = 0.0060), 383^FLP∩R26E01 (*P = 0.0147), 447^FLP∩R26E01 (**P = 0.0159), and 529^FLP∩R26E01 (**P = 0.0094) flies compared with controls. Dunn’s multiple-comparison post hoc tests determined these values. (D) Number of head butts in the 10 min after the initial head butt (Kruskal–Wallis, H = 61.15, P < 0.0001; n = 12 to 17 pairs) during thermogenetic activation of R26E01-Gal4 intersected with FLP^#∩R26E01-positive neurons. 210^FLP∩R26E01 (***P = 0.0007), 357^FLP∩R26E01 (****P < 0.0001), and 383^FLP∩R26E01 (****P < 0.0001) pairings showed more head butting compared with control pairings. Dunn’s multiple-comparison post hoc tests determined these values. (E) An increase in the number of times flies displayed wings up and charging during the 10-min fight (Kruskal–Wallis, H = 39.34, P < 0.0001, n = 11 to 17 pairs). Fight pairings 210^FLP∩R26E01 (****P < 0.0001), 357^FLP∩R26E01 (*P = 0.0261), and 383^FLP∩R26E01 (***P = 0.0002) held up their wings during charging more often compared with control pairings. Dunn’s multiple-comparison post hoc tests determined these values. (F) Confocal Z-stack images of intersectional expression of TrpPA1^myc in female brains of R26E01-Gal4 with 357^FLP∩R26E01. (F′) A male brain and female brain immunostained with anti-myc antibody (green). (Scale bars: 50 µm.) Data in C to E: center line, median; boxes, first and third quartiles; whiskers, range. Circles represent individual values. NS, not significant at P > 0.05. Aggressive behaviors were measured over a 40-min period.

Fig. 3.

Sexually dimorphic pC1 dsx neurons. (A and B) Confocal Z-stack images of UAS > stop > mCD8::GFP; R26E01-Gal4/dsx^FLP anterior (A) and posterior (B) female adult brain with bilateral GFP expression in dsx neurons, as visualized with anti-mCD8 (membrane-bound GFP) (green) and neuropil counterstained with nc82 (magenta). The number of neurons varied from 2 to 4 pairs of neurons bilaterally. (C) Some neurons were also expressed in the abdominal ganglion in females. (D and E) Their presynaptic expression in the anterior (D) and posterior (E) regions of the brain, expressing UAS > stop > synaptotagmin (presynaptic marker) tagged with GFP (green). (F) Schematic of pC1–3 neuronal clusters found in the posterior region of the female brain (–31, 33, 36, 37).

Fig. 4.

pC1 dsx neurons are cholinergic. (A) Immunostaining of GFP in adult female genotype UAS > stop > mCD8::GFP;R26E01-Gal4/dsx^FLP counterstained with dsx. (B and C) Higher-magnification (60× objective) of anti-mCD8 (green) (B₁ and C₁), anti-dsx (red) (B₂ and C₂), and a merger of B₁ and C₁ and B₂ and C₂ (B₃ and C₃). (D) Immunostaining of cell membrane in the adult female genotype UAS > stop > mCD8::GFP;R26E01-Gal4/dsx^FLP counterstained with ChAT. (E and F) Higher-magnification (60× objective) views of anti-mCD8 (green) (E₁ and F₁), anti-ChAT (red) (E₂ and F₂), and a merger of E₁ and F₁ and E₂ and F₂ (E₃ and F₃). (Scale bars: 50 µm in A and D; 25 µm in B, C, E, and F.)

Fig. 5.

pC1 dsx sexually dimorphic neurons are required for induction of female hyperaggressive behavior. (A) dsx^FLP∩R26E01 females attacked (Kruskal–Wallis, H = 22.43, P < 0.0001; n = 4 to 11 flies) their opponents sooner compared with wild-type (**P = 0.0011), UAS > stop > TrpRPA1^myc/+;R26E01-Gal4/+ (*P = 0.0156), and dsx^FLP/+ (**P = 0.0070) control pairings. Dunn’s multiple-comparison post hoc tests determined these values. (B) Increased head butting was observed (Kruskal–Wallis, H = 22.39, P < 0.0001; n = 7 to 11 pairs) in dsx^FLP∩R26E01 fight pairings compared with wild-type (***P = 0.0004), UAS > stop > TrpRPA1^myc/+;R26E01-Gal4/+ (**P = 0.0060), and dsx^FLP/+ (***P = 0.0004) control pairings. Dunn’s multiple-comparison post hoc tests determined these values. (C) No differences were observed in the number of wings up and charging in female fights (n = 7 to 11 pairs). (D–F) Inactivation of dsx neurons does not enhance female aggression. No differences in their latency to attack (D) or in the number of head butts (E) and wings up/charging (F) in dsx^FLP∩R26E01 > Kir2.1 pairings compared with control genotype UAS > stop > Kir2.1/+;R26E01/dsx^FLP or dsx^FLP/+ female fight pairings. Data in A to F: center line, median; boxes, first and third quartiles; whiskers, range. Circles represent individual values. NS, not significant at P > 0.05. Aggressive behaviors were measured over a 40-min period.