Female contact modulates male aggression via a sexually dimorphic GABAergic circuit in Drosophila

Abstract

Intraspecific male-male aggression, which is important for sexual selection, is regulated by environment, experience and internal states through largely undefined molecular and cellular mechanisms. To understand the basic neural pathway underlying the modulation of this innate behavior, we established a behavioral assay in Drosophila melanogaster and investigated the relationship between sexual experience and aggression. In the presence of mating partners, adult male flies exhibited elevated levels of aggression, which was largely suppressed by prior exposure to females via a sexually dimorphic neural mechanism. The suppression involved the ability of male flies to detect females by contact chemosensation through the pheromone-sensing ion channel ppk29 and was mediated by male-specific GABAergic neurons acting on the GABAA receptor RDL in target cells. Silencing or activating this circuit led to dis-inhibition or elimination of sex-related aggression, respectively. We propose that the GABAergic inhibition represents a critical cellular mechanism that enables prior experience to modulate aggression.

Figure 1. Prior female experience inhibits sex-related male-male aggression

(a) Scheme for the aggression assay, four conditions are shown with color coding as naïve male only (blue), experienced male only (purple), naïve male + virgin female (red), experienced male + virgin female (green). (b) Timeline of the average aggression duration of wild-type Canton-S (cs) males within 2 hours. Each data point represents average aggression duration quantified in 5 min intervals. The lack of aggression within the first 30 min reflects courtship behavior. (n = 4 pairs for each condition). (c) Average aggression duration of cs and w¹¹¹⁸ males under different conditions. Both genotypes showed female experience induced inhibition of aggression in pair-or single-housed settings. (n = 11 and 6 for cs and w1118 in pair-housed, 8 and 7 in single-housed). Aggression interactions 60-90 min after the onset were quantified. p <0.0001 (***), =0.019 (*), =0.0084 (**), =0.0059 (**), <0.0001 (***) . (d) The inhibition index, defined as (Aggression^N – Aggression^E) / Aggression^N, for pair- and single-housed settings. (n = 11 and 8 for pair and single.) p = 0.1989. Student's t-test. *: p<0.05, **: p <0.01, ***: p <0.001. Error bars denote s.e.m.

Figure 2. Prior female-contacts dependent inhibition of aggression is long term and requires chemosensation through the pheromone sensing channel ppk29

(a) The inhibitory effect of prior female experience on aggression lasted up to two days. The experienced (E) male flies were housed with females for 24 hours and were then separated from the females and reared for 0, 2, 3 or 5 days until behavior assay. (n = 11, 6, 7 and 5 for each manipulation). (b) Memory mutants had no defects in female experience induced inhibition. Inhibition index was comparable among cs, w1118, rut, amn and Orb2^k/o flies. (n = 11, 6 ,4, 4 and 4 for each genotype). (c) Inhibition index for various conditioning paradigms. (n = 11, 6, 5, 6, 5, 5 and 7). (d) Inhibition of aggression by female experience was impaired specifically in mutants lacking ppk29. Compared to genotypes with defects in pheromone sensation, vision or olfaction, only ppk29^–/–, ppk29-Gal4>Kir and ppk29-Gal4, fru^FLP, UAS>stop>TNTact flies showed significant reduction of inhibition index (n = 5, 4, 4, 4, 6, 5, 5 and 5 for each genotype). Genotypes tested were as indicated. TNTina and TNTact denote the inactive and active forms of TNT transgenes respectively. (a-d) One-way ANOVA followed by Bonferroni's multiple comparison test. (d, last two genotypes) Student's t-test. p = 0.0013 (**). **: p<0.01, ***: p<0.001. Error bars denote s.e.m.

Figure 3. fru⁺, d5-HT1B⁺ sexually dimorphic neurons mediate female experience dependent inhibition of aggression

(a) Temperature shift induced silencing of d5-HT1B⁺ neurons resulted in dis-inhibition of aggression, without affecting baseline aggression. (n = 5, 5 and 6 for each genotype). (b) fru⁺, d5-HT1B⁺ neurons in the adult brain labeled by mCD8::GFP and Dscam17.1::GFP, including γ-neurons of the mushroom bodies and a cluster with soma located between antennal lobes and the SOG region. The sexual dimorphism was evident with more cells and more elaborated arbors in male brains as compared to female brains. nc82 co-staining (red) showed the neuropil. Scale bar = 40 μm. (c) Blocking chemical transmission by a TNT transgene or electrically silencing by a Kir2.1 transgene expression in the fru⁺, d5-HT1B⁺ neurons led to strong dis-inhibition of aggression. (n = 6, 6, 5 and 7 for each genotype). p = 0.0012 (**), =0.0099 (**). (d) Inducible activation of fru⁺, d5-HT1B⁺ neurons by a heat-activated channel dTrpA1 led to reduced baseline aggression at 29°C as compared to the 20°C control. This reduction of baseline aggression persisted when the mushroom body neurons were excluded using MB-Gal80. The genotype for the manipulation is w+; UAS>stop>dTrpA1; fru^FLP/d5-HT1B-Gal4, without or with MB-Gal80. (n = 5 for each genotype). p <0.0001 (***), =0.0049 (**). (b upper) One-way ANOVA followed by Bonferroni's multiple comparison test. (b lower, d, e) Student's t-test. *: p<0.05, **: p<0.01, ***: p<0.001. Error bars denote s.e.m.

Figure. 4. Ectopic activation of the serotoninergic circuit elevates baseline aggression without affecting female-contacts dependent inhibition of aggression

5-HTP treatment elevated baseline sex-related aggression without affecting the female-contacts dependent inhibition of aggression. (n = 11 and 5 for each condition). *: p = 0.0424. Student's t-test. Error bars denote s.e.m.

Figure 5. Female experience acts through GABAergic neurotransmission in fru⁺, GABA⁺ and d5-HT1B⁺ neurons to inhibit aggression

(a) fru⁺, GABA⁺ neurons display sexual dimorphism in the adult brain, especially in the region above the SOG, that show difference both in the cell number and the projection pattern in female and male brains. nc82 co-staining (red) showed the neuropil. Scale bar = 40 μm. (b) A subpopulation of the fru⁺, d5-HT1B⁺ neurons (green) above the SOG region is labeled with anti-GAD antibody (red), indicated by arrows, suggesting these neurons are GABAergic. Top three panels are representative max projection images and the bottom panel is an image from a single confocal optical section. Scale bar = 10 μm. (c) Blocking neurotransmission by TNT in GABA⁺, fru⁺ neurons led to dis-inhibition of aggression. Whereas inhibition of fru⁺, d5-HT1B⁺ neurons by TNT resulted in dis-inhibition (see Fig. 3c), adding Gad-Gal80 abolished this effect, suggesting that the GABAergic fru⁺, d5-HT1B⁺ neurons are responsible for the dis-inhibition of aggression. (n = 5 for each genotype). (d) Activation of GABA⁺ neurons or GABA⁺, fru⁺ neurons by dTrpA1 at 29°C suppressed the baseline aggression. Whereas activation of fru⁺, d5-HT1B⁺ neurons by dTrpA1 suppressed the baseline aggression (see Fig. 3d), adding Gad-Gal80 abolished this reduction, suggesting that the GABAergic fru⁺, d5-HT1B⁺ neurons are responsible for the reduction of baseline aggression. (n = 5, 5, 5, 6, 7 and 4 for each genetype). ***: p<0.001. Student's t-test. Error bars denote s.e.m.

Figure 6. GABA neurotransmission, the GABA-a receptor RDL and Rdl⁺, fru⁺ sexually dimorphic neurons mediate female experience dependent inhibition of aggression

(a) Blocking GABA neurotransmission in d5-HT1B⁺ neurons using RNAi against GAD or VGAT led to dis-inhibition of aggression. (n = 5, 4, 5 and 4 for each genotype). p = 0.0359 (*), =0.0139 (*). (b) Inhibition of aggression was reduced in flies with Rdl knockdown pan-neuronally (elav-Gal4>UAS-Rdl RNAi), or with the hypomorphic allele Rdl^MB08800 caused by P-element insertion. (n = 4, 4, 5 and 5 for each genotype). Activation of fru⁺, Rdl⁺ neurons by dTrpA1 at 29°C resulted in dis-inhibition of male-male aggression induced by female contacts. (n = 8 and 6). (c) Neurons labeled by Rdl-Gal4 are widely distributed in the brain. Scale bar = 40 μm. (d) Sexual dimorphisms of fru⁺, Rdl⁺ neurons in the fly brain. Representative images of the male and female brains are shown. nc82 co-staining (red) showed the neuropil. Scale bar = 40 μm. (a, b, last two genotypes) Student's t-test, (b) One-way ANOVA followed by Bonferroni's multiple comparison test. *: p<0.05, **: p<0.01, ***: p<0.001. Error bars denote s.e.m.