Targeted manipulation of serotonergic neurotransmission affects the escalation of aggression in adult male Drosophila melanogaster

Abstract

Dopamine (DA) and serotonin (5HT) are reported to serve important roles in aggression in a wide variety of animals. Previous investigations of 5HT function in adult Drosophila behavior have relied on pharmacological manipulations, or on combinations of genetic tools that simultaneously target both DA and 5HT neurons. Here, we generated a transgenic line that allows selective, direct manipulation of serotonergic neurons and asked whether DA and 5HT have separable effects on aggression. Quantitative morphological examination demonstrated that our newly generated tryptophan hydroxylase (TRH)-Gal4 driver line was highly selective for 5HT-containing neurons. This line was used in conjunction with already available Gal4 driver lines that target DA or both DA and 5HT neurons to acutely alter the function of aminergic systems. First, we showed that acute impairment of DA and 5HT neurotransmission using expression of a temperature sensitive form of dynamin completely abolished mid- and high-level aggression. These flies did not escalate fights beyond brief low-intensity interactions and therefore did not yield dominance relationships. We showed next that manipulation of either 5HT or DA neurotransmission failed to duplicate this phenotype. Selective disruption of 5HT neurotransmission yielded flies that fought, but with reduced ability to escalate fights, leading to fewer dominance relationships. Acute activation of 5HT neurons using temperature sensitive dTrpA1 channel expression, in contrast, resulted in flies that escalated fights faster and that fought at higher intensities. Finally, acute disruption of DA neurotransmission produced hyperactive flies that moved faster than controls, and rarely engaged in any social interactions. By separately manipulating 5HT- and DA- neuron systems, we collected evidence demonstrating a direct role for 5HT in the escalation of aggression in Drosophila.

Figure 1. Simultaneous disruption of serotonergic and dopaminergic neurotransmission results in increased numbers of low intensity encounters.

(A) The numbers of encounters were significantly increased in DDC-Gal4/UAS-sh^ts1 flies at the restrictive temperature (gray striped bar) during the first 20 min of the fight compared to the same genotype of flies at the permissive temperature (gray bar), or genetic controls at the restrictive temperature (striped bar). Effects were less pronounced and not statistically significant during the remaining 40 min of a fight. (B) The average time of each encounter was shortened in DDC-Gal4/UAS-shi^ts1 flies at the restrictive temperature (gray striped bars) for the entire fight. (C) The encounters between pairs of DDC-Gal4/UAS-shi^ts1 males at the restrictive temperature consisted mainly of fencing behavior. As above, results were significant only for the first 20 min.*p<0.05; **p<0.01; ***p<0.001, analyzed by nonparametric two-independent-sample Mann-Whitney U-test.

Figure 2. Effects of disrupted amine neuron function on locomotion and courtship behavior.

(Ai,Aii) Flies of all genotypes examined showed parallel temperature-induced increases in the numbers of midline crossings in a locomotion assay. (Bi,Bii) A temperature shift from 19°C to 30°C increased the time that flies spent moving during the locomotion assay in all groups except in males with disrupted dopaminergic neurotransmission (TH/Shi^ts1, 30°C). (Ci) In courtship assays, genetic controls (+/Shi^ts1, 30°C) showed a significant increase in courtship index at the elevated temperature. Flies with lowered function of both amine systems (DDC/Shi^ts1, 30°C) had a slight decrease rather than an increase in CI as seen in controls. With selective interference with dopaminergic function (TH/Shi^ts1, 30°C), however, male flies showed a significant reduction in courtship index. (Cii) By contrast, disruption of serotonergic neurotransmission had no effect on the courtship index. **p<0.01; . ***p<0.001 vs. same genotype at the permissive temperature (19°C), analyzed by nonparametric two-independent-sample Mann-Whitney U-test.

Figure 3. A comparison of TRH-Gal4 driven GFP expression and 5HT immunostaining in male Drosophila brains.

(A–C) TRH-Gal4 (3^rd chromosome line) driven mCD8∶GFP signal (A), 5HT immunostaining (B) and overlay (C) of the staining patterns in the brain and the ventral cord of an adult male. (D–F) Anterior (D) and posterior (E) adult male brain 5HT clusters visualized by TRH-Gal4 driven nuclear nls∶GFP (green) expression and 5-HT immunostaining (red). Note that anterior AMP cells are visible with UAS-nls∶GFP (D), but not with UAS-mCD8∶GFP (A). (F) The absence of overlap between TRH-Gal4 driven UAS-nls∶GFP (green) and DA-containing neurons visualized by TH immunostaining (red).

Figure 4. Selective disruption of serotonergic synaptic transmission results in less aggressive flies.

No significant differences were seen in the numbers of encounters (A) or in the average duration of each encounter (B) throughout a fight (shown for the first 20 min only). (C) TRH-Gal4/UAS-shi^ts1 flies at the restrictive temperature (gray striped bars), however, did show decreased numbers of lunges compared to the experimental males at the permissive temperature (gray bars). This effect was most pronounced at the 20–40 and 40–60 min time windows. Decreases also were seen in the numbers of lunges in genetic control flies, but these were not significant, and were related to switching flies to an elevated temperature. *p<0.05 vs. same genotype at the permissive temperature (19°C), analyzed by nonparametric two-independent-sample Mann-Whitney U-test.

Figure 5. Selective activation of serotonergic neurons increases the intensity of aggression.

TRH-Gal4/UAS-dTrpA1 flies after 15 min at 26°C had a significantly shorter latency to the first lunge (A), and kept lunging more then controls after dominance status was established (B). *p<0.05 vs. genetic control group, analyzed by nonparametric two-independent-sample Mann-Whitney U-test.