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

Alekseyenko OV, Lee C, Kravitz EA - PLoS ONE (2010)

Bottom Line: These flies did not escalate fights beyond brief low-intensity interactions and therefore did not yield dominance relationships.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.

View Article: PubMed Central - PubMed

Affiliation: Neurobiology Department, Harvard Medical School, Boston, Massachusetts, USA. olga_alekseenko@hms.harvard.edu

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.

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A comparison of TRH-Gal4 driven GFP expression and 5HT immunostaining in male Drosophila brains.(A–C) TRH-Gal4 (3rd 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).
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pone-0010806-g003: A comparison of TRH-Gal4 driven GFP expression and 5HT immunostaining in male Drosophila brains.(A–C) TRH-Gal4 (3rd 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).

Mentions: Towards this goal, we cloned short and long regulatory sequences of the Trh gene that encodes the nervous system specific tryptophan hydroxylase enzyme (CG9122, [12], [13]) upstream of a Gal4 coding region. Twelve viable TRH-Gal4 fly lines were generated, and Gal4 expression patterns were visualized for each line by crossing these flies to UAS-nls∶GFP (for nuclear labeling) or UAS-mCD8∶GFP (for membrane labeling). The GFP fluorescence in progeny was examined in brains and in ventral nerve cords (VNC). Ten lines derived from the short regulatory sequence showed variable and partial overlap between TRH-Gal4 driven GFP expression and immunostaining for 5HT (Fig. S1, D–I). Two lines, derived from the long regulatory sequence of Trh, had more specific patterns of GFP expression that almost completely overlapped with 5HT immunostaining (Fig. 3, A–C for the line on 3rd chromosome; Fig. S1, A–C for the line on 2nd chromosome). For the TRH-Gal4 line on the 3rd chromosome, nuclear GFP labeling was seen in all of the previously reported adult brain 5HT clusters (Fig. 3, D–E): LP2, SE1, SE2 and SE3 anterior clusters [17]; the more recently classified AMP and ALP anterior clusters [26]; and groups of cells forming the PMP and PLP posterior clusters [26]. Co-labeling between GFP and 5HT immunostaining was present in 75–100% of the neurons forming each 5HT cluster. See Table S2 for numbers of labeled serotonergic neurons in each cluster in comparison with previously used genetic approaches. Also see Fig. S2 for close-up views of 5HT clusters visualized by TRH-Gal4 and TPH-Gal4 [23]. We did not observe co-localization between GFP-expressing cells and tyrosine hydroxylase antibody staining, indicating that the new line does not drive expression in dopaminergic neurons (Fig 3, F). This TRH-Gal4 line, mapped to the third chromosome, was used in the behavioral experiments that follow.


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

Alekseyenko OV, Lee C, Kravitz EA - PLoS ONE (2010)

A comparison of TRH-Gal4 driven GFP expression and 5HT immunostaining in male Drosophila brains.(A–C) TRH-Gal4 (3rd 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).
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2875409&req=5

pone-0010806-g003: A comparison of TRH-Gal4 driven GFP expression and 5HT immunostaining in male Drosophila brains.(A–C) TRH-Gal4 (3rd 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).
Mentions: Towards this goal, we cloned short and long regulatory sequences of the Trh gene that encodes the nervous system specific tryptophan hydroxylase enzyme (CG9122, [12], [13]) upstream of a Gal4 coding region. Twelve viable TRH-Gal4 fly lines were generated, and Gal4 expression patterns were visualized for each line by crossing these flies to UAS-nls∶GFP (for nuclear labeling) or UAS-mCD8∶GFP (for membrane labeling). The GFP fluorescence in progeny was examined in brains and in ventral nerve cords (VNC). Ten lines derived from the short regulatory sequence showed variable and partial overlap between TRH-Gal4 driven GFP expression and immunostaining for 5HT (Fig. S1, D–I). Two lines, derived from the long regulatory sequence of Trh, had more specific patterns of GFP expression that almost completely overlapped with 5HT immunostaining (Fig. 3, A–C for the line on 3rd chromosome; Fig. S1, A–C for the line on 2nd chromosome). For the TRH-Gal4 line on the 3rd chromosome, nuclear GFP labeling was seen in all of the previously reported adult brain 5HT clusters (Fig. 3, D–E): LP2, SE1, SE2 and SE3 anterior clusters [17]; the more recently classified AMP and ALP anterior clusters [26]; and groups of cells forming the PMP and PLP posterior clusters [26]. Co-labeling between GFP and 5HT immunostaining was present in 75–100% of the neurons forming each 5HT cluster. See Table S2 for numbers of labeled serotonergic neurons in each cluster in comparison with previously used genetic approaches. Also see Fig. S2 for close-up views of 5HT clusters visualized by TRH-Gal4 and TPH-Gal4 [23]. We did not observe co-localization between GFP-expressing cells and tyrosine hydroxylase antibody staining, indicating that the new line does not drive expression in dopaminergic neurons (Fig 3, F). This TRH-Gal4 line, mapped to the third chromosome, was used in the behavioral experiments that follow.

Bottom Line: These flies did not escalate fights beyond brief low-intensity interactions and therefore did not yield dominance relationships.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.

View Article: PubMed Central - PubMed

Affiliation: Neurobiology Department, Harvard Medical School, Boston, Massachusetts, USA. olga_alekseenko@hms.harvard.edu

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.

Show MeSH
Related in: MedlinePlus