Single dopaminergic neurons that modulate aggression in Drosophila

Abstract

Monoamines, including dopamine (DA), have been linked to aggression in various species. However, the precise role or roles served by the amine in aggression have been difficult to define because dopaminergic systems influence many behaviors, and all can be altered by changing the function of dopaminergic neurons. In the fruit fly, with the powerful genetic tools available, small subsets of brain cells can be reliably manipulated, offering enormous advantages for exploration of how and where amine neurons fit into the circuits involved with aggression. By combining the GAL4/upstream activating sequence (UAS) binary system with the Flippase (FLP) recombination technique, we were able to restrict the numbers of targeted DA neurons down to a single-cell level. To explore the function of these individual dopaminergic neurons, we inactivated them with the tetanus toxin light chain, a genetically encoded inhibitor of neurotransmitter release, or activated them with dTrpA1, a temperature-sensitive cation channel. We found two sets of dopaminergic neurons that modulate aggression, one from the T1 cluster and another from the PPM3 cluster. Both activation and inactivation of these neurons resulted in an increase in aggression. We demonstrate that the presynaptic terminals of the identified T1 and PPM3 dopaminergic neurons project to different parts of the central complex, overlapping with the receptor fields of DD2R and DopR DA receptor subtypes, respectively. These data suggest that the two types of dopaminergic neurons may influence aggression through interactions in the central complex region of the brain involving two different DA receptor subtypes.

Fig. 1.

Dopaminergic neurons identified by the et-FLP screen. (A) A schematic representation of the intersectional approach to restrict the numbers of targeted DA neurons. (B) Example of dopaminergic neuron-specific TH immunostaining pattern in Drosophila brain (the full z stack is shown). (Scale bar, 100 μm.) (C) Example of a line that targets most of the DA neurons in the fly brain. The GFP signal is driven by a combination of FLP³⁸³, TH-Gal4, and UAS>stop>GFP transgenes (the full z stack is shown). (Scale bar, 100 μm.) (D–F) Individual DA neurons targeted by the use of different et-FLP lines. The GFP signals restricted by et-FLPs are shown in green, neuropil areas stained by an nc82 neuronal marker are shown in gray, and anti-TH immunostaining is shown in magenta. Dotted boxes indicate the magnification fields shown in lower panels. Different frontal z stacks through either anterior or posterior areas of the same triple-stained brains were created when needed to demonstrate either processes or cell bodies. The full frontal z stacks are shown in Fig. S1. (Scale bars, 50 μm.) (D) FLP²⁴³ line restricts GFP expression to a bilateral pair of neurons from the T1 cluster (green) that arborize in the protocerebral bridge (gray) (also see Fig. S1A). (E) FLP⁴⁴⁷ line restricts GFP expression to one or two bilateral neurons from the PPM3 cluster (green) that arborize in the fan-shaped body and the noduli (gray) (also see Fig. S1B). (F) FLP³⁴⁶ line restricts GFP expression to two bilateral neurons from the PPL1 cluster (green) that arborize in the heel, lower stalk, and junction regions of the mushroom bodies (gray) (also see Fig. S1C).

Fig. 2.

TNT inactivation of DA neurons has selective effects on behavior. (A) Inactivation of large numbers of DA neurons (light gray bar) results in very low levels of locomotion, whereas inactivation of individual DA neurons (dark gray bars) has either a small or no effect on locomotion. Data are presented as means ± SEM; ***P < 0.001 vs. corresponding control (white bar), analyzed by nonparametric two-independent-sample Mann–Whitney U test. (B) Males carrying one copy of the recombinase transgene from different et-FLP lines (light and dark gray bars) have the same levels of locomotion as wild-type Canton-S males (white bar). Data are presented as means ± SEM. (C) Numbers of lunges between pairs of males carrying one copy of various et-FLP transgenes. None of the transgenic et-FLP lines crossed to the wild-type Canton-S strain replicated the aggression phenotypes observed with experimental flies (Fig. 3). Each dot represents the lunge count for an individual pair of flies. The data are presented as boxplots with a median line. The lower and upper parts of the boxes are 25th and 75th percentiles, respectively. *P < 0.05 vs. Canton-S control (white dots), analyzed by nonparametric two-independent-sample Mann–Whitney U test.

Fig. 3.

Manipulation of individual T1 and PPM3 DA neurons targeted by FLP²⁴³ and FLP⁴⁴⁷ increases aggression. (A) Total numbers of lunges performed by pairs of males with TNT-inactivated subsets of DA neurons (chronic inactivation). Also see Fig. S3A for statistical analysis without outliers. (B) Latency to the first lunge and to the establishment of dominance in flies with TNT-inactivated subsets of DA neurons (chronic inactivation). (C) Total numbers of lunges performed by pairs of males with dTrpA1-activated subsets of DA neurons (acute, short-term activation). Also see Fig. S3B for statistical analysis without outliers. (D) Latency to the first lunge and to the establishment of dominance in flies with dTrpA1-activated subsets of DA neurons (acute, short-term activation). Each dot in A and C represents the lunge count for an individual pair of flies. Data are presented as boxplots with a median line. The lower and upper parts of the boxes show 25th and 75th percentiles, respectively. Latencies in B and D are presented as means ± SEM; *P < 0.05, ***P < 0.001 vs. corresponding control (white bar or white dots), analyzed by nonparametric two-independent-sample Mann–Whitney U test.

Fig. 4.

Putative targets of aggression-modulating dopaminergic neurons in the central complex. (A) The polarity of the dopaminergic T1 neurons. (Left) Arborization pattern of the T1 neurons visualized by membrane-bound CD8::GFP. (Center) Presynaptic terminals of the T1 neurons revealed by the presynaptic marker nsyb::GFP. (Right) The dendritic arbors of the T1 neurons visualized by expression of the postsynaptic marker DsCam:GFP. (B) An overlap between presynaptic terminals of the T1 dopaminergic neurons (green) and anti-DD2R antibody staining (magenta) in the protocerebral bridge region of the central complex (60× objective). See z stack in Movie S1. (C) The polarity of the dopaminergic PPM3 neurons. (Left) Arborization pattern of the PPM3 neurons visualized by membrane-bound CD8::GFP. (Center) Presynaptic terminals of the PPM3 neurons revealed by the presynaptic marker nsyb::GFP. (Right) The dendritic arbors of the PPM3 neurons visualized by expression of the postsynaptic marker DsCam:GFP. (D) A partial overlap between presynaptic terminals of the PPM3 dopaminergic neurons (green) and anti-DopR antibody staining (magenta) in the fan-shaped body and the noduli of the central complex (60× objective). See z stack in Movie S2. (Scale bars, 20 μm.)