Electrical synapses mediate synergism between pheromone and food odors in Drosophila melanogaster

Abstract

In Drosophila melanogaster, the sex pheromone produced by males, cis-vaccenyl acetate (cVA), evokes a stereotypic gender-specific behavior in both males and females. As Drosophila adults feed, mate, and oviposit on food, they perceive the pheromone as a blend against a background of food odors. Previous studies have reported that food odors enhance flies' behavioral response to cVA, specifically in virgin females. However, how and where the different olfactory inputs interact has so far remained unknown. In this study, we elucidated the neuronal mechanism underlying the response at an anatomical, functional, and behavioral level. Our data show that in virgin females cVA and the complex food odor vinegar evoke a synergistic response in the cVA-responsive glomerulus DA1. This synergism, however, does not appear at the input level of the glomerulus, but is restricted to the projection neuron level only. Notably, it is abolished by a mutation in gap junctions in projection neurons and is found to be mediated by electrical synapses between excitatory local interneurons and projection neurons. As a behavioral consequence, we demonstrate that virgin females in the presence of vinegar become receptive more rapidly to courting males, while male courtship is not affected. Altogether, our results suggest that lateral excitation via gap junctions modulates odor tuning in the antennal lobe and drives synergistic interactions between two ecologically relevant odors, representing food and sex.

Keywords: courtship behavior; electrical synapse; functional imaging; mixture synergism; sex pheromone.

Fig. 1.

PNs in the glomerulus DA1 reveal synergistic responses to the mixture of cVA and vinegar specifically in virgin females. (A) Schematic of the experimental approach: UAS-GCaMP3 was expressed in PNs (green) using GH146-Gal4 in virgin female flies. (B) Representative odor-evoked calcium responses of PNs in the AL of a virgin female to cVA, vinegar, and their binary mixture (10⁻¹ concentration). (C) Box plots display ΔF/F responses in glomerulus DA1 in virgin females to vinegar (orange), cVA (blue), and their binary mixture (striped) at three different concentrations. The white line in the box represents the median. The mixture evokes a significantly enhanced response (***P < 0.001; **P < 0.01; Wilcoxon matched paired test). (D) Schematic of the experimental approach: UAS-GCaMP3 was expressed in PNs (green) using GH146-Gal4 in virgin male flies. (E) Representative odor-evoked calcium responses of PNs in the AL of a virgin male to cVA, vinegar, and their binary mixture (10⁻¹ concentration). (F) Box plots display ΔF/F in DA1 in virgin males to vinegar (orange), cVA (blue), and their mixture (striped) at three different concentrations. The mixture evokes a similar response as cVA (P > 0.05; Wilcoxon matched paired test). (G) Schematic of the experimental approach: UAS-GCaMP3 was expressed in PNs (green) using GH146-Gal4 in mated female flies. (H) Representative odor-evoked calcium responses of PNs in the AL of a mated female to cVA, vinegar, and their mixture (10⁻¹ concentration). (I) Box plots display ΔF/F in DA1 in mated females to vinegar (orange), cVA (blue), and their mixture (striped) at three different concentrations. The mixture evokes a similar response as cVA (P > 0.05; Wilcoxon matched paired test). (J) Representative odor-evoked calcium responses of PNs in the AL of a virgin female to limonene (lim), 1-hexanol (hex), acetic acid (aca), and their individual binary mixtures with cVA (10⁻¹ concentration). (K) Box plots represent ΔF/F responses of PNs in DA1 to limonene (lim, yellow), 1-hexanol (hex, indigo), acetic acid (aca, brown), and cVA (blue), and the mixtures of cVA with the individual odors (striped boxes) at 10⁻¹ concentration. None of the mixtures evokes a synergistic response (P > 0.05; Wilcoxon matched paired test). (Magnification in B, E, H, and J, 200×.)

Fig. 2.

Mixture synergism does not occur at the sensory level. In vivo extracellular SSRs from the at1 sensillum expressing OR67d. (A, Left) Representative traces display the response of OR67d ORNs in virgin females to vinegar, cVA and their binary mixture (10⁻¹ concentration). (Right) Line curves represent the averaged neuronal activity (spikes per second) to vinegar (orange), cVA (blue), and their binary mixture (striped) at three different concentrations (P > 0.05; Wilcoxon matched paired test). (B) Schematic of the experimental approach: UAS-GCaMP3 was expressed in the majority of ORNs (green) using Orco-Gal4 in virgin females. (C) Representative odor-evoked calcium responses of ORNs in the AL of a virgin female to cVA, vinegar, and their binary mixture (10⁻¹ concentration). (D) Box plots represent ΔF/F responses of ORNs in the glomerulus DA1 in virgin females to vinegar (orange), cVA (blue), and their binary mixture (striped boxes). The white line in the box represents the median. The ORN response to the mixture is equal to the response to the stronger component (i.e., cVA) (P > 0.05; Wilcoxon matched paired test). (E) Schematic of the experimental approach: UAS-GCaMP3 was expressed in ORNs expressing IRs (green) using IR8a-Gal4 in virgin females. (F) Representative odor-evoked calcium responses of IR8a-expressing ORNs in the AL of a virgin female to cVA, vinegar, and their binary mixture (10⁻¹ concentration). (G) Box plots represent ΔF/F responses of IR8a-expressing ORNs in different vinegar-responsive glomeruli in virgin females to vinegar (orange), cVA (blue), and their binary mixture (striped boxes) at 10⁻¹ concentration. The ORN response to the mixture is equal to the response to the stronger component (i.e., vinegar) (P > 0.05; Wilcoxon matched paired test). (Magnification in C and F, 200×.)

Fig. 3.

Excitatory local interneurons do not reveal a synergistic mixture response. (A) Schematic of the experimental approach: UAS-GCaMP3 was expressed in eLNs (green) using Krasavietz-Gal4 in virgin female flies. (B, Left) Representative odor-evoked calcium responses of eLNs in the AL in the background of END1-2 (elav-n-synaptobrevin:DsRed) of a virgin female to cVA, vinegar, and their binary mixture (10⁻¹ concentration). (Right) Box plots display ΔF/F responses in the glomerulus DA1 in virgin females to vinegar (orange), cVA (blue), and their binary mixture (striped) at three different concentrations. The white line in the box represents the median. The eLN response to the mixture is significantly higher than the response to the stronger component (i.e., vinegar) at 10⁻¹ concentration. (P < 0.05; Wilcoxon matched paired test). *P = 0.03. (Magnification in B, 200×.)

Fig. 4.

Gap junctions between PNs and eLNs are necessary and sufficient to induce mixture synergism. (A) Schematic of the experimental approach: UAS-GCaMP3 was expressed in PNs (green) using GH146-Gal4 in virgin females. (B) Box plots display ΔF/F responses in the glomerulus DA1 in virgin females, in the background of the shakB² mutant to vinegar (orange), cVA (blue), and their binary mixture (striped) at two different concentrations (10⁻² and 10⁻¹). The white line in the box represents the median. The mixture does not evoke a synergistic response (P > 0.05; Wilcoxon matched paired test). (C) Box plots display ΔF/F responses in the glomerulus DA1 in virgin females to vinegar (orange), cVA (blue), and their binary mixture (striped) at two different concentrations (10⁻² and 10⁻¹). Gap junctions have been blocked in PNs using RNAi against inx8. The mixture does not evoke a synergistic response (P > 0.05; Wilcoxon matched paired test). (D) Schematic of the experimental approach: UAS-GCaMP6s was expressed in PNs and eLNs (green) using GH146-Gal4 and Krasavietz-Gal4 in virgin females. (E) Box plots display ΔF/F responses in the glomerulus DA1 in virgin females to vinegar (orange), cVA (blue), and their binary mixture (striped) at 10⁻² and 10⁻¹ concentration. Genotypes are as follows: control line, GH146-Gal4; Krasavietz-Gal4; mutant line, shakB²; GH146-Gal4; Krasavietz-Gal4; rescue line, UAS- shakB.neural/GH146-Gal4; Krasavietz-Gal4 in the shakB² mutant background. The control and rescue lines show a synergistic mixture response at both concentrations (*P < 0.05, **P < 0.01; Wilcoxon matched paired test).

Fig. 5.

Vinegar modulates copulation latency in females, which requires gap junctions in PNs. Courtship behavior assays performed with wild-type and different mutant flies in the presence of water (gray) or vinegar (10⁻³, orange). (A and A′) Histograms represent copulation success and the box plots show the copulation latency of wild-type pairs of D. melanogaster. The presence of vinegar significantly reduces copulation latency, while copulation success is unaffected (*P < 0.05; Mann–Whitney test; n = 24). (A′′) Box plots reveal courtship index of wild-type pairs. The presence of vinegar does not significantly affect the courtship index (n = 12). (B and B′) Histograms represent copulation success and the box plots show the copulation latency of wild-type males (Canton-s) and OR67d mutant females. Neither copulation success nor latency are influenced by the presence of vinegar (P > 0.05; Mann–Whitney test; n = 24). (C and C′) Histograms represent copulation success and the box plots show the copulation latency of wild-type males (Canton-s) and mutant females in which gap junctions have been blocked in PNs (GH146-Gal4 > UAS-inx8-RNAi), as well as the parental control lines (UAS-inx8-RNAi/+ and GH146-Gal4/+). Only the parental lines show a reduced copulation latency in the presence of vinegar (*P < 0.05; Mann–Whitney test; n = 24). χ² Test with Yates correction was used for copulation success and Mann–Whitney test was used for copulation latency.

Fig. 6.

Circuit model for mixture synergism. Proposed mechanism underlying the observed synergism in virgin females to the mixture of the sex pheromone cVA and the complex food odor vinegar. (Left) The sole cVA stimulation, which is detected by ORNs expressing OR67d that target glomerulus DA1 in the AL. As a result, PNs in glomerulus DA1 are activated, which transfer the cVA response to higher brain centers promoting courtship and virgin female receptivity. (Right) Illustration of how the simultaneous stimulation with vinegar and cVA enhances the activity of DA1 in a synergistic manner. Vinegar activates specific vinegar-responsive glomeruli which convey this input through eLNs to the DA1 and other glomeruli via electrical synapses. Since DA1 receives a stronger lateral excitation by vinegar (thick line) than other glomeruli (thin line), the PNs of DA1 are stronger activated. As glomerulus DA1 possesses a large number of electrically coupled sister PNs, the signal gets further amplified and leads to the observed synergistic mixture response. The resultant synergistic activity of DA1 is reflected behaviorally by a faster receptivity of virgin females to courting males in the presence of vinegar. As previously shown, in the mated female glomerulus DL3 suppresses the cVA response in glomerulus DA1 via inhibitory LNs; as a result, the synergism cannot occur in this scenario (32).