Drosophila insulin-producing cells are differentially modulated by serotonin and octopamine receptors and affect social behavior

Abstract

A set of 14 insulin-producing cells (IPCs) in the Drosophila brain produces three insulin-like peptides (DILP2, 3 and 5). Activity in IPCs and release of DILPs is nutrient dependent and controlled by multiple factors such as fat body-derived proteins, neurotransmitters, and neuropeptides. Two monoamine receptors, the octopamine receptor OAMB and the serotonin receptor 5-HT1A, are expressed by the IPCs. These receptors may act antagonistically on adenylate cyclase. Here we investigate the action of the two receptors on activity in and output from the IPCs. Knockdown of OAMB by targeted RNAi led to elevated Dilp3 transcript levels in the brain, whereas 5-HT1A knockdown resulted in increases of Dilp2 and 5. OAMB-RNAi in IPCs leads to extended survival of starved flies and increased food intake, whereas 5-HT1A-RNAi produces the opposite phenotypes. However, knockdown of either OAMB or 5-HT1A in IPCs both lead to increased resistance to oxidative stress. In assays of carbohydrate levels we found that 5-HT1A knockdown in IPCs resulted in elevated hemolymph glucose, body glycogen and body trehalose levels, while no effects were seen after OAMB knockdown. We also found that manipulations of the two receptors in IPCs affected male aggressive behavior in different ways and 5-HT1A-RNAi reduced courtship latency. Our observations suggest that activation of 5-HT1A and OAMB signaling in IPCs generates differential effects on Dilp transcription, fly physiology, metabolism and social interactions. However the findings do not support an antagonistic action of the two monoamines and their receptors in this particular system.

Figure 1. Branches of octopaminergic neurons superimpose those of insulin producing cells (IPCs).

A–C Dissected adult brains were stained with rabbit anti-DILP2 (magenta) and octopaminergic neurons were visualized by Tdc2-Gal4 driving GFP (green). Cb, cell bodies of IPCs; Dendr1 and dendr2, presumed dendrites of IPCs. Scale bar = 20 µm. A1–C1 Magnification of regions of interest in A–C. D and E The efficiencies of the UAS-5-HT_1A- and UAS-OAMB-RNAi (UAS-OAMB-RNAi GD) were determined by using the pan-neuronal Elav-Gal4 driver and monitoring extracted RNA by qPCR. The transcript value for Elav-Gal4>w¹¹¹⁸ was set as 1 in both assays. A significant knockdown (about 60–70% reduction) of 5-HT_1A and OAMB transcript was seen (***p<0.001 to both parental controls, One-way ANOVA, n = 2 samples for each genotype).

Figure 2. Dilp transcript levels in the brain are affected by 5-HT_1A and OAMB knockdown in IPCs.

A–C Relative expression of Dilp2, 3 and 5 transcripts after 5-HT_1A-RNAi and OAMB-RNAi (UAS-OAMB-RNAi GD) in IPCs was measured from head extracts of fed flies. The value for w¹¹¹⁸>5-HT_1A-RNAi was set to 1. The Dilp2 and 5 transcripts increase significantly after 5-HT_1A knockdown, and Dilp3 transcript increases after OAMB knockdown (one way ANOVA; **p<0.01, *p<0.05, ns, not significant; n = 3 samples for each genotype).

Figure 3. Stress responses after knockdown of octopamine receptor OAMB and serotonin receptor 5-HT_1A in IPCs.

A Reduction of octopamine receptor OAMB level in IPCs increases resistance to starvation (seen by extended survival). OAMB was diminished in IPCs by driving two different OAMB-RNAi lines with Dilp2-Gal4. The Dilp2-Gal4>OAMB-RNAi GD flies showed about 50% percent increase of median lifespan compared with controls (***p<0.001 to both parental controls; Log rank test, Mantel-Cox; n = 120 for each genotype). We used the OAMB-RNAi GD lines hereafter for all other experiments. In an earlier study it was shown that 5-HT_1A knockdown decreased starvation resistance . B Reduction of octopamine receptor OAMB or serotonin receptor 5-HT_1A levels in IPCs increases resistance to oxidative stress induced by feeding male flies 20mM paraquat in standard food. Both receptor knockdowns result in flies that display significantly increased survival under paraquat exposure (***p<0.001 to both parental controls, Log rank test, Mantel-Cox; n = 60–90 for each genotype).

Figure 4. Knockdown of OAMB or 5-HT_1A in IPCs resulted in opposite phenotypes in a capillary feeding assay (CAFE).

A Diminishing 5-HT_1A in IPCs (Dilp2-Gal4>5-HT_1A-RNAi) significantly decreases food intake only during day 2. The units in the Y-axis are mm food consumed from the calibrated capillary tube (total volume 5 µL; see methods). The graph shows cumulative intake of food over 4 days (*p<0.05, n = 5-10 flies for each genotype in 3 replicates, One-way ANOVA). B Reduction of OAMB level in IPCs (Dilp2-Gal4>OAMB-RNAi) significantly increases food intake from the second day onwards (**p<0.01, ***p<0.001, n = 5–10 flies for each genotype in 3 replicates, One-way ANOVA).

Figure 5. Levels of carbohydrates were affected by knockdown of 5-HT_1A in IPCs while no changes were observed for OAMB knockdown.

A Hemolymph glucose levels in normally fed flies were higher after 5-HT_1A-RNAi in the IPCs (Dilp2>5HT_1A-Ri) than controls, while no changes were observed for OAMB knockdown (Dilp2>OAMB-Ri). Data were analyzed with one way ANOVA, **p<0.01, n = 60-90 for each genotype, and experiments were performed in 6 independent replicates. B No significant changes of hemolymph trehalose levels were observed after 5HT_1A-RNAi or OAMB-RNAi in the IPCs. One-way ANOVA, ns, not significant; n = 60-90 for each genotype, and experiments were performed in 6 independent replicates. C Body trehalose levels in normally fed flies are higher after 5HT_1A-RNAi in the IPCs (Dilp2>5HT_1A-Ri) than controls, but were not significantly changed after OAMB-RNAi in the same cells (Dilp2>OAMB-Ri). One way ANOVA, ***p<0.001, n = 60-90 for each genotype, and experiments were performed in 6 independent replicates. D Glycogen levels (whole body) in normally fed flies are higher after 5HT_1A-RNAi in the IPCs (Dilp2>5HT_1A-Ri) than controls, but were not significantly changed after OAMB-RNAi in the same cells. One way ANOVA, ***p<0.001, n = 60–90 for each genotype, and experiments were performed in 6 independent replicates.

Figure 6. Social behaviors are affected by manipulations of 5-HT_1A and OAMB.

A Aggressive interactions in controls, 5-HT1A-RNAi and OAMB-RNAi knockdown males were determined. All males were between 5–7 days old. The types of behaviors were distributed into three categories, high intensity fighting (HIF), low intensity fighting (LIF) and courtship behavior (Courtship) and the percentage of each type is represented. In all instances the assay was repeated at least 10 times. (n = 20 males/treatment; * P<0.05, ** P<0.005 compared with controls, two-way ANOVA with Bonferroni post hoc test for multiple comparisons). B The percentage of time a male spent actively courting a 3–4 day old wild-type virgin female was determined for control, 5-HT1A-RNAi and OAMB-RNAi knockdown males. C The amount of time, in seconds, before the first courtship behavior, or latency, was determined for control, 5-HT1A-RNAi and OAMB-RNAi knockdown males introduced to 3–4 day old wild-type virgin females. In B and C n = 10 males per genotype; * P<0.05 ** P<0.005 compared with controls (one-way ANOVA with Bonferroni post hoc test for multiple comparisons).