Fig 1.
(A, B) Expression pattern of 891-Gal4 (A), and MB021B-Gal4 (B), containing the OA-VPM4 cluster (UAS-mCD8GFP, green). Scale = 100 μm. (C) Single labeled OA-VPM4 cell generated by Multi-Color-Flip-Out (MCFO) using MB021B-Gal4. Scale = 50μm. (D) Schematic showing OA-VPM4 arbors, with arrows denoting cell bodies and box showing SEZ. Magenta indicates neuropil, labeled with nc82 (A-D).
Fig 2.
Inducible activation or inactivation of OA-VPM4 alters the threshold for proboscis extension.
(A-C, E-G) PER was tested to four different concentration of sucrose (10mM, 100mM, 350mM, 1M) while neurons in 891-Gal4 (A) or MB021B-Gal4 (E) were thermogenetically activated with UAS-dTrpA1. Flies starved for 24 hours were heated to 31°C for ~5 min before and during testing to activate dTrpA1, or were kept at room temperature (21°C) as controls. Genetic controls for each Gal4 line and UAS-dTrpA1 line were also tested in the same condition (B, C, F, G). n = 86 (A), 86 (B), 67 (C), 60 (E), 22 (F) and 26 (G) flies, mean ± SEM, Mann-Whitney-U test, *p<0.05, **p<0.01, ***p<0.001. (D, H) PER was tested to four different concentration of sucrose (10mM, 100mM, 350mM, 1M) while neurons in 891-Gal4 (D) or MB021B-Gal4 (H) were optogenetically silenced with the chloride channel, GtACR1. Flies starved for 24 hours were stimulated with 530nm light to activate GtACR1. n = 118 (D) and 122 (H) flies, mean ± SEM, Kruskal-Wallis 1-way ANOVA with Bonferroni correction. *p<0.05, **p<0.01, ***p<0.001. Pairwise comparisons to each control, colors of asterisks indicate which genetic controls were compared to the experimental group.
Fig 3.
Octopamine is required for proboscis extension modulation by OA-VPM4.
(A) Double labeling of neurons in 891-Gal4 (magenta, CD8::tdTomato) with OA neurons driven by Tdc2-LexA (green, CD2::GFP). Arrows point to OA-VPM4 cell bodies in the SEZ, showing co-labeling with the Tdc2-LexA reporter. Scale = 20 μm. (B, C) Flies expressing Tβh RNAi (B) or Tdc2 RNAi (C) in 891-Gal4 neurons, were tested for PER to 10mM, 100mM, 350mM, 1M sucrose. n = 155 (B) and 120 (C) flies, mean ± SEM, Kruskal-Wallis 1-way ANOVA with Bonferroni correction. *p<0.05, **p<0.01, ***p<0.001. Pairwise comparisons to each control, asterisk colors indicate which genetic controls were compared to the experimental group.
Fig 4.
OA-VPM4 axons arborize near sugar sensory fibers.
(A) Double labeling of 891-Gal4 (magenta, CD8-tdTOMATO) and sugar-sensing GRNs (green, CD4-GFP) indicates that axons of sugar-sensing GRNs anatomically overlap with neural processes in 891-Gal4. Shown is SEZ as in Fig 1D, where gustatory axons project. Single optical slice (2 μm) in region of maximum overlap is shown. (B) 891-Gal4 axons (magenta, synaptotagmin::eGFP) and sugar-sensing GRNs (green, CD8-tdTOMATO) overlap in the SEZ. Single optical slice (2 μm) in region of maximum overlap is shown. (C) Reconstitution of GFP in flies expressing spGFP(1–10) in 891-Gal4 (co-labeled with CD8-tdTOMATO, magenta) and spGFP(11) in sugar-sensing GRNs (Gr5a-LexA). Scale = 20μm.
Fig 5.
The octopamine receptor OAMB modulates sugar-sensing GRN responses.
(A-C) Flies expressing distinct OAMB-RNAi (UAS-OAMB-RNAi #1: BDSC #31233; UAS-OAMB-RNAi #2: BDSC #2861) in sugar-sensing GRN neurons were tested for PER to 10mM, 100mM, 350mM, 1M sucrose. n = 102 (E, F) and 80 (G) flies, mean ± SEM, Kruskal-Wallis 1-way ANOVA with Bonferroni correction. *p<0.05, **p<0.01, ***p<0.001. Pairwise comparisons to each control, asterisk colors indicate which genetic controls were compared to the experimental group.
Fig 6.
Octopamine potentiates sugar-sensing GRNs responses in satiated flies.
(A) Schematic of the calcium imaging experiments monitoring sensory responses to sucrose before and after OA application to the whole brain in a live fly preparation. First, calcium increases in response to 100mM sucrose solution on the proboscis were examined [PRE]. Then, the fly rested for 10 min and 10μM OA was provided to the whole brain at the 5 min time point. Finally, calcium responses to 100mM sucrose on the proboscis [POST] were examined. (B) In fed flies, OA enhanced calcium responses in sugar-sensing GRNs to 100mM sucrose, while control flies with no OA application showed no difference in [PRE] and [POST]. Co-application of 10μM mianserin, an OA receptor antagonist, and OA abolished the potentiation. Applying OA did not induce potentiation in starved flies. Each data point represents the max ΔF/F of a single neuron. n = 7–12 flies each. Wilcoxon signed-rank test for clustered data. **p<0.01.
Fig 7.
Model: OA-VPM4 enhances feeding by potentiating sugar-sensing GRNs via an octopaminergic pathway.
Schematic of the model. OA-VPM4 releases OA in response to physiological signals (i.e., hunger or arousal). OA, in turn, activates OAMB on sugar-sensing GRNs and potentiates their responses.