Mechanosensory neurons control sweet sensing in Drosophila

Abstract

Animals discriminate nutritious food from toxic substances using their sense of taste. Since taste perception requires taste receptor cells to come into contact with water-soluble chemicals, it is a form of contact chemosensation. Concurrent with that contact, mechanosensitive cells detect the texture of food and also contribute to the regulation of feeding. Little is known, however, about the extent to which chemosensitive and mechanosensitive circuits interact. Here, we show Drosophila prefers soft food at the expense of sweetness and that this preference requires labellar mechanosensory neurons (MNs) and the mechanosensory channel Nanchung. Activation of these labellar MNs causes GABAergic inhibition of sweet-sensing gustatory receptor neurons, reducing the perceived intensity of a sweet stimulus. These findings expand our understanding of the ways different sensory modalities cooperate to shape animal behaviour.

Figure 1. Food hardness preference.

(a,b) Effect of food hardness on food preference. Flies were given a choice between (a) 0.5 mM sucrose in 0.2% agarose and 1 mM sucrose in varying concentrations of agarose (0.2–2%) or (b) 0.5 mM sucrose in varying concentrations of agarose (2–0.2%) and 1 mM sucrose in 2% agarose. The red dashed line indicates no preference. n=4. (c) Effect of sweetness on food hardness-based preference. Flies were given a choice between 0.5 mM sucrose in 0.2% agarose and varying concentrations of sucrose (0.5–5 mM) in 2% agarose. n=4. All data are presented as means±s.e.m.

Figure 2. Characterization of labellar MN GAL4 drivers.

(a) Expression of MN drivers in the labellum and the leg. Confocal images of labella and legs expressing mCD8::GFP driven by VT2692-GAL4, R41E11-GAL4 and R55B01-GAL4. Labella were stained with a rabbit anti-GFP. GFP fluorescence superimposed on a transmitted light image. Scale bars, 50 μm. (b–d) Co-localization of R41E11 cells and cell-type specific markers; (b) MNs, Fru^LexA (c) sweet GRNs, Gr64f^LexA (d) bitter GRNs, Gr66a-I-GFP. Labellum of UAS-mCD8::RFP/LexAOp-rCD2::GFP;Fru^LexA/R41E11-GAL4 flies stained with a rat anti-CD8 and a rabbit anti-GFP. Labellum of UAS-mCD8::RFP/LexAOp-GCaMP3;Gr64f^LexA/R41E11-GAL4 and UAS-mCD8::RFP/+;Gr66a-I-GFP/R41E11-GAL4 flies stained with a mouse anti-GFP and a rabbit anti-DsRed. The white boxes indicate the insets shown at higher magnification below. Scale bars, 50 μm. (e) Comparison of the dendritic morphology of MNs and sweet GRNs. Labellum of UAS-DsRed/+;NOMPA-GFP/ R41E11-GAL4 (up) or UAS-DsRed/Gr64f-GAL4;NOMPA-GFP/+ (bottom) flies stained with rabbit anti-DsRed. The arrowheads indicate the extension of sweet neuron dendrites beyond the bristle base. Scale bars, 10 μm. We used NOMPA as a marker for the bristle base. Note that fluorescent signals from the GAL4 and NOMPA were pseudocoloured green and magenta, respectively, to improve clarity. (f) Expression of labellar MN drivers in the central nervous system. Confocal images of brains and ventral nerve cords expressing mCD8::GFP driven by R41E11-GAL4, VT2692-GAL4, and R55B01-GAL4. Samples were stained with rabbit anti-GFP and nc82. Scale bar, 50 μm. All confocal images are maximal intensity projections of z-stacks.

Figure 3. Food hardness-mediated preference is regulated by labellar MNs.

(a) Food preference on silencing of MNs. Flies were given a choice between 0.5 mM sucrose in 0.2% agarose and 1 mM sucrose in 2% agarose. Flies were raised at 21 °C and shifted to the indicated temperature for 3 days before assaying. Unpaired Student's t-tests, **P<0.01. (b,c) Mechanosensor screen. Flies were given a choice between 0.5 mM sucrose in 0.2% agarose and 1 mM sucrose in 2% agarose. ANOVA with Tukey post-hoc tests, **P<0.01. n is indicated in parentheses. All data are presented as means±s.e.m.

Figure 4. Functional interaction between MNs and sweet GRNs in the SEZ.

(a) PER on activation of MNs. PER assays were performed at the indicated temperature. ANOVA with Tukey post-hoc. **P<0.01. (b) Double-labelling of labellar MN and sweet GRN projections in the SEZ. Brains of Gr5a-LexA/+;UAS-mCD8::GFP/+;VT2692-GAL4/LexAOp-mCherryHA flies were stained with a rabbit anti-GFP (green) and the neuropil marker nc82 (blue). Scale bar, 50 μm. (c) GRASP between MNs labelled with VT2692-GAL4 and sweet GRNs labelled with Gr5a-LexA. The genotypes are Gr5a-LexA/+;LexAOp-CD4::spGFP₁₁/+;UAS-CD4::spGFP_1-10/VT2692-GAL4 (GRASP, left), Gr5a-LexA/+;LexAOp-CD4::spGFP₁₁/+;UAS-CD4::spGFP_1-10/+ (LexA only, middle), and LexAOp-CD4::spGFP₁₁/+;UAS-CD4::spGFP_1-10/VT2692-GAL4 (GAL4 only, right). Brains were stained with the neuropil marker nc82 (magenta). A schematic for GRASP is shown below. Scale bar, 50 μm. (d–f) Measurement of sweet GRN activity in the SEZ on MN activation. GCaMP3 was expressed in Gr5a-LexA neurons for calcium imaging and dTrpA1 was expressed in MNs (VT2692-GAL4 and R41E11-GAL4) for artificial neuronal activation. 100 mM sucrose was applied to the labellum. (d) Representative pseudocoluor images showing sweet GRN activity. Scale bar, 20 μm. SEZ ROIs are outlined in white. Arrows point to the corresponding traces in e. (e) Representative traces of fluorescence intensity changes evoked by 100 mM sucrose in sweet GRN termini on MN activation. (f) Mean maximal fluorescent intensity changes. Each trial was carried out at the indicated temperature. Repeated measure ANOVAs with post-hoc Mann–Whitney U-tests, *P<0.05, **P<0.01. n is indicated in parentheses. All data are presented as means±s.e.m.

Figure 5. GABAergic inhibition of sweet GRNs by MNs.

(a) PER in flies with knockdown of GABA-related genes in MNs. Each RNAi line crossed to UAS-dTrpA1;R41E11-GAL4 was subjected to the PER assay. PER was carried out at the indicated temperature. ANOVAs with Tukey post-hoc tests. *P<0.05 (b,c) Food preference of flies with (b) knockdown of GABA-related genes in MNs and (c) knockdown of GABA receptors in sweet GRNs. Flies were given a choice between 0.5 mM sucrose in 0.2% agarose and 1 mM sucrose in 2% agarose. UAS-Dcr2;R41E11-GAL4 (b) and Gr64f-GAL4;UAS-Dcr2 (c) were crossed to each RNAi line. The red dashed line at 0.5 represents no preference. ANOVAs with Tukey post-hoc tests. *P<0.05, **P<0.01. (d,e) Effect of pharmacological GABA_BR blockade in sweet GRN termini on MN-mediated PER inhibition. (d) Representative traces for fluorescence intensity changes in sweet GRN termini after stimulation with 100 mM sucrose and MN activation in the presence of the GABA_BR antagonist CGP54626. Gr5a-LexA/+;LexAOp-GCaMP3/+;UAS-dTrpA1/VT2692-GAL4 were used for calcium imaging. Brains were incubated in artificial haemolymph containing 5 μM CGP54626 for 5 min. (e) Normalized inhibition ratio of maximal ΔF/F₀. Unpaired Student's t-tests, *P<0.05, **P<0.01. n is indicated in parentheses. All data are presented as means±s.e.m.