sd-L neurons signal to the sweet-sensing neurons to control hardness preference.

a GRASP signal (green, anti-GFP) between iav-LexA and Gr5a-gal4 neurons (iav-LexA > lexAop-CD4-spGFP11 and Gr5a-gal4 > UAS-CD4-spGFP1-10) in the SEZ. Scale bar, 50 μm. b Co-localization signal (green, anti-GFP; magenta, anti-Brp) between iav-LexA and Gr5a-gal4 neurons (iav-LexA > 8×LexAop2-FLPL and Gr5a-gal4 > 20XUAS> dsFRT>chrimson-mVenus) in the SEZ. Scale bar, 50 μm. Magenta: nc82. c, d GRASP signal (green, anti-GFP) between Gr5aLexA and sd-L neurons in the SEZ. (c) Gr5aLexA > lexAop-CD4-spGFP11, UAS-CD4-spGFP1-10; (d) sd-L spilt gal4 > UAS-CD4-spGFP1-10, Gr5aLexA > lexAop-CD4-spGFP11. Scale bar, 50 μm. e-i Calcium imaging of the sweet-sensing neurons in response to the activation of sd-L neurons. (g) An image of fly preparation for calcium imaging. Red arrow pointed to SEZ region (top) and labellum (bottom). Labellum and brain were connected (circled by a dotted line). Sweet neurons express GCaMP6m under the control of Gr64f > LexA. Representative imaging of Ca2+ responses in the SEZ before (left) and after (right) the application of ATP or AHL were shown. Sd-L-SS drove the expression of P2X2, with 5 mM ATP (upper panels) or equal volume of AHL (lower panels) (e); Scale bar, 50 μm. Parental control (sd-L-SS only (upper panels) or UAS-P2X2 only (lower panels)) were given 5 mM ATP (f); Scale bar, 50 μm. Changes of fluorescence intensity along the recording time window (h), the purple arrow pointed the time when ATP or AHL were added in. Summary of maximum calcium responses of Gr64f neurons to sd-L neurons’ activation and controls (i); n = 5, 5, 5 and 8 for each group. Statistical test: one-way ANOVA with Dunnett’s correction for multiple comparisons against the sd-L-SS > P2X2 with ATP group; mean ± SEM. j, k FlyPAD assay of fly feeding when Gr64f neurons were silenced with UAS-kir2.1. Flies were all starved for 24 h before assay. Both 0.25% and 0.4% agarose containing 10 mM sucrose. Cumulative sips numbers of Gr64f-Gal4 flies, n = 15 for each group (j). Cumulative sips numbers of Gr64f-Gal4 > UAS-kir2.1 flies, n = 15 for each group (k). For all analyses, statistical differences are represented as follows: ns, not significant, p > 0.05; *, p < 0.05; **, p < 0.01; ***, p < 0.001; ****, p < 0.0001.