(a) Schematic of the Drosophila head (viewed from above) illustrating the projection of Ir68a-Gal4-labeled neurons (green) from the sacculus to the antennal lobes in the brain, visualized through a hole cut in the head cuticle. (b) Left panel: Raw fluorescence image of Ir68a-labeled axons (in an Ir68a-Gal4;UAS-GCaMP6m(III) animal) innervating the antennal lobe. The circle indicates the position of the ROI used for quantification. Middle and right panels: color-coded images (reflecting GCaMP6m relative fluorescence intensity changes) of responses to a switch from 7% to 90% RH (‘Moist response’) and to a switch from 90% to 7% RH (‘Dry response’). Scale bar is 10 µm. (c,d) Moist-elicited (c) and dry-elicited (d) fluorescence changes in the region of interest in panel (b) (moist = 90% RH, dry = 7% RH). Left panels: Traces represent mean ± SEM. Right panels: Quantification of responses. Letters denote statistically distinct groups (p≤0.05) Steel-Dwass. Data obtained using UAS-GCaMP6m(II) and UAS-GCaMP6m(III) were analyzed separately. Moist-responses were calculated as [F/F0 at 90% RH (average F/F0 from 4.5 to 6.5 s after shift to 90% RH)] - [F/F0 at 7% RH (average F/F0 from 3.5 to 1 s prior to shift to 90% RH)]. Dry-responses were quantified using the converse calculation. Violin plots: internal white circles show median; black boxes denote 25th to 75th percentiles; whiskers extend 1.5x the interquartile range. Genotypes: wild type (UAS-GCaMP6m(II)): Ir68a-Gal4,UAS-GCaMP6m(II) (n = 7 animals). Ir68a-/-: UAS-GCaMP6m(II);Ir68a-Gal4,Ir68ac04139/Ir68ac04139 (n = 9). Ir68a rescue: Ir68a+ rescue transgene(II)/UAS-GCaMP6m(II);Ir68a-Gal4,Ir68ac04139/Ir68ac04139 (n = 8). Ir93a-/-: UAS-GCaMP6m(II);Ir68a-Gal4,Ir93aMI05555/Ir93aMI05555 (n = 8). Ir93a rescue: UAS-GCaMP6m(II);Ir68a-Gal4,Ir93aMI05555/UAS-mCherry:Ir93a,Ir93aMI05555) (n = 6). wild type (UAS-GCaMP6m(III)): Ir68a-Gal4,UAS-GCaMP6m(III) (n = 10 animals). Ir25a-/-: Ir25a2;Ir68a-Gal4,UAS-GCaMP6m(III) (n = 8). Ir25a rescue: Ir25a2,Ir25aBAC/Ir25a2;Ir68a-Gal4,UAS-GCaMP6m(III) (n = 8). Ir40-/-: Ir40a1;Ir68a-Gal4,UAS-GCaMP6m(III) (n = 8). (e,f) Moist-elicited (e) and dry-elicited (f) fluorescence changes in Ir40a-Gal4-labeled dry receptors, as in panels c-d. Genotypes: wild type: Ir40a-Gal4/UAS-GCaMP6m (n = 9). Ir68a-/-: Ir40aGal4/UAS-GCaMP6m;Ir68ac04139 (n = 9). Ir68a mutant alleles and genomic rescue fragment are shown in Figure 1—figure supplement 1. Source data for summary graphs are provided in Figure 2—source data 1.