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Extended data figures and tables
Extended Data Figure 1 Odour screen for Ir40a neuron agonists.
Top, representative wide-field fluorescence image of the antennal lobes of an Ir40a-Gal4;UAS-GCaMP3 fly, highlighting the region of interest (marked by a black dashed circle) used for quantification of stimulus-evoked calcium responses in Ir40a neuron axon termini in the ‘column’ glomerulus. Bottom, mean response amplitude ± s.e.m. (n =3–6 flies) for the indicated odours (10% v/v in paraffin oil or water solvent, presented for 1 s as in Fig. 1d). GCaMP3 fluorescence changes during these recordings were not bleach-corrected; thus, the decreases in fluorescence intensity quantified for the vast majority of odours do not reflect stimulus-evoked inhibitory responses.
Extended Data Figure 2 Generation and characterization of an Ir40a mutant.
a, Schematic of the Ir40a locus (exons in green), showing the position of the CRISPR target sequence, and the sequence of the Ir40a1 allele in this region, which contains a 10-base-pair frameshift deletion. PAM, protospacer adjacent motif. b, Immunofluorescence with anti-Ir40a (green) and anti-Ir25a (magenta) on antennal cryosections from wild-type (w1118) and Ir40a1 mutant flies. The merged channels are shown on a bright-field background to provide anatomical landmarks. The arrowhead marks the sensory dendrites where Ir40a and Ir25a co-localize in wild-type flies. The same result was obtained in at least five independent experiments. The scale bar is 20 μm. c, Top: Calcium responses of Ir40a neurons to 3% ammonia in the antennal lobes of control (Ir40a-Gal4;UAS-GCaMP3/+), Ir40a mutant (Ir40a-Gal4,Ir40a1;UAS-GCaMP3/+) and Ir40a rescue (Ir40a-Gal4,Ir40a1;UAS-GCaMP3,UAS-Ir40a/+) animals (n = 9, 11 and 8 flies for control, mutant and rescue, respectively). Bottom: Calcium responses of Ir40a neurons to 3% ammonia in the antennal lobes of control (Ir40a-LexA,LexAop-GCaMP3), Ir25a mutant (Ir25a2;Ir40a-LexA,LexAop-GCaMP3) and Ir25a rescue (Ir25a2,Ir25a-BAC;Ir40a-LexA,LexAop-GCaMP3) animals (n = 8, 7 and 7 flies for control, mutant and rescue, respectively). The bar amplitude corresponds to the median percentage ΔF/F of the peak of the response across flies; the error bars indicate the range between the first and third quartiles of the distribution. All pairwise comparisons were performed using the Wilcoxon Test corrected for multiple testing with Benjamini and Hochberg’s method; *P < 0.05.
Supplementary information
Supplementary Information
This file contains Supplementary Methods, Supplementary References and Acknowledgements. (PDF 252 kb)
Video 1: Responses of IR40a neurons to air stimulation
Raw changes in fluorescence of IR40a OSN soma expressing GCaMP3 (imaged in the antenna by confocal microscopy at 4 Hz) in response to a puff of air (blank control) delivered manually. The ROI is marked by a red circle and the time of stimulation is indicated by the label appearing in the bottom right corner of the image during the corresponding frames. Reproduction rate is 7 fps. (MOV 2341 kb)
Video 2: Responses of IR40a neurons to DEET stimulation
Raw changes in fluorescence of IR40a OSN soma expressing GCaMP3 (imaged in the antenna by confocal microscopy at 4 Hz) in response to a puff of 100% DEET delivered manually. The ROI is marked by a red circle and the time of stimulation is indicated by the label appearing in the bottom right corner of the image during the corresponding frames. Reproduction rate is 7 fps. (MOV 2284 kb)
Video 3: Responses of IR40a neurons to DMSO stimulation
Raw changes in fluorescence of IR40a OSN soma expressing GCaMP3 (imaged in the antenna by confocal microscopy at 4 Hz) in response to a puff of DMSO delivered manually. The ROI is marked by a red circle and the time of stimulation is indicated by the label appearing in the bottom right corner of the image during the corresponding frames. Reproduction rate is 7 fps. (MOV 2243 kb)
Video 4: Responses of IR40a neurons to BA stimulation
Raw changes in fluorescence of IR40a OSN soma expressing GCaMP3 (imaged in the antenna by confocal microscopy at 4 Hz) in response to a puff of 50% BA delivered manually. The ROI is marked by a red circle and the time of stimulation is indicated by the label appearing in the bottom right corner of the image during the corresponding frames. Reproduction rate is 7 fps. (MOV 2266 kb)
Video 5: Responses of IR40a neurons to EA stimulation
Raw changes in fluorescence of IR40a OSN soma expressing GCaMP3 (imaged in the antenna by confocal microscopy at 4 Hz) in response to a puff of 50% EA delivered manually. The ROI is marked by a red circle and the time of stimulation is indicated by the label appearing in the bottom right corner of the image during the corresponding frames. Reproduction rate is 7 fps. (MOV 2314 kb)
Video 6: Responses of IR40a neurons to MDA stimulation
Raw changes in fluorescence of IR40a OSN soma expressing GCaMP3 (imaged in the antenna by confocal microscopy at 4 Hz) in response to a puff of 50% MDA delivered manually. The ROI is marked by a red circle and the time of stimulation is indicated by the label appearing in the bottom right corner of the image during the corresponding frames. Reproduction rate is 7 fps. (MOV 2263 kb)
Video 7: Responses of IR40a neurons to H2O stimulation
Raw changes in fluorescence of IR40a OSN soma expressing GCaMP3 (imaged in the antenna by confocal microscopy at 4 Hz) in response to a puff of H2O delivered manually. The ROI is marked by a red circle and the time of stimulation is indicated by the label appearing in the bottom right corner of the image during the corresponding frames. Reproduction rate is 7 fps. (MOV 2392 kb)
Video 8: Responses of IR40a neurons to ammonia stimulation
Raw changes in fluorescence of IR40a OSN soma expressing GCaMP3 (imaged in the antenna by confocal microscopy at 4 Hz) in response to a puff of 10% ammonia delivered manually. The ROI is marked by a red circle and the time of stimulation is indicated by the label appearing in the bottom right corner of the image during the corresponding frames. Reproduction rate is 7 fps. (MOV 2285 kb)
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Silbering, A., Bell, R., Münch, D. et al. Ir40a neurons are not DEET detectors. Nature 534, E5–E7 (2016). https://doi.org/10.1038/nature18321
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DOI: https://doi.org/10.1038/nature18321
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