Sensilla of targeted ORNs were identified using 10x and 50x objectives with an optovar 1.6x attachment (Zeiss, EC Epiplan-Neofluar 10x, LC EC Epiplan-Neofluar 50x and Optovar Module 1.6x P&C ACR) on a Zeiss AxioExaminer D1 compound microscope, using a light source and eGFP filter cube (FL Filter Set 38 HE GFP shift free). Green fluorescence signals in flies were visualized from OrX-Gal4 and 10xUAS-IVS-mCD8GFP (Bloomington Stock #32186; for the OrX-Gal4 on Chr. II) or 15xUAS-IVS-mCD8GFP (Bloomington Stock #32193; for the OrX-Gal4 on Chr. III). The representative images shown in Fig 2A–2C and S1 Fig were taken on the recording rig. The suggested mounting positions of antenna are shown in Fig 3. The electrode was filled with Beadle-Ephrussi ringers solution (7.5g of NaCl+0.35g of KCl+0.279g of CaCl2-2H2O in 1L of H2O). Extracellular activity was recorded by inserting a glass electrode into the base of the sensillum of 4–8 day-old flies. Signals were amplified 100X (USB-IDAC System; Syntech, Hilversum, The Netherlands), inputted into a computer via a 16-bit analog-digital converter and analyzed off-line with AUTOSPIKE software (USB-IDAC System; Syntech). The low cutoff filter setting was 50Hz, and the high cutoff was 5kHz. Stimuli consisted of 1000 ms air pulses passed over odorant sources. The Δspikes/second was obtained by counting the spikes in a 1000ms window from 500 ms after odor stimuli were triggered, subtracting the spikes in a 1000ms window prior to stimulation. 10 standard odors for identification of sensillar types are acquired at highest purity and listed as follows: Ethyl acetate (Sigma #270989), Pentyl acetate (Sigma #109584), Ethyl butyrate (Sigma #W242705), Methyl salicylate (Sigma #76631), Hexanol (Sigma #471402), 1-octen-3-ol (Sigma #68225), E2-hexenal (Sigma #W256005), 2,3-butanedione (Sigma #B85307), Geranyl acetate (Sigma #45896), 2-heptanone (Sigma #537683), 11-cis vaccenyl acetate (50mg in 1ml ethanol, Cayman Chemical Company #0424297–6). The odors except cVA were diluted in mineral oil (Sigma #330779) at 1:100 and 30 μl was used for stimulation. Odors were delivered to the antenna as previously described [4 (link), 6 (link), 10 (link)]. Stimuli were delivered by placing the tip of an odor Pasteur pipette through a hole in a pipette (Denville Scientific Inc, 10ml pipette) that carried a purified continuous air stream (8.3 ml/s) directed at the antenna. A solenoid valve (Syntech) diverted delivery of a 1 s pulse of charcoal-filtered air (5 ml/s) to a Pasteur pipette containing odorant dissolved onto filter paper. Fresh odorant pipettes were used after no more than 3 odor presentations.
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