Flies were reared on standard cornmeal agar medium. We used the Gal4/UAS system 41 (link) to direct the expression of the calcium sensors to PNs. GH146-Gal4 flies were a gift from L. Luo (Stanford University, Stanford, CA). UAS-GCaMP1.6 flies were a gift from D. Reiff and A. Borst (MPI, Martinsried, Germany). All experimental animals were adult females, 3–5 days after eclosion. Adult flies were dissected using previously described methods 11 (link). Flies were anaesthetized in a vial on ice just until movement stopped (<15 second) and then gently inserted into a hole in a piece of aluminum foil. Small drops of wax (55°C) were used to suspend the fly in the hole, with the edge of foil defining a horizontal plane around the head and thorax, from the first antennal segment anteriorly to the scutellum posteriorly. The dorsal side of the foil was bathed in saline, while the ventral side (including antennae and maxillary palps) remained dry and accessible to odours. A window was cut in the dorsal head cuticle between the eyes, extending from the ocelli to the first antennal segment. Fat and air sacs dorsal and anterior to the brain were removed, but the perineural sheath was left intact. The proboscis was affixed with a small drop of wax to a strand of human hair to limit brain movement. Spontaneous leg movements were typically observed in this preparation for the duration of the recording (1.5–3 hr). The saline composition used in all experiments was 42 (link) (in mM): 103 NaCl,3 KCl, 5 N-tris(hydroxymethyl) methyl-2-aminoethane-sulfonicacid, 10 trehalose, 10 glucose, 2 sucrose, 26 NaHCO3, 1 NaH2PO4,1.5 CaCl2, and 4 MgCl2, adjusted to 275 mOsm, pH 7.3 when bubbled with 95% O2/5%CO2.
Odours (cis-3-hexen-1-ol (cis), and isoamyl acetate (ia)) were delivered using a custom-made odour-delivery system and a Teflon nozzle (entry diameter 1/8″) directed towards the antennae. Odours were delivered in a constant stream of air (1 l/min) at final concentrations of ca. 15%. Odour delivery times were measured using a mini-PID (Aurora Scientific Inc., Ontario, Canada). Odours were presented for either 3s or 5s. All comparisons of sensor performance were made using experiments with identical odour presentation times. The results reported are based on data obtained from 3 GCaMP1.6-expressing flies (4 ALs) and 4 GCaMP3-expressing flies (4 ALs).
Odours (cis-3-hexen-1-ol (cis), and isoamyl acetate (ia)) were delivered using a custom-made odour-delivery system and a Teflon nozzle (entry diameter 1/8″) directed towards the antennae. Odours were delivered in a constant stream of air (1 l/min) at final concentrations of ca. 15%. Odour delivery times were measured using a mini-PID (Aurora Scientific Inc., Ontario, Canada). Odours were presented for either 3s or 5s. All comparisons of sensor performance were made using experiments with identical odour presentation times. The results reported are based on data obtained from 3 GCaMP1.6-expressing flies (4 ALs) and 4 GCaMP3-expressing flies (4 ALs).
