*2.5. Electrophysiology*

Electroantennograms (EAGs) were recorded with Ringer-filled capillary glass electrodes (NaCl 120 mM; KCl 5 mM; CaCl2 1 mM; MgCl2 4 mM; HEPES 10 mM; pH 7.2) from 4- to 6-day-old adult male flies [58]. Immobilized flies were placed in a 1000 μL micropipet-tip, the extremity of the tip was filled with cotton and flies were blocked by dental wax. The ground electrode (tip diameter = 2–3 μm) was introduced into the right eye and the recording electrode (tip diameter = 10 μm) was placed on the distolateral surface of the third antennal segment. Recorded signals were amplified (×100) and low-pass filtered at 1 kHz using an Axopatch 200B amplifier (Molecular Devices, San José, USA) and monitored on a computer using a Digidata 1440A acquisition board (Molecular Devices, San José, USA) driven by Clampex 10 software (Molecular Devices, San José, USA).

Odorants were diluted 1/1000 (v/v) in para ffin oil and delivered from Pasteur pipettes containing 10 μL of diluted odorant deposited on a strip of filter paper. Stimulation with sex-specific odors was obtained by blowing a flow of humidified air (37.5 mL/s) for 1 s through a Pasteur pipette (inner diameter 5 mm, length 250 mm) containing 25 same-sex flies (4- to 6-day-old) [59]. Flies were placed in the tube one hour prior to experiments.

Flies were first stimulated with 2-Heptanone (2-H), then with odors of living males and finally with odors of living females. Flies were allowed to recover at least 2 min between each stimulation. The odorant 2-H molecule, which elicits a robust electrophysiological response, was chosen to normalize the EAG responses to male and female odors.
