2.2. Electroantennograms (EAGs) Recordings

Electroantennogram (EAG) recordings were performed in vivo as previously described [47, 48].

By taking into account the circadian cycle in olfactory sensitivity [49], WT and mutant flies were always tested in parallel. Briefly, living 10- to 15-day-old male flies were singly inserted in a 100 μL truncated plastic pipette, with the head protruding at the tip. The preparation was fixed with dental wax on a microscope slide and positioned under the viewer of an Olympus BX51WI light microscope (Olympus, Tokyo, Japan). Glass capillaries with a silver wire were filled with a conductive 0.15 M NaCl solution. The recording glass electrode was gently placed on the tip of the antennal funiculus, whereas the reference electrode was inserted in the compound eye. The EAG signal was amplified with an AC/DC probe and then acquired with an IDAC-4 interface board (Syntech, Hilversum NL). A charcoal purified and humidified airflow was constantly blown over the antennae (speed 0.5 m/s) via a glass tube, placed approximately 1 cm from the antenna. The tip of a Pasteur pipette containing an odour-loaded filter paper (5 mm × 25 mm) was inserted into a small hole in the glass tube. Odour stimulation was administered by injecting a puff of purified air (0.5 s at 10 mL/s airflow) through the pipette using the stimulus delivery controller (Syntech).

Odour stimuli tested were dissolved in hexane and presented in series from minor to higher concentration (resp., 0.01, 0.1, 1, and 10% v/v). 1-Hexanol was chosen for its well-known stimulant activity in Drosophila [50–52], and 1-linalool, a terp commonly found in plants, was chosen for its capability to excite olfactory sensory neurons in different species of insects [53]. As the standard reference, 1-hexanol was administered at the 10% v/v dilution at the beginning of the experiments, to confirm the activity of the antenna. Both 1-hexanol and 1-linalool were purchased from Sigma-Aldrich (Milan, Italy).

Mean values of EAG amplitude were calculated and then analysed by comparing the results obtained in LRRK^WD40 mutant groups with the age-matched WT control group.

The significance of differences was tested by repeated-measures ANOVA or one-way ANOVA (followed by HSD post hoc test) with a threshold level of statistical significance set at P ≤ 0.05 (statistical software package). EAG results are expressed as average values + SEM and represented by histograms.