Drosophila stocks were reared on standard cornmeal agar medium supplemented with dry yeast for 24 h at 60% relative humidity. α-Syn-expressing Drosophila were modified by microinjection and expression of A53T α-Syn transgenes was driven by the elav-GAL4 expression system. In brief, the α-Syn clone was constructed using the elav-GAL4 expression system, then this was injected into the eggs of white eye Drosophila (control flies). After hybridization of the red eye flies, α-Syn flies were created. Elav-GAL4 flies were crossed with UAS-A53T flies (provided by the Biochemistry and Cell Institute of Shanghai Life Science Research Institute of the Chinese Academy of Sciences, Shanghai, China), to produce the A53T flies.

The cerebral proteins of α-Syn Drosophila brains were extracted from tissue lysate with the protease inhibitor phenylmethylsulfonyl fluoride (Sigma-Aldrich, St. Louis, MO, USA). The solution was sonicated and ultracentrifuged for 30 min at 4695 × g at below 4°C. The resulting supernatant was boiled for 15 min in a water bath. Adult fly heads of the appropriate genotypes were prepared and analyzed using standard SDS-PAGE with 10% Tris-glycine gradient gels. The primary α-Syn antibody (cat. no. 610786) was developed by BD Biosciences (Mountain View, CA, USA) and was diluted to 1:1,000 prior to use. The goat anti-mouse secondary antibody (cat. no. 81-6511; Thermo Fisher Scientific, San Francisco, CA, USA) was then diluted to 1:10,000 and applied, and an X-ray film exposure was conducted.

Flies were divided into random groups of 20–30 individuals per vial and then assessed for geotaxis as described previously (8). Briefly, groups of 10 flies were placed in a 95×27 mm empty vial. Flies were gently taped to the bottom of the vial and the number of flies crossing an 8 cm mark was recorded after 10 sec. Researchers were blinded to the corresponding genotype and condition of the flies.

Hungry flies (80–100), which had been in the dark for 30 min prior to the experiment were placed in the maze. After 90 sec, smell A was released in one side of the T maze and at the same time, twelve 1.5 sec of the 70 volt DC electrical stimulation was applied over 60 sec. Flies were then allowed to breathe air for 45 sec. Subsequently, smell B was released for 60 sec in the other side of the T maze followed by 45 sec air. The flies were then allowed to move around the maze freely for 2 min, after which the numbers of flies at the sides of the maze with smells A and B were counted.

The flies brains were stripped and placed on a slide. The slide was rinsed with 1% PBS (Sigma-Aldrich) three times, blocked and incubated in a blocking buffer (0.1 M PBS, 0.1% Triton X-100 and 1% BSA; Sigma-Aldrich) for 3 h at room temperature. The α-synuclein antibody (1:250; 610786; BD Biosciences) was then applied and washed by PBS for 5 min, three times. Subsequently, the rabbit anti-human IgG secondary antibody (BA1020; Boster, Wuhan, China) was applied. Following incubation, the brain was washed three times with PBS with 5 min intervals and fixed with a coverslip. The brains of flies were observed with an immunofluorescence laser scanning confocal microscope (Zeiss LSM710; Carl Zeiss, Oberkochen, Germany).

In order to identify and confirm that the cells recorded were PNs, cells recorded were stained by biocytin (Sigma-Aldrich). While recording, cells were injected with biocytin through a recording pipette filled with internal solution containing biocytin in whole-cell recording mode for a minimum of 30 min. After recording, the brains were collected and fixed in 4% formaldehyde (Sigma-Aldrich) in phosphate-buffered saline (PBS; Sigma-Aldrich) at 4°C for 10 h. The brain was then rinsed in 1% PBS three times, blocked and incubated in a blocking buffer (0.1 M PBS, 0.1% Triton X-100, and 1% bovine serum albumin; Sigma-Aldrich) containing streptavidin-Cy3 (Molecular Devices Ltd., Wokingham, UK) for 3 h at room temperature. After incubation, the brain was washed three times with 5 min intervals in PBS. A BX51WI microscope (Olympus, Tokyo, Japan) with a ×40 objective and confocal camera was used to capture images of dendritic arborization of the PNs in the antennal lobe.

Adult Drosophila brains were fixed in 4% paraformaldehyde and embedded in paraffin. The paraffin-embedded brains were then cut into sections. The α-Syn antibody (1:250; BD Biosciences) was used to detect the expression of α-Syn.

All brains were obtained from female flies two days prior to eclosion. The entire brain, including optic lobes, was removed from the head and prepared for recordings in standard external solution containing 20 U/ml papain (Sigma-Aldrich) with 1 mM l-cysteine. Subsequently, the dissected brains were mounted in an RC-26 perfusion chamber (Sigma-Aldrich) containing the recording solution bubbled with 95% O₂ and 5% CO₂ (2 ml/min) throughout the experiments, with the ventral surface of the brain facing up. Pipettes were targeted to PNs in the dorsal neuron cluster in the antennal lobe. For measurements of cholinergic miniature excitatory postsynaptic currents (mEPSCs), tetrodotoxin (TTX; 1 μM) was added to the external solution to block voltage-gated sodium currents and γ-aminobutyric (GABA)ergic synaptic currents, and picrotoxin (PTX; 10 μM; Sigma-Aldrich) was added to block GABAergic synaptic currents. CaCl₂ was omitted and tetraethylammonium (TEA; 10 mM; Sigma-Aldrich) and 4-aminopyridine (4-AP; 1 mM) were added to the external solution to measure Ca²⁺ currents. For Ca²⁺ current measurements, the external solution consisted of 101 mM NaCl, 1.8 mM CaCl₂, 0.8 mM MgCl₂, 5.4 mM KCl, 5 mM glucose, 1.25 mM NaH₂PO₄, 20.7 mM NaHCO₃, 1 μM TTX, 10 mM TEA and 1 mM 4-AP.

Statistical analysis was conducted using SPSS software, version 11.5 (SPSS, Inc., Chicago, IL, USA) Comparison of the magnitude of the calcium currents prior to and following application of permethrin was made using an unpaired t-test. Comparison of the area below the baseline of sodium currents prior to and following application of permethrin was made using a paired t-test. Comparisons of more than two treatments were made with analysis of variance (ANOVA) and the Bonferroni post hoc correction.

The pan-neuronal driver elav-GAL4 was used to drive expression of normal and mutant (A53T) human α-Syn throughout the nervous system. Western blot analysis was performed to verify the expression of α-Syn (Fig. 1).

Previous studies have demonstrated that a decrease in the ability of flies to climb up the wall of a plastic vial is associated with the progression of the pathology in PD (15,16). However, in the present study, no significant decrease in climbing ability was observed (Fig. 2).

Compared with CS flies, the learning and memory ability of α-Syn wild-type and A53T mutation flies decreased exhibiting a reduction of ~18% (Fig. 3) as elucidated through a T maze experiment.

The whole brain of the Drosophila were observed with an immunofluorescence laser scanning confocal microscopy, and it was identified that the levels of green fluorescence were greater in the α-Syn Drosophila compared with the control saline group. In addition, green fluorescence was particularly evident in the mushroom body (Fig. 4).

Previous studies have demonstrated that cac-encoded voltage-gated calcium channels are important in regulating the spontaneous release of neurotransmitter at excitatory cholinergic synapses and mEPSC frequency in the PNs of Drosophila (7,17,18). Recordings were performed at room temperature for a minimum of 1 min in the presence of TTX to block sodium channels and PTX to block GABA receptors. The dorsal antennal lobe glomeruli is the location of the PNs, with two main branches projecting to the lateral horn and the local mushroom body. The PNs were identified morphologically from biocytin fills and their specific electrical activities (Fig. 5). The PNs were selected for analysis as they were cholinergic and cholinoceptive. PNs firing spontaneously were recorded in α-Syn Drosophila in the presence of TTX and PTX. The experiments indicated that the frequency of PNs was reduced. Sodium AP-independent mEPSCs in the antennal lobe PNs were monitored in the whole brain, which was isolated from female fly pupae two days prior to eclosion. mEPSCs were directly monitored by whole cell patch-clamp recordings from PNs. The average mEPSC frequency was 1.92±0.3 Hz under normal conditions. The average mEPSC frequency decreased significantly by 0.62±0.11 Hz in the α-Syn Drosophila. The present results demonstrated that the mEPSC frequency in the α-Syn Drosophila was decreased (Fig. 6).

Cholinergic mEPSCs were recorded from single PNs following the addition of TTX and PTX to the external solution. The mean frequency of mEPSCs was significantly reduced by α-Syn (ANOVA, Bonferroni post hoc test; P<0.01). No significant difference was identified between the mean mEPSC amplitude of the α-Syn and A53T groups and that of the CS group.

Calcium currents have been revealed to have broad effects on neuronal function, particularly in presynaptic terminals for neurotransmitter release, electrical excitability and activity-dependent gene regulation (19,20). Calcium currents were directly calculated in antennal lobe PNs isolated from the whole brain of fly pupae two days prior to eclosion. Using whole cell voltage-clamp recordings, the inward calcium current was isolated in PNs as previously described (21). The average calcium current in normal PNs and α-Syn PNs were 5.57±1.94 and 3.03±0.85 pA/pF, respectively (Fig. 7).