Synaptopathy induced by α-syn accumulation causes progressive motor impairment and akinetic behaviour prior to age-related dopaminergic neurodegeneration. (A) The DART system was used to measure spontaneous activity of individual flies that were continuously recorded at five frames per second for 2 h, as set by the DART software using a USB-webcam. (B) Top: spontaneous activity is reduced in 3-day-old flies (3-DO) expressing WT-α-syn-EGFP compared with controls; ****P < 0.0001, **P = 0.0071 (n = 90–100 flies/genotype). Bottom: spontaneous activity is further reduced in 20-day-old (20-DO) TH>WT-α-syn-EGFP when compared with controls; ****P < 0.0001 and ***P = 0.0002 (n = 30 flies/genotype). (C) Active speed of 3-day-old flies (3-DO) (top) and 20-day-old flies (20-DO) (top) is impaired by WT-α-syn-EGFP expression; ****P < 0.0001. (D) Schematic of the activity metrics units. The initiation of a locomotor action (red arrows) is called action initiation. Bout length is the length of a motor action (dark grey boxes); and the pause between the end and beginning of a new motor action is called inter-bout interval (white boxes). (E) Top: the number of locomotor actions initiated by 3-day-old (3-DO) TH>WT-α-syn-EGFP flies was reduced compared with the control group TH/+; ****P < 0.0001 and ns—not significant. Bottom: the locomotor actions initiated by 20-day-old (20-DO) TH>WT-α-syn-EGFP flies was reduced when compared with both controls; **P < 0.0011 compared with TH/+ and **P = 0.0097 compared with WT-α-syn-EGFP/+ control. (F) The length of each bout of activity was shorter in TH>WT-α-syn-EGFP flies at 3 (top) and 20 (bottom) days of age, depicting their impaired ability of sustaining a locomotor action; **P = 0.0053; ****P < 0.0001. (G) The length between each bout of activity was longer in flies expressing WT-α-syn-EGFP at 3 (top) and 20 (bottom) days; *P = 0.0108, **P < 0.0014, ****P < 0.0001. Box-and-whisker plots represent the median (horizontal line), 25 and 75% quartiles (box), and 5 and 95% quartiles (whiskers); statistical analyses were performed using Kruskal−Wallis test with Dunn’s multiple comparison post hoc test. (H) Custom-made apparatus used to perform startle induced negative geotaxis (SING) assay which allows all fly genotypes to be probed under equal conditions simultaneously. (I) A group of 10 flies were placed in an assay tube containing 1 cm of fresh media and then allocated back in the fly holder. Next, the holder was gently tapped allowing all the flies to reach the bottom of the tubes (t = 0 s). (J) After 10 s, the number of flies that successfully climbed above the 7 cm line is recorded. (K) Cohorts of flies were analysed at 3, 10, 20, 30 and 40 days of age. The analysis showed an age-related deficiency in their climbing performance which was further enhanced by the overexpression of WT-α-syn-EGFP; ****P < 0.0001; mean ± SEM are shown, n = 9–13 groups of 10 flies. PER assay evaluated the ability of flies to respond to sucrose offer after starvation. (L) Flies were fixed in a card (grey bar) with rubber cement and were left to recover for 3 h. (M) Starved flies were presented with a droplet of 100 mM of sucrose to the legs and then immediately scored; (N) flies that extended or not their proboscis in response to sucrose were scored. (O) Young flies (5–8-day-old) expressing WT-α-syn-EGFP under control of TH-Gal4 driver showed a reduced response to sucrose compared with controls flies, resembling an akinetic behaviour; *P = 0.03 and ****P < 0.0001; mean ± SEM shown for each genotype (n = 13–14 flies/genotype).