Pattern of rest/activity cycles at different times during adulthood evaluated in control lines. (A) Representative double plotted actograms from pdf-gal4/+ flies of increasing age are shown. Each panel depicts the activity of a single fly throughout the experiment. Age at the onset of the experiment is indicated at the bottom of each panel. White, grey and black boxes indicate day, subjective day and night, respectively; arrows represent the transfer to constant darkness (DD). (B) Expression pattern of pdf-gal4 driving a UAS-CD8-GFP reporter gene in the adult brain. GFP recreates the entire PDF circuit. Several relevant landmarks in the adult brain are indicated: small and large LNvs (s-LNvs and l-LNvs, respectively), posterior optic tract (POT), dorsal protocerebrum (dp). (C) Percentage of rhythmic flies for control genotypes (Canton S, CS, and pdf-gal4/+). No significant differences were found through the evaluated timescale. Data represent the mean±S.E.M. Statistical analysis was performed as described in experimental procedures. Three independent experiments were carried out. Additional details are included in Table S1. (D–G) A misexpression screen in young and aged flies reveals progressive behavioral phenotypes. (D) Schematic diagram of the misexpression screen. The pdf-gal4 line was crossed to a number of independent target P[UAS] lines. In the progeny containing both elements the GAL4 transcription factor binds to the UAS within the P[UAS] transposon, inducing the misexpression of the gene immediately adjacent to it. (E–F) APP overexpression leads to progressive arrhythmicity. (E) Representative double plotted actograms of progressively older pdf>APP and control (pdf-gal4/+) flies. (F) The percentage of rhythmic flies for each strain is shown. Aged pdf>APP flies show decreased rhythmicity, which is significantly different from aged controls (***), and its younger counterparts (*) . (* p<0.05 and *** p<0.001). Three independent experiments including forty to seventy flies were carried out. (G) A direct comparison of rhythmicity for each misexpressed line in young and old flies. Misexpression of most P[UAS] lines do not result in a progressive phenotype (╦). Each dot represents the average rhythmicity for a certain insertion line tested at a young and older age. Flies that were highly rhythmic when young but whose rhythmicity decreased severely as they aged were considered as potential neurodegenerative mutants and further retested (indicated by •). This was the case with P[UAS]¹¹⁷ (○). Control flies such as pdf-gal4/+ (□) and pdf-gal4, UAS-CD8-GFP/++ (▪) describe the behavior of the majority of the lines tested (╦).