Versatile Method to measure locomotion

Abstract

Many studies require the ability to quantify locomotor behavior over time. The 9 list of tracking softwares and their capabilities are constantly growing. At the 2019 10CanFly Conference we presented preliminary results from an investigation of the effects 11 of expressing polyglutamine repeats in fly muscles on longevity, locomotion and protein 12 aggregation and received a lot of inquiries about our protocol to measure locomotion 13 and how to use the FlyTracker MatLab software. This report describes a versatile 14 locomotion measuring device and custom MatLab scripts for the extraction and 15 analysis, and compilation of FlyTracker data in a format compatible with spreadsheet 16 softwares. The measurement and analysis of multiple genotypes and both sexes across 17 age shows that this method yields reproducible results that confirm that normal aging is 18 associated with a progressive decline in locomotion as indicated by increased 19 immobility and reduced velocity. 20 22Here, we report a locomotion measuring device that can accommodate 5 to 50 flies and 50 custom MatLab scripts for the extraction and analysis, and compilation of FlyTracker 51 data in a format compatible with spreadsheet softwares. Multiple genotypes, both sex 52 and two experimental replicates across age demonstrate the reproducibility achieved. 53 54 Materials and Methods 55 Construction of the arena 56A drill press was used to drill 2.5cm circles into thin (3mm), clear plexi glass in 57 rows of 5. The rows were cut into strips ( Figure 1A). A piece of clear plexi glass the 58 same dimensions as the strip was adhered to the back of the strip with superglue. By 59 constructing chambers in strips the arena can be adapted to accommodate experiments 60 requiring 5 chambers and up to 50. A base was made from another piece of plexi glass, 61 large enough to fit all 10 strips ( Figure 1A). A small hole is drilled near the edge of the 62 base to load the flies. Three small pieces of plexi glass were cut and glued to the base 63 to use as a guide for positioning the strips consistently. Legs were made for the base 64 out of cuvettes. 65 Fly Husbandry 66Crosses were set up in bottles to obtain desired genotypes. Four different wild-67 type genotypes were obtained by crossing male w 1118 with UAS-Httex1-Q72-eGFP and 68 UAS-Httex1-Q25-eGFP[21] and female w 1118 with DJ694 [22] and MHC-Geneswitch 69[23]. After allowing the parents to mate for 2 days, they were then transferred to a 70 second set of bottles to generate a replicate. The second set of bottles was emptied 71 after two days. Staged flies (0-2 days) were collected under nitrogen anesthesia. A 72 minimum of 4 sample vials for each sex of each genotype containing a minimum of 25 73 flies were collected. This was repeated for the replicate crosses. Flies were allowed to 74 recover for at least 1 day before being recorded. Flies were maintained on standard fly 75 food (0.01% molasses, 8.2% cornmeal, 3.4% yeast, 0.94% agar, 0.18% benzoic acid, 76 0.66% propionic acid) at 23-26˚C for the duration of...