Drosophila melanogaster were maintained on diet containing yeast (35 g/l), cornmeal (50 g/l), and molasses (5%). Temperature was maintained at 25 ± 1°C with a 12:12 h light/dark cycle with fluorescent light of luminous energy of 8 ± 2 μmol m−2s−1. We used mated males in all experiments to minimize differences in lifespan, which may vary with sex and mating status. Males were aged in groups of 50 in polypropylene cages (Genesee Scientific, San Diego, CA) inverted over 35 mm petri dish (BD Falcon, San Jose, CA) containing 15 mL of diet. Diet dishes were replaced every 2–3 days. Young (day 5) and old (day 55) males expressing nuclear GFP in specific glial cell subtypes were obtained by crossing w;UAS-GFP with nuclear localization signal (Bloomington Drosophila Stock Center stock 4775) males with females carrying GAL4 drivers expressing in the following glia types: perineurial glia, NP6293-GAL4 (Awasaki et al., 2008 (link)); subperineurial glia moody-GAL4 (Schwabe et al., 2005 (link)); cortex glia NP577-GAL4 and NP2222-GAL4; ensheathing glia NP6520-GAL4 (Awasaki et al., 2008 (link)) and mz0709-GAL4 (Ito et al., 1995 (link)); astrocytes alrm-GAL4 (Doherty et al., 2009 (link)). UAS-GFP with the nuclear localization signal was chosen to clearly discern nuclear overlap between GFP and PER protein; however, some GFP was also visible in the cytoplasm of glial cells.
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