Surface glia consist of two distinct glial subtypes namely perineurial and subperineurial glia. Perineurial glial cells were labeled by crossing UAS-GFP to NP6293-GAL4 driver line, which specifically marks this layer of glia (Awasaki et al., 2008). All GFP-positive nuclei of cells on the dorsal surface of the brain showed PER signal at ZT22 but not at ZT10, suggesting rhythmic expression in the perineurial glia in young brains (Figures 1A,B). PER expression persisted in the brains of old flies but the average signal at ZT22 was significantly reduced (Figure 1C, p-value < 0.0001, t-value = 8.928, df = 9.171). Subperineurial glial cells were visualized via moody-GAL4 driver line (Schwabe et al., 2005) combined with UAS-GFP. We observed that GFP-labeled cells surrounded the entire brain in both young and old flies. PER signal was quantified in GFP positive subperineurial glia surrounding the optic lobe (Figures 2A,B). Although some GFP leaked and was observed in the cytoplasm of the subperineurial glia predominant signal came from their large nuclei. Subperineurial glia showed nuclear PER signal at ZT22 (Figure 2A, arrowheads) but not at ZT10 in brains of young flies suggesting rhythmic expression of this protein. PER was discernible from the background in old brains at ZT22 but the average PER signal was significantly reduced compared to young flies (Figure 2C, p-value ≤ 0.0031, t-value = 3.719, df = 11.56).