In order to quantify the relative fluorescence of PER signal at ZT10 and ZT22 in each glia subtype, images were reviewed and maximum intensity projections were created using ZEN 2012 software (Zeiss). Due to their location on the outer surface of the adult Drosophila brain, in order to capture a sufficient number of surface glial cells for measurement, multiple images of non-overlapping 6 μm stacks were captured in several regions of each brain. The area of focus for perineurial glia was the dorsal brain while the surface of the optic lobes was used for subperineurial glial cells. For all other glial subtypes, a single 11 μm thick stack from each brain was used for PER signal quantification. PER levels were evaluated by measuring the fluorescence intensity in an average of 15 GFP-positive cell nuclei located in the region of interest specified below. After converting the mean level of fluorescence to the Mean Gray Value the intensity was quantified using Fiji ImageJ software (Schindelin et al., 2012). For each stack, measurements of non-specific background fluorescence were taken from the adjacent areas of similar size as glial cell nuclei (avoiding non-specific red speckles). The background values were averaged and subtracted from the averaged PER measurements obtained from that stack. Five to seven brains were used to measure PER at given time point and age. Statistical significance for average intensity of PER staining between young and old brains at ZT22 was calculated by unpaired t-test with Welch's correction using GraphPad Prism 6 (GraphPad Prism v6.0;GraphPad Software Inc. San Diego, CA). The p- and t-values and the degrees of freedom (df) from these measurements are provided in the results section and in the figure captions.