Analysis of axonal morphology in constant darkness was performed on the second day after switching to DD (DD2). For the analysis of activity-dependent changes in axonal morphology, yw; Pdf-Gal4, UAS-mCD8GFP /UAS-TrpA1 and yw; Pdf-Gal4, UAS-mCD8GFP /UAS-TrpA1; UAS-Mef2RNAi/+ flies were entrained for 3 days using a 12:12 LD cycle at 21°C and collected for dissection at ZT14 immediately after

a 2 hr temperature elevation to 29°C. Imaging was performed with a Leica TCS SP2 confocal microscope using a 20× objective and a 4× digital zoom. Axons were traced using the Simple Neurite Tracer plugin for Fiji software VX-770 ( Longair et al., 2011). Quantitative analysis was performed with ImageJ 1.40 from NIH (http://rsb.info.nih.gov/ij). Axons of all s-LNv neurons in each brain hemisphere were analyzed as a group ( Fernández et al., 2008). For the Sholl’s analysis, 15 concentric circles spaced 10 μm apart were centered on the point where dorsal ramification opens. Total number of intersections selleck compound between axon branches and the concentric circles was computed using Sholl Analysis Plugin for ImageJ (Ghosh laboratory, UCSD). We have also modified this plugin

to additionally detect a 15° cone containing most of the intersections and to compute the fraction of the intersections outside of that “main projection direction” cone. Nearly identical results were seen when brains were stained with anti-GFP antibody using a standard immunohistochemistry

protocol. Immunostaining was performed as previously described in Tang et al. (2010). Briefly, fly heads were removed, fixed in 4% paraformaldehyde for 45 min at 4°C, and brains were dissected Astemizole in PBS. Brains were blocked in 10% normal goat serum (Jackson Immunoresearch) and subsequently incubated with primary antibodies at 4°C for 48 hr. Primary antibodies and their dilutions used were as follows: rabbit anti-GFP at 1:500 (Invitrogen), mouse anti-mCherry at 1:100 (Clontech), and mouse anti-PDF at 1:10 (from Developmental Studies Hybridoma Bank, University of Iowa). For detection of primary antisera, Alexa 488 goat anti-rabbit, Alexa 488 goat anti-mouse, and Alexa 633 goat anti-mouse (Invitrogen) were used at a dilution of 1:200. Brains were mounted in Vectashield Mounting Medium (Vector Laboratories). Locomotor rhythms of individual male flies were monitored for 4 days in LD conditions (12:12 LD intervals) followed by 4–9 days in DD conditions (constant darkness) using Trikinetics Drosophila Activity Monitors. Analyses of period length and rhythmic strength (assessed by by rhythmicity index [RI]; Levine et al., 2002) were performed with MATLAB-based software ( Donelson et al., 2012). Flies with an RI > 0.15 were considered rhythmic, with an RI = 0.1–0.15 weakly rhythmic, and with an RI < 0.1 arrhythmic.