Oscillating dCREB2 and NF-kB Activity Figure 1. Developing a spatially restricted reporter system. (A) A cartoon of the reporter system. Three DNA binding sites are placed upstream of the CaSpeR TATAA sequence, followed by transcription (arrow) and translation (arrowhead) initiation sites, an FRT-flanked open reading frame for mCherry (ORF) including two tandem stop codons, and the luciferase ORF (#1). Targeted GAL4 expression (#3) drives the expression of UAS-FLP (#2), which catalyzes site-specific recombination at the FRT sites, activating the reporter. (B) CRE-F-luc reporter activity is FLP-dependent. Singly (CRE-F-luc) or doubly transgeneic flies (hs-FLP/+;CRE-F-luc/+) are maintained under 12:12 LD conditions. Flies are exposed to heat-shock (+HS) or not (2HS), and measured for in vivo luminescence. The relative luminescence is plotted as a function of time, with daytime (white bars) and nighttime (black bars) durations indicated below the graph. Each data point represents the average hourly luminescence counts in relative light units (RLU) of 24 flies. (C) FLP protein expressed using the GAL4-UAS system can activate the reporter. Reporter activity (Y-axis, the mean hourly relative light units [RLUs] over a 4-day window) is plotted as a function of genotype (indicated with different colored bars) and treatment (+/2 HS, heat shock). Error bars = S.E.M. *** p,0.001, Student’ T-test, n = 24; n.s. signifies not significant. Similar statistical comparisons are made for the remaining figures. (D) Anatomical specificity of gmrlong-GAL4 driven reporters. In vitro luciferase activity measured in extracts made from isolated heads and bodies. The relative light units (Y-axis) are plotted as a function of the genotype (shown in gray [UAS-FLP/+; CRE-F-luc/+ or black [UAS-FLP/gmrlong- GAL4; CRE-F-luc/+]) or tissue source (head versus body). doi:10.1371/journal.pone.0045130.g001 NF-kB signaling via stabilization of I-kBa and inhibition of the ubiquitin-proteasome pathway [41–46]. Feeding of this drug has been used in Drosophila to suppress NF-kB activity [47]. Female UAS-FLP/+;NFkB-F-luc/actin-GAL4 flies were maintained for 24 h on yeast paste containing PDTC and luciferin, and luciferase activity was measured 1 h later in vivo. Reporter activity is reduced in a dose-dependent manner (Fig. 2E). At the highest concentra- tion (400 mg/L), significant suppression of reporter activity lasts for 24 h (Fig. S3). To measure the temporal pattern of dCREB2 and NF-kB activity in the nervous system, we activated the reporter under the control of the pan-neuronal elavc155-GAL4 (Fig. 3A, E), the pan- glial repo-Gal4 (Fig. 3B,F), or the MB-specific ok107-GAL4 drivers (Fig. 3C,G). For both reporters, we observe significant signal with each of the three drivers when compared to flies carrying the reporter and UAS-FLP transgenes alone (Fig. 3D,H). CRE-F-luc activity in neurons, glia, and the MB shows a similar diurnal pattern, with broad peaks during the middle of the day and the middle of the night (Fig. 3A–C). NF-kB reporter activity shows a similar oscillating pattern in neurons, glia, and the MB, with a major peak shortly after lights on (Fig. 3E–G). Interestingly, unlike with the actin-GAL4 driver (Fig. 2B), a secondary peak in NF-kB reporter activity in the middle of the nighttime period is also present in neurons, glia, and the MB (Fig. 3E–G). In general, there is some variability in the presence of the secondary peak (data not shown), consistent with mixed reports of secondary peaks in per reporter activity [27,28,48,49]. This issue requires further investigation. Because NF-kB reporter activity shows a robust rhythmic activity, we asked whether the reporter oscillates under circadian control. Robust oscillations in neurons, glia, and the MB are maintained in constant darkness after light:dark entrainment (Fig. 4A). Under LL conditions (see Methods), oscillations are maintained, but dampen over the course of light exposure (Fig. 4B). The persistence of oscillating activity in light-entrained flies maintained in constant darkness is strong behavioral evidence for circadian control of reporter activity. To determine if this activity is genetically under circadian control, we asked if PLOS ONE | www.plosone.org 3 October 2012 | Volume 7 | Issue 10 | e45130