Oscillating dCREB2 and NF-kB Activity Figure 5. Oscillations in dCREB2 and NF-kB activity persist after luciferin removal. (A) Reporter activity in UAS-FLP/+;CRE-F-luc/actin-GAL4 flies starting ,1 h following luciferin removal, plotted as a function of time. The inset shows a scaled view of the same data, with the first 24 h dropped. (B) Reporter activity in UAS-FLP/+;NFkB-F-luc/actin-GAL4 flies starting ,1 h following luciferin removal. Inset shows a scaled view of the same data, with the first 24 h dropped. n = 24 flies for each group. doi:10.1371/journal.pone.0045130.g005 under circadian regulation. However, dCREB2 and NF-kB reporter oscillations do not appear to be explained by diurnal variations in substrate ingestion (Fig. 5, S5). Furthermore, the relative changes in reporter activity that we observe are stronger than the low-amplitude changes (#0.25 fold) reported in hsp-luc control reporters [28], and in another transcription factor reporter that we have analyzed (data not shown). Finally, under light:dark conditions, the dCREB2 and NF-kB reporters show different kinetics, with the onset and peak of NF-kB activity (ZT<1) occurring during a relative trough in dCREB2 activity (Fig. 1B, 2B, 3, S2). It seems most likely, therefore, that dCREB2 and NF- kB reporter oscillations represent genuine changes in transcrip- tional activity, rather than downstream artifacts. We detect oscillatory dCREB2 and NF-kB reporter activity in neurons, glia, and the MB (Fig. 3). While the specific roles of circadian activity in these particular tissues are unclear, oscillating transcription may be relevant to circadian-regulated functions in these areas. For example, a role for glial function in circadian behavior has recently emerged [52,53]. This raises the question of whether these oscillations in glia are required for any behavioral rhythmicity or if they modulate other physiological functions. In Drosophila, the MB is important for sleep regulation, a process that is an output of both circadian control and homeostatic drive [54,55]. dCREB2 has been linked to the clock [13] and has a non- circadian role in sleep-wake homeostasis [56]. In addition, manipulation of PKA activity in MB affects sleep [54]. Further work is needed to determine if dCREB2 oscillation in the MB relates to the sleep-wake cycle. The MB is also an important learning and memory center. Although the role of dCREB2 in the MB for olfactory long-term memory formation is controversial [57], a long-term molecular memory trace may require dCREB2 in MB [58]. Given recent mammalian data that suggest a role for circadian MAPK oscillations in memory maintenance [59], the possibility that dCREB2 activity cycling in the MB could be functionally important for memory processing is particularly intriguing. In mammals, NF-kB plays roles in both pathological conditions of neuronal disease [60] and activity-dependent neural plasticity [61]. The functions of NF-kB in the Drosophila nervous system have not been extensively studied, although previous work shows that this family of transcription factors is important in immune function. Our NF-kB reporter fly provides another tool to study its function in both pathological and physiological condi- tions. Our results suggest that the overall activities of these transcrip- tion factors have relatively simple diurnal patterns of activation in vivo, and that this is downstream of circadian oscillators. It is also commonly understood that both of these transcription factor families respond to a variety of acute and extra-cellular stimuli in cells. There are a number of non-mutually exclusive possibilities for how the overall patterns of activity that we observe could relate to acute responses that are occurring in the organism. First, there may be some stimuli in vivo, downstream of circadian activity, that are especially dominant during the mid-day and nighttime periods, producing peaks. The stimuli at these particular times may be targeted to most if not all cells, thus producing a strong signal. These possibilities are especially intriguing given that much of the reporter activity we observe occurs during time periods when flies typically sleep [62,63], a state characterized by neural synchro- nization in mammals [64]. Whether fly brain neurons show sleep- dependent synchronization is unknown, but there is some evidence that there are changes in neuronal activity during sleep [65]. How the patterned activity of these transcription factors relates to sleep state is an interesting question for future studies. Another possibility is that the other types of cues that can activate these transcription factors may be less global and more cell and tissue specific, thus producing limited activation. The resulting peaks in activation may be obscured beneath a background level of net circadian activity. Finally, dCREB2 and NF-kB may indeed respond to a plethora of signals, but in vivo these signals are synchronized by the circadian system to occur mostly during the two periods of peak activity. This last possibility implies that many physiological processes and their intercellular signals may be under circadian control in the animal, and occur at common times. In this study, we focus primarily on the circadian pattern of dCREB2 and NF-kB activity in the nervous system. However, these factors are also involved in many other processes, including PLOS ONE | www.plosone.org 7 October 2012 | Volume 7 | Issue 10 | e45130