Circadian rhythms give the sense of time to cyanobacteria and most eukaryotes, so that these organisms can adapt their physiology and behavior to daily environmental variations. These rhythms are generated by an endogenous, self-sustained molecular pacemaker (Dunlap, 1999). In Drosophila, this pacemaker is a transcriptional feedback loop (Hardin, 2005). Two proteins, PER and TIM, repress their own gene transcription by blocking the activity of two transcription factors: CLK and CYC. A set of kinases (DBT, CKII, SGG) and a phosphatase (PP2A) regulate PER and TIM phosphorylation, and therefore their stability and activity, so that the cycle lasts 24 hours. A second feedback loop regulates CLK expression. PDP1 and VRI are positive and negative transcriptional regulators of the clk gene, respectively, while CLK regulates positively their circadian expression (Hardin, 2005). The first loop is absolutely essential for circadian rhythms, but the function of the second loop still needs to be established. It might be important for the robustness of circadian rhythms, or their stability (Emery and Reppert, 2004). A strikingly similar molecular architecture that involves two interlocked feedback loops is also found in mammals (Shearman et al., 2000).
Recent studies have begun to elucidate the neural circuitry underlying circadian rhythms in Drosophila. This crepuscular animal shows two peaks of activity: around dawn and before dusk. Two separate groups of cells control these two peaks of activity: the ventral Lateral Neurons (LNvs) - that express the neuropeptide PDF - control the morning peak of activity, while the dorsal Lateral Neurons (LNds) and possibly two Dorsal Neurons (DNs) control the evening peak (Grima et al., 2004; Stoleru et al., 2004). A recent study suggests that a specific LNv that does not express PDF might also contribute to the evening peak (Rieger et al., 2006). The PDF positive LNvs have another crucial function: they maintain circadian rhythms in constant environmental conditions (constant darkness and constant temperature to be precise; Renn et al., 1999). These cells are believed to synchronize the other groups of circadian neurons through the rhythmic secretion of PDF (Park et al., 2000; Stoleru et al., 2005). The absence of the LNvs or of PDF results in rapid loss of behavioral rhythmicity under constant darkness, severely reduced amplitude of tim mRNA oscillations, and desynchronization of PER cycling within different groups of circadian neurons (Lin et al., 2004; Peng et al., 2003; Renn et al., 1999).
Since the LNvs control the oscillations of other circadian cells, they must be properly synchronized with the environment. The light:dark (LD) cycle is a crucial environmental cue. The LNvs receive two kinds of photic input. First, these cells are directly blue-light sensitive because they express the photoreceptor CRY (Emery et al., 2000b). Second, photoreceptive organs that express rhodopsins (eyes, ocelli, Hofbauer-Buchner eyelets) all contribute to a certain degree to the synchronization of the LNvs (Helfrich-Forster et al., 2001; Rieger et al., 2003).
CRY is thought to be the primary circadian photoreceptor, because it functions within circadian neurons (Emery et al., 2000b). Flies defective for CRY show very severe circadian photoreceptive defects. They cannot respond to short light pulses, while pulses as short as 1 minute can change the phase of circadian rhythms by several hours in wild-type flies (Egan et al., 1999; Stanewsky et al., 1998). They also react abnormally to constant light. Under these conditions, wild-type flies are arrhythmic, but flies without a functional CRY input pathway have a 24-hr period rhythm, as if they were in constant darkness (Emery et al., 2000a). Rescuing CRY function only in the LNvs is sufficient to significantly restore circadian behavioral light responses (Emery et al., 2000b). This indicates an important autonomous role of the LNvs in CRY dependent light responses. However, since these responses are not completely restored to normal, there might be other cells that contribute to CRY photoreception.
Here, we show that a gain-of-function mutation in the circadian pacemaker can also protect flies from the disruptive effects of constant light. Indeed, flies overexpressing the key pacemaker gene per are robustly rhythmic under constant illumination. Interestingly, our results demonstrate that the cells maintaining these behavioral rhythms are not the LNvs, but a subset of Dorsal Neurons of the DN1 group. Thus, these poorly characterized neurons play a central role in the control of circadian rhythms and the modulation of circadian responses to constant light.
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