Discussion

- A subset of dorsal neurons modulates circadian behavior and light responses in Drosophila

Recent studies have shown that two groups of cells control circadian behavior. The PDF positive LNvs are called morning cells (M-cells), and the LNds evening cells (E-cells), because they control the anticipatory behavior observed before dawn and dusk respectively (Grima et al., 2004; Stoleru et al., 2004). In addition, the LNvs are the cells maintaining circadian behavior in constant darkness and controlling the phase of most circadian neurons of the brain (Lin et al., 2004; Peng et al., 2003; Renn et al., 1999; Stoleru et al., 2005). In their absence, circadian behavior rhythms are lost after a few days in DD. Surprisingly, our results show that a functional circadian clock in the LNvs is actually not necessary for long-term behavioral rhythms. In flies overexpressing PER, the LNvs are no longer circadianly functional under constant illumination. No oscillation of the circadian protein PDP1 can be detected and yet these flies remain rhythmic for at least 7 days. Moreover, limiting per overexpression to circadian neurons that do not express PDF is sufficient to obtain circadian behavioral rhythms under constant environmental conditions.

We believe that the neurons maintaining circadian behavior independently of the LNvs are not the E-cells. Indeed, when per is overexpressed, we did not see any sign of circadian oscillation in the neurons that are thought to control the evening activity: the LNds (Grima et al., 2004; Stoleru et al., 2004). In addition, the PDF negative LNv that might also contribute to the evening activity (Rieger et al., 2006) did not cycle in LL when morgue was overexpressed. Moreover, flies with per overexpression driven by cry-GAL4 were completely arrhythmic under constant light (Table I). cry-GAL4 is one of the critical GAL4 driver used to define the E-cells (Stoleru et al., 2004). Importantly, we actually detected molecular circadian oscillations in only one group of cells when per was overexpressed: the DN1s. Due to the high number of DN3s, we cannot rule out that a few cells in the DN3 groups also oscillate. Interestingly, Veleri et al. (2003) have previously shown that a subset of DN3 neurons can maintain their own circadian oscillations in DD, in the absence of circadianly functional LNvs. However, these DN3 cells were not able to generate rhythmic behavior in DD. While it is possible that light is a necessary co-factor for these self-sustained DN3s to participate in the control circadian behavior, we favor the hypothesis that it is the DN1s that maintain circadian rhythmicity in LL. This idea is strongly supported by several additional findings. First, the phase of PDP1 molecular oscillations in the DN1s on the 3^rd day of LL fits well with the long period of the circadian behavior observed under these conditions in per overexpressing flies. Second, the behavioral observations made with morgue overexpression also suggest that the critical cells for rhythmicity are not the LNvs, and PER staining in morgue overexpressing flies gave us an independent confirmation that robust circadian molecular oscillations are restricted to the DN1s in LL. Finally, in LNv-rescued cry^b flies, only the DN1s show robust, coherent circadian rhythms in phase with the behavioral rhythms. Remarkably, the DN1s can maintain circadian behavior in LL even when PDF is absent. This indicates that they can work autonomously of LNv output. Interestingly, not all DN1s do oscillate in LL, only about 6–7 cells most likely. This shows that the DN1 group is heterogeneous. This is not surprising, since the different groups of circadian neurons were named based on their location in the brain, not on their function or developmental lineage. There is ample evidence for heterogeneity of morphology, gene expression and behavior within these different groups of cells, including the DN1s (see for example Rieger et al., 2006; Shafer et al., 2006).

Thus, a subset of DN1s can control and generate circadian behavioral rhythms. They must therefore play an important role in the circadian neuronal circuits. Since ablation of the M cells and E cells results in flies with no morning and evening activity, and no self-sustained rhythms in DD (Stoleru et al., 2004), this could mean that the DN1s are usually functioning downstream of the M and E cells. This is further supported by the fact that in the absence of the neuropeptide PDF - believed to be the critical synchronizing signal secreted by the M cells – the DN1s cannot maintain their circadian rhythms in the long run in DD (Lin et al., 2004; Peng et al., 2003). The DN1s can thus probably function as a relay connecting the LNvs with the neurosecretory cells of the pars intercerebralis (PI), believed to play an important role in the control of locomotor behavior (Helfrich-Foerster et al., 1998: Kaneko and Hall, 2000). A LNvs-DN1-PI pathway has also been suggested based on the anatomical studies of the projections of the small LNvs and the DN1s (Kaneko and Hall, 2000). The expression of the receptor for PDF in at least a subset of DN1s also supports the existence of a functional connection between them and the LNvs (Hyun et al., 2005; Lear et al., 2005; Mertens et al., 2005). The implication of this connection is that in wild-type flies under LL, the LNvs should constantly send a disruptive signal to the DN1s, presumably the non-oscillating secretion of PDF.

This leaves us with the following question: if the LNvs and rhythmic PDF secretion are normally required for the DN1s to be rhythmic, why are the DN1s able to free themselves from the disruptive effects of constant light, while at the same time becoming independent of the LNvs? Our results show that an important mechanism is the inhibition of the CRY-dependent light input pathway. Indeed, morgue overexpressing flies are defective in the CRY-dependent behavioral responses to short light pulses, and cry loss-of-function mutations also result in rhythms driven by the DN1s. In the case of per overexpression, we presume that TIM role is reduced, since one of its major functions is to protect PER from proteasomal degradation (Grima et al., 2002; Ko et al., 2002; Price et al., 1995). TIM is the target of CRY, thus its reduced importance would result in DN1s that are less sensitive to the CRY input pathway. In addition, overexpression of SHAGGY, which inhibits CRY signaling, also results in LL rhythms driven by dorsal neurons (Stoleru et al., 2007). However, under natural environmental conditions, inhibition of the CRY input pathway is probably not required for the DN1s to participate in the control circadian rhythms. Indeed, even in the polar regions of the globe that experience constant light conditions during the summer, the elevation of the sun varies during the day, and this should result in variations of temperature sufficient to synchronize the DN1 circadian clock (Yoshii et al., 2005).

The mechanism by which the DN1s avoid to become arrhythmic in LL as a result of the molecular arrhythmicity of the LNvs, which should result in constant PDF secretion, is not clear yet. It is possible that the presence of light inhibits PDF signaling and thus promotes the role of the DN1s. Light input could come from the eyes, ocelli, or from the DN1s themselves (Rieger et al., 2003). Alternatively, as mentioned in the result section, the DN1s could induce rhythmic PDF secretion. The fact that PDF is not required for LL behavioral rhythms does not exclude this possibility, particularly since the robustness of the rhythms is improved by the presence of PDF.

Interestingly, per and morgue overexpression results in a very similar long period phenotype under LL, which could suggest that these two molecules coincidentally affect the period length of the circadian molecular pacemaker in the same way. In DD however, per overexpression does affect behavioral period length, while morgue does not. The long period phenotype observed in LL actually probably reflects the fact that the CRY input pathway is not completely blocked in the DN1s of per or morgue overexpressing flies. Indeed, under very low light intensity, wild-type flies exhibit a long period phenotype as well (Konopka et al., 1989). In addition, morgue overexpression does not completely block the CRY-dependent responses to short light pulses (figure 5A). Finally and most importantly, LNv-rescued cry^b flies - in which the CRY input pathway is completely non-functional in the DN1s – have 24-hr period rhythms. The LNv-rescued cry^b flies show nevertheless a higher degree of arrhythmicity than normal cry^b flies, or than flies overexpressing morgue or per. This might be due to the desynchronization observed within the DN3 group of circadian neurons. Indeed, the DN3s do not appear to be desynchronized in per or morgue overexpressing flies.

A previous report had already shown that LNv-rescued cry^b flies are partially rhythmic (Emery et al., 2000b), and this was interpreted as evidence for a functional role of CRY directly in the LNvs. Our new results show that expression of CRY in the LNvs is probably not very important for the response to constant light. The DN1s are the important cells for this response. Does this mean that CRY is not a photoreceptor in the LNvs? We believe it actually does function as a photoreceptor in the LNvs as well. CRY is expressed in these cells (Emery et al., 2000b; Klarsfeld et al, 2004), and LNv-rescued cry^b flies show very significantly rescued responses to short light pulses. Preliminary experiments with morgue overexpression limited to the LNvs confirm a predominant role of these cells for light pulse responses (A. M. and P. E., unpublished data). Thus, the CRY input pathway might mediate response to short light pulses by its action in the LNvs, and constant light responses by its action in the DN1s.

In summary, our work underscores the importance of the DN1s in the control of circadian behavior and responses to light. Earlier genetic studies have indicated that the DN1s modulate the sensitivity of the circadian network to light:dark cycles of very low light intensity (Klarsfeld et al., 2004). Our results significantly extend this observation by showing the profound impact the DN1s have on the response to constant light and by demonstrating that these cells not only modulate circadian light responses, but can also become the driving force controlling circadian locomotor behavior, and this in the absence of environmental cues and functional LNvs. This confers upon them a unique status among non-PDF circadian neurons. One of our striking results is that genetically identical flies rely either on the LNvs or the DN1s for the control of their circadian rhythms, depending on the presence or absence of light. Indeed, the LNvs determine period length in our experiments with per overexpression in DD, but in LL the DN1s set the pace. That the presence or the absence of light can so remarkably shift the dominance from one cell group to the other strongly suggests that the relative contribution of the LNvs and DN1s to the control of circadian rhythms change during the course of the year, particularly at high latitude. The DN1s, which interestingly generate evening activity (figure 1,4 and 5), would play a more prominent role in the control of circadian behavior during the long days of the summer, while the LNvs would be more important when photoperiods are shorter (see also Stoleru et al., 2007).