Elisabeth Chélot

Morning and evening peaks of activity rely on different clock neurons of the Drosophila brain

In Drosophila, a ‘clock’ situated in the brain controls circadian rhythms of locomotor activity. This clock relies on several groups of neurons that express the Period (PER) protein, including the ventral lateral neurons (LNvs), which express the Pigment-dispersing factor (PDF) neuropeptide, and the PDF-negative dorsal lateral neurons (LNds). In normal cycles of day and night, adult flies exhibit morning and evening peaks of activity; however, the contribution of the different clock neurons to the rest–activity pattern remains unknown. Here, we have used targeted expression of PER to restore the clock function of specific subsets of lateral neurons in arrhythmic per0 mutant flies. We show that PER expression restricted to the LNvs only restores the morning activity, whereas expression of PER in both the LNvs and LNds also restores the evening activity. This provides the first neuronal bases for ‘morning’ and ‘evening’ oscillators in the Drosophila brain. Furthermore, we show that the LNvs alone can generate 24 h activity rhythms in constant darkness, indicating that the morning oscillator is sufficient to drive the circadian system.

The F-box protein slimb controls the levels of clock proteins period and timeless.

The Drosophila circadian clock is driven by daily fluctuations of the proteins Period and Timeless, which associate in a complex and negatively regulate the transcription of their own genes. Protein phosphorylation has a central role in this feedback loop, by controlling Per stability in both cytoplasmic and nuclear compartments as well as Per/Tim nuclear transfer. However, the pathways regulating degradation of phosphorylated Per and Tim are unknown. Here we show that the product of the slimb (slmb) gene—a member of the F-box/WD40 protein family of the ubiquitin ligase SCF complex that targets phosphorylated proteins for degradation—is an essential component of the Drosophila circadian clock. slmb mutants are behaviourally arrhythmic, and can be rescued by targeted expression of Slmb in the clock neurons. In constant darkness, highly phosphorylated forms of the Per and Tim proteins are constitutively present in the mutants, indicating that the control of their cyclic degradation is impaired. Because levels of Per and Tim oscillate in slmb mutants maintained in light:dark conditions, light- and clock-controlled degradation of Per and Tim do not rely on the same mechanisms.

Defining the role of Drosophila lateral neurons in the control of circadian rhythms in motor activity and eclosion by targeted genetic ablation and PERIOD protein overexpression.

The ventral lateral neurons (LNvs) of the Drosophila brain that express the period (per) and pigment dispersing factor (pdf) genes play a major role in the control of circadian activity rhythms. A new P‐gal4 enhancer trap line is described that is mostly expressed in the LNvs This P‐gal4 line was used to ablate the LNvs by using the pro‐apoptosis gene bax, to stop PER protein oscillations by overexpressing per and to block synaptic transmission with the tetanus toxin light chain (TeTxLC). Genetic ablation of these clock cells leads to the loss of robust 24‐h activity rhythms and reveals a phase advance in light–dark conditions as well as a weak short‐period rhythm in constant darkness. This behavioural phenotype is similar to that described for disconnected1 (disco1) mutants, in which we show that the majority of the individuals have a reduced number of dorsally projecting lateral neurons which, however, fail to express PER. In both LNv‐ablated and disco1 flies, PER cycles in the so‐called dorsal neurons (DNs) of the superior protocerebrum, suggesting that the weak short‐period rhythm could stem from these PDF‐negative cells. The overexpression of per in LNs suppresses PER protein oscillations and leads to the disruption of both activity and eclosion rhythms, indicating that PER cycling in these cells is required for both of these rhythmic behaviours. Interestingly, flies overexpressing PER in the LNs do not show any weak short‐period rhythms, although PER cycles in at least a fraction of the DNs, suggesting a dominant role of the LNs on the behavioural rhythms. Expression of TeTxLC in the LNvs does not impair activity rhythms, which indicates that the PDF‐expressing neurons do not use synaptobrevin‐dependent transmission to control these rhythms.

Novel Features of Cryptochrome-Mediated Photoreception in the Brain Circadian Clock of Drosophila

In Drosophila, light affects circadian behavioral rhythms via at least two distinct mechanisms. One of them relies on the visual phototransduction cascade. The other involves a presumptive photopigment, cryptochrome (cry), expressed in lateral brain neurons that control behavioral rhythms. We show here that cry is expressed in most, if not all, larval and adult neuronal groups expressing the PERIOD (PER) protein, with the notable exception of larval dorsal neurons (DN2s) in which PER cycles in antiphase to all other known cells. Forcing cry expression in the larval DN2s gave them a normal phase of PER cycling, indicating that their unique antiphase rhythm is related to their lack of cry expression. We were able to directly monitor CRY protein in Drosophila brains in situ. It appeared highly unstable in the light, whereas in the dark, it accumulated in both the nucleus and the cytoplasm, including some neuritic projections. We also show that dorsal PER-expressing brain neurons, the adult DN1s, are the only brain neurons to coexpress the CRY protein and the photoreceptor differentiation factor GLASS. Studies of various visual system mutants and their combination with the cryb mutation indicated that the adult DN1s contribute significantly to the light sensitivity of the clock controlling activity rhythms, and that this contribution depends on CRY. Moreover, all CRY-independent light inputs into this central behavioral clock were found to require the visual system. Finally, we show that the photoreceptive DN1 neurons do not behave as autonomous oscillators, because their PER oscillations in constant darkness rapidly damp out in the absence of pigment-dispersing-factor signaling from the ventral lateral neurons.

PDF-modulated visual inputs and cryptochrome define diurnal behavior in Drosophila

Morning and evening circadian oscillators control the bimodal activity of Drosophila in light-dark cycles. The lateral neurons evening oscillator (LN-EO) is important for promoting diurnal activity at dusk. We found that the LN-EO autonomously synchronized to light-dark cycles through either the cryptochrome (CRY) that it expressed or the visual system. In conditions in which CRY was not activated, flies depleted for pigment-dispersing factor (PDF) or its receptor lost the evening activity and displayed reversed PER oscillations in the LN-EO. Rescue experiments indicated that normal PER cycling and the presence of evening activity relied on PDF secretion from the large ventral lateral neurons and PDF receptor function in the LN-EO. The LN-EO thus integrates light inputs and PDF signaling to control Drosophila diurnal behavior, revealing a new clock-independent function for PDF.

A Role for Blind DN2 Clock Neurons in Temperature Entrainment of the Drosophila Larval Brain

Circadian clocks synchronize to the solar day by sensing the diurnal changes in light and temperature. In adult Drosophila, the brain clock that controls rest–activity rhythms relies on neurons showing Period oscillations. Nine of these neurons are present in each larval brain hemisphere. They can receive light inputs through Cryptochrome (CRY) and the visual system, but temperature input pathways are unknown. Here, we investigate how the larval clock network responds to light and temperature. We focused on the CRY-negative dorsal neurons (DN2s), in which light–dark (LD) cycles set molecular oscillations almost in antiphase to all other clock neurons. We first showed that the phasing of the DN2s in LD depends on the pigment-dispersing factor (PDF) neuropeptide in four lateral neurons (LNs), and on the PDF receptor in the DN2s. In the absence of PDF signaling, these cells appear blind, but still synchronize to temperature cycles. Period oscillations in the DN2s were stronger in thermocycles than in LD, but with a very similar phase. Conversely, the oscillations of LNs were weaker in thermocycles than in LD, and were phase-shifted in synchrony with the DN2s, whereas the phase of the three other clock neurons was advanced by a few hours. In the absence of any other functional clock neurons, the PDF-positive LNs were entrained by LD cycles but not by temperature cycles. Our results show that the larval clock neurons respond very differently to light and temperature, and strongly suggest that the CRY-negative DN2s play a prominent role in the temperature entrainment of the network.

CULLIN-3 Controls TIMELESS Oscillations in the Drosophila Circadian Clock

Eukaryotic circadian clocks rely on transcriptional feedback loops. In Drosophila, the PERIOD (PER) and TIMELESS (TIM) proteins accumulate during the night, inhibit the activity of the CLOCK (CLK)/CYCLE (CYC) transcriptional complex, and are degraded in the early morning. The control of PER and TIM oscillations largely depends on post-translational mechanisms. They involve both light-dependent and light-independent pathways that rely on the phosphorylation, ubiquitination, and proteasomal degradation of the clock proteins. SLMB, which is part of a CULLIN-1-based E3 ubiquitin ligase complex, is required for the circadian degradation of phosphorylated PER. We show here that CULLIN-3 (CUL-3) is required for the circadian control of PER and TIM oscillations. Expression of either Cul-3 RNAi or dominant negative forms of CUL-3 in the clock neurons alters locomotor behavior and dampens PER and TIM oscillations in light-dark cycles. In constant conditions, CUL-3 deregulation induces behavioral arrhythmicity and rapidly abolishes TIM cycling, with slower effects on PER. CUL-3 affects TIM accumulation more strongly in the absence of PER and forms protein complexes with hypo-phosphorylated TIM. In contrast, SLMB affects TIM more strongly in the presence of PER and preferentially associates with phosphorylated TIM. CUL-3 and SLMB show additive effects on TIM and PER, suggesting different roles for the two ubiquitination complexes on PER and TIM cycling. This work thus shows that CUL-3 is a new component of the Drosophila clock, which plays an important role in the control of TIM oscillations.

Four of the six Drosophila rhodopsin-expressing photoreceptors can mediate circadian entrainment in low light.

Light is the major stimulus for the synchronization of circadian clocks with day–night cycles. The light‐driven entrainment of the clock that controls rest–activity rhythms in Drosophila relies on different photoreceptive molecules. Cryptochrome (CRY) is expressed in most brain clock neurons, whereas six different rhodopsins (RH) are present in the light‐sensing organs. The compound eye includes outer photoreceptors that express RH1 and inner photoreceptors that each express one of the four rhodopsins RH3–RH6. RH6 is also expressed in the extraretinal Hofbauer–Buchner eyelet, whereas RH2 is only found in the ocelli. In low light, the synchronization of behavioral rhythms relies on either CRY or the canonical rhodopsin phototransduction pathway, which requires the phospholipase C‐β encoded by norpA (no receptor potential A). We used norpAP24 cry02 double mutants that are circadianly blind in low light and restored NORPA function in each of the six types of photoreceptors, defined as expressing a particular rhodopsin. We first show that the NORPA pathway is less efficient than CRY for synchronizing rest–activity rhythms with delayed light–dark cycles but is important for proper phasing, whereas the two light‐sensing pathways can mediate efficient adjustments to phase advances. Four of the six rhodopsin‐expressing photoreceptors can mediate circadian entrainment, and all are more efficient for advancing than for delaying the behavioral clock. In contrast, neither RH5‐expressing retinal photoreceptors nor RH2‐expressing ocellar photoreceptors are sufficient to mediate synchronization through the NORPA pathway. Our results thus reveal different contributions of rhodopsin‐expressing photoreceptors and suggest the existence of several circuits for rhodopsin‐dependent circadian entrainment. J. Comp. Neurol. 524:2828–2844, 2016.

Identifying Specific Light Inputs for Each Subgroup of Brain Clock Neurons in Drosophila Larvae

In Drosophila, opsin visual photopigments as well as blue-light-sensitive cryptochrome (CRY) contribute to the synchronization of circadian clocks. We focused on the relatively simple larval brain, with nine clock neurons per hemisphere: five lateral neurons (LNs), four of which express the pigment-dispersing factor (PDF) neuropeptide, and two pairs of dorsal neurons (DN1s and DN2s). CRY is present only in the PDF-expressing LNs and the DN1s. The larval visual organ expresses only two rhodopsins (RH5 and RH6) and projects onto the LNs. We recently showed that PDF signaling is required for light to synchronize the CRY− larval DN2s. We now show that, in the absence of functional CRY, synchronization of the DN1s also requires PDF, suggesting that these neurons have no direct connection with the visual system. In contrast, the fifth (PDF−) LN does not require the PDF-expressing cells to receive visual system inputs. All clock neurons are light-entrained by light–dark cycles in the rh52;cryb, rh61 cryb, and rh52;rh61 double mutants, whereas the triple mutant is circadianly blind. Thus, any one of the three photosensitive molecules is sufficient, and there is no other light input for the larval clock. Finally, we show that constant activation of the visual system can suppress molecular oscillations in the four PDF-expressing LNs, whereas, in the adult, this effect of constant light requires CRY. A surprising diversity and specificity of light input combinations thus exists even for this simple clock network.

Reconfiguration of a Multi-oscillator Network by Light in the Drosophila Circadian Clock

The brain clock that drives circadian rhythms of locomotor activity relies on a multi-oscillator neuronal network. In addition to synchronizing the clock with day-night cycles, light also reformats the clock-driven daily activity pattern. How changes in lighting conditions modify the contribution of the different oscillators to remodel the daily activity pattern remains largely unknown. Our data in Drosophila indicate that light readjusts the interactions between oscillators through two different modes. We show that a morning s-LNv > DN1p circuit works in series, whereas two parallel evening circuits are contributed by LNds and other DN1ps. Based on the photic context, the master pacemaker in the s-LNv neurons swaps its enslaved partner-oscillator-LNd in the presence of light or DN1p in the absence of light-to always link up with the most influential phase-determining oscillator. When exposure to light further increases, the light-activated LNd pacemaker becomes independent by decoupling from the s-LNvs. The calibration of coupling by light is layered on a clock-independent network interaction wherein light upregulates the expression of the PDF neuropeptide in the s-LNvs, which inhibits the behavioral output of the DN1p evening oscillator. Thus, light modifies inter-oscillator coupling and clock-independent output-gating to achieve flexibility in the network. It is likely that the light-induced changes in the Drosophila brain circadian network could reveal general principles of adapting to varying environmental cues in any neuronal multi-oscillator system.