Setting the clock by nature: Circadian rhythm in the fruitfly Drosophila melanogaster

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1 FEBS Letters 585 (2011) journal homepage: Review Setting the clock by nature: Circadian rhythm in the fruitfly Drosophila melanogaster Nicolai Peschel, Charlotte Helfrich-Förster Biozentrum Am Hubland, Universität Würzburg, D Würzburg, Germany article info abstract Article history: Received 19 October 2010 Revised 8 February 2011 Accepted 21 February 2011 Available online 25 February 2011 Edited by Martha Merrow and Michael Brunner Keywords: Circadian clock Period Timeless Clock Cycle Cryptochrome Light Temperature Adaptation Drosophila melanogaster Nowadays humans mainly rely on external, unnatural clocks such as of cell phones and alarm clocks driven by circuit boards and electricity. Nevertheless, our body is under the control of another timer firmly anchored in our genes. This evolutionary very old biological clock drives most of our physiology and behavior. The genes that control our internal clock are conserved among most living beings. One organism that shares this ancient clock mechanism with us humans is the fruitfly Drosophila melanogaster. Since it turned out that Drosophila is an excellent model, it is no surprise that its clock is very well and intensely investigated. In the following review we want to display an overview of the current understanding of Drosophila s circadian clock. Ó 2011 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved. 1. Introduction Evolution has shaped and fine-tuned all living beings to their current existence. Adaptations to different environments like the sea, the woods, or the mountains yielded in fins, wings or tails and thus allowing creatures to conquer new territories and to find their vacant ecological niche. One parameter of our environment is so obvious that it is often neglected or taken for granted this parameter is the daily changing of day and night. The turning of the earth around its own axis once every 24 h causes a daily rhythmical change of light and temperature. Virtually all living beings on this planet are exposed to this light and temperature changes and hence it is not surprising that they adapted to this 24 h rhythm and found by doing so a new vacant ecological niche not in terms of space, but in terms of time. There are for example nocturnal, diurnal or crepuscular (i.e., mainly active in dusk or dawn) animals all living in the same habitat, but the different activity times allows them to live happily together. Crepuscular insects like Corresponding author. Address: Lehrstuhl für Genetik und Neurobiologie, Universität Würzburg, Biozentrum Am Hubland, D Würzburg, Germany. address: nicolaipeschel@biozentrum.uni-wuerzburg.de (N. Peschel). Drosophila for instance are only active when it is not too hot and dry on the one hand and when it is not too cold on the other hand. One could argue now that this just reflects a direct response to the changing temperature or light, but when this organism is isolated from all environmental cues, like light, food or temperature it still keeps the same times of activity with a period of close to but not exactly 24 h. In this case one speaks of a circadian period (Latin: circa = about and dies = day), i.e., an approximately 24 h cycle that is endogenously generated by an organism [1]. The phenomenon of circadian rhythms is known since hundreds of years [2], but the question what actually drives those rhythms was not answered until the end of the last century and Drosophila played the starring role. 2. Why Drosophila? The circadian clock is investigated in many different model organisms from fungi to cyanobacteria, zebra fish or mice. So, why Drosophila? Several facts speak in the little fly s favor. Drosophila s genes are examined now since almost a century so that many sophisticated molecular, genetical and biochemical techniques and tools are now available to study complex behavior on /$36.00 Ó 2011 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved. doi: /j.febslet

2 1436 N. Peschel, C. Helfrich-Förster / FEBS Letters 585 (2011) the molecular basis. The fruit fly shows many different, and easily in large scale measurable circadian patterns of behavior. Especially the locomotor activity should be mentioned here. As a crepuscular animal Drosophila shows two activity peaks one in the morning and one in the evening. This rhythmic activity persists under constant conditions. Other clock regulated behavior includes eclosion [3], olfactory sensitivity [4], egg laying (reviewed in [5]), courtship [6,101], gustatory sensitivity [102] or learning and memory [7]. Another important factor is the simplicity of Drosophila s neuronal network organization. About 150 clock cells per brain hemisphere are yet manageable and therefore permit dissection of the function of single cells or cell clusters in the brain. A combination of the described advantages caused scientists in the last century to take a closer look at Drosophila s circadian rhythm. 3. The discovery of the first genes The late Seymore Benzer ( ) and his student Ron Konopka were working with the fruit fly in the beginning of the 1970s. After chemical mutagenesis they screened for flies with abnormal circadian behavior. They could isolate several different fly strains showing unusual endogenous eclosion periods. One strain had a longer period (29 h), another strain had a shorter period (19 h) and a third strain did not show rhythmic eclosion at all. The milestone discovery was though that all three fly strains had a mutation in the same gene locus, located on the X-chromosome. This locus was called period with mutants designated period Long, period- Short or period 01 and the first so called clock gene was revealed [3]. Many more clock genes followed in the next decades and it turned out that period was not restricted to Drosophilidae but was also well conserved in mouse, zebrafish, and humans. But what is period s contribution to the clock s mechanism? The current model (Fig. 1) predicts that the helix-loop-helix transcription factors clock (Clk) and cycle (Cyc) bind as heterodimers to E-Box sequences (CACGTG) at midday in the genome of the fly [8 10]. E-Boxes are found in the promoter region of many circadian regulated genes like period (per), timeless (tim), vrille (vri) or PAR domain protein 1e (pdp1e) [11], but the very center of the clock represents the activation of per and tim. The subsequent rise of the per and tim mrna in the evening/night leads to accumulation of Per and Tim in the cytoplasm but only after dark, because of the light sensitivity of Tim and the fact that Tim stabilizes Per. Without Tim s protection Per is phosphorylated by the Double-Time (Dbt) kinase [12,13] and afterward ubiquitinated by the F-Box protein Slimb and then degraded in the proteasome [14]. Period s degradation is counterbalanced by the protein phosphatase 2A (PP2A) [15]. Tim and Per then enter the nucleus alone or as a heterodimer [16,17] thus allowing Per associated Dbt to coenter the nucleus. This transport is mediated through Per phosphorylation by Casein Kinase 2 (CK2) [18,19]. Inside the nucleus the Per-Dbt-Tim complex accumulates and binds to Clk/Cyc dimers via a Per-Clk interaction. This interaction causes hyperphosphorylation of clock and thus prevents Clk/Cyc dimers from binding to the DNA and inhibits the transcription of tim and per [20,21]. Period s inhibition of its own transcription generates a negative feedback loop and thus cyclic expression of Period and Timeless protein and mrna. A second feedback loop regulates the transcription of Clk and thereby of course interlocks with the first loop. Clk mrna is transcribed in a reciprocal way to tim/per mrna, showing peak times in the late evening, early morning. As already mentioned Clk/Cyc dimers activate the transcription of two basic leucine zipper proteins, Vrille and Pdp1e [22,23]. Both proteins bind to so called V/P boxes in the promoter region of Clk. While Vri is inhibiting the transcription of Clk, Pdp1e activates Clk s transcription. Fig. 1. Molecular mechanisms of the Drosophila circadian clock. The main text describes the details. The figure displays a clock cell at different times of the day. The neuron is compartmented in nucleus and cytoplasm. Each quarter of the cell roughly represents six hours of the circadian day, while the right side (0 12) is in light and the left side (12 24) in darkness. P indicates phosphate/phosphorylation, the accompanying arrow indicates transfer of phosphate. Dashed arrows or dashed proteins symbols indicate proteasomal degradation. Angular arrows display transcription, sinuous lines mark mrnas. Normal arrows indicate movement from the cytoplasm to the nucleus.

3 N. Peschel, C. Helfrich-Förster / FEBS Letters 585 (2011) However, recent results indicate that altering the VRI/PDP1e ratio by reducing or increasing PDP1e levels has little effect on Clk transcription or VRI cycling. So another role for Pdp1e might be found in the regulation of the circadian locomotor output behavior [24,25]. Another recently discovered clock gene is the basic Helix- Loop-Helix orange-domain putative transcription factor clockwork orange (cwo) [26 29]. Cwo is a transcriptional repressor of the Clk target genes, like per, tim, vri, pdp1e, thus acting as a transcriptional and behavioral rhythm amplifier. 4. The location of the circadian clock Drosophila s clock is comprised of just 150 neurons per hemisphere. These clock neurons are divided into seven major groups, named after their anatomical position. Three neuronal groups are located more dorsally and are thus called dorsal neurons 1 3 (DN 1 3 ), the other four groups are located more laterally and are therefore called lateral neurons (LN d, l-ln v, LPN, and s-ln v ). Additionally there are a few hundred glia cells expressing clock proteins like Per or Tim in the fly s brain [30 32]. This classification is of course very crude and does not always reflect functional unity among one cluster. Therefore clock neurons can be further subdivided into different subgroups according to their protein content, size and/or function (Fig. 2). The DN 1 cells consist of about 16 cells. Two of those cells, the DN 1a do not express the transcription factor Glass, but do express the neuropeptide IPN-amide and the blue light photoreceptor Cryptochrome [33]. Cryptochrome is expressed in two to six other DN 1 cells, that are located more posterior, namely the DN 1p [34]. With only two cells, the DN 2 cluster is the smallest among the clock neurons, while the 40 DN 3 neurons form the largest group. Again DN 3 neurons show a variety in cell body sizes and can be subgrouped as well [33]. The about four l-ln v s and four of the five s-ln v s are expressing the neuropeptide pigment-dispersing factor (PDF). A 5th s-ln v is located in close proximity to the l-ln v s and is lacking PDF [35,36]. The LN d s are located more dorsally. This heterogeneous cell cluster comprises six cells, all expressing different neurotransmitters, like acetylcholine (as judged from the presence of the choline acetyltransferase), ion transporter peptide, the long or the short form of neuropeptide F [37]. The last lateral neuronal group is the LPN cluster. Those neurons seem to be tightly connected with the temperature entrainment of the circadian clock [33,38,39]. 5. Adaption to the environment The sun does not rise and set each subsequent day of the year at the same time. During the short winter days the sun rises not earlier than 8 a.m., while during long summer days the first light appears at 5 a.m. already. If a circadian clock would not be flexible to adapt to different environments or photoperiods it would lack an important ability. Therefore circadian clocks evolved sophisticated molecular mechanisms to react to new environments. Several input factors so called Zeitgebers harmonize the clock neurons to the environment [40]. Light, temperature, social cues [41] or even magnetism [42] can influence the circadian clock, whereupon light is the most important input factor. How does the light reach the clock neurons, and what is the molecular reaction of the clock? The fruit fly has three different photoreceptive organs, containing different rhodopsins. Those organs are the complex eyes, the ocelli and the Hofbauer Buchner eyelets (HB-eyelets). Furthermore, there are two or more photopigments not restricted to those photoreceptive organs [43]. The response to daily or seasonal changes in light is mainly caused by the degradation of Tim protein. If a fly is briefly exposed to light at night its onset of activity on the next day will be advanced or delayed, depending on the timing of the given light pulse. If a fly is in constant light (LL) conditions, it becomes arrhythmic presumably as a consequence to the lack of Tim [44]. Timeless protein itself is not responsive to light. So another protein must act as circadian photoreceptor. The identity of this photoreceptor was unknown for a long time, until it was shown that animals with a mutated cryptochrome (cry) gene, like cry b hypomorphs or cry 0 nulls still behave rhythmic in LL and show abnormalities in their behavioral responses to light [45 48]. Cry is a blue-light photopigment that is expressed in specific subsets of the clock neurons (Fig. 2) and in the compound eyes [34,49]. This protein is activated in the light, most probably because of a conformational change. Light activated Cryptochrome can bind to Tim and thus triggers Tim degradation (Fig. 3). As a consequence to the lack of Tim, Period is now vulnerable to Dbt phosphorylation and subsequently degraded (and the clock reset). How Cry induces Tim degradation is not revealed in detail. Tyrosine phosphorylated Tim protein is recognized by light-activated Cry [50]. This complex is detected by the F-Box protein Jetlag (Jet) and as a consequence Tim is ubiquitinated and degraded in the proteasome [51,52]. Not only Tim is target of Jet, but Cry as well. Jet binds light-dependently to Cry and promotes Cry s degradation but only after Tim is present at very low level, thus allowing a new start of a circadian cycle [53]. Another interesting part in the light dependent degradation of Tim, and the adaptation of the fly to its environment assumes the GSK3-beta ortholog Shaggy (Sgg). Overexpression of Sgg causes a similar phenotype under LL, like cry b. Furthermore it was shown, that Sgg can bind to Cry and thus dramatically stabilizes Cryptochrome and hinders somehow Cry from triggering Tim degradation [54]. Even though the direct interaction between Sgg and Tim was never revealed, Sgg seems to phosphorylate Tim, thus allowing Tim to enter the nucleus [55]. Protein Phosphatase 1 (PP1) is dephosphorylating Tim, but this dephosphorylation is not affecting the nuclear entry of Tim. Hence it is possible that Sgg and PP1 target different phosphorylation sites [56]. 6. Tim the long and the short of it Another way of fine-tuning circadian photoreception was revealed in several recent publications [52,57,58]. Natural polymorphisms in the timeless gene generate different isoforms of TIM. A single nucleotide insertion, for instance, leads to a haplotype (lstim) that has two in-frame start codons, producing a long TIM isoform (l-tim) from the upstream and a shorter isoform (s-tim) from the downstream ATG. Animals without this insertion are only able to produce the s-tim isoform. Interestingly the two different naturally occurring polymorphisms lead to a different photosensitivity. The long isoform that contains 23 extra N-terminal amino acids is much less sensitive to light because it binds poorly to CRY. The s-tim isoform on the other hand is very light sensitive and strongly binds CRY. Hence those animals adapted to different light intensities in their environment by changing the sensitivity of clock neurons. Another important Zeitgeber is temperature. The daily cycle of cold and warm can properly entrain the fly, while a change of about 3 C is enough to adapt Drosophila s circadian clock to the environment [59]. Temperature cycles entrain the clock under constant dark or constant light conditions, implying that there must be an override of light dependent degradation of Tim normally LL renders fruit flies arrhythmic [38]. The molecular mechanism how temperature entrains the clock is still not known, though recent publications shed some light on this pathway. Temperature can

4 1438 N. Peschel, C. Helfrich-Förster / FEBS Letters 585 (2011) Fig. 2. A basic overview of clock-gene-expressing neurons and their neurochemical characterization. The different neuronal subgroups are described in more detail in the text. The different colors of the neurons indicate the peptides/proteins that are expressed in those cells. Namely Cryptochrome (Cry) in yellow, Pigment dispersing factor (PDF) in green, short Neuropeptide F (snpf) in blue, Neuropeptide F (NPF) in red, ion transport peptide (ITP) in gray, choline acetyltransferase (Cha) in purple and the IPNamide in orange. Cells with unknown peptidergic content are colored in black. Aborization of PDF is indicated in green. Fig. 3. An overview of how light regulates adaption of the circadian clock to its environment. Details are described in the main text. The figure shows a Cry expressing clock neuron. For simplicity there is no differentiation between nucleus and cytoplasm. The left side is in the absence of light (night) the right side in the presence of light (day). Inactive Cryptochrome including the FAD and MHTF domain is colored in blue, while active Cry is yellow. Arrows indicate interaction of proteins, blunted arrows indicate no interaction. Dashed arrows or proteins indicate degradation events. Bended arrows indicate transmission of phosphate (P), ubiquitin (U) or light (sun).

5 N. Peschel, C. Helfrich-Förster / FEBS Letters 585 (2011) be perceived similar as light in cultures of isolated body parts, like wings, legs or heads indicating that the circadian temperature sensor is tissue-autonomous [60]. On the other hand a very big difference between light/dark and cold/warm entrainment can be observed looking at the receptors. The circadian photoreceptor Cry can be found in many clock neurons in the brain, thus directly responding to light stimuli [34,61]. The circadian thermoreceptor is not located in the brain or clock neurons. Isolated brains are not able to synchronize to temperature cycles, showing that the brain is dependent on temperature information from the periphery [62]. Two genes are known so far to influence the circadian temperature reception. One is the norpa gene that encodes for the Phospholipase C. Animals carrying a mutated norpa gene are not able to synchronize to temperature cycles [60]. This indicates that a G-protein mediated signal transduction might be involved in circadian temperature reception. The second gene is nocte, which encodes a large glutamine-rich protein with unknown function. A mutated nocte gene does not allow proper entrainment of the Drosophila circadian clock by temperature [60,62]. Interestingly, downregulation of nocte in peripheral tissues is enough to prevent circadian temperature entrainment in the fly. Further narrowing of the peripheral tissue revealed that specific sensory structures the so called chordotonal organs are necessary for behavioral temperature entrainment [62]. A current model predicts that the chordotonal organ neurons, which do not possess a functional clock, send temperature information to peripheral clock neurons in the thoracic CNS, or directly to the clock neurons within the brain. Which clock neurons receive the temperature information from the periphery first or if there are some specific temperature neurons in the brain is not revealed yet. But it was shown, that some Crynegative neurons are very sensitive to temperature, namely the DN 2s and the LPNs [39,63]. It is still under debate how the temperature information from the periphery is causing a change in the expression of Per and Tim in the clock neurons. What is known so far is that an 89 bp sized intron of period is spliced alternatively, depending on the temperature. At low temperatures, more of the spliced per variant is produced (type B) than of the unspliced variant (type A). As a consequence to an up regulation of type B spliced per, Per level is increasing earlier and so is the locomotor activity [64,65]. Different levels of spliced per cannot be observed in norpa mutants here type B is always high. nocte mutants on the other hand display a wildtype like splicing [60]. All those results are obtained from experiments performed under laboratory conditions, i.e., for example a change of temperature, while the light conditions are constant. Under natural conditions light and temperature interact in entraining the clock. A first simulation of quasi natural conditions in the lab shows that the molecular cycling in the different groups of clock neurons occurs rather synchronously and with high amplitude under the combination of light and temperature cycles [63]. Under temperature cycles solely some clock neurons were not entrained at all, and under LD cycles solely others were only slightly entrained and cycled with low amplitude. 7. Morning vs. evening Most animals display a bimodal peak of their activity, e.g., Drosophila shows one peak in the morning and one in the evening [66]. Therefore Pittendrigh and Daan proposed, back in the seventies, a model explaining this bimodal activity pattern. They predicted that there is not only one circadian oscillator, but two. One oscillator the so called morning oscillator (M) is responsible for the activity in the morning and is accelerated by light. The other oscillator the evening oscillator (E) is inducing activity in the evening and is slowed down by light. Both oscillators are coupled [67]. The reason for this bimodal activity is the adaptation to different photoperiods. In summertime the sun rises each day slightly earlier and sets slightly later. The M-oscillator responds to this changed environment with an advanced activity whilst the E- oscillator delays the onset of activity. A first hint that those two different oscillators really exist was revealed in Drosophila cry b mutants. As mentioned above wild-type Drosophila is rendered arrhythmic in LL. But when cry b flies or occasionally even wildtype animals were exposed to very low levels of constant light they started to display a split activity rhythm. One rhythm was faster than normal, i.e., about 22 h and one rhythm was slower i.e., 25 h [36,68]. This phenomenon can be explained by the two oscillator model and the claim that light is causing acceleration (M-oscillator) or deceleration (E-oscillator) depending on the oscillator. Two further studies unraveled the location of the E- and M-oscillator. By manipulating different subsets of clock neurons they showed that the LN v s function as M-oscillator while the LN d s and DNs function as E-oscillator [69,70]. Further studies restricted the location of the M-oscillator to the s-ln v s and revealed that the 5th, Pdf-negative, s-ln v is part of the E-oscillator [36,70]. The s-ln v s are considered to be the main circadian pacemaker cells, because they are mandatory to sustain rhythmic locomotor behavior under constant darkness (DD). Recent studies revealed though that under certain conditions the E-oscillator cells drive circadian behavior in constant light (LL) [71 73]. This was possible after reducing the light-sensitivity of the neurons or under dim LL even in the absence of a functional clock in the s-ln v s. A current model predicts that the E-oscillator cells rather maintain circadian rhythm in LL or under long summerday conditions, while the M-oscillator cells maintain circadian rhythm under DD conditions or in short winterdays [54,71]. As appealing as this model of an E and M oscillator may be, it is just a model and there are of course findings that do not fit. In a recent study animals were investigated that only expressed Period protein in a small subset of clock neurons. The authors of this study restricted period gene expression to four lateral neurons the 5th s-ln v and three LN d s and therefore restricted a functional clock to four cells of the E-oscillator. The interesting discovery was that those animals still showed a bimodal activity pattern under certain environmental light regimes. This suggests that these four neurons can behave as M and E oscillator under these conditions. Their results imply that the M or E characteristic of clock neurons is rather flexible. M and E oscillator function may not be restricted to certain anatomically defined groups of clock neurons but instead may depend on the environmental conditions [73,74]. 8. Cross talk between the clock neurons After realizing that different neuronal groups and different oscillators exist in the fly s brain the question arose, how all the diverse cells and groups communicate with each other and whether there is a certain hierarchy in the clock network. Recent evidence suggests that interneuronal communication may be required to sustain basic molecular rhythms. Altering pacemaker membrane excitability or disrupting peptide signaling between clock neurons induces disruption or dampening of molecular rhythms [75]. Overexpression of potassium channels (e.g., inward-rectifier K+ channel, Kir2.1) in the LN v s abolishes behavioral rhythms and also molecular rhythms in those cells [75]. Interestingly, electrical silencing of LN v s phenocopies PDF null mutants (Pdf 01 ) and PDF-receptor null mutants (e.g., han 5304 ) at both behavioral and molecular levels. Pdf 01 and PDF-receptor null mutants show severe defects in their circadian behavior [76 78]. The morning activity peak is absent, the evening activity peak is advanced by 2 h and when released in DD conditions

6 1440 N. Peschel, C. Helfrich-Förster / FEBS Letters 585 (2011) Pdf 01 and han 5304 mutants have a short period and become arrhythmic after a few days. Pdf 01 mutants have been further investigated. The reason for their arrhythmic behavior is that the flies main pacemaker neurons, the small LN v s (plus some DNs) become asynchronous in DD [79 81]. The short period of Pdf 01 mutants derives from the LN d s and the 5th s-ln v that cycle nicely with short period in Pdf 01 mutants [82,83], but are not able to drive robust activity rhythms in DD [71]. PDF peptide is expressed in the large and small LN v s(fig. 2). PDF from the small LN v s may connect the lateral neurons to the more dorsally located clock cells and may transfer circadian signals to other neurons. Indeed, PDF-release from the small LN v s seems to occur rhythmical [84], and down regulation of PDF in these neurons results in arrhythmic behavior comparable to that of Pdf 01 mutants [85]. In addition, PDF seems to influence the period of other clock neurons in a way that is independent of rhythmic release [83,86]. When PDF-action is prolonged artificially a fast- and a slow-period component appear in the free-running rhythm (as already described above for cry b mutants under constant light). Such a behavior can be provoked by the following genetic and biochemical manipulations (1) ectopic overexpression of PDF [87], (2) overexcitation of the PDF-neurons by expressing a Na+ channel or a spider toxin [82,88,89], or (3) forcing PDF to remain permanently in the synaptic cleft by expressing a membrane-tethered PDF in all clock neurons [86]. The same happens, when the large LN v s project ectopically into the dorsal brain, e.g., in neural mutants with largely reduced optic lobes [90]. The large LN v s are supposed to constantly release highly active PDF under DD conditions [83,91]. In such flies, PDF seems to shorten the period of the small LN v s and certain DNs (that are equivalent to the M cells) and to lengthen the period of the 5th s-ln v, the 3 CRY-positive LN d s and other DNs (that are equivalent to the E cells) [83]. Thus, PDF seems to have a strong influence on the period of the clock neurons under DD conditions. PDF seems to have a similar strong influence on their phase under LD conditions as can be already seen for the advanced E peak of Pdf 01 mutants. When light inputs from CRY are absent, the effects of PDF on the phase of the E cells and the E activity peak become very evident: The E peak is advanced by 12 h in absence of PDF [92,93]. The location of the PDF-receptor confirms the strong effects of PDF on the clock neurons: It is located on most clock neurons [94 96], but preferentially on those that are strongly responsive to PDF as the small LN v s, the 5th LN v, the CRY-positive LN d s and subsets of the DNs [97]. PDF signaling therefore represents an important synchronizing pathway regardless of which E and M model is favored. The large LN v s have further important functions in LD: recent work has implicated them in the control of sleep and arousal and in the transduction of light-input into the clock neuron circuit [89,92,98 100]. So the function of PDF may be fourfold: (1) to work as output transmitter of the circadian clock, (2) to coordinate and synchronize the oscillations of the clock neurons, when external signals (like LD cycles) are absent, thus functioning as an internal Zeitgeber, (3) to phase the oscillations of the clock neurons and the activity peaks to the right time of the day under LD and (4) to work as light-input and arousal factor of the clock. Under natural conditions, the main role of PDF may be to keep the oscillations in the different subsets of clock neurons at appropriate phases. By altering the coupling between the clock neurons PDF may help the system to adapt to seasonal variations in the photoperiod. Pdf 01 mutants are indeed unable to adapt their activity pattern to long photoperiods [83], confirming the role of PDF in the seasonal adaptation of the clock. 9. Conclusion After almost forty years of investigating the important molecules of Drosophila s clock, the animal has not lost its appeal to scientists. Though a lot of important facts were revealed molecular genetic analysis has made circadian rhythms in Drosophila one of the best understood behavior at the molecular level a lot more is hidden inside the fly s brain. The ever growing Drosophila community and the consistent availability of new sophisticated methods will allow scientists to further unravel Drosophila s circadian clock. Hence it is very likely that Drosophila will still play an important role in the circadian rhythm field in the future. 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