per 01 and quasimodo

Almost 40 years ago the period⁰¹ phenotype was described for the very first time.

Period is at the very core of the circadian clock – without a functional period gene a fly looses its rhythmicity (Konopka and Benzer, 1971). Our findings that per⁰¹;;qsmRNAi(16) animals still exhibit some rhythmicity caused us to reconsider this hallmark of circadian clocks. Clearly those animals anticipate the lights-off in the evening (Figure 3-20). Even though an experiment using the different photoperiods suggests the involvement of an hourglass mechanism (Figure 3-21), the result that the rhythmicity endures even under constant light conditions argues for a real endogenous rhythm (Figure 3-22). Recent observations demonstrate that in Drosophila not only one oscillator contributes to the circadian behavior of the fly, but that different oscillators act together to control this rhythm. In 2004 two different groups reported independently about the existence of a morning and evening oscillator in the flies brain (Grima et al., 2004; Stoleru et al., 2004). Distinct cell groups produce different locomotor behavior (see introduction). A current paper enhances this theory and discusses the possibility of a Dark and Light oscillator. One oscillator that regulates activity in constant darkness or the dark part of one day - the other oscillator

controlling activity in constant light or the light part of the day (Picot et al., 2007).

Our current findings suggest that under illumination (LL or the Light part of LD) Period protein is not mandatory for circadian rhythms (Figure 3-22). Without illumination though the animal looses its rhythmicity (in DD or in the Dark part of LD) (Figure 3-22). If we now combine our findings (Chapter 3.3.6) with the Dark and Light oscillator paradigm (Picot et al., 2007) we suggest a modificated model:

In a flies‟ brain two individual oscillators coexist. One „Dark oscillator‟ that is located in the small and large LNvs, generates the activity peak in the morning (Figure 3-44 right). In the night time (or in constant darkness) those cells induce activity in the fly.

After illumination/lights-on the activity ceases (Figure 3-44 right).

Contrary the „Light oscillator‟ in the LNds, the 5^th s-LNv, and the DNs is important for the activity in the evening or in LL. When the darkness starts again, the activity stops (Figure 3-44 left). In the Light oscillator the Period protein seems to be not compulsory. The question remains, why per⁰¹ animals behave arrhythmic in LL conditions and do not exhibit evening anticipation – while per⁰¹;;qsmRNAi(16) animals do behave rhythmic in LL and exhibit evening anticipation? The answer can be found in the connection of the Dark oscillator and the Light oscillator. The dominant Dark oscillator determines the period of the entire system by providing a daily signal to the Light oscillator and is therefore a true cellular Zeitgeber (Stoleru et al., 2005). In wild-type flies the resetting signal from the Dark oscillator may be required to adapt the Light oscillator to different photoperiods. In per⁰¹ animals on the

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Figure 3-44 The Light and Dark oscillator

The neurons of the so called evening cells contribute to the activity in the light. In those cells light has an activatory role – darkness inhibits the activity (left side). The cells of the so called morning oscillator contribute to the activity in the darkness. Here illumination has an inhibitory effect on the activity (right side).

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other hand the information from the Dark to the Light oscillator renders the flies arrhythmic. In per⁰¹;;qsmRNAi(16) animals this signal (from the Dark to the Light oscillator) seems to be impaired. As a consequence to this the evening activity can not be adapted to different photoperiods (Figure 3-21) and the Dark oscillator can not render the animals arrhythmic in LL conditions thus allowing the Light oscillator to free run (Figure 3-22).

Another important clock protein, Tim is intimately involved in the circadian light response. Therefore we investigated if Tim protein alone can drive the behavior in the Light oscillator in per⁰¹ mutant flies. Under LD conditions we still could detect light dependent degradation of Tim – as well in per⁰¹ as in per⁰¹;;qsmRNAi(16) flies. But we were not able to see a significant difference in the phosphorylation. Along with this observation we were not able to detect Tim in the nucleus in wholemount stainings, which implies that Tim phosphorylation – that can be determined by Western blot in form of slower migrating bands (Martinek et al., 2001)- is important for the nuclear localization of Tim, but is not necessary for the light dependent degradation of the protein. This raises the question, if a mainly cytoplasmatic Tim contributes to the evening and/or constant light activity of the per⁰¹;qsmRNAi(16) animals. On the other hand Collins et al. were able to demonstrate the necessity of Tim for at least the evening activity in per⁰¹ flies; per⁰¹;cry^b mutants display evening anticipation, while per⁰¹;tim⁰¹;cry^b animals do not (Collins et al., 2005). When we compared the Tim level in adult brains from per⁰¹ and per⁰¹;;qsmRNAi(16) animals we could see some minor differences in the degradation and spacial and temporal localization of Tim. Under LD conditions we could detect a decrease of Tim at ZT9 – this result was revealed by Western Blot and by staining of the brains. Furthermore additionally Tim cells could be observed that might be the previously described Tim (+) Per (-) cells

The fact that only four different time points were investigated does not allow a clear answer about Timeless in per⁰¹ animals. In particular we can not rule out that Tim is localized to the nucleus only during a very narrow time window. Additionally other clock proteins like Pdp1 should be investigated to find out if we still can see a circadian rhythm on a molecular basis in those animals.