The neuropeptide pigment-dispersing factor (PDF) is a key transmitter in the circadian clock of Drosophila melanogaster. PDF is necessary for robust activity rhythms and is thought to couple the circadian oscillations of the clock neurons. However, little is known about the action of PDF on individual clock neurons. Here, we combined the period-luciferase reporter system with immunolabeling of clock proteins in wild-type and Pdf 01 mutants to dissect the effects of PDF on specific subgroups of clock neurons. Additionally, PDF levels were elevated to higher than normal levels using specific neural mutants, and a correlation analysis of locomotor activity and clock protein staining served to determine the periods of specific clock cells. We found that PDF has multiple effects on the clock neurons: In some groups of clock neurons, PDF was required for maintaining the oscillations of individual cells, and in others, PDF was required for synchronous cycling of the individual members. Other clock neurons cycled with high amplitude in absence of PDF, but PDF affected their intrinsic clock speed. Sometimes PDF shortened and sometimes PDF lengthened period. Our observations indicate that PDF is crucial for adjusting cycling amplitude, period, and phase of the different players in the circadian clock. Under natural conditions PDF may be required for adapting Drosophila's clock to varying photoperiods. Indeed, we show here that Pdf 01 mutants are not able to adapt their activity to long photoperiods in a wild-type manner.

Cryptochrome (CRY) is intimately associated with the circadian clock of many organisms. In the fruit fly Drosophila melanogaster, CRY seems to be involved in photoreception as well as in the core clockwork. In spite of the critical role of CRY for the clock of Drosophila, it was not quite clear whether CRY is expressed in every clock cell. With the help of a new antibody and a mutant that lacks CRY, we show here that CRY is expressed in specific subsets of Drosophila's pacemaker neurons and in the photoreceptor cells of the compound eyes. In the pacemaker neurons, CRY levels and kinetics under light-dark cycles are quite different from each other. High-amplitude oscillations are observed in only three groups of clock neurons, suggesting that these three groups are strongly receptive to light. The different CRY kinetics may account for phase differences in oscillations of the clock proteins observed in these three groups in earlier studies. The molecular clock of the neurons that contain lower CRY levels or are completely CRY negative can still be synchronized by light, probably via intercellular communication with the CRY-positive neurons as well as via external photoreceptors.

While the rapid development of personal computers and high-throughput recording systems for circadian rhythms allow chronobiologists to produce huge amounts of data, the software to analyze them often lags behind. Here, we announce newly developed chronobiology software that is easy to use, compatible with many different systems, and freely available. Our system can perform the most frequently used analyses: actogram drawing, periodogram analysis, and waveform analysis. The software is distributed as a pure Java plug-in for ImageJ and so works on the 3 main operating systems: Linux, Macintosh, and Windows. We believe that this free software raises the speed of data analyses and makes studying chronobiology accessible to newcomers.

Since 1960, magnetic fields have been discussed as Zeitgebers for circadian clocks, but the mechanism by which clocks perceive and process magnetic information has remained unknown. Recently, the radical-pair model involving light-activated photoreceptors as magnetic field sensors has gained considerable support, and the blue-light photoreceptor cryptochrome (CRY) has been proposed as a suitable molecule to mediate such magnetosensitivity. Since CRY is expressed in the circadian clock neurons and acts as a critical photoreceptor of Drosophila's clock, we aimed to test the role of CRY in magnetosensitivity of the circadian clock. In response to light, CRY causes slowing of the clock, ultimately leading to arrhythmic behavior. We expected that in the presence of applied magnetic fields, the impact of CRY on clock rhythmicity should be altered. Furthermore, according to the radical-pair hypothesis this response should be dependent on wavelength and on the field strength applied. We tested the effect of applied static magnetic fields on the circadian clock and found that flies exposed to these fields indeed showed enhanced slowing of clock rhythms. This effect was maximal at 300 μT, and reduced at both higher and lower field strengths. Clock response to magnetic fields was present in blue light, but absent under red-light illumination, which does not activate CRY. Furthermore, cryb and cryOUT mutants did not show any response, and flies overexpressing CRY in the clock neurons exhibited an enhanced response to the field. We conclude that Drosophila's circadian clock is sensitive to magnetic fields and that this sensitivity depends on light activation of CRY and on the applied field strength, consistent with the radical pair mechanism. CRY is widespread throughout biological systems and has been suggested as receptor for magnetic compass orientation in migratory birds. The present data establish the circadian clock of Drosophila as a model system for CRY-dependent magnetic sensitivity. Furthermore, given that CRY occurs in multiple tissues of Drosophila, including those potentially implicated in fly orientation, future studies may yield insights that could be applicable to the magnetic compass of migratory birds and even to potential magnetic field effects in humans.