Taishi Yoshii

A new ImageJ plug-in "ActogramJ" for chronobiological analyses.

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.

Temperature cycles drive Drosophila circadian oscillation in constant light that otherwise induces behavioural arrhythmicity

The fruit fly, Drosophila melanogaster, shows a clear circadian locomotor rhythm in light cycles and constant darkness. Although the rhythm disappears in constant light, we found that temperature cycles drive the circadian rhythm both in locomotor activity and molecular abundance of PERIOD (PER) and TIMELESS (TIM). The thermoperiodically induced locomotor rhythm entailed an anticipatory activity at the late thermophase, which required several transient cycles to establish a steady‐state entrainment, suggesting that the rhythm is endogenous and driven by a circadian clock. Western blot analysis revealed that PER and TIM increased during the cryophase, peaking at the middle to late cryophase. PER was also cyclically expressed under the temperature cycle in the known per‐expressing neurons, i.e. so‐called lateral (LNs) and dorsal neurons (DNs), and two pairs of cells (LPNs) that were located in the lateral posterior protocerebrum. It is thus suggested that the temperature cycle induces the cycling of PER and TIM either by blocking somewhere in the photic entrainment pathway during the cryophase or temporally activating their translation to sufficient protein levels to drive a circadian oscillation. In flies lacking pigment‐dispersing factor (PDF) or PDF‐expressing cells, the anticipatory activity was relatively dispersed. disco2 mutant flies lacking the lateral neurons still showed an anticipatory activity, but with dispersed activity. These behavioural results suggest that not only LNs but also DNs and LPNs can, at least, partially participate in regulating the thermoperiodically induced rhythm.

Peripheral circadian rhythms and their regulatory mechanism in insects and some other arthropods: a review.

Many physiological functions of insects show a rhythmic change to adapt to daily environmental cycles. These rhythms are controlled by a multi-clock system. A principal clock located in the brain usually organizes the overall behavioral rhythms, so that it is called the “central clock”. However, the rhythms observed in a variety of peripheral tissues are often driven by clocks that reside in those tissues. Such autonomous rhythms can be found in sensory organs, digestive and reproductive systems. Using Drosophila melanogaster as a model organism, researchers have revealed that the peripheral clocks are self-sustained oscillators with a molecular machinery slightly different from that of the central clock. However, individual clocks normally run in harmony with each other to keep a coordinated temporal structure within an animal. How can this be achieved? What is the molecular mechanism underlying the oscillation? Also how are the peripheral clocks entrained by light–dark cycles? There are still many questions remaining in this research field. In the last several years, molecular techniques have become available in non-model insects so that the molecular oscillatory mechanisms are comparatively investigated among different insects, which give us more hints to understand the essential regulatory mechanism of the multi-oscillatory system across insects and other arthropods. Here we review current knowledge on arthropod’s peripheral clocks and discuss their physiological roles and molecular mechanisms.

Drosophila Clock Neurons under Natural Conditions

The circadian clock modulates the adaptive daily patterns of physiology and behavior and adjusts these rhythms to seasonal changes. Recent studies of seasonal locomotor activity patterns of wild-type and clock mutant fruit flies in quasi-natural conditions have revealed that these behavioral patterns differ considerably from those observed under standard laboratory conditions. To unravel the molecular features accompanying seasonal adaptation of the clock, we investigated Drosophila’s neuronal expression of the canonical clock proteins PERIOD (PER) and TIMELESS (TIM) in nature. We find that the profile of PER dramatically changes in different seasons, whereas that of TIM remains more constant. Unexpectedly, we find that PER and TIM oscillations are decoupled in summer conditions. Moreover, irrespective of season, PER and TIM always peak earlier in the dorsal neurons than in the lateral neurons, suggesting a more rapid molecular oscillation in these cells. We successfully reproduced most of our results under simulated natural conditions in the laboratory and show that although photoperiod is the most important zeitgeber for the molecular clock, the flies’ activity pattern is more strongly affected by temperature. Our results are among the first to systematically compare laboratory and natural studies of Drosophila rhythms.

Two clocks in the brain: an update of the morning and evening oscillator model in Drosophila.

Circadian clocks play an essential role in adapting the activity rhythms of animals to the day-night cycles on earth throughout the four seasons. In many animals, including the fruit fly Drosophila melanogaster, two separate but mutually coupled clocks in the brain -morning (M) and evening (E) oscillators- control the activity in the morning and evening. M and E oscillators are thought to track dawn and dusk, respectively. This alters the phase-angle between the two oscillators under different day lengths, optimally adapting the animals activity pattern to colder short and warmer long days. Using excellent genetic tools, Drosophila researchers have addressed the neural basis of the two oscillators and could partially track these to distinct clock cells in the brain. Nevertheless, not all data are consistent with each other and many questions remained open. So far, most studies about M and E oscillators focused on the influence of light (photoperiod). Here, we will review the effects of light and temperature on the two oscillators, will update the present knowledge, discuss the limitations of the model, and raise questions that have to be addressed in the future.

Laboratory versus nature: The two sides of the Drosophila circadian clock

The daily pattern of animal behavior is thought to be of potential enormous importance for survival. Here, we compared the daily activity pattern of Drosophila melanogaster wild-type flies and the clock-impaired mutants, per01 and ClkJrk, under pseudo-natural conditions and laboratory conditions with natural-like temperature profiles. We found that clock-impaired flies respond stronger to changes in the environment, namely temperature increases, than wild-type flies. We hypothesize that the circadian clock may suppress unproductive activity in response to temperature fluctuations but that such suppression can be overcome in extreme conditions that are likely life-threatening for the flies. Thus, possessing a clock seems to be of adaptive significance.

Induction of Drosophila Behavioral and Molecular Circadian Rhythms by Temperature Steps in Constant Light

In constant light, where Drosophila rhythms are normally disrupted, temperature cycles induce circadian rhythms at both the molecular and behavioral level. The authors investigated the process by which the thermoperiod induces the rhythms using temperature steps. A 10 °C temperature step-up induced a single locomotor activity peak ca 9 h after the temperature transition, whereas a 10 °C step-down induced a strong activity peak ca 24 h after the transition, and the peak recurred for several cycles, suggesting that the underlying clock is reset. Arrhythmic per01 , tim 01 , dClkJrk , and cyc01 mutant flies failed to show the rhythm after the step-down, suggesting that per, tim, dClk, and cyc are necessary for the step-down—induced rhythm. After the step-up, per01 flies exhibited an activity peak similar to that of wild-type flies, suggesting that the peak can be induced by the step-up in absence of PER. mRNA levels of per, tim , dClk, vri, and Pdp1ε were changed in response to the temperature steps, but the changes differed depending on the direction of temperature steps, suggesting that steps-up and steps-down have different roles in the initiation of the oscillation. Probably, alternating 12-h temperature steps-up and steps-down will induce opposite changes in mRNA levels of clock genes, eventually producing stable molecular oscillations. Although TIM shows responses to temperature consistent with the changes of its mRNA, this is not the case for PER, consistent with posttranscriptional regulation. Changes of the mRNA levels were significantly altered but still observed in per 01 flies but not observed in dClkJrk flies, except for per mRNA. This suggests that dCLK is involved in the temperature-induced changes in the levels of most clock gene mRNA but that per is regulated via a different mechanism.

Cryptochrome-Dependent and -Independent Circadian Entrainment Circuits in Drosophila

Entrainment to environmental light/dark (LD) cycles is a central function of circadian clocks. In Drosophila, entrainment is achieved by Cryptochrome (CRY) and input from the visual system. During activation by brief light pulses, CRY triggers the degradation of TIMELESS and subsequent shift in circadian phase. This is less important for LD entrainment, leading to questions regarding light input circuits and mechanisms from the visual system. Recent studies show that different subsets of brain pacemaker clock neurons, the morning (M) and evening (E) oscillators, have distinct functions in light entrainment. However, the role of CRY in M and E oscillators for entrainment to LD cycles is unknown. Here, we address this question by selectively expressing CRY in different subsets of clock neurons in a cry-null (cry0) mutant background. We were able to rescue the light entrainment deficits of cry0 mutants by expressing CRY in E oscillators but not in any other clock neurons. Par domain protein 1 molecular oscillations in the E, but not M, cells of cry0 mutants still responded to the LD phase delay. This residual light response was stemming from the visual system because it disappeared when all external photoreceptors were ablated genetically. We concluded that the E oscillators are the targets of light input via CRY and the visual system and are required for normal light entrainment.

GABA(B) receptors play an essential role in maintaining sleep during the second half of the night in Drosophila melanogaster.

SUMMARY GABAergic signalling is important for normal sleep in humans and flies. Here we advance the current understanding of GABAergic modulation of daily sleep patterns by focusing on the role of slow metabotropic GABAB receptors in the fruit fly Drosophila melanogaster. We asked whether GABAB-R2 receptors are regulatory elements in sleep regulation in addition to the already identified fast ionotropic Rdl GABAA receptors. By immunocytochemical and reporter-based techniques we show that the pigment dispersing factor (PDF)-positive ventrolateral clock neurons (LNv) express GABAB-R2 receptors. Downregulation of GABAB-R2 receptors in the large PDF neurons (l-LNv) by RNAi reduced sleep maintenance in the second half of the night, whereas sleep latency at the beginning of the night that was previously shown to depend on ionotropic Rdl GABAA receptors remained unaltered. Our results confirm the role of the l-LNv neurons as an important part of the sleep circuit in D. melanogaster and also identify the GABAB-R2 receptors as the thus far missing component in GABA-signalling that is essential for sleep maintenance. Despite the significant effects on sleep, we did not observe any changes in circadian behaviour in flies with downregulated GABAB-R2 receptors, indicating that the regulation of sleep maintenance via l-LNv neurons is independent of their function in the circadian clock circuit.

Exquisite Light Sensitivity of Drosophila melanogaster Cryptochrome

Drosophila melanogaster shows exquisite light sensitivity for modulation of circadian functions in vivo, yet the activities of the Drosophila circadian photopigment cryptochrome (CRY) have only been observed at high light levels. We studied intensity/duration parameters for light pulse induced circadian phase shifts under dim light conditions in vivo. Flies show far greater light sensitivity than previously appreciated, and show a surprising sensitivity increase with pulse duration, implying a process of photic integration active up to at least 6 hours. The CRY target timeless (TIM) shows dim light dependent degradation in circadian pacemaker neurons that parallels phase shift amplitude, indicating that integration occurs at this step, with the strongest effect in a single identified pacemaker neuron. Our findings indicate that CRY compensates for limited light sensitivity in vivo by photon integration over extraordinarily long times, and point to select circadian pacemaker neurons as having important roles.