Are competing intermolecular and intramolecular interactions of PERIOD protein important for the regulation of circadian rhythms in Drosophila?

Genetic analysis is revealing molecular components of circadian rhythms. The gene products of the period gene in Drosophila and the frequency gene in Neurospora oscillate with a circadian rhythm. A recent paper⁽¹⁾ has shown that the PERIOD protein can undergo both intermolecular and intramolecular interactions in vitro. The effects of temperature and two period mutations on these molecular interactions were compared to the effects of the mutations and temperature on the in vivo period length of circadian rhythms. The results suggest that the molecular interactions may compete to maintain a rhythm with a constant period over a wide temperature range.     

Simulated microgravity influences circadian rhythm of NIH3T3 cells

Gravity heavily influences living organisms on earth including their circadian rhythm, which is fundamentally important for coordinately physiology in organisms as diverse as cyanobacteria, fungus and humans. Numerous researches have revealed that microgravity in outer space can affect circadian rhythm of astronauts and rodent animals, but the mechanism remains unknown. Using rotary cell culture system to simulate microgravity environment, we investigated the role of simulated microgravity in regulating the circadian rhythm of NIH3T3 cells. Our experiments found that simulated microgravity can not only influence the mRNA level of some core circadian genes, but also modify the circadian rhythm of Per1 and Per2 synchronized after phorbol myristate acetate treatment. Remarkably, MEK/ERK pathway was transiently activated after a 2-h simulated microgravity treatment, with a significant upregulation of Kras, Raf1 and p-ERK1/ERK2. Moreover, U0126, a selective inhibitor of MEK/ERK pathway, could disrupt the circadian rhythm of Per1 and Per2 synchronized after simulated microgravity treatment. Together, our results unveil that simulated microgravity could act like a zeitgeber to influence the circadian rhythm of NIH3T3 by acting on MEK/ERK pathway, indicating that MEK/ERK pathway may act as a bridge which connects cells mechanotransduction pathway and circadian rhythm regulation.     

Modeling Temperature Compensation in Chemical and Biological Oscillators

All physicochemical and biological oscillators maintain a balance between destabilizing reactions (as, for example, intrinsic autocatalytic or amplifying reactions) and stabilizing processes. These two groups of processes tend to influence the period in opposite directions and may lead to temperature compensation whenever their overall influence balances. This principle of “antagonistic balance” has been tested for several chemical and biological oscillators. The Goodwin negative feedback oscillator appears of particular interest for modeling the circadian clocks in Neurospora and Drosophila and their temperature compensation. Remarkably, the Goodwin oscillator not only gives qualitative, correct phase response curves for temperature steps and temperature pulses, but also simulates the temperature behavior of Neurospora frq and Drosophila per mutants almost quantitatively. The Goodwin oscillator predicts that circadian periods are strongly dependent on the turnover of the clock mRNA or clock protein. A more rapid turnover of clock mRNA or clock protein results, in short, a slower turnover in longer period lengths. (Chronobiology International, 14(5), 499–510, 1997)     

Egg-laying rhythm in <Emphasis Type="Italic">Drosophila melanogaster</Emphasis>

Extensive research has been carried out to understand how circadian clocks regulate various physiological processes in organisms. The discovery of clock genes and the molecular clockwork has helped researchers to understand the possible role of these genes in regulating various metabolic processes. In Drosophila melanogaster, many studies have shown that the basic architecture of circadian clocks is multi-oscillatory. In nature, different neuronal subgroups in the brain of D. melanogaster have been demonstrated to control different circadian behavioural rhythms or different aspects of the same circadian rhythm. Among the circadian phenomena that have been studied so far in Drosophila, the egg-laying rhythm is unique, and relatively less explored. Unlike most other circadian rhythms, the egg-laying rhythm is rhythmic under constant light conditions, and the endogenous or free-running period of the rhythm is greater than those of most other rhythms. Although the clock genes and neurons required for the persistence of adult emergence and activity/rest rhythms have been studied extensively, those underlying the circadian egg-laying rhythm still remain largely unknown. In this review, we discuss our current understanding of the circadian egg-laying rhythm in D. melanogaster, and the possible molecular and physiological mechanisms that control the rhythmic output of the egg-laying process.     

Effect of Spaceflight on the Circadian Rhythm,Lifespan and Gene Expression of Drosophila melanogaster

Space travelers are reported to experience circadian rhythm disruption during spaceflight. However, how the space environment affects circadian rhythm is yet to be determined. The major focus of this study was to investigate the effect of spaceflight on the Drosophila circadian clock at both the behavioral and molecular level. We used China’s Shenzhou-9 spaceship to carry Drosophila. After 13 days of spaceflight, behavior tests showed that the flies maintained normal locomotor activity rhythm and sleep pattern. The expression level and rhythm of major clock genes were also unaffected. However, expression profiling showed differentially regulated output genes of the circadian clock system between space flown and control flies, suggesting that spaceflight affected the circadian output pathway. We also investigated other physiological effects of spaceflight such as lipid metabolism and lifespan, and searched genes significantly affected by spaceflight using microarray analysis. These results provide new information on the effects of spaceflight on circadian rhythm, lipid metabolism and lifespan. Furthermore, we showed that studying the effect of spaceflight on gene expression using samples collected at different Zeitgeber time could obtain different results, suggesting the importance of appropriate sampling procedures in studies on the effects of spaceflight.     

Circadian Modulation of Acute Alcohol Sensitivity But Not Acute Tolerance in Drosophila

An increased understanding of the factors affecting behavioral and neurological responses to alcohol and alcohol physiology is necessary given the tremendous toll alcohol abuse and alcoholism exert on individuals and society. At the behavioral and molecular levels, the response to alcohol appears remarkably conserved from Drosophila to humans, suggesting that investigations across model species can provide insight into the identification of common modulatory factors. We investigated the interaction between the circadian clock and alcohol sensitivity, alcohol tolerance, and alcohol absorbance in Drosophila melanogaster. Using a loss-of-righting reflex (LoRR) assay, we found that flies exhibit a circadian rhythm in the LoRR, with the greatest sensitivity to alcohol occurring from mid to late night, corresponding to the flies' inactive phase. As predicted, a circadian rhythm in the LoRR was absent in circadian mutant flies and under conditions in which the circadian clock was nonfunctional. Circadian modulation of the response to alcohol was not due to circadian regulation of alcohol absorbance. Similar to other animals, Drosophila develop acute and chronic tolerance to alcohol upon repeat exposures. We found that the circadian clock did not modulate the development of acute alcohol tolerance measured as the difference in sensitivity to alcohol between naïve and pre-exposed flies. Thus, the circadian clock modulates some, but not all, of the behavioral responses to alcohol exposure, suggesting that specific mechanisms underlie the observed circadian modulation of LoRR rather than global cellular circadian regulation. This study provides valuable new insights in our understanding of the circadian modulation of alcohol-induced behaviors that ultimately could facilitate preventative measures in combating alcohol abuse and alcoholism. (Author correspondence: lyons@bio.fsu.edu)     

Phase Shift Effects in the Kalanchoë Petal Rhythm due to Two or More Light Pulses

The circadian petal rhythm of Kalanchoë blossfeldiana Poellniz was studied theoretically and experimentally. Results of experiments in which (i) two light pulses and (ii) repeated light pulses were given to the flowers are compared with predictions based on a previously published feedback model. In this model both the amplitude and the phase of the rhythm are affected by light pulses. Results from the present phase shift studies are shown to be in good agreement with the model. The results are also discussed in relation to a constant amplitude model like that suggested by Pittendrigh for the eclosion rhythm of Drosophila.     

Circadian rhythms

The neurobiological substratum of circadian rhythmicity encompasses three levels of integration: firstly, generation of time signals by circadian pacemakers; secondly, entrainment of pacemakers by environmental influences; thirdly, coupling of circadian pacemakers among themselves and with target systems responsible for the expression of overt rhythms. From recent contributions, the notion that circadian organization results from the interaction of independent oscillators and pathways has been strengthened. In addition, recent evidence supports the existence of circadian rhythmicity in single isolated neurons. New information was produced on the gene control of circadian rhythm generation in Drosophila, as well as interesting advances in the understanding of neuronal mechanisms involved in the generation, entrainment and coupling of circadian rhythms in various species.     

Pigment‐dispersing factor affects circadian molecular oscillations in the cricket Gryllus bimaculatus

Pigment‐dispersing factor (PDF) is an important neurotransmitter in insect circadian systems. In the cricket Gryllus bimaculatus, it affects nocturnal activity, the free‐running period and photic entrainment. In this study, to investigate whether these effects of PDF occur through a circadian molecular machinery, we measured mRNA levels of clock genes period (per) and timeless (tim) in crickets with pdf expression knocked‐down by pdf RNAi. The pdf RNAi decreased per and tim mRNA levels during the night to reduce the amplitude of their oscillation. The phase of the rhythm advanced by about 4 h in terms of trough and/or peak phases. On the other hand, pdf mRNA levels were little affected by per and tim RNAi treatment. These results suggest that PDF affects the circadian rhythm at least in part through the circadian molecular oscillation while the circadian clock has little effect on the pdf expression.     

研究报告：果蝇习得性无助影响空间位置记忆

目的 习得性无助（learned helplessness）是指动物经历了不可逃避性刺激后而产生的无助状态。它对随后实验动物的运动能力、生理状态等多方面都会产生影响,也是人类抑郁症的有效动物模型之一。目前已在多种动物模型中建立了研究习得性无助神经机制的行为实验范式,包括黑腹果蝇（Drosophila melanogaster）。之前的研究发现,经历过不可逃避高温惩罚的果蝇在活跃程度等方面有显著改变。然而,果蝇的习得性无助状态是否也影响学习认知能力,目前仍然未知。本文对果蝇空间位置学习能力是否受到习得性无助状态影响进行了研究。方法 此研究中,我们完善实验范式,分别检测果蝇在新范式中的空间位置学习行为和习得性无助行为,以及经过长时程随机刺激后果蝇空间位置学习能力的变化。结果 野生型果蝇可在此范式中展现空间位置学习能力与习得性无助状态,而经过长时程不可逃避刺激训练的果蝇,空间位置记忆有显著下降。结论 此研究完善了果蝇习得性无助行为实验范式,实验结果表明习得性无助对果蝇空间位置记忆存在影响。这将对进一步加深对动物习得性无助行为的理解,进而其发生机制研究起到推进作用。     

Effect of different light regimes on fitness of two species of Drosophila montium subgroup

Most organisms possess “biological chronometers” in the form of circadian clocks. Organism possessing circadian clock gains fitness advantage in two ways, by synchronizing its behavior through physiological process and secondly by coordinating its internal metabolic process. Environmental manipulations of circadian clocks have been shown to affect many life-history-related traits. Life-history traits are important components of fitness. To enhance individual fitness, organism has to synchronize the physiology with the surrounding environment. The present investigations were made to understand whether rhythm changes affect fitness of two co-existing species of montium a subgroup of Drosophila. The stocks were maintained at 20 ± 1 °C with 75% RH. Fitness such as fecundity, male lifetime fertility, female lifetime fertility, and longevity was assessed in LD (light/dark), LL (continuous light), and DD (continuous dark) for 15 and 30th generations. Fecundity was assessed in 25 pairs of flies for 20 days, and fertility and longevity was assessed in 10 pairs of flies until lifetime. The result revealed differential effect of light regimes on the two different species of Drosophila. Although the two species are related, effect of the three light regimes, LD, LL, and DD on them was different. It is evident that these two species although genetically related exhibit different responses to different light regimes.     

Assaying Locomotor Activity to Study Circadian Rhythms and Sleep Parameters in Drosophila

Most life forms exhibit daily rhythms in cellular, physiological and behavioral phenomena that are driven by endogenous circadian (≡24 hr) pacemakers or clocks. Malfunctions in the human circadian system are associated with numerous diseases or disorders. Much progress towards our understanding of the mechanisms underlying circadian rhythms has emerged from genetic screens whereby an easily measured behavioral rhythm is used as a read-out of clock function. Studies using Drosophila have made seminal contributions to our understanding of the cellular and biochemical bases underlying circadian rhythms. The standard circadian behavioral read-out measured in Drosophila is locomotor activity. In general, the monitoring system involves specially designed devices that can measure the locomotor movement of Drosophila. These devices are housed in environmentally controlled incubators located in a darkroom and are based on using the interruption of a beam of infrared light to record the locomotor activity of individual flies contained inside small tubes. When measured over many days, Drosophila exhibit daily cycles of activity and inactivity, a behavioral rhythm that is governed by the animal''s endogenous circadian system. The overall procedure has been simplified with the advent of commercially available locomotor activity monitoring devices and the development of software programs for data analysis. We use the system from Trikinetics Inc., which is the procedure described here and is currently the most popular system used worldwide. More recently, the same monitoring devices have been used to study sleep behavior in Drosophila. Because the daily wake-sleep cycles of many flies can be measured simultaneously and only 1 to 2 weeks worth of continuous locomotor activity data is usually sufficient, this system is ideal for large-scale screens to identify Drosophila manifesting altered circadian or sleep properties.     

Temperature sensitivity of circadian clocks is conserved across Drosophila species melanogaster,malerkotliana and ananassae

Light and temperature are the major environmental cycles that can synchronize circadian rhythms in a variety of organisms. Previously, we have shown that under light/dark cycles of various photoperiods, the Drosophila species ananassae exhibits unimodal activity pattern with a prominent morning activity peak in contrast with Drosophila melanogaster and Drosophila malerkotliana, which show bimodal activity pattern with morning and evening activity peaks. Here we report that circadian clocks controlling activity/rest rhythm of these two less-studied species D. malerkotliana and D. ananassae can be synchronized by temperature cycles and that even under temperature cycles D. ananassae exhibits only a pronounced morning (thermophase onset) activity peak. Although D. melanogaster and D. ananassae exhibit differences in the phase of activity/rest rhythm under temperature cycles, circadian clocks of both show similar sensitivity to warm temperature pulses. Circadian period of activity/rest rhythm of D. ananassae differs from the other two species at some moderate-range temperatures; however, in conditions that are more extreme, circadian clocks of D. melanogaster, D. malerkotliana and D. ananassae appear to be largely temperature compensated.     

Circadian control of heat tolerance in stationary phase cultures of Schizosaccharomyces pombe

The capacity of stationary phase cultures of Schizosaccharomyces pombe to survive a heat treatment at 55°C is controlled by a circadian rhythm. In a synchronizing light-dark-cycle this rhythm shows a stable phase relationship to the onset of light. In continuous darkness it persists for several cycles without marked damping. The free-running period of about 27 h at 30°C is only slightly longer at 20°C, hence temperature-compensated. These results indicate that S. pombe is a suitable experimental organism for further research into both heat tolerance and circadian rhythms.