Circadian clock of Drosophila montana is adapted to high variation in summer day lengths and temperatures prevailing at high latitudes

Photoperiodic regulation of the circadian rhythms in insect locomotor activity has been studied in several species, but seasonal entrainment of these rhythms is still poorly understood. We have traced the entrainment of activity rhythm of northern Drosophila montana flies in a climate chamber mimicking the photoperiods and day and night temperatures that the flies encounter in northern Finland during the summer. The experiment was started by transferring freshly emerged females into the chamber in early and late summer conditions to obtain both non-diapausing and diapausing females for the studies. The locomotor activity of the females and daily changes in the expression levels of two core circadian clock genes, timeless and period, in their heads were measured at different times of summer. The study revealed several features in fly rhythmicity that are likely to help the flies to cope with high variation in the day length and temperature typical to northern summers. First, both the non-diapausing and the diapausing females showed evening activity, which decreased towards the short day length as observed in the autumn in nature. Second, timeless and period genes showed concordant daily oscillations and seasonal shifts in their expression level in both types of females. Contrary to Drosophila melanogaster, oscillation profiles of these genes were similar to each other in all conditions, including the extremely long days in early summer and the cool temperatures in late summer, and their peak expression levels were not locked to lights-off transition in any photoperiod. Third, the diapausing females were less active than the non-diapausing ones, in spite of their younger age. Overall, the study showed that D. montana clock functions well under long day conditions, and that both the photoperiod and the daily temperature cycles are important zeitgebers for seasonal changes in the circadian rhythm of this species.     

Drosophila Free-Running Rhythms Require Intercellular Communication

Robust self-sustained oscillations are a ubiquitous characteristic of circadian rhythms. These include Drosophila locomotor activity rhythms, which persist for weeks in constant darkness (DD). Yet the molecular oscillations that underlie circadian rhythms damp rapidly in many Drosophila tissues. Although much progress has been made in understanding the biochemical and cellular basis of circadian rhythms, the mechanisms that underlie the differences between damped and self-sustaining oscillations remain largely unknown. A small cluster of neurons in adult Drosophila brain, the ventral lateral neurons (LN_vs), is essential for self-sustained behavioral rhythms and has been proposed to be the primary pacemaker for locomotor activity rhythms. With an LN_v-specific driver, we restricted functional clocks to these neurons and showed that they are not sufficient to drive circadian locomotor activity rhythms. Also contrary to expectation, we found that all brain clock neurons manifest robust circadian oscillations of timeless and cryptochrome RNA for many days in DD. This persistent molecular rhythm requires pigment-dispersing factor (PDF), an LN_v-specific neuropeptide, because the molecular oscillations are gradually lost when Pdf⁰¹ mutant flies are exposed to free-running conditions. This observation precisely parallels the previously reported effect on behavioral rhythms of the Pdf⁰¹ mutant. PDF is likely to affect some clock neurons directly, since the peptide appears to bind to the surface of many clock neurons, including the LN_vs themselves. We showed that the brain circadian clock in Drosophila is clearly distinguishable from the eyes and other rapidly damping peripheral tissues, as it sustains robust molecular oscillations in DD. At the same time, different clock neurons are likely to work cooperatively within the brain, because the LN_vs alone are insufficient to support the circadian program. Based on the damping results with Pdf⁰¹ mutant flies, we propose that LN_vs, and specifically the PDF neuropeptide that it synthesizes, are important in coordinating a circadian cellular network within the brain. The cooperative function of this network appears to be necessary for maintaining robust molecular oscillations in DD and is the basis of sustained circadian locomotor activity rhythms.     

Drosophila free-running rhythms require intercellular communication

Robust self-sustained oscillations are a ubiquitous characteristic of circadian rhythms. These include Drosophila locomotor activity rhythms, which persist for weeks in constant darkness (DD). Yet the molecular oscillations that underlie circadian rhythms damp rapidly in many Drosophila tissues. Although much progress has been made in understanding the biochemical and cellular basis of circadian rhythms, the mechanisms that underlie the differences between damped and self-sustaining oscillations remain largely unknown. A small cluster of neurons in adult Drosophila brain, the ventral lateral neurons (LN_vs), is essential for self-sustained behavioral rhythms and has been proposed to be the primary pacemaker for locomotor activity rhythms. With an LN_v-specific driver, we restricted functional clocks to these neurons and showed that they are not sufficient to drive circadian locomotor activity rhythms. Also contrary to expectation, we found that all brain clock neurons manifest robust circadian oscillations of timeless and cryptochrome RNA for many days in DD. This persistent molecular rhythm requires pigment-dispersing factor (PDF), an LN_v-specific neuropeptide, because the molecular oscillations are gradually lost when Pdf⁰¹ mutant flies are exposed to free-running conditions. This observation precisely parallels the previously reported effect on behavioral rhythms of the Pdf⁰¹ mutant. PDF is likely to affect some clock neurons directly, since the peptide appears to bind to the surface of many clock neurons, including the LN_vs themselves. We showed that the brain circadian clock in Drosophila is clearly distinguishable from the eyes and other rapidly damping peripheral tissues, as it sustains robust molecular oscillations in DD. At the same time, different clock neurons are likely to work cooperatively within the brain, because the LN_vs alone are insufficient to support the circadian program. Based on the damping results with Pdf⁰¹ mutant flies, we propose that LN_vs, and specifically the PDF neuropeptide that it synthesizes, are important in coordinating a circadian cellular network within the brain. The cooperative function of this network appears to be necessary for maintaining robust molecular oscillations in DD and is the basis of sustained circadian locomotor activity rhythms.     

Relationships between the Circadian System and Alzheimer's Disease-Like Symptoms in Drosophila

Circadian clocks coordinate physiological, neurological, and behavioral functions into circa 24 hour rhythms, and the molecular mechanisms underlying circadian clock oscillations are conserved from Drosophila to humans. Clock oscillations and clock-controlled rhythms are known to dampen during aging; additionally, genetic or environmental clock disruption leads to accelerated aging and increased susceptibility to age-related pathologies. Neurodegenerative diseases, such as Alzheimer''s disease (AD), are associated with a decay of circadian rhythms, but it is not clear whether circadian disruption accelerates neuronal and motor decline associated with these diseases. To address this question, we utilized transgenic Drosophila expressing various Amyloid-β (Aβ) peptides, which are prone to form aggregates characteristic of AD pathology in humans. We compared development of AD-like symptoms in adult flies expressing Aβ peptides in the wild type background and in flies with clocks disrupted via a null mutation in the clock gene period (per⁰¹). No significant differences were observed in longevity, climbing ability and brain neurodegeneration levels between control and clock-deficient flies, suggesting that loss of clock function does not exacerbate pathogenicity caused by human-derived Aβ peptides in flies. However, AD-like pathologies affected the circadian system in aging flies. We report that rest/activity rhythms were impaired in an age-dependent manner. Flies expressing the highly pathogenic arctic Aβ peptide showed a dramatic degradation of these rhythms in tune with their reduced longevity and impaired climbing ability. At the same time, the central pacemaker remained intact in these flies providing evidence that expression of Aβ peptides causes rhythm degradation downstream from the central clock mechanism.     

Silencing the circadian clock gene Clock using RNAi reveals dissociation of the circatidal clock from the circadian clock in the mangrove cricket

Whether a clock that generates a circatidal rhythm shares the same elements as the circadian clock is not fully understood. The mangrove cricket, Apteronemobius asahinai, shows simultaneously two endogenous rhythms in its locomotor activity; the circatidal rhythm generates active and inactive phases, and the circadian rhythm modifies activity levels by suppressing the activity during subjective day. In the present study, we silenced Clock (Clk), a master gene of the circadian clock, in A. asahinai using RNAi to investigate the link between the circatidal and circadian clocks. The abundance of Clk mRNA in the crickets injected with double-stranded RNA of Clk (dsClk) was reduced to a half of that in control crickets. dsClk injection also reduced mRNA abundance of another circadian clock gene period (per) and weakened diel oscillation in per mRNA expression. Examination of the locomotor rhythms under constant conditions revealed that the circadian modification was disrupted after silencing Clk expression, but the circatidal rhythm remained unaffected. There were no significant changes in the free-running period of the circatidal rhythm between the controls and the crickets injected with dsClk. Our results reveal that Clk is essential for the circadian clock, but is not required for the circatidal clock. From these results we propose that the circatidal rhythm of A. asahinai is driven by a clock, the molecular components of which are distinct from that of the circadian clock.     

Circadian Regulation of Glutathione Levels and Biosynthesis in Drosophila melanogaster

Circadian clocks generate daily rhythms in neuronal, physiological, and metabolic functions. Previous studies in mammals reported daily fluctuations in levels of the major endogenous antioxidant, glutathione (GSH), but the molecular mechanisms that govern such fluctuations remained unknown. To address this question, we used the model species Drosophila, which has a rich arsenal of genetic tools. Previously, we showed that loss of the circadian clock increased oxidative damage and caused neurodegenerative changes in the brain, while enhanced GSH production in neuronal tissue conferred beneficial effects on fly survivorship under normal and stress conditions. In the current study we report that the GSH concentrations in fly heads fluctuate in a circadian clock-dependent manner. We further demonstrate a rhythm in activity of glutamate cysteine ligase (GCL), the rate-limiting enzyme in glutathione biosynthesis. Significant rhythms were also observed for mRNA levels of genes encoding the catalytic (Gclc) and modulatory (Gclm) subunits comprising the GCL holoenzyme. Furthermore, we found that the expression of a glutathione S-transferase, GstD1, which utilizes GSH in cellular detoxification, significantly fluctuated during the circadian day. To directly address the role of the clock in regulating GSH-related rhythms, the expression levels of the GCL subunits and GstD1, as well as GCL activity and GSH production were evaluated in flies with a null mutation in the clock genes cycle and period. The rhythms observed in control flies were not evident in the clock mutants, thus linking glutathione production and utilization to the circadian system. Together, these data suggest that the circadian system modulates pathways involved in production and utilization of glutathione.     

Circadian rhythms in the morphology of neurons in <Emphasis Type="Italic">Drosophila</Emphasis>

Neurons have an enormous capacity to adapt to changing conditions through the regulation of gene expression, morphology, and physiology. In the fruit fly Drosophila melanogaster, this plasticity includes recurrent changes taking place within intervals of a few hours during the day. The rhythmic alterations in the morphology of neurons described so far include changes in axonal diameter, branching complexity, synapse numbers, and the number of synaptic vesicles. The cycles of these changes have larger amplitude when the fly is exposed to light, but they persist in constant darkness and require the expression of the clock genes period and timeless, leading to the concept of circadian plasticity. The molecular mechanisms driving these cycles appear to require the expression of these genes either inside the neurons themselves or in other peripheral pacemaker cells. Loss-of-function mutations in period and timeless not only abolish the morphological rhythms, but also often cause abnormal axonal branching suggesting that circadian plasticity is relevant for the maintenance of normal morphology. Research into whether (1) circadian plasticity is a common feature of neurons in all animals and (2) our own neurons change shape between day and night will be of interest.     

A Circadian Rhythm in Mice that is Unaffected by the Period Mutation at Clock

Circadian rhythms of locomotor behavior were induced in mice using chronic d-amphetamine. The periods of the rhythms at various doses of amphetamine were unaffected by a mutation at the clock locus that lengthens circadian period. Amphetamine-induced rhythms were superimposed on the hostÕs natural circadian rhythm that is driven by the pacemaker in the suprachiasmatic nucleus. The results demonstrate the existence of an alternative mechanism for generating circadian rhythms that does not require the activity of canonical clock genes.     

Circadian regulation of response to oxidative stress in Drosophila melanogaster

Circadian rhythms are fundamental biological phenomena generated by molecular genetic mechanisms known as circadian clocks. There is increasing evidence that circadian synchronization of physiological and cellular processes contribute to the wellness of organisms, curbing pathologies such as cancer and premature aging. Therefore, there is a need to understand how circadian clocks orchestrate interactions between the organism’s internal processes and the environment. Here, we explore the nexus between the clock and oxidative stress susceptibility in Drosophila melanogaster. We exposed flies to acute oxidative stress induced by hydrogen peroxide (H₂O₂), and determined that mortality rates were dependent on time at which exposure occurred during the day/night cycle. The daily susceptibility rhythm was abolished in flies with a null mutation in the core clock gene period (per) abrogating clock function. Furthermore, lack of per increased susceptibility to H₂O₂ compared to wild-type flies, coinciding with enhanced generation of mitochondrial H₂O₂ and decreased catalase activity due to oxidative damage. Taken together, our data suggest that the circadian clock gene period is essential for maintaining a robust anti-oxidative defense.     

Entrainment of eclosion and preliminary ontogeny of circadian clock gene expression in the flesh fly,Sarcophaga crassipalpis

Timing of circadian activities is controlled by rhythmic expression of clock genes in pacemaker neurons in the insect brain. Circadian behavior and clock gene expression can entrain to both thermoperiod and photoperiod but the availability of such cues, the organization of the brain, and the need for circadian behavior change dramatically during the course of insect metamorphosis. We asked whether photoperiod or thermoperiod entrains the clock during pupal and pharate adult stages by exposing flies to different combinations of thermoperiod and photoperiod and observing the effect on the timing of adult eclosion. This study used qRT-PCR to examine how entrainment and expression of circadian clock genes change during the course of development in the flesh fly, Sarcophaga crassipalpis. Thermoperiod entrains expression of period and controls the timing of adult eclosion, suggesting that the clock gene period may be upstream of the eclosion pathway. Rhythmic clock gene expression is evident in larvae, appears to cease during the early pharate adult stage, and resumes again by the time of adult eclosion. Our results indicate that both patterns of clock gene expression and the cues to which the clock entrains are dynamic and respond to different environmental signals at different developmental stages in S. crassipalpis.     

Drosophilarhythms: from brain to behavior

Theperiod(per) gene and thetimeless(tim) gene are essential components of the circadian clock inDrosophila melanogaster. Both gene products interact in interdependent feedback loops, producing a self-sustained cellular rhythmin situ. Several oscillating cells are combined to discrete pacemaker centers that control rhythmic behavior. This paper reviews the work on localizing the circadian pacemaker neurons controlling activity and eclosion, leading to questions about how these pacemaker cells are synchronized to the external light–dark cycle, and how they impose periodicity on behavior. The circadian system ofDrosophilais also compared with that of other arthropods.     

Rhythm-Specific Mutations in Drosophila rajasekari Altered Adult Locomotor Activity Rhythm but Not Eclosion Rhythm

The early and late strains for phase angle difference (Φ) of adult locomotor activity in Drosophila rajasekari were developed by artificial selection; these strains differed in Φ, activity pattern, activity level, free-running period (τ) in constant darkness (DD) and light induced phase shifts from those of the wild type (Joshi, 1998). The present studies were designed to determine whether or not the psi-mutations for adult locomotor activity rhythm had also altered the fundamental properties of the eclosion rhythms in these strains. The circadian rhythms of eclosion have been studied in the wild type, the early and late strains. In contrast to the effects on the locomotor activity rhythms in the early and late strains, the psi-mutations have no apparent effect on the eclosion median in light-dark cycles of 12 : 12 h, on τ in DD, light induced phase shifts or subjective light sensitivity in these strains. Thus the psi-mutations for the adult locomotor activity rhythms in D. rajasekari appear to be rhythm-specific mutations altering the locomotor rhythms but not the eclosion rhythms.     

Blocking synaptic transmission with tetanus toxin light chain reveals modes of neurotransmission in the PDF-positive circadian clock neurons of Drosophila melanogaster

Circadian locomotor rhythms of Drosophila melanogaster are controlled by a neuronal circuit composed of approximately 150 clock neurons that are roughly classified into seven groups. In the circuit, a group of neurons expressing pigment-dispersing factor (PDF) play an important role in organizing the pacemaking system. Recent studies imply that unknown chemical neurotransmitter(s) (UNT) other than PDF is also expressed in the PDF-positive neurons. To explore its role in the circadian pacemaker, we examined the circadian locomotor rhythms of pdf-Gal4/UAS-TNT transgenic flies in which chemical synaptic transmission in PDF-positive neurons was blocked by expressed tetanus toxin light chain (TNT). In constant darkness (DD), the flies showed a free-running rhythm, which was similar to that of wild-type flies but significantly different from pdf null mutants. Under constant light conditions (LL), however, they often showed complex rhythms with a short period and a long period component. The UNT is thus likely involved in the synaptic transmission in the clock network and its release caused by LL leads to arrhythmicity. Immunocytochemistry revealed that LL induced phase separation in TIMELESS (TIM) cycling among some of the PDF-positive and PDF-negative clock neurons in the transgenic flies. These results suggest that both PDF and UNT play important roles in the Drosophila circadian clock, and activation of PDF pathway alone by LL leads to the complex locomotor rhythm through desynchronized oscillation among some of the clock neurons.     

The Drosophila dCREB2 gene affects the circadian clock

We report the role of dCREB2, the Drosophila homolog of CREB/CREM, in circadian rhythms. dCREB2 activity cycles with a 24 hr rhythm in flies, both in a light:dark cycle and in constant darkness. A mutation in dCREB2 shortens circadian locomotor rhythm in flies and dampens the oscillation of period, a known clock gene. Cycling dCREB2 activity is abolished in a period mutant, indicating that dCREB2 and Period affect each other and suggesting that the two genes participate in the same regulatory feedback loop. We propose that dCREB2 supports cycling of the Period/Timeless oscillator. These findings support CREB's role in mediating adaptive behavioral responses to a variey of environmental stimuli (stress, growth factors, drug addiction, circadian rhythms, and memory formation) in mammals and long-term memory formation and circadian rhythms in Drosophila.     

Effect of the apterous 56f mutation on N -acetyltransferase and alkaline phosphatase activities in Drosophila melanogaster females

Alkaline phosphatase (AP) and N-acetyltransferase (NAT) activities were studied in 1-day-old Drosophila melanogaster females of the apterous ^56f (ap ^56f ) strain, having an decreased level of the juvenile hormone (JH) and a increased level of dopamine as a result of the mutation, and in the Canton S ancestral wild-type strain in the normal conditions and upon an experimental increase in JH titer. The AP and NAT activities in ap ^56f females were significantly lower than in Canton S females in the norm. JH application increased the AP activity of mutant females to the level characteristic to JH-treated wild-type females. Original Russian Text ? E.V. Bogomolova, N.V. Adonyeva, N.E. Gruntenko, I.Yu. Rauschenbach, 2008, published in Genetika, 2008, Vol. 44, No. 5, pp. 710–712.     

A role for the Drosophila fragile X-related gene in circadian output

Mutations that abolish expression of an X-linked gene, FMR1, result in the pathogenesis of fragile X syndrome, the most common form of inherited mental retardation. To understand the normal function of the FMR1 protein, we have produced fly strains bearing deletions in a Drosophila homolog of FMR1 (dfmr1). Since fragile X patients show a number of abnormal behaviors including sleep problems, we investigated whether a loss-of-function mutation of dfmr1 affect circadian behavior. Here we show that under constant darkness (DD), a lack of dfmr1 expression causes arrhythmic locomotor activity, but in light:dark cycles, their behavioral rhythms appear normal. In addition, the clock-controlled eclosion rhythm is normal in DFMR1-deficient flies. These results suggest that DFMR1 plays a critical role in the circadian output pathway regulating locomotor activity in Drosophila.