rdgB knockdown in neurons reduced nocturnal sleep in Drosophila melanogaster.

Recent studies revealed behaviorally defined sleep is conserved across broad species from insect to human. For evolutional analysis, it is critical to determine how homologous genes regulate the homologous function among species. Drosophila melanogaster shares numerous sleep related genes with mammals including Sik3, salt-inducible kinase 3, whose mutation caused long sleep both in mouse and fruit fly. The Drosophila rdgB (retinal degeneration B) encodes a membrane-associated phosphatidylinositol transfer protein and its mutation caused light-induced degeneration of photoreceptor cells. rdgB mutation also impaired phototransduction and olfactory behavior, indicating rdgB is involved in the normal neural transmission. Mammalian rdgB homologue, Pitpnm2 (phosphatidylinositol transfer protein membrane-associated 2) was discovered as one of SNIPPs (sleep-need index phosphoproteins), suggesting its role in sleep. Here, we show that rdgB is involved in sleep regulation in Drosophila. Pan-neuronal and mushroom body (MB) specific rdgB knockdown decreased nocturnal sleep. MB neurons play a dominant role, since the rescue of rdgB expression only in MB neurons in pan-neuronal knockdown reversed the sleep reducing effect of rdgB knockdown. These results revealed the sleep-related function of rdgB in Drosophila which may be conserved across species.

Insulin signaling in clock neurons regulates sleep in Drosophila.

Sleep relates to numerous biological functions, including metabolism. Both dietary conditions and genes related to metabolism are known to affect sleep behavior. Insulin signaling is well conserved across species including the fruit fly and relates to both metabolism and sleep. However, the neural mechanism of sleep regulation by insulin signaling is poorly understood. Here, we report that insulin signaling in specific neurons regulates sleep in Drosophila melanogaster. We analyzed the sleep behavior of flies with the mutation in insulin-like ligands expressed in the brain and found that three insulin-like ligands participate in sleep regulation with some redundancy. We next used 21 Gal4 drivers to express a dominant-negative form of the insulin receptor (InR DN) in various neurons including circadian clock neurons, which express the clock gene, and the pars intercerebralis (PI). Inhibition of insulin signaling in the anterior dorsal neuron group 1 (DN1a) decreased sleep. Additionally, the same manipulation in PI also decreased sleep. Pan-neuronal induced expression of InR DN also decreased sleep. These results suggested that insulin signaling in DN1a and PI regulates sleep.

Effects of D-amino acids on sleep in Drosophila.

Sleep and metabolism are closely related and nutritional elements such as sugars and amino acids are known to regulate sleep differently. Here we comprehensively investigated the effects of D-amino acids fed in the diet on the sleep of Drosophila melanogaster. Among 19 amino acids examined, both D-serine (Ser) and D-glutamine (Gln) induced a significant increase in sleep amount and the effect of D-Ser was the largest at the same concentration of 1% of the food. The effects were proportional to its concentration and significant above 0.5% (about 50 mM). D-Ser is known to bind NR1 subunit of NMDA type glutamate receptor (NMDAR) and activate it. D-Ser did not increase the sleep of the NR1 hypomorphic mutant flies indicating its effects on sleep is mediated by NMDAR. In addition, hypomorphic mutants of D-amino acid oxidase (Daao1), which catabolizes D-amino acids and its disruption is known to increase D-Ser in the brain, showed increase in sleep. These results altogether suggested that D-Ser activated NMDAR in the brain thus increase sleep, and that D-Ser work physiologically to regulate sleep.

Forward-genetics analysis of sleep in randomly mutagenized mice.

Sleep is conserved from invertebrates to vertebrates, and is tightly regulated in a homeostatic manner. The molecular and cellular mechanisms that determine the amount of rapid eye movement sleep (REMS) and non-REMS (NREMS) remain unknown. Here we identify two dominant mutations that affect sleep and wakefulness by using an electroencephalogram/electromyogram-based screen of randomly mutagenized mice. A splicing mutation in the Sik3 protein kinase gene causes a profound decrease in total wake time, owing to an increase in inherent sleep need. Sleep deprivation affects phosphorylation of regulatory sites on the kinase, suggesting a role for SIK3 in the homeostatic regulation of sleep amount. Sik3 orthologues also regulate sleep in fruitflies and roundworms. A missense, gain-of-function mutation in the sodium leak channel NALCN reduces the total amount and episode duration of REMS, apparently by increasing the excitability of REMS-inhibiting neurons. Our results substantiate the use of a forward-genetics approach for studying sleep behaviours in mice, and demonstrate the role of SIK3 and NALCN in regulating the amount of NREMS and REMS, respectively.

The initiation of nocturnal dormancy in Synechococcus as an active process.

BACKGROUND: Most organisms, especially photoautotrophs, alter their behaviours in response to day-night alternations adaptively because of their great reliance on light. Upon light-to-dark transition, dramatic and universal decreases in transcription level of the majority of the genes in the genome of the unicellular cyanobacterium, Synechococcus elongatus PCC 7942 are observed. Because Synechococcus is an obligate photoautotroph, it has been generally assumed that repression of the transcription in the dark (dark repression) would be caused by a nocturnal decrease in photosynthetic activities through the reduced availability of energy (e.g. adenosine triphosphate (ATP)) needed for mRNA synthesis. RESULTS: However, against this general assumption, we obtained evidence that the rapid and dynamic dark repression is an active process. Although the addition of photosynthesis inhibitors to cells exposed to light mimicked transcription profiles in the dark, it did not significantly affect the cellular level of ATP. By contrast, when ATP levels were decreased by the inhibition of both photosynthesis and respiration, the transcriptional repression was attenuated through inhibition of RNA degradation. This observation indicates that Synechococcus actively downregulates genome-wide transcription in the dark. Even though the level of total mRNA dramatically decreased in the dark, Synechococcus cells were still viable, and they do not need de novo transcription for their survival in the dark for at least 48 hours. CONCLUSIONS: Dark repression appears to enable cells to enter into nocturnal dormancy as a feed-forward process, which would be advantageous for their survival under periodic nocturnal conditions.

Preference of position in the proximity of various sugars revealed by location analysis of Drosophila melanogaster.

Feeding behaviors are determined by two main factors. One is the internal state, such as hunger or previous experiences; the other is external factors, such as sensory stimulation. During starvation, animals must balance food-seeking behavior with energy conservation. The fruit fly, Drosophila melanogaster, serves as a useful model for studying food selectivity and various behaviors related to food intake. However, few studies have directly connected food selectivity with other behaviors, such as locomotor activity and sleep. In this study, we report that flies exhibited a preference for specific positions and spent more time in the proximity of sweet sugars, such as sucrose and sucralose, but not non-sweet and nutritious sugars like xylitol and sorbitol. On the other hand, prolonged exposure to sorbitol increased the staying time of flies in the proximity of sorbitol. Additionally, after starvation, flies immediately exhibited a position preference in the proximity of sorbitol. These findings suggest that flies prefer the proximity of sweet food, and starvation alters their preference for nutritious food, which may be beneficial for their survival.

Temporal organization of rest defined by actigraphy data in healthy and childhood chronic fatigue syndrome children.

BACKGROUND: Accumulating evidence has shown a universality in the temporal organization of activity and rest among animals ranging from mammals to insects. Previous reports in both humans and mice showed that rest bout durations followed long-tailed (i.e., power-law) distributions, whereas activity bouts followed exponential distributions. We confirmed similar results in the fruit fly, Drosophila melanogaster. Conversely, another report showed that the awakening bout durations, which were defined by polysomnography in bed, followed power-law distributions, while sleeping periods, which may correspond to rest, followed exponential distributions. This apparent discrepancy has been left to be resolved. METHODS: Actigraphy data from healthy and disordered children were analyzed separately for two periods: time out of bed (UP period) and time in bed (DOWN period). RESULTS: When data over a period of 24 h were analyzed as a whole, rest bouts showed a power law distribution as previously reported. However, when UP and DOWN period data were analyzed separately, neither showed power law properties. Using a newly developed strict method, only 30% of individuals satisfied the power law criteria, even when the 24 h data were analyzed. The human results were in contrast to the Drosophila results, which revealed clear power-law distributions for both day time and night time rest through the use of a strict method. In addition, we analyzed the actigraphy data from patients with childhood type chronic fatigue syndrome (CCFS), and found that they showed differences from healthy controls when their UP and DOWN data were analyzed separately. CONCLUSIONS: These results suggested that the DOWN sleep, the bout distribution of which showed exponential properties, contributes to the production of long-tail distributions in human rest periods. We propose that separate analysis of UP and DOWN period data is important for understanding the temporal organization of activity.

A phosphorylation-deficient mutant of Sik3, a homolog of Sleepy, alters circadian sleep regulation by PDF neurons in Drosophila.

Sleep behavior has been observed from non-vertebrates to humans. Sleepy mutation in mice resulted in a notable increase in sleep and was identified as an exon-skipping mutation of the salt-inducible kinase 3 (Sik3) gene, conserved among animals. The skipped exon includes a serine residue that is phosphorylated by protein kinase A. Overexpression of a mutant gene with the conversion of this serine into alanine (Sik3-SA) increased sleep in both mice and the fruit fly Drosophila melanogaster. However, the mechanism by which Sik3-SA increases sleep remains unclear. Here, we found that Sik3-SA overexpression in all neurons increased sleep under both light-dark (LD) conditions and constant dark (DD) conditions in Drosophila. Additionally, overexpression of Sik3-SA only in PDF neurons, which are a cluster of clock neurons regulating the circadian rhythm, increased sleep during subjective daytime while decreasing the amplitude of circadian rhythm. Furthermore, suppressing Sik3-SA overexpression specifically in PDF neurons in flies overexpressing Sik3-SA in all neurons reversed the sleep increase during subjective daytime. These results indicate that Sik3-SA alters the circadian function of PDF neurons and leads to an increase in sleep during subjective daytime under constant dark conditions.

Pan-neuronal knockdown of calcineurin reduces sleep in the fruit fly, Drosophila melanogaster.

Sleep is a unique physiological state, which is behaviorally defined, and is broadly conserved across species from mammals to invertebrates such as insects. Because of the experimental accessibility provided by various novel animal models including the fruit fly, Drosophila melanogaster, there have been significant advances in the understanding of sleep. Although the physiological functions of sleep have not been fully elucidated, accumulating evidence indicates that sleep is necessary to maintain the plasticity of neuronal circuits and, hence, is essential in learning and memory. Calcineurin (Cn) is a heterodimeric phosphatase composed of CnA and CnB subunits and known to function in memory consolidation in the mammalian brain, but its neurological functions in the fruit fly are largely unknown. Here, we show that Cn is an important regulator of sleep in Drosophila. A pan-neuronal RNA interference-mediated knockdown of Cn expression resulted in sleep loss, whereas misexpression of the constitutively active form of a CnA protein led to increased sleep. Furthermore, CnA knockdown also impaired the retention of aversive olfactory memory. These results indicate a role for Cn and calcium-dependent signal transduction in sleep and memory regulation and may bring insight into the relationship between them.

Pan-neuronal knockdown of the c-Jun N-terminal Kinase (JNK) results in a reduction in sleep and longevity in Drosophila.

Sleep is a unique behavioral state that is conserved between species, and sleep regulation is closely associated to metabolism and aging. The fruit fly, Drosophila melanogaster has been used to study the molecular mechanism underlying these physiological processes. Here we show that the c-Jun N-terminal Kinase (JNK) gene, known as basket (bsk) in Drosophila, functions in neurons to regulate both sleep and longevity in Drosophila. Pan-neuronal knockdown of JNK mRNA expression by RNA interference resulted in a decrease in both sleep and longevity. A heterozygous knockout of JNK showed similar effects, indicating the molecular specificity. The JNK knockdown showed a normal arousal threshold and sleep rebound, suggesting that the basic sleep mechanism was not affected. JNK is known to be involved in the insulin pathway, which regulates metabolism and longevity. A JNK knockdown in insulin-producing neurons in the pars intercerebralis had slight effects on sleep. However, knocking down JNK in the mushroom body had a significant effect on sleep. These data suggest a unique sleep regulating pathway for JNK.

High calorie diet augments age-associated sleep impairment in Drosophila.

The fruit fly, Drosophila melanogaster is an established model used for aging and longevity studies and more recently for sleep studies. Mammals and Drosophila share various physiological, pathological, pharmacological and genetic similarities in these processes. In particular, sleep is essential for survival in both species and both have age-associated sleep quality alterations. Here we report that a high calorie diet, which accelerates the aging process and reduces lifespan across species, also accelerates age-associated sleep changes in Drosophila. These changes are more evident in the dopamine transporter mutant, fumin, that displays a short sleep phenotype due to enhanced dopaminergic signaling. With normal food, fumin mutants sleep for only one third of the time that the control flies do, but still show equivalent longevity. However, when on a mildly high calorie diet, their sleep length shows a marked decrease and they have a reduced longevity. These data indicate that the age-associated change in sleep in Drosophila is a physiologically regulated aging process that is tightly linked to calorie intake and that the dopamine level plays an important role. In addition, this provides another evidence that sleep is essential for the longevity of Drosophila.

Cyanobacterial daily life with Kai-based circadian and diurnal genome-wide transcriptional control in Synechococcus elongatus.

In the unicellular cyanobacterium Synechococcus elongatus PCC 7942, essentially all promoter activities are under the control of the circadian clock under continuous light (LL) conditions. Here, we used high-density oligonucleotide arrays to explore comprehensive profiles of genome-wide Synechococcus gene expression in wild-type, kaiABC-null, and kaiC-overexpressor strains under LL and continuous dark (DD) conditions. In the wild-type strains, >30% of transcripts oscillated significantly in a circadian fashion, peaking at subjective dawn and dusk. Such circadian control was severely attenuated in kaiABC-null strains. Although it has been proposed that KaiC globally represses gene expression, our analysis revealed that dawn-expressed genes were up-regulated by kaiC-overexpression so that the clock was arrested at subjective dawn. Transfer of cells to DD conditions from LL immediately suppressed expression of most of the genes, while the clock kept even time in the absence of transcriptional feedback. Thus, the Synechococcus genome seems to be primarily regulated by light/dark cycles and is dramatically modified by the protein-based circadian oscillator.

The regulation of circadian rhythm by insulin signaling in Drosophila.

Circadian rhythm is well conserved across species and relates to numerous biological functions. Circadian misalignment impairs metabolic function. Insulin signaling is a key modulator of metabolism in the fruit fly as well as mammals and its defects cause metabolic disease. Daily diet timing affects both circadian rhythmicities of behavior and metabolism. However, the relationship between the circadian clock and insulin signaling is still elusive. Here, we report that insulin signaling regulates circadian rhythm in Drosophila melanogaster. We found the insulin receptor substrate mutant, chico1, showed a shorter free-running circadian period. The knockdown of insulin receptor (InR), or another signaling molecule downstream of InR, dp110, or the expression of a dominant-negative form of InR resulted in the shortening of the circadian period and diminished its amplitude. The impairment of insulin signaling both in all neurons and restricted circadian clock neurons altered circadian period length, indicating that the insulin signaling plays a role in the regulation of circadian rhythm in clock cells. Among 3 insulin-like ligands expressed in the brain, dilp5 showed the largest effect on circadian phenotype when deleted. These results suggested that insulin signaling contributes to the robustness of the circadian oscillation and coordinates metabolism and behavior.

Interneurons of fan-shaped body promote arousal in Drosophila.

Sleep is required to maintain physiological functions and is widely conserved across species. To understand the sleep-regulatory mechanisms, sleep-regulating genes and neuronal circuits are studied in various animal species. In the sleep-regulatory neuronal circuits in Drosophila melanogaster, the dorsal fan-shaped body (dFB) is a major sleep-promoting region. However, other sleep-regulating neuronal circuits were not well identified. We recently found that arousal-promoting T1 dopamine neurons, interneurons of protocerebral bridge (PB) neurons, and PB neurons innervating the ventral part of the FB form a sleep-regulatory circuit, which we named "the PB-FB pathway". In the exploration of other sleep-regulatory circuits, we found that activation of FB interneurons, also known as pontine neurons, promoted arousal. We then found that FB interneurons had possible connections with the PB-FB pathway and dFB neurons. Ca2+ imaging revealed that FB interneurons received excitatory signals from the PB-FB pathway. We also demonstrated the possible role of FB interneurons to regulate dFB neurons. These results suggested the role of FB interneurons in sleep regulation.

Biological functions of α2-adrenergic-like octopamine receptor in Drosophila melanogaster.

Octopamine regulates various physiological phenomena including memory, sleep, grooming and aggression in insects. In Drosophila, four types of octopamine receptors have been identified: Oamb, Oct/TyrR, OctßR and Octα2R. Among these receptors, Octα2R was recently discovered and pharmacologically characterized. However, the effects of the receptor on biological functions are still unknown. Here, we showed that Octα2R regulated several behaviors related to octopamine signaling. Octα2R hypomorphic mutant flies showed a significant decrease in locomotor activity. We found that Octα2R expressed in the pars intercerebralis, which is a brain region projected by octopaminergic neurons, is involved in control of the locomotor activity. Besides, Octα2R hypomorphic mutants increased time and frequency of grooming and inhibited starvation-induced hyperactivity. These results indicated that Octα2R expressed in the central nervous system is responsible for the involvement in physiological functions.

Protocerebral Bridge Neurons That Regulate Sleep in Drosophila melanogaster.

The central complex is one of the major brain regions that control sleep in Drosophila. However, the circuitry details of sleep regulation have not been elucidated yet. Here, we show a novel sleep-regulating neuronal circuit in the protocerebral bridge (PB) of the central complex. Activation of the PB interneurons labeled by the R59E08-Gal4 and the PB columnar neurons with R52B10-Gal4 promoted sleep and wakefulness, respectively. A targeted GFP reconstitution across synaptic partners (t-GRASP) analysis demonstrated synaptic contact between these two groups of sleep-regulating PB neurons. Furthermore, we found that activation of a pair of dopaminergic (DA) neurons projecting to the PB (T1 DA neurons) decreased sleep. The wake-promoting T1 DA neurons and the sleep-promoting PB interneurons formed close associations. Dopamine 2-like receptor (Dop2R) knockdown in the sleep-promoting PB interneurons increased sleep. These results indicated that the neuronal circuit in the PB, regulated by dopamine signaling, mediates sleep-wakefulness.

A Randomized Trial of Home Blood-Pressure Reduction by Alcohol Guidance During Outpatient Visits: OSAKE Study.

BACKGROUND: To evaluate the effectiveness of the nurse-led alcohol guidance to control home blood pressure (HBP) in the morning among male patients with hypertension during outpatient visits. METHODS: We enrolled 53 male patients with an HBP of ≥135/85 mm Hg with excessive drinking (alcohol ≥210 g/week or ≥60 g/day habitually) among outpatients in a randomized trial. Patients were assigned to a nurse-led alcohol guidance intervention or to the control. The primary outcomes were the mean HBP of 5 consecutive days at 6 months and alcohol consumption. RESULTS: Twenty-eight and 25 patients were randomized to intervention and control groups, respectively (mean age; 62.7 years old and 64.5, respectively). At baseline, the groups were well balanced across most characteristics. At 6 months, the mean HBP was 131/82 mm Hg in the intervention group vs. 145/87 mm Hg in the control group (SBP <0.001, DBP = 0.09). An HBP level of less than 135/85 mm Hg was achieved among 55.6% of the participants in the intervention group vs. 16.7% in the control group (P = 0.004). The alcohol consumption at 6 months was 256 ± 206 g/w vs. 413 ± 260 g/w, respectively (P = 0.020). CONCLUSIONS: We confirmed the effectiveness of the nurse-led alcohol guidance to control the HBP in male patients with hypertension during outpatient visits. PUBLIC TRIALS REGISTRY NUMBER: UMIN000017454 (UMIN Clinical Trials Registry).

Fruit Fly, Drosophila melanogaster, as an In Vivo Tool to Study the Biological Effects of Proton Irradiation.

The clinical superiority of proton therapy over photon therapy has recently gained recognition; however, the biological effects of proton therapy remain poorly understood. The lack of in vivo evidence is especially important. Therefore, the goal of this study was to validate the usefulness of Drosophila melanogaster as an alternative tool in proton radiobiology. To determine whether the comparative biological effects of protons and X rays are detectable in Drosophila, we assessed their influence on survival and mRNA expression. Postirradiation observation revealed that protons inhibited their development and reduced the overall survival rates more effectively than X rays. The relative biological effectiveness of the proton beams compared to the X rays estimated from the 50% lethal doses was 1.31. At 2 or 24 h postirradiation, mRNA expression analysis demonstrated that the expression patterns of several genes (such as DNA-repair-, apoptosis- and angiogenesis-related genes) followed different time courses depending on radiation type. Moreover, our trials suggested that the knockdown of individual genes by the GAL4/UAS system changes the radiosensitivity in a radiation type-specific manner. We confirmed this Drosophila model to be considerably useful to evaluate the findings from in vitro studies in an in vivo system. Furthermore, this model has a potential to elucidate more complex biological mechanisms underlying proton irradiation.

Fruit Fly, Drosophila melanogaster, as an In Vivo Tool to Study the Biological Effects of Proton Irradiation.

The clinical superiority of proton therapy over photon therapy has recently gained recognition; however, the biological effects of proton therapy remain poorly understood. The lack of in vivo evidence is especially important. Therefore, the goal of this study was to validate the usefulness of Drosophila melanogaster as an alternative tool in proton radiobiology. To determine whether the comparative biological effects of protons and X rays are detectable in Drosophila, we assessed their influence on survival and mRNA expression. Postirradiation observation revealed that protons inhibited their development and reduced the overall survival rates more effectively than X rays. The relative biological effectiveness of the proton beams compared to the X rays estimated from the 50% lethal doses was 1.31. At 2 or 24 h postirradiation, mRNA expression analysis demonstrated that the expression patterns of several genes (such as DNA-repair-, apoptosis- and angiogenesis-related genes) followed different time courses depending on radiation type. Moreover, our trials suggested that the knockdown of individual genes by the GAL4/UAS system changes the radiosensitivity in a radiation type-specific manner. We confirmed this Drosophila model to be considerably useful to evaluate the findings from in vitro studies in an in vivo system. Furthermore, this model has a potential to elucidate more complex biological mechanisms underlying proton irradiation.

The clock components Period2, Cryptochrome1a, and Cryptochrome2a function in establishing light-dependent behavioral rhythms and/or total activity levels in zebrafish.

The circadian clock generates behavioral rhythms to maximize an organism's physiological efficiency. Light induces the formation of these rhythms by synchronizing cellular clocks. In zebrafish, the circadian clock components Period2 (zPER2) and Cryptochrome1a (zCRY1a) are light-inducible, however their physiological functions are unclear. Here, we investigated the roles of zPER2 and zCRY1a in regulating locomotor activity and behavioral rhythms. zPer2/zCry1a double knockout (DKO) zebrafish displayed defects in total locomotor activity and in forming behavioral rhythms when briefly exposed to light for 3-h. Exposing DKO zebrafish to 12-h light improved behavioral rhythm formation, but not total activity. Our data suggest that the light-inducible circadian clock regulator zCRY2a supports rhythmicity in DKO animals exposed to 12-h light. Single cell imaging analysis revealed that zPER2, zCRY1a, and zCRY2a function in synchronizing cellular clocks. Furthermore, microarray analysis of DKO zebrafish showed aberrant expression of genes involved regulating cellular metabolism, including ATP production. Overall, our results suggest that zPER2, zCRY1a and zCRY2a help to synchronize cellular clocks in a light-dependent manner, thus contributing to behavioral rhythm formation in zebrafish. Further, zPER2 and zCRY1a regulate total physical activity, likely via regulating cellular energy metabolism. Therefore, these circadian clock components regulate the rhythmicity and amount of locomotor behavior.