Jun Tomita

Identification of a dopamine pathway that regulates sleep and arousal in Drosophila

Sleep is required to maintain physiological functions, including memory, and is regulated by monoamines across species. Enhancement of dopamine signals by a mutation in the dopamine transporter (DAT) decreases sleep, but the underlying dopamine circuit responsible for this remains unknown. We found that the D1 dopamine receptor (DA1) in the dorsal fan-shaped body (dFSB) mediates the arousal effect of dopamine in Drosophila. The short sleep phenotype of the DAT mutant was completely rescued by an additional mutation in the DA1 (also known as DopR) gene, but expression of wild-type DA1 in the dFSB restored the short sleep phenotype. We found anatomical and physiological connections between dopamine neurons and the dFSB neuron. Finally, we used mosaic analysis with a repressive marker and found that a single dopamine neuron projecting to the FSB activated arousal. These results suggest that a local dopamine pathway regulates sleep.

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.

Dopamine Modulates Metabolic Rate and Temperature Sensitivity in Drosophila melanogaster

Homeothermal animals, such as mammals, maintain their body temperature by heat generation and heat dissipation, while poikilothermal animals, such as insects, accomplish it by relocating to an environment of their favored temperature. Catecholamines are known to regulate thermogenesis and metabolic rate in mammals, but their roles in other animals are poorly understood. The fruit fly, Drosophila melanogaster, has been used as a model system for the genetic studies of temperature preference behavior. Here, we demonstrate that metabolic rate and temperature sensitivity of some temperature sensitive behaviors are regulated by dopamine in Drosophila. Temperature-sensitive molecules like dTrpA1 and shi ts induce temperature-dependent behavioral changes, and the temperature at which the changes are induced were lowered in the dopamine transporter-defective mutant, fumin. The mutant also displays a preference for lower temperatures. This thermophobic phenotype was rescued by the genetic recovery of the dopamine transporter in dopamine neurons. Flies fed with a dopamine biosynthesis inhibitor (3-iodo-L-tyrosine), which diminishes dopamine signaling, exhibited preference for a higher temperature. Furthermore, we found that the metabolic rate is up-regulated in the fumin mutant. Taken together, dopamine has functions in the temperature sensitivity of behavioral changes and metabolic rate regulation in Drosophila, as well as its previously reported functions in arousal/sleep regulation.

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.

The NMDA Receptor promotes sleep in the fruit fly, drosophila melanogaster

Considerable evidence indicates that sleep is essential for learning and memory. Drosophila melanogaster has emerged as a novel model for studying sleep. We previously found a short sleeper mutant, fumin (fmn), and identified its mutation in the dopamine transporter gene. We reported similarities in the molecular basis of sleep and arousal regulation between mammals and Drosophila. In aversive olfactory learning tasks, fmn mutants demonstrate defective memory retention, which suggests an association between sleep and memory. In an attempt to discover additional sleep related genes in Drosophila, we carried out a microarray analysis comparing mRNA expression in heads of fmn and control flies and found that 563 genes are differentially expressed. Next, using the pan-neuronal Gal4 driver elav-Gal4 and UAS-RNA interference (RNAi) to knockdown individual genes, we performed a functional screen. We found that knockdown of the NMDA type glutamate receptor channel gene (Nmdar1) (also known as dNR1) reduced sleep. The NMDA receptor (NMDAR) plays an important role in learning and memory both in Drosophila and mammals. The application of the NMDAR antagonist, MK-801, reduced sleep in control flies, but not in fmn. These results suggest that NMDAR promotes sleep regulation in Drosophila.

High calorie diet augments age-associats 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.

Sweetness induces sleep through gustatory signalling independent of nutritional value in a starved fruit fly

Starvation reduces sleep in various animal species including humans and fruit flies. Immediate hunger and the following insufficient nutritional status resulting from starvation may affect sleep and arousal differently. In order to clarify the mechanism underlying the relationship between diet and sleep, we analysed the sleep behaviour of Drosophila melanogaster that were either starved or fed with different types of sugars. Starved flies showed longer activity bouts, short sleep bouts and a decreased arousal threshold. Non-nutritive sweeteners such as sucralose and arabinose, which are sweet but not nutritive, induced sleep in starved flies, but sleep bout length and the arousal threshold was short and decreased, respectively. On the other hand, sorbitol, which is not sweet but nutritive, did not induce sleep, but slightly increased the lowered arousal threshold. Activation of sweetness receptor expressing neurons induced sleep in starved flies. These results suggest that sweetness alone is sufficient to induce sleep in starved flies and that the nutritional status affects sleep homeostasis by decreasing the arousal threshold, which resulted in short sleep bouts in Drosophila.

Genetic analysis of the relationship between high caloric diet and sleep regulation in Drosophila melanogaster

asmatic nucleus (SCN), which is known to house the mammalian circadian pacemaker. The intracellular signaling pathway in GRP-R-mediated resetting in the SCN, however, remains to be clarified. Previously, Tei and his colleague established an immortalized SCN progenitor cell line, RS182 cells, from rat carrying a luciferase reporter gene under control of the Period1 promoter (kawaguchi et al., 2007). In this study, we examined whether RS182 cells could be used as a model cell for studying GRP-R-mediated resetting in the dorsal SCN and tried to clarify the intracellular mechanism of GRP-Rmediated resetting using this cell line. We first observed the GRP-R mRNA was expressed in RS182 cells. Furthermore, RS182 cells strongly expressed the arginine-vasopressin, which was known to be expressed in the dorsal SCN. The treatment with GRP caused the transient increase in the intracellular Ca2+ levels and this was completely blocked by either GRP-R antagonist or a Gq inhibitor, YM-254890. We also found that GRP increased mRNA levels of Period2 and this induction was suppressed by the pretreatment with a blocker of GRP-R. Finally, we found that GRP delayed the bioluminescence rhythm of RS182 cells when treated 7 hr after the bioluminescence peak. These results suggest that RS182 cells can be used as a model cell for studying GRP-R-mediated resetting in the dorsal SCN and Gq signaling pathway mediates GRP-induced resetting in RS182 cells.