The NMDA Receptor Promotes Sleep in the Fruit Fly, Drosophila melanogaster

May The NMDA Receptor Promotes Sleep in the Fruit Fly, Drosophila melanogaster Jun Tomita 0 1 Taro Ueno 0 1 Madoka Mitsuyoshi 0 1 Shoen Kume 0 1 Kazuhiko Kume 0 1 0 1 Institute of Molecular Embryology and Genetics, Kumamoto University , Kumamoto , Japan , 2 Department of Sensory and Motor Systems, Tokyo Metropolitan Institute of Medical Science , Tokyo , Japan , 3 Department of Neuropharmacology, Graduate School of Pharmaceutical Sciences, Nagoya City University , Nagoya , Japan 1 Academic Editor: Björn Brembs , Universitaet Regensburg, GERMANY 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. - Competing Interests: The authors have declared that no competing interests exist. Sleep is a physiological state with unique characteristics. Behaviorally, sleep constitutes a consolidated period of rest and immobility without bulky movements, accompanied by an apparent reduced responsiveness to outside stimuli. The amount of sleep is regulated by both circadian and homeostatic processes [1]. Scientifically, sleep has been defined by electroencephalogram criteria in humans [2] and thus is mainly described in mammalian and avian species. In insects, sleep-like resting states were described first in the cockroach [3] and in the fruit fly, Drosophila melanogaster [4, 5]. We discovered a mutant with a reduced amount of these sleep-like states, and named it fumin, meaning 'insomnia' in Japanese and identified the mutation in the dopamine transporter gene [6]. This was a striking finding, since dopamine is also used to maintain wakefulness in the mammalian brain. Thus, it demonstrated the similarity between the molecular mechanism for regulating sleep-like states in Drosophila to that of the mammalian system [6]. Since then, cumulative evidence has continued to reveal similarities with mammalian sleep, so the sleep-like state of Drosophila is now simply called “Drosophila sleep”. In addition to similar behavioral characteristics and molecular mechanisms, mammalian sleep and Drosophila sleep share common physiological traits. Many short sleeping mutants have a reduced life span [7–10], but despite their short sleeper phenotype, fmn have an equivalent life span to control flies [6]. In addition, together with other short sleep mutants, fmn have a memory retention defect and deprivation of sleep impairs their memory [11]. These findings indicate that sleep plays an important role in lifespan and memory in Drosophila and may provide an insight into why sleep evolves in a broad range of species. In order to further elucidate the molecular mechanisms regulating sleep in Drosophila, we attempted to isolate more sleep related genes using the fmn mutant. In this report, we describe a gene expression analysis of fmn, followed by the successful isolation of the N-methyl-D-aspartic acid type glutamate receptor channel (NMDAR) gene, Nmdar1, as a novel sleep related gene and its function in promoting sleep. Materials and Methods Fly strains and culture conditions Flies were reared at 25°C in 50–60% relative humidity on standard fly food consisting of corn meal, yeast, glucose, wheat germ and agar. They were kept under 12 h light (zeitgeber time, ZT 0–12) followed by 12 h dark (ZT 12–24) cycles defined as the LD conditions. The transgenic RNA interference (RNAi) lines for Nmdar1 (VDRC 37333 and 104773), UAS-Dicer-2 flies (60008) and the w1118 (60000) which is the genetic background of the RNAi line were obtained from the Vienna Drosophila RNAi Center (VDRC) [12]. MB247 was provided by Dr. Hiromu Tanimoto. 11Y, 30Y, 201Y, 7Y, 104Y, 121Y, c232 and c767 were a gift from Dr. J. Douglas Armstrong. c747 and c772 were provided by Dr. Toshiro Aigaki. TH-Gal4 was a gift from Dr. Jay Hirsh. GAD-Gal4 was provided by Dr. Takaomi Sakai. OK371-Gal4 was a gift from Dr. Hermann Aberle. dilp2-Gal4 was a gift from Dr. Linda Partridge. npf-Gal4 was a gift from Dr. Ping Shen. OK307-Gal4 and c17 were a gift from Dr. Tanja Godenschwege. tsh-Gal4 was provided by Dr. Julie H. Simpson. The following stocks were ordered from the Bloomington Stock Center, Indiana University: elav-Gal4 (Stock number: 458), c309 (6906), c739 (7362), DdcGal4 (7009), Tdc2-Gal4 (9313), Cha-Gal4 (6793), dimm-Gal4 (25373), per-Gal4 (7127), timGal4 (7126), repo-Gal4 (7415) and D42 (8816). NP3529, NP6510, NP10, NP1004 and NP5103 were obtained from the Drosophila Genetics Resource Center, Kyoto Institute of Technology, Japan. The dopamine transporter mutant fumin (fmn) flies were isolated in our laboratory in a stock of y w flies, and backcrossed over five generations to the control strain (w1118) [6]. The backcrossed w; fmn line and the original y w; fmn line showed similar short sleeping phenotypes. Knockdown and control flies were obtained by crossing the pan-neuronal Gal4 driver elav-Gal4 with the UAS-Dicer-2 on the second chromosome to each of the RNAi lines and w1118, respectively. The 2- to 4-d-old post-eclosion male flies were used in this study. Control (w1118) and fmn flies subjected to four LD cycles were harvested every 4 h at seven time points under the following LD conditions (ZT 0, 4, 8, 12, 16, 20 and 24) and immediately frozen in liquid nitrogen. Frozen flies were vigorously shaken and sieved to collect the heads which had separated from the bodies. Total RNA was extracted from approximately 400 male and female fly heads homogenized in TRIzol reagent (Invitrogen) and its quality was assessed with an RNA 600 Nano Assay Kit using the Agilent 2100 Bioanalyzer (Agilent Technologies). Double-stranded cDNA was synthesized from 5 μg of total RNA using a One-Cycle cDNA Synthesis Kit (Affymetrix) and served as a template to synthesize biotin-labeled cRNA using a GeneChip IVT Labeling Kit (Affymetrix). Biotin-lab (...truncated)