睡眠、生体リズム Sleep and Biological Rhythms
P1-2-157 ラット海馬スライスにおいて測定される、カルバコール誘導θ振動、てんかん波の時空間パターン The spatiotemporal pattern recorded in carbachol-induced theta oscillation and epileptic discharges in rat hippocampal slices ○蔭山逸行1, 夏目季代久1 ○Itsuki Kageyama1, Kiyohisa Natsume1 九州工業大学大学院 生命体工学研究科1 Dept. of Brain Sci. and Eng., Grad. Sch. of Life Sci. and Sys. Eng., Kyusyu Inst. of Tech., Kitakyusyu, Japan1 Theta rhythm is related to memory and learning process. During the generation of the rhythm, pyramidal and inhibitory cells fire synchronously.Hippocampus has another rhythm in which the pyramidal cells fire synchronously.It is an epileptic discharges. These rhythms are induced in rat hippocampal slices.In the present study,the spatiotemporal pattern of the action potential and synaptic potential was recorded and compared between these rhythms.The theta rhythms were induced with the application of the cholinergic agent carbachol.The epileptic discharges were induced with the application of GABAA receptor antagonist picrotoxin.The spatiotemporal pattern was recorded in the CA3 region using 8×8 multi-electrode array system MED64.After the spatiotemporal pattern of potential was recorded,current source density(CSD) analysis was carried out using Laplacian filter in the neighboring electrodes.Carbachol-induced theta oscillation(CITO) was induced intermittently.The phase of the recorded potential in CITO and picrotoxin-induced epileptic discharges(PIED) at stratum radiatum was reversed in the distal dendrite of the pyramidal cells.The amplitude of CITO was smaller than that of PIED.CSD analysis clarified that the current sink of CITO propagated along the pyramidal cells layer from CA3b to CA3a with the same distance as that of PIED did. It occurred simultaneously with a population spike in a burst of CITO and PIED.After that,the current sink at stratum radiatum and current source at stratum pyramidale were induced in CITO for shorter time,and then diminished immediately.On the other hand,they were induced at wider area in PIED,and they continued for a longer time.The flowing sink and source current were larger in PIED than in CITO.These results suggest that while the spatial synchronized neurons are almost the same between in CITO and PIED,the flowing current can be larger in PIED than in CITO,and the magnitude can reflect the larger amplitude of PIED.	P1-2-158 Insomniac相同遺伝子による哺乳類睡眠制御 Involvement of insomniac homolog in mammalian sleep homeostasis ○田中進1, 本多芳子1, 本多和樹1, 児玉亨1 ○Susumu Tanaka1, Yoshiko Honda1, Kazuki Honda1, Tohru Kodama1 都医学研・精神行動医学・睡眠1 SLEEP Proj, Tokyo Metropolitan Inst of Med Sci, Tokyo1 A forward genetic study in Drosophila have shown a mutation in insomniac gene that represents insomnia (Stavropoulos N et al. Neuron. 2011). Restricted insomniac expression with pars intercerebralis driver in a mutant indicated behavioral rescue. The insomniac protein is a member of the BTB/POZ superfamily and is evolutionary conserved in mammalians. The BTB/POZ superfamily includes the adaptors for the Cullin-3 ubiquitin ligase complex and RNAi for cullin-3 or Nedd8 exhibited a decrease in sleep duration, suggesting that Ub-proteasome pathway regulates sleep/wakefulness in Drosophila.In this study, we examined the insomniac homolog expression in mice hypothalamus using quantitative RT-PCR and in situ hybridization. One of the three homologs showed specific expression in anterior hypothalamus compared with the expression in pituitary. The expression is restricted in anterior hypothalamus compared with other region of hypothalamus and is changed in sleep/wake cycle. The cRNA probe for the insomniac homolog hybridized with brain transcripts approximately 1.6 kb by Northern hybridization. We have confirmed the expression of some proteasome pathway genes including E3 ligases in anterior hypothalamus and the interaction of E3 ligases with insomniac homolog. Consequently, it is proposed that cullin-dependent protein degradation pathways are required even in mammalian sleep.
P1-2-159 新規orexin-Creマウス,ラットの作成 Generation of new orexin-Cre transgenic mice and rats ○犬束歩1, 乾あずさ1, 常松友美2, 冨田江一2, 平林真澄2今吉格4, 影山龍一郎4, 山中章弘1,2,5 ○Ayumu Inutsuka1, Azusa Inui1, Tomomi Tsunematsu2, Koichi Tomita2, Masumi Hirabayashi2, Michael Lazarus3, Itaru Imayoshi4, Ryoichiro Kageyama4, Akihiro Yamanaka1,2,5 名大・環研・神経系分野II1, 生理学研究所2, 大阪バイオサイエンス研究所3, 京大・ウイルス研4, さきがけ5 Research Institute of Environmental Medicine, Nagoya University, Nagoya, JAPAN1, National Institute for Physiological Sciences, Okazaki, JAPAN2, Osaka Bioscience Institute, Osaka, JAPAN3, Institute for Virus Research, Kyoto University, Kyoto, JAPAN4, PRESTO, Tokyo, JAPAN5 The hypothalamic neuropeptides, orexins/hypocretins are a key regulator of sleep and wakefulness. Prepro-orexin knockout mice, orexin 2 receptor knockout mice, and orexin neuron-ablated transgenic mice all show severe fragmentation of sleep and wakefulness, which is similar to a human sleep disorder, narcolepsy. It is also reported that orexin peptide levels in the cerebrospinal fluid are decreased, and that the number of orexin neurons is highly reduced in narcoleptic patients. Because orexin neurons show scattered and sparse distribution within the lateral hypothalamic area (LHA), genetic approaches are required to manipulate the activity of orexin neurons specifically.To address this, we generated new orexin-Cre transgenic mice and rats in which Cre recombinase is exclusively expressed in orexin neurons. For this purpose, a transgene was constructed with the 3.2-kb fragment of the 5'-upstream region of the human prepro-orexin gene as a promoter, which drives the expression of the Cre recombinase connected with EGFP via viral 2A peptide (EGFP-2A-Cre). Using this transgene, several orexin-Cre transgenic mouse and rat lines were established. We carried out immunohistochemistry using an anti-Cre antibody and found that Cre immunoreactivity was observed in most of the nucleus of orexin-ir neurons. Expression of EGFP could be also detected without enhancement by immunohistochemistry. Then we examine if functional Cre protein is expressed in orexin neurons by infecting adeno-associated virus (AAV) vectors including double floxed transgenes such as channelrhodopsin-2-EYFP and archearhodopsin-tdTomato. This confirmed the Cre activity as the fluorescence of transgenes was restricted to orexin neurons. These observations suggest that our new orexin-Cre mice and rats can be used to express genes of interest exclusively in orexin neurons by breeding with reporter mice line or injecting AAV vectors.	P1-2-160 ショウジョウバエのLIMホメオボックス遺伝子apterousは睡眠/覚醒の調節に関与する The Drosophila LIM homeobox gene, apterous, is involved in the regulation of sleep/arousal ○嶋田直人1, 坂井貴臣1 ○Naoto Shimada1, Takaomi Sakai1 首都大学東京大学院 理工学研究科 生命科学専攻1 Department of biological science, Tokyo Metropolitan Univ, Tokyo, Japan1 Sleep is crucial for proper brain function and plays a role in the processing of information acquired from waking experience. The fruit fly, Drosophila melanogaster, shows sleep-like state and the so-called "Drosophila sleep" shares many of the features of mammalian sleep. In Drosophila, induction of sleep after training leads to enhancement of long-term memory (LTM), while sleep deprivation after training inhibits LTM formation. Thus, it is possible that Drosophila sleep is required for converting initial short-lasting memory into long-lasting form (memory consolidation). However, the molecular and physiological mechanisms of sleep-dependent memory consolidation are largely unknown. Previously, we demonstrated that one of the Drosophila LIM homeodomain transcriptional factors, Apterous (Ap), is required for LTM formation (The 32th and 35th annual meeting of the Japan neuroscience society). ap mutants are defective for long-term memory, using a courtship-conditioning assay, where previous experiences with mated females causes males to subsequently reduce their courtship of virgin females. Here, there is an intriguing possibility that the defective LTM in ap mutants results from the dysfunction of memory consolidation induced by reduction of sleep. In this current study, we examined whether Ap plays a role in the regulation of Drosophila sleep/arousal. Males heterozygous for ap showed abnormal sleep pattern and reduction of daytime sleep. Pan-neural knockdown of ap expression by RNA interference (RNAi) reduced daytime and nighttime sleep, respectively. Targeted expression of ap RNAi in the mushroom bodies (MBs), a brain region important for Drosophila memory and sleep, reduced daytime sleep. These results suggest that Ap expression in the MB neurons is involved in the regulation of sleep/arousal during daytime.
P1-2-161 視交叉上核神経回路網の大規模カルシウムイメージング解析 Large-scale calcium imaging in the neuronal network of the suprachiasmatic nucleus ○榎木亮介1,2, 三枝理博3, 黒田茂4, 小野大輔1, マザヒルハサン5, 上田哲男6, 本間さと2, 本間研一2 ○Ryosuke Enoki1,2, Michihiro Mieda3, Shigeru Kuroda4, Daisuke Ono1, Hasan Mazahir5, Tetsuo Ueda6, Sato Honma2, Ken-ichi Honma2 北大院・医・光バイオイメージング1, 北大院・医・時間医学2, 金沢大院・医・分子神経―統合生理3, はこだて未来大学・複雑系知能4, マックスプランク医学研究所5, 北大・電子研6 Hokkaido Univ, Grad Sch of Med, Photonic Bioimaging Sec, Sapporo, Japan1, Hokkaido Univ, Grad Sch of Med, Dep of Chronomedicine, Sapporo, Japan2, Kanazawa Univ, Fac of Med, Dep of Mol Neurosci & Integrative Physiol, Kanazawa, Japan3, Hakodate Future Univ. Sch of Systems Information Sci, Hakodate, Japan4, Max Planck Institute for Medical Research, Hiderberg, Germany5, Hokkaido Univ, RIES, Sapporo, Japan6 The circadian pacemaker in the hypothalamic suprachiasmatic nucleus (SCN) is a hierarchical multi-oscillator system in which neuronal networks play crucial roles in expressing coherent rhythms in physiology and behavior. Using a unique large-scale calcium imaging method with genetically-encoded calcium sensors, we visualized intracellular calcium from the entire SCN neuronal network in culture. We found circadian calcium rhythms at a single-cell level in the SCN, which were topologically specific with a larger amplitude and more delayed phase in the ventral region than the dorsal. The robustness of the rhythm was reduced but persisted even after blocking the neuronal firing with tetrodotoxin (TTX). TTX dissociated the circadian calcium rhythms between the dorsal and ventral SCN. In contrast, a blocker of gap junctions, carbenoxolone, had only a minor effect on the calcium rhythms at both the single-cell and network levels. Using a novel dual-color fluorescence imaging, we successfully visualized expression patterns of Per1 and calcium in the SCN at single cell resolution. These results reveal the topological specificity of the circadian calcium rhythm in the SCN and the presence of coupled regional pacemakers in the dorsal and ventral regions. Our rhythm analysis also showed the topologically organized patterns of circadian rhythms of Per1 and calcium in the SCN.	P1-2-162 ラット海馬におけるシータ波は外側手綱核から正中縫線核のセロトニンを介する経路によって維持される Hippocampal theta oscillation is maintained by LHb via serotonergic median raphe in rat ○柳原真1, 相澤秀紀2, 小林恵実1岡本仁1 ○Shin Yanagihara1, Hidenori Aizawa2, Megumi Kobayashi1, Joshua Johansen1, Hitoshi Okamoto1 理化学研究所・脳センター1, 東京医歯大・難治研2 RIKEN Brain Science Institute, Saitama1, Tokyo Medical and Dental University Medical Research Institute, Tokyo2 A considerable body of evidence indicates that the serotonin-containing median raphe suppresses the theta and desynchronizes local field potential (LFP) in the hippocampus (Vertes 2005). Since the lateral habenula (LHb) specifically and directly projects to the median raphe, we questioned whether LHb needs activity in the serotonergic raphe to modulate theta oscillation in the hippocampus. We examined the hippocampal theta oscillation of the LHb-lesioned rats in which the serotonergic neurons in the median raphe were destroyed by a neurotoxin, 5,7-dihydroxytryptamine (5,7-DHT). We observed a significant reduction in the number of serotonergic neurons in the median raphe one week after the 5,7-DHT injection by serotonin immunohistochemistry. In addition, with reduced serotonergic activity following the injection of 5,7-DHT, an LHb lesion could not affect the total duration of theta oscillation or the duration of a single theta bout in the LFP of hippocampus. This implies that the effect of LHb lesions on theta maintenance depends on the intact serotonergic median raphe. We further examined effect of pharmacological inactivation of serotonergic median raphe on the hippocampal theta. Since injection of 5-HT1A agonist, 8-hydroxy-2(di-n-propylamino) tetralin hydrobromide (8-OH-DPAT), into the median raphe selectively inhibits activity of serotonergic neurons and that produces continuous hippocampal theta in urethane anesthetized rats (Vertes et al. 1994), we tested whether the comparable effect could be observed in LHb-lesioned rats. As expected, LHb lesion did not affect the pharmacological effect of 8-OH-DPAT injection on the hippocampal theta. Injection of 8-OH-DPAT into the median raphe generated continuous hippocampal theta in the LHb-lesioned rats for a duration of several tens of minutes. Taken together, these results suggest that LHb plays a critical role in maintaining the hippocampal theta oscillation via the serotonergic median raphe.
P1-2-163 サル大脳皮質における睡眠紡錘波の時間空間的分布 Spatiotemporal organization of sleep spindles in the primate cerebral cortex ○竹内佐織1, 村井理絵1, 島津秀紀2, 礒村宜和3, 逵本徹1 ○Saori Takeuchi1, Rie Murai1, Hideki Shimazu2, Yoshikazu Isomura3, Toru Tsujimoto1 生理研1, マサチューセッツ工科大学2, 玉川大学3 NIPS, Okazaki1, MIT, Cambridge, USA2, Tamagawa Univ. Machida3 Sleep spindles are reported to appear sequentially along the posterior-anterior axis of the human cerebral cortex. However, the significance of spindles still remains unclear because of the difficulties of invasive study. In order to investigate the detailed spatiotemporal distribution and oscillatory characteristics of spindles, we recorded cortical field potentials using electrodes implanted in various cortical areas of the monkey during sleep.The experiment was carried out on three Japanese monkeys using a telemetry system.We identified significant current sources of spindles in cortical areas 46, 9, 8, 6, 5, and 7, as well as the primary motor, somatosensory, and anterior cingulate cortices during stages NREM2-4. The number of identified spindles was relatively small in the medial frontal cortex as compared to the dorsolateral frontal and parietal cortices.We also noted a significant difference in the frequencies of spindle oscillations between cortical areas. The frequency in the dorsolateral prefrontal cortex was higher than that in the premotor, medial frontal, and parietal cortices; the former was 17-19 Hz in the spectral peak, and the latter 15-16Hz. We observed that spindles of different frequencies often occurred concurrently in distinct areas. We found a significant correlation in the timing of spindle occurrences across different cortical areas. Spindles in the dorsolateral prefrontal cortex tended to occur around -0.1 s to 0.3 s using parietal spindles as a reference, preceding those in the medialfrontal and premotor areas by a time lag of about 0.1-2 s.These results indicate that spindles that have different frequencies and occur in distinct cortical areas are not independent but organized in time; this suggests the existence of underlying neural mechanisms to synchronize the timing of spindles across distinct cortical areas. Such mechanisms may serve as a functional basis to link and integrate information distributed in separate cortical areas.	P1-2-164 視索前野のGABA作動性ニューロンによるオレキシンニューロンの制御 GABAergic transmission from preoptic area to orexin neurons ○齊藤夕貴1, 辻野なつ子1, 三枝理博1, 明石馨2, 阿部学2, 崎村建司2, 櫻井武1 ○Yuki Saito1, Natsuko Tsujino1, Michihiro Mieda1, Kaori Akashi2, Manabu Abe2, Kenji Sakimura2, Takeshi Sakurai1 金沢大院・医・分子神経科学・統合生理学1, 新潟大・脳研究所・細胞神経生物学2 Dept Molecular Neuroscience and Integrative Physiol, Univ of Kanazawa, Ishikawa, Japan1, Dept Cellular Neurobiol, Brain Research Institute, Univ of NIigata, NIigata, Japan2 Wakefulness is associated with increase in neuronal discharge rates of several chemically defined neuronal populations reside in the hypothalamus and brain stem. Conversely, populations of neurons in the hypothalamic preoptic area fire rapidly during sleep, exhibiting sleep/waking state-dependent firing patterns that are the reciprocal of that observed in the arousal systems. The majority of these preoptic sleep regulatory neurons contains inhibitory neurotransmitter GABA, and locates in the median preoptic nucleus (MnPN) and ventrolateral preoptic area (VLPO). Conversely, a population of neurons in the lateral hypothalamic area (LHA) contains neuropetide orexin, which send strong excitatory influence to arousal-related neurons. It is important to know anatomical and functional interactions between the POA sleep-active neurons and orexin neurons. In this study, we examined direct connectivity between these neuronal populations using channelrhodopsin 2 (ChR2) as an anterograde tracer and optogenetic tool. We expressed ChR2 selectively in GABAergic neurons in the POA by AAV-mediated gene transfer, and examined the projection sites of ChR2 expressing axons, and effects of optogenetic stimulation of ChR2 in vitro and in vivo. We found that these neurons send direct projections to the LHA, where these fibers make apposition to orexin neurons. Optogenetic stimulations of these fibers resulted in strong inhibition of orexin neurons both in vitro and in vivo.
P1-2-165 明暗サイクルによって調節されるラット海馬スライスにおける神経リズムとシナプス可塑性 Neuronal rhythm and synaptic plasticity modulated in light-dark cycle in rat hippocampal slices ○中司弘樹1, 夏目季代久1 ○Hiroki Nakatsuka1, Kiyohisa Natsume1 九州工業大学大学院　生命体工学研究科　脳情報専攻1 Dept Brain Sci and Tech, Grad School of LSSE, KIT, Kyusyu1 Brain has a circadian rhythm and the rhythm affects the neuronal activities. Cholinergic receptor agonist carbachol induces β oscillation intermittently in rat hippocampal slices. In the present paper, whether the carbachol-induced β oscillation and the synaptic potentiation are modulated in the light and dark cycle was studied using rat hippocampal slices. First, carbachol induced β oscillations were compared in dark- and light-phase slices. The inter-burst interval (IBI) in dark-phase slices was significantly shorter than that in light-phase slices. A GABAA receptor antagonist SR95531 significantly shortened IBI of the oscillation in the light-phase slices, while it did not change it in the dark-phase slices. Melatonin also shortened IBI in the light-phase slices. Because carbachol-induced β oscillation generates in CA3 neuronal network, these results suggest that disinhibition will occur in hippocampal CA3 network in the light phase by melatonin secreted from pineal gland, and can modulate the neuronal rhythm induced in hippocampus. Next whether long-term potentiation (LTP) induced by theta burst stimulation was altered in the light and dark cycle was studied. Population spike (PS) amplitudes were not different in dark- and light-phase slices. The magnitude of LTP of PS amplitude in the dark-phase hippocampal slice was significantly larger than that in the light-phase slice. The magnitude of LTP of population excitatory postsynaptic potential (pEPSP) slopes was not different in dark- and light-phase slices. In hippocampal network, there are two types of inhibitory neurons, one is feed-forward type, and the other is feedback type. The obtained results suggest that the disinhibition of the feedback network will induce the facilitation of LTP in dark-phase slices. Therefore, induced neuronal rhythms and synaptic plasticity were modulated in light-dark cycle in hippocampus.	P1-2-166 セロトニン5HT1A受容体によるオレキシンニューロンの抑制性制御は正常な睡眠覚醒維持において重要である Reguation of orexin neurons via 5HT1A receptors plays an important role in sleep/wake behavior ○餌取慶史1, 齊藤夕貴1, 辻野なつ子1, 山崎真弥2, 阿部学2, 崎村建司2, 櫻井武1 ○Keishi Etori1, Yuki Saito1, Natsuko Tsujino1, Maya Yamazaki2, Manabu Abe2, Kenji Sakimura2, Takeshi Sakurai1 金沢大学 医学系研究科 分子神経科学・統合生理学1, 新潟大・脳研究所・細胞神経生物学2 Dept Molecular Neuroscience and Integrative Physiol, Univ of Kanazawa, Ishikawa, Japan1, Dept Cellular Neurobiol, Brain Research Institute, Univ of NIigata, NIigata, Japan2 Orexin A and orexin B are lateral hypothalamic neuropeptides implicated in a variety of functions. A series of studies have suggested that orexin-deficiency causes narcolepsy in humans and other mammalian species, highlighting roles of this hypothalamic neuropeptide in the regulation of sleep and wakefulness. Orexins were shown to have a strong excitatory influence on serotonergic neurons in the raphe nuclei through both orexin 1 and orexin 2 receptors. Conversely, orexin neurons receive abundant input from the serotonergic neurons in the raphe nuclei. We also found serotonin potently inhibited orexin neurons through 5HT1A receptors, implying the negative feedback regulation. This linkage might play an important role in the regulation of sleep/wakefulness, because both orexin and serotonin have been implicated in the sleep/wake regulation. To evaluate this hypothesis, we generated mice in which orexin neurons specifically lack expression of 5HT1A receptors utilizing Cre-loxP mediated deletion of 5HT1A gene. We made mice with 5HT1A floxed alles and crossed them with orexin-Cre transgenic mice, in which orexin neurons highly specifically express Cre recombinase. Histological studies showed specific disruption of 5HT1A receptor expression in orexin neurons. We examined sleep/wake characteristics of these mice, and found that these mice exhibited several abnormality in sleep/wake architecture, including severe fragmentation of sleep states. This observation suggests that serotonergic inhibitory regulation of orexin neurons play an important role in normal maintenance of sleep/wake behavior.
P1-2-167 ヒト睡眠覚醒リズムの動物モデルーメタンフェタミン投与ラットにおける脳内振動機構とサーカディアンリズム An animal model of human sleep-wake cycle: methamphetamine-induced oscillation and circadian behavioral rhythm in rats ○夏堀晃世1,2, 本間研一1, 本間さと1 ○Akiyo Natsubori1,2, Ken-ichi Honma1, Sato Honma1 北海道大院・医・時間医1, 北海道大院・医・神経薬理2 Dept Chronomed. Univ of Hokkaido, Sapporo1, Dept Neuropharmaco. Univ of Hokkaido, Sapporo2 Chronic Methamphetamine (MAP) treatment to rats and mice induces behavioral rhythm independent of the suprachiasmatic nucleus (SCN), master oscillation. The MAP-induced behavioral rhythm is regarded as an animal model of human's sleep-wake rhythm because of the several common features such as an internal desynchronization. An extra-SCN brain oscillation, called as MAP-induced oscillation (MAO), is strongly suggested to regulate the MAP-induced behavioral rhythm. But neither the site nor mechanism of MAO has been clarified. In this study, we used SCN-lesioned as well as -intact transgenic rats carrying a Period2-dLuciferase (Per2-dLuc) reporter. Rats were administered MAP-contained water (0.005%) for 4 h from 10:00 (restricted-MAP: R-MAP) under the LD cycle (lights on 06:00 - 18:00) for 14 days. In control rats, water supply was restricted to 4 h (R-water). After 14 days of the schedule, one group was administered MAP-contained water continuously, while the other was sacrificed for bioluminescence monitoring in brain cultured tissues. The behavioral rhythms appeared at the treatment time under R-MAP and started to free-run from this phase after the termination of R-MAP, suggesting the entrainment of MAO to the treatment time. Circadian Per2-dLuc rhythms were measured in sliced cultured tissues containing one of the olfactory bulb, caudate-putamen, parietal cortex, substantia nigra and SCN. The Per2-dLuc rhythms in all the brain areas examined was not different between R-MAP and R-water groups in SCN-intact rats. Whereas in SCN-lesioned rats, Per2-dLuc rhythm in the olfactory bulb and caudate-putamen markedly phase-shifted in both R-MAP and R-water groups, but not in the substantia nigra. These results suggest that the Per2-dLuc rhythm is differentially affected by the MAO among the brain areas. Furthermore, the Per2 expressing rhythms in the extra-SCN brain areas examined seem to be more strongly regulated by the SCN than by the MAO under R-MAP.	P1-2-168 メタンフェタミン慢性飲水中投与によるラット脳内振動機構の部位特異的変化 Differential responses of circadian Per2 expression rhythms in discrete cultured brain areas from rats under chronic methamphetamine treatment ○本間さと1, 夏堀晃世1,2, 本間研一1 ○Sato Honma1, Akiyo Natsubori1,2, Ken-ichi Honma1 北海道大院・医・時間医1, 北海道大院・医・神経薬理2 Dept Chronomed, Univ of Hokkaido, Spaaoro1, Dept Neuropharmaco, Univ of Hokkaido, Spaaoro2 Chronic methamphetamine (MAP) treatment to rats desynchronizes the behavioral rhythm from light-dark (LD) cycles in spite that the master pacemaker in the suprachiasmatic nucleus (SCN) remains to be entrained by LD cycle. The results suggest that behavioral rhythms are regulated by an extra-SCN brain oscillator which desynchronizes from the SCN pacemaker under MAP treatment. Although the involvement of the dopaminergic systems is suggested, the site and mechanism of this oscillator remain to be clarified. The previous study using in situ hybridization showed phase-shifts of clock gene expression rhythms in several brain areas in parallel with their behavioral rhythms under chronic MAP treatment. However, it is still unclear whether the phase-shifts of gene expression rhythms were oscillatory phenomena or a secondary response to behavior. In the present study, we measured clock gene expression rhythms in cultured brain slices of Period2-dLuciferase (Per2-dLuc) transgenic rats. They were treated with MAP via drinking water for about 1 month and their spontaneous locomotor activity was recorded. When the behavioral rhythms were desynchronized from LD cycle and phase-reversed, rats were decapitated and brain slices were cultured to measure Per2-dLuc rhythms in the olfactory bulb (OB), caudate-putamen (CPU), parietal cortex (PC), substantia nigra (SN) or SCN for 5 days. In MAP-treated rats, circadian phases of Per2-dLuc rhythms in the SCN and CPU were not different from those in the controls. By contrast, the rhythms in the PC and SN significantly phase-advanced by 8.0 h and 6.3 h, respectively, compared with controls, while that in the OB phase-delayed by 7.8 h. These results indicate that some of dopaminergic brain areas exhibit circadian oscillation which is desynchronized from the SCN circadian rhythms by MAP treatment. Furthermore, the responses of circadian Per2-dLuc rhythms to MAP treatment are different among the brain areas examined.
P1-2-169 オレキシンニューロン欠損による麻酔覚醒の遷延には低体温の影響が大きい Impact of hypothermia on emergence from anesthesia in orexin neuron-ablated mice ○黒木千晴1,2, 高橋佳子1,2, 上村裕一1, 桑木共之2 ○Chiharu Kuroki1,2, Yoshiko Takahashi1,2, Yuichi Kanmura1, Tomoyuki Kuwaki2 鹿児島大院・医歯学・侵襲制御1, 鹿児島大院・医歯学・統合分子生理2 Dept Anesthesiology, Kagoshima Univ, Kagoshima1, Dept Physiology, Kagoshima Univ, Kagoshima2 BACKGROUND: Orexin neurons have been shown to drive emergence from general anesthesia. However, orexin neurons regulate not only the sleep/wake cycle, but also body temperature. We hypothesized that orexin neurons do not directly regulate emergence from anesthesia, but instead affect emergence indirectly through thermoregulation because anesthesia-induced hypothermia can greatly influence emergence time. METHODS: To test our hypothesis, we used simultaneous measurement of body temperature and locomotor activity in orexin neuron-ablated mice (ORX-AB) and their wild-type (WT) littermates. Induction of anesthesia and emergence from anesthesia were defined behaviorally as loss and return, respectively, of body movement. Mice received general anesthesia with 1.5% isoflurane in 100% oxygen for 30 min under three temperature conditions.RESULTS: In the warmed (32°C) condition, ensuring a constant body temperature of animals during anesthesia, there were no significant differences between the ORX-AB and control mice with respect to body temperature, locomotor activity, induction time, or emergence time. In the room temperature (25°C) condition, allowing body temperature to fluctuate, anesthesia-induced hypothermia was greater and longer lasting in ORX-AB than that in WT mice. Emergence time in ORX-AB was significantly prolonged from the warmed condition (14.2±0.8 vs. 6.0±1.1 min) while that in WT mice was not different (7.4±0.8 vs. 4.9±0.2 min). Finally, body temperature in WT mice was decreased by cooling (23°C) to the comparable value to that obtained in the ORX-AB during room temperature condition. Emergence time in WT mice was prolonged to 12.4±1.3 min. Induction time did not differ among temperature conditions or genotypes.CONCLUSIONS: The effect of orexin-deficiency to impair thermoregulation during general anesthesia is of sufficient magnitude that body temperature must be appropriately controlled when studying the role of orexin neurons in emergence from anesthesia.	P1-2-170 オレキシン神経は、2つの異なる経路を介して覚醒状態を強化しカタプレキシーを抑制する Orexin neurons consolidate wakefulness and inhibit cataplexy in narcoleptic mice through two distinct pathways ○長谷川恵美1, 柳沢正史2, 櫻井武1, 三枝理博1 ○Emi Hasegawa1, Masashi Yanagisawa2, Takeshi Sakurai1, Michihiro Mieda1 金沢大学大学院　医学系研究科　分子神経科学・統合生理学1, テキサス大・サウスウエスタンメディカルセンター2 Fac. Med. Kanazawa Univ., Kanazawa, Japan1, UTSW, Dallas, USA2 Loss of orexin neurons in humans is associated with narcolepsy, a sleep disorder characterized by excessive daytime sleepiness and cataplexy. Mice lacking orexin peptides, as well as those lacking orexin receptors (OX1R-/-;OX2R-/- mice), display a phenotype similar to narcolepsy, highlighting a critical role of orexin signaling in the maintenance of wakefulness. However, the precise neural mechanisms downstream of orexin neurons remain uncertain. We found that targeted restoration of orexin receptor expression in noradrenergic neurons of the locus coeruleus and in serotonergic neurons of the dorsal raphe in OX1R-/-;OX2R-/- mice differentially inhibited pathological fragmentation of wakefulness (i.e., sleepiness) and direct transitions from wakefulness to REM sleep (cataplexy-like episodes), respectively. Furthermore, pharmacogenetic activation of these neurons using DREADD technology significantly ameliorated narcolepsy of mice lacking orexin neurons. These results suggest that orexin neurons consolidate wakefulness and suppress cataplexy by activating locus coeruleus noradrenergic and dorsal raphe serotonergic neurons, respectively.
P1-2-171 時差症候群モデルマウスにおける分子機構の解析 Molecular, cellular, and physiological response in a mouse model of jet-lag ○鈴木暢1, 山口賀章1, 岡村均1 ○Toru Suzuki1, Yoshiaki Yamaguchi1, Hitoshi Okamura1 京都大院・薬・システムバイオロジー1 Department of Systems Biology, Graduate School of Pharmaceutical Sciences, Kyoto University, Kyoto1 Modern society has now become the 24-hour society that allows people to do everything at any hour of the day or night in economic and productive activities as well as in individual behaviours. Consequently, the number of shift workers including extended nighttime workers has been increasing dramatically. Their daily rhythm of life is quite different from natural light and dark environment. As a result, their body clock system is severely disrupted, leading to perturbed balance in the control of physiological rhythms, such as blood pressure, body temperature, endocrine systems, and energy homeostasis. Actually, recent epidemiological studies reported that shift workers are at increased risks of developing many chronic diseases such as nighttime insomnia, gastrointestinal disturbances, breast cancer, type 2 diabetes, obesity, and heart diseases. Indeed, experimental animal model of chronic jet-lag can induce variety of pathological symptoms including cardiomyopathy, accelerated tumor growth, and hastened death upon aging. However, the molecular mechanisms responsible for these symptoms are poorly understood. Human as mammals has a circadian clock system which is generated basically by the transcription-(post)translational feedback loop of clock genes. The transcriptional activators CLOCK and BMAL1 drive transcription of Period and Cryptochrome genes. The PERs are made in the cytoplasm, translocate into the nucleus, and form a negative complex with CRYs, and repress their own expression by inhibiting the CLOCK/BMAL1 complexes. Virtually all cells throughout the body have this circadian clock oscillating system, and they are dominated by the master clock in the suprachiasmatic nucleus of the hypothalamus. Here, we have characterized the molecular, cellular, and physiological response in the mouse under experimental paradigm for jet-lag whereby mice entrained to a 12-hour light/12-hour dark cycle undergo light phase advancement by 8 hours.	P1-2-172 CRYPTOCHROMEは生後発達における視交叉上核の概日リズムの細胞間カップリングに重要である CRYPTOCHROMES are critical for the development of coherent circadian rhythms in the suprachiasmatic nucleus ○小野大輔1, 本間さと2, 本間研一2 ○Daisuke Ono1, Sato Honma2, Ken-ichi Honma2 北大院・医・光バイオイメージング1, 北大院・医・時間医学2 Photonic Bioimaging Sec, Hokkaido Univ, Grad Sch of Med, Sapporo, Japan1, Dep of Physiol Chronomed, Hokkaido Univ, Grad Sch of Med, Sapporo, Japan2 In mammals, the central circadian clock is located the suprachiasmatic nucleus (SCN) of the hypothalamus. Cell autonomous circadian rhythms are generated by a transcription and translational negative feedback loop involving clock genes and their protein products. Cry1 and Cry2 have been regarded as crucial components of the molecular feedback loop. However, we recently observed robust circadian PER2::LUC rhythms in the cultured SCN from neonatal mice, suggesting that SCN cells can exhibit synchronized circadian rhythms. The discrepancy between arrhythmic behavior and rhythmic clock gene expression in the SCN may be due to the change in the synchronization mechanism depending on age. In order to access role of CRYs in the development of SCN circadian system, we measured PER2::LUC bioluminescence in the SCN of Cry1 and Cry2 double deficient (Cry1-/-/Cry2-/-) mice by using a Photomultiplier tube and CCD camera, respectively. Slice culture of the SCN was made at postnatal day 1, 7, 14, 21, and adult (>8week). At the tissue level, circadian rhythms were observed in neonatal but not in adult SCN. Whereas at the cellular level, rhythms were detected in both SCNs. Cellular circadian rhythms were well synchronized in neonates, but not in adults, indicating a loss of rhythm synchrony in the course of development. Next, we examined roles of neuronal and humoral communications in the synchronization of cellular rhythms. The SCN from Cry1-/-/Cry2-/- mice were treated with tetrodotoxin to block the synaptic communication, while co-cultured with wild type SCN from different ages to examine the contribution of a diffusible factor(s) to the coupling of cellular rhythms. We will discuss the mechanisms of this CRY independent coupling in the SCN.
P1-2-173 時期特異的オレキシン神経運命制御によるナルコレプシーの症状発現メカニズム解析 Timing controlled specific ablation of orexin neurons for new narcolepsy model mice ○田淵紗和子1,2,3, 常松友美2,3, ブラックサラ5, キルドフトーマス5, 富永真琴1,2, 山中章弘4,6 ○Sawako Tabuchi1,2,3, Tomomi Tsunematsu2,3, Sarah W Black5, Thomas S Kilduff5, Makoto Tominaga1,2, Akihiro Yamanaka4,6 総研大・生命科学1, 生理学研究所・細胞生理2, 日本学術振興会3, 神経2・環境医・名大4, SRI International5, さきがけ6 Life Science, SOKENDAI, Okazaki1, Cell Signaling, NIPS, Okazaki2, JSPS, Tokyo3, RIEM, Nagoya univ.,Nagoya4, SRI International, Melon Park, USA5, PRESTO research, JST, Saitama6 Orexin is a neuropeptide which is produced in small number of neurons located in the hypothalamus, orexin neurons. Previous studies revealed that orexin neurons are important for regulation of sleep and wakefulness. It is reported that specific loss of orexin neurons causes sleep disorder "narcolepsy". Narcolepsy typically has onset in adolescence or early adulthood. However, it takes about a decade from onset to correct diagnosis. This delay makes it difficult to follow the progress of the symptoms which appear in the early stage of narcolepsy. There is no perfect mice model for narcolepsy to address this matter so far. Here we used tet-off gene expression system to control gene expression specifically in orexin neurons in the presence or absence of doxycycline (DOX). Orexin-tTA mice, which express tTA in orexin neurons, were bred with TetO diphtheria toxin A fragment (DTA) mice to generate orexin-tTA; TetO DTA mice. In these mice, in the absence of DOX, DTA is expressed in the orexin neurons and induced cell death. Thus, orexin neurons were specifically ablated at any timing by replacing chow containing doxycycline (DOX (+), 100 mg/kg) to that without DOX (DOX(-)). An immunohistochemical study revealed that 95% of orexin-immunoreactive neurons were ablated at 2 weeks after DOX(-). Sleep/wakefulness pattern was analyzed by continuous recording of EEG and EMG during ablation of orexin neurons. We revealed the relationship between number of orexin neurons and progress of symptoms. Progress of symptom was highly related with the number of orexin neurons. 85% and 95% loss of orexin neurons induced fragmentation of wakefulness and cataplexy-like behavioral arrest, respectively. These results suggest that orexin-tTA mice are useful tool for studying its physiological role in vivo.	P1-2-174 NIH3T3細胞においてサーカディアン転写リズムを制御する細胞内情報伝達経路の同定 Intracellular signal transduction pathways regulating circadian rhythms of CLOCK/BMAL1-mediated transcription in NIH3T3 cell ○森下良一1, 三浦大樹1, 喜田聡1,2 ○Yoshikazu Morishita1, Daiki Miura1, Satoshi Kida1,2 東京農業大学大学院　農学研究科　バイオサイエンス専攻1, 科学技術振興機構2 Dept. of Bioscience, Tokyo univ. of Agriculture, Tokyo1, CREST, JST2 Previous studies have shown that circadian transcription rhythms mediated by CLOCK/BMAL1 play essential roles in behavioral circadian rhythms. Importantly, circadian transcription rhythms generated by CLOCK/BMAL1 are observed not only in the suprachiasmatic nuclei (SCN), but also in the peripheral tissues. Although many types of genes displaying critical roles in circadian rhythms have been identified, functions of intracellular signal transduction pathways on the regulation of circadian transcription rhythms are still unclear. Therefore, we have tried to identify intracellular signal transduction pathways that play regulatory roles in CLOCK/BMAL1-mediated transcription in NIH3T3 cells. To do this, we examined effects of various selective inhibitors of intracellular signal transduction pathways on circadian transcription rhythms of Dbp (albumin D-element binding protein) gene, a target of CLOCK/BMAL1, following the application of high concentrations of serum (50% horse serum) for 2 hrs (serum shock), that induces circadian transcription rhythms in NIH3T3 cells. Consistent with previous observations, we observed a robust circadian rhythm of Dbp mRNA levels following the serum shock. Therefore, we next examined effects of selective inhibitors such as U0126 (MEK), SP600125 (JNK), SB203580 (p38), H89 (PKA), and rapamycin (mTORC1) on the expression level of Dbp mRNA. We found that PKA-specific inhibitor H89 and JNK-specific inhibitor SP600125 significantly decreased the level of Dbp mRNA. Consistently, PKA-specific inhibitor H89 also inhibited the transcription activation from Dbp reporter gene. These results suggest that PKA signaling pathway regulates Dbp expression in NIH3T3 cells. We are now trying to examine effects of other selective inhibitors on expression levels of other target genes of CLOCK/BMAL1 such as Period and Cryptochrome.

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