睡眠、生体リズム Sleep and Biological Rhythms
P2-1-158 オレキシンによる呼吸神経回路の調節 Modulation of orexin on respiratory neuron in parabrachial nucleus ○荒田晶子1, 下村英毅2, 谷澤隆邦2, 山中章弘3 ○Akiko Arata1, Hideki Shimomura2, Takakuni Tanizawa2, Akihiro Yamanaka3 兵庫医科大学　生理学・生体機能部門1, 兵庫医科大学　小児科学2, 名古屋大学　環境医学研究所3 Dept Physiol, Hyogo College of Med1, Dept of Pediatrics, Hyogo College of Medicine2, Dept of Neuroscience, Research Institute of Environmental Medicine, Nagoya University3 Orexin is neuropeptide involved in the regulation of wakefulness and sleep cycle. Especially, orexin plays to maintain wakefulness through the orexin receptor 2 (OX2R). Dysfunction of OX2R results in the fragmentation of wakefulness and the cataplexy-like behavioral arrest which is similar to narcolepsy. Moreover, pontine structures are important for switch of wakefulness and sleep. However, the effects of orexin to the relationship between respiration and sleep/wakefulness in fetal and neonatal stage in pons had not been investigated. The parabrachial nucleus (PBN) which is relating to an autonomic function participates in coordinating the motion output from the sentience input. According to our previous research from the brainstem-spinal cord preparation, the active inspiratory-expiratory phase switching mechanism existed in PBN. In fetal and neonatal stage, we examined the effects of orexin for the respiratory-related neuron in PBN; and we examined participation of the orexin in the medulla-spinal cord preparation with the pons and without the pons to investigate what kinds of role PBN played about sleep awakening mechanism. As a result, a preparation with a pons significantly increased respiratory rhythm than without the pons, and it became clear that the firing rate of the inspiratory-expiratory phase switching neuron (I-E neuron), which fires from late inspiratory to early expiratory phase, increased by orexin. Orexin increased to the firing rate of the I-E neurons located in PBN, and this thereby promoted the switching from inspiratory phase to expiratory phase in respiratory-related neuron network in the medulla. These results suggested that orexin excited the respiratory rhythm and might maintain wakefulness mode.	P2-1-159 光で慨日リズムを調節する The circadian clock can be phase-reset by photo-switched channels ○沼野利佳1, 松尾美奈子 ○Rika Numano1, Minako Matsuo 豊橋技術科学大学・エレクトロニクス先端融合研究所1 EIIRIS, TOYOHASHI Univ of Tech, Japan1 Intact biostructure ionotropic glutamate receptors (iGluR6) is normally expressed in synaptic neural processes in mammalian brain. To control any neural activity remotely and reversibly, photoswitchable nanomachine LiGluR was developed based on iGluR6 and operated using photoiosomerizable new chemicals, maleimde-azobenzene-glutamate (MAG). This iGluR6 mutants could be photo-switched using MAG compounds, which dangled 2R,4R-allyi glutamate (G) from a linker containing the photoisomerizable azobenzene (A) that was attached to the introduced cysteines via maleimide (M). LiGluR(with L439C mutation): opening in UV light and closing in visible light by all MAGs. In neural cells with LigluR, action potentials were optimally evoked and extinguished by UV and visible light, respectively. Mammalian clock gene Period1 (Per1) is an important member of the central clock system to maintain the autonomous oscillator and synchronize environmental light cycle after transient Per1 induction in central clock: in the suprachiasmatic nucleus (SCN). The circadian clock in both fibroblast and neural cell can be reset through LigluR and MAG by UV light and be compared each other.
P2-1-160 VSFPを用いたマウス全半球膜電位計測系における麻酔下伝搬波の解析 Analysis of membrane potential waves during recovery from anesthesia in mice using VSFP-based imaging ○嶋岡大輔1,2,3, , 武藤弘樹2 ○Daisuke Shimaoka1,2,3, Walther Akemann2, Hiroki Mutoh2, Thomas Knopfel2 東大・総文・広域科学1, 理研BSI2, 眼科学研　UCL3 Dept Basic Sci, Univ of Tokyo, Tokyo1, RIKEN BSI, Saitama2, IoO, UCL, UK3 During sleep, cortical neurons involve characteristic slow oscillation that propagates across cortical areas. Currently, detailed analysis of the spatio-temporal organization of the slow oscillation has been limited by the low spatial resolution of standard EEG technique. Here, we used voltage-sensitive fluorescent protein (VSFP) -based imaging to map the spatial dynamics of slow wave oscillation across mouse cortex in different sedative brain states from deep anesthesia to the awake state. VSFP-Butterfly 1.2 was expressed in layer 2/3 pyramidal cells over large portions of the dorsal aspect of one hemisphere using in utero electroporation. At the beginning of each experimental session, deep anesthesia was induced with pentobarbital. Cortical population activity was subsequently imaged over one hemisphere until spontaneous motor activity commenced (Recording sessions lasting of up to two hours). Using this experimental protocol, we observed slow oscillatory activity with a characteristic frequency which gradually rose from 2 to 10Hz, consistent with the simultaneously recorded EEG. Moreover, we observed a shift in propagation direction depending on the animal's anesthetized state. Next, we asked whether the shift in direction is explained by a monotonous change of individual oscillatory events in propagation direction, or by increase or decrease in incidence among different propagating patterns. To address this question, we calculated the propagation direction for each wave event. Analysis of individual waves revealed several distinct patterns of propagation with sporadic transitions from one pattern to another. Prevalence of each pattern changed depending on anesthesia level. The approach described here may provide novel insight into the spatial dynamics of the slow oscillation during anesthesia and probably also during sleep.	P2-1-161 位相反応曲線による概日リズム特異点の探索 Singular point of the circadian rhythm investigated with a phase response curve ○重吉康史1, 鯉沼聡1, 郡宏2 ○Yasufumi Shgeyoshi1, Satoshi Koinuma1, Hiroshi Kori2 近畿大学医学部1, お茶の水女子大学2 Dept Neurobiol, Anatomy, Univ of Kinki, Osaka1, Division of Advanced Sciences, Ochadai Academic Production, Ochanomizu University2 Phased response curve (PRC) shows the amount of phase shift caused by stimulation. As for the circadian rhythm, photic stimulation always draws PRCs showing smooth curves without break point called type 1 PRC. However, PRC shows type0 with a break point when we stimulate cell lines generating a circadian rhythm. Numerical simulation resuming limit cycles demonstrated that PRC progresses from type0 to type1 by reducing the amount of phase-shifting stimulation. Here we tried to see whether the cell lines shows transition from Type0 to Type1 and shows a singular point predicted by numerical simulations. We stimulated circadian rhythm-generating C6 cell line derived from a glioma. After the administration of dexamethasone that made the cell lines show a circadian rhythm, we added 10micorM Forskolin into the medium and obtained type0 PRC with a break point. Then we gradually reduced FK concentration and obtained a PRC of type 1.In addition, we predicted a singular point by detecting the largest variation of shift by a certain concentration of FK application.
P2-1-162 強制脱同調プロトコルは表情刺激呈示時の扁桃体活動を変容させた Forced Desynchronization Protocol altered Amygdala Activation to Emotional Face Stimulus ○元村祐貴1, 北村真悟1, 田村美由紀1, 片寄泰子1, 野崎健太郎1, 榎本みのり1, 有竹清夏1, 渡辺真紀子1, 肥田昌子1, 守口善也1, 樋口重和1, 三島和夫1 ○Yuki Motomura1, Shingo Kitamura1, Miyuki Tamura1, Yasuko Katayose1, Kentaro Nozaki1, Minori Enomoto1, Sayaka Aritake-Okada1, Makiko Watanabe1, Akiko Hida1, Yoshiya Moriguchi1, Shigekazu Higuchi1, Kazuo Mishima11 国立精神・神経医療研究センター精神保健研究所1 National Institute of Mental Health, National Center of Neurology and Psychiatry, Tokyo1 Due to increase in the shift work population in today's 24 hour society, a lot of people are suffering from internal desynchonization (social jet-lag), i.e., the misalignment between sleep schedule and biological rhythms in various physiological functions (e.g. core body temperature). Considering the high prevalence of depressive episodes in shift workers, it is possible that internal desynchronization causes the emotional dysregulation resulting in increased vulnerability to mood disorders. Twelve healthy male participants underwent emotional face viewing task in MRI before and after the 13-day forced desynchrony protocol (FD). Before the FD, first constant routine protocol (CR1; 38.67-hour sustained wakefulness) was applied to evaluate the circadian phase of biological rhythm in these subjects (melatonin, body temperature). After that, participants got through 28-hour sleep-wake schedule (18.67h wake and 9.33h sleep) for 7 days. On the 11th day, the second CR (CR2) was conducted. We compared pre- and post-FD brain activity while viewing fearful, happy, and neutral face stimuli. As to fMRI results, we found significant Time(pre- or post-FD) x Emotional category (Fear, Happy, or Neutral) interaction in amygdala activation. The response to fear faces increased while response to happy faces decreased. Furthermore, the pre-post change of the amygdala activation to fearful faces correlated with the individual shift of circadian phase indicated by body temperature and melatonin between CR1 and CR2. These findings suggest that internal desynchronization alters amygdala response to emotional face stimuli. The shift of sleep wake schedule from intrinsic biological rhythm may attenuate the sensitivity to happy, and enhance the sensitivity to fear. This reads the possible mechanism of depression which occurs frequently in shift workers.	P2-1-163 ショウジョウバエの雄どうしの個体間相互作用による睡眠量の増加 Male-male interaction-induced sleep in a fruit fly, Drosophila melanogaster ○冨田淳1, 上野太郎1, 坂本枝里菜1, 粂昭苑1, 粂和彦1 ○Jun Tomita1, Taro Ueno1, Erina Sakamoto1, Shoen Kume1, Kazuhiko Kume1 熊本大・発生研・多能性幹細胞1 Dept Stem Cell Biol, IMEG, Kumamoto Univ, Kumamoto1 Intraspecific social interaction modulates behavior of exposed animals. The fruit fly Drosophila melanogaster normally occurs as a group, and social-experience dependent change in behaviors such as courtship, aggression, circadian locomotion and sleep has been studied. When the individual locomotor activities of flies exposed to either social enrichment or isolation are measured, socially enriched individuals sleep significantly more during daytime than socially deprived siblings. In this study, we assessed sleep in the flies during the simple social interaction. Single or two male flies were placed in glass tubes (length, 65 mm; inside diameter, 2 mm) containing food at one end and entrained to 12 h light/dark cycles. Their sleep were monitored in constant darkness. In wild-type strain, there was no significant difference in the amount of total daily sleep between single and two files. Interestingly, in short sleeper mutant, fumin (fmn) flies that carry a mutation in the dopamine transporter gene, two males showed a 2- to 3-fold increase in sleep compared with single males. The male-male interaction-induced sleep in fmn mutants was predominantly observed during subjective night and required olfactory input. We found that male-male interaction in fmn flies grown on a minimal medium containing only sucrose and agarose without odors did not increase sleep. Moreover, we identified Odorant receptor 42a (Or42a) as significantly up-regulated gene in the heads of fmn flies by microarray analysis. Or42a is a generalist odorant receptor which is sensitive to a large number of fruit odors. Male-male interaction in Or42a-,fmn double-mutants did not increase sleep. These results suggest that food-derived odors received by Or42a were required for the increase in sleep in interacting fmn males.
P2-1-164 Coupled Oscillations in Primate Hippocampus during Rapid Eye Movement Sleep Coupled Oscillations in Primate Hippocampus during Rapid Eye Movement Sleep ○美馬達哉1, 竹内佐織2, 村井理絵2, 島津秀紀3, 礒村宜和4, 逵本徹2 ○Tatsuya Mima1, Saori Takeuchi2, Rie Murai2, Hideki Shimazu3, Yoshikazu Isomura4, Toru Tsujimoto2 京都大学医学研究科附属脳機能総合研究センター1, 生理学研究所2, マサチューセッツ工科大学3, 玉川大学4 HBRC, Kyoto University Graduate School of Medicine1, NIPS, Japan2, MIT, USA3, Tamagawa Univ, Japan4 REM sleep is a normal stage of sleep, and characterized by a unique mixture of physiological, behavioral and cognitive states; saccadic eye movement, muscle relaxation, increased autonomic function, low-amplitude fast activities in electroencephalogram (EEG), memory consolidation, and dreaming. Animal studies suggested that the hippocampal rhythmic slow activity (RSA) is a key feature of REM sleep. Moreover, in a phase-dependent way, RSA modulates the gamma band neural oscillation, which might represent cell assemblies. However, this state-dependent alteration of cross-frequency coupling in hippocampus has rarely been studied in primates. To investigate this, we analyzed the coupling of local field potential (LFP) oscillations between RSA and gamma band activity in the hippocampus of 3 freely behaving monkeys during wakefulness, REM sleep and non-REM sleep conditions. We applied the Hilbert-Huang transform (HHT) to LFP oscillations for spectral analysis. The HHT is an empirically based data analysis method and can achieve a high spectral, as well as temporal, resolution for extracting features of nonlinear and nonstationary signals. The most outstanding characteristic of the state-dependent change in hippocampal LFP oscillation was the increased gamma band activity (30-80 Hz) during REM sleep. The RSA-like slow activities in the theta and delta range appeared in short bursts during all conditions, and most prominently during non-REM sleep. The amplitude of the gamma band was modulated by the RSA phase.Our findings suggest that the hippocampal RSA might not be a ubiquitous feature of REM sleep but that its pattern of occurrence can vary across species. Since the gamma band oscillation is thought to be the physiologic basis of visual awareness, its enhancement during REM sleep in primates might represent the internal processing of consciousness during sleep, such as dreaming.	P2-1-165 Withdrawn
P2-1-166 電位依存性カルシウムチャネル制御ユニットα2δ3の生体リズム調節機構に関する研究 Role of α2δ3 subunit of voltage-dependent calcium channels for the generation of circadian rhythms in the suprachiasmatic nucleus ○松尾雅博1, 瀬尾和志1土居雅夫1, 山口賀章1, 岡村均1 ○Masahiro Matsuo1, Kazuyuki Seo1, Jean-Michel Fustin1, Masao Doi1, Yoshiaki Yamaguchi1, Hitoshi Okamura1 京都大学大学院　薬学研究科1 Department of Systems Biology, Graduate School of Pharmaceutical Sciences, Kyoto University, Kyoto, Japan1 The voltage-dependent Ca2+ channels (VDCCs) are transmembrane proteins act as transducers of electrical signals into numerous intracellular activities including muscular contraction, hormone secretion, release of neurotransmitters and gene expression. On the basis of their electrophysiological properties they are classified as high- and low-voltage-activated calcium channels. Among them, high-voltage-activated calcium channels are heterooligomeric proteins consisting of a pore-forming α1 subunit and accessory β, γ, and α2δ subunits. Structural studies have shown that the extracellular α2 domain provides the crucial elements required for channel stimulation and that the δ domain harbors the regions important for the shift in voltage-dependent activation, steady-state inactivation, and the modulation of the inactivation kinetics. Although previous studies have demonstrated the important role of VDCC for generation and entrainment of circadian rhythms, the role of α2δ subunits on the circadian rhythms have not been clarified. We found the strong expression of α2δ3 in the suprachiasmatic nucleus (SCN). Gene ablation of Cacna2d3 led to the decrease of circadian rhythmicity, and in constant-light conditions, rhythm is severely disrupted. Furthermore, Cacna2d3 deficiency abolishes the spatiotemporal expression pattern of Per1 in the SCN in constant-light conditions. The α2δ3 subunits of VDCC form the binding site for an important therapeutic class of drugs, and this study will be beneficial to develop a new drug for a circadian rhythm sleep disorders.	P2-1-167 Ampakine rescues chronic intermittent hypoxia-induced impairment in hippocampal long-term synaptic plasticity and long-term memory in mouse ○Wing-Ho Yung1, Hui Xie1, Linhao Xu1, Ya Ke1 School of Biomedical Sciences, The Chinese University of Hong Kong1 Obstructive sleep apnea (OSA) is a common sleep disorder characterized by repeated episodes of airway obstruction during sleep resulting in intermittent hypoxia (IH) and intermittent hypoxemia. OSA is often accompanied by neurocognitive deficits including impairment in perception, attention and memory. It is well known that learning and memory involves a cellular process known as long-term potentiation (LTP). Our recent work has shown that IH impaired hippocampal synaptic plasticity in mouse, both on early phase LTP (E-LTP) and late phase LTP (L-LTP), which were caused by a reduction in the expression of brain-derived neurotrophic factor (BDNF), in the hippocampus. Ampakine is a class of AMPA receptor modulator reported to elevate the BDNF level after short-term administration. To explore novel ways to alleviate cognitive symptoms of OSA subjects, we designed experiments to investigate the effect of ampakines on IH-induced LTP impairment. Two groups of adult male mice were exposed to 1 to 2 weeks of IH and each received either vehicle or ampakine injection. Another group under nomoxia was used as control. The paradigm of IH consisted of cycles of oxygen levels between 10% and 21% every 90 s during the daytime for 8 hrs. We found that there was a significant restoration of E-LTP in ampakine injection group compared with the vehicle-treated group, and was not significantly different from that of the normoxia group. Ampakine treatment also restored the decreased level of hippocampal BDNF in the IH-treated group. Furthermore, IH-induced impairment in long-term reference memory was also alleviated by ampakine treatment. Together, these data suggest that, by targeting at the level of BDNF expression, ampakine administration could be a potential therapeutic treatment for the neurocognitive symptoms of OSA subjects.
P2-1-168 Withdrawn	P2-1-169 Withdrawn
P2-1-170 給餌時刻の調節によるClockミュータントマウスの睡眠相後退傾向の緩和 Time-restricted feeding improves delayed sleep-phase syndrome-like behavior in Clock mutant mice ○筋野貢1, 小西啓悦2, 重吉康史1 ○Mitsugu Sujino1, Hiroyoshi Konishi2, Yasufumi Shigeyoshi1 近畿大学医学部　解剖学講座1, 四條畷学園大学2 Department of Anatomy and Neurobiology, Kinki University School of Medicine, Osaka, Japan1, Shijonawate Gakuen University, Osaka, Japan2 Environmental light/dark cycles are the main stimulation that entrains circadian biological clock of mammals. However, feeding behavior also affects daily activity patterns. Nocturnal rodents mainly eat during the night when food is ad libitum. In contrast, in a case that food is only available during the day, various physiological and metabolic circadian rhythms are entrained by this feeding time. In this restricted feeding (RF) conditions, food anticipatory activity (FAA) precedes several hours daily food presentation. Clock mutant mice have an abnormal biological clock which exhibits a long free-running period of about 27 hours. Moreover, their activity onset tends to be delayed compared with wild-type mice under a steady light:dark condition (L:D = 12h:12h). Therefore, the activity pattern of Clock mutant mice has been regarded as one of the model mouse of delayed sleep-phase syndrome (DSPS) of human. In the present study, we performed RF on the Clock mutant mice to see if it improves their DSPS-like behavior. In the present RF condition, food is available only during the first half of the night and the mice did not show loss of body weight. Clock mutant mice showed not only FAA but also a repression of activity during the latter half of the night which the period of their main activity before RF. Further, to analyze effects of RF on other physiological events, we measured various physiological rhythms such as core body temperature and sleep-wake rhythm in the RF condition.	P2-1-171 微小区画培養法を用いて空間的に隔離した視交叉上核シングルニューロンの光イメージング解析 Optical imaging of spatially isolated solitary suprachiasmatic neuron using micloisland culture method ○平田快洋1, 本間さと2, 本間研一2 ○Yoshihiro Hirata1, Sato Honma2, Ken-ichi Honma2 北海道大学大学院　医学研究科　光バイオイメージング部門1, 北海道大学大学院　医学研究科　時間医学講座2 Photonic Bioimaging Sect., Hokkaido Univ. Grad. Sch. Med. , Sapporo Japan1, Dept. Chronomedicine, Hokkaido Univ. Grad. Sch. Med., Sapporo Japan2 In mammals, a master circadian clock is located in the hypothalamic suprachiasmatic nucleus (SCN) which is composed of multiple, single-neuron circadian oscillator cells. Recent studies suggest that the SCN neurons are heterogeneous in not only cytochemical but also oscillatory properties. In the present study, we examined circadian properties of the individual SCN neurons using dissociated culture of isolate individual SCN neurons on spatially isolated small spots, i.e., microislands. Culture dish were prepared by opening a hole in the bottom of a 35 mm Petri dish and attaching ITO (Indium Tin Oxide) coated glass slide. Low-melting-point agarose thin layer was made above the collagen layer which was formed on the ITO surface. Collagen microislands were made by melting agarose with an infrared laser beam. Dissociated SCN neurons were derived from transgenic mice carrying a bioluminescent reporter for Per1-expression (Per1-luc) or PER2::LUC knock-in mice, and cultured on the microislands. Bioluminescence from each single cell was measured by an EMCCD camera every hour for at least 5 days. We succeeded to demonstrate that spatially dissociated solitary SCN neurons exhibit circadian rhythms of Per1 expression or PER2. We also detected circadian oscillation of clock gene/protein of glial cells on microisland with/without SCN neurons. The circadian oscillations of these cells were not affected with carbenoxolone, a gap junction blocker. In some microislands, circadian bioluminescence rhythms of glial cells and the single SCN neuron were synchronized each other. These results suggest that each of single SCN neurons exhibits cell-autonomous circadian oscillation. Further, the rhythmic activity of glial cells may affect circadian expression of clock gene/protein in each SCN neurons, or vice versa, the mechanism of which remains to be studied.
P2-1-172 光誘導相は主観的夜の限局された時刻に存在する The photoinducible phase is in the restricted time of the subjective late night under the short day condition ○升本宏平1, 長野護1, 重吉康史1 ○Kohei Masumoto1, Mamoru Nagano1, Yasufumi Shigeyoshi1 近畿大学医学部解剖学教室1 Department of Anatomy and Neurobiology, Kinki University Faculty of Medicine, Osaka, Japan1 Living organisms detect seasonal changes in day length (photoperiod) and alter their physiological functions accordingly to fit seasonal environmental changes. Thyroid stimulating hormone beta subunit (TSHβ), induced in the pars tuberalis (PT), plays a key role in the pathway that regulates vertebrate photoperiodism that governs many physiological responses such as maturation of the reproduction system. Light stimulation at the photoinducible phase of the circadian rhythm causes photoperiodism to organisms. To identify the genes that initiate TSHβ induction, we performed genome-wide expression analysis of the PT under chronic short-day and long-day conditions in melatonin-proficient CBA/N mice, in which the photoperiodic TSHβ expression response is preserved. As a result, we identified the eyes absent 3 (Eya3) gene as a key molecule that leads to TSHβ gene induction. In the study, we also found the essential role of the light exposure during the late night, however, the precise time point that effectively induces the genes, or photoinducible phase, was unclear. Here we exposed CBA/N mice to light at various time points during the night to see if there is induction of Eya3 mRNA in the PT. Consequently we determined the precise time of the phtoinducible phase during the late night.

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