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Genome-wide profiling of the core clock protein BMAL1 targets reveals strict relationship with metabolism
Abstracts / Neuroscience Research 71S (2011) e46–e107
O2-E-2-1 Reversible gene expression control of serotonin receptor 1A in orexin neurons reveals physiological significance of serotonergic inhibitory inputs to orexin neurons Sawako Tabuchi 1,2 , Tomomi Tsunematsu 1 , Shouta Sugio 1,2 , Kenji Tanaka 1,2 , Makoto Tominaga 1,2 , Akihiro Yamanaka 1,2 1
Cell Signaling, NIPS, Okazaki, Japan Okazaki, Japan
2
Physiological Science, SOKENDAI,
The neurons producing neuropeptide orexin (orexin neurons) have an important role in the maintenance of arousal. It is reported that serotonergic neurons in the raphe nucleus are densely innervated by orexin neurons. These serotonergic neurons express both orexin receptors, OX1R and OX2R, and are activated by orexin. On the other hand, orexin neurons are innervated by serotonergic neurons in the raphe nucleus, and are inhibited by serotonin through the serotonin 1A receptor (Htr1a). Although these results suggest that serotonergic input forms a negative feedback circuit, its physiological role has not been completely understood. To reveal this, we generated transgenic mice in which orexin neurons specifically express tetracycline transactivator orexin/tTA. tTA enable to induce gene expression to bind tetracycline responsive element in the absence of doxycycline (DOX). In orexin/tTA; Tet-O Htr1a (Htr1a over expression) mice, in situ hybridization revealed that Htr1a mRNA was reversibly regulated in the orexin neurons by applying or removing DOX from chew. Electrophysiological analysis of orexin neurons revealed that inhibitory effect by serotonin application was approximately 2-hold prolonged using Htr1a over expression mice compare with control mice. EEG and EMG recording from Htr1a over expression mice revealed that these mice showed fragmentation of wakefulness in active period, the early dark period. DOX application for 5 days consolidated wakefulness in the early dark period. It was fragmented again by removing DOX for 14 days. Although stage transition frequency was significantly increased in Htr1a over expression mice in the early dark period, sleep/wakefulness pattern in the light period was not significantly different from control mice. These results suggest that inhibitory inputs from serotonergic neurons to orexin neurons function as negative feedback circuitry to preserve the activity of orexin neurons in moderate range in the early dark period. Research fund: 20680021. doi:10.1016/j.neures.2011.07.223
O2-E-2-2 Bmal1 in the nervous system is indispensable for normal adaptation of circadian locomotor activity and food intake to periodic feeding Michihiro Mieda , Takeshi Sakurai Dep. of Mol. Neurosci. & Integr. Physiol., Kanazawa Univ, Kanazawa, Japan Temporal restriction of feeding (RF) can entrain circadian behavioral and physiological rhythms in mammals. These changes in biological rhythms are postulated to be brought about by a food-entrainable oscillator (FEO) that is independent of the suprachiasmatic nucleus (SCN). However, the anatomical and molecular machineries of FEO have remained elusive. Moreover, despite well-characterized circadian nature of food entrainable rhythms, involvement of canonical clock genes, such as Bmal1, Per1, Per2, Cry1, and Cry2, remains controversial. We report here that mice with a nervous system-specific deletion of Bmal1 (N-Bmal1−/− mice) had a severe deficit in entrainment of locomotor activity by periodic feeding, accompanied by reduced food intake and subsequent loss of body weight. Control mice started to exhibit significant increase in locomotor activity during 2 h preceding food availability (food anticipatory activity: FAA, or food-seeking activity) on day 4 under RF, which reached a plateau on day 8. In clear contrast, acquisition of FAA was dramatically delayed in N-Bmal1−/− mice. They did not exhibit significant FAA until day 13, which reached to the level comparable to those in control mice on day 15. This delayed FAA in N-Bmal1−/− mice was not circadian since it did not free-run under food deprivation following RF. NBmal1−/− mice also showed reduction of food intake and subsequent loss of body weight under RF. On the other hand, these mice exhibited relatively normal light-entrainable activity rhythm driven by the SCN due to inefficient deletion of Bmal1 gene in the SCN. These findings suggest that an SCN-independent FEO in the nervous system requires Bmal1 and plays a critical role for adaptation of circadian locomotor activity and food intake to periodic feeding. Research fund: KAKENHI (20390056), HFSP Career Development Award. doi:10.1016/j.neures.2011.07.224
e53
O2-E-2-3 Time-lapse confocal imaging of clock gene expression and calcium in neuronal networks of suprachiasmatic nucleus Ryosuke Enoki 1,2 , Shigeru Kuroda 4 , Daisuke Ono 2 , Tetsuo Ueda 4 , Hasan Mazhir 5 , Sato Honma 1,2 , Ken-ichi Honma 1,3
1 Photo Bioimag Sec., Cent for Coop Proj., Grad Sch of Med., Hokkaido Univ., Sapporo, Japan 2 Dep of Chronophysiol., Grad Sch of Med, Hokkaido Univ., Sapporo, Japan 3 Dep of Chronomed, Grad Sch of Med, Hokkaido Univ., Sapporo, Japan 4 Lab of Cellu Informa., Res Int for Elec Sci., Hokkaido Univ., Sapporo, Japan 5 Max Planck Int for Med Res, Hidelberg, Germany
The suprachiasmatic nucleus (SCN) is the primary circadian pacemaker in mammals. Individual SCN neurons can generate independent circadian oscillations of clock gene expression, neuronal firing and cytosolic calcium. However, neuronal network properties as multicellular system are largely not known yet. In the present study, to monitor the activities of large neuronal population at high spatio-temporal resolution for many days, we developed a novel time-lapse imaging system using Nipkow-disk spinning confocal unit, EM-CCD camera and a microscope with XY movable stage and autofocus system. Using SCN slicess derived from Per1::d2EGFP transgenic mice and slices expressing a genetically encoded calcium calcium probe, Yellow Cameleon 3.60, we successfully monitored circadian gene expression and cytosolic calcium change in large population of neurons (>100 neurons) at single cell resolution without noticeable photo-toxicity and focus drift. We analyzed spatio-tempral dynamics of clock gene expression and cytosolic calcium change, and the cellular interaction in entire neuronal network in SCN. Research fund: KAKENHI (21890003). doi:10.1016/j.neures.2011.07.225
O2-E-2-4 Genome-wide profiling of the core clock protein BMAL1 targets reveals strict relationship with metabolism Fumiyuki Hatanaka 1,2 , Chiaki Matsubara 2 , Jihwan Myung 1,2 , Takashi Yoritaka 1 , Naoko Kamimura 3 , Shuichi Tsutsumi 3 , Akinori Kanai 4 , Yutaka Suzuki 4 , Hiroyuki Aburatani 3 , Sumio Sugano 4 , Toru Takumi 1,2 1 Lab. Integrative Bio., Grad. Sch. Biomed. Sci., Hiroshima Univ., Hiroshima, Japan 2 Osaka Biosci. Inst., Osaka, Japan 3 Genome Science Div., RCAST, Univ. of Tokyo, Tokyo, Japan 4 Dept. of Frontier Sci., Univ. of Tokyo, Tokyo, Japan
Circadian rhythms are common to most organisms and govern much of homeostasis and physiology. In addition, the circadian clock is recently implicated in control of brain-based activities, such as memory formation and the regulation of emotions. Since a significant fraction of the mammalian genome is controlled by the clock machinery, understanding the genome-wide signaling and epigenetic basis of circadian gene expression is essential. BMAL1 is a critical circadian transcription factor that regulates genes via E-box elements in their promoters. Here we use multiple high-throughput approaches including chromatin immunoprecipitation (ChIP)-based systematic analyses and DNA microarrays combined with bioinformatics, to generate genomewide profiles of BMAL1 target genes. We reveal that, in addition to E-boxes, CCAATG element contributes to elicit robust circadian expression. BMAL1 occupancy is found in more than 150 sites, including all known clock genes. Importantly, a significant proportion of BMAL1 targets include genes that encode central regulators of metabolic processes. The database generated in this study constitutes a useful resource to decipher the network of circadian gene control and its intimate links with several fundamental physiological functions. Research fund: JST, CREST. doi:10.1016/j.neures.2011.07.226
O2-F-1-1 Astrocyte lesion and secondary demyelination: The lesson from neuromyelitis optica Tatsuro Misu 1 , Hans Lassmann 3 , Yoshiki Takai 2 , Shuhei Nishiyama 2 , Toshiyuki Takahashi 2 , Ichiro Nakashima 2 , Masashi Aoki 2 , Yasuto Itoyama 4 , Kazuo Fujihara 1,2 1
Dep. of Multiple Sclerosis Therapeutics, Tohoku University, Sendai, Japan Dep. of Neurology, Tohoku University, Sendai, Japan 3 Dep. Neuroimmunol, Vienna University 4 National Center of Neurology and Psychiatry NCNP
2
Background: Neuromyelitis optica (NMO) has originally been defined as an acute inflammatory demyelinating disease, predominantly affecting the optic nerves and spinal cord. A major breakthrough in our understanding of
O2-E-2-1 Reversible gene expression control of serotonin receptor 1A in orexin neurons reveals physiological significance of serotonergic inhibitory inputs to orexin neurons Sawako Tabuchi 1,2 , Tomomi Tsunematsu 1 , Shouta Sugio 1,2 , Kenji Tanaka 1,2 , Makoto Tominaga 1,2 , Akihiro Yamanaka 1,2 1
Cell Signaling, NIPS, Okazaki, Japan Okazaki, Japan
2
Physiological Science, SOKENDAI,
The neurons producing neuropeptide orexin (orexin neurons) have an important role in the maintenance of arousal. It is reported that serotonergic neurons in the raphe nucleus are densely innervated by orexin neurons. These serotonergic neurons express both orexin receptors, OX1R and OX2R, and are activated by orexin. On the other hand, orexin neurons are innervated by serotonergic neurons in the raphe nucleus, and are inhibited by serotonin through the serotonin 1A receptor (Htr1a). Although these results suggest that serotonergic input forms a negative feedback circuit, its physiological role has not been completely understood. To reveal this, we generated transgenic mice in which orexin neurons specifically express tetracycline transactivator orexin/tTA. tTA enable to induce gene expression to bind tetracycline responsive element in the absence of doxycycline (DOX). In orexin/tTA; Tet-O Htr1a (Htr1a over expression) mice, in situ hybridization revealed that Htr1a mRNA was reversibly regulated in the orexin neurons by applying or removing DOX from chew. Electrophysiological analysis of orexin neurons revealed that inhibitory effect by serotonin application was approximately 2-hold prolonged using Htr1a over expression mice compare with control mice. EEG and EMG recording from Htr1a over expression mice revealed that these mice showed fragmentation of wakefulness in active period, the early dark period. DOX application for 5 days consolidated wakefulness in the early dark period. It was fragmented again by removing DOX for 14 days. Although stage transition frequency was significantly increased in Htr1a over expression mice in the early dark period, sleep/wakefulness pattern in the light period was not significantly different from control mice. These results suggest that inhibitory inputs from serotonergic neurons to orexin neurons function as negative feedback circuitry to preserve the activity of orexin neurons in moderate range in the early dark period. Research fund: 20680021. doi:10.1016/j.neures.2011.07.223
O2-E-2-2 Bmal1 in the nervous system is indispensable for normal adaptation of circadian locomotor activity and food intake to periodic feeding Michihiro Mieda , Takeshi Sakurai Dep. of Mol. Neurosci. & Integr. Physiol., Kanazawa Univ, Kanazawa, Japan Temporal restriction of feeding (RF) can entrain circadian behavioral and physiological rhythms in mammals. These changes in biological rhythms are postulated to be brought about by a food-entrainable oscillator (FEO) that is independent of the suprachiasmatic nucleus (SCN). However, the anatomical and molecular machineries of FEO have remained elusive. Moreover, despite well-characterized circadian nature of food entrainable rhythms, involvement of canonical clock genes, such as Bmal1, Per1, Per2, Cry1, and Cry2, remains controversial. We report here that mice with a nervous system-specific deletion of Bmal1 (N-Bmal1−/− mice) had a severe deficit in entrainment of locomotor activity by periodic feeding, accompanied by reduced food intake and subsequent loss of body weight. Control mice started to exhibit significant increase in locomotor activity during 2 h preceding food availability (food anticipatory activity: FAA, or food-seeking activity) on day 4 under RF, which reached a plateau on day 8. In clear contrast, acquisition of FAA was dramatically delayed in N-Bmal1−/− mice. They did not exhibit significant FAA until day 13, which reached to the level comparable to those in control mice on day 15. This delayed FAA in N-Bmal1−/− mice was not circadian since it did not free-run under food deprivation following RF. NBmal1−/− mice also showed reduction of food intake and subsequent loss of body weight under RF. On the other hand, these mice exhibited relatively normal light-entrainable activity rhythm driven by the SCN due to inefficient deletion of Bmal1 gene in the SCN. These findings suggest that an SCN-independent FEO in the nervous system requires Bmal1 and plays a critical role for adaptation of circadian locomotor activity and food intake to periodic feeding. Research fund: KAKENHI (20390056), HFSP Career Development Award. doi:10.1016/j.neures.2011.07.224
e53
O2-E-2-3 Time-lapse confocal imaging of clock gene expression and calcium in neuronal networks of suprachiasmatic nucleus Ryosuke Enoki 1,2 , Shigeru Kuroda 4 , Daisuke Ono 2 , Tetsuo Ueda 4 , Hasan Mazhir 5 , Sato Honma 1,2 , Ken-ichi Honma 1,3
1 Photo Bioimag Sec., Cent for Coop Proj., Grad Sch of Med., Hokkaido Univ., Sapporo, Japan 2 Dep of Chronophysiol., Grad Sch of Med, Hokkaido Univ., Sapporo, Japan 3 Dep of Chronomed, Grad Sch of Med, Hokkaido Univ., Sapporo, Japan 4 Lab of Cellu Informa., Res Int for Elec Sci., Hokkaido Univ., Sapporo, Japan 5 Max Planck Int for Med Res, Hidelberg, Germany
The suprachiasmatic nucleus (SCN) is the primary circadian pacemaker in mammals. Individual SCN neurons can generate independent circadian oscillations of clock gene expression, neuronal firing and cytosolic calcium. However, neuronal network properties as multicellular system are largely not known yet. In the present study, to monitor the activities of large neuronal population at high spatio-temporal resolution for many days, we developed a novel time-lapse imaging system using Nipkow-disk spinning confocal unit, EM-CCD camera and a microscope with XY movable stage and autofocus system. Using SCN slicess derived from Per1::d2EGFP transgenic mice and slices expressing a genetically encoded calcium calcium probe, Yellow Cameleon 3.60, we successfully monitored circadian gene expression and cytosolic calcium change in large population of neurons (>100 neurons) at single cell resolution without noticeable photo-toxicity and focus drift. We analyzed spatio-tempral dynamics of clock gene expression and cytosolic calcium change, and the cellular interaction in entire neuronal network in SCN. Research fund: KAKENHI (21890003). doi:10.1016/j.neures.2011.07.225
O2-E-2-4 Genome-wide profiling of the core clock protein BMAL1 targets reveals strict relationship with metabolism Fumiyuki Hatanaka 1,2 , Chiaki Matsubara 2 , Jihwan Myung 1,2 , Takashi Yoritaka 1 , Naoko Kamimura 3 , Shuichi Tsutsumi 3 , Akinori Kanai 4 , Yutaka Suzuki 4 , Hiroyuki Aburatani 3 , Sumio Sugano 4 , Toru Takumi 1,2 1 Lab. Integrative Bio., Grad. Sch. Biomed. Sci., Hiroshima Univ., Hiroshima, Japan 2 Osaka Biosci. Inst., Osaka, Japan 3 Genome Science Div., RCAST, Univ. of Tokyo, Tokyo, Japan 4 Dept. of Frontier Sci., Univ. of Tokyo, Tokyo, Japan
Circadian rhythms are common to most organisms and govern much of homeostasis and physiology. In addition, the circadian clock is recently implicated in control of brain-based activities, such as memory formation and the regulation of emotions. Since a significant fraction of the mammalian genome is controlled by the clock machinery, understanding the genome-wide signaling and epigenetic basis of circadian gene expression is essential. BMAL1 is a critical circadian transcription factor that regulates genes via E-box elements in their promoters. Here we use multiple high-throughput approaches including chromatin immunoprecipitation (ChIP)-based systematic analyses and DNA microarrays combined with bioinformatics, to generate genomewide profiles of BMAL1 target genes. We reveal that, in addition to E-boxes, CCAATG element contributes to elicit robust circadian expression. BMAL1 occupancy is found in more than 150 sites, including all known clock genes. Importantly, a significant proportion of BMAL1 targets include genes that encode central regulators of metabolic processes. The database generated in this study constitutes a useful resource to decipher the network of circadian gene control and its intimate links with several fundamental physiological functions. Research fund: JST, CREST. doi:10.1016/j.neures.2011.07.226
O2-F-1-1 Astrocyte lesion and secondary demyelination: The lesson from neuromyelitis optica Tatsuro Misu 1 , Hans Lassmann 3 , Yoshiki Takai 2 , Shuhei Nishiyama 2 , Toshiyuki Takahashi 2 , Ichiro Nakashima 2 , Masashi Aoki 2 , Yasuto Itoyama 4 , Kazuo Fujihara 1,2 1
Dep. of Multiple Sclerosis Therapeutics, Tohoku University, Sendai, Japan Dep. of Neurology, Tohoku University, Sendai, Japan 3 Dep. Neuroimmunol, Vienna University 4 National Center of Neurology and Psychiatry NCNP
2
Background: Neuromyelitis optica (NMO) has originally been defined as an acute inflammatory demyelinating disease, predominantly affecting the optic nerves and spinal cord. A major breakthrough in our understanding of