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Junichi Nakai

Section

Graduate School of Dentistry

Job title

Professor

Degree

医学博士（京都大学）

researchmap

https://researchmap.jp/junichinakai

J-GLOBAL ID

200901020977429020

Research History 7

2019/04 - Present

Tohoku University　Graduate School of Dentistry, Oral physiology　professor
2009/02 - 2019/03

Saitama University
2008/04 - 2009/01

RIKEN
2004/06 - 2008/03

RIKEN
2003/04 - 2004/05

RIKEN
1996/09 - 2003/03

岡崎国立共同研究機構生理学研究所　液性情報部門　助教
1991/04 - 1996/08

Kyoto University　Faculty of Medicine

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Research Interests 18

包括脳ネットワーク
統合脳・神経回路機能
海馬
カルシウム放出チャンネル
ニューロン
神経回路
蛍光
カルシウム放出チャネル
ジヒドロピリジン受容体
電位依存性カルシウムチャネル
カルシウム
バイオセンサー
cDNA発現
分子生物学
カルシウムチャネル
GFP
興奮収縮連関
リアノジン受容体

Research Areas 3

Life sciences / Neuroscience - general /
Life sciences / Clinical pharmacy /
Life sciences / Physiology /

Papers 99

Frequency-Regulated Repeated Micro-Vibration Promotes Osteoblast Differentiation Through BMP Signaling in MC3T3-E1 Cells

Ayumu Matsushita, Tada-aki Kudo, Kanako Tominami, Yohei Hayashi, Takuya Noguchi, Takakuni Tanaka, Satoshi Izumi, Keiko Gengyo-Ando, Atsushi Matsuzawa, Guang Hong, Junichi Nakai

Life　15　(4)　588-588　2025/04/03
Publisher: MDPI AG
DOI： 10.3390/life15040588 　

eISSN： 2075-1729

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Physical stimulation, which is a key factor affecting the metabolism of osteoblasts and their precursor cells, plays an important role in bone remodeling; however, the role of micro-vibrations in osteoblast differentiation is unclear. In the present study, we determined the effects of frequency-regulated repeated micro-vibration (FRMV) on cell proliferation and established a method to induce osteoblast differentiation through FRMV using the mouse pre-osteoblast-like cell line MC3T3-E1, which is widely used in bone metabolism research. The results indicated that FRMV significantly influenced the proliferation of MC3T3-E1 cells in a normal growth medium. FRMV at 42.2 Hz significantly promoted proliferation, whereas FRMV at 92.1 Hz showed no effect on the proliferation rate. Moreover, FRMV at 42.2 Hz significantly increased alkaline phosphatase (ALP) enzyme activity and ALP gene expression in MC3T3-E1 cells. Treatment with LDN193189, a bone morphogenetic protein (BMP) signaling inhibitor, revealed that the FRMV-induced upregulation in ALP enzyme activity and ALP gene expression were significantly suppressed in MC3T3-E1 cells. The results suggest that the FRMV protocol developed in the present study induces osteoblast differentiation through the BMP signaling pathway. Thus, FRMV may contribute to the development of effective bone regeneration technologies.
Physical Stimulation Methods Developed for In Vitro Neuronal Differentiation Studies of PC12 Cells: A Comprehensive Review

Kanako Tominami, Tada-aki Kudo, Takuya Noguchi, Yohei Hayashi, You-Ran Luo, Takakuni Tanaka, Ayumu Matsushita, Satoshi Izumi, Hajime Sato, Keiko Gengyo-Ando, Atsushi Matsuzawa, Guang Hong, Junichi Nakai

International Journal of Molecular Sciences　25　(2)　772-772　2024/01/07
Publisher: MDPI AG
DOI： 10.3390/ijms25020772 　

eISSN： 1422-0067

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PC12 cells, which are derived from rat adrenal pheochromocytoma cells, are widely used for the study of neuronal differentiation. NGF induces neuronal differentiation in PC12 cells by activating intracellular pathways via the TrkA receptor, which results in elongated neurites and neuron-like characteristics. Moreover, the differentiation requires both the ERK1/2 and p38 MAPK pathways. In addition to NGF, BMPs can also induce neuronal differentiation in PC12 cells. BMPs are part of the TGF-β cytokine superfamily and activate signaling pathways such as p38 MAPK and Smad. However, the brief lifespan of NGF and BMPs may limit their effectiveness in living organisms. Although PC12 cells are used to study the effects of various physical stimuli on neuronal differentiation, the development of new methods and an understanding of the molecular mechanisms are ongoing. In this comprehensive review, we discuss the induction of neuronal differentiation in PC12 cells without relying on NGF, which is already established for electrical, electromagnetic, and thermal stimulation but poses a challenge for mechanical, ultrasound, and light stimulation. Furthermore, the mechanisms underlying neuronal differentiation induced by physical stimuli remain largely unknown. Elucidating these mechanisms holds promise for developing new methods for neural regeneration and advancing neuroregenerative medical technologies using neural stem cells.
Functional analysis of epilepsy-linked pathogenic variants of the Munc18-1 gene in the inhibitory nervous system of Caenorhabditis elegans. International-journal

Keiko Gengyo-Ando, Akane Osawa-Noguchi, Hideki Ando, Junichi Nakai

microPublication biology　2024　2024

DOI： 10.17912/micropub.biology.001174 　

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Heterozygous de novo mutations in Munc18-1, which is essential for neurotransmitter release, cause early infantile epileptic encephalopathy. Munc18-1-linked epilepsy is currently an untreatable disorder and its precise disease mechanism remains elusive. Here, we investigated how Munc18-1 pathogenic variants affect inhibitory neurons using Caenorhabditis elegans . Expression analysis revealed that three missense mutant proteins form aggregates in the cell body of gamma-aminobutyric-acid (GABA)-ergic motoneurons, resulting in a strong reduction of their expression in axons. Their defects of axonal expression correlated closely with pentylenetetrazol-induced convulsions, suggesting that the degree of instability of each mutant protein account for the severity of the epileptic phenotypes.
Virtual reality-based real-time imaging reveals abnormal cortical dynamics during behavioral transitions in a mouse model of autism. International-journal

Nobuhiro Nakai, Masaaki Sato, Okito Yamashita, Yukiko Sekine, Xiaochen Fu, Junichi Nakai, Andrew Zalesky, Toru Takumi

Cell reports　112258-112258　2023/03/24

DOI： 10.1016/j.celrep.2023.112258 　

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Functional connectivity (FC) can provide insight into cortical circuit dysfunction in neuropsychiatric disorders. However, dynamic changes in FC related to locomotion with sensory feedback remain to be elucidated. To investigate FC dynamics in locomoting mice, we develop mesoscopic Ca2+ imaging with a virtual reality (VR) environment. We find rapid reorganization of cortical FC in response to changing behavioral states. By using machine learning classification, behavioral states are accurately decoded. We then use our VR-based imaging system to study cortical FC in a mouse model of autism and find that locomotion states are associated with altered FC dynamics. Furthermore, we identify FC patterns involving the motor area as the most distinguishing features of the autism mice from wild-type mice during behavioral transitions, which might correlate with motor clumsiness in individuals with autism. Our VR-based real-time imaging system provides crucial information to understand FC dynamics linked to behavioral abnormality of neuropsychiatric disorders.
A humanized Caenorhabditis elegans model for studying pathogenic mutations in VPS45, a protein essential for membrane trafficking, associated with severe congenital neutropenia. International-journal

Keiko Gengyo-Ando, Minoru Tateyama, Shohei Mitani, Hideki Ando, Junichi Nakai

microPublication biology　2023　2023

DOI： 10.17912/micropub.biology.001052 　

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VPS45, one of the essential membrane trafficking factors, has been identified as a cause of severe congenital neutropenia 5 (SCN5), but its pathophysiological role remains unknown. Here, we developed a humanized C. elegans model for three pathogenic VPS45 variants. We found that wild-type human VPS45 functionally complemented the loss of C. elegans VPS-45 , and the pathogenic human VPS45 variants functioned almost normally with respect to larval development and endocytosis in C. elegans . These results suggest that SCN5-associated mutations have little effect on the core function of VPS45, and/or that the degree of VPS45 requirement varies, depending on the cell/tissue.
SP600125 Enhances Temperature-Controlled Repeated Thermal Stimulation-Induced Neurite Outgrowth in PC12-P1F1 Cells

You-Ran Luo, Tada-aki Kudo, Kanako Tominami, Satoshi Izumi, Takakuni Tanaka, Yohei Hayashi, Takuya Noguchi, Atsushi Matsuzawa, Junichi Nakai, Guang Hong, Hang Wang

International Journal of Molecular Sciences　23　(24)　15602-15602　2022/12/09
Publisher: MDPI AG
DOI： 10.3390/ijms232415602 　

eISSN： 1422-0067

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This study evaluated the mechanism of temperature-controlled repeated thermal stimulation (TRTS)-mediated neuronal differentiation. We assessed the effect of SP600125, a c-Jun N-terminal kinase (JNK) inhibitor, on neuronal differentiation of rat PC12-P1F1 cells, which can differentiate into neuron-like cells by exposure to TRTS or neurotrophic factors, including bone morphogenetic protein (BMP) 4. We evaluated neuritogenesis by incubating the cells under conditions of TRTS and/or SP600125. Cotreatment with SP600125 significantly enhanced TRTS-mediated neuritogenesis, whereas that with other selective mitogen-activated protein kinase (MAPK) inhibitors did not—e.g., extracellular signal-regulated kinase (ERK)1/2 inhibitor U0126, and p38 MAPK inhibitor SB203580. We tried to clarify the mechanism of SP600125 action by testing the effect of U0126 and the BMP receptor inhibitor LDN193189 on the SP600125-mediated enhancement of intracellular signaling. SP600125-enhanced TRTS-induced neuritogenesis was significantly inhibited by U0126 or LDN193189. Gene expression analysis revealed that TRTS significantly increased β3-Tubulin, MKK3, and Smad7 gene expressions. Additionally, Smad6 and Smad7 gene expressions were substantially attenuated through SP600125 co-treatment during TRTS. Therefore, SP600125 may partly enhance TRTS-induced neuritogenesis by attenuating the negative feedback loop of BMP signaling. Further investigation of the mechanisms underlying the effect of SP600125 during TRTS-mediated neuritogenesis may contribute to the future development of regenerative neuromedicine.
Development of microglia-targeting adeno-associated viral vectors as tools to study microglial behavior in vivo

Yukihiro Okada, Nobutake Hosoi, Yasunori Matsuzaki, Yuuki Fukai, Akito Hiraga, Junichi Nakai, Keisuke Nitta, Yoichiro Shinohara, Ayumu Konno, Hirokazu Hirai

Communications Biology　5　(1)　2022/11/11
Publisher: Springer Science and Business Media LLC
DOI： 10.1038/s42003-022-04200-3 　

eISSN： 2399-3642

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Abstract Here we describe the microglia-targeting adeno-associated viral (AAV) vectors containing a 1.7-kb putative promoter region of microglia/macrophage-specific ionized calcium-binding adaptor molecule 1 (Iba1), along with repeated miRNA target sites for microRNA (miR)-9 and miR-129-2-3p. The 1.7-kb genomic sequence upstream of the start codon in exon 1 of the Iba1 (Aif1) gene, functions as microglia preferential promoter in the striatum and cerebellum. Furthermore, ectopic transgene expression in non-microglial cells is markedly suppressed upon adding two sets of 4-repeated miRNA target sites for miR-9 and miR-129-2-3p, which are expressed exclusively in non-microglial cells and sponged AAV-derived mRNAs. Our vectors transduced ramified microglia in healthy tissues and reactive microglia in lipopolysaccharide-treated mice and a mouse model of neurodegenerative disease. Moreover, live fluorescent imaging allowed the monitoring of microglial motility and intracellular Ca²⁺ mobilization. Thus, microglia-targeting AAV vectors are valuable for studying microglial pathophysiology and therapies, particularly in the striatum and cerebellum.
HOTARU: Automatic sorting system for large scale calcium imaging data

Takashi Takekawa, Masanori Nomoto, Hirotaka Asai, Noriaki Ohkawa, Reiko Okubo-Suzuki, Khaled Ghandour, Masaaki Sato, Masamichi Ohkura, Junichi Nakai, Shin-ichi Muramatsu, Yasunori Hayashi, Kaoru Inokuchi, Tomoki Fukai

2022/04/07
Publisher: Cold Spring Harbor Laboratory
DOI： 10.1101/2022.04.05.487077 　

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Currently, calcium imaging allows for the long-term recording of large-scale neuronal activity in diverse states. However, it remains difficult to extract neuronal dynamics from recorded imaging data. In this study, we propose an improved CNMF-based algorithm and an effective method for extracting cell shapes with fewer false positives and false negatives caused by image processing. We also showed that the values obtained during image processing can be combined and used for false positive determination of cells. For the CNMF algorithm, we combined cell-by-cell regularization and baseline shrinkage estimation, which greatly improved its stability and robustness. We applied these methods to artificial and real data and confirmed their effectiveness. Our method is simpler and faster, detects more cells with lower firing rates and signal-to-noise ratios, and enhance the quality of the extracted cell signals. These advances can improve the standard of downstream analysis and contribute to progress in neuroscience.
Low computational-cost cell detection method for calcium imaging data

Tsubasa Ito, Keisuke Ota, Kanako Ueno, Yasuhiro Oisi, Chie Matsubara, Kenta Kobayashi, Masamichi Ohkura, Junichi Nakai, Masanori Murayama, Toru Aonishi

Neuroscience Research　2022/04
Publisher: Elsevier BV
DOI： 10.1016/j.neures.2022.02.008 　

ISSN： 0168-0102
Effect of Beta 2-Adrenergic Receptor Gly16Arg Polymorphism on Taste Preferences in Healthy Young Japanese Adults

Kohei Narita, Tada-aki Kudo, Guang Hong, Kanako Tominami, Satoshi Izumi, Yohei Hayashi, Junichi Nakai

Nutrients　14　(7)　1430-1430　2022/03/29
Publisher: MDPI AG
DOI： 10.3390/nu14071430 　

eISSN： 2072-6643

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The Gly16Arg polymorphism results in a G to C nucleotide mutation in the human beta 2-adrenergic receptor (ADRB2) gene and has a relationship with obesity; however, this substitution’s effects on food preferences are unclear. Therefore, we determined this relationship among healthy young adults (mean age, 23.4; n = 52). To evaluate food preferences, four categories of food (sweet, salty, sour, and bitter) along with high-fat foods were evaluated using a self-reporting questionnaire. Male (n = 26) and female subjects (n = 26) were genotyped for the polymorphism and further divided into three groups (two homozygous groups, GG, CC; and a heterozygous group, GC). Preference for sour foods in the GG group was higher compared with that in the CC group in females (p < 0.05). When sweet foods were classified into low- and high-fat subgroups, preference for high-fat sweet foods in the GG group was higher than that for low-fat sweet foods in all subjects (p < 0.05). The degree of preference for high-fat foods in the GG group was higher than other groups for males (p < 0.05). These results suggest that ADRB2 polymorphism is associated with food preference. Understanding the relationship of ADRB2 substitution to food preference will be valuable for designing individualized anti-obesity strategies.
GABAergic neuron-specific whole-brain transduction by AAV-PHP.B incorporated with a new GAD65 promoter

Chiaki Hoshino, Ayumu Konno, Nobutake Hosoi, Ryosuke Kaneko, Ryo Mukai, Junichi Nakai, Hirokazu Hirai

Molecular Brain　14　(1)　2021/12
Publisher: Springer Science and Business Media LLC
DOI： 10.1186/s13041-021-00746-1 　

eISSN： 1756-6606

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<title>Abstract</title>GABAergic interneurons play a critical role in tuning neural networks in the central nervous system, and their defects are associated with neuropsychiatric disorders. Currently, the mDlx enhancer is solely used for adeno-associated virus (AAV) vector-mediated transgene delivery into cortical interneurons. Here, we developed a new inhibitory neuron-specific promoter (designated as the mGAD65 promoter), with a length of 2.5 kb, from a mouse genome upstream of exon 1 of the <italic>Gad2</italic> gene encoding glutamic acid decarboxylase (GAD) 65. Intravenous infusion of blood–brain barrier-penetrating AAV-PHP.B expressing an enhanced green fluorescent protein under the control of the mGAD65 promoter transduced the whole brain in an inhibitory neuron-specific manner. The specificity and efficiency of the mGAD65 promoter for GABAergic interneurons, which was assessed at the motor cortex, were almost identical to or slightly higher than those of the mDlx enhancer. Immunohistochemical analysis revealed that the mGAD65 promoter preferentially transduced parvalbumin (PV)-expressing interneurons. Notably, the mGAD65 promoter transduced chandelier cells more efficiently than the mDlx enhancer and robustly labeled their synaptic boutons, called the cartridge, targeting the axon initial segments of excitatory pyramidal neurons. To test the ability of the mGAD65 promoter to express a functional molecule, we virally expressed G-CaMP, a fluorescent Ca²⁺ indicator, in the motor cortex, and this enabled us to monitor spontaneous and drug-induced Ca²⁺ activity in GABAergic inhibitory neurons. These results suggest that the mGAD65 promoter is useful for AAV-mediated targeting and manipulation of GABAergic neurons with the dominance of cortical PV-expressing neurons, including chandelier cells.
Global knockdown of glutamate decarboxylase 67 elicits emotional abnormality in mice

Shigeo Miyata, Toshikazu Kakizaki, Kazuyuki Fujihara, Hideru Obinata, Touko Hirano, Junichi Nakai, Mika Tanaka, Shigeyoshi Itohara, Masahiko Watanabe, Kenji F. Tanaka, Manabu Abe, Kenji Sakimura, Yuchio Yanagawa

Molecular Brain　14　(1)　2021/12
Publisher: Springer Science and Business Media LLC
DOI： 10.1186/s13041-020-00713-2 　

eISSN： 1756-6606

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<title>Abstract</title>Reduced expression of glutamate decarboxylase 67 (GAD67), encoded by the <italic>Gad1</italic> gene, is a consistent finding in postmortem brains of patients with several psychiatric disorders, including schizophrenia, bipolar disorder and major depressive disorder. The dysfunction of GAD67 in the brain is implicated in the pathophysiology of these psychiatric disorders; however, the neurobiological consequences of GAD67 dysfunction in mature brains are not fully understood because the homozygous <italic>Gad1</italic> knockout is lethal in newborn mice. We hypothesized that the tetracycline-controlled gene expression/suppression system could be applied to develop global GAD67 knockdown mice that would survive into adulthood. In addition, GAD67 knockdown mice would provide new insights into the neurobiological impact of GAD67 dysfunction. Here, we developed <italic>Gad1</italic>^{tTA/STOP−tetO} biallelic knock-in mice using <italic>Gad1</italic>^STOP−tetO and <italic>Gad1</italic>^tTA knock-in mice, and compared them with <italic>Gad1</italic>^+/+ mice. The expression level of GAD67 protein in brains of <italic>Gad1</italic>^{tTA/STOP−tetO} mice treated with doxycycline (Dox) was decreased by approximately 90%. The GABA content was also decreased in the brains of Dox-treated <italic>Gad1</italic>^{tTA/STOP−tetO} mice. In the open-field test, Dox-treated <italic>Gad1</italic>^{tTA/STOP−tetO} mice exhibited hyper-locomotor activity and decreased duration spent in the center region. In addition, acoustic startle responses were impaired in Dox-treated <italic>Gad1</italic>^{tTA/STOP−tetO} mice. These results suggest that global reduction in GAD67 elicits emotional abnormalities in mice. These GAD67 knockdown mice will be useful for elucidating the neurobiological mechanisms of emotional abnormalities, such as anxiety symptoms associated with psychiatric disorders.
Fast, cell-resolution, contiguous-wide two-photon imaging to reveal functional network architectures across multi-modal cortical areas. International-journal

Keisuke Ota, Yasuhiro Oisi, Takayuki Suzuki, Muneki Ikeda, Yoshiki Ito, Tsubasa Ito, Hiroyuki Uwamori, Kenta Kobayashi, Midori Kobayashi, Maya Odagawa, Chie Matsubara, Yoshinori Kuroiwa, Masaru Horikoshi, Junya Matsushita, Hiroyuki Hioki, Masamichi Ohkura, Junichi Nakai, Masafumi Oizumi, Atsushi Miyawaki, Toru Aonishi, Takahiro Ode, Masanori Murayama

Neuron　109　(11)　1810-1824　2021/06/02

DOI： 10.1016/j.neuron.2021.03.032 　

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Fast and wide field-of-view imaging with single-cell resolution, high signal-to-noise ratio, and no optical aberrations have the potential to inspire new avenues of investigations in biology. However, such imaging is challenging because of the inevitable tradeoffs among these parameters. Here, we overcome these tradeoffs by combining a resonant scanning system, a large objective with low magnification and high numerical aperture, and highly sensitive large-aperture photodetectors. The result is a practically aberration-free, fast-scanning high optical invariant two-photon microscopy (FASHIO-2PM) that enables calcium imaging from a large network composed of ∼16,000 neurons at 7.5 Hz from a 9 mm2 contiguous image plane, including more than 10 sensory-motor and higher-order areas of the cerebral cortex in awake mice. Network analysis based on single-cell activities revealed that the brain exhibits small-world rather than scale-free behavior. The FASHIO-2PM is expected to enable studies on biological dynamics by simultaneously monitoring macroscopic activities and their compositional elements.
Modality specific impairment of hippocampal CA1 neurons of Alzheimer’s disease model mice

Risa Takamura, Kotaro Mizuta, Yukiko Sekine, Tanvir Islam, Takashi Saito, Masaaki Sato, Masamichi Ohkura, Junichi Nakai, Toshio Ohshima, Takaomi C Saido, Yasunori Hayashi

The Journal of Neuroscience　41　(24)　JN-RM　2021/05/12
Publisher: Society for Neuroscience
DOI： 10.1523/jneurosci.0208-21.2021 　

ISSN： 0270-6474

eISSN： 1529-2401
Shared and unique properties of place cells in anterior cingulate cortex and hippocampus

Ayaka Bota, Akihiro Goto, Suzune Tsukamoto, Alexander Schmidt, Fred Wolf, Alessandro Luchetti, Junichi Nakai, Hajime Hirase, Yasunori Hayashi

2021/03/30
Publisher: Cold Spring Harbor Laboratory
DOI： 10.1101/2021.03.29.437441 　

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<title>Abstract</title>In the brain, spatial information is represented by neurons that fire when an animal is at specific locations, including place cells in hippocampus and grid cells in entorhinal cortex. But how this information is processed in downstream brain regions still remains elusive. Using chronic Ca²⁺ imaging, we examined the activity of neurons in anterior cingulate cortex (ACC), a brain region implicated in memory consolidation, and found neurons that fire in a manner consistent with the properties of place cells. While the ACC place cells showed stability, location and context specificity similar to the hippocampal counterparts, they also have unique properties. Unlike hippocampal place cells that immediately formed upon exposure to a novel environment, ACC place cells increased over days. Also, ACC place cells tend to have additional place fields whereas typical hippocampal place cells have only one. Hippocampal activity is required for the formation of ACC place cells, but once they are established, hippocampal inactivation did not have any impact on ACC place cell firing. We thus identified features of ACC place cells that carry spatial information in a unique fashion.
Multiple coordinated cellular dynamics mediate CA1 map plasticity. International-journal

Kotaro Mizuta, Junichi Nakai, Yasunori Hayashi, Masaaki Sato

Hippocampus　31　(3)　235-243　2021/03

DOI： 10.1002/hipo.23300 　

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In the hippocampus, spatial and nonspatial information are jointly represented as a neural map in which locations associated with salient features are over-represented by increased densities of relevant place cells. Although we recently demonstrated that experience-dependent establishment of these disproportionate maps is governed by selective stabilization of salient place cells following their conversion from non-place cells, the underlying mechanism for pre-established map reorganization remained to be understood. To this end, we investigated the changes in CA1 functional cellular maps imaged using two-photon calcium imaging in mice performing a reward-rearrangement task in virtual reality. Mice were pre-trained on a virtual linear track with a visual landmark and a reward in two distinct locations. Then, they were re-trained on the same track with the exception that the location of reward was shifted to match the landmark location. We found that, in contrast to de novo map formation, robust map reorganization occurred through parallel coordination of new place field formation, lateral shifting of existing place fields, and selective stabilization of place fields encoding salient locations. Our findings demonstrate that intricate interplay between multiple forms of cellular dynamics enables rapid updating of information stored in hippocampal maps.
Characterization of PC12 Cell Subclones with Different Sensitivities to Programmed Thermal Stimulation. International-journal Peer-reviewed

Tada-Aki Kudo, Kanako Tominami, Satoshi Izumi, Yohei Hayashi, Takuya Noguchi, Atsushi Matsuzawa, Guang Hong, Junichi Nakai

International journal of molecular sciences　21　(21)　8356-8356　2020/11/07
Publisher: MDPI AG
DOI： 10.3390/ijms21218356 　

eISSN： 1422-0067

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Neuritogenesis is the process underling nervous system regeneration; however, optimal extracellular signals that can promote neuronal regenerative activities require further investigation. Previously, we developed a novel method for inducing neuronal differentiation in rat PC12 cells using temperature-controlled repeated thermal stimulation (TRTS) with a heating plate. Based on neurogenic sensitivity to TRTS, PC12 cells were classified as either hyper- or hyposensitive. In this study, we aimed to investigate the mechanism of hyposensitivity by establishing two PC12-derived subclones according to TRTS sensitivity during differentiation: PC12-P1F1, a hypersensitive subclone, and PC12-P1D10, a hyposensitive subclone. To characterize these subclones, cell size and neuritogenesis were evaluated in subclones treated with nerve growth factor (NGF), bone morphogenetic protein (BMP), or various TRTS. No significant differences in cell size were observed among the parental cells and subclones. BMP4- or TRTS-induced neuritogenesis was increased in PC12-P1F1 cells compared to that in the parental cells, while no neuritogenesis was observed in PC12-P1D10 cells. In contrast, NGF-induced neuritogenesis was observed in all three cell lines. Furthermore, a BMP inhibitor, LDN-193189, considerably inhibited TRTS-induced neuritogenesis. These results suggest that the BMP pathway might be required for TRTS-induced neuritogenesis, demonstrating the useful aspects of these novel subclones for TRTS research.
Machine learning enables improved runtime and precision for bio-loggers on seabirds. International-journal

Joseph Korpela, Hirokazu Suzuki, Sakiko Matsumoto, Yuichi Mizutani, Masaki Samejima, Takuya Maekawa, Junichi Nakai, Ken Yoda

Communications biology　3　(1)　633-633　2020/10/30

DOI： 10.1038/s42003-020-01356-8 　

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Unravelling the secrets of wild animals is one of the biggest challenges in ecology, with bio-logging (i.e., the use of animal-borne loggers or bio-loggers) playing a pivotal role in tackling this challenge. Bio-logging allows us to observe many aspects of animals' lives, including their behaviours, physiology, social interactions, and external environment. However, bio-loggers have short runtimes when collecting data from resource-intensive (high-cost) sensors. This study proposes using AI on board video-loggers in order to use low-cost sensors (e.g., accelerometers) to automatically detect and record complex target behaviours that are of interest, reserving their devices' limited resources for just those moments. We demonstrate our method on bio-loggers attached to seabirds including gulls and shearwaters, where it captured target videos with 15 times the precision of a baseline periodic-sampling method. Our work will provide motivation for more widespread adoption of AI in bio-loggers, helping us to shed light onto until now hidden aspects of animals' lives.
Cellular identity and Ca2+ signaling activity of the non-reproductive GnRH system in the Ciona intestinalis type A (Ciona robusta) larva. International-journal

Nanako Okawa, Kotaro Shimai, Kohei Ohnishi, Masamichi Ohkura, Junichi Nakai, Takeo Horie, Atsushi Kuhara, Takehiro G Kusakabe

Scientific reports　10　(1)　18590-18590　2020/10/29

DOI： 10.1038/s41598-020-75344-7 　

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Tunicate larvae have a non-reproductive gonadotropin-releasing hormone (GnRH) system with multiple ligands and receptor heterodimerization enabling complex regulation. In Ciona intestinalis type A larvae, one of the gnrh genes, gnrh2, is conspicuously expressed in the motor ganglion and nerve cord, which are homologous structures to the hindbrain and spinal cord, respectively, of vertebrates. The gnrh2 gene is also expressed in the proto-placodal sensory neurons, which are the proposed homologue of vertebrate olfactory neurons. Tunicate larvae occupy a non-reproductive dispersal stage, yet the role of their GnRH system remains elusive. In this study, we investigated neuronal types of gnrh2-expressing cells in Ciona larvae and visualized the activity of these cells by fluorescence imaging using a calcium sensor protein. Some cholinergic neurons and dopaminergic cells express gnrh2, suggesting that GnRH plays a role in controlling swimming behavior. However, none of the gnrh2-expressing cells overlap with glycinergic or GABAergic neurons. A role in motor control is also suggested by a relationship between the activity of gnrh2-expressing cells and tail movements. Interestingly, gnrh2-positive ependymal cells in the nerve cord, known as a kind of glia cells, actively produced Ca2+ transients, suggesting that active intercellular signaling occurs in the glia cells of the nerve cord.
Encoding of social exploration by neural ensembles in the insular cortex. International-journal Peer-reviewed

Isamu Miura, Masaaki Sato, Eric T N Overton, Nobuo Kunori, Junichi Nakai, Takakazu Kawamata, Nobuhiro Nakai, Toru Takumi

PLoS biology　18　(9)　e3000584-e3000584　2020/09
Publisher: Public Library of Science (PLoS)
DOI： 10.1371/journal.pbio.3000584 　

eISSN： 1545-7885

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The insular cortex (IC) participates in diverse complex brain functions, including social function, yet their cellular bases remain to be fully understood. Using microendoscopic calcium imaging of the agranular insular cortex (AI) in mice interacting with freely moving and restrained social targets, we identified 2 subsets of AI neurons-a larger fraction of "Social-ON" cells and a smaller fraction of "Social-OFF" cells-that change their activity in opposite directions during social exploration. Social-ON cells included those that represented social investigation independent of location and consisted of multiple subsets, each of which was preferentially active during exploration under a particular behavioral state or with a particular target of physical contact. These results uncover a previously unknown function of AI neurons that may act to monitor the ongoing status of social exploration while an animal interacts with unfamiliar conspecifics.
Distinct Mechanisms of Over-Representation of Landmarks and Rewards in the Hippocampus. International-journal Peer-reviewed

Masaaki Sato, Kotaro Mizuta, Tanvir Islam, Masako Kawano, Yukiko Sekine, Takashi Takekawa, Daniel Gomez-Dominguez, Alexander Schmidt, Fred Wolf, Karam Kim, Hiroshi Yamakawa, Masamichi Ohkura, Min Goo Lee, Tomoki Fukai, Junichi Nakai, Yasunori Hayashi

Cell reports　32　(1)　107864-107864　2020/07/07
Publisher: Elsevier BV
DOI： 10.1016/j.celrep.2020.107864 　

ISSN： 2211-1247

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In the hippocampus, locations associated with salient features are represented by a disproportionately large number of neurons, but the cellular and molecular mechanisms underlying this over-representation remain elusive. Using longitudinal calcium imaging in mice learning to navigate in virtual reality, we find that the over-representation of reward and landmark locations are mediated by persistent and separable subsets of neurons, with distinct time courses of emergence and differing underlying molecular mechanisms. Strikingly, we find that in mice lacking Shank2, an autism spectrum disorder (ASD)-linked gene encoding an excitatory postsynaptic scaffold protein, the learning-induced over-representation of landmarks was absent whereas the over-representation of rewards was substantially increased, as was goal-directed behavior. These findings demonstrate that multiple hippocampal coding processes for unique types of salient features are distinguished by a Shank2-dependent mechanism and suggest that abnormally distorted hippocampal salience mapping may underlie cognitive and behavioral abnormalities in a subset of ASDs.
Role of Ca2+ transients at the node of the mouse embryo in breaking of left-right symmetry. International-journal

Katsutoshi Mizuno, Kei Shiozawa, Takanobu A Katoh, Katsura Minegishi, Takahiro Ide, Yayoi Ikawa, Hiromi Nishimura, Katsuyoshi Takaoka, Takeshi Itabashi, Atsuko H Iwane, Junichi Nakai, Hidetaka Shiratori, Hiroshi Hamada

Science advances　6　(30)　eaba1195　2020/07

DOI： 10.1126/sciadv.aba1195 　

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Immotile cilia sense extracellular signals such as fluid flow, but whether Ca2+ plays a role in flow sensing has been unclear. Here, we examined the role of ciliary Ca2+ in the flow sensing that initiates the breaking of left-right (L-R) symmetry in the mouse embryo. Intraciliary and cytoplasmic Ca2+ transients were detected in the crown cells at the node. These Ca2+ transients showed L-R asymmetry, which was lost in the absence of fluid flow or the PKD2 channel. Further characterization allowed classification of the Ca2+ transients into two types: cilium-derived, L-R-asymmetric transients (type 1) and cilium-independent transients without an L-R bias (type 2). Type 1 intraciliary transients occurred preferentially at the left posterior region of the node, where L-R symmetry breaking takes place. Suppression of intraciliary Ca2+ transients delayed L-R symmetry breaking. Our results implicate cilium-derived Ca2+ transients in crown cells in initiation of L-R symmetry breaking in the mouse embryo.
An aspherical microlens assembly for deep brain fluorescence microendoscopy. International-journal

Masaaki Sato, Shotaro Sano, Hiroki Watanabe, Yoko Kudo, Junichi Nakai

Biochemical and biophysical research communications　527　(2)　447-452　2020/06/25

DOI： 10.1016/j.bbrc.2020.04.009 　

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Fluorescence microendoscopy is becoming a standard technique in neuroscience for visualizing neuronal activity in the deep brain. Gradient refractive index (GRIN) lenses are increasingly used for fluorescence microendoscopy; however, they inherently suffer from strong aberrations and distortion. Aspherical lenses change their radius of curvature with distance from the optical axis and can effectively eliminate spherical aberrations. The use of these lenses has not been fully explored in deep brain fluorescence microendoscopy due to technical difficulties in manufacturing miniature aspherical lenses. In this study, we fabricated a novel microendoscope lens assembly comprised two nested pairs of aspherical microlenses made by precision glass molding. This assembly, which was 0.6 mm in diameter and 7.06 mm in length, was assembled in a stainless steel tube of 0.7 mm outer diameter. This assembly exhibited marked improvements in monochromatic and chromatic aberrations compared with a conventional GRIN lens, and is useful for deep brain fluorescence microendoscopy, as demonstrated by two-photon microendoscopic calcium imaging of R-CaMP1.07-labeled mouse hippocampal CA1 neurons. Our aspherical-lens-based approach offers a non-GRIN-lens alternative for fabrication of microendoscopic lenses.
Role of somite patterning in the formation of Weberian apparatus and pleural rib in zebrafish. International-journal

Kagari Akama, Kanami Ebata, Akiteru Maeno, Tomohito Taminato, Shiori Otosaka, Keiko Gengyo-Ando, Junichi Nakai, Kyo Yamasu, Akinori Kawamura

Journal of anatomy　236　(4)　622-629　2020/04

DOI： 10.1111/joa.13135 　

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In the vertebrate body, a metameric structure is present along the anterior-posterior axis. Zebrafish tbx6-/- larvae, in which somite boundaries do not form during embryogenesis, were shown to exhibit abnormal skeletal morphology such as rib, neural arch and hemal arch. In this study, we investigated the role of somite patterning in the formation of anterior vertebrae and ribs in more detail. Using three-dimensional computed tomography scans, we found that anterior vertebrae including the Weberian apparatus were severely affected in tbx6-/- larvae. In addition, pleural ribs of tbx6 mutants exhibited severe defects in the initial ossification, extension of ossification, and formation of parapophyses. Two-colour staining revealed that bifurcation of ribs was caused by fusion or branching of ribs in tbx6-/- . The parapophyses in tbx6-/- juvenile fish showed irregular positioning to centra and abnormal attachment to ribs. Furthermore, we found that the ossification of the distal portion of ribs proceeded along myotome boundaries even in irregularly positioned myotome boundaries. These results provide evidence of the contribution of somite patterning to the formation of the Weberian apparatus and rib in zebrafish.
Confocal and multiphoton calcium imaging of the enteric nervous system in anesthetized mice. International-journal Peer-reviewed

Yuki Motegi, Masaaki Sato, Kazuhide Horiguchi, Masamichi Ohkura, Keiko Gengyo-Ando, Yuji Ikegaya, Yasuyuki Fusamae, Yoshie Hongo, Minoru Suzuki, Koichi Ogawa, Miyako Takaki, Junichi Nakai

Neuroscience research　151　53-60　2020/02

DOI： 10.1016/j.neures.2019.02.004 　

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Most imaging studies of the enteric nervous system (ENS) that regulates the function of the gastrointestinal tract are so far performed using preparations isolated from animals, thus hindering the understanding of the ENS function in vivo. Here we report a method for imaging the ENS cellular network activity in living mice using a new transgenic mouse line that co-expresses G-CaMP6 and mCherry in the ENS combined with the suction-mediated stabilization of intestinal movements. With confocal or two-photon imaging, our method can visualize spontaneous and pharmacologically-evoked ENS network activity in living animals at cellular and subcellular resolutions, demonstrating the potential usefulness for studies of the ENS function in health and disease.
Orchestrated ensemble activities constitute a hippocampal memory engram. International-journal Peer-reviewed

Khaled Ghandour, Noriaki Ohkawa, Chi Chung Alan Fung, Hirotaka Asai, Yoshito Saitoh, Takashi Takekawa, Reiko Okubo-Suzuki, Shingo Soya, Hirofumi Nishizono, Mina Matsuo, Makoto Osanai, Masaaki Sato, Masamichi Ohkura, Junichi Nakai, Yasunori Hayashi, Takeshi Sakurai, Takashi Kitamura, Tomoki Fukai, Kaoru Inokuchi

Nature communications　10　(1)　2637-2637　2019/06/14
Publisher: Springer Science and Business Media LLC
DOI： 10.1038/s41467-019-10683-2 　

eISSN： 2041-1723

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The brain stores and recalls memories through a set of neurons, termed engram cells. However, it is unclear how these cells are organized to constitute a corresponding memory trace. We established a unique imaging system that combines Ca2+ imaging and engram identification to extract the characteristics of engram activity by visualizing and discriminating between engram and non-engram cells. Here, we show that engram cells detected in the hippocampus display higher repetitive activity than non-engram cells during novel context learning. The total activity pattern of the engram cells during learning is stable across post-learning memory processing. Within a single engram population, we detected several sub-ensembles composed of neurons collectively activated during learning. Some sub-ensembles preferentially reappear during post-learning sleep, and these replayed sub-ensembles are more likely to be reactivated during retrieval. These results indicate that sub-ensembles represent distinct pieces of information, which are then orchestrated to constitute an entire memory.
Wide and Deep Imaging of Neuronal Activities by a Wearable NeuroImager Reveals Premotor Activity in the Whole Motor Cortex. International-journal Peer-reviewed

Takuma Kobayashi, Tanvir Islam, Masaaki Sato, Masamichi Ohkura, Junichi Nakai, Yasunori Hayashi, Hitoshi Okamoto

Scientific reports　9　(1)　8366-8366　2019/06/10

DOI： 10.1038/s41598-019-44146-x 　

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Wearable technologies for functional whole brain imaging in freely moving animals would advance our understanding of cognitive processing and adaptive behavior. Fluorescence imaging can visualize the activity of individual neurons in real time, but conventional microscopes have limited sample coverage in both the width and depth of view. Here we developed a novel head-mounted laser camera (HLC) with macro and deep-focus lenses that enable fluorescence imaging at cellular resolution for comprehensive imaging in mice expressing a layer- and cell type-specific calcium probe. We visualized orientation selectivity in individual excitatory neurons across the whole visual cortex of one hemisphere, and cell assembly expressing the premotor activity that precedes voluntary movement across the motor cortex of both hemispheres. Including options for multiplex and wireless interfaces, our wearable, wide- and deep-imaging HLC technology could enable simple and economical mapping of neuronal populations underlying cognition and behavior.
Self-organizing cell tactile perception which depends on mechanical stimulus history Peer-reviewed

Masahiro Shimizu, Kosuke Minzan, Hiroki Kawashima, Kota Miyasaka, Takuya Umedachi, Toshihiko Ogura, Junichi Nakai, Masamichi Ohkura, Koh Hosoda

Advanced Robotics　33　(5)　232-242　2019/03/04

DOI： 10.1080/01691864.2019.1590232 　

ISSN： 0169-1864

eISSN： 1568-5535
ELKS/Voltage-Dependent Ca2+ Channel-β Subunit Module Regulates Polarized Ca2+ Influx in Pancreatic β Cells. International-journal Peer-reviewed

Mica Ohara-Imaizumi, Kyota Aoyagi, Hajime Yamauchi, Masashi Yoshida, Masayuki X Mori, Yamato Hida, Ha Nam Tran, Masamichi Ohkura, Manabu Abe, Yoshihiro Akimoto, Yoko Nakamichi, Chiyono Nishiwaki, Hayato Kawakami, Kazuo Hara, Kenji Sakimura, Shinya Nagamatsu, Yasuo Mori, Junichi Nakai, Masafumi Kakei, Toshihisa Ohtsuka

Cell reports　26　(5)　1213-1226　2019/01/29

DOI： 10.1016/j.celrep.2018.12.106 　

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Pancreatic β cells secrete insulin by Ca2+-triggered exocytosis. However, there is no apparent secretory site similar to the neuronal active zones, and the cellular and molecular localization mechanism underlying polarized exocytosis remains elusive. Here, we report that ELKS, a vertebrate active zone protein, is used in β cells to regulate Ca2+ influx for insulin secretion. β cell-specific ELKS-knockout (KO) mice showed impaired glucose-stimulated first-phase insulin secretion and reduced L-type voltage-dependent Ca2+ channel (VDCC) current density. In situ Ca2+ imaging of β cells within islets expressing a membrane-bound G-CaMP8b Ca2+ sensor demonstrated initial local Ca2+ signals at the ELKS-localized vascular side of the β cell plasma membrane, which were markedly decreased in ELKS-KO β cells. Mechanistically, ELKS directly interacted with the VDCC-β subunit via the GK domain. These findings suggest that ELKS and VDCCs form a potent insulin secretion complex at the vascular side of the β cell plasma membrane for polarized Ca2+ influx and first-phase insulin secretion from pancreatic islets.
Super-wide-field two-photon imaging with a micro-optical device moving in post-objective space. International-journal Peer-reviewed

Shin-Ichiro Terada, Kenta Kobayashi, Masamichi Ohkura, Junichi Nakai, Masanori Matsuzaki

Nature communications　9　(1)　3550-3550　2018/09/03

DOI： 10.1038/s41467-018-06058-8 　

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Wide-field imaging of neural activity at a cellular resolution is a current challenge in neuroscience. To address this issue, wide-field two-photon microscopy has been developed; however, the field size is limited by the objective size. Here, we develop a micro-opto-mechanical device that rotates within the post-objective space between the objective and brain tissue. Two-photon microscopy with this device enables sub-second sequential calcium imaging of left and right mouse sensory forelimb areas 6 mm apart. When imaging the rostral and caudal motor forelimb areas (RFA and CFA) 2 mm apart, we found high pairwise correlations in spontaneous activity between RFA and CFA neurons and between an RFA neuron and its putative axons in CFA. While mice performed a sound-triggered forelimb-movement task, the population activity between RFA and CFA covaried across trials, although the field-averaged activity was similar across trials. The micro-opto-mechanical device in the post-objective space provides a novel and flexible design to clarify the correlation structure between distant brain areas at subcellular and population levels.
アルツハイマー病モデルマウス海馬CA1領域における神経機能回路の段階的破綻

高村理沙, 水田恒太郎, 関根友紀子, イスラム・タンビル, 斉藤貴志, 竹川高志, 大倉正道, 深井朋樹, 中井淳一, 大島登志男, 西道隆臣, 林康紀

Dementia Japan　32　(3)　415-415　2018/09
Publisher: (一社)日本認知症学会
ISSN： 1342-646X
Higher visual responses in the temporal cortex of mice. International-journal

Nana Nishio, Hiroaki Tsukano, Ryuichi Hishida, Manabu Abe, Junichi Nakai, Meiko Kawamura, Atsushi Aiba, Kenji Sakimura, Katsuei Shibuki

Scientific reports　8　(1)　11136-11136　2018/07/24

DOI： 10.1038/s41598-018-29530-3 　

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The visual cortex of mice is a useful model for investigating the mammalian visual system. In primates, higher visual areas are classified into two parts, the dorsal stream ("where" pathway) and ventral stream ("what" pathway). The ventral stream is known to include a part of the temporal cortex. In mice, however, some cortical areas adjacent to the primary visual area (V1) in the occipital cortex are thought to be comparable to the ventral stream in primates, although the whole picture of the mouse ventral stream has never been elucidated. We performed wide-field Ca2+ imaging in awake mice to investigate visual responses in the mouse temporal cortex, and found that the postrhinal cortex (POR), posterior to the auditory cortex (AC), and the ectorhinal and temporal association cortices (ECT), ventral to the AC, showed clear visual responses to moving visual objects. The retinotopic maps in the POR and ECT were not clearly observed, and the amplitudes of the visual responses in the POR and ECT were less sensitive to the size of the objects, compared to visual responses in the V1. In the ECT, objects of different sizes activated different subareas. These findings strongly suggest that the mouse ventral stream extends to the ECT ventral to the AC, and that it has characteristic response properties that are markedly different from the response properties in the V1.
A Critical Neurodevelopmental Role for L-Type Voltage-Gated Calcium Channels in Neurite Extension and Radial Migration. International-journal Peer-reviewed

Satoshi Kamijo, Yuichiro Ishii, Shin-Ichiro Horigane, Kanzo Suzuki, Masamichi Ohkura, Junichi Nakai, Hajime Fujii, Sayaka Takemoto-Kimura, Haruhiko Bito

The Journal of neuroscience : the official journal of the Society for Neuroscience　38　(24)　5551-5566　2018/06/13

DOI： 10.1523/JNEUROSCI.2357-17.2018 　

ISSN： 0270-6474

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Despite many association studies linking gene polymorphisms and mutations of L-type voltage-gated Ca2+ channels (VGCCs) in neurodevelopmental disorders such as autism and schizophrenia, the roles of specific L-type VGCC during brain development remain unclear. Calcium signaling has been shown to be essential for neurodevelopmental processes such as sculpting of neurites, functional wiring, and fine tuning of growing networks. To investigate this relationship, we performed submembraneous calcium imaging using a membrane-tethered genetically encoded calcium indicator (GECI) Lck-G-CaMP7. We successfully recorded spontaneous regenerative calcium transients (SRCaTs) in developing mouse excitatory cortical neurons prepared from both sexes before synapse formation. SRCaTs originated locally in immature neurites independently of somatic calcium rises and were significantly more elevated in the axons than in dendrites. SRCaTs were not blocked by tetrodoxin, a Na+ channel blocker, but were strongly inhibited by hyperpolarization, suggesting a voltage-dependent source. Pharmacological and genetic manipulations revealed the critical importance of the Cav1.2 (CACNA1C) pore-forming subunit of L-type VGCCs, which were indeed expressed in immature mouse brains. Consistently, knocking out Cav1.2 resulted in significant alterations of neurite outgrowth. Furthermore, expression of a gain-of-function Cav1.2 mutant found in Timothy syndrome, an autosomal dominant multisystem disorder exhibiting syndromic autism, resulted in impaired radial migration of layer 2/3 excitatory neurons, whereas postnatal abrogation of Cav1.2 enhancement could rescue cortical malformation. Together, these lines of evidence suggest a critical role for spontaneous opening of L-type VGCCs in neural development and corticogenesis and indicate that L-type VGCCs might constitute a perinatal therapeutic target for neuropsychiatric calciochannelopathies.SIGNIFICANCE STATEMENT Despite many association studies linking gene polymorphisms and mutations of L-type voltage-gated Ca2+ channels (VGCCs) in neurodevelopmental disorders such as autism and schizophrenia, the roles of specific L-type VGCCs during brain development remain unclear. We here combined the latest Ca2+ indicator technology, quantitative pharmacology, and in utero electroporation and found a hitherto unsuspected role for L-type VGCCs in determining the Ca2+ signaling landscape of mouse immature neurons. We found that malfunctional L-type VGCCs in immature neurons before birth might cause errors in neuritic growth and cortical migration. Interestingly, the retarded corticogenesis phenotype was rescued by postnatal correction of L-type VGCC signal aberration. These findings suggest that L-type VGCCs might constitute a perinatal therapeutic target for neurodevelopment-associated psychiatric disorders.
In vivo wide-field calcium imaging of mouse thalamocortical synapses with an 8 K ultra-high-definition camera. International-journal Peer-reviewed

Eriko Yoshida, Shin-Ichiro Terada, Yasuyo H Tanaka, Kenta Kobayashi, Masamichi Ohkura, Junichi Nakai, Masanori Matsuzaki

Scientific reports　8　(1)　8324-8324　2018/05/29

DOI： 10.1038/s41598-018-26566-3 　

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In vivo wide-field imaging of neural activity with a high spatio-temporal resolution is a challenge in modern neuroscience. Although two-photon imaging is very powerful, high-speed imaging of the activity of individual synapses is mostly limited to a field of approximately 200 µm on a side. Wide-field one-photon epifluorescence imaging can reveal neuronal activity over a field of ≥1 mm2 at a high speed, but is not able to resolve a single synapse. Here, to achieve a high spatio-temporal resolution, we combine an 8 K ultra-high-definition camera with spinning-disk one-photon confocal microscopy. This combination allowed us to image a 1 mm2 field with a pixel resolution of 0.21 µm at 60 fps. When we imaged motor cortical layer 1 in a behaving head-restrained mouse, calcium transients were detected in presynaptic boutons of thalamocortical axons sparsely labeled with GCaMP6s, although their density was lower than when two-photon imaging was used. The effects of out-of-focus fluorescence changes on calcium transients in individual boutons appeared minimal. Axonal boutons with highly correlated activity were detected over the 1 mm2 field, and were probably distributed on multiple axonal arbors originating from the same thalamic neuron. This new microscopy with an 8 K ultra-high-definition camera should serve to clarify the activity and plasticity of widely distributed cortical synapses.
Dynamic embedding of salience coding in hippocampal spatial maps Peer-reviewed

Sato M, Mizuta K, Islam T, Kawano M, Takekawa T, Gomez-Dominguez D, Kim K, Yamakawa H, Ohkura M, Fukai T, Nakai J, Hayashi Y

2018/02

DOI： 10.1101/266767 　
Common Defects of Spine Dynamics and Circuit Function in Neurodevelopmental Disorders: A Systematic Review of Findings From in Vivo Optical Imaging of Mouse Models. International-journal

Nobuhiro Nakai, Toru Takumi, Junichi Nakai, Masaaki Sato

Frontiers in neuroscience　12　412-412　2018

DOI： 10.3389/fnins.2018.00412 　

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In vivo optical imaging is a powerful tool for revealing brain structure and function at both the circuit and cellular levels. Here, we provide a systematic review of findings obtained from in vivo imaging studies of mouse models of neurodevelopmental disorders, including the monogenic disorders fragile X syndrome, Rett syndrome, and Angelman syndrome, which are caused by genetic abnormalities of FMR1, MECP2, and UBE3A, as well as disorders caused by copy number variations (15q11-13 duplication and 22q11.2 deletion) and BTBR mice as an inbred strain model of autism spectrum disorder (ASD). Most studies visualize the structural and functional responsiveness of cerebral cortical neurons to sensory stimuli and the developmental and experience-dependent changes in these responses as a model of brain functions affected by these disorders. The optical imaging techniques include two-photon microscopy of fluorescently labeled dendritic spines or neurons loaded with fluorescent calcium indicators and macroscopic imaging of cortical activity using calcium indicators, voltage-sensitive dyes or intrinsic optical signals. Studies have revealed alterations in the density, stability, and turnover of dendritic spines, aberrant cortical sensory responses, impaired inhibitory function, and concomitant failure of circuit maturation as common causes for neurological deficits. Mechanistic hypotheses derived from in vivo imaging also provide new directions for therapeutic interventions. For instance, it was recently demonstrated that early postnatal administration of a selective serotonin reuptake inhibitor (SSRI) restores impaired cortical inhibitory function and ameliorates the aberrant social behaviors in a mouse model of ASD. We discuss the potential use of SSRIs for treating ASDs in light of these findings.
Role of tyramine in calcium dynamics of GABAergic neurons and escape behavior in Caenorhabditis elegans. International-journal Peer-reviewed

Yuko Kagawa-Nagamura, Keiko Gengyo-Ando, Masamichi Ohkura, Junichi Nakai

Zoological letters　4　19-19　2018

DOI： 10.1186/s40851-018-0103-1 　

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Background: Tyramine, known as a "trace amine" in mammals, modulates a wide range of behavior in invertebrates; however, the underlying cellular and circuit mechanisms are not well understood. In the nematode Caenorhabditis elegans (C. elegans), tyramine affects key behaviors, including foraging, feeding, and escape responses. The touch-evoked backward escape response is often coupled with a sharp omega turn that allows the animal to navigate away in the opposite direction. Previous studies have showed that a metabotropic tyramine receptor, SER-2, in GABAergic body motor neurons controls deep body bending in omega turns. In this study, we focused on the role of tyramine in GABAergic head motor neurons. Our goal is to understand the mechanism by which tyraminergic signaling alters neural circuit activity to control escape behavior. Results: Using calcium imaging in freely moving C. elegans, we found that GABAergic RME motor neurons in the head had high calcium levels during forward locomotion but low calcium levels during spontaneous and evoked backward locomotion. This calcium decrease was also observed during the omega turn. Mutant analyses showed that tbh-1 mutants lacking only octopamine had normal calcium responses, whereas tdc-1 mutants lacking both tyramine and octopamine did not exhibit the calcium decrease in RME. This neuromodulation was mediated by SER-2. Moreover, tyraminergic RIM neuron activity was negatively correlated with RME activity in the directional switch from forward to backward locomotion. These results indicate that tyramine released from RIM inhibits RME via SER-2 signaling. The omega turn is initiated by a sharp head bend when the animal reinitiates forward movement. Interestingly, ser-2 mutants exhibited shallow head bends and often failed to execute deep-angle omega turns. The behavioral defect and the abnormal calcium response in ser-2 mutants could be rescued by SER-2 expression in RME. These results suggest that tyraminergic inhibition of RME is involved in the control of omega turns. Conclusion: We demonstrate that endogenous tyramine downregulates calcium levels in GABAergic RME motor neurons in the head via the tyramine receptor SER-2 during backward locomotion and omega turns. Our data suggest that this neuromodulation allows deep head bending during omega turns and plays a role in the escape behavior in C. elegans.
Two-photon calcium imaging of the medial prefrontal cortex and hippocampus without cortical invasion. International-journal Peer-reviewed

Masashi Kondo, Kenta Kobayashi, Masamichi Ohkura, Junichi Nakai, Masanori Matsuzaki

eLife　6　e26839　2017/09/25

DOI： 10.7554/eLife.26839 　

ISSN： 2050-084X
Fast varifocal two-photon microendoscope for imaging neuronal activity in the deep brain. International-journal Peer-reviewed

Masaaki Sato, Yuki Motegi, Shogo Yagi, Keiko Gengyo-Ando, Masamichi Ohkura, Junichi Nakai

Biomedical optics express　8　(9)　4049-4060　2017/09/01

DOI： 10.1364/BOE.8.004049 　

ISSN： 2156-7085
A new platform for long-term tracking and recording of neural activity and simultaneous optogenetic control in freely behaving Caenorhabditis elegans. International-journal Peer-reviewed

Keiko Gengyo-Ando, Yuko Kagawa-Nagamura, Masamichi Ohkura, Xianfeng Fei, Min Chen, Koichi Hashimoto, Junichi Nakai

Journal of neuroscience methods　286　56-68　2017/07/15

DOI： 10.1016/j.jneumeth.2017.05.017 　

ISSN： 0165-0270

eISSN： 1872-678X
Calcium dynamics regulating the timing of decision-making in C. elegans. International-journal

Yuki Tanimoto, Akiko Yamazoe-Umemoto, Kosuke Fujita, Yuya Kawazoe, Yosuke Miyanishi, Shuhei J Yamazaki, Xianfeng Fei, Karl Emanuel Busch, Keiko Gengyo-Ando, Junichi Nakai, Yuichi Iino, Yuishi Iwasaki, Koichi Hashimoto, Koutarou D Kimura

eLife　6　2017/05/23

DOI： 10.7554/eLife.21629 　

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Brains regulate behavioral responses with distinct timings. Here we investigate the cellular and molecular mechanisms underlying the timing of decision-making during olfactory navigation in Caenorhabditis elegans. We find that, based on subtle changes in odor concentrations, the animals appear to choose the appropriate migratory direction from multiple trials as a form of behavioral decision-making. Through optophysiological, mathematical and genetic analyses of neural activity under virtual odor gradients, we further find that odor concentration information is temporally integrated for a decision by a gradual increase in intracellular calcium concentration ([Ca2+]i), which occurs via L-type voltage-gated calcium channels in a pair of olfactory neurons. In contrast, for a reflex-like behavioral response, [Ca2+]i rapidly increases via multiple types of calcium channels in a pair of nociceptive neurons. Thus, the timing of neuronal responses is determined by cell type-dependent involvement of calcium channels, which may serve as a cellular basis for decision-making.
An R-CaMP1.07 reporter mouse for cell-type-specific expression of a sensitive red fluorescent calcium indicator. International-journal Peer-reviewed

Philipp Bethge, Stefano Carta, Dayra A Lorenzo, Ladan Egolf, Despoina Goniotaki, Linda Madisen, Fabian F Voigt, Jerry L Chen, Bernard Schneider, Masamichi Ohkura, Junichi Nakai, Hongkui Zeng, Adriano Aguzzi, Fritjof Helmchen

PloS one　12　(6)　e0179460　2017

DOI： 10.1371/journal.pone.0179460 　

ISSN： 1932-6203
Olfactory receptor for prostaglandin F-2 alpha mediates courtship behavior of male zebrafish Peer-reviewed

Yabuki Yoichi, Koide Tetsuya, Miyasaka Nobuhiko, Wakisaka Noriko, Masuda Miwa, Ohkura Masamichi, Nakai Junichi, Tsuge Kyoshiro, Tsuchiya Soken, Sugimoto Yukihiko, Yoshihara Yoshihiro

CHEMICAL SENSES　41　(9)　E143　2016/11

ISSN： 0379-864X
Distinct roles of the two VPS33 proteins in the endolysosomal system in Caenorhabditis elegans. International-journal

Keiko Gengyo-Ando, Eriko Kage-Nakadai, Sawako Yoshina, Muneyoshi Otori, Yuko Kagawa-Nagamura, Junichi Nakai, Shohei Mitani

Traffic (Copenhagen, Denmark)　17　(11)　1197-1213　2016/11

DOI： 10.1111/tra.12430 　

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Sec1/Munc-18 (SM) family proteins are essential regulators in intracellular transport in eukaryotic cells. The SM protein Vps33 functions as a core subunit of two tethering complexes, class C core vacuole/endosome tethering (CORVET) and homotypic fusion and vacuole protein sorting (HOPS) in the endocytic pathway in yeast. Metazoan cells possess two Vps33 proteins, VPS33A and VPS33B, but their precise roles remain unknown. Here, we present a comparative analysis of Caenorhabditis elegans null mutants for these proteins. We found that the vps-33.1 (VPS33A) mutants exhibited severe defects in both endocytic function and endolysosomal biogenesis in scavenger cells. Furthermore, vps-33.1 mutations caused endocytosis defects in other tissues, and the loss of maternal and zygotic VPS-33.1 resulted in embryonic lethality. By contrast, vps-33.2 mutants were viable but sterile, with terminally arrested spermatocytes. The spermatogenesis phenotype suggests that VPS33.2 is involved in the formation of a sperm-specific organelle. The endocytosis defect in the vps-33.1 mutant was not restored by the expression of VPS-33.2, which indicates that these proteins have nonredundant functions. Together, our data suggest that VPS-33.1 shares most of the general functions of yeast Vps33 in terms of tethering complexes in the endolysosomal system, whereas VPS-33.2 has tissue/organelle specific functions in C. elegans.
Allogeneic transplantation of iPS cell-derived cardiomyocytes regenerates primate hearts. International-journal

Yuji Shiba, Toshihito Gomibuchi, Tatsuichiro Seto, Yuko Wada, Hajime Ichimura, Yuki Tanaka, Tatsuki Ogasawara, Kenji Okada, Naoko Shiba, Kengo Sakamoto, Daisuke Ido, Takashi Shiina, Masamichi Ohkura, Junichi Nakai, Narumi Uno, Yasuhiro Kazuki, Mitsuo Oshimura, Itsunari Minami, Uichi Ikeda

Nature　538　(7625)　388-391　2016/10/20

DOI： 10.1038/nature19815 　

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Induced pluripotent stem cells (iPSCs) constitute a potential source of autologous patient-specific cardiomyocytes for cardiac repair, providing a major benefit over other sources of cells in terms of immune rejection. However, autologous transplantation has substantial challenges related to manufacturing and regulation. Although major histocompatibility complex (MHC)-matched allogeneic transplantation is a promising alternative strategy, few immunological studies have been carried out with iPSCs. Here we describe an allogeneic transplantation model established using the cynomolgus monkey (Macaca fascicularis), the MHC structure of which is identical to that of humans. Fibroblast-derived iPSCs were generated from a MHC haplotype (HT4) homozygous animal and subsequently differentiated into cardiomyocytes (iPSC-CMs). Five HT4 heterozygous monkeys were subjected to myocardial infarction followed by direct intra-myocardial injection of iPSC-CMs. The grafted cardiomyocytes survived for 12 weeks with no evidence of immune rejection in monkeys treated with clinically relevant doses of methylprednisolone and tacrolimus, and showed electrical coupling with host cardiomyocytes as assessed by use of the fluorescent calcium indicator G-CaMP7.09. Additionally, transplantation of the iPSC-CMs improved cardiac contractile function at 4 and 12 weeks after transplantation; however, the incidence of ventricular tachycardia was transiently, but significantly, increased when compared to vehicle-treated controls. Collectively, our data demonstrate that allogeneic iPSC-CM transplantation is sufficient to regenerate the infarcted non-human primate heart; however, further research to control post-transplant arrhythmias is necessary.
A New In Vitro Co-Culture Model Using Magnetic Force-Based Nanotechnology. International-journal

Hiroki Takanari, Keiko Miwa, XianMing Fu, Junichi Nakai, Akira Ito, Kousuke Ino, Hiroyuki Honda, Wataru Tonomura, Satoshi Konishi, Tobias Opthof, Marcel Ag van der Heyden, Itsuo Kodama, Jong-Kook Lee

Journal of cellular physiology　231　(10)　2249-56　2016/10

DOI： 10.1002/jcp.25342 　

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Skeletal myoblast (SkMB) transplantation has been conducted as a therapeutic strategy for severe heart failure. However, arrhythmogenicity following transplantation remains unsolved. We developed an in vitro model of myoblast transplantation with "patterned" or "randomly-mixed" co-culture of SkMBs and cardiomyocytes enabling subsequent electrophysiological, and arrhythmogenic evaluation. SkMBs were magnetically labeled with magnetite nanoparticles and co-cultured with neonatal rat ventricular myocytes (NRVMs) on multi-electrode arrays. SkMBs were patterned by a magnet beneath the arrays. Excitation synchronicity was evaluated by Ca(2+) imaging using a gene-encoded Ca(2+) indicator, G-CaMP2. In the monoculture of NRVMs (control), conduction was well-organized. In the randomly-mixed co-culture of NRVMs and SkMBs (random group), there was inhomogeneous conduction from multiple origins. In the "patterned" co-culture where an en bloc SKMB-layer was inserted into the NRVM-layer, excitation homogenously propagated although conduction was distorted by the SkMB-area. The 4-mm distance conduction time (CT) in the random group was significantly longer (197 ± 126 ms) than in control (17 ± 3 ms). In the patterned group, CT through NRVM-area did not change (25 ± 3 ms), although CT through the SkMB-area was significantly longer (132 ± 77 ms). The intervals between spontaneous excitation varied beat-to-beat in the random group, while regular beating was recorded in the control and patterned groups. Synchronized Ca(2+) transients of NRVMs were observed in the patterned group, whereas those in the random group were asynchronous. Patterned alignment of SkMBs is feasible with magnetic nanoparticles. Using the novel in vitro model mimicking cell transplantation, it may become possible to predict arrhythmogenicity due to heterogenous cell transplantation. J. Cell. Physiol. 231: 2249-2256, 2016. © 2016 Wiley Periodicals, Inc.
The integrative role of orexin/hypocretin neurons in nociceptive perception and analgesic regulation. International-journal Peer-reviewed

Ayumu Inutsuka, Akira Yamashita, Srikanta Chowdhury, Junichi Nakai, Masamichi Ohkura, Toru Taguchi, Akihiro Yamanaka

Scientific reports　6　29480-29480　2016/07/07

DOI： 10.1038/srep29480 　

ISSN： 2045-2322
Olfactory receptor for prostaglandin F2α mediates male fish courtship behavior. International-journal Peer-reviewed

Yoichi Yabuki, Tetsuya Koide, Nobuhiko Miyasaka, Noriko Wakisaka, Miwa Masuda, Masamichi Ohkura, Junichi Nakai, Kyoshiro Tsuge, Soken Tsuchiya, Yukihiko Sugimoto, Yoshihiro Yoshihara

Nature neuroscience　19　(7)　897-904　2016/07

DOI： 10.1038/nn.4314 　

ISSN： 1097-6256
G-CaMP7発現マウスを用いた大脳皮質神経活動のミクロ、マクロ解析法

茂木優貴, 安藤恵子, 沖篤志, 岩井陽一, 毛内拡, 平瀬肇, 大倉正道, 中井淳一

日本生理学雑誌　78　(3)　56-56　2016/05
Publisher: (一社)日本生理学会
ISSN： 0031-9341
Calcium imaging reveals glial involvement in transcranial direct current stimulation-induced plasticity in mouse brain. International-journal Peer-reviewed

Hiromu Monai, Masamichi Ohkura, Mika Tanaka, Yuki Oe, Ayumu Konno, Hirokazu Hirai, Katsuhiko Mikoshiba, Shigeyoshi Itohara, Junichi Nakai, Youichi Iwai, Hajime Hirase

Nature communications　7　11100-11100　2016/03/22

DOI： 10.1038/ncomms11100 　

ISSN： 2041-1723
G-CaMP7発現マウス大脳皮質脳細胞活動の細胞-領野スケール長期イメージング実験解析法

茂木優貴, 安藤恵子, 沖篤志, 岩井陽一, 毛内拡, 平瀬肇, 大倉正道, 中井淳一

日本生化学会大会・日本分子生物学会年会合同大会講演要旨集　88回・38回　[1LBA113]-[1LBA113]　2015/12
Publisher: (公社)日本生化学会
REI-1 Is a Guanine Nucleotide Exchange Factor Regulating RAB-11 Localization and Function in C. elegans Embryos. International-journal

Aisa Sakaguchi, Miyuki Sato, Katsuya Sato, Keiko Gengyo-Ando, Tomohiro Yorimitsu, Junichi Nakai, Taichi Hara, Ken Sato, Ken Sato

Developmental cell　35　(2)　211-21　2015/10/26

DOI： 10.1016/j.devcel.2015.09.013 　

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The small GTPase Rab11 dynamically changes its location to regulate various cellular processes such as endocytic recycling, secretion, and cytokinesis. However, our knowledge of its upstream regulators is still limited. Here, we identify the RAB-11-interacting protein-1 (REI-1) as a unique family of guanine nucleotide exchange factors (GEFs) for RAB-11 in Caenorhabditis elegans. Although REI-1 and its human homolog SH3-binding protein 5 do not contain any known Rab-GEF domains, they exhibited strong GEF activity toward Rab11 in vitro. In C. elegans, REI-1 is expressed in the germline and co-localizes with RAB-11 on the late-Golgi membranes. The loss of REI-1 specifically impaired the targeting of RAB-11 to the late-Golgi compartment and the recycling endosomes in embryos and further reduced the RAB-11 distribution to the cleavage furrow, which resulted in cytokinesis delay. These results suggest that REI-1 is a GEF specifically regulating the RAB-11 localization and functions in early embryos.
JSAP1/JIP3 and JLP regulate kinesin-1-dependent axonal transport to prevent neuronal degeneration. Peer-reviewed

Sato T, Ishikawa M, Mochizuki M, Ohta M, Ohkura M, Nakai J, Takamatsu N, Yoshioka K

Cell death and differentiation　22　(8)　1260-1274　2015/08

DOI： 10.1038/cdd.2014.207 　

ISSN： 1350-9047
A new transgenic mouse model revealed a role of astrocytic calcium in vivo

Mika Tanaka, Pei-Yu Shih, Hiroshi Gomi, Takamasa Yoshida, Junichi Nakai, Reiko Ando, Teiichi Furuichi, Katsuhiko Mikoshiba, Alexey Semyanov, Shigeyoshi Itohara

JOURNAL OF PHARMACOLOGICAL SCIENCES　128　(3)　S38-S38　2015/07

ISSN： 1347-8613

eISSN： 1347-8648
A Top-Down Cortical Circuit for Accurate Sensory Perception. International-journal Peer-reviewed

Satoshi Manita, Takayuki Suzuki, Chihiro Homma, Takashi Matsumoto, Maya Odagawa, Kazuyuki Yamada, Keisuke Ota, Chie Matsubara, Ayumu Inutsuka, Masaaki Sato, Masamichi Ohkura, Akihiro Yamanaka, Yuchio Yanagawa, Junichi Nakai, Yasunori Hayashi, Matthew E Larkum, Masanori Murayama

Neuron　86　(5)　1304-16　2015/06/03

DOI： 10.1016/j.neuron.2015.05.006 　

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A fundamental issue in cortical processing of sensory information is whether top-down control circuits from higher brain areas to primary sensory areas not only modulate but actively engage in perception. Here, we report the identification of a neural circuit for top-down control in the mouse somatosensory system. The circuit consisted of a long-range reciprocal projection between M2 secondary motor cortex and S1 primary somatosensory cortex. In vivo physiological recordings revealed that sensory stimulation induced sequential S1 to M2 followed by M2 to S1 neural activity. The top-down projection from M2 to S1 initiated dendritic spikes and persistent firing of S1 layer 5 (L5) neurons. Optogenetic inhibition of M2 input to S1 decreased L5 firing and the accurate perception of tactile surfaces. These findings demonstrate that recurrent input to sensory areas is essential for accurate perception and provide a physiological model for one type of top-down control circuit.
Comparison of genetically encoded calcium indicators for monitoring action potentials in mammalian brain by two-photon excitation fluorescence microscopy. International-journal Peer-reviewed

Borbala Podor, Yi-Ling Hu, Masamichi Ohkura, Junichi Nakai, Roger Croll, Alan Fine

Neurophotonics　2　(2)　021014-021014　2015/04

DOI： 10.1117/1.NPh.2.2.021014 　

ISSN： 2329-423X 2329-4248

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Imaging calcium transients associated with neuronal activity has yielded important insights into neural physiology. Genetically encoded calcium indicators (GECIs) offer conspicuous potential advantages for this purpose, including exquisite targeting. While the catalogue of available GECIs is steadily growing, many newly developed sensors that appear promising in vitro or in model cells appear to be less useful when expressed in mammalian neurons. We have, therefore, evaluated the performance of GECIs from two of the most promising families of sensors, G-CaMPs [Nat. Biotechnol.2(2), 137-141 (2001)] and GECOs [Science2(2), 1888-1891 (2011)], for monitoring action potentials in rat brain. Specifically, we used two-photon excitation fluorescence microscopy to compare calcium transients detected by G-CaMP3; GCaMP6f; G-CaMP7; Green-GECO1.0, 1.1 and 1.2; Blue-GECO; Red-GECO; Rex-GECO0.9; Rex-GECO1; Carmine-GECO; Orange-GECO; and Yellow-GECO1s. After optimizing excitation wavelengths, we monitored fluorescence signals associated with increasing numbers of action potentials evoked by current injection in CA1 pyramidal neurons in rat organotypic hippocampal slices. Some GECIs, particularly Green-GECO1.2, GCaMP6f, and G-CaMP7, were able to detect single action potentials with high reliability. By virtue of greatest sensitivity and fast kinetics, G-CaMP7 may be the best currently available GECI for monitoring calcium transients in mammalian neurons.
RING finger protein 121 facilitates the degradation and membrane localization of voltage-gated sodium channels. International-journal

Kazutoyo Ogino, Sean E Low, Kenta Yamada, Louis Saint-Amant, Weibin Zhou, Akira Muto, Kazuhide Asakawa, Junichi Nakai, Koichi Kawakami, John Y Kuwada, Hiromi Hirata

Proceedings of the National Academy of Sciences of the United States of America　112　(9)　2859-64　2015/03/03

DOI： 10.1073/pnas.1414002112 　

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Following their synthesis in the endoplasmic reticulum (ER), voltage-gated sodium channels (NaV) are transported to the membranes of excitable cells, where they often cluster, such as at the axon initial segment of neurons. Although the mechanisms by which NaV channels form and maintain clusters have been extensively examined, the processes that govern their transport and degradation have received less attention. Our entry into the study of these processes began with the isolation of a new allele of the zebrafish mutant alligator, which we found to be caused by mutations in the gene encoding really interesting new gene (RING) finger protein 121 (RNF121), an E3-ubiquitin ligase present in the ER and cis-Golgi compartments. Here we demonstrate that RNF121 facilitates two opposing fates of NaV channels: (i) ubiquitin-mediated proteasome degradation and (ii) membrane localization when coexpressed with auxiliary NaVβ subunits. Collectively, these results indicate that RNF121 participates in the quality control of NaV channels during their synthesis and subsequent transport to the membrane.
Mitochondrial Ca(2+) uptake by the voltage-dependent anion channel 2 regulates cardiac rhythmicity. International-journal

Hirohito Shimizu, Johann Schredelseker, Jie Huang, Kui Lu, Shamim Naghdi, Fei Lu, Sarah Franklin, Hannah Dg Fiji, Kevin Wang, Huanqi Zhu, Cheng Tian, Billy Lin, Haruko Nakano, Amy Ehrlich, Junichi Nakai, Adam Z Stieg, James K Gimzewski, Atsushi Nakano, Joshua I Goldhaber, Thomas M Vondriska, György Hajnóczky, Ohyun Kwon, Jau-Nian Chen

eLife　4　2015/01/15

DOI： 10.7554/eLife.04801 　

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Tightly regulated Ca(2+) homeostasis is a prerequisite for proper cardiac function. To dissect the regulatory network of cardiac Ca(2+) handling, we performed a chemical suppressor screen on zebrafish tremblor embryos, which suffer from Ca(2+) extrusion defects. Efsevin was identified based on its potent activity to restore coordinated contractions in tremblor. We show that efsevin binds to VDAC2, potentiates mitochondrial Ca(2+) uptake and accelerates the transfer of Ca(2+) from intracellular stores into mitochondria. In cardiomyocytes, efsevin restricts the temporal and spatial boundaries of Ca(2+) sparks and thereby inhibits Ca(2+) overload-induced erratic Ca(2+) waves and irregular contractions. We further show that overexpression of VDAC2 recapitulates the suppressive effect of efsevin on tremblor embryos whereas VDAC2 deficiency attenuates efsevin's rescue effect and that VDAC2 functions synergistically with MCU to suppress cardiac fibrillation in tremblor. Together, these findings demonstrate a critical modulatory role for VDAC2-dependent mitochondrial Ca(2+) uptake in the regulation of cardiac rhythmicity.
Optogenetic manipulation and probing Peer-reviewed

Masamichi Ohkura, Junko Sadakari, Junichi Nakai

Optogenetics: Light-Sensing Proteins and their Applications　133-148　2015/01/01
Publisher: Springer Japan
DOI： 10.1007/978-4-431-55516-2_9 　
Rational design of a high-affinity, fast, red calcium indicator R-CaMP2. International-journal Peer-reviewed

Masatoshi Inoue, Atsuya Takeuchi, Shin-ichiro Horigane, Masamichi Ohkura, Keiko Gengyo-Ando, Hajime Fujii, Satoshi Kamijo, Sayaka Takemoto-Kimura, Masanobu Kano, Junichi Nakai, Kazuo Kitamura, Haruhiko Bito

Nature methods　12　(1)　64-70　2015/01

DOI： 10.1038/nmeth.3185 　

ISSN： 1548-7091

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Fluorescent Ca(2+) reporters are widely used as readouts of neuronal activities. Here we designed R-CaMP2, a high-affinity red genetically encoded calcium indicator (GECI) with a Hill coefficient near 1. Use of the calmodulin-binding sequence of CaMKK-α and CaMKK-β in lieu of an M13 sequence resulted in threefold faster rise and decay times of Ca(2+) transients than R-CaMP1.07. These features allowed resolving single action potentials (APs) and recording fast AP trains up to 20-40 Hz in cortical slices. Somatic and synaptic activities of a cortical neuronal ensemble in vivo were imaged with similar efficacy as with previously reported sensitive green GECIs. Combining green and red GECIs, we successfully achieved dual-color monitoring of neuronal activities of distinct cell types, both in the mouse cortex and in freely moving Caenorhabditis elegans. Dual imaging using R-CaMP2 and green GECIs provides a powerful means to interrogate orthogonal and hierarchical neuronal ensembles in vivo.
Generation and Imaging of Transgenic Mice that Express G-CaMP7 under a Tetracycline Response Element. International-journal Peer-reviewed

Masaaki Sato, Masako Kawano, Masamichi Ohkura, Keiko Gengyo-Ando, Junichi Nakai, Yasunori Hayashi

PloS one　10　(5)　e0125354　2015

DOI： 10.1371/journal.pone.0125354 　

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The spatiotemporally controlled expression of G-CaMP fluorescent calcium indicator proteins can facilitate reliable imaging of brain circuit activity. Here, we generated a transgenic mouse line that expresses G-CaMP7 under a tetracycline response element. When crossed with a forebrain-specific tetracycline-controlled transactivator driver line, the mice expressed G-CaMP7 in defined cell populations in a tetracycline-controlled manner, notably in pyramidal neurons in layer 2/3 of the cortex and in the CA1 area of the hippocampus; this expression allowed for imaging of the in vivo activity of these circuits. This mouse line thus provides a useful genetic tool for controlled G-CaMP expression in vivo.
Reward-timing-dependent bidirectional modulation of cortical microcircuits during optical single-neuron operant conditioning. International-journal Peer-reviewed

Riichiro Hira, Fuki Ohkubo, Yoshito Masamizu, Masamichi Ohkura, Junichi Nakai, Takashi Okada, Masanori Matsuzaki

Nature communications　5　5551-5551　2014/11/24

DOI： 10.1038/ncomms6551 　

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Animals rapidly adapt to environmental change. To reveal how cortical microcircuits are rapidly reorganized when an animal recognizes novel reward contingency, we conduct two-photon calcium imaging of layer 2/3 motor cortex neurons in mice and simultaneously reinforce the activity of a single cortical neuron with water delivery. Here we show that when the target neuron is not relevant to a pre-trained forelimb movement, the mouse increases the target neuron activity and the number of rewards delivered during 15-min operant conditioning without changing forelimb movement behaviour. The reinforcement bidirectionally modulates the activity of subsets of non-target neurons, independent of distance from the target neuron. The bidirectional modulation depends on the relative timing between the reward delivery and the neuronal activity, and is recreated by pairing reward delivery and photoactivation of a subset of neurons. Reward-timing-dependent bidirectional modulation may be one of the fundamental processes in microcircuit reorganization for rapid adaptation.
Excitation propagation in three-dimensional engineered hearts using decellularized extracellular matrix. International-journal

Haruyo Yasui, Jong-Kook Lee, Akira Yoshida, Teruki Yokoyama, Hiroyuki Nakanishi, Keiko Miwa, Atsuhiko T Naito, Toru Oka, Hiroshi Akazawa, Junichi Nakai, Shigeru Miyagawa, Yoshiki Sawa, Yasushi Sakata, Issei Komuro

Biomaterials　35　(27)　7839-50　2014/09

DOI： 10.1016/j.biomaterials.2014.05.080 　

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Engineering of three-dimensional (3D) cardiac tissues using decellularized extracellular matrix could be a new technique to create an "organ-like" structure of the heart. To engineer artificial hearts functionally comparable to native hearts, however, much remain to be solved including stable excitation-propagation. To elucidate the points, we examined conduction properties of engineered tissues. We repopulated the decellularized hearts with neonatal rat cardiac cells and then, we observed excitation-propagation of spontaneous beatings using high resolution cameras. We also conducted immunofluorescence staining to examine morphological aspects. Live tissue imaging revealed that GFP-labeled-isolated cardiac cells were migrated into interstitial spaces through extravasation from coronary arteries. Engineered hearts repopulated with Ca(2+)-indicating protein (GCaMP2)-expressing cardiac cells were subjected to optical imaging experiments. Although the engineered hearts generally showed well-organized stable excitation-propagation, the hearts also demonstrated arrhythmogenic propensity such as disorganized propagation. Immunofluorescence study revealed randomly-mixed alignment of cardiomyocytes, endothelial cells and smooth muscle cells. The recellularized hearts also showed disarray of cardiomyocytes and markedly decreased expression of connexin43. In conclusion, we successfully demonstrated that the recellularized hearts showed dynamic excitation-propagation as a "whole organ". Our strategy could provide prerequisite information to construct a 3D-engineered heart, functionally comparable to the native heart.
Large-scale imaging of subcellular calcium dynamics of cortical neurons with G-CaMP6-actin. International-journal Peer-reviewed

Chiaki Kobayashi, Masamichi Ohkura, Junichi Nakai, Norio Matsuki, Yuji Ikegaya, Takuya Sasaki

Neuroreport　25　(7)　501-6　2014/05/07
Publisher: 7
DOI： 10.1097/WNR.0000000000000126 　

ISSN： 0959-4965

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Understanding the information processing performed by a single neuron requires the monitoring of physiological dynamics from a variety of subcellular compartments including dendrites and axons. In this study, we showed that the expression of a fusion protein, consisting of a Ca²⁺ indicator protein (G-CaMP6) and a cytoskeleton protein (actin), enabled large-scale recording of Ca²⁺ dynamics from hundreds of postsynaptic spines and presynaptic boutons in a cortical pyramidal cell. At dendritic spines, G-CaMP6-actin had the potential to detect localized Ca²⁺ activity triggered by subthreshold synaptic inputs. Back-propagating action potentials reliably induced Ca²⁺ fluorescent increases in all spines. At axonal boutons, G-CaMP6-actin reported action potential trains propagating along axonal collaterals. The detectability of G-CaMP6-actin should contribute toward a deeper understanding of neural network architecture and dynamics at the level of individual synapses.
Toll-like receptor 9 protects non-immune cells from stress by modulating mitochondrial ATP synthesis through the inhibition of SERCA2. International-journal

Yasunori Shintani, Hannes C A Drexler, Hidetaka Kioka, Cesare M N Terracciano, Steven R Coppen, Hiromi Imamura, Masaharu Akao, Junichi Nakai, Ann P Wheeler, Shuichiro Higo, Hiroyuki Nakayama, Seiji Takashima, Kenta Yashiro, Ken Suzuki

EMBO reports　15　(4)　438-45　2014/04

DOI： 10.1002/embr.201337945 　

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Toll-like receptor 9 (TLR9) has a key role in the recognition of pathogen DNA in the context of infection and cellular DNA that is released from damaged cells. Pro-inflammatory TLR9 signalling pathways in immune cells have been well investigated, but we have recently discovered an alternative pathway in which TLR9 temporarily reduces energy substrates to induce cellular protection from stress in cardiomyocytes and neurons. However, the mechanism by which TLR9 stimulation reduces energy substrates remained unknown. Here, we identify the calcium-transporting ATPase, SERCA2 (also known as Atp2a2), as a key molecule for the alternative TLR9 signalling pathway. TLR9 stimulation reduces SERCA2 activity, modulating Ca(2+) handling between the SR/ER and mitochondria, which leads to a decrease in mitochondrial ATP levels and the activation of cellular protective machinery. These findings reveal how distinct innate responses can be elicited in immune and non-immune cells--including cardiomyocytes--using the same ligand-receptor system.
Electrical Propagation of Three-Dimensional Engineered Hearts using Decellularized Extracellular Matrix Peer-reviewed

Haruyo Yasui, Jong-Kook Lee, Akira Yoshida, Teruki Yokoyama, Junichi Nakai, Issei Komuro

BIOPHYSICAL JOURNAL　106　(2)　630A-630A　2014/01

DOI： 10.1016/j.bpj.2013.11.3486 　

ISSN： 0006-3495

eISSN： 1542-0086
Imaging of astrocytic activity in living rodents Peer-reviewed

Norio Takata, Yoshiaki Shinohara, Masamichi Ohkura, Tsuneko Mishima, Junichi Nakai, Hajime Hirase

Neuromethods　85　191-207　2014
Publisher: Humana Press Inc.
DOI： 10.1007/978-1-62703-785-3_12 　

ISSN： 1940-6045 0893-2336
Genetically encoded probes provide a window on embryonic arrhythmia. International-journal

Yvonne Norine Tallini, Kai Su Greene, Bo Shui, Calum William Russell, Jane Constance Lee, Robert Michael Doran, Junichi Nakai, Michael I Kotlikoff

Methods in molecular biology (Clifton, N.J.)　1092　195-219　2014

DOI： 10.1007/978-1-60327-292-6_13 　

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Supraventricular tachycardias are the most prevalent group of arrhythmias observed in the fetus and infant and their incidence increases through early childhood. The molecular pathogenesis of embryonic cardiac dysfunction is poorly understood, due in part to the absence of imaging techniques that provide functional information at the cellular and molecular levels in the developing mammalian heart, particularly during early heart formation. The combination of protein engineering, genetic specification, and high-resolution optical imaging enables new insights into cardiac function and dysfunction during cardiac development. Here we describe the use of GCaMP2, a genetically encoded Ca(2+) indicator (GECI), to determine the processes of cardiac electrical activation during cardiac organogenesis. Transgenic specification of GCaMP2 in mice allows sufficient expression for Ca(2+) imaging as early as embryonic day (e.d.) 9.5, just after the heart begins to function at e.d. 8.5. Crosses with knockout lines in which lethality occurs due to cardiac dysfunction will enable precise determination of the conduction or excitation-contraction coupling phenotypes and thereby improve the understanding of the genetic basis of heart development and the consequence of gene mutations. Moreover, lineage-specific targeting of these sensors of cell signaling provides a new window on the molecular specification of the heart conduction system. We describe mouse lines and imaging methods used to examine conduction in the pre-septated heart (e.d. 10.5), which occurs through dramatically slowed atrioventricular (AV) canal conduction, producing a delay between atrial and ventricular activation prior to the development of the AV node. Genetic constructs including single and bi-allelic minimal promoter systems, and single allele BAC transgenes, enable general or lineage-specific targeting of GCaMP2. High-resolution imaging of embryonic heart conduction provides a new window on one of the most complex events in the mammalian body plan.
Imaging of neuronal activity in zebrafish during visual perception using G-CaMP7a, an improved genetically encoded green fluorescent calcium indicator Peer-reviewed

Masamichi Ohkura, Akira Muto, Gembu Abe, Koichi Kawakami, Junichi Nakai

JOURNAL OF PHARMACOLOGICAL SCIENCES　124　(1)　120P-120P　2014

ISSN： 1347-8613

eISSN： 1347-8648
Principal component analysis of odor coding at the level of third-order olfactory neurons in Drosophila. International-journal Peer-reviewed

Makoto Hiroi, Masamichi Ohkura, Junichi Nakai, Naoki Masuda, Koichi Hashimoto, Kiichi Inoue, André Fiala, Tetsuya Tabata

Genes to cells : devoted to molecular & cellular mechanisms　18　(12)　1070-81　2013/12

DOI： 10.1111/gtc.12094 　

ISSN： 1356-9597

eISSN： 1365-2443
Real-time visualization of neuronal activity during perception. International-journal Peer-reviewed

Akira Muto, Masamichi Ohkura, Gembu Abe, Junichi Nakai, Koichi Kawakami

Current biology : CB　23　(4)　307-11　2013/02/18

DOI： 10.1016/j.cub.2012.12.040 　

ISSN： 0960-9822

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To understand how the brain perceives the external world, it is desirable to observe neuronal activity in the brain in real time during perception. The zebrafish is a suitable model animal for fluorescence imaging studies to visualize neuronal activity because its body is transparent through the embryonic and larval stages. Imaging studies have been carried out to monitor neuronal activity in the larval spinal cord and brain using Ca(2+) indicator dyes and DNA-encoded Ca(2+) indicators, such as Cameleon, GFP-aequorin, and GCaMPs. However, temporal and spatial resolution and sensitivity of these tools are still limited, and imaging of brain activity during perception of a natural object has not yet been demonstrated. Here we demonstrate visualization of neuronal activity in the optic tectum of larval zebrafish by genetically expressing the new version of GCaMP. First, we demonstrate Ca(2+) transients in the tectum evoked by a moving spot on a display and identify direction-selective neurons. Second, we show tectal activity during perception of a natural object, a swimming paramecium, revealing a functional visuotopic map. Finally, we image the tectal responses of a free-swimming larval fish to a paramecium and thereby correlate neuronal activity in the brain with prey capture behavior.
Astrocytic Ca2+ signals are required for the functional integrity of tripartite synapses. International-journal

Mika Tanaka, Pei-Yu Shih, Hiroshi Gomi, Takamasa Yoshida, Junichi Nakai, Reiko Ando, Teiichi Furuichi, Katsuhiko Mikoshiba, Alexey Semyanov, Shigeyoshi Itohara

Molecular brain　6　6-6　2013/01/28

DOI： 10.1186/1756-6606-6-6 　

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BACKGROUND: Neuronal activity alters calcium ion (Ca2+) dynamics in astrocytes, but the physiologic relevance of these changes is controversial. To examine this issue further, we generated an inducible transgenic mouse model in which the expression of an inositol 1,4,5-trisphosphate absorbent, "IP3 sponge", attenuates astrocytic Ca2+ signaling. RESULTS: Attenuated Ca2+ activity correlated with reduced astrocytic coverage of asymmetric synapses in the hippocampal CA1 region in these animals. The decreased astrocytic 'protection' of the synapses facilitated glutamate 'spillover', which was reflected by prolonged glutamate transporter currents in stratum radiatum astrocytes and enhanced N-methyl-D-aspartate receptor currents in CA1 pyramidal neurons in response to burst stimulation. These mice also exhibited behavioral impairments in spatial reference memory and remote contextual fear memory, in which hippocampal circuits are involved. CONCLUSIONS: Our findings suggest that IP3-mediated astrocytic Ca2+ signaling correlates with the formation of functional tripartite synapses in the hippocampus.
Cilia at the node of mouse embryos sense fluid flow for left-right determination via Pkd2. International-journal

Satoko Yoshiba, Hidetaka Shiratori, Ivana Y Kuo, Aiko Kawasumi, Kyosuke Shinohara, Shigenori Nonaka, Yasuko Asai, Genta Sasaki, Jose Antonio Belo, Hiroshi Sasaki, Junichi Nakai, Bernd Dworniczak, Barbara E Ehrlich, Petra Pennekamp, Hiroshi Hamada

Science (New York, N.Y.)　338　(6104)　226-31　2012/10/12

DOI： 10.1126/science.1222538 　

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Unidirectional fluid flow plays an essential role in the breaking of left-right (L-R) symmetry in mouse embryos, but it has remained unclear how the flow is sensed by the embryo. We report that the Ca(2+) channel Polycystin-2 (Pkd2) is required specifically in the perinodal crown cells for sensing the nodal flow. Examination of mutant forms of Pkd2 shows that the ciliary localization of Pkd2 is essential for correct L-R patterning. Whereas Kif3a mutant embryos, which lack all cilia, failed to respond to an artificial flow, restoration of primary cilia in crown cells rescued the response to the flow. Our results thus suggest that nodal flow is sensed in a manner dependent on Pkd2 by the cilia of crown cells located at the edge of the node.
Expanding the repertoire of optogenetically targeted cells with an enhanced gene expression system. International-journal

Kenji F Tanaka, Ko Matsui, Takuya Sasaki, Hiromi Sano, Shouta Sugio, Kai Fan, René Hen, Junichi Nakai, Yuchio Yanagawa, Hidetoshi Hasuwa, Masaru Okabe, Karl Deisseroth, Kazuhiro Ikenaka, Akihiro Yamanaka

Cell reports　2　(2)　397-406　2012/08/30

DOI： 10.1016/j.celrep.2012.06.011 　

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Optogenetics has been enthusiastically pursued in recent neuroscience research, and the causal relationship between neural activity and behavior is becoming ever more accessible. Here, we established knockin-mediated enhanced gene expression by improved tetracycline-controlled gene induction (KENGE-tet) and succeeded in generating transgenic mice expressing a highly light-sensitive channelrhodopsin-2 mutant at levels sufficient to drive the activities of multiple cell types. This method requires two lines of mice: one that controls the pattern of expression and another that determines the protein to be produced. The generation of new lines of either type readily expands the repertoire to choose from. In addition to neurons, we were able to manipulate the activity of nonexcitable glial cells in vivo. This shows that our system is applicable not only to neuroscience but also to any biomedical study that requires understanding of how the activity of a selected population of cells propagates through the intricate organic systems.
Direct monitoring of mitochondrial calcium levels in cultured cardiac myocytes using a novel fluorescent indicator protein, GCaMP2-mt. International-journal

Moritake Iguchi, Masashi Kato, Junichi Nakai, Toshihiro Takeda, Madoka Matsumoto-Ida, Toru Kita, Takeshi Kimura, Masaharu Akao

International journal of cardiology　158　(2)　225-34　2012/07/12

DOI： 10.1016/j.ijcard.2011.01.034 　

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BACKGROUND: An opening of the mitochondrial permeability transition pore (MPTP), which leads to loss of mitochondrial membrane potential (ΔΨ(m)), is the earliest event that commits a cell to death. Mitochondrial matrix calcium ([Ca(2+)](m)) is considered to be a critical regulator of MPTP, but direct monitoring of [Ca(2+)](m) is difficult with previously-reported sensors. We developed a novel fluorescent indicator for [Ca(2+)](m), GCaMP2-mt, by adding a mitochondrial targeting sequence to a high signal-to-noise Ca(2+) sensor protein GCaMP2, and monitored dynamic changes in oxidant-induced cardiac myocyte death. METHODS AND RESULTS: GCaMP2-mt was transduced into neonatal rat cardiac myocytes using a recombinant adenovirus. We confirmed that GCaMP2-mt colocalized with tetramethylrhodamine ethyl-ester, a fluorescent indicator of ΔΨ(m). We monitored oxidant-induced responses of [Ca(2+)](m) and ΔΨ(m) using time-lapse confocal microscopy. The response of [Ca(2+)](m) was synchronous with that of cytosolic calcium and was divided into three kinetically-distinct phases; the first phase, during which [Ca(2+)](m) maintained its baseline level; the second phase, during which [Ca(2+)](m) showed a rapid and sudden increase; and the third phase, during which [Ca(2+)](m) continued to increase at a slower rate until the collapse of ΔΨ(m). The third phase was likely to be mediated through a mitochondrial Ca(2+) uniporter, because it was modulated by uniporter-acting drugs. Importantly, there was a remarkable cellular heterogeneity in the third phase, and ΔΨ(m) loss occurred in an all-or-none manner depending on the cellular [Ca(2+)](m) level with a clear cut-off value. CONCLUSIONS: Direct monitoring of [Ca(2+)](m) using GCaMP2-mt provides deeper insight into the mechanism of cardiac myocyte death.
LIS1-dependent retrograde translocation of excitatory synapses in developing interneuron dendrites. International-journal Peer-reviewed

Izumi Kawabata, Yutaro Kashiwagi, Kazuki Obashi, Masamichi Ohkura, Junichi Nakai, Anthony Wynshaw-Boris, Yuchio Yanagawa, Shigeo Okabe

Nature communications　3　722-722　2012/03/06

DOI： 10.1038/ncomms1736 　

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Synaptic remodelling coordinated with dendritic growth is essential for proper development of neural connections. After establishment of synaptic contacts, synaptic junctions are thought to become stationary and provide fixed anchoring points for further dendritic growth. However, the possibility of active translocation of synapses along dendritic protrusions, to guide the proper arrangement of synaptic distribution, has not yet been fully investigated. Here we show that immature dendrites of γ-aminobutyric acid-positive interneurons form long protrusions and that these protrusions serve as conduits for retrograde translocation of synaptic contacts to the parental dendrites. This translocation process is dependent on microtubules and the activity of LIS1, an essential regulator of dynein-mediated motility. Suppression of this retrograde translocation results in disorganized synaptic patterns on interneuron dendrites. Taken together, these findings suggest the existence of an active microtubule-dependent mechanism for synaptic translocation that helps in the establishment of proper synaptic distribution on dendrites.
Genetically encoded green fluorescent Ca2+ indicators with improved detectability for neuronal Ca2+ signals. International-journal Peer-reviewed

Masamichi Ohkura, Takuya Sasaki, Junko Sadakari, Keiko Gengyo-Ando, Yuko Kagawa-Nagamura, Chiaki Kobayashi, Yuji Ikegaya, Junichi Nakai

PloS one　7　(12)　e51286　2012
Publisher: 12
DOI： 10.1371/journal.pone.0051286 　

ISSN： 1932-6203

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Imaging the activities of individual neurons with genetically encoded Ca(2+) indicators (GECIs) is a promising method for understanding neuronal network functions. Here, we report GECIs with improved neuronal Ca(2+) signal detectability, termed G-CaMP6 and G-CaMP8. Compared to a series of existing G-CaMPs, G-CaMP6 showed fairly high sensitivity and rapid kinetics, both of which are suitable properties for detecting subtle and fast neuronal activities. G-CaMP8 showed a greater signal (F(max)/F(min) = 38) than G-CaMP6 and demonstrated kinetics similar to those of G-CaMP6. Both GECIs could detect individual spikes from pyramidal neurons of cultured hippocampal slices or acute cortical slices with 100% detection rates, demonstrating their superior performance to existing GECIs. Because G-CaMP6 showed a higher sensitivity and brighter baseline fluorescence than G-CaMP8 in a cellular environment, we applied G-CaMP6 for Ca(2+) imaging of dendritic spines, the putative postsynaptic sites. By expressing a G-CaMP6-actin fusion protein for the spines in hippocampal CA3 pyramidal neurons and electrically stimulating the granule cells of the dentate gyrus, which innervate CA3 pyramidal neurons, we found that sub-threshold stimulation triggered small Ca(2+) responses in a limited number of spines with a low response rate in active spines, whereas supra-threshold stimulation triggered large fluorescence responses in virtually all of the spines with a 100% activity rate.
An improved genetically encoded red fluorescent Ca2+ indicator for detecting optically evoked action potentials. International-journal Peer-reviewed

Masamichi Ohkura, Takuya Sasaki, Chiaki Kobayashi, Yuji Ikegaya, Junichi Nakai

PloS one　7　(7)　e39933　2012
Publisher: 7
DOI： 10.1371/journal.pone.0039933 　

ISSN： 1932-6203

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Genetically encoded Ca(2+) indicators (GECIs) are powerful tools to image activities of defined cell populations. Here, we developed an improved red fluorescent GECI, termed R-CaMP1.07, by mutagenizing R-GECO1. In HeLa cell assays, R-CaMP1.07 exhibited a 1.5-2-fold greater fluorescence response compared to R-GECO1. In hippocampal pyramidal neurons, R-CaMP1.07 detected Ca(2+) transients triggered by single action potentials (APs) with a probability of 95% and a signal-to-noise ratio >7 at a frame rate of 50 Hz. The amplitudes of Ca(2+) transients linearly correlated with the number of APs. The expression of R-CaMP1.07 did not significantly alter the electrophysiological properties or synaptic activity patterns. The co-expression of R-CaMP1.07 and channelrhodpsin-2 (ChR2), a photosensitive cation channel, in pyramidal neurons demonstrated that R-CaMP1.07 was applicable for the monitoring of Ca(2+) transients in response to optically evoked APs, because the excitation light for R-CaMP1.07 hardly activated ChR2. These technical advancements provide a novel strategy for monitoring and manipulating neuronal activity with single cell resolution.
Genetic visualization with an improved GCaMP calcium indicator reveals spatiotemporal activation of the spinal motor neurons in zebrafish. International-journal Peer-reviewed

Akira Muto, Masamichi Ohkura, Tomoya Kotani, Shin-ichi Higashijima, Junichi Nakai, Koichi Kawakami

Proceedings of the National Academy of Sciences of the United States of America　108　(13)　5425-30　2011/03/29

DOI： 10.1073/pnas.1000887108 　

ISSN： 0027-8424

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Animal behaviors are generated by well-coordinated activation of neural circuits. In zebrafish, embryos start to show spontaneous muscle contractions at 17 to 19 h postfertilization. To visualize how motor circuits in the spinal cord are activated during this behavior, we developed GCaMP-HS (GCaMP-hyper sensitive), an improved version of the genetically encoded calcium indicator GCaMP, and created transgenic zebrafish carrying the GCaMP-HS gene downstream of the Gal4-recognition sequence, UAS (upstream activation sequence). Then we performed a gene-trap screen and identified the SAIGFF213A transgenic fish that expressed Gal4FF, a modified version of Gal4, in a subset of spinal neurons including the caudal primary (CaP) motor neurons. We conducted calcium imaging using the SAIGFF213A; UAS:GCaMP-HS double transgenic embryos during the spontaneous contractions. We demonstrated periodic and synchronized activation of a set of ipsilateral motor neurons located on the right and left trunk in accordance with actual muscle movements. The synchronized activation of contralateral motor neurons occurred alternately with a regular interval. Furthermore, a detailed analysis revealed rostral-to-caudal propagation of activation of the ipsilateral motor neuron, which is similar to but much slower than the rostrocaudal delay observed during swimming in later stages. Our study thus demonstrated coordinated activities of the motor neurons during the first behavior in a vertebrate. We propose the GCaMP technology combined with the Gal4FF-UAS system is a powerful tool to study functional neural circuits in zebrafish.
マウス海馬神経回路の構造と機能のin vivo二光子イメージング(In vivo two-photon imaging of hippocampal circuit structure and function in mice)

佐藤正晃, 河野真子, 中井淳一, 林康紀

神経化学　49　(2-3)　774-774　2010/08
Publisher: (一社)日本神経化学会
ISSN： 0037-3796
Tissue-tissue interaction-triggered calcium elevation is required for cell polarization during Xenopus gastrulation. International-journal Peer-reviewed

Asako Shindo, Yusuke Hara, Takamasa S Yamamoto, Masamichi Ohkura, Junichi Nakai, Naoto Ueno

PloS one　5　(2)　e8897　2010/02/02

DOI： 10.1371/journal.pone.0008897 　

ISSN： 1932-6203
Derived (mutated)-types of TRPV6 channels elicit greater Ca²+ influx into the cells than ancestral-types of TRPV6: evidence from Xenopus oocytes and mammalian cell expression system. Peer-reviewed

Yuka Sudo, Kiyotaka Matsuo, Tomoyuki Tetsuo, Satoshi Tsutsumi, Masamichi Ohkura, Junichi Nakai, Yasuhito Uezono

Journal of pharmacological sciences　114　(3)　281-91　2010
Publisher: 3
DOI： 10.1254/jphs.10169FP 　

ISSN： 1347-8613

eISSN： 1347-8648

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The frequency of the allele containing three derived nonsynonymous SNPs (157C, 378M, 681M) of the gene encoding calcium permeable TRPV6 channels expressed in the intestine has been increased by positive selection in non-African populations. To understand the nature of these SNPs, we compared the properties of Ca²+ influx of ancestral (in African populations) and derived-TRPV6 (in non-African populations) channels with electrophysiological, Ca²+-imaging, and morphological methods using both the Xenopus oocyte and mammalian cell expression systems. Functional electrophysiological and Ca²+-imaging analyses indicated that the derived-TRPV6 elicited more Ca²+ influx than the ancestral one in TRPV6-expressing cells where both channels were equally expressed in the cells. Ca²+-inactivation properties in the ancestral- and derived-TRPV6 were almost the same. Furthermore, fluorescence resonance energy transfer (FRET) analysis showed that both channels have similar multimeric formation properties, suggesting that derived-TRPV6 itself could cause higher Ca²+ influx. These findings suggest that populations having derived-TRPV6 in non-African areas may absorb higher Ca²+ from the intestine than ancestral-TRPV6 in the African area.
Radially expanding transglial calcium waves in the intact cerebellum. International-journal Peer-reviewed

Tycho M Hoogland, Bernd Kuhn, Werner Göbel, Wenying Huang, Junichi Nakai, Fritjof Helmchen, Jane Flint, Samuel S-H Wang

Proceedings of the National Academy of Sciences of the United States of America　106　(9)　3496-501　2009/03/03

DOI： 10.1073/pnas.0809269106 　

ISSN： 0027-8424
Encoding gender and individual information in the mouse vomeronasal organ. International-journal Peer-reviewed

Jie He, Limei Ma, Sangseong Kim, Junichi Nakai, C Ron Yu

Science (New York, N.Y.)　320　(5875)　535-8　2008/04/25

DOI： 10.1126/science.1154476 　

ISSN： 0036-8075
Characterization and subcellular targeting of GCaMP-type genetically-encoded calcium indicators. International-journal Peer-reviewed

Tianyi Mao, Daniel H O'Connor, Volker Scheuss, Junichi Nakai, Karel Svoboda

PloS one　3　(3)　e1796　2008/03/19

DOI： 10.1371/journal.pone.0001796 　

ISSN： 1932-6203
Propagated endothelial Ca2+ waves and arteriolar dilation in vivo: measurements in Cx40BAC GCaMP2 transgenic mice. International-journal Peer-reviewed

Yvonne N Tallini, Johan Fredrik Brekke, Bo Shui, Robert Doran, Seong-min Hwang, Junichi Nakai, Guy Salama, Steven S Segal, Michael I Kotlikoff

Circulation research　101　(12)　1300-9　2007/12/07

DOI： 10.1161/CIRCRESAHA.107.149484 　

ISSN： 0009-7330

eISSN： 1524-4571
Local subplasma membrane Ca2+ signals detected by a tethered Ca2+ sensor. International-journal Peer-reviewed

Moo Yeol Lee, Hong Song, Junichi Nakai, Masamichi Ohkura, Michael I Kotlikoff, Stephen P Kinsey, Vera A Golovina, Mordecai P Blaustein

Proceedings of the National Academy of Sciences of the United States of America　103　(35)　13232-7　2006/08/29

DOI： 10.1073/pnas.0605757103 　

ISSN： 0027-8424
Imaging cellular signals in the heart in vivo: Cardiac expression of the high-signal Ca2+ indicator GCaMP2. International-journal Peer-reviewed

Yvonne N Tallini, Masamichi Ohkura, Bum-Rak Choi, Guangju Ji, Keiji Imoto, Robert Doran, Jane Lee, Patricia Plan, Jason Wilson, Hong-Bo Xin, Atsushi Sanbe, James Gulick, John Mathai, Jeffrey Robbins, Guy Salama, Junichi Nakai, Michael I Kotlikoff

Proceedings of the National Academy of Sciences of the United States of America　103　(12)　4753-8　2006/03/21

DOI： 10.1073/pnas.0509378103 　

ISSN： 0027-8424
Genetically encoded bright Ca2+ probe applicable for dynamic Ca2+ imaging of dendritic spines. International-journal Peer-reviewed

Masamichi Ohkura, Masanori Matsuzaki, Haruo Kasai, Keiji Imoto, Junichi Nakai

Analytical chemistry　77　(18)　5861-9　2005/09/15

DOI： 10.1021/ac0506837 　

ISSN： 0003-2700
Activation of cerebellar parallel fibers monitored in transgenic mice expressing a fluorescent Ca2+ indicator protein. International-journal Peer-reviewed

Javier Díez-García, Shinichi Matsushita, Hiroki Mutoh, Junichi Nakai, Masamichi Ohkura, Jennifer Yokoyama, Dimitar Dimitrov, Thomas Knöpfel

The European journal of neuroscience　22　(3)　627-35　2005/08

DOI： 10.1111/j.1460-9568.2005.04250.x 　

ISSN： 0953-816X
Insulin induces internalization of the 5-HT2A receptor expressed in HEK293 cells. International-journal Peer-reviewed

Masamichi Ohkura, Naoko Tanaka, Hideyuki Kobayashi, Akihiko Wada, Junichi Nakai, Ryuichi Yamamoto

European journal of pharmacology　518　(1)　18-21　2005/07/25

DOI： 10.1016/j.ejphar.2005.06.011 　

ISSN： 0014-2999
In vivo performance of genetically encoded indicators of neural activity in flies. International-journal Peer-reviewed

Dierk F Reiff, Alexandra Ihring, Giovanna Guerrero, Ehud Y Isacoff, Maximilian Joesch, Junichi Nakai, Alexander Borst

The Journal of neuroscience : the official journal of the Society for Neuroscience　25　(19)　4766-78　2005/05/11

DOI： 10.1523/JNEUROSCI.4900-04.2005 　

ISSN： 0270-6474

eISSN： 1529-2401
Methodologic advancements in the study of airway smooth muscle. International-journal Peer-reviewed

Michael I Kotlikoff, Mathur S Kannan, Julian Solway, Ke-Yu Deng, Deepak A Deshpande, Maria Dowell, Morris Feldman, Kai Su Green, Guangju Ji, Robyn Johnston, Oren Lakser, Jane Lee, Frances E Lund, Carlos Milla, Richard W Mitchell, Junichi Nakai, Mark Rishniw, Timothy F Walseth, Thomas A White, Jason Wilson, Hong-Bo Xin, Prescott G Woodruff

The Journal of allergy and clinical immunology　114　(2 Suppl)　S18-31-31　2004/08
Publisher: 2 Suppl
DOI： 10.1016/j.jaci.2004.04.040 　

ISSN： 0091-6749

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The study of isolated airway myocytes has provided important information relative to specific processes that regulate contraction, proliferation, and synthetic properties of airway smooth muscle (ASM). To place this information in physiological context, however, improved methods to examine airway biology in vivo are needed. Advances in genetic, biochemical, and optical methods provide unprecedented opportunities to improve our understanding of in vivo physiology and pathophysiology. This article describes 4 important methodologic advances in the study of ASM: (1) the development of transgenic mice that could be used to investigate ASM proliferation and phenotype switching during the development of hypersensitivity, and to investigate excitation-contraction coupling; (2) the use of CD38-deficient mice to confirm the role of CD38-dependent, cyclic adenosine diphosphate-ribose-mediated calcium release in airway responsiveness; (3) investigation of the role of actin filament length and p38 mitogen-activated protein kinase activity in regulating the mechanical plasticity-elasticity balance in contracted ASM; and (d) the use of bronchial biopsies to study ASM structure and phenotype in respiratory science.
Ca2+-sensing transgenic mice: postsynaptic signaling in smooth muscle. International-journal Peer-reviewed

Guangju Ji, Morris E Feldman, Ke-Yu Deng, Kai Su Greene, Jason Wilson, Jane C Lee, Robyn C Johnston, Mark Rishniw, Yvonne Tallini, Jin Zhang, Winthrop G Wier, Mordecai P Blaustein, Hong-Bo Xin, Junichi Nakai, Michael I Kotlikoff

The Journal of biological chemistry　279　(20)　21461-8　2004/05/14

DOI： 10.1074/jbc.M401084200 　

ISSN： 0021-9258
Targeted gene expression using the GAL4/UAS system in the silkworm Bombyx mori. International-journal Peer-reviewed

Morikazu Imamura, Junichi Nakai, Satoshi Inoue, Guo Xing Quan, Toshio Kanda, Toshiki Tamura

Genetics　165　(3)　1329-40　2003/11

ISSN： 0016-6731
Degeneration of the amygdala/piriform cortex and enhanced fear/anxiety behaviors in sodium pump alpha2 subunit (Atp1a2)-deficient mice. International-journal Peer-reviewed

Keiko Ikeda, Tatsushi Onaka, Makoto Yamakado, Junichi Nakai, Tomo-o Ishikawa, Makoto M Taketo, Kiyoshi Kawakami

The Journal of neuroscience : the official journal of the Society for Neuroscience　23　(11)　4667-76　2003/06/01

ISSN： 0270-6474

eISSN： 1529-2401
Probing calcium ions with biosensors. International-journal Peer-reviewed

Junichi Nakai, Masamichi Ohkura

Biotechnology & genetic engineering reviews　20　3-21　2003

ISSN： 0264-8725
Multiple regions of RyR1 mediate functional and structural interactions with alpha(1S)-dihydropyridine receptors in skeletal muscle. International-journal Peer-reviewed

Feliciano Protasi, Cecilia Paolini, Junichi Nakai, Kurt G Beam, Clara Franzini-Armstrong, Paul D Allen

Biophysical journal　83　(6)　3230-44　2002/12

DOI： 10.1016/S0006-3495(02)75325-3 　

ISSN： 0006-3495
Identification of a region of RyR1 that participates in allosteric coupling with the alpha(1S) (Ca(V)1.1) II-III loop. International-journal Peer-reviewed

Catherine Proenza, Jennifer O'Brien, Junichi Nakai, Santwana Mukherjee, Paul D Allen, Kurt G Beam

The Journal of biological chemistry　277　(8)　6530-5　2002/02/22

DOI： 10.1074/jbc.M106471200 　

ISSN： 0021-9258

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Misc. 102

Astrocytes in Atp1a2-deficient heterozygous mice exhibit hyperactivity after induction of cortical spreading depression. International-journal

Hiroki Sugimoto, Masaaki Sato, Junichi Nakai, Kiyoshi Kawakami

FEBS open bio　10　(6)　1031-1043　2020/06
Publisher: Wiley
DOI： 10.1002/2211-5463.12848 　

ISSN： 2211-5463

eISSN： 2211-5463

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The ATP1A2 coding α2 subunit of Na,K-ATPase, which is predominantly located in astrocytes, is a causative gene of familial hemiplegic migraine type 2 (FHM2). FHM2 model mice (Atp1a2tmCKwk/+ ) are susceptible to cortical spreading depression (CSD), which is profoundly related to migraine aura and headache. However, astrocytic properties during CSD have not been examined in FHM2 model mice. Using Atp1a2tmCKwk/+ crossed with transgenic mice expressing G-CaMP7 in cortical neurons and astrocytes (Atp1a2+/- ), we analyzed the changes in Ca2+ concentrations during CSD. The propagation speed of Ca2+ waves and the percentages of astrocytes with elevated Ca2+ concentrations in Atp1a2+/- were higher than those in wild-type mice. Increased percentages of astrocytes with elevated Ca2+ concentrations in Atp1a2+/- may contribute to FHM2 pathophysiology.
甦れβ細胞よ! 女性研究者が糖尿病を克服するアクティブゾーンタンパク質ELKSのインスリン分泌における役割

今泉美佳, 青柳共太, 吉田昌史, 飛田耶馬人, 大倉正道, 山内肇, 崎村建司, 中井淳一, 加計正文, 永松信哉, 大塚稔久

糖尿病　61　(Suppl.1)　S-68　2018/04
Publisher: (一社)日本糖尿病学会
ISSN： 0021-437X

eISSN： 1881-588X
Ultra-deep brain imaging with a novel two-photon varifocal microendoscope

Masaaki Sato, Yuki Motegi, Keiko Gengyo-Ando, Masamichi Ohkura, Junichi Nakai

JOURNAL OF PHARMACOLOGICAL SCIENCES　133　(3)　S135-S135　2017/03

ISSN： 1347-8613

eISSN： 1347-8648
高性能な赤色蛍光Ca²⁺プローブタンパク質 (特集革新的ライフサイエンスの展望)

大倉正道, 中井淳一

化學工業　67　(7)　489-496　2016/07
Publisher: 小峰工業出版
ISSN： 0451-2014
G-CaMP7発現マウスを用いた大脳皮質神経活動のミクロ,マクロ解析法

茂木優貴, 茂木優貴, 安藤恵子, 安藤恵子, 沖篤志, 沖篤志, 岩井陽一, 毛内拡, 平瀬肇, 平瀬肇, 平瀬肇, 大倉正道, 大倉正道, 中井淳一, 中井淳一

日本生理学雑誌(Web)　78　(3)　2016

ISSN： 0031-9341
【細胞シグナル操作法】分子からみたシグナル操作法セカンドメッセンジャーカルシウムシグナル

安藤恵子, 中井淳一

生体の科学　66　(5)　388-389　2015/10
Publisher: (公財)金原一郎記念医学医療振興財団
ISSN： 0370-9531
Rational design of a high-affinity, fast, red calcium indicator R-CaMP2 (vol 12, pg 64, 2015)

Masatoshi Inoue, Atsuya Takeuchi, Shin-ichiro Horigane, Masamichi Ohkura, Keiko Gengyo-Ando, Hajime Fujii, Satoshi Kamijo, Sayaka Takemoto-Kimura, Masanobu Kano, Junichi Nakai, Kazuo Kitamura, Haruhiko Bito

NATURE METHODS　12　(9)　893-893　2015/09

DOI： 10.1038/nmeth0915-893b 　

ISSN： 1548-7091

eISSN： 1548-7105
Rational design of a high-affinity, fast, red calcium indicator R-CaMP2

Masatoshi Inoue, Atsuya Takeuchi, Shin-ichiro Horigane, Masamichi Ohkura, Keiko Gengyo-Ando, Hajime Fujii, Satoshi Kamijo, Sayaka Takemoto-Kimura, Masanobu Kano, Junichi Nakai, Kazuo Kitamura, Haruhiko Bito

JOURNAL OF PHARMACOLOGICAL SCIENCES　128　(3)　S92-S92　2015/07

ISSN： 1347-8613

eISSN： 1347-8648
G-CaMP7を大脳皮質に発現するマウスを用いた神経活動のミクロ,マクロ解析法

茂木優貴, 茂木優貴, 安藤恵子, 安藤恵子, 沖篤志, 沖篤志, 岩井陽一, 毛内拡, 平瀬肇, 平瀬肇, 平瀬肇, 大倉正道, 大倉正道, 中井淳一, 中井淳一

日本薬理学会関東部会プログラム・要旨集　132nd　2015
G-CaMP7発現マウス大脳皮質脳細胞活動の細胞-領野スケール長期イメージング実験解析法

茂木優貴, 茂木優貴, 安藤恵子, 安藤恵子, 沖篤志, 沖篤志, 岩井陽一, 毛内拡, 平瀬肇, 平瀬肇, 平瀬肇, 大倉正道, 大倉正道, 中井淳一, 中井淳一

日本生化学会大会(Web)　88th　2015
蛍光カルシウムプローブG‐CaMPによるin vivoカルシウムイメージング

中井淳一, 大倉正道, 安藤恵子

日本薬学会関東支部大会講演要旨集　58th　82　2014/09/30
細胞触覚センサのための小型蛍光観察システムの開発

民山浩輔, 清水正宏, 宮坂恒太, 小椋利彦, 中井淳一, 大倉正道, 細田耕

日本ロボット学会学術講演会予稿集(CD-ROM)　32nd　ROMBUNNO.1P1-05　2014/09/04
ホヤ幼生の遊泳運動神経回路の構造と生理機能の解析

堀江健生, 大倉正道, 日下部岳広, 中井淳一, 中川将司

日本動物学会大会予稿集　85th　41　2014/08/07
じっくり学ぶ組織細胞化学の基礎と応用(2)Ca²⁺イメージング Invited

大倉正道, 中井淳一

組織細胞化学　2014　161-168　2014/07
線虫受精卵においてダイナミックに変化するRAB-11の動態制御機構の解析

坂口愛沙, 佐藤美由紀, 安藤恵子, 佐藤克哉, 中井淳一, 佐藤健

日本細胞生物学会大会講演要旨集　66回　117-117　2014/05
Publisher: (一社)日本細胞生物学会
[Ca(2+) imaging of neurons and astrocytes with genetically encoded Ca(2+) indicators]. Invited

Masamichi Ohkura, Junichi Nakai

Nihon yakurigaku zasshi. Folia pharmacologica Japonica　142　(5)　226-30　2013/11

DOI： 10.1254/fpj.142.226 　

ISSN： 0015-5691
1P1-C12 Cell tactile sensor that self-organizes induced from mechanical stimulations(Bio Assembler for 3D Cellular System Innovation)

MINZAN Kosuke, SHIMIZU Masahiro, MIYASAKA Kota, OGURA Toshihiko, Nakai Junichi, Ohkura Masamichi, HOSODA Koh

2013　"1P1-C12(1)"-"1P1-C12(4)"　2013/05/22
Publisher: The Japan Society of Mechanical Engineers

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By applying cells, and/or tissues to engineering devices, these devices are expected to have intrinsic prominent functions of living organisms such as environment adaptability, self-repair and self-assembly. In this study, we proposed integrated tactile receptors built by cultured cells (Cell Tactile Sensor). We exploit that the cells cultured on the PDMS membrane as the force receptors. At first, we designed a fine configuration structure of cell tactile sensor by self-organization induced from mechanical stimulations, which is applied to cells during culturing. Then, we visualized the mechanical stimulation response of cells by introducing G-CaMP, and finally obtained tactile information. Thus, we realized a novel tactile sensor which has environment adaptability.
[Development of the genetically encoded red fluorescent Ca(2+) indicator R-CaMP1.07]. Invited

Masamichi Ohkura, Junichi Nakai

Nihon yakurigaku zasshi. Folia pharmacologica Japonica　141　(3)　175-175　2013/03

DOI： 10.1254/fpj.141.175 　

ISSN： 0015-5691
Imaging of neuronal activities at the single-cell and the single-synapse levels using high-sensitivity and fast-responsivity G-CaMP-type genetically encoded calcium indicators

Masamichi Ohkura, Junko Sadakari, Takuya Sasaki, Keiko Gengyo-Ando, Yuko Kagawa-Nagamura, Chiaki Kobayashi, Yuji Ikegaya, Junichi Nakai

JOURNAL OF PHARMACOLOGICAL SCIENCES　121　129P-129P　2013

ISSN： 1347-8613
Comprehensive Odor mapping in the Mushroom Body neurons in Drosophila melanogaster

Yutaro Ueoka, Makoto Hiroi, Masamichi Ohkura, Junichi Nakai, Tetsuya Tabata

JOURNAL OF PHYSIOLOGICAL SCIENCES　63　S163-S163　2013

ISSN： 1880-6546
Imaging of neuronal spikes evoked by photo-stimulation of channelrhodopsin-2 using R-CaMP1.07, an improved genetically encoded red fluorescent calcium indicator

Masamichi Ohkura, Takuya Sasaki, Chiaki Kobayashi, Yuji Ikegaya, Junichi Nakai

JOURNAL OF PHARMACOLOGICAL SCIENCES　121　130P-130P　2013

ISSN： 1347-8613
細胞の機械刺激応答を利用した生体機械融合システムの開発

清水正宏, 民山浩輔, 宮坂恒太, 小椋利彦, 中井淳一, 大倉正道, 細田耕

自動制御連合講演会(CD-ROM)　56th　ROMBUNNO.521　2013
難治性てんかんの責任遺伝子Munc18‐1変異の機能解析

大澤明香音, 安藤恵子, 永村ゆう子, 大倉正道, 中井淳一

日本分子生物学会年会プログラム・要旨集(Web)　36th　WEB ONLY 1P-0915　2013
ホヤ幼生の遊泳運動神経回路のin vivo Ca2+イメージング

堀江健生, 大倉正道, 中井淳一, 中川将司

日本分子生物学会年会プログラム・要旨集(Web)　36th　WEB ONLY 2P-0736　2013
タンパク質でできた高性能な蛍光カルシウムセンサー Invited

大倉正道, 貞苅純子, 中井淳一

ケミカルエンジニヤリング　57　(12)　926-931　2012/12
Publisher: 化学工業社
ISSN： 0387-1037
香りの評価法の検討―バイオアッセイを用いた香り評価の試み― Invited

中井淳一, 永村ゆう子, 大倉正道, 清宮はるな, 藤田孝, 松永祐, 長谷川登志夫, 安藤恵子

Aroma Res　13　(2)　126-131　2012/05

ISSN： 1345-4722
改良型G‐CaMPを用いた線虫運動出力系の神経調節機構の解明

宇佐美篤, 安藤恵子, 大倉正道, 池谷裕二, 中井淳一, 松木則夫

日本薬学会年会要旨集　132nd　(3)　117　2012/03/05

ISSN： 0918-9823
Visualization of spatiotemporal activity of the spinal motor neurons in zebrafish with a genetically-encoded calcium indicator

Masamichi Ohkura, Akira Muto, Tomoya Kotani, Shin-ichi Higashijima, Koichi Kawakami, Junichi Nakai

JOURNAL OF PHARMACOLOGICAL SCIENCES　118　94P-94P　2012

ISSN： 1347-8613
Dynamic neuromuscular regulation in freely crawling C. elegans: High-resolution and large-scale in vivo Ca2+ imaging

Atsushi Usami, Keiko Gengyo-Ando, Yuko Nagamura, Yuka Yoshida, Norio Matsuki, Yuji Ikegaya, Junichi Nakai

NEUROSCIENCE RESEARCH　71　E242-E242　2011

DOI： 10.1016/j.neures.2011.07.1058 　

ISSN： 0168-0102
Genetic dissection of the brain function by transposon-mediated gene and enhancer trapping

Koichi Kawakami, Gembu Abe, Kazuhide Asakawa, Ryuichi Fukuda, Pradeep Lal, Akira Muto, Junichi Nakai

NEUROSCIENCE RESEARCH　71　E23-E23　2011

DOI： 10.1016/j.neures.2011.07.095 　

ISSN： 0168-0102
Calcium imaging of the zebrafish visual system with the GCaMP

Akira Muto, Junichi Nakai, Koichi Kawakami

NEUROSCIENCE RESEARCH　71　E68-E69　2011

DOI： 10.1016/j.neures.2011.07.292 　

ISSN： 0168-0102
In vivo calcium imaging of C. elegans body wall muscles with genetically encoded calcium probe G-CaMP

Junichi Nakai, Keiko Gengyo-Ando, Atsushi Usami, Masamichi Ohkura, Yuji Ikegaya, Norio Matsuki

JOURNAL OF PHARMACOLOGICAL SCIENCES　115　111P-111P　2011

ISSN： 1347-8613
Visualization of tissue-tissue interaction-triggered Ca2+ elevation for cell polarization during Xenopus gastrulation with improved GCaMP-type genetic Ca2+ probes

Masamichi Ohkura, Asako Shindo, Yusuke Hara, Takamasa Yamamoto, Naoto Ueno, Junichi Nakai

JOURNAL OF PHARMACOLOGICAL SCIENCES　115　78P-78P　2011

ISSN： 1347-8613
In vivo two-photon imaging of neuronal circuit activity using transgenic mice that express fluorescent calcium sensor proteins in the brain

Masaaki Sato, Masako Kawamo, Tanvir Islam, Yoko Yamaguchi, Masamichi Ohkura, Junichi Nakai, Yasunori Hayashi

NEUROSCIENCE RESEARCH　71　E309-E309　2011

DOI： 10.1016/j.neures.2011.07.1347 　

ISSN： 0168-0102
In vivo imaging of neural activities along the olfactory circuitry in transgenic zebrafish

Tetsuya Koide, Masamichi Ohkura, Junichi Nakai, Yoshihiro Yoshihara

NEUROSCIENCE RESEARCH　71　E357-E357　2011

DOI： 10.1016/j.neures.2011.07.1567 　

ISSN： 0168-0102
蛍光カルシウムセンサーG-CaMP4を用いたin vivoとin vitroイメージング(In vivo or in vitro imaging of calcium dynamics with genetically encoded sensor G-CaMP4)

宇佐美篤, 安藤恵子, 大倉正道, 松木則夫, 池谷裕二, 中井淳一

神経化学　49　(2-3)　772-772　2010/08
Publisher: 日本神経化学会
ISSN： 0037-3796
Astrocytic IP3-mediated Ca2+ signaling is required for functional integrity of tripartite synapse

Mika Tanaka, Hiroshi Gomi, Junichi Nakai, Alexander Lebedinskiy, Pei-Yu Shih, Reiko Ando, Katsuhiko Mikoshiba, Alexey Semyanov, Shigeyoshi Itohara

NEUROSCIENCE RESEARCH　68　E243-E243　2010

DOI： 10.1016/j.neures.2010.07.1076 　

ISSN： 0168-0102
In vivo two-photon imaging of hippocampal circuit structure and function in mice

Masaaki Sato, Masako Kawano, Junichi Nakai, Yasunori Hayashi

NEUROSCIENCE RESEARCH　68　E446-E446　2010

DOI： 10.1016/j.neures.2010.07.1978 　

ISSN： 0168-0102
In vivo or in vitro imaging of calcium dynamics with genetically encoded sensor G-CaMP4

Atsushi Usami, Keiko Ando, Masamichi Ohkura, Norio Matsuki, Yuji Ikegaya, Junichi Nakai

NEUROSCIENCE RESEARCH　68　E443-E443　2010

DOI： 10.1016/j.neures.2010.07.1966 　

ISSN： 0168-0102
Brain Imaging with Improved GCaMPs in Zebrafish

Akira Muto, Junichi Nakai, Koichi Kawakami

NEUROSCIENCE RESEARCH　68　E65-E65　2010

DOI： 10.1016/j.neures.2010.07.053 　

ISSN： 0168-0102
Transglial calcium waves in the intact cerebellum

Junichi Nakai, Tycho M. Hoogland, Bernd Kuhn, Werner Gobel, Wenying Huang, Fritjof Helmchen, Jane Flint, Samuel. S-H Wang

JOURNAL OF PHARMACOLOGICAL SCIENCES　112　91P-91P　2010

ISSN： 1347-8613
In vivo Ca[2+] imaging using novel fluorescent Ca[2+] sensor G-CaMP

61　(1)　86-92　2010/01
Publisher: 金原一郎記念医学医療振興財団
DOI： 10.11477/mf.2425100967 　

ISSN： 0370-9531
Recognition of Sexual Cues in the Urine by Mouse Vomeronasal Organ

Ron Yu, Jie He, Limei Ma, Sangseong Kim, Junichi Nakai

CHEMICAL SENSES　34　(7)　A4-A4　2009/09

ISSN： 0379-864X
Reduced astrocytic IP3-mediated Ca2+ signaling results in behavioral abnormality in mice

Mika Tanaka, Junichi Nakai, Alexander Lebedinskiy, Hiroshi Gomi, Katsuhiko Mikoshiba, Alexey Semyanov, Shigeyoshi Itohara

NEUROSCIENCE RESEARCH　65　S148-S148　2009

DOI： 10.1016/j.neures.2009.09.752 　

ISSN： 0168-0102
Visualization of functional neural circuits using the GAL4-UAS system in zebrafish

Akira Muto, Junichi Nakai, Koichi Kawakami

NEUROSCIENCE RESEARCH　65　S69-S69　2009

DOI： 10.1016/j.neures.2009.09.227 　

ISSN： 0168-0102
AN IMPROVED GENETICALLY-ENCODED FLUORESCENT Ca2+ PROBE THAT LESS AFFECTS CALCINEURIN SIGNALING Peer-reviewed

Ohkura Masamichi, Kitahara Tatsuro, Norota Ikuo, Nakai Junichi, Kubota Isao, Ishii Kuniaki

JOURNAL OF PHYSIOLOGICAL SCIENCES　59　528　2009

ISSN： 1880-6546
Pheromone reception of the vomeronasal neuron

Junichi Nakai, Jie He, Limer Ma, SangSeong Kim, Ron Yu

JOURNAL OF PHARMACOLOGICAL SCIENCES　109　108P-108P　2009

ISSN： 1347-8613
Development of improved G-CaMP2-type genetic Ca2+ probe, which minimally enhances cardiac hypertrophic signaling

Masamichi Ohkura, Ikuo Norota, Junichi Nakai, Kuniaki Ishii

JOURNAL OF PHARMACOLOGICAL SCIENCES　109　118P-118P　2009

ISSN： 1347-8613
Development of improved G-CaMP2-type genetic Ca2+ probe, which minimally enhances cardiac hypertrophic signaling

M. Ohkura, I. Norota, J. Nakai, K. Ishii

Meeting abstract　109　118　2009
Direct monitoring of mitochondrial calcium levels in cultured cardiomyocytes using a novel fluorescent indicator protein, GCaMP2-mt

17　(2)　238-239　2008/10/30

ISSN： 0919-4916
Local subplasma membrane Ca2+ signals detected by a tethered Ca sensor (G-CaMP2)

Nakai J, Ohkura M, Lee MY, Song H, Kotlikoff MI, Kinsey SP, Golovina VA, Blaustein MP

J Pharmacol Sci Mtg Abst　106　97P　2008
Imaging with genetically encoded Ca[2+] sensors

Microscopy　43　(2)　121-124　2008
Publisher: 日本顕微鏡学会
ISSN： 1349-0958
蛋白質でできた蛍光分子センサー (特集革新的センシング技術)

中井淳一

ケミカルエンジニヤリング　52　(9)　700-705　2007/09
Publisher: 化学工業社
ISSN： 0387-1037
Imaging cellular Ca2+ signals in the heart in vivo

Junichi Nakai, Masamichi Ohkura, Keiji Imoto, Yvonne N. Tallini, Bum-Rak Choi, Guangju Ji, Robert Doran, Jane Lee, Patricia Plan, Jason Wilson, Hong-Bo Xin, Atsush Sanbe, James Gulick, John Mathai, Jeffrey Robbins, Guy Salama, Michael L. Kotlikoff

JOURNAL OF PHARMACOLOGICAL SCIENCES　103　89P-89P　2007

ISSN： 1347-8613
news 蛍光タンパク質で心臓の活動を可視化

DayCharles, 中井淳一

パリティ　21　(9)　38-41　2006/09
Publisher: 丸善
ISSN： 0911-4815
Calcium imaging of neurons with an improved G-CaMP

J Nakai, M Ohkura, K Imoto, M Matsuzaki, H Kasai, D Reiff

JOURNAL OF PHARMACOLOGICAL SCIENCES　100　102P-102P　2006

ISSN： 1347-8613
Na+ pump alpha 1 and alpha 2 isoform targeting of Ca2+ reporter protein reveals spatially distinct sub-plasma membrane Ca2+ signals

MY Lee, H Song, J Nakai, M Ohkura, MI Kotlikoff, MP Blaustein

JOURNAL OF GENERAL PHYSIOLOGY　126　(1)　70A-71A　2005/07

ISSN： 0022-1295
Measurement of Ca2+ localization in distinct compartments between plasma membrane and 'junctional' sarco-/endoplasmic reticulum

MY Lee, H Song, J Nakai, MI Kotlikoff, MP Blaustein

FASEB JOURNAL　19　(4)　A664-A664　2005/03

ISSN： 0892-6638
Ca2+ sensing transgenic mice: Postsynaptic signaling in smooth muscle

J Nakai, G Ji, ME Feldman, KY Deng, KS Greene, J Wilson, JC Lee, RC Johnston, M Rishniw, Y Tallini, J Zhang, WG Wier, MP Blaustein, HB Xin, MI Kotlikoff

JOURNAL OF PHARMACOLOGICAL SCIENCES　97　74P-74P　2005

ISSN： 1347-8613
Near-membrane Ca2+ measurement with novel fluorochromes in arterial myocytes

M Cavalli, MY Lee, M Ohkura, H Song, J Zhang, SP Kinsey, J Nakai, MI Kotlikoff, MP Blaustein

FASEB JOURNAL　18　(5)　A1240-A1240　2004/03

ISSN： 0892-6638
Visualization of cellular and subcellular [Ca2+] with an improved GFP-based Ca2+ probe, G-CaMP2

M Ohkura, K Imoto, J Nakai

JOURNAL OF PHARMACOLOGICAL SCIENCES　94　118P-118P　2004

ISSN： 1347-8613
Measurements of [Ca2+](i) in cells and tissues of transgenic mice expressing the calcium sensitive GFP analogue G-CaMP restricted to smooth muscle.

MI Kotlikoff, J Nakai, K Su, KY Deng, JC Lee, J Wilson, ME Feldman, GJ Ji

BIOPHYSICAL JOURNAL　84　(2)　144A-144A　2003/02

ISSN： 0006-3495
GFPを用いた蛍光カルシウムプローブG-CaMPの開発

中井淳一, 大倉正道

比較生理生化学　19　(2)　135-145　2002
Publisher: 日本比較生理生化学会
DOI： 10.3330/hikakuseiriseika.19.135 　

ISSN： 0916-3786 1881-9346
A high signal-to-noise Ca2+ probe composed of a single green fluorescent protein

J Nakai, M Ohkura, K Imoto

NATURE BIOTECHNOLOGY　19　(2)　137-141　2001/02

DOI： 10.1038/84397 　

ISSN： 1087-0156
Two independent regions of RyR1 (1837-2154 and 2659-3720) restore skeletal type e-c coupling and DHPR junctional tetrads.

F Protasi, J Nakai, KG Beam, H Takekura, C Franzini-Armstrong, PD Allen

BIOPHYSICAL JOURNAL　80　(1)　330A-330A　2001/01

ISSN： 0006-3495
A novel high signal-to-noise Ca2+ probe composed of a single green fluorescent protein.

Nakai J, Ohkura M, Imoto K

Biophys J Mtg Abst　80　168A-168A　2001
A high signal-to-noise ca2+ probe composed of a single green fluorescent protein

J. Nakai, M. Ohkura, K. Imoto

Nature Biotechnology　19　(2)　137-141　2001

DOI： 10.1038/84397 　

ISSN： 1087-0156
Role of RyR1 residues 1837-2154 in reciprocal communication with alpha(1).

JJ O'Brien, C Proenza, J Nakai, PD Allen, KG Beam

BIOPHYSICAL JOURNAL　78　(1)　428A-428A　2000/01

ISSN： 0006-3495
Sorting signal sequence of triadin, an associated protein of ryanodine receptor Ca2+ release channel

M Ohkura, K Imoto, J Nakai

BIOPHYSICAL JOURNAL　78　(1)　430A-430A　2000/01

ISSN： 0006-3495
Evidence for a role of C-terminus in Ca2+ inactivation of skeletal muscle Ca2+ release channel (ryanodine receptor)

J Nakai, L Gao, L Xu, CL Xin, DA Pasek, G Meissner

FEBS LETTERS　459　(2)　154-158　1999/10

DOI： 10.1016/S0014-5793(99)01232-6 　

ISSN： 0014-5793
Functional reconstitution of a RyR1-RyR2 chimera (R9) in a homologous RyR-deficient muscle cell line.

JD Fessenden, RA Moore, Y Wang, J Nakai, C Franzini-Armstrong, F Protasi, PD Allen, IN Pessah

BIOPHYSICAL JOURNAL　76　(1)　A394-A394　1999/01

ISSN： 0006-3495
Localization in the II-III loop of the dihydropyridine receptor of a sequence critical for excitation-contraction coupling

J Nakai, T Tanabe, T Konno, B Adams, KG Beam

JOURNAL OF BIOLOGICAL CHEMISTRY　273　(39)　24983-24986　1998/09

DOI： 10.1074/jbc.273.39.24983 　

ISSN： 0021-9258
Two regions of the ryanodine receptor involved in coupling with L-type Ca2+ channels

J Nakai, N Sekiguchi, TA Rando, PD Allen, KG Beam

JOURNAL OF BIOLOGICAL CHEMISTRY　273　(22)　13403-13406　1998/05

DOI： 10.1074/jbc.273.22.13403 　

ISSN： 0021-9258
Protein-protein interactions of dihydropyridine and ryanodine receptors.

C Proenza, J Nakai, KG Beam

BIOPHYSICAL JOURNAL　74　(2)　A164-A164　1998/02

ISSN： 0006-3495
Two regions of the ryanodine receptor involved in coupling with L-type Ca2+ channels.

J Nakai, N Sekiguchi, TM Rando, PD Allen, KG Beam

BIOPHYSICAL JOURNAL　74　(2)　A142-A142　1998/02

ISSN： 0006-3495
Expression of rabbit skeletal and cardiac ryanodine receptors in HEK 293 cells.

L Gao, C Xin, L Xu, A Tripathy, J Nakai, G Meissner

BIOPHYSICAL JOURNAL　74　(2)　A60-A60　1998/02

ISSN： 0006-3495
Molecular machinery of excitation-contraction coupling in striated muscle

KG Beam, J Nakai, RT Dirksen, T Ogura, F Protasi, HT Nguyen, IN Pessah, CF FranziniArmstrong, PD Allen

JOURNAL OF PHYSIOLOGY-LONDON　504P　S11-S12　1997/11

ISSN： 0022-3751
Molecular cloning and characterization of a human brain ryanodine receptor

Y Nakashima, S Nishimura, A Maeda, EL Barsoumian, Y Hakamata, J Nakai, PD Allen, K Imoto, T Kita

FEBS LETTERS　417　(1)　157-162　1997/11

DOI： 10.1016/S0014-5793(97)01275-1 　

ISSN： 0014-5793
The S5-S6 linker of repeat I is a critical determinant of L-type Ca2+ channel conductance

RT Dirksen, J Nakai, A Gonzalez, K Imoto, KG Beam

BIOPHYSICAL JOURNAL　73　(3)　1402-1409　1997/09

ISSN： 0006-3495
Role of S4 segments and the leucine heptad motif in the activation of an L-type calcium channel

J Garcia, J Nakai, K Imoto, KG Beam

BIOPHYSICAL JOURNAL　72　(6)　2515-2523　1997/06

DOI： 10.1016/S0006-3495(97)78896-9 　

ISSN： 0006-3495
The S5-S6 linker of repeat I is a critical determinant of L-type Ca2+ channel unitary conductance.

RT Dirksen, J Nakai, A Gonzalez, K Beam

BIOPHYSICAL JOURNAL　72　(2)　WPMD7-WPMD7　1997/02

DOI： 10.1016/S0006-3495(97)78172-4 　

ISSN： 0006-3495

eISSN： 1542-0086
Functional nonequality of the cardiac and skeletal ryanodine receptors

J Nakai, T Ogura, F Protasi, C FranziniArmstrong, PD Allen, KG Beam

PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA　94　(3)　1019-1022　1997/02

DOI： 10.1073/pnas.94.3.1019 　

ISSN： 0027-8424
Functional non-equality of the cardiac and skeletal ryanodine receptors.

J Nakai, T Ogura, F Protasi, C FranziniArmstrong, PD Allen, KG Beam

BIOPHYSICAL JOURNAL　72　(2)　MAMH3-MAMH3　1997/02

DOI： 10.1073/pnas.94.3.1019 　

ISSN： 0006-3495

eISSN： 1542-0086
The S5-S6 linker of repeat I is a critical determinant of L-type Ca2+ channel unitary conductance.

RT Dirksen, J Nakai, A Gonzalez, K Beam

BIOPHYSICAL JOURNAL　72　(2)　WPMD7-WPMD7　1997/02

ISSN： 0006-3495

eISSN： 1542-0086
リアノジン受容体(RyR-1)による電位依存性L型カルシウムチャネルの活性化

中井淳一, DIRKSENR. t, NGUYENH. t, PESSAHI. n, BEAMK. g, ALLENP. d

日本分子生物学会年会プログラム・講演要旨集　19　313-313　1996/08/01
Enhanced dihydropyridine receptor channel activity in the presence of ryanodine receptor

J Nakai, RT Dirksen, HT Nguyen, IN Pessah, KG Beam, PD Allen

NATURE　380　(6569)　72-75　1996/03

DOI： 10.1038/380072a0 　

ISSN： 0028-0836
Altered voltage dependence of L-type calcium current by S4 segment and leucine-heptad repeat mutations.

J Garcia, J Nakai, K Imoto, KG Beam

BIOPHYSICAL JOURNAL　70　(2)　TUAM5-TUAM5　1996/02

ISSN： 0006-3495

eISSN： 1542-0086
Restoration of excitation-contraction coupling and slow calcium current by expression of ryanodine receptor.

J Nakai, RT Dirksen, HT Nguyen, IN Pessah, KG Beam, PD Allen

BIOPHYSICAL JOURNAL　70　(2)　MPMB7-MPMB7　1996/02

ISSN： 0006-3495

eISSN： 1542-0086
INVOLVEMENT OF THE BRAIN-TYPE OF RYANODINE RECEPTOR IN T-CELL PROLIFERATION

Y HAKAMATA, S NISHIMURA, J NAKAI, Y NAKASHIMA, T KITA, K IMOTO

FEBS LETTERS　352　(2)　206-210　1994/09

DOI： 10.1016/0014-5793(94)00955-4 　

ISSN： 0014-5793
MULTIPLE TYPES OF RYANODINE RECEPTOR CA2+ RELEASE CHANNELS ARE DIFFERENTIALLY EXPRESSED IN RABBIT BRAIN

T FURUICHI, D FURUTAMA, Y HAKAMATA, J NAKAI, H TAKESHIMA, K MIKOSHIBA

JOURNAL OF NEUROSCIENCE　14　(8)　4794-4805　1994/08

ISSN： 0270-6474
CRITICAL ROLES OF THE S3 SEGMENT AND S3-S4 LINKER OF REPEAT-I IN ACTIVATION OF L-TYPE CALCIUM CHANNELS

J NAKAI, BA ADAMS, K IMOTO, KG BEAM

PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA　91　(3)　1014-1018　1994/02

DOI： 10.1073/pnas.91.3.1014 　

ISSN： 0027-8424
CRITICAL ROLES OF THE S3 SEGMENT AND S3-S4 LINKER OF REPEAT-I IN ACTIVATION OF L-TYPE CALCIUM CHANNELS

J NAKAI, BA ADAMS, K IMOTO, KG BEAM

BIOPHYSICAL JOURNAL　66　(2)　A129-A129　1994/02

ISSN： 0006-3495
PRIMARY STRUCTURE AND FUNCTIONAL EXPRESSION OF THE OMEGA-CONOTOXIN-SENSITIVE N-TYPE CALCIUM-CHANNEL FROM RABBIT BRAIN

Y FUJITA, M MYNLIEFF, RT DIRKSEN, MS KIM, T NIIDOME, J NAKAI, T FRIEDRICH, N IWABE, T MIYATA, T FURUICHI, D FURUTAMA, K MIKOSHIBA, Y MORI, KG BEAM

NEURON　10　(4)　585-598　1993/04

DOI： 10.1016/0896-6273(93)90162-K 　

ISSN： 0896-6273
PRIMARY STRUCTURE AND DISTRIBUTION OF A NOVEL RYANODINE RECEPTOR CALCIUM RELEASE CHANNEL FROM RABBIT BRAIN

Y HAKAMATA, J NAKAI, H TAKESHIMA, K IMOTO

FEBS LETTERS　312　(2-3)　229-235　1992/11

DOI： 10.1016/0014-5793(92)80941-9 　

ISSN： 0014-5793
EXPRESSION OF CA-2+-INDUCED CA-2+ RELEASE CHANNEL ACTIVITY FROM CARDIAC RYANODINE RECEPTOR CDNA IN CHINESE-HAMSTER OVARY CELLS

T IMAGAWA, J NAKAI, H TAKESHIMA, Y NAKASAKI, M SHIGEKAWA

JOURNAL OF BIOCHEMISTRY　112　(4)　508-513　1992/10

ISSN： 0021-924X
PRIMARY STRUCTURE AND FUNCTIONAL EXPRESSION FROM COMPLEMENTARY-DNA OF A BRAIN CALCIUM-CHANNEL

Y MORI, T FRIEDRICH, MS KIM, A MIKAMI, J NAKAI, P RUTH, E BOSSE, F HOFMANN, FLOCKERZI, V, T FURUICHI, K MIKOSHIBA, K IMOTO, T TANABE, S NUMA

NATURE　350　(6317)　398-402　1991/04

DOI： 10.1038/350398a0 　

ISSN： 0028-0836
PRIMARY STRUCTURE AND FUNCTIONAL EXPRESSION FROM CDNA OF THE CARDIAC RYANODINE RECEPTOR CALCIUM RELEASE CHANNEL

J NAKAI, T IMAGAWA, Y HAKAMATA, M SHIGEKAWA, H TAKESHIMA, S NUMA

FEBS LETTERS　271　(1-2)　169-177　1990/10

DOI： 10.1016/0014-5793(90)80399-4 　

ISSN： 0014-5793
LOCATION OF A REGION OF THE MUSCARINIC ACETYLCHOLINE-RECEPTOR INVOLVED IN SELECTIVE EFFECTOR COUPLING

T KUBO, H BUJO, AKIBA, I, J NAKAI, M MISHINA, S NUMA

FEBS LETTERS　241　(1-2)　119-125　1988/12

DOI： 10.1016/0014-5793(88)81043-3 　

ISSN： 0014-5793
DIFFERENT SENSITIVITIES TO AGONIST OF MUSCARINIC ACETYLCHOLINE-RECEPTOR SUBTYPES

H BUJO, J NAKAI, T KUBO, K FUKUDA, AKIBA, I, A MAEDA, M MISHINA, S NUMA

FEBS LETTERS　240　(1-2)　95-100　1988/11

DOI： 10.1016/0014-5793(88)80346-6 　

ISSN： 0014-5793
RINGS OF NEGATIVELY CHARGED AMINO-ACIDS DETERMINE THE ACETYLCHOLINE-RECEPTOR CHANNEL CONDUCTANCE

K IMOTO, C BUSCH, B SAKMANN, M MISHINA, T KONNO, J NAKAI, H BUJO, Y MORI, K FUKUDA, S NUMA

NATURE　335　(6191)　645-648　1988/10

DOI： 10.1038/335645a0 　

ISSN： 0028-0836
PRIMARY STRUCTURE OF PORCINE MUSCARINIC ACETYLCHOLINE RECEPTOR-III AND ANTAGONIST BINDING-STUDIES

AKIBA, I, T KUBO, A MAEDA, H BUJO, J NAKAI, M MISHINA, S NUMA

FEBS LETTERS　235　(1-2)　257-261　1988/08

DOI： 10.1016/0014-5793(88)81274-2 　

ISSN： 0014-5793
MOLECULAR-BASIS OF THE FUNCTIONAL-HETEROGENEITY OF THE MUSCARINIC ACETYLCHOLINE-RECEPTOR

S NUMA, K FUKUDA, T KUBO, A MAEDA, AKIBA, I, H BUJO, J NAKAI, M MISHINA, H HIGASHIDA

COLD SPRING HARBOR SYMPOSIA ON QUANTITATIVE BIOLOGY　53　295-301　1988

DOI： 10.1101/SQB.1988.053.01.036 　

ISSN： 0091-7451

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Books and Other Publications 1

ニューロンの生理学

京都大学学術出版会　2009

Industrial Property Rights 2

Method of producing a biosensor protein capable of regulation a fluorescence property of Green Fluorescent Protein, and the biosensor protein produced by the method.

Property Type: Patent

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US 6,783,958 B2
緑色蛍光蛋白質の蛍光強度を効率的に制御する方法と緑色蛍光蛋白を用いたバイオセンサーの遺伝子の開発

Property Type: Patent

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3650815

Research Projects 49

Novel molecular mechanism of oral cancer pain focused attention on plastic changes in intercellular network in the trigeminal ganglion

Offer Organization: Japan Society for the Promotion of Science

System: Grants-in-Aid for Scientific Research

Category: Grant-in-Aid for Scientific Research (B)

Institution: Nihon University

2023/04/01 - 2028/03/31
マルチスケール脳機能イメージングによる神経障害性疼痛の情動変容機序の解明

水田健太郎, 篠田雅路, 佐々木啓一, 中井淳一, 小山内実

Offer Organization: 日本学術振興会

System: 科学研究費助成事業

Category: 基盤研究(B)

Institution: 東北大学

2021/04/01 - 2025/03/31

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神経障害性疼痛は、末梢神経の炎症や傷害を契機として侵害情報伝達経路が持続的な可塑的変化を起こすことで生じる難治性疼痛である。一旦、中枢神経系に可塑的変化が生じると、鎮痛薬や神経ブロック療法が奏功し難くなり難治化するだけでなく、情動を司る中枢領域にも可塑的変化をきたし、抑うつや不安亢進といった情動変容が生じると考えられる。しかし、神経障害性疼痛に随伴する情動変容に上位中枢のどの脳領域が関与するのか、またどのような機序で情動変容が生じるかは不明であり、治療法を開発する上での壁になっている。本研究は、マルチスケール脳機能イメージングで上位中枢の神経活動を可視化し、神経障害性疼痛に随伴する情動変容の機序を解明するものである。本年度は、篠田雅路教授が確立した低侵襲に眼窩下神経結紮モデルを作成する方法を東北大学でも安定して作成できる体制を確立した。次いで、神経障害性疼痛の難治化に関与する上位中枢の神経活動を可視化するため、ラットの全脳レベルでの脳神経活動の履歴をMRIで捉える手法である定量的活動依存性マンガン造影MRI（qAIM-MRI）の撮像条件設定を行った。動物用MRIは実験計画立案当初は東北大学医学系研究科共通機器室の小動物用MRIを使用予定であったが、動物実験施設の改修工事により使用不可となったため、川島隆太教授（東北大学加齢医学研究所）の協力を得て、小動物用7T-MRI装置を使用することとし、新たにラット脳の撮影条件設定を行った。ラットを眼窩下神経結紮手術前１週、手術後1週、及び手術後3週にMRIを撮像するプロトコルを組み、それぞれの撮像日の24時間もしくは48時間前に塩化マンガンを腹腔内投与し、撮像した画像を比較検討した。その結果、当該モデルラットにおいて、MRI撮像48時間前に塩化マンガンを腹腔内投与するプロトコールが最も適していることが明らかになった。
マルチスケール脳機能イメージングによる神経障害性疼痛の情動変容機序の解明

水田健太郎, 篠田雅路, 佐々木啓一, 中井淳一, 小山内実

Offer Organization: 日本学術振興会

System: 科学研究費助成事業

Category: 基盤研究(B)

Institution: 東北大学

2021/04/01 - 2025/03/31
Study on neural mechanism of swallowing by imaging

Offer Organization: Japan Society for the Promotion of Science

System: Grants-in-Aid for Scientific Research

Category: Grant-in-Aid for Scientific Research (B)

Institution: Tohoku University

2020/04/01 - 2023/03/31
System Identification of Bio-Navigation and Development of Innovative Logging Device

Hashimoto Koichi

Offer Organization: Japan Society for the Promotion of Science

System: Grants-in-Aid for Scientific Research

Category: Grant-in-Aid for Scientific Research on Innovative Areas (Research in a proposed research area)

Institution: Tohoku University

2016/06/30 - 2021/03/31

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Research was conducted to develop advanced small multidimensional data loggers to measure animal behavior and to analyze and understand the navigation data collected from the loggers. By developing Logbot (an AI logger equipped with GPS, camera, accelerometer, etc.), previously unknown behaviors were recorded, making it effective for behavior analysis. In addition, microphone-equipped, camera-integrated, camera-separated, and intervention stimulators were realized. Data acquisition continues with new animal species such as bears, dogs, and bats. Furthermore, predictive and analytical models of animal and human behavior have been developed, and results such as hierarchy and clustering of migratory behavior have been achieved.
Development of Fluorescent Probes with Molecular Evolution Engineering

NAKAI JUNICHI

Offer Organization: Japan Society for the Promotion of Science

System: Grants-in-Aid for Scientific Research

Category: Grant-in-Aid for Scientific Research (S)

2015/05/29 - 2020/03/31

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Up until now, gene-encoded imaging probes were created using known protein fragments that bind to the molecule of interest to be detected. In this study, we applied molecular evolution engineering to develop a technology to create a new imaging probe using a peptide aptamer library or a small molecule antibody with a completely new sequence. In connection with this, we have developed technology for visualizing biological phenomena and evolutionary engineering technology such as immuno-PCR method. The developed technology is useful for elucidation of biological phenomena and medical treatment.
New development of clarification and control on mechanism of neurogenesis of fluorescent enteric neurons responding to stimuli.

Takaki Miyako

Offer Organization: Japan Society for the Promotion of Science

System: Grants-in-Aid for Scientific Research

Category: Grant-in-Aid for Scientific Research (B)

Institution: Nara Medical University

2015/04/01 - 2018/03/31

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The reconstruct of impaired enteric neural circuits at the thick granulation tissue at anastomosis in small intestine of H-line: Thy1 promoter GFP mouse was observed by two-photon microscopy a week after drinking a 5-HT4-receptor agonist, mosapride citrate (MOS) 100 microM solution. In Thy1-promoter YFP mouse after gut transection and anastomosis, the fetal brain-derived neural stem cells (NSC) with red fluorescent cell linker were transplanted from the tail vein. 5-HT4 receptor-mediated facilitation of neurogenesis was confirmed by clear three-dimensional in vivo imaging of newborn enteric neurons generated from enteric neural progenitors (host NSC; green fluorescence) and those from transplant NSC (red fluorescence). The facilitating effect of MOS and the distribution of new neurons were similar between the transplant and host NSC. Finally, though preliminary, we succeeded in in vivo Ca2+ imaging of myenteric neuronal activity in the normal ileum of Thy1-G6-mCherry transgenic mouse.
Study of neuronal network mechanism for habit learning using highly sensitive calcium probe

OHKURA Masamichi

Offer Organization: Japan Society for the Promotion of Science

System: Grants-in-Aid for Scientific Research

Category: Grant-in-Aid for Scientific Research (B)

Institution: Saitama University

2014/04/01 - 2018/03/31

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To enable visualization and manipulation of neuronal network activities in zebrafish, we developed transgenic zebrafish expressing probes such as high-performance genetically-encoded fluorescent calcium indicators (improved G-CaMPs). We established an experimental system for quantitative calcium imaging of zebrafish brain region in response to visual stimuli, using transgenic fish expressing an improved green calcium probe G-CaMP and a red fluorescent protein.
A study of neural circuit mechanism of voluntary behavior in C. elegans

GENGYO-ANDO Keiko, NAKAI Junichi, OHKURA Masamichi, TAKAGI Shin

Offer Organization: Japan Society for the Promotion of Science

System: Grants-in-Aid for Scientific Research

Category: Grant-in-Aid for Challenging Exploratory Research

Institution: Saitama University

2015/04/01 - 2017/03/31

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Animals exhibit voluntary behaviors, but their neural mechanisms are not well understood. In this study, we developed a new imaging system, ICaST, which combines an automatic tracking system and a fast-scanning laser confocal microscope, to image neurons of interest in freely behaving C. elegans. Using ICaST in conjunction with an improved G-CaMP7, we conducted Ca2+ imaging of a freely moving animal at high magnification while automatically tracking the target in the microscope's field of view. We identified several neurons in the head ganglia that act before or during spontaneous backward locomotion. We also developed a system for optogenetic stimulation and simultaneous imaging by concurrent use of G-CaMP and ChR2. Our new approach will provide a powerful platform for studying the neural circuit mechanisms of C. elegans behaviors and has potential for use in other animals.
運動学習における長期記憶機構の研究

中井淳一

Offer Organization: 日本学術振興会

System: 科学研究費助成事業

Category: 新学術領域研究(研究領域提案型)

Institution: 埼玉大学

2014/04/01 - 2016/03/31

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記憶の定着がどのように起こるのか、また、良く知られた記憶の中枢である海馬と運動学習における中枢である小脳で、記憶の定着のメカニズムの違いや共通点は何かなど、記憶の定着についてはまだよく分かっていないことが多い。運動学習の短期記憶について、小脳における運動学習では記憶が小脳皮質でおこると考えられている。しかし、記憶の定着がどのように起こるのか、そのメカニズムはまだよくわかっていない。本研究では、運動学習に関する短期記憶のメカニズムの解明を行う。小脳学習の定着のメカニズムが解明される。記憶の理解が進むことにより、短期記憶と長期記憶の分子機構の違いが明らかになる。また、この成果は海馬などの記憶の定着のメカニズムとの相同性や、違いを知ることにつながり、脳における長期記憶の全体像の理解が深まる。老化で記憶力が落ちるメカニズムの解明やその対処法、記憶に関するヒトの疾患の原因解明、脳卒中後のリハビリの効率化や、スポーツ強化にもつながる可能性がある。ゼブラフィッシュを用いて、optokinetic response（OKR）用視覚刺激装置を作成し、サッケードの角度、速度を解析した。この装置を用いて、OKRの学習を行わせ、学習後のサッカードの角度、速度を解析した。エンハンサートラップラインを用いてG-CaMPを小脳顆粒細胞、下オリーブ核、Eurydendroid cellに発現させ、その際の小脳の顆粒細胞、下オリーブ核の細胞活動をG-CaMPを用いたカルシウムイメージングを用いて解析を行った。今後はプルキンエ細胞にG-CaMPを発現するトランスジェニックラインを用いたカルシウムイメージングによりさらに解析を行っていく必要がある。
Development of whole heart calcium imaging system with two different waves using modified G-CaMP-DsRed-coexpressed transgenic rat model in the blood-perfused excised heart preparation

Obata Koji, Takaki Miyako, Nakai Junichi, Ohkura Masamichi

Offer Organization: Japan Society for the Promotion of Science

System: Grants-in-Aid for Scientific Research

Category: Grant-in-Aid for Scientific Research (C)

Institution: Gifu University

2013/04/01 - 2016/03/31

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I attempted to develop a whole heart calcium imaging system with two different waves using modified G-CaMP-DsRed-coexpressed transgenic rat model to investigate the relationship among cardiac contractility, oxygen consumption, and intracellular calcium handling in the blood-perfused excised heart preparation. I just have to overcome a couple of problems for measuring accurate calcium transient. Now, I try to find the effective breakthrough.
Development of novel molecular probes toward bio-imaging and photopharmacology of neural systems

OBA Toru, NAKAI Junichi, OHKURA Masamichi

Offer Organization: Japan Society for the Promotion of Science

System: Grants-in-Aid for Scientific Research

Category: Grant-in-Aid for Scientific Research (C)

Institution: Utsunomiya University

2013/04/01 - 2016/03/31

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This study aims at development of novel molecular probes for researches of neural systems. (1) We successfully developed novel voltage sensitive fluorescent probes. (2) We developed selective iodination of the vinyl group and meso-position of a chlorophyll derivative and a natural porphyrin. We also developed facile, green conversion reactions of vinyl group into epoxy, formyl, and acetyl groups via the iodination. (3) We established design principles of pyridyl-pyrrole-based fluorescent probes. (4) We successfully developed novel environment-responsive fluorescent probes.
Toward Brain Census: Construction of a method for revealing the memory mechanism at the molecular level

NISHIGAKI Koichi, KOBAYASHI Tetsuya, YAMASU Kyo, NAKAI Junichi, OHKURA Masamichi

Offer Organization: Japan Society for the Promotion of Science

System: Grants-in-Aid for Scientific Research

Category: Grant-in-Aid for Scientific Research (A)

Institution: Saitama University

2012/04/01 - 2016/03/31

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For understanding the memory mechanism, not only neuron cell networks and their activities but also dynamic molecular systems should be elucidated. However, the latter could not be performed due to the lack of methodology. We have developed a novel approach to clarify the molecular system within an extremely small region (1mm3 of zebrafish brain) by dissecting it into minute masses and orderly arranging them in an MMV microarray, enabling them to be subjected to parallel and multistep analysis. On the other hand, a highly sensitive and parallel detection method was developed by employing the peptide ELISA (ELISA is usually antibody-based detection method) facilitated with an MMV microarray. Five pairs of novel peptides for each of MGMT, Aβ42, p38α, CREB1a and NM23 were identified and implemented to make up pepELISA array. Thus, we have succeeded in building up a novel methodology for the analysis of brain molecular systems, expecting future fruits.
Development of a fast photo-stimulation device

NAKAI Junichi, GENGYO-ANDO Keiko

Offer Organization: Japan Society for the Promotion of Science

System: Grants-in-Aid for Scientific Research

Category: Grant-in-Aid for Challenging Exploratory Research

Institution: Saitama University

2014/04/01 - 2015/03/31

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Recently, optogenetics is getting popular for studying neuronal circuits. In this research, we developed a photostimulation device that used a fast XY scanner with potassium tantalate niobate (KTN) crystal. World-fast two photon excitation of fluorescein could be achieved with this newly developed photostimulation device with KTN scanner. Scan speed of the KTN scanner is more than 10 times faster than that of the galvano scanner. This research will advance brain science and natural science in which optical technique is more important.
蛍光プローブによる細胞移動の予測と移動秩序の分子機構の解明

中井淳一

Offer Organization: 日本学術振興会

System: 科学研究費助成事業

Category: 新学術領域研究(研究領域提案型)

Institution: 埼玉大学

2013/04/01 - 2015/03/31

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発生初期には多くの細胞が目的の位置に移動する現象が頻繁に見られ、細胞移動にはガイダンス分子や細胞内シグナル伝達系が関与するが、細胞内カルシウムイオンの変化が深くかかわっていることが示されている。本研究ではゼブラフィッシュ初期胚を用いて、細胞移動とカルシウムイオンの関係について検討した。実験はゼブラフィッシュの受精卵に蛍光カルシウムプローブ（G-CaMPまたはR-CaMP）を発現させ、レーザー顕微鏡を用いて、発生中の細胞（epiboly中のenvelope細胞）の細胞移動と細胞内カルシウム変動を経時的に計測した。その結果、envelop細胞の細胞内カルシウム濃度が高いグループと、低いグループの２つのタイプがあることが分かった。これらの２つの細胞種を詳しく観察したところ、カルシウム濃度が高いグループの細胞は非常に活発に周りの細胞をかき分けて「能動的に」移動していることがわかった。一方、細胞内カルシウム濃度が低いグループの細胞は周りの細胞との位置関係が比較的保たれて移動しており、「受動的」または周囲よりの力に「流されて」移動していると考えられた。また、細胞移動中のカルシウム計測をより高感度に行う事ができるよう赤色の蛍光を持つ蛍光カルシウムセンサーR-CaMP2を開発した。線虫、マウスの神経、筋細胞にR-CaMP2とG-CaMPおよび/またはチェネルロドプシン2を発現させin vivoカルシウムイメージングを行い2波長でのカルシウム同時イメージング、光遺伝学との併用も可能であることを示した。
Imaging analysis of neural circuit involved in voluntary behaviors in C. elegans

GENGYO-ANDO Keiko, NAKAI Junichi, OHKURA Masamichi, HASHIMOTO Kouichi

Offer Organization: Japan Society for the Promotion of Science

System: Grants-in-Aid for Scientific Research

Category: Grant-in-Aid for Challenging Exploratory Research

Institution: Saitama University

2013/04/01 - 2015/03/31

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Most Animals can initiate and regulate voluntary behaviors in the absence of external stimuli, but the mechanism underlying decision-making is largely unknown. To elucidate the molecular and neural mechanism of these behaviors, we focused on C. elegans, which have a simple brain consisting of only 302 neurons and transparent body. Using our recently developed G-CaMP6-8, green genetically encoded calcium indicators (GECIs), we developed a system that allowed systematic imaging and analysis of the neural activity during spontaneous locomotion. We further designed R-CaMP2, a high-affinity, fast, red GECI, and successfully achieved dual-color imaging and optical stimulation in combination with green GECIs and channelrhodopshin 2 in freely moving C. elegans.
Clarification and control on mechanism of neurogenesis of fluorescent enteric neurons.

TAKAKI Miyako, KUNIYASU Hiroki, NABEKURA Junichi, NAKAI Junichi

Offer Organization: Japan Society for the Promotion of Science

System: Grants-in-Aid for Scientific Research

Category: Grant-in-Aid for Scientific Research (B)

Institution: Nara Medical University

2011/04/01 - 2015/03/31

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The reconstruct of impaired enteric neural circuits at the thick granulation tissue at anastomosis in small intestine of H-line: Thy1 promoter GFP mouse was observed by two-photon microscopy (2PM) a week after drinking a 5-HT4-receptor agonist, mosapride citrate (MOS) 100 microM solution. In Thy1-promoter YFP mouse after gut transection and anastomosis, the fetal brain-derived neural stem cells (NSC) with red fluorescent cell linker were transplanted from the tail vein. 5-HT4 receptor-mediated facilitation of neurogenesis was confirmed by clear three-dimensional in vivo imaging of newborn enteric neurons generated from enteric neural progenitors (host NSC; green fluorescence) and those from transplant NSC (red fluorescence). The facilitating effect of MOS and the distribution of new neurons were similar between the transplant and host NSC. Finally, though preliminary, we succeeded in in vivo Ca2+ imaging of myenteric neuronal activity in the normal ileum of GCaMP6 transgenic mouse.
Next generation photostimulation system using tunable laser

NAKAI Junichi, ANDO Keiko

Offer Organization: Japan Society for the Promotion of Science

System: Grants-in-Aid for Scientific Research

Category: Grant-in-Aid for Challenging Exploratory Research

Institution: Saitama University

2013/04/01 - 2014/03/31

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In this study we developed a tunable wavelength photo-stimulation equipment. We could generate second harmonic waves ranging from ~400 nm to ~500 nm with a fun-out type periodically polarized crystal. So this equipment can photo-stimulate GFP, channelrhodopsin, and many other photosensitive molecules. In the system we also equipped a pair of potassium tantalate niobate (KTN) crystals as a tunable lens, an electro-optic device that changes its refractive index by voltage. By changing the voltage which applied to the tunable KTN lens, focus changed within ~20 microseconds. The speed of focus change was about 500~1000 times faster than that of the conventional piezo focuser. This technique can be applicable not only to scientific researches including brain science but also industrial field such as laser processing and laser measurement.
Looking into living enteric neurons

TAKAKI Miyako, KUNIYASU Hiroki, NABEKURA Junichi, NAKAI Junichi

Offer Organization: Japan Society for the Promotion of Science

System: Grants-in-Aid for Scientific Research

Category: Grant-in-Aid for Challenging Exploratory Research

Institution: Nara Medical University

2012/04/01 - 2014/03/31

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In the present study, using transgenic mice (TG) expressing GFP in neurons we succeeded in in vivo imaging of enteric neurons with 2 photon microscopy (2PM). Then, the process of neurogenesis of injured enteric nervous system in TG mouse transected and re-astomosed gut was revealed by in vivo imaging with 2PM. We determined that a 5-HT4 receptor agonist facilitated to generate newborn enteric neurons at the anastomosis mediated via 5-HT4 receptor from neural stem cells. The neurogenesis of enteric neurons would give an improvement of dysfunction of gut function and defecation function. This study is revolutionary and unexplored in medical field of rehabilitation.
Functional analyses of newborn neurons in CNS by Cereblon overexpression

ANDO Hideki, HANDA Hiroshi, NAKAI Juniti, ANDO Keiko

Offer Organization: Japan Society for the Promotion of Science

System: Grants-in-Aid for Scientific Research

Category: Grant-in-Aid for Challenging Exploratory Research

2012/04/01 - 2014/03/31

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Zebrafish embryos overexpressing Cereblon (CRBN) showed enlarged brain. They have excess number of neurons as well as glia cells with spatially normal pattern.In this study, we investigated whether those increased neurons actually function in higher behavior of the animal by the following approaches. 1)Measurement of neural activity by the use of calcium sensor: (Results) Neural activities in the telencephalon were increased to 140% of normal ones in CRBN-overexpressed larvae at 6 days after fertilization both in resting condition and in light-stimulated condition. 2)Measurement of learning potential by the 2-directional active avoidance assay using combination of light stimulation and electrical shock: (Results) In this experiment, CRBN-overexpressed larvae showed higher score than normal ones.
Development of a next-generation photo-stimulation device

NAKAI Junichi, ANDO Keiko, OHKURA Masamichi

Offer Organization: Japan Society for the Promotion of Science

System: Grants-in-Aid for Scientific Research

Category: Grant-in-Aid for Challenging Exploratory Research

Institution: Saitama University

2012 - 2012

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In this study we developed a microscope equipment with which photosensitive molecules can be stimulated. In the equipment a pair of potassium tantalate niobate (KTN) crystals were used as a tunable lens, an electro-optic device that changes its refractive index by voltage. By changing the voltage which applied to the tunable KTN lens focus changed within ~23 microseconds. The speed of focus change was about 500~1000 times faster than that of the conventional piezo focuser. This technique can be applicable not only to scientific researches including neuroscience but also industrial field such as laser processing.
NETWORK ANALYSIS AMONG HARD TISSUE CELLS USING MULTI-PHOTON MICROSCOPE

WAKAMORI Minoru, NAKAI Jyunichi

Offer Organization: Japan Society for the Promotion of Science

System: Grants-in-Aid for Scientific Research

Category: Grant-in-Aid for Challenging Exploratory Research

Institution: Tohoku University

2010 - 2012

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Osteoblasts and bone cells detect mechanical stimuli, and it is thought that their networks share information. The final goal of this project is to establish the method to analyze the network function among the cells by measuring intracellular Ca^<2+> concentration as an index. Ca^<2+> influx was observed in the osteoblast-like cells after extension. Moreover, Ca^<2+> fluorescent proteins worked like Ca^<2+> indicator fura-2.
Functional study of Munc18 mutations associated with epilepsy

ANDO Keiko, NAKAI Junichi, OHKURA Masamichi

Offer Organization: Japan Society for the Promotion of Science

System: Grants-in-Aid for Scientific Research

Category: Grant-in-Aid for Scientific Research (C)

Institution: Saitama University

2010 - 2012

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To elucidate the molecular mechanism of intractable epilepsy, we developed C. elegans models having Munc18-1 mutations associated with Ohtahara syndrome (OS) known as early infantile epileptic encephalopathy (EIEE). We found that the Munc18-1 mutations resulted in behavioral defects such as paralysis and epileptic-like convulsions, and abnormalities in synaptic transmission. We also developed the optical measurement system of neural function using in vivo imaging and optogenetics. These disease models and optical technique can contribute to the understanding of the molecular pathogenesis and drug discovery for epilepsy.
STUDY OF SYNAPTIC MODULATORY MECHANISMS IN RESPONSE TO STRESS MEDIATED BY 5-HT2A RECEPTOR

OHKURA Masamichi, NAKAI Junichi, ANDO Keiko

Offer Organization: Japan Society for the Promotion of Science

System: Grants-in-Aid for Scientific Research

Category: Grant-in-Aid for Scientific Research (C)

Institution: Saitama University

2010 - 2012

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To enable detection of small calcium signals of neurons in response to single action potentials or synaptic inputs, we developed high-performance genetically-encoded fluorescent calcium indicators (improved G-CaMPs and R-CaMPs). We also established an experimental system for simultaneous photo-stimulation and fluorescence imaging of C. elegans expressing channelrhodopsin-2 in the HSN neuron and GFP-labeled 5-HT^<2A> or an improved G-CaMP in the vulval muscle.
ゼブラフィッシュの覚醒・睡眠の分子機構に関する研究 Competitive

中井淳一

Offer Organization: 文部科学省

System: 科学研究費補助金(新学術領域研究(研究領域提案型))

Category: 新学術領域研究(研究領域提案型)

Institution: 埼玉大学

2009 - 2009

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多くの動物は眠るが、睡眠の分子メカニズムはまだ良くわかっていない。この状況は、Hypocretin/Orexin(Hcrt)とその受容体が発見され大きく進展した。本研究は、ゼブラフィッシュを用いて動物の睡眠と覚醒の分子機構を明らかにすることを目的としている。そこで蛍光カルシウムプローブG-CaMPを神経細胞に発現させ神経細胞の機能とゼブラフィッシュの行動との関係を探ることを計画した。本研究において蛍光カルシウムプローブG-CaMP2を改良したG-CaMP-HSを新たに開発した。G-CaMP-HSはG-CaMP2と比較して高感度であるとともに、細胞内でのタンパク質の安定性が増しており、安静時の蛍光強度が高いという特徴を持つ。これに関連して、蛍光カルシウムプローブの性能向上に関する特許を申請した。次にG-CaMP-HSをGal4-UASの下流につないだコンストラクトを作成し、ゼブラフィッシュに導入することにより遺伝子改変ゼブラフィッシュを作成した。このゼブラフィッシュと運動ニューロン(Caudal primary neuron : CaPニューロン)にGal4を発現するゼブラフィッシュ(SAIGFF213A)とを掛け合わせることによりCaPニューロンにG-CaMP-HSを発現するゼブラフィッシュを作成した。CaPニューロンの活動をイメージングにより解析したところ、CaPニューロンのバースト発火に伴い、G-CaMP-HSの蛍光強度が変化することが観察された。この成果はProc Natl Acad Sci USAに報告した。ゼブラフィッシュの神経細胞からG-CaMP-HSを用いて神経活動をイメージングにて解析することが可能となったことにより、今後HcrtニューロンにG-CaMP-HSを発現させ解析することにより睡眠覚醒のメカニズムの解明が進むと考えられる。
Development of a red fluorescent Ca^<2+> sensor Competitive

NAKAI Junichi

Offer Organization: Japan Society for the Promotion of Science

System: Grants-in-Aid for Scientific Research

Category: Grant-in-Aid for Scientific Research (C)

Institution: Saitama University

2009 - 2009

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The purpose of this study is to develop a fluorescent protein-based red fluorescent Ca^<2+> sensor which can be excited by red light and emit red fluorescence. We generated a prototype red fluorescent Ca^<2+> sensor by replacing the circularly permuted GFP domain of G-CaMP2, which is our original green fluorescent Ca^<2+> sensor, with the circularly permuted RFP, then randomly mutagenized this prototype sensor and screened variants with high performance. As a result, we successfully found the sensor termed R-CaMP, which showed a great red fluorescence change upon Ca^<2+> binding.
ケージドグルタミン酸を用いた多点同時刺激による海馬ニューロンの長期増強の解析 Competitive

中井淳一, 平瀬肇, 小島比呂志, 塚田稔

Offer Organization: 文部科学省

System: 科学研究費補助金(特定領域研究)

Category: 特定領域研究

Institution: 独立行政法人理化学研究所

2005 - 2005

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この研究の目的はケイジドグルタミン酸と高速多点光刺激法を用いて海馬CA1ニューロンにおけるヘテロシナプス性の長期増強を研究することにある。新開発の高速光刺激レーザー顕微鏡システムの調整に当初予想以上の時間を取られたが、ラットの海馬スライスを作製し、開発したレーザー顕微鏡を用いて2光子励起レーザーにより高速に多点刺激を行い、CA1ニューロンからカルシウム濃度の変化を計測した。また、我々が開発したカルシウムセンサー蛋白質であるG-CaMPを改良し、これまでのG-CaMPより約40倍明るいG-CaMP1.6を作製した。このG-CaMP1.6を遺伝子銃を用いてラット海馬スライスに発現させることにより、2光子励起レーザー顕微鏡を用いてスパインでのカルシウムの変化を捉えることができた。このシステムを用いて、ヘテロシナプス性の長期増強の研究に応用可能であることが明らかとなった。また、Gal4-UASによる発現系を用いて、G-CaMP1.6を発現する遺伝子改変ショウジョウバエを作製し、幼虫の運動ニューロン終末におけるカルシウム変動をin vivoで観察することが可能となった。G-CaMP1.6の蛍光変化量と反応速度は、比較検討したセンサーの中で最も性能が高いものに属していた。さらにG-CaMP1.6を改良したG-CaMP2を小脳顆粒細胞に発現する遺伝子導入マウスを作製し、平行線維および顆粒細...
グルタミン酸受容体のリン酸化と神経回路形成の分子生物学的研究 Competitive

中井淳一

Offer Organization: 文部科学省

System: 科学研究費補助金(特定領域研究)

Category: 特定領域研究

Institution: 岡崎国立共同研究機構->独立行政法人理化学研究所

2003 - 2004

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グルタミン酸受容体は興奮性神経伝達に関与し、シナプスの長期増強(LTP)のメカニズムとしてAMPA型グルタミン酸受容体のリン酸化により膜への受容体の組み込みやチャネル活性の増加が関与しているという報告がある。また脱リン酸化によるLTDも報告されている。この研究ではAMPA型グルタミン酸受容体のリン酸化を可視化するための蛍光センサーを試作し、試作したセンサーを用いてグルタミン酸受容体の活動を時間的空間的に捉え、神経回路に及ぼす作用を明らかにすることを目的とする。 AMPA型グルタミン酸受容体のC末端の細胞内領域に存在するカルシウムカルモジュリン依存性キナーゼII (CaMKII)およびAキナーゼによりリン酸化部位を含むアミノ酸配列の前後に、GFPの変異体であるYFPおよびCFPを付加した蛍光センサー分子のcDNAを試作した。試作したセンサーの機能を調べるため、センサーcDNAをHEK293細胞に発現させ、リン酸化を即す刺激により蛍光変化を測定した。Aキナーゼを活性化するForskolinおよびdibutyryl cAMPによる刺激ではYFP/CFPの蛍光比の微弱な増加が観測された。またCaMKIIを刺激するためIonomycinを投与した結果、明らかなYFP/CFPの蛍光比の増加が観測された。今回、グルタミン酸受容体のリン酸化のFRETを用いた蛍光センサーを作成した。リン酸化...
Development of CFP-based calcium probes that emit blue fluorescence Competitive

NAKAI Junichi

Offer Organization: Japan Society for the Promotion of Science

System: Grants-in-Aid for Scientific Research

Category: Grant-in-Aid for Scientific Research (C)

2002 - 2003

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Calcium ions play important roles as an intracellular mediator of muscle contraction, neuronal activity and hormone secretion. Therefore, it is useful to measure calcium ion to study cellular function. The purpose of this research is to develop new Green fluorescent protein (GFP) based calcium sensors that emit blue fluorescence. This type of sensors are genetically-encoded, therefore, it can be easily expressed in specific cells or tissues using a specific promoter. This sensor may become a useful tool for in vivo calcium measurements. To make such kind of sensors we fused a cyan version of GFP to calmodulin and the M13 fragment from myosin light chain kinase. When the DNA ~vas expressed HEK cells or PC12 cells. we could monitor fluorescence changes in the cells with a CCD camera or a laser confocal microscope. Interestingly, the fluorescence was brighter at the resting condition and it became dimmer when the cells were activated. There are 4 calcium binding sites in a calcium sensor. To reduce the calcium buffering effect and to change the calcium sensitivity of the sensor, we next introduced mutations in there calcium binding sites. When we destroyed more than 3 calcium binding sites, the sensor was severely affected and it was not useful as a calcium sensor any more. On the other hand, mutating one or two calcium binding sites is tolerable and those mutants were still useful as calcium sensors. To be surprised, although their calcium sensitivities were affected by the mutations, the effects were not as large as we expected. These sensors which have mutations in calcium binding sites may be good tools because they have reduced calcium buffering effects.
DEVELOPMENT OF VOLTAGE-SENSING FLUORESCENT PROTEIN BASED ON GFP Competitive

NAKAI Junichi

Offer Organization: Japan Society for the Promotion of Science

System: Grants-in-Aid for Scientific Research

Category: Grant-in-Aid for Scientific Research (B)

Institution: OKAZAKI NATIONAL RESEARCH INSTITUTES

2001 - 2002

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Measurement of membrane voltage is one of the most important techniques to understand function of excitable cells. The purpose of this project is developing fluorescent protein probes which can sense membrane voltage, using Green Fluorescent Protein (GFP). To develop voltage-sensing fluorescent probes, one of the voltage-gated potassium channels were used as a base structure, and GFP was fused to the region near the S4 segment of this protein by means of molecular biological techniques. cDNAs were transfected to HEK293 cells and it was tested whether the fluorescenee from voltage-sensing probes change in response to the membrane voltage with the CCD camera system equipped on the fluorescent microscope. Unfortunately, the probes which had GFP near the C-terminal side of the S4 segment did not become fluorescent. Then, GFP was inserted at the N-terminus of S1, or the N-terminus of S3. These proteins became fluorescent. Parts of potassium channel (S1-S4, S3-S4, and S4-S6) were also used for fusion protein. All these truncated channels became fluorescent. One of the probes which used the S3-S4 region remained in cytoplasm and others was transported to the membrane. To improve the transportation of the proteins to the plasma membrane, the probes were fused to another protein which belongs to the ammo-acid transporters. The fused protein is transported to the plasma membrane better than the original protein. At this moment, the signals from the probes are so small to use as probes. We are trying to improve the signal/noise ration by changing the connecting sequences and the position of the GFP.
Rho蛋白質と神経細胞のリモデリングに関する研究 Competitive

中井淳一

Offer Organization: 文部科学省

System: 科学研究費補助金(特定領域研究(A), 特定領域研究)

Category: 特定領域研究(A), 特定領域研究

Institution: 岡崎国立共同研究機構

2001 - 2002

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低分子量G蛋白質のRhoは細胞の形態や細胞膜のラッフリング、平滑筋の収縮、細胞凝集、細胞運動、細胞分裂を制御している蛋白質である。この研究は低分子量G蛋白質RhoAとそのRhoAのシグナル伝達経路に関与する蛋白(特にRho Kinase)の活性を生細胞でリアルタイムに視覚化し、低分子量G蛋白質とそのカスケードの機能をin vivoで解析することを目的としている。計画は2年度にまたがっており、本年度はRho Kinaseに加えて、RhoA蛋白質と相互作用をすることが知られているRhoGDI, Rhotekin, Citron K蛋白質をクローン化し、Rho蛋白質との相互作用を視覚化することを試みた。RhoGDIなどの蛋白質にYFPを付加した遺伝子を作成し、CFPを付加したRhoAの活性型変異体であるRhoA-V14または不活性型変異体であるRhoA-N19を同時に細胞に発現させCFPとYFPの間のFRET効果を指標に蛋白間の相互作用を観察した。その結果RhotekinにおいてRhoA-V14とRhoA-N19の間で少ないながらFRETの差が見られた。今後蛍光変化が大きくなるようにデザインを検討しDNAの配列を調節していく。また神経細胞での視覚化に応用していく。また本研究領域のメンバーである京都大学の根岸教授と共同研究を行うこととなり、細胞の突起形成に関与すると考えられている新...
蛋白性蛍光センサープローブによるショウジョウバエ脳の情報処理経路の研究 Competitive

中井淳一

Offer Organization: 文部科学省

System: 科学研究費補助金(特定領域研究(A))

Category: 特定領域研究(A)

Institution: 岡崎国立共同研究機構

2000 - 2001

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本研究はGreen Fluorescent Protein(GFP)を用いてカルシウムイオンを持つプローブを開発し、ショウジョウバエの脳に発現させて臭覚系および味覚系の神経回路の同定とその情報処理の過程を明らかにすることを目標とする。 GFPにカルモジュリンとそれに結合するミオシン軽鎖キナーゼのM13配列をfusionさせることによりカルシウムイオンに対する蛋白性蛍光プローブの作成を行った。このプローブは1波長励起(489nm)1波長測光(509nm)のプローブであり、その蛍光変化量が最大で4.5と大きいため、この遺伝子を筋肉細胞に発現させたとき顕微鏡を通して肉眼的にもその蛍光の変化を観察できるほどであった。このプローブに関して特許を申請した。 G-CaMPには温度感受性があるため、培養細胞に発現させ37度で培養したとき蛍光団を形成できないという問題があった。この点を改良するために蛋白質のfoldingを安定化することが知られている点突然変異をG-CaMPに導入して新たにG-CaMP1.6を作成した。G-CaMP1.6は残念ながら37度では蛍光団を形成できなかったが、28度で蛍光団を形成しモル吸光係数、量子収率ともにG-CaMPより大きな値を示した。その結果G-CaMP1.6はG-CaMPに比較して約40倍明るいことが明らかとなった。またG-CaMP1.6は蛍光団を形成する速...
Dynamic analysis of subcellular ionic signalling in neurons Competitive

IMOTO Keiji, WAKAMORI Minoru, NAKAI Junichi, MORI Yasuo

Offer Organization: Japan Society for the Promotion of Science

System: Grants-in-Aid for Scientific Research

Category: Grant-in-Aid for Scientific Research (B).

Institution: Okazaki National Research Institutes

1999 - 2000

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We investigated dynamic aspect of local ionic signaling en central neurons, by comparing wild-type and mutant functional molecules or mouse strains. More precisely, we studied molecular biological and electrophysilogical analyses of voltage-gated calcium channels and receptor-activated calcium permeable cation channels. We clarified some steps of the pathogenic mechanism how the calcium channel mutations lead to cerebellar ataxia. Also we showed that receptor-activated calcium permeable channels are expressed in a wide range of tissues including brain, and are critically involved in various aspects of cell regulation. To uncover the pathogenic mechanism of human spinocerebellar ataxia type 6, which has been shown to be associated with CAG nucleotide repeat extension in the P/Q-type calcium channel, we analyzed functional properties of the calcium channel with the extension in a recombinant expression system, to obtain the negative shift of voltage-dependent inactivation. This change is consistent with the idea that reduced calcium influx can be a cause of the cerebellar neuronal disorder. We identified the mutation in the calcium channel α1A gene of the mutant ataxic mouse rolling nagoya. The mutation was located in the voltage sensing region. In fact, the calcium channel was shown to have a reduced voltage sensitivity in addition to reduced current amplitude. This study showed how a single mutation leads to abnormal development and function of the neuronal circuit. The TRP channel is a representative of receptor-activated channels. We conducted molecular analyses of the members of the TRP channel family. The functional properties of TRP channels are characterized using recombinant expression systems. In particular, TRP7, which is constitutively activated without external stimulation, was shown to be functionally similar to the channel previously identified as a calcium permeable non-selective cation channel. The members of TRP channel family may contribute to sustained depolarization of neurons when then receive repetitive stimulations.
EXCITATION-CONTRUCTION COUPLING AND TETRAD STRUCTURE OF L-TYPE CACLIUM CHANNEL. Competitive

NAKAI Junichi

Offer Organization: Japan Society for the Promotion of Science

System: Grants-in-Aid for Scientific Research

Category: Grant-in-Aid for Scientific Research (C)

Institution: OKAZAKI NATIONAL RESEARCH INSTITUTES

1999 - 2000

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Voltage-gated L-type calcium channels (Dihydropyridine receptors) and ryanodine receptors (calcium release channels) play important roles in muscle contruction. In this research, chimeras of the skeletal muscle (RyR1) and cardiac muscle (RyR2) ryanodine receptor were expressed in HEK293 cells to assess their calcium dependence in [^3H] ryanodine binding and single channel measurements. The results indicate that the C-terminal one-fourth has a major role in calcium activation and inactivation of RyR1. Further, our results show that replacement of RyR1 regions corresponding RyR2 regions can result in loss and/or reduction of [^3H] ryanodine binding affinity while maintaining channel activity.
ショウジョウバエ脳の知覚情報処理経路の研究 Competitive

中井淳一, 宍戸恵美子

Offer Organization: 文部科学省

System: 科学研究費補助金(特定領域研究(A))

Category: 特定領域研究(A)

Institution: 岡崎国立共同研究機構

1999 - 1999

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本研究は知覚情報がショウジョウバエの脳内でどのような回路を通って処理されるのかを神経回路網レベルで明らかにし、情報処理統合システムの機構を解明することを目的とし、Green Fluorescent Protein(GFP)とその派生物(YFP,CFP)、カルシウム感受性蛍光タンパク質プローブ(cameleon)と電位依存性の蛍光タンパク質プローブ(FlaSh)のtransgenic flyを作成し、ショウジョウバエのエンハンサートラップラインと掛け合わせることにより細胞特異的に発現を行い、in vivoで神経細胞の興奮の指標となる膜電位と細胞内のカルシウムを測定することにより知覚回路の同定を試みた。その結果、GFP,YFP,CFP,yellow cameleon2(YC2)およびFlaShのtransgenic flyを作成できた。GFP,YFP,CFPによりin vivoで神経細胞を同定できるようになった。またpH感受性があるYFPを細胞特異的に発現させることによりin vivoでの細胞内のpHの変化を測定することができた。YC2を発現させたハエは成虫にまでなったが、YC2自体のカルシウム感受性が低くin vivoでカルシウム変化を捕らえることはできなかった。次にYC2よりカルシウムに対して感受性が高いことが報告されていた改良型のyellow cameleon2.1(YC2...
Production & analysis of Ca^<2+> channel subunit-defecient mice. Competitive

IMOTO Keiji, BEAM Kurt G, SCHWARTZ Arnold, WAKAMORI Minoru, NAKAI Junichi, MORI Yasuo

Offer Organization: Japan Society for the Promotion of Science

System: Grants-in-Aid for Scientific Research

Category: Grant-in-Aid for international Scientific Research

Institution: National Institute for Physiological Sciences

1997 - 1998

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We have characterized functional defects of the P/Q-type Ca2^<2+> channels induced by the ataxic mutations tottering (tg) and leaner (tg-la) in the pore-forming alpha_<14> (BI) subunit. Of these two mutations, tg-la which causes more sever ataxia affected biophysical properties of the P/Q-typeCa2^<2+> channel more seriously than the other. The stargazer gene whose mutation cause also epilepsy and ataxia was shown to encode a novel neuronal gamma isoform gamma_2. The interaction sites of the G protein subunits and intracellular Ca2^<2+> release channel (ryanodine receptor)were mapped in the cytoplasmic regions of the Ca2^<2+> channel alpha1 subunits. In addition, we isolated a novel receptor-activated channel TRP5 from mouse brain. TRP5 was activated by receptor stimulation via a mechanism unrelated to store depletion.
Protein-protein interaction between voltage-gateal calcium channel and ryanodine recegtor. Competitive

NAKAI Junichi

Offer Organization: Japan Society for the Promotion of Science

System: Grants-in-Aid for Scientific Research

Category: Grant-in-Aid for Scientific Research (C)

Institution: Okazaki National Research Institutes

1997 - 1998

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In skeletal muscle, voltage-gated calcium channel (dihyrdopyridine receptor or DHPR) and ryanodine receptor (RyR) play critical roles for excitation-contraction coupling.The voltage-gated calcium channel is a sensor molecule of the membrane voltage and controls the opening of the ryanodine receptor.Although these two molecules are thought to interact directly, there is little evidence of the physical protein-protein interaction.The aim of this project is to study the protein-protein interaction between the DHPR and the RyR.For this purpose, we used the yeast two-hybrid system. Project 1 : Test for the interaction between DHPR and RyR. A small piece of the DHPRalpha1s subunit (amino acid number ; 719-768) was inserted into the DNA-binding (DB) plasmid to yield s53.Likewise, a piece of the type-1 ryanodine receptor (RyR-1) and the type-2 ryanodine receptor (RyR-2) were inserted into the activation-domain (AD) plasmid to yield sRl6(RyR-1 ; 1837-2168) and cRl6(RyR-2 ; 1817-2142), respectively.These plasmids were co-transfected and His^+ cells were selected.Results showed that only the yeast co-transfected with s53 and sRl6 could grow in synthetic minimum medium.However, this combination did not show positive beta-galactosidase activity.Because of the discrepancy between the nutrition-requirement test and the beta-galactosidase-activity test, we could not get clear evidence of the physical interaction between the DHPR and the RyR. Project 2 : Screening of new proteins which may bind to the DHPR. To screen new proteins which may bind to the DHPR, we constructed a cDNA library from the skeletal muscle which was inserted into the AD plasmid and tested interaction with five DB plasmids which include internal loops of the DHPR.First, using the I-II loop of the DHPR, we isolated the beta subunit of the DHPR.This is consistent with previous results and indicates that the screening system works well.Next, we found that the troponin T binds to the C-terminal potion of the DHPR.The function of the troponin T to the DHPR is not clear so far.However, there is the possibility of the troponin T to localize the ion channels.
興奮収縮連関におけるリアノジン受容体の構造と機能の研究 Competitive

中井淳一

Offer Organization: 文部科学省

System: 科学研究費補助金(重点領域研究)

Category: 重点領域研究

Institution: 岡崎国立共同研究機構

1997 - 1997

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骨格筋において細胞膜の電位依存性L型カルシウムチャネル(ジヒドロピリジン受容体、DHPR)と筋小胞体のリアノジン受容体は興奮収縮連関の中心的機能を果たしている。この2つの分子は蛋白どうしが結合して情報伝達を行っていると考えられているが、その分子機構は明らかになっていない。先に我々は骨格筋型リアノジン受容体(RyR-1)はDHPRと相互に作用しあうが、心筋型リアノジン受容体(RyR-2)はDHPRと相互に作用しないことを見い出した。この実験の目的はRyR-1とRyR-2のこの機能の差に着目し、分子生物学的方法を用いて興奮収縮連関におけるリアノジン受容体とDHPRの相互の活性化機構を明らかにすることである。実験方法:RyR-1とRyR-2のキメラcDNAを作製し、RyR-1遺伝子を欠くdyspedic mouseの培養骨格筋細胞に発現させた。パッチクランプ法と蛍光カルシウム測定法によりカルシウム電流とカルシウム放出をそれぞれ測定した。その結果、foot領域にRyR-1のアミノ酸配列を含むキメラR1を発現させた細胞では骨格筋型の興奮収縮連関もカルシウムチャネルの機能もどちらも回復していた。次にキメラのRyR-1の領域をさらに狭めていき、最終的に1635-2636番目のアミノ酸にのみRyR-1の配列を含むキメラR10を発現させた。このR10を発現させた細胞は骨格筋型の興奮収縮連関...
Studies on molecular mechanism of neural calcium signaling Competitive

IMOTO Keiji, SCHWARTZ Aenold, SAKMANN Bert, BEAM Kurt G, NAKAI Junichi, MORI Yasuo

Offer Organization: Japan Society for the Promotion of Science

System: Grants-in-Aid for Scientific Research

Category: Grant-in-Aid for international Scientific Research

Institution: Okazaki National Research Institutes

1996 - 1996

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Intracellular calcium ion plays critical roles in neural signaling and neural plasticity. Recently, application of molecular biological techniques into neurobiology has dramatically accelerated studies on molecular mechanism of calcium metabolism in the nervous system, and led to elucidation of functions and properties of proteins which work as functional elements. On the other hand, our knowledge of higher brain functions is also rapidly increasing, especially owing to non-invasive measurement methods. However, there still remains a big gap between understanding molecules in brain and understanding higher brain functions. In order to fill the gap, we studied following subject in this project. We studied molecular mechanism of modulation of voltage-dependent calcium channels through GTP-binding proteins, by electrophysiological-and pharmacological measurements of recombinant calcium channels expressed in cultured cells. We obtained novel observation indicating showing multiple mode of G-protein modulation. We investigated molecular interaction between voltage-dependent calcium channels and ryanodine receptors expressed in skeletal muscle cells derived from ryanodine receptor-deficient mice. We demonstrated functional non-equality between skeletal type and cardiac type of ryanodine receptors in the excitation-contraction coupling mechanism. We have been trying to clone a family of cDNAs encoding a novel type of non-voltage-dependent calcium permeable channels. So far we have obtained cDNAs of several members of this family, and now are now studying tissue distribution and functional properties. To meet the need for calcium imaging of mouse brain, we have made a setup designed for brain slice preparation and developed a software environment. The basic data of calcium metabolism of mouse neurons corresponds well to those of rat neurons, however, the small size of mouse brain makes it difficult to handle smaller structures or immature brains.
電位依存性カルシムチャネルの不活性化機構と不整脈の研究 Competitive

中井淳一

Offer Organization: 文部科学省

System: 科学研究費補助金(基盤研究(C))

Category: 基盤研究(C)

Institution: 岡崎国立共同研究機構

1996 - 1996

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Lonf QT症候群(LQT)は遺伝性の心臓不整脈の1つで電位依存性カルシウムチャネルやナトリウムチャネルの異常が報告され、現在までのところ、LQT3でナトリウムチャネルの不活性化領域の異常であることが明らかにされている。また最近、低カリウム性周期性四肢麻痺の原因遺伝子が骨格筋の電位依存性カルシウムチャネルであることがあきらかにされ、カルシムチャネルの不活性化との関係が示唆された。本研究では電位依存性カルシムチャネルの不活性化機構を分子レベルで解析し心臓不整脈の成因を明らかにすることを目的としている。具体的には低カリウム性周期性四肢麻痺で見つかった異常がヒト低カリウム性周期性四肢麻痺の原因として見つかった変異(アルギニンがヒスチジンに置換)を人工的にウサギL型カルシムチャネルに導入し、異変カルシウムチャネルcDNA(R528HとR1239H)を作製した。このcDNAを先天的にL型カルシウムチャネルを欠くマウス(dysgenic mouse)の培養骨格筋細胞にマイクロインジェクション法により導入し、変異カルシウムチャネルの性質をパッチクランプを用いた電気生理学的方法とFluo3を用いた細胞内カルシウムイオン測定法を用いた同時測定により調べた。その結果、R528HとR1239Hのいずれの変異によってもgating current、カルシウム電流および細胞内カルシウム濃度の増加に変...
カルシウムチャネル、カルシウムATPaseおよびリアノジン受容体の機能協関 Competitive

中井淳一

Offer Organization: 文部科学省

System: 科学研究費補助金(重点領域研究)

Category: 重点領域研究

Institution: 岡崎国立共同研究機構

1996 - 1996

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骨格筋においてカルシウムチャネルとリアノジン受容体は興奮収縮連関の中心的役割を果たしている。この2つの分子は細胞内で直接カップリングし情報伝達を行っている。本研究では骨格筋型リアノジン受容体(RyR-1)遺伝子を人工的に破壊したマウス(dyspedic mouse)から骨格筋細胞を単離・培養し、心筋型リアノジン受容体(RyR-2)cDNAをこの細胞に発現させて、RyR-2が興奮収縮連関に及ぼす影響とカルシウムチャネルのチャネル活性に及ぼす影響をパッチクランプ法と蛍光カルシウム測定法を用いて調べた。 RyR-2を発現した細胞では自発性の収縮が見られ,収縮にともない細胞内カルシウムの増加が観察された。RyR-2を発現した細胞をwhole-cellパッチクランプ法により電位固定しカルシウム電流と細胞内カルシウムの同時測定を行った結果、カルシウム電流密度は低く、RyR-1の発現で見られるようなカルシウム電流の増加はRyR-2では見られなかった。またこのときdepolarization-induced calcium releaseは観察されなかった。L型カルシウムチャネルのキメラであるCSk3(リピ-トIIとリピ-トIIIの間の細胞内ル-プにのみ骨格筋型のアミノ酸反列を持つ)をRyR-2とともに発現させると,もカルシウム電流を生成しcalcium-induced calcium re...
Studies on the molecular mechanism for calcium signaling Competitive

IMOTO Keiji, NAKAI Junichi, STUHMER Walter, BEAM Kurt G., MORI Yasuo, FUJITA Yoshihiko

Offer Organization: Japan Society for the Promotion of Science

System: Grants-in-Aid for Scientific Research

Category: Grant-in-Aid for international Scientific Research

Institution: Kyoto University

1993 - 1993

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Previous studies demonstrated that the difference in speed of activation between the cardiac and skeletal muscle calcium channels can be attributed to the difference in amino acid sequence of the first repeat of the four repeated units of homology. To identify the region in repeat I responsible for the different activation kinetics, we have made series of 'repeat I chimera' DNA constructs systematically, and expressed them in cultured skeletal muscle cells derived from mice with muscular dysgenesis. Analyses of speed of activation have revealed that the third putative transmembrane region (S3) and the linker region between S3 and S4 is critical to determine the speed of activation. Together with the notion that S4 functions as a voltage sensor, we speculate that a change in membrane potential triggers movement of S4, which is transmitted by affecting S3 and its adjacent portion, finally leading to channel openings. Voltage-gated calcium channels contain smaller subunits. When the main subunit (alpha1 subunit) is coexpressed with smaller subunits, functional activity is markedly increased. To understand the molecular mechanism of small subunit function, we have expressed cardiac calcium channel alpha1 subunit alone or together with small subunits. From the results of Northern blots, protein gel electrophoresis, dihydropyridine binding assay, and electrophysiological measurements, we concluded that the small subunits do not affect metabolism of the main subunit, and that they are necessary for proper functional conformation. From cDNA libraries of rabbit brain, we have cloned cDNA encoding a new calcium channel (named BIII). By sequencing the cDNA, we have elucidated the primary structure of BIII calcium channel. This calcium channel showed homology to other voltage-dependent calcium channel. Especially, BIII showed higher similarity to BI and BII brain calcium channel. These three types of calcium channel form a subgroup distinct from dihydropyridine-sensitive L-type calcium channel. When BIII channel is expressed by injecting DNA into cultured skeletal muscle cells derived from mice with muscular dysgenesis, it showed omega-conotoxin sensitive calcium channel activity. Its kinetics and voltage-dependence were similar to those reported for N-type calcium channels. Since the N-type calcium channel is important for neurotransmitter release, cloning of an N-type calcium channel BIII will contribute to future studies of brain function.
Studies on the structural basis for ion channel functions Competitive

IMOTO Keiji, TSIEN Richard W., BEAM Kurt G., MICHEL Hartmut, MORI Yasuo, NAKAI Junichi, FUKUDA Kazuhiro, STUMER Walter

Offer Organization: Japan Society for the Promotion of Science

System: Grants-in-Aid for Scientific Research

Category: Grant-in-Aid for international Scientific Research

Institution: Kyoto University

1992 - 1992

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Bovine skeletal muscle nicotinic acetylcoline receptor alpha-subunit was expressed using baculovirus-infected insect cells. This alpha-subunit protein was 2-3% of total cellular protein. The protein could be solubilized and purifeid. Purified protein was shown to be glycosilated, and showed a high-affinity activity to alpha-bungarotoxin. By analyzing ion permeation properties of site-specifically mutated rat brain sodium channel ll, amino acid residues that from part of the selectivity filter of the sodium channel were identified. Those acid residues are located in the SS2 regions. Substitution of glutamic acid residues for lysine and alanine in these critical regions conferred calcium channel properties on the sodium channel. The channel pore size was estimated for site-specifically mutated nicotinic acetylcholine receptor channel. The results suggested that both of the pore size and the fixed charge are determinant of permeability, end that changes in charge may affect permeability by altering the pore size. Two new types of barin calcium channel (Bll and Blll) were cloned. They are similar to the Bl calcium channel in amino acid seqence, and those three calcium channel from a subfamily which is distinct from dihydropyridine-sensitive L-type calcium. The Blll calcium was expressed using myotube from mice with muscular dysgenesis. The current was sensitive to omega-conotoxin, and its single-channel conductance was about 16 PS. These results indicated that Blll calcium channel is N-type. A novel type of ryanodine receptor/calcium release channel was cloned, and its primary structure was determined. This receptor is strusturally similar to the skeletal muscle and cardiac conuterparts. The brain type ryanodine receptor is abundantly expressed in hippocampus, striate body, and thalamus, while the cardiac type of ryanodine receptor is more diffusely distributed in the brain. The brain type was expressed in tissues containing smooth muslce.
興奮収縮連関の分子機構に関する研究 Competitive

中井淳一

Offer Organization: 文部科学省

System: 科学研究費補助金(一般研究(C))

Category: 一般研究(C)

Institution: 京都大学

1992 - 1992

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ジヒドロピリジン受容体とリアノジン受容体(カルシウム放出チャンネル)は骨格筋・心筋における興奮収縮連関の中心的役割を果たすことが知られており、ジヒドロピリジン受容体の分子内繰り返し単位(リピ-ト)IIとIIIの間にある推定細胞内領域が興奮収縮連関において重要であることが明らかされた。本研究ではこれまでの研究をさらに発展させ、リピ-トIIとIIIの間の領域をさらに詳細に検討し、興奮収縮連関に関与するアミノ酸残基を同定することを目的としている。リピ-トIIとIIIの間の領域の一部が骨格筋ジヒドロピリジン受容体に由来し、他の部分は心筋ジヒドロピリジン受容体に由来するような6種類のキメラジヒドロピリジン受容体cDNAを遺伝子工学的技術を用いて作成した。そして興奮収縮連関を欠く変異であるmuscular dysgenesisを持つマウスの骨格筋細胞の核にマイクロインジェクション法を用いてキメラ受容体cDNAを導入した。cDNAを導入された骨格筋細胞の電気刺激に対する収縮を検討した結果、導入された6種類のジヒドロピリジン受容体cDNAはいずれも機能的なキメラ受容体を発現していることが明らかになり、興奮収縮連関に関与する領域を漸次限定していくことができた。骨格筋型興奮収縮連関に関与する部位は骨格筋ジヒドロピリジン受容体のリピ-トIIとIIIの間の領域のほぼ中央に位置する約3分の1の長さの領...
イオンチャンネルの立体構造と作用機序に関する研究 Competitive

Offer Organization: 文部科学省

System: 科学研究費補助金(国際学術研究)

Category: 国際学術研究

Institution: 京都大学

1990 - 1991

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cDNAと発現ベクタ-を用いてCHO細胞にニコチン性アセチルコリン受容体(nAChR)サブユニットあるいはムスカリン性アセチルコリン受容体(nAChR I)を発現させ、これらの蛋白を産生する細胞株を得た。遺伝子増幅などの技術を用いて産生蛋白量の増加を図るとともに、大量培養を行いこれらの膜蛋白の精製を検討した。またBaculoウィルス発現系を用いて培養昆虫細胞にnAChRサブユニットを発現させた結果、CHO細胞を用いる発現系に比べて発現効率のよいことが明かとなった。さらに昆虫細胞から精製したnAChRαサブユニットあるいはαサブユニットとδサブユニットからなる複合体を用いて、結晶化のための界面活性剤、pH、温度、沈澱剤などの条件検討を行い、cDNAより大量に発現させた蛋白を用いてX線結晶解析を行うことが現実的に十分可能であることを示した。変異を導入したnAChRチャンネルの解析から、イオン透過速度を決定する中間環のアミノ酸残基がイオン選択に重要であることを見いだした。また内壁形成が予測される部位の電荷を持たない極性アミノ酸残基が環状様構造をなし、近接する中間環と共に短いチャンネル狭小部を構成すると結論した。ウシ骨格筋nAChRα、β、δサブユニットより構成されるチャンネルがアゴニストなしに高頻度に開き、γあるいはεサブユニットを含むチャンネルと比較してイオンの透過特性が異なる...
モデル生物（線虫等）を用いた研究 Competitive
functional neuronal network study Competitive
カルシウムイオン濃度測定 Competitive
神経回路機能の研究 Competitive

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