一般口演

一般口演7

2021/10/1　10:00～11:00　オンデマンド D会場 O7-1 軸索の直径に依存してミエリン形成を制御する力学的因子: 蛍光共鳴エネルギー移動に基づく張力センサーの解析 Tension sensor based on fluorescence resonance energy transfer reveals fiber diameter-dependent mechanical factors during myelination ^○清水健史¹, Hideji Murakoshi^2,3, Hidetoshi Matsumoto³, Akimasa Ishida¹, Shinya Ueno¹, Naoki Tajiri¹, Hideki Hida¹ 1.Department of Neurophysiology and Brain Science, Graduate School of Medical Sciences, Nagoya City University, 2.Supportive Center for Brain Research, National Institute for Physiological Sciences, 3.Department of Physiological Sciences, The Graduate University for Advanced Studies ^○Takeshi Shimizu¹, Hideji Murakoshi^2,3, Hidetoshi Matsumoto³, Akimasa Ishida¹, Shinya Ueno¹, Naoki Tajiri¹, Hideki Hida¹ 1.Department of Neurophysiology and Brain Science, Graduate School of Medical Sciences, Nagoya City University, 2.Supportive Center for Brain Research, National Institute for Physiological Sciences, 3.Department of Physiological Sciences, The Graduate University for Advanced Studies Oligodendrocytes (OLs) form a myelin sheath around neuronal axons to increase conduction velocity of action potential. Although both large and small diameter axons are intermingled in the central nervous system, the number of myelin wrapping is related to the axon diameter, such that the ratio of the diameter of the axon to that of the entire myelinated-axon unit is optimal for each axon, which is required for exerting higher brain functions. This indicates there are unknown axon diameter-dependent factors that control myelination. We tried to investigate physical factors to clarify the mechanisms underlying axon diameter-dependent myelination. To visualize OL-generating forces during myelination, a tension sensor based on fluorescence resonance energy transfer (FRET) was used. Polystyrene nanofibers with varying diameters similar to neuronal axons were prepared to investigate biophysical factors regulating the OL-axon interactions. We found that higher tension was generated at OL processes contacting larger diameter fibers compared with smaller diameter fibers. Additionally, OLs formed longer focal adhesions (FAs) on larger diameter axons and shorter FAs on smaller diameter axons. Furthermore, distal FAs far from the OL processes exhibit lowered tension generation. These results suggest that OLs respond to the fiber diameter and activate mechanotransduction initiated at FAs, which controls their cytoskeletal organization and myelin formation. This study leads to the novel and interesting idea that physical factors are involved in myelin formation in response to axon diameter.		2021/10/1　10:00～11:00　オンデマンド D会場 O7-2 超解像陰影法による複数オルガネラの同時可視化 Super-resolution shadow imaging of intracellular space reveals various organelle dynamics in living cells ^○野住素広¹, Michihiro Igarashi¹, U. Valentin Nägerl² 1.Department of Neurochemistry, School of Medicine Niigata University, 2.Interdisciplinary Institute for Neuroscience, University of Bordeaux / CNRS, Bordeaux, France ^○Motohiro Nozumi¹, Michihiro Igarashi¹, U. Valentin Nägerl² 1.Department of Neurochemistry, School of Medicine Niigata University, 2.Interdisciplinary Institute for Neuroscience, University of Bordeaux / CNRS, Bordeaux, France Eukaryotic cells have many different types of membranous organelles, which enable them to use the compartmentalized signaling of various cell-biological processes. The precise spatial arrangement and dynamics of organelles are essential to the proper cytoskeletal organization and the protein trafficking in neurons, and hence ultimately also for the formation and maintenance of neural circuits. In addition, some organelle dysfunctions are associated with developmental brain disorders; thus, there is a growing need for improved imaging tools to visualize the spatio-temporal dynamics and interactions of intracellular organelles. With this interest in mind, we have adopted the recent super-resolution shadow imaging technique (SUSHI), which was originally developed to image the extracellular space of living brain tissue using 3D-STED microscopy. As in the case of SUSHI (Tønnesen J et al. Cell 172: 1108[2018]), membrane-delimited subcellular structures like organelles will cast tell-tale"shadows" if surrounded by a bright sea of fluorescence and imaged with sufficiently high spatial resolution. Using cultured cells that cytosolically expressed the fluorescent protein Citrine, we could readily discriminate various organelles in the inverted images, such as mitochondria and endoplasmic reticulum. In addition, this method enabled us to distinguish them without specific labeling and to monitor their movements and morphological changes over time. Currently, we are optimizing the labeling and imaging conditions for cultured neurons, paving the way to “intracellular SUSHI” of organelle dynamics and interactions in in vitro model systems of developmental brain disorders.

2021/10/1　10:00～11:00　オンデマンド D会場 O7-3 退色の軽減と効果的な免疫染色のための透明化手法の開発 Advanced whole-mount immunostaining and organic solvent-based tissue clearing technology ^○Tao Lu¹, Munehisa Shinozaki¹, Narihito Nagoshi², Masaya Nakamura², Hideyuki Okano¹ 1.慶應義塾大学大学院, 2.Department of Orthopaedic Surgery, Keio University Graduate School of Medicine. ^○Tao Lu¹, Munehisa Shinozaki¹, Narihito Nagoshi², Masaya Nakamura², Hideyuki Okano¹ 1.Department of Physiology, Keio University Graduate School of Medicine., 2.Department of Orthopaedic Surgery, Keio University Graduate School of Medicine. Three-dimensional (3D) reconstruction and analysis of whole organs have been broadly conducted in biomedical research. With the development of tissue-clearing and 3D imaging techniques, samples can be comprehensively observed with cellular to subcellular resolution over single organs. Among those protocols, organic solvent-based clearing protocols, such as 3D imaging of solvent-cleared organs (3DISCO), provide the advantages of high clearing efficiency and tissue shrinkage for imaging of large samples such as whole brain. Fluorescent labeling is one of the biggest issues in clearing techniques. Besides endogenous markers, whole-mount staining has been used for the observation of animal organs and bodies, and human specimens. However, organic solvent quickly quenches fluorescent proteins (within 48 hours), which has limited its application. In addition, the insufficient penetration of antibodies remains a crucial challenge in 3D staining situations. Here, we propose an optimized technique to overcome these limitations. First, we modified 3DISCO, and preserved the endogenous fluorescent proteins of EGFP and mCherry with a long storage time of weeks and potent clearing ability. Second, we developed whole-mount staining technique, which permitted volume imaging of large cleared samples of adult organs, such as whole brains or hearts. This demonstrated the visualization of vasculature system of various organs. We also revealed novel aspects of chronic glial scars with incomplete injured spinal cords. This advanced technique not only enables direct imaging of endogenous fluorescent signals, but also visualization of spatial connections of immunolabeled structures and cellular types in tissue blocks and organs, providing access to 3D visualization in various biomedical fields.		2021/10/1　10:00～11:00　オンデマンド D会場 O7-4 脳深部回路を可視化するPETレポーターイメージング技術 Genetically targeted PET reporter imaging of deep neuronal circuit in the mammalian brain ^○下條雅文¹, Maiko Ono¹, Hiroyuki Takuwa¹, Koki Mimura¹, Yuji Nagai¹, Masayuki Fujinaga², Tatsuya Kikuchi², Maki Okada², Chie Seki¹, Yuhei Takado¹, Manami Takahashi¹, Takeharu Minamihisamatsu¹, Ming-Rong Zhang², Yutaka Tomita³, Norihiro Suzuki³, Anton Maximov⁴, Tetsuya Suhara¹, Takafumi Minamimoto¹, Naruhiko Sahara¹, Makoto Higuchi¹ 1.Department of Functional Brain Imaging, National Institutes for Quantum and Radiological Science and Technology, 2.Department of Radiopharmaceuticals Development, National Institutes for Quantum and Radiological Science and Technology, 3.Department of Neurology, Keio University School of Medicine, 4.Department of Neuroscience, The Scripps Research Institute ^○Masafumi Shimojo¹, Maiko Ono¹, Hiroyuki Takuwa¹, Koki Mimura¹, Yuji Nagai¹, Masayuki Fujinaga², Tatsuya Kikuchi², Maki Okada², Chie Seki¹, Yuhei Takado¹, Manami Takahashi¹, Takeharu Minamihisamatsu¹, Ming-Rong Zhang², Yutaka Tomita³, Norihiro Suzuki³, Anton Maximov⁴, Tetsuya Suhara¹, Takafumi Minamimoto¹, Naruhiko Sahara¹, Makoto Higuchi¹ 1.Department of Functional Brain Imaging, National Institutes for Quantum and Radiological Science and Technology, 2.Department of Radiopharmaceuticals Development, National Institutes for Quantum and Radiological Science and Technology, 3.Department of Neurology, Keio University School of Medicine, 4.Department of Neuroscience, The Scripps Research Institute A gene reporter imaging is a fundamental technology for real-time tracking of protein dynamics in the mammalian brain. Among various imaging modalities, positron emission tomography (PET) offers a powerful approach to monitor a biosynthesized molecule in living animals and humans. However, the visualization of a genetically targeted reporter protein in the nervous system by PET has been hampered due to the lack of radioactive ligand capable of penetrating the blood-brain barrier. Here, we demonstrate that E.coli dihydrofolate reductase (ecDHFR) and its small antagonist trimethoprim (TMP) serve a solid technical platform for in vivo brain reporter imaging in living animals. In mice, individual neurons expressing ecDHFR can be visualized by two-photon microscopy after systemic administration of fluorescent TMP-HEX, whereas the intravenous injection with radioactive [11C]TMP or [18F]fluoroethoxy-TMP followed by dynamic PET scan enabled macroscopic assessment of ecDHFR distribution. We also demonstrate the utility of TMP analogs for PET analysis of aggregation and turnover of proteins tagged with wild-type ecDHFR or its mutant that mediates protein decay in the absence of the ligand. Moreover, in combination with immediate early gene promoter, neuronal ensemble activities elicited by chemogenetic manipulation could be successfully captured in the hippocampal dentate gyrus. Finally, we show that ecDHFR/TMP systems can be used to illustrate neuronal tracts in deep brain regions of non-human primates. These new technologies facilitate a broad spectrum of optical and PET-based cellular and molecular assays that were not previously applied for technical reasons.