一般口演
画像法と可視化
Imaging and Visualization
座長：上口 裕之（理化学研究所 脳神経科学研究センター） |
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| 2022年6月30日 17:10～17:25 沖縄コンベンションセンター 会議場B2 第5会場
1O05e2-01
透明化脳で加速するセルオミクスと神経疾患への展開
Cellomics accelerated by cleared organs with an application for neurology
*三谷 智樹(1,2)、松本 桂彦(1)、上田 泰己(3,1,2)
1. 理化学研究所生命機能科学研究センター、2. 大阪大学大学院医学系研究科、3. 東京大学大学院医学系研究科
*Tomoki T Mitani(1,2), Katsuhiko Matsumoto(1), Hiroki R Ueda(3,1,2)
1. RIKEN Center for Biosystems Dynamics Research, Osaka, Japan, 2. Osaka University Graduate School of Medicine, Osaka, Japan, 3. Grad Sch Med, Univ of Tokyo, Tokyo, Japan
Keyword: CELLOMICS, TISSUE CLEARING, LIGHT SHEET MICROSCOPY, NEURODEGENERATIVE
The genome project around the year 2000 has dramatically accelerated the study of the hierarchy from molecules to cells in life science and medical research. The reference genome sequence and high-throughput next-generation sequencing have made it possible to compare an enormous number of samples at the single nucleotide level, which has attracted attention as data-driven research. On the other hand, complex organs, such as the brain, have a variety of functions that can arise from intercellular connections of multiple neurons. Therefore, it is essential to take a systems science approach not only from molecules to cells but also from cells to organs in order to elucidate the details of biological phenomena and diseases. In our group, we have combined tissue clearing and light-sheet fluorescence microscopy techniques to enable visualization of whole organs at a single-cell resolution [1,2]. Recently, a whole mouse brain atlas consisting of 100,120,098 cells has been established, and the high-speed and high-quality light-sheet microscopy has enabled whole-organ cell profiling in a few hours[3,4]. These are equivalent to reference whole genome sequences and next-generation sequencing in genomics. In addition, three-dimensional immunostaining [5] and a data platform that can organize and analyze all cells by representing a single cell as a point in the 3D point cloud[6] have been established, and thus it has become feasible to perform cellomics, which is an omics of organs with a single cell as the basic unit. In this study, we report a study of the application of cellomics to neurological diseases. The quantification of neurons and microglia in the whole mouse brain has the potential to provide a much more detailed pathological evaluation, including the pathological progression in the very early stages of the disease and the relationship with symptoms in each region. [Reference] [1] Cell. 2014;157(3):726-739, [2] Cell. 2014;159(4):911-924, [3] Nat Neurosci. 2018;21(4):625-637 [4] Nat Protoc. 2019;14(12):3506-3537 [5] Nat Commun. 2020;11(1):1982 [6] Cell Reports Methods. 2021;1 (2): 2667-2375 | | 2022年6月30日 17:25～17:40 沖縄コンベンションセンター 会議場B2 第5会場
1O05e2-02
Spatiotemporal dynamics of dopamine and acetylcholine
*Julie A. Chouinard(1), Akash Pal(2), Sakiko Takahashi(1), Kiyoto Kurima(1), Nobuyoshi Kitamura(1), Philip M. Borden(3), Loren L. Looger(3), Lin Tian(2), Jeffery R. Wickens(1)
1. Okinawa Institute of Science and Technology Graduate University, Onna-son, Okinawa, Japan, 2. University of California Davis, Davis, California, USA, 3. Janelia Research Campus, Howard Hughes Medical Institute, Ashburn, Virginia, USA
Keyword: Dopamine, Acetylcholine, Voltammetry, Fluorescence Microscopy
The growing availability and quality of biosensors able to report the spatiotemporal dynamics of neurotransmitter levels opens new avenues for studying chemical signaling in the brain. These sensors directly and specifically report synaptic signals as they are received by their receptors. Sensors for dopamine (dLight) and acetylcholine (iAChSnFR) have high affinity, millisecond response times, good signal-to-noise ratio, and expression and targeting suitable for use in vitro and in vivo. We created Cre-dependent viruses optimized to express these genetically encoded sensors pre- or post-synaptically in transgenic animals. Dopamine imaging with dLight1.3b and dLight2.1 was performed alongside simultaneously recorded fast-scan voltammetry traces with electrical stimulation in the presence and absence of the dopamine reuptake inhibitor methylphenidate. Contrasting results from these techniques indicate they do not report dopamine levels from the same compartment, in that the sensors report (synaptic) dopamine release, whereas voltammetry detects dopamine diffusion away from release sites into the extracellular space. In the presence of methylphenidate, dopamine signals measured by voltammetry were increased and prolonged by reduced reuptake, in contrast to that measured by dLight, which showed a decreased amplitude, but still prolonged time course. Acetylcholine could be detected by iAChSnFR expressed in acute brain slices after exposure to increasing [acetylcholine], KCl, the GABA antagonist bicuculline, choline esterase inhibitors and electrical stimulation. The sensor revealed complicated spatiotemporal dynamics of acetylcholine signaling. Sensor responses were most intense in regions that were presumably axonal, displaying high extracellular acetylcholine levels that were extremely responsive to our pharmacological manipulations. Our data show that iAChSnFR and dLight pave the way toward a more complete understanding of neurotransmitter dynamics in the basal ganglia circuitry and beyond. With improved imaging and analysis methods, both biosensors could be useful tools to decipher neural activity into its composite molecular signaling events. | | 2022年6月30日 17:40～17:55 沖縄コンベンションセンター 会議場B2 第5会場
1O05e2-03
蛍光分子インプリント高分子ナノ粒子での神経細胞染色による伝達物質分泌イメージング
Imaging of transmitter secretion by neuronal staining with fluorescent molecularly imprinted polymeric nanoparticles
*吉見 靖男(1)、遠藤 健太(1)、飯島 柊(1)
1. 芝浦工業大学
*Yasuo YOSHIMI(1), Kenta ENDO(1), Shu IIJIMA(1)
1. Shibaura Institute of Technology
Keyword: Molecularly Imprinted Polymer, Serotonin, Imaging, Aplysia
Analysis of the secretion of neurotransmitters in the nervous system is essential for elucidating the mechanism of the neural network in the nervous system. Thus, developing a probe that can track a specified neurotransmitter in real-time with high selectivity has been required. Molecularly imprinted polymer (MIP), a molecular recognition material obtained by polymerization with a template-effect of the target molecule, may be usable for the probe. In this study, a nanoparticle of MIP including fluorescent group (fMIP-NP) was developed as the probe of serotonin. The serotonin, as the template, was immobilized on glass beads by using mixed silane couplers with different chain lengths. The template-immobilized beads were fluidized in a hybrid solution of a fluorescent monomer, a template-affinity monomer, an aminated monomer, a crosslinking monomer, and a photoinitiator of radical polymerization under UV irradiation. The colloidal fMIP-NP was collected from the surface of the beads by washing with dimethylformamide. And the dispersion medium of the colloidal fMIP-NP was replaced with physiological phosphate buffer saline. The fluorescent intensity and the radius of the fMIP-NP were increased by the addition of serotonin but were insensitive to L-tryptophan. Buccal mass, a cerebral ganglion, and buccal ganglion were sampled from Aplysia sea snail with keeping the nervous connection. The fMIP-NP was allowed to adsorb on the cerebral ganglion covalently via poly (glycidyl methacrylate -co- methacrylamide). Fluorescent intensity change was not observed during administration of Nori seaweed extraction, which has the preferred taste, or of distilled water which has the unpreferred taste. The taste preference was modified by administration of distilled water immediately after giving the Nori extraction. The spike response in the fluorescence of the stained the cerebral ganglion during the administration of Nori extraction after the preference modification. The result indicates that fMIP-NP is a potential probe to detect serotonin secretion concerning the Aplysia’s learning. The imaging with fMIP-NP will reveal the neurotransmitter networks which control animals’ learning and memory. | | 2022年6月30日 17:55～18:10 沖縄コンベンションセンター 会議場B2 第5会場
1O05e2-04
高感度cAMPプローブの開発と応用
Development and application of a supersensitive indicator for cAMP
*横山 達士(1)、坂本 雅行(1)
1. 京都大学
*Tatsushi Yokoyama(1), Masayuki Sakamoto(1)
1. Kyoto University
Keyword: cAMP, Imaging, genetically encoded indicator
Information processing in the brain is mediated by many types of neurotransmitters and the downstream signaling as well as electrical signals. Deciphering the interplay between these signals is one of the central goals of neuroscience. The technologies for this goal have been extensively developed so far. The development of green genetically encoded calcium indicators (GECIs) in the last two decades now permits decoding of electrical signals in hundreds or thousands of neurons in vivo. More recently, as well as color variants of calcium indicators, the indicators for other signals including voltage, glutamate, dopamine and noradrenaline have been developed. These indicators have visualized the spatial and temporal distribution of each signal in vivo. In addition, the simultaneous use of multiple indicators with different colors is revealing how the information encoded through different signals is integrated. Cyclic adenosine monophosphate (cAMP) is a downstream integrator of several kinds of neurotransmitters and intracellular signaling cascades in the brain, regulating synaptic and membrane properties. Thus, visualizing cAMP dynamics in vivo would further support the understanding of the information processing in the brain. However, although several studies have reported cAMP imaging in the mouse brain, the number of recorded cells or temporal resolution was limited due to sensitivity of the existing cAMP indicators. To overcome these limitations, here, we introduce a supersensitive genetically encoded cAMP indicator, named cAMPinG1. cAMPinG1 much outperformed the existing cAMP sensors in many respects, including the dynamic range, fluorescent properties, affinity for cAMP, and usability. In our presentation, we will show the applications of this sensitive cAMP sensor to neuroscience research. | | 2022年6月30日 18:10～18:25 沖縄コンベンションセンター 会議場B2 第5会場
1O05e2-05
Super-multicolour labelling of neurons for automated circuit reconstructions
*Marcus Leiwe(1), Satoshi Fujimoto(1), Toshikazu Baba(1), Takeshi Imai(1)
1. Department of Developmental Neurophysiology, Graduate School of Medical Sciences, Kyushu University, Fukuoka, Japan.
Keyword: Connectomics, Neurite Tracing, Machine Learning
The brain is made up of dense networks of interconnected neurons. Mapping the anatomy of these dense networks is one of the biggest challenges in neuroscience. Electron microscopy provides the highest resolution and is used as a gold standard in connectomics; however, its data size hampers large-scale circuit reconstruction at the millimetre scale. Light microscopy combined with tissue clearing is a new emerging approach for mesoscopic circuit mapping. However, the reconstruction of densely labelled circuits is challenging as its limited resolution hinders the discrimination of different neurons. Stochastic multicolour labelling strategies, such as Brainbow, utilise a combination of 3 fluorescent proteins (XFPs) to create different colour hues. Allowing for the reconstruction of densely labelled circuits. However, these tools only produce ~20 colour hues, which is not enough to reconstruct neuronal circuits at sufficient density. Moreover, manual circuit tracing based on the colour hue is a rate limiting step in this strategy.
We aimed to solve these issues by increasing the number of colour hues available, then use machine learning to automatically reconstruct neurons based on their colour hue alone. Firstly, we increased the number of colour hues by stochastically expressing a combination of 7 different fluorescent proteins, then separating the spectral overlap through linear unmixing. Our modelling suggests that this can generate ~1,200 different colour hues. Secondly, as our eyes are limited to trichromatic vision, we developed a pipeline to automatically recognize the combination of >3 colours. This pipeline includes a newly developed unsupervised clustering algorithm, named the “Euclidean Crawler”, which classifies data points in N-dimensional space purely based on the threshold Euclidean distance. It holds an advantage over other distance-based clustering algorithms as we do not need to specify the number of clusters (like K-means), nor the density of clusters (like mean-shift clustering). As proof of concept, first, we successfully reconstructed densely labelled layer 2/3 neurons in S1 (~300 neurons). Secondly, we automatically reconstructed long range (>2 mm) axonal terminals of mitral and tufted cell axons in the olfactory cortex. Finally, we used our automatic circuit reconstruction pipeline to register neighbouring brain sections: this was done by identifying neurites that go between sections by their colour hue, then performing piece-wise linear mapping for registration. Thus, super multi-colour labelling is a powerful tool for highly multiplexed circuit reconstruction on the mesoscopic scale. | | | |
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