公募シンポジウム

公募シンポジウム2【先端的イメージング技術で読み解く高次脳機能の仕組み】

2021/9/30　10:00～12:00　ZOOM B会場 S2-1 合理的な膜電位プローブの設計と応用 Rational design of genetically encoded voltage indicators and their application. ^○坂本雅行¹^,² 1.京都大学大学院生命科学研究科,2.JST さきがけ ^○Masayuki Sakamoto Graduate School of Biostudies, Kyoto University JST PRESTO Imaging membrane potential in neurons has become a fruitful approach to study neural circuits. Recently, the high performance of genetically encoded voltage indicators has been developed to monitor millisecond-scale neuronal dynamics. A microbial rhodopsin proton pump, Archaerhodopsin-3 (Arch), has been introduced as a basic structure of fluorescent voltage indicator that accurately tracked changes in neuronal membrane potentials. However, Arch fluorescence is relatively dim and imperfect membrane localization. Here, to develop new opsin-based voltage indicators that overcome these problems, we comparatively evaluated the fluorescence properties of 30 microbial rhodopsins. As a result, we found several rhodopsins which showed voltage-dependent fluorescent change and brighter basal fluorescence than Arch in primary cultured neurons. Then, by utilizing these new rhodopsins, we engineered a new genetically encoded voltage indicator. The electrophysiological recording revealed that a new opsin-based indicator could detect action potentials from the soma and dendritic spines with a high signal-to-noise ratio and fast kinetics. Simultaneous optical stimulation with channelrhodopsin and measurement of with voltage indicator faithfully induced and reported action potentials and subthreshold events with cross-talk-free. Lastly, we applied this indicator in vivo and successfully recorded visual stimulus-induced fluorescent response in the visual cortex with single-cell resolution in single trials. These results demonstrate that our new opsin-based voltage indicator allows the investigation of neuronal activity in defined populations and will notably facilitate dissecting functional relationships of neural networks.		2021/9/30　10:00～12:00　ZOOM B会場 S2-2 ニューロン－グリア間情報伝達の可視化 Visualization of activities and transmitters for neuron-glia interaction ^○繁冨英治¹^,²、小泉修一¹^,² 1.山梨大学医学部,2.山梨GLIAセンター ^○Eiji Shigetomi¹^,², Schuichi Koizumi¹^,² 1.Interdisciplinary Graduate School of Medicine University of Yamanashi 2.Yamanashi GLIA center Emerging evidence suggests that astrocytes regulate neurons contributing to the brain functions through bidirectional communication at synapses. Among the transmitters for the bidirectional communication, we focused on extracellular purines which are major transmitters for both neuron-glia and glia-glia communications. P2Y1 receptor, one of major purinergic receptors, plays a central role in Ca²⁺ signals and is upregulated in astrocytes in neurological disorders, such as epilepsy and Alzheimer’s disease. However, the significance of P2Y1 receptor upregulation in astrocytes is unknown. To reveal P2Y1 receptor-mediated signaling and its significance, we used transgenic mice in which astrocytes overexpress P2Y1 receptor specifically using Tet-Off system (P2Y1OE) and performed dual-color Ca²⁺ imaging of both astrocytes and neurons in the hippocampal slices using GCaMP6f, a green genetically encoded Ca²⁺ indicator (GECI) and jRGECO1a, a red GECI, respectively. Then, we found that electrical stimulation of the Schaffer collateral resulted in fast Ca²⁺ rise in dendrites of neurons followed by slow-onset Ca²⁺ rise in astrocytes. Slow-onset Ca²⁺ rise was mediated by P2Y1 receptor activation by ATP or ADP released from neurons, while fast Ca²⁺ rise in dendrites was augmented in P2Y1OE and mediated by ionotropic glutamate receptor, which is activated by neuronal but astrocytic glutamate. Transcriptome analysis of isolated astrocytes from P2Y1OE revealed a novel candidate molecule X as an astrocyte-derived excitatory signals which could contribute to augmented fast Ca²⁺ signal. Overall, the data suggest that astrocyte P2Y1 receptor-mediated signals enhanced glutamatergic synaptic transmission through a novel candidate molecule contributing to the disease pathogenesis.

2021/9/30　10:00～12:00　ZOOM B会場 S2-3 単一細胞解像度を有した高速・広視野2光子顕微鏡FASHIO-2PM によって明らかにされた大脳皮質の機能的ネットワーク特性 Cortical functional network proprieties revealed by “FASHIO-2PM”, a fast scanning and wide-field two-photon microscope with single-cell resolution ^○太田桂輔東京大学大学院医学系研究科・理化学研究所脳神経科学研究センター ^○Keisuke Ota 1.Graduate School of Medicine, University of Tokyo 2.RIKEN Center for Brain Science The information processing by cortical networks involving functionally diverse multiple areas is thought to surpass the simple integration of each processing in single brain areas. Thus, monitoring network activity consisting of cooperative and interactive activities via mid/long-range connections among numerous neurons is necessary for a comprehensive understanding of the mechanisms of brain functions. To monitor a large number of neurons and their network structure across multi-modal brain areas, a microscope must simultaneously achieve a high spatial resolution and a wide field-of-view (FOV). Additionally, to clearly monitor neural activities in vivo, the microscope must collect fluorescence with both fast sample rate and high signal-to-noise ratio. However, such monitoring is challenging because of the inevitable tradeoffs among these parameters. To overcome the tradeoffs, we developed a practically aberration-free, fast-scanning high optical invariant two-photon microscopy (FASHIO-2PM) which equips a large-angled resonant scanning system, a large objective with low magnification and high numerical aperture (0.8 NA, 56 mm pupil diameter), and large-aperture GaAsP photodetectors with high current output (14 mm square aperture, 50 μA). FASHIO-2PM enabled us to monitor more than 16,000 neural activities of L2 excitatory neurons at 7.5 Hz from a 9mm² FOV, including more than 10 sensory-motor and higher-order areas of the cerebral cortex in awake mice. Then, we assessed the functional network properties of the cortical layer 2. The cortical network structure was characterized by both a high clustering coefficient and short average path length, which indicated small-world properties.		2021/9/30　10:00～12:00　ZOOM B会場 S2-4 1分子イメージングによる脳神経疾患へのアプローチ Approaches to neurological diseases by single molecule imaging ^○坂内博子早稲田大学先進理工学部 ^○Hiroko Bannai School of Advanced Science and Engineering, Waseda University In order to understand the pathogenesis of neurological diseases, it is necessary to identify abnormalities that appear on neurons as early as possible. Here, we will introduce a novel approach to find cellular level phenotype pathology in neurons focusing on membrane molecules. According to the fluid mosaic model, plasma membrane molecules such as lipids and transmembrane proteins have the ability to undergo lateral diffusion freely within the cell membrane. Using a single particle tracking technique with quantum dots (QD-SPT), we found that the mobility of some membrane molecules became abnormal in cellular models of epilepsy and Alzheimer’s disease. We also found that cells isolated from animals doomed to develop neuronal disorders preserved such abnormality in membrane molecules behavior, even though they were isolated before symptom onset. Based on these findings, here we discuss the possibility that the behavior of membrane molecules reflects the destiny and properties of individual cells. These results suggest that abnormalities in the dynamics of membrane molecules may be the primary phenotype of neurological diseases, appearing in cells at a very early stage.

2021/9/30　10:00～12:00　ZOOM B会場 S2-5 高精細全脳イメージングから読み解く情動制御系回路 An emotional control system deciphered by whole-brain imaging at subcellular resolution ^○笠井淳司大阪大学大学院薬学研究科 ^○Atsushi Kasai Graduate School of Pharmaceutical Sciences, Osaka University Recently, various high-resolution whole-brain imaging techniques have been developed to capture all the cells in the brain. Studies using large-scale information on brain states or circuits are being conducted to elucidate the mechanisms controlling brain functions. Here, using an immediate early gene reporter system (Arc-dVenus reporter mice) and our whole-brain imaging system, FAST, we investigated the brain-wide neuronal activation patterns following restraint stress or social defeat stress, both of which induce anxiety-related behaviors. Then, we performed machine learning-based analyses to identify previously uncharacterized brain regions and neuronal ensembles implicated in emotional control, and identified a new ensemble that connects to the basolateral amygdala and medial prefrontal cortex and mediates bidirectional and reversible control of stress-induced emotional behaviors. In this symposium, we would like to introduce the latest research using a whole-brain imaging system, and discuss the possibility of contributing to the creation of interdisciplinary researches integrating neurochemistry, optics, and data science.