一般口演（Oral）

Oral Molecular Imaging 一般口演分子イメージング

7月27日（土）14:20～14:35　第10会場（万代島ビル　6F　会議室） 3O-10a1-1 F-18標識AMPA受容体PET薬剤の合成とその機能評価 Tetsu Arisawa（有澤哲），Kimito Kimura（木村キミト），Yuuki Takada（髙田由貴），Tomoyuki Miyazaki（宮﨑智之），Takuya Takahashi（高橋琢哉）横浜市大院生理学 The AMPA receptor is an ionotropic transmembrane receptor for glutamate that is widely distributed in central nervous system, and related with memory and learning. But, AMPA receptor distribution in vivo was not revealed clearly. For this issue, we have already developed C-11 labelled PET tracer, [C-11]K-2, for AMPA receptor, which could visualize distribution of AMPA receptor in vivo. However, a C-11 radiopharmaceutical is very inconvenient because C-11 has very short half life (about 20 minutes). Because of the restriction, C-11 PET tracer have to be synthesized in clinical site. Widespread PET tracers in the world, such as FDG, FLT, and some amyloid tracer, are labelled with F-18 which has tractable half life (about 110 minutes). So, it is preferable that AMPA-PET tracer is also labelled with F-18. Now, we synthesized dozens of F-18 labelled K-2 derivatives, and evaluated efficiency of these compounds as a AMPA-PET tracer. Concretely, synthesized F-18 labelled compound were injected to rat, and these rats were scanned by micro-PET camera. The efficiencies were determined by correlation of AMPA receptor distribution by western blotting, and conformity of [C-11]K-2 which is leading AMPA-PET tracer. As a result, a few promising candidates were found. Throughout this screening, structure-activity relationship as AMPA-PET tracer is also revealed in K-2 derivatives. In this screening, metabolite analysis of K-2 was also utilized. We report these screening approaches and structure-activity relation ship as AMPA-PET tracer in detail.		7月27日（土）14:35～14:50　第10会場（万代島ビル　6F　会議室） 3O-10a1-2 超小型３D蛍光イメージングシステム Yuichiro Hayashi（林勇一郎）関西医科大学　附属生命医学研究所 Miniaturized fluorescence microscopes (Ghosh et al., 2011 Nat. Meth. 8 871) are becoming more important for deciphering neural codes underlying perception, memory and action. With a combination of genetically-engineered calcium indicators, these devices enable long-term recording of neural activity at single-cell resolution in freely moving animals. Considering the three-dimensional structure of neural circuits in the brain, volumetric imaging capability would increase the number of recorded neurons and enable simultaneous observation of multiple neuronal populations located in different depths. In addition, 3D imaging would improve the accuracy of cell tracking over multiple imaging sessions. Here, I developed a head-mount miniaturized microscope for volumetric fluorescence imaging from mice. The microscope was made from custom 3D printed parts, off-the-shelf optical components and CMOS image sensor electronics designed by an open-source miniature microscope project (UCLA miniscope). By introducing an electrically tunable lens for axial scanning, the device captures the fluorescence from a calcium sensor protein GCaMP6f within a volume of ~1200 x 800 x 400 micrometers at 5Hz from awake behaving mice. The overall weight of the microscope is under 4 grams, which have a negligible effect on the animal's behavior. This easily constructed device with volumetric imaging capability enables large-scale and precise recording of ensemble neural activities from freely moving animals.

7月27日（土）14:50～15:05　第10会場（万代島ビル　6F　会議室） 3O-10a1-3 合理的設計による生体内における脳神経回路網ダイナミクスイメージングに向けた多色カルシウムセンサーXCaMPの開発 Masatoshi Inoue（井上昌俊）^1,2，Atsuya Takeuchi（竹内敦也）³，Satoshi Manita（真仁田聡）⁴，Shin-ichiro Horigane（堀金慎一郎）^1,5，Masayuki Sakamoto（坂本雅行）¹，Ryang Kim（金亮）¹，Tatsushi Yokoyama（横山達士）¹，Sayaka Takemoto-Kimura（竹本-木村さやか）⁵，Manabu Abe（阿部学）⁶，Sean Quirin（Quirin Sean）²，Michiko Okamura（岡村理子）¹，Kenji Sakimura（崎村建司）⁶，Masanobu Kano（狩野方伸）^3,7，Hajime Fujii（藤井哉）¹，Deisseroth Karl（Karl Deisseroth）²，Kazuo Kitamura（喜多村和郎）⁴，Haruhiko Bito（尾藤晴彦）^1,7 ¹東京大院医神経生化学 ²Dept BioE Stanford Univ, Stanford, USA ³東京大院医神経生理 ⁴山梨大院医神経生理 ⁵名古屋大環境医神経系１ ⁶新潟大脳研基礎神経科学細胞神経生物 ⁷東京大ニューロインテリジェンス国際研究機構 Genetically encoded calcium indicators (GECIs) are widely used to monitor orchestrated information processing in active sets of neurons and their substructures. However, current GECIs are yet limited in single action potential (AP) detection speed, combinatorial spectral compatibility, and two-photon imaging depth, to resolve complex circuit properties in vivo. To address this, here we rationally engineered a next-generation quadricolor (B, G, Y, R) GECI suite, XCaMPs, for enhanced and combinatorial in vivo AP detection. Using a green color XCaMP-Gf, which resolved a single AP within 3-10 msec of stimulus onset in vivo, we recorded fast-spiking parvalbumin (PV)-positive interneurons in the mouse barrel cortex, and identified non-random, biased spatial firing sequences during spontaneous activity. Fiber photometric imaging in 3 distinct neuronal cell types (two inhibitory and one excitatory) reveals dynamic succession of inhibition and disinhibition pre-motion activity in freely moving mice. In vivo paired recording of pre- and postsynaptic firing revealed spatiotemporal constraints of dendritic inhibition in layer 1 in vivo, between axons of somatostatin (SST)-positive interneurons and apical tufts dendrites of excitatory pyramidal neurons. Taken together, the XCaMP indicators provide optical access to a much broader bandwidth of neuronal signaling, while significantly augmenting multiplexity, thus offering a critical enhancement of solution space in studies of complex neuronal circuit dynamics.		7月27日（土）15:05～15:20　第10会場（万代島ビル　6F　会議室） 3O-10a1-4 転写因子CREBリン酸化のin vivo可視化蛍光プローブ開発 Tatsushi Yokoyama（横山達士），Masatoshi Inoue（井上昌俊），Takuya Ito（伊藤拓也），Yayoi Kondo（近藤弥生），Masayuki Sakamoto（坂本雅行），Hajime Fujii（藤井哉），Haruhiko Bito（尾藤晴彦）東京大院医神経生化学 Deciphering mechanisms of long-term plasticity in complex neural networks of live animals is one of the major goals in modern neuroscience. It is widely known that the late phase of long-term plasticity requires "excitation-transcription coupling", or a privileged communication between electrical signals and nuclear gene expression via intracellular biochemical reactions. Recent technological advances in genetically encoded calcium indicators (GECIs) now permit tracking of electrical signals in hundreds of neurons in vivo. On the other hand, visualizing dynamics of biochemical signals in a large neuronal population in vivo remains challenging due to lack of sensitive indicators. Here, we designed and validated a supersensitive, genetically encoded green fluorescent indicator, XCREB-G, which faithfully reported the phosphorylation state of cyclic AMP-responsive element binding protein (CREB), a key transcription factor required for excitation-transcription coupling, at single cell resolution in vivo. XCREB-G was expressed in layer 2/3 neurons of the mouse primary visual cortex using an adeno-associated virus (AAV) vector or via in utero electroporation (IUE). Using in vivo two-photon microscopy, we directly tested how rapidly and selectively CREB phosphorylation was triggered by visual input-selective stimuli, in hundreds of visual cortical neurons responding to orientation-selective moving gratings. The fastest CREB activation was shown within 10 sec, and the in vivo dynamics of CREB activation was overall consistent with previous reports using cultured neurons. Simultaneous dual-color imaging of calcium (using a red GECI XCaMP-R) and CREB phosphorylation activity (using XCREB-G) demonstrated that a sizable number of neurons showed an orientation-selective CREB activation, following a burst of orientation-selective neuronal activation. More interestingly, we found evidence that a subthreshold CREB activation after a first bout of orientation-selective neuronal activity could lead to a suprathreshold CREB activation upon a second round of repetitive visual stimuli. Taken together, our findings suggest that nuclear CREB phosphorylation state indeed extracts and integrates information contained in synaptic input events in vivo. This technological advance will pave the way to better understand the complex dynamics and information processing elicited during excitation-transcription coupling in living animals.