染色、トレーサー、画像化技術
Staining, Tracing, and Imaging Techniques
P3-2-233
神経回路解析に適した新規狂犬病ウイルスベクターの開発
Development of recombinant rabies virus vectors with neuron-specific, highly-efficient retrograde gene transfer
○井上謙一1, 藤原真紀1, 奥田泰宏1, 高田昌彦1
○Kenichi Inoue1, Maki Fujiwara1, Yasuhiro Okuda1, Masahiko Takada1
京都大学霊長類研究所　統合脳システム部門1
Sys Neurosci Sec, Primate Res Inst, Kyoto Univ, Inuyama1

The central nervous system is composed of synaptically connected neurons to form complex networks and achieve diverse functions. Transneuronal tracing with neurotropic viruses is a powerful technique for revealing neural circuits linking multiple regions. These viruses, such as rhabdoviruses and α-herpesviruses, can expand infection across synapses and amplify themselves in infected neurons, resulting in intense transsynaptic labeling of neurons along the circuits. Among them, the challenge virus standard (CVS) strain of rabies virus (RV) is a very useful virus, because it is taken up exclusively by axon terminals and moves in the retrograde direction to cross individual synapses in a time-dependent manner. Unlike other neurotropic viruses, RV does not cause cell lysis in infected neurons, and, therefore, neither local spillage of viral particles nor infection to functionally non-related nearby neurons occurs. This feature makes RV a promising candidate for producing vectors that transfer foreign genes into given neural circuits.Here, we report a newly-developed RV vector based on the CVS strain. The RV genome was manipulated to add the ability to express foreign genes efficiently onto the backbone strain. An additional transcription unit was created in the genome, and genes of fluorescence proteins were inserted as a marker. We found that this vector retains the infectious property of the parental CVS strain though its propagation rate is slightly decreased, and exhibits brilliant labeling of infected neurons. The present results indicate that the recombinant RV vector can be used as novel transsynaptic tracers that permits multicolor labeling, and that it is a potential candidate as the backbone vector for propagation-incompetent (glycoprotein-deleted) RV vectors.
 P3-2-234
TET2重感染法(TEDI)を使った大脳皮質投射ニューロンサブタイプの同時標識
Differential labeling of the cortical projection neuron subpopulations by TET-double infection method (TEDI)
○渡我部昭哉1,2, 加藤成樹3, 小林和人3, 水上浩明4, 小澤敬也4, 山森哲雄1,2
○Akiya Watakabe1,2, Shigeki Kato3, Kazuto Kobayashi3, Hiroaki Mizukami4, Keiya Ozawa4, Tetsuo Yamamori1,2
基生研・脳生物1, 総研大2, 福島県立医科大・医・附属生体情報伝達研3, 自治医大・分子病態治療・遺伝子治療4
Dev Brain Biol, Natl Inst Basic Biol, Okazaki1, Sokendai, Hayama2, Inst Biomedical Sciences, Fukushima medical Univ, Fukushima3, Div Genetic Therap, Ctr Molecular Medicine, Jichi Medical Univ., Tochigi4

To identify and characterize various subtypes of cortical projection neurons, we developed TET-double infection method (TEDI) for their retrograde labeling. The principle of TEDI is to infect the target cells with two components of the TET-system. That is, the coding sequences for the TET-activator and the transgene under the TET Responsive Element (TRE) are packaged in two viral vectors: one in a rabies-G protein coated retrograde lentiviral vector and the other in a VSV-G coated lentiviral vector or adenoassociated viral vector (AAV). By infecting these vectors at the terminal and the origin, respectively, high-level expression of the transgene occurs only in the neurons that connect the two points of infection. We now enhanced the technique by incorporating two fluorescent proteins to label distinct populations of projection neurons simultaneously. For example, we were able to differentially label the corticothalamic and corticopontine cells in the barrel cortex from the cells of origins to their terminals. We were also able to label corticocortical and corticothalamic cells of the barrel cortex projecting to the motor cortex and the thalamus (including reticular nucleus, VPm and PO), respectively. By simultaneous labeling, TEDI provides a novel means to analyze the subtle lamina structure of the mammalian neocortex composed of dendritic and axonal processes.
P3-2-235
マカクサル脳の拡散テンソルアトラスと線維束に基づく空間的統計
Diffusion tensor atlas and tract-based spatial statistics in macaque monkey brain
○林拓也1, 浦山慎一2, 渡部浩司2, 合瀬恭幸1, 村田弓4, 肥後範行4, 尾上浩隆1
○Takuya Hayashi1, Shin-ichi Urayama2, Hiroshi Watabe2, Takayuki Ose1, Yumi Murata4, Noriyuki Higo4, Hirotaka Onoe1
理化学研究所分子イメージング科学研究センター1, 京都大学医学研究科脳機能総合研究センター2, 大阪大学医学研究科医薬分子イメージング講座3, 産業技術総合研究所ヒューマンライフテクノロジー部門4
RIKEN CMIS, Kobe1, Kyoto Univ HBRC, Kyoto2, Dept of Mol Imag in Med, Osaka Univ, Suita3, Hum Tech Res Inst, AIST, Tsukuba4

The diffusion tensor imaging visualizes microarchitecture in the brain white matter. Fractional anisotropy (FA), analyzed on the diffusion tensor model, is recently used for spatially localizing FA changes related to skills, plasticity and neurodegeneration. Studies using non-human primates are also increasing; however, analysis has not been well standardized. Here, we present a population-average FA atlas for macaque monkeys in MNI space and the potential of the tract-based spatial statistics using the atlas. We collected diffusion weighted imaging (DWI) data from rhesus monkey. Using DWI, fieldmap and T1 images, FA atlas was constructed by multiple steps including brain extraction, distortion correction, FA calculation, registration between DWI and T1 images, averaging FA images across subjects, and iterative non-linear registrations. The FA atlas and skeleton were clearly visible for white matter details such as the Muratoff's bundle, external/extreme capsule, cortico-bulbar and cortico-spinal tracts (CBT/CST), cingulate bundle and fronto-orbital fascicules. Within- and between-subject variability was smaller in skeletonized than in non-skeletonized FA in most of locations, as well as in global median values. The percentage of non-normality voxels was less in skeletonized than in non-skeletonized FA. Finally, when voxel-wise statistics were applied to 5 animals injured in the primary motor cortical digit area, significant FA decrease specifically located in the CBT/CST in the injured animals by analyzing skeletonized FA (threshold-free cluster corrected P <0.05), while additional non-tract areas was also significant by non-skeletonized FA. These findings indicate that constructed FA atlas provides high-resolution anatomical details and could be used for spatial statistics in monkey MNI space. In addition, overall results of reliability, normality, and applicative tests showed better performance of tract-based spatial statistics than non-tract-based analysis.
 P3-2-236
マーモセット大脳皮質におけるスパインの生体内可視化
Visualization of dendritic spines in marmoset neocortex in vivo
○定金理1, 渡我部昭哉1, 大塚正成1, 高司雅史1, 佐々木哲也2, 笠井昌俊3, 伊佐正3, 加藤剛4, 鍋倉淳一4, 水上浩明5, 小澤敬也5, 河崎洋志6, 山森哲雄1
○Osamu Sadakane1, Akiya Watakabe1, Masanari Ohtsuka1, Masafumi Takaji1, Tetsuya Sasaki2, Masatoshi Kasai3, Tadashi Isa3, Go Kato4, Junichi Nabekura4, Hiroaki Mizukami5, Keiya Ozawa5, Hiroshi Kawasaki66, Tetsuo Yamamori1
基生研・脳生物1, 国立精神・神経セ神経研・微細構造2, 生理研・認知行動発達3, 生理研・生体恒常機能4, 自治医大・分子病態治セ5, 東京大院・医・神経機能解明ユニット6
Div. Brain Biology, NIBB, Aichi, Japan1, Dept. Ultrastructural Research, NIN, NCNP, Tokyo, Japan2, Div. Behavioral Development, NIPS, Aichi, Japan3, Div. Homeostatic Development, NIPS, Aichi, Japan4, Div. Genetic Therapeutics, Center for Molecular Medicine, Jichi Medical Univ., Tochigi, Japan5, Dept. Molecular and Systems Neurobiology, Grad. Sch. of Medicine, Univ. of Tokyo, Tokyo, Japan6

The dynamics of dendritic spines is thought to represent the rewiring of neural circuits, and hence, to be the basis of learning and memory. To understand the neural basis of learning and memory associated with higher cognitive process, it is crucial to investigate the spine plasticity in the primate neocortex, which has the most specialized cortical areas among mammals. Our specific aim in this study is to develop the method to observe the spine plasticity in the neurons of the primate neocortex. We chose the marmoset as a model animal for the study of primate brain, because the relatively flat surface of the marmoset neocortex allowed us to observe various neocortical regions, some of which are difficult to access in larger monkeys with more distinct gyrus and sulcus. We used the AAV vector to express hrGFP and successfully observed the spiny structures of neurons repeatedly from anesthetized animals using two-photon microscopy. To observe each dendritic spine clearly, strong and sparse expression of fluorescent protein was required. We achieved this requirement by amplifying the hrGFP expression using the TET-Off system, while titrating the amount of TET-activator driven by the Thy1S promoter (Ako et al., Mol. Cell. Neurosci., 2011) for sparse expression. The method we developed here will be useful to answer the questions whether and how the plastic state of neural circuits differ in specific cortical areas in the primate neocortex, which questions may be related to the higher order cognitive function or the malfunction of it.
P3-2-237
新規PETトレーサー[11C]-DPA713による神経炎症の生体内イメージング
In vivo imaging of neuroinflammation using a new PET tracer [11C]-DPA713
○横倉正倫1, 尾内康臣2, 竹林淳和1, 岩田泰秀1, 森則夫1
○Masamichi Yokokura1, Yasuomi Ouchi2, Kiyokazu Takebayashi1, Yasuhide Iwata1, Norio Mori1
浜松医科大学精神科1, 浜松医科大学生体機能イメージング研究室2
Deapartment of Psyhiatry and Neurology, Hamamatsu University School of Medicine, Hamamatsu, Japan1, Department of Biofunctional Imaging, Hamamatsu University School of Medicine, Hamamatsu, Japan2

Introduction Translocator protein (TSPO) is established as an important marker of neuroinflammation in the brain. [11C]-PK11195, the most common TSPO PET ligand, suffers from high nonspecific binding and low brain uptake. [11C]-DPA713, recently developed as a new PET tracer, is shown to have a higher affinity for TSPO than [11C]-PK11195 in humans. Binding potential (BP) of [11C]-DPA713 is based on the invasive Logan plot model, which needs arterial blood sampling. In contrast to this invasive method, a simplified reference tissue model (SRTM) is also available without arterial blood collection. In clinical circumstances, a non-invasive method may be preferable. Here, we compare the BP values estimated from these methods and evaluate differences in the BP levels of [11C]-DPA713 and [11C]-PK11195.
Methods Nine healthy males underwent [11C]-DPA713 PET with arterial blood sampling. The values of BP were estimated in the Logan plot analysis and SRTM model. In several brain regions, we examined correlations between BPs from these two methods. Secondary, 12 healthy males underwent [11C]-PK11195 PET. Estimation of BP was based on the SRTM. To evaluate BP levels of each subject, a semi-quantitative index was calculated as binding potential ratio (BPR), where the BP of each brain region was divided by the BP of the cerebellar hemisphere within the same subject. Then, we compared the BPR levels of [11C]-DPA713 with those of [11C]-PK11195.
Results There were positive correlations on BP between two analytic methods in several brain regions. BPR values of [11C]-DPA713 was higher than [11C]-PK11195.
Discussion Positive correlations in [11C]-DPA713 PET measures between invasive and non-invasive methods indicate that the non-invasive technique is satisfactorily applicable to any patients with neurological or psychiatric disorders. A higher BPR in [11C]-DPA713 PET suggests a greater advantage of [11C]-DPA713 than [11C]-PK11195 in depicting neuroinflammation in the living brain.
 P3-2-238
末梢神経選択的刺激を用いたマウス脳機能MRI
functional MRI with specific frequency stimulations for mouse brain mapping
○小牧裕司1,2,3, 疋島啓吾1,3, 許斐恒彦2, 芝田晋介1, 山田雅之4, 宮坂尚幸5, 藤吉兼浩2, 八木一夫6, 百島祐貴7, 中村雅也2, 岡野 J洋尚1,8, 岡野栄之1
○Yuji Komaki1,2,3, Keigo Hikishima1,3, Tsunehiko Konomi2, Shinsuke Shibata1, Masayuki Yamada4, Naoyuki Miyasaka5, Kanehiro Fujiyoshi2, Kazuo Yagi6, Suketaka Momoshima7, Masaya Nakamura2, Hirotaka Okano1,8, Hideyuki Okano11
慶應大院・医・生理学1, 慶應大・医・整形2, 実験動物中央研究所3, 藤田保健衛生大4, 東京医科歯科大5, 首都大学東京6, 慶應大・医・放射線7, 慈恵会医大8
Dept Physiol, Keio Univ, Tokyo1, Dept Ortho Surge, Keio Univ, Tokyo2, CIEA, Kanagawa3, Fujita Health Univ, Aichi4, TMDU, Tokyo5, TMU, Tokyo6, Dept Radiology, Keio Univ, Tokyo7, Jikei Univ, Tokyo8

We are committed to translational researches for the treatment of spinal cord injury by using methodsof behavior, histology and MRI. Although previous study succeeded in recovering motor function, a study about allodynia (experiencing pain by non-painful stimulation) caused by spinal cord injury donot reveal successful result yet. The purpose of this study is creating the path to develop the treatment for allodynia by revealing the mechanism of brain with fMRI technology for mice.
Mouse received three different kinds of stimuli in this study in order to specify the activated brainregion when stimulation is given. Stimulus of 2,000Hz (Aβ fiber, sense of touch) induced activation only in contralateral somatosensory cortex (S1). Stimulatus of 250Hz (Aδ fiber, Primary pain) induced activation in contralateral S1, secondary somatosensory cortex (S2), anterior cingulate cortex (ACC). Furthermore, stimulus of 5Hz (C fiber, Second pain and sense of warmth) induced activation in S1, ACC.
The stimulation of 2,000Hz to healthy mice elicited activation only in contralateral S1, whereas thestimulation to neuropathic pain model mice elicited activation in ACC besides S1.
We established functinal MRI for mice and succeeded in clarifying brain function localization.Examining fMRI when each peripheral nerve fibers received specific frequency stimulations showedthe relation with allodynia and brain.
P3-2-239
AMPA型受容体のエンドサイトーシス新規可視化法
Visualization of AMPA type receptors endocytosis
○林亜矢子1, 浅沼大祐2, 神谷真子3, 浦野泰照3, 岡部繁男1
○Ayako Hayashi1, Daisuke Asanuma2, Mako Kamiya3, Yasuteru Urano3, Shigeo Okabe1
東京大学大学院　医学系研究科　神経細胞生物学1, 東京大学大学院　医学系研究科　 神経生物学2, 東京大学大学院　医学系研究科　生体情報学3
Dept Cellular Neurobiol, Univ of Tokyo, Tokyo1, Dept Neurobiol, Univ of Tokyo, Tokyo2, Lab of Chem Biol and Molecular Imaging, Univ of Tokyo, Tokyo3

Endocytosis is a fundamental mechanism by which neurons control distribution of surface receptors. We developed a new method of monitoring endocytosis of AMPA-type glutamate receptors GluR1 and 2. Receptor subunits were tagged with ZIP-binding cassette at the N-terminal extracellular domain and labeled by ZIP peptides conjugated with pH sensitive dye RhPM. RhPM-labeled GluRs increased its fluorescence after endocytosis, due to the acidification in the endosomal compartment. Because the labeling complex was small, this technique has an advantage in monitoring native receptor behavior. Here we report rapid endocytosis of GluRs in both dendritic spines and dendritic shafts in a time scale of minutes. The rapid endocytosis of GluRs in spines was prominent at 18 DIV (days in vitro) and most PSD-95-positive mushroom-type spines showed internalization of GluRs. Immature neurons (10 DIV) showed GluR endocytosis mainly in dendritic shafts and the kinetics and distribution of endocytotic events were similar to those of RhPM-labeled transferrin. We propose the presence of local endocytotic machinery within mature dendritic spines, which serves to regulate the content of synaptic AMPA receptors.

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