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ポスター H. 方法論 H. Methodology

2022年7月2日　11:00～12:00　沖縄コンベンションセンター展示棟　ポスター会場1 3P-312 Suppression of resting-state connectivity induced by transcranial static magnetic stimulation to the precuneus Mohammad Amin Abdollahi(1), Ali Moharramipour(1), Shigeru Kitazawa(1,2,3) 1. Graduate School of Frontier Biosciences, Osaka University, 2. Graduate School of Medicine, Osaka University, 3. Center for Information and Neural Networks (CiNet), National Institute of Information and Communications Technology Keyword:* transcranial static magnetic stimulation (tSMS), resting-state functional magnetic resonance imaging (rs-fMRI), precuneus, brain stimulation Transcranial static magnetic stimulation (tSMS) is a non-invasive method to suppress neural activities. However, the effects on the human cerebral cortex remain controversial and not established yet. In the present study, we applied tSMS to the precuneus, a central hub of the default mode network (DMN) and the entire cortical network, in an expectation that it would induce clear and maximum suppression to the entire cortical network. We recruited sixteen neurologically normal subjects and exposed them to the tSMS by placing a neodymium magnet (45 mm in diameter, 1 Tesla in the center) over the precuneus for one hour. We recorded their resting brain activity using the functional MRI (fMRI) before and immediately after the exposure. We used sham (non-magnet metal) as a control in comparison with the tSMS. The experiment was conducted on two separate days, on each day exposing to a sham or magnet. We found suppression of the connectivity between the precuneus and the hippocampus, thalamus, and parahippocampal regions. Since these regions are involved in the formation and retrieval of memory, our result might implicate that the tSMS on the precuneus should somehow disturb the memory-related functions. Last we noticed that regardless of the exposure to the sham or magnet, there was an increase in the functional connectivity of the DMN on the first day compared to the second day. This might be associated with the surprise due to the novelty of the experimental setup on the first day.		2022年7月2日　11:00～12:00　沖縄コンベンションセンター展示棟　ポスター会場1 3P-313 音響圧分布とin vitroマウス脳スライスの皮質回路網を反映した超音波誘発応答の時空間的解析 A spatiotemporal analysis of ultrasound-driven responses reflecting acoustic pressure distributions and cortical circuits in mouse brain slices in vitro 古川凌(1)、金田弘貴(1)、舘野高(2) 1. 北海道大学大学院情報科学院、2. 北海道大学大学院情報科学研究院 Ryo Furukawa(1), Hiroki Kaneta(1), Takashi Tateno(2) 1. Graduate School of Information Science and Technology, Hokkaido University, 2. Faculty of Information Science and Technology, Hokkaido University Keyword: CORTICAL SLICE, MULTIELECTRODE ARRAY, PATTERN CLASSIFICATION, ULTRASOUND STIMULATION Ultrasound stimulation has been promising because it could noninvasively stimulate the local and deep area of the brain, which is difficult to achieve using conventional electromagnetic stimulation. However, the pressure distribution of ultrasound stimulation can induce more complex responses in brain tissue than electromagnetic stimulation. Little has been known about the detailed spatial irradiation properties of ultrasound waves through brain tissue, and extensive analyses of ultrasound-driven responses have not been reported. Here, we attempt to characterize spatiotemporal responses reflecting cortical circuit properties and acoustic pressure distributions. We performed electrical and ultrasound stimulations to mouse brain slices including the auditory cortex on a 64-channel multielectrode array (MEA) substrate and recorded the evoked activity over the area of 2.1 × 2.1 mm². We also measured the fluid pressure in the MEA chamber using a hydrophone located 1.0-mm away from the surface of the MEA with the same setup. In response to the 0.5-MHz ultrasound stimulation from the smallest (110 kPa) to largest pressure (410 kPa) levels with a 100-kPa step and a 200-ms duration, the peak amplitudes of the ultrasound-driven local field potential (LFP) responses were significantly increased with the intensities. In contrast, as the pressure levels of the ultrasound increased with the same 200-ms duration, the latencies of the large negative peak were around 100 ms and remained over the pressure levels. Further, the duration of the ultrasound stimulation (100 ms vs. 200 ms) influenced that of the LFP response. Our experimental results indicate the ultrasound-evoked responses in mouse brain slices depended on the intensities and durations of the stimulation. Additionally, we numerically and experimentally examined how discrepancies in the center position of an ultrasound transducer could affect pressure distributions and similarities between response patterns and the pressure distributions. Our analysis of the similarities among current- or ultrasound-driven response patterns and the ultrasound pressure distributions indicated the laminar properties of cortical slices and/or spatial patterns of ultrasound stimulation might profoundly influence ultrasound-driven response patterns. Our methodology combining the MEA with ultrasound stimulation has the potential to examine detailed mechanisms for spatiotemporal activation of neural circuits to ultrasound stimulation.

2022年7月2日　11:00～12:00　沖縄コンベンションセンター展示棟　ポスター会場1 3P-314 毒性の低い新しい経シナプス性ウイルストレーサーの開発 Development of a new trans-synaptic viral tracing system with low cytotoxicity 近藤邦生(1,2)、井上謙一(2,3)、箕越靖彦(1) 1. 自然科学研究機構生理学研究所、2. JST さきがけ、3. 京都大学霊長類研究所 Kunio Kondoh(1,2), Ken-ichi Inoue(2,3), Yasuhiko Minokoshi(1) 1. National Institute for Physiological Sciences, National Institute of Natural Sciences, 2. JST PRESTO, 3. Primate Research Institute, Kyoto University Keyword: trans-synaptic virus, neural circuit tracing, pseudorabies virus The nervous system consists of an immense number of neurons that are connected with highly specific and complex manners. Diverse types of neurons are intermingled in the same brain area and differently connected to specific target neurons in different brain areas. To understand the functions of neural circuits, it is necessary to understand their neural connectivity within the neural circuits. For this purpose, trans-synaptic viruses, such as rabies virus (RV) and pseudorabies virus (PRV), have been used for mapping neural circuitry. These viruses can infect neurons and travel to other neurons through synaptic connections, making it possible to label neurons that are specifically connected to targeted neurons. Although trans-synaptic viral tracers are powerful tools, there is a problem that viral infection has cytotoxicity which affects the physiological states of infected neurons and ultimately causes cell death. Therefore, it is difficult to image or manipulate virus-infected neurons and thus the use of trans-synaptic viruses is mostly restricted to anatomical applications. To overcome this problem, we have developed a new trans-synaptic viral tracing system by using PRV lacking an immediate early gene IE180. Our modified IE180-deleted PRVs successfully expressed exogenous genes of interest for a long time without any obvious cytotoxicity. Furthermore, with co-infection of helper viruses that express IE180, these PRVs were able to be retrogradely transported to upstream neurons, suggesting that they worked as a monosynaptic tracer. These results indicated that our new tracing system can be combined with other neural imaging or manipulation methods, providing new tools to analyze neural circuits.		2022年7月2日　11:00～12:00　沖縄コンベンションセンター展示棟　ポスター会場1 3P-315 In vivo近赤外光神経刺激によって誘発されるマウス下丘での双方向性の神経活動変調効果 Bidirectional neural modulation effects induced by near-infrared light neural stimulation in mice inferior colliculus in vivo 佐藤広務(1)、舘野高(2) 1. 北海道大学大学院情報科学院、2. 北海道大学大学院情報科学研究院 Hiromu Sato(1), Takashi Tateno(2) 1. Graduate School of Information Science and Technology, Hokkaido University, 2. Faculty of Information Science and Technology, Hokkaido University Keyword: INFERIOR COLLICULUS, INFRARED LIGHT, NEURAL STIMULATION, PHOTOTHERMAL EFFECTS The spatial and temporal distribution of temperature in the central nervous system and its maintenance are critical in many neurophysiological processes including brain homeostasis. Diagnostic and therapeutic tools introducing thermal changes in neural tissue are intensively studied; some brain stimulation methods such as electromagnetic stimulation are partially owing to the heating effects when neural responses are evoked with a large stimulus intensity. Among brain stimulation techniques, a novel optogenetic method inevitably requires prior genetic incorporation of light-sensitive opsins, while infrared-light neural stimulation (INS) relying on thermal effect intervention can provide a more convenient way to modify neural activity without such genetic modification. To generate the temperature changes in neurons, INS typically uses pulsed infrared (IR) light (between the wavelength λ of 1400–2100 nm), inducing neural activity modulations. During photothermal stimulation to neural tissue, local temperature increases are primarily responsible for neural excitation and/or inhibition in a matter that depends on stimulation power, temporal patterns, and brain regions. The inferior colliculus (IC) is the main receiving station for the ascending auditory pathway from lower stages of the brainstem, forming the primary site of convergence of sound processing streams. Here, we examined the INS-induced effects on IC responses in anesthetized mice in vivo, which have not been reported before. We observed the neural responses of mouse IC driven by a pulsed infrared light (λ = 1,940 nm) radiated from a diode laser via a multimode optical fiber (diameter = 200 µm; numerical aperture = 0.22) placed at 1-2 mm away from the IC surface. Using a linear multielectrode array, local field potential (LFP) responses in IC to INS stimulation showed positive and negative voltage changes depending on the power intensities and temporal stimulation patterns of INS. The INS with different levels of irradiation power induced the decreased or increased changes of responsive LFPs, although the variances around the means were relatively large. The result suggests the bidirectional effects may be used as a technique for an excitatory or inhibitory stimulation to modify neural activity in an irradiation power-dependent manner. Thus, INS could open up a new therapeutic tool to modify neural activity in the auditory pathway and other brain regions.

2022年7月2日　11:00～12:00　沖縄コンベンションセンター展示棟　ポスター会場1 3P-316 霊長類脳への高効率な導入遺伝子発現と神経細胞特異的な感染を実現するモザイクAAVベクターの開発 A mosaic adeno-associated virus vector as a versatile tool displaying high levels of transgene expression and neuron specificity in the primate brain 木村慧(1)、永井裕司(2)、畑中岳(3,4)、Yang Fang(3,4)、田辺創思(1)、Andi Zheng(1)、藤原真紀(1)、中野真由子(1)、堀由紀子(2)、竹内遼介(6)、稲垣未来男(3,4)、南本敬史(2)、藤田一郎(3,4)、井上謙一(1,5)、高田昌彦(1) 1. 京都大学霊長類研究所神経科学研究部門統合脳システム分野、2. 国立研究開発法人量子科学技術研究開発機構脳機能イメージング研究部、3. 大阪大学大学院生命機能研究科認知脳科学研究室、4. 大阪大学・情報通信研究機構脳情報通信融合研究センター、5. 国立研究開発法人科学技術振興機構戦略的創造研究推進事業（さきがけ）、6. 名古屋大学大学院創薬科学研究科 Kei Kimura(1), Yuji Nagai(2), Gaku Hatanaka(3,4), Yang Fang(3,4), Soshi Tanabe(1), Andi Zheng(1), Maki Fujiwara(1), Mayuko Nakano(1), Yukiko Hori(2), Ryosuke Takeuchi(6), Mikio Inagaki(3,4), Takafumi Minamimoto(2), Ichiro Fujita(3,4), Ken-ichi Inoue(1,5), Masahiko Takada(1) 1. Systems Neuroscience Section, Department of Neuroscience, Primate Research Institute, Kyoto University, 2. Department of Functional Brain Imaging, National Institutes for Quantum and Radiological Science and Technology, 3. Laboratory for Cognitive Neuroscience, Graduate School of Frontier Biosciences, Osaka University, 4. Center for Information and Neural Networks, National Institute of Information and Communications Technology and Osaka University, 5. PRESTO, Japan Science and Technology Agency, 6. Graduate School of Pharmaceutical Science, Nagoya University Keyword: Primate, AAV, DREADDs, Calcium imaging Recent emphasis has been placed on gene transduction mediated through recombinant adeno-associated virus (AAV) vector for manipulating activity of neurons and their circuits in the primate brain. Although the use of different serotypes of AAV vectors for optogenetic and chemogenetic approaches has demonstrated the validity of this strategy, no reliable vector is yet available, at least in primates, to achieve modulation of neuronal activity or animal’s behavior. Some vectors exhibit high-level expression of target genes but cause inflammation due to infection to glial cells or transgene expression therein, while others have high-level neuron specificity with relatively low expression level. It is conceivable that there is usually a trade-off relationship between the transgene expression capacity and the neuron-specific infectivity. For improving the quality of gene transduction into the primate brain for diverse purposes, it should be essential to overcome this issue, creating a novel AAV vector with marked superiority in both aspects. In the present study, we have developed mosaic AAV vectors (termed AAV2.1-A and AAV2.1-B), composed of AAV1 and AAV2 capsids. We injected these vectors into the cerebral cortex of macaque monkeys and examined the pattern and efficiency of their transgene expression by comparing with those via the original AAV1 and AAV2 vector. We found that the AAV2 and AAV2.1-A vectors showed higher neuron specificity, whereas the AAV1 and AAV2.1-B vectors had higher glial infectivity. On the other hand, it was revealed that both the AAV2.1-A and the AAV2.1-B vectors displayed high-level transgene expression which was equivalent to that of the AAV1 vector and 3-4 times higher than that of the AAV2 vector. These results indicate that the AAV2.1-A vector possesses both high-level transgene expression capacity and neuron-specific infectivity. To explore its applicability to chemogenetic manipulation and in vivo calcium imaging, the AAV2.1-A vector expressing excitatory DREADDs or GCaMP was injected into the striatum or the visual cortex of macaque monkeys, respectively. Our results have defined that this novel vector secures intense and long-term expression of the target proteins and produces conspicuous modulation and imaging of neuronal activity. Thus, our newly-developed AAV vector could be most suitable for effective and stable gene transduction into neurons of the primate brain.		2022年7月2日　11:00～12:00　沖縄コンベンションセンター展示棟　ポスター会場1 3P-317 サル脳内DREADD発現と効果の長期的評価 Longitudinal assessment of DREADD expression and efficacy in the monkey brain 永井裕司(1)、堀由紀子(1)、井上謙一(2)、小山佳(1)、宮川尚久(1)、平林敏行(1)、三村喬生(1)、須原哲也(1)、高田昌彦(2)、樋口真人(1)、南本敬史(1) 1. 量子科学技術研究開発機構脳機能イメージング研究部、2. 京都大霊長研統合脳システム Yuji Nagai(1), Yukiko Hori(1), Ken-ichi Inoue(2), Kei Oyama(1), Naohisa Miyakawa(1), Toshiyuki Hirabayashi(1), Koki Mimura(1), Tetsuya Suhara(1), Masahiko Takada(2), Makoto Higuchi(1), Takafumi Minamimoto(1) 1. Dept Functional Brain Imag, QST, Chiba, JAPAN, 2. Systems Neurosci Section, PRI, Kyoto Univ, Inuyama, Japan Keyword: chemogenetics, PET, DREADD, in vivo imaging Designer Receptors Exclusively Activated by Designer Drugs (DREADDs) are the cutting-edge chemogenetic technology that offers a means to reversibly control the activity of a neuronal population expressing a designer receptor by systemic administration of an inert actuator. Muscarinic-based DREADDs, hM3Dq (excitatory) and hM4Di (inhibitory), are widely used in rodent models, but reports available on monkey models are still limited. Given that the chemogenetic controllability depends on the expression levels of DREADDs, understanding of their temporal trends is highly valuable, especially for planning long-term experiments in monkeys. In this study, we longitudinally quantified in vivo DREADD expression in macaque monkeys using positron emission tomography (PET) with DREADD-selective tracers, [¹¹C]deschloroclozapine (DCZ) and [¹¹C]clozapine (CLZ). A total of 12 macaques that had received injections of adeno-associated virus vectors expressing hM4Di or hM3Dq was evaluated. DREADD expression levels were estimated based on the increase in binding potential and the uptake from baseline for [¹¹C]DCZ and [¹¹C]CLZ, respectively. The expression levels of hM4Di and hM3Dq reached peaks at about 60 days post-injection and remained at near-peak levels for about a year. In the meantime, the expression level of hM3Dq was decreased at around 120 days post-injection and then recovered. At about two years post-injection, the expression levels were decreased to approximately 80% for hM4Di and 70% for hM3Dq. Despite these reductions, activation of DREADDs was effective in changing neuronal activity and behavior; for example, at 2.3 years after the vector injection, activation of hM4Di expressed in the dorsolateral prefrontal cortex impaired working memory. Importantly, repetitive DREADD activation by optimum doses of agonists (DCZ or CNO) did not have a strong impact on the expression level of hM4Di or hM3Dq, while over-activation of hM3Dq by high agonist doses reduced its expression level even after one-time activation. These results provide valuable information and promise the use of DREADDs in long-term experiments in monkeys.

2022年7月2日　11:00～12:00　沖縄コンベンションセンター展示棟　ポスター会場1 3P-318 光刺激にマイクロLEDアレイデバイスを用いたマウスVTA内のGABAニューロンとドーパミンニューロンの関係についての考察 A study on the relationship between GABAergic and dopaminergic neurons in the VTA of a mouse using micro-LED array devices for photo-stimulation 太田安美(1)、邑上貴秋(1)、 Mark Guinto(1)、河原麻実子(1)、Yasemin Akay(2)、竹原浩成(1)、春田　牧人(1)、田代　洋行(1)、笹川清隆(1)、Metin Akay(2)、太田　淳(1) 1. 奈良先端科学技術⼤学院⼤学、2. ヒューストン大学 Yasumi Ohta(1), Takaaki E. Murakami(1), Mark Christian Guinto(1), Mamiko Kawahara(1), Yasemin M. Akay(2), Hironari Takehara(1), Makito Haruta(1), Hiroyuki Tashiro(1), Kiyotaka Sasagawa(1), Metin Akay(2), Jun Ohta(1) 1. Nara Institute of Science and Technology, 2. University of Huston Keyword: optogenetics, dopamine neuron, GABAergic neuron, microdialysis Optogenetics is a powerful tool for the control of biological functions using light. We have developed a micro-LED array device for optical stimulation, and evaluated the device's operation, light intensity, and safety. We confirmed the effectiveness of the developed micro-LED array device by photostimulating the ventral tegmental area (VTA) of mice and measuring dopamine concentration in the nucleus accumbens shell (NAcShell) by microdialysis. In this study, we also used this device in the mouse brain to investigate the role of GABAergic neurons in the DA neuron system of the mesolimbic system, which is involved in the reward system. Neurons in the VTA of DA-specific ChrimsonR-expressing Dopamine transporter (DAT)-cre mice or DA-non-specific ChrimsonR-expressing WT type mice were photo-stimulated and dopamine release in the NAcShell was measured to evaluate the differences between the two. As a result, when comparing the optical stimulation frequency of 2 Hz and 20 Hz, the changes in DA concentration in NAcShell were both larger at 20 Hz. DA-specific expression of ChrimsonR increased the release of DA in NAcShell in response to photo stimulation to VTA. On the other hand, when ChrimsonR was expressed non-specifically for DA, the amount of DA released at NAcShell by photo-stimulation for VTA was almost unchanged. However, even when ChrimsonR was expressed DA-nonspecifically, intraperitoneal administration of bicuculline, which competitively antagonizes the GABA-binding site of the GABAA receptor, significantly increased the release of DA in response to light stimulation of VTA in NAcShell. Immunochemical staining confirmed that GABAergic neurons in the VTA inhibit DA activation, indicating that alterations in GABAergic neurons may have severe downstream effects on DA activity, NAcShell release, and neural adaptation of the VTA. This study confirms the importance of optogenetic techniques to study the relationship between mesolimbic dopaminergic and GABAergic neurons in a neuro-specific manner.		2022年7月2日　11:00～12:00　沖縄コンベンションセンター展示棟　ポスター会場1 3P-319 高密度多点電極アレイ上でのモジュール構造型培養神経回路のパターニング Micropatterned culture of modular neuronal networks on high-density microelectrode arrays 佐藤有弥(1,2)、山本英明(1)、住拓磨(1,2)、谷井孝至(3)、佐藤茂雄(1)、平野愛弓(1,2,4) 1. 東北大学電気通信研究所、2. 東北大学大学院医工学研究科、3. 早稲田大学理工学術院、4. 東北大学材料科学高等研究所 Sato Yuya(1,2), Hideaki Yamamoto(1), Takuma Sumi(1,2), Takasi Tanii(3), Sigeo Sato(1), Ayumi Hirano-Iwata(1,2,4) 1. RIEC, Tohoku Univ, Sendai, Japan, 2. Grad Sch Biomed Eng, Tohoku Univ, Sendai, Japan, 3. Fac Sci Eng, Waseda Univ, Tokyo, Japan, 4. AIMR, Tohoku Univ, Sendai, Japan Keyword: Neuronal network, Multielectrode array, Microfluidic device, Complex networks An in vitro reconstitution of biological functions with cultured cells plays an important role in understanding the neural basis of brain functions. However, there is a significant difference between the activities of cultured neuronal networks with randomly connected structures and those of the neuronal networks in the brain. By applying microfabrication techniques, we have reconstructed modular neuronal networks that generates spatiotemporally complex neural activity in culture [1, 2] and measured their neural activity using fluorescence calcium imaging. However, the temporal resolution of the calcium imaging technique is limited to a few hundred Hz. In the present study, we reconstructed the modular neuronal network on a high-density microelectrode array, measured its activity with high temporal resolution, and compared it with a homogeneous cultured neuronal network. As observed in calcium imaging, the modular networks produced a richer repertoire of dynamics, which was now captured with sub-millisecond (0.05 ms) temporal resolution. The work was supported by MEXT KAKENHI Grant-in-Aid for Transformative Research Areas (B) “Multicellular Neurobiocomputing” (21H05164), JSPS KAKENHI (18H03325), JST-PRESTO (JMPJPR18MB), JST-CREST (JPMJCR19K3), and the Cooperative Research Project Program of the Research Institute of Electrical Communication of Tohoku University. References: [1] H. Yamamoto et al., Sci. Adv. 4, eaau4914 (2018). [2] T. Takemuro et al., Jpn. J. Appl. Phys. 59, 117001 (2020).

2022年7月2日　11:00～12:00　沖縄コンベンションセンター展示棟　ポスター会場1 3P-320 2領域から単シナプス入力を受ける神経細胞の標識・操作法 Viral targeting of the neurons receiving monosynaptic inputs from two upstream regions 北西卓磨(1,2)、田代麻里子(1)、北西なおみ(1)、水関健司(1) 1. 大阪市立大学大学院医学研究科、2. 科学技術振興機構さきがけ Takuma Kitanishi(1,2), Mariko Tashiro(1), Naomi Kitanishi(1), Kenji Mizuseki(1) 1. Osaka City Univ Grad Sch Med, Osaka, Japan, 2. PRESTO, JST Keyword: Transsynaptic tracing, AAV1, PHP.eB, INTRSECT The brain consists of many distinct regions with corresponding functions. A single brain region generally receives synaptic inputs from multiple upstream areas and distributes the information processed within the region to multiple downstream areas. Such integration and distribution are fundamental interregional interactions that support various brain functions. The neurons that distribute information to multiple downstream areas through collateral projections can be targeted using retrograde tracers/viral vectors. However, no method is currently available to selectively access neurons that receive monosynaptic inputs from two upstream regions. Here, we devised a method to genetically label such neurons with a single gene of interest in mice by combining the anterograde transsynaptic spread of adeno-associated virus serotype 1 (AAV1) with intersectional gene expression. Injections of AAV1s expressing either Cre or Flpo recombinases and the Cre/Flpo double-dependent AAV into two upstream regions and the downstream region, respectively, were used to label the postsynaptic neurons receiving inputs from the two upstream regions. We demonstrated this labelling in two distinct circuits: the retina/primary visual cortex to the superior colliculus and the bilateral motor cortex to the dorsal striatum. Systemic delivery of the intersectional AAV-PHP.eB allowed for unbiased detection of the labelled neurons throughout the brain. This strategy may help analyse the interregional integration of information in the brain.		2022年7月2日　11:00～12:00　沖縄コンベンションセンター展示棟　ポスター会場1 3P-321 ミクログリア動態と神経細胞活動をサブ秒の時間分解能で同時に解析するin vivoイメージング技術 In vivo imaging technique for simultaneous analysis of microglial dynamics and neuronal activity with a sub-second temporal resolution 丸岡久人(1)、亀井亮佑(1)、水谷俊介(1)、劉慶瑞(1)、岡部繁男(1) 1. 東京大学 Hisato Maruoka(1), Ryosuke Kamei(1), Shunsuke Mizutani(1), Qingrui Liu(1), Shigeo Okabe(1) 1. The University of Tokyo Keyword: In vivo imaging, Microglia, Neuron The accumulating evidence suggests that microglia, resident immune cells in the brain, are involved in the development and plasticity of neural circuits. However, how microglial dynamics are coordinated with neuronal activity on time scales of sub-second to a few tens of seconds remains unclear. Here, we developed a technique for simultaneously imaging microglial dynamics and neuronal activity in awake mice with sub-second temporal resolution. First, we evaluated the stability of our surgical preparation and imaging setup. A cranial window and a head-plate were installed on the skull over the neocortex using transgenic mice expressing EGFP in layer 5 excitatory neurons. Mice were placed under a two-photon excitation microscope and allowed to run freely on the treadmill during imaging. In vivoimaging using this setup demonstrated that mouse movement did not affect the stability of EGFP intensities in neurons, suggesting that the motion artifacts are negligible. Subsequently, adeno-associated virus encoding a red fluorescent calcium indicator protein, R-CaMP1.07, was injected into the primary visual cortex (V1) of transgenic mice with microglia-specific expression of EGFP (CX3CR1-EGFP transgenic mice). In vivo imaging of microglial dynamics and neuronal activity was performed 4 weeks after the craniotomy with the frame rate of 30 Hz. Analysis revealed that dynamic changes of microglial processes took place on time scales of a few seconds, with simultaneous calcium response of neurons to visual grating stimuli with a specific orientation. This in vivo simultaneous imaging technique enables us to clarify the coordination between microglial dynamics and neuronal activity induced by sensory stimuli with sub-second temporal resolution.

2022年7月2日　11:00～12:00　沖縄コンベンションセンター展示棟　ポスター会場1 3P-322 神経の高速・高分解能な３次元光計測 Fast, high-resolution 3D optical measurement of nerves 星野鉄哉(1)、青木貞雄(1)、伊藤雅英(1) 1. 筑波大学 Tetsuya Hoshino(1), Sadao Aoki(1), Masahide Itoh(1) 1. University of Tsukuba Keyword: scatterometry, neural signal, 3D shape, neuron When the influence of absorption and scattering is large, scatterometry can improve the resolution by two orders of magnitude in the three-dimensional measurement of a simple shape as compared with lens imaging. In addition, it is possible to measure with a time resolution of milliseconds or less. It is a non-destructive, non-contact, simple measurement method. High-resolution and high-speed detection of structural changes in the refractive index associated with neural signal transduction is possible [1]. Recent advances in optical calculation technology have made it possible to quantitatively analyze shapes and sizes. In this report, we will introduce this technique. We also show scatterometry measurement of preparation specimens of brains. If the relationship between the refractive index distribution in the cross section and the scattering pattern in the distance can be calculated accurately, the refractive index distribution can be clarified. As a method for calculating the scattered light angle distribution, rigorous coupled-wave analysis (RCWA) is used here because the internal structure can be easily considered and the scattering pattern in the far field can be directly calculated. RCWA treats only the periodic structure, however, it can also be applied to isolated systems by devising the calculation method [2]. In addition, light focusing is required to select a specific structure, and the setting of the positional relationship between the light focusing point and the sample greatly affects the reproducibility, but the contrast of the scattering pattern can be adjusted to ensure reproducibility [3]. This method is suitable for the analysis of simple three-dimensional shapes such as ellipses and rectangles, and true cross-sectional shapes without projection are obtained. The resolution and the measurement size can be reduced by double-digits when compared to holography and laser microscopy. If a wide dynamic range and high sensitivity of the image sensor can be secured, measurement can be performed at that frame rate. First, as a model shape, we examined the relationship between the scattering pattern of a lattice and its cross-sectional shape, using not only visible light but also soft X-rays. Furthermore, we observed preparation specimen of Golgi-stained cerebrum of cat and Weigert stained cerebellum of human. We measured the diffraction pattern of the neuron by visible light and estimated its cross-sectional shape. [Reference] [1] R.A.Stepnoski, et al. PNAS 88.21 9382-9386 (1991) [2] T.Hoshino, T.Yatagai, M.Itoh Opt. Express 20.4 3954-3966 (2012) [3] T.Hoshino, N.Watanabe, S.Aoki, K.Sakurai, M.Itoh Opt. Express 25.21 26329-26348 (2017)		2022年7月2日　11:00～12:00　沖縄コンベンションセンター展示棟　ポスター会場1 3P-323 脳内オキシトシン動態のリアルタイム計測を実現する蛍光センサーの開発 A fluorescent sensor for the real-time measurement of extracellular oxytocin dynamics in the brain 稲生大輔(1)、日比野浩(1) 1. 大阪大学大学院医学系研究科 Daisuke Ino(1), Hiroshi Hibino(1) 1. Grad Sch Med, Osaka Univ, Osaka, Japan Keyword: Oxytocin, Fluorescent probe Oxytocin (OT), a hypothalamic neuropeptide that acts as a neuromodulator in the brain, orchestrates a variety of animal behaviors. However, the relationship between brain OT dynamics and complex animal behaviors remains largely elusive, partly because of the lack of a suitable technique for its real-time recording in vivo. Here, we developed an ultrasensitive fluorescent OT sensor named MTRIA_OT, which is composed of a medaka OTR and a circularly permutated green fluorescent protein (cpGFP)-based fluorescent module named MTRIA (Modular fluorescence unit fused with TRansmembrane region-to-IntrAcellular loop linkers). MTRIA_OT displayed excellent properties for measuring extracellular OT; it had a large dynamic range (~720% ΔF/F₀), an optimal affinity to OT (EC₅₀ of ~20 nM), ligand specificity to OT orthologs, minimal effects on cellular signaling, and long-term fluorescence stability. We demonstrated that MTRIA_OT can be used to analyze changes in brain OT levels following exogenous OT application in anesthetized mice. Moreover, we conducted the real-time measurement of brain OT dynamics in freely behaving adult mice. To our surprise, our measurement revealed the existence of “OT oscillation,” in which brain OT levels oscillate at approximately 2-hour intervals. We also demonstrated that the patterns of OT oscillation were altered by anesthesia, food deprivation, and aging. Finally, we also demonstrated the utility of MTRIA, the fluorescent module of MTRIA_OT, for the efficient development of a variety of GPCR-based fluorescent sensors. Together, our findings indicate that MTRIA_OT offers opportunities for the real-time detection of OT dynamics in the living brain, and potentially expands the repertoire of GPCR-based fluorescent sensors for extracellular ligands.

2022年7月2日　11:00～12:00　沖縄コンベンションセンター展示棟　ポスター会場1 3P-324 海馬周辺領域における電圧感受性色素イメージング（VSDI）を用いたin vitro痙攣リスク評価法の開発 In vitro assay for seizure liability using voltage-sensitive dye imaging (VSDI) in broad areas around hippocampal system 内海雄一(1,2)、竹歳麻紀子(2)、三輪倫子(3)、冨永洋子(2)、冨永貴志(1,2,3) 1. 徳島文理大学大学院薬学研究科、2. 徳島文理大学神経科学研究所、3. 徳島文理大学香川薬学部 Yuichi Utsumi(1,2), Makiko Taketoshi(2), Michiko Miwa(3), Yoko Tominaga(2), Takashi Tominaga(1,2,3) 1. Graduate School of Pharmaceutical Sciences, Tokushima Bunri University, Kagawa, Japan, 2. Institute of Neuroscience, Tokushima Bunri University, Kagawa, Japan, 3. Kagawa School of Pharmaceutical Sciences, Tokushima Bunri University, Kagawa, Japan Keyword: in vitro, Seizure, Voltage-sensitive-dye The central nervous system (CNS) toxicity is a critical factor to consider when considering possible adverse effects during non-clinical and clinical trials in drug development. Seizure and convulsions frequently appear in CNS toxicity and are the leading cause of drug development attrition. Seizure liability assessment relies on conventional behavioral batteries to detect seizure and convulsions susceptibility. We aim to establish a novel in vitro assay system using voltage-sensitive dye (VSD) imaging. VSD allows us to record the membrane to monitor potential changes in the neuron's entire membrane components under a field of view. We have already reported some results using electrically evoked responses in hippocampal slices (350 μm thick) from the two age groups (four to seven and eight to ten weeks). Electrical stimulation was applied to the Schaffer collateral pathway from a stimulation electrode near the CA1-CA3 border. The responses were recorded every 30 seconds using an imaging system (MiCAM02, Brainvision Ltd.), along with simultaneous field potential recordings from stratum radiatum. Four convulsive compounds of picrotoxin (PiTX; 1, 10, and 100 μM), SR95331 (Gz; 0.1, 1, and 10 μM), 4-aminopyridine (4AP; 1, 40, and 100 μM), and pilocarpine (Pilo; 10, 30, and 100 μM) were tested. We applied the drugs with cumulative ascending concentrations for 20 minuites per concentration. We also designed the tests to include the stimulus-response relationships during perfusion. PiTX and Gz (GABA receptor antagonist) enhanced the duration of the response in the stratum radiatum (SR). An application of 4AP induced significant slow components in the SR. Pilo suppressed EPSP and thus action potential. PiTX and Gz enhanced the PR ratio (ratio of the response in SP and SR), and 4AP and Pilo did not enhance it. To seek a better assay for seizure liability, we tested the same drugs on the perirhinal cortex and the entorhinal cortex. We found that 4AP induced seizure-like responses at 10 to 20 Hz in the perirhinal cortex (PC), while these were less noticeable in the entorhinal cortex (EC). The result shows the importance of a broader swath of examination on CNS toxicity (seizure liability).		2022年7月2日　11:00～12:00　沖縄コンベンションセンター展示棟　ポスター会場1 3P-325 ナノボディをチラミドシグナル増感法により検出する三次元免疫組織化学法の開発 Nanobody-based Three-dimensional Immunohistochemical Detection with a Tyramide Signal Amplification Method, FT-GO 山内健太(1,2)、星野希沙良(1,2)、石田葉子(1,2)、小池正人(2)、日置寛之(1,2) 1. 順天堂大学大学院医学研究科脳回路形態学講座、2. 順天堂大学医学部　神経生物学・形態学講座　 Kenta Yamauchi(1,2), Kisara Hoshino(1,2), Yoko Ishida(1,2), Masato Koike(2), Hiroyuki Hioki(1,2) 1. Dept Neuroanat, Juntendo Univ Grad Sch Med, Japan, 2. Dept Cell Biol Neurosci, Juntendo Univ Grad Sch Med, Japan Keyword: Three dimensional immunohistochemistry, Nanobody, Tyramide signal amplification Visualization of cellular morphology and molecular composition within large tissues via three dimensional immunohistochemistry (3D-IHC) would advance understanding of biological systems. However, 3D-IHC has been hampered by limited penetration of conventional immunoglobulin G (IgG) and immunoglobulin Y (IgY) antibodies into large tissues. Nanobodies, variable heavy chain domains of camelid heavy chain IgG antibodies, should be suitable for 3D-IHC because of their much smaller in size than conventional IgG and IgY antibodies. Here, we developed a nanobody-based 3D-IHC that enables high-speed and sensitive detection of target molecules within large tissues. We combined horseradish peroxidase-containing nanobodies, P-RANbodies (Yamagata and Sanes, PNAS 115: 2126-31), with an originally developed fluorescent tyramide signal amplification (TSA) technique, Fluorochromized Tyramide-Glucose Oxidase (FT-GO). Our nanobody-based 3D-IHC technique is based on AbScale method (Hama et al., Nat Neurosci 18:1518-29) and incorporates three main components: 1) treatment with a permeabilization reagent, 2) incubation with P-RANbodies and 3) detection of the P-RANbodies with FT-GO reaction. Using the 3D-IHC method, we succeeded in visualizing morphologies and subcellular structures of neuronal cells in 1-mm-thick mouse brain slices within three days. Given its simplicity, swiftness and high sensitivity, our nanobody-based 3D-IHC would provide a versatile tool for histochemical analysis in three dimensions.

2022年7月2日　11:00～12:00　沖縄コンベンションセンター展示棟　ポスター会場1 3P-326 ベイズ推定による神経細胞イメージングデータのパラメータ推定 Parameter estimation of neuroimaging data through Bayesian estimation 斎藤陽平(1)、竹田晃人(1) 1. 茨城大学大学院理工学研究科 Yohei Saito(1), Koujin Takeda(1) 1. Grad Sch Sci and Eng, Ibaraki Univ, Ibaraki, Japan Keyword: Bayesian inference, Calcium imaging The function of neurons can be inferred by their responses to stimuli. In calcium imaging, we can track changes in calcium ion concentration associated with spike generation using fluorescent proteins, which enables us to measure a wide range of neural activity including response to stimuli. However, the spike train cannot be measured directly. Therefore, we must estimate the spike train of each neuron from time series of fluorescence intensity at each pixel in calcium imaging. In previous studies [1, 2], the dynamics of calcium ion concentration are described by the autoregressive (AR) model, and fluorescence intensity is approximated by the linear function of the calcium concentration with Gaussian noise. In Ref. [1], maximum a posteriori estimation and the particle filter are employed to estimate the spike train as an integer sequence. However, since the average of multiple fluorescence intensities is used rather than pixel-by-pixel intensity, we cannot associate each pixel with a neuron. In Ref. [2], pixel-by-pixel fluorescence intensity is used, and the spike train is estimated by nonnegative matrix factorization. However, spikes are approximated by real numbers from the viewpoint of calculation cost. In our work, we attempt to improve Ref. [1] by using fluorescence intensity at each pixel. Before inferring the spike train, the model parameter must be estimated. Since many neurons in the imaging data show diverse dynamics, there is no guarantee that point estimation for model parameters will work effectively. Thus, we employ Bayesian inference for the posterior distribution of model parameters. However, it is difficult to evaluate the posterior distribution due to the computational cost of the normalization constant. Therefore, we perform Stein’s method [3] to obtain samples from the posterior distribution. In this method, we must evaluate the gradient of the logarithm of the posterior distribution with high speed. We employ a technique called Paris [4] for evaluation, which uses a particle filter and smoothing. Reference [1] J. T. Vogelstein, et al., Biophys. J 97, 636, (2009). [2] E. A. Pnevmatikakis, et al., Neuron 89, 285, (2016). [3] Q. Liu, and D. Wang, Adv. Neural Inf. Process Syst. 29. [4] J. Olsson, and J. Westerborn, Bernoulli 23, 1251, (2017).		2022年7月2日　11:00～12:00　沖縄コンベンションセンター展示棟　ポスター会場1 3P-327 CMOS Implantable Imaging Device and Mircrodialysis System for Observing Serotonergic Neurons During Nociception in Freely Moving Mice Latiful Akbar(1), Yasumi Ohta(1), Mamiko Kawahara(1), Makito Haruta(1), Hironari Takehara(1), Hiroyuki Tashiro(1), Kiyotaka Sasagawa(1), Jun Ohta(1) 1. Grad Sch of Sci and Tech, Nara Inst of Sci and Tech, Nara, Japan Keyword:* calcium imaging, image sensor, microdialysis, serotonin Pain is one of the major health problems globally which involves multidimensional aspects in a higher brain region. Amygdala as a center of emotion is responsible for the affective component of pain, alongside with other areas, such as anterior cingulate cortex (ACC). Given that the complexity of pain processing in the brain, an approach which includes various observation is indispensable in elucidating the phenomena. In this research, we combined imaging and microdialysis system to concurrently visualize neural activity and measure serotonin release. Serotonin is a neurotransmitter known to have an important role in pain transmission. We implanted an imaging device in the dorsal raphe nuclei (DRN), the main source of serotonin, and microdialysis probes were implanted both in ACC and the capsular-lateral subsection of central amygdala (CeLC). The microdialysis samples were measured by high-performance liquid chromatographic column-electrochemical detector (HPLC-ECD). Our implantable imaging device consists of CMOS image sensor and micro-LED (λ=470 nm) embedded on the flexible printed circuit (FPC) substrate. The absorption filter also applied to attenuate blue excitation light. The device was coated by Parylene-C for waterproofing and biocompatibility. Compared to others, our CMOS imaging device is thinner and lighter (0.02 gram), enables us to conduct triple implantation in GCaMP6 mice under freely moving condition. To induce nociception in the mouse hindpaw, we injected 2% paraformaldehyde (PFA). The results depicted that the imaging device was able to detect neural activity in DRN. The fluorescence intensity was observably apparent after PFA stimulation. It indicates that our device could visualize the activity and sensitive enough to image the changing due to the nociception. Our results showed that PFA injection can elevate serotonin release in ACC and CeLC, as reflected by the increase of serotonin concentration in those areas. The data also suggested that contralateral shows higher serotonin release compared to ipsilateral, particularly this difference was clearly observed in CeLC. In this study, we demonstrated the utility of our device in conducting multiple implantations with less invasiveness and did not hinder the mice movement. It allows us to collect the data of different activities from distinct locations simultaneously, providing us a better understanding in monitoring the causal relation of behavior and brain activity.

2022年7月2日　11:00～12:00　沖縄コンベンションセンター展示棟　ポスター会場1 3P-328 An optimized protocol for physiological recordings in dorsal forebrain organoids Zeynep Yentur(1,2,3), Jasmin Treu(4), Shokoufeh Khakipoor(1), Kseniia Sarieva(1,2), Theresa Kagermeier(1,4), Thomas Euler(5,6,7), Simone Mayer(1,3) 1. Hertie Institute for Clinical Brain Research, University of Tübingen, Tübingen, Germany, 2. International Max Planck Research School, Graduate Training Centre of Neuroscience, University of Tübingen, Tübingen, Germany, 3. Heidelberg Academy of Sciences and Humanities, Heidelberg, Germany, 4. Graduate Training Centre of Neuroscience, University of Tübingen, Tübingen, Germany, 5. Institute for Ophthalmic Research, University of Tübingen, Tübingen, Germany, 6. Centre for Integrative Neuroscience, University of Tübingen, Tübingen, Germany, 7. Bernstein Centre for Computational Neuroscience, Tübingen, Germany Keyword:* Calcium imaging, Multielectrode arrays, Neural activity, Dorsal forebrain organoids Neocortical organoids generated from human pluripotent stem cells mimic the complexity of human brain development in vitro. Cortical organoids recapitulate early stages of human neocortical development including stem cell proliferation, differentiation, and neuronal migration in a timely manner. However, later events such as synaptogenesis, neural network formation, and maturation are yet to be studied. Until recently, many studies using cortical organoids focused on the cellular and molecular complexity of these organoids, leaving functional recordings underinvestigated. Thus, investigation of physiological properties of brain organoids is essential to support that this model system also recapitulates the in vivo development of the neural circuits. Different approaches can be used in parallel for physiological characterization of brain organoids. Calcium imaging can be complemented with multielectrode arrays (MEA) recordings to investigate neural activity. The aim of this study is to establish a robust protocol for calcium imaging and electrophysiological recordings in dorsal forebrain organoids to study its neural activity. For this purpose, we generated, sectioned and subsequently cultured dorsal forebrain organoids as organotypic air-liquid-interface cultures. We have adopted calcium imaging with epifluorescent and two-photon microscopes using membrane permeable calcium indicators as well as genetically encoded calcium indicators transduced virally. For electrophysiological recordings, the organoid sections were placed on different types of MEAs (e.g. planar MEA and 3D MEA). Both optical and electrophysiological approaches allow the detection of spontaneous activity in dorsal forebrain organoids. The experimental pipeline that was used for calcium imaging can be further optimized to other genetically encoded indicators or dyes that allow for optical imaging of brain organoids. In conclusion, our protocol can be used to study the physiological activity in neocortical organoids on single cell as well as on network level to gain functional insight. Understanding the physiological properties of human pluripotent cell derived brain organoids could permit the advancement of this new model system in research.		2022年7月2日　11:00～12:00　沖縄コンベンションセンター展示棟　ポスター会場1 3P-329 シナプス解像度を持つ高速スキャンレス３次元２光子顕微鏡の開発 Development of fast scanless 3D two-photon imaging system at synaptic resolution 根東覚(1)、瀧口優(2) 1. 東京大学、2. 浜松ホトニクス株式会社 Satoru Kondo(1), Yu Takiguchi(2) 1. The University of Tokyo, 2. Hamamatsu Photonics K.K Keyword: two-photon imaging, temporal-focusing, scanless imaging, synapse Recent advances in the optical measurement methods of neural activity made it possible to record brain activity with high temporal and spatial resolution in living animals. In particular, two-photon imaging technique even allows micro-measurements at the synaptic level. A single neuron receives a large number of synaptic inputs, but the mechanisms by which it selectively integrates and reads-out information from inputs remain largely unexplored. Detailed functional dendritic map of synaptic inputs, i.e., which spine received what input, when and where, is essential to reveal the computational mechanisms in single neuron. However, the number of synaptic inputs that single neuron receives is so large (thousands) that it is pretty hard to record all synaptic responses using current two-photon imaging systems, making the development of new imaging system essential. The current two-photon microscopy usually uses point-scanning systems to obtain a single frame image and the scanning process is the rate-limiting factor for the image acquisition rate. Thus, it is ideal to obtain a single frame image without scanning. However, the density of excitation photon is reduced in the scanless two-photon imaging compared to conventional point-scanning imaging. As the power of laser increased, strong fluorescence signal that mostly occur at the boundary of specimen or glass cover become prominent and behave as serious background noise and drastically reduces the s/n ratio of the image. To overcome this problem, we developed a new imaging system based on the temporal focusing wide-field microscopy that efficiently reduces such noise. A diffraction grating separates the ultra-short pulse into its constituent wavelengths. A focusing lens after the grating will then converts the spectrum into a wavelength-dependent positional offset by its Fourier transform at the back-focal plane of the objective lens. The objective lens will collimate and recombines the various spectral components only within the focus volume. Therefore, ultrashort pulse will be reconstructed only at the focus of objective lens and two-photon excitation at out of focus can be suppressed. Our new imaging system with temporal-focusing technique will be useful to understand not only the principles of single neuronal computation but also the dynamic properties of synapses, such as the reorganization of synaptic inputs during development, pathological changes of synaptic inputs in psychiatric disorders, and reorganization of synaptic inputs at learning.

2022年7月2日　11:00～12:00　沖縄コンベンションセンター展示棟　ポスター会場1 3P-330 Neuroimaging and modulation of the septo-hippocampal circuit using novel brain-implantable CMOS image sensors and micro-LEDs during seizure and epilepsy Olorocisimo Joshua(1)、Ohta Yasumi(1)、Regonia Paul(2,3)、Takehara Hironari (1)、Haruta Makito(1)、Tashiro Hiroyuki (1,4)、Sasagawa Kiyotaka(1)、Yoshimoto Junichiro(2)、Ikeda Kazushi(2)、Ohta Jun(1) Joshua Philippe Olorocisimo(1), Yasumi Ohta(1), Paul Rossener Regonia(2,3), Hironari Takehara(1), Makito Haruta(1), Hiroyuki Tashiro(1,4), Kiyotaka Sasagawa(1), Junichiro Yoshimoto(2), Kazushi Ikeda(2), Jun Ohta(1) 1. Photonic Device Science Lab, Nara Institute of Science and Technology, Japan, 2. Mathematical Informatics Lab, Nara Institute of Science and Technology, Japan, 3. Department of Computer Science, College of Engineering, UP Diliman, Philippines, 4. Department of Health Sciences, Faculty of Medical Sciences, Kyushu University, Japan Keyword: Implantable devices, Calcium imaging, Optogenetics, Epilepsy Several neuroscience principles and neuropsychiatric disorders are not yet fully understood. Thus, international brain research programs encourage the development of new methods and techniques for basic neuroscience research. Findings using these new technologies can hopefully help us better understand brain disorders. For instance, over 50 million people worldwide suffer from epilepsy, which is one of the most prevalent neurological diseases. The most common type of focal epilepsy is temporal lobe epilepsy (TLE); however, the mechanism and treatment of this condition remain unresolved. Therefore, new technologies to study epilepsy must be developed. Here, we present a novel biophotonic system for studying and monitoring epilepsy in freely-behaving mice. We developed a brain implantable CMOS image sensor (CIS) for neuronal calcium imaging. The device is 450 µm x 1500 µm in size and 0.05 g in weight. The image sensor is coated with a biocompatible polymer and implanted directly into the brain tissue. Due to its vertical orientation and wide field-of-view, multiple layers of the hippocampus can be imaged simultaneously. Then, we designed implantable photostimulation devices consisting of micro-LEDs to optogenetically modulate cholinergic neurons in the medial septum (MS). Therefore, by using our system, we can image hippocampal neurons expressing GCaMP6s and activate MS neurons expressing ChRmine in mice injected with kainic acid. We found differential calcium activity across the multiple layers of the hippocampus during seizures. Furthermore, different epileptiform wavetypes were characterized which coincided with previously published results. These were then correlated with behavioral phenotypes based on the Racine seizure scale. Preliminary data suggest higher activity in the dentate gyrus compared to CA1 during epilepsy. In addition, using our devices, we are determining the parameters and mechanism of MS cholinergic neuronal activation to prevent seizure. We plan to investigate which hippocampal neurons are modulated by MS cholinergic activation and whether this results in a net feedforward inhibition of epileptic neurons. In conclusion, using our small and lightweight brain-implantable devices, we can uncover circuit mechanisms of focal epilepsy that may hopefully guide treatment options in the future.		2022年7月2日　11:00～12:00　沖縄コンベンションセンター展示棟　ポスター会場1 3P-331 非接続挿入型コイルを用いたヒト用7T-MRIシステムによるMRマイクロスコピー Non-wired insert coils for high-resolution MR microscopy using a human 7T-MRI system. 岡田知久(1)、浦山慎一(1)、赤坂太(1)、尾上浩隆(1)、伊佐正(1) 1. 京都大学大学院医学研究科 Tomo Okada(1), Shin-ichi Urayama(1), Thai Akasaka(1), Hirotaka Onoe(1), Tadashi Isa(1) 1. Grad Sch Med, Kyoto University, Kyoto, Japan Keyword: MR microscopy, Non-wired coil The number of the human 7T-MRI system is increasing world-wide including Japan. The system enables functional and neurochemical imaging as well as high-resolution anatomical imaging. It can also be used for animal models and specimen to investigate pathophysiological conditions. However, additional coils that fit to the sizes of the animals and specimen is required to realize high-resolution imaging at mesoscopic level, i.e., around 100 micrometers. Fabrication of such coils and plugging them into the human 7T-MRI system is not always feasible, and the new coil may damage the MRI system. On the other hand, the latest MRI systems comes with multi-element coils that have high signal-to-noise ratios (SNR). These elements can be coupled with other coils without wiring. Using a knee coil with 28 elements as the mother coil, we fabricated non-wired insertion coils with diameters of 26 mm and 64 mm. They had relative SNR around 12 and 6, respectively, compared to that of the scan using only the knee coil. Combined use of insert coils, the mouse and macaque brains could be measured in isotropic 50 and 160 micrometers, respectively, in ex vivo with high SNR without averaging. The scans took 2 – 6 hours depending on the specimen size and resolution. Human brain specimen can also be scanned using these coils. In addition to ex vivo imaging, in vivo imaging of small animals will be feasible when a non-wired insert-type coils that can be aligned parallel to the static magnetic field is fabricated. MR microimaging will be much facilitated using non-wired insert coils.

2022年7月2日　11:00～12:00　沖縄コンベンションセンター展示棟　ポスター会場1 3P-332 (Simultaneous Two-Photon Voltage or Calcium Imaging and Multi-Channel Local Field Potential Recordings in Barrel Cortex of Awake and Anesthetized Mice) Claudia Cecchetto(1,2), Bernd Kuhn(3), Stefano Vassanelli(1,2) 1. Department of Biomedical Sciences, University of Padova (UNIPD), Padua, Italy, 2. Padova Neuroscience Center (PNC), University of Padova (UNIPD), Padua, Italy, 3. Okinawa Institute of Science and Technology Graduate University (OIST), Okinawa, Japan Keyword:* voltage imaging with ANNINE-6, local field potentials, neuroimaging, combined approach Neuronal population activity, both spontaneous and sensory-evoked, is known to generate propagating waves in cortex. However, high spatiotemporal-resolution mapping of these waves is difficult as calcium imaging, the work horse of current imaging techniques, does not reveal subthreshold activity. Here, we present a platform that combines voltage or calcium two-photon imaging with multi-channel local field potential (LFP) recordings in different layers of the barrel cortex from anesthetized and awake head-restrained mice. A chronic cranial window with access port allows injecting a viral vector expressing GCaMP6f or the voltage-sensitive dye (VSD) ANNINE-6plus, as well as entering the brain with a multi-channel neural probe. We present both average spontaneous activity and average evoked signals in response to multi-whisker air-puff stimulations. Time domain analysis shows the dependence of the evoked responses on the cortical layer and on the state of the animal, here separated into anesthetized, awake but resting, and running. The simultaneous data acquisition allows to compare the average membrane depolarization measured with ANNINE-6plus with the amplitude and shape of the LFP recordings. The calcium imaging data connects these datasets to the large existing database of this important second messenger. Interestingly, in the calcium imaging data, we found a few cells which showed a decrease in calcium concentration in response to vibrissa stimulation in awake mice. This system offers a multimodal technique to study the spatiotemporal dynamics of neuronal signals through a 3D architecture in vivo. It will provide novel insights on sensory coding, closing the gap between electrical and optical recordings. This work was carried out between OIST Graduate University (Okinawa, Japan) and University of Padova (Padua, Italy) and was supported by the European Commission, GRACE project, H2020-MSCA-IF-2017, GA number: 796177, and 2018 Canon Foundation Research Fellowship.		2022年7月2日　11:00～12:00　沖縄コンベンションセンター展示棟　ポスター会場1 3P-333 単語理解時のMEG電流源推定および電流信号伝達のグループ解析における再現性 Reproducibility of group analysis of MEG source current reconstruction and source current transmission during word comprehension 山下宙人(1)、武田祐輔(1,2)、廣江総雄(1)、井原綾(3) 1. 国際電気通信基礎技術研究所、2. 理化学研究所革新知能統合研究センター、3. 脳情報通信融合研究センター Okito Yamashita(1), Yusuke Takeda(1,2), Nobuo Hiroe(1), Aya Ihara(3) 1. ATR, 2. RIKEN AIP, 3. CINET Keyword: MEG, current source reconstruction, reproducibility, word comprehension In recent years, the EEG and/or MEG current source estimation method has been utilized as a unique method for non-invasively investigating brain activity with sub-second resolution. It elucidates where and how brain information processing is performed by associating the estimated spatiotemporal pattern of the current source to task conditions. Previously we have proposed the fMRI-informed MEG current source estimation algorithm (Sato et al, 2004, NeuroImage) and current signal transmission analysis based on the connectome dynamics identification (Fukushima et al, 2015, NeuroImage). However, the validation of these approach in experimental data, in particular, for the cognitive task is still lacking. In this study we have investigated reproducibility of current source reconstruction and current source transmission through the structure connections during word comprehension in the group level. We have conducted MEG and EEG simultaneous recording during the word priming task (Ihara et al, 2007, NeuroImage) from twenty subjects in two different days. To investigate effect of the prime word on the second target word, we have compared MEG data of related words condition with that of unrelated word conditions in the group level. We used the max-T statistics for controlling the family-wise error rate (correction for multiple comparison). The sensor space analysis showed the significant difference around frontal and parietal sensors in the left hemisphere at the latency 400msec after the second word presentation. The current source analysis using fMRI information showed the significant difference in the left superior temporal cortex at the same latency. These results are highly consistent across two days. Finally the current signal transmission analysis identified the reproducible current signal transmission between the left superior temporal and transverse temporal cortex around 400~500 msec. Our results suggested feasibility of our proposed approach to the cognitive task.

2022年7月2日　11:00～12:00　沖縄コンベンションセンター展示棟　ポスター会場1 3P-334 FRETを利用したインジケーターを用いたクラスター型プロトカドヘリンホモフィリックtrans相互作用の神経細胞での検出 Detection of clustered protocadherin homophilic trans interactions in neurons using a FRET-based indicator 星野七海(1)、京卓志(2)、足澤悦子(1)、金子涼輔(1)、井上（上野）由紀子(3)、井上高良(3)、松田知己(2)、永井健治(2)、八木健(1) 1. 大阪大学大学院生命機能研究科、2. 大阪大学産業科学研究所、3. 国立精神・神経医療研究センター疾病研究第６部 Natsumi Hoshino(1), Takashi Kanadome(2), Etsuko Tarusawa(1), Ryosuke Kaneko(1), Yukiko U. Inoue(3), Takayoshi Inoue(3), Tomoki Matsuda(2), Takeharu Nagai(2), Takeshi Yagi(1) 1. Grad School of Frontier Biosciences, Osaka Univ, Suita, Japan, 2. SANKEN, Osaka Univ, Ibaraki, Japan, 3. Dep of Biochem and Cellular Biol, National Institute of Neurosci, Tokyo, Japan Keyword: FRET-based indicator, protein-protein interaction, cell-cell recognition, self-avoidance Cell surface recognition molecules are required to form the specific neural connections. Clustered protocadherin (Pcdh) family is one of the key recognition molecules, which consists of 58 isoforms and can work through the strict homophilic interaction in trans. To date, there has been no direct evidence of the homophilic interaction of Pcdh proteins due to a technical difficulty. To visualize it, we developed a Förster resonance energy transfer (FRET) -based indicator, in which CFP and YFP were fused in a PcdhγB2 isoform which is one of 22 Pcdhγ isoforms. We confirmed the specificity of the PcdhγB2-FRET signal as a homophilic interaction in trans using a cultured cell line (Kanadome T, Hoshino N et al. Sci Rep 2021). Here we first observed the PcdhγB2-FRET signal in neurons of primary hippocampal culture. We next generated a transgenic mouse line, which overexpresses the PcdhγB2-FRET indicator in a Cre-dependent manner. To confirm the function of the PcdhγB2-FRET in the transgenic mice, we analyzed dendritic arborization of Purkinje cells, which is patterned by a process of self-avoidance. Previous studies showed that the deletion of Pcdhγ increases dendritic self-crossings of Purkinje cells. We prepared parvalbumin-Cre mice, Pcdhγ conditional knockout mice and the PcdhγB2-FRET indicator conditional overexpression mice to generate mice whose Cre positive cells express only the PcdhγB2-FRET indicator as a Pcdhγ isoform. In a Purkinje cell of the genotype, the Pcdhγ knockout phenotype was rescued, suggesting that the single isoform of PcdhγB2 is sufficient for the self-avoidance process, and that the PcdhγB2-FRET indicator retains the native function. PcdhγB2-FRET signals were further detected in Purkinje cells of the transgenic mice, highlighting that we succeeded, for the first time, in directly confirming the Pcdh homophilic interaction in neurons in vivo.		2022年7月2日　11:00～12:00　沖縄コンベンションセンター展示棟　ポスター会場1 3P-335 電位感受性色素（VSD)イメージングによる海馬スライスへのビスフェノールA関連物質の急性適用の影響の検出 Detection of the effects of the acute application of bisphenol-A-related substances on hippocampal slices with voltage-sensitive dye imaging 冨永洋子(1)、竹歳麻紀子(1)、種村健太郎(3)、冨永貴志(1,2) 1. 徳島文理大学神経科学研究所、2. 徳島文理大学香川薬学部、3. 東北大学大学院農学研究科 Yoko Tominaga(1), Makiko Taketoshi(1), Kentaro Tanemura(3), Takashi Tominaga(1,2) 1. Inst Neurosci, Tokushima Bunri Univ, 2. Kagawa Sch Pharm Sci, Tokushima Bunri Univ, 3. Grad Sch of Agr Sci, Tohoku Univ Keyword: voltage sensitive dye, hippocampal slice, bisphenol, tri-synaptic circuit Subtle internal and external causes can skew the central nervous circuit function. For example, vulnerability to exposure during early developmental stages raises the risk for various neuropsychiatric disorders, including Asperger spectrum syndrome. The neural circuit-level assay is essential to fill a gap between the cellular assay using primary and cell lines and in vivo assay.[WE1] However, effects on neural circuit activity are challenging to detect using conventional electrophysiological methods. We developed a voltage-sensitive dye (VSD; Di-4-ANEPPS) imaging system to detect subtle neural activity modification in the neural circuits quantitatively on a large scale. Here, we used the hippocampal slice preparation as a representative neural circuit and tested acute effects of the environmental chemical bisphenol-A (BPA) and its related substances BBMTBP and MBMTBP (5 ppm in perfusate). We tested the hippocampal tri-synaptic responses by applying electrical stimulation to Schaffer collateral to CA1, mossy fiber to CA3, and perforant path to DG. The three different regions' responses in the hippocampus were accumulated across five to eight different slices. We assessed the effects on excitatory and inhibitory networks using a GABA-A receptor blocker (SR95331) to the system (+Gz). First, we tested with adult mice (eight weeks old). We evaluated the VSD signals with the amplitude of early response (ER, peak values within 40 ms from the stimulation), which corresponds to excitatory post-synaptic potential (EPSP) and action potentials (AP). We used selective ROI to evaluate the response corresponding to CA1, CA3, and DG regions. BBM and MBM reduced CA1-ER in the normal solution (-Gz) and CA3-EPSP components in +Gz. We also evaluated the late phase (40–500 ms) of the response as a time-integral response (LR). In LR, CA1 and CA3 were reduced by BBM in -Gz and -Gz. We also used young (two-week-old) mice to assess the vulnerability of circuit activity. To our surprise, the young group was more-or-less resistant to those chemicals, while still BBM and MBM showed significant suppression of CA3-EPSP components +Gz. In LR, CA1 responses were affected by BBM and MBM. The overall result indicates higher risks with BBMTBP and MBMTBP than with BPA. The result also highlights the usefulness of the imaging assay to evaluate the changes in circuit modifications.

2022年7月2日　11:00～12:00　沖縄コンベンションセンター展示棟　ポスター会場1 3P-336 Three Protocols Associated with Long-term Two-photon Microscopy in Head-fixed Mice: Chronic Cranial Window Surgery, Intrinsic Optical Signal Imaging and Targeted Injections through the Modified Cranial Window Sigita Augustinaite(1), Bernd Kuhn(1) 1. OIST, Okinawa, Japan Keyword:* two-photon, mouse, imaging, cortex In vivo two-photon microscopy is a powerful technique which can be combined with other physiological, pharmacological, and/or behavioral methods. However, it requires scrupulous preparation and planning. Here, we present three protocols associated with in vivo two-photon microcopy: (i) chronic cranial window surgery, (ii) intrinsic optical signal imaging and (iii) targeted injections through the access port of the chronic cranial window. These procedures can be used separately or in combination, enabling flexibility of the experiment design and timeline. In our surgery protocol we present several subtle improvements that increase the success rate significantly. We provide instructions for craniotomy, synthetic dye or viral-vector injection and mounting of a chronic cranial window with the headplate. The window allows high-quality imaging in head-fixed behaving mice within the first week after the surgical procedure and remains clear for months. We used this procedure to prepare mice for intrinsic signal imaging and two-photon imaging of layer 6 neurons in primary visual cortex. The intrinsic optical signal through the cranial window can be imaged at different times, allowing to map the functional cortical areas soon- or long after the window surgery. Here we provide instructions for mapping of primary visual cortex and other sensory cortical areas, such as somatosensory or posterior-parietal cortex. If the chronic cranial window has an access port, the generated maps can be used for two-photon imaging combined with targeted electrophysiological, pharmacological, or labeling procedures. Like intrinsic imaging, the manipulations can be made soon (few days) or late (few weeks or months) after the surgery. The procedure might be especially useful for behavioral two-photon imaging experiments, when expression of the fluorescent agent must be delayed because of long-lasting training of the animals. Intrinsic signal imaging and injections through the access port of the cranial window can be performed on the same imaging setup by adding sensory stimulation equipment, a stable, red-light source, a suitable camera, a body temperature control system, and a micromanipulator system with injection apparatus. It can be also combined with a two-photon setup.		2022年7月2日　11:00～12:00　沖縄コンベンションセンター展示棟　ポスター会場1 3P-337 Simultaneous imaging of hippocampal subfields in freely moving GCaMP transgenic mice using a novel implantable micro-imager Mark Christian Guinto(1), Yasumi Ohta(1), Mamiko Kawahara(1), Hironari Takehara(1), Makito Haruta(1), Hiroyuki Tashiro(1,2), Kiyotaka Sasagawa(1), Jun Ohta(1) 1. Div Materials Science, NAIST, Nara, Japan, 2. Div Med Tech, Kyushu Univ, Kyushu, Japan Keyword:* implantable device, calcium imaging, hippocampus, microendoscopy Investigating the cooperative interactions between the subfields of the hippocampus requires not only the ability to track the spatiotemporal dynamics of ensemble activity but also the capacity to conduct simultaneous monitoring across different layers, especially during their concerted recruitment under freely behaving conditions. In this regard, implantable fluorescence imaging platforms, whose ultra-small form factor enables translaminar imaging, are particularly suited for providing both the necessary precision and scope to analyze the flow of information in the neural network. Furthermore, tracking activity across multiple layers of the hippocampus along the dorsoventral axis provides the opportunity to assess place field sizes of putative place cells as a function of depth in the hippocampus. To this end, our laboratory has been developing a CMOS-based micro-imaging device that can be chronically implanted into the brains of transgenic mice for monitoring GCaMP-related fluorescence changes in specific neuronal populations. We have implanted the micro-imaging device with an optimal size into hippocampal subfields (CA1-DG, CA3-DG, CA2-CA3-DG). By simultaneously imaging using our device together with a microendoscope (Miniscope) on the same mouse, we could benchmark the performance of our device alongside a relatively standard system for calcium imaging. Since our device is conformable, compact and weighs only 20 mg, we can install both the microendoscope, including the microprism-coupled GRIN lens, and our device in a small region with unprecedented ease. Intra- and inter-hemispheric implantation of lenses and micro-imagers allows comparisons of activity and imaging quality within the local circuitry and in bilateral structures, respectively. Using the novel object recognition (NOR) test as a demonstration, we also visualize hippocampal subfield activity of unrestrained mice across various phases (habituation, training, and testing) of the NOR test, which is a standard behavioral assay for memory. Our work lays the foundation for ultralight, lensless imaging systems, opening avenues to optically investigate the relationship of laminar activity patterns with animal behavior and contribute to a more complete understanding of the neuronal substrate of memory.

2022年7月2日　11:00～12:00　沖縄コンベンションセンター展示棟　ポスター会場1 3P-338 後眼窩注入法を用いた生体内二光子顕微鏡記録のための脳細胞の散在ラベリング Retro-orbital virus injection allows sparse labelling of neurons for in vivo 2-photon microscopy 森一夫(1)、来間清人(1)、Jeffery Wickens(1)、Bernd Kuhn(1) 1. 沖縄科学技術大学院大学 Kazuo Mori(1), Kiyoto Kurima(1), Jeffery Wickens(1), Bernd Kuhn(1) 1. Okinawa Institute of Science and Technology, Okinawa, Japan Keyword: retro-orbital injection, 2 photon imaging, AAV Adeno-associated viral vectors (AAVs) are widely used to label populations of neurons for imaging experiments by delivering genes encoding protein fluorophores or sensors. We use AAV to express Ca²⁺ indicators in neurons for imaging their activities in vivo. However, local AAV application by injection directly into the brain risks causing local damage. Additionally, the labeled neurons are highly inhomogeneously distributed, being densely labelled at the injection site and fading with distance. Here, for homogeneous but sparse labeling of neurons, we injected AAV into the bloodstream via the retro-orbital sinus instead of injecting directly into the brain. We used an AAV capsid pseudotyped with serotype 9 variant PHP.eB which makes the AAV highly able to permeate the blood brain barrier. We used the strong, tetracycline-dependent TET-off promotor to drive expression. We injected 0.1 ml of the AAV solution with different titers higher than 10¹⁰ GC/ml behind the right eye of the mouse using a 0.3 ml insulin syringe with a 30G needle. One week after the injection, we mounted a chronic cranial window for 2-photon imaging over the cerebral or cerebellar cortex and observed the labeling two weeks and four weeks after the injection. We sectioned the whole brain post mortem to observe the label distribution. We succeeded in homogeneous and sparse labeling of neurons with AAV concentrations of 10¹¹ GC/ml. This technique will be particularly useful for studying dendritic information processing in single neurons, neuronal population dynamics of somata and dendrites, and synaptic communication in vivo.		2022年7月2日　11:00～12:00　沖縄コンベンションセンター展示棟　ポスター会場1 3P-339 ArcLightを用いたゼブラフィッシュにおけるニューロン集団の膜電位イメージング Population imaging of neurons in the zebrafish brain by genetically encoded voltage indicator, ArcLight 白石飛鳥(1)、福田成美(1)、菱沼まり(1)、宮澤碩彬(1)、日吉加菜映(1)、弥益恭(1)、津田佐知子(1) 1. 埼玉大学大学院理工学研究科 Asuka Shiraishi(1), Narumi Fukuda(1), Mari Hishinuma(1), Hiroaki Miyazawa(1), Kanae Hiyoshi(1), Kyo Yamasu(1), Sachiko Tsuda(1) 1. Grad Sch of Sci and Eng, Saitama Univ, Saitama, Japan Keyword: ArcLight, zebrafish, Genetically encoded voltage indicator Voltage imaging is a powerful approach for functional analysis of neural circuits since it enables fast and direct recording of neural activity from neuronal populations. In recent years, remarkable advances have been made in the development of voltage sensors, especially genetically encoded voltage indicators (GEVIs), with the improved signal-to-noise ratio, brightness, and speed. Here, we applied ArcLight, a voltage sensitive phosphatase based GEVI, to zebrafish, which is known to have brain circuitry similar to that of mammals and whose transparency and small size provide a significant advantage in applying optical techniques. First, we established a transgenic fish line that expresses ArcLight in neurons by the Gal4-UAS system and confirmed the membrane localization of ArcLight in the zebrafish brain. By high-speed in vivo imaging of the spinal cord, which is known to have characteristic spontaneous firing, we detected periodic changes in the fluorescence intensity in the spinal cord neurons at the single-cell resolution. These signals were abolished by tetrodotoxin treatment. During the period of spinal cord formation (21-28 hpf), various patterns of activity were observed, including depolarization, hyperpolarization, and subthreshold signals, which resembled previously reported electrophysiological recordings. We also succeeded in detecting voltage changes in axons. We will discuss the quantitative analysis of the spatiotemporal dynamics of spinal cord neuron populations. Furthermore, we applied ArcLight to the zebrafish cerebellum. We showed that ArcLight could be properly expressed specifically in Purkinje cells, the primary neurons in the cerebellum. By mosaically expressing ArcLight in Purkinje cell, we detected depolarization signals which might be complex spikes. Our voltage imaging approach with ArcLight would enable a spatiotemporal analysis of the network dynamics in the various brain regions at a high temporal and spatial resolution.

2022年7月2日　11:00～12:00　沖縄コンベンションセンター展示棟　ポスター会場1 3P-340 Application of Expansion Microscopy for 3D Reconstruction of Purkinje Neurons after Functional Imaging in Awake Animals. Mykola Medvidov(1), Christopher J Roome(1), Bernd Kuhn(1) 1. Okinawa Institute of Science and Technology Graduate University, Okinawa, Japan Keyword:* Expansion Microscopy, 3D reconstruction Neurons are the basic building blocks of the nervous system. Understanding their function in the living animal is essential for understanding the nervous system and curing neurological diseases. In our previous work we were able to image voltage and calcium signals in cerebellar Purkinje neurons in awake animals with unprecedented spatial and temporal resolution, using the voltage-sensitive dye (ANNINE-6plus) and the Ca²⁺ indicator (GCaMP6f) (1). To interpret and simulate these recordings, structural information on a spine level is indispensable, including the position of excitatory and inhibitory synapses. Previously, we used 3D electron microscopy to reconstruct the dendritic tree of Purkinje neurons. However, this approach is extremely time consuming. To significantly reduce reconstruction time, we applied Expansion Microscopy (ExM). Briefly, after fixing and slicing the brain, we searched for the targeted cell by matching the morphology of the ANNINE-6plus-labeled cell with the morphology of GCaMP6f labelled cells in the fixed sections. This step was necessary because ANNINE-6plus fluorescence disappears during the fixation process. The section with the targeted Purkinje cell was stained with fluorescent labelled antibody against GCaMP6f, because the fluorescence of GCaMP6f completely disappears after expansion due to protein degradation. Next, the tissue was expanded with a 10x factor in all 3 dimensions as described (4). After expansion, the slice was imaged with conventional confocal microscopy. The received imaging dataset was adjusted using ImageJ and Imaris software. To extract the structural information from the image stacks we applied the ilastik toolkit (https://www.ilastik.org/). 3D reconstruction was performed with a marching cube algorithm (https://github.com/topics/marching-cubes) and the received 3D mesh was adjusted with MeshLab software (https://www.meshlab.net). Finally, the reconstructed cell was downscaled according to an expansion factor that was estimated previously. To summarize, application of the Expansion Microscopy to large scale high resolution dendritic reconstruction is a fast and reliable alternative to conventional 3D electron microscopy. 1. Roome CJ & Kuhn B (2018) Simultaneous dendritic voltage and calcium imaging and somatic recording from Purkinje neurons in awake mice. Nat Commun 9(1):3388. 2. Truckenbrodt S at al., (2019) A practical guide to optimization in X10 expansion microscopy. Nat Protoc 14(3):832-863.		2022年7月2日　11:00～12:00　沖縄コンベンションセンター展示棟　ポスター会場1 3P-341 安静時にヒト全脳で自発的に生じる信号伝達 Whole-brain signal flows in human resting-state brain activities 武田祐輔(1,2)、廣江総雄(2)、山下宙人(1,2) 1. 理化学研究所革新知能統合研究センター、2. ATR脳情報解析研究所 Yusuke Takeda(1,2), Nobuo Hiroe(2), Okito Yamashita(1,2) 1. RIKEN Center for Advanced Intelligence Project, 2. ATR Neural Information Analysis Laboratories Keyword: resting-state, magnetoencephalography (MEG), electroencephalography (EEG), whole-brain signal flow Repetitive spatiotemporal patterns in resting-state brain activities have been widely observed in various species and regions. Since they resemble the preceding brain activities during tasks, they are assumed to reflect past experiences embedded in neuronal circuits. Moreover, spatiotemporal patterns involving whole-brain activities may also reflect a process that integrates information distributed over the entire brain, such as motor and visual information. Therefore, revealing signal flows in the patterns may elucidate how the information is integrated to generate consciousness. In this study, we reveal whole-brain signal flows from human resting-state magnetoencephalography (MEG) and electroencephalography (EEG) data. We simultaneously recorded the MEGs and EEGs and estimated the source currents from both measurements. Then using our recently proposed algorithm, we extracted repetitive spatiotemporal patterns from the source currents. Based on the reproducibility of the estimated patterns, their lengths were determined to be 0.1–0.3 sec. The estimated patterns consisted of multiple frequency components, each of which transiently exhibited the frequency-specific resting-state networks (RSNs) of functional MRIs, such as the default mode and sensorimotor networks. To reveal signal flows in the spatiotemporal patterns, we examined the sequence of the RSNs appearing in the patterns. As a result, the sensorimotor network tended to appear at the beta band (13–30 Hz) component before the default mode network (DMN) appeared at the alpha band (8–13 Hz) component, and the DMN tended to appear before the visual network at the alpha band component. This result suggests that the spatiotemporal patterns reflected transient signal flows across the frequencies and networks: from the beta band activation in the sensorimotor network to the alpha band activation in the DMN and from the DMN to the visual network. These signal flows may reflect a process to integrate sensorimotor and visual information via the DMN.

2022年7月2日　11:00～12:00　沖縄コンベンションセンター展示棟　ポスター会場1 3P-342 Super-resolution analysis of the impact of chemical fixation on brain tissue Idziak Agata(1)、Arizono Misa(1)、Nägerl U.(1) Agata Idziak(1), Misa Arizono(1), U. Valentin Nägerl(1) 1. Interdisciplinary Institute for Neuroscience, Univ of Bordeaux/CNRS, Bordeaux, France Keyword: Chemical fixation, SUSHI, STED Chemical fixation is a commonly used method to prepare biological samples for immunohistochemical and electron microscopic (EM) analysis. Chemical fixatives like paraformaldehyde (PFA) work by covalently cross-linking proteins, protecting the tissue against subsequent preparatory steps, such as tissue slicing, membrane permeabilization (for immunohistochemistry) or sample dehydration and embedding (for EM). The question of how well the micro-architecture and ultrastructure of brain tissue are actually preserved during chemical fixation was recently revisited by EM studies, raising concerns about the use of standard chemical fixation protocols in high-resolution anatomical studies of brain tissue. We addressed this issue of tissue with the benefit of super-resolution fluorescence microscopy, which makes it possible to monitor directly over time the effects of the fixatives on tissue integrity, permitting ‘before-and-after’ observations of identified structures of interest, not just statistical comparisons of different tissue samples. Specifically, we used STED microscopy to image fluorescently labeled neurons and astrocytes in organotypic hippocampal brain slices and the recent SUSHI technique to visualize the ECS before and after adding PFA to the microscopy chamber. For the structural analysis, we applied image processing to segment the images, identify structures of interest and extract commonly studied morphological parameters as well as the volume fraction of the ECS. We could readily image the brain slices for up to 90 minutes during PFA fixation without prohibitive bleaching or loss of image contrast. The macro-anatomical organization of the brain slices remained unaffected by the PFA treatment and no changes in ECS volume fraction or in the morphology of astrocytes, including their fine processes were detected. However, we observed a decrease in dendritic spine head area and an increase in spine neck width after PFA. Importantly, our findings indicate that PFA by itself is not sufficient to explain the depletion of brain ECS typically observed in transcardiacally perfused animals. By applying our super-resolution imaging approach to the intact brain in vivo, we seek to understand the causes of brain tissue shrinkage and distortions, allowing us to develop improved fixation protocols that minimize these effects.		2022年7月2日　11:00～12:00　沖縄コンベンションセンター展示棟　ポスター会場1 3P-343 活動依存性マンガン造影 MRI のための塩化マンガン腹腔投与後のマウス脳内マンガン動態 Manganese dynamics in mouse brain after intraperitoneal MnCl₂ administration for activation-induced manganese-enhanced MRI 小山内実(1,2,3)、谷平大樹(2)、藤原智徳(4,5)、菊田里美(2,6)、本間経康(2,7) 1. 大阪大学大学院医学系研究科、2. 東北大学大学院医学系研究科、3. 情報通信研究機構・大阪大学脳情報通信融合研究センター、4. 埼玉医科大学保健医療学部、5. 杏林大学医学部、6. 国立精神・神経医療研究センター神経研究所、7. 東北大学大学院医工学研究科 Makoto Osanai(1,2,3), Hiroki Tanihira(2), Tomonori Fujiwara(4,5), Satomi Kikuta(2,6), Noriyasu Homma(2,7) 1. Osaka Univ Grad Sch Med, Suita, Japan, 2. Tohoku Univ Grad Sch Med, Sendai, Japan, 3. CiNet, NICT, Suita, Japan, 4. Fac Health Med Care, Saitama Medical Univ, Hidaka, Japan, 5. Fac Med, Kyorin Univ, Mitaka, Japan, 6. Natl Inst Neurosci, NCNP, Kodaira, Japan, 7. Grad Sch Biomed Eng, Tohoku Univ, Sendai, Japan Keyword: MRI, manganese, activity mapping, whole-brain The first step in understanding the expression mechanisms of brain functions and pathophysiological mechanisms of neurological disorders is to understand which brain regions are associated with those functions and diseases. To address this issue, we need methods to measure and analyze neural activities within the entire brain volume. Activation-induced manganese-enhanced MRI (AIM-MRI) is an attractive tool for noninvasively mapping whole-brain activities. Manganese ions (Mn²⁺) enter and accumulate in active neurons via calcium channels. Mn²⁺ shortens the longitudinal relaxation time (T1) of H⁺, and the longitudinal relaxation rate R1 (1/T1) is proportional to Mn²⁺ concentration. Thus, AIM-MRI can map neural activities throughout the brain by assessing the R1 map. However, AIM-MRI is still not widely used, partially due to a lack the information regarding Mn²⁺ dynamics in the brain. To solve this issue, we conducted a longitudinal study to elucidate the manganese dynamics after intraperitoneal administration of MnCl₂ by AIM-MRI with quantitative analysis. In the ventricle, Mn²⁺ increased rapidly within 1 h, remained high for 3 h, and returned to near control levels by 24 h after administration. Microdialysis showed that extracellular Mn returned to control levels by 4 h after administration, indicating a high concentration of extracellular Mn²⁺ lasts at least about 3 h after administration. In the brain parenchyma, Mn²⁺ increased slowly, peaked 24-48 h after administration, and returned to control level by 5 days after a single administration and by 2 weeks after a double administration with a 24-h interval. These time courses suggest that AIM-MRI records neural activity 1-3 h after MnCl₂ administration, appropriate timing of the MRI scan is in the range of 24-48 h following systemic administration, and at least an interval of 5 days or a couple of weeks for single or double administrations, respectively, is needed for a repeat AIM-MRI experiment.

2022年7月2日　11:00～12:00　沖縄コンベンションセンター展示棟　ポスター会場1 3P-344 Simultaneous dual color imaging in freely-behaving animal using a head-mountable miniscope Kevin Zitelli(1), Srishti Gulati(1), Douglas Ollerenshaw(1), Alice Stamatakis(1) 1. Inscopix Inc., Mountain View, California Keyword:* Miniscope, Calcium imaging, Dual color, Blood flow We have developed a one-photon miniscope platform nVueTM that enables dual color imaging in freely-behaving animals, thus greatly expanding the current range of in vivo imaging applications. With the use of two excitation LEDs, the nVue system is capable of imaging green and red fluorescent indicators without optical or biological crosstalk. These LEDs are multiplexed rapidly during imaging, enabling simultaneous visualization of green and red fluorescent signals. To correct for chromatic shifts in green and red focal planes, we have also developed novel GRIN lenses that minimize axial and lateral chromatic aberrations. Furthermore, the electronic focusing capabilities of the miniscope are used to automatically correct for any residual color aberration. Newly developed algorithms in our data processing software also enable additional types of analysis, such as measurement of blood vessel diameter and detection of colocalized red and green cells. Here, we present several examples of applications enabled by the nVue miniscope system: 1) Simultaneous imaging of neuronal calcium activity alongside blood flow, 2) Simultaneous imaging of neuronal calcium activity alongside neurotransmitter release, and 3) imaging neuronal calcium activity along with a static marker identifying a subpopulation of neurons. These applications allow researchers to measure direct correlations between multiple signals in a freely-behaving context, enabling deeper insight into central nervous system function.		2022年7月2日　11:00～12:00　沖縄コンベンションセンター展示棟　ポスター会場1 3P-345 経頭蓋直流電気刺激が脳脊髄液と間質液の交換に与える影響の解析 Transcranial direct current stimulation alters cerebrospinal fluid-interstitial fluid exchanges in mouse brain 王岩(1)、毛内拡(1,2) 1. お茶の水女子大学大学院人間文化創成科学研究科、2. お茶の水女子大学理学部生物学科 Yan Wang(1), Hiromu Monai(1,2) 1. Graduate School of Humanities and Sciences, Ochanomizu University, 2. Department of Biology, Faculty of Science, Ochanomizu University Keyword: brain stimulation, neuromodulation, stroke, rehabilitation Cerebrovascular diseases, including ischemic stroke, are among the most severe conditions. Not only is the mortality rate high, but the secondary effects such as brain dysfunction, motor and language problems are not negligible and result in long-term nursing and rehabilitation. In clinical research, transcranial direct current stimulation (tDCS), which involves passing a very weak direct current through the skull or scalp for 10-30 minutes, has been increasingly investigated as an adjunct to facilitate the rehabilitation of diseases including stroke. Many studies have shown that tDCS has positive therapeutic effects on chronic and acute stroke, but the mechanisms are still not clearly understood. The cellular mechanisms of anodal tDCS have been suggested by Monai et al. (2016), in which activation of adrenergic receptors has a significant role in the mouse brain. In contrast, Monai et al. (2019) found that adrenergic receptor blockade also facilitated the recovery from motor dysfunctions after acute ischemic stroke in mice. The results suggested that the adrenergic receptor blockade facilitated the normalization of the brain extracellular ion milieu by the cerebrospinal fluid (CSF) and interstitial fluid (ISF) exchange. However, it is still unclear how tDCS affects the dynamics of CSF-ISF exchange in the brain. Therefore, in this study we will directly visualize brain fluid dynamics using immunohistochemistry and demonstrate that tDCS can alter CSF-ISF exchange rates in mouse brain.

2022年7月2日　11:00～12:00　沖縄コンベンションセンター展示棟　ポスター会場1 3P-346 組織透明化による頭足類の全身・全脳イメージング Whole body/brain imaging of the cephalopod nervous system using tissue clearing methods 真野智之(1)、南部美友(1)、Reiter Samuel(1) 1. 沖縄科学技術大学院大学 Tomoyuki Mano(1), Miyu F Nambu(1), Samuel Reiter(1) 1. Okinawa Institute of Science and Technology Keyword: Tissue clearing, Cephalopod, Squid, Imaging Cephalopods, such as squid, octopus and cuttlefish, have the largest central nervous systems among all invertebrate species. Their rich and complex behaviors, involving systems such as vision and memory, often resemble those of vertebrates, suggesting the convergent evolution of neural circuits. Further, cephalopods are characterized by their unique ability to change their skin patterns through a neurally-controlled array of pigment cells. These features make the cephalopods highly attractive organisms for investigation of potentially universal neural mechanisms for high-level cognition, as well as for elucidation of species-specific circuitry underlying their unique camouflage ability. However, the anatomy of the cephalopod brains has scarcely been updated since the days of J.Z. Young, half a century ago. Inspired by the recent success of the Allen Brain Atlas project serving as an integrative platform for cellular and molecular understanding of the mouse brain, here we aim to develop the modern digital atlas of the cephalopod brains by employing state-of-the-art tissue clearing and light-sheet imaging techniques. First, we modified existing clearing methods, such as CUBIC and DISCO, to solve the challenges unique to cephalopod tissues (e.g. body pigmentation). Second, we constructed a custom-built light-sheet microscope capable of acquiring high resolution (<1 um in lateral and <2.5 um in axial) 3D images. The microscope features an axially-sweeping mechanism, as well as automatic focus correction, to achieve high image quality throughout the centimeter-sized specimen. We used these techniques to analyze the anatomy of the Sepioteuthis lessoniana (oval squid; local to Okinawa). Combined with histological staining methods, we successfully imaged the entire body, revealing the complex central and peripheral nervous systems. We will showcase the images obtained by this technique and present the ongoing efforts to extract anatomical foundations for the squid's advanced visual processing. Our technique will make it possible to construct a modern 3D digital atlas of the cephalopod brain, providing common means to integrate molecular and physiological observations. The time to upgrade a cephalopod brain atlas is near.		2022年7月2日　11:00～12:00　沖縄コンベンションセンター展示棟　ポスター会場1 3P-347 複数実験課題を用いたOPM-MEGシステムの検証 Validation of OPM-MEG system using multi-task experiments 鈴木啓大(1)、武田祐輔(1,2)、廣江総雄(1)、山下宙人(1,2) 1. ATR脳情報解析研究所、2. 理研AIP Keita Suzuki(1), Yusuke Takeda(1,2), Nobuo Hiroe(1), Okito Yamashita(1,2) 1. ATR Neural Information Analysis Laboratories, Kyoto, Japan, 2. RIKEN Center for Advanced Intelligence Project, Kyoto, Japan Keyword: Magnetoencephalography (MEG), Optically Pumped Magnetometer (OPM), Current source reconstruction Background Magnetoencephalography (MEG) is one of the noninvasive ways to measure human brain activity. Thanks to its milli-second order temporal resolution, it is employed by various studies. However, the measuring cost of a conventional device using SQUID-type sensors is expensive due to a liquid helium requirement, and it is the primary bottleneck. Therefore, we built a cerebral magnetic field measuring system using optically pumped magnetometer (OPM) devices, requiring no expensive medium. This report compares the SQUID-MEG and the OPM-MEG using multi-task experiments of several subjects. Experimental equipment We used 10 channels of OPM device (QZFM Gen-2) manufactured by QuSpin. Because OPM-MEG measurement requires a relatively low magnetic field environment, we installed a magnetic field canceling coil in a magnetically shielded room. It controlled the magnetic field around the subject's head to less than 1 nT. We employed a 400 channels system (PQ1400RM) manufactured by Yokogawa Electric Corporation for the SQUID-MEG measurement. Experimental tasks This report shows the results of sensory-motor and auditory stimulation tasks. Four subjects participated in the experiments with both OPM-MEG and SQUID-MEG recordings. Sensory-motor stimulation: The median nerve in the right wrist was stimulated electrically to induce activations on the left somatosensory cortex. For OPM-MEG, 10 channels were placed around the left temporal cortex. Auditory stimulation: Pure tones in both ears were presented to stimulate the auditory cortex. Although auditory responses could be observed in both hemispheres, we focused on the left hemisphere. Hence, OPM-MEG channels were placed on the left temporal cortex as well as the sensory-motor task. Results We observed well-known waveforms for both OPM-MEG and SQUID-MEG recordings for each task in accordance with the previous SQUID-MEG studies. Importantly, the number of channels of the OPM-MEG was much smaller than that of the SQUID-MEG. As a result of the current source reconstruction, sources were localized at places consistent with the conventional knowledge of each task for both devices. These results confirm that our OPM-MEG system records the cerebral magnetic field with low-cost and sufficient quality. The high flexibility of the OPM-MEG device is expected to contribute to the experiments with high mobility task design. The data shown in this report will be opened as the publicly available dataset.

2022年7月2日　11:00～12:00　沖縄コンベンションセンター展示棟　ポスター会場1 3P-348 Bicistronicベクターはリークする：Cre依存性ベクターがリークするメカニズムの解明 Leaky transgene expression in Cre-inducible vectors: causes and solutions 長内康幸(1,2)、Yao Lulu Xing(2)、小林憲太(3)、Jihane Homman-Ludiye(2)、Amali Cooray(2)、Jasmine Poh(2)、大野伸彦(1,4)、Tobias D Merson(2) 1. 自治医科大学解剖学講座組織学部門、2. モナッシュ大学オーストラリア再生医療研究所、3. 生理学研究所ウイルスベクター開発室、4. 生理学研究所超微形態研究部門 Yasuyuki Osanai(1,2), Yao Lulu Xing(2), Kenta Kobayashi(3), Jihane Homman-Ludiye(2), Amali Cooray(2), Jasmine Poh(2), Nobuhiko Ohno(1,4), Tobias D Merson(2) 1. Dept Anatomy, Jichi Medical University, 2. ARMI, Monash University, 3. Sec Viral Vector Dev, NIPS, 4. Div Ultrast Res, NIPS Keyword: Cre, AAV, Bicistronic vector, leak Cre-inducible gene expression systems such as lox-STOP-lox or FLEX cassette are widely used for spatiotemporal regulation of transgene expression, and multiple gene expression can be achieved by a self-cleaving 2A peptide. Using lox-STOP-lox and P2A peptide, we generated a Cre-responsive bicistronic vector, pCAG-lox-GFP-STOP-lox-rtTA-P2A-mCherry (here after, CAG-LGL-rtTA-P2A-mC), expressing nuclear-targeted GFP in the absence of Cre, and expressing the reverse tetracycline-controlled transactivator (rtTA) plus membrane-targeted mCherry after Cre-mediated recombination. Unexpectedly, upon transfection into cells, > 7% of transfected cells misexpressed mCherry (3’ transgene) without Cre recombination, whereas rtTA (5’ transgene) was expressed in a strictly Cre-dependent manner. When the order of mCherry and rtTA coding sequences was swapped, mCherry (5’ transgene) expression was tightly regulated but rtTA (3’ transgene) expression became leaky, suggesting that expression of the 3’ transgene is not efficiently blocked by the upstream STOP cassette. Surprisingly, removing the upstream promoter did not prevent the leaky expression of the 3’ transgene in the bicistronic construct, indicating 5’ transgene coding sequence drives 3’ transgene expression. Inserting an additional lox-STOP-lox cassette after the 2A sequence dramatically reduced the leaky expression. We found that popular bicistronic AAV-FLEX constructs from addgene also express 3’ transgene without Cre activity. This study indicates that inducible bicistronic construct will exhibit Cre-independent (leaky) expression due to promoter activity of the 5’ protein coding sequence.		2022年7月2日　11:00～12:00　沖縄コンベンションセンター展示棟　ポスター会場1 3P-349 接触依存的なウイルス感染制御(CONVIRT)による順行性経シナプス標識法の確立 Establishment of contact-mediated viral targeting (CONVIRT) system for anterograde transsynaptic tracing 山本悠(1)、Ole S Schwartz(1)、米原圭祐(1,2,3) 2. 国立遺伝学研究所、3. 総研大院遺伝学 Haruka Yamamoto(1), Ole S Schwartz(1), Keisuke Yonehara(1,2,3) 1. DANDRITE, Dept Biomed, Aarhus Univ, Aarhus, Denmark, 2. National Institute of Genetics, Mishima, Japan, 3. The Graduate University for Advanced Studies (SOKENDAI), Mishima, Japan Keyword: Anterograde tracing, Proximity labeling, Rabies virus Trans-synaptic viral tracers have been widely used for investigating the structure and function of neural circuits. Trans-synaptic tracing can be categorized into retrograde and anterograde tracing depending on the transport direction of virus. Rabies virus-based retrograde synaptic tracing revolutionalized how we map synaptic connectivity and link the gained insights into circuit function. On the other hand, anterograde trans-synaptic viral tracers are less developed. Here, we try to establish a novel strategy for tracking trans-cellular physical interactions by utilizing a bacteria-derived enzyme and viral targeting. Next, we applied this strategy for developing anterograde monosynaptic tracing, which comprises two experimental steps. In the first step, the receptor (i.e. TVA receptor) for EnvA-pseudotyped virus is spread from genetically labeled starter cells to synaptically connected neurons. In the second step, the network of TVA-labeled neurons is selectively infected with EnvA-coated rabies or lenti virus for fluorescent marker expression. We first confirmed TVA translocation and rabies infection into neighboring cells in culture cells. Next, as a proof of concept of anterograde tracing in vivo, we used the retina-superior colliculus circuit in mice as a model because the superior colliculus is the major target area of retinal output cells and this circuit is not reciprocal. We successfully observed some rabies-infected neurons in the superior colliculus after starting the tracing from genetically-labeled retinal ganglion cell types. This novel transsynaptic labeling method would serve as a powerful tool for investigating not only neural circuit connections but also other types of cellular interactions such as dynamic neuron-glia interactions.

2022年7月2日　11:00～12:00　沖縄コンベンションセンター展示棟　ポスター会場1 3P-350 In vivo条件で発火活動を記録した神経細胞の遺伝子発現解析 Gene expression profiles in neurons recorded by in vivo extracellular recordings 柳下晴也(1,2)、岡本和樹(3)、郷康広(4)、池谷裕二(1,5,6)、佐々木拓哉(1,2) 1. 東京大学大学院薬学系研究科、2. 東北大学大学院薬学研究科、3. 順天堂大学大学院医学研究科、4. 自然科学研究機構生命創成探究センター、5. 情報通信研究機構脳情報通信融合研究センター、6. 東京大学Beyond AI 研究推進機構 Haruya Yagishita(1,2), Kazuki Okamoto(3), Yasuhiro Go(4), Yuji Ikegaya(1,5,6), Takuya Sasaki(1,2) 1. Grad Sch Pharm Sci, Univ of Tokyo, Tokyo, Japan, 2. Grad Sch Pharm Sci, Tohoku Univ, Miyagi, Japan, 3. Grad Sch Med, Juntendo Univ, Tokyo, Japan, 4. ExCELLS, NINS, Aichi, Japan, 5. CiNet, NICT, Osaka, Japan, 6. Inst AI Beyond, Univ of Tokyo, Tokyo, Japan Keyword: electrophysiology, single cell RNA sequencing, juxtacellular recording, gene expression Neurons in the brain exhibit diverse electrophysiological, morphological, and molecular characteristics. The neuronal circuit including these diverse neurons underlies a variety of brain functions and behavior. However, experimental methods to comprehensively analyze these properties at multiple levels from a single neuron are still lacking. To address this issue, we developed an experimental method to identify firing properties and gene expression profiles in the same neuron by serially combining existing techniques, including in vivo unit recordings, cell labeling, brain slicing, patch-clamping, and RNA sequencing. In detail, we recorded spike train patterns of a cortical neuron from a living mouse by a juxtacellular recording method using a sharp glass electrode. After performing the spike recording, we electroporated Alexa 488/594 hydrazide, a soluble fluorophore, into the recorded neuron for cell labeling. Subsequently, we prepared acute cortical slices with a thickness of 200 µm from the same mouse and detected and collected the labeled neuron by a glass pipette in the slice under a microscopy. Finally, we applied single-cell RNA sequencing to the collected cell to identify its gene expression profiles. So far, we confirmed that our method is applicable to anesthetized mice and hippocampal pyramidal neurons and interneurons exhibit distinct spike train patterns and gene expression patterns, which potentially enable to link molecular insights with physiological characteristics. As juxtacellular recordings can be applied to freely moving animals (Tang et al. 2015), we now apply our methodological idea to animals engaging in various sensory stimulus responses and behavioral patterns.		2022年7月2日　11:00～12:00　沖縄コンベンションセンター展示棟　ポスター会場1 3P-351 分子イメージングへの応用を指向したシナプス形成分子に対する新規VHH抗体の取得 Acquisition of novel VHH antibodies to synaptic organizers for application to molecular imaging 横尾尚典(1)、中木戸誠(1,2)、松田恵子(3)、柚﨑通介(3)、津本浩平(1,2,4) 1. 東京大学大学院工学系研究科化学生命工学専攻、2. 東京大学大学院工学系研究科バイオエンジニアリング専攻、3. 慶應義塾大学医学部生理学、4. 東京大学医科学研究所 Takanori Yokoo(1), Makoto Nakakido(1,2), Keiko Matsuda(3), Michisuke Yuzaki(3), Kouhei Tsumoto(1,2,4) 1. Dept of Chem &Biotech, Sch of Eng, Univ of Tokyo, 2. Dept of Bioeng, Sch of Eng, Univ of Tokyo, 3. Dept of Physiol, Keio Univ Sch of Med, 4. Inst of Med Sci, Univ of Tokyo Keyword: antibody, VHH, molecular imaging, synaptic organizer Synapse formation is induced through selective interactions between synaptic organizers, but their molecular mechanisms remain unclear. Molecular imaging technology using antibodies is expected to be a powerful tool for elucidation of the mechanisms. It has been shown, however, that molecular epitopes localized at synaptic sites cannot be easily detected by IgG and Fab antibodies due to the large molecular size and molecular crowding in the synaptic cleft. To overcome this challenge and apply antibodies for synaptic imaging, we are trying to take advantage of single-domain antibodies, called VHH antibodies, derived from camelids. In this research, we generated novel VHH antibodies toward two different extracellular scaffold proteins, Cbln1 (Cerebellin 1) and NP2 (Neuronal pentraxin 2) for the use of molecular imaging and analyzed their interactions with antigens by biophysical techniques. Each target protein was prepared as a recombinant protein and used for immunization to alpaca. We then isolated lymphocytes from the alpaca after 5 weeks of immunization and constructed an immune library from total RNA utilizing phage display technology. We subsequently selected VHH antibodies to each target protein by several rounds of bio-panning and evaluated the binding by phage ELISA. As a result, we have obtained 5 VHH clones for NP2 and 3 clones for Cbln1, respectively. As for VHH clones, confirmed to bind to the targets, we prepared them as recombinant proteins. The interactions between each VHH and Cbln1 / NP2 were quantitatively evaluated with Surface plasmon resonance (SPR).We have determined the affinity of each anti-Cbln1 VHH clone (VHHCA1, 6, and 10) and Cbln1. VHHCA1 bound to Cbln1 with high affinity (0.831 nM) with slow dissociation. The other clones, VHHCA6 and CA10 have relatively high affinity (189 nM and 162 nM, respectively) with fast association and dissociation. The difference of affinity and kinetics of VHHs suggest that the molecular mechanism by which these antibodies recognize Cbln1 would be considerably different. Also, we have analyzed the interaction between each anti-NP2 VHH clones and NP2 as well. All of them have the affinity of about 100 nM or higher to NP2. We also prepared NP2 PTX domain to identify the epitope of VHHs roughly and conducted SPR as well. As a result, all VHHs recognized PTX domain of NP2. Among them, VHHN1 showed high affinity (1.37 nM). Moreover, VHHN1 recognized NP2 on cell surface that overexpressing NP2 with high affinity and with no cross reactivity to other NP family.We plan to obtain additional clones binding to each target. Also, we sequentially identify epitope using crystallography and assess if obtained VHHs interfere with the interaction between the antigens and their own receptors. In parallel, we plan to apply it to imaging.

2022年7月2日　11:00～12:00　沖縄コンベンションセンター展示棟　ポスター会場1 3P-352 新規高感度オキシトシンELISA系の開発 Development of novel highly sensitive oxytocin ELISA 杉野史朗(1)、小島正章(2)、阿久津史絵(2)、西部隆宏(1) 1. 富士フイルム和光純薬株式会社、2. 富士フイルムワコーシバヤギ株式会社 Shiro Sugino(1), Masaaki Kojima(2), Fumie Akutsu(2), takahiro nishibu(1) 1. FUJIFILM Wako Pure Chemical Corporation, 2. FUJIFILM Wako Shibayagi Corporation Keyword: Oxytocin, ELISA, assay Oxytocin is a peptide hormone comprising nine amino acids. It is produced in the hypothalamus and released mainly from the posterior pituitary gland following childbirth and during lactation, and promotes uterine contraction and milk production. It also has stress-relieving, anxiolytic, and fear-reducing effects and plays a role in the development of maternal behavior. Due to these properties, oxytocin is often referred to as the "happy hormone" or "love hormone". Recently, oxytocin has attracted attention in treating mental disorders such as depression and autism or developing functional food materials. We investigated several commercially available oxytocin ELISA kits, but the existing kits require a large volume of sample and complicated pretreatment using a C18 column and organic solvent. Consequently, we embarked on developing a novel ELISA for oxytocin that only needed simple pretreatment and a small sample. First, we constructed an oxytocin competition ELISA, which has much higher sensitivity (4 pg/mL) than the commercial kit, by using oxytocin antibody, biotin-label oxytocin, streptavidin conjugated HRP, and chemiluminescent substrate. It has been reported that oxytocin binds proteins and other molecules in the sample and these can affect measurement by inhibiting oxytocin and antibody interaction. We accordingly investigated various methods for removing the impurities in the sample and found a novel pretreatment method for adding acid and gel to the sample and centrifuge. In conventional methods, the required sample volume is 250-1000 μL and the pretreatment time is from two hours to overnight. However, with our method, measurement with a 50 μL sample can be completed in 30 minutes by a simple manipulation. In addition, our method also demonstrated a good recovery rate in a dilution linearity test and spike-recovery test (90-120%) using human plasma, serum, urine, and saliva, and mouse/rat plasma and serum. In conclusion, our novel ELISA is expected to be a useful tool in basic research on oxytocin using human subjects, mice, rats, and other animals.		2022年7月2日　11:00～12:00　沖縄コンベンションセンター展示棟　ポスター会場1 3P-353 Tagging active neurons by soma-targeted Cal-Light Jung Ho Hyun(1,2,5), Kenichiro Nagahama(1), Ho Namkung(4), Neymi Mignocchi(5), Patrick Hannan(1,5), Sarah Krüssel(1,5), Chuljung Kwak(1), Dongmin Lee(3), Richard L Huganir(1), Akira Sawa(4), Hyung-Bae Kwon(1,5) 1. Department of Neuroscience, Johns Hopkins University School of Medicine, Baltimore, USA, 2. Department of Brain Sciences, Daegu Gyeongbuk Institute of Science & Technology (DGIST), Daegu, S.Korea, 3. Department of Anatomy, Korea University, Seoul, S.Korea , 4. Department of Psychiatry, Johns Hopkins University School of Medicine, Baltimore, USA, 5. Max Planck Florida Institute for Neuroscience, Jupiter, USA Keyword:* Cal-Light, Soma-targeted, KA2, in vivo labeling Verifying causal effects of neural circuits is essential for proving direct a circuit-behavior relationship. However, techniques for tagging only active neurons with high spatiotemporal precision remain at the beginning stages. Here we developed the soma-targeted Cal-Light (STCal-Light) which selectively converts somatic calcium rise triggered by action potentials into gene expression. Such modification simultaneously increases the signal-to-noise ratio (SNR) of reporter gene expression and reduces the light requirement for successful labeling. Because of the enhanced efficacy, the ST-Cal-Light enables the tagging of functionally engaged neurons in various forms of behaviors, including context-dependent fear conditioning, leverpressing choice behavior, and social interaction behaviors. We also targeted kainic acid-sensitive neuronal populations in the hippocampus which subsequently suppressed seizure symptoms, suggesting ST-Cal-Light’s applicability in controlling disease-related neurons. Furthermore, the generation of a conditional ST-Cal-Light knock-in (KI) mouse provides an opportunity to tag active neurons in a region- or cell-type specific manner via crossing with other Cre-driver lines. Thus, the versatile ST-Cal-Light system links somatic action potentials to behaviors with high temporal precision, and ultimately allows functional circuit dissection at a single cell resolution.

2022年7月2日　11:00～12:00　沖縄コンベンションセンター展示棟　ポスター会場1 3P-354 hTERT遺伝子の機能と神経疾患に対する細胞治療への応用 Development of hTERT-expanded cell-based regenerative medicine in neurological diseases and spinal cord injury 池野正史(1)、笹倉寛之(1)、武内恒成(1) 1. 愛知医科大学 Masashi Ikeno(1), Hiroyuki Sasakura(1), Kosei Takeuchi(1) 1. Aichi Medical University Keyword: hTERT, regenerative medicine, stem cell Stem/progenitor cells are useful in regenerative medicine and cell therapy. Spinal cord injury (SCI) is a common neurological disease that results in loss of sensory function and mobility. Regenerative medicine and cell therapy are well studied for a potential therapeutic strategy for treatment of SCI. Transplantation of human neural stem/progenitor cells functionally promoted functional recovery from SCI in rodents and monkeys. However, recovery mechanism from SCI and tumorigenicity of human induced pluripotent stem cell derived-stem/progenitor cells is in the process of research and development. Separately, application of readily available human mesenchymal stem cell (hMSC) or stem cells from human exfoliated deciduous teeth (SHED) is making progress for regenerative medicine and cell therapy. Limited life span and donor-dependent variation of primary cells such as hMSC and SHED present hurdles to reproducible experiments and applications. Therefore, our major aim was establishment of cell lines from primary cells that provide steady supply of homogeneous cells while retain essential features of primary cells for neurological disease including SCI. Limited replication capacity of primary cells was overcome by introduction of the human telomerase reverse transcriptase (hTERT). We demonstrated that transfection of hTERT alone was sufficient to extend the life span of hMSC and SHED without significantly perturbing their phenotype or biological behavior. However, p16 gene, a marker of cellular senescence was up-regulated in hTERT-expanded cells. Senescence may be advantageous by promoting tissue repair and regeneration. Therefore, we examined senescent state of hTERT-expanded SHED. The availability of hTERT-expanded and functionally identified cells will be helpful for expansion of cells for regenerative medicine and cell therapy. To validate recovery from SCI, we are now undergoing transplantation experiment to mouse SCI model using hTERT-expanded hMSC or SHED.		2022年7月2日　11:00～12:00　沖縄コンベンションセンター展示棟　ポスター会場1 3P-355 ラットのプラットフォーム事業-ナショナルバイオリソースプロジェクトと先端動物モデル支援- The platform projects for rats-National Bio Resource Project and Advanced Animal Model Support- 服部晃佑(1)、石田紗恵子(1)、星美穂(1)、王金曦(1)、吉見一人(1,2)、真下知士(1,2) 1. 東京大学医科学研究所実験動物研究施設先進動物ゲノム研究分野、2. 東京大学医科学研究所システム疾患モデル研究センターゲノム編集研究分野 Kosuke Hattori(1), Saeko Ishida(1), Miho Hoshi(1), Jinxi Wang(1), Kazuto Yoshimi(1,2), Tomoji Mashimo(1,2) 1. Division of Animal Genetics, Laboratory Animal Research Center, The Institute of Medical Science, The University of Tokyo, 2. Division of Genome Engineering, Center for Experimental Medicine and Systems Biology, The Institute of Medical Science, The University of Tokyo Keyword: NBRP-Rat, genome editing , Platform of Advanced Animal Model Support Rat (Rattus norvgicus) has been widely applied in various studies including neurology. Taking the advantage of larger body size than mouse, rat is easier to operate and can provide larger volume of samples and detailed image of brain. As the development of genome editing technology, the gene modification in rats has become much easier than before and the laboratory rats are more worthwhile in nowadays. Since the National Bio-Resource Project for rat (NBRP-Rat) has been set up in 2002, more than 800 rat strains have been collected and kept. The NBRP-Rat has become one of the top research-resource in the world. Since the year of 2017, we became one of the members of the NBRP-Rat project and provide three immunodeficient rat models, including F344-Il2rg^em1Iexas (NBRP Rat No.0883), F344-Rag2^em1Iexas (NBRP Rat No.0894) and F344-Il2rg/Rag2^em1Iexas (NBRP Rat No.0895). These immunodeficient rats have large applicability and potential in the translational researches and are supplying to researchers for all over the world. During 2017~2019, we provide total 128 immunodeficient rat to 8 institutes (7 domestic institutes and 1 oversea institute) in Osaka University, via MTA agreement. After moving to The University of Tokyo in April 2020, we have signed another 20 agreements with 7 institutes, provided 107 immunodeficient rats to them. We are also the member of Platform of advanced Animal Model Support established by MEXT in Japan since 2016. This platform aims to support researchers who want to generate and to analyze various gene-editing animals. We are supporting to provide genetically modified rats upon requests with our unique genome editing technologies. Here we will introduce the outline of ‘NBRP-immunodeficient rats’ and ‘Platform of Advanced Animal Model Support’ with our genome editing technology.