脊髄，リズム運動 Spinal Cord, Rhythmic Motor Control
O3-9-3-1 重症慢性期脊髄損傷モデルラットに対するコンドロイチナーゼABCとリハビリテーションの併用療法 Combination therapy of CABC and rehabilitation for severe chronic spinal cord injury rats ○篠崎宗久1, 中村雅也2, 藤吉兼浩2, 田代祥一3, 戸山芳昭2, 岡野栄之1 ○Munehisa Shinozaki1, Masaya Nakamura2, Kanehiro Fujiyoshi2, Syouichi Tashiro3, Yoshiaki Toyama2, Hideyuki Okano1 慶應義塾大学　医学部　生理学1, 慶應義塾大学　医学部　整形外科2, 慶應義塾大学　医学部　リハビリテーション科3 Dept Physiol, Keio Univ, Tokyo1, Dept Orthopedic Surgery, Keio Univ, Tokyo2, Dept Rehabilitation, Keio Univ, Tokyo3 [Background]It is well known that regeneration-ability after spinal cord injury (SCI) is limited. Glial scar formation after SCI inhibits axonal regeneration, and therapeutic strategy using chondroitinase-ABC (CABC), which dissolves chondroitin sulfate proteoglycan (CSPG) has been investigated. Nevertheless, there has been no report to examine the effect of combined therapy of CABC and rehabilitation on severe chronic SCI. [Object] To determine the effect of combined therapy of CABC and conventional rehabilitation on severe chronic SCI model-rats.[Methods]We made severe contusion injury model-rats, and treated them with combination of the CABC and the rehabilitation. Spinal cord injury of 250 kdyn on Th10 was created with IH impactor. At 6 weeks after SCI, CABC or inactivated CABC as control was intrathecally administrated for 1 week. At the same time, the rehabilitation was started in both groups with body weight supporting treadmill for 30 minutes a day, 4 days a week until 14 weeks. Hindlimbs function was scored with Basso, Beattie, and Bresnahan (BBB) score and von Frey test. [Results]BBB scores decreased to 0, followed by a slight recovery and reached a plateau at 4weeks after SCI (2.78 in the CABC group and 2.50 in the control group). After the rehabilitation started, both groups showed further functional recovery. The control rats reached a plateau around 4.32 at 9 weeks, whereas the CABC treated animals showed gradual improvement until 14 weeks (5.67). Histological evaluation using hematoxylin-eosin staining suggested larger residual area in axial sections of CABC treated rats. They also showed significantly more neural fibers at the distal spinal cord compared with the control group. [Conclusion]In the chronic severe SCI model-rats, motor function is improved by treadmill exercise, and CABC boosts the motor function, and increases axonal fibers in the spinal cord.	O3-9-3-2 マーモセット損傷脊髄内の微小環境の網羅的解析 Exhaustive analyses of microenvironments of injured spinal cord in adult marmosets ○西村空也1,2, 岩井宏樹1,2, 小林喜臣1,2, 吉田賢司3, 芝田晋介2, 海老瀬速雄3, 古家育子4, 佐々木貴史4, 清水厚志4, 工藤純4, 戸山芳昭1, 中村雅也1, 岡野栄之2 ○Soraya Nishimura1,2, Hiroki Iwai1,2, Yoshiomi Kobayashi1,2, Kenji Yoshida3, Shinsuke Shibata2, Hayao Ebise3, Ikuko Koya4, Takashi Sasaki4, Atsushi Shimizu4, Jun Kudoh4, Yoshiaki Toyama1, Masaya Nakamura11, Hideyuki Okano2 慶大整形1, 慶大生理2, 大日本住友製薬株式会社ゲノム科学研究所3, 慶大生命情報学センター4 Dept Orthop Surg, Keio Univ Sch of Med, Tokyo1, Dept Physiol, Keio Univ Sch of Med, Tokyo2, Genomic Science Laboratories, Dainippon Sumitomo Pharma Co., Ltd., Osaka3, Center for Bioinformatics, Keio Univ Sch of Med, Tokyo4 The microenvironment of injured spinal cord significantly affects the fate of neural stem cells (NSCs) transplanted into the spinal cord. Analyses of microenvironmental changes of injured spinal cord, which are critical to determine a therapeutic time-window of NSCs transplantation for spinal cord injury (SCI), was previously reported in rodent SCI models, but not in a non-human primate SCI model. Here, we examined the global gene expression analyses to investigate microenvironment of injured spinal cord in adult common marmosets to determine an optimal time-window of NSC transplantation in primate SCI. Contusive SCI was induced at C5 level in adult female common marmoset. The injured spinal cord samples were harvested from each experimental animal at 1, 2, 4 and 6 WPI. Transcriptome analyses, such as microarray and next generation sequencing and histological analyses were performed using these samples. Transcriptome analyses revealed that the expressions of genes associated with inflammatory cytokines or reactive oxygen were significantly up-regulated at 1 WPI and gradually decreased at 2 WPI and thereafter. In contrast, the expressions of genes about synaptic transmission were down-regulated at 1 WPI, slightly elevated at 2 WPI and reached a plateau at 4 WPI. Furthermore, the expressions of chondroitin sulfate proteogricans (CSPG) increased at 2 WPI and thereafter. Histological analysis revealed that macrophage infiltration around the lesion site was significant at 1 WPI and mostly disappeared 6 WPI. In contrast, immunohistology for GFAP and CSPG showed that the glial scar formation and the CSPG accumulation became prominent at 6 WPI. Taken together, the acute inflammatory response after SCI was diminished at 2 WPI, however the glial scar formation and the CSPG accumulation were already irreversible at 6 WPI. These findings suggested that the optimal time-window of NSCs transplantation might be around 2 to 4 WPI at the sub-acute phase of SCI in non-human primates
O3-9-3-3 ショウジョウバエ幼虫運動回路における介在神経細胞による運動速度制御機構 Segmental control of motor output by pre-motor inhibitory interneurons for generating innate speed of larval locomotion in Drosophila ○高坂洋史1, 能瀬聡直1,2 ○Hiroshi Kohsaka1, Akinao Nose1,2 東京大院・新領域・複雑理工1, 東京大院・理・物理2 Dept Complex Sci Eng, Univ of Tokyo, Kashiwa, Chiba1, Dept physics, Univ of Tokyo, Tokyo2 The speed of locomotion is a predominant parameter in animal behavior and believed to be controlled by the neural circuits that generate specific spatio-temporal activity of motor neurons. While some interneurons involved in locomotion have been identified, the mechanisms of the speed control remain unclear. We use Drosophila larval crawling locomotion as a model system to dissect motor circuits. The behavior is generated by propagation of local muscle contraction from the posterior to anterior segments. We have previously identified a class of local interneurons, termed period-positive median segmental interneurons, or PMSIs, as inhibitory pre-motor interneurons. When the activity of PMSIs was silenced by halorhodopsin, or a temperature-sensitive dynamin mutant, the speed of locomotion was greatly decreased, indicating that these neurons play crucial roles in speed control of larval locomotion.To understand the mechanisms of the speed control by PMSIs, we examined the temporal relationship between the activity of PMSIs and that of motor neurons. By expressing a Ca-sensor, GCaMP, in both PMSIs and motor neurons, we simultaneously imaged activity of the two neural populations. We found that during a motor wave, PMSIs and motor neurons in the same segment are active at a similar timing,, suggesting that PMSIs shape the temporal pattern of motor neuronal firing through local inhibition. To test this possibility, we acutely blocked the activity of PMSIs with halorhodopsin, while measuring the activity of motor neurons by extracellular nerve recording. We found that the manipulation prolonged the duration of segmental motor bursting activity. Furthermore, it took longer for the peak of the bursting to travel along the segments. These results suggest that PMSIs control the speed of locomotion by regulating, through direct inhibition, the duration of motor neuronal bursting in each segment.	O3-9-3-4 オプトジェネティクスと神経活動可視化による運動回路の活動伝播ダイナミクスの解析 Simultaneous application of optogenetics and calcium imaging for system-level analyses of motor circuits ○松永光幸1, 高坂洋史1, 能瀬聡直1,2 ○Teruyuki Matsunaga1, Hiroshi Kohsaka1, Akinao Nose1,2 東京大学大学院新領域創成科学研究科複雑理工学1, 東京大学大学院理学系研究科物理学2 Department of Complexity Sciences and Engineering, Graduate School of Frontier Science, The University of Tokyo1, Department of Physics, Graduate School of Science, The University of Tokyo2 Towards system level analyses of motor circuits, we aim to establish a new experimental system that enables recording of the response of a population of neurons upon local stimulation/silencing of the same or other population of neurons. Here, we report on the successful use of the system to simultaneously record and manipulate motor neurons (MNs) in Drosophila, which revealed a novel property of the motor circuits. Peristaltic locomotion of Drosophila larvae is achieved by propagation of muscle contraction from tail to head, which is in turn generated by sequential activation of MNs in the corresponding neuromeres. Previous studies suggested that MNs transmit neuronal signals not only to muscles but also within the central nervous system. To test this possibility further, we studied the effects of local MN activation on the propagation of motor wave. By simultaneously expressing channelrhodopsin2 (ChR2) and GCaMP3 in MNs, we optically manipulated the activities of MNs while measuring the activities of these neurons using calcium imaging. To avoid interference with calcium imaging, we applied focused and transient laser light to motor nerves in the periphery to retrogradely stimulate motor neurons. A raise in calcium concentration was observed in the neurites of motor neurons in the corresponding neuromere, indicating that we successfully activated and recorded MNs activities. We then systematically studied the effects of the activity manipulation on motor wave propagation. We found that the local and transient activation of MNs slowed down the wave in the manipulated segment but not in neighboring segments. Furthermore, this phenomenon was observed even when the activity manipulation was applied only to one hemisegment. These results suggest for the presence of neuronal signal(s) from MNs that regulate the propagation of motor wave. Furthermore, the signal(s) appear to be shared between the right and left hemisegment but closed within the segment.

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