脊髄、運動細胞、筋肉 Spinal cord Motor Neurons and Muscle
P1-1-68 サル頸髄前角におけるレンショウ細胞の分布：免疫組織学的解析 Rostrocaudal distribution of calbindin D28k positive cells in the ventral horn of the cervical spinal cord in monkeys ○西丸広史1, 柿崎美代1, 関和彦2 ○Hiroshi Nishimaru1, Miyo Kakizaki1, Kazuhiko Seki2 筑波大・医学医療系・生命医科学域1, 国立精神・神経セ神経研モテル動物2 Div Biomed, Fac Medicine, Univ Tsukuba, Tsukuba1, Dept Neurophysiol, Natl Inst Neurosci, NCNP, Tokyo, Japan2 Renshaw cells (RCs) are excited by axon collaterals of motoneurons (MNs), and in turn, provide recurrent inhibition to MNs. Although this circuit has been studied intensively, its role in motor control remains elusive. In particular, little is known about the functional and anatomical organization of the RCs inhibiting the MNs in the cervical spinal cord which innervate the upper limb muscles in primates. In monkeys, motoneurons innervating hand muscles are mainly located between cervical segment 8 (C8) and thoracic segment 1 (T1) while those controlling the arm and wrist are located in the rostral region (Jenny and Inukai , J. Neurosci., 1983). RCs that are located in the ventral medial region are known to express calbindin D28k, a calcium binding protein in rats (Carr et al. Neuroreport 1998). In this study, we examined the rostrocaudal distribution of calbindin-postive cells in the ventromedial region of the cervical cord of the monkey (macaca fuscata; n =3) to examine whether there is a difference in numbers of RCs along the rostrocaudal axis. Cell bodies immunoreactive to calbindin D28k were distributed along the cervical ventral horn. However, the number of immunopositive cells was higher in the rostral segments (C5-C6) compared to the caudal region (C8-T1). These results indicate that recurrent inhibition is more prominent in regions controlling the proximal muscles than the regions controlling the distal muscles in the upper limb of the monkey. Moreover, similar results were obtained in the cerviacal spinal cord of the rat (n = 3) indicating that such rostrocaudal gradient of the recurrent inhibitory circuit is preserved across different species.	P1-1-69 摘出脳幹脊髄標本における胎動性活動のオレキシンによる調節 Orexinergic modulation on fetal movemnet in rat brainstem-spinal cord preparation ○下村英毅1, 西山紋惠2, 谷澤隆邦1, 山中章弘3, 荒田晶子2 ○Hideki Shimomura1, Ayae Nishiyama2, Takakuni Tanizawa1, Akihiro Yamanaka3, Akiko Arata2 兵庫医科大学　小児科学1, 兵庫医大医生理（生体機能）2, 名古屋大学　環境医学研究所3 Dept. Pediatrics. Hyogo College of Med. Nishinomiya, Japan1, Div Physiome, Dept Physiol, Hyogo College of Med, Hyogo, Japan2, Dept Neurosci II, Research Institute of Environmental Medicine Nagoya Univ, Nagoya, Japan3 Orexin deficiency results in narcolepsy in humans, dogs, and rodents, suggesting that the orexin system is particularly important for maintenance of wakefulness. However, the effects of orexin to the relationship between fetal movement and sleep/wakefulness in fetal stage had not been investigated. In late fetal stage, fetal movement in sleep begins to be synchronous with rapid eye movement, and this state is called the prototype of REM sleep. In fetal rat isolated brainstem-spinal cord preparation, we already observed two kinds of activities recorded from 4th cervical nerve root (C4); one is respiratory activity that is comparatively rhythmic activity corresponding to phrenic nerve activity, and the other is non-respiratory activity (NRA) which is not rhythmic, accompanied by high amplitude. To further investigate the NRA, we developed unilateral forelimb intact preparation to understand correlation between the NRA and the fetal movement. The forelimb movement which was corresponding to a radial nerve activity was synchronized with the NRA. Thus, this movement might be thought to the fetal movement. Additionally in after birth, we could not observe the NRA, but it was induced by strychnine application. Therefore, the critical point of the disappearance of the NRA existed between E19 and E20. The NRA was depressed by application of an antagonist of the NMDA receptor glycine binding site. Thus the NRA depends on the NMDA receptor glycine binding site. In fetal rat, orexin application abolished the NRA and potentiated respiratory activity. Conversely, application to the strychnine induced NRA in neonatal rat preparation showed no alteration. These results showed that there are developmental changes of orexin function to the circuit using glycinergic system in brainstem-spinal cord between fetus and neonate. In addition, orexin might form sleep and wakefulness circuit by modulation of the fetal movement with the NMDA receptor glycine binding site.
P1-1-70 ラット脊髄内における発達期の皮質－運動神経直接シナプスの形態学的解析 Morphological analysis of direct cortico-motoneuronal synapses in the developing rat spinal cord ○亀田浩司1, 福田諭1, 前田仁士1, 村部直之1, 高橋一郎1, 桜井正樹1 ○Hiroshi Kameda1, Satoshi Fukuda1, Hitoshi Maeda1, Naoyuki Murabe1, Ichiro Takahashi1, Masaki Sakurai1 帝京大学 医学部医学科 生理学講座1 Dept Physiol, Teikyo Univ, Tokyo, Japan1 It is well known that corticospinal (CS) neurons make direct synaptic contact with motor neurons in primates, but there is still controversy as to whether this direct contact exists in rodents. Our previous electrophysiological study with optogenetic methods showed that stimulation of CS axons elicited monosynaptic EPSCs in motor neurons innervating forelimb muscles in the infant rat spinal cord. In the present study, we investigated the direct synaptic contact in a morphological manner. We first injected adeno-associated virus vectors expressing channelrhodopsin-2 (ChR2)-EYFP into the newborn rat cerebral cortex to label CS axons. We used ChR2-EYFP because ChR2 is the transmembrane protein, and this tagged EYFP is located just beneath the plasma membranes of CS axons. The virus vectors were stereotaxically injected into the deep layers of the motor area at postnatal day zero. Five days after the injection, we then injected AlexaFluor 594-conjugated cholera toxin β subunit (CTB-Alexa594) into forelimb-muscle groups to retrogradely label motor-neuron pools in the cervical spinal cord. After 72 hours survival, the rats were terminated, and 50-μm-thick coronal sections of the cervical cord were prepared. Double-immunofluorescence labeling for EYFP and CTB revealed that the distributed area of motoneuronal dendrites overlapped with that of CS axons, and close appositions of the dendrites and the axons were frequently observed under a confocal laser-scanning microscopy, indicating the direct synaptic contact between these neuron groups in the infant rat spinal cord. We further investigated the ultrastructure of these close appositions by immunoelectron-microscopic technique to confirm the existence of the direct cortico-motoneuronal synaptic connections.	P1-1-71 マカクザル脊髄損傷後の損傷周囲部にはRGMaが過剰発現される RGMa expression is increased in the peri-lesional sites after spinal cord injury in macaques ○中川浩1,3, 二宮太平2,3, 高田昌彦2,3, 山下俊英1,3 ○Hiroshi Nakagawa1,3, Taihei Ninomiya2,3, Masahiko Takada2,3, Toshihide Yamashita1,3 大阪大学大学院　医学系研究科　分子神経科学1, 京都大学霊長類研究所　統合脳システム2 Dep.of Mol. Neurosci., Grad. Sch. of Med., Osaka Univ., Osaka, Japan1, Sys Neurosci, Primate Res Inst, Kyoto Univ, Aichi, Japan2, Core Research for Evolutional Science and Technology (CREST), Japan Science and Technology Agency (JST)3 Impairment of dexterous finger movements after spinal cord injury (SCI) is often caused by disruption of corticospinal tract projection. Axonal plasticity and/or regeneration after SCI is considered critical for recovery of motor function; however, plastic changes in the adult mammalian central nervous system (CNS) are prevented by the obstacles such as myelin, glial scar, and so on. Repulsive guidance molecule a (RGMa) has been originally identified as an axon guidance molecule in the visual system. We previously reported that RGMa expression is increased around the lesion sites after SCI and is involved in inhibition of axon regeneration in adult rat. Here, we show the expression pattern of RGMa in the spinal cord after injury in adult primates. We made incomplete unilateral lesion at the cervical level of C7/8 in rhesus monkeys. At 10 and 150 days after SCI, we perfused the monkey transcardially with 10% formalin in PB and prepared the cryosection from the spinal cords. Immunohistochemical analysis revealed that RGMa expression was increased around the lesion sites compared with the control. We performed additional immunohistochemical experiments to characterize the cell type expression of RGMa. We found that RGMa was expressed in Iba1 (marker for microglia/macrophages) and MOSP (marker for myelin/oligodendrocyte)-positive cells around the lesion sites. We also examined the expression of neogenin, a receptor for RGMa, in pyramidal neurons in layer V (SMI32) of primary motor cortex. These results indicate that RGMa expression is increased in microglia/macrophages and oligodendrocyte in the adult CNS after SCI. RGMa might be also involved in the axonal regeneration after CNS injury in macaques.
P1-1-72 脊髄交叉型抑制性細胞の役割 The role of commissural inhibitory neurons in spinal cord for locomotion ○佐藤千恵1, 木村有希子1, 東島眞一1 ○Chie Satou1, Yukiko Kimura1, Shinichi Higashijima1 岡崎統合バイオサイエンスセンター1 Okazaki Institute for Integrative Bioscience1 Vertebrates can produce movements of widely varying strengths and speeds such as walking, running or swimming at slow/fast frequency. These rhythmical movements are created by spinal neural networks which consist of various types of excitatory and inhibitory interneurons. Some of the most important inhibitory interneurons in spinal motor networks are commissural ones because they are directly responsible for the patterns of alternation during the bending movements that underlie axial behaviors. Although commissural inhibitory interneurons show morphological and functional diversity, their details are not well understood.In the vertebrate spinal cord, the first step toward achieving the diversity generation occurs early in development with the division of neuronal progenitor cells into distinct domains along dorsoventral axis. Each domain is thought to produce different types of neurons. Therefore, we hypothesized that diversity of commissural inhibitory neurons could, in part, be attributed to their different developmental origins. To examine this hypothesize, we first explore the origin of commissural inhibitory neurons. .Using transgenic fish which label neurons that derived from different domains, we found that commissural inhibitory neurons are produced from several different domains. Among them, we found that two types of neurons, each derived from different domain, rhythmically fire during locomotion. Although axonal length is not significantly different between these two types, their functions appear to be distinct. One class of neurons rhythmically fired consistently at all the swimming cycle, from high frequency swimming to low frequency swimming. By contrast, the other class of neurons was only active at fast swimming frequencies: they did not fire at slow swimming speeds. These data show that the diversity of inhibitory commissural neurons is, at least in part, attributed to their different developmental origins.	P1-1-73 糖尿病性多発性神経炎はラットの後肢の遠位筋を支配するγ運動ニューロンを標的とする Diabetic polyneuropathy targets gamma motoneurons of the distal muscle of hindlimb in STZ rats ○村松憲1, 丹羽正利2, 石黒友康1, 長谷川達也3, 佐々木誠一4 ○Ken Muramatsu1, Masatoshi Niwa2, Tomoyasu Ishiguro1, Tatsuya Hasegawa3, Sei-Ichi Sasaki4 健康科学大学 理学療法学科1, 杏林大学 作業療法学科2, 山梨県環境科学研究所 環境生化学研究室3, 茨城県立医療大学 医科学センター4 Dept Physical Therapy, Health Science Univ, Yamanashi1, Dept. Occupational Therapy, Kyorin Univ, Tokyo2, Dept. of Environmental biochemistry, Yamanashi Institute of Environmental Sciences, Yamanashi3, Center for Medical Sciences, Ibaraki Prefectural Univ of Health Sciences, Ibaraki4 Alterations in the number and size of motoneurons were studied in the medial gastrocnemius (MG) and biceps femoris (BF) motor nucleus of diabetic rats (12 or 22 weeks after injection of storeptozotocin) and age-matched controls. MG and BF motoneurons were retrogradely labeled by dextran, and the number and size of cell bodies were examined. Additionally, morphological alterations of muscle fibers of MG and BF muscles were examined. Significantly fewer labeled MG motoneurons were found in the diabetic rats as compared with BF motoneurons of diabetic animals and age-matched control animals. The mean soma diameters of MG and BF motoneurons were significantly smaller in the diabetic animals. The distribution of average soma diameters in the motoneurons of control animals and BF motoneurons of diabetic animal was bimodal; cells with larger average diameter were presumed to be alpha-motoneurons and those with smaller diameters were presumed to be gamma. On the other hand, the number of smaller MG motoneurons was reduced in 12-week diabetic animals. By 22 weeks, diabetic animals had virtually no small MG motoneurons and the size distribution became unimodal. Abnormal intrafusal muscle fibers were observed in MG muscle of diabetic animals, although structures of extrafusal muscle fibers were preserved. We conclude that it is highly possible that diabetic polyneuropathy predominantly targets gamma motoneurons of the distal muscle in diabetic rats.
P1-1-74 サルの皮質脊髄路損傷後の手指巧緻性回復における脊髄固有ニューロンの寄与の検証 Verified contribution of propriospinal neurons to recovery of hand dexterity after a corticospinal tract lesion in the monkey ○當山峰道1,2, 木下正治1, 松井亮介3, 加藤成樹4, 長谷川拓3, 笠原洋紀3, 伊佐かおる1, 渡邉大3, 小林和人4, 里宇明元5, 伊佐正1,6 ○Takamichi Tohyama1,2, Masaharu Kinoshita1, Ryosuke Matsui3, Shigeki Kato4, Taku Hasegawa3, Hironori Kasahara3, Kaoru Isa1, Dai Watanabe3, Kazuto Kobayashi4, Meigen Liu5, Tadashi Isa1,6 生理研・認知行動発達1, 慶應大・院医・リハ2, 京都大・院医・生体情報3, 福島医大・医・生体機能4, 慶應大・医・リハ5, 総研大6 Dept Dev Physiol, NIPS, Okazaki1, Dept Reha Med, Grad Sch Med, Keio Univ, Tokyo2, Dept Mol and Sys Bio, Grad Sch Bio, Kyoto Univ, Kyoto3, Dept Mol Gene, Inst Bio Sci, Fukushima Med Univ, Fukushima4, Dept Reha Med, Keio Univ Sch Med, Tokyo5, The Grad Univ for Adv Stud, Hayama6 It is generally believed that the direct cortico-motoneuronal connection is essential for the control of dexterous finger movements, like a precision grip, in primates. It has been reported, however, that even after the lesion of corticospinal tract (CST) at C5 segment the precision grip behavior was restored within one to three months, but not restored after CST lesion at C2. These findings suggest that the recovery could be due to neural transmission mediated by neurons located between C2 and C5, presumably the C3-C4 propriospinal neurons (PNs). To show the direct evidence of contribution of the PNs to the recovery, in this study, we applied the pathway-specific and reversible blocking method using double viral vectors (Kinoshita et al. 2012) for the PNs, to one monkey after the C5 CST lesion. We injected retrograde vector (NeuRet) into the C6-T1, where the PNs axon terminates and injected anterograde vector (AAV) into the C3-C5, where the PNs cell body exists. Two months after the injections, we started to administrate doxycycline (Dox) to induce the expression of tetanus neurotoxin under the control of Tet-ON system in the double infected PNs for selective blockade of their synaptic transmission. Two weeks after the start of Dox, we made the CST lesion at C4/C5. We assessed the extent of recovery of a precision grip under continuous administration of Dox for 4.5 months. As a result, recovery of a precision grip was partial, less than in the other monkeys with C5 CST lesion in our previous studies. At the end point, by recording presumably PN-mediated field potential, partial blockade of synaptic transmission through the PNs might be demonstrated. This result suggests that the PNs contribute to recovery of hand dexterity after the C5 CST lesion.

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