Name: the preparation of oblique spinal cord slices for ventral root stimulation
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Description: Watch this Scientific Journal Video about The Preparation of Oblique Spinal Cord Slices for Ventral Root Stimulation at JoVE.com

作者感谢马林·曼努埃尔和Olivia高盛-Szwajkajzer他们在拍摄照片的帮助。作者还感谢阿琼Masukar和托拜厄斯·博克校对稿件。金融支持是由国民法新社提供的倒拉RECHERCHE（HYPER-MND，ANR-2010-汪曾祺，1429至1401年），美国国立卫生研究院，NINDS（R01NS077863）时，蒂埃里Latran基金会（OHEX项目），法国协会肌病（授权编号16026）和Target ALS是表示感谢。菲利克斯乐华是一个"成功承接博士"，从巴黎高等师范学校，卡尚的收件人。

实验是按照欧盟指令（86/609 / CEE和2010-63-UE）和法国的立法进行，由巴黎第五大学伦理委员会的批准。 1.脊髓切片准备<ol><li>每天准备了以下解决方案或提前一天。如果保持过夜，用95％ 的 O 2和5％CO 2的气泡并保持冷藏在密闭瓶。 <ol><li> 制备低钠离子的人工脑脊液（ACSF）:3毫米氯化钾，1mM的的NaH 2 PO 4，230 mM的蔗糖，26毫碳酸氢钠 ，0.8毫米氯化钙2,8毫氯化镁 ，25mM的葡萄糖，0.4毫摩尔抗坏血酸， 1mM的钠kynurenate，2毫丙酮酸钠。气泡以95％ 的 O 2和5％的CO 2（pH 7.4）中。由于钠kynurenate通常很难溶解，一定要在购买材料表中列出的之一。 </li><li> 准备葡萄糖酸钾SOLU灰:130毫葡萄糖酸钾，15mM的氯化钾，0.05毫EGTA，20mM的HEPES，25mM的葡萄糖，1毫钠kynurenate，2毫丙酮酸钠，调节至pH 7.4，用KOH。 </li><li> 制备ACSF:130 mM氯化钠，2.5mM的氯化钾，2mM的氯化钙 ，1mM的MgCl 2的，1毫米的NaH 2 PO 4，26 mM的碳酸氢钠 ，25mM的葡萄糖，0.4毫摩尔抗坏血酸，2mM的丙酮酸钠。气泡以95％ 的 O 2和5％的CO 2（pH 7.4）中。 </li><li>到清扫开始之前，溶解2％琼脂在80毫升的K葡糖溶液，并在60℃下保温。 </li></ol></li><li> 心腔内灌注 <ol><li>执行对雌性和雄性小鼠这种准备，年龄从P2到P11。 </li><li>麻醉小鼠以0.1毫升的25mM戊巴比妥钠（50毫克/千克）腹膜内注射。 </li><li>用针或磁带，固定鼠标在其上的大的陪替氏培养皿填充有硅背面。使用解剖显微镜E对于解剖的其余部分。 </li><li>按住胸骨尖，挺胸，切用细剪刀隔膜。然后通过经由肋切削，露出心脏打开双方的胸部。 </li><li>用27G针的穿刺左心室之前切断右心房。 </li><li>用冰冷的低的 Na +学联灌注。 30秒后，低的 Na +学联应该被看作流出心房。低量钠防止扣球细胞和解剖过程中减少细胞死亡。 </li><li>继续保持在心脏针的解剖显微镜下，直到血的时候排出肝脏变为黄色。 </li></ol></li><li> 脊髓解剖 <ol><li>斩首动物，并把它放在它的肚子。 </li><li>快速去除背面（ 图1A1）的皮肤。提出两个切过肩部和下降胸部笼（ 图1A2）。然后剪断脐带为L流中，以便将脊柱与来自动物的下部肋的开头隔离尾部分尽可能。再次翻转动物和删除仍连接到肋骨内脏。 </li><li>脊柱转移到另一个，更小的硅填充培养皿，并使用4昆虫针将其保持背侧（ 图1A）。 </li><li>连续灌注与卡波金鼓泡ACSF的动物（在约4℃），同时执行背侧的椎板，并从延髓端（ 图1B）暴露脊髓。要做到这一点，插入骨和脊髓之间的细微剪刀尖端和从吻端切成末骨头一点一点，并确保从白质望而却步。而用镊子备用两边各远离骨乐队已经削减（ 图1B1）。 </li><li>使用可用最小的弹簧剪刀和镊子，抬起硬脑膜和削减在两侧的同时，为了避免与剪刀破坏脊髓保持硬脑膜的松散部分。沿着rostro - 尾轴切割。 注:硬脑膜是一个半透明连续膜;在这个年龄段的软脑膜太脆弱，解剖和切片（ 图1B2）时会散开。 </li><li>一旦硬脑膜是除去使用钝的玻璃或塑料尖端轻轻推由半切割脊柱形成的槽的左侧的线，并切在右侧的腹侧和背侧的根，从延髓侧开始，从那里进入帘线（几个毫米至少， 图1B2）最远。 </li><li>重复左侧的操作，总是会从吻端到尾。如果左撇子，从左侧开始，然后移动到右侧。 </li></ol></li><li> 嵌入在琼脂 <ol><li>滑索出了脊柱。使用较小的昆虫针牵制用背索面和微妙删除任何一块膜仍然连接到它（ 图1C1）。 </li><li>一旦清洗，修剪两端（ 图1C2）。插入在帘线操纵线，并注意其方向的前部的弯曲昆虫针（在图1C2的箭头）。然后脊髓转移到冰冷的K-葡糖溶液。 注意:此方法模仿CSF的细胞成分，并防止细胞渗透休克而死亡，一旦运动神经元将被削减26。 </li><li>一旦脊髓是细胞内溶液内，取与琼脂烧杯出干浴并冷却下来的冰和水的混合物中。 </li><li>保持在测量温度下搅拌。当温度达到38℃，浸入脊髓，由昆虫销保持它和喙侧放置下来。确保脊髓是STraight尽量远离墙壁，尾椎部分略微向上（ 图1D1）。 </li><li>留在冰和水的混合物的烧杯，以使琼脂尽快固化。确保它留在地方，该线是尽量伸直（ 图1D1）。 </li></ol></li><li> 切片 <ol><li>凝固后，切割含有以这样的方式，该块的基是在一个35°的角度与电线的腰部脊髓琼脂块（在图1D2的箭头）。背表面应从底部（ 图1D2）面临的路程。 注意:这是在过程中的关键步骤，用于保持运动神经元池的连续性腹侧根部从它们退出。 </li><li>胶块到使用氰基丙烯酸酯胶的vibratome的室。沉浸它在K-葡糖溶液和添加slushed冷冻葡萄糖酸钾溶液保持浴中冷却（2℃以下）。 </li><li>削减350 - 400微米腰部区域的厚片（由它的曲率和直径较大的识别）。在合适的方向（见1.4.1。），使用刀片从背切向腹面并且使切片连续多喙。通常情况下，有4-5延伸2mm或更前根合适的切片。使用以下参数:10°角，70赫兹的振动频率和切片的10毫米/分钟的速度。 </li></ol></li><li> 孵化 <ol><li>转移切片学联在34℃。经过大约30分钟，冷却切片至RT，并开始记录会话。 </li></ol></li></ol><img alt="图1" src="/files/ftp_upload/54525/54525fig1.jpg" /> 图1.解剖 A1:背面以暴露背柱的皮肤去除A2:肩膀和肋骨的切割释放背柱B1:固定到硅填充培养皿脊柱（背侧，尾侧左）B2:同样的，揭露和解剖脊髓C1:脊髓孤立（喙侧左）C2:脊髓可以直接嵌入（腹侧，喙侧左）。注意在喙侧的小昆虫针D1:。在琼脂烧杯脊髓（喙侧道，腹面面朝下）D2:与嵌入式脊髓切琼脂块。注意35度角与该块的基部和腰膨大（箭头）脊髓的形式。比例尺1厘米。 <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/54525/54525fig1large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> 2.放置切成商会注:Ve的ntral根是大小可变的。 <ol><li>准备各种吸管的盒子尖直径范围从40到170微米提前。为了准备吸吸管，用长锥形准备许多吸管。使用钻石刀，使在不同位置切割。然后在解剖显微镜下，通过敲击用镊子的尖端打破它。 </li><li>从记录显微镜移除腔室，并将其放置在解剖显微镜下。 </li><li>选择包含足够的长度（2mm以上）的前根被安装在吸入刺激电极片。选择与前根的切片向上（ 图2A）的适当的取向和微妙切断周围的腹侧根部琼脂同时留下琼脂的其余部分的片（ 图2B）左右。 注意:由于琼脂比切片坚挺，这将允许该切片的锚的螺纹休息的琼脂而不是在切片，因而一个nchor不会损伤组织。确保片的锚固件的螺纹上方的运动神经元池（在图2C中红色示出）。 </li><li>装入室放回显微镜和以1:1的速度连续灌注与ACSF录音室 - 2毫升/分钟，在RT。使用玻璃吸管充满ACSF并连接到注射器，吸腹侧根（在图2C的箭头）中的一个。为了实现腹侧根的良好的刺激，枪头需要周围的腹侧根紧。一个极应该在刺激电极，另一个在浴（或连接到膜片钳电极参考）。 </li><li>实现所需的细胞类型的膜片钳记录和previsouly 10描述记录前根刺激的效果。 <ol><li>这里，使用用于数据采集的放大器。在3 kHz滤波器全细胞记录。数字化在10千赫。补偿真正水流桥电阻NT-钳模式。 </li></ol></li></ol><img alt="图2" src="/files/ftp_upload/54525/54525fig2.jpg" /> 图2.前根准备 答 :嵌入琼脂与前根朝上腰脊髓切片B:腰脊髓切片与琼脂Ç释放了前根:腰脊髓切片紧紧周围放置前根（箭头）刺激电极。注意表达的chrna2 Cre重组鼠28鼠标记者R26 汤姆17。比例尺1毫米越过红色荧光的细胞伦肖的位置。 <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/54525/54525fig2large.jpg" target="_blank">请点击此处查看该图的放大版本。</a>

<ol>
	<li>Brooks, C. M., Downman, C. B., Eccles, J. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=After-potentials+and+excitability+of+spinal+motoneurones+following+antidromic+activation.">After-potentials and excitability of spinal motoneurones following antidromic activation.</a> J Neurophysiol. 13 (1), 9-38 (1950).</li><li>Bories, C., Amendola, J., Lamotte d'Incamps, B., Durand, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Early+electrophysiological+abnormalities+in+lumbar+motoneurons+in+a+transgenic+mouse+model+of+amyotrophic+lateral+sclerosis.">Early electrophysiological abnormalities in lumbar motoneurons in a transgenic mouse model of amyotrophic lateral sclerosis.</a> Eur J Neurosci. 25 (2), 451-459 (2007).</li><li>Takahashi, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Membrane+currents+in+visually+identified+motoneurones+of+neonatal+rat+spinal+cord.">Membrane currents in visually identified motoneurones of neonatal rat spinal cord.</a> J Physiol. 423, 27-46 (1990).</li><li>Hori, N., Tan, Y., Strominger, N. L., Carpenter, D. O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intracellular+activity+of+rat+spinal+cord+motoneurons+in+slices.">Intracellular activity of rat spinal cord motoneurons in slices.</a> J Neurosci Methods. 112 (2), 185-191 (2001).</li><li>Arai, Y., Mentis, G. Z., Wu, J. Y., O'Donovan, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ventrolateral+origin+of+each+cycle+of+rhythmic+activity+generated+by+the+spinal+cord+of+the+chick+embryo.">Ventrolateral origin of each cycle of rhythmic activity generated by the spinal cord of the chick embryo.</a> PLoS One. 2 (5), e417 (2007).</li><li>Cullheim, S., Lipsenthal, L., Burke, R. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Direct+monosynaptic+contacts+between+type-identified+alpha-motoneurons+in+the+cat.">Direct monosynaptic contacts between type-identified alpha-motoneurons in the cat.</a> Brain Res. 308 (1), 196-199 (1984).</li><li>Cullheim, S., Kellerth, J. O., Conradi, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evidence+for+direct+synaptic+interconnections+between+cat+spinal+alpha-motoneurons+via+the+recurrent+axon+collaterals:+a+morphological+study+using+intracellular+injection+of+horseradish+peroxidase.">Evidence for direct synaptic interconnections between cat spinal alpha-motoneurons via the recurrent axon collaterals: a morphological study using intracellular injection of horseradish peroxidase.</a> Brain Res. 132 (1), 1-10 (1977).</li><li>Gogan, P., Gueritaud, J. P., Horcholle-Bossavit, G., Tyc-Dumont, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Direct+excitatory+interactions+between+spinal+motoneurones+of+the+cat.">Direct excitatory interactions between spinal motoneurones of the cat.</a> J Physiol. 272 (3), 755-767 (1977).</li><li>Ichinose, T., Miyata, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Recurrent+excitation+of+motoneurons+in+the+isolated+spinal+cord+of+newborn+rats+detected+by+whole-cell+recording.">Recurrent excitation of motoneurons in the isolated spinal cord of newborn rats detected by whole-cell recording.</a> Neurosci Res. 31 (3), 179-187 (1998).</li><li>Lamotte d'Incamps, B., Ascher, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Four+excitatory+postsynaptic+ionotropic+receptors+coactivated+at+the+motoneuron-Renshaw+cell+synapse.">Four excitatory postsynaptic ionotropic receptors coactivated at the motoneuron-Renshaw cell synapse.</a> J Neurosci. 28 (52), 14121-14131 (2008).</li><li>Renshaw, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Central+effects+of+centripetal+impulses+in+axons+of+spinal+ventral+roots.">Central effects of centripetal impulses in axons of spinal ventral roots.</a> J Neurophysiol. 9, 191-204 (1946).</li><li>Renshaw, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Interaction+of+nerve+impulses+in+the+gray+matter+as+a+mechanism+in+central+inhibition.">Interaction of nerve impulses in the gray matter as a mechanism in central inhibition.</a> Fed Proc. 5 (1 Pt 2), 86 (1946).</li><li>Renshaw, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Observations+on+interaction+of+nerve+impulses+in+the+gray+matter+and+on+the+nature+of+central+inhibition).">Observations on interaction of nerve impulses in the gray matter and on the nature of central inhibition).</a> Am J Physiol. 146, 443-448 (1946).</li><li>Pambo-Pambo, A., Durand, J., Gueritaud, J. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Early+excitability+changes+in+lumbar+motoneurons+of+transgenic+SOD1G85R+and+SOD1G(93A-Low)+mice.">Early excitability changes in lumbar motoneurons of transgenic SOD1G85R and SOD1G(93A-Low) mice.</a> J Neurophysiol. 102 (6), 3627-3642 (2009).</li><li>Quinlan, K. A., Schuster, J. E., Fu, R., Siddique, T., Heckman, C. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Altered+postnatal+maturation+of+electrical+properties+in+spinal+motoneurons+in+a+mouse+model+of+amyotrophic+lateral+sclerosis.">Altered postnatal maturation of electrical properties in spinal motoneurons in a mouse model of amyotrophic lateral sclerosis.</a> J Physiol. 589 (Pt 9), 2245-2260 (2011).</li><li>Martin, E., Cazenave, W., Cattaert, D., Branchereau, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Embryonic+alteration+of+motoneuronal+morphology+induces+hyperexcitability+in+the+mouse+model+of+amyotrophic+lateral+sclerosis.">Embryonic alteration of motoneuronal morphology induces hyperexcitability in the mouse model of amyotrophic lateral sclerosis.</a> Neurobiol Dis. 54, 116-126 (2013).</li><li>Hadzipasic, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Selective+degeneration+of+a+physiological+subtype+of+spinal+motor+neuron+in+mice+with+SOD1-linked+ALS.">Selective degeneration of a physiological subtype of spinal motor neuron in mice with SOD1-linked ALS.</a> Proc Natl Acad Sci U S A. 111 (47), 16883-16888 (2014).</li><li>Wichterle, H., Lieberam, I., Porter, J. A., Jessell, T. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Directed+differentiation+of+embryonic+stem+cells+into+motor+neurons.">Directed differentiation of embryonic stem cells into motor neurons.</a> Cell. 110 (3), 385-397 (2002).</li><li>Tallini, Y. N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=BAC+transgenic+mice+express+enhanced+green+fluorescent+protein+in+central+and+peripheral+cholinergic+neurons.">BAC transgenic mice express enhanced green fluorescent protein in central and peripheral cholinergic neurons.</a> Physiol Genomics. 27 (3), 391-397 (2006).</li><li>Manuel, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fast+kinetics,+high-frequency+oscillations,+and+subprimary+firing+range+in+adult+mouse+spinal+motoneurons.">Fast kinetics, high-frequency oscillations, and subprimary firing range in adult mouse spinal motoneurons.</a> J Neurosci. 29 (36), 11246-11256 (2009).</li><li>Obeidat, A. Z., Nardelli, P., Powers, R. K., Cope, T. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Modulation+of+motoneuron+firing+by+recurrent+inhibition+in+the+adult+rat+in+vivo.">Modulation of motoneuron firing by recurrent inhibition in the adult rat in vivo.</a> J Neurophysiol. 112 (9), 2302-2315 (2014).</li><li>Leroy, F., Lamotte d'Incamps, B., Imhoff-Manuel, R. D., Zytnicki, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Early+intrinsic+hyperexcitability+does+not+contribute+to+motoneuron+degeneration+in+amyotrophic+lateral+sclerosis.">Early intrinsic hyperexcitability does not contribute to motoneuron degeneration in amyotrophic lateral sclerosis.</a> Elife. 3, (2014).</li><li>Leroy, F., Lamotte d'Incamps, B., Zytnicki, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Potassium+currents+dynamically+set+the+recruitment+and+firing+properties+of+F-type+motoneurons+in+neonatal+mice.">Potassium currents dynamically set the recruitment and firing properties of F-type motoneurons in neonatal mice.</a> J Neurophysiol. 114 (3), 1963-1973 (2015).</li><li>Lamotte d'Incamps, B., Ascher, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Subunit+composition+and+kinetics+of+the+Renshaw+cell+heteromeric+nicotinic+receptors.">Subunit composition and kinetics of the Renshaw cell heteromeric nicotinic receptors.</a> Biochem Pharmacol. 86 (8), 1114-1121 (2013).</li><li>Lamotte d'Incamps, B., Krejci, E., Ascher, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanisms+shaping+the+slow+nicotinic+synaptic+current+at+the+motoneuron-renshaw+cell+synapse.">Mechanisms shaping the slow nicotinic synaptic current at the motoneuron-renshaw cell synapse.</a> J Neurosci. 32 (24), 8413-8423 (2012).</li><li>Dugue, G. P., Dumoulin, A., Triller, A., Dieudonne, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Target-dependent+use+of+co-released+inhibitory+transmitters+at+central+synapses.">Target-dependent use of co-released inhibitory transmitters at central synapses.</a> J Neurosci. 25 (28), 6490-6498 (2005).</li><li>Mentis, G. Z., Siembab, V. C., Zerda, R., O'Donovan, M. J., Alvarez, F. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Primary+afferent+synapses+on+developing+and+adult+Renshaw+cells.">Primary afferent synapses on developing and adult Renshaw cells.</a> J Neurosci. 26 (51), 13297-13310 (2006).</li><li>Perry, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Firing+properties+of+Renshaw+cells+defined+by+Chrna2+are+modulated+by+hyperpolarizing+and+small+conductance+ion+currents+Ih+and+ISK.">Firing properties of Renshaw cells defined by Chrna2 are modulated by hyperpolarizing and small conductance ion currents Ih and ISK.</a> Eur J Neurosci. 41 (7), 889-900 (2015).</li><li>Thurbon, D., Luscher, H. R., Hofstetter, T., Redman, S. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Passive+electrical+properties+of+ventral+horn+neurons+in+rat+spinal+cord+slices.">Passive electrical properties of ventral horn neurons in rat spinal cord slices.</a> J Neurophysiol. 79 (5), 2485-2502 (1998).</li><li>Zengel, J. E., Reid, S. A., Sypert, G. W., Munson, J. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Membrane+electrical+properties+and+prediction+of+motor-unit+type+of+medial+gastrocnemius+motoneurons+in+the+cat.">Membrane electrical properties and prediction of motor-unit type of medial gastrocnemius motoneurons in the cat.</a> J Neurophysiol. 53 (5), 1323-1344 (1985).</li><li>Cooper, S., Sherington, C. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gower's+tract+and+spinal+border+cells.">Gower's tract and spinal border cells.</a> Brain. 63, 123-124 (1940).</li><li>Morin, F., Schwartz, H. G., O'Leary, J. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Experimental+study+of+the+spinothalamic+and+related+tracts.">Experimental study of the spinothalamic and related tracts.</a> Acta Psychiatr Neurol Scand. 26 (3-4), 371-396 (1951).</li><li>Sengul, G., Fu, Y., Yu, Y., Paxinos, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Spinal+cord+projections+to+the+cerebellum+in+the+mouse.">Spinal cord projections to the cerebellum in the mouse.</a> Brain Struct Funct. 220 (5), 2997-3009 (2015).</li><li>Russier, M., Carlier, E., Ankri, N., Fronzaroli, L., Debanne, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A-,+T-,+and+H-type+currents+shape+intrinsic+firing+of+developing+rat+abducens+motoneurons.">A-, T-, and H-type currents shape intrinsic firing of developing rat abducens motoneurons.</a> J Physiol. 549 (Pt 1), 21-36 (2003).</li><li>Dourado, M., Sargent, P. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Properties+of+nicotinic+receptors+underlying+Renshaw+cell+excitation+by+alpha-motor+neurons+in+neonatal+rat+spinal+cord).">Properties of nicotinic receptors underlying Renshaw cell excitation by alpha-motor neurons in neonatal rat spinal cord).</a> J Neurophysiol. 87 (6), 3117-3125 (2002).</li><li>Mitra, P., Brownstone, R. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+in+vitro+spinal+cord+slice+preparation+for+recording+from+lumbar+motoneurons+of+the+adult+mouse.">An in vitro spinal cord slice preparation for recording from lumbar motoneurons of the adult mouse.</a> J Neurophysiol. 107 (2), 728-741 (2012).</li><li>Rothman, S. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+neurotoxicity+of+excitatory+amino+acids+is+produced+by+passive+chloride+influx.">The neurotoxicity of excitatory amino acids is produced by passive chloride influx.</a> J Neurosci. 5 (6), 1483-1489 (1985).</li><li>Olney, J. W., Price, M. T., Samson, L., Labruyere, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+role+of+specific+ions+in+glutamate+neurotoxicity.">The role of specific ions in glutamate neurotoxicity.</a> Neurosci Lett. 65 (1), 65-71 (1986).</li></ol>

The authors have nothing to disclose.

脊髓倾斜切片是重要的，因为它允许在一个可靠的，全面和具体的方式的单个椎骨段运动神经元池和伦肖细胞单方面刺激。此外，它允许一个快速的，优雅的和无歧义的识别记录的运动神经元。下一步，我们将突出相比其他片制备方法这一技术的优势，然后我们将强调出最常见的陷阱，以避免在执行此过程。 使用横切片大多数研究的基础上固有的电性能3,14,15,29鉴定。然而，这些差异很大的运动神经元池之间以及运动神经元亚型之间一个给定的池（α-，β-和γ-运动神经元30）内。因此，使用尺寸参数的研究都可能排除较小的伽玛运动神经元。在另一方面，库珀和谢林顿ð在1940年一旁切大组神经细胞，其中已经越过上升轴突31,32猴子和猫的脊髓的腹外侧灰质。除了是接近运动神经元胞体，他们指出，他们似乎从组织学上运动神经元无法区分。最近的一篇文章证实在小鼠33这样的细胞的存在。由于大尺寸的这些细胞和它们的位置，他们可能被错误地列入前研究。我们的鉴定标准不包括假定脊髓边缘细胞，因为它们的轴突不腹侧根项目，同时允许所有运动神经元的可靠识别，无论大小。此外，在分析本征电性能，（ 例如 ，输入电阻），更耗时比逆向动作电位的简单观察。 除了使用固有的电性能，一些studie■使用横切片，通过执行对运动神经元的分子标记的事后分析（如胰岛-1/2 16）或使用生物胞素标号14,17可视化形态确认他们的身份。这样的标识不过是乏味和并不提供直接的鉴定。因此，他们很少有系统地进行。 一些研究试图利用表达基因编码的运动神经元的荧光标记（HB9-GFP小鼠线18鼠标线，聊天-EGFP转基因小鼠，其表达eGFP的胆碱能细胞，包括运动神经元19，或G85R SOD1-YFP的转基因小鼠，其中强烈表达运动神经元17 YFP融合蛋白）。然而，由于遗传标志物表达在时间上，并且由于片通常空调通常是从胚胎或少年采取，标志物的表达可能是不够的年龄在Study进行。其他研究也采用逆行剂喷射到可视化的运动神经元池34。我们还进行了霍乱毒素注射公测到比目鱼或EDL（个人观察）。这种标记技术是很有价值的标记特定的运动神经元池，但需要繁琐的手术实验前几天。此外，在该片的实验正在开展的年龄小，使得它更难显著专门针对肌肉。最后，有通过​​注入外源试剂扰动运动神经元的一个真正的问题。 在最佳条件下，我们能够在几乎所有记录的运动神经元来获得逆向动作电位。所用的35°角度是临界获得足够长的前根。总之，倾斜切片制备提供了一种快速和可靠的方式来确定每个运动神经元，这是优于其他识别技术。如在引言中所述，只有两个研究报道在横切片腹侧根成功刺激。第一项研究记录的颈椎神经元在大鼠和刺激，其中前根桩出现4白色物质。他们成功地诱导在所记录的神经元的85％逆向动作电位。我们相信它们的高的成功率依赖于一个事实，即在颈部水平，轴突和运动神经元的胞体是在同一平面上。这不是在更尾片的情况。即使在颈部水平，它们的横切片仅保留了前根短截线，这是较不可靠的刺激。第二项研究刺激鸡胚后背成腰段5。然而，它们使用电压敏感染料记录，它缺乏空间分辨率，以指示在单细胞水平诱导逆向动作电位的成功。 我们的片preparat离子还提供了刺激伦肖细胞在最近的一篇文章指出28优越的方式。使用倾斜切片，他们能够在一种情况下，这是由10％的成功率的主要改进了46％，记录单突触Renshaw的细胞反应用横切片35遇到。虽然我们无法找到运动神经元池的刺激比较类似，这种观察在伦肖细胞使我们相信，运动神经元还保留更多的连接。 脊髓斜切片是一种要求很高的技术，它需要获得可靠的准备从中可以录制之前大量的练习。使用非常精细和锋利的镊子和微型剪刀是必不可少的。作为整个过程在冷却卡波金鼓泡夹层介质进行花费解剖时间可能很长（达1小时），为长。尽管有一些学者不承认腔内perfusio任何好处n的切片36的制备中，我们决定维持过程的这一步。这是很难客观地测量心内灌注的利益为记录的质量，特别是因为它与年龄而异。 最关键的因素是鼠标的年龄。我们从使用上述协议的P2和P11之间的小鼠成功地记录。过去的这个年龄段，脊髓髓鞘变得过于密集和最大的细胞（运动神经元）因缺氧切片死之前，最有可能的。该窗口后，我们发现越来越难获得健康的切片。近来，新技术已被报道在老年动物17,36以获得健康切片。它们用于额外的添加剂，以它们的解剖和记录介质诸如能量（乙基丙酮酸盐）的补充来源，低级Na +和Cl -的浓度，以防止胀亡细胞37,38和聚乙二醇的限制EFF切割过程中发生的广泛膜横断的学分。通过这样的改进，他们能够从多达6个月大的动物进行录制。 在未来这将是有趣的尝试成年动物的倾斜切片准备的两种技术的优点结合起来。结合这两个协议应该被证明相对容易，并提供一个可靠的工具，以确认运动神经元的身份并研究通过在成年小鼠的运动神经元的轴突络目标人口。此外，一个可以尝试在胚胎小鼠获得斜切片。值得注意的是，我们的制剂也保留了背根，其可以被刺激，研究激活突触我一个支配的运动神经元，以及背侧脊髓神经元群体的活化。总之，前根刺激提供了可靠的工具来确认身份的运动神经元，并研究通过运动神经元轴突络的目标人群。&lt;/ P&gt;

从历史上看，使用锋利的电极运动神经元录音被体内的大型动物，如猫或大鼠1或对小鼠2孤立全脊髓进行。视觉指导下，要达到的膜片钳技术在80年代的出现，要求直接进入体细胞运动神经元的密封需要。因此，脊髓切片准备一直以来随手20世纪90年代3月初实现。然而，早期的切片准备往往没有允许前根的刺激。据我们所知，只有两个研究报道在横切片腹侧根成功刺激，从小鼠4,5-得到无。 在这篇文章中，我们提出了一种技术来实现新生小鼠的存活脊髓切片（P2 - P11），其中运动神经元池保留其神经前根轴突出发。发泄拉尔根刺激引发逆向动作电位放回池中运动神经元从相同的前根离开的肉体。这也激发了运动神经元抵押的目标，其他的运动神经元6-10和伦肖细胞11-13。因为只有运动神经元发送它们的轴突沿着前根中，我们使用逆向动作电位的记录作为一个简单的和明确的方式来physiologicaly确定运动神经元10。 除了使用潜在的非包容性或误导性的电生理和形态准则确认身份的运动神经元，近期对脊髓运动神经元的研究还依赖于繁琐和耗时的事后染色16。这种识别通常只有在记录细胞的样品进行的。其他标识的策略依赖于它的运动神经元表达内源性荧光鼠标线<sup&gt; 17-19。然而，当标志物的表达仍是变量，如果这项研究已经要求使用的转基因小鼠行中使用基因编码的标记可能难以在年轻的时候。另外，逆向动作电位的录音可以从常规细胞记录的发生进行的所有鼠标。在猫，大鼠和小鼠完整脊髓的准备工作实验者，都可靠地使用，因为1950年的1,2,20,21等识别技术。在最佳条件下，我们能够从几乎所有记录的运动神经元的诱发逆向动作电位。 此外，前根刺激可以用于可靠地激发其它运动神经元22,23或它们的靶标。在伦肖细胞10,24,25。我们在这里提出在逆向动作电位记录从运动神经元胞体的形式腹侧根刺激的应用，以及伦肖细胞的激发。

<table><tbody><tr><td>Na-kynurenate</td><td>ABCAM</td><td>ab120256</td><td>dissolves better then other brands</td></tr><tr><td>KCl</td><td>Sigma</td><td>P3911</td><td></td></tr><tr><td>NaH&lt;sub&gt;2&lt;/sub&gt;PO&lt;sub&gt;4&lt;/sub&gt;</td><td>Sigma</td><td>P5655</td><td></td></tr><tr><td>sucrose&amp;nbsp;</td><td>Sigma</td><td>S9378</td><td></td></tr><tr><td>NaHCO&lt;sub&gt;3&lt;/sub&gt;&amp;nbsp;</td><td>Sigma</td><td>S6014</td><td></td></tr><tr><td>CaCl&lt;sub&gt;2&lt;/sub&gt;&amp;nbsp;</td><td>G Biosciences</td><td>R040</td><td></td></tr><tr><td>MgCl&lt;sub&gt;2&lt;/sub&gt;&amp;nbsp;</td><td>Quality Biological</td><td>351-033-721</td><td></td></tr><tr><td>glucose&amp;nbsp;</td><td>Sigma</td><td>G5767</td><td></td></tr><tr><td>ascorbic acid&amp;nbsp;</td><td>Sigma</td><td>A5960</td><td></td></tr><tr><td>Na-pyruvate&amp;nbsp;</td><td>Sigma</td><td>P2250</td><td></td></tr><tr><td>K-gluconate&amp;nbsp;</td><td>Sigma</td><td>P1847</td><td></td></tr><tr><td>EGTA&amp;nbsp;</td><td>Sigma</td><td>E3889</td><td></td></tr><tr><td>HEPES&amp;nbsp;</td><td>Sigma</td><td>H4034</td><td></td></tr><tr><td>NaCl</td><td>Sigma</td><td>S9888</td><td></td></tr><tr><td>Agar</td><td>Sigma</td><td>A9799</td><td></td></tr><tr><td>QX-314</td><td>Alomone</td><td>Q150</td><td></td></tr><tr><td>Mg-ATP</td><td>Sigma</td><td>A9187</td><td></td></tr><tr><td>CsOH</td><td>Sigma</td><td>232041</td><td></td></tr><tr><td>Na-GTP</td><td>Sigma</td><td>51120</td><td></td></tr><tr><td>gluconic acid</td><td>Sigma</td><td>G1951</td><td></td></tr><tr><td>Cesium hydroxide solution</td><td>Sigma</td><td>232041</td><td></td></tr><tr><td>KOH</td><td>Sigma</td><td>P5958</td><td></td></tr><tr><td>Vannas Spring Scissors - 2.5mm&amp;nbsp;</td><td>FST</td><td>15000-08</td><td>only use for cutting the dura, might get damaged if cutting bones</td></tr><tr><td>Stimulator</td><td>A-M Systems</td><td>Isolated Pulse Stimulator Model 2100</td><td></td></tr><tr><td>Vibratome</td><td>Campden</td><td>Vibrating Microtome 7000 - Model 7000smz-2</td><td></td></tr></tbody></table>

the preparation of oblique spinal cord slices for ventral root stimulation

从脊髓切片电生理记录已经被证明是调查范围广泛的问题，对网络性能的有价值的技术，从细胞。我们展示如何准备年轻小鼠（P2 - P11）脊髓可行的斜片。在此制备方法中，运动神经元保留它们的轴突从脊髓的腹侧根部出来。这些轴突的刺激引起背传播动作电位侵入脊髓中运动神经元胞体和令人兴奋的运动神经元的抵押品。逆向动作电位记录是表征运动神经元的身份，这超过了其他的识别方法的直接，明确的和优雅的方式。此外，刺激运动神经元络是一种简单而可靠的方式来激发运动神经元的脊髓内抵押品目标，如其它运动神经元或Renshaw细胞。在这个协议中，我们提出从运动神经元胞体以及伦肖细胞激发逆向录音，腹侧根刺激所致。

采用逆向动作电位运动神经元身份确认 细胞靶向 运动神经元在腹角（在红色可见于图2C）中找到。从形成前根轴突束，至多直至完全捆绑分散一开始看到的大细胞（SOMA长轴线，高于20微米）。实现使用3初始电阻的电极至4MΩ圆形健康寻找细胞的全细胞记录。所使用的内部溶液含有:140毫K -葡糖酸盐，6毫米氯化钾，10mM的HEPES，1mM的EGTA，0.1mM的氯化钙 ，4毫镁ATP，0.3毫的 Na 2 GTP。通过加入蒸馏水295毫渗量 - 调节pH值至7.3，用KOH，和渗透压至285。 <strong&gt;的运动神经元身份确认一旦全细胞记录，实现，应用前根的单个双相刺激（1 - 50伏，0.1 - 0.3毫秒）在腹侧脊髓的记录细胞以引起逆向动作电位图3A1和3A2显示逆向动作。电位分别电压钳或电流钳。当从1到5伏（ 图3A1），并从提高刺激强度10到15 V（ 图3A2）的逆向动作电位出现在一个全有或全无的方式分别与0.9毫秒，1.1毫秒，一个延迟。鉴于只有运动神经元通过前根送他们的轴突，逆行的动作电位是细胞身份的证明。 在运动神经元（约10％）的少数，前根刺激未能引起逆向动作电位而引起的顺动作电位。 图3B1和3B2分别表示电压钳或电流钳顺动作电位。当从20增加的刺激至30 V（ 图3B1）和从25到40 V（ 图3B2），将出现一个动作电位，以下以前兴奋性突触后电流（EPSC）或兴奋性突触后电位（EPSP）。在动作电位的潜伏期较长（分别为5.1毫秒，5.3毫秒，）。在这两种细胞中，刺激神经前根引起了前馈激励（运动神经元轴突发送络对自己和其他运动神经元），这是强大到足以引起的顺动作电位。在这些细胞中，以引发逆向动作电位的失败（特点是它的全有或全无的方式和潜伏期短于5毫秒）不允许我们得出结论，这些细胞AR<html> Ë运动神经元。注意，所要求的刺激强度以引发前馈激励比以引起逆向动作电位所需要的一种高级。这里所示的所有刺激是0.1毫秒，但有时，较长的刺激（高达0.3毫秒），可能需要以引起逆向动作电位。 <img alt="图3" src="/files/ftp_upload/54525/54525fig3.jpg" /> 图3.运动神经元回应前根刺激 A1:1 V（红色曲线）和5 V（黑色线）刺激A2:10 V（红色曲线）和15 V（黑色线）刺激B1:20 V（红色曲线）和30 V（黑色线）刺激。B2:25 V（红色曲线）和40 V（黑色线）刺激。黑点表示刺激时机。双箭头强调邻和逆向之间的尖峰延迟的差异。M /文件/ ftp_upload / 54525 / 54525fig3large.jpg"目标="_空白"&gt;点击此处查看该图的放大版本。 伦肖细胞刺激 细胞靶向 伦肖细胞腹角27,28被发现。可视化与使用20X和63X一个目标微分干涉对比（DIC）成像轴突束。从形成腹侧根轴突束就上去直到轴突开始分散，但仍然明显的（ 图4A，箭头）。瞄准中间尺寸的细胞（约10 - 15微米的直径， 图4A中 ，箭头）。实现使用5初始电阻的电极7MΩ圆看起来健康的细胞的全细胞记录。根据不同的实验中，我们使用EI疗法铯基于或K +的内部解决方案10。在基于CS的解决方案，防止大的钾电流的是大约-45 mV的可见。在一些实验中，我们还增加了的5mM QX-314阻断负责在记录单元扣球钠通道。在Cs +的溶液含有:125毫铯葡糖酸盐，5mM的QX-314氯，10毫米的HEPES，10mM的EGTA，1mM的氯化钙 ，4毫镁ATP和0.4mM的钠-GTP，用pH值调至7.3用CsOH。在K +的溶液应被用于分析，无论是在电压和电流钳，伦肖细胞应答前根刺激的条件近似尽可能生理的。此溶液含有:125毫K -葡糖酸盐，10mM的HEPES，1mM的EGTA，0.1mM的氯化钙 ，4毫镁ATP，0.4mM的钠GTP，以调节至7.3，用KOH将pH。 10和100伏，其持续时间之间变化的刺激强度50和300微秒之间变化。 Bipo<html> LAR脉冲，在所有情况下使用。 在伦肖细胞的响应前根刺激 前根的刺激引发谷氨酸和运动神经元络乙酰胆碱到伦肖细胞10 corelease。通过GABA和甘氨酸介导的前馈抑制还招募10。 图4B显示向腹侧根的单一刺激的反应。细胞被记录在与基于铯溶液的电压钳模式，并维持在-45 mV的电压。 QX-314细胞内液从发射和GABA防止细胞并阻止分别为3μMgabazine和1μM士的宁，甘氨酸的反应。在图4B的向内的突触电流是谷氨酸和烟碱电流的总和。 <html> NT"FO:保together.within页="1"&gt; <img alt="图4" src="/files/ftp_upload/54525/54525fig4.jpg" /> 来自前根图4.伦肖细胞刺激 使用两个不同的放大倍数（20X和40X的目标）采取了同样的片斜冷凝器收购伦肖细胞池图像: 一个 。注意运动神经元轴突合并到前根（箭头）。假定伦肖细胞以表示箭头。比例尺:100微米在A1中，50微米A2 B:一个伦肖细胞的电压钳记录下列腹侧根（黑点）的刺激。红色曲线是灰色的人的平均水平。保持电压设定为-45毫伏。 QX-314在细胞内的解决方案，从发射和GABA防止细胞并阻止分别为3μMgabazine和1μM士的宁，甘氨酸的反应。黑点表示刺激时机。COM /文件/ ftp_upload / 54525 / 54525fig4large.jpg"目标="_空白"&gt;点击此处查看该图的放大版本。

Watch this Scientific Journal Video about The Preparation of Oblique Spinal Cord Slices for Ventral Root Stimulation at JoVE.com

The Preparation of Oblique Spinal Cord Slices for Ventral Root Stimulation

我们显示如何在年轻小鼠制备脊髓倾斜切片。该制剂允许前根的刺激。

斜脊髓切片用于神经前根刺激的制备

Electrophysiological recordings from spinal cord slices have proven to be a valuable technique to investigate a wide range of questions, from cellular to ...

the-preparation-oblique-spinal-cord-slices-for-ventral-root

Universidad San Sebastian

Research

JoVE Journal

Neuroscience

9.4K 次观看.  Centre de Neurophysique, Physiologie et Pathologie, Université Paris Descartes. 我们显示如何在年轻小鼠制备脊髓倾斜切片。该制剂允许前根的刺激。 

视频: The Preparation of Oblique Spinal Cord Slices for Ventral Root Stimulation

Dissection and Culture of Commissural Neurons from Embryonic Spinal Cord

Commissural neurons have been widely used to investigate the mechanisms underlying axon guidance during embryonic spinal cord development. The cell bodies of these neurons are located in the dorsal spinal cord and their axons follow stereotyped trajectories during embryonic development. Commissural axons initially project ventrally towards the floorplate. After crossing the midline, these axons turn anteriorly and project towards the brain. Each of these steps is regulated by the action of several guidance cues. Cultures highly enriched in commissural neurons are ideally suited for many experiments addressing the mechanisms of axon pathfinding, including turning assays, immunochemistry and biochemistry. Here, we describe a method to dissect and culture commissural neurons from E13 rat dorsal spinal cord. First, the spinal cord is isolated and dorsal strips are dissected out. The dorsal tissue is then dissociated into a cell suspension by trypsinization and mechanical disruption. Neurons are plated onto poly-L-lysine-coated glass coverslips or tissue-culture dishes. After 30 hours in vitro, most neurons have extended an axon. The purity of the culture (Yam et al. 2009), typically over 90%, can be assessed by immunolabeling with the commissural neuron markers DCC, LH2 and TAG1 (Helms and Johnson, 1998). This neuronal preparation is a useful tool for in vitro studies of the cellular and molecular mechanisms of commissural axon growth and guidance during spinal cord development.

This video demonstrates a method to dissect and culture commissural neurons from E13 rat dorsal spinal cord. Dissociated commissural neurons are useful to study the cellular and molecular mechanisms of axon growth and guidance.

从胚胎脊髓​​连合神经的解剖和文化

Commissural neurons have been widely used to investigate the mechanisms underlying axon guidance during embryonic spinal cord development. The cell bodies ...

科研

神经科学

Spinal Cord Electrophysiology

The neonatal mouse spinal cord is a model for studying the development of neural circuitries and locomotor movement. We demonstrate the spinal cord dissection and preparation of recording bath artificial cerebrospinal fluid used for locomotor studies. Once dissected, the spinal cord ventral nerve roots can be attached to a recording electrode to record the electrophysiologic signals of the central pattern generating circuitry within the lumbar cord.

A demonstration of the isolation of neonatal mouse spinal cord for electrophysiologic studies.

脊髓电

The neonatal mouse spinal cord is a model for studying the development of neural circuitries and locomotor movement. We demonstrate the spinal cord ...

生物学

Acute Dissociation of Lamprey Reticulospinal Axons to Enable Recording from the Release Face Membrane of Individual Functional Presynaptic Terminals

Synaptic transmission is an extremely rapid process. Action potential driven influx of Ca2+&#160;into the presynaptic terminal, through voltage-gated calcium channels (VGCCs) located in the release face membrane, is the trigger for vesicle fusion and neurotransmitter release. Crucial to the rapidity of synaptic transmission is the spatial and temporal synchrony between the arrival of the action potential, VGCCs and the neurotransmitter release machinery. The ability to directly record Ca2+&#160;currents from the release face membrane of individual presynaptic terminals is imperative for a precise understanding of the relationship between presynaptic Ca2+&#160;and neurotransmitter release. Access to the presynaptic release face membrane for electrophysiological recording is not available in most preparations and presynaptic Ca2+&#160;entry has been characterized using imaging techniques and macroscopic current measurements &#8211; techniques that do not have sufficient temporal resolution to visualize Ca2+ entry. The characterization of VGCCs directly at single presynaptic terminals has not been possible in central synapses and has thus far been successfully achieved only in the calyx-type synapse of the chick ciliary ganglion and in rat calyces. We have successfully addressed this problem in the giant reticulospinal synapse of the lamprey spinal cord by developing an acutely dissociated preparation of the spinal cord that yields isolated reticulospinal axons with functional presynaptic terminals devoid of postsynaptic structures. We can fluorescently label and identify individual presynaptic terminals and target them for recording. Using this preparation, we have characterized VGCCs directly at the release face of individual presynaptic terminals using immunohistochemistry and electrophysiology approaches. Ca2+ currents have been recorded directly at the release face membrane of individual presynaptic terminals, the first such recording to be carried out at central synapses.

Recording Ca2+ currents at the presynaptic release face membrane is key to a precise understanding of Ca2+ entry and neurotransmitter release. We present an acute dissociation of the lamprey spinal cord that yields functional isolated reticulospinal axons, permitting recording directly from the release face membrane of individual presynaptic terminals.

七鳃鳗网状脊髓轴突急性分解到从单个功能突触前终端剥离面膜启用记录

Synaptic transmission is an extremely rapid process. Action potential driven influx of Ca2+&#160;into the presynaptic terminal, through voltage-gated ...

Intramuscular Injections Along the Motor End Plates: A Minimally Invasive Approach to Shuttle Tracers Directly into Motor Neurons

Diseases affecting the integrity of spinal cord motor neurons are amongst the most debilitating neurological conditions. Over the last decades, the development of several animal models of these neuromuscular disorders has provided the scientific community with different therapeutic scenarios aimed at delaying or reversing the progression of these conditions. By taking advantage of the retrograde machinery of neurons, one of these approaches has been to target skeletal muscles in order to shuttle therapeutic genes into corresponding spinal cord motor neurons. Although once promising, the success of such gene delivery approach has been hampered by the sub-optimal number of transduced motor neurons it has so far shown to yield. Motor end plates (MEPs) are highly specialized regions on the skeletal musculature that are in direct synaptic contact to the spinal cord &#945; motor neurons. In this regard, it is important to note that, so far, the efforts to retrogradely transfer genes into motor neurons were made without reference to the location of the MEP region in the targeted muscles. Here, we describe a simple protocol 1) to reveal the exact location of the MEPs on the surface of skeletal muscles and 2) to use this information to guide the intramuscular delivery and subsequent optimal retrograde transport of retrograde tracers into motor neurons. We hope to utilize the results from these tracing experiments in further studies into investigating retrograde transport of therapeutic genes to spinal cord motor neurons through the targeting of MEPs.

The efficacy of intramuscular uptake and retrograde transport of molecules to corresponding motor neurons depends on the location of the injection sites with respect to the motor end plates (MEPs). Here, we describe how to locate MEPs on skeletal muscles to optimise retrograde transport of tracers into motor neurons.

肌肉注射沿运动终板:微创方法来接送示踪剂直接为运动神经元

Diseases affecting the integrity of spinal cord motor neurons are amongst the most debilitating neurological conditions. Over the last decades, the ...

Zebrafish In Situ Spinal Cord Preparation for Electrophysiological Recordings from Spinal Sensory and Motor Neurons

Zebrafish, first introduced as a developmental model, have gained popularity in many other fields. The ease of rearing large numbers of rapidly developing organisms, combined with the embryonic optical clarity, served as initial compelling attributes of this model. Over the past two decades, the success of this model has been further propelled by its amenability to large-scale mutagenesis screens and by the ease of transgenesis. More recently, gene-editing approaches have extended the power of the model.
For neurodevelopmental studies, the zebrafish embryo and larva provide a model to which multiple methods can be applied. Here, we focus on methods that allow the study of an essential property of neurons, electrical excitability. Our preparation for the electrophysiological study of zebrafish spinal neurons involves the use of veterinarian suture glue to secure the preparation to a recording chamber. Alternative methods for recording from zebrafish embryos and larvae involve the attachment of the preparation to the chamber using a fine tungsten pin1,2,3,4,5. A tungsten pin is most often used to mount the preparation in a lateral orientation, although it has been used to mount larvae dorsal-side up4. The suture glue has been used to mount embryos and larvae in both orientations. Using the glue, a minimal dissection can be performed, allowing access to spinal neurons without the use of an enzymatic treatment, thereby avoiding any resultant damage. However, for larvae, it is necessary to apply a brief enzyme treatment to remove the muscle tissue surrounding the spinal cord. The methods described here have been used to study the intrinsic electrical properties of motor neurons, interneurons, and sensory neurons at several developmental stages6,7,8,9.

Zebrafish In Situ Spinal Cord Preparation for Electrophysiological Recordings from Spinal Sensory and Motor Neurons

This manuscript describes methods for electrophysiological recordings from spinal neurons of zebrafish embryos and larvae. The preparation maintains neurons in situ and often involves minimum dissection. These methods allow for the electrophysiological study of a variety of spinal neurons, from the initial electrical excitability acquisition through the early larval stages.

斑马鱼<em&gt;原位</em&gt;脊髓制备用于脊髓感觉和运动神经元的电生理记录

Zebrafish, first introduced as a developmental model, have gained popularity in many other fields. The ease of rearing large numbers of rapidly developing ...

Preparation of Acute Spinal Cord Slices for Whole-cell Patch-clamp Recording in Substantia Gelatinosa Neurons

Recent whole-cell patch-clamp studies from substantia gelatinosa (SG) neurons have provided a large body of information about the spinal mechanisms underlying sensory transmission, nociceptive regulation, and chronic pain or itch development. Implementations of electrophysiological recordings together with morphological studies based on the utility of acute spinal cord slices have further improved our understanding of neuronal properties and the composition of local circuitry in SG. Here, we present a detailed and practical guide for the preparation of spinal cord slices and show representative whole-cell recording and morphological results. This protocol permits ideal neuronal preservation and can mimic in vivo conditions to a certain extent. In summary, the ability to obtain an in vitro preparation of spinal cord slices enables stable current- and voltage-clamp recordings and could thus facilitate detailed investigations into the intrinsic membrane properties, local circuitry and neuronal structure using diverse experimental approaches.

Here, we describe the essential steps for whole-cell patch-clamp recordings made from substantia gelatinosa (SG) neurons in the in vitro spinal cord slice. This method allows the intrinsic membrane properties, synaptic transmission and morphological characterization of SG neurons to be studied.

凝胶神经元全细胞斑块夹的急性脊髓切片的制备

Recent whole-cell patch-clamp studies from substantia gelatinosa (SG) neurons have provided a large body of information about the spinal mechanisms ...

Preparation of Rhythmically-active In Vitro Neonatal Rodent Brainstem-spinal Cord and Thin Slice

Mammalian inspiratory rhythm is generated from a neuronal network in a region of the medulla called the preB&#246;tzinger complex (pBC), which produces a signal driving the rhythmic contraction of inspiratory muscles. Rhythmic neural activity generated in the pBC and carried to other neuronal pools to drive the musculature of breathing may be studied using various approaches, including en bloc nerve recordings and transverse slice recordings. However, previously published methods have not extensively described the brainstem-spinal cord dissection process in a transparent and reproducible manner for future studies. Here, we present a comprehensive overview of a method used to reproducibly cut rhythmically-active brainstem slices containing the necessary and sufficient neuronal circuitry for generating and transmitting inspiratory drive. This work builds upon previous brainstem-spinal cord electrophysiology protocols to enhance the likelihood of reliably obtaining viable and rhythmically-active slices for recording neuronal output from the pBC, hypoglossal premotor neurons (XII pMN), and hypoglossal motor neurons (XII MN). The work presented expands upon previous published methods by providing detailed, step-by-step illustrations of the dissection, from whole rat pup, to in vitro slice containing the XII rootlets.

This protocol both visually communicates the brainstem-spinal cord preparation and clarifies the preparation of brainstem transverse slices in a comprehensive step-by-step manner. It was designed to increase reproducibility and enhance the likelihood of obtaining viable, long lasting, rhythmically-active slices for recording neural output from the respiratory regions of the brainstem.

头脊髓和薄片体外制备心律失常活性

Mammalian inspiratory rhythm is generated from a neuronal network in a region of the medulla called the preB&#246;tzinger complex (pBC), which produces a ...

In Vitro Wedge Slice Preparation for Mimicking In Vivo Neuronal Circuit Connectivity

In vitro slice electrophysiology techniques measure single-cell activity with precise electrical and temporal resolution. Brain slices must be relatively thin to properly visualize and access neurons for patch-clamping or imaging, and in vitro examination of brain circuitry is limited to only what is physically present in the acute slice. To maintain the benefits of in vitro slice experimentation while preserving a larger portion of presynaptic nuclei, we developed a novel slice preparation. This &#8220;wedge slice&#8221; was designed for patch-clamp electrophysiology recordings to characterize the diverse monaural, sound-driven inputs to medial olivocochlear (MOC) neurons in the brainstem. These neurons receive their primary afferent excitatory and inhibitory inputs from neurons activated by stimuli in the contralateral ear and corresponding cochlear nucleus (CN). An asymmetrical brain slice was designed which is thickest in the rostro-caudal domain at the lateral edge of one hemisphere and then thins towards the lateral edge of the opposite hemisphere. This slice contains, on the thick side, the auditory nerve root conveying information about auditory stimuli to the brain, the intrinsic CN circuitry, and both the disynaptic excitatory and trisynaptic inhibitory afferent pathways that converge on contralateral MOC neurons. Recording is performed from MOC neurons on the thin side of the slice, where they are visualized using DIC optics for typical patch-clamp experiments. Direct stimulation of the auditory nerve is performed as it enters the auditory brainstem, allowing for intrinsic CN circuit activity and synaptic plasticity to occur at synapses upstream of MOC neurons. With this technique, one can mimic in vivo circuit activation as closely as possible within the slice. This wedge slice preparation is applicable to other brain circuits where circuit analyses would benefit from preservation of upstream connectivity and long-range inputs, in combination with the technical advantages of in vitro slice physiology.

Integration of diverse synaptic inputs to neurons is best measured in a preparation that preserves all pre-synaptic nuclei for natural timing and circuit plasticity, but brain slices typically sever many connections. We developed a modified brain slice to mimic in vivo circuit activity while maintaining in vitro experimentation capability.

体外楔形切片准备模拟在Vivvo神经元电路连接

In vitro slice electrophysiology techniques measure single-cell activity with precise electrical and temporal resolution. Brain slices must be relatively ...

Recording Network Activity in Spinal Nociceptive Circuits Using Microelectrode Arrays

The roles and connectivity of specific types of neurons within the spinal cord dorsal horn (DH) are being delineated at a rapid rate to provide an increasingly detailed view of the circuits underpinning spinal pain processing. However, the effects of these connections for broader network activity in the DH remain less well understood because most studies focus on the activity of single neurons and small microcircuits. Alternatively, the use of microelectrode arrays (MEAs), which can monitor electrical activity across many cells, provides high spatial and temporal resolution of neural activity. Here, the use of MEAs with mouse spinal cord slices to study DH activity induced by chemically stimulating DH circuits with 4-aminopyridine (4-AP) is described. The resulting rhythmic activity is restricted to the superficial DH, stable over time, blocked by tetrodotoxin, and can be investigated in different slice orientations. Together, this preparation provides a platform to investigate DH circuit activity in tissue from na&#239;ve animals, animal models of chronic pain, and mice with genetically altered nociceptive function. Furthermore, MEA recordings in 4-AP-stimulated spinal cord slices can be used as a rapid screening tool to assess the capacity of novel antinociceptive compounds to disrupt activity in the spinal cord DH.

The combined use of microelectrode array technology and 4-aminopyridine-induced chemical stimulation for investigating network-level nociceptive activity in the spinal cord dorsal horn is outlined.

使用微电极阵列记录脊髓伤害感受回路中的网络活动

The roles and connectivity of specific types of neurons within the spinal cord dorsal horn (DH) are being delineated at a rapid rate to provide an ...

了解运动神经元对脊髓刺激的反应

The Ex vivo Preparation of Spinal Cord Slice for the Whole-Cell Patch-Clamp Recording in Motor Neurons During Spinal Cord Stimulation

Spinal cord stimulation (SCS) can effectively restore locomotor function after spinal cord injury (SCI). Because the motor neurons are the final unit to execute sensorimotor behaviors, directly studying the electrical responses of motor neurons with SCS can help us understand the underlying logic of spinal motor modulation. To simultaneously record diverse stimulus characteristics and cellular responses, a patch-clamp is a good method to study the electrophysiological characteristics at a single-cell scale. However, there are still some complex difficulties in achieving this goal, including maintaining cell viability, quickly separating the spinal cord from the bony structure, and using the SCS to successfully induce action potentials. Here, we present a detailed protocol using patch-clamp to study the electrical responses of motor neurons to SCS with high spatiotemporal resolution, which can help researcher improve their skills in separating the spinal cord and maintaining the cell viability at the same time to smoothly study the electrical mechanism of SCS on motor neuron and avoid unnecessary trial and mistake.

作者聚焦:推进脊髓刺激 - 通过膜片钳电生理学探索运动神经元的细胞反应 

This protocol describes a method using a patch-clamp to study the electrical responses of motor neurons to spinal cord stimulation (SCS) with high spatiotemporal resolution, which can help researchers improve their skills in separating the spinal cord and maintaining cell viability simultaneously.

脊髓切片 的离体 制备，用于脊髓刺激过程中运动神经元的全细胞膜片钳记录

Spinal cord stimulation (SCS) can effectively restore locomotor function after spinal cord injury (SCI). Because the motor neurons are the final unit to ...

斜脊髓切片用于神经前根刺激的制备

摘要

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此视频中的章节

从胚胎脊髓连合神经的解剖和文化

脊髓电

七鳃鳗网状脊髓轴突急性分解到从单个功能突触前终端剥离面膜启用记录

肌肉注射沿运动终板:微创方法来接送示踪剂直接为运动神经元

斑马鱼

凝胶神经元全细胞斑块夹的急性脊髓切片的制备

头脊髓和薄片体外制备心律失常活性

体外楔形切片准备模拟在Vivvo神经元电路连接

使用微电极阵列记录脊髓伤害感受回路中的网络活动

脊髓切片的离体制备，用于脊髓刺激过程中运动神经元的全细胞膜片钳记录

斜脊髓切片用于神经前根刺激的制备

摘要

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此视频中的章节

从胚胎脊髓​​连合神经的解剖和文化

脊髓电

七鳃鳗网状脊髓轴突急性分解到从单个功能突触前终端剥离面膜启用记录

肌肉注射沿运动终板:微创方法来接送示踪剂直接为运动神经元

斑马鱼

凝胶神经元全细胞斑块夹的急性脊髓切片的制备

头脊髓和薄片体外制备心律失常活性

体外楔形切片准备模拟在Vivvo神经元电路连接

使用微电极阵列记录脊髓伤害感受回路中的网络活动

脊髓切片 的离体 制备，用于脊髓刺激过程中运动神经元的全细胞膜片钳记录

从胚胎脊髓连合神经的解剖和文化

脊髓切片的离体制备，用于脊髓刺激过程中运动神经元的全细胞膜片钳记录