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/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/files/ftp_upload/54525/54525fig2large.jpg" target="_blank">请点击此处查看该图的放大版本。</a>

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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?...

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 >NaH2PO4</td>
			<td >Sigma</td>
			<td >P5655</td>
			<td></td>
		</tr>
		<tr >
			<td >sucrose&#160;</td>
			<td >Sigma</td>
			<td >S9378</td>
			<td></td>
		</tr>
		<tr >
			<td >NaHCO3&#160;</td>
			<td >Sigma</td>
			<td >S6014</td>
			<td></td>
		</tr>
		<tr >
			<td >CaCl2&#160;</td>
			<td >G Biosciences</td>
			<td >R040</td>
			<td></td>
		</tr>
		<tr >
			<td >MgCl2&#160;</td>
			<td >Quality Biological</td>
			<td >351-033-721</td>
			<td></td>
		</tr>
		<tr >
			<td >glucose&#160;</td>
			<td >Sigma</td>
			<td >G5767</td>
			<td></td>
		</tr>
		<tr >
			<td >ascorbic acid&#160;</td>
			<td >Sigma</td>
			<td >A5960</td>
			<td></td>
		</tr>
		<tr >
			<td >Na-pyruvate&#160;</td>
			<td >Sigma</td>
			<td >P2250</td>
			<td></td>
		</tr>
		<tr >
			<td >K-gluconate&#160;</td>
			<td >Sigma</td>
			<td >P1847</td>
			<td></td>
		</tr>
		<tr >
			<td >EGTA&#160;</td>
			<td >Sigma</td>
			<td >E3889</td>
			<td></td>
		</tr>
		<tr >
			<td >HEPES&#160;</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&#160;</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 colspan="2">Isolated Pulse Stimulator Model 2100</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?...

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

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

Spinal Cord Electrophysiology II: Extracellular Suction Electrode Fabrication

Development of neural circuitries and locomotion can be studied using neonatal rodent spinal cord central pattern generator (CPG) behavior. We demonstrate a method to fabricate suction electrodes that are used to examine CPG activity, or fictive locomotion, in dissected rodent spinal cords. The rodent spinal cords are placed in artificial cerebrospinal fluid and the ventral roots are drawn into the suction electrode. The electrode is constructed by modifying a commercially available suction electrode. A heavier silver wire is used instead of the standard wire given by the commercially available electrode. The glass tip on the commercial electrode is replaced with a plastic tip for increased durability. We prepare hand drawn electrodes and electrodes made from specific sizes of tubing, allowing consistency and reproducibility. Data is collected using an amplifier and neurogram acquisition software. Recordings are performed on an air table within a Faraday cage to prevent mechanical and electrical interference, respectively.

A demonstration of the fabrication and use of an extracellular suction electrode used to measure electrophysiological recordings of neonatal rodent spinal cords in vitro

脊髓电二:外吸电极的制作

Development of neural circuitries and locomotion can be studied using neonatal rodent spinal cord central pattern generator (CPG) behavior. We demonstrate ...

科研

神经科学

Whole Mount Preparation of the Adult Drosophila Ventral Nerve Cord for Giant Fiber Dye Injection

To analyze the axonal and dendritic morphology of neurons, it is essential to obtain accurate labeling of neuronal structures. Preparing well labeled samples with little to no tissue damage enables us to analyze cell morphology and to compare individual samples to each other, hence allowing the identification of mutant anomalies.
In the demonstrated dissection method the nervous system remains mostly inside the adult fly. Through a dorsal incision, the abdomen and thorax are opened and most of the internal organs are removed. Only the dorsal side of the ventral nerve cord (VNC) and the cervical connective 
(CvC) containing the big axons of the giant fibers (GFs)1 are exposed, while the brain containing the GF cell body and dendrites remains2 in the 
intact head. In this preparation most nerves of the VNC should remain attached to their muscles. 
Following the dissection, the intracellular filling of the giant fiber (GF) with a fluorescent dye is demonstrated. In the CvC the GF axons are located at the dorsal surface and thus can be easily visualized under a microscope with differential interference contrast (DIC) optics. This allows the injection of the GF axons with dye at this site to label the entire GF including the axons and their terminals in the VNC. This method results in reliable and strong staining of the GFs allowing the neurons to be imaged immediately after filling with an epifluorescent microscope. Alternatively, the fluorescent signal can be enhanced using standard immunohistochemistry procedures3 suitable for high resolution confocal microscopy.

Whole Mount Preparation of the Adult Drosophila Ventral Nerve Cord for Giant Fiber Dye Injection

An in vivo dissection of the adult Drosophila ventral nerve cord (VNC) is demonstrated. This particular dissection method causes little damage to the VNC allowing the subsequent labeling of the giant fiber neurons with fluorescent dye for high resolution imaging.

成人整装准备果蝇巨纤维染料注射腹侧神经索

To analyze the axonal and dendritic morphology of neurons, it is essential to obtain accurate labeling of neuronal structures. Preparing well labeled ...

Preparation of Parasagittal Slices for the Investigation of Dorsal-ventral Organization of the Rodent Medial Entorhinal Cortex

Computation in the brain relies on neurons responding appropriately to their synaptic inputs. Neurons differ in their complement and distribution of membrane ion channels that determine how they respond to synaptic inputs. However, the relationship between these cellular properties and neuronal function in behaving animals is not well understood. One approach to this problem is to investigate topographically organized neural circuits in which the position of individual neurons maps onto information they encode or computations they carry out1. Experiments using this approach suggest principles for tuning of synaptic responses underlying information encoding in sensory and cognitive circuits2,3.
The topographical organization of spatial representations along the dorsal-ventral axis of the medial entorhinal cortex (MEC) provides an opportunity to establish relationships between cellular mechanisms and computations important for spatial cognition. Neurons in layer II of the rodent MEC encode location using grid-like firing fields4-6. For neurons found at dorsal positions in the MEC the distance between the individual firing fields that form a grid is on the order of 30 cm, whereas for neurons at progressively more ventral positions this distance increases to greater than 1 m. Several studies have revealed cellular properties of neurons in layer II of the MEC that, like the spacing between grid firing fields, also differ according to their dorsal-ventral position, suggesting that these cellular properties are important for spatial computation2,7-10.
Here we describe procedures for preparation and electrophysiological recording from brain slices that maintain the dorsal-ventral extent of the MEC enabling investigation of the topographical organization of biophysical and anatomical properties of MEC neurons. The dorsal-ventral position of identified neurons relative to anatomical landmarks is difficult to establish accurately with protocols that use horizontal slices of MEC7,8,11,12, as it is difficult to establish reference points for the exact dorsal-ventral location of the slice. The procedures we describe enable accurate and consistent measurement of location of recorded cells along the dorsal-ventral axis of the MEC as well as visualization of molecular gradients2,10. The procedures have been developed for use with adult mice (&gt; 28 days) and have been successfully employed with mice up to 1.5 years old. With adjustments they could be used with younger mice or other rodent species. A standardized system of preparation and measurement will aid systematic investigation of the cellular and microcircuit properties of this area.

We describe procedures for preparation and electrophysiological recording from brain slices that maintain the dorsal-ventral axis of the medial entorhinal cortex (MEC). Because neural encoding of location follows a dorsal-ventral organization within the MEC, these procedures facilitate investigation of cellular mechanisms important for navigation and memory.

背腹鼠​​类内侧嗅皮层组织的调查矢状窦片的制备

Computation in the brain relies on neurons responding appropriately to their synaptic inputs. Neurons differ in their complement and distribution of ...

Spinal Cord Transection in the Larval Zebrafish

Mammals fail in sensory and motor recovery following spinal cord injury due to lack of axonal regrowth below the level of injury as well as an inability to reinitiate spinal neurogenesis. However, some anamniotes including the zebrafish Danio rerio exhibit both sensory and functional recovery even after complete transection of the spinal cord. The adult zebrafish is an established model organism for studying regeneration following spinal cord injury, with sensory and motor recovery by 6&#160;weeks post-injury. To take advantage of in vivo analysis of the regenerative process available in the transparent larval zebrafish as well as genetic tools not accessible in the adult, we use the larval zebrafish to study regeneration after spinal cord transection. Here we demonstrate a method for reproducibly and verifiably transecting the larval spinal cord. After transection, our data shows sensory recovery beginning at 2&#160;days post-injury (dpi), with the C-bend movement detectable by 3 dpi and resumption of free swimming by 5 dpi. Thus we propose the larval zebrafish as a companion tool to the adult zebrafish for the study of recovery after spinal cord injury.

After spinal transection, adult zebrafish have functional recovery by six weeks post-injury. To take advantage of larval transparency and faster recovery, we present a method for transecting the larval spinal cord. After transection, we observe sensory recovery beginning at 2&#160;days post-injury, and C-bend movement by 3 days post-injury.

在斑马鱼幼虫脊髓横断

Mammals fail in sensory and motor recovery following spinal cord injury due to lack of axonal regrowth below the level of injury as well as an inability ...

A Procedure for Implanting a Spinal Chamber for Longitudinal In Vivo Imaging of the Mouse Spinal Cord

Studies in the mammalian neocortex have enabled unprecedented resolution of cortical structure, activity, and response to neurodegenerative insults by repeated, time-lapse in vivo imaging in live rodents. These studies were made possible by straightforward surgical procedures, which enabled optical access for a prolonged period of time without repeat surgical procedures. In contrast, analogous studies of the spinal cord have been previously limited to only a few imaging sessions, each of which required an invasive surgery. As previously described, we have developed a spinal chamber that enables continuous optical access for upwards of 8 weeks, preserves mechanical stability of the spinal column, is easily stabilized externally during imaging, and requires only a single surgery. Here, the design of the spinal chamber with its associated surgical implements is reviewed and the surgical procedure is demonstrated in detail. Briefly, this video will demonstrate the preparation of the surgical area and mouse for surgery, exposure of the spinal vertebra and appropriate tissue debridement, the delivery of the implant and vertebral clamping, the completion of the chamber, the removal of the delivery system, sealing of the skin, and finally, post-operative care. The procedure for chronic in vivo imaging using nonlinear microscopy will also be demonstrated. Finally, outcomes, limitations, typical variability, and a guide for troubleshooting are discussed.

A Procedure for Implanting a Spinal Chamber for Longitudinal In Vivo Imaging of the Mouse Spinal Cord

In this video, we describe a procedure for implanting a chronic optical imaging chamber over the dorsal spinal cord of a live mouse. The chamber, surgical procedure, and chronic imaging are reviewed and demonstrated.

一个程序，植入脊柱商会纵<em&gt;在体内</em&gt;鼠标脊髓成像

Studies in the mammalian neocortex have enabled unprecedented resolution of cortical structure, activity, and response to neurodegenerative insults by ...

Preparation of Primary Mixed Glial Cultures from Adult Mouse Spinal Cord Tissue

It has been well-accepted that spinal cord glial responses contribute significantly to the development of neuropathic pain. Tremendous information regarding glial activities at the cellular and molecular levels has been obtained through in vitro cell culture systems. The in vitro systems utilized, mainly include primary glia derived from neonatal brain cortical tissue and immortalized cell lines. However, these systems may not reflect the characteristics of spinal cord glial cells in vivo. In order to further investigate the roles of spinal cord glial cells in the development of peripheral nerve injury-induced neuropathic pain using a culture system that better reflects the in vivo condition, our laboratory has developed a method to establish primary spinal cord mixed glial cultures from adult mice. Briefly, spinal cords are collected from adult mice and processed through papain digestion followed by myelin removal with a density-gradient medium. Single cell suspensions are cultured in complete Dulbecco&#39;s modified Eagle media (cDMEM) supplemented with 2-mercaptoethanol (2-ME) at 35.9 oC. These culture conditions were optimized specifically for the growth of mixed glial cells. Under these conditions, cells are ready to be used for experimentation between 12 - 14 d&#160;(cells are usually in log phase during this time) after the establishment of the culture (D 0) and can be kept in culture conditions up to D 21. This culture system can be used to investigate the responses of spinal cord glial cells upon stimulation with various substances and agents. Besides neuropathic pain, this system can be used to study glial responses in other diseases that involve pathological changes of spinal cord glial cells.

The development of neuropathic pain involves pathological changes of spinal cord glial cells. A reliable glial culture system derived from adult spinal cord tissue and designed for studying these cells in vitro is lacking. Therefore, we show here how&#160;to establish primary mixed glial cultures from adult mouse spinal cord tissue.

第一次混合胶质细胞培养的成年小鼠脊髓组织准备

It has been well-accepted that spinal cord glial responses contribute significantly to the development of neuropathic pain. Tremendous information ...

Hydraulic Extrusion of the Spinal Cord and Isolation of Dorsal Root Ganglia in Rodents

Traditionally, the spinal cord is isolated by laminectomy, i.e. by breaking open the spinal vertebrae one at a time. This is both time consuming and may result in damage to the spinal cord caused by the dissection process. Here, we show how the spinal cord can be extruded using hydraulic pressure. Handling time is significantly reduced to only a few minutes, likely decreasing protein damage. The low risk of damage to the spinal cord tissue improves subsequent immunohistochemical analysis. By performing hydraulic spinal cord extrusion instead of traditional laminectomy, the rodents can further be used for DRG isolation, thereby lowering the number of animals and allowing analysis across tissues from the same rodent. We demonstrate a consistent method to identify and isolate the DRGs according to their localization relative to the costae. It is, however, important to adjust this method to the particular animal used, as the number of spinal cord segments, both thoracic and lumbar, may vary according to animal type and strain. In addition, we illustrate further processing examples of the isolated tissues.

Here, we present a protocol for hydraulic extrusion of the spinal cord as well as identification and isolation of specific dorsal root ganglia (DRGs) in the same rodent. Compared to standard spinal cord isolation methods, this method is significantly faster and reduces the risk of tissue damage.

背根神经节的啮齿动物的脊髓和隔离的液压挤压

Traditionally, the spinal cord is isolated by laminectomy, i.e. by breaking open the spinal vertebrae one at a time. This is both time consuming and may ...

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 ...

Imaging Neural Activity in the Primary Somatosensory Cortex Using Thy1-GCaMP6s Transgenic Mice

The mammalian brain exhibits marked symmetry across the sagittal plane. However, detailed description of neural dynamics in symmetric brain regions in adult mammalian animals remains elusive. In this study, we describe an experimental procedure for measuring calcium dynamics through dual optical windows above bilateral primary somatosensory corticies (S1) in Thy1-GCaMP6s transgenic mice using 2-photon (2P) microscopy. This method enables recordings and quantifications of neural activity in bilateral mouse brain regions one at a time in the same experiment for a prolonged period in vivo. Key aspects of this method, which can be completed within an hour, include minimally invasive surgery procedures for creating dual optical windows, and the use of 2P imaging. Although we only demonstrate the technique in the S1 area, the method can be applied to other regions of the living brain facilitating the elucidation of structural and functional complexities of brain neural networks.

Imaging Neural Activity in the Primary Somatosensory Cortex Using Thy1-GCaMP6s Transgenic Mice

We describe an experimental procedure for measuring neuronal activity through dual optical windows above bilateral primary somatosensory corticies (S1) in Thy1-GCaMP6s transgenic mice using 2-photon (2P) microscopy in vivo.

胸腺1-gmcmp6 转基因小鼠在原代体血源皮质中的成像神经活性

The mammalian brain exhibits marked symmetry across the sagittal plane. However, detailed description of neural dynamics in symmetric brain regions in ...

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 ...

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

脊髓电二:外吸电极的制作

成人整装准备果蝇巨纤维染料注射腹侧神经索

背腹鼠类内侧嗅皮层组织的调查矢状窦片的制备

在斑马鱼幼虫脊髓横断

一个程序，植入脊柱商会纵

第一次混合胶质细胞培养的成年小鼠脊髓组织准备

背根神经节的啮齿动物的脊髓和隔离的液压挤压

斑马鱼

胸腺1-gmcmp6 转基因小鼠在原代体血源皮质中的成像神经活性

凝胶神经元全细胞斑块夹的急性脊髓切片的制备

斜脊髓切片用于神经前根刺激的制备

摘要

探索更多视频

此视频中的章节

脊髓电二:外吸电极的制作

成人整装准备果蝇巨纤维染料注射腹侧神经索

背腹鼠​​类内侧嗅皮层组织的调查矢状窦片的制备

在斑马鱼幼虫脊髓横断

一个程序，植入脊柱商会纵

第一次混合胶质细胞培养的成年小鼠脊髓组织准备

背根神经节的啮齿动物的脊髓和隔离的液压挤压

斑马鱼

胸腺1-gmcmp6 转基因小鼠在原代体血源皮质中的成像神经活性

凝胶神经元全细胞斑块夹的急性脊髓切片的制备

背腹鼠类内侧嗅皮层组织的调查矢状窦片的制备