这项工作是由国家健康与医疗研究中心（NHMRC）项目赠款支持RM

这里介绍的所有实验程序符合澳大利亚新南威尔士大学的动物护理和伦理委员会，并按照国家卫生和医学研究理事会澳大利亚规定动物实验进行。在这个协议中的所有程序应按照有关动物护理和伦理委员会的要求进行。 1.乙酰胆碱酯酶组织化学染色<ol><li>准备乙酰胆碱酯酶反应混合物<ol><li>添加290毫克碘化乙酰硫胆碱的200毫升的0.1M磷酸盐缓冲液（PB）。混合用磁力搅拌器在900rpm下。 </li><li>添加600毫克甘氨酸下连续搅拌。 </li><li>慢慢在连续搅拌下添加420毫克硫酸铜到反应混合物中，直到产品完全溶解。 </li></ol></li><li>卸下动物胴体皮肤通过切口（通过组织共享获得）成灌注大鼠或小鼠的皮肤，抓从颈部区域的皮肤和拉过去它的脚。确保筋膜覆盖各肌肉是删除或者显著穿孔以确保肌肉纤维在反应溶液的充足的曝光。 </li><li>浸入整个身体或感兴趣肢到反应混合物中，并孵育O / N在4℃。 </li><li>洗胎体为在蒸馏水中2分钟。注意:在此阶段，肌肉呈现蓝色着色和电动机的端板（的MEP）可以被看作是白点。 </li><li>暴露胴体为3-5秒的10％硫化铵溶液。 注:肌纤维会迅速变成褐​​色和欧洲议会议员将观察到的黑色斑点点。如果发生太快的反应，并且肌纤维被染色太暗的MEP和肌肉纤维之间进行区分尝试使用较低的铵的硫化物溶液（ 例如 ，5％）的浓度。反应时间，以获得的MEP和肌纤维之间最佳的对比度变化从一个样品到另一个。因此它是难以定义的精确曝光时间到试剂。该反应应密切监测和时的对比度被视为足够停止。 </li><li>洗胎体两次，同时搅拌在蒸馏水，轻轻拍打胎体干燥。 </li><li>照片两个外侧和内侧的肢体的各方面，确保维护端点对所有感兴趣的肌肉被捕获。 </li></ol> 2.肌内注射的运动终板<ol><li>拉玻璃微有微量拉马（采用分级用微量柱塞的建议）。用解剖显微镜的帮助下，打破微量的尖端具有一对钳子的，使得微量的腔的内直径大约为0.5毫米。 </li><li>确保使用的所有外科手术器械灭菌的新鲜和TH在外科手术区域是无菌的。 </li><li>填充氟金（5％的蒸馏水）的微量。 </li><li>诱导anaesthetise在动物中的感应室与异氟烷（在O 2 4％）。检查翻正反射，并确保从感应室后，再取出它的缺席。固定鼻锥上的大鼠的口鼻部和递送异氟烷（在O 2 2％）维持麻醉的。捏动物的脚趾和轻轻触摸动物的眼睛，以确保两个踏板撤出和角膜反射消失。注意:氯胺酮和xylazil的混合物也可以使用（80和10毫克/公斤分别腹膜内递送）。氯胺酮是一种附表8（"控制"）的药物和药物的采购，使用，保管和处置相关的都必须坚持必要的协议。 </li><li>敷眼润滑剂，以防止在过程期间的眼睛干燥。 </li><li>刮胡子的Targeted肢体和使用纱布洗必泰三个交替磨砂和70％的酒精擦拭剃光区域。将动物转移到手术区。 </li><li>将动物放在一个干净的底垫，并适当地定位，以保证良好的访问目标肌肉（S）。 </li><li>使用一对镊子与齿夹具解除皮肤在目标肌肉远离底层肌肉，并在与外科剪刀的皮肤切口。确保切口足够大以完全暴露感兴趣的肌肉（多个）。确保有到筋膜最小的中断。 </li><li>使用MEP照片精神上调换MEP区域的位置和形状上的肌肉（S）。 注:细毛毡标记可以协助重现从照片环境保护部地区到感兴趣的肌肉（S）。 </li><li>沿着该MEP区域的全长进行多次注射（对于荧光金，1-2微升3至4次注射各sHOULD足够了）。轻轻擦拭肌肉（S），以消除任何渗漏。 </li><li>把皮肤切开的两端钝钳并拢并关闭与手术夹伤。渗透0.1毫升布比卡因（0.5％，在水中）（或其它局部麻醉药）沿着伤口的整个跨度。 </li><li>打开麻醉机关闭并监测动物，直到它已完全从麻醉中恢复。 </li><li>等待的肌内注射给药和动物的灌注之间14天。 </li></ol> 3.灌注<ol><li>通过腹膜内注射;（150毫克/千克例如 Lethabarb）给动物施用致死剂量的戊巴比妥钠溶液。 <ol><li>密切监测动物，直到麻醉的深层次到达时，证实了没有踏板撤出和角膜反射。 </li></ol></li><li>在定位上的动物其解剖板放置在perfusio回ñ下沉。 </li><li>使用一对镊子与齿夹具解除皮肤覆盖胸骨的基础。做一个小的皮肤切口用一双有力的手术剪立即胸骨下方，然后用钳子夹住胸骨剑突软骨。按住剑突软骨，扩大对胸腔两侧的切口，从胸骨向腋下。 </li><li>切割膜片暴露心脏。 <ol><li>注射肝素0.ml直接进入心脏的顶点，以防止血液凝固。 </li><li>切的顶点，以允许套管插入到左心室，然后迅速夹紧套管就位用止血剂的帮助。 </li><li>使在右心房中的切口具有一对细剪刀并立即启动蠕动泵。灌注用0.1M PB直至流出心房的液体几乎是自由的血液和肝脏的颜色变成浅棕色。然后，灌注了SOLU多聚甲醛（在0.1M PB 4％），直到动物的整个身体和灰变得僵硬。 </li></ol></li></ol> 4.颈脊髓解剖和准备组织学<ol><li>莱在其腹部的动物尸体。 </li><li>切开皮肤在人体的中线用解剖刀从颅底到髂骨的水平反映它。 </li><li>切穿并反映椎旁肌肉以暴露脊柱的背部方面。 </li><li>确定地图集，第二颈椎节段（ 即 C2）的骨棘突，并与一对手术咬骨钳或镊子取出的是找到相应的底层背根。 <ol><li>用一个永久性标记毛毡它着色识别正确的C2根。 </li><li>由一个除去在下椎骨之一，并标记右背根交替的颜色（ 即 ，C2，C4，C6和C8可以在绿色和C3着色，C5，C7，T1可以是山口在或运算蓝色）。 </li></ol></li><li>使用小型手术针（ 例如 ，地下30号针）轻轻刺穿硬脑膜覆盖脊髓的下端。朝上针的斜面，从帘线抬起硬脑膜远离移动的同时将针吻侧使一个纵向缝隙。 <ol><li>反映硬脑膜。 </li></ol></li><li>切断脊髓横向成一个或两个段块具有一个新的手术刀刀片。 <ol><li>离开原位的脊髓节段，并且对于每个块，小心地使在与两个相邻根部之间的手术刀刀片中途各块的左侧的小基准标记。确保该基准标记是穿过组织足够深为可从该块的腹侧可见。使基准标记以大约45°的角度，指点要么前方或后方相对于动物解剖，在组织段的以下夹层的定向，以帮助。 </li></ol></li><li>收集各块到含有多聚甲醛（在0.1M PB 4％）溶液小，清楚地标明瓶子，并在室温O / N离开。 </li><li>传输块到含有蔗糖（在0.1M PB 30％）的溶液中清洁，明确标示瓶子和保持在4℃下为至少两天。 </li><li>发生在低温的模具段，都用组织冷冻介质。对于纵向组织编制，东方与背侧块朝上，使用基准标记来定位，在低温的模具块。 </li><li>冻结的低温模具，在-20℃，并切断与一个低温恒温器在50微米厚的切片。注意:这些部分可以直接安装在粘附显微镜载玻片和放置干燥O / N。或者，所述部分可以浮动在48孔板填充用0.1M PB和随后使用基准标记来定向在幻灯片上的组织切片安装。 </li></ol> 5.腰椎有限公司RD解剖和准备组织学<ol><li>莱动物尸体上回来。 </li><li>沿着动物的腹部进行中线切开和除去内脏。解剖出来后腹壁的肌肉以暴露脊柱的腹侧。 </li><li>找到短小尾椎，肋骨大部分及其邻近椎体T13所在的T13前根退出骨。 <ol><li>连续由一个除去一个的两个或三个椎骨喙和T13（ 即 ，T12，T11和，如果需要的话，T10）用细外科骨钳跟随T13前根直到其在腹侧脊髓的腹侧进入点和它具有永久性标记毛毡标记。 </li><li>从这点上，重复相同的过程，以确定的腹侧根入口点L1，L2，L3，L4，L5，L6和S1的位置，与交替的颜色着色它们。 </li></ol></li><li>如第4.5-4.10描述LUM遵循相同的程序酒吧脊髓清扫。 </li></ol>

<ol>
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B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Targeted+delivery+of+TrkB+receptor+to+phrenic+motoneurons+enhances+functional+recovery+of+rhythmic+phrenic+activity+after+cervical+spinal+hemisection.">Targeted delivery of TrkB receptor to phrenic motoneurons enhances functional recovery of rhythmic phrenic activity after cervical spinal hemisection.</a> PLoS ONE. 8 (5), e64755 (2013).</li></ol>

作者宣称，他们有没有竞争的财务权益。

肌内靶向和治疗性转基因的后续逆行转移到相应的α运动神经元的实验性治疗的神经肌肉病症是不是一个新的策略。例如，这种递送方法已被用来延迟神经肌肉变性在ALS的进展在SOD1小鼠和大鼠1,9-12不同阶段以及在小鼠中有SMA 13。尽管有希望，这些基因治疗方案的功效受到了限制。在这方面，我们提出了转基因通过脊髓运动神经元的吸收可以通过在肌纤维中的运动终板的选择性靶向得到改善。要注意的是，在上述的研究中，没有提及在目标肌肉进行到该MEP区域的位置的病毒载体的肌肉内注射是很重要的。 在我们的运动终板的分布的分析后肢和前肢（MEPS），几个原则已经出现。首先，该MEP区域跨越肌的整个宽度跨越。此外，该MEP将始终对准正交与问候myofibres的方向。值得注意的是，该MEP区域不总是位于肌肉的最厚部分内，区域经常被称为"肚子"，这是经常的丸药肌内注射的目标。值得注意的是，目前的分析在C57BL6小鼠22-23和Long埃文斯进行大鼠21和发现维护端点的分布在每个这两种菌株的动物的保守的。然而，在同一物种内的动物的不同菌株间该MEP分配是否是保守的，还有待证明。 本来，我们已经证明了环境保护部区域的位置数肌肉前肢及后肢，以引导小，MUL逆行神经示踪氟黄金tiple肌内注射。这些MEP地图可以用来梭转基因成运动神经元。在这样的实例，但是，它根据所用的病毒的类型（ 例如 ，腺病毒，慢病毒， 等等 ）其血清型，以及其滴度调整注射剂的体积是重要的。保持筋膜完整是保持所感兴趣的肌肉（多个）的范围内限制在病毒的好方法。另一种方式，以避免运动神经元的假染支配邻居肌肉是在注射后轻轻擦拭肌肉的表面上。这些预防措施可能是无关紧要的一些实验设计中实现高水平转导能比运动神经元提供了有针对性的肌肉池中包含它更有意义。然而，他们是显而易见的安全原因很好的做法。如果一个以上的孤立肌肉需要被注入，这可能是明智的，如果可能的话，要选择一组肌肉是彼此接近，以尽量减少皮肤切口的大小。大鼠和小鼠具有非常松弛的皮肤（ 即 ，未连接到肌肉），所以它是比较容易的目标数的肌肉，而无需执行巨大的皮肤切口。 有效的基因治疗，使用神经元的逆行机械ALS和其他神经肌肉条件仍然是一个挑战为，理想的是，这种疗法将整个脊髓运动神经元的整个人口转移有益基因。然而，这是没有必要的目标的所有的运动神经元来实现转导的可持续的水平，如果感兴趣的治疗性基因编码一种分泌蛋白，如神经营养蛋白。的确，非转导的运动神经元在那些已经转导的附近已被证明通过旁分泌机制24内化的神经营养因子的外源性分子。这种"旁观者效应"极大地增强了艾菲cacy肌肉注射靶向穿梭分泌蛋白编码基因导入相应的运动神经元。另一方面，鉴于呼吸控制的丧失是死亡的ALS的最终原因，治疗性病毒构建体的肌肉内注射可以在肋间肌1来执行或胸腔内25保护参与呼吸的运动神经元。通过描述的方法可以轻松地突出显示骨骼肌的欧洲议会议员的位置和安排，本协议将被证明是一个有价值的工具，探索新的基因治疗策略，神经肌肉功能障碍。该协议还提供了一种微创方式保留这些下运动神经元疾病的神经肌肉接头的完整性。

自主运动而导致的神经系统疾病，如运动神经元病和spinal-以及假肥大型肌萎缩的控制权的丧失是一个衰弱的条件，即对每一天的生活受影响的个人的高持久的影响。在过去的十年中，旨在阻止或至少延迟这些神经肌肉疾病的有害影响的研究工作已经有很多临床医生和科学家在世界各地的一个优先事项。在这方面，最近一代模仿这些神经肌肉疾病的动物模型一直在获得基本见解底层的这些条件1-13的发展和进展的生理机制。这些神经肌肉疾病的治疗需要直接访问脊髓，并且可以通过脊髓注射14,15来实现。在基因治疗的最新进展已经还针对性鞋面的横纹肌和下肢穿梭治疗基因为相应的α运动神经元位于脊髓1,9-13的腹角内。然而，这一次希望的策略已经失败，以提高这些神经疾病的结果。虽然它是公平的结论是，这些不良后果可能是，至少部分是由于这些保护性基因的疗效低，不能排除这些基因传递方法的疗效低。 运动终板（MEPS）是由大周边运动纤维从α运动神经元发起轴突缩进骨骼myofibres专门的区域。总之，外周神经纤维末梢和欧洲议会议员组成的神经肌肉接头处， 即 ，在突触脉冲通过顺行释放神经递质乙酰胆碱引发了现场。重要的是，外周神经纤维和MEP之间的关系是双向的直销tional，虽然不同的马达分别负责分子和细胞器的朝向以及远离神经元的传输胞体16-18。鉴于这些解剖考虑，维护端点似乎是所选择的目标的递送和遗传物质的后续逆行运输到相应的运动神经元。在这种情况下，它是不奇怪的运动神经元传导的成功在很大程度上依赖于注射病毒载体和肌肉的维护端点19-20的肌内之间的距离。令人惊讶的是，然而，在实验室大鼠和小鼠的myofibres该MEP区域的准确位置，这两个物种的选择来建模神经肌肉疾病，没有可用的直到最近。 我们公司生产的MEP地区综合地图数前肢肌肉在大鼠和小鼠21-22。最近，我们已经表明了环境保护部R的组织的详细资料egion几个肌肉鼠标后肢23，我们目前正在分析对大鼠后肢的欧洲议会议员的特点。在我们的手中，定向到这些肌肉在整个MEP区逆行示踪剂肌内注射引起人们正在跨越多个脊髓节段比以前更多的报道标记的运动神经元。这里，我们提出已经开发了在过去的几年中，为揭示的MEP的位置上的外表面以及整个后肢的深度和前肢肌肉同时在小鼠和大鼠的协议。

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>Fluoro-Gold</td>
			<td>Fluorochrome, LLC</td>
			<td>Nil</td>
			<td>Diluted to 5%</td>
		</tr>
		<tr>
			<td>Drummond PCR Micropipets 1-10&#181;l</td>
			<td>Drummond Scientific</td>
			<td>5-000-1001-X10</td>
			<td>accompanied with plungers</td>
		</tr>
		<tr>
			<td>Acetylthiocholine Iodide</td>
			<td>Sigma Life Science</td>
			<td>A5751-25G</td>
			<td></td>
		</tr>
		<tr>
			<td>Copper(II) Sulfate Anhydrous</td>
			<td>Sigma-Aldrich</td>
			<td>61230-500G-F</td>
			<td></td>
		</tr>
		<tr>
			<td>Tissue-Tek O.C.T Compound</td>
			<td>Sakura Finetek</td>
			<td>25608-930</td>
			<td></td>
		</tr>
		<tr>
			<td>Glycine</td>
			<td>Ajax Finechem</td>
			<td>1083-500G</td>
			<td></td>
		</tr>
		<tr>
			<td>Dextran, Tetramethylrhodamine and biotin</td>
			<td>Life Technologies</td>
			<td>D-3312</td>
			<td>Diliuted in distilled water</td>
		</tr>
		<tr>
			<td>Isothesia</td>
			<td>Provet</td>
			<td>ISOF00</td>
			<td>1000mg/g Isoflurane inhalation vapour</td>
		</tr>
		<tr>
			<td>Autoclip 9mm Wound Clips</td>
			<td>Texas Scientific Instruments, LLC</td>
			<td align="right">205016</td>
			<td></td>
		</tr>
		<tr>
			<td>Lethabarb Enthanasia Injection</td>
			<td>Virbac (Australia) Pty Ltd.</td>
			<td>LETHA450</td>
			<td></td>
		</tr>
		<tr>
			<td>Formaldehyde Solution</td>
			<td>Ajax Finechem</td>
			<td>A809-2.5L PL</td>
			<td></td>
		</tr>
		<tr>
			<td>SuperFrost Plus glass slides</td>
			<td>Menzel-Glaser</td>
			<td>J1800AMNZ</td>
			<td></td>
		</tr>
		<tr>
			<td>Ammonium Sulphide</td>
			<td>Sigma-Aldrich</td>
			<td>A1952</td>
			<td>Diluted to 10%</td>
		</tr>
		<tr>
			<td>Marcain Spinal 0.5% (Bupivacaine hydrochloride)</td>
			<td>Astrazenca</td>
			<td></td>
			<td>Diluted to 0.25%</td>
		</tr>
	</tbody>
</table>

intramuscular injections along the motor end plates: a minimally invasive approach to shuttle tracers directly into motor neurons

影响脊髓运动神经元疾病的诚信是最有神经衰弱的条件。在过去的几十年中，这些神经肌肉疾病的几种动物模型的发展提供了科学界与旨在延缓或逆转这些条件的级数不同的治疗方案。通过采取神经元逆行机械的优点，这些方法之一是对目标骨骼肌以穿梭治疗基因为对应脊髓运动神经元。虽然曾经有希望的，这样的基因递送方法的成功已经阻碍了转导的运动神经元它迄今所示，得到的亚最佳数目。马达的端板（的MEP）是在骨骼肌高度专业化的区域是在直接的突触接触到脊髓α运动神经元。在这方面，值得注意的是，重要的是，到目前为止，在努力逆行基因转移到运动神经元作了不参照该MEP区域的位置在目标肌肉。这里，我们描述一个简单的协议1）揭示骨骼肌的表面上，该MEP的确切位置和2）以使用该信息来指导肌内递送和逆行示踪剂的后续最佳逆行运输成运动神经元。我们希望利用来自于进一步研究这些追踪实验的结果通过进入欧洲议会议员的调查靶向治疗基因的逆行运输到脊髓运动神经元。

乙酰胆碱酯酶组织化学染色揭示了在整个肌肉的宽度的马达端板的位置。 图1示出在整个大鼠前肢执行这种染色的结果。因此建议以优化铵硫化物溶液的浓度（ 如 ，5-7％，而不是10％），以及检体浸渍在溶液中的时间，如果对肌肉纤维的非特异性背景染色过过量。 图2示出了注射逆行神经示踪剂沿着鼠标肱三头肌运动终板区域的长度后标记运动神经元中的颈脊髓的一列。的逆行标记运动神经元通常形成一个纵向的列横跨脊髓的多个段延伸。 图3示出了通过SEL得到的不同数目的标记的脊髓运动神经元的ective荧光金运动终板区的肱三头肌目标。最大摄取后观察到递送到该MEP区域的整个跨度，相对于靶向该MEP区域的特定区室。 <img alt="图1" src="/files/ftp_upload/52846/52846fig1.jpg" /> 图1:查看乙酰胆碱酯酶组织化学染色后的大鼠的运动终板的横向前肢的前（A）和（b）后暴露于硫化铵溶液。在（A）中 ，电机端板是明显的，因为其形成穿过肌肉的整个宽度，而肌肉纤维采用蓝绿色调的连续线的白色斑点的圆点。在（B）中 ，相同的前肢被浸没在铵的硫化物溶液后呈现。发展中的铵的硫化物溶液后，电动机端板涂RN在黑褐色的肌肉纤维。在图中这两个部分中的箭头指向运动终板为（1）Acromiotrapezius，（2）Spinodeltoideus和（3）肱三头肌的位置。 <img alt="图2" src="/files/ftp_upload/52846/52846fig2.jpg" /> 图2:50微米通过脊髓前角纵科颈椎水平揭示荧光金标记的运动神经元 WM 显微照片表示的白质和通用代表脊髓灰质。虚线表示在该图中观察到的脊髓截面内的灰质和白质之间的边界。显微照片是采取一个10X的目标过滤DAPI而下的插图是根据相同的过滤器采取了与20X目标。在两个面板，荧光金标记所用的运动神经元细胞质被观察以及近端轴突/ dendritiÇ过程。图为最初发表于Tosolini 等 。 （2013年）针对在小鼠前肢运动终板区的完整长度的增加，荧光金的摄取相应的脊髓运动神经元的接待。神经学。 4:58。 DOI:10.3389 / fneur.2013.00058 <img alt="图3" src="/files/ftp_upload/52846/52846fig3.jpg" /> 图3:选择荧光金瞄准运动终板区的肱三头肌和脊髓运动神经元产生的标签的（A）是肱三头肌纤维的运动终板位置的示意图。该MEP区域的那个被单独靶向的三个不同的部分是由绿色，红色和蓝色点表示。绿色和红色的点表示整个MEP区域的前部和后部两半分别，而蓝点represe新台币推注FG的位置进入肌肉的腹部。双箭头orientates在前后轴上的肌肉。 （B）是一种复合示出以下的肱三头肌的MEP区域的不同部分的选择性靶向得到的标签，如（A）表示。在（B）中的每个点代表一个单一标记的运动神经元。黑色柱代表典型标签后观察注射FG进入肌肉的MEP区域的整个跨度。红色运动神经元列以下FG注射到肌肉的后半获得，而以下FG注射入肌肉的前一半的绿色的运动神经元的列被获得。注射限于肌肉的"肚子"得到的运动神经元中只有少数脊髓节段的，这表现在蓝色列。图最初发布编在Tosolini 等 。 （2013年）针对在小鼠前肢运动终板区的完整长度的增加，荧光金的摄取相应的脊髓运动神经元的接待。神经学。 4:58。 DOI:10.3389 / fneur.2013.00058

Watch this Scientific Journal Video about Intramuscular Injections Along the Motor End Plates: A Minimally Invasive Approach to Shuttle Tracers Directly into Motor Neurons at JoVE.com

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

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 ...

intramuscular-injections-along-motor-end-plates-minimally-invasive

Nanyang Technological University

Research

JoVE Journal

Neuroscience

12.5K 次观看.  University of New South Wales. 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.

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

Extracellularly Identifying Motor Neurons for a Muscle Motor Pool in Aplysia californica

In animals with large identified neurons (e.g. mollusks), analysis of motor pools is done using intracellular techniques1,2,3,4. Recently, we developed a technique to extracellularly stimulate and record individual neurons in Aplysia californica5. We now describe a protocol for using this technique to uniquely identify and characterize motor neurons within a motor pool.
This extracellular technique has advantages. First, extracellular electrodes can stimulate and record neurons through the sheath5, so it does not need to be removed. Thus, neurons will be healthier in extracellular experiments than in intracellular ones. Second, if ganglia are rotated by appropriate pinning of the sheath, extracellular electrodes can access neurons on both sides of the ganglion, which makes it easier and more efficient to identify multiple neurons in the same preparation. Third, extracellular electrodes do not need to penetrate cells, and thus can be easily moved back and forth among neurons, causing less damage to them. This is especially useful when one tries to record multiple neurons during repeating motor patterns that may only persist for minutes. Fourth, extracellular electrodes are more flexible than intracellular ones during muscle movements. Intracellular electrodes may pull out and damage neurons during muscle contractions. In contrast, since extracellular electrodes are gently pressed onto the sheath above neurons, they usually stay above the same neuron during muscle contractions, and thus can be used in more intact preparations.
To uniquely identify motor neurons for a motor pool (in particular, the I1/I3 muscle in Aplysia) using extracellular electrodes, one can use features that do not require intracellular measurements as criteria: soma size and location, axonal projection, and muscle innervation4,6,7. For the particular motor pool used to illustrate the technique, we recorded from buccal nerves 2 and 3 to measure axonal projections, and measured the contraction forces of the I1/I3 muscle to determine the pattern of muscle innervation for the individual motor neurons.
We demonstrate the complete process of first identifying motor neurons using muscle innervation, then characterizing their timing during motor patterns, creating a simplified diagnostic method for rapid identification. The simplified and more rapid diagnostic method is superior for more intact preparations, e.g. in the suspended buccal mass preparation8 or in vivo9. This process can also be applied in other motor pools10,11,12 in Aplysia or in other animal systems2,3,13,14.

Extracellularly Identifying Motor Neurons for a Muscle Motor Pool in Aplysia californica

In animals with large identified neurons (e.g. mollusks), analysis of motor pools is done using intracellular techniques1,2,3,4. Recently, we developed a technique to extracellularly stimulate and record individual neurons in Aplysia californica5. We now describe a protocol for using this technique to uniquely identify and characterize motor neurons within a motor pool.

运动神经元细胞外识别的肌肉运动游泳池<em&gt;海兔夜蛾</em

In animals with large  identified neurons (e.g. mollusks), analysis of motor pools is done using  intracellular techniques1,2,3,4. Recently,  we developed ...

科研

神经科学

Minimally-invasive Technique for Injection into Rat Optic Nerve

The rat optic nerve is a useful model for stem cell regeneration research. Direct injection into the rat optic nerve allows delivery into the central nervous system in a minimally-invasive surgery without bone removal. This technique describes an approach to visualization and direct injection of the optic nerve following minor fascial dissection from the orbital ridge, using a conjunctival traction suture to gently pull the eye down and out. Representative examples of an injected optic nerve show successful injection of dyed beads.

Direct injection into the rat optic nerve is useful for regenerative research. We demonstrate a minimally-invasive technique for direct injection into a rat optic nerve that does not involve opening the skull. Using this method, surgical complications are minimized and recovery is more rapid.

微创技术注入到老鼠视神经

The rat optic nerve is a useful model for stem cell regeneration research. Direct injection into the rat optic nerve allows delivery into the central ...

Functional and Morphological Assessment of Diaphragm Innervation by Phrenic Motor Neurons

This protocol specifically focuses on tools for assessing phrenic motor neuron (PhMN) innervation of the diaphragm at both the electrophysiological and morphological levels. Compound muscle action potential (CMAP) recording following phrenic nerve stimulation can be used to quantitatively assess functional diaphragm innervation by PhMNs of the cervical spinal cord in vivo in anesthetized rats and mice. Because CMAPs represent simultaneous recording of all myofibers of the whole hemi-diaphragm, it is useful to also examine the phenotypes of individual motor axons and myofibers at the diaphragm NMJ in order to track disease- and therapy-relevant morphological changes such as partial and complete denervation, regenerative sprouting and reinnervation. This can be accomplished via whole-mount immunohistochemistry (IHC) of the diaphragm, followed by detailed morphological assessment of individual NMJs throughout the muscle. Combining CMAPs and NMJ analysis provides a powerful approach for quantitatively studying diaphragmatic innervation in rodent models of CNS and PNS disease.

Compound muscle action potential recording quantitatively assesses functional diaphragm innervation by phrenic motor neurons. Whole-mount diaphragm immunohistochemistry assesses morphological innervation at individual neuromuscular junctions. The goal of this protocol is to demonstrate how these two powerful methodologies can be used in various rodent models of spinal cord disease.

隔膜的神经支配膈运动神经元的功能和形态评估

This protocol specifically focuses on tools for assessing phrenic motor neuron (PhMN) innervation of the diaphragm at both the electrophysiological and ...

Why Quantification Matters: Characterization of Phenotypes at the Drosophila Larval Neuromuscular Junction

Most studies on morphogenesis rely on qualitative descriptions of how anatomical traits are affected by the disruption of specific genes and genetic pathways. Quantitative descriptions are rarely performed, although genetic manipulations produce a range of phenotypic effects and variations are observed even among individuals within control groups.&#160;Emerging evidence shows that morphology, size and location of organelles play a previously underappreciated, yet fundamental role in cell function and survival. Here we provide step-by-step instructions for performing quantitative analyses of phenotypes at the Drosophila larval neuromuscular junction (NMJ). We use several reliable immuno-histochemical markers combined with bio-imaging techniques and morphometric analyses to examine the effects of genetic mutations on specific cellular processes. In particular, we focus on the quantitative analysis of phenotypes affecting morphology, size and position of nuclei within the striated muscles of Drosophila larvae. The Drosophila larval NMJ is a valuable experimental model to investigate the molecular mechanisms underlying the structure and the function of the neuromuscular system, both in health and disease. However, the methodologies we describe here can be extended to other systems as well.

Why Quantification Matters: Characterization of Phenotypes at the Drosophila Larval Neuromuscular Junction

Morphology, size and location of intracellular organelles are evolutionarily conserved and appear to directly affect their function. Understanding the molecular mechanisms underlying these processes has become an important goal of modern biology. Here we show how these studies can be facilitated by the application of quantitative techniques.

为什么量化事项:表型的表征在<em&gt;果蝇</em&gt;幼虫神经肌肉接头

Most studies on morphogenesis rely on qualitative descriptions of how anatomical traits are affected by the disruption of specific genes and genetic ...

Visualization of the Axonal Projection Pattern of Embryonic Motor Neurons in Drosophila

The establishment of functional neuromuscular circuits relies on precise connections between developing motor axons and target muscles. Motor neurons extend growth cones to navigate along specific pathways by responding to a large number of axon guidance cues that emanate from the surrounding extracellular environment. Growth cone target recognition also plays a critical role in neuromuscular specificity. This work presents a standard immunohistochemistry protocol to visualize motor neuron projections of late stage-16 Drosophila melanogaster embryos. This protocol includes a few key steps, including a genotyping procedure, to sort the desired mutant embryos; an immunostaining procedure, to tag embryos with fasciclin II (FasII) antibody; and a dissection procedure, to generate filleted preparations from fixed embryos. Motor axon projections and muscle patterns in the periphery are much better visualized in flat preparations of filleted embryos than in whole-mount embryos. Therefore, the filleted preparation of fixed embryos stained with FasII antibody provides a powerful tool to characterize the genes required for motor axon pathfinding and target recognition, and it can also be applied to both loss-of-function and gain-of-function genetic screens.

Visualization of the Axonal Projection Pattern of Embryonic Motor Neurons in Drosophila

This work details a standard immunohistochemistry method to visualize motor neuron projections of late stage-16 Drosophila melanogaster embryos. The filleted preparation of fixed embryos stained with FasII antibody provides a powerful tool to characterize the genes required for motor axon pathfinding and target recognition during neural development.

胚胎运动神经元轴突投影模式的可视化<em&gt;果蝇</em

The establishment of functional neuromuscular circuits relies on precise connections between developing motor axons and target muscles. Motor neurons ...

Endoscopic Endonasal Trans-sphenoidal Approach: Minimally Invasive Surgery for Pituitary Adenomas

Endoscopic endonasal trans-sphenoidal surgery has become the gold standard for the surgical treatment of pituitary adenomas and many other pituitary lesions. Refinements in surgical techniques, technological advancements, and incorporation of neuronavigation have rendered this surgery minimally invasive. The complication rates of this surgery are very low while excellent results are consistently obtained through this approach.
This paper focuses on the step-by-step surgical approach to pituitary adenomas, which is based on personal experience, and details the results obtained with this minimally invasive surgery.

The aim of this paper is to describe the different steps of the endoscopic endonasal approach to the sella turcica.

内镜下鼻跨蝶入路: 垂体腺瘤的微创手术

Endoscopic endonasal trans-sphenoidal surgery has become the gold standard for the surgical treatment of pituitary adenomas and many other pituitary ...

Retrograde Neuroanatomical Tracing of Phrenic Motor Neurons in Mice

Phrenic motor neurons are cervical motor neurons originating from C3 to C6 levels in most mammalian species. Axonal projections converge into phrenic nerves innervating the respiratory diaphragm. In spinal cord slices, phrenic motor neurons cannot be identified from other motor neurons on morphological or biochemical criteria. We provide the description of procedures for visualizing phrenic motor neuron cell bodies in mice, following intrapleural injections of cholera toxin subunit beta (CTB) conjugated to a fluorophore. This fluorescent neuroanatomical tracer has the ability to be caught up at the diaphragm neuromuscular junction, be carried retrogradely along the phrenic axons and reach the phrenic cell bodies. Two methodological approaches of intrapleural CTB delivery are compared: transdiaphragmatic versus transthoracic injections. Both approaches are successful and result in similar number of CTB-labeled phrenic motor neurons. In conclusion, these techniques can be applied to visualize or quantify the phrenic motor neurons in various experimental studies such as those focused on the diaphragm-phrenic circuitry.

Here, we describe a protocol for identifying phrenic motor neurons in mice after intrapleural delivery of fluorophore conjugated cholera toxin subunit beta. Two techniques are compared to inject the pleural cavity: transdiaphragmatic versus transthoracic approaches.

小鼠膈运动神经元逆行神经解剖学追踪的研究

Phrenic motor neurons are cervical motor neurons originating from C3 to C6 levels in most mammalian species. Axonal projections converge into phrenic ...

Conversion of Human Induced Pluripotent Stem Cells (iPSCs) into Functional Spinal and Cranial Motor Neurons Using PiggyBac Vectors

We describe here a method to obtain functional spinal and cranial motor neurons from human induced pluripotent stem cells (iPSCs). Direct conversion into motor neuron is obtained by ectopic expression of alternative modules of transcription factors, namely Ngn2, Isl1 and Lhx3 (NIL) or Ngn2, Isl1 and Phox2a (NIP). NIL and NIP specify, respectively, spinal and cranial motor neuron identity. Our protocol starts with the generation of modified iPSC lines in which NIL or NIP are stably integrated in the genome via a piggyBac transposon vector. Expression of the transgenes is then induced by doxycycline and leads, in 5 days, to the conversion of iPSCs into MN progenitors. Subsequent maturation, for 7 days, leads to homogeneous populations of spinal or cranial MNs. Our method holds several advantages over previous protocols: it is extremely rapid and simplified; it does not require viral infection or further MN isolation; it allows generating different MN subpopulations (spinal and cranial) with a remarkable degree of maturation, as demonstrated by the ability to fire trains of action potentials. Moreover, a large number of motor neurons can be obtained without purification from mixed populations. iPSC-derived spinal and cranial motor neurons can be used for in vitro modeling of Amyotrophic Lateral Sclerosis and other neurodegenerative diseases of the motor neuron. Homogeneous motor neuron populations might represent an important resource for cell type specific drug screenings.

Conversion of Human Induced Pluripotent Stem Cells iPSCs into Functional Spinal and Cranial Motor Neurons Using PiggyBac Vectors

This protocol allows rapid and efficient conversion of induced pluripotent stem cells into motor neurons with a spinal or cranial identity, by ectopic expression of transcription factors from inducible piggyBac vectors.

利用 PiggyBac 载体将人诱导的多能干细胞 (Ipsc) 转化为功能性脊柱和颅内运动神经元

We describe here a method to obtain functional spinal and cranial motor neurons from human induced pluripotent stem cells (iPSCs). Direct conversion into ...

Non-Invasive Modulation and Robotic Mapping of Motor Cortex in the Developing Brain

Mapping the motor cortex with transcranial magnetic stimulation (TMS) has potential to interrogate motor cortex physiology and plasticity but carries unique challenges in children. Similarly, transcranial direct current stimulation (tDCS) can improve motor learning in adults but has only recently been applied to children. The use of tDCS and emerging techniques like high&#8211;definition tDCS (HD-tDCS) require special methodological considerations in the developing brain. Robotic TMS motor mapping may confer unique advantages for mapping, particularly in the developing brain. Here, we aim to provide a practical, standardized approach for two integrated methods capable of simultaneously exploring motor cortex modulation and motor maps in children. First, we describe a protocol for robotic TMS motor mapping. Individualized, MRI-navigated 12x12 grids centered on the motor cortex guide a robot to administer single-pulse TMS. Mean motor evoked potential (MEP) amplitudes per grid point are used to generate 3D motor maps of individual hand muscles with outcomes including map area, volume, and center of gravity. Tools to measure safety and tolerability of both methods are also included. Second, we describe the application of both tDCS and HD-tDCS to modulate the motor cortex and motor learning. An experimental training paradigm and sample results are described. These methods will advance the application of non-invasive brain stimulation in children.

We demonstrate protocols for the modulation (tDCS, HD-tDCS) and mapping (robotic TMS) of the motor cortex in children.

发育中大脑运动皮质的非侵入性调制和机器人映射

Mapping the motor cortex with transcranial magnetic stimulation (TMS) has potential to interrogate motor cortex physiology and plasticity but carries ...

A Minimally Invasive Lesion Technique for Muscles Intrinsic to the Odontophore of Aplysia californica

Aplysia californica is a model system for studying the neural control of learning and behavior. This animal has a semi-open circulatory system, making it possible to access many of its internal structures without causing any significant damage. Many manipulations can be easily performed both in vivo and in vitro, making it a highly tractable model for the analysis of behavior and neural circuitry. To better understand the functions of muscles within the feeding grasper, we have developed a technique for lesioning them without opening the main body cavity of the animal or damaging the outer layers of the feeding organ (i.e., the buccal mass). In this technique, the grasper is partially everted, allowing direct access to the musculature. This procedure allows animals to recover quickly and reliably. This has made it possible to lesion the I7 muscles and sub-radular fibers, allowing us to show that both muscles significantly contribute to the opening in vivo.

A Minimally Invasive Lesion Technique for Muscles Intrinsic to the Odontophore of Aplysia californica

Here we present a protocol for minimally invasive surgical lesioning of muscles intrinsic to the feeding apparatus of the marine mollusk Aplysia californica to understand the roles of these muscles during feeding behavior.

阿普利西亚的奥多托福雷内在肌肉的微创病变技术

Aplysia californica is a model system for studying the neural control of learning and behavior. This animal has a semi-open circulatory system, making it ...