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>

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<li>Gransee, H. M., Zhan, W. -Z., Sieck, G. C., Mantilla, C. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Targeted+delivery+of+TrkB+receptor+to+phrenic+motoneurons+enhances+functional+recovery+of+rhythmic+phrenic+activity+after+cervical+spinal+hemisection%5BTitle%5D)+AND+%22PLoS+ONE%22%5BJournal%5D)">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><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 &amp;micro;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>1000 mg/g Isoflurane inhalation vapour</td></tr><tr><td>Autoclip 9 mm Wound Clips</td><td>Texas Scientific Instruments, LLC</td><td>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

12.6K 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

Subcutaneous Administration of Muscarinic Antagonists and Triple-Immunostaining of the Levator Auris Longus Muscle in Mice

Hind limb muscles of rodents, such as gastrocnemius and tibialis anterior, are frequently used for in vivo pharmacological studies of the signals essential for the formation and maintenance of mammalian NMJs. However, drug penetration into these muscles after subcutaneous or intramuscular administration is often incomplete or uneven and many NMJs can remain unaffected. Although systemic administration with devices such as mini-pumps can improve the spatiotemporal effects, the invasive nature of this approach can cause confounding inflammatory responses and/or direct muscle damage. Moreover, complete analysis of the NMJs in a hind limb muscle is challenging because it requires time-consuming serial sectioning and extensive immunostaining. 
The mouse LAL is a thin, flat sheet of muscle located superficially on the dorsum of the neck. It is a fast-twitch muscle that functions to move the pinna. It contains rostral and caudal portions that originate from the midline of the cranium and extend laterally to the cartilaginous portion of each pinna. The muscle is supplied by a branch of the facial nerve that projects caudally as it exits the stylomastoid foramen. We and others have found LAL to be a convenient preparation that offers advantages for the investigation of both short and long-term in vivo effects of drugs on NMJs and muscles. First, its superficial location facilitates multiple local applications of drugs under light anesthesia. Second, its thinness (2-3 layers of muscle fibers) permits visualization and analysis of almost all the NMJs within the muscle. Third, the ease of dissecting it with its nerve intact together with the pattern of its innervation permits supplementary electrophysiological analysis in vitro9,5. Last, and perhaps most importantly, a small applied volume (&tilde;50&mu;l) easily covers the entire muscle surface, provides a uniform and prolonged exposure of all its NMJs to the drug and eliminates the need for a systemic approach1,8.

We describe procedures for repeated administration of inhibitors of muscarinic signaling to the levator auris longus (LAL) muscle of young adult mice and for subsequent immunostaining of its neuromuscular junctions (NMJs) in wholemounts. The LAL muscle has unique advantages for revealing in vivo pharmacological effects on NMJs.

皮下给药的毒蕈碱受体拮抗剂和上睑提的Auris在小鼠体内长肌的三重免疫染色

Hind limb muscles of rodents, such as gastrocnemius and tibialis anterior, are frequently used for in vivo  pharmacological studies of the signals ...

科研

神经科学

Revealing Neural Circuit Topography in Multi-Color

Neural circuits are organized into functional topographic maps. In order to visualize complex circuit architecture we developed an approach to reliably label the global patterning of multiple topographic projections. The cerebellum is an ideal model to study the orderly arrangement of neural circuits. For example, the compartmental organization of spinocerebellar mossy fibers has proven to be an indispensable system for studying mossy fiber patterning. We recently showed that wheat germ agglutinin (WGA) conjugated to Alexa 555 and 488 can be used for tracing spinocerebellar mossy fiber projections in developing and adult mice (Reeber et al. 2011). We found three major properties that make the WGA-Alexa tracers desirable tools for labeling neural projections. First, Alexa fluorophores are intense and their brightness allows for wholemount imaging directly after tracing. Second, WGA-Alexa tracers label the entire trajectory of developing and adult neural projections. Third, WGA-Alexa tracers are rapidly transported in both retrograde and anterograde directions. Here, we describe in detail how to prepare the tracers and other required tools, how to perform the surgery for spinocerebellar tracing and how best to image traced projections in three dimensions. In summary, we provide a step-by-step tracing protocol that will be useful for deciphering the organization and connectivity of functional maps not only in the cerebellum but also in the cortex, brainstem, and spinal cord.

We provide a practical guide for delivering tracers in vivo and use the spinocerebellar pathway as a model system to demonstrate essential steps for successful neuronal circuit analysis in mice. We describe in detail our versatile tracing protocol that exploits wheat germ agglutinin (WGA) conjugated to Alexa fluorophores.

多色揭示神经回路地形

Neural circuits are organized into functional topographic maps. In order to visualize complex circuit architecture we developed an approach to reliably ...

Simultaneous Intracellular Recording of a Lumbar Motoneuron and the Force Produced by its Motor Unit in the Adult Mouse In vivo

The spinal motoneuron has long been a good model system for studying neural function because it is a neuron of the central nervous system with the unique properties of (1) having readily identifiable targets (the muscle fibers) and therefore having a very well-known function (to control muscle contraction); (2) being the convergent target of many spinal and descending networks, hence the name of "final common pathway"; and (3) having a large soma which makes it possible to penetrate them with sharp intracellular electrodes. Furthermore, when studied in vivo, it is possible to record simultaneously the electrical activity of the motoneurons and the force developed by their muscle targets. Performing intracellular recordings of motoneurons in vivo therefore put the experimentalist in the unique position of being able to study, at the same time, all the compartments of the "motor unit" (the name given to the motoneuron, its axon, and the muscle fibers it innervates1): the inputs impinging on the motoneuron, the electrophysiological properties of the motoneuron, and the impact of these properties on the physiological function of the motoneurons, i.e. the force produced by its motor unit. However, this approach is very challenging because the preparation cannot be paralyzed and thus the mechanical stability for the intracellular recording is reduced. Thus, this kind of experiments has only been achieved in cats and in rats. However, the study of spinal motor systems could make a formidable leap if it was possible to perform similar experiments in normal and genetically modified mice.
For technical reasons, the study of the spinal networks in mice has mostly been limited to neonatal in vitro preparations, where the motoneurons and the spinal networks are immature, the motoneurons are separated from their targets, and when studied in slices, the motoneurons are separated from most of their inputs. Until recently, only a few groups had managed to perform intracellular recordings of motoneurons in vivo2-4 , including our team who published a new preparation which allowed us to obtain very stable recordings of motoneurons in vivo in adult mice5,6. However, these recordings were obtained in paralyzed animals, i.e. without the possibility to record the force output of these motoneurons. Here we present an extension of this original preparation in which we were able to obtain simultaneous recordings of the electrophysiological properties of the motoneurons and of the force developed by their motor unit. This is an important achievement, as it allows us to identify the different types of motoneurons based on their force profile, and thereby revealing their function. Coupled with genetic models disturbing spinal segmental circuitry7-9, or reproducting human disease10,11, we expect this technique to be an essential tool for the study of spinal motor system.

Simultaneous Intracellular Recording of a Lumbar Motoneuron and the Force Produced by its Motor Unit in the Adult Mouse In vivo

This new method permits the simultaneous intracellular recording of a single adult mouse motoneuron and the measurement of the force produced by its muscle fibers. The combined investigation of the electrical and mechanical properties of motor units in normal and genetically modified animals is a breakthrough for the study of the neuromuscular system.

同时细胞内记录的车组在成年小鼠的腰椎运动神经元产生的力<em在体内</em

The  spinal motoneuron has long been a good model system for studying neural  function because it is a neuron of the central nervous system with the ...

Transsynaptic Tracing from Peripheral Targets with Pseudorabies Virus Followed by Cholera Toxin and Biotinylated Dextran Amines Double Labeling

Transsynaptic tracing has become a powerful tool used to analyze central efferents that regulate peripheral targets through multi-synaptic circuits. This approach has been most extensively used in the brain by utilizing the swine pathogen pseudorabies virus (PRV)1. PRV does not infect great apes, including humans, so it is most commonly used in studies on small mammals, especially rodents. The pseudorabies strain PRV152 expresses the enhanced green fluorescent protein (eGFP) reporter gene and only crosses functional synapses retrogradely through the hierarchical sequence of synaptic connections away from the infection site2,3. Other PRV strains have distinct microbiological properties and may be transported in both directions (PRV-Becker and PRV-Kaplan)4,5&#160;.&#160;This protocol will deal exclusively with PRV152. By delivering the virus at a peripheral site, such as muscle, it is possible to limit the entry of the virus into the brain through a specific set of neurons. The resulting pattern of eGFP signal throughout the brain then resolves the neurons that are connected to the initially infected cells. As the distributed nature of transsynaptic tracing with pseudorabies virus makes interpreting specific connections within an identified network difficult, we present a sensitive and reliable method employing biotinylated dextran amines (BDA) and cholera toxin subunit b (CTb) for confirming the connections between cells identified using PRV152. Immunochemical detection of BDA and CTb with peroxidase and DAB (3, 3&#39;-diaminobenzidine) was chosen because they are effective at revealing cellular processes including distal dendrites6-11.

Transsynaptic tracing has become a powerful tool for analyzing central efferents regulating peripheral targets through multi-synaptic circuits. Here we present a protocol that exploits the transsynaptic pseudorabies virus to identify and localize a functional brain circuit, followed by classical tract tracing techniques to validate specific connections in the circuit between identified groups of neurons.

从外围目标跨突触跟踪与伪狂犬病病毒其次是霍乱毒素和生物素葡聚糖胺双标

Transsynaptic tracing has become a powerful tool used to analyze central efferents that regulate peripheral targets through multi-synaptic circuits. This ...

Implantation of Miniosmotic Pumps and Delivery of Tract Tracers to Study Brain Reorganization in Pathophysiological Conditions

Pharmacological treatment in animal models of cerebral disease imposes the problem of repeated injection protocols that may induce stress in animals and result in impermanent tissue levels of the drug. Additionally, drug delivery to the brain is delicate due to the blood brain barrier (BBB), thus significantly reducing intracerebral concentrations of selective drugs after systemic administration. Therefore, a system that allows both constant drug delivery without peak levels and circumvention of the BBB is in order to achieve sufficiently high intracerebral concentrations of drugs that are impermeable to the BBB. In this context, miniosmotic pumps represent an ideal system for constant drug delivery at a fixed known rate that eludes the problem of daily injection stress in animals and that may also be used for direct brain delivery of drugs. Here, we describe a method for miniosmotic pump implantation and post operatory care that should be given to animals in order to successfully apply this technique. We embed the aforementioned experimental paradigm in standard procedures that are used for studying neuroplasticity within the brain of C57BL6 mice. Thus, we exposed animals to 30 min brain infarct and implanted with miniosmotic pumps connected to the skull via a cannula in order to deliver a pro-plasticity drug. Behavioral testing was done during 30 days of treatment. After removal the animals received injections of anterograde tract tracers to analyze neuronal plasticity in the chronic phase of recovery. Results indicated that neuroprotection by the delivered drug was accompanied with increase in motor fibers crossing the midline of the brain at target structures. The results affirm the value of these techniques for drug administration and brain plasticity studies in modern neuroscience.

In order to study brain reorganization under pathological conditions we used miniosmotic pumps for direct protein delivery into the brain circumventing the blood brain barrier. Tract tracers are then injected to study alterations in brain connectivity under the influence of the protein.

Miniosmotic植入泵和交货道示踪研究在病理情况下脑重组

Pharmacological treatment in animal models of cerebral disease imposes the problem of repeated injection protocols that may induce stress in animals and ...

Laser-guided Neuronal Tracing In Brain Explants

We present a technique which combines an in vitro tracer injection protocol, which uses a series of electrical and pressure pulses to increase dye uptake through electroporation in brain explants with targeted laser illumination and matching filter goggles during the procedure. The described technique of in vitro electroporation by itself yields relatively good visual control for targetting certain areas of the brain. By combining it with laser excitation of fluorescent genetic markers and their read-out through band-passing filter goggles, which can pick up the emissions of the genetically labeled cells/nuclei and the fluorescent tracing dye, a researcher can substantially increase the accuracy of injections by finding the area of interest and controlling for the dye-spread/uptake in the injection area much more efficiently. In addition, the laser illumination technique allows to study the functionality of a given neurocircuit by providing information about the type of neurons projecting to a certain area in cases where the GFP expression is linked to the type of transmitter expressed by a subpopulation of neurons.

We describe a technique to label neurons and their processes via anterograde or retrograde tracer injections into brain nuclei using an in vitro preparation. We modified an existing method of in vitro tracer electroporation by taking advantage of fluorescently labeled mouse mutants and basic optical equipment in order to increase labeling accuracy.

激光制导神经跟踪在脑外植体

We present a technique which combines an in vitro tracer injection protocol, which uses a series of electrical and pressure pulses to increase dye uptake ...

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 ...

Expanding the Toolkit for In Vivo Imaging of Axonal Transport

Axonal transport maintains neuronal homeostasis by enabling the bidirectional trafficking of diverse organelles and cargoes. Disruptions in axonal transport have devastating consequences for individual neurons and their networks, and contribute to a plethora of neurological disorders. As many of these conditions involve both cell autonomous and non-autonomous mechanisms, and often display a spectrum of pathology across neuronal subtypes, methods to accurately identify and analyze neuronal subsets are imperative.
This paper details protocols to assess in vivo axonal transport of signaling endosomes and mitochondria in sciatic nerves of anesthetized mice. Stepwise instructions are provided to 1) distinguish motor from sensory neurons in vivo, in situ, and ex vivo by using mice that selectively express fluorescent proteins within cholinergic motor neurons; and 2) separately or concurrently assess in vivo axonal transport of signaling endosomes and mitochondria. These complementary intravital approaches facilitate the simultaneous imaging of different cargoes in distinct peripheral nerve axons to quantitatively monitor axonal transport in health and disease.

Expanding the Toolkit for In Vivo Imaging of Axonal Transport

Using transgenic fluorescent mice, detailed protocols are described to assess in vivo axonal transport of signaling endosomes and mitochondria within motor and sensory axons of the intact sciatic nerve in live animals.

扩展轴突转运体内成像工具包

Axonal transport maintains neuronal homeostasis by enabling the bidirectional trafficking of diverse organelles and cargoes. Disruptions in axonal ...

Lumbar Intrathecal Injection of Gene Therapy Vectors for Central Nervous System Targeting in Mice and Rats

One method to target the central nervous system for treating neurological diseases involves utilizing the lumbar intrathecal route of administration. This approach bypasses the blood-brain barrier to directly access the cerebrospinal fluid and preferentially target cells within the central nervous system. Multiple published preclinical studies employing the lumbar intrathecal injection route have contributed to the development of gene therapy clinical trials; however, the described protocols are variable and dispersed across multiple resources. Here, a comprehensive set of protocols for lumbar intrathecal injections in neonatal, juvenile, and adult mice and rats for preclinical gene therapy studies is presented. With proper training, this injection technique can be performed quickly and reliably. In addition to detailing the injection protocol at each developmental stage, associated parameters, such as injection volume, that can influence study outcomes are discussed. To demonstrate the application of lumbar intrathecal injections for targeting the central nervous system, the expression of adeno-associated virus serotype 9 in the brain, spinal cord, and peripheral tissues is presented following a successful or unsuccessful injection.

A lumbar intrathecal injection represents a translationally relevant route of administration for delivering gene therapy to the central nervous system. This comprehensive standardized protocol for lumbar intrathecal injections in neonatal, juvenile, and adult mice and rats aims to guide researchers in adopting this technique for preclinical gene therapy studies.

腰椎鞘内注射基因治疗载体靶向小鼠和大鼠中枢神经系统

One method to target the central nervous system for treating neurological diseases involves utilizing the lumbar intrathecal route of administration. This ...

Intraspinal Cavity Injection of Human Mesenchymal Stem Cells and Tracking their Migration into the Rat Brain

Mesenchymal stem cells (MSCs) have been studied for the treatment of various diseases. In neurodegenerative diseases involving defects in both the brain and the spinal cord, the route of administration is very important, because MSCs must migrate to both the brain and the spinal cord. This paper describes a method for administering MSCs into the spinal canal (intraspinal cavity injection) that can target the brain and spinal cord in a rat model. One million MSCs were injected into the spinal canals of rats at the level of lumbar vertebrae 2-3. After administration, the rats were euthanized at 0, 6, and 12 h post-injection. Optical imaging and quantitative real-time polymerase chain reaction (qPCR) were used to track the injected MSCs. The results of the present study demonstrated that MSCs administered via the spinal cavity could be detected subsequently in both the brain and spinal cord at 12 h. Intraspinal cavity injection has the advantage of not requiring general anesthesia and has few side effects. However, the drawback of the low migration rate of MSCs to the brain must be overcome.

<meta charset="utf8"></head><body>人间充质干细胞的椎管腔内注射并追踪它们迁移到大鼠大脑中

Several routes of administration can be used to deliver mesenchymal stem cells (MSCs) to the brain. In the present study, MSCs were delivered throughout the neuraxis and brain via intra-spinal cavity injection. MSCs were injected into the spinal cavities of rats, and stem cell migration was tracked and quantified.

人间充质干细胞的椎管腔内注射并追踪它们迁移到大鼠大脑中

Mesenchymal stem cells (MSCs) have been studied for the treatment of various diseases. In neurodegenerative diseases involving defects in both the brain ...