我们想了很多精辟的讨论和B Novitch抗体的一种恩赐，感谢沙龙大话FOXP1。资助这项工作从威康信托基金会的助学金KCT和SRP（的格兰特参考号094399/B/10/Z）从威康信托基金会的项目资助。

1。制作所需的解决方案<ol><li> 1 M磷酸盐缓冲液：称取的109.4克Na 2 HPO 4和32克的NaH 2 PO 4 H 2 O中 ，混合和溶解在水中，至总体积为1升。 </li><li> 磷酸盐缓冲盐水（PBS）中：准备的0.1M磷酸盐缓冲液，0.15M NaCl的。 100毫升PBS中应足以从10胚胎的处理的片。 </li><li> 琼脂糖：热的PBS溶液至约70℃，并缓慢加入固体低熔点琼脂糖同时不断搅拌。使该溶液至终浓度为4％（重量/体积）的琼脂糖。使50毫升该溶液中的库存。发布此解决方案水浴中，保持在48℃，直到需要。实验后的未使用的任何的琼脂糖溶液在4℃下，可以存储为几个星期。 </li><li> 抗体块解决方法：添加小牛血清和Triton X-100的到最后concentr的梁漱溟的1％（V / V）胎牛血清，0.1％（体积/体积）的Triton X-100的PBS。 5片冰冻切片的免疫块解决方案的10毫升就足够了。 </li><li> 固色剂：准备0.75毫米氢氧化钠，4％（W / V）多聚甲醛（小心）。为了制备固定液热所需的量的水至70℃，添加浓NaOH到所需的最终浓度，添加固体多聚甲醛和混合直至溶解。冷却冰至4℃。 10毫升该溶液就足够了的20口油井的胚胎切片。 </li><li> 蔗糖溶液：准备10毫升含0.1M磷酸盐缓冲液的30％（重量/体积）蔗糖溶液。 10毫升该溶液就足够了胚胎切片和10口井。 </li><li> 70％（体积/体积）乙醇，70毫升无水乙醇稀释，用30毫升的水。 </li><li> 培养基：准备的溶液的1％鸡胚胎提取物，1％青霉素/链霉素，0.35％L-谷氨酰胺，0.1％2 - mercaptoethanol，4％鸡血清，2％B27补充剂和100纳克/毫升ciliaryneurotrophic的生长因子（CNTF）所有在neurobasal培养基稀释;制备在冰上，并用的同一天。 10毫升培养基所需的20井胚胎切片。 </li></ol> 2。鸡胚制备<ol><li>地点受精母鸡鸡蛋的最长边的水平在强制通风孵化器在38°C，直到他们开发阶段15 24。这通常需要大约4天的潜伏期。 </li><li>在培养的第二天，灭菌的蛋壳，浸泡在70％乙醇用纸巾擦拭。小心地刺进使用21号针头连接到一个5毫升注射器取出5毫升白蛋白的蛋壳的扁平端。这降低了胚胎离壳所以胚胎的外壳被打开时，不被损坏。返回蛋的孵化器和孵化他们的另外2天。 </li><li>用迟钝杜蒙特＃5镊子Çarefully捅破了约9厘米2的外壳顶部中间的外壳和删除。周围的胚胎切割，并小心的胚胎，并在HBSS剥离时。胚胎应根据汉堡和汉密尔顿（1992）15上演。所有的胚胎应该是在近阶段24。任何的胚胎表现出任何异常迹象，应被丢弃。 </li><li>删除羊膜，绒毛膜尿囊膜，头部和尿囊两个杜蒙特＃5镊子。剔骨的胚胎取出所有内脏器官。要做到这一点，剖开腹中的胚胎微解剖剪刀，一个钳子举行的胚胎周围的喙主干区域，抢头端的一部分，肠道和心脏和尾部拉。重要的是，这样做是干净的。你应该能够清楚地看到胚胎体节。一半，横向切胚胎之间的上肢和下肢buds.Now修剪胸体采用显微切割剪刀墙。 </li></ol> 3。胚胎切片的制备<ol><li>卸下的琼脂糖溶液从水浴中。一次性3毫升的塑胶巴斯德吸管的底部切5厘米。 </li><li>轻轻地取出腰椎的解剖胚胎用两个镊子摇篮的胚胎解剖的解决方案，并将其放置在一个干燥的培养皿中的一半。使用巴斯德移液管，取出约2毫升的琼脂糖，吸移管约0.5毫升的胚胎的顶部，然后进入移液管吸胚胎切片。在胚胎转移和琼脂糖进行剥离，远离塑料模具照顾，不引入任何气泡在琼脂糖。轻轻地将胚胎在塑料模具的底部的琼脂糖与胸部朝下。胚胎应定位要直接使用21号针头。切片也将包含在显影肢体的一部分，它是重要的，在琼脂的胚胎的取向OSE会允许这样做。将艇上冰的琼脂糖。要小心，胚胎不改变方向在此期间（约2分钟）。 </li><li>徕卡：VT1000S vibratome（速度，频率，400微米厚的片），应设置与切片室充满了HBSS，周围环绕着冰冷的水。琼脂糖块，然后粘超级胶水的vibratome平台。在琼脂糖块修整用剃刀刀片与它周围的琼脂糖1-3毫米离开胚片。选择，包括肢芽的部分有明显的顶外胚层rigde切片，这些切片，然后放置在HBSS。一般来说，有只有一到两个合适的片每个胚胎。轻轻地取出琼脂糖周围的组织切片，用两针。至关重要的是，这样做是彻底和仔细。 </li></ol> 4。培养胚胎切片<ol><li>加入500毫升培养液的必需参数（如上所述）24孔超低附件处理的组织培养板的孔的数目。片小心地从入井的HBSS溶液转移。在一般情况下，我们尝试在各孔中有两到三个切片。 24孔组织培养板在培养箱中在37℃下放置24小时，用5％的CO 2。 </li></ol> 5。切片的切片，免疫组化染色<ol><li>文化时期，加入500μl的无缓冲修复，每口井的胚胎切片，留在冰上20分钟。总溶液然后小心地除去和3的PBS洗涤，用5分钟的时间间隔进行。的切片，然后留在蔗糖溶液，在4℃，直到切片水槽中，约6小时，但可以离开的时间（ 例如过夜）。 </li><li>约5分钟，取出切片，然后轻拍掉额外的蔗糖溶液中平衡约1毫升OCT在20°C每片安装在一个充满OCT-剥离的塑料模具持平。安装后，将模具垂直上干冰巩固。 </li><li>安装OCT块，在-24°C的低温恒温器和平衡块约20分钟。将每个切片，用15毫米的部分安装对的Superfrost加幻灯片。幻灯片可以储存于-80℃直到需要它们。 </li></ol> 6。免疫<ol><li>移液器1-2毫升PBS持在潮湿的腔室与从腔室底部（我们使用1或2毫升塑料移液器用高压釜磁带固定）提出的幻灯片上水平滑动。孵育在PBS中的部分在20℃下5分钟，以除去过量的OCT取出PBS溶液从滑动和块溶液加入500毫升的部分，温育30分钟，在20℃下删除的块的解决方案，并立即加入500毫升稀释的抗体添加到幻灯片，在4°C孵育18-22小时。 </li><li>移除第一抗体溶液，并立即h的幻灯片用1ml PBS中5分钟，3次，在20℃，以除去过量的第一抗体溶液。加入500毫升稀释后的二抗的部分。在20°C 30分钟的幻灯片，然后离开。的二次抗体溶液，然后除去和滑动洗涤3次，在20℃下在PBS中的5分钟</li><li>这些清洗后，取出最后PBS冲洗，DAB的幻灯片边缘以去除多余的PBS，加两滴vectashield幻灯片上的，轻轻打下了的盖玻片超过的部分，避免泡沫的形成。删除多余的Vectashield印迹从之前的幻灯片的边缘成像。 </li></ol> 7。成像<ol><li>图像的免疫染色的部分。我们使用了尼康的Eclipse E80i配备了一个Hamammatsu ORCA ER数码相机，10倍，20倍和40 X物镜的荧光显微镜。 </li></ol>

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的作者都没有相互竞争的利益或利益冲突。

这里所描述的协议允许的鸡胚胎切片保温一天以上，同时保持运动神经元的存活，并继续培养期间迁移。在阐明的条件，以保持运动神经元的存活，我们已经确定了几个主要特点。高达4％的小鸡血清它似乎是关键的运动神经元的生存和它的替换与来自不同物种的血清是不能容忍的。有趣的是，我们发现，较高浓度的血清的存在下，不利的，因为它们促进导致严重扭曲切片中的形状的组织过度生长的切片。神经营养因子cilliary的存在下，更重要的是，也被证明是至关重要的。它仍然是一个明显的可能性，可能可以相当长的时间，比只是24小时进行培养，甚至允许可视化感觉传入输入到脊髓切片。此外，CULTURE条件，也可能是有用的切片文化的发展脑干和中脑/小脑。 或许这些切片的培养条件下，在使用的最大的潜在的好处是，它们促进蛋白质的功能，同时尽量减少潜在的胚胎的其余部分上的不利影响，如心脏药理操纵的可能性。大量的不同的信号通路抑制剂和激动剂可用于评估不同的脊髓神经元亚型的迁移和潜在的，他们在开发过程中形成的局部脊髓电路的影响。此外，一些基因蛋白的表达，如那些利用siRNA和吗啉代寡核苷酸，受到扰动的事实，他们只能在开发初期推出（中在OVO电过程碍于）和效果只持续短期内添E（通常为一天）。我们的片文化在稍后阶段开始，并让使用这种技术的可能性在发展期间，文化的切片切片阶段，以查看效果。 片培养协议，也可以让双方脊髓运动神经元，以及作为背interneuron的迁移时间的推移实时通过视频显微镜的可视化。这可以实现白色光下使用相差显微镜或通过使用荧光成像。如果后者的技术被使用，则需要引入荧光标记。这可能通过引入如荧光染料DiI荧光或DIO来实现，也可以通过任逆行标记从肢体或顺行标签从心室或卵内的电驱动荧光蛋白，在神经元的一个子集的结构。

在正常胚胎发育过程中，许多不同类型的细胞生成，必须从他们的角度原产地迁移，在那里他们将最终在成熟的有机体的功能。脊髓内的显影，在中线位于脑室区祖细胞，并生成许多亚型的有丝分裂后的神经元和神经胶质细胞，这些细胞必须迁移到融入功能的神经元的感官处理所需的电路和电机输出1。脊髓运动神经元控制四肢肌肉的收缩已被广泛研究，多是已知的分子和机制，推动形成不同的子类。然而，更何况是运动神经元迁移，分离和接收突触输入的机制。 运动神经元亚型的身份获得在开发过程中在一个分层的方式。运动神经元亚型可大致归我置身于不同的列，被定义为他们的轴突预测的部门和游泳池。汽车列项目轴突为轴向，内脏或四肢的肌肉。汽车部门细分的肢体投影到那些投射到腹部或背部肌肉的运动神经元电机外侧柱（LMC）。运动神经元群内的部门，被称为电机池项目单独的肌肉，肢体的2，3。肢体突出的运动神经元中定义的由它们的表达的转录因子FOXP1 4，5，而分割的身份是其特征在于，表达的同源结构域的转录因子胰岛-1（腹侧凸）或LHX-1（背侧凸）6。事实上，LHX-1的表达所需的适当的背突起的运动神经元7。这些亚型占据不同的位置，在脊髓腹侧突出的运动神经元来居住内侧背投影法纳克运动神经元。结果被隔离在大麦哲伦星云所谓的LMCmedial（LMCm）和LMClateral（LMCl）部门。池组织部门和电机的收购分为两个阶段8。在第一阶段中，分割偏析实现通过侧向时尚内侧的特性的运动神经元的迁移。 LMCl细胞后，将产生的LMCm细胞等LMCl的通过的LMCm移动实现其最终停留位置。第二阶段运动神经元内的分工和集群成运动神经元池，大概是通过一个背腹排序，运动神经元。已经示出连环蛋白依赖的古典钙粘蛋白家族的成员，是至关重要的两个阶段的运动神经元组织8-10。然而，钙粘蛋白功能如何控制细胞运动知之甚少。 收购柱状，部门和池标识需要外在的信号，这种模式本质安全C表达的转录因子11。此外，约50％的运动神经元死亡通过细胞凋亡在正常发展的一个过程，需要从肢体的外在信号，主要是通过神经营养支持的运动神经元生存。因此，运动神经元的发展，需要适当的环境，以保持在一个相对长的时间过程。出于这个原因，产生脊髓切片培养的实用性，以保持运动神经元存活所需要的适当的培养条件下的澄清。往前的胚胎切片培养已被用于跟踪的行为非本质加入纯化的神经元群体的12-14。然而，据我们所知，在片本身在原地运动神经元的存活和迁移的培养条件，促进尚未公布。我们修改一个标准的文化条件，有利于生存的纯化，分离颅运动神经元，以方便米OTOR神经元内的胚胎切片文化系统的发展。我们还监测了定位尚存的运动神经元的，并表明主要为正常的位置的运动神经元师培养期间获取。

<table border="1"><tbody><tr><td>鸡蛋</td><td>英国亨利斯图亚特农场， </td><td></td><td> Fertilised布朗Bovan金母鸡的蛋（亨利·斯图尔特农场，英国），保存在14°C，直到孵化</td></tr><tr><td> 21号针</td><td> BD-Microlance 3 </td><td> 304432 </td><td></td></tr><tr><td> 5毫升注射器</td><td> BD Plastipak公司</td><td> 302187 </td><td></td></tr><tr><td> ＃5杜蒙钳</td><td> Roboz </td><td> RS 5045 </td><td></td></tr><tr><td>微解剖剪</td><td> Roboz </td><td> RS 5910SC </td><td> （3.5中直） </td></tr><tr><td>胚胎嵌入模具：剥离模具</td><td>琼脂科学</td><td> G3764 </td><td>金字塔形22毫米至12毫米</td></tr><tr><td>超能胶</td><td></td><td></td><td>电源的Flex，乐泰</td></tr><tr><td>乙醇</td><td> SIGMA </td><td> 32221 </TD&gt; <td></td></tr><tr><td>钠，二氢磷酸盐，单水合物</td><td> BDH </td><td> 102454R </td><td></td></tr><tr><td>迪单钠，磷酸氢</td><td> VWR / BDH </td><td> 102494C </td><td></td></tr><tr><td>氯化钠</td><td> VWR / BDH </td><td> 27810.295 </td><td></td></tr><tr><td>低熔点琼脂糖</td><td> Invitrogen公司</td><td> 16520 </td><td></td></tr><tr><td>胎牛血清</td><td> Gibco公司</td><td> 10500-064 </td><td></td></tr><tr><td>的Triton X-100的</td><td> SIGMA </td><td> T8787 </td><td></td></tr><tr><td>氢氧化钠</td><td> FLUKA </td><td> 71689 </td><td></td></tr><tr><td>多聚甲醛</td><td> VWR / BDH </td><td> 28794.295 </td><td></td></tr><tr><td>一次性塑料巴氏吸管</td><td> ELKAY精密实验室消耗品</td><td> 127-P503-000 </td><td> Liquipette 3毫升的量</td></tr><tr><td> Hanks平衡盐溶液（HBSS）中</td><td> Gibco公司</td><td> 14170 </td><td></td></tr><tr><td>蔗糖</td><td> SIGMA </td><td> S0389 </td><td></td></tr><tr><td>碳酸氢钠</td><td> SIGMA </td><td> S5761 </td><td></td></tr><tr><td>鸡胚提取物</td><td> Seralabs </td><td> CE650-J </td><td></td></tr><tr><td>青霉素/链霉素溶液</td><td> Gibco公司</td><td> 15070 </td><td></td></tr><tr><td> L-谷氨酰胺溶液</td><td> Gibco公司</td><td> 25030 </td><td></td></tr><tr><td> 2 - 巯基乙醇</td><td> Gibco公司</td><td> 21985 </td><td></td></tr><tr><td>马血清</td><td>南方集团实验室</td><td> 9028B </td><td></td></tr><tr><td> B27的补充</td><td> Invitrogen公司</td><td> 17504-044 &lt;/ TD&gt; <td></td></tr><tr><td> Neurobasal培养基</td><td> Gibco公司</td><td>猫〜2103 </td><td></td></tr><tr><td> CilliaryNeurotrophic因子（CNTF） </td><td> R和D系统</td><td> 557-NT-010 </td><td></td></tr><tr><td>鸡血清</td><td> SIGMA </td><td> C5405 </td><td></td></tr><tr><td>一抗</td><td></td><td></td><td>购自发展研究杂交银行，除反FOXP1抗体，这是一种从Bennett Novitch加州大学洛杉矶分校的礼物。初级抗体稀释兔抗-FOXP1（1/32000），小鼠的抗-LHX1 / 2（4F2）（1/50），豚鼠的抗FOXP1（1/1000）。 所有的抗体在4℃下存储</td></tr><tr><td>第二抗体</td><td></td><td></td><td>杰克逊Immunoreseach Laboratories公司的驴抗小鼠（的Alexa Fluor 488）（1:1000），反主动购买的驴抗，豚鼠（CY3）（1:1000）EY（抗兔的Alexa Fluor 594）购自Invitrogen（1:1000） 所有的抗体在4℃下存储</td></tr><tr><td> 24 - 孔板</td><td>康宁COSTAR </td><td> 3473 </td><td>超低附件24 - 孔板</td></tr><tr><td> OCT </td><td>组织康，樱花</td><td> 4583 </td><td>最佳的切削温度</td></tr><tr><td> Vectashield </td><td>矢量实验室</td><td> H1000 </td><td> Vectashield荧光安装介质</td></tr><tr><td>幻灯片</td><td> Thermo Scientific的</td><td></td><td>的Superfrost的带正电荷的幻灯片，25×75×1.0毫米</td></tr><tr><td>盖玻片</td><td> VWR国际</td><td> 631-0167 </td><td> 25x60毫米</td></tr><tr><td>强制通风孵化器： </td><td>里昂电气公司</td><td></td><td>保持湿润​​，确保总是有足够的水位，保持在38°C </td></tr><tr><td>低温恒温器</td><td>布里特，亨廷顿，英国</td><td></td><td> -36°C </td></tr><tr><td>组织培养箱</td><td> DH自动流</td><td></td><td> nuaire 38℃，5％CO 2的 </td></tr><tr><td> Vibratome </td><td>徕卡</td><td></td><td>徕卡VT1000-S </td></tr><tr><td>显微镜</td><td>尼康</td><td></td><td>尼康的Eclipse的E80i荧光显微镜</td></tr><tr><td>数码相机</td><td>尼康</td><td></td><td>尼康DS5M和Hamammatsu ORCA ER </td></tr></tbody></table>

chicken embryo spinal cord slice culture protocol

切片培养的胚胎发育可以方便的操作都通过药理学和基因操作。这降低了系统潜在的致命的副作用，由于全身药物的应用是可以克服的。然而，文化条件必须确保正常发展所得的片内减少环境。我们一直专注于脊髓的发展，尤其是脊髓运动神经元。我们系统地不同的培养条件的鸡胚胎切片，的点最脊髓运动神经元诞生了。我们测定存活培养期间和运动神经元的位置的那些相比， 在体内的运动神经元的数目和类型。我们发现，培养期间需要血清型和神经营养因子，并能够保持为至少24小时，并允许这些运动神经元迁移到适当的位置中的运动神经元存活脊髓。我们提出这些培养条件和方法，准备使用嵌入在琼脂糖全净膛鸡胚胎的胚胎切片文化和切片用一个vibratome。

使用培养的片一直遵循的发展interneuron和投射神经元影响力的进展我们对组织产生的内皮层16。在脊髓运动神经元的发展一直遵循文化的整个斑马鱼胚胎17。然而，在斑马鱼的脊髓运动神经元组织是相对简单的（由于缺乏鱼的大型四肢肌肉）。该驱动器的脊髓运动神经元组织层次的高等脊椎动物的发展过程中的分子机制知之甚少。在高等脊椎动物的胚胎切片文化，促进神经元的存活和细胞体迁移，因此需要培养条件。目前，脊髓切片培养的高等脊椎动物已被用于种子解离的神经元的顶部片12-14，调查荧光标记的切片的外周中的神经轴突18，或祖细胞早期脊髓发育行为19。片培养条件后脊髓，尤其是那些保持运动神经元发展目前所缺乏的。 为了试图脊髓运动神经元脊髓神经元的发育，特别是，我们研究了各种文化条件，可能会促进运动神经元的存活和运动组织通过横向迁移的神经元的初始订单收购。使用阶段18阶段20鸡胚脊髓切片的初步试验证明无果而终，我们不能够保持运动神经元存活超过几个小时的时间，并不能证明代相当数量的LMCl细胞的培养周期（数据未示出）。因此，我们研究阶段，24胚片文化的一个时间点时，大多数的LMCl和LMCm神经元已经产生，但是当LMCl神经元的迁移仍然是在我nfancy（ 图1A-C）。这种横向的LMCl神经元的迁移，主要是完成阶段27，约24至36小时后（ 图1 DF）。因此，我们的分析可以被简化为能够保持活着的神经元，以协助他们迁移到腹角横向。我们开始我们的实验中，使用相对简单的培养条件下，只包含Hank氏平衡盐溶液的鸡血清或鸡胚提取物带或不带。在所有情况下，尽管我们能够保持LMC切片中的神经元，我们发现几乎没有证据表明LMCl神经元保持（至少LHX-1的表达）。此外，我们发现不适当表达的基因，如HB9在脊髓背角， 在体内的 （数据未示出）的情况下，永远不会发生。因此，这些简单的文化条件不适合的收购运动神经元组织的调查。 因此，我们找到了更复杂的条件[NS和使用的碱的配方已发布，以方便分离的鸡胚脑神经运动神经元的存活。此条件20（1％鸡胚提取物，1％笔链球菌，0.35％L-谷氨酰胺，0.1％2 -巯基乙醇，2％马血清，2％B27补充剂和50纳克/毫升ciliaryneurotrophic的生长因子（CNTF）在neurobasal培养基（这里称为格思里中等），并保持活着比更简单的媒体更多的运动神经元。此外，它没有导致在杂散表达的转录因子在脊髓背角。然而，在生存的LMCl神经元是穷人（ 图1 GI， p）的，因此，我们改变我们认为是关键因素的Guthrie介质，即动物血清的来源，血清中的浓度和睫状神经营养因子在培养基中的浓度，我们发现，改变从马血清的血清小鸡血清和CNTF的浓度增加，从50毫微克/毫升至100毫微克/毫升的大幅增加吨他的的LMCl和LMCm细胞的生存，也允许他们迁移到脊髓前角超过24小时的培养条件（ 图1 JL，P）。运动神经元的总数，比LMCm LMCl和，的确，迁移的LMCl细胞到腹侧角出现非常相似的部分的胚胎被允许开发卵内 ，而不是在该切片培养（ 图1 KO，P）。因此，我们认为，基于转录因子的表达，细胞数量和位置的运动神经元，切片培养条件概括正常的脊髓运动神经元的发展至少在24小时内。 <img alt="图1" fo:content-width="2in" fo:src="/files/ftp_upload/50295/50295fig1highres.jpg" src="/files/ftp_upload/50295/50295fig1.jpg" /> 图1。 A - C。代LMC的状态（FOXP1染色b）和LMCl（LHX-1染色在 a）中，在阶段24。 c是一个合并的两个通道。中线作为a和D中的虚线所示，V表示的脊髓背腹轴（D，V）的取向。D - F。 。G - 我的状况：LMC（d） 中 FOXP1染色和LMCl（LHX-1染色在 e）组织阶段27。f是一个合并的两个通道。 LHX-1（g）和FOXP1（ 小时 ）在部分支持颅运动神经元细胞的存活（我们的“初始培养基”的Guthrie介质，见参考文献20），在培养基中培养的切片中表达。 LHX-1的是，不再在运动神经元中表达，虽然有一些LMC证明FOXP1表达细胞的生存。 ，D，V（g）中示出的脊髓背腹轴（D，V）的取向。 ML显示的正中侧面（M，L）的s轴的方向皮纳尔线的J - 升 。 LMCl细胞（LHX在腹侧角在 j-1）和LMC一般（FOXP1 在 k）中可以观察到在含有4％鸡血清和100 ng / ml的睫状神经营养因子的培养基中培养的切片。请注意，在腹侧角的横向位置被发现在一些LMCl细胞。 （j）条中的箭头示出了一些的LMCl细胞的横向位置。 （L）是一个合并的两个通道。 米- O。 LHX-1（m）和FOXP1（n）中的胚胎内的胚胎在 j - 升所示的切片培养过程中保持在卵的表达状况。 （O）是一个合并的两个通道。定量的百分比的LMCl（FOXP1 + VE / LHX1 +）细胞与LMC（FOXP1 + VE / LHX1-VE）在片文化在不同条件下的细胞中发现的Çompared控制卵 （占100％） 中保持的胚胎。学生的t检验***表示p &lt;0.01。 <a href="https://www.jove.com/files/ftp_upload/50295/50295fig1large.jpg" target="_blank">点击此处查看大图</a> 。

Watch this Scientific Journal Video about Chicken Embryo Spinal Cord Slice Culture Protocol at JoVE.com

Chicken Embryo Spinal Cord Slice Culture Protocol

切片文化促进胚胎发育的基因和药理学的扰动操作。然而，文化条件必须确保可以继续正常发展的片内减少环境。我们说明了一个协议，有利于脊髓发育正常进行至少24小时。

鸡胚胎脊髓片培养协议

Slice cultures can facilitate the manipulation of  embryo development both pharmacologically and through gene manipulations. In  this reduced system, ...

chicken-embryo-spinal-cord-slice-culture-protocol

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JoVE Journal

Neuroscience

21.9K 次观看.  University College London. 切片文化促进胚胎发育的基因和药理学的扰动操作。然而，文化条件必须确保可以继续正常发展的片内减少环境。我们说明了一个协议，有利于脊髓发育正常进行至少24小时。

视频: Chicken Embryo Spinal Cord Slice Culture Protocol

Dissection and Culture of Commissural Neurons from Embryonic Spinal Cord

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

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

从胚胎脊髓​​连合神经的解剖和文化

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

科研

神经科学

A Functional Motor Unit in the Culture Dish: Co-culture of Spinal Cord Explants and Muscle Cells

Human primary muscle cells cultured aneurally in monolayer rarely contract spontaneously because, in the absence of a nerve component, cell differentiation is limited and motor neuron stimulation is missing1. These limitations hamper the in vitro study of many neuromuscular diseases in cultured muscle cells. Importantly, the experimental constraints of monolayered, cultured muscle cells can be overcome by functional innervation of myofibers with spinal cord explants in co-cultures.
Here, we show the different steps required to achieve an efficient, proper innervation of human primary muscle cells, leading to complete differentiation and fiber contraction according to the method developed by Askanas2. To do so, muscle cells are co-cultured with spinal cord explants of rat embryos at ED 13.5, with the dorsal root ganglia still attached to the spinal cord slices. After a few days, the muscle fibers start to contract and eventually become cross-striated through innervation by functional neurites projecting from the spinal cord explants that connecting to the muscle cells. This structure can be maintained for many months, simply by regular exchange of the culture medium.
The applications of this invaluable tool are numerous, as it represents a functional model for multidisciplinary analyses of human muscle development and innervation. In fact, a complete de novo neuromuscular junction installation occurs in a culture dish, allowing an easy measurement of many parameters at each step, in a fundamental and physiological context.
Just to cite a few examples, genomic and/or proteomic studies can be performed directly on the co-cultures. Furthermore, pre- and post-synaptic effects can be specifically and separately assessed at the neuromuscular junction, because both components come from different species, rat and human, respectively. The nerve-muscle co-culture can also be performed with human muscle cells isolated from patients suffering from muscle or neuromuscular diseases3, and thus can be used as a screening tool for candidate drugs. Finally, no special equipment but a regular BSL2 facility is needed to reproduce a functional motor unit in a culture dish. This method thus is valuable for both the muscle as well as the neuromuscular research communities for physiological and mechanistic studies of neuromuscular function, in a normal and disease context.

Cultured muscle cells are an inadequate model to recapitulate innervated muscle in vivo. A functional motor unit can be reproduced in vitro by innervation of differentiated human primary muscle cells using rat embryo spinal cord explants. This article describes how co-cultures of spinal cord explants and muscle cells are established.

一个在培养皿中的功能电机组：脊髓植和肌肉细胞共培养

Human primary muscle cells cultured  aneurally in monolayer rarely contract spontaneously because, in the absence of  a nerve component, cell ...

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 ...

Complete Spinal Cord Injury and Brain Dissection Protocol for Subsequent Wholemount In Situ Hybridization in Larval Sea Lamprey

After a complete spinal cord injury, sea lampreys at first are paralyzed below the level of transection. However, they recover locomotion after several weeks, and this is accompanied by short distance regeneration (a few mm) of propriospinal axons and spinal-projecting axons from the brainstem. Among the 36 large identifiable spinal-projecting neurons, some are good regenerators and others are bad regenerators. These neurons can most easily be identified in wholemount CNS preparations. In order to understand the neuron-intrinsic mechanisms that favor or inhibit axon regeneration after injury in the vertebrates CNS, we determine differences in gene expression between the good and bad regenerators, and how expression is influenced by spinal cord transection. This paper illustrates the techniques for housing larval and recently transformed adult sea lampreys in fresh water tanks, producing complete spinal cord transections under microscopic vision, and preparing brain and spinal cord wholemounts for in situ hybridization. Briefly, animals are kept at 16&#160;&#176;C and anesthetized in 1% Benzocaine in lamprey Ringer. The spinal cord is transected with iridectomy scissors via a dorsal approach and the animal is allowed to recover in fresh water tanks at 23 &#176;C. For in situ hybridization, animals are reanesthetized and the brain and cord removed via a dorsal approach.

Complete Spinal Cord Injury and Brain Dissection Protocol for Subsequent Wholemount In Situ Hybridization in Larval Sea Lamprey

Lampreys recover locomotion after a complete spinal cord injury. However, some spinal-projecting neurons are good regenerators and others are not. This paper illustrates the techniques for housing sea lamprey larvae (and recently transformed adults), producing complete spinal cord transections and preparing wholemount brains and spinal cords for in situ hybridization.

完全性脊髓损伤和脑解剖协议后续Wholemount<em&gt;原位</em&gt;杂交苗种海七鳃鳗

After a complete spinal cord injury, sea lampreys at first are paralyzed below the level of transection. However, they recover locomotion after several ...

An Ex Vivo Laser-induced Spinal Cord Injury Model to Assess Mechanisms of Axonal Degeneration in Real-time

Injured CNS axons fail to regenerate and often retract away from the injury site. Axons spared from the initial injury may later undergo secondary axonal degeneration. Lack of growth cone formation, regeneration, and loss of additional myelinated axonal projections within the spinal cord greatly limits neurological recovery following injury. To assess how central myelinated axons of the spinal cord respond to injury, we developed an ex vivo living spinal cord model utilizing transgenic mice that express yellow fluorescent protein in axons and a focal and highly reproducible laser-induced spinal cord injury to document the fate of axons and myelin (lipophilic fluorescent dye Nile Red) over time using two-photon excitation time-lapse microscopy. Dynamic processes such as acute axonal injury, axonal retraction, and myelin degeneration are best studied in real-time. However, the non-focal nature of contusion-based injuries and movement artifacts encountered during in vivo spinal cord imaging make differentiating primary and secondary axonal injury responses using high resolution microscopy challenging. The ex vivo spinal cord model described here mimics several aspects of clinically relevant contusion/compression-induced axonal pathologies including axonal swelling, spheroid formation, axonal transection, and peri-axonal swelling providing a useful model to study these dynamic processes in real-time. Major advantages of this model are excellent spatiotemporal resolution that allows differentiation between the primary insult that directly injures axons and secondary injury mechanisms; controlled infusion of reagents directly to the perfusate bathing the cord; precise alterations of the environmental milieu (e.g., calcium, sodium ions, known contributors to axonal injury, but near impossible to manipulate in vivo); and murine models also offer an advantage as they provide an opportunity to visualize and manipulate genetically identified cell populations and subcellular structures. Here, we describe how to isolate and image the living spinal cord from mice to capture dynamics of acute axonal injury.

An Ex Vivo Laser-induced Spinal Cord Injury Model to Assess Mechanisms of Axonal Degeneration in Real-time

We present a protocol utilizing two-photon excitation time-lapse microscopy to simultaneously visualize the dynamics of axon and myelin injuries in real time. This proposed protocol permits studies of both intrinsic and extrinsic factors which can influence central myelinated axon fate after injury and contribute to permanent clinical disability.

一个<em&gt;离体</em&gt;激光诱导脊髓损伤模型，以评估轴索变性的机制在实时

Injured CNS axons fail to regenerate and often retract away from the injury site. Axons spared from the initial injury may later undergo secondary axonal ...

Spinal Cord Neurons Isolation and Culture from Neonatal Mice

We present a protocol for the isolation and culture of spinal cord neurons. The neurons are obtained from neonatal C57BL/6 mice and are isolated on postnatal day 1-3. A mouse litter, usually 4-10 pups born from one breeding pair, is gathered for one experiment, and spinal cords are collected individually from each mouse after euthanasia with isoflurane. The spinal column is dissected out and then the spinal cord is released from the column. The spinal cords are then minced to increase the surface area of delivery for an enzymatic protease that allows for the neurons and other cells to be released from the tissue. Trituration is then used to release the cells into solution. This solution is subsequently fractionated in a density gradient to separate the various cells in solution, allowing for neurons to be isolated. Approximately 1-2.5 x 106 neurons can be isolated from one litter group. The neurons are then seeded onto wells coated with adhesive factors that allow for proper growth and maturation. The neurons take approximately 7 days to reach maturity in the growth and culture medium and can be used thereafter for treatment and analysis.

This study presents a technique for the isolation of neurons from WT neonatal mice. It requires the careful dissection of the spinal cord from the neonatal mouse, followed by the separation of neurons from the spinal cord tissue through mechanical and enzymatic cleavage.

脊髓神经元从新生小鼠中分离和培养

We present a protocol for the isolation and culture of spinal cord neurons. The neurons are obtained from neonatal C57BL/6 mice and are isolated on ...

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

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

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

头脊髓和薄片体外制备心律失常活性

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

Intra-Arterial Delivery of Neural Stem Cells to the Rat and Mouse Brain: Application to Cerebral Ischemia

Neural stem cell (NSC) therapy is an emerging innovative treatment for stroke, traumatic brain injury and neurodegenerative disorders. As compared to intracranial delivery, intra-arterial administration of NSCs is less invasive and produces a more diffuse distribution of NSCs within the brain parenchyma. Further, intra-arterial delivery allows the first-pass effect in the brain circulation, lessening the potential for trapping of cells in peripheral organs, such as liver and spleen, a complication associated with peripheral injections. Here, we detail the methodology, in both mice and rats, for delivery of NSCs through the common carotid artery (mouse) or external carotid artery (rat) to the ipsilateral hemisphere after an ischemic stroke. Using GFP-labeled NSCs, we illustrate the widespread distribution achieved throughout the rodent ipsilateral hemisphere at 1 d, 1 week and 4 weeks after postischemic delivery, with a higher density in or near the ischemic injury site. In addition to long-term survival, we show evidence of differentiation of GFP-labeled cells at 4 weeks. The intra-arterial delivery approach described here for NSCs can also be used for administration of therapeutic compounds, and thus has broad applicability to varied CNS injury and disease models across multiple species.

A method for delivering neural stem cells, adaptable for injecting solutions or suspensions, through the common carotid artery (mouse) or external carotid artery (rat) after ischemic stroke is reported. Injected cells are distributed broadly throughout the brain parenchyma and can be detected up to 30 d after delivery.

神经干细胞向大鼠和小鼠大脑的动脉内传递：对脑缺血的应用

Neural stem cell (NSC) therapy is an emerging innovative treatment for stroke, traumatic brain injury and neurodegenerative disorders. As compared to ...

A Minimally Invasive, Fast Spinal Cord Lateral Hemisection Technique for Modeling Open Spinal Cord Injuries in Rats

Open spinal cord injury techniques modeling laceration-like injuries are time-consuming and invasive because they involve laminectomy. This new technique eliminates laminectomy by removing two spinous processes and lifting, then tilting the caudal vertebral arch. The surgical area opens up without the need for laminectomy. Lateral hemisection is then performed with direct visible control under a microscope. The trauma is minimized, requiring only a small bone wound. 
This technique has several advantages: it is faster and, therefore, less of a burden for the animal, and the bone wound is smaller. Because the laminectomy is eliminated, there is less chance for unwanted injury to the spinal cord, and there are no bone splinters that can cause problems (bone splinters embedded in the spinal cord can cause swelling and secondary damage). The vertebral canal remains intact. The main limitation is that the hemisection can only be performed in the intervertebral spaces. 
The results show that this technique can be performed much faster than the traditional surgical approach, using laminectomy (11 min vs. 35 min). This technique can be useful for researchers working with animal models of open spinal cord injury as it is widely adaptable and does not require any additional specialized instrumentation.

Here, we describe a new, fast technique modeling open spinal cord injury in rats that eliminates laminectomy. Lateral hemisection is performed while viewing through a microscope. The technique is versatile and can also be used in the cervical, thoracic, and lumbar regions of the spinal cord of other animals.

一种微创、快速脊髓侧向半切技术，用于模拟大鼠开放性脊髓损伤

Open spinal cord injury techniques modeling laceration-like injuries are time-consuming and invasive because they involve laminectomy. This new technique ...