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在我们的实验室，我们用了两个略有不同的变体的单神经元标记法。差异有关胚胎收集，dechorionisation，devitellinisation和胚胎圆角步骤。 图1给出了一个变体的共同和不同的步骤概述。 1。微针的制备染料注射胚胎解剖和微量<ol><li>胚胎解剖的玻璃针都来自玻璃毛细管（直径1 mm和0.1 mm壁厚）与萨特拉出器（科学仪器）或类似的设备。或者，一个电解削尖0.15毫米的钨丝安装在缝合针夹持器是用于清扫。 （说明书为电解锐化给出9）。 </li><li>与萨特拉出器（科学染料注射微量的来自薄壁玻璃毛细管内丝（科学GB 100 TF 8P）仪器）或类似的设备。微吸管尖端必须是清晰的，循序渐进，均匀锥度的刀柄，以帮助通过的胚胎组织。所需的微量移液器是“高电阻微电极，夏普和龙”的品种，如第20页的萨特仪器移液器食谱的手册（ <a href="http://www.sutter.com/contact/faqs/pipette_cookbook.pdf" target="_blank">http://www.sutter.com/contact/faqs/pipette_cookbook.pdf</a> ）。一般情况下，微量不久被拉到注射前进行，以确保他们的秘诀保持锋利和清洁。 </li></ol> 2。收集，装配和胚胎解剖<ol><li> 胚胎收集，我们已成功地应用于神经元的注射方法在胚胎级12至17日3。胚胎逐步发展在第一阶段17角质层，和解剖变得越来越困难。两种可供选择的方法是获得EM胚在一个特定的发育阶段。 <ol class="ualpha"><li>允许苍蝇产卵酵母膏涂有苹果汁琼脂平板上过夜。鸡蛋收集的温度调整，以适应在计划时间注射第二天。收集所有的鸡蛋第二天和选择在所需阶段利用形态学标准3的胚胎。 </li><li>允许苍蝇趴在琼脂平板上面一样，但换了琼脂平板上用新鲜每1.5小时，在25°C孵育每块板的一个定义的时间周期，直到的胚胎已达到所需的发育阶段。 </li></ol></li><li><ol class="ualpha"><li> 化学Dechorionation。使用金属刮刀刮胚胎关闭琼脂，并将它们传送到含有约10ml 果蝇林格氏或磷酸盐缓冲盐水（PBS）溶液的玻璃容器（ 例如陪替氏培养皿或空腔块）。加入几滴浓漂白剂，搅拌几分钟就一个旋转平台，直到关闭胚胎绒毛膜泥坑。洗胚胎中的振铃/ PBS的一些变化，直到有没有残留的漂白剂气味。在这些洗涤，确保胚胎不与液体表面接触到。否则，他们将浮动和淹没后的操作变得困难。或者也可以进行使用购物篮4所描述的技术，化学dechorionation。 </li><li> 机械dechorionation（参见图2，步骤1-4）。移植胚胎的琼脂板双面胶带覆盖到幻灯片。避免琼脂与胚胎转移，因为它会影响到磁带上的附着力。允许胚胎干5至10分钟，直到绒毛膜变得稍脆。 （在等候时，外套盖玻片所需要的以后devitellinisation用胶水 - 23亿以下）。用针轻轻地触摸每个胚胎。绒毛膜会分裂和胚胎将继续坚持东北edle。立即移植胚胎的琼脂块，以防止干燥和约10的一排对齐，用自己的腹侧朝上。 </li></ol></li></ol> devitellinisation和圆角手动完成，使用在2.3A及2.3B描述的两种方法中的任一。 <ol start="3"><li><ol class="ualpha"><li>林格氏液的菜数滴林格在硅大坝上事先准备好的显微镜幻灯片传输单一dechorionated的胚胎适当的发展阶段。 （做一个3厘米见方的大坝显微镜载片上涂抹一层薄薄的有机硅密封胶，再加入0.01％的聚-L-赖氨酸溶液滴在大坝的中心。允许硅胶固化24小时）。胚胎转移用玻璃巴斯德吸管，确保胚胎仍然在传输过程中的表面之下的振铃。 振奋的胚胎的后端与一对的细钳子，同时轻轻squeezING的胚胎一对细虹膜切除术剪刀，大约有四分之一的方式从前端。不要切断通过胚胎。其目的是，破解打开卵黄膜而导致对胚胎的损害降到最低。镊子的位置，使用钨针，以纾缓胚胎腹侧面打开的膜和位置在幻灯片上。 ，的胚胎要坚持多聚赖氨酸涂层。 圆角的胚胎削尖的钨针。轻轻穿孔，然后沿背中线体​​壁撕裂。用针，体壁向下推，这样坚持到幻灯片。为了防止损坏的体壁，推和针之间的肠道的体壁。然后用针通过肠道撕裂，在其前，后两端，然后将它带走。如果需要，额外的胚胎可能会被转移到同一个幻灯片，devitellinised和鱼片，小心不要损坏其他胚胎已经在在幻灯片上。 </li><li>外套一个24 x60毫米的盖玻片，用庚烷，胶水（ 见表1）通过将一小滴胶水在中心的滑动和传播它与一个18×18毫米到一个非常薄的膜的盖玻片。盖玻片放置在显微镜载玻片上，并围绕其边缘的帧1的双面粘合带。剪去多余的琼脂块行10个胚胎，轻轻触摸的胚胎庚烷胶盖玻片盖玻片。他们现在应该坚持盖玻片腹侧朝下。大方量的PBS的胚胎（参见图2，步骤4-6）。 穿透卵黄膜用玻璃或靠近其后端的每个胚胎的背侧上的钨针。撕开膜沿背中线，并拖动出膜的胚胎。东方胚胎腹侧下降上其他地方使用的方法相同的方法中描述的2.3A的庚烷胶基板和圆角）（见图2，步骤7-8）。由于圆角的胚胎将同一张幻灯片上，这是有利于自己的定位是非常准确的，或在幻灯片上画了自己的方向。 </li></ol></li><li>经过圆角，胚胎可轻易固定在7.4％甲醛的PBS 10-15分钟，其次是4 PBS洗涤。必须采取特别小心不接触带来的胚胎与该溶液的表面在此过程中，作为表面张力会破坏胚胎。此预先固定步骤不是绝对必要的，但是有用的，以防止在后期胚胎和体壁肌肉收缩，以帮助稳定年轻阶段的胚胎组织。请记住，固定的胚胎组织更不透明，所以有些妥协下的图像质量DIC观察。 </li></ol> 3。灌装注射微量半填了0.5毫升的Eppendorf离心管中，有0.1％的carbocynanine染料DII（Molecular Probes公司，尤金，俄勒冈州）的100％乙醇（请务必使用“干”的无水乙醇，以避免DiI荧光沉淀）解决方案。一个狭窄的孔在盖子中的管，并通过在盖子中的孔插入的微量吸移管的钝端。允许的的DII提升了至少5分钟的灯丝。 （孔的盖子盖住的微管不灌装，以避免蒸发的乙醇）。 设备所需的神经元，染料注射液（图3）。 显微镜。一个固定阶段显微镜神经元染料注射液，以避免必须使用聚焦期间的垂直运动的胚胎。任何这样的运动将位移后的吸移管已经接触到与胚胎。我们发现蔡司Axioskop FS和奥林巴斯AX50/BX50的固定台上的模特是非常适合用于此目的。应配备显微镜透射光DI​​C的可视化的光学医师协会Journal of P. Medical Prog附加染色前。该显微镜还应该是一个直立的，而不是一个倒模型。随着一个直立显微镜，是作为观看目标的胚胎的同一侧上的微量吸移管的前端，而与两个倒置显微镜的相对侧上的胚胎。后者的安排妥协DIC的光学质量。应设置在显微镜的荧光显微镜，DiI荧光观​​察与一个合适的过滤器组。 DiI荧光有同等广泛的激发和发射最大值在549和565 nm处的过滤器设置在此范围内工作， 例如 ，Alexa的568，CY3，罗丹明B，得克萨斯红或TRITC。虽然它是方便的荧光光源路径中的适合的电子控制快门，以允许快门的操作，而不用手接触显微镜，我们还有效地只配备了一个手动快门上设置标记。最后，高倍率，高数值apertu在注射过程中，再用水浸泡客观需要观察。这个目标应该是设计用于无盖玻片。我们已经成功地使用的蔡司Achroplan 100x/1.0W，奥林巴斯LUMPlan FL 100x/1.0W，和奥林巴斯的LUMPlan FL / IR 60x/0.9的W目标。 一台显微神经元染料注射过程中需要定位和移动微管。我们都用了的徕卡显微操作的舞台装Narashige 3轴液压显微操纵器。 一种细胞内的直流放大器 ，与注入电流的设施，从微管的离子电渗疗法的DiI注射所需的。 4。染料注入程序<ol><li>将注射显微镜在舞台上（ 图3）与所安装的胚胎（s）滑动。使用10x物镜定位的胚胎，并把它到的视场的中心。 </li><li>瑞士法郎ING的高功率目标的观看位置和使胚胎成为关注的焦点。 DIC的光学元件（或使用，如果靶细胞表达绿色荧光蛋白的荧光）下找到感兴趣的小区，并把它到的视场的中心。确保在显微镜的视场光阑打开的视场的边缘，没有超出。一条狭窄的照明光束，将有助于微管的位置（参见下面的步骤4.4）。提高目标，直到它上面的胚胎是尽可能高的接触的同时，仍保持与林格氏/ PBS溶液。 </li><li>将浴电极到PBS以及清楚的胚胎和的微量吸移管的路径的直流放大器。 </li><li>注射微量吸管插入到它的保持器，它已被用0.1M LiCl溶液填充。将微吸管持有人的微操作机器人。使用显微操作器的粗控制，带来的微量吸移管的前端之间的空间中的目标和胚胎。确保它是级以上的胚胎。由于工作距离短的高功率目标，微量吸移管将被保持在一个浅的角度，以使其成为焦点（参见图3）。通过试验和错误中的第一个实例，你将不得不建立适当的角度。如果角度太陡，你将不能够得到微吸管尖端，成为关注的焦点。如果太浅，微吸管轴将靠在墙上的大坝沐浴液犯规。 </li><li>中心的视场中的微量吸移管的前端。要做到这一点，使你的眼睛到胚胎的水平和向后和向前移动微吸管的显微镜载物台的Y轴。当它的尖端穿过光路，你会看到明亮的反射的光线从它。向前移动的微量吸移管的前端，在X轴，这样它冲出的光束。这将是更容易找到的小微管如果你正在寻找它的轴，而不是它的尖领域的高功率目标。现在看，通过显微镜目镜，直到轴的微管是成为关注的焦点，逐渐降低高功率的目标。向后和向前的微量吸移管移动的Y轴与显微操作器控制有助于找到它，因为它逐渐进入焦点。当轴处于焦点时，在X-轴的微量吸移管移动，直到它的端部位于视场的中心。在这个阶段，微吸管尖要的水平之上的胚胎。切换到荧光照明和检查提示，检查是否有泄漏的直接投资收益。调节的直流电流，以防止任何DiI荧光晶体的尖端处形成。 </li><li>使用显微操作器控制，降低在Z轴的微量吸移管。把它带回成为关注的焦点，在显微镜聚焦控制。逐步降低对胚胎的微管成这个样子。首先，你可以做到这一点粗Z-轴控制的显微操作显微镜。然而，作为的胚胎成为关注的焦点，您应该切换到很好的控制旋钮。 </li><li>当表面的胚胎进入焦点，移动的微量吸移管的前端的显微操作的方向，朝向的视场的边缘。专注于细胞被注入，并检查它是否是在视场中心的。重新聚焦于微吸管尖端和在Y-轴移动，直到它位于Y轴作为细胞中的同一级别。向细胞中的X轴移动的微量吸移管的前端，作为降低它在Z-轴。如果使用徕卡显微操纵器，你可能会发现，在显微镜载物台控制给你更好地控制运动的X-轴比微操作机器人控制，切换到舞台控制的细胞如针尖接近。 </li><li>带来的微量吸移管的前端接触到与该单元格，并在其表面上的凹陷。通过几个纳安depolarizing的电流为几秒钟。您应该看到一个小水晶的直接投资收益形成的细胞。要确认已被标记细胞，短暂打开快门日光灯照亮胚胎，然后再切换回DIC。如果身体的关注单元的标签有迹象显示，施加电流为几秒钟以上。关闭电流，并迅速拉胚胎远离使用的显微镜​​载物台控制在X-轴的微量吸移管。然后撤回从该溶液中的微量吸移管。 DiI荧光标记如果没有很明显，微管可能被堵塞，需要更换一个新的微量移液器。您可能会发现，微吸管尖端的途中细胞的利息已陷入僵局，其他组织和本组织的染色，而不是细胞。在这种情况下，尝试稍微撤回的微量吸移管，并重新接近细胞。这可能是必要的替换的微量吸移管，并再次尝试。 </li></ol><ol start="9"><li>如果需要的话，可以是多个单元格的个体加盟标记在同一个胚胎。 </li><li>除去大部分的喷射室的溶液中，然后由4用PBS洗涤10分钟，然后加入7.4％甲醛/ PBS固定胚胎。胚胎，然后在室温下放置至少4小时（或在4℃下过夜），在黑暗，潮湿的腔室（以避免漂白或干下降沿）允许DiI荧光扩散到整个神经元的过程。在这个阶段，DiI荧光标记的细胞可被光转换或直接在共焦显微镜下观察。 </li></ol> 5。光转化为DIC光学检查光转化的原理是由荧光染色的细胞与适当的波长的照射过程中发出的光通过的DAB（照片）的氧化。这意味着明亮的细胞在较短的时间内弱阳性细胞相比，光转换。如果多个小区在一个试样标记相差太多关于它们的亮度，这可能导致困难lties，在photoconverting所有的人都在相同的质量（ 见图4C）：在这种情况下，可能会决定忽略较弱的细胞（使用简短照明）之间的一种折衷或接受光明的细胞开始膨胀（使用更长的照明） 。如果所有的细胞都太弱标记的（因此，需要很长的照明），由内源性过氧化物酶引起的背景可以成为一个问题。由于DAB是有毒的，应该小心处理。废物可以“停用”用7％的氯漂白。 <ol><li>必须进行光转化为每个胚胎单独。在每张幻灯片的情况下，只有一个胚胎被注入，在PBS溶液覆盖的胚胎被替换为一个DAB溶液（3毫克的DAB /毫升Tris缓冲液），用100倍的目标观察荧光显微镜和填充的电池放置在滑动。 （使用正置显微镜的客观逢低到民建联解决方案和应后用清水清洗几次，光转化procedure完成，这可避免如果使用倒置显微镜）。一个Cy3的过滤器组和一个100W汞灯和红色的激发波长，直到棕色的DAB反应产物的细胞标记的神经元变得明显（切换到明视场照明，定期检查）。最优DAB染色所花费的时间是可变的，但需要超越阶段已经完全褪色荧光的激发光的曝光。光电转换完成后，多次用PBS洗制剂。更换下降70％甘油的PBS，刮掉的有机硅用手术刀刀片，更换环凡士林，加盖玻片。 </li><li>当多个胚胎已填充上的一个滑动时，每个胚胎转移到一小滴的PBS对一个单独的24×60毫米盖玻片与腹侧朝向胚胎玻璃。将每个胚胎周围创造一个良好的框架胶带和覆盖的编制与PBS。避免在潮湿的光电转换，以防止他们从干燥室前的幻灯片。交流PBS的DAB溶液的胚胎。立即将幻灯片上的倒置显微镜配备了一个100W的水银灯和使用Cy3的激发DII的过滤器，用斧头50的目标。经光电转换后，转移胚胎到一个新的幻灯片中下降了70％甘油，加盖玻片并加盖指甲油。 </li></ol> 6。激光共聚焦显微镜检查<ol><li>在步骤4.10，转移胚胎到一个新的24×60毫米盖玻片上滴一小滴PBS。我们达到最佳的光学系统，通过安装与背面朝向盖玻片（盖玻片和胚胎之间只留下少量的PBS）的扁平的胚胎。 </li><li>将一帧胶带缠绕的胚胎和覆盖的幻灯片时，扩大PBS下降，以填满整个画面。将一个幻灯片上，并修复它的指甲油。应该是充满神经元考试激光共聚焦显微镜（或荧光）尽快独立非执行董事。 </li></ol>

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M., Seifert, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Axon+growth+from+limb+motorneurons+in+the+locust+embryo:+the+effect+of+target+limb+removal+on+the+pattern+of+axon+branching+in+the+periphery.">Axon growth from limb motorneurons in the locust embryo: the effect of target limb removal on the pattern of axon branching in the periphery.</a> Developmental Biology. 106, 438-449 (1984).</li><li>Whitington, P. M., Leach, D., Sandeman, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evolutionary+change+in+neural+development+within+the+arthropods:+axonogenesis+in+the+embryos+of+two+crustaceans.">Evolutionary change in neural development within the arthropods: axonogenesis in the embryos of two crustaceans.</a> Development. 118, 449-461 (1993).</li><li>Williams, D. 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作者宣称，他们没有竞争的金融利益。

果蝇作为一个模型系统的一个主要优点是，它允许单细胞水平上的发展和功能的分析。有关的神经系统，其中的细胞类型的多样性是非常高的，周边小区的功能和形态，可以是完全不同的，这是特别有用的。 我们在这里提出的方法允许单个神经元的标记的染料，可以被转化为一个永久染色或直接用荧光显微镜检查。它揭示了神经过程非常详细的形态（ 图4）。</s...

在单个细胞水平的神经元形态的知识是理解神经元的连接和中枢神经系统功能的一个重要先决条件。因此，从最早的天，神经科学，研究人员试图开发单细胞标记技术（参见图7为处理这一问题的历史）。经典方法，如高尔基染色的神经元的形态，但提供了极好的分辨率，是不适合的，如果一个人来标记特定类型的神经元的定向的方式，作为染色以随机方式发生。染色单细胞细胞内或juxtacellular的注射染料从一个微电极的方法的发展，处理特定的标记的要求。 单个神经元的染料注射到果蝇的应用提出了重大挑战，因为规模小的有机体，它的神经元。然而，单一的胚胎果蝇的神经元染色</em&gt;神经元实现了在20世纪80年代中期，在实验室中科古德曼15。虽然长足的方法有用的，在过去的20-30年（ 例如 ，10）在果蝇神经元的发育机制，提供了一些重要的见解，很多工人纷纷避开它，主要是因为它的技术要求。 基因技术在果蝇的神经元标签的情况在最近几年也促进了不受欢迎的单个神经元的染料注入。 GAL4表达的膜有针对性的GFP结构，可提供出色的分辨率神经形态1，20。然而，这种方法有一定的局限性：经常不可避免的多个单元格中的GFP表达，可以掩盖单个神经元的结构和一个GAL4驱动线可能不是可用来驱动在感兴趣的特定的神经元中的表达。 MARCM（马赛克分析与代理商ressible细胞标记物）方法8基本上任何神经元标记可以提供在单个细胞水平，但不能被成功地用于在胚胎和早期幼虫，因为的GAL80蛋白的周转缓慢。 由于这些遗传标记的局限性，我们相信在果蝇胚胎，单个神经元的染料注入仍然是一个有价值的技术，值得更广泛的应用。为了促进这一目标，我们在这里提供的方法的详细说明。说明它的权力是由我们最近帐户的形态一套完整的interneurons在腹部neuromeres的果蝇胚胎后期14。本研究揭示的形态变异的个体的神经细胞类型和组织在中枢神经系统中的胚胎neuromere的原则，将不可能有任何其他现有的标记方法。

<table border="1"><tbody><tr><td> 的试剂/材料名称 </td><td> 公司 </td><td> 目录编号 </td><td> 评论 </td></tr><tr><td></td><td></td><td></td><td> 试剂 </td></tr><tr><td> DAB </td><td> Sigma-Aldrich公司</td><td> <a href="http://www.sigmaaldrich.com/catalog/product/sigma/d5905" target="_blank">D-5905</a> </td><td>在100mM的Tris-HCl，pH值7.4的2-3毫克/毫升</td></tr><tr><td> DiI荧光</td><td> Invitrogen公司/分子探针</td><td> <a href="http://products.invitrogen.com/ivgn/product/D282" target="_blank">D-282</a> </td><td>在乙醇中的1毫克/毫升</td></tr><tr><td> <a href="http://www.merckmillipore.com/germany/chemicals/formaldehydloesung-min-37-prozent/MDA_CHEM-104002/p_Wt6b.s1L6pAAAAEWpOEfVhTl" target="_blank">甲醛</a> </td><td>默克密理博</td><td></td><td>在PBS 7.4％ </td> &lt;/ TR&gt; <tr><td>甘油</td><td> <a href="http://www.carlroth.com/catalogue/" target="_blank">罗斯</a> </td><td> 3783 </td><td>在PBS中的70％的</td></tr><tr><td>正庚烷胶</td><td>拜尔斯道夫公司</td><td>大提琴31-39-30 * </td><td>稀释约。 1为1，n-庚烷</td></tr><tr><td> PBS </td><td></td><td></td><td> 1个</td></tr><tr><td> TRIS </td><td> <a href="http://www.carlroth.com/catalogue/" target="_blank">罗斯</a> </td><td> 4855 </td><td></td></tr><tr><td> Vectashield </td><td>矢量实验室</td><td> <a href="http://www.vectorlabs.com/catalog.aspx?prodID=427" target="_blank">H1000</a> </td><td></td></tr><tr><td></td><td></td><td></td><td> 设备 </td></tr><tr><td>共聚焦显微镜</td><td>徕卡</td><td> TCS SP2 </td><td>视图和文档标号的荧光</td></tr><tr><td> DC -mplifier </td><td>德拉甘公司</td><td>基础ION-100 </td><td>执行标号</td></tr><tr><td>盖玻片</td><td> <a href="http://www.menzel.de/" target="_blank">门泽尔</a> </td><td> BB018018A1 </td><td> 18×18毫米</td></tr><tr><td>盖玻片</td><td> <a href="http://www.menzel.de/" target="_blank">门泽尔</a> </td><td> BB024060A1 </td><td> 24×60毫米</td></tr><tr><td>解剖显微镜</td><td>徕卡</td><td> MZ8 </td><td>准备，剖析胚胎的</td></tr><tr><td>平板毛细管</td><td> <a href="http://www.hilgenberg-gmbh.de/" target="_blank">Hilgenberg</a> </td><td></td><td>外直径1毫米; GLAS厚度为0.1毫米</td></tr><tr><td>注射毛细管</td><td>科学产品</td><td> GB 100 TF 8P </td><td></td></tr><tr><td>微操作机器人ŕ </td><td>徕卡</td><td></td><td>执行标号</td></tr><tr><td>型号：P-97 </td><td>萨特仪器</td><td></td><td>拉毛细血管</td></tr><tr><td>对象的幻灯片</td><td> <a href="http://www.marienfeld-superior.com/" target="_blank">马林费尔德高级</a> </td><td> 1000000 </td><td></td></tr><tr><td>科学显微镜</td><td>蔡司</td><td> Axioskop 2电机</td><td>查看标号后光转化</td></tr><tr><td>科学显微镜</td><td>奥林巴斯</td><td> BX50 </td><td>执行标号</td></tr><tr><td>索尼MC3255摄像机</td><td>索尼/ AVT喇叭</td><td>索尼MC3255 </td><td>光电转换后记录标号</td></tr></tbody></table> 表1中。

labeling of single cells in the central nervous system of drosophila melanogaster

在这篇文章中，我们将介绍如何单独标记的亲脂性荧光膜标记DII juxtacellular注射在果蝇胚胎中枢神经系统的神经元。这种方法可以非常详细的神经细胞形态的可视化。这是可能的标记在中枢神经系统中的任何单元格：在DIC的光学系统或通过表达的荧光（如GFP）的遗传标记的目标神经元的细胞体的可视化。 DiI荧光标记后，可以被转化为一个永久褐变光转化用透射光与DIC光学允许可视化的细胞形态。或者，可以直接观察到DiI荧光标记的细胞用激光共聚焦显微镜，使要colocalised基因引入荧光报告蛋白。这项技术可以被用在任何动物，不论基因型，使得有可能在单细胞分辨率分析突变体的表型。

图4示出了该技术的典型的结果，在这里，我们将描述图4A示出一个DiI荧光填充的单interneuron，干净光转换的一个例子。它很好地体现了这些准备工作提供大量的细节。下观看时，DIC的光学标记的单元格内的非标记的周围组织的空间范围内变为可见的， 例如 ，所述单元主体的内皮层和内神经纤维的纤维突起的位置。 图4B是染料下降...

Watch this Scientific Journal Video about Labeling of Single Cells in the Central Nervous System of Drosophila melanogaster at JoVE.com

Labeling of Single Cells in the Central Nervous System of Drosophila melanogaster

我们提出了一种技术，用于标识单个神经元在中枢神经系统（CNS）的<em&gt;果蝇</em&gt;，胚胎，它允许由任一透射光或激光共聚焦显微镜的神经元的形态学分析。

标记的单细胞在中枢神经系统<em&gt;果蝇</em

In this  article we describe how to individually label neurons in the embryonic CNS of Drosophila melanogaster by juxtacellular injection of the ...

labeling-single-cells-central-nervous-system-drosophila

Research

JoVE Journal

Neuroscience

Labeling of Single Cells in the Central Nervous System of Drosophila melanogaster

12.6K 次观看.  University of Mainz. 我们提出了一种技术，用于标识单个神经元在中枢神经系统（CNS）的果蝇，胚胎，它允许由任一透射光或激光共聚焦显微镜的神经元的形态学分析。

视频: Labeling of Single Cells in the Central Nervous System of Drosophila melanogaster

Systemic and Local Drug Delivery for Treating Diseases of the Central Nervous System in Rodent Models

Thorough preclinical testing of central nervous system (CNS) therapeutics includes a consideration of routes of administration and agent biodistribution in assessing therapeutic efficacy. Between the two major classifications of administration, local vs. systemic, systemic delivery approaches are often preferred due to ease of administration. However, systemic delivery may result in suboptimal drug concentration being achieved in the CNS, and lead to erroneous conclusions regarding agent efficacy. Local drug delivery methods are more invasive, but may be necessary to achieve therapeutic CNS drug levels. Here, we demonstrate proper technique for three routes of systemic drug delivery: intravenous injection, intraperitoneal injection, and oral gavage. In addition, we show a method for local delivery to the brain: convection-enhanced delivery (CED). The use of fluorescently-labeled compounds is included for in vivo imaging and verification of proper drug administration. The methods are presented using murine models, but can easily be adapted for use in rats.

Thorough preclinical testing of drugs that act in the central nervous system often involves assessing and comparing drug biodistribution in association with specific routes of administration. Here, three commonly used methods of systemic delivery (intravenous, intraperitoneal, and oral) as well as a method for local delivery (convection-enhanced delivery) are demonstrated in mice.

全身和局部给药治疗的中枢神经系统疾病的啮齿动物模型

Thorough preclinical testing of central nervous system (CNS) therapeutics includes a consideration of routes of administration and agent biodistribution ...

科研

神经科学

Visualization of the Embryonic Nervous System in Whole-mount Drosophila Embryos

The Drosophila embryo is an attractive model system for investigating the cellular and molecular basis of neuronal development. Here we describe the procedure for the visualization of Drosophila embryonic nervous system using antibodies to neuronal proteins. Since the entire embryonic peripheral nervous and central nervous systems are well characterized at the level of individual cells (Dambly-Chaudi&egrave;re et al., 1986; Bodmer et al., 1987; Bodmer et al., 1989), any aberrations to these systems can be easily identified using antibodies to different neuronal proteins. The developing embryos are collected at certain times to ensure that the embryos are in the proper developmental stages for visualization. After collection, the outer layers of the embryo, the chorion membrane and the vitelline envelope that surrounds the embryo, are removed before fixation. Embryos are then incubated with neuronal antibodies and visualized using fluorescently labeled secondary antibodies. Embryos at stages 12-17 are visualized to access the embryonic nervous system. At stage 12 the CNS germ band starts shortening and by stage 15 the definitive pattern of the commissure has been achieved. By stage 17 the CNS contracts and the PNS is fully developed (Campos-Ortega et al. 1985). Thus changes in the pattern of the PNS and CNS can be easily observed during these developmental stages.

Visualization of the Embryonic Nervous System in Whole-mount Drosophila Embryos

We describe the procedure to prepare staged Drosophila embryos for the visualization of the embryonic nervous system during embryogenesis.

在全安装的胚胎神经系统的可视化果蝇胚胎

The Drosophila embryo is an attractive model system for investigating the cellular and molecular basis of neuronal development. Here we describe the ...

Optic Nerve Transection: A Model of Adult Neuron Apoptosis in the Central Nervous System

Retinal ganglion cells (RGCs) are CNS neurons that output visual information from the retina to the brain, via the optic nerve. 
The optic nerve can be accessed within the orbit of the eye and completely transected (axotomized), cutting the axons of the entire RGC population. Optic nerve 
transection is a reproducible model of apoptotic neuronal cell death in the adult CNS 1-4. This model is particularly attractive because the vitreous
 chamber of the eye acts as a capsule for drug delivery to the retina, permitting experimental manipulations via intraocular injections. The diffusion of chemicals 
through the vitreous fluid ensures that they act upon the entire RGC population. Moreover, RGCs can be selectively transfected by applying short interfering RNAs 
(siRNAs), plasmids, or viral vectors to the cut end of the optic nerve 5-7 or injecting vectors into their target, the superior colliculus 8. 
This allows researchers to study apoptotic mechanisms in the desired neuronal population without confounding effects on other bystander neurons or surrounding glia. 
An additional benefit is the ease and accuracy with which cell survival can be quantified after injury. The retina is a flat, layered tissue and RGCs are localized in 
the innermost layer, the ganglion cell layer. The survival of RGCs can be tracked over time by applying a fluorescent tracer (3% Fluorogold) to the cut end of the 
optic nerve at the time of axotomy, or by injecting the tracer into the superior colliculus (RGC target) one week prior to axotomy. The tracer is retrogradely transported, labeling 
the entire RGC population. Because the ganglion cell layer is a monolayer (one cell thick), RGC densities can be quantified in flat-mounted tissue, without the need 
for stereology. Optic nerve transection leads to the apoptotic death of 90% of injured RGCs within 14 days postaxotomy 9-11. RGC apoptosis has a 
characteristic time-course whereby cell death is delayed 3-4 days postaxotomy, after which the cells rapidly degenerate. This provides a time window for 
experimental manipulations directed against pathways involved in apoptosis.

Optic Nerve transection is a widely used model of adult CNS injury. Ninety percent of retinal ganglion cells (RGCs) whose axons are completely transected (axotomy) die within 14 days after axotomy. This model is easily amenable to experimental manipulations and highly reproducible.

视神经横断：一个成人在中枢神经系统的神经元凋亡模型

Retinal ganglion cells (RGCs) are CNS neurons that output visual information from the retina to the brain, via the optic nerve. 
The optic nerve can be ...

Multimodal Imaging of Stem Cell Implantation in the Central Nervous System of Mice

During the past decade, stem cell transplantation has gained increasing interest as primary or secondary therapeutic modality for a variety of diseases, both in preclinical and clinical studies. However, to date results regarding functional outcome and/or tissue regeneration following stem cell transplantation are quite diverse. Generally, a clinical benefit is observed without profound understanding of the underlying mechanism(s)1. Therefore, multiple efforts have led to the development of different molecular imaging modalities to monitor stem cell grafting with the ultimate aim to accurately evaluate survival, fate and physiology of grafted stem cells and/or their micro-environment. Changes observed in one or more parameters determined by molecular imaging might be related to the observed clinical effect. In this context, our studies focus on the combined use of bioluminescence imaging (BLI), magnetic resonance imaging (MRI) and histological analysis to evaluate stem cell grafting.
BLI is commonly used to non-invasively perform cell tracking and monitor cell survival in time following transplantation2-7, based on a biochemical reaction where cells expressing the Luciferase-reporter gene are able to emit light following interaction with its substrate (e.g. D-luciferin)8, 9. MRI on the other hand is a non-invasive technique which is clinically applicable10 and can be used to precisely locate cellular grafts with very high resolution11-15, although its sensitivity highly depends on the contrast generated after cell labeling with an MRI contrast agent. Finally, post-mortem histological analysis is the method of choice to validate research results obtained with non-invasive techniques with highest resolution and sensitivity. Moreover end-point histological analysis allows us to perform detailed phenotypic analysis of grafted cells and/or the surrounding tissue, based on the use of fluorescent reporter proteins and/or direct cell labeling with specific antibodies.
In summary, we here visually demonstrate the complementarities of BLI, MRI and histology to unravel different stem cell- and/or environment-associated characteristics following stem cell grafting in the CNS of mice. As an example, bone marrow-derived stromal cells, genetically engineered to express the enhanced Green Fluorescent Protein (eGFP) and firefly Luciferase (fLuc), and labeled with blue fluorescent micron-sized iron oxide particles (MPIOs), will be grafted in the CNS of immune-competent mice and outcome will be monitored by BLI, MRI and histology (Figure 1).

This article describes an optimized sequence of events for multimodal imaging of cellular grafts in rodent brain using: (i) in vivo bioluminescence and magnetic resonance imaging, and (ii) post mortem histological analysis. Combining these imaging modalities on a single animal allows cellular graft evaluation with high resolution, sensitivity and specificity.

在中央小鼠中枢神经系统的干细胞移植的多模态成像

During the past decade, stem cell transplantation has gained increasing interest as primary or secondary therapeutic modality for a variety of diseases, ...

Production of Lentiviral Vectors for Transducing Cells from the Central Nervous System

Efficient gene delivery in the central nervous system (CNS) is important in studying gene functions, modeling neurological diseases and developing therapeutic approaches. Lentiviral vectors are attractive tools in transduction of neurons and other cell types in CNS as they transduce both dividing and non-dividing cells, support sustained expression of transgenes, and have relatively large packaging capacity and low toxicity 1-3. Lentiviral vectors have been successfully used in transducing many neural cell types in vitro 4-6 and in animals 7-10.
Great efforts have been made to develop lentiviral vectors with improved biosafety and efficiency for gene delivery. The current third generation replication-defective and self-inactivating (SIN) lentiviral vectors are depicted in Figure 1. The required elements for vector packaging are split into four plasmids. In the lentiviral transfer plasmid, the U3 region in the 5' long terminal repeat (LTR) is replaced with a strong promoter from another virus. This modification allows the transcription of the vector sequence independent of HIV-1 Tat protein that is normally required for HIV gene expression 11. The packaging signal (&Psi;) is essential for encapsidation and the Rev-responsive element (RRE) is required for producing high titer vectors. The central polypurine tract (cPPT) is important for nuclear import of the vector DNA, a feature required for transducing non-dividing cells 12. In the 3' LTR, the cis-regulatory sequences are completely removed from the U3 region. This deletion is copied to 5' LTR after reverse transcription, resulting in transcriptional inactivation of both LTRs. Plasmid pMDLg/pRRE contains HIV-1 gag/pol genes, which provide structural proteins and reverse transcriptase. pRSV-Rev encodes Rev which binds to the RRE for efficient RNA export from the nucleus. pCMV-G encodes the vesicular stomatitis virus glycoprotein (VSV-G) that replaces HIV-1 Env. VSV-G expands the tropism of the vectors and allows concentration via ultracentrifugation 13. All the genes encoding the accessory proteins, including Vif, Vpr, Vpu, and Nef are excluded in the packaging system. The production and manipulation of lentiviral vectors should be carried out according to NIH guidelines for research involving recombinant DNA (<a href="http://oba.od.nih.gov/oba/rac/Guidelines/NIH_Guidelines.pdf" target="_blank">http://oba.od.nih.gov/oba/rac/Guidelines/NIH_Guidelines.pdf</a>). An approval from individual Institutional Biological and Chemical Safety Committee may be required before using lentiviral vectors. Lentiviral vectors are commonly produced by cotransfection of 293T cells with lentiviral transfer plasmid and the helper plasmids encoding the proteins required for vector packaging. Many lentiviral transfer plasmids and helper plasmids can be obtained from Addgene, a non-profit plasmid repository (<a href="http://www.addgene.org/" target="_blank">http://www.addgene.org/</a>). Some stable packaging cell lines have been developed, but these systems provide less flexibility and their packaging efficiency generally declines over time 14, 15. Commercially available transfection kits may support high efficiency of transfection 16, but they can be very expensive for large scale vector preparations. Calcium phosphate precipitation methods provide highly efficient transfection of 293T cells and thus provide a reliable and cost effective approach for lentiviral vector production.
In this protocol, we produce lentiviral vectors by cotransfection of 293T cells with four plasmids based on the calcium phosphate precipitation principle, followed by purification and concentration with ultracentrifugation through a 20% sucrose cushion. The vector titers are determined by fluorescence- activated cell sorting (FACS) analysis or by real time qPCR. The production and titration of lentiviral vectors in this protocol can be finished with 9 days. We provide an example of transducing these vectors into murine neocortical cultures containing both neurons and astrocytes. We demonstrate that lentiviral vectors support high efficiency of transduction and cell type-specific gene expression in primary cultured cells from CNS.

In this protocol we describe production, purification and titration of lentiviral vectors. We provide an example of lentiviral vector-mediated gene delivery in primary cultured neurons and astrocytes. Our methods may also apply to other cell types in vitro and in vivo.

从中枢神经系统转导细胞的慢病毒载体生产

Efficient gene delivery in the central nervous system (CNS) is important in studying gene functions, modeling neurological diseases and developing ...

An Injury Paradigm to Investigate Central Nervous System Repair in Drosophila

An experimental method has been developed to investigate the cellular responses to central nervous system (CNS) injury using the fruit-fly Drosophila. Understanding repair and regeneration in animals is a key question in biology. The damaged human CNS does not regenerate, and understanding how to promote the regeneration is one of main goals of medical neuroscience. The powerful genetic toolkit of Drosophila can be used to tackle the problem of CNS regeneration.
A lesion to the CNS ventral nerve cord (VNC, equivalent to the vertebrate spinal cord) is applied manually with a tungsten needle. The VNC can subsequently be filmed in time-lapse using laser scanning confocal microscopy for up to 24 hr to follow the development of the lesion over time. Alternatively, it can be cultured, then fixed and stained using immunofluorescence to visualize neuron and glial cells with confocal microscopy. Using appropriate markers, changes in cell morphology and cell state as a result of injury can be visualized. With ImageJ and purposely developed plug-ins, quantitative and statistical analyses can be carried out to measure changes in wound size over time and the effects of injury in cell proliferation and cell death. These methods allow the analysis of large sample sizes. They can be combined with the powerful genetics of Drosophila to investigate the molecular mechanisms underlying CNS regeneration and repair.

An Injury Paradigm to Investigate Central Nervous System Repair in Drosophila

An injury paradigm using the Drosophila larval ventral nerve cord to investigate central nervous system regeneration and repair is described. Stabbing followed by laser scanning confocal microscopy in time-lapse and fixed specimens, combined with quantitative analysis with purposefully developed software and genetics, are used to investigate the molecular mechanisms of CNS regeneration and repair.

调查中枢神经系统修复中受伤范式<em&gt;果蝇</em

An experimental method has been developed to investigate the cellular responses to central nervous system (CNS) injury using the fruit-fly Drosophila. ...

Direct Intraventricular Delivery of Drugs to the Rodent Central Nervous System

Due to an inability to cross the blood brain barrier, certain drugs need to be directly delivered into the central nervous system (CNS). Our lab focuses specifically on antisense oligonucleotides (ASOs), though the techniques shown in the video here can also be used to deliver a plethora of other drugs to the CNS. Antisense oligonucleotides (ASOs) have the capability to knockdown sequence-specific targets 1 as well as shift isoform ratios of specific genes 2. To achieve widespread gene knockdown or splicing in the CNS of mice, the ASOs can be delivered into the brain using two separate routes of administration, both of which we demonstrate in the video.
The first uses Alzet osmotic pumps, connected to a catheter that is surgically implanted into the lateral ventricle. This allows the ASOs to be continuously infused into the CNS for a designated period of time. The second involves a single bolus injection of a high concentration of ASO into the right lateral ventricle. Both methods use the mouse cerebral ventricular system to deliver the ASO to the entire brain and spinal cord, though depending on the needs of the study, one method may be preferred over the other.

We describe a method to target drugs to the central nervous system by either implanting a catheter or performing a bolus injection into the right lateral ventricle in mice. We focus specifically on the delivery of antisense oligonucleotides. This technique is readily adaptable to other drugs and to rats.

啮齿动物的中枢神经系统药物直接脑室交货

Due to an inability to cross the blood brain barrier, certain drugs need to be directly delivered into the central nervous system (CNS). Our lab focuses ...

In vivo Interrogation of Central Nervous System Translatome by Polyribosome Fractionation

Multiple processes are involved in gene expression including transcription, translation and stability of mRNAs and proteins. Each of these steps are tightly regulated, affecting the final dynamics of protein abundance. Various regulatory mechanisms exist at the translation step, rendering mRNA levels alone an unreliable indicator of gene expression. In addition, local regulation of mRNA translation has been particularly implicated in neuronal functions, shifting &#39;translatomics&#39; to the focus of attention in neurobiology. The presented method can be used to bridge transcriptomics and proteomics.
Here we describe essential modifications to the technique of polyribosome fractionation, which interrogates the translatome based on the association of actively translated mRNAs to multiple ribosomes and their differential sedimentation in sucrose gradients. Traditionally, working with in vivo samples, particularly of the central nervous system (CNS), has proven challenging due to the restricted amounts of material and the presence of fatty tissue components. In order to address this, the described protocol is specifically optimized for use with minimal amount of CNS material, as demonstrated by the use of single mouse spinal cord and brain. Briefly, CNS tissues are extracted and translating ribosomes are immobilized on mRNAs with cycloheximide. Myelin flotation is then performed to remove lipid rich components. Fractionation is performed on a sucrose gradient where mRNAs are separated according to their ribosomal loading. Isolated fractions are suitable for a range of downstream assays, including new genome wide assay technologies.

In vivo Interrogation of Central Nervous System Translatome by Polyribosome Fractionation

This protocol illustrates essential modifications of polyribosome fractionation in order to study the translatome of in vivo CNS samples. It allows global assessment of translation and transcription regulation through the isolation and comparison of total RNA to ribosome bound RNA fractions.

在中枢神经系统Translatome由多核糖体分馏体内拷问

Multiple processes are involved in gene expression including transcription, translation and stability of mRNAs and proteins. Each of these steps are ...

In vivo Optogenetic Stimulation of the Rodent Central Nervous System

The ability to probe defined neural circuits in awake, freely-moving animals with cell-type specificity, spatial precision, and high temporal resolution has been a long sought tool for neuroscientists in the systems-level search for the neural circuitry governing complex behavioral states. Optogenetics is a cutting-edge tool that is revolutionizing the field of neuroscience and represents one of the first systematic approaches to enable causal testing regarding the relation between neural signaling events and behavior. By combining optical and genetic approaches, neural signaling can be bi-directionally controlled through expression of light-sensitive ion channels (opsins) in mammalian cells. The current protocol describes delivery of specific wavelengths of light to opsin-expressing cells in deep brain structures of awake, freely-moving rodents for neural circuit modulation. Theoretical principles of light transmission as an experimental consideration are discussed in the context of performing in vivo optogenetic stimulation. The protocol details the design and construction of both simple and complex laser configurations and describes tethering strategies to permit simultaneous stimulation of multiple animals for high-throughput behavioral testing.

In vivo Optogenetic Stimulation of the Rodent Central Nervous System

Optogenetics has become a powerful tool for use in behavioral neuroscience experiments. This protocol offers a step-by-step guide to the design and set-up of laser systems, and provides a full protocol for carrying out multiple and simultaneous in vivo optogenetic stimulations compatible with most rodent behavioral testing paradigms.

在啮齿动物中枢神经系统的体内光遗传学刺激

The ability to probe defined neural circuits in awake, freely-moving animals with cell-type specificity, spatial precision, and high temporal resolution ...

Optical Clearing of the Mouse Central Nervous System Using Passive CLARITY

Traditionally, tissue visualization has required that the tissue of interest be serially sectioned and imaged, subjecting each tissue section to unique non-linear deformations, dramatically hampering one's ability to evaluate cellular morphology, distribution and connectivity in the central nervous system (CNS). However, optical clearing techniques are changing the way tissues are visualized. These approaches permit one to probe deeply into intact organ preparations, providing tremendous insight into the structural organization of tissues in health and disease. Techniques such as Clear Lipid-exchanged Acrylamide-hybridized Rigid Imaging-compatible Tissue-hYdrogel (CLARITY) achieve this goal by providing a matrix that binds important biomolecules while permitting light-scattering lipids to freely diffuse out. Lipid removal, followed by refractive index matching, renders the tissue transparent and readily imaged in 3 dimensions (3D). Nevertheless, the electrophoretic tissue clearing (ETC) used in the original CLARITY protocol can be challenging to implement successfully and the use of a proprietary refraction index matching solution makes it expensive to use the technique routinely. This report demonstrates the implementation of a simple and inexpensive optical clearing protocol that combines passive CLARITY for improved tissue integrity and 2,2&#8242;-thiodiethanol (TDE), a previously described refractive index matching solution.

Optical clearing techniques are revolutionizing the way tissues are visualized. In this report we describe modifications of the original Clear Lipid-exchanged Acrylamide-hybridized Rigid Imaging-compatible Tissue-hYdrogel (CLARITY) protocol that yields more consistent and less expensive results.

鼠标中枢神经系统的光透明使用被动CLARITY

Traditionally, tissue visualization has required that the tissue of interest be serially sectioned and imaged, subjecting each tissue section to unique ...