在Hammarlund实验室的工作是由美国国立卫生研究院拨款R01 NS066082 - 01和T32GM007223，贝克曼基金会和埃利森医学基金会。

1。组装系统下面列出了我们系统的具体组成部分。正确组装时，用户应该能够集中精力，用一只手的显微镜，将与其他阶段（或者用鼠标或操纵杆），激活与脚踏板的激光，并有一个明确的说法屏幕图像。 <ol><li> MicroPoint激光装到一个复式显微镜落射荧光港口。我们使用尼康80I，但任何研究级直立或倒置的范围应工作。 MicroPoint系统将包括一个自定义的分色，这应该与荧光标记的细胞，你希望削减。切割与替代荧光团标记的细胞额外dichroics。此外，脉冲发生器和脚踏板来与MicroPoint系统。 </li><li> 100倍，1.4 NA手术和评估再生石油的目标，再加上必要的额外的目标定位在幻灯片上的样品。我们对手术中使用尼康计划阿婆VC，发现粗的重点，发现样品，4X是有用的。 </li><li>机动与操纵杆的阶段。具有极高的精度和可重复性的一个阶段是不必要的，因为只为目标阶段。我们使用之前OPTISCAN II（见讨论）。 </li><li>相机。相机的需求，以提供足够的帧速率，标记的突起与100X目标，重点和目标，而可视化在计算机显示器上的形象。我们喜欢干切断过程的衰减过程中的照明灯，从照片褪色或损坏（见，低于1.7），以避免潜在的混杂影响。因此，相机的需求不够敏感，以适应这种减少光照水平。我们使用滨松ORCA 05G，并发现，在16.3赫兹成像足够。同样的相机可以被替换。 </li><li>计算机，显示器和图像处理软件，可以驱动摄像头。此外，我们使用图像处理软件，用鼠标操作的机动阶段（见讨论）。 </li><li>空气球台。此要求可能会有所不同位置而有所不同，但因为手术是用100X目标，我们发现振动隔离是必不可少的。 </li><li>光照明的荧光光导和快门机制供应。光导会重视的MicroPoint。快门机制是很有用的独立的激光，以减少光照强度的。我们使用一个200流明之前或徕卡EL6000，衰减高达80％的总的光强度为干切断。 </li></ol> 2。激光设置 MicroPoint激光系统提供一个详细的协议，初步调整和重点激光。我们假设一直遵循这一程序在初始安装成功。这包括使在目镜的十字线激光。在这里，我们提供了一个协议定期保养激光的重点和力度。 在MicroPoint手册中指出，脉冲氮激光是严重损害人的眼睛的能力。应小心，切勿直视激光束直接。一旦安装完毕，应安全地阻止屏障过滤器通过目镜辐射。 一粒沙子的大小，切割在一个生物体的神经细胞可以是具有挑战性。由于激光所造成的损害的面积很小，这是必须要知道，正是以目标样本三维激光的焦点位置。激光的重点是发现使用镜像幻灯片。首先，我们检查，激光聚焦的Z位置相匹配的成像路径的焦点。下一步，XY位置的损害的重点是与成像软件十字线标记。 重点在z平面<ol><li>推中性密度滤光镜ND16（图1a）。我们使用中性密度滤光片，在外延照明路径衰减的激光，同时注重。更精细的重点，推动两个ND16和ND4基因荧光过滤器。 </li><li>与交换机的设置MicroPoint 1个脉冲的激光打开"选择"（图1b），然后移动过滤器轮适当的通长的屏障过滤器（图1c）。 </li><li>将在舞台上的镜像的幻灯片和4X使用透射光的目标集中在它的针孔。 </li><li>浸油下降到镜像的幻灯片，并重新调整与100X目标的针孔。打开相机的快门与光开关杆（图1d）和激光快门（图1e）的路径。如果有必要，减少透射光强度，使针孔边缘清晰:这将有助于完全集中。 </li><li>按下脚踏板。这应该产生一个在镜子里的小洞，如果激光聚焦正确。 </li><li>略高于平面镜像幻灯片聚焦显微镜，并再次发射激光。这应该使一个更小的孔比第一枪，或没有留下任何印记。重复下面的镜像幻灯片的重点。 </li><li>如果没有一个洞是在2.5，如果高于或低于重点镜面时产生一个更大的洞，重新调整与激光消融头的对焦环。验证该洞仍然是正确与目镜十字线对准。 z平面的重点一致的使用，很少需要进行调整。 </li></ol>聚焦在XY平面上<ol start="8"><li>按照2.1到2.3以上的步骤，并按下脚踏板，使镜中的幻灯片的一个小孔。 </li><li>启动"活"捕捉模式从要素"收购"菜单。在"测量"工具栏上选择"投资回报率主编的"，然后选择"双十字"工具拖动十字线，以配合新孔的中心。点击"保存的投资回报率"，然后"退出编辑器"，并恢复实时捕捉模式。 </li><li>激活元素中的"设置鼠标的XY"，用鼠标操作的阶段。 </li><li>中性密度​​滤光镜（S）拉回到原来的位置（S），到10集的激光脉冲，并删除镜像幻灯片。 </li></ol>调整激光功率<ol start="12"><li>应调整，以减少您所选择的神经元所需的最低功率的激光功率。调整移动衰减器板（图1F），同时削减生活蠕虫（如下所述）的功率。如果激光功率过高，它会引起周围的损害或爆炸蠕虫洞。如果激光功率过低，不会切断神经元。一旦系统已成立，衰减器的位置很少需要改变。如果激光变弱，需要衰减器板块移动继续切割，它可能是一个迹象，需要更换（见讨论），染料在染料细胞。 </li></ol> 3。固定的蠕虫<ol><li>准备了3％M9的熔融在琼脂糖（磷酸二氢钾22MM，42MM的Na2HPO4，85MM氯化钠，硫酸镁1MM）的解决方案。分送熔化琼脂糖5毫升到15毫升猎鹰管。填写另一个管水。放置在一个模块化的加热元件管设置为55 ° C装满水的加热元件来创建每管的微型水浴。注:琼脂糖可以提前准备大量的后续实验重新回炉。 </li><li>每两张幻灯片，这些幻灯片之间的一个干净的幻灯片放在两层标签磁带。分配一个干净的幻灯片和另一张幻灯片的第一次，造成的琼脂糖凝胶垫使是两件磁带的厚度（图2）垂直的中间用一个塑料球吸管琼脂糖下降。从垫在30秒后取出两张幻灯片。冲洗和存放在装满水的猎鹰管与随后的幻灯片使用的塑料吸管。 </li><li>下一步，放置3-5微升0.10微米或0.05微米的聚苯乙烯珠垫的中心。添加感兴趣的神经元中表达荧光蛋白的5-10蠕虫。如果感兴趣的神经元是不对称分布的右边或左边的蠕虫，使用铂金拿起，翻转，使正确的一面是朝上的蠕虫。蠕虫必须放在准备琼脂糖垫在10分钟的幻灯片。 </li><li>小心地将盖玻片上的蠕虫。放置盖玻片后，不要动它相对的琼脂糖凝胶垫，因为这会破坏角质层和破坏的蠕虫。放置在显微镜舞台上的准备幻灯片。 </li></ol> 4。切断神经元<ol><li>光线直接进入光路切换杆的目镜。重点要削减与4X目标的蠕虫，地方的盖玻片上的油下降，并切换到100X的目标。返回到相机的光路。 </li><li>使用鼠标移动到您所选择的神经元，下十字线的中心。按脚踏火激光切断的神经元的过程。如果需要，可以使用两套脉冲切断的神经元。首先，它是有帮助的精确跟踪神经元有个别蠕虫切断，以评估后续再生。当正确执行，激光干切断生产不造成重大损害周围的皮肤组织或其他神经元（见图3）的情况下一个神经元的小破。在某些情况下，可能会形成一个小的疤痕周围损伤的部位。 </li><li>收回的蠕虫取出盖玻片直接向上运动，小心蠕虫琼脂糖垫。不要滑动盖玻片。然后，切开尖嘴钳的蠕虫周围垫和放置新鲜种子NGM板OP50 8节琼脂糖。放在垫10μL无菌M9摆脱珠的蠕虫和去除琼脂糖。 </li></ol> 5。分数再生神经元<ol><li>在步骤2.2中所述，准备琼脂糖垫。 </li><li>在3-5及广场的蠕虫万亩; L的聚苯乙烯珠。应用盖玻片。可以按顺序准备幻灯片，或者所有的蠕虫可以在推进，并保持在10厘米的培养皿中，每个都含有保持琼脂糖湿润潮湿Kimwipe幻灯片准备。 </li><li>使用100X的目标形象化切断的神经元。在许多神经元，神经断端远端切站点将保持不变。我们使用这残余的存在，作为一个神经元确实切断（图4）的指示。这个残端的存在，使我们能够区分出已被切断，从一个不会削减再生神经元。注:无论是远端和近端的树桩最初被切断后收回。 </li><li>评估再生。各种参数才能确定。一个简单的实验是受伤的神经元是否已形成一个生长锥（图4a）和再生的，或不（图4b）。此外，突起的长度可以被追踪和比较。 </li></ol> 6。代表性的成果作为一个例子，我们描述的γ-氨基丁酸（GABA）的运动神经元再生的特性。这些神经元，其中居住在腹神经索和扩展过程圆周背神经索，是必要的适当运动9。 GABA的神经细胞可以表达一个基因编码的荧光基团，如绿色荧光蛋白（GFP），可视化，控制下的联合国军司令部 -47或UNC的-25促销员（株EG1285和CZ1200， 分别可从线虫遗传中心）。 山L4阶段蠕虫描述的那样，翻转蠕虫，所以在他们的左右两侧的GABA神经元朝上，并将其放置盖玻片。切割后，在每一个蠕虫病毒，背，腹线之间的中途commissures 1-3。避免削减commissures交叉或与其他commissures，因为这些fasciculate以后难以得分。恢复所述的蠕虫。 一个成功的干切断后18至24小时，蠕虫会从手术中恢复，并表现出正常的locomotory和产蛋行为。丢弃死亡或生病的蠕虫。重新装入其余部分所描述的那样，评估再生。我们通常的目的是评估至少有30％的实验条件下切断神经元。 oxIs12动物，切断L4 GABA的神经元通常是60-70％，将发起一个生长锥的结构，扩大膜的一角和几个突起分行的延伸，并从削减站点（图4a）迁移证明。在许多情况下，生长锥​​将迁移到重新与背神经索，相邻的神经元合缝，或远侧断端。其余30％的神经元将发起没有增长，形成一个树桩近端干切断网站，或将延长只小的丝状伪足（图4b）。 <img src="/files/ftp_upload/2707/2707fig1.jpg" alt="图1"/> 图1。紫外线脉冲激光干切断系统 。具体内容是:（一）ND滤镜（二）脉冲选择（C）滤光轮杆（D）光路开关杆（E）激光快门和（F）衰减器板。 <img src="/files/ftp_upload/2707/2707fig2.jpg" alt="图2"/> 图2。准备琼脂糖垫 。为了准备所需的厚度的琼脂糖垫，两层胶带（绿色）被放置在两个幻灯片。前两个幻灯片之间放置，和琼脂糖下降，然后添加到干净的幻灯片。最后，第四个幻灯片是垂直放置的前三个，导致在一垫就是两件磁带的厚度。 <img src="/files/ftp_upload/2707/2707fig3.jpg" alt="图3"/> 图3。代表GABA的神经元axotomies。在一个典型的实验中，GABA的神经元commissures蠕虫的侧面中间被切断。一个割断合缝立即前（左图）和（右图）干切断后。 100X油目标所采取的所有图像。 <img src="/files/ftp_upload/2707/2707fig4.jpg" alt="图4"/> 图4。 GABA的神经元axotomies代表性的结果 。 24小时后干切断切断轴突再生取得。与再生轴突的生长锥（箭头）（a）在非再生轴突近端残端（箭头）（b）中所示。每个切断轴突的远侧断端显示在每个面板（箭头），是轴突被切断的迹象。

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F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sub-cellular+precision+on-chip+small-animal+immobilization,+multi-photon+imaging+and+femtosecond-laser+manipulation.">Sub-cellular precision on-chip small-animal immobilization, multi-photon imaging and femtosecond-laser manipulation.</a> Lab on a Chip. 8, 653-656 (2008).</li><li>Rohde, C. B., Zeng, F., Gonzalez-Rubio, R., Angel, M., Yanik, M. F., F, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microfluidic+system+for+on-chip+high-throughput+whole-animal+sorting+and+screening+at+subcellular+resolution.">Microfluidic system for on-chip high-throughput whole-animal sorting and screening at subcellular resolution.</a> Proceedings of the National Academy of Sciences. 104, 13891-13895 (2007).</li><li>Hulme, S. E., Shevkoplyas, S. S., Apfeld, J., Fontana, W., Whitesides, G. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+microfabricated+array+of+clamps+for+immobilizing+and+imaging+C.+elegans.">A microfabricated array of clamps for immobilizing and imaging C. elegans.</a> Lab on a Chip. 7, 1515-1523 (2007).</li><li>Ben-Yakar, A., Chronis, N., Lu, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microfluidics+for+the+analysis+of+behavior,+nerve+regeneration,+and+neural+cell+biology+in+C.+elegans.">Microfluidics for the analysis of behavior, nerve regeneration, and neural cell biology in C. elegans.</a> Current Opinion in Neurobiology. 19, 561-567 (2009).</li><li>Samara, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Large-scale+in+vivo+femtosecond+laser+neurosurgery+screen+reveals+small-molecule+enhancer+of+regeneration.">Large-scale in vivo femtosecond laser neurosurgery screen reveals small-molecule enhancer of regeneration.</a> Proceedings of the National Academy of Sciences. , (2010).</li><li>Nelson, F. K., Riddle, D. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functional+study+of+the+Caenorhabditis+elegans+secretory-excretory+system+using+laser+microsurgery.">Functional study of the Caenorhabditis elegans secretory-excretory system using laser microsurgery.</a> Journal of Experimental Zoology. 231, 45-56 (1984).</li><li>Sulston, J. E., White, J. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regulation+and+cell+autonomy+during+postembryonic+development+of+Caenorhabditis+elegans.">Regulation and cell autonomy during postembryonic development of Caenorhabditis elegans.</a> Developmental Biology. 78, 577-597 (1980).</li><li>Kimble, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Alterations+in+cell+lineage+following+laser+ablation+of+cells+in+the+somatic+gonad+of+Caenorhabditis+elegans.">Alterations in cell lineage following laser ablation of cells in the somatic gonad of Caenorhabditis elegans.</a> Developmental Biology. 87, 286-300 (1981).</li><li>Allen, P. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Single-synapse+ablation+and+long-term+imaging+in+live+C.+elegans.">Single-synapse ablation and long-term imaging in live C. elegans.</a> Journal of Neuroscience Methods. 173, 20-26 (2008).</li><li>Pujol, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Distinct+Innate+Immune+Responses+to+Infection+and+Wounding+in+the+C.+elegans+Epidermis.">Distinct Innate Immune Responses to Infection and Wounding in the C. elegans Epidermis.</a> Current biology. CB 18, 481-489 (2008).</li></ol>

各种激光系统已被用于削减在 C突起在详细3,7,10,11研究他们的表现， 线虫和一些研究。 MicroPoint在我们​​的协议中使用的激光是一个交钥匙系统，容易设置和维护，并以较低的成本相比，一个钛蓝宝石激光系统的研究人员提供。然而，一个钛蓝宝石系统相比，预计MicroPoint激光造成损坏面积较大，这可能对一些应用程序不利。如果需要一个钛蓝宝石系统，建设这样一个系统的一个很好的协议是提供12。 目前的协议，可以执行各种不同的神经元在C 线虫 ，但是，我们注意到，在不同类型的神经元之间的再生能力的差异，预计13。此外，不同的基因背景可以影响再生成功。虽然GABA的神经元再生的比例是相当不同的转基因标记菌株之间的一致，我们已经注意到在再生的整体 略有增加， 与 oxIs12 15蠕虫juIs76 14。触摸神经元的标记之间的差异也被描述2。 我们喜欢固定与微球，而不是麻醉药的蠕虫，如珠导致更快和更一致的固定16。这也是有利的，因为我们能够观察到任何可能由于麻醉17混杂影响的再生。另一种麻醉无固定的方法是使用微流体装置。已被广泛使用干切断微流体描述17-22。 我们发现，一致的使用，激光功能的最佳时香豆素染料单元440改为每周一次以下MicroPoint手册中所述的程序。如果有必要，可以使用衰减滑块，增加功率，但是这可能是一个旧的染料或激光错位的迹象。此外，染料的细胞有一个有限的寿命，并最终将需要进行重建或替换（请参阅排除）。 它可能很难操纵下十字线，标志着激光聚焦的目标突起，特别是如果动物是不完全瘫痪。我们发现，手工阶段是不为这一目的的最佳，但它肯定是可用。一个操纵杆控制机动阶段是更精确，我们发现，使用的软件，支持图像拖动鼠标移动的机动阶段是最好的。尼康元素提供此功能，并在本协议中所述，但事无巨细的免费软件包，以及其他图像处理软件，可能也有类似的功能。精细定位的一个不同的方式，是将激光聚焦，而不是动物。阿振光束控制机制可作为一个MicroPoint激光添加，如果这种方法是首选。 除了 ​​神经再生的研究及其应用，激光可用于消融其他类型的细胞，如皮肤，肌肉，或专门的细胞，或破坏特定的神经元突触23-27。此外，规范附带伤害或疾病的神经元退化的途径，这个系统可以进行调查。因此，使用脉冲激光器将继续揭示遗传因素和细胞生物学的变化，促进神经再生和其他相关进程。 故障排除: 这里描述的一些常见问题及其相关的解决方案。 <ol type="a"><li> 激光切割不正确 。这可能是因为无论是激光的重点（见第2节），或染料在染料细胞需要更换（见MicroPoint手册）。另外，染料电池可能需要更换或重建。 </li><li> 蠕虫正在琼脂糖垫 。这通常是一个符号，琼脂糖垫太潮湿。我们发现垫需要约30秒钟，而后虫放在他们绕过这个问题。您也可以尝试使用较小的尺寸（0.05微米）微球。 </li><li> 蠕虫的恢复是困难和效率低下。这可能是造成不正确的盖玻片拆除或干琼脂糖垫。如果垫过于干燥，您可能会注意到轴突发展串珠的外观。在某些情况下，这是因为蠕虫不放在垫后，它是很快。另外，老股票的琼脂糖溶液可能有反复融化后的浓度高于3％。 </li></ol>

<table border="1"><tbody><tr><th>试剂名称</th><th>公司</th><th>目录编号</th><th>评论</th></tr><tr><td> 0.05微米的聚苯乙烯珠</td><td> Polysciences，公司</td><td> 08691 </td><td></td></tr><tr><td> 0.10微米的聚苯乙烯珠</td><td> Polysciences，公司</td><td> 00876 </td><td></td></tr><tr><td>琼脂糖的GPG / LE </td><td>美国生物分析</td><td> 00972 </td><td>超纯水</td></tr><tr><td>猎鹰14毫升聚苯乙烯圆底管</td><td> BD公司</td><td> 352057 </td><td> 17 × 100毫米的风格，nonpyrogenic </td></tr><tr><td> Thermo Scientific的平原precleaned显微镜幻灯片</td><td>伊利科学公司</td><td> 420 - 004T </td><td> 3"× 1"x 1毫米</td></tr><tr><td>盖玻片</td><td> VWR </td><td> 48366 205 </td><td> 18毫米× 18毫米号1 1 / 2 </td></tr><tr><td> KIMTECH科学Kimwipes </td><td>金佰利</td><td> 34155 </td><td></td></tr><tr><td>屋宇署猎鹰100 × 15mm的风格培养皿</td><td> BD公司</td><td> 351029 </td><td></td></tr><tr><td>录像带的空白3/4w x 500L </td><td> TimeMed </td><td> T - 512 </td><td></td></tr><tr><td>浸油</td><td>尼康</td><td></td><td>键入一个，ND = 1.515 </td></tr><tr><td> EG1285或CZ1200 </td><td> 线虫遗传中心</td><td> http://www.cbs.umn.edu/CGC/strains/ </td><td></td></tr><tr><td> OPTISCAN二</td><td>事先科学</td><td></td><td></td></tr><tr><td> NIS元素氩或Br </td><td>尼康</td><td></td><td></td></tr><tr><td> MicroPoint烧蚀激光系统</td><td>光子科学</td><td></td><td></td></tr><tr><td>复式显微镜</td><td>尼康</td><td> Eclipse的80I </td><td></td></tr><tr><td>滨松相机</td><td>日本滨松光子学</td><td>型号C8484 - 05G01 </td><td></td></tr><tr><td>戴尔Precision T3400与英特尔Core 2 Duo的PC </td><td>戴尔</td><td></td><td></td></tr><tr><td> Windows XP专业版</td><td></td><td>微软</td><td> 2002版系列包3 </td></tr><tr><td>双干浴孵化器</td><td> Fisher Scientific则</td><td></td><td>模拟控制</td></tr><tr><td> 4X计划福陆公司的目标</td><td>尼康</td><td></td><td></td></tr><tr><td> 100X计划APO VC油目标</td><td>尼康</td><td></td><td></td></tr><tr><td>杜蒙＃5 / 45钳</td><td>杜蒙</td><td> 11251-35 </td><td> Dumoxel标准提示，也可以使用＃7 </td></tr><tr><td>解剖显微镜</td><td>尼康</td><td> SMZ800 </td><td>随着NI - 150高强度照明</td></tr></tbody></table>

in vivo laser axotomy in c. elegans

神经元与其他细胞通过轴突和树突，修长的膜扩展，包含前或突触后专业沟通。如果一个神经元受到伤害或疾病受损，可能会再生。细胞内在和外在因素的影响，神经元的再生能力和恢复功能。近日，线虫C.线虫已经成为一个很好的模式生物基因识别和信号转导通路，影响神经元1-6的再生。启动C.神经再生的主要途径线虫是介导的激光切割，或干切断。干切断期间，一个荧光标记的神经元过程中使用高能量的脉冲被切断。起初， 在 C中的神经再生线虫是研究用放大飞秒激光5。然而，随后的再生的研究表明，传统的脉冲激光可以用于准确地切断神经元在体内，并引起了类似的再生反应1,3,7。 我们目前在体内的激光干切断表演的协议，在使用MicroPoint脉冲激光，交钥匙系统，是现成的，并已被广泛用于细胞进行有针对性的消融的蠕虫。我们描述的激光对准，安装蠕虫，切割特定神经元，并评估后续再生。该系统提供的能力在一项实验中削减多个蠕虫的神经元大量。因此，激光干切断本文所述的是一个高效的系统启动和分析的再生过程。

Watch this Scientific Journal Video about In vivo Laser Axotomy in C. elegans at JoVE.com

In vivo Laser Axotomy in C. elegans

减少神经元的协议<em&gt; C。线虫</em&gt;与MicroPoint脉冲激光。我们描述了建立该系统，固定蠕虫，并切断标记的神经元。优势包括成本相对较低的系统和能力，切断神经元突起或消融细胞<em&gt;在体内</em&gt;。

在 C 在体内激光干切断线虫

Neurons communicate with other cells via axons and dendrites, slender membrane extensions that contain pre- or post-synaptic specializations. If a neuron ...

Research

JoVE Journal

Neuroscience

In vivo Laser Axotomy in C. elegans

18.2K 次观看.  Yale University School of Medicine. 减少神经元的协议 C。线虫与MicroPoint脉冲激光。我们描述了建立该系统，固定蠕虫，并切断标记的神经元。优势包括成本相对较低的系统和能力，切断神经元突起或消融细胞在体内。

视频: In vivo Laser Axotomy in C. elegans

Transfection of Mouse Retinal Ganglion Cells by in vivo Electroporation

The targeting and refinement of RGC projections to the midbrain is a popular and powerful model system for studying how precise patterns of neural connectivity form during development. In mice, retinofugal projections are arranged in a topographic manner and form eye-specific layers in the Lateral Geniculate Nucleus (dLGN) of the thalamus and the Superior Colliculus (SC). The development of these precise patterns of retinofugal projections has typically been studied by labeling populations of RGCs with fluorescent dyes and tracers, such as horseradish peroxidase1-4. However, these methods are too coarse to provide insight into developmental changes in individual RGC axonal arbor morphology that are the basis of retinotopic map formation. They also do not allow for the genetic manipulation of RGCs.
Recently, electroporation has become an effective method for providing precise spatial and temporal control for delivery of charged molecules into the retina5-11. Current retinal electroporation protocols do not allow for genetic manipulation and tracing of retinofugal projections of a single or small cluster of RGCs in postnatal mice. It has been argued that postnatal in vivo electroporation is not a viable method for transfecting RGCs since the labeling efficiency is extremely low and hence requires targeting at embryonic ages when RGC progenitors are undergoing differentiation and proliferation6. 
In this video we describe an in vivo electroporation protocol for targeted delivery of genes, shRNA, and fluorescent dextrans to murine RGCs postnatally. This technique provides a cost effective, fast and relatively easy platform for efficient screening of candidate genes involved in several aspects of neural development including axon retraction, branching, lamination, regeneration and synapse formation at various stages of circuit development. In summary we describe here a valuable tool which will provide further insights into the molecular mechanisms underlying sensory map development.

Transfection of Mouse Retinal Ganglion Cells by in vivo Electroporation

We demonstrate an in vivo electroporation protocol for transfecting single or small clusters of retinal ganglion cells (RGCs) and other retinal cell types in postnatal mice over a wide range of ages. The ability to label and genetically manipulate postnatal RGCs in vivo is a powerful tool for developmental studies.

小鼠视网膜神经节细胞的转染在体内电穿孔

The targeting and refinement of RGC projections to the midbrain is a popular and powerful model system for studying how precise patterns of neural ...

科研

神经科学

Retrograde Fluorescent Labeling Allows for Targeted Extracellular Single-unit Recording from Identified Neurons In vivo

The overall goal of this method is to record single-unit responses from an identified population of neurons. In vivo electrophysiological recordings from individual neurons are critical for understanding how neural circuits function under natural conditions. Traditionally, these recordings have been performed 'blind', meaning the identity of the recorded cell is unknown at the start of the recording. Cellular identity can be subsequently determined via intracellular1, juxtacellular2 or loose-patch3 iontophoresis of dye, but these recordings cannot be pre-targeted to specific neurons in regions with functionally heterogeneous cell types. Fluorescent proteins can be expressed in a cell-type specific manner permitting visually-guided single-cell electrophysiology4-6. However, there are many model systems for which these genetic tools are not available. Even in genetically accessible model systems, the desired promoter may be unknown or genetically homogenous neurons may have varying projection patterns. Similarly, viral vectors have been used to label specific subgroups of projection neurons7, but use of this method is limited by toxicity and lack of trans-synaptic specificity. Thus, additional techniques that offer specific pre-visualization to record from identified single neurons in vivo are needed. Pre-visualization of the target neuron is particularly useful for challenging recording conditions, for which classical single-cell recordings are often prohibitively difficult8-11. The novel technique described in this paper uses retrograde transport of a fluorescent dye applied using tungsten needles to rapidly and selectively label a specific subset of cells within a particular brain region based on their unique axonal projections, thereby providing a visual cue to obtain targeted electrophysiological recordings from identified neurons in an intact circuit within a vertebrate CNS.
The most significant novel advancement of our method is the use of fluorescent labeling to target specific cell types in a non-genetically accessible model system. Weakly electric fish are an excellent model system for studying neural circuits in awake, behaving animals12. We utilized this technique to study sensory processing by "small cells" in the anterior exterolateral nucleus (ELa) of weakly electric mormyrid fish. "Small cells" are hypothesized to be time comparator neurons important for detecting submillisecond differences in the arrival times of presynaptic spikes13. However, anatomical features such as dense myelin, engulfing synapses, and small cell bodies have made it extremely difficult to record from these cells using traditional methods11, 14. Here we demonstrate that our novel method selectively labels these cells in 28% of preparations, allowing for reliable, robust recordings and characterization of responses to electrosensory stimulation.

Retrograde Fluorescent Labeling Allows for Targeted Extracellular Single-unit Recording from Identified Neurons In vivo

Retrograde transport of fluorescent dye labels a sub-population of neurons based on anatomical projection. Labeled axons can be visually targeted in vivo, permitting extracellular recording from identified axons. This technique facilitates recording when neurons cannot be labeled through genetic manipulation or are difficult to isolate using 'blind' in vivo approaches.

逆行荧光染料标记允许有针对性的外单细胞记录技术鉴定神经元<em&gt;在体内</em

The overall goal of this method is to record single-unit responses from an identified population of neurons. In vivo electrophysiological recordings from ...

Functional Neuroimaging Using Ultrasonic Blood-brain Barrier Disruption and Manganese-enhanced MRI

Although mice are the dominant model system for studying the genetic and molecular underpinnings of neuroscience, functional neuroimaging in mice remains technically challenging. One approach, Activation-Induced Manganese-enhanced MRI (AIM MRI), has been used successfully to map neuronal activity in rodents 1-5. In AIM MRI, Mn2+ acts a calcium analog and accumulates in depolarized neurons 6,7. Because Mn2+ shortens the T1 tissue property, regions of elevated neuronal activity will enhance in MRI. Furthermore, Mn2+ clears slowly from the activated regions; therefore, stimulation can be performed outside the magnet prior to imaging, enabling greater experimental flexibility. However, because Mn2+ does not readily cross the blood-brain barrier (BBB), the need to open the BBB has limited the use of AIM MRI, especially in mice.
One tool for opening the BBB is ultrasound. Though potentially damaging, if ultrasound is administered in combination with gas-filled microbubbles (i.e., ultrasound contrast agents), the acoustic pressure required for BBB opening is considerably lower. This combination of ultrasound and microbubbles can be used to reliably open the BBB without causing tissue damage 8-11.
Here, a method is presented for performing AIM MRI by using microbubbles and ultrasound to open the BBB. After an intravenous injection of perflutren microbubbles, an unfocused pulsed ultrasound beam is applied to the shaved mouse head for 3 minutes. For simplicity, we refer to this technique of BBB Opening with Microbubbles and UltraSound as BOMUS 12. Using BOMUS to open the BBB throughout both cerebral hemispheres, manganese is administered to the whole mouse brain. After experimental stimulation of the lightly sedated mice, AIM MRI is used to map the neuronal response.
To demonstrate this approach, herein BOMUS and AIM MRI are used to map unilateral mechanical stimulation of the vibrissae in lightly sedated mice 13. Because BOMUS can open the BBB throughout both hemispheres, the unstimulated side of the brain is used to control for nonspecific background stimulation. The resultant 3D activation map agrees well with published representations of the vibrissae regions of the barrel field cortex 14. The ultrasonic opening of the BBB is fast, noninvasive, and reversible; and thus this approach is suitable for high-throughput and/or longitudinal studies in awake mice.

A technique is described for broadly opening the blood-brain barrier in the mouse using microbubbles and ultrasound. Using this technique, manganese can be administered to the mouse brain. Because manganese is an MRI contrast agent that accumulates in depolarized neurons, this approach enables imaging of neuronal activity.

使用超声波血脑屏障破坏和锰增强MRI的脑功能成像

Although mice are the dominant model system for studying the genetic and molecular underpinnings of neuroscience, functional neuroimaging in mice remains ...

Automated Quantification of Synaptic Fluorescence in C. elegans

Synapse strength refers to the amplitude of postsynaptic responses to presynaptic neurotransmitter release events, and has a major impact on overall neural circuit function. Synapse strength critically depends on the abundance of neurotransmitter receptors clustered at synaptic sites on the postsynaptic membrane. Receptor levels are established developmentally, and can be altered by receptor trafficking between surface-localized, subsynaptic, and intracellular pools, representing important mechanisms of synaptic plasticity and neuromodulation. Rigorous methods to quantify synaptically-localized neurotransmitter receptor abundance are essential to study synaptic development and plasticity. Fluorescence microscopy is an optimal approach because it preserves spatial information, distinguishing synaptic from non-synaptic pools, and discriminating among receptor populations localized to different types of synapses. The genetic model organism Caenorhabditis elegans is particularly well suited for these studies due to the small size and relative simplicity of its nervous system, its transparency, and the availability of powerful genetic techniques, allowing examination of native synapses in intact animals.
Here we present a method for quantifying fluorescently-labeled synaptic neurotransmitter receptors in C. elegans. Its key feature is the automated identification and analysis of individual synapses in three dimensions in multi-plane confocal microscope output files, tabulating position, volume, fluorescence intensity, and total fluorescence for each synapse. This approach has two principal advantages over manual analysis of z-plane projections of confocal data. First, because every plane of the confocal data set is included, no data are lost through z-plane projection, typically based on pixel intensity averages or maxima. Second, identification of synapses is automated, but can be inspected by the experimenter as the data analysis proceeds, allowing fast and accurate extraction of data from large numbers of synapses. Hundreds to thousands of synapses per sample can easily be obtained, producing large data sets to maximize statistical power. Considerations for preparing C. elegans for analysis, and performing confocal imaging to minimize variability between animals within treatment groups are also discussed. Although developed to analyze C. elegans postsynaptic receptors, this method is generally useful for any type of synaptically-localized protein, or indeed, any fluorescence signal that is localized to discrete clusters, puncta, or organelles.
The procedure is performed in three steps: 1) preparation of samples, 2) confocal imaging, and 3) image analysis. Steps 1 and 2 are specific to C. elegans, while step 3 is generally applicable to any punctate fluorescence signal in confocal micrographs.

Automated Quantification of Synaptic Fluorescence in C. elegans

The abundance of neurotransmitter receptors clustered at synapses strongly influences synaptic strength. This method quantifies fluorescently-labeled neurotransmitter receptors in three dimensions with single-synapse resolution in C. elegans, allowing hundreds of synapses to be rapidly characterized within a single sample without distortions introduced by z-plane projection.

突触荧光自动定量<em&gt; C。线虫</em

Synapse strength refers to the amplitude of postsynaptic responses to presynaptic neurotransmitter release events, and has a major impact on overall ...

C. elegans Tracking and Behavioral Measurement

We have developed instrumentation, image processing, and data analysis techniques to quantify the locomotory behavior of C. elegans as it crawls on the surface of an agar plate. For the study of the genetic, biochemical, and neuronal basis of behavior, C. elegans is an ideal organism because it is genetically tractable, amenable to microscopy, and shows a number of complex behaviors, including taxis, learning, and social interaction1,2. Behavioral analysis based on tracking the movements of worms as they crawl on agar plates have been particularly useful in the study of sensory behavior3, locomotion4, and general mutational phenotyping5. Our system works by moving the camera and illumination system as the worms crawls on a stationary agar plate, which ensures no mechanical stimulus is transmitted to the worm. Our tracking system is easy to use and includes a semi-automatic calibration feature. A challenge of all video tracking systems is that it generates an enormous amount of data that is intrinsically high dimensional. Our image processing and data analysis programs deal with this challenge by reducing the worms shape into a set of independent components, which comprehensively reconstruct the worms behavior as a function of only 3-4 dimensions6,7. As an example of the process we show that the worm enters and exits its reversal state in a phase specific manner.

C. elegans Tracking and Behavioral Measurement

We have developed a video-rate tracking microscope system that can record and quantify C. elegans behavior at high resolution and high speeds. We have also developed computational methods to reduce the dimensionality of the worm images to a fundamental set of measurements that completely describe the shape of the worm.

线虫跟踪和行为测量

We have developed instrumentation, image processing, and data analysis techniques to quantify the locomotory behavior of C. elegans as it crawls on the ...

A Molecular Readout of Long-term Olfactory Adaptation in C. elegans

During sustained stimulation most sensory neurons will adapt their response by decreasing their sensitivity to the signal. The adaptation response helps shape attention and also protects cells from over-stimulation. Adaptation within the olfactory circuit of C. elegans was first described by Colbert and Bargmann1,2. Here, the authors defined parameters of the olfactory adaptation paradigm, which they used to design a genetic screen to isolate mutants defective in their ability to adapt to volatile odors sensed by the Amphid Wing cells type C (AWC) sensory neurons. When wildtype C. elegans animals are exposed to an attractive AWC-sensed odor3 for 30 min they will adapt their responsiveness to the odor and will then ignore the adapting odor in a chemotaxis behavioral assay for ~1 hr. When wildtype C. elegans animals are exposed to an attractive AWC-sensed odor for ~1 hr they will then ignore the adapting odor in a chemotaxis behavioral assay for ~3 hr. These two phases of olfactory adaptation in C. elegans were described as short-term olfactory adaptation (induced after 30 min odor exposure), and long-term olfactory adaptation (induced after 60 min odor exposure). Later work from L'Etoile et al.,4 uncovered a Protein Kinase G (PKG) called EGL-4 that is required for both the short-term and long-term olfactory adaptation in AWC neurons. The EGL-4 protein contains a nuclear localization sequence that is necessary for long-term olfactory adaptation responses but dispensable for short-term olfactory adaptation responses in the AWC4. By tagging EGL-4 with a green fluorescent protein, it was possible to visualize the localization of EGL-4 in the AWC during prolonged odor exposure. Using this fully functional GFP-tagged EGL-4 (GFP::EGL-4) molecule we have been able to develop a molecular readout of long-term olfactory adaptation in the AWC5. Using this molecular readout of olfactory adaptation we have been able to perform both forward and reverse genetic screens to identify mutant animals that exhibit defective subcellular localization patterns of GFP::EGL-4 in the AWC6,7. Here we describe: 1) the construction of GFP::EGL-4 expressing animals; 2) the protocol for cultivation of animals for long-term odor-induced nuclear translocation assays; and 3) the scoring of the long-term odor-induced nuclear translocation event and recovery (re-sensitization) from the nuclear GFP::EGL-4 state.

A Molecular Readout of Long-term Olfactory Adaptation in C. elegans

Here we describe a molecular readout of long-term olfactory adaptation in Caenorhabditis elegans. The Protein Kinase G, EGL-4, is necessary for stable adaptation responses in the primary sensory neuron pair called AWC. During prolonged odor exposure EGL-4 translocates from the cytosol to nucleus of the AWC.

长期的嗅觉适应性的分子读数<em&gt; C.线虫</em

During sustained stimulation most sensory neurons will adapt their response by decreasing their sensitivity to the signal. The adaptation response helps ...

In vivo Neuronal Calcium Imaging in C. elegans

The nematode worm C. elegans is an ideal model organism for relatively simple, low cost neuronal imaging in vivo. Its small transparent body and simple, well-characterized nervous system allows identification and fluorescence imaging of any neuron within the intact animal. Simple immobilization techniques with minimal impact on the animal's physiology allow extended time-lapse imaging. The development of genetically-encoded calcium sensitive fluorophores such as cameleon 1 and GCaMP 2 allow in vivo imaging of neuronal calcium relating both cell physiology and neuronal activity. Numerous transgenic strains expressing these fluorophores in specific neurons are readily available or can be constructed using well-established techniques. Here, we describe detailed procedures for measuring calcium dynamics within a single neuron in vivo using both GCaMP and cameleon. We discuss advantages and disadvantages of both as well as various methods of sample preparation (animal immobilization) and image analysis. Finally, we present results from two experiments: 1) Using GCaMP to measure the sensory response of a specific neuron to an external electrical field and 2) Using cameleon to measure the physiological calcium response of a neuron to traumatic laser damage. Calcium imaging techniques such as these are used extensively in C. elegans and have been extended to measurements in freely moving animals, multiple neurons simultaneously and comparison across genetic backgrounds. C. elegans presents a robust and flexible system for in vivo neuronal imaging with advantages over other model systems in technical simplicity and cost.

In vivo Neuronal Calcium Imaging in C. elegans

With its small transparent body, well-documented neuroanatomy and a host of amenable genetic techniques and reagents, C. elegans makes an ideal model organism for in vivo neuronal imaging using relatively simple, low-cost techniques. Here we describe single neuron imaging within intact adult animals using genetically encoded fluorescent calcium indicators.

C.在体内神经元钙成像技术线虫

The nematode worm C. elegans is an ideal model organism  for relatively simple, low cost neuronal imaging in vivo. Its small transparent body and simple, ...

Visualizing Neuroblast Cytokinesis During C. elegans Embryogenesis

This protocol describes the use of fluorescence microscopy to image dividing cells within developing Caenorhabditis elegans embryos. In particular, this protocol focuses on how to image dividing neuroblasts, which are found underneath the epidermal cells and may be important for epidermal morphogenesis. Tissue formation is crucial for metazoan development&#160;and relies on external cues from neighboring tissues. C. elegans is an excellent model organism to study tissue morphogenesis in vivo due to its transparency and simple organization, making its tissues easy to study via microscopy. Ventral enclosure is the process where the ventral surface of the embryo is covered by a single layer of epithelial cells. This event is thought to be facilitated by the underlying neuroblasts, which provide chemical guidance cues to mediate migration of the overlying epithelial cells. However, the neuroblasts are highly proliferative and also may act as a mechanical substrate for the ventral epidermal cells. Studies using this experimental protocol could uncover the importance of intercellular communication during tissue formation, and could be used to reveal the roles of genes involved in cell division within developing tissues.

Visualizing Neuroblast Cytokinesis During C. elegans Embryogenesis

This protocol describes how to image dividing cells within a tissue in Caenorhabditis elegans embryos. While several protocols describe how to image cell division in the early embryo, this protocol describes how to image cell division within a developing tissue during mid late embryogenesis.

可视化的神经细胞胞质分裂过程中<em&gt; C。线虫</em&gt;胚胎

This protocol describes the use of fluorescence microscopy to image dividing cells within developing Caenorhabditis elegans embryos. In particular, this ...

Laser Nanosurgery of Cerebellar Axons In Vivo

Only a few neuronal populations in the central nervous system (CNS) of adult mammals show local regrowth upon dissection of their axon. In order to understand the mechanism that promotes neuronal regeneration, an in-depth analysis of the neuronal types that can remodel after injury is needed. Several studies showed that damaged climbing fibers are capable of regrowing also in adult animals1,2. The investigation of the time-lapse dynamics of degeneration and regeneration of these axons within their complex environment can be performed by time-lapse two-photon fluorescence (TPF) imaging in vivo3,4. This technique is here combined with laser surgery, which proved to be a highly selective tool to disrupt fluorescent structures in the intact mouse cortex5-9.
This protocol describes how to perform TPF time-lapse imaging and laser nanosurgery of single axonal branches in the cerebellum in vivo. Olivocerebellar neurons are labeled by anterograde tracing with a dextran-conjugated dye and then monitored by TPF imaging through a cranial window. The terminal portion of their axons are then dissected by irradiation with a Ti:Sapphire laser at high power. The degeneration and potential regrowth of the damaged neuron are monitored by TPF in vivo imaging during the days following the injury.

Laser Nanosurgery of Cerebellar Axons In Vivo

Two-photon imaging, coupled to laser nanodissection, are useful tools to study degenerative and regenerative processes in the central nervous system with subcellular resolution. This protocol shows how to label, image, and dissect single climbing fibers in the cerebellar cortex in vivo.

小脑轴突的激光Nanosurgery<em&gt;在体内</em

Only a few neuronal populations in the central nervous system (CNS) of adult mammals show local regrowth upon dissection of their axon. In order to ...

In Vivo Imaging of Dauer-specific Neuronal Remodeling in C. elegans

The mechanisms controlling stress-induced phenotypic plasticity in animals are frequently complex and difficult to study in vivo. A classic example of stress-induced plasticity is the dauer stage of C. elegans. Dauers are an alternative developmental larval stage formed under conditions of low concentrations of bacterial food and high concentrations of a dauer pheromone. Dauers display extensive developmental and behavioral plasticity. For example, a set of four inner-labial quadrant (IL2Q) neurons undergo extensive reversible remodeling during dauer formation. Utilizing the well-known environmental pathways regulating dauer entry, a previously established method for the production of crude dauer pheromone from large-scale liquid nematode cultures is demonstrated. With this method, a concentration of 50,000 - 75,000 nematodes/ml of liquid culture is sufficient to produce a highly potent crude dauer pheromone. The crude pheromone potency is determined by a dose-response bioassay. Finally, the methods used for in vivo time-lapse imaging of the IL2Qs during dauer formation are described.

In Vivo Imaging of Dauer-specific Neuronal Remodeling in C. elegans

Following exposure to specific environmental stressors, the nematode Caenorhabditis elegans undergoes extensive phenotypic plasticity to enter into a stress-resistant &#8216;dauer&#8217; juvenile stage. We present methods for the controlled induction and imaging of neuroplasticity during dauer.

在永久性幼虫特有的神经重构的C语言活体成像线虫

The mechanisms controlling stress-induced phenotypic plasticity in animals are frequently complex and difficult to study in vivo. A classic example of ...