在这种方法的发展，我们非常感谢博士罗伯特E巴克斯鲍姆的重要贡献。

1。使玻璃针。 <ol><li>一个可调的微针车夫是用来制造一个约4毫米长，已关闭了坚实的梁锥尖的针。相对于长期的灵活的提示，这短短的4毫米的长度将限制在实验过程中针尖的振动。在4毫米的光纤近端地区，针锥迅速从玻璃管的直径在1毫米至15微米，而最远端1毫米的纤维直径为2.5微米。我们使用的R - 6玻璃毛细管，外径为0.9毫米，编号0.6毫米8“长度和BB - CH车夫。如果研究者感兴趣的是在测量不同的弹簧属性（即不同的长度和或其他力量的范围，针应该拉到厚度），每个毛细管拉成4个“针。针是储存在两个橡皮泥“蛇”到其中的针轻轻按下覆盖160毫米培养皿。 </li><li>的玻璃针，以及一个被描述的线针，基本上是梁用于适当刚度的弯曲弹簧力所需的测量。请注意，作为其长度立方体光束尺度的弯曲力，无论组成。因此，下面描述的校准步骤，针/梁刚度不足可以提高通过缩短少量（例如缩短10％，产生一个刚性增加28％，即0.9 3） 。 </li></ol> 2。制作和校准线针<ol><li>通过玻璃针内（第1.1节）为0.001的“镍铬丝，其尖端折断插入一根电线校准针的线拉出来的尖端在长度26毫米和胶水超级胶水。弯曲成钩远端1毫米的导线。 </li><li>使微重量从0.003“康铜导线1米。措施的电线，磁带到一米棒，以协助它尽量伸直。称量的1米的电线，准确地切1厘米的片，和弯曲到VS的几件</li><li>校准线针，需要一个测量光罩和显微解剖显微镜。该显微镜是在其一侧倾斜90 °，使用一个普遍繁荣的立场，例如，可以观察到的线针向下偏转。一个刀架在显微迷上地区是显微镜的光轴垂直安装到2.1节的线针。一个十字线标记线钩线针。挂起勾上1厘米的微型重量，并记录偏转。弯曲常数的参考线（μdynes/微米）除以所观察到的偏转（微米）的微重量比（注：1毫克= 0.98达因）计算。要准备，为下一步的线针，用手术刀在权衡点切断钩。钩的存在是额外的重量，弯曲的线，以一个小的程度。因此，它的影响光罩线的初始位置前的体重是补充。丝弹簧常数的测量是从线的挠度时的重量将被添加到弯曲点（线未来的提示）。当钩子是切断它不会改变春天的电线不断。 </li></ol> 3。校准中间的玻璃针<ol><li>中间的玻璃针的校准需要使用眼光罩倒置显微镜。在显微镜阶段的一侧是一个机械的显微操纵器，用于在光学领域的中心举行​​的第2条线针。在另一边则是一个用于移动中间的玻璃针校准（见设备表）三轴液压显微。使用micromanipulators，在光学领域的中心带来两针一起〜45 °角成160毫米× 30毫米的Petri水盘，在10倍的领域。 </li><li>第一次产生了一系列使用不同的设置吸管拉马拉针进行评估。我们的目标是使中间的玻璃针弯曲的弹簧常数为20-30μdynes/微米。刚度的测定是通过测量中间针偏转的比例相比，线针偏转。例如，电线和玻璃针触摸，玻璃针移动偏转线针的距离光罩5马克，通过眼片。侧身移动的玻璃针分离针。线针，然后返回到原来的起始位置，但现在的玻璃针将在一个新的位置。玻璃针的负载，然后计算出其新的零力具有同等效力的负载（两个针头从事）减去两针位置的位置。在这个例子中，如果两个针偏转法郎（从零光罩），线针将返回到零的GLASS针弹簧至25日，如果是这样，它弯曲具有同等效力的，弯曲的线针5分20分。因此，两针之间的弯曲的比例给人的玻璃针的校准，它是¼线针僵硬。由于弹簧的弹性系数在3.4节计算使用的距离，它们是独立的放大或光罩大小划分比例。唯一的限制的考虑是，变形小到足以在针的线性弹性范围。 </li><li>做的挠度一系列5,10,15,3,7,12,17,5,10,15光罩上的标记的位置。如果任何变形释放过早地重复。挠度之间保持在零线针对齐。对于这些变形，注意的偏转量和最终零负载释放后的玻璃针的位置。这将创建10个数据点对。其他序列可以使用，但这个系列的目的是给内部的一致性，线性和滞后的早期迹象（挠度结果5 + 10 = 15; 3 +7 = 10;释放后5,10,15挠度应约相同的开头和结尾）。 </li><li>从3.3节的记录挠度线性回归，给出了一个比一个单一的偏转比例的中间的玻璃针弯曲常数更准确的量化。它还提供了一个针和质量较高的R值接近回归线为零的截距表示校准线性的评估。从3.3节准备的10个数据对回归，第二个数字录得双，最终零负载释放后的玻璃针的位置，必须有相应的初始挠度减去，这是第一个数字对。一旦执行此操作包括设置新的电话号码对初始挠度和相应的差额计算。新增5个控制对0,0这10个实验数据对锚定在零零偏转力的线性回归。这不是绝对必要的，但增加了一个额外的准确性程度。锚固在0,0针没有弯曲武力时是不适用的原则基础上。有了这15个数据对计算线性回归。不断校准的玻璃针是弯曲的弹簧钢丝针的弯曲常数除以此回归线的斜率。 </li></ol> 4。校准工作的玻璃针用来操纵神经元<ol><li>玻璃针充分遵​​守，2-15μdynes/微米，可用于对神经元的实验是一个类似的方式进行校准第3条所述的对中间的玻璃针。也就是说，候选人的工作针偏转已知刚度中间线针的位置摆在第3针。 ，小于2μdynes/微米的玻璃针可能会略有缩短，使他们在适当的刚度范围。 </li></ol> 5。针是带有附件的因素预处理<ol><li>压入一侧以上的附件因素的解决方案，如只有针尖在溶液中浸泡10毫升的烧杯中设置一个钳举行的粘土一次性针头处理。浸入毛细管轴是不可取的，因为它提高了涂层的积累随着时间的推移，这会改变的刚度和减少针疗效的附着尖。只能用于校准的针约4倍，可用于重新校准精度。针蘸依次在0.1％多聚赖氨酸为30分钟和30分钟，然后刀豆蛋白A（1毫克/毫升的PBS）解决方案。的多聚赖氨酸可反复使用几个星期，并储存在-20 ° C ConA的新鲜每周和存储4 ° C。对于其他类型的细胞，其表面的分子针可以直接调查的过程中。 </li></ol> 6。设备设置了<ol><li>牵引工作，站上设置隔振表。它由倒置显微镜装有沿着舞台上方的左侧和液压机械臂连接到它，我们平衡的自定义扩展，在其中插入双刀架酒吧。沿栏液压显微定位，将其持有人一针显微镜的光路，而在其范围为中心的机械臂的控制。针是带有附件的因素时，打开Ringcubator或其他显微镜阶段加热系统，因此在实验开始阶段，在热平衡。 </li></ol> 7。调心针<ol><li>我们的目标是获得两针（参考和工作针），在显微镜的视野安排，以便他们的秘诀是相距约50微米，除了上述培养皿中的参考，因此有些失焦​​，当牵引针盘上的重点是。安装到徕卡双刀架，它本身就是一个小显微持针两针。使用双刀架显微，两个提示被带进平行排列。参考针插入一个持针器，只在短距离，足以让收紧领握，并放置在右侧的双刀架本次大会。插入持针器的处理工作针其长度的一半，并放置在左侧的双刀架。当两个针尖带来相同的前沿阵地，一字排开，在其持有的针头长度不同错开的领子，所以他们不妨碍获得非常接近的提示。第7.2至7.4没有放大倍率。第7.5-7.8下进行显微观察。 </li><li>预先设定的垂直控制液压显微所以一路。设置其他控制其活动范围的中心，也是前进/后退刀架螺丝。 </li><li>带两针的提示一起使用右侧刀架一侧到另一侧的调整螺丝。然后推持针双刀架在自己向前或向后需要带来的针尖相同的前沿阵地。检查从侧面对使用右侧的向上/向下拧，使其在同一平面上提示。在刀架旋转针持有人关闭的差距，如果在其持针一针的角度。 </li><li>摆动刀架，​​并放入上述介质的表面光路的秘诀，钓鱼他们急剧下降。在基地的球关节螺钉拧紧之前，你移动你的手推开。 </li><li>在显微镜的视野中找到您的针尖之前他们带来太远向下突破或犯规培养皿底部的碎片。在一个60毫米的菜，15毫升的中型盘底部以上的安全保证金提供足够的深度。不要与未校准针的初步实践，得到这一步的感觉。由以上的细胞a10x目标盘的底部向上重点开始。面积的菜远离光敏感或惩戒性的实验细胞。降低到媒体的针头。移动的针一边到另一边寻找自己的影子。如果你看不出来，降低一点，向前移动的菜，重复一边到另一边通过。查找提示，搬回（或转发）上下。 </li><li>打开一侧到另一侧的螺丝，看看你发现的针。如果你是在寻找参考针用螺丝的动作，如果没有它的校准针。校准针，使用侧面侧面的螺丝推向参考针。移动前进和后退参考的影子。如果它是你找到参考针，拧到左边，而移动的液压寻找校准针的权利。 </li><li>当两个位于针使用的精细控制的双刀架行。未来针头在盘边缘的陡峭的角度向上/向下控制的原因也有向前，向上调整，缩短与上下延长组件。左侧，前进/后退螺丝控制，相反，推动前进的提示也下降了入菜，拔回撤回针，同时提高针尖的位置。因此，提高的参考和推动校准针将延长既能使他们即使在提示的同时，也带来了成以上菜的最佳高度关系。如果参考针延伸远远超出了工作的针，你可以螺丝/工作针向下和向前推而摆动参考，并缩短，直到两个甚至工作针是较低的。倍螺丝范围不会带来针，所以你可能需要轻轻滑动的持有人本身，确保球关节螺钉以及收紧，用手指对刀架的边缘楔形使用武力滑动轴。像以前一样，您也可以在刀架旋转持针，而在媒体和观察的效果，看到的，如果他们越来越近。 </li><li>记录下这最后的调整位置的图像，应用没有任何力量的针头分离是零的参考距离。提高试样的平面以上和“公园”他们的视野边缘针。 </li><li>提高通过半月板，一针的碎片可能会被删除。第一招参考远离其他针针到对方锁定，以避免两个提示。可能需要多次传递。 </li> &lt;/ OL&gt; 8。附加到细胞<ol><li>这一点，已针直接观察到通过显微镜。从细胞附着，意见将通过显微镜和视频屏幕上。左/右的方面是相同的，但在视频屏幕上的形象向上/向下方向是不一样的（引力）显微镜上的向上和向下。为了澄清向上/向下直接观察，通过显微镜和视频屏幕上观察到的方向之间的差异，后者将被转介和引号“&#39;”了下来，“即</li><li>拖曳的过程是通过改变两针的视频屏幕上从左至右（“块块”）位置。因此，选择实验轴突部分，在相同的“横向”方向延伸的轴突。轴突可以“横向”偏离“向下”的高达15 °，因为这将是补偿由8.5节的“推”演习。此外，应选择操纵轴突细胞体的长度或更多，与活跃的生长锥。轴突延伸至细胞体的权利，是首选，由于安装参考针左侧的工作针双刀架，紧张，在牵引应用，扩大了两针之间的差距。 </li><li>一个主要的轴突延伸到细胞体的左，可以被拖走而重新调整针之间的差距扩大和提高针一点点参考的双刀架。这将允许更多的空间牵引针弯曲走向的参考，如有必要，它下面通。拖车将提前到左，牵引针弯曲走向参考针张力增加。在紧张的参考距离的关系的改变，当分析数据输入到电子表格中减去从零参考距离，而不是从测量的零距离针分离测量。 </li><li>移动接近轴突的生长锥针，再次记录零距离，提高针，隔振表了空气。 </li><li>干脆把促进生长锥前的工作针，可能会产生自发的生长锥的进步附件。要积极主动地操纵生长锥的附件，将牵引针降“下面的”生长锥，然后降低对盘底部的针。这将导致针，以“转移”，弯曲和滑动沿生长锥的菜，撞出和移动“向上”。按对盘底部的针足以使周围生长锥针尖挂钩，但到目前为止，它比单针和幻灯片落后。等待20分钟，让抓地力成为建立。随着经验的积累，成功率可高达90％高，但比较典型的是在75％范围内。起初的成功率可能接近25％。开始的时候是“操纵”生长锥的主要缺陷。在“推步”的初始互动需要几秒钟。生长锥，然后被单独留在家中，形成一个坚定执着。然后耐心的行使必须在以下步骤。 </li><li>当生长锥，针，通过移动针的权利上略有紧张，提高针一点点。补偿针针位，而提高的“向下”移动。分阶段提高，直到你有附加的进程，扩大针和上面的菜表面的垂直。轴突的垂直安排延长拖带已经开始从针一次，即需要准确的测力，沿轴突的轴是唯一适用于力矢量。如果远离针牵引过程的角度，力矢量是沿轴针，而不是弯曲的针，总兵力不测量出生。从初始菜附件当场了提高持久附件针的前景。提示可能是上面的菜的表面（可见小的震动或从盘的重点不同的平面）或触摸它以最小的力量。太多拖动表面会歪斜的测量。针坚持的菜，或重新连接到基板的细胞的过程会导致强制加载，如果你继续试图将它移到，然后突然“持久性有机污染物松”附件可能发生的损失。要摆脱这种症结针，将针头和背面（“起来”和“倒”在屏幕平面），而不是添加更多的力量（右）。 </li></ol> 9。扩展轴突或细胞的过程<ol><li>牵引是通过运用武力，通过移动针远离细胞体小的增量。部队是观测VED作为针之间的差距日益扩大。虽然逐渐增加了紧张的情绪，仔细观察支队从生长锥针的任何迹象。 </li><li>生长锥分离，如果不能迅速收回，略有下降的紧张等待，往往生长锥将“追赶”和再次抢尖上。如果轴突脱落针对盘针，它通过捕获并重复之前捕获它缩回“推”或一个“下拉”把它一点点紧张，等待大约10分钟再附着。经过两次失败的尝试后，我们建议使用不同的细胞。 </li><li>加水，每小时以抵消蒸发，使用的菜端上的一个标志，以表明最初的深度保持在菜的媒体水平。有了一个长远的巴斯德吸管，慢慢加水的菜一边远离针。扩展过程中，往往一个“摊位”，如果媒体变成高渗。可以减少蒸发量与介质的表面上涂油，但这个针后是在媒体上，都必须加入。如果通过油针传递，生长锥是不太可能附加到针。 </li><li>在一个实验结束时，你解除前的菜，复检针零距离。 </li></ol> 10。数据分析<ol><li>陪读针的位置，在屏幕上的横轴，细胞体的位置，并记录时间。分离针针弯曲常数乘以减去卸载零距离距离等于所适用的武力μdynes。使用一个阶段千分尺校准屏幕图像的距离。 </li><li>不会改变在实验过程中非常重要的是确保质量数据的参考距离（零武力）针之间的分离。温度变化会影响到微米尺度上的设备，即不打开空调在一个实验。此外，如果您的显微镜加热系统有一个滞后的时间等到你的热稳定性。 Ringcubator跌幅滞后时间也隔离在盘子里的温度变化，其余的显微镜和操纵系统，减少零温漂和重点的变化。另一种力量，改变针的卸载分离针轴的表面张力。负载的变化与液体深度菜。检查备份针的参考距离和暂时无零距离武力在一个实验中偶尔看到提高数据的质量，即使你注意到一个变化中分离。 </li></ol> 11。代表性的成果： 校准玻璃针可以很容易地应用到任何蜂窝地区，通常生长锥，可视光镜直接的力量。随着针获得细胞附着适当的治疗，机械张力实验可以应用到蜂窝地区的利益，或该地区所产生的力量可以测量。一个有代表性的神经牵引试验是在图 1 所示 ，其中已被拖走轴突的长度增加了一倍，在两小时内。 <img src="/files/ftp_upload/2509/2509fig1.jpg" alt="图1" /> 图1。一个培养的鸡背根神经节细胞与牵引的首选方向选择。前开始实验操作（一），针之间最初的零参考距离记录。 “推”演习是适用于生长锥（二）。牵引开始增加武力针（三）分离加载。牵引延长的轴突，拖走轴突和工作针“的水平和垂直对齐，有利于良好的测力（D）。酒吧= 40微米。针校准= /μdyne6.9微米。

<ol>
	<li>Zheng, J., Buxbaum, R. E., Heidemann, S. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Measurements+of+growth+cone+adhesion+to+culture+surfaces+by+micromanipulation.">Measurements of growth cone adhesion to culture surfaces by micromanipulation.</a> J Cell Biol. 127, 2049-2060 (1994).</li><li>Chada, S., Lamoureux, P., Buxbaum, R. E., Heidemann, S. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cytomechanics+of+neurite+outgrowth+from+chick+brain+neurons.">Cytomechanics of neurite outgrowth from chick brain neurons.</a> J Cell Sci. 110, 1179-1186 (1997).</li><li>Heidemann, S. R., Lamoureux, P., Buxbaum, R. E., Haynes, L. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> The Neuron in Tissue Culture. , 105-119 (1999).</li><li>Lamoureux, P., Altun-Gultekin, Z. F., Lin, C., Wagner, J. A., Heidemann, S. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rac+is+required+for+growth+cone+function+but+not+neurite+assembly.">Rac is required for growth cone function but not neurite assembly.</a> J Cell Sci. 110, 635-641 (1997).</li><li>Lamoureux, P., Ruthel, G., Buxbaum, R. E., Heidemann, S. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanical+tension+can+specify+axonal+fate+in+hippocampal+neurons.">Mechanical tension can specify axonal fate in hippocampal neurons.</a> J Cell Biol. 159, 499-508 (2002).</li><li>Lamoureux, P., Heidemann, S. R., Martzke, N. R., Miller, K. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Growth+and+elongation+within+and+along+the+axon.">Growth and elongation within and along the axon.</a> Dev Neurobiol. 70, 135-149 (2010).</li><li>Dennerll, T. J., Lamoureux, P., Buxbaum, R. E., Heidemann, S. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+cytomechanics+of+axonal+elongation+and+retraction.">The cytomechanics of axonal elongation and retraction.</a> J Cell Biol. 109, 3073-3083 (1989).</li><li>Heidemann, S. R., Kaech, S., Buxbaum, R. E., Matus, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Direct+observations+of+the+mechanical+behaviors+of+the+cytoskeleton+in+living+fibroblasts.">Direct observations of the mechanical behaviors of the cytoskeleton in living fibroblasts.</a> J Cell Biol. 145, 109-122 (1999).</li><li>Yoneda, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Force+Exerted+by+a+Single+Cilium+of+Mytilus-Edulis+.1.">Force Exerted by a Single Cilium of Mytilus-Edulis .1.</a> Journal of Experimental Biology. 37, (1960).</li><li>Bray, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanical+Tension+Produced+by+Nerve-Cells+in+Tissue-Culture.">Mechanical Tension Produced by Nerve-Cells in Tissue-Culture.</a> Journal of Cell Science. 37, 391-410 (1979).</li><li>Pfister, B. J., Iwata, A., Meaney, D. F., Smith, D. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Extreme+stretch+growth+of+integrated+axons.">Extreme stretch growth of integrated axons.</a> J Neurosci. 24, 7978-7983 (2004).</li><li>Fass, J. N., Odde, D. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tensile+force-dependent+neurite+elicitation+via+anti-beta1+integrin+antibody-coated+magnetic+beads.">Tensile force-dependent neurite elicitation via anti-beta1 integrin antibody-coated magnetic beads.</a> Biophys J. 85, 623-636 (2003).</li><li>Yang, S., Saif, M. T. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microfabricated+Force+Sensors+and+Their+Applications+in+the+Study+of+Cell+Mechanical+Response.">Microfabricated Force Sensors and Their Applications in the Study of Cell Mechanical Response.</a> Exp Mech. 49, 135-151 (2009).</li><li>Bernal, R., Melo, F., Pullarkat, P. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Drag+Force+as+a+Tool+to+Test+the+Active+Mechanical+Response+of+PC12+Neurites.">Drag Force as a Tool to Test the Active Mechanical Response of PC12 Neurites.</a> Biophysical Journal. 98, 515-523 (2010).</li><li>Lamoureux, P., Buxbaum, R. E., Heidemann, S. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Direct+evidence+that+growth+cones+pull.">Direct evidence that growth cones pull.</a> Nature. 340, 159-162 (1989).</li><li>Heidemann, S. R., Lamoureux, P., Buxbaum, R. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Growth+cone+behavior+and+production+of+traction+force.">Growth cone behavior and production of traction force.</a> J Cell Biol. 111, 1949-1957 (1990).</li><li>O'Toole, M., Lamoureux, P., Miller, K. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+physical+model+of+axonal+elongation:+force,+viscosity,+and+adhesions+govern+the+mode+of+outgrowth.">A physical model of axonal elongation: force, viscosity, and adhesions govern the mode of outgrowth.</a> Biophys J. 94, 2610-2620 (2008).</li><li>Bray, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Axonal+growth+in+response+to+experimentally+applied+mechanical+tension.">Axonal growth in response to experimentally applied mechanical tension.</a> Dev Biol. 102, 379-389 (1984).</li><li>Heidemann, S. R., Lamoureux, P., Ngo, K., Reynolds, M., Buxbaum, R. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Open-dish+incubator+for+live+cell+imaging+with+an+inverted+microscope.">Open-dish incubator for live cell imaging with an inverted microscope.</a> Biotechniques. 35, 708-708 (2003).</li></ol>

技术应用和测量细胞的力量，有着悠久的历史9。我们的方法最初是由丹尼斯布雷，谁使用玻璃针，类似我们的“拖”的神经元在一个恒定的速率使用机动液压设备10的工作积极性。有许多细胞，其中包括应用力量的替代手段：步进电机11，磁珠12日，13微束和流体流动14。后者是类似于我们的做法，在蜂窝探头最终是对一个已知的刚度弯曲梁校准和蜂窝测量?...

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		<div>
		<table border="1">
		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>R-6 cap. Tube</td>
<td>&nbsp;</td>
<td>Drummond Scientific Co., Broomall, PA, USA</td>
<td>9-000-3111</td>
<td>R-6 glass OD 0.9mm, ID 0.6 mm, 8"</td>
</tr>
<tr>
<td>BB-CH puller</td>
<td>&nbsp;</td>
<td>Mecanex S.A., Geneva, Switzerland</td>
<td>BB-CH puller</td>
<td>Use Mode 4 Alt by CP=100, PP=10, SP1=1000, SP2=1000</td>
</tr>
<tr>
<td>0.001" Chromel wire</td>
<td>&nbsp;</td>
<td>Omega Engineering, Stamford, CT, USA</td>
<td>SPCH-001-50</td>
<td>unsheathed, themocouple wire, 50ft spool now called Chromega</td>
</tr>
<tr>
<td>0.003" Constatan wire</td>
<td>&nbsp;</td>
<td>Omega Engineering, Stamford, CT, USA</td>
<td>SPCI-003-50</td>
<td>unsheathed, themocouple wire, 50 ft spool</td>
</tr>
<tr>
<td>fine forceps</td>
<td>&nbsp;</td>
<td>Fine Science Tools, USA</td>
<td>91150-20</td>
<td>Dumont Inox #5</td>
</tr>
<tr>
<td>universal microscope boom stand</td>
<td>&nbsp;</td>
<td>Nikon</td>
<td>76135 or 90430</td>
<td>most brands or types of boom stand will work for this use</td>
</tr>
<tr>
<td>mechanical micromanipulator</td>
<td>&nbsp;</td>
<td>Narishige </td>
<td>M-152</td>
<td>three-axis direct-drive coarse micromanipulator</td>
</tr>
<tr>
<td>hydraulic micromanipulator</td>
<td>&nbsp;</td>
<td>Narishige </td>
<td>MO-203</td>
<td>now available as MMO-203, three movable axis type</td>
</tr>
<tr>
<td>needle holder</td>
<td>&nbsp;</td>
<td>Leica Microsystems</td>
<td>11520145</td>
<td>set of 3</td>
</tr>
<tr>
<td>single instrument holder</td>
<td>&nbsp;</td>
<td>Leica Microsystems</td>
<td>11520142</td>
<td>&nbsp;</td>
<tr>
<td>double instrument holder</td>
<td>&nbsp;</td>
<td>Leica Microsystems</td>
<td>11520143</td>
<td>&nbsp;</td>
<tr>
<td>mechanical micromanipulator</td>
<td>&nbsp;</td>
<td>Leica Microsystems</td>
<td>39430001</td>
<td>post mount,1 prob holder, RH Model 430001</td>
</tr>
<tr>
<td>joystick mech. micromanipulator</td>
<td>&nbsp;</td>
<td>Leica Microsystems</td>
<td>11520137</td>
<td>&nbsp;</td>
<tr>
<td>Leica DM IRB</td>
<td>&nbsp;</td>
<td>Leica Microsystems</td>
<td>&nbsp;</td>
<td>inverted microscope</td>
</tr>
<tr>
<td>Vibraplane isolation table</td>
<td>&nbsp;</td>
<td>Kinetic System, Boston, MA, USA</td>
<td>1200 series</td>
<td>ours is model 1201-02-12</td>
</tr>
<tr>
<td>Ringcubator</td>
<td>&nbsp;</td>
<td>self manufactured see reference 19</td>
<td>&nbsp;</td>
<td>reference 19, requires updated controller listed below</td>
</tr>
<tr>
<td>programable temperature controller</td>
<td>&nbsp;</td>
<td>Instrumart.com</td>
<td>Fuji Electric PXR3</td>
<td>replaces the retired PXV3 temperature controller</td>
</tr>
<tr>
<td>Nikon Diaphot TMD</td>
<td>&nbsp;</td>
<td>Nikon Instruments, Inc.</td>
<td>&nbsp;</td>
<td>inverted microscope, circa 1980</td>
</tr>
<tr>
<td>Nikon SMZ-10 binocular dissecting </td>
<td>&nbsp;</td>
<td>Nikon Instruments, Inc.</td>
<td>&nbsp;</td>
<td>other dissecting microscopes will work</td>
</tr>		</tbody>
		</table>
		</div>

mechanical manipulation of neurons to control axonal development

细胞操作和神经轴突的延伸，可以校准玻璃微纤维能够测量和应用在10-1000μdyne范围1,2的力量来完成。力的测量是通过观察Hookean弯曲的玻璃针，这是直接和实证方法3校准获得。设备制造，校准，处理和使用的针头对细胞的要求和程序进行了全面介绍。力制度与以前使用的不同的细胞类型，这些技术已应用示范的方法的灵活性，并为今后的调查4-6的例子给出。技术优势是连续的“可视化”的操作所产生的力量和能力在各种细胞活动的直接干预。这些措施包括直接刺激和调节轴突生长和回缩 7，以及分队，对任何类型的培养细胞 8机械测量。

Watch this Scientific Journal Video about Mechanical Manipulation of Neurons to Control Axonal Development at JoVE.com

Mechanical Manipulation of Neurons to Control Axonal Development

应用在2-1000 microdyne范围内的神经元上的力量和直接测量是实现与高精密校准的玻璃针。这种方法可以用来控制和测量轴突发展的几个方面，包括轴突起始，轴突紧张，轴突伸长速度，和力矢量。

机械操纵控制神经元轴突的发展

Cell manipulations and extension of neuronal axons can be accomplished with calibrated glass micro-fibers capable of measuring and applying forces in the ...

mechanical-manipulation-of-neurons-to-control-axonal-development

Research

JoVE Journal

Neuroscience

10.4K 次观看.  Michigan State University, East Lansing. 应用在2-1000 microdyne范围内的神经元上的力量和直接测量是实现与高精密校准的玻璃针。这种方法可以用来控制和测量轴突发展的几个方面，包括轴突起始，轴突紧张，轴突伸长速度，和力矢量。

视频: Mechanical Manipulation of Neurons to Control Axonal Development

Lentivirus-mediated Genetic Manipulation and Visualization of Olfactory Sensory Neurons in vivo

Development of a precise olfactory circuit relies on accurate projection of olfactory sensory neuron (OSN) axons to their synaptic 
targets in the olfactory bulb (OB). The molecular mechanisms of OSN axon growth and targeting are not well understood. Manipulating gene expression and subsequent 
visualizing of single OSN axons and their terminal arbor morphology have thus far been challenging. To study gene function at the single cell level within a specified 
time frame, we developed a lentiviral based technique to manipulate gene expression in OSNs in vivo. Lentiviral particles are delivered to OSNs by microinjection 
into the olfactory epithelium (OE). Expression cassettes are then permanently integrated into the genome of transduced OSNs. Green fluorescent protein expression 
identifies infected OSNs and outlines their entire morphology, including the axon terminal arbor. Due to the short turnaround time between microinjection and 
reporter detection, gene function studies can be focused within a very narrow period of development. With this method, we have detected GFP expression within as 
few as three days and as long as three months following injection. We have achieved both over-expression and shRNA mediated knock-down by lentiviral microinjection. 
This method provides detailed morphologies of OSN cell bodies and axons at the single cell level in vivo, and thus allows characterization of candidate gene function 
during olfactory development.

Lentivirus-mediated Genetic Manipulation and Visualization of Olfactory Sensory Neurons in vivo

We present a lentiviral technique for genetic manipulation and visualization of single olfactory sensory neuron axon and its terminal arborization in vivo.

嗅觉感官神经元的慢病毒介导的遗传操作和可视化在体内</em

Development of a precise olfactory circuit relies on accurate projection of olfactory sensory neuron (OSN) axons to their synaptic 
targets in the ...

科研

神经科学

DiI-Labeling of DRG Neurons to Study Axonal Branching in a Whole Mount Preparation of Mouse Embryonic Spinal Cord

Here we present a technique to label the trajectories of small groups of DRG neurons into the embryonic spinal cord by diffusive staining using the lipophilic tracer 1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate (DiI)1. The comparison of axonal pathways of wild-type with those of mouse lines in which genes are mutated allows testing for a functional role of candidate proteins in the control of axonal branching which is an essential mechanism in the wiring of the nervous system. Axonal branching enables an individual neuron to connect with multiple targets, thereby providing the physical basis for the parallel processing of information. Ramifications at intermediate target regions of axonal growth may be distinguished from terminal arborization. Furthermore, different modes of axonal branch formation may be classified depending on whether branching results from the activities of the growth cone (splitting or delayed branching) or from the budding of collaterals from the axon shaft in a process called interstitial branching2 (Fig. 1).
The central projections of neurons from the DRG offer a useful experimental system to study both types of axonal branching: when their afferent axons reach the dorsal root entry zone (DREZ) of the spinal cord between embryonic days 10 to 13 (E10 - E13) they display a stereotyped pattern of T- or Y-shaped bifurcation. The two resulting daughter axons then proceed in rostral or caudal directions, respectively, at the dorsolateral margin of the cord and only after a waiting period collaterals sprout from these stem axons to penetrate the gray matter (interstitial branching) and project to relay neurons in specific laminae of the spinal cord where they further arborize (terminal branching)3. DiI tracings have revealed growth cones at the dorsal root entry zone of the spinal cord that appeared to be in the process of splitting suggesting that bifurcation is caused by splitting of the growth cone itself4 (Fig. 2), however, other options have been discussed as well5.
This video demonstrates first how to dissect the spinal cord of E12.5 mice leaving the DRG attached. Following fixation of the specimen tiny amounts of DiI are applied to DRG using glass needles pulled from capillary tubes. After an incubation step, the labeled spinal cord is mounted as an inverted open-book preparation to analyze individual axons using fluorescence microscopy.

The stereotyped projections of sensory afferents into the rodent spinal cord offer an easily accessible experimental system to study axonal branching through the tracing of single axons.

DII的标签DRG神经元的研究轴突分支在小鼠胚胎脊髓整装准备

Here we  present a technique to label the trajectories of small groups of DRG neurons  into the embryonic spinal cord by diffusive staining using the ...

Single-cell Profiling of Developing and Mature Retinal Neurons

Highly specialized, but exceedingly small populations of cells play important roles in many tissues. The identification of cell-type specific markers and gene expression programs for extremely rare cell subsets has been a challenge using standard whole-tissue approaches. Gene expression profiling of individual cells allows for unprecedented access to cell types that comprise only a small percentage of the total tissue1-7. In addition, this technique can be used to examine the gene expression programs that are transiently expressed in small numbers of cells during dynamic developmental transitions8.
This issue of cellular diversity arises repeatedly in the central nervous system (CNS) where neuronal connections can occur between quite diverse cells9. The exact number of distinct cell types is not precisely known, but it has been estimated that there may be as many as 1000 different types in the cortex itself10. The function(s) of complex neural circuits may rely on some of the rare neuronal types and the genes they express. By identifying new markers and helping to molecularly classify different neurons, the single-cell approach is particularly useful in the analysis of cell types in the nervous system. It may also help to elucidate mechanisms of neural development by identifying differentially expressed genes and gene pathways during early stages of neuronal progenitor development.
As a simple, easily accessed tissue with considerable neuronal diversity, the vertebrate retina is an excellent model system for studying the processes of cellular development, neuronal differentiation and neuronal diversification. However, as in other parts of the CNS, this cellular diversity can present a problem for determining the genetic pathways that drive retinal progenitors to adopt a specific cell fate, especially given that rod photoreceptors make up the majority of the total retinal cell population11. Here we report a method for the identification of the transcripts expressed in single retinal cells (Figure 1). The single-cell profiling technique allows for the assessment of the amount of heterogeneity present within different cellular populations of the retina2,4,5,12. In addition, this method has revealed a host of new candidate genes that may play role(s) in the cell fate decision-making processes that occur in subsets of retinal progenitor cells8. With some simple adjustments to the protocol, this technique can be utilized for many different tissues and cell types.

A method for the isolation of single retinal cells and subsequent amplification of their cDNAs is described. Single-cell transcriptomics reveals the degree of cellular heterogeneity present in a tissue and uncovers new marker genes for rare cell populations. The accompanying protocol can be adjusted to suit many different cell types.

视网膜神经细胞发育和成熟的单细胞的剖析

Highly specialized, but exceedingly small populations of cells play important roles in many tissues. The identification of cell-type specific markers and ...

A Method for High Fidelity Optogenetic Control of Individual Pyramidal Neurons In vivo

Optogenetic methods have emerged as a powerful tool for elucidating neural circuit activity underlying a diverse set of behaviors across a broad range of species. Optogenetic tools of microbial origin consist of light-sensitive membrane proteins that are able to activate (e.g., channelrhodopsin-2, ChR2) or silence (e.g., halorhodopsin, NpHR) neural activity ingenetically-defined cell types over behaviorally-relevant timescales. We first demonstrate a simple approach for adeno-associated virus-mediated delivery of ChR2 and NpHR transgenes to the dorsal subiculum and prelimbic region of the prefrontal cortex in rat. Because ChR2 and NpHR are genetically targetable, we describe the use of this technology to control the electrical activity of specific populations of neurons (i.e., pyramidal neurons) embedded in heterogeneous tissue with high temporal precision. We describe herein the hardware, custom software user interface, and procedures that allow for simultaneous light delivery and electrical recording from transduced pyramidal neurons in an anesthetized in vivo preparation. These light-responsive tools provide the opportunity for identifying the causal contributions of different cell types to information processing and behavior.

A Method for High Fidelity Optogenetic Control of Individual Pyramidal Neurons In vivo

The recent development of neuroscience tools that combine genetics and optics, termed "optogenetics", enables control over neural circuit activity with an unprecedented level of spatial and temporal resolution. Here we provide a protocol for integrating in vivo recording with optogenetic manipulation of genetically-defined subsets of prefrontal cortical and subicular pyramidal neurons.

单个锥体神经元的高保真光遗传学控制方法<em&gt;在体内</em

Optogenetic methods  have emerged as a powerful tool for elucidating neural circuit activity  underlying a diverse set of behaviors across a broad range ...

Genetic Manipulation of Cerebellar Granule Neurons In Vitro and In Vivo to Study Neuronal Morphology and Migration

Developmental events in the brain including neuronal morphogenesis and migration are highly orchestrated processes. In vitro and in vivo analyses allow for an in-depth characterization to identify pathways involved in these events. Cerebellar granule neurons (CGNs) that are derived from the developing cerebellum are an ideal model system that allows for morphological analyses. Here, we describe a method of how to genetically manipulate CGNs and how to study axono- and dendritogenesis of individual neurons. With this method the effects of RNA interference, overexpression or small molecules can be compared to control neurons. In addition, the rodent cerebellar cortex is an easily accessible in vivo system owing to its predominant postnatal development. We also present an in vivo electroporation technique to genetically manipulate the developing cerebella and describe subsequent cerebellar analyses to assess neuronal morphology and migration.

Genetic Manipulation of Cerebellar Granule Neurons In Vitro and In Vivo to Study Neuronal Morphology and Migration

Neuronal morphogenesis and migration are crucial events underlying proper brain development. Here, we describe methods to genetically manipulate cultured cerebellar granule neurons and the developing cerebellum for the assessment of morphology and migratory characteristics of neurons.

小脑颗粒神经元的遗传操作<em&gt;体外</em&gt;和<em&gt;在体内</em&gt;为研究神经元形态和迁移

Developmental events in the brain including neuronal morphogenesis and migration are highly orchestrated processes. In vitro and in vivo analyses allow ...

Real-time Imaging of Axonal Transport of Quantum Dot-labeled BDNF in Primary Neurons

BDNF plays an important role in several facets of neuronal survival, differentiation, and function. Structural and functional deficits in axons are increasingly viewed as an early feature of neurodegenerative diseases, including Alzheimer&#8217;s disease (AD) and Huntington&#8217;s disease (HD). As yet unclear is the mechanism(s) by which axonal injury is induced. We reported the development of a novel technique to produce biologically active, monobiotinylated BDNF (mBtBDNF) that can be used to trace axonal transport of BDNF. Quantum dot-labeled BDNF (QD-BDNF) was produced by conjugating quantum dot 655 to mBtBDNF. A microfluidic device was used to isolate axons from neuron cell bodies. Addition of QD-BDNF to the axonal compartment allowed live imaging of BDNF transport in axons. We demonstrated that QD-BDNF moved essentially exclusively retrogradely, with very few pauses, at a moving velocity of around 1.06 &#956;m/sec. This system can be used to investigate mechanisms of disrupted axonal function in AD or HD, as well as other degenerative disorders.

Axonal transport of BDNF, a neurotrophic factor, is critical for the survival and function of several neuronal populations. Some degenerative disorders are marked by disruption of axonal structure and function. We demonstrated the techniques used to examine live trafficking of QD-BDNF in microfluidic chambers using primary neurons.

初级量子神经元轴突运输的实时成像点标记的BDNF

BDNF plays an important role in several facets of neuronal survival, differentiation, and function. Structural and functional deficits in axons are ...

Optical Control of a Neuronal Protein Using a Genetically Encoded Unnatural Amino Acid in Neurons

Photostimulation is a noninvasive way to control biological events with excellent spatial and temporal resolution. New methods are desired to photo-regulate endogenous proteins expressed in their native environment. Here, we present an approach to optically control the function of a neuronal protein directly in neurons using a genetically encoded unnatural amino acid (Uaa). By using an orthogonal tRNA/aminoacyl-tRNA synthetase pair to suppress the amber codon, a photo-reactive Uaa 4,5-dimethoxy-2-nitrobenzyl-cysteine (Cmn) is site-specifically incorporated in the pore of a neuronal protein Kir2.1, an inwardly rectifying potassium channel. The bulky Cmn physically blocks the channel pore, rendering Kir2.1 non-conducting. Light illumination instantaneously converts Cmn into a smaller natural amino acid Cys, activating Kir2.1 channel function. We express these photo-inducible inwardly rectifying potassium (PIRK) channels in rat hippocampal primary neurons, and demonstrate that light-activation of PIRK ceases the neuronal firing due to the outflux of K+ current through the activated Kir2.1 channels. Using in utero electroporation, we also express PIRK in the embryonic mouse neocortex in vivo, showing the light-activation of PIRK in neocortical neurons. Genetically encoding Uaa imposes no restrictions on target protein type or cellular location, and a family of photoreactive Uaas is available for modulating different natural amino acid residues. This technique thus has the potential to be generally applied to many neuronal proteins to achieve optical regulation of different processes in brains. The current protocol presents an accessible procedure for intricate Uaa incorporation in neurons in vitro and in vivo to achieve photo control of neuronal protein activity on the molecular level.

Here, a procedure to selectively activate a neuronal protein with a short pulse of light by genetically encoding a photo-reactive unnatural amino acid into a target neuronal protein expressed in neurons in culture or in vivo is presented.

一个神经元蛋白在神经元使用遗传编码的非天然氨基酸的光控制

Photostimulation is a noninvasive way to control biological events with excellent spatial and temporal resolution. New methods are desired to ...

Insect-machine Hybrid System: Remote Radio Control of a Freely Flying Beetle (Mercynorrhina torquata)

The rise of radio-enabled digital electronic devices has prompted the use of small wireless neuromuscular recorders and stimulators for studying in-flight insect behavior. This technology enables the development of an insect-machine hybrid system using a living insect platform described in this protocol. Moreover, this protocol presents the system configuration and free flight experimental procedures for evaluating the function of the flight muscles in an untethered insect. For demonstration, we targeted the third axillary sclerite (3Ax) muscle to control and achieve left or right turning of a flying beetle. A thin silver wire electrode was implanted on the 3Ax muscle on each side of the beetle. These were connected to the outputs of a wireless backpack (i.e., a neuromuscular electrical stimulator) mounted on the pronotum of the beetle. The muscle was stimulated in free flight by alternating the stimulation side (left or right) or varying the stimulation frequency. The beetle turned to the ipsilateral side when the muscle was stimulated and exhibited a graded response to an increasing frequency. The implantation process and volume calibration of the 3 dimensional motion capture camera system need to be carried out with care to avoid damaging the muscle and losing track of the marker, respectively. This method is highly beneficial to study insect flight, as it helps to reveal the functions of the flight muscle of interest in free flight.

Insect-machine Hybrid System: Remote Radio Control of a Freely Flying Beetle Mercynorrhina torquata

This protocol describes the process of constructing an insect-machine hybrid system and carrying out wireless electrical stimulation of the flight muscles required to control the turning motion of a flying insect.

昆虫机的混合动力系统：一个自由飞翔的甲壳虫无线电远程控制（<em&gt; Mercynorrhina torquata</em&gt;）

The rise of radio-enabled digital electronic devices has prompted the use of small wireless neuromuscular recorders and stimulators for studying in-flight ...

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

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

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

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

胚胎运动神经元轴突投影模式的可视化<em&gt;果蝇</em

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

Visualizing Axonal Growth Cone Collapse and Early Amyloid &#946; Effects in Cultured Mouse Neurons

Amyloid-&#946; (A&#946;) causes memory impairments in Alzheimer's disease (AD). Although therapeutics have been shown to reduce A&#946; levels in the brains of AD patients, these do not improve memory functions. Since A&#946; aggregates in the brain before the appearance of memory impairments, targeting A&#946; may be inefficient for treating AD patients who already exhibit memory deficits. Therefore, downstream signaling due to A&#946; deposition should be blocked before AD development. A&#946; induces axonal degeneration, leading to the disruption of neuronal networks and memory impairments. Although there are many studies on the mechanisms of A&#946; toxicity, the source of A&#946; toxicity remains unknown. To help identify the source, we propose a novel protocol that uses microscopy, gene transfection, and live cell imaging to investigate early changes caused by A&#946; in axonal growth cones of cultured neurons. This protocol revealed that A&#946; induced clathrin-mediated endocytosis in axonal growth cones followed by growth cone collapse, demonstrating that inhibition of endocytosis prevents A&#946; toxicity. This protocol will be useful in studying the early effects of A&#946; and may lead to more efficient and preventative AD treatment.

Visualizing Axonal Growth Cone Collapse and Early Amyloid &amp;#946; Effects in Cultured Mouse Neurons

Here a protocol to investigate the early effects of amyloid-&#946; (A&#946;) in the brain is presented. This shows that A&#946; induces clathrin-mediated endocytosis and collapse of axonal growth cones. The protocol is useful in studying early effects of A&#946; on axonal growth cones and may facilitate prevention of Alzheimer's disease.

在培养的小鼠神经元中可视化轴突生长锥塌陷和早期淀粉样β效应

Amyloid-&#946; (A&#946;) causes memory impairments in Alzheimer's disease (AD). Although therapeutics have been shown to reduce A&#946; levels in the ...