作者感谢詹姆斯Bamburg博士和他在这些研究中的合作的实验室的成员。这项工作是由美国国立卫生研究院补助HD19950，EY07133从美国明尼苏达州医学基金会的资助。

罗丹明-肌动蛋白标签，在35毫米的塑料菜底部，或盖玻片粘到“视频”菜放在玻片上培养神经元。 1。制备盖玻片或“视频”菜<ol><li>许多神经类型不佳胶粘剂体外基板。盖玻片，此过程需要的，可能不允许，除非他们有足够的清理和准备足够的神经底材的附着力。酸洗涤和清洁玻片一个详细的过程描述在书中培养的神经细胞，由银行家和Goslin编辑。另外，盖玻片，如饥似渴地结合底物分子，硝化棉涂料是在下面的步骤1.7和1.8。 </li><li>盖玻片（如18平方毫米或24毫米的圆圈）卫生署2 O冲洗和烘烤干热（≥225 ° C）只要尽可能（数天的最低可达3个月或更长的时间），去除有机的痕迹化合物。转到步骤1.6或视频菜肴进行1.3。 </li><li>要创造一个清晰的玻璃基板薄高分辨率videomicroscopy，适当直径的圆形孔钻成35或50毫米的塑料的Petri或组织培养皿的底部使用电动软木螟（或类似的文书）。钻探是慢慢与轻的压力，以避免开裂的塑料。钻孔的边缘刮用剪刀，双方创建一个光滑的表面。 </li><li>组织培养用玻片清洗，如1.1或1.2中所述。盖玻片粘在孔，采用无毒硅胶水族馆水泥（18x18mm盖玻片，直径12mm位）。水泥固化24小时。 </li><li> （以下步骤使用无菌条件下进行）视频菜用无菌的dh 2 O冲洗3次，并允许空气干燥。 </li><li>盖玻片上都涂有神经元文化的底物分子。首先，盖玻片表面涂（250μL18 × 18盖玻片）100μg/ mL的聚- D -赖氨酸PBS为4-8小时（或过夜）在饱和湿度38℃孵化器的解决方案。盖玻片，然后用无菌DH 2 O冲洗3次，以去除未结合的D -聚赖氨酸，并允许在空气中晾干。许多神经类型与D -聚赖氨酸单独涂的，如果在培养基中的血清蛋白的盖玻片上培养。然而，结果更具相关性，在体内条件下，使用天然的底物分子获得。的解决方案，如层粘连蛋白，纤维连接蛋白或L1 CAM（1-20微克/毫升的PBS），这些分子可直接应用D -聚赖氨酸涂布盖玻片为4-8小时或过夜，但在我们的实验室中，我们经常大衣与薄膜的细胞粘附分子在提出申请前增加蛋白底物结合的硝化棉的准备盖玻片。 </li><li>通过了1％硝酸纤维素溶液溶解在5毫升100％戊酯​​5克硝化棉。应用10μL解决盖玻片，轻轻地分布在表面上。空气干燥10分钟。 </li><li>应用250μL25微克/毫升层粘连蛋白或4微克/毫升L1 CAM在PBS和硝酸纤维素表面传播的解决方案。在饱和湿度38 ° C培养箱中培养，吸基板孵育4小时（或过夜），并立即加入培养基，使基材不燥。 </li></ol> 2。神经元文化的制备<ol><li>从胚胎第7天的鸡胚蛋，放置在100毫米培养皿盘含有10％的夹层血清培养基中删除从胸部和腹部的内脏器官，使用立体显微镜。 </li><li>胚胎冲洗中和转移到与液体介质中盘60毫米，并进一步解剖，删除和准备背根神经节（DRG）或神经视网膜组织的外植体。一个详细清扫描述，可以发现在细胞生物学，71卷，神经元：细胞生物学家，编辑霍伦贝克和Bamburg方法和应用方法。背根神经节植1/2-whole神经节神经视网膜植或直径小于1毫米。 </li><li>清洗后的多余组织植，个别植移动到这是已经在培养基的培养皿中的盖玻片，镊子制表大师。在解剖显微镜下，外植体，轻轻压盖玻片，用镊子而运送到孵化器，以防止运动的中心。 F12培养基中培养外植体与B27的添加剂，与10毫米HEPES pH值7.4的缓冲和放置在饱和湿度38℃培养箱中过夜。必要时，可以添加生长因子，作为培养基。神经营养因子往往添加到DRG的年纪较大的胚胎培养，尽管E7的DRG神经元轴突将扩大从没有补充神经营养因子的植。神经视网膜植延长没有在此培养基中的轴突dding增长的因素。 </li><li>文化孵育至少18小时，以便在开始以下步骤之前，有足够的轴突生长。 </li></ol> 3。罗丹明肌动蛋白的制备通透缓冲<ol><li>一个通透含有138毫米氯化钾，10毫米的管道，3毫米EGTA，4MM MgCl 2的，和1％BSA（pH值= 6.9）缓冲库存解决方案可以保持2个星期在4 ° C11。使用前，加入终浓度为以下几部分组成：0.025％，皂甙，0.1毫米ATP和100 nm的Alexa的福陆公司的350鬼笔环肽。 </li><li>一个单独的解决方案是也只是之前使用这些相同的组件加上0.45μm的罗丹明非肌动蛋白，解决的办法是交替振荡5分钟解散罗丹明-肌动蛋白团块tritrated新鲜配制，振荡1分钟，在最初的通透性一步管，以防止聚合。如果尽管这样，在管的灯丝大会仍然是一个问题，该解决方案可以事先离心使用。 </li></ol> 4。神经元文化的通透性和罗丹明-肌动蛋白结合到F -肌动蛋白刺完<ol><li>一个背根神经节或视网膜植培养皿仔细从孵化器中删除，并在室温下执行以下步骤。 </li><li>解决方案的所有更改必须小心。枪头应放置在盖玻片边缘的解决方案应该除掉，并缓慢加入，用最少的力。 </li><li>培养基通过吸管小心被删除，但稳步增长，和足够的通透缓冲盖细胞添加1分钟（18毫米x 18毫米盖玻片，〜60μL）。文化不与任何其他的解决方案之前加入通透缓冲冲洗，并盖玻片不允许晾干后方可加入通透缓冲区。 </li><li>通透缓冲区吸管轻轻地去除和替换包含4分钟0.45微米罗丹明非肌动蛋白的通透性缓冲区。 </li><li>罗丹明-肌动蛋白含有缓冲区是通过吸管轻轻除去，神经元是固定的，加入4％多聚甲醛（实验室级，用磷酸盐缓冲液，pH值7.3），0.05％戊二醛和10％的蔗糖溶液。 5分钟后固定在盖玻片，用PBS轻轻冲洗，并安装在Slowfade 3英寸的载玻片上。 </li><li>不断变化的解决方案的另一种方法是使用流动池。这可能是一个市售的流动池，或可以由一个简单的流动细胞间隔在盖玻片的角落放置小的塑料件（≤1毫米厚），然后安装上的前一个同​​等大小的盖玻片盖玻片。可以通过流动池的一侧放置的吸管和撤回解决方案，在对方芯（棉或Kimwipe）交换解决方案。 </li><li>罗丹明-肌动蛋白纳入到F -肌动蛋白刺结束，鬼笔环肽荧光标签的F - actin的生长锥是通过60X油浸物镜落射荧光光学可视化，图像收集，使用冷却CCD相机。共聚焦显微镜可以提供更好的成像。 </li><li>为了获得最佳的量化罗丹明-肌动蛋白掺入的所有图像应收集在一个会话中，使用相同的增益和曝光设置为每个图像的数码相机的灵敏度。标签分析，应该从每个图像相当于地区; Metamorph，图像J或类似的计算机化图像分析工具可以使用。 </li></ol>此过程的变异 5。变化之一：罗丹明肌动蛋白标签之前收集活细胞图像<ol><li>视频菜肴被放置到一个温暖的显微镜阶段，并收集现场生长锥图像。可用于网格盖玻片，或蚀刻和/或盖玻片上的笔标记可以更容易地找到特定的细胞后，标签，或视频盘可以固定在显微镜舞台上过程的持续时间。 </li><li>通透，掺入罗丹明-肌动蛋白和细胞固定进行视频菜，打开或关闭在显微镜阶段。固定后，PBS添加到立即成像的视频菜。要保留的样本以供日后成像，slowfade安装介质添加到盖玻片和同等大小的盖玻片轻轻地放置在上面（避免气泡），和边缘，然后用透明指甲油密封。 </li></ol> 6。两个变化：合作标签附加蛋白的免疫组织化学<ol><li>纳入罗丹明肌动蛋白和固定，可进行对在盖玻片上或视频菜培养神经元以上（4.2-4.4）所述。 </li><li>固定后，PBS漂洗，细胞治疗与0.1 M甘氨酸的PBS 15分钟，然后提取0.1％的Triton X - 100的2％山羊血清和1小时1％BSA的PBS（TX - 100）盖玻片PBS稀释的抗体，1小时含1％BSA孵育在盖玻片，然后冲洗和TX - 100的PBS 2％山羊血清和1％的牛血清白蛋白1小时，在提出申请前1小时1％BSA的荧光抗体1:1000稀释的PBS孵育0.1％，冲洗后，再次培养盖玻片在0.1％TX - 100的PBS，2％山羊血清和1％BSA 30分钟，冲洗，并安装在抗衰落介质中。一些蛋白质的本地化被打乱步骤（4.3）由最初的通透性。可以添加到要保留这些蛋白抗体的本地化，0.05％和0.05％的多聚甲醛戊二醛通透缓冲1分（即前加入罗丹明-肌动蛋白），而不会影响罗丹明-肌动蛋白的刺末端标记。 </li><li>三重标记的生长锥的荧光或共聚焦显微镜图像采集。 </li></ol> 7。变化之三：评估F - actin的免费刺完的指导线索的影响<ol><li>一种生长因子或指导提示，是在几分钟的几个毫微克/毫升（或适当的治疗时间）的浓度在孵化器的培养皿前从孵化器中删除的菜，并开始通透过程。这使得生长因子或F - actin的自由刺完的指导线索的短期效应的评估。 </li><li>一个视频与神经元的文化菜，可以放在一个温暖的显微镜阶段，并在附近生长锥开始之前的通透性和罗丹明-肌动蛋白掺入的梯度，生长因子或指导线索可以从微发布。例如，NGF可以释放为背根神经节神经视网膜神经节细胞神经元或netrin。移液器可引入短1-2分钟才开始的通透性程序。这允许指导提示梯度上形成的F -肌动蛋白在生长锥的利润（见图像的参考引文10）刺完当地的影响进行评估。 </li><li>这些变化可在6.2以上所述，与其他神经元组件的抗体介导的标签相结合。 </li></ol> 8。变化之四：F - actin的免费转染神经元刺末端标记<ol><li>组成性激活或显性负构造或蛋白敲除利用RNAi（用荧光标记识别）的使用，可用于评估一种蛋白质在参与调节F -肌动蛋白刺结束。根据生长锥内的蛋白质，其本土化，而且，如果它是融合一个荧光标记，荧光蛋白表达在转染细胞后可通透丢失。如果是这样的情况下，视频菜可以用来识别显微镜前一个转染的细胞通透性，并可以执行的通透性标记转染细胞的位置后，在显微镜舞台上。另外，GFP -肌动蛋白的构造，可以共同转染GFP的构建利益。 GFP -肌动蛋白在这种情况下，将被纳入F -肌动蛋白，并不会丢失的通透性，从而使转染细胞的识别后通透性。 </li><li>这种变化可以结合另外的指导线索，在7.1-7.3以上所述。 </li></ol> 9。代表性的成果<img src="/files/ftp_upload/2409/2409fig1.jpg" alt="图1" /> 图1。 NGF的全球除了增加总的F -肌动蛋白和F -肌动蛋白在生长锥的领先保证金刺完 ，当一个背根神经节生长锥是5分钟的神经生长因子（NGF）的刺激，肌动蛋白聚合是在领先的保证金的刺激，罗丹明-肌动蛋白标签的亮带周围可见生长锥外围。图1比较罗丹明-肌动蛋白掺入到未刺激背根神经节生长锥和一个神经生长因子刺激的背​​根神经节生长锥。在合并后的图像中的绿色荧光鬼笔环肽标记F -肌动蛋白在生长锥的红色罗丹明-肌动蛋白标签。刺末端标记的一个很好的控制是添加在步骤4.2和4.3通透缓冲区10-6 M或更高的细胞松弛素B或D。 Cytochalasins第F -肌动蛋白刺完，抑制刺完罗丹明-肌动蛋白结合。这严重降低或消除掺入罗丹明-肌动蛋白进入细胞。比例尺，10微米。 <img src="/files/ftp_upload/2409/2409fig2.jpg" alt="图2" /> 图2。一种神经生长因子的梯度本地增加F -肌动蛋白刺完一个释放NGF的微量F -肌动蛋白标签免费刺完，一个2分钟的背根神经节生长锥的一侧。下面的图片显示，暴露于梯度本地的NGF刺激增加F -肌动蛋白在生长锥地区接近移液器刺完。合并后的图像显示在绿色的鬼笔环肽红色罗丹明-肌动蛋白。比例尺，10微米。 <img src="/files/ftp_upload/2409/2409fig3.jpg" alt="图3" /> 图3。激活ERM蛋白积聚在生长锥领先的利润率。phospho-Ezrin/Radixin/Moesin免疫组织化学标记（PERM）与F - actin的自由刺完。 Gluteraldehyde（0.05％）和多聚甲醛（0.05％），分别加入1分钟，以保持企业风险管理本地化的初步通透缓冲。罗丹明-肌动蛋白，红烫发，绿色;鬼笔环肽，蓝。比例尺，10微米。 <img src="/files/ftp_upload/2409/2409fig4.jpg" alt="图4" /> 图4。肌动蛋白聚合的刺激，需要积极的ERM蛋白。游离病种付费，共转染GFP -肌动蛋白和GFP控制质粒或一个显性负Ezrin / Radixin / Moesin（DN ERM）的构造。使用GFP -肌动蛋白的允许后，确定转染细胞通透性。这里，神经生长因子增加了5分钟，F -肌动蛋白刺结束标签之前。注意：生长锥的转染DN的企业风险管理，减少生长锥保证金（较低的中间面板），这与附近的未转染生长锥（下板）对比，或与GFP控制（中上部面板）刺年底的水平。若丹明-肌动蛋白，红色; GFP -肌动蛋白，绿色。比例尺，10微米。

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	<li>Lowery, L. A., Van Vactor, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+trip+of+the+tip:+Understanding+the+growth+cone+machinery.">The trip of the tip: Understanding the growth cone machinery.</a> Nat Rev Mol Cell Biol. 10, 332-343 (2009).</li><li>Letourneau, P., Squire, L. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Axonal+Pathfinding:+Extracellular+Matrix+Role.">Axonal Pathfinding: Extracellular Matrix Role.</a> Encyclopedia of Neuroscience. , 1139-1145 (2008).</li><li>Kalil, K., Dent, E. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Touch+and+go:+Guidance+cues+signal+to+the+growth+cone+cytoskeleton.">Touch and go: Guidance cues signal to the growth cone cytoskeleton.</a> Curr Opin Neurobio. 15, 521-526 (2005).</li><li>Dent, E. W., Gertler, F. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cytoskeletal+dynamics+and+transport+in+growth+cone+motility+and+axon+guidance.">Cytoskeletal dynamics and transport in growth cone motility and axon guidance.</a> Neuron. 40, 209-227 (2003).</li><li>Pak, C. W., Flynn, K. C., Bamburg, J. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Actin-binding+proteins+take+the+reins+in+growth+cones.">Actin-binding proteins take the reins in growth cones.</a> Nat Rev Neurosci. 9, 136-147 (2008).</li><li>Guan, K. L., Rao, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Signaling+mechanisms+mediating+neuronal+responses+to+guidance+cues.">Signaling mechanisms mediating neuronal responses to guidance cues.</a> Nat Rev Neurosci. 4, 941-956 (2003).</li><li>Gallo, G., Letourneau, P. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regulation+of+growth+cone+actin+filaments+by+guidance+cues.">Regulation of growth cone actin filaments by guidance cues.</a> J. Neurobiology. 58, 92-102 (2004).</li><li>Fass, J., Gehler, S., Sarmiere, P., Letourneau, P., Bamburg, J. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regulating+filopodial+dynamics+through+actin-depolymerizing+factor/cofilin.">Regulating filopodial dynamics through actin-depolymerizing factor/cofilin.</a> Anat Sci Inter. 79, 173-183 (2004).</li><li>Bernstein, B. W., Bamburg, J. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=ADF/cofilin:+A+functional+node+in+cell+biology.">ADF/cofilin: A functional node in cell biology.</a> Trends Cell Biol. 20, 187-195 (2010).</li><li>Marsick, B. M., Flynn, K. C., Santiago, M., Bamburg, J. R., Letourneau, P. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Activation+of+ADF/cofilin+mediates+attractive+growth+cone+turning+toward+nerve+growth+factor+and+netrin-1.">Activation of ADF/cofilin mediates attractive growth cone turning toward nerve growth factor and netrin-1.</a> Dev Neurobiol. 70, 565-588 (2010).</li><li>Symons, M. H., Mitchison, T. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Control+of+actin+polymerization+in+live+and+permeabilized+fibroblasts.">Control of actin polymerization in live and permeabilized fibroblasts.</a> J Cell Biol. 114, 503-513 (1991).</li><li>Chan, A. Y., Raft, S., Bailly, M., Wyckoff, J. B., Segall, J. E., Condeelis, J. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=EGF+stimulates+an+increase+in+actin+nucleation+and+filament+number+at+the+leading+edge+of+the+lamellipod+in+mammary+adenocarcinoma+cells.">EGF stimulates an increase in actin nucleation and filament number at the leading edge of the lamellipod in mammary adenocarcinoma cells.</a> J Cell Sci. 111, 199-211 (1998).</li></ol>

这里介绍的方法允许在生长锥的迁移领先保证金，参与肌动蛋白细胞骨架的动态重塑的细胞成分的时间和空间分辨率。诱食分子，如神经生长因子或netrin，行动迅速刺激肌动蛋白丝聚合透露，作为在当地增加肌动蛋白刺完由肌动蛋白丝切断激活ADF / cofilin 10，如在图1和2所示。该方法允许介导生长锥chemotropic反应，如radixin，图，在图3和4的ERM蛋白，参与其他蛋白质的本地化。这些方法也可以...

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		<th>Material Name</th>
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		<th>Catalogue Number</th>
		<th>Comment</th>
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<tr>
<td>18x18 mm coverslip</td>
<td>&nbsp;</td>
<td>Gold Seal</td>
<td>3305</td>
<td>&nbsp;</td>
<tr>
<td>35mm Petri dishes</td>
<td>&nbsp;</td>
<td>Falcon</td>
<td>351008</td>
<td>&nbsp;</td>
<tr>
<td>Aquarium cement</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>DAP 100% silicone aquarium sealant</td>
<td>Any hardware store</td>
</tr>
<tr>
<td>Poly-D-lysine (mw &gt; 300,000)</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>P1024</td>
<td>&nbsp;</td>
<tr>
<td>Natural mouse laminin</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>23017-015</td>
<td>&nbsp;</td>
<tr>
<td>L1 CAM</td>
<td>&nbsp;</td>
<td>R &amp; D systems</td>
<td>777-NC</td>
<td>&nbsp;</td>
<tr>
<td>Alexa-fluor 350 phalloidin</td>
<td>&nbsp;</td>
<td>Invitrogen (Molecular Probes)</td>
<td>A22281</td>
<td>&nbsp;</td>
<tr>
<td>Rhodamine non-muscle actin</td>
<td>&nbsp;</td>
<td>Cytoskeleton, Inc.</td>
<td>APHR-A</td>
<td>&nbsp;</td>
<tr>
<td>F12 Culture medium</td>
<td>&nbsp;</td>
<td>Invitrogen (Gibco)</td>
<td>21700-075</td>
<td>&nbsp;</td>
<tr>
<td>B27</td>
<td>&nbsp;</td>
<td>Invitrogen (Gibco)</td>
<td>17504-044</td>
<td>&nbsp;</td>
<tr>
<td>Slowfade </td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>536937</td>
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labeling f-actin barbed ends with rhodamine-actin in permeabilized neuronal growth cones

轴突生长的能动的小技巧，被称为生长锥。生长锥导致导航轴突通过发展组织与当地表达的分子的指导线索，结合生长锥的受体和调节的动态和组织的生长锥细胞骨架3-6交互。这些导航信号的主要目标是肌动蛋白丝小梁，填补了生长锥外围和驱动器的增长，通过不断的肌动蛋白聚合和动态重构7锥蠕动。正或有吸引力的指导线索诱导生长锥转向通过刺激肌动蛋白丝（F - actin的）聚合在生长锥的外围接近源引诱提示地区。此肌动蛋白聚合驱动本地生长锥突领先的利润率和走向的诱食性轴索伸长率，附着力强。 肌动蛋白丝聚合取决于可用性和足够的肌动蛋白单体聚合单体此外细胞核或肌动蛋白丝刺结束。肌动蛋白单体是十分小鸡视网膜和背根神经节（DRG）生长锥。因此，聚合迅速增加F -肌动蛋白刺结束时成为单体此外的。出现这种情况通过当地的大义灭亲“F -肌动蛋白的蛋白质因子（ADF / cofilin）肌动蛋白解聚在生长锥地区接近一个引诱 8-10激活在鸡背根神经节和视网膜生长锥。这加剧ADF / cofilin活动中断微丝创造新的F -肌动蛋白聚合刺完。下面的方法演示了这种机制。 F -肌动蛋白总含量是荧光灯的鬼笔环肽染色观察。 F -肌动蛋白刺两端增长以下，这是适应从其他运动型细胞11，12以前的研究中所描述的程序透视锥细胞内的肌动蛋白罗丹明纳入可视化。当罗丹明-肌动蛋白浓度高于临界浓度的肌动蛋白单体除刺完补充说，罗丹明-肌动蛋白组装到免费的刺完。如果有吸引力的线索是在梯度，如被释放从定位到一个生长锥的一侧一个微量，纳入到F -肌动蛋白刺结 ​​束罗丹明-肌动蛋白在生长锥端提高对微量10 。 生长锥，小巧精致的细胞结构。通透，罗丹明-肌动蛋白的法团，固定和荧光可视化的程序都用心去做，可在倒置显微镜阶段进行。这些方法可以适用于研究当地的肌动蛋白聚合中迁移神经元，其他基层组织细胞或细胞株。

Watch this Scientific Journal Video about Labeling F-actin Barbed Ends with Rhodamine-actin in Permeabilized Neuronal Growth Cones at JoVE.com

Labeling F-actin Barbed Ends with Rhodamine-actin in Permeabilized Neuronal Growth Cones

描述的方法来可视化和量化，F -肌动蛋白在神经生长锥刺完。盖玻片上培养神经细胞后，细胞透用含皂甙的解决方案。然后，潜伏期短皂苷含有若丹明-肌动蛋白的缓冲区纳入到免费的肌动蛋白刺完肌动蛋白的荧光。

罗丹明-肌动蛋白标记F -肌动蛋白在通透的神经生长锥刺完

The motile tips of growing axons are called growth cones. Growth cones lead navigating axons through developing tissues by interacting with locally ...

labeling-f-actin-barbed-ends-with-rhodamine-actin-permeabilized

Research

JoVE Journal

Neuroscience

14.7K 次观看.  University of Minnesota. 描述的方法来可视化和量化，F -肌动蛋白在神经生长锥刺完。盖玻片上培养神经细胞后，细胞透用含皂甙的解决方案。然后，潜伏期短皂苷含有若丹明-肌动蛋白的缓冲区纳入到免费的肌动蛋白刺完肌动蛋白的荧光。

视频: Labeling F-actin Barbed Ends with Rhodamine-actin in Permeabilized Neuronal Growth Cones

DiOLISTIC Labeling of Neurons from Rodent and Non-human Primate Brain Slices

DiOLISTIC staining uses the gene gun to introduce fluorescent dyes, such as DiI, into neurons of brain slices (Gan et al., 2009; O'Brien and Lummis, 2007; Gan et al., 2000). Here we provide a detailed description of each step required together with exemplary images of good and bad outcomes that will help when setting up the technique. In our experience, a few steps proved critical for the successful application of DiOLISTICS. These considerations include the quality of the DiI-coated bullets, the extent of fixative exposure, and the concentration of detergent used in the incubation solutions. Tips and solutions for common problems are provided. This is a versatile labeling technique that can be applied to multiple animal species at a wide range of ages. Unlike other fluorescent labeling techniques that are limited to preparations from young animals or restricted to mice because they rely on the expression of a fluorescent transgene, DiOLISTIC labeling can be applied to animals of all ages, species and genotypes and it can be used in combination with immunostaining to identify a specific subpopulation of cells. Here we demonstrate the use of DiOLISTICS to label neurons in brain slices from adult mice and adult non-human primates with the purpose of quantifying dendrite branching and dendritic spine morphology.

We demonstrate the use of the gene gun to introduce fluorescent dyes, such as DiI, into neurons in brain slices from rodents and non-human primates of different ages. In this particular case, we use adult mice (3-6 months old) and adult cynomologus monkeys (9-15 years old). This technique, originally described by the laboratory of Dr. Lichtman (Gan et al., 2000), is well suited for the study of dendritic branching and dendritic spine morphology and can be combined with traditional immunostaining, if detergents are kept at a low concentration.

从啮齿动物的神经元和非人类灵长类动物的脑片DiOLISTIC标签

DiOLISTIC staining uses the gene gun to introduce fluorescent dyes, such as DiI, into neurons of brain slices (Gan et al., 2009; O'Brien and Lummis, 2007; ...

科研

神经科学

Efficient Derivation of Human Neuronal Progenitors and Neurons from Pluripotent Human Embryonic Stem Cells with Small Molecule Induction

There is a large unfulfilled need for a clinically-suitable human neuronal cell source for repair or regeneration of the damaged central nervous system (CNS) structure and circuitry in today's healthcare industry. Cell-based therapies hold great promise to restore the lost nerve tissue and function for CNS disorders. However, cell therapies based on CNS-derived neural stem cells have encountered supply restriction and difficulty to use in the clinical setting due to their limited expansion ability in culture and failing plasticity after extensive passaging1-3. Despite some beneficial outcomes, the CNS-derived human neural stem cells (hNSCs) appear to exert their therapeutic effects primarily by their non-neuronal progenies through producing trophic and neuroprotective molecules to rescue the endogenous cells1-3. Alternatively, pluripotent human embryonic stem cells (hESCs) proffer cures for a wide range of neurological disorders by supplying the diversity of human neuronal cell types in the developing CNS for regeneration1,4-7. However, how to channel the wide differentiation potential of pluripotent hESCs efficiently and predictably to a desired phenotype has been a major challenge for both developmental study and clinical translation. Conventional approaches rely on multi-lineage inclination of pluripotent cells through spontaneous germ layer differentiation, resulting in inefficient and uncontrollable lineage-commitment that is often followed by phenotypic heterogeneity and instability, hence, a high risk of tumorigenicity7-10. In addition, undefined foreign/animal biological supplements and/or feeders that have typically been used for the isolation, expansion, and differentiation of hESCs may make direct use of such cell-specialized grafts in patients problematic11-13. To overcome these obstacles, we have resolved the elements of a defined culture system necessary and sufficient for sustaining the epiblast pluripotence of hESCs, serving as a platform for de novo derivation of clinically-suitable hESCs and effectively directing such hESCs uniformly towards clinically-relevant lineages by small molecules14 (please see a schematic in Fig. 1). Retinoic acid (RA) does not induce neuronal differentiation of undifferentiated hESCs maintained on feeders1, 14. And unlike mouse ESCs, treating hESC-differentiated embryoid bodies (EBs) only slightly increases the low yield of neurons1, 14, 15. However, after screening a variety of small molecules and growth factors, we found that such defined conditions rendered retinoic acid (RA) sufficient to induce the specification of neuroectoderm direct from pluripotent hESCs that further progressed to neuroblasts that generated human neuronal progenitors and neurons in the developing CNS with high efficiency (Fig. 2). We defined conditions for induction of neuroblasts direct from pluripotent hESCs without an intervening multi-lineage embryoid body stage, enabling well-controlled efficient derivation of a large supply of human neuronal cells across the spectrum of developmental stages for cell-based therapeutics.

We have established a protocol for induction of neuroblasts direct from pluripotent human embryonic stem cells maintained under defined conditions with small molecules, which enables derivation of a large supply of human neuronal progenitors and neuronal cell types in the developing CNS for neural repair.

人类神经祖细胞和神经元小分子诱导人类胚胎干细胞的多能干高效的推导

There is a large unfulfilled need for a clinically-suitable human neuronal cell source for repair or regeneration of the damaged central nervous system ...

Recording Large-scale Neuronal Ensembles with Silicon Probes in the Anesthetized Rat

Large scale electrophysiological recordings from neuronal ensembles offer the opportunity to investigate how the brain orchestrates the wide variety of behaviors from the spiking activity of its neurons. One of the most effective methods to monitor spiking activity from a large number of neurons in multiple local neuronal circuits simultaneously is by using silicon electrode arrays1-3.
Action potentials produce large transmembrane voltage changes in the vicinity of cell somata. These output signals can be measured by placing a conductor in close proximity of a neuron. If there are many active (spiking) neurons in the vicinity of the tip, the electrode records combined signal from all of them, where contribution of a single neuron is weighted by its 'electrical distance'. Silicon probes are ideal recording electrodes to monitor multiple neurons because of a large number of recording sites (+64) and a small volume. Furthermore, multiple sites can be arranged over a distance of millimeters, thus allowing for the simultaneous recordings of neuronal activity in the various cortical layers or in multiple cortical columns (Fig. 1). Importantly, the geometrically precise distribution of the recording sites also allows for the determination of the spatial relationship of the isolated single neurons4. Here, we describe an acute, large-scale neuronal recording from the left and right forelimb somatosensory cortex simultaneously in an anesthetized rat with silicon probes (Fig. 2).

Extracellular recordings of neuronal activity using silicon probes in the anesthetized rat will be described. This technique allows information to be obtained across multiple brain areas from more than 100 neurons simultaneously. It provides information with single cell resolution about neuronal ensembles dynamics in multiple local circuits.

在麻醉大鼠的硅探头录制的大型神经元合唱

Large scale electrophysiological recordings from neuronal ensembles offer the opportunity to investigate how the brain orchestrates the wide variety of ...

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 ...

Dual Electrophysiological Recordings of Synaptically-evoked Astroglial and Neuronal Responses in Acute Hippocampal Slices

Astrocytes form together with neurons tripartite synapses, where they integrate and modulate neuronal activity. Indeed, astrocytes sense neuronal inputs through activation of their ion channels and neurotransmitter receptors, and process information in part through activity-dependent release of gliotransmitters. Furthermore, astrocytes constitute the main uptake system for glutamate, contribute to potassium spatial buffering, as well as to GABA clearance. These cells therefore constantly monitor synaptic activity, and are thereby sensitive indicators for alterations in synaptically-released glutamate, GABA and extracellular potassium levels. Additionally, alterations in astroglial uptake activity or buffering capacity can have severe effects on neuronal functions, and might be overlooked when characterizing physiopathological situations or knockout mice. Dual recording of neuronal and astroglial activities is therefore an important method to study alterations in synaptic strength associated to concomitant changes in astroglial uptake and buffering capacities. Here we describe how to prepare hippocampal slices, how to identify stratum radiatum astrocytes, and how to record simultaneously neuronal and astroglial electrophysiological responses. Furthermore, we describe how to isolate pharmacologically the synaptically-evoked astroglial currents.

The preparation of acute brain slices from isolated hippocampi, as well as the simultaneous electrophysiological recordings of astrocytes and neurons in stratum radiatum during stimulation of schaffer collaterals is described. The pharmacological isolation of astroglial potassium and glutamate transporter currents is demonstrated.

双突触诱发的星形胶质细胞和神经元的反应，在急性海马脑片电生理记录

Astrocytes form together with neurons tripartite synapses, where they integrate and modulate neuronal activity. Indeed, astrocytes sense neuronal inputs ...

Fast Micro-iontophoresis of Glutamate and GABA: A Useful Tool to Investigate Synaptic Integration

One of the fundamental interests in neuroscience is to understand the integration of excitatory and inhibitory inputs along the very complex structure of the dendritic tree, which eventually leads to neuronal output of action potentials at the axon. The influence of diverse spatial and temporal parameters of specific synaptic input on neuronal output is currently under investigation, e.g. the distance-dependent attenuation of dendritic inputs, the location-dependent interaction of spatially segregated inputs, the influence of GABAergig inhibition on excitatory integration, linear and non-linear integration modes, and many more.
With fast micro-iontophoresis of glutamate and GABA it is possible to precisely investigate the spatial and temporal integration of glutamatergic excitation and GABAergic inhibition. Critical technical requirements are either a triggered fluorescent lamp, light-emitting diode (LED), or a two-photon scanning microscope to visualize dendritic branches without introducing significant photo-damage of the tissue. Furthermore, it is very important to have a micro-iontophoresis amplifier that allows for fast capacitance compensation of high resistance pipettes. Another crucial point is that no transmitter is involuntarily released by the pipette during the experiment.
Once established, this technique will give reliable and reproducible signals with a high neurotransmitter and location specificity. Compared to glutamate and GABA uncaging, fast iontophoresis allows using both transmitters at the same time but at very distant locations without limitation to the field of view. There are also advantages compared to focal electrical stimulation of axons: with micro-iontophoresis the location of the input site is definitely known and it is sure that only the neurotransmitter of interest is released. However it has to be considered that with micro-iontophoresis only the postsynapse is activated and presynaptic aspects of neurotransmitter release are not resolved. In this article we demonstrate how to set up micro-iontophoresis in brain slice experiments.

In this article we introduce fast micro-iontophoresis of neurotransmitters as a technique to investigate integration of postsynaptic signals with high spatial and temporal precision.

谷氨酸和GABA快速微离子导入：一个有用​​的工具，调查突触整合

One of the fundamental interests in neuroscience is to understand the integration of excitatory and inhibitory inputs along the very complex structure of ...

Retrograde Labeling of Retinal Ganglion Cells in Adult Zebrafish with Fluorescent Dyes

As retrograde labeling retinal ganglion cells (RGCs) can isolate RGCs somata from dying sites, it has become the gold standard for counting RGCs in RGCs survival and regeneration experiments. Many studies have been performed in mammalian animals to research RGCs survival after optic nerve injury. However, retrograde labeling of RGCs in adult zebrafish has not yet been reported, though some alternative methods can count cell numbers in retinal ganglion cell layers (RGCL). Considering the small size of the adult zebrafish skull and the high risk of death after drilling on the skull, we open the skull with the help of acid-etching and seal the hole with a light curing bond, which could significantly improve the survival rate. After absorbing the dyes for 5 days, almost all the RGCs are labeled. As this method does not need to transect the optic nerve, it is irreplaceable in the research of RGCs survival after optic nerve crush in adult zebrafish. Here, we introduce this method step by step and provide representative results.

We introduce an efficient method to retrograde label retinal ganglion cells (RGCs) in adult zebrafish.

视网膜神经节细胞逆行标记的成年斑马鱼与荧光染料

As retrograde labeling retinal ganglion cells (RGCs) can isolate RGCs somata from dying sites, it has become the gold standard for counting RGCs in RGCs ...

Preparation of Neuronal Co-cultures with Single Cell Precision

Microfluidic embodiments of the Campenot chamber have attracted great interest from the neuroscience community. These interconnected co-culture platforms can be used to investigate a variety of questions, spanning developmental and functional neurobiology to infection and disease propagation. However, conventional systems require significant cellular inputs (many thousands per compartment), inadequate for studying low abundance cells, such as primary dopaminergic substantia nigra, spiral ganglia, and Drosophilia melanogaster neurons, and impractical for high throughput experimentation. The dense cultures are also highly locally entangled, with few outgrowths (&#60;10%) interconnecting the two cultures. In this paper straightforward microfluidic and patterning protocols are described which address these challenges: (i) a microfluidic single neuron arraying method, and (ii) a water masking method for plasma patterning biomaterial coatings to register neurons and promote outgrowth between compartments. Minimalistic neuronal co-cultures were prepared with high-level (&#62;85%) intercompartment connectivity and can be used for high throughput neurobiology experiments with single cell precision.

Protocols for single neuron microfluidic arraying and water masking for the in-chip plasma patterning of biomaterial coatings are described. Highly interconnected co-cultures can be prepared using minimal cell inputs.

神经元共培养与单细胞精度的制备

Microfluidic embodiments of the Campenot chamber have attracted great interest from the neuroscience community. These interconnected co-culture platforms ...

Inducing Plasticity of Astrocytic Receptors by Manipulation of Neuronal Firing Rates

Close to two decades of research has established that astrocytes in situ and in vivo express numerous G protein-coupled receptors (GPCRs) that can be stimulated by neuronally-released transmitter. However, the ability of astrocytic receptors to exhibit plasticity in response to changes in neuronal activity has received little attention. Here we describe a model system that can be used to globally scale up or down astrocytic group I metabotropic glutamate receptors (mGluRs) in acute brain slices. Included are methods on how to prepare parasagittal hippocampal slices, construct chambers suitable for long-term slice incubation, bidirectionally manipulate neuronal action potential frequency, load astrocytes and astrocyte processes with fluorescent Ca2+ indicator, and measure changes in astrocytic Gq GPCR activity by recording spontaneous and evoked astrocyte Ca2+ events using confocal microscopy. In essence, a &#8220;calcium roadmap&#8221; is provided for how to measure plasticity of astrocytic Gq GPCRs. Applications of the technique for study of astrocytes are discussed. Having an understanding of how astrocytic receptor signaling is affected by changes in neuronal activity has important implications for both normal synaptic function as well as processes underlying neurological disorders and neurodegenerative disease.

Here we describe an adaptation of protocols used to induce homeostatic plasticity in neurons for the study of plasticity of astrocytic G protein-coupled receptors. Recently used to examine changes in astrocytic group I mGluRs in juvenile mice, the method can be applied to measure scaling of various astrocytic GPCRs, in tissue from adult mice in situ and in vivo, and to gain a better appreciation of the sensitivity of astrocytic receptors to changes in neuronal activity.

诱导星形胶质细胞受体的可塑性由神经元的放电频率操作

Close to two decades of research has established that astrocytes in situ and in vivo express numerous G protein-coupled receptors (GPCRs) that can be ...

Quantification of Filamentous Actin (F-actin) Puncta in Rat Cortical Neurons

Filamentous actin protein (F-actin) plays a major role in spinogenesis, synaptic plasticity, and synaptic stability. Changes in dendritic F-actin rich structures suggest alterations in synaptic integrity and connectivity. Here we provide a detailed protocol for culturing primary rat cortical neurons, Phalloidin staining for F-actin puncta, and subsequent quantification techniques. First, the frontal cortex of E18 rat embryos are dissociated into low-density cell culture, then the neurons grown in vitro for at least 12-14 days. Following experimental treatment, the cortical neurons are stained with AlexaFluor 488 Phalloidin (to label the dendritic F-actin puncta) and microtubule-associated protein 2 (MAP2; to validate the neuronal cells and dendritic integrity). Finally, specialized software is used to analyze and quantify randomly selected neuronal dendrites. F-actin rich structures are identified on second order dendritic branches (length range 25-75 &#181;m) with continuous MAP2 immunofluorescence. The protocol presented here will be a useful method for investigating changes in dendritic synapse structures subsequent to experimental treatments.

Quantification of Filamentous Actin F-actin Puncta in Rat Cortical Neurons

Filamentous actin (F-actin) plays an important role in spinogenesis, synaptic plasticity, and synaptic stability. Quantification of F-actin puncta is therefore a useful tool to study the integrity of synaptic structures. This protocol describes the procedures of quantifying F-actin puncta labeled with Phalloidin in low-density primary cortical neuronal cultures.

大鼠皮层神经元丝状肌动蛋白（F-肌动蛋白）泪点的量化

Filamentous actin protein (F-actin) plays a major role in spinogenesis, synaptic plasticity, and synaptic stability. Changes in dendritic F-actin rich ...