Name: utilizing combined methodologies to define the role of plasma membrane delivery during axon branching and neuronal morphogenesis
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Description: Watch this Scientific Journal Video about Utilizing Combined Methodologies to Define the Role of Plasma Membrane Delivery During Axon Branching and Neuronal Morphogenesis at JoVE.com

RO1 - GM108970（SLG）和F31-NS087837（CW）:这项工作是由美国国立卫生研究院的支持。

研究伦理的声明 :所有涉及此详述动物实验是受规则和UNC委员会动物护理的法规和标准NIH为照顾和使用实验动物。 1.准备和游离皮层神经元的电镀<ol><li>通过CO安乐死定时怀孕女2吸入颈椎脱位。从每个半球胚胎天15.5（E15.5）小鼠和microdissect皮质的头骨取下整个大脑。有关胚胎小鼠皮层的显微请Viesselmann 等 8详细说明。 </li><li>放置在1ml​​解剖媒体的不超过4皮质在无菌1.6毫升微型管。加入120微升10X胰蛋白酶和翻转试管3 - 5次混合。 </li><li>使用血球，细胞计数种子细胞培养皿。注意事项:1半球皮质APPR提供oximately 3000000细胞。 <ol><li>对于耐SDS SNARE复合生化检测，每板35mm培养皿6000000细胞，并利用每个条件两道菜。注:这是通过比较多个条件时，使用6孔板简化。 </li><li>为支化测定法，对硝酸板神经元在15万的密度以每35mm培养皿250,000个细胞的密度洗涤圆形盖玻片，对轴突分枝板的DIC成像。这些密度避免拥挤和简化分析。 </li></ol></li><li>活细胞的TIRF显微镜，悬浮每转2000000神经元在RT核转染溶液。 <ol><li>加入100微升细胞悬浮液，以微管，根据制造商协议9含有VAMP2-pHlourin表达质粒电穿孔的10微克带有nucleofector。 </li><li>转染后立即加入500μl胰蛋白酶淬火介质，在书房移除试管和细胞板停牌每35毫米PDL镀膜玻璃底菜400,000个单元格的减到。 </li></ol></li><li>板上的所有皮层神经元在2毫升无血清培养基。 </li></ol> 2. SNARE复合体形成分析注:耐SDS SNARE复合物加工成最初描述与10下文详述的修改进行分析。为了证实有效的替代抗体，在这里使用的那些，请参阅材料部分。 <ol><li> E15.5刺激皮层神经元2天体外 （DIV）250毫微克/毫升导蛋白1或假控制裂解前1小时。 </li><li>前处理的样品，凉爽离心机至4℃，并设置水浴温度至37℃，并加热块到100℃。 </li><li>从孵化器中取出细胞的菜肴，置于冰上。 <ol><li>从细胞中吸出培养基，替换为约2ml冰冷的磷酸盐缓冲盐水（PBS）轻轻2次，1 - 每次洗涤2分钟。 </li><li>对于该测定，使用每个COND 2孔银行足球比赛。 </li><li>抽吸PBS从条件的第一阱和替换用250微升匀浆缓冲液。留在冰上5分钟，然后使用细胞升降机，匀化细胞在冰上。 </li><li>从相同的条件下的下一个孔吸出PBS和最近匀浆混合物添加到孔中。这会增加蛋白浓度。重复所有条件。 </li><li>完成后，吸管匀浆溶液到预先冷却1.6毫升微型管在冰上，加入20％的TritonX-100，达到1％的TritonX-100的最终浓度。磨碎，同时尽量减少泡沫与1000微升吸管搅拌了10倍。 </li></ol></li><li>在冰上孵育管2分钟以溶解的蛋白质。在6,010 RCF在4℃下培养后，离心10分钟以沉淀非溶解材料。移动裂解上清液进入冰冷冻新1.6毫升管。 </li><li>用Bradford法进行11蛋白浓度分析和稀释样品到国际泳联3 L蛋白浓度 - 5毫克/毫升剩余补充有1％的TritonX-100和5×样品缓冲液补充有B-巯基乙醇（BME）的匀浆缓冲液。 </li><li>平均分配每个实验样品放入试管2。在37℃下进行30分钟（SNARE复合物），以及其他的在100℃下进行30分钟（SNARE单体）孵育一个样本。反转管间歇性地将样本保持在溶液中。 </li><li>在-20℃冷冻样品直至准备通过SDS-PAGE分离。前用标准技术通过电泳样品，再沸腾先前煮沸样品在100℃5分钟，但在RT解冻37℃的样品。 </li><li>样品的运行重复（30 - 40微克每个样品的蛋白质）在两个8％和15％的凝胶;的8％提供了复杂的理想分离，而15％被用于量化SNARE蛋白单体（SNAP-25〜25kDa，VAMP2〜18kDa，突触-1〜35kDa的）。在70 v使电源直到染料行是通过STAC王露和增加电压90V。运行凝胶，直到染料逃跑。 </li><li>为了保持单体，使用冷转移缓冲液，用20％甲醇（MeOH）加入，在70 V代表45分钟传送在冰上蛋白至0.2μm硝酸纤维素膜上。 </li><li>在盖菜2小时到0干膜/ N在室温（O / N提供了最好的结果）。 </li><li>块SNARE，在10％牛血清白蛋白（BSA）复合膜在RT 1小时。 </li><li>以1:1的稀释度制备一抗（识别特定SNARE复合组件）:1000和探针O / N在4℃的旋转振荡器上。 <ol><li>冲洗在Tris缓冲盐水加0.1％吐温20（TBS-T），每次5分钟，3次印迹。 </li></ol></li><li>以1:1的稀释度制备的荧光第二抗体的解决方案:在TBS-T 20,000在1％BSA和探头1小时，覆盖在RT。 1小时后，重复步骤2.12.1。 </li><li>荧光扫描机的图像印迹配有软件套件和定量两种SNARE蛋白合作mplex（以上40kDa免疫反应条带）和单体谱带（25 kDa的，18kDa的，35kDa的用于SNAP-25，VAMP2免疫反应条带和突触-1，分别地）使用制造商的说明用于绘制矩形的ROI。 </li></ol> 3.成像胞吐通过TIRF显微镜活动注意:此协议需要专门显微镜设备，包括一个环境室，以保持温度，湿度和CO 2，配备有表面荧光照明，高倍率/高数值孔径（NA）的TIRF目标，一个自动XYZ台一个倒置的TIRF显微镜，和敏感电荷耦合器件（CCD）检测器。该协议使用配备了100倍1.49NA TIRF目标固态491 nm激光和电子倍增CCD（EM-CCD）的全自动倒置显微镜。所有设备由成像和激光控制软件控制。此前开始的ENVIR成像协议电源onmental室，舞台，灯，计算机和摄像机。 <ol><li>成像软件中选择目标。一旦该目标是到位，拧紧领围绕目标加热器。 </li><li>确认目标是在舞台插槽固定阶段孵化器之前完全降低。倒入蒸馏水注入阶段培养箱入口均匀，以防止溢出。 </li><li>打开孵化系统。打开阀门到CO 2罐和确认的压力是适当的每个制造商的说明该系统。允许腔一些时间来达到5％的CO 2和37℃。之前加入的摄像培养皿，放置一个空盘在孵化器，以避免对目标冷凝水。 </li><li>功率激光源。 </li><li>开阶段培养箱和浸油添加到该透镜。将样品中的孵化器，并与稳定的武器或一盘菜的重量稳定。提高物镜直到油使与样品的底部接触。 SWITCh至透射光照明，并通过目镜发现神经焦平面。 </li><li>启动激光软件并连接到激光控制软件。设置照度广角并选择正在使用的目标（建议100X 1.49 NA TIRF）。样品的设定折射率（细胞〜1.38）。通过取消选中"TTL"为491 nm激光调节激光强度。调整滑块到100，然后把它回落到一个值之间20 - 40％。重新检查"TTL"。 </li><li>在透射光照明再次聚焦样本。转到成像软件，并选择491 nm激光照明和开启快门。细调整天花板上的激光的焦点和中心的点与除去冷凝器闭合场光阑的中心。将聚光光学板凳上倒挂，以免划伤镜头。 </li><li>更换冷凝器和去TIRF软件，并设置穿透深度（PD）到110纳米。从切换广角照度TIRF ILLUM萌发的模式进行成像。 </li><li>查找VAMP2-phluorin采用宽视场落射荧光与啶光源表达通过目镜细胞。 <ol><li>调整摄像参数（​​曝光时间，增益和激光功率），使用最小的曝光时间和激光强度以减少漂白和光毒性（例如最大化信噪比和动态范围:50之间的曝光 - 100毫秒，用15 - 30获得30％的最大激光功率）。 </li><li>每单元设置连续自动对焦。获取图像时光倒流与并购发生的每0.5秒5分钟设置。为的netrin-1刺激的情况下，1的netrin-加入500纳克/毫升的细胞的培养皿在层流罩和盘返回到培养箱中成像前1小时。 </li></ol></li><li>要在开放的ImageJ图像堆栈通过将文件拖动到窗口或将文件&gt;打开&gt;文件名 ​​（ 图量化每个单元面积和时间归胞吐事件的频率0; 2A插图1）。 <ol><li>要除去稳定的荧光信号，不代表囊泡融合事件，创建一个使用图像&gt;栈&gt; Z-项目&gt;投影类型整个堆栈的平均ž预测:平均强度。从每个图像使用过程中的缩时​​&gt;图像计算器减去这意味着图像&gt; Image1的:你的栈操作:减去IMAGE2新创建的平均ž投影。这强调胞吐事件（ 图2A插图2 - 3）。 </li><li>由伯爵眼外融合的事件。胞吐事件被定义为受衍射限制的荧光信号，从而迅速扩散作为VAMP2-phluorin质膜（ 图2A插图6）内扩散的外观。 </li><li>使用阈值命令用图片来突出第一图像&gt;调整&gt;阈值&gt;直到整个细胞是门槛凸显（ 图2A插图7 - 8）调整滑块。 </li><li>在安娜lyze，选择SetMeasurements和检查区和显示标签。选择使用棒跟踪工具，然后按"M"来衡量（ 图2A插图7 - 8）阈值的蜂窝区。 </li><li>转让双方外融合事件计数，细胞面积测量，并经过成像时间到电子表格来计算（ 图2A插图9）的胞吐事件/毫米2 /次号码。 </li></ol></li></ol> 4.微分干涉对比（DIC）轴突分支的显微缩时注:完整的协议与示范的一般方法DIC成像可用12。虽然这种协议利用DIC时，也可以使用其它的透射光显微术的方法为相同的目的（例如:相衬）。 <ol><li>在2 DIV，包含预热的环境室未转染的神经元发生玻璃底菜成像，以保持潮湿ENV用5％的CO 2和37℃ironment。利用最佳的图像质量和分辨率装有60X计划复消色差透镜的显微镜，1.4的NA DIC物镜和高NA冷凝器。 </li><li>调整焦平面发现通过使用透射光照明目镜神经元。 </li><li>在使用图像软件多区域采集功能，找到并节省至少6个细胞的XYZ位置。阶段的位置，使的视图以取得最佳效果的领域内感兴趣拟合神经元。 <ol><li> 250纳克刺激/ ml的导蛋白1，然后按"启动多区域获取"。在每个位置顺序地获取图像每20秒24小时，暂停采集和再聚焦是必要的。 </li></ol></li><li>查看使用应用程序中的图像处理软件图像&gt;查看多维数据&gt;打开的文件名。鉴定稳定轴突分支（20微米长）的成像会议期间的形式。用画线工具来测量一个stableaxon branc从在轴突到尖端的基部小时以确保在20微米长度资格得到满足。 <ol><li>在电子表格中，记录帧号的netrin刺激后当膜的突起中时新生的分支达到20微米的长度，其中分支以后形成以及netrin的刺激后的帧号码的区域开始。 </li><li>由20秒乘以突起和分支地层之间的帧的数目来计算每个分支的形成时间。 </li></ol></li></ol> 5.毒素操作和固定细胞免疫荧光<ol><li>在2 DIV，治疗中的实验条件下的神经元用250纳克/毫升的netrin-1和/或10nM的A型肉毒毒素（BONTA），其裂解SNARE蛋白SNAP25和有效地抑制SNARE介导的胞吐13，再加上250纳克/毫升netrin- 1。留下一组神经元未处理作为控制条件的。 注意:BONTA需要用眼和呼吸道的防护的准备和使用时，解决方案。尽快维护所有被污染的材料的生物危险材料和高压灭菌。 </li><li>在3 DIV（24小时后处理）抽吸介质，并立即与至少1毫升PHEM修复（详情见表1）更换加热到37℃。孵育在室温黑暗中20分钟。 </li><li>用PBS冲洗3倍（以备将来使用密封用封口膜和储存在4°C）。 </li><li>在黑暗中，加湿染色室地方盖玻片和透化细胞膜在新鲜稀释的0.2％的TritonX-100的PBS 10分钟（从10％的TritonX-100的库存。制造）。 </li><li>除去透化溶液，并用50微升1×的用PBS冲洗。块，用于在30分钟内约10％牛血清白蛋白的50μl的在PBS（BSA-PBS），或10％在RT水煮驴血清在PBS中。 </li><li>用1X PBS冲洗一遍。孵育盖玻片在50μl初级抗体（1:1000βIII微管蛋白，以确认神经同一性）在1％BSA-PBS在RT 1小时， 在黑暗中。 </li><li>在PBS进行3次5分钟洗涤。孵育盖玻片在50μl光谱上不同的第二抗体对微管蛋白（1:400），和荧光鬼笔环肽（通过激发而异:488鬼笔环肽在1:400，647鬼笔环肽在1:100），在1％BSA-PBS中1小时。 </li><li>在1X PBS洗涤3次5分钟并安装到盖玻片幻灯片安装介质。 </li><li>从盖玻片和密封用透明指甲油的边缘多余的真空安装介质。 </li><li>在具有40X 1.4NA目标，啶能力和EM-CCD倒置显微镜收集广角落射荧光图像。 <ol><li>手动分析使用ImageJ分支。在开放的ImageJ图像堆栈通过将文件拖动到窗口或将文件&gt;打开&gt;文件名 ​​（ 图4A插图1）。 </li><li>使用线&gt;分割线，微量轴突，定义为从体细胞（ 图4A插图2 - 3）延伸的最长的神经突。 </li><li>使用分析&gt;工具&gt;投资回报率经理，保存轴突作为跟踪感兴趣的区域。跟踪和保存每个分支轴突，定义为突起≥20微米长。包括在分析（ 图4A插图3 - 4）只有小学支（副产物从轴突发芽直接）。 </li><li>按"M"来测量感兴趣的保存区域，并且以像素为单位的长度传输到电子表格，计算在微米的长度。 </li><li>划分轴突分支≥20微米长度的次数，通过在微米轴突除以100得到每100μm长度轴突分枝的数目的长度。 </li></ol></li></ol>

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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+rapid+and+sensitive+method+for+the+quantitation+of+microgram+quantities+of+protein+utilizing+the+principle+of+protein-dye+binding.">A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding.</a> Analytical Biochemistry. 72, 248-254 (1976).</li><li>Centonze Frohlich, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Phase+Contrast+and+Differential+Interference+Contrast+(DIC)+Microscopy.">Phase Contrast and Differential Interference Contrast (DIC) Microscopy.</a> Journal of Visualized Experiments. (17), (2008).</li><li>Blasi, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Botulinum+neurotoxin+A+selectively+cleaves+the+synaptic+protein+SNAP-25.">Botulinum neurotoxin A selectively cleaves the synaptic protein SNAP-25.</a> Nature. 365 (6442), 160-163 (1993).</li></ol>

The authors have nothing to disclose.

Axon branching is a fundamental neurodevelopmental process and underpins the vast neuroconnectivity of the mammalian nervous system. Understanding the mechanisms involved in localized plasma membrane expansion is integral to our understanding of both normal and pathological neurodevelopment. The use of a multipronged approach incorporating both population level and single cell level methodologies enhances reproducibility and increases spatial and temporal resolution without compromising population level analysis. At the ...

Recent estimates suggest that the human brain contains 1011 neurons with 1014 synaptic connections1, highlighting the importance of axon branching in vivo. Extracellular axon guidance cues such as netrin-1 guide axons to appropriate synaptic partners and stimulate axonal branching, thereby increasing synaptic capacity2-5. Netrin-1-dependent axonal arborization involves substantial plasma membrane expansion6, which we hypothesized requires delivery of additional membrane components via SNARE complex dependent exocytic vesicle fusion7. 
Investigating the role of SNARE-mediated exocytosis in netrin-1 dependent axon branching is complicated by several factors. First, the heterogeneity of cortical neurons increases the sample size required to identify significant effects, complicating single cell techniques like imaging. Second, although biochemical techniques permit observation of changes that occur at the population level, they lack the temporal and spatial resolution necessary to localize plasma membrane expansion to the axon in the time frame of axon branching. Lastly, although axon branches form over hours, the cellular changes that contribute to axonal extension may begin within minutes and occur on the order of seconds, thus extending the temporal scope for experimental consideration. 
We outline a multi-technique approach that addresses these diverse temporal and spatial scales of exocytosis and axon branching, and thus enhances our understanding of the fundamental cellular mechanisms. Utilizing these approaches provides evidence that supports a critical role for SNARE-mediated exocytosis in axon branching.

<table><tbody><tr><td>6-well tissue culture treated plates</td><td>Olympus Plastics</td><td>25-105</td><td></td></tr><tr><td>glass coverslips</td><td>Fisher scientific</td><td>12-545-81</td><td>12CIR-1.5; must be nitric acid treated for 24 hours, rinsed in DI water 2x, and dried prior to use. Must be coated with 1 mg/ml Poly-d-lysine and rinsed prior to plating cells.</td></tr><tr><td>Amaxa nucleofection solution</td><td>Lonza</td><td>VPG-1001</td><td>100 ml/transfection</td></tr><tr><td>Amaxa Nucleofector/electroporator</td><td>Lonza</td><td></td><td>program O-005</td></tr><tr><td>35 mm Glass bottom live cell imaging dishes</td><td>Matek Corporation</td><td>p356-1.5-14-C</td><td>must be coated with 1 mg/ml Poly-d-lysine and rinsed prior to plating cells</td></tr><tr><td>Olympus IX81-ZDC2 inverted microscope</td><td>Olympus</td><td></td><td></td></tr><tr><td>Lambda LS xenon lamp</td><td>Sutter Instruments Company</td><td></td><td></td></tr><tr><td>Environmental Stage top incubator</td><td>Tokai Hit</td><td></td><td></td></tr><tr><td>100x 1.49 NA TIRF objective</td><td>Olympus</td><td></td><td></td></tr><tr><td>Andor iXon EM-CCD</td><td>Andor</td><td></td><td></td></tr><tr><td>Odyssey Licor Infrared Imaging System</td><td>LI-COR</td><td>Odyssey CL-X</td><td>Used for scanning blots</td></tr><tr><td>Image studio software suite</td><td>LI-COR</td><td></td><td>Used for scanning on the Odyssey Infrared system; Image studio lite used for offline analysis of blots</td></tr><tr><td>Metamorph for Olympus</td><td>Molecular devices, LLC</td><td>version 7.7.6.0</td><td>Software used for all imaging and the analysis of DIC timelapse</td></tr><tr><td>CELL TIRF control software</td><td>Olympus</td><td></td><td>Software used to control lasers for TIRF imaging</td></tr><tr><td>Fiji (Image J)</td><td>NIH</td><td>ImageJ Version 1.49t</td><td></td></tr><tr><td>60x Plan Apochromat 1.4 NA objective</td><td>Olympus</td><td></td><td></td></tr><tr><td>40x 1.4 NA Plan Apochromat objective</td><td>Olympus</td><td></td><td></td></tr><tr><td>Neurobasal media</td><td>GIBCO</td><td>21103-049</td><td>Base solution for both serum free and trypsin quenching media</td></tr><tr><td>Supplement B27</td><td>GIBCO</td><td>17504-044</td><td>500 ml/50 ml Serum free media and Trypsin Quenching media</td></tr><tr><td>L-Glutamine</td><td></td><td>35050-061</td><td>1 ml/50 ml Serum free media</td></tr><tr><td>Bovine serum albumin</td><td>Bio Basic Incorporated</td><td>9048-46-8</td><td>10% solution in 1x PBS for blocking coverslips; 5% solution in TBS-T for blocking nitrocellulose membranes.</td></tr><tr><td>10x&amp;nbsp; trypsin</td><td>Sigma</td><td>59427C</td><td></td></tr><tr><td>HEPES</td><td>CELLGRO</td><td>25-060-Cl</td><td></td></tr><tr><td>Dulbecco&#039;s Phosphate Buffered Saline (DPBS)+ Ca + Mg</td><td>Corning</td><td>21-030-cm</td><td></td></tr><tr><td>Fetal bovine serum</td><td>Corning/CELLGRO</td><td>35-010-CV</td><td></td></tr><tr><td>Hank&#039;s Balanced Salt Solution (HBSS)</td><td>Corning/CELLGRO</td><td>20-021-CV</td><td></td></tr><tr><td>NaCl</td><td>Fisher scientific</td><td>BP358-10</td><td></td></tr><tr><td>EGTA</td><td>Fisher scientific</td><td>CAS67-42-5</td><td></td></tr><tr><td>MgCl&lt;sub&gt;2&lt;/sub&gt;</td><td>Fisher scientific</td><td>BP214-500</td><td></td></tr><tr><td>TRIS HCl</td><td>Sigma</td><td>T5941-500</td><td></td></tr><tr><td>TRIS base</td><td>Fisher scientific</td><td>BP152-5</td><td></td></tr><tr><td>N-Propyl Gallate</td><td>MP Biomedicals</td><td>102747</td><td></td></tr><tr><td>Glycerol Photometric grade</td><td>Acros Organics</td><td>18469-5000</td><td></td></tr><tr><td>Glycerol (non optics grade)</td><td>Fisher scientific</td><td>CAS56-81-5</td><td></td></tr><tr><td>B-mercaptoethonal</td><td>Fisher scientific</td><td>BP176-100</td><td></td></tr><tr><td>SDS</td><td>Fisher scientific</td><td>BP166-500</td><td></td></tr><tr><td>Distilled Water&amp;nbsp;</td><td>GIBCO</td><td>152340-147</td><td></td></tr><tr><td>Poly-D-Lysine</td><td>Sigma</td><td>p-7886</td><td>Dissolved in sterile water at 1 mg/ml</td></tr><tr><td>Botulinum A toxin BoNTA</td><td>List Biological Laboratories</td><td>128-A</td><td></td></tr><tr><td>Rabbit polyclonal anti human VAMP2</td><td>Cell signaling</td><td>11829</td><td></td></tr><tr><td>Mouse monoclonal anti rat Syntaxin1A</td><td>Santa Cruz Biotechnology</td><td>sc-12736</td><td></td></tr><tr><td>Goat polyclonal anti human SNAP-25</td><td>Santa Cruz Biotechnology</td><td>sc-7538</td><td></td></tr><tr><td>Mouse monoclonal anti human &amp;beta;III-tubulin&amp;nbsp;</td><td>Covance</td><td>MMS-435P</td><td></td></tr><tr><td>Alexa Fluor 568 and Alexa Fluor 488 phalloidin, or Alexa Fluor 647</td><td>Invitrogen</td><td></td><td></td></tr><tr><td>LI-COR IR-dye secondary antibodies</td><td>LI-COR</td><td>P/N 925-32212,P/N 925-68023, P/N 926-68022</td><td>800 donkey anti-mouse, 680 donkey anti rabbit, 680 donkey anti goat</td></tr><tr><td>0.2 &amp;#956;m pore size nitrocellulose membrane</td><td>Biorad</td><td>9004-70-0</td><td></td></tr><tr><td>Tween-20</td><td>Fisher scientific</td><td>BP337-500</td><td></td></tr><tr><td>Methanol</td><td>Fisher scientific</td><td>S25426A</td><td></td></tr><tr><td>Bromphenol Blue</td><td>Sigma</td><td>B5525-5G</td><td></td></tr><tr><td>Sucrose</td><td>Fisher scientific</td><td>S6-212</td><td></td></tr><tr><td>Paraformaldehyde</td><td>Fisher scientific</td><td>O-4042-500</td><td></td></tr><tr><td>Triton-X100</td><td>Fisher scientific</td><td>BP151-500</td><td></td></tr><tr><td>TEMED</td><td>Fisher scientific</td><td>BP150-20</td><td></td></tr><tr><td>40% Bis-Acrylimide</td><td>Fisher scientific</td><td>BP1408-1</td><td></td></tr><tr><td>&lt;strong&gt;Name&lt;/strong&gt;</td><td>&lt;strong&gt;Company&lt;/strong&gt;</td><td>&lt;strong&gt;Catalog Number&lt;/strong&gt;</td><td>&lt;strong&gt;Comments&lt;/strong&gt;</td></tr><tr><td>&lt;strong&gt;Alternative Validated Antibodies&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Mouse Monoclonal Anti-Syntaxin HPC-1 clone</td><td>Sigma Aldrich</td><td>S0664</td><td></td></tr><tr><td>&amp;nbsp;Mouse Monoclonal Synaptobrevin 2 (VAMP2)</td><td>Synaptic Systems</td><td>104-211</td><td></td></tr><tr><td>Mouse Monoclonal SNAP25</td><td>Synaptic Systems</td><td>111-011</td><td></td></tr></tbody></table>

utilizing combined methodologies to define the role of plasma membrane delivery during axon branching and neuronal morphogenesis

During neural development, growing axons extend to multiple synaptic partners by elaborating axonal branches. Axon branching is promoted by extracellular guidance cues like netrin-1 and results in dramatic increases to the surface area of the axonal plasma membrane. Netrin-1-dependent axon branching likely involves temporal and spatial control of plasma membrane expansion, the components of which are supplied through exocytic vesicle fusion. These fusion events are preceded by formation of SNARE complexes, comprising a v-SNARE, such as VAMP2 (vesicle-associated membrane protein 2), and plasma membrane t-SNAREs, syntaxin-1 and SNAP25 (synaptosomal-associated protein 25). Detailed herein isa multi-pronged approach used to examine the role of SNARE mediated exocytosis in axon branching. The strength of the combined approach is data acquisition at a range of spatial and temporal resolutions, spanning from the dynamics of single vesicle fusion events in individual neurons to SNARE complex formation and axon branching in populations of cultured neurons. This protocol takes advantage of established biochemical approaches to assay levels of endogenous SNARE complexes and Total Internal Reflection Fluorescence (TIRF) microscopy of cortical neurons expressing VAMP2 tagged with a pH-sensitive GFP (VAMP2-pHlourin) to identify netrin-1 dependent changes in exocytic activity in individual neurons. To elucidate the timing of netrin-1-dependent branching, time-lapse differential interference contrast (DIC) microscopy of single neurons over the order of hours is utilized. Fixed cell immunofluorescence paired with botulinum neurotoxins that cleave SNARE machinery and block exocytosis demonstrates that netrin-1 dependent axon branching requires SNARE-mediated exocytic activity.

利用体外生物化学技术以测定神经元群体SDS抗性SNARE复合物的量。 图1示出探查SNAP-25，syntaxin1A和VAMP2的耐SDS SNARE复合测定的所得印迹以下完成。 在基底细胞膜TIRF显微镜提供了在单个细胞个体胞吐融合事件的高清晰度的图像。 图2A展示了用于识别VAMP2-phluorin介导的胞吐事件图像分析方法。插图显?...

Watch this Scientific Journal Video about Utilizing Combined Methodologies to Define the Role of Plasma Membrane Delivery During Axon Branching and Neuronal Morphogenesis at JoVE.com

Utilizing Combined Methodologies to Define the Role of Plasma Membrane Delivery During Axon Branching and Neuronal Morphogenesis

Light microscopy techniques coupled with biochemical assays elucidate the involvement of SNARE-mediated exocytosis in netrin-dependent axon branching. This combination of techniques permits identification of molecular mechanisms controlling axon branching and cell shape change.

采用组合运用各种方法，定义质膜交付在轴突分支和神经元形态发生中的作用

During neural development, growing axons extend to multiple synaptic partners by elaborating axonal branches. Axon branching is promoted by extracellular ...

utilizing-combined-methodologies-to-define-role-plasma-membrane

Research

JoVE Journal

Neuroscience

6.1K 次观看.  The University of North Carolina at Chapel Hill. Light microscopy techniques coupled with biochemical assays elucidate the involvement of SNARE-mediated exocytosis in netrin-dependent axon branching. This combination of techniques permits identification of molecular mechanisms controlling axon branching and cell shape change. 

视频: Utilizing Combined Methodologies to Define the Role of Plasma Membrane Delivery During Axon Branching and Neuronal Morphogenesis

Methods for Study of Neuronal Morphogenesis: Ex vivo RNAi Electroporation in Embryonic Murine Cerebral Cortex

The cerebral cortex directs higher cognitive functions. This six layered structure is generated in an inside-first, outside-last manner, in which the first born neurons remain closer to the ventricle while the last born neurons migrate past the first born neurons towards the surface of the brain1. In addition to neuronal migration2, a key process for normal cortical function is the regulation of neuronal morphogenesis3. While neuronal morphogenesis can be studied in vitro in primary cultures, there is much to be learned from how these processes are regulated in tissue environments.
We describe techniques to analyze neuronal migration and/or morphogenesis in organotypic slices of the cerebral cortex4,6. A pSilencer modified vector is used which contains both a U6 promoter that drives the double stranded hairpin RNA and a separate expression cassette that encodes GFP protein driven by a CMV promoter7-9. Our approach allows for the rapid assessment of defects in neurite outgrowth upon specific knockdown of candidate genes and has been successfully used in a screen for regulators of neurite outgrowth8. Because only a subset of cells will express the RNAi constructs, the organotypic slices allow for a mosaic analysis of the potential phenotypes. Moreover, because this analysis is done in a near approximation of the in vivo environment, it provides a low cost and rapid alternative to the generation of transgenic or knockout animals for genes of unknown cortical function. Finally, in comparison with in vivo electroporation technology, the success of ex vivo electroporation experiments is not dependant upon proficient surgery skill development and can be performed with a shorter training time and skill.

Methods for Study of Neuronal Morphogenesis: Ex vivo RNAi Electroporation in Embryonic Murine Cerebral Cortex

To conduct a rapid assessment of the function of genes in the development of cerebral cortex, we describe methods involving the ex vivo electroporation of plasmids co-expressing inhibitory RNA (RNAi) and GFP in murine embryonic cortex. This protocol is amenable to the study of various aspects of neurodevelopment such as neurogenesis, neuronal migration and neuronal morphogenesis including dendrite and axon outgrowth.

神经元形态的研究方法：<em&gt;体外</em&gt; RNAi技术在胚胎小鼠大脑皮质的电穿孔

The cerebral cortex directs higher cognitive functions. This six layered structure is generated in an inside-first, outside-last manner, in which the ...

科研

神经科学

In ovo Electroporation of miRNA-based Plasmids in the Developing Neural Tube and Assessment of Phenotypes by DiI Injection in Open-book Preparations

Commissural dI1 neurons have been extensively studied to elucidate the mechanisms underlying axon guidance during development1,2. These neurons are located in the dorsal spinal cord and send their axons along stereotyped trajectories. Commissural axons initially project ventrally towards and then across the floorplate. After crossing the midline, these axons make a sharp rostral turn and project longitudinally towards the brain. Each of these steps is regulated by the coordinated activities of attractive and repulsive guidance cues. The correct interpretation of these cues is crucial to the guidance of axons along their demarcated pathway. Thus, the physiological contribution of a particular molecule to commissural axon guidance is ideally investigated in the context of the living embryo. Accordingly, gene knockdown in vivo must be precisely controlled in order to carefully distinguish axon guidance activities of genes that may play multiple roles during development.
Here, we describe a method to knockdown gene expression in the chicken neural tube in a cell type-specific, traceable manner. We use novel plasmid vectors3 harboring cell type-specific promoters/enhancers that drive the expression of a fluorescent protein marker, followed directly by a miR30-RNAi transcript4 (located within the 3'-UTR of the cDNA encoding the fluorescent protein) (Figure 1). When electroporated into the developing neural tube, these vectors elicit efficient downregulation of gene expression and express bright fluorescent marker proteins to enable direct tracing of the cells experiencing knockdown3. Mixing different RNAi vectors prior to electroporation allows the simultaneous knockdown of two or more genes in independent regions of the spinal cord. This permits complex cellular and molecular interactions to be examined during development, in a manner that is fast, simple, precise and inexpensive. In combination with DiI tracing of commissural axon trajectories in open-book preparations5, this method is a useful tool for in vivo studies of the cellular and molecular mechanisms of commissural axon growth and guidance. In principle, any promoter/enhancer could be used, potentially making the technique more widely applicable for in vivo studies of gene function during development6.
This video first demonstrates how to handle and window eggs, the injection of DNA plasmids into the neural tube and the electroporation procedure. To investigate commissural axon guidance, the spinal cord is removed from the embryo as an open-book preparation, fixed, and injected with DiI to enable axon pathways to be traced. The spinal cord is mounted between coverslips and visualized using confocal microscopy.

In ovo Electroporation of miRNA-based Plasmids in the Developing Neural Tube and Assessment of Phenotypes by DiI Injection in Open-book Preparations

A method by which gene expression in the neural tube can be downregulated in a cell type-specific, traceable manner is described. We demonstrate how in ovo electroporation of microRNA-based plasmids that elicit spatiotemporally controlled RNA interference can be used to investigate commissural axon guidance in the developing neural tube.

基于miRNA的质粒在发展中的神经管和表型的评估DiI荧光注射开卷准备在卵的电

Commissural dI1 neurons have been extensively studied to elucidate the mechanisms underlying axon guidance during development1,2. These neurons are ...

Use of pHluorin to Assess the Dynamics of Axon Guidance Receptors in Cell Culture and in the Chick Embryo

During development, axon guidance receptors play a crucial role in regulating axons sensitivity to both attractive and repulsive cues. Indeed, activation of the guidance receptors is the first step of the signaling mechanisms allowing axon tips, the growth cones, to respond to the ligands. As such, the modulation of their availability at the cell surface is one of the mechanisms that participate in setting the growth cone sensitivity. We describe here a method to precisely visualize the spatio-temporal cell surface dynamics of an axon guidance receptor both in vitro and in vivo in the developing chick spinal cord. We took advantage of the pH-dependent fluorescence property of a green fluorescent protein (GFP) variant to specifically detect the fraction of the axon guidance receptor that is addressed to the plasma membrane. We first describe the in vitro validation of such pH-dependent constructs and we further detail their use in vivo, in the chick spinal chord, to assess the spatio-temporal dynamics of the axon guidance receptor of interest.

We describe here the use of a pH-sensitive green fluorescent protein variant, pHluorin, to study the spatio-temporal dynamics of axon guidance receptors trafficking at the cell surface.&#160;The pHluorin-tagged receptor is expressed both in cell culture and in vivo, using electroporation of the chick embryo.

使用普洛林来评估阿克森指导受体在细胞培养和小鸡胚胎中的动力学

During development, axon guidance receptors play a crucial role in regulating axons sensitivity to both attractive and repulsive cues. Indeed, activation ...

Deciphering Axonal Pathways of Genetically Defined Groups of Neurons in the Chick Neural Tube Utilizing in ovo Electroporation

Employment of enhancer elements to drive expression of reporter genes in neurons is a widely used paradigm for tracking axonal projection. For tracking axonal projection of spinal interneurons in vertebrates, germ line-targeted reporter genes yield bilaterally symmetric labeling. Therefore, it is hard to distinguish between the ipsi- and contra-laterally projecting axons. Unilateral electroporation into the chick neural tube provides a useful means to restrict expression of a reporter gene to one side of the central nervous system, and to follow axonal projection on both sides 1 ,2-5. This video demonstrates first how to handle the eggs prior to injection. At HH stage 18-20, DNA is injected into the sacral level of the neural tube, then tungsten electrodes are placed parallel to the embryo and short electrical pulses are administered with a pulse generator. The egg is sealed with tape and placed back into an incubator for further development. Three days later (E6) the spinal cord is removed as an open book preparation from embryo, fixed, and processed for whole mount antibody staining. The stained spinal cord is mounted on slide and visualized using confocal microscopy.

Deciphering Axonal Pathways of Genetically Defined Groups of Neurons in the Chick Neural Tube Utilizing in ovo Electroporation

This video demonstrates how to visualize axonal pathways of genetically defined groups of neurons in the embryonic chick spinal cord utilizing in ovo electroporation of reporter genes under the control of specific enhancer elements.

利用鸡胚神经管破译基因定义的组的神经元轴突途径在OVO电穿孔

Employment of enhancer elements to drive expression of reporter genes in neurons is a widely used paradigm for tracking axonal projection. For tracking ...

生物学

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 ...

The Olfactory System as a Model to Study Axonal Growth Patterns and Morphology In Vivo

The olfactory system has the unusual capacity to generate new neurons throughout the lifetime of an organism. Olfactory stem cells in the basal portion of the olfactory epithelium continuously give rise to new sensory neurons that extend their axons into the olfactory bulb, where they face the challenge to integrate into existing circuitry. Because of this particular feature, the olfactory system represents a unique opportunity to monitor axonal wiring and guidance, and to investigate synapse formation. Here we describe a procedure for in vivo labeling of sensory neurons and subsequent visualization of axons in the olfactory system of larvae of the amphibian Xenopus laevis. To stain sensory neurons in the olfactory organ we adopt the electroporation technique. In vivo electroporation is an established technique for delivering fluorophore-coupled dextrans or other macromolecules into living cells. Stained sensory neurons and their axonal processes can then be monitored in the living animal either using confocal laser-scanning or multiphoton microscopy. By reducing the number of labeled cells to few or single cells per animal, single axons can be tracked into the olfactory bulb and their morphological changes can be monitored over weeks by conducting series of in vivo time lapse imaging experiments. While the described protocol exemplifies the labeling and monitoring of olfactory sensory neurons, it can also be adopted to other cell types within the olfactory and other systems.

The Olfactory System as a Model to Study Axonal Growth Patterns and Morphology In Vivo

We describe a protocol for in vivo labeling of olfactory sensory neurons by electroporation and subsequent confocal laser-scanning or multiphoton microscopy to visualize neuronal morphology and its development over time.

嗅觉系统作为模型来研究轴突生长的模式和形态<em&gt;在体内</em

The olfactory system has the unusual capacity to generate new neurons throughout the lifetime of an organism. Olfactory stem cells in the basal portion of ...

An Approach to Enhance Alignment and Myelination of Dorsal Root Ganglion Neurons

Axon regeneration is a chaotic process due largely to unorganized axon alignment. Therefore, in order for a sufficient number of regenerated axons to bridge the lesion site, properly organized axonal alignment is required. Since demyelination after nerve injury strongly impairs the conductive capacity of surviving axons, remyelination is critical for successful functioning of regenerated nerves. Previously, we demonstrated that mesenchymal stem cells (MSCs) aligned on a pre-stretch induced anisotropic surface because the cells can sense a larger effective stiffness in the stretched direction than in the perpendicular direction. We also showed that an anisotropic surface arising from a mechanical pre-stretched surface similarly affects alignment, as well as growth and myelination of axons. Here, we provide a detailed protocol for preparing a pre-stretched anisotropic surface, the isolation and culture of dorsal root ganglion (DRG) neurons on a pre-stretched surface, and show the myelination behavior of a co-culture of DRG neurons with Schwann cells (SCs) on a pre-stretched surface.

This protocol describes the isolation of dorsal root ganglion (DRG) neurons isolated from rats and the culture of DRG neurons on a static pre-stretched cell culture system to enhance axon alignment, with subsequent co-culture of Schwann Cells (SCs) to promote myelination.

一种方法来提高背根神经节的对齐和髓鞘

Axon regeneration is a chaotic process due largely to unorganized axon alignment. Therefore, in order for a sufficient number of regenerated axons to ...

Rewiring Neuronal Circuits: A New Method for Fast Neurite Extension and Functional Neuronal Connection

Brain and spinal cord injury may lead to permanent disability and death because it is still not possible to regenerate neurons over long distances and accurately reconnect them with an appropriate target. Here a procedure is described to rapidly initiate, elongate, and precisely connect new functional neuronal circuits over long distances. The extension rates achieved reach over 1.2 mm/h, 30-60 times faster than the in vivo rates of the fastest growing axons from the peripheral nervous system (0.02 to 0.04 mm/h)28 and 10 times faster than previously reported for the same neuronal type at an earlier stage of development4. First, isolated populations of rat hippocampal neurons are grown for 2-3 weeks in microfluidic devices to precisely position the cells, enabling easy micromanipulation and experimental reproducibility. Next, beads coated with poly-D-lysine (PDL) are placed on neurites to form adhesive contacts and pipette micromanipulation is used to move the resulting bead-neurite complex. As the bead is moved, it pulls out a new neurite that can be extended over hundreds of micrometers and functionally connected to a target cell in less than 1 h. This process enables experimental reproducibility and ease of manipulation while bypassing slower chemical strategies to induce neurite growth. Preliminary measurements presented here demonstrate a neuronal growth rate far exceeding physiological ones. Combining these innovations allows for the precise establishment of neuronal networks in culture with an unprecedented degree of control. It is a novel method that opens the door to a plethora of information and insights into signal transmission and communication within the neuronal network as well as being a playground in which to explore the limits of neuronal growth. The potential applications and experiments are widespread with direct implications for therapies that aim to reconnect neuronal circuits after trauma or in neurodegenerative diseases.

This procedure describes how to rapidly initiate, extend and connect neurites organized in microfluidic chambers using poly-D-lysine-coated beads fixed to micropipettes that guide neurite elongation.

重建神经元电路:快速神经延伸和功能性神经元连接的新方法

Brain and spinal cord injury may lead to permanent disability and death because it is still not possible to regenerate neurons over long distances and ...

Visualization of Thalamocortical Axon Branching and Synapse Formation in Organotypic Cocultures

Axon branching and synapse formation are crucial processes for establishing precise neuronal circuits. During development, sensory thalamocortical (TC) axons form branches and synapses in specific layers of the cerebral cortex. Despite the obvious spatial correlation between axon branching and synapse formation, the causal relationship between them is poorly understood. To address this issue, we recently developed a method for simultaneous imaging of branching and synapse formation of individual TC axons in organotypic cocultures.
This protocol describes a method which consists of a combination of an organotypic coculture and electroporation. Organotypic cocultures of the thalamus and cerebral cortex facilitate gene manipulation and observation of axonal processes, preserving characteristic structures such as laminar configuration. Two distinct plasmids encoding DsRed and EGFP-tagged synaptophysin (SYP-EGFP) were co-transfected into a small number of thalamic neurons by an electroporation technique. This method allowed us to visualize individual axonal morphologies of TC neurons and their presynaptic sites simultaneously. The method also enabled long-term observation which revealed the causal relationship between axon branching and synapse formation.

This protocol describes a method for simultaneous imaging of thalamocortical axon branching and synapse formation in organotypic cocultures of the thalamus and cerebral cortex. Individual thalamocortical axons and their presynaptic terminals are visualized by a single cell electroporation technique with DsRed and GFP-tagged synaptophysin.

脊髓 Cocultures 丘脑-皮质轴突分支和突触形成的可视化研究

Axon branching and synapse formation are crucial processes for establishing precise neuronal circuits. During development, sensory thalamocortical (TC) ...

3D Particle Tracking for Noninvasive In Vivo Analysis of Synaptic Microtubule Dynamics in Dendrites and Neuromuscular Junctions of Drosophila

Microtubules (MTs) play critical roles in neuronal development, but many questions remain about the molecular mechanisms of their regulation and function. Furthermore, despite progress in understanding postsynaptic MTs, much less is known about the contributions of presynaptic MTs to neuronal morphogenesis. In particular, studies of in vivo MT dynamics in Drosophila sensory dendrites yielded significant insights into polymer-level behavior. However, the technical and analytical challenges associated with live imaging of the fly neuromuscular junction (NMJ) have limited comparable studies of presynaptic MT dynamics. Moreover, while there are many highly effective software strategies for automated analysis of MT dynamics in vitro and ex vivo, in vivo data often necessitate significant operator input or entirely manual analysis due to inherently inferior signal-to-noise ratio in images and complex cellular morphology. &#160;To address this, this study optimized a new software platform for automated and unbiased in vivo particle detection. Multiparametric analysis of live time-lapse confocal images of EB1-GFP labeled MTs was performed in both dendrites and the NMJ of Drosophila larvae and found striking differences in MT behaviors. MT dynamics were furthermore analyzed following knockdown of the MT-associated protein (MAP) dTACC, a key regulator of Drosophila synapse development, and identified statistically significant changes in MT dynamics compared to wild type. These results demonstrate that this novel strategy for the automated multiparametric analysis of both pre- and postsynaptic MT dynamics at the polymer-level significantly reduces human-in-the-loop criteria. The study furthermore shows the utility of this method in detecting distinct MT behaviors upon dTACC-knockdown, indicating a possible future application for functional screens of factors that regulate MT dynamics in vivo. Future applications of this method may also focus on elucidating cell type and/or compartment-specific MT behaviors, and multicolor correlative imaging of EB1-GFP with other cellular and subcellular markers of interest.&#160;

This study presents a noninvasive intravital neuronal imaging strategy combined with a new software strategy to achieve automated, unbiased tracking and analysis of in vivo microtubule (MT) plus-end dynamics in the sensory dendrites and the neuromuscular junctions of Drosophila.

Microtubules (MTs) play critical roles in neuronal development, but many questions remain about the molecular mechanisms of their regulation and function. ...

采用组合运用各种方法，定义质膜交付在轴突分支和神经元形态发生中的作用

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