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>

<ol>
	<li>Drachman, D. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Do+we+have+brain+to+spare?.">Do we have brain to spare?.</a> Neurology. 64 (12), 2004-2005 (2005).</li><li>Serafini, T., 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=Netrin-1+Is+Required+for+Commissural+Axon+Guidance+in+the+Developing+Vertebrate+Nervous+System.">Netrin-1 Is Required for Commissural Axon Guidance in the Developing Vertebrate Nervous System.</a> Cell. 87 (6), 1001-1014 (1996).</li><li>M&#233;tin, C., Del&#233;glise, D., Serafini, T., Kennedy, T. E., Tessier-Lavigne, 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+role+for+netrin-1+in+the+guidance+of+cortical+efferents.">A role for netrin-1 in the guidance of cortical efferents.</a> Development. 124 (24), 5063-5074 (1997).</li><li>Kennedy, T. E., Serafini, T., de la Torre, J. R., Tessier-Lavigne, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Netrins+are+diffusible+chemotropic+factors+for+commissural+axons+in+the+embryonic+spinal+cord.">Netrins are diffusible chemotropic factors for commissural axons in the embryonic spinal cord.</a> Cell. 78 (3), 425-435 (1994).</li><li>Sun, K. L. W., Correia, J. P., Kennedy, T. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Netrins:+versatile+extracellular+cues+with+diverse+functions.">Netrins: versatile extracellular cues with diverse functions.</a> Development. 138 (11), 2153-2169 (2011).</li><li>Pfenninger, K. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Plasma+membrane+expansion:+a+neuron's+Herculean+task.">Plasma membrane expansion: a neuron's Herculean task.</a> Nature Reviews Neuroscience. 10 (4), 251-261 (2009).</li><li>Winkle, C. C., McClain, L. M., Valtschanoff, J. G., Park, C. S., Maglione, C., Gupton, S. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+novel+Netrin-1-sensitive+mechanism+promotes+local+SNARE-mediated+exocytosis+during+axon+branching.">A novel Netrin-1-sensitive mechanism promotes local SNARE-mediated exocytosis during axon branching.</a> The Journal of Cell Biology. 205 (2), 217-232 (2014).</li><li>Viesselmann, C., Ballweg, J., Lumbard, D., 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=Nucleofection+and+Primary+Culture+of+Embryonic+Mouse+Hippocampal+and+Cortical+Neurons.">Nucleofection and Primary Culture of Embryonic Mouse Hippocampal and Cortical Neurons.</a> Journal of Visualized Experiments. (47), e2373 (2011).</li><li>Miesenb&#246;ck, G., De Angelis, D. A., Rothman, J. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Visualizing+secretion+and+synaptic+transmission+with+pH-sensitive+green+fluorescent+proteins.">Visualizing secretion and synaptic transmission with pH-sensitive green fluorescent proteins.</a> Nature. 394 (6689), 192-195 (1998).</li><li>Hayashi, T., 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=Synaptic+vesicle+membrane+fusion+complex:+action+of+clostridial+neurotoxins+on+assembly.">Synaptic vesicle membrane fusion complex: action of clostridial neurotoxins on assembly.</a> The EMBO Journal. 13 (21), 5051-5061 (1994).</li><li>Bradford, M. 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 border="0" cellpadding="0" cellspacing="0" width="539">
	<tbody>
		<tr height="40">
			<td height="40" style="height:40px;width:179px;">6-well tissue culture treated plates</td>
			<td style="width:99px;">Olympus Plastics</td>
			<td style="width:76px;">25-105</td>
			<td style="width:187px;"></td>
		</tr>
		<tr height="141">
			<td height="141" style="height:141px;width:179px;">glass coverslips</td>
			<td style="width:99px;">Fisher scientific</td>
			<td style="width:76px;">12-545-81</td>
			<td style="width:187px;">12CIR-1.5;must be nitric acid treated for 24 hours, rinsed in DI H2O 2X, and dried prior to use. Must be coated with 1mg/mL Poly-d-lysine and rinsed prior to plating cells</td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:179px;">Amaxa nucleofection solution</td>
			<td style="width:99px;">Lonza</td>
			<td style="width:76px;">VPG-1001</td>
			<td style="width:187px;">100ml/transfection</td>
		</tr>
		<tr height="61">
			<td height="61" style="height:61px;width:179px;">Amaxa Nucleofector/electroporator</td>
			<td style="width:99px;">Lonza</td>
			<td style="width:76px;"></td>
			<td style="width:187px;">program O-005</td>
		</tr>
		<tr height="81">
			<td height="81" style="height:81px;width:179px;">35mm Glass bottom live cell imaging dishes</td>
			<td style="width:99px;">Matek Corporation</td>
			<td style="width:76px;">p356-1.5-14-C</td>
			<td style="width:187px;">must be coated with 1mg/mL Poly-d-lysine and rinsed prior to plating cells</td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:179px;">Olympus IX81-ZDC2 inverted microscope</td>
			<td style="width:99px;">Olympus</td>
			<td style="width:76px;"></td>
			<td style="width:187px;"></td>
		</tr>
		<tr height="61">
			<td height="61" style="height:61px;width:179px;">Lambda LS xenon lamp</td>
			<td style="width:99px;">Sutter Instruments Company</td>
			<td style="width:76px;"></td>
			<td style="width:187px;"></td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:179px;">Environmental Stage top incubator</td>
			<td style="width:99px;">Tokai Hit</td>
			<td style="width:76px;"></td>
			<td style="width:187px;"></td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:179px;">100x 1.49 NA TIRF objective</td>
			<td style="width:99px;">Olympus</td>
			<td style="width:76px;"></td>
			<td style="width:187px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:179px;">Andor iXon EM-CCD</td>
			<td style="width:99px;">Andor</td>
			<td style="width:76px;"></td>
			<td style="width:187px;"></td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:179px;">Odyssey Licor Infrared Imaging System</td>
			<td style="width:99px;">LI-COR</td>
			<td style="width:76px;">Odyssey CL-X</td>
			<td style="width:187px;">Used for scanning blots</td>
		</tr>
		<tr height="81">
			<td height="81" style="height:81px;width:179px;">Image studio software suite</td>
			<td style="width:99px;">LI-COR</td>
			<td style="width:76px;"></td>
			<td style="width:187px;">Used for scanning on the Odyssey Infrared system; Image studio lite used for offline analysis of blots</td>
		</tr>
		<tr height="61">
			<td height="61" style="height:61px;width:179px;">Metamorph for Olympus</td>
			<td style="width:99px;">Molecular devices, LLC</td>
			<td style="width:76px;">version 7.7.6.0</td>
			<td style="width:187px;">Software used for all imaging and the analysis of DIC timelapse</td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:179px;">CELL TIRF control software</td>
			<td style="width:99px;">Olympus</td>
			<td style="width:76px;"></td>
			<td style="width:187px;">Software used to control lasers for TIRF imaging</td>
		</tr>
		<tr height="61">
			<td height="61" style="height:61px;width:179px;">Fiji (Image J)</td>
			<td style="width:99px;">NIH</td>
			<td style="width:76px;">ImageJ Version 1.49t</td>
			<td style="width:187px;"></td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:179px;">60x Plan Apochromat 1.4 NA objective</td>
			<td style="width:99px;">Olympus</td>
			<td style="width:76px;"></td>
			<td style="width:187px;"></td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:179px;">40x 1.4 NA Plan Apochromat objective</td>
			<td style="width:99px;">Olympus</td>
			<td style="width:76px;"></td>
			<td style="width:187px;"></td>
		</tr>
		<tr height="61">
			<td height="61" style="height:61px;width:179px;">Neurobasal media</td>
			<td style="width:99px;">GIBCO</td>
			<td style="width:76px;">21103-049</td>
			<td style="width:187px;">Base solution for both serum free and trypsin quenching media</td>
		</tr>
		<tr height="62">
			<td height="62" style="height:62px;width:179px;">Supplement B27</td>
			<td style="width:99px;">GIBCO</td>
			<td style="width:76px;">17504-044</td>
			<td style="width:187px;">500ml/50mLs Serum free media and Trypsin Quenching media</td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:179px;">L-Glutamine</td>
			<td style="width:99px;"></td>
			<td>35050-061</td>
			<td style="width:187px;">1mL/50mLs Serum free media</td>
		</tr>
		<tr height="101">
			<td height="101" style="height:101px;width:179px;">Bovine serum albumin</td>
			<td style="width:99px;">Bio Basic Incorporated</td>
			<td style="width:76px;">9048-46-8</td>
			<td style="width:187px;">10% solution in 1XPBS for blocking coverslips; 5% solution in TBS-T for blocking nitrocellulose membranes.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:179px;">10X&#160; trypsin</td>
			<td style="width:99px;">Sigma</td>
			<td style="width:76px;">59427C</td>
			<td style="width:187px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:179px;">HEPES</td>
			<td style="width:99px;">CELLGRO</td>
			<td colspan="2">25-060-Cl</td>
		</tr>
		<tr height="61">
			<td height="61" style="height:61px;width:179px;">Dulbecco&#39;s Phosphate Buffered Saline (DPBS)+ Ca + Mg</td>
			<td style="width:99px;">Corning</td>
			<td colspan="2">21-030-cm</td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:179px;">Fetal bovine serum</td>
			<td style="width:99px;">Corning/CELLGRO</td>
			<td style="width:76px;">35-010-CV</td>
			<td style="width:187px;"></td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:179px;">Hank&#39;s Balanced Salt Solution (HBSS)</td>
			<td style="width:99px;">Corning/CELLGRO</td>
			<td colspan="2">20-021-CV</td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:179px;">NaCL</td>
			<td style="width:99px;">Fisher scientific</td>
			<td style="width:76px;">BP358-10</td>
			<td style="width:187px;"></td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:179px;">EGTA</td>
			<td style="width:99px;">Fisher scientific</td>
			<td style="width:76px;">CAS67-42-5</td>
			<td style="width:187px;"></td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:179px;">MgCl2</td>
			<td style="width:99px;">Fisher scientific</td>
			<td style="width:76px;">BP214-500</td>
			<td style="width:187px;"></td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:179px;">TRIS HCl</td>
			<td style="width:99px;">Sigma</td>
			<td style="width:76px;">T5941-500</td>
			<td style="width:187px;"></td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:179px;">TRIS base</td>
			<td style="width:99px;">Fisher scientific</td>
			<td style="width:76px;">BP152-5</td>
			<td style="width:187px;"></td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:179px;">N-Propyl Gallate</td>
			<td style="width:99px;">MP Biomedicals</td>
			<td style="width:76px;">102747</td>
			<td style="width:187px;"></td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:179px;">Glycerol Photometric grade</td>
			<td style="width:99px;">Acros Organics</td>
			<td style="width:76px;">18469-5000</td>
			<td style="width:187px;"></td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:179px;">Glycerol (non optics grade)</td>
			<td style="width:99px;">Fisher scientific</td>
			<td style="width:76px;">CAS56-81-5</td>
			<td style="width:187px;"></td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:179px;">B-mercaptoethonal</td>
			<td style="width:99px;">Fisher scientific</td>
			<td style="width:76px;">BP176-100</td>
			<td style="width:187px;"></td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:179px;">SDS</td>
			<td style="width:99px;">Fisher scientific</td>
			<td style="width:76px;">BP166-500</td>
			<td style="width:187px;"></td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:179px;">Distilled Water&#160;</td>
			<td style="width:99px;">GIBCO</td>
			<td style="width:76px;">152340-147</td>
			<td style="width:187px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:179px;">Poly-D-Lysine</td>
			<td style="width:99px;">Sigma</td>
			<td style="width:76px;">p-7886</td>
			<td style="width:187px;">Dissolved in sterile water at 1mg/mL</td>
		</tr>
		<tr height="81">
			<td height="81" style="height:81px;width:179px;">Botulinum A toxin BoNTA</td>
			<td style="width:99px;">List Biological Laboratories</td>
			<td style="width:76px;">128-A</td>
			<td style="width:187px;"></td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:179px;">Rabbit polyclonal anti human VAMP2</td>
			<td style="width:99px;">Cell signaling</td>
			<td style="width:76px;">11829</td>
			<td style="width:187px;"></td>
		</tr>
		<tr height="61">
			<td height="61" style="height:61px;width:179px;">Mouse monoclonal anti rat Syntaxin1A</td>
			<td style="width:99px;">Santa Cruz Biotechnology</td>
			<td>sc-12736</td>
			<td style="width:187px;"></td>
		</tr>
		<tr height="61">
			<td height="61" style="height:61px;width:179px;">Goat polyclonal anti human SNAP-25</td>
			<td style="width:99px;">Santa Cruz Biotechnology</td>
			<td>sc-7538</td>
			<td style="width:187px;"></td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:179px;">Mouse monoclonal anti human &#946;III-tubulin&#160;</td>
			<td style="width:99px;">Covance</td>
			<td style="width:76px;">MMS-435P</td>
			<td style="width:187px;"></td>
		</tr>
		<tr height="81">
			<td height="81" style="height:81px;width:179px;">Alexa Fluor 568 and Alexa Fluor 488 phalloidin, or Alexa Fluor 647</td>
			<td style="width:99px;">Invitrogen</td>
			<td style="width:76px;"></td>
			<td style="width:187px;"></td>
		</tr>
		<tr height="121">
			<td height="121" style="height:121px;width:179px;">LI-COR IR-dye secondary antibodies</td>
			<td style="width:99px;">LI-COR</td>
			<td style="width:76px;">P/N 925-32212,P/N 925-68023, P/N 926-68022</td>
			<td style="width:187px;">800 donkey anti-mouse, 680 donkey anti rabbit, 680 donkey anti goat</td>
		</tr>
		<tr height="61">
			<td height="61" style="height:61px;width:179px;">0.2um pore size nitrocellulose membrane</td>
			<td style="width:99px;">Biorad</td>
			<td style="width:76px;">9004-70-0</td>
			<td style="width:187px;"></td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:179px;">Tween-20</td>
			<td style="width:99px;">Fisher scientific</td>
			<td style="width:76px;">BP337-500</td>
			<td style="width:187px;"></td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:179px;">Methanol</td>
			<td style="width:99px;">Fisher scientific</td>
			<td style="width:76px;">S25426A</td>
			<td style="width:187px;"></td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:179px;">Bromphenol Blue</td>
			<td style="width:99px;">Sigma</td>
			<td style="width:76px;">B5525-5G</td>
			<td style="width:187px;"></td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:179px;">Sucrose</td>
			<td style="width:99px;">Fisher scientific</td>
			<td style="width:76px;">S6-212</td>
			<td style="width:187px;"></td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:179px;">Paraformaldehyde</td>
			<td style="width:99px;">Fisher scientific</td>
			<td style="width:76px;">O-4042-500</td>
			<td style="width:187px;"></td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:179px;">Triton-X100</td>
			<td style="width:99px;">Fisher scientific</td>
			<td style="width:76px;">BP151-500</td>
			<td style="width:187px;"></td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:179px;">TEMED</td>
			<td style="width:99px;">Fisher scientific</td>
			<td style="width:76px;">BP150-20</td>
			<td style="width:187px;"></td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:179px;">40% Bis-Acrylimide</td>
			<td style="width:99px;">Fisher scientific</td>
			<td style="width:76px;">BP1408-1</td>
			<td style="width:187px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:179px;">Name</td>
			<td style="width:99px;">Company</td>
			<td style="width:76px;">Catalog Number</td>
			<td style="width:187px;">Comments</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:179px;">Alternative Validated Antibodies</td>
			<td style="width:99px;"></td>
			<td style="width:76px;"></td>
			<td style="width:187px;"></td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:179px;">Mouse Monoclonal Anti-Syntaxin HPC-1 clone</td>
			<td style="width:99px;">Sigma Aldrich</td>
			<td style="width:76px;">S0664</td>
			<td style="width:187px;"></td>
		</tr>
		<tr height="61">
			<td height="61" style="height:61px;width:179px;">&#160;Mouse Monoclonal Synaptobrevin 2 (VAMP2)</td>
			<td style="width:99px;">Synaptic Systems</td>
			<td style="width:76px;">104-211</td>
			<td style="width:187px;"></td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:179px;">Mouse Monoclonal SNAP25</td>
			<td style="width:99px;">Synaptic Systems</td>
			<td style="width:76px;">111-011</td>
			<td style="width:187px;"></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

Imaging Analysis of Neuron to Glia Interaction in Microfluidic Culture Platform (MCP)-based Neuronal Axon and Glia Co-culture System

Proper neuron to glia interaction is critical to physiological function of the central nervous system (CNS). This bidirectional communication is sophisticatedly mediated by specific signaling pathways between neuron and glia1,2 . Identification and characterization of these signaling pathways is essential to the understanding of how neuron to glia interaction shapes CNS physiology. Previously, neuron and glia mixed cultures have been widely utilized for testing and characterizing signaling pathways between neuron and glia. What we have learned from these preparations and other in vivo tools, however, has suggested that mutual signaling between neuron and glia often occurred in specific compartments within neurons (i.e., axon, dendrite, or soma)3. This makes it important to develop a new culture system that allows separation of neuronal compartments and specifically examines the interaction between glia and neuronal axons/dendrites. In addition, the conventional mixed culture system is not capable of differentiating the soluble factors and direct membrane contact signals between neuron and glia. Furthermore, the large quantity of neurons and glial cells in the conventional co-culture system lacks the resolution necessary to observe the interaction between a single axon and a glial cell.
	In this study, we describe a novel axon and glia co-culture system with the use of a microfluidic culture platform (MCP). In this co-culture system, neurons and glial cells are cultured in two separate chambers that are connected through multiple central channels. In this microfluidic culture platform, only neuronal processes (especially axons) can enter the glial side through the central channels. In combination with powerful fluorescent protein labeling, this system allows direct examination of signaling pathways between axonal/dendritic and glial interactions, such as axon-mediated transcriptional regulation in glia, glia-mediated receptor trafficking in neuronal terminals, and glia-mediated axon growth. The narrow diameter of the chamber also significantly prohibits the flow of the neuron-enriched medium into the glial chamber, facilitating probing of the direct membrane-protein interaction between axons/dendrites and glial surfaces.

Imaging Analysis of Neuron to Glia Interaction in Microfluidic Culture Platform MCP-based Neuronal Axon and Glia Co-culture System

This study describes the procedures of setting up a novel neuronal axon and (astro)glia co-culture platform. In this co-culture system, manipulation of direct interaction between a single axon (and single glial cell) becomes feasible, allowing mechanistic analysis of the mutual neuron to glial signaling.

神经元对神经胶质细胞在微流控文化互动平台（MCP）的神经元轴突和神经胶质细胞共培养系统的影像学分析

Proper  neuron to glia interaction is critical to physiological function of the central  nervous system (CNS). This bidirectional communication is ...

Imaging pHluorin-tagged Receptor Insertion to the Plasma Membrane in Primary Cultured Mouse Neurons

A better understanding of the mechanisms governing receptor trafficking between the plasma membrane (PM) and intracellular compartments requires an experimental approach with excellent spatial and temporal resolutions. Moreover, such an approach must also have the ability to distinguish receptors localized on the PM from those in intracellular compartments. Most importantly, detecting receptors in a single vesicle requires outstanding detection sensitivity, since each vesicle carries only a small number of receptors. Standard approaches for examining receptor trafficking include surface biotinylation followed by biochemical detection, which lacks both the necessary spatial and temporal resolutions; and fluorescence microscopy examination of immunolabeled surface receptors, which requires chemical fixation of cells and therefore lacks sufficient temporal resolution1-6 . To overcome these limitations, we and others have developed and employed a new strategy that enables visualization of the dynamic insertion of receptors into the PM with excellent spatial and temporal resolutions 7-17 . The approach includes tagging of a pH-sensitive GFP, the superecliptic pHluorin 18, to the N-terminal extracellular domain of the receptors. Superecliptic pHluorin has the unique property of being fluorescent at neutral pH and non-fluorescent at acidic pH (pH &lt; 6.0). Therefore, the tagged receptors are non-fluorescent when within the acidic lumen of intracellular trafficking vesicles or endosomal compartments, and they become readily visualized only when exposed to the extracellular neutral pH environment, on the outer surface of the PM. Our strategy consequently allows us to distinguish PM surface receptors from those within intracellular trafficking vesicles. To attain sufficient spatial and temporal resolutions, as well as the sensitivity required to study dynamic trafficking of receptors, we employed total internal reflection fluorescent microscopy (TIRFM), which enabled us to achieve the optimal spatial resolution of optical imaging (~170 nm), the temporal resolution of video-rate microscopy (30 frames/sec), and the sensitivity to detect fluorescence of a single GFP molecule. By imaging pHluorin-tagged receptors under TIRFM, we were able to directly visualize individual receptor insertion events into the PM in cultured neurons. This imaging approach can potentially be applied to any membrane protein with an extracellular domain that could be labeled with superecliptic pHluorin, and will allow dissection of the key detailed mechanisms governing insertion of different membrane proteins (receptors, ion channels, transporters, etc.) to the PM.

By tagging the extracellular domains of membrane receptors with superecliptic pHluorin, and by imaging these fusion receptors in cultured mouse neurons, we can directly visualize individual vesicular insertion events of the receptors to the plasma membrane. This technique will be instrumental in elucidating the molecular mechanisms governing receptor insertion to the plasma membrane.

成像pHluorin标记插入到质膜受体原代培养的小鼠神经元

A better understanding of the mechanisms governing receptor trafficking between the plasma membrane (PM) and intracellular compartments requires an ...

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 ...

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 ...

Preparation of Synaptic Plasma Membrane and Postsynaptic Density Proteins Using a Discontinuous Sucrose Gradient

Neuronal subcellular fractionation techniques allow the quantification of proteins that are trafficked to and from the synapse. As originally described in the late 1960&#8217;s, proteins associated with the synaptic plasma membrane can be isolated by ultracentrifugation on a sucrose density gradient. Once synaptic membranes are isolated, the macromolecular complex known as the post-synaptic density can be subsequently isolated due to its detergent insolubility. The techniques used to isolate synaptic plasma membranes and post-synaptic density proteins remain essentially the same after 40 years, and are widely used in current neuroscience research. This article details the fractionation of proteins associated with the synaptic plasma membrane and post-synaptic density using a discontinuous sucrose gradient. Resulting protein preparations are suitable for western blotting or 2D DIGE analysis.

This article details the enrichment of proteins associated with the synaptic plasma membrane by ultracentrifugation on a discontinuous sucrose gradient. The subsequent preparation of post-synaptic density proteins is also described. Protein preparations are suitable for western blotting or 2D DIGE analysis.

突触质膜和突触后密度蛋白采用不连续蔗糖梯度准备

Neuronal subcellular fractionation techniques allow the quantification of proteins that are trafficked to and from the synapse. As originally described in ...

Surgical Method for Virally Mediated Gene Delivery to the Mouse Inner Ear through the Round Window Membrane

Gene therapy, used to achieve functional recovery from sensorineural deafness, promises to grant better understanding of the underlying molecular and genetic mechanisms that contribute to hearing loss. Introduction of vectors into the inner ear must be done in a way that widely distributes the agent throughout the cochlea while minimizing injury to the existing structures. This manuscript describes a post-auricular surgical approach that can be used for mouse cochlear therapy using molecular, pharmacologic, and viral delivery to mice postnatal day 10 and older via the round window membrane (RWM). This surgical approach enables rapid and direct delivery into the scala tympani while minimizing blood loss and avoiding animal mortality. This technique involves negligible or no damage to essential structures of the inner and middle ear as well as neck muscles while wholly preserving hearing. To demonstrate the efficacy of this surgical technique, the vesicular glutamate transporter 3 knockout (VGLUT3 KO) mice will be used as an example of a mouse model of congenital deafness that recovers hearing after delivery of VGLUT3 to the inner ear using an adeno-associated virus (AAV-1).

The described post-auricular surgical approach allows rapid and direct delivery into the mouse cochlear scala tympani while minimizing blood loss and animal mortality. This method can be used for cochlear therapy using molecular, pharmacologic and viral delivery to postnatal mice through the round window membrane.

通过圆窗膜病毒介导的基因传递给鼠标内耳手术方法

Gene therapy, used to achieve functional recovery from sensorineural deafness, promises to grant better understanding of the underlying molecular and ...

Live Imaging of Nicotine Induced Calcium Signaling and Neurotransmitter Release Along Ventral Hippocampal Axons

Sustained enhancement of axonal signaling and increased neurotransmitter release by the activation of pre-synaptic nicotinic acetylcholine receptors (nAChRs) is an important mechanism for neuromodulation by acetylcholine (ACh). The difficulty with access to probing the signaling mechanisms within intact axons and at nerve terminals both in vitro and in vivo has limited progress in the study of the pre-synaptic components of synaptic plasticity. Here we introduce a gene-chimeric preparation of ventral hippocampal (vHipp)&#8211;accumbens (nAcc) circuit in vitro that allows direct live imaging to analyze both the pre- and post-synaptic components of transmission while selectively varying the genetic profile of the pre- vs post-synaptic neurons. We demonstrate that projections from vHipp microslices, as pre-synaptic axonal input, form multiple, reliable glutamatergic synapses with post-synaptic targets, the dispersed neurons from nAcc. The pre-synaptic localization of various subtypes of nAChRs are detected and the pre-synaptic nicotinic signaling mediated synaptic transmission are monitored by concurrent electrophysiological recording and live cell imaging. This preparation also provides an informative approach to study the pre- and post-synaptic mechanisms of glutamatergic synaptic plasticity in vitro.

We developed a gene-chimeric preparation of ventral hippocampal &#8211; accumbens circuit in vitro that allows direct live imaging to analyze presynaptic mechanisms of nicotinic acetylcholine receptors (nAChRs) mediated synaptic transmission. This preparation also provides an informative approach to study the pre- and post-synaptic mechanisms of synaptic plasticity.

尼古丁诱导的钙信号和神经递质的释放了沿着腹侧海马轴突实时成像

Sustained enhancement of axonal signaling and increased neurotransmitter release by the activation of pre-synaptic nicotinic acetylcholine receptors ...

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) ...

Morphological and Functional Evaluation of Axons and their Synapses during Axon Death in Drosophila melanogaster

Axon degeneration is a shared feature in neurodegenerative disease and when nervous systems are challenged by mechanical or chemical forces. However, our understanding of the molecular mechanisms underlying axon degeneration remains limited. Injury-induced axon degeneration serves as a simple model to study how severed axons execute their own disassembly (axon death). Over recent years, an evolutionarily conserved axon death signaling cascade has been identified from flies to mammals, which is required for the separated axon to degenerate after injury. Conversely, attenuated axon death signaling results in morphological and functional preservation of severed axons and their synapses. Here, we present three simple and recently developed protocols that allow for the observation of axonal morphology, or axonal and synaptic function of severed axons that have been cut-off from the neuronal cell body, in the fruit fly Drosophila. Morphology can be observed in the wing, where a partial injury results in axon death side-by-side of uninjured control axons within the same nerve bundle. Alternatively, axonal morphology can also be observed in the brain, where the whole nerve bundle undergoes axon death triggered by antennal ablation. Functional preservation of severed axons and their synapses can be assessed by a simple optogenetic approach coupled with a post-synaptic grooming behavior. We present examples using a highwire loss-of-function mutation and by over-expressing dnmnat, both capable of delaying axon death for weeks to months. Importantly, these protocols can be used beyond injury; they facilitate the characterization of neuronal maintenance factors, axonal transport, and axonal mitochondria.

Morphological and Functional Evaluation of Axons and their Synapses during Axon Death in Drosophila melanogaster

Here, we provide protocols to perform three simple injury-induced axon degeneration (axon death) assays in Drosophila melanogaster to evaluate the morphological and functional preservation of severed axons and their synapses.

在果蝇黑色无gaster的Axon死亡期间，对Axon及其突触的形态和功能评估

Axon degeneration is a shared feature in neurodegenerative disease and when nervous systems are challenged by mechanical or chemical forces. However, our ...