笔者想承认UNIVERSITA德利阿布鲁Studi住宅米兰向埃利安娜迪卡伊拉诺（博士后奖学金）和斯特凡莫雷蒂（博士奖学金）的支持。这项工作是由大学研究计划PUR到CP支持我们要感谢教授杰里米·M.亨利，生物化学学院，布里斯托，英国的大学，为pHluorin建设和Dotti弗朗西斯博士在数据分析的援助，和Silvia Marsicano技术援助。

1.细胞培养和转染<ol><li> SH-SY5Y细胞培养 注意:使用人神经母细胞瘤SH-SY5Y（ATCC＃CRL-2266）15实验已经完成。 SH-SY5Y细胞成长为浮动集群和贴壁细胞的混合物。按照报告的协议（细胞密度，分光比等 ），以有生长牢固地附着在玻璃盖，其用于TIRFM关键细胞说明书。 <ol><li>在开始之前，层流生物安全柜下，使无菌磷酸盐缓冲生理盐水（PBS）和培养基的时机音量。 <ol><li>制成50毫升的PBS以150 mM氯化钠，24 mM磷酸盐缓冲液，pH 7.4中的浓度。过滤该溶液。 </li><li>使从Dulbecco改进的Eagle具有高葡萄糖，10％热灭活的小牛血清（FBS），青霉素（100单位/毫升），链霉素（100微克/毫升），L-谷氨酰胺（培养基（DMEM）50 ml的细胞培养基2毫摩尔），和丙酮酸钠（1毫米）。过滤该溶液。 </li></ol></li><li>除去完全生长培养基，并用3毫升的PBS洗涤细胞。 </li><li>孵育细胞用2ml的0.05％胰蛋白酶-乙二胺四乙酸（EDTA）（对于6cm培养皿中）中5分钟，在37℃，5％的CO 2，并使用移液管分离的细胞。 </li><li>灭活胰蛋白酶通过加入2ml DMEM中，并通过离心收集细胞，在300×g离心5分钟。 </li><li>除去上清液，加入1 ml DMEM中，以沉淀和吸取的溶液上下充分分散细胞分化成单细胞悬浮液。 </li><li>他们分成1:4在一个新的直径6厘米的Petri装有3毫升完全培养基菜。在一个5％CO 2培养箱保持培养中的细胞在直径6cm的培养皿，在37℃下。子培养，每周一次，或当它们已经覆盖80 - 90％的表面区域。 </li></ol></li><li> SH-SY5Y细胞接种于摄像 <ol><li>对于TIRFM实验，板细胞在玻璃盖。采用玻璃覆盖与0.17±0.005毫米的厚度和1.5255±0.00015折射率。开始前，准备盖玻片如下: <ol><li>干净的玻璃覆盖90％的乙醇，O / N。 </li><li>在蒸馏水（蒸馏水三个转变）彻底冲洗。干燥时的玻璃覆盖在干燥炉中。 </li><li>地方覆盖在玻璃培养皿和消毒在预热烘箱中在200℃下进行3小时。 </li></ol></li><li>在转染前一天，将每个盖玻片在3.5公分培养皿，加入1 ml培养基并在5％CO 2的培养箱中孵育在37℃。 </li><li>如在步骤1.1.3描述Trypsinize细胞 - 1.1.5，暂停将细胞沉淀在1ml完全培养基和计数。计算细胞悬浮液的正确体积添加至每个培养皿中，得到3×10 5细胞/孔。此密度是必需的以获得最佳的细胞生长和有效转染。孵育在37℃下在一个5％CO 2培养箱O / N。 </li></ol></li><li> SH-SY5Ytransfection由聚乙烯亚胺（PEI） 注意:为了形象化突触小泡动力学，已使用含突触-pHluorin pCB6载体。所述突触-pHluorin已在绿色荧光蛋白（GFP）16和小泡膜蛋白突触2.构建体已被广泛用于研究神经元9内突触小泡特性的PH敏感的变体的框内融合生成由。 <ol><li>在开始转染前，打10毫升以下的解决方案。保持解决方案，最大为1个月。 <ol><li>做一个150毫米的NaCl溶液。调节至pH 5.5 0.01N HCl中。 </li><li>使在10％的聚乙烯亚胺（PEI; 25kDa的线性）一个PEI溶液在150mM的NaCl溶液。溶液的pH升高到8.8。调节pH至7.8用0.01N HCl中。 </li></ol></li><li>铺板后24小时，去除培养基，并用1.5ml的刷新完全培养基。保持所述细胞在37℃，在5％CO 2的培养箱中培养。 </li><li>下的层流生物安全柜，在1.5ml微量离心管中，加入3微克质粒DNA到25微升150 mM氯化钠溶液和100微升每3.5厘米培养皿的PEI溶液。 </li><li>涡旋10秒，然后孵育该DNA / PEI混合物进行30分钟，在RT。 </li><li>小心将DNA / PEI混合物添加到含有盖玻片与细胞培养皿并轻轻摇动，以平均分配，试剂中的培养皿。 </li><li> 4小时后，改变培养基，并在5％CO 2的培养箱中孵育细胞O / N在37℃。转染后48小时 - 进行成像试验24。 </li></ol></li></ol>通过全内反射荧光显微镜2.细胞成像（TIRFM） <ol><li> 成像设置 <ol><li>与图1中描述的设置执行的TIRF成像，它包括一个机动倒置显微镜（ 图1，小图a），激光源（ 图1，小图B）和TIRF滑块（ 图1，小图C）。通过高数值孔径（NA 1.45阿尔法普兰-Fluar）100X油，浸泡的目的达到TIRFM照明。 </li><li>对于TIRFM照明，采用多线（458/488/514纳米）100毫瓦氩离子激光。使用单模式光纤，引入线偏振激光光进入光束路径，经由TIRF滑块。插入的TIRF滑块成反射光束路径的发光场光阑平面。 <ol><li>对宽视场照明，显微镜连接到常规汞短弧灯HBO白光。在滑块的偏振波保持双棱镜可以确保的TIRF照明和白光的同时的联合。 </li></ol></li><li>过滤该激光光用激发滤波器（频带宽度十分之四百八十八纳米）安装在滤光轮中，被引入激光路径。聘用高速，软件控制，快门，以便快速控制激光照射。对于pHluorin分析，安装一个带通五十分之五百二十五nm发射过滤器。在快速冷却的CCD摄像头，图像PROPLUS软件捕捉数字影像（512×512像素）。 </li></ol></li><li> 实现的TIRF照明（图2） <ol><li>打开激光，计算机，照相机，滤光轮，并且快门控制器;然后，等待20分钟开始实验的激光器需预热和稳定之前。 </li><li>影像之前，请通过以下方法解决的时机音量。 <ol><li>制成50毫升克雷布斯（KRH）溶液在125 mM氯化钠，5mM的氯化钾，1.2mM的MgSO 4干燥，1.2毫KH 2 PO 4，25mM的4-（2-羟乙基）哌嗪-1-乙磺酸（HEPES）（缓冲至pH为7.4），2mM的氯化钙 ，以及6毫米的葡萄糖。 </li><li>使溶于10ml氯化钾-KRH溶液（pH 7.4）中，在80毫米的NaCl，50mM的氯化钾，1.2mM的MgSO 4干燥，1.2毫磷酸二氢钾，25毫米的HEPES（缓冲到pH 7.4），2mM的氯化钙 ，以及6毫米的葡萄糖。 </li></ol></li><li>与转染细胞中取出玻璃盖，并在适当的成像室插入。装配该室，并在玻璃的中心加入500微升的KRH溶液。 </li><li>加油过的目标。将成像室在显微镜的阶段，并在该玻璃盖玻片定位目标。定位安全盖在样本。 </li><li>在落射荧光模式，专注于盖玻片（上面），然后选择转染细胞放置在室内中心。选择单元格，其荧光信号可以用低于80​​毫秒的曝光时间可以清楚地记录下来。 </li><li>在软件控制下，切换到TIRF照明在实时模式。 </li><li>要设置的TIRF配置，检查浮现出的目标，在样品上覆盖（ 图2B）的光束的位置。当光束被定位在吨的中心他物镜（ 图2A，左），一个光点是在TIRF样品盖的中心可见（ 图2B，左）和该信元被成像在落射荧光模式（几个聚焦平面，高背景荧光; 图2C，左） 。 </li><li>使用上的TIRF滑块（ 图1C）的角度调整螺钉;达到临界角，将聚焦光点在Y方向（ 图2B中，中心向前或向后）。当光束以一个角度大于临界角大（ 图2A，右）会聚在样品平面，光点消失，一个直的，薄的，聚焦线是显而易见的，在样品盖的中部（ 图2B，右）。 </li><li>要微调的TIRF角度使用细胞样本（ 图2C）。观看的视频的荧光图像，在这个阶段中，落射荧光状图象仍是可见的。轻轻地，将螺丝，直到TIRF CONDITION实现:仅一个光电池的平面是在聚焦（ 即，在与盖滑动接触质膜），这导致在一个平面图像具有高对比度（ 图2C，右）。 </li></ol></li><li> 样品成像 <ol><li>设置单声道的时间推移的实验。为了最大限度地减少漂白，使用低曝光时间和高增益捕获图像。适当的曝光时间之间的40 - 80毫秒。在采集的图像1 - 2 Hz的采样频率。囊泡动力学可能是在更高的频率（10赫兹）更好地理解采样。观察常规时间一般为2分钟的。 </li><li>加入500μlKRH解决方案和记录细胞的TIRFM模式。这是静止状态。节省时间序列图像。 </li><li>着眼于静息的相同的条件（激光功率，时间曝光，帧号）根据相同的小区和记录。经过五年的框架，加入500μl的氯化钾，KRH的解决方案，并保持在氯化钾室。这是受激状态;节省时间序列图像。 </li></ol></li></ol> 3.图像分析和数据处理注意:要分析的图像，宏已经开发了在实验室中，基于该图像分析软件的现有功能;类似宏可在网上（见表材料和设备提供URL）。 <ol><li> 荧光强度量化 <ol><li>使用"序列的荧光强度"宏观为荧光强度定量中的图像的感兴趣区域（ROI）的区域中，在电影的过程。 </li><li>打开时间序列图像。转到宏观菜单，然后选择"序列荧光强度"。在"分析"窗口出现"选择ROI"。 </li><li>选择的选择的工具之一，在菜单中创建的投资回报率。放置3感兴趣区在细胞膜的区域无斑点（背景ROI）。聘请THIs"背景的ROI"来评估漂白和设定的阈值的融合事件的分析（ 图3A）。 </li></ol></li><li> 漂白校正和阈值判定（图3B） <ol><li>评估漂白，打开荧光强度行"背景的投资回报"，（ 图3BA）。正常化在每个帧的初始强度值（F 0）（F / F 0）（ 图3BB）的荧光强度值。平均化的值。 </li><li>突出的平均数据，并使用图表菜单选项的线图。 </li><li>从数据分析菜单中选择"趋势线"，打开图分析对话框。选择回归的类型。设置"指数"的回归。然后选择"在图表上显示方程"。在图形窗口中，指数方程出现的参数值将被自动分配，（ 图3BC </strong&gt;的）。 </li><li>指数修正适用于每个帧如下的强度值: FN（修正）= FN / EXP（N *一） FN =在帧n实验测量荧光强度; N =帧数;一个=漂白因子（恒定表达强度的损失是由于光漂白速率; &#160; 图3BD）。 </li><li>以设定阈值，打开一个规格化和校正"背景的ROI"，计算平均荧光信号，其标准偏差（SD）。平均值加上3 SD代表的阈值（ 图3BE）。使用此阈值进行数据分析。 </li></ol></li><li> 使用半自动程序的融合活动选择 <ol><li>打开用图像分析软件的时间序列图像。应用高斯滤波器活动图像序列。 </li><li>使用"计算对象"工具或宏允许选择分析图像的一个对象，其像素具有一个限定的范围内的平均荧光强度。设置强度范围手动，使用阈值函数（去酒吧的菜单，设置测量→门槛，突出感兴趣的区域）。适当的阈值是在本地的荧光背景信号的30％。 </li><li>应用宏"过滤器对象"选择符合以下条件的唯一对象: <ol><li>应用范围选项（最小和最大含）方面。一方面报告长轴和等同的对象的椭圆的短轴之间的比率。看点永远是≥1。适当的值是分= 1，最大值= 3。 </li><li>适用范围的直径。 直径报道在2度间隔连接两个轮廓点并穿过对象的质心测量的直径的平均长度。设置在像素的范围内（或者在微米，如果使用校准系统）。 </li><li>定义prelimin最佳范围元实验:手动选择感兴趣的点，然后使用积曲线函数测量它们的直径。 </li></ol></li><li>选择"显示对象":选择的对象会出现叠加到TIRFM图像（ 图4B）。 </li><li>包括在分析中只有那些表现出短（一到三个帧）点的瞬态荧光强度增加，紧接着的信号（瞬时点）的显着损失。采用圆形选择大约一个光斑直径径向创建一个ROI围绕选定的囊泡/点（实验的ROI）。手动执行此步骤。 </li><li>与感兴趣区选择，计算在电影的过程中的每个ROI的平均荧光强度。 </li></ol></li><li> 数据分析（图3C-D）的 <ol><li>导出的时间过程中的每一个"实验的ROI"到电子表格测得的荧光变化; （ 图3DA）。正常化在每一帧的初始荧光强度（F / F 0），（ 图3分贝 ）的强度值。 </li><li>所报告的步骤3.2.4（ 图3Dc的 ）中的每个帧的指数应用校正强度值。 </li><li>计算的融合事件的总数（峰数），每个聚变发生的时间（峰宽）和荧光峰（峰高和AUC）的振幅申请使用的电子表格或数学软件包逻辑功能。使用逻辑公式聚变事件分析的一个例子报道于图的3DD和3DE。 </li><li>假设荧光强度超过阈值（平均背景荧光强度±3SD），为小泡的融合到质膜的增加，将所得的峰为融合事件。 </li><li>计算峰宽作为最后的和每个峰的第一x值之间的差异。这个值乘以1 /（采样频率）。考虑这个值š囊泡融合和粘附在质膜囊泡重新酸化和回收（ 图的3DD）之前的时间。 </li><li>计算的全细胞的AUC作为值超过阈值的总和。考虑这个值作为在记录时间由于自发（休息）净荧光改变或诱发（刺激）突触活动。 </li><li>计算峰高为每个峰和阈的最大y值之间的差。考虑这个值作为指示融合型（单与同时/顺序融合或瞬态主场迎战全融合）的。 </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=Balice-Gordon+R.J.+Heterogeneity+in+Synaptic+Vesicle+Release+at+Neuromuscular+Synapses+of+Mice+Expressing+SynaptopHluorin.">Balice-Gordon R.J. Heterogeneity in Synaptic Vesicle Release at Neuromuscular Synapses of Mice Expressing SynaptopHluorin.</a> J. Neurosci. 28 (1), 325-335 (2008).</li><li>Tsuboi, T., Rutter, G. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multiple+forms+of+&#34;kiss-and-run&#34;+exocytosis+revealed+by+evanescent+wave+microscopy.">Multiple forms of &#34;kiss-and-run&#34; exocytosis revealed by evanescent wave microscopy.</a> Curr Biol. 13, 563-567 (2003).</li><li>Miloso, M., 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=Retinoic+Acid-Induced+Neuritogenesis+of+Human+Neuroblastoma+SH-SY5Y+Cells+Is+ERK+Independent+and+PKC+Dependent.">Retinoic Acid-Induced Neuritogenesis of Human Neuroblastoma SH-SY5Y Cells Is ERK Independent and PKC Dependent.</a> J. Neurosci. Res. 75, 241-252 (2004).</li><li>Jaskolski, F., Mayo-Martin, B., Jane, D., Henley, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dynamin-dependent+Membrane+Drift+Recruits+AMPA+Receptors+to+Dendritic+Spines.">Dynamin-dependent Membrane Drift Recruits AMPA Receptors to Dendritic Spines.</a> J Biol Chem. 284 (18), 12491-12503 (2009).</li><li>Treccani, G., 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=Stress+and+corticosterone+rapidly+increase+the+readily+releasable+pool+of+glutamate+vesicles+in+synaptic+terminals+of+prefrontal+and+frontal+cortex.">Stress and corticosterone rapidly increase the readily releasable pool of glutamate vesicles in synaptic terminals of prefrontal and frontal cortex.</a> Mol Psychiatry. 19 (4), 433-443 (1038).</li><li>D'Amico, A., 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=The+surface+density+of+the+glutamate+transporter+EAAC1+is+controlled+by+interactions+with+PDZK1+and+AP2+adaptor+complexes.">The surface density of the glutamate transporter EAAC1 is controlled by interactions with PDZK1 and AP2 adaptor complexes.</a> Traffic. 11 (11), 1455-1470 (2010).</li><li>Perego, C., Di Cairano, E. S., Ballabio, M., Magnaghi, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neurosteroid+allopregnanolone+regulates+EAAC1-mediated+glutamate+uptake+and+triggers+actin+changes+in+Schwann+cells.">Neurosteroid allopregnanolone regulates EAAC1-mediated glutamate uptake and triggers actin changes in Schwann cells.</a> J Cell Physiol. 227 (4), 1740-1751 (2012).</li><li>Bergeron, A., Pucci, L., Bezzi, P., Regazzi, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Analysis+of+synaptic-like+microvesicles+exocytosis+of+B-cells+using+a+live+imaging+technique.">Analysis of synaptic-like microvesicles exocytosis of B-cells using a live imaging technique.</a> PlosOne. 9, e87758 (2014).</li><li>Encinas, M., 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=Sequential+treatment+of+SH-Sy5Y+cells+with+retinoic+acid+and+brain-derived+neurotrophic+factor+gives+rise+to+fully+differentiated,+neutrophic+factor-dependent,+human+neuron-like+cells.">Sequential treatment of SH-Sy5Y cells with retinoic acid and brain-derived neurotrophic factor gives rise to fully differentiated, neutrophic factor-dependent, human neuron-like cells.</a> J. Neurochem. 75 (3), 991-1003 (2000).</li><li>Kume, 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=Dibutyryl+cyclic+AMP+induces+differentiation+of+human+neuroblastoma+SH-SY5Y+cells+into+a+noradrenergic+phenotype.">Dibutyryl cyclic AMP induces differentiation of human neuroblastoma SH-SY5Y cells into a noradrenergic phenotype.</a> Neurosci Lett. 443 (3), 199-203 (2008).</li><li>Diaspro, A., Chirico, G., Usai, C., Ramoino, P., Dobrucki, J., Pawley, J. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Photobleaching.">Photobleaching.</a> Handbook of Biological Confocal Microscopy. , 690-699 (2006).</li><li>Rossano, A. J., Chouhan, A. K., Macleod, G. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genetically+encoded+pH-indicators+reveal+activity-dependent+cytosolic+acidification+of+Drosophila+motor+nerve+termini+in+vivo.">Genetically encoded pH-indicators reveal activity-dependent cytosolic acidification of Drosophila motor nerve termini in vivo.</a> J Physiol. 591 (7), 1691-1706 (2013).</li></ol>

The authors declare that they have no competing financial interests.

本文提出了一种协议，以图像和分析囊泡动力学分泌细胞，采用荧光基因编码向量和TIRFM。成功的成像通过TIRFM关键要素是细胞模型和细胞转染与囊泡释放和回收的基因编码的光学指标的选择。 TIRFM非常适合于细胞生长附着于玻璃盖和足够平坦以使膜融合事件的稳定可视化。囊泡最好应分散在细胞使它们的贩卖，融合和胞吞作用可以被成像和量化在单囊泡水平。不幸的是，神?...

神经元之间的化学突触传递是通信中的神经系统的主要机制。它依赖于神经递质通过囊泡融合和检索的动态循环的释放在突触前部位。许多参与囊泡动力学的蛋白质已被鉴定;然而，它们对这一现象的具体贡献仍有待阐明1。 我们的理解是由以下事实最广泛使用的测定法外/胞吞作用并不总是最合适的部分的限制。相关囊泡融合一些研究和动态依靠电生理技术。这种技术提供了一个最佳的时间分辨率和优良调查囊泡至质膜的初始熔化，但无法检测到许多支持突触功能的基本分子事件。电子显微镜，在另一侧，提供了最好的morphologica每个奇异步骤，但是事件的动态方面的升说明不能被捕获，因为样品必须固定以便进行分析。 的新的光记录技术2,3，结合在荧光分子探针发展4-6进步的出现，使得在活细胞中胞吐过程的可视化，从而提供约突触的结构和功能的信息的新的水平。 最初的研究开发活动相关的苯乙烯染料（FM1 - 43和相关有机染料）7,8。国家的最先进的成像技术采用了绿色荧光蛋白（GFP）（pHluorin）拴管腔小泡蛋白9的pH敏感的变体。这些探针通常被关闭，因为低pH值管腔的囊泡当存在时。融合与质膜后，囊泡内部暴露于中性细胞外空间中，pH<html>突然增加，缓解pHluorin的质子依赖淬火和荧光信号迅速出现。如pHluorin的变化的速度比的融合事件，通过监测荧光的增加，囊泡融合与膜可以测量和分析。因为表面pHluorin标签的分子被内吞，所述荧光信号随后返回到基础水平，因此也可以使用相同的构建体并监控囊泡再循环9。 而囊泡标记的pH传感器可以确保只有那些囊泡真正融合与质膜的可视化，成像在高空间和时间分辨率是必需的，以详细描述了所涉及的外/内吞过程的步骤。的光学技术，它提供了必要的时空分辨率为全内反射荧光显微镜（TIRFM），荧光显微镜10的应用程序。 "&gt;全内反射发生在玻璃盖玻片和样品。当光路到达玻璃盖玻片用的入射角比临界角大的界面，激发光不被传递到样品，但完全反射回去。在这些条件下，一个瞬逝光波的形式在界面处，并传播与较少的光密度（样品）的平台。由于渐逝场的强度呈指数衰减的距离从接口（具有穿透深度约100nm）只在最接近于盖滑移的荧光团可被激发而更远离边界都没有。在转染的细胞用GFP-构建体，该深度对应于表示在质膜上的蛋白质或在囊泡结构接近它。如在细胞内部的荧光团可以不被激励时，所述背景荧光最小化，并具有非常高的信号/背景的大鼠的图像IO形成11。 有几个特点使选择的TIRFM技术监测囊泡动态。完美的对比度和高的信号 - 噪声比允许非常低的信号从单个囊泡导出的检测。在各帧的基于芯片的图像采集提供必要检测高动态过程的时间分辨率。最后，细胞的最小曝光的样品中任何其他飞机大大地减少光毒性，并能持久延时录制12。 数据分析仍然这种技术的最具挑战性的和关键的方面。监视囊泡融合的最简单的方法是测量报告荧光蛋白在细胞表面的积累，随着时间的推移13。作为融合的增加，净荧光信号也增加。然而，这种方法可能会低估的过程中，特别是在大的细胞和在静止的条件下，因为内吞作用和漂白过程偏移由于囊泡的胞吐作用的增加的荧光强度。另一种方法是按照每个单个融合事件14。这后一种方法是很敏感的，并且可以揭示关于融合的机制的重要细节。然而，它要求手动选择单一活动，因为完全自动化程序遵循囊泡和登记他们的荧光信号的波动并不总是可用。囊泡动力学的观察要求采样单元在高频率。这会产生大量的数据，可以几乎不进行手动分析。 本文的建议是优化TIRFM成像技术用于监测基底和刺激的神经递质释放，在SH-5YSY神经母细胞瘤细胞系，并描述，一步一步，在实验室中开发的程序来分析数据，既在全细胞和单囊泡水平。

<table><tbody><tr><td colspan="4">Equipment</td></tr><tr><td>Axio Observer Z1</td><td><a href="http://www.zeiss.com/microscopy/en_de/products/light-microscopes/axio-observer-for-biology.html#introduction" target="_blank"><img src="/img/new_window.png" /> Zeiss</a></td><td>491912-9850-000</td><td>inverted microscope</td></tr><tr><td>Multiline Argon Laser Lasos 77</td><td><a href="http://www.lasos.com/products/argon-ion-laser" target="_blank"><img src="/img/new_window.png" /> Lasos</a></td><td>00000-1312-752</td><td>multi-line (458/488/514 nm), 100&amp;nbsp;mW argon-ion laser</td></tr><tr><td>Laser TIRF slider</td><td><a href="http://www.zeiss.com/microscopy/en_de/products/imaging-systems/single-molecular-imaging-laser-tirf-3.html" target="_blank"><img src="/img/new_window.png" /> Zeiss</a></td><td>423681-9901-000</td><td></td></tr><tr><td>100X Objective</td><td><a href="https://www.micro-shop.zeiss.com/?l=en&amp;amp;p=us&amp;amp;f=o&amp;amp;a=v&amp;amp;m=a&amp;amp;id=421190-9900-000&amp;amp;ss=1" target="_blank"><img src="/img/new_window.png" /> Zeiss</a></td><td>421190-9900-000</td><td>Oil, NA 1.45 Alpha-Plan</td></tr><tr><td>CCD Camera RetigaSRV Fast 1394</td><td><a href="http://www.qimaging.com/products/datasheets/Retiga-SRV.pdf" target="_blank"><img src="/img/new_window.png" /> QImaging</a></td><td></td><td></td></tr><tr><td>LAMBDA 10-3 optical filter changer with SmartShutter</td><td><a href="http://www.sutter.com/IMAGING/lambda103.html" target="_blank"><img src="/img/new_window.png" /> Sutter Instrument Company</a></td><td></td><td></td></tr><tr><td colspan="4">Software</td></tr><tr><td>Image ProPlus 6.3 Software</td><td><a href="http://www.mediacy.com/index.aspx?page=IPP" target="_blank"><img src="/img/new_window.png" /> Media Cybernetics</a></td><td></td><td>spot selection, ROI selection, fluorescence intensity determination</td></tr><tr><td>Excel</td><td><a href="http://office.microsoft.com/it-it/excel/" target="_blank"><img src="/img/new_window.png" /> Microsoft</a></td><td></td><td>photobleaching correction, whole-cell and single-vesicle analyses</td></tr><tr><td>GraphPad Prism 4.00</td><td><a href="http://www.graphpad.com/scientific-software/prism/" target="_blank"><img src="/img/new_window.png" /> GraphPad Software, Inc.</a></td><td></td><td>statistical analysis</td></tr></tbody></table>

tirfm and ph-sensitive gfp-probes to evaluate neurotransmitter vesicle dynamics in sh-sy5y neuroblastoma cells: cell imaging and data analysis

突触囊泡融合，通过和检索的动态循环释放神经递质的化学突触。 实时监测突触活动和解剖的不同步骤外-内吞作用的单囊泡水平对于理解在健康和疾病突触功能至关重要。 遗传编码的PH敏感的探针直接靶向突触小泡和全内反射荧光显微镜（TIRFM）提供必要遵循囊泡动力学的时空分辨率。通过全内反射所产生的渐逝场只能激发置于一薄层（&lt;150纳米），其上的细胞粘附在玻璃盖之上的荧光团，正是外 - 内吞作用的过程发生。所得高对比度的图像非常适合囊泡跟踪和融合事件的定量分析。 在这个协议中，SH-SY5Y人N-euroblastoma细胞被提出作为一种有价值的模型为研究，因为他们的平坦面神经递质释放在通过TIRFM单囊泡水平，并分散囊泡的存在。被提供的方法用于生长的SH-SY5Y以贴壁细胞和与突触-pHluorin染它们，以及执行TIRFM和成像技术。最后，战略目标选择，计数和分析，在全细胞和单囊泡融合水平的事件呈现。 为了验证成像过程和数据分析方法，pHluorin标记的囊泡的动力学下休息了分析和刺激（去极化钾浓度）的条件。膜去极化增加的融合事件的频率，并导致记录在全细胞的净荧光信号的并行加注。单囊泡分析揭示聚变事件行为（上升峰的高度和宽度）的修饰。这些数据表明，第在钾去极化不仅诱导大量神经递质释放，而且修改囊泡融合和再循环的机制。 用适当的荧光探针，该技术可以在不同的蜂窝系统可以采用解剖构和刺激分泌的机制。

描述的全内反射荧光成像和数据分析程序的目的是研究在蜂窝系统囊泡动态。这种技术可以被用于确定信号分子和药物对聚变事件和神经递质的囊泡动力学17的影响。使用GFP标记质膜蛋白，所述TIRFM分析已被用来表征在胶质GFP标记谷氨酸转运和上皮细胞18,19的构贩卖。 为了验证报告的成像过程和数据分析策略，融合事件被记录在基础和刺激条件（钾引起的去极?...

Watch this Scientific Journal Video about TIRFM and pH-sensitive GFP-probes to Evaluate Neurotransmitter Vesicle Dynamics in SH-SY5Y Neuroblastoma Cells: Cell Imaging and Data Analysis at JoVE.com

TIRFM and pH-sensitive GFP-probes to Evaluate Neurotransmitter Vesicle Dynamics in SH-SY5Y Neuroblastoma Cells: Cell Imaging and Data Analysis

This paper provides a method for investigating neurotransmitter vesicle dynamics in neuroblastoma cells, using a synaptobrevin2-pHluorin construct and Total Internal Reflection Fluorescence Microscopy. The strategy developed for image processing and data analysis is also reported.

TIRFM和pH敏感的GFP-探针来评估神经递质囊泡动力学SH-SY5Y神经母细胞瘤:细胞成像和数据分析

Synaptic vesicles release neurotransmitters at chemical synapses through a dynamic cycle of fusion and retrieval. Monitoring synaptic activity in real ...

tirfm-ph-sensitive-gfp-probes-to-evaluate-neurotransmitter-vesicle

Research

JoVE Journal

Neuroscience

10.7K 次观看.  Università degli Studi di Milano. This paper provides a method for investigating neurotransmitter vesicle dynamics in neuroblastoma cells, using a synaptobrevin2-pHluorin construct and Total Internal Reflection Fluorescence Microscopy. The strategy developed for image processing and data analysis is also reported.

视频: TIRFM and pH-sensitive GFP-probes to Evaluate Neurotransmitter Vesicle Dynamics in SH-SY5Y Neuroblastoma Cells: Cell Imaging and Data Analysis

Quantitative Analysis of Synaptic Vesicle Pool Replenishment in Cultured Cerebellar Granule Neurons using FM Dyes

After neurotransmitter release in central nerve terminals, SVs are rapidly retrieved by endocytosis. Retrieved SVs are then refilled with neurotransmitter and rejoin the recycling pool, defined as SVs that are available for exocytosis1,2. The recycling pool can generally be subdivided into two distinct pools - the readily releasable pool (RRP) and the reserve pool (RP). As their names imply, the RRP consists of SVs that are immediately available for fusion while RP SVs are released only during intense stimulation1,2. It is important to have a reliable assay that reports the differential replenishment of these SV pools in order to understand 1) how SVs traffic after different modes of endocytosis (such as clathrin-dependent endocytosis and activity-dependent bulk endocytosis) and 2) the mechanisms controlling the mobilisation of both the RRP and RP in response to different stimuli.
FM dyes are routinely employed to quantitatively report SV turnover in central nerve terminals3-8. They have a hydrophobic hydrocarbon tail that allows reversible partitioning in the lipid bilayer, and a hydrophilic head group that blocks passage across membranes. The dyes have little fluorescence in aqueous solution, but their quantum yield increases dramatically when partitioned in membrane9. Thus FM dyes are ideal fluorescent probes for tracking actively recycling SVs. The standard protocol for use of FM dye is as follows. First they are applied to neurons and are taken up during endocytosis (Figure 1). After non-internalised dye is washed away from the plasma membrane, recycled SVs redistribute within the recycling pool. These SVs are then depleted using unloading stimuli (Figure 1). Since FM dye labelling of SVs is quantal10, the resulting fluorescence drop is proportional to the amount of vesicles released. Thus, the recycling and fusion of SVs generated from the previous round of endocytosis can be reliably quantified.
Here, we present a protocol that has been modified to obtain two additional elements of information. Firstly, sequential unloading stimuli are used to differentially unload the RRP and the RP, to allow quantification of the replenishment of specific SV pools. Secondly, each nerve terminal undergoes the protocol twice. Thus, the response of the same nerve terminal at S1 can be compared against the presence of a test substance at phase S2 (Figure 2), providing an internal control. This is important, since the extent of SV recycling across different nerve terminals is highly variable11.
Any adherent primary neuronal cultures may be used for this protocol, however the plating density, solutions and stimulation conditions are optimised for cerebellar granule neurons (CGNs)12,13.

A live fluorescence imaging technique to quantify the replenishment and mobilisation of specific synaptic vesicle (SV) pools in central nerve terminals is described. Two rounds of SV recycling are monitored in the same nerve terminals providing an internal control.

使用调频染料培养的小脑颗粒神经突触小泡池补充的定量分析

After neurotransmitter release in central nerve terminals, SVs are rapidly retrieved by endocytosis.  Retrieved SVs are then refilled with ...

科研

神经科学

Lateral Diffusion and Exocytosis of Membrane Proteins in Cultured Neurons Assessed using Fluorescence Recovery and Fluorescence-loss Photobleaching

Membrane proteins such as receptors and ion channels undergo active trafficking in neurons, which are highly polarised and morphologically complex. This directed trafficking is of fundamental importance to deliver, maintain or remove synaptic proteins.
Super-ecliptic pHluorin (SEP) is a pH-sensitive derivative of eGFP that has been extensively used for live cell imaging of plasma membrane proteins1-2. At low pH, protonation of SEP decreases photon absorption and eliminates fluorescence emission. As most intracellular trafficking events occur in compartments with low pH, where SEP fluorescence is eclipsed, the fluorescence signal from SEP-tagged proteins is predominantly from the plasma membrane where the SEP is exposed to a neutral pH extracellular environment. When illuminated at high intensity SEP, like every fluorescent dye, is irreversibly photodamaged (photobleached)3-5. Importantly, because low pH quenches photon absorption, only surface expressed SEP can be photobleached whereas intracellular SEP is unaffected by the high intensity illumination6-10. 
FRAP (fluorescence recovery after photobleaching) of SEP-tagged proteins is a convenient and powerful technique for assessing protein dynamics at the plasma membrane. When fluorescently tagged proteins are photobleached in a region of interest (ROI) the recovery in fluorescence occurs due to the movement of unbleached SEP-tagged proteins into the bleached region. This can occur via lateral diffusion and/or from exocytosis of non-photobleached receptors supplied either by de novo synthesis or recycling (see Fig. 1). The fraction of immobile and mobile protein can be determined and the mobility and kinetics of the diffusible fraction can be interrogated under basal and stimulated conditions such as agonist application or neuronal activation stimuli such as NMDA or KCl application8,10. 
We describe photobleaching techniques designed to selectively visualize the recovery of fluorescence attributable to exocytosis. Briefly, an ROI is photobleached once as with standard FRAP protocols, followed, after a brief recovery, by repetitive bleaching of the flanking regions. This 'FRAP-FLIP' protocol, developed in our lab, has been used to characterize AMPA receptor trafficking at dendritic spines10, and is applicable to a wide range of trafficking studies to evaluate the intracellular trafficking and exocytosis.

This report describes the use of live cell imaging and photobleach techniques to determine the surface expression, transport pathways and trafficking kinetics of exogenously expressed, pH-sensitive GFP-tagged proteins at the plasma membrane of neurons.

横向扩散和培养的神经元膜蛋白的胞吐评估使用荧光恢复和荧光损失漂白

Membrane proteins such as receptors and ion channels undergo active trafficking in neurons, which are highly polarised and morphologically complex. This ...

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 ...

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 ...

Visualizing Intracellular SNARE Trafficking by Fluorescence Lifetime Imaging Microscopy

Soluble N-ethylmaleimide sensitive fusion protein (NSF) attachment protein receptor (SNARE) proteins are key for membrane trafficking, as they catalyze membrane fusion within eukaryotic cells. The SNARE protein family consists of about 36 different members. Specific intracellular transport routes are catalyzed by specific sets of 3 or 4 SNARE proteins that thereby contribute to the specificity and fidelity of membrane trafficking. However, studying the precise function of SNARE proteins is technically challenging, because SNAREs are highly abundant and functionally redundant, with most SNAREs having multiple and overlapping functions. In this protocol, a new method for the visualization of SNARE complex formation in live cells is described. This method is based on expressing SNARE proteins C-terminally fused to fluorescent proteins and measuring their interaction by F&#246;rster resonance energy transfer (FRET) employing fluorescence lifetime imaging microscopy (FLIM). By fitting the fluorescence lifetime histograms with a multicomponent decay model, FRET-FLIM allows (semi-)quantitative estimation of the fraction of the SNARE complex formation at different vesicles. This protocol has been successfully applied to visualize SNARE complex formation at the plasma membrane and at endosomal compartments in mammalian cell lines and primary immune cells, and can be readily extended to study SNARE functions at other organelles in animal, plant, and fungal cells.

This protocol describes a new method allowing for the quantitative visualization of complex formation of SNARE proteins, based on F&#246;rster resonance energy transfer, and fluorescence lifetime imaging microscopy.

荧光寿命成像显微镜观察细胞内圈套的贩运

Soluble N-ethylmaleimide sensitive fusion protein (NSF) attachment protein receptor (SNARE) proteins are key for membrane trafficking, as they catalyze ...

免疫与感染

Quantification of Endosome and Lysosome Motilities in Cultured Neurons Using Fluorescent Probes

In the brain, membrane trafficking systems play important roles in regulating neuronal functions, such as neuronal morphology, synaptic plasticity, survival, and glial communications. To date, numerous studies have reported that defects in these systems cause various neuronal diseases. Thus, understanding the mechanisms underlying vesicle dynamics may provide influential clues that could aid in the treatment of several neuronal disorders. Here, we describe a method for quantifying vesicle motilities, such as motility distance and rate of movement, using a software plug-in for the ImageJ platform. To obtain images for quantification, we labeled neuronal endosome-lysosome structures with EGFP-tagged vesicle marker proteins and observed the movement of vesicles using a time-lapse microscopy. This method is highly useful and simplify measuring vesicle motility in neurites, such as axons and dendrites, as well as in the soma of both neurons and glial cells. Furthermore, this method can be applied to other cell lines, such as fibroblasts and endothelial cells. This approach could provide a valuable advancement of our understanding of membrane trafficking.

The investigation of membrane trafficking is crucial for understanding neuronal functions. Here, we introduce a method for quantifying vesicle motility in neurons. This is a convenient method that can be adapted to the quantification of membrane trafficking in the nervous system.

使用荧光探针定量培养的神经元中的内体和溶酶体运动

In the brain, membrane trafficking systems play important roles in regulating neuronal functions, such as neuronal morphology, synaptic plasticity, ...

Measuring Synaptic Vesicle Endocytosis in Cultured Hippocampal Neurons

During endocytosis, fused synaptic vesicles are retrieved at nerve terminals, allowing for vesicle recycling and thus the maintenance of synaptic transmission during repetitive nerve firing. Impaired endocytosis in pathological conditions leads to decreases in synaptic strength and brain functions. Here, we describe methods used to measure synaptic vesicle endocytosis at the mammalian hippocampal synapse in neuronal culture. We monitored synaptic vesicle protein endocytosis by fusing a synaptic vesicular membrane protein, including synaptophysin and VAMP2/synaptobrevin, at the vesicular lumenal side, with pHluorin, a pH-sensitive green fluorescent protein that increases its fluorescence intensity as the pH increases. During exocytosis, vesicular lumen pH increases, whereas during endocytosis vesicular lumen pH is re-acidified. Thus, an increase of pHluorin fluorescence intensity indicates fusion, whereas a decrease indicates endocytosis of the labelled synaptic vesicle protein. In addition to using the pHluorin imaging method to record endocytosis, we monitored vesicular membrane endocytosis by electron microscopy (EM) measurements of Horseradish peroxidase (HRP) uptake by vesicles. Finally, we monitored the formation of nerve terminal membrane pits at various times after high potassium-induced depolarization. The time course of HRP uptake and membrane pit formation indicates the time course of endocytosis.

Synaptic vesicle endocytosis is detected by light microscopy of pHluorin fused with synaptic vesicle protein and by electron microscopy of vesicle uptake.

海马神经元中突触囊泡吞的测量

During endocytosis, fused synaptic vesicles are retrieved at nerve terminals, allowing for vesicle recycling and thus the maintenance of synaptic ...

An Optical Assay for Synaptic Vesicle Recycling in Cultured Neurons Overexpressing Presynaptic Proteins

At active presynaptic nerve terminals, synaptic vesicles undergo cycles of exo- and endocytosis. During recycling, the luminal domains of SV transmembrane proteins become exposed at the cell surface. One of these proteins is Synaptotagmin-1 (Syt1). An antibody directed against the luminal domain of Syt1, once added to the culture medium, is taken up during the exo-endocytotic cycle. This uptake is proportional to the amount of SV recycling and can be quantified through immunofluorescence. Here, we combine Syt1 antibody uptake with double transfection of cultured hippocampal neurons. This allows us to (1) localize presynaptic sites based on expression of recombinant presynaptic marker Synaptophysin, (2) determine their functionality using Syt1 uptake, and (3) characterize the targeting and effects of a protein of interest, GFP-Rogdi.

We describe an optical assay for synaptic vesicle (SV) recycling in cultured neurons. Combining this protocol with double transfection to express a presynaptic marker and protein of interest allows us to locate presynaptic sites, their synaptic vesicle recycling capacity, and determine the role of the protein of interest.

Overexpressing 突触前蛋白培养神经元突触囊泡再生的光学检测

At active presynaptic nerve terminals, synaptic vesicles undergo cycles of exo- and endocytosis. During recycling, the luminal domains of SV transmembrane ...

Real-Time Fluorescent Measurement of Synaptic Functions in Models of Amyotrophic Lateral Sclerosis

Before neuronal degeneration, the cause of motor and cognitive deficits in patients with amyotrophic lateral sclerosis (ALS) and/or frontotemporal lobe dementia (FTLD) is dysfunction of communication between neurons and motor neurons and muscle. The underlying process of synaptic transmission involves membrane depolarization-dependent synaptic vesicle fusion and the release of neurotransmitters into the synapse. This process occurs through localized calcium influx into the presynaptic terminals where synaptic vesicles reside. Here, the protocol describes fluorescence-based live-imaging methodologies that reliably report depolarization-mediated synaptic vesicle exocytosis and presynaptic terminal calcium influx dynamics in cultured neurons.
Using a styryl dye that is incorporated into synaptic vesicle membranes, the synaptic vesicle release is elucidated. On the other hand, to study calcium entry, Gcamp6m is used, a genetically encoded fluorescent reporter. We employ high potassium chloride-mediated depolarization to mimic neuronal activity. To quantify synaptic vesicle exocytosis unambiguously, we measure the loss of normalized styryl dye fluorescence as a function of time. Under similar stimulation conditions, in the case of calcium influx, Gcamp6m fluorescence increases. Normalization and quantification of this fluorescence change are performed in a similar manner to the styryl dye protocol. These methods can be multiplexed with transfection-based overexpression of fluorescently tagged mutant proteins. These protocols have been extensively used to study synaptic dysfunction in models of FUS-ALS and C9ORF72-ALS, utilizing primary rodent cortical and motor neurons. These protocols easily allow for rapid screening of compounds that may improve neuronal communication. As such, these methods are valuable not only for the study of ALS but for all areas of neurodegenerative and developmental neuroscience research.

Two related methods are described to visualize subcellular events required for synaptic transmission. These protocols enable the real-time monitoring of the dynamics of presynaptic calcium influx and synaptic vesicle membrane fusion using live-cell imaging of in vitro cultured neurons.

肌萎缩性侧索硬化症模型中突触功能的实时荧光测量

Before neuronal degeneration, the cause of motor and cognitive deficits in patients with amyotrophic lateral sclerosis (ALS) and/or frontotemporal lobe ...

Utilizing pHluorin-tagged Receptors to Monitor Subcellular Localization and Trafficking

Understanding membrane protein trafficking, assembly, and expression requires an approach that differentiates between those residing in intracellular organelles and those localized on the plasma membrane. Traditional fluorescence-based measurements lack the capability to distinguish membrane proteins residing in different organelles. Cutting edge methodologies transcend traditional methods by coupling pH-sensitive fluorophores with total internal reflection fluorescence microscopy (TIRFM). TIRF illumination excites the sample up to approximately 150 nm from the glass-sample interface, thus decreasing background, increasing the signal to noise ratio, and enhancing resolution. The excitation volume in TIRFM encompasses the plasma membrane and nearby organelles such as the peripheral ER. Superecliptic pHluorin (SEP) is a pH sensitive version of GFP. Genetically encoding SEP into the extracellular domain of a membrane protein of interest positions the fluorophore on the luminal side of the ER and in the extracellular region of the cell. SEP is fluorescent when the pH is greater than 6, but remains in an off state at lower pH values. Therefore, receptors tagged with SEP fluoresce when residing in the endoplasmic reticulum (ER) or upon insertion in the plasma membrane (PM) but not when confined to a trafficking vesicle or other organelles such as the Golgi. The extracellular pH can be adjusted to dictate the fluorescence of receptors on the plasma membrane. The difference in fluorescence between TIRF images at neutral and acidic extracellular pH for the same cell corresponds to a relative number of receptors on the plasma membrane. This allows a simultaneous measurement of intracellular and plasma membrane resident receptors. Single vesicle insertion events can also be measured when the extracellular pH is neutral, corresponding to a low pH trafficking vesicle fusing with the plasma membrane and transitioning into a fluorescent state. This versatile technique can be exploited to study localization, expression, and trafficking of membrane proteins.

Labeling the extracellular domain of a membrane protein with a pH sensitive fluorophore, superecliptic pHluorin (SEP), allows subcellular localization, expression, and trafficking to be determined. Imaging SEP-labeled proteins with total internal reflection fluorescence microscopy (TIRFM) enables the quantification of protein levels in the peripheral ER and plasma membrane.

利用pHluorin标记的受体监视亚细胞定位和贩运

Understanding membrane protein trafficking, assembly, and expression requires an approach that differentiates between those residing in intracellular ...