作者感谢原田实验室的成员在整个这项工作的执行有益的讨论。这项工作是由美国心脏协会，肌张力障碍医学研究基金会，爱德华马林克罗特，小基金会，美国国家科学基金会和白厅基金，以资助NCH

1。神经元从哺乳动物脑原代培养在这项研究中进行的所有动物的程序是由爱荷华大学的机构动物护理和使用委员会批准。 <ol><li>海马准备从小鼠海马CA3-CA1区或大鼠分离的细胞培养在出生后0-1天19,20。镀上12毫米的盖玻片（厚度数0）预置与大鼠神经胶质饲养层，在24孔培养皿中，并以12,000个细胞/孔的密度将海马细胞。 </li><li>培养海马神经元至少8天为功能性神经末梢发展21。我们使用的神经元在11-14天在体外 ，当个人的神经末梢是相对孤立的没有太多的集群。神经元可用于实验最多体外例如威利格等人 22和Nosyreva和Kavalali 23大约21-28天。 </li><li>为功能性研究，评估盖玻片（染色前）利用透射光显微镜（ 例如相衬光学元件在10-20X的放大倍率）健康的神经元的指标。身体健康的指标包括:一个统一的胶质细胞层，周围的神经元细胞清晰保证金，延长树突无串珠结构，缺乏集群胞体，以及缺乏捆绑突起的。 </li></ol> 2。加载突触小泡与FM染料（染色） <ol><li>染色由于受电场刺激诱发的突触活动（包括动作电位） <ol><li>准备由FM染料添加到HEPES为基础的无染料染色解决方案解决方案1， 如台氏液（见解决方案的详细信息）。调频染料的浓度为10μMFM1-43和2.5μMFM4-64。典型浓度范围如下:2-15微米FM1-43，或2.5-20微米FM4-64 3,6。对于需要经常性的抑制实验谷氨酸能突触活动和突触可塑性的诱导，进一步添加离子型谷氨酸受体的拮抗剂:AMPA受体（ 例如，10μMCNQX）和NMDA受体（ 例如，50μMAP5）。 </li><li>与细胞转移盖玻片从培养介质中预先填充与普通台氏液成像室。我们使用的成像腔室与连接到所述侧壁（铂丝分离6.3毫米，RC-21BRFS）的刺激电极。此腔室被设计成一个封闭的成像室中，但我们使用它作为一个开放的腔室（ 即没有顶部盖玻片），用〜200微升的浴体积。在转移过程中，切勿使培养的细胞在空气中，并避免与镊子（挑盖玻片从外围而不是靠近中心）捏培养细胞。 </li><li>灌注成像室中，在室温（23-25​​℃）纯台氏液。 </li><li>申请者共同染色液1nstant灌注。 </li><li>通过施加电脉冲激发突触活动。对于海马神经元突触小泡的总回收池的最大染色，刺激文化在10 Hz为60-120秒24。当试图达到场刺激的最大效率，保持几个特点在心。 1）浴溶液的高度应尽可能地低，同时保持了神经元和神经刺激电极完全浸没在一层薄薄的台氏溶液（以保持神经元存活并得到均匀的电场）。 2）电流强度和持续时间，应使用神经元兴奋性的指标， 如荧光Ca 2 +的影像进行优化，在我们的例子中，30毫安强度和1毫秒持续时间的恒定电流给出可靠的结果。 3）槽液应以恒定流量的刺激过程中应用;电刺激导致电解，导致质子（H <s向上&gt; +）积聚在带正电的阳极和阳极附近的pH为在静态浴迅速降低。通过含有pH指示剂（ 例如汉克斯用酚红平衡盐溶液）中的溶液通过多于几百个脉冲，检查在一个单独的实验的酸化。 </li><li>离开神经元染色液1额外60秒后场刺激终止，从而使刺激后细胞内吞作用将完成25。 </li><li>用冲洗液1，无染料台氏液加AMPA受体和NMDA受体（参见第2.1.1节）的拮抗剂，和电压依赖性钠通道的阻断剂灌注洗染色液出了成像室（ 例如 0.5-1微米河豚毒素，TTX）。受体阻滞剂的组合将通过自发突触活动抑制调频染料的损失。为了最大限度地提高洗涤效率，增加灌注率（&lt;em&gt;的如高达5-6毫升/分钟）1-2分钟，并加入直接漂洗溶液1手动打开的成像室中，用移液管的大丸剂（ 例如 1ml）中。手动控制输液的强度和方向，以免打扰培养的神经元。 </li></ol></li><li>染色由于高-K +解决方案诱发突触活动（不涉及动作电位）。 <ol><li>准备染色液2由FM染料加入到高-K +台氏液。具体来说，准备修改台氏液用45毫米氯化钾，氯化钾通过对氯化钠等摩尔取代。如果需要，添加AMPA和NMDA受体的拮抗剂。这种高-K +解决方案将不断神经元去极化，使钙离子流入神经 ​​终端，并启动突触囊泡的胞吐到最大程度26。高浓度（70〜90毫米）的其他报告也使用了如 Klingauf 等人 27的ð理查兹等人 28 </li><li>用培养的神经元传输盖玻片从培养介质中预先填充与普通台氏液成像室。 </li><li>通过将染色溶液2以恒定的灌注，或用移液管26,28染色的神经元在室温下搅拌1-2分钟。在后一种情况下，最终的染料浓度应通过加入染色溶液2至无染料溶液的已知容积的已知容积来控制。 </li><li>洗染色液出成像室，如在2.1.7节中描述。 </li></ol></li><li>染色由于自发和微型突触活动。 <ol><li> Prewarm碳酸氢盐为基础的解决方案（ 例如最小必需培养基，MEM），以37℃放置在培养箱内培养超过60分钟。 </li><li>基于自发突触活性染色，由FM染料加入到MEM制备染色溶液3。用于染色基于米iniature突触活动，加入TTX的染色液3准备染色液4。 </li><li>染色的神经元通过将盖玻片用从培养液中培养的神经元染色溶液3或4，并且让它们在37℃的同一染色溶液10分钟。 </li><li>在清洗液1简要冲洗盖玻片。 </li><li>盖玻片转移到清洗液1在成像室。请记住以下几点。 1）细胞不应该暴露在空气中，如有必要，直接将盖玻片成像室不漂洗。 2）神经元可以通过更换染色液3或4用HEPES-基溶液在室温下被染色。 3）神经元可被染色以使用这种方法在高-K +溶液的上下文诱发突触活动，通过更换染色液3或4与染色溶液2，并进行在室温下将剩余的步骤。 </li><li>洗染色液出成像室如第2.1.7所述。 </li></ol></li></ol> 3。洗出的FM染料<ol><li>神经细胞的染色和初期洗涤通过任何上述方法（部分2.1.7）后，继续灌注成像室中，在室温漂洗溶液1 5-10分钟。这会从质膜和细胞外溶液中除去调频染料。在我们的成像腔室的情况下，浴灌注速率为600-1,200微升/分钟。洗，也可以在不存在细胞外的Ca 2 +的抑制调频染料损失是由于突触活动17进行。洗涤液可通过ADVASEP-7，改性环糊精，作为从质膜29-31调频染料清除剂的简要应用增强。关键是要保持任何ADVASEP-7曝光短（ 例如 5秒单层培养的情况下），以避免既降低调频泪点31的强度和损害HEA第l个细胞。从FM1-43在细胞膜上的荧光也可以通过猝灭磺酰罗丹明101（50-100微米），不进入突触小泡32的应用受到抑制。 </li></ol> 4。寻找最佳的图像场在洗涤<ol><li>验证该细胞中该字段将被成像是健康的，用透射光显微镜用高倍率和高数值孔径的物镜（ 例如 40X）。从本节开始，我们继续使用倒置显微镜（Eclipse中的Ti，尼康）配有相衬或微分干涉对比光学系统。这种显微镜还配备了完美的对焦系统（PFS），使​​连续，实时对焦校正，克服了显微镜焦点漂移。这个功能是必不可少的延时成像具有相同的聚焦平面脱色过程。 </li><li>验证染色是有效的，通过使用荧光光学。 AV调频泪点的OID聚集体，除非它们是研究的目标，因为个别boutons将是难以辨别。要注意的FM泪点的形状:如果大部分染色boutons的显示为圆形串珠，他们可以代表不健康的神经末梢。尽量减少神经元的暴露在强烈的荧光激发，以避免光漂白。 </li></ol> 5。从突触小泡卸载调频染料（脱色） <ol><li>由于脱色由电场刺激诱发的突触活动。 <ol><li>洗TTX通过广泛灌注神经元用冲洗液2（同清洗液1，但没有TTX）。确认洗脱的TTX在单独的实验中的效力，使用相同的洗涤参数（灌注速率和持续时间）， 例如 ，通过荧光的Ca 2 +成像的诱导场的刺激细胞质的Ca 2 +瞬变，或由伏的膜片钳记录年龄依赖性Na +电流。 </li><li>启动成像调频染料。用于成像的FM1-43或FM4-64中，使用520纳米或650纳米长通发射滤波器，分别和一个490nm的激发滤光器。采集图像每隔1-2秒，使用短的曝光时间（ 例如 10〜20毫秒），弱激发强度（ 如 LED电源的5-10％）和高灵敏度（ 例如，使用EM增益）。通过减少曝光时间和荧光激发的强度最小化在FM染料的光致漂白。通过打开完美的对焦系统减低显微镜焦点漂移。 </li><li>刺激神经 ​​元的冲洗液2，使用现场的刺激（ 如在10 Hz 120秒）20。 </li><li>应用多轮的刺激，以1-2分钟间隔休息期，以消耗调频染料全部回收利用突触小泡。评估的染色带FM染料在日突触小泡的总回收池的大小时，这是很重要的e之前的状态20,33。 </li></ol></li><li>脱色是由于突触活动诱发在没有动作电位。 <ol><li>启动成像调频染料。 </li><li>申请溶解在漂洗溶液1或2所述刺激的试剂。这样的试剂包括:氯化钾（高-K +溶液， 例如 45毫米）26，离子霉素（一个Ca 2 +的离子载体，通过允许的Ca 2 +流入细胞从细胞外溶液中，使用例如在引发细胞内Ca 2 +浓度5微米）20,34，和蔗糖（高渗溶液，刺激突触小泡从易释放池， 如使用500毫米）35,36释放。使用一个可靠的时间控制一个快速，局部灌注系统中应用的试剂， 例如从华纳仪器37，或Y型管系统38-40的SF-77B系统。 </li></ol></li><li>脱色由于自发和最小iature突触活动。 <ol><li>对于脱色由于自发突触活动，洗出河豚毒素上文（5.1.1节），用冲洗液2。启动成像调频染料灌注时神经元的冲洗液2。对脱色由于微型突触活动，开始成像调频染料，同时继续灌注神经元的漂洗溶液1（含TTX）。 </li><li>脱色的基础上无论是自发或微型活动后，透过诱发突触活动脱色识别功能神经末梢。活性可以通过任何上述的方法来诱发。这个识别过程是必要的，因为被污染的结构可以是那些比官能神经末梢（ 如缓慢地循环核内体或星形细胞囊泡）和大量脱色的由在此过程中诱发的活性助剂等。 </li></ol></li></ol> 6。评估调频染料的光漂白率<ol><li>染色与醛可以解决的调频染料（ 如 FM1-43FX，FM4-64FX）神经末梢。这样做可以用上述诱发的，自发的，或者微型染色方法，和通过用可固定的调频染料代替调频染料。 </li><li>如第2.1.7所述洗出可修复的调频染料。 </li><li>化学通过将盖玻片固定液固定的神经元:4％多聚甲醛和4％的蔗糖在台氏在4℃下30分钟，溶液可定影调频染料的荧光强度将化学固定后保留。 </li><li>洗出固定液在台氏液10分钟，通过将盖玻片新鲜台氏液两次。 </li><li>转移到盖玻片在成像室新鲜台氏液。 </li><li>开始使用一组相同的成像参数，作为活的神经元成像的可定影调频染料。它使用任何化学固定标本后冲洗成像室非常好是很重要的，因为任何剩余的固定剂可能会影响在同一室内进行后续的活细胞成像实验。可替换地，以固定细胞成像，染色的活细胞可能被成像以评估光漂白速率。然而，值得记住，这是很难清楚地阻止了微型突触活动，例如，微型活动的频率可以降低到约50％通过除去细胞外Ca 2 +的23，但它不是一个完整的封锁。 </li><li>在每个成像会话结束时，获取用于评估调频荧光强度的绝对值的背景图像。调频泪点的强度不仅代表从神经末梢的调频染料的真实信号，而且从1）神经胶质细胞所依据的神经细胞的单层培养物，2）将盖玻片和光学元件（ 例如，物镜，过滤器和背景噪声镜），和3）的检测器（EMCCD相机）。背景噪声ASSEssed在成像领域的nonstained地区。当这样的区域中的强度或空间异构的限制，与图像替换它与照相机快门关闭，并获取来自检测器的噪声，尽管它不是一种理想的方法。使用相同的成像参数获得约10张图像。 </li></ol> 7。图像分析<ol><li>导入所获取的数据转换为图像分析软件作为堆叠时间序列图像的。我们使用ImageJ的（WS Rasband，美国国立卫生研究院）和相关的插件， 例如图像稳定器插件（康丽），用于校正小程度的运动之中调频泪点，和时间序列分析仪V2.0（巴拉吉Jayaprakash）分析的时间序列。 </li><li>计算的变化调频强度在串联（ΔFM）的开始和结束。为此，脱色开始前和脱色（ 例如 5图像中的每个帧）结束后的平均化图像，并通过subtractin生成的差分图像克他们。 </li><li>识别潜在的功能性神经末梢通过施加强度阈值的差分图像，并且检测像素的强度都高于阈值。设定阈值的一种方法是选择从裸盖玻片面积得到的背景强度的标准偏差。 </li><li>识别孤立的，功能性神经末梢作为进行检测，并满足一个标准大小（以邻接像素的最小和最大的数字）的相邻像素。这个过程消除背景噪声和分析汇总神经末梢。 </li><li>指定感兴趣区域的所检测到的像素群（与对应于个别神经末梢个体群）（感兴趣区域）。脱色（ΔFM）过程中的强度变化总量是分析的典型参数。这代表诱发的，自发的或微型突触活动的累计金额。排除的ROI，如果它们表现出下列任何根据目的的实验强度的变化:增加了录音过程中，刺激前突然下降，或刺激后很长的等待时间。 </li><li>通过导入所获得的时间序列数据，并将其图像分析软件评估光漂白速率。平均的背景图像（ 例如，具有封闭快门），并从各个图像中减去它。分配投资回报调频泪点的推定对应神经末梢，并测量变化的投资回报率的强度随着时间的推移。 </li></ol>解决方案<ol><li>台氏液<ul><li>组合物（以mM计）:125氯化钠，氯化钾2，2的CaCl 2，2的MgCl 2，30葡萄糖，25 HEPES，310毫渗透摩尔浓度，pH值7.4。 </li></ul></li><li>染色液1 <ul><li>台氏液加调频染料。 </li><li>该溶液被用于染色，基于由电刺激诱发的突触活性。 </li><li>添加AMPA受体和NMDA的拮抗剂recepto如有必要，RS。 </li></ul></li><li>染色液2 <ul><li>高-K +台氏液加调频 ​​染料。 </li><li>这个修改​​过的台氏液是通过增加KCl浓度至45毫米，由氯化钾对NaCl的摩尔取代制备。 </li><li>该溶液被用于染色的基础上，通过连续的去极化诱发的突触活性。 </li><li>如果需要的话添加AMPA受体和NMDA受体的拮抗剂。 </li></ul></li><li>染色液3 <ul><li> MEM液加调频染料。 </li><li>该溶液被用于染色，根据自发突触活动。 </li></ul></li><li>染色液4 <ul><li> MEM液加调频染料和TTX。 </li><li>该溶液被用于染色，根据微型突触活动。 </li></ul></li><li>清洗液1 <ul><li>台氏液加AMPA受体和NMDA受体的拮抗剂，和TTX。 </li></ul></li><li>清洗液2 <ul><li>台氏液加AMPA受体和NMDA受体的拮抗剂，但没有河豚毒素。 </li></ul></li></ol>

作者宣称没有竞争的财务权益。

我们已经描述了协议的染色和脱色突触小泡响应引起的，自发的和微型突触活动，并为在脱色相位成像。除了现有的协议，我们已观察的基础上微型突触活动的调频脱色的一个新的协议。使用这些协议，我们先前确定的运动障碍肌张力障碍的小鼠模型中培养的神经元异常。相较于他们的同行在野生型小鼠中，这些神经元后行加速在Ca 2 +的依赖性突触囊泡的胞吐的时候用高活性20刺?...

突触小泡的功能是突触传递的重要决定因素。这些囊泡释放神经递质，当他们与突触前质膜（胞吐作用）熔化，并且它们成为准备发布的另一周期从质膜（内吞作用）进行再生和重新装载神经递质之后。研究的动态及相关突触囊泡循环机制已被引入苯乙烯调频染料1大大加快。这些两亲性分子，其具有带正电荷的亲水性头部基团和疏水性尾（多种染料在图1A中，FM1-43的图1B中stereoview），能可逆地进入和退出脂膜不渗透其中。调频染料组共享影响的光，它们发出的一系列类似的功能。例如，FM2-10，FM1-43，和FM1-84有两个环状化合物和升之间的一个双键瓦特绿色发光。它们之间的区别是疏水尾，这决定了其疏水性，因此，出口处的从膜的速率（departitioning）的长度。在FM5-95和FM4-64的情况下，三个双键连接的环状化合物，它们显示红色发射。这些染料差异相对于它们的亲水部分。在所有的FM染料，当它们被插入到生物膜，由于增加量子产率在相对于所述亲水性环境的疏水性环境中的荧光强度增加。因此，改变FM强度代表了改变膜的营业额。不同的颜色（发射光谱）和疏水性使调频染料一种多用途的研究工具，在突触囊泡循环。 基于这些特性，调频染料大多根据在分析突触小泡循环利用下面的方案（ 图2）中使用。神经元S是沐浴在包含FM染料的细胞外液，使其能够被带进突触小泡（SVS），因为它们通过内吞作用（染色）形成。的染料，然后洗出通过应用染料-自由胞外溶液，这表明功能神经末梢， 即只有那些积极进行内吞作用将包含突触小泡被装入染料（ 图2底部）的群集。随后的胞吐作用导致的调频染料的损失到细胞外空间以及随之而来的损失荧光（脱色;由于两者departitioning到亲水环境和扩散远离胞吐作用的位点）。因此，改变FM的荧光强度是突触囊泡外切和内吞作用的指标。 调频染料已被用于染色和脱色的各种生物和制剂2,3突触囊泡。例子包括哺乳动物neurONAL培养4-9，哺乳动物的脑片10,11，神经肌肉接头12,13，视网膜双极神经细胞14,15和16耳蜗毛细胞。 通常，在这样的实验中，无论染色和脱色是通过广泛刺激神经元（诱发活性）触发。然而，最近，突触小泡循环响应于弱刺激也被分析，因为在没有外部刺激（自发和微型突触活动）9,17-19具有回收利用。自发和微型突触活动被定义为那些发生在没有外界刺激的，前者涉及动作电位（ 图3）的自发发射。这些微弱的突触活动与调频信号比由粗放刺激引发的较小的变化相关联。测量要求的变化，调频fluorescENCE强度准确反映突触囊泡的胞吐或胞吞作用，但强度不是伪迹的变化。伪像的原因之一是通过调频染料质膜的非特异性染色的存在。此组件的逐步洗脱将导致一个逐渐变化的测得的荧光强度，这将被错误地归因于突触活动。这个因子可以通过适当的方法来减少（见协议）。神器的最显着的原因是调频染料突触小泡内保留的漂白。在调频强度的光漂白相关的变更必须在比较所测得的生物（突触）的变化很小。敏感的摄像机的最新发展， 例如在电子倍增电荷耦合器件（EMCCD）相机，使得它可以最小化，通过缩短曝光时间，削弱用于激发荧光团的光的强度的光漂白。神器的另一个原因是我的漂移n个光显微镜的聚焦水平。成像会话期间的焦点漂移可以通过机械或热效应引起，并且将错误导致所测量的荧光强度的变化。 这里我们描述的协议和设备，使得有可能使用调频染料来分析突触小泡循环，即使在弱的或无刺激的情况下，特别是在微型突触活动。我们发现囊泡的染色和脱色的例子中诱发和自发突触活动，利用培养的啮齿动物的海马神经元，并且成像脱色阶段。我们还说明了如何评估的调频染料光漂白的程度，在没有任何调频染料损失是由于突触活动。

<table border="0" cellpadding="0" cellspacing="0" width="1767">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr>
			<td>Pulse generator</td>
			<td>AMPI</td>
			<td>Master-8</td>
			<td></td>
		</tr>
		<tr>
			<td>Isolated stimulator</td>
			<td>Digitimer</td>
			<td>DS3</td>
			<td></td>
		</tr>
		<tr>
			<td>Inverted microscope</td>
			<td>Nikon</td>
			<td>Eclipse TS100</td>
			<td>This is used for assessing the cell morphology at low magnification.</td>
		</tr>
		<tr>
			<td>Inverted microscope</td>
			<td>Nikon</td>
			<td>Eclipse TiE</td>
			<td>This is used for high-resolution fluorescence and transmitted light imaging, with minimal focus drift.</td>
		</tr>
		<tr>
			<td>Objective lens</td>
			<td>Nikon</td>
			<td></td>
			<td>Water-immersion lens is recommended. Oil-immersion lens is usable unless an imaged structure is deep from the coverslip surface (e.g. &#62;10 &#956;m).</td>
		</tr>
		<tr>
			<td>Filter cube</td>
			<td>Nikon</td>
			<td>77032509</td>
			<td>490/20-nm ex, 510-nm dclp, 520-nm-LP em for FM1-43</td>
		</tr>
		<tr>
			<td>Filter cube</td>
			<td>Nikon</td>
			<td>77032809</td>
			<td>490/20-nm ex, 510-nm dclp, 650-nm-LP em for FM4-64.&#160;</td>
		</tr>
		<tr>
			<td>EMCCD camera</td>
			<td>Andor Technology</td>
			<td>iXon EM+ DU-860</td>
			<td>This EMCCD camera is used for high-sensitivity detection of fluorescence.</td>
		</tr>
		<tr>
			<td>Liquid recirculating chiller</td>
			<td>Solid State Cooling Systems</td>
			<td>Oasis 160</td>
			<td>This is used for continuously perfusing the camera with chilled water for maintaining a temperature of -80&#176;C, and thereby reducing noise.</td>
		</tr>
		<tr>
			<td>LED</td>
			<td>CoolLED-Custom Interconnect</td>
			<td>490 nm&#160;</td>
			<td>This light source is used for rapid on/off control of fluorescence excitation.</td>
		</tr>
		<tr>
			<td>Image acquisition software</td>
			<td>Andor Technology</td>
			<td>Solis</td>
			<td></td>
		</tr>
		<tr>
			<td>Imaging chamber</td>
			<td>Warner Instruments</td>
			<td>RC-21BRFS</td>
			<td></td>
		</tr>
		<tr>
			<td>Fast perfusion system</td>
			<td>Warner Instruments</td>
			<td>SF-77B</td>
			<td></td>
		</tr>
		<tr>
			<td>CNQX</td>
			<td>Tocris Bioscience</td>
			<td>1045</td>
			<td></td>
		</tr>
		<tr>
			<td>D,L-AP5</td>
			<td>Tocris Bioscience</td>
			<td>0106</td>
			<td></td>
		</tr>
		<tr>
			<td>Tetrodotoxin</td>
			<td>Tocris Bioscience</td>
			<td>1069</td>
			<td>Caution: toxic reagent. Handle with care.</td>
		</tr>
		<tr>
			<td>FM1-43</td>
			<td>Invitrogen</td>
			<td>T35356</td>
			<td></td>
		</tr>
		<tr>
			<td>Aldehyde-fixable FM1-43 (FM1-43FX)</td>
			<td>Invitrogen</td>
			<td>F35355</td>
			<td></td>
		</tr>
		<tr>
			<td>FM4-64</td>
			<td>Invitrogen</td>
			<td>T13320</td>
			<td></td>
		</tr>
		<tr>
			<td>Aldehyde-fixable FM4-64 (FM4-64FX)</td>
			<td>Invitrogen</td>
			<td>F34653</td>
			<td></td>
		</tr>
		<tr>
			<td>Ionomycin</td>
			<td>Sigma-Aldrich</td>
			<td>I0634</td>
			<td></td>
		</tr>
		<tr>
			<td>Hanks&#8217; balanced salt</td>
			<td>Sigma-Aldrich</td>
			<td>H2387</td>
			<td></td>
		</tr>
		<tr>
			<td>Minimum Essential Medium</td>
			<td>Invitrogen</td>
			<td>51200-038</td>
			<td>This solution does not contain phenol red that will interfere with fluorescence imaging.</td>
		</tr>
		<tr>
			<td>Paraformaldehyde</td>
			<td>Electron Microscopy Sciences</td>
			<td>15710</td>
			<td>Caution: toxic reagent. Handle with care.</td>
		</tr>
		<tr>
			<td>Sucrose</td>
			<td>Sigma-Aldrich</td>
			<td>S7903</td>
			<td></td>
		</tr>
	</tbody>
</table>

examination of synaptic vesicle recycling using fm dyes during evoked, spontaneous, and miniature synaptic activities

在功能神经末梢突触囊泡的胞吐接受和内吞作用。这种突触囊泡循环可以使用苯乙烯调频染料，它揭示了膜有效的营业额分析。对于使用调频染料的传统协议的设计分析神经元以下的刺激（诱发）突触活动。最近，协议，成为可用于分析伴随弱突触活动，如自发或微型突触活动的调频信号。在FM信号的这些小的变化分析，需要对成像系统是足够敏感以检测强度的微小变化，但振幅较大的那个伪迹变化被抑制。在这里，我们描述了可应用于诱发的，自发的和微型的突触活动，并使用培养的海马神经元作为一个例子的协议。这个协议还包含评估调频染料的光漂白速率的一种手段，因为这是一个显著源成像强度的微小变化，当文物。

作为一个例子，我们展示代表结果的突触小泡的脱色时间过程（ 图4）。培养海马神经元采用自发突触活动（步骤2.3）沾上FM4-64，用无染料溶液（冲洗液2）。成像显示了使用自发活动（步骤5.3）（连续线的起始部分， 图4A）初始脱色时间过程。这后面是脱色时间过程使用三轮诱发活性与10赫兹场刺激为120秒每个（步骤5.1）。测量诱发脱色量的一种方法是说明与对应于囊泡?...

Watch this Scientific Journal Video about Examination of Synaptic Vesicle Recycling Using FM Dyes During Evoked, Spontaneous, and Miniature Synaptic Activities at JoVE.com

在诱发、自发和微型突触活动期间使用 FM 染料检查突触小泡回收 |神经科学 |视频

Examination of Synaptic Vesicle Recycling Using FM Dyes During Evoked, Spontaneous, and Miniature Synaptic Activities

我们描述了使用的苯乙烯调频染料图像突触囊泡循环的功能神经末梢。这个协议可以不仅诱发，也自发和微型突触活动的应用。该协议扩展了多种可有效评估的突触事件。

使用调频染料诱发过程中，自发的，微型突触活动检查突触囊泡循环的

Synaptic vesicles in functional nerve terminals undergo exocytosis and endocytosis. This synaptic vesicle recycling can be effectively analyzed using ...

examination-synaptic-vesicle-recycling-using-fm-dyes-during-evoked

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21.0K 次观看.  University of Iowa Carver College of Medicine. 我们描述了使用的苯乙烯调频染料图像突触囊泡循环的功能神经末梢。这个协议可以不仅诱发，也自发和微型突触活动的应用。该协议扩展了多种可有效评估的突触事件。 

视频: Examination of Synaptic Vesicle Recycling Using FM Dyes During Evoked, Spontaneous, and Miniature Synaptic Activities

Studying Synaptic Vesicle Pools using Photoconversion of Styryl Dyes

The fusion of synaptic vesicles with the plasma membrane (exocytosis) is a required step in neurotransmitter release and neuronal communication. The vesicles are then retrieved from the plasma membrane (endocytosis) and grouped together with the general pool of vesicles within the nerve terminal, until they undergo a new exo- and endocytosis cycle (vesicle recycling). These processes have been studied using a variety of techniques such as electron microscopy, electrophysiology recordings, amperometry and capacitance measurements. Importantly, during the last two decades a number of fluorescently labeled markers emerged, allowing optical techniques to track vesicles in their recycling dynamics. One of the most commonly used markers is the styryl or FM dye 1; structurally, all FM dyes contain a hydrophilic head and a lipophilic tail connected through an aromatic ring and one or more double bonds (Fig. 1B). A classical FM dye experiment to label a pool of vesicles consists in bathing the preparation (Fig. 1Ai) with the dye during the stimulation of the nerve (electrically or with high K+). This induces vesicle recycling and the subsequent loading of the dye into recently endocytosed vesicles (Fig. 1Ai-iii). After loading the vesicles with dye, a second round of stimulation in a dye-free bath would trigger the FM release through exocytosis (Fig. 1Aiv-v), process that can be followed by monitoring the fluorescence intensity decrease (destaining).

Although FM dyes have contributed greatly to the field of vesicle recycling, it is not possible to determine the exact localization or morphology of individual vesicles by using conventional fluorescence microscopy. For that reason, we explain here how FM dyes can also be used as endocytic markers using electron microscopy, through photoconversion. The photoconversion technique exploits the property of fluorescent dyes to generate reactive oxygen species under intense illumination. Fluorescently labeled preparations are submerged in a solution containing diaminobenzidine (DAB) and illuminated. Reactive species generated by the dye molecules oxidize the DAB, which forms a stable, insoluble precipitate that has a dark appearance and can be easily distinguished in electron microscopy 2,3. As DAB is only oxidized in the immediate vicinity of fluorescent molecules (as the reactive oxygen species are short-lived), the technique ensures that only fluorescently labeled structures are going to contain the electron-dense precipitate. The technique thus allows the study of the exact location and morphology of actively recycling organelles.

FM dyes have been of invaluable help in the understanding of synaptic dynamics. FMs are normally followed under the fluorescent microscope during different stimulation conditions. However, photoconversion of FM dyes combined with electron microscopy allows the visualization of distinct synaptic vesicle pools, among other ultrastructure components, in synaptic boutons.

突触小泡池，利用苯乙烯染料的光转化的研究

The fusion of synaptic vesicles with the plasma membrane (exocytosis) is a required step in neurotransmitter release and neuronal communication. The ...

科研

神经科学

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 ...

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 ...

Deriving the Time Course of Glutamate Clearance with a Deconvolution Analysis of Astrocytic Transporter Currents

The highest density of glutamate transporters in the brain is found in astrocytes. Glutamate transporters couple the movement of glutamate across the membrane with the co-transport of 3 Na+ and 1 H+ and the counter-transport of 1 K+. The stoichiometric current generated by the transport process can be monitored with whole-cell patch-clamp recordings from astrocytes. The time course of the recorded current is shaped by the time course of the glutamate concentration profile to which astrocytes are exposed, the kinetics of glutamate transporters, and the passive electrotonic properties of astrocytic membranes. Here we describe the experimental and analytical methods that can be used to record glutamate transporter currents in astrocytes and isolate the time course of glutamate clearance from all other factors that shape the waveform of astrocytic transporter currents. The methods described here can be used to estimate the lifetime of flash-uncaged and synaptically-released glutamate at astrocytic membranes in any region of the central nervous system during health and disease.

We describe an analytical method to estimate the lifetime of glutamate at astrocytic membranes from electrophysiological recordings of glutamate transporter currents in astrocytes.

星形细胞转运电流派生谷氨酸间隙的时间进程与反卷积分析

The highest density of glutamate transporters in the brain is found in astrocytes. Glutamate transporters couple the movement of glutamate across the ...

High-Resolution Quantitative Immunogold Analysis of Membrane Receptors at Retinal Ribbon Synapses

Retinal ganglion cells (RGCs) receive excitatory glutamatergic input from bipolar cells. Synaptic excitation of RGCs is mediated postsynaptically by NMDA receptors (NMDARs) and AMPA receptors (AMPARs). Physiological data have indicated that glutamate receptors at RGCs are expressed not only in postsynaptic but also in perisynaptic or extrasynaptic membrane compartments. However, precise anatomical locations for glutamate receptors at RGC synapses have not been determined. Although a high-resolution quantitative analysis of glutamate receptors at central synapses is widely employed, this approach has had only limited success in the retina. We developed a postembedding immunogold method for analysis of membrane receptors, making it possible to estimate the number, density and variability of these receptors at retinal ribbon synapses. Here we describe the tools, reagents, and the practical steps that are needed for: 1) successful preparation of retinal fixation, 2) freeze-substitution, 3) postembedding immunogold electron microscope (EM) immunocytochemistry and, 4) quantitative visualization of glutamate receptors at ribbon synapses.

The postembedding immunogold method is one of the most effective ways to provide high-resolution analyses of the subcellular localization of specific molecules. Here we describe a protocol to quantitatively analyze glutamate receptors at retinal ribbon synapses.

高分辨率定量免疫膜受体在视网膜带状突触分析

Retinal ganglion cells (RGCs) receive excitatory glutamatergic input from bipolar cells. Synaptic excitation of RGCs is mediated postsynaptically by NMDA ...

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 ...

Culturing In Vivo-like Murine Astrocytes Using the Fast, Simple, and Inexpensive AWESAM Protocol

The AWESAM (a low-cost easy stellate astrocyte method) protocol entails a fast, simple, and inexpensive way to generate large quantities of in vivo-like mouse and rat astrocyte monocultures: Brain cells can be isolated from different brain regions, and after a week of cell culture, non-astrocytic cells are shaken off by placing the culture dishes on a shaker for 6 h in the incubator. The remaining astrocytes are then passaged into new plates with an astrocyte-specific medium (termed NB+H). NB+H contains low concentrations of heparin-binding EGF-like growth factor (HBEGF), which is used in place of serum in medium. After growing in NB+H, AWESAM astrocytes have a stellate morphology and feature fine processes. Moreover, these astrocytes have more in vivo-like gene expression than astrocytes generated by previously published methods. Ca2+ imaging, vesicle dynamics, and other events close to the membrane can thus be studied in the fine astrocytic processes in vitro, e.g., using live cell confocal or TIRF microscopy. Notably, AWESAM astrocytes also exhibit spontaneous Ca2+ signaling similar to astrocytes in vivo.

Culturing In Vivo-like Murine Astrocytes Using the Fast, Simple, and Inexpensive AWESAM Protocol

The AWESAM protocol described here is optimal for culturing murine astrocytes in isolation from other brain cells in a fast, simple, and inexpensive manner. AWESAM astrocytes exhibit spontaneous Ca2+ signaling, morphology, and gene expression profiles similar to astrocytes in vivo.

使用快速、简单且便宜的 AWESAM 协议培养在体内像小鼠星形胶质细胞

The AWESAM (a low-cost easy stellate astrocyte method) protocol entails a fast, simple, and inexpensive way to generate large quantities of in vivo-like ...

Pentylenetetrazole-Induced Kindling Mouse Model

Pentylenetetrazole (PTZ) is a GABA-A receptor antagonist. An intraperitoneal injection of PTZ into an animal induces an acute, severe seizure at a high dose, whereas sequential injections of a subconvulsive dose have been used for the development of chemical kindling, an epilepsy model. A single low-dose injection of PTZ induces a mild seizure without convulsion. However, repetitive low-dose injections of PTZ decrease the threshold to evoke a convulsive seizure. Finally, continuous low-dose administration of PTZ induces a severe tonic-clonic seizure. This method is simple and widely applicable to investigate the pathophysiology of epilepsy, which is defined as a chronic disease that involves repetitive seizures. This chemical kindling protocol causes repetitive seizures in animals. With this method, vulnerability to PTZ-mediated seizures or the degree of aggravation of epileptic seizures was estimated. These advantages have led to the use of this method for screening anti-epileptic drugs and epilepsy-related genes. In addition, this method has been used to investigate neuronal damage after epileptic seizures because the histological changes observed in the brains of epileptic patients also appear in the brains of chemical-kindled animals. Thus, this protocol is useful for conveniently producing animal models of epilepsy.

This protocol describes a method of chemical kindling with pentylenetetrazole and provides a mouse model of epilepsy. This protocol can also be used to investigate vulnerability to seizure induction and pathogenesis after epileptic seizures in mice.

戊四氮诱导点燃小鼠模型

Pentylenetetrazole (PTZ) is a GABA-A receptor antagonist. An intraperitoneal injection of PTZ into an animal induces an acute, severe seizure at a high ...

Combining Optogenetics with Artificial microRNAs to Characterize the Effects of Gene Knockdown on Presynaptic Function within Intact Neuronal Circuits

The purpose of this protocol is to characterize the effect of gene knockdown on presynaptic function within intact neuronal circuits. We describe a workflow on how to combine artificial microRNA (miR)-mediated RNA interference with optogenetics to achieve selective stimulation of manipulated presynaptic boutons in acute brain slices. The experimental approach involves the use of a single viral construct and a single neuron-specific promoter to drive the expression of both an optogenetic probe and artificial miR(s) against presynaptic gene(s). When stereotactically injected in the brain region of interest, the expressed construct makes it possible to stimulate with light exclusively the neurons with reduced expression of the gene(s) under investigation. This strategy does not require the development and maintenance of genetically modified mouse lines and can in principle be applied to other organisms and to any neuronal gene of choice. We have recently applied it to investigate how the knockdown of alternative splice isoforms of presynaptic P/Q-type voltage-gated calcium channels (VGCCs) regulates short-term synaptic plasticity at CA3 to CA1 excitatory synapses in acute hippocampal slices. A similar approach could also be used to manipulate and probe the neuronal circuitry in vivo.

This protocol provides a workflow on how to combine artificial microRNA-mediated RNA interference with optogenetics to stimulate specifically presynaptic boutons with reduced expression of selective gene(s) within intact neuronal circuits.

光遗传学与人工 microRNAs 相结合对完整神经元回路中基因击倒对突触前功能的影响

The purpose of this protocol is to characterize the effect of gene knockdown on presynaptic function within intact neuronal circuits. We describe a ...

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 ...