这项研究是由赠款德国科学基金会（DFG，Ro2327 / 6-1），以CRR作者要感谢南Durry和C罗德利哥专家技术援助和M.Dübbert（电子实验室，动物学研究所的支持，科隆大学，德国）在实施普克尔盒控制器和高频开关的帮助。

这项研究进行了严格按照海涅大学杜塞尔多夫，德国的制度指引，以及欧共体理事会指令（86/609 / EEC）。 （:O52 / 05机构行为号）所有实验均在海涅大学杜塞尔多夫，德国的动物护理和使用设备来传达并经动物福利办公室。按照德国的动物福利法（Tierschutzgesetz，第4和第7），没有正式的额外批准的尸检切除脑组织是必要的。 对于代急性切片，小鼠麻醉与CO 2，迅速 ​​断头（按照欧盟委员会的建议发表于:安乐死实验动物，卢森堡:欧洲共同体，1997年正式出版，国际标准书号92-827-9694 -9）。 1，制备的解决方案<ol><li>制备含有（以mM计）夹层人工脑脊液（ACSF）:125氯化钠，氯化钾2.5，0.5的CaCl 2，6的MgCl 2，1.25和 NaH 2 PO 4，26的NaHCO 3和20葡萄糖，通入95％O 2和5％的CO 2，从而在pH值为7.4。 </li><li>制备ACSF含有（以mM计）的实验:125氯化钠，氯化钾2.5，2的CaCl 2，1 MgCl 2的，1.25的NaH 2 PO 4，26的NaHCO 3和20葡萄糖，通入95％O 2和5％CO 2下，得到的在pH值为7.4。 </li><li>制备含有（以mM计）的细胞内溶液（ICS）:150 KMeSO 3，12.5羟乙基哌嗪乙磺酸（HEPES），40的KCl，5的NaCl，1.25乙二醇四乙酸（EGTA），5的Mg-ATP，0.5 NA- GTP。调节pH值至7.3。 1毫升，在-20℃下存储区等分。 </li><li>稀钠结合的苯并呋喃间苯二甲酸酯（SBFI） - 盐在双蒸水中，制备的5μl的等分的10mM储备溶液。 </li><li>解冻ICS和添加SBFI原液在1mM的最终浓度。涡旋和微过滤（0.22微米）。保持在4℃直至在实验中使用。不重新冻结，并且只使用一昼夜。 </li><li>制备50mM的MNI谷氨酸原液溶解在双蒸水中的化合物。稀MNI谷氨酸原液至5mM的正常ACSF的最终浓度。保持在4℃直至在实验中使用。不重新冻结，并且只使用一昼夜。商店等份，其中未立即使用在该实验中，在-20℃下。 </li></ol> 2，解剖组织注:啮齿动物的大脑急性海马脑片的制备在前面详细13,14被描述。简而言之，以下方案被用于本研究。 <ol><li>断头后，迅速从颅骨取出大脑。 </li><li>立即将大脑中的培养皿用冰冷的解剖和学联通过沿着中线矢状切解剖半球。 </li><li>在所需方向的阻挡表面进行第二次切割和连接这与强力胶Ävibratome的切割阶段。 </li><li>取切割室和冷却元件（均保持在-20℃）的vibratome出来的冷冻和地方切割阶段，在该腔室的大脑部分的。然后将冷却元件在所述腔室与浸没组织在冰冷的解剖ACSF。以稳定的制备中，人们可能想要以对抗与琼脂凝胶的组织块。 </li><li>切断与vibratome海马250微米厚，矢状窦旁片。确保所有的ACS流体鼓泡时刻。 </li><li>切片后，保持组织上的网格与正常学联烧杯和孵化在34℃，30分钟。然后在室温下保存。 </li></ol> 3，准备硬件 图1计划表示的光路和实验控制由多光子成像，激光扫描UV光解，和电生理的多光子束（红色） 的钻机的是通过脉冲，可调谐红外（IR）激光产生（TISA）。它通过一个机械快门，泡克耳斯细胞，并且IR检测器（检测光束强度的），所有这些都使激光功率和它的给药持续时间的控制。翻盖式/翻转出可选的激光二极管被用于激光束的基本取向。扩束器可以组合使用目标具有非常宽的后焦平面。远程控制的ND滤光轮可以代替泡克耳斯细胞，以控制激光束的功率相加或使用。通过扫描头后，脉冲的红外光被引导到被检体。发射泰德荧光光（绿色）是由外部或内部的光电倍增管检测器（光电倍增管）收集的任一。外部检测器通过高频开关（光电倍增管控制器）内部的光电倍增管完全同步。的解笼锁梁（浅蓝色）由紫外光的固态激光器（DPSS紫外激光）产生的。它然后被引导至检流计驱动的扫描单元的回顶部落射荧光冷凝器的由导光。精确定位的解笼锁点或区域（成像和解笼锁梁之间的色差）是通过从图像处理软件的图像导出启用。用于同步成像，电生理和闪光光解的时间管理是电子控制的。在透射检测器（TL-PMT）是需要在组织内的移液管的定位的文件。所述控制单元和成像系统，该系统已被​​修改的软件，用于控制和同步所有其它设备运行该系统所需的。作为"定制"的设计和建造/改编，作者系统组件标记。一些组件进行了调整，以满足定制打造多光子系统的要求，并标有"修改"。 <a href="https://jove.unicartagenaproxy.elogim.com/files/ftp_upload/52038/52038fig1highres.jpg" target="_blank">请点击这里查看该图的放大版本。</a> <ol><li>开关上的多光子系统的组件。测试和调整红外激光束对准。 <ol><li>控制光束定位通过翻转在定心棱镜而不是一个目标。如果激光束错位，通过在潜望镜镜重新调整。请注意，棱镜的中心平面必须在相同的水平，而成像在物镜的后焦平面中。 </li><li>开关上的光谱仪，并检查该光束的多光子特性。 </li><li>设置在成像软件的参数为下列值:选择的512×512像素的的小区（小夹拖曳以2.5为高帧率和高空间分辨率，分别缩放因子）概览图像的帧大小。设置成像速度快模式和扫描模式XYT。 Z型步进应为1微米的概述栈和0.2微米的详细协议栈，以匹配后进行反褶积空间采样要求。 </li></ol></li><li>与光电倍增器（光电倍增管） - :通过改变泡克耳斯细胞的设置（16μW≈14根据目标最终功率）调节多光子激光（790 nm）的强度。 </li><li>开机和通过将荧光样品玻片物镜下校准解笼锁系统。 <ol><li>使用双筒望远镜，调整UV光学器件的焦点和空间通过采用聚焦单元在UGA扫描头限制紫外激光点到2微米直径的尺寸为最大。 </li><li>启动解笼锁单元控制仿真​​软件的校准程序ê。 <ol><li>紫外激光设置为在其扫描范围的几个点，而荧光捕获用CCD照相机。通过点击在每个点处的紫外激光点，调整的电扫描镜的位置，以对应于一定的软件坐标。 </li></ol></li></ol></li></ol><img alt="图2" fo:content-width="5in" src="/files/ftp_upload/52038/52038fig2highres.jpg" width="500" /> 图2详细的方案示出激发，发射和解笼锁束路径的特性。红外激发光束（红）通过电子束在显微镜的落射荧光冷凝器和目的来合成分色镜在水平向文件管理器转台到达样品。的解笼锁梁（蓝色）经由石英光纤光光纤准直和束递送聚焦单元，并且随后被引导到扫描英里在扫描单元rrors，通过二向色镜的光束合成分色镜。在这里，它是结合激发扫描光束。从样品发出的光遵循朝向成像扫描头的相反方向的励磁扫描光束路径上的路径。相机被用于补丁吸管的视觉控制并且用于与所述激光扫描共聚焦显微镜组合的解笼锁梁的定位（未示出）。绿色的光路表示可以与其他应用程序使用的一个可选落射荧光照明。 <ol start="3"><li><ol start="2"><li><ol start="2"><li>利用闪光光解系统中的光斑模式获得焦点的应用程序。确保解笼锁梁（平均功率客观下方:0.55毫瓦）的焦距调整对应于业绩在1.5微米直径（XY扩展名）的光斑尺寸为透露，目前荧光滑动定位在Z-级组织片（未示出）。 </li></ol></li><li>的解笼锁点的准确定位是通过成像系统的图像的进口通过uncaging-和成像计算机之间的网络连接来实现的。 <ol><li>使用屏幕抓取软件，连续读出的图像处理软件（而不是相机饲料）的框架和同成分调整成像和解笼锁帧。导出每10 个帧，从所述成像软件作为参考图像到快闪单元，以确保在整个实验过程中适当调整。 </li></ol></li></ol></li></ol><img alt="图3" fo:content-width="6.5in" src="/files/ftp_upload/52038/52038fig3highres.jpg" width="600" /> 图3调整的解笼锁现货:精确定位解笼锁点，屏幕截图的图像处理软件（黄框）导入到解笼锁软件（绿框）中。该黄色框内左边的图像代表了高低编码图像的整个CA1锥体细胞（蓝色:饱和像素:黑色像素，红色）。该图像由解笼锁的软件（绿色的"X"）中的校准网格覆盖。到右边的单元格的放大部分所示的叠加解笼锁点（红十字会）。黄斑是解笼锁梁自动叠加图像。 <a href="https://jove.unicartagenaproxy.elogim.com/files/ftp_upload/52038/52038fig3highres.jpg" target="_blank">请点击这里查看该图的放大版本。</a> <ol start="4"><li>打开显微操作器，电组件，以及加压装置，用于输送的笼锁化合物的靶区域的。 </li><li>对于笼化合物的局部灌注安装一个焦点施加装置。相比于这些物质的浴灌注，这将降低成本巨大。调整保压给定的大气压力，以防止交流电的阻力顺丰进入或从应用吸管笼物质的泄漏。 注:施加装置承载高精度和超微型阀移液器支架。这使得采用最小的应用程序的压力，从而减少运动伪影，以最低的局部灌注的笼锁化合物中。 </li><li>拔用火抛光的硼硅玻璃毛细管吸液管为全细胞膜片钳和局部灌注。移液器应该有〜1微米的尖端直径情况R≈3MΩ（使用K-MESO 3系的ICS确定的值）的电阻。 </li><li>片放置在实验浴缸和一个网格贴上它（白金框250微米厚，跨区手术细丝，外圆40微米）（ 图4A，B）。使用倒置，尖折断，火抛光的巴斯德吸液管（吸球的破碎前端侧）的切片转移。避免弯腰和切片的任何其他恶劣的处理。 </li></ol&gt; <img alt="图4" fo:content-width="5in" src="/files/ftp_upload/52038/52038fig4highres.jpg" width="500" /> 图4的组件试验浴和水浴定位在显微镜载物台。（一）实验浴缸由洗浴室本身（蓝色方块），它是由磁性金属环包围。管确保了浴用盐水（ACSF）连续灌注。按住并固定片的网格放入浴室（B）急性切片准备定位在浴室中。在实验过程中的片被固定在网格的线（C）的定位和实验浴中在显微镜载物台的几何形状。 <ol start="8"><li>将沐浴在显微镜载物台，并永久灌注片与学联。 </li></ol> 4，全细胞膜片钳闭<ol><li>加载补丁吸管含SBFI和加载局部灌注吸管笼锁化合物的ICS。移液管连接到相应的显微操作。将参比电极的浴室。请确保您永久接地，以避免损坏磁头阶段（参见图4C）。 </li><li>放下两个吸管进浴室，并把他们的海马CA1区的上方。轻轻推压到补丁吸管（40毫巴），以避免稀释学联在ICS的。 </li><li>补偿使用电软件补丁吸管的偏移电位。 </li><li>接近补丁吸管采用IR-DIC的视频显微镜中的CA1锥体细胞。直到获得千兆密封适用于温和的吸力。选择的细胞胞体，其中位于30 -70微米的切片的表面之下，以确保在另一方面侧的解笼锁梁一方面侧和低散射和衰减完整的细胞形态。 </li><li>补偿快的能力。歇卷材胶粘e和开孔以获得全细胞构型。 </li><li>弥补缓慢的容量和串联电阻。 </li><li>允许细胞在开始成像实验之前进行透析与SBFI-ICS为至少30分钟。 </li></ol> 5，多光子成像和刺激<img alt="图5" fo:content-width="6in" src="/files/ftp_upload/52038/52038fig5highres.jpg" width="600" /> 图5的实验协议。要初始化一个实验中，触发脉冲（由红色标志（ᴨ）和红色线表示）被设置为同时开始成像（蓝色）时，电生理学（绿），和笼的管理化合物（黄色）在时间点0秒。在这个第一周期中，在成像光束应该完全变暗（浅蓝色）。经过4.5秒（虚线），该笼形化合物的局部灌注终止和成像光束应被设置为它的窝王强度的数据采集（蓝色）。由黄色闪光指示; 300毫秒:1.5秒后（时刻6秒），第二触发脉冲发出，初始化UV闪光灯（闪光的持续时间（以红色符号（ᴨ）和红色线表示） ），并设置在电生理学和成像协议标记。 <ol><li>添加河豚毒素（TTX，500纳米），以ACSF防止激活电压门控钠通道的动作电位的产生。 </li><li>可视化细胞形态使用多光子激发和产生SBFI荧光，然后选择一个带刺的树枝晶的实验​​。放大的图像在一个更高的分辨率，并将剪辑框周围的枝蔓。 </li><li>填写标准插吸管含谷氨酸笼10微升学联。移液管连接到压力应用系统，并将它附加到微操作。 </li><li>放置吸管与邻近的枝晶笼化合物（〜30微米）。放置吸管，让选择的枝晶的有效局部灌注。调整解笼锁激光:紧密地定位解笼锁点（〜1 - 2微米）到感兴趣的结构。 </li><li>对所选择的枝晶的兴趣，并使用图像处理软件相邻刺组区域。 </li><li>接近感兴趣的区域和灶性几秒钟以低压（&lt;3 PSI）注入的笼锁化合物。开始通过触发信号膜片钳和荧光录音（在790 nm的多光子激发）。 注:在笼形化合物的局部灌注，激发光束被完全变暗以防止褪色。 </li><li>停止笼化合物的局部灌注。提高双光子激光的强度，使SBFI有效激发并应用UV闪光初始化解笼锁（解笼锁的持续时间300毫秒）。 </li><li>监测变化SBFI荧光。停止录音后SBFI荧光已经恢复回基准。 </li></ol> 6，药理学<ol><li>来测试离子型谷氨酸受体在通过UV闪光笼谷氨酸的光分解引起的电流和/或钠的信号的生成的介入，采用受体阻滞剂。 <ol><li>切换到包含AMPA受体阻断氰基硝基喹喔啉二酮（CNQX，10μM）和NMDA受体阻断氨基phosphonopentanoate（APV，50μM）除了河豚毒素（见上文）ACSF和灌注切片，至少10分钟。 </li><li>重复刺激的过程（见5.4和5.5）。 </li></ol></li><li>的抑制剂"可逆性的影响<ol><li>切换到只含河豚毒素正常学联。 </li><li>灌注切片20分钟。 </li><li>重复刺激的过程（见5.4和5.5）。 </li></ol></li></ol> 7，形态<ol><li>录制XYZ堆栈的进行测量夹中区一套。确保这叠过采样ð空间（每个像素至少为0.2微米），以使最佳的图像去卷积，并提高图像质量和分辨率。 </li><li>录制XYZ栈的整个小区，以评估细胞形态。 </li><li>运行反卷积算法。 </li></ol>

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作者宣称没有利益冲突。笔者收到的财政支持拉普光电（威德尔，德国），产生在视频文章中使用的工具能够开放获取出版。该公司既不参与了实验，也没有在数据处理，也没有在手稿的写作。

本研究表明，SBFI非常适合在小细胞区室的细胞内钠瞬态双光子成像。它必须牢记，但是，SBFI的量子效率是相当低的15和相对变化的钠浓度是相当小的生理活性。因此， 钠离子瞬变精细工艺高分辨率测量是相对繁琐的任务，和分级的几次试验或平均化可能是必要的，以获得满意的信号。此外，钠的瞬变动力学是惊人的慢，使得它强制记录更长的时间内。这个观察对应于那些在早期的研究中，其中在神经元树突和星形胶质细胞钠瞬变单指数衰减，在室温下1,2,6其特征在于大的衰减时间常数在10秒的范围内。钠瞬变似乎从而发挥慢得多的时间合作URSE和更大的衰减时间常数相比，钙瞬变16。 抛开这些缺点，钠成像证明是神经元的子域生理特性的调查，一个有价值的工具。例如，钠成像可以用于在或接近活性突触并监控兴奋性突触活动。由于钠基本上没有缓冲8,17，活动引起的瞬态钠是线性相关的一系列突触谷氨酸释放或外源性应用谷氨酸。因此，他们代表神经元谷氨酸的活动直接和公正的指标。此外，钠指示剂染料表现出高K值d的（SBFI为其k d为25毫米范围1）。甚至在用于实现足够的亮度相对较高的细胞内的染料浓度（通常为0.5 - 1毫米）时，高K值d的暗示，染料本身不充当缓冲器š裂果。因此，他们不扭曲的钠瞬变幅度也不时程，这始终是一个问题时，钙敏感染料引入细胞18。因此，可以假设检测出的信号代表的胞内钠的"真实"的变化的良好量度。他们缓慢的时程则意味着速度为细胞内的扩散对钠的神经元比以前承担19小得多。 为成功执行实验针对的兴奋性突触传递和钠内流的途径进入棘和树突的性能的关键要求是突触结构的快速和高度本地化的活化诱导。这可以通过对谷氨酸或谷氨酸激动剂在脊柱的直接附近或枝状晶体通过笼化合物的闪光光解的快速和局部应用而得到。闪光光解不机械地按需分配GE的组织，是自由流动的文物，并确立对相关问题进行调查（ 如本地钙信号）。的解笼锁点的直径是1.5微米，在xy平面上。解笼锁接近刺枝晶致钠信号在两个刺及家长枝蔓。而该信号往往是最大的最接近解笼锁点刺，在父枝晶的振幅和棘较远仍然非常类似于那些在解笼锁点的附近。解笼锁下进行300毫秒期间内向电流的幅值增加。在同一时间段出笼谷氨酸可能扩散到组织中，激活离子型谷氨酸受体和钠涌入对树突状区域和棘进一步远离解笼锁点。 在使用紫外线激光器通过细胞沐浴液投笼化合物光解时，会出现一个固有的问题是"内在的过滤"。因为保持器的高吸收率，UV光沿着从物镜到的刺激部位20,21的方式强烈地衰减。为了避免这一个可行的办法是局部灌注与只局限于刺激部位，这进一步减小所需的最小笼锁化合物的量的笼子。相比于紫外激光扫描介导的解笼锁，近红外双光子解笼锁22是空间上更精确，这主要是因为刺激的精度在z轴增加。另一方面，由于通常使用的笼子的如采用与双光子激发，或者以高浓度的笼形化合物的或非常高，则需要潜在的光毒性的光强度波长较长的小横截面。此外，双光子解笼锁就必须更高投资设备;在其他中，一个附加的红外激光加所需的光学元件，还需要。 既SBFI和MNI-谷氨酸是可激发的在相同的波长范围内。这可能会导致紫外线解笼锁激光和SBFI或红外激光成像和MNI-谷氨酸的非预期的相互依赖性，导致SBFI漂白的解笼锁闪光和/或谷氨酸的由红外线光束的连续解笼锁。然而， 如图6C所示 ，无论其本身也不多光子成像的UV闪光灯引起我们的实验条件下，这种相互影响。 类似于此处所描述的一种UV-闪光系统通常用于在许多其他实验室，并可以相对容易地被结合到任何现有的多光子成像显微镜。它提供了使激光束为光解可自由在视场定位的优点。此外，该系统使激光束的快速和自动的重新定位和释放体积，分别在视场中的实验过程中。欧ř修改简化和改进了的解笼锁点的准确定位，以使得成像软件的帧可用于同成分调整成像和解笼锁帧。 两者合计，全细胞膜片钳和多光子钠成像在树突和中枢神经元的棘，结合谷氨酸的紫外光诱导的解笼锁的变形过程中，使谷氨酸受体在组织中的可靠和焦活化。因此，它可以用来分析突触后信号钠兴奋性突触传递和性能的神经元在完整的组织。

在诸如多光子显微术的光显微技术的最新改进已启用的脑细胞的形态和生理参数的研究，在完整的组织具有较高的空间和时间分辨率。联合电生理学，这些技术现在被广泛用于分析活动有关的神经元的电信号，以及随之而来的钙信号中的小的亚细胞区室，即在细树突和树突棘。除了钙瞬变，突触活动也诱导树突和棘钠的信号，其中的性能是基本未开发。这样的信号可以通过细胞内钠的双光子成像（[Na +]为ⅰ）它使在线测量[的Na +] i的瞬时长时间不显著染料漂白或光损伤1,2进行分析。 对于[娜+] i的成像</子&gt;，只有少数化学指示剂染料可用， 如电晕绿色或阿散蒂绿钠3,4。最常用的荧光探针的Na +成像是钠结合的苯并呋喃间苯二甲酸酯（SBFI）。它是一种比例，UV激发类似于众所周知的钙敏感染料呋喃-2染料，并已用于常规的Na +成像在许多细胞类型（ 例如，5,6）。有令人兴奋的染料和收集其荧光的不同的可能性。如果高时间分辨率（ 即高的成像帧速率）是必需的，具有空间信息结合，SBFI可兴奋与高速充电检测的氙弧灯或高功率发光二极管（LED）器件和其发射耦联器件（CCD）摄像机7,8。为深的组织最大空间分辨率，多光子激光扫描显微术是选择9的方法。相对较低的量子efficieSBFI的NCY就必须相对较高的染料浓度（0.5 - 2毫米），并通过一个尖锐的微电极10或膜片电极1直接加载SBFI的不可透过膜的形式的。 使用SBFI，在啮齿动物海马急性组织切片进行的早期工作证明了这些主要是通过离子型NMDA受体引起钠涌入受体1,2树突CA1锥体神经元和刺活动相关的钠瞬变。为更详细地，例如本地钠信号的特性的研究中，突触后受体通过应用受体激动剂中的特定激活是一个选择的非常适合的方法。为了模拟突触前活性和递质释放，应用程序应该是比较简短，重点突出，使局部刺激。然而，这被证明是在完整的组织相当有挑战性的。用细尖的吸管受体激动剂的局部压力应用，使非常焦距AP褶皱，但承载用于产生感兴趣结构的运动（ 例如，诸如枝状晶体或树突）的潜在风险，并因此阻碍了高分辨率成像。的神经活性物质的离子电渗疗法的适合性取决于它们的电性能和高的电流幅值可能产生蜂窝工件为好。 绕过这些障碍的方法之一是光激活化合物和其光解就业。基本上，用于光激活笼物质的两种不同的原则:1）宽视场光解11和II）焦解笼锁采用扫描模块与激光器12的组合。而宽视场光解被用于激活放大感兴趣的区域， 例如 ，一个完整的细胞，焦解笼锁被用来特异性刺激小细胞区室。在本研究中，我们证明了全细胞膜片钳和多页记载的方法HOTON钠成像在树突和中枢神经元的棘，结合对紫外光诱导的解笼锁谷氨酸，它允许谷氨酸受体在组织中的可靠和焦活化的改性过程。

<table><tbody><tr><td>Borosilicate-glass capillaries</td><td>Hilgenberg</td><td>1405059</td><td>75 x 2 mm, wall thickness 0.3 mm</td></tr><tr><td>Camera</td><td>Jenoptik</td><td>ProgRes MF</td><td>IR-DIC compatible camera</td></tr><tr><td>Deconvolution software</td><td>SVI</td><td>Huygens Pofessional X11</td><td></td></tr><tr><td>Fiber optic spectrometer</td><td>Ocean Optics</td><td>USB 4000</td><td>Miniature fiber optic spectrometer</td></tr><tr><td>Filter tubes</td><td>VWR</td><td></td><td>cenrifugal filter, 0.2 &amp;micro;m</td></tr><tr><td>Imaging software</td><td>Olympus Europe</td><td>Flowview Ver 5.0c, Tiempo</td><td></td></tr><tr><td>IR beam power controller</td><td>Custom built</td><td></td><td>Laser beam intensity control + fast switch to zero on flyback via pockels cell</td></tr><tr><td>IR laser</td><td>SpectraPhysics</td><td>Mai-Tai</td><td>fs-pulsed Ti:Sa tunable laser (710 - 990 nm); CAUTION</td></tr><tr><td>IR power monitor</td><td>Custom built</td><td></td><td></td></tr><tr><td>Micromanipulator</td><td>Burleigh</td><td>PCS 5000</td><td></td></tr><tr><td>Microscope/Imaging System</td><td>Olympus Europe</td><td>BX51WI/FV300</td><td>Both, the microscope and the imaging systems were strongly modified</td></tr><tr><td>ND filter wheel</td><td>Newport</td><td>50Q04AV.2</td><td>UV fused silica, optical density 0.05 - 2.0</td></tr><tr><td>Objective</td><td>Nikon Instruments Europe</td><td>NIR Apo 60x/1.0w</td><td></td></tr><tr><td>Patch clamp amplifier</td><td>HEKA</td><td>EPC-10</td><td></td></tr><tr><td>Pipette puller</td><td>Narishige</td><td>Model PP-830</td><td></td></tr><tr><td>Pneumatic drug ejection system</td><td>NPI Electronic</td><td>PDES-DXH</td><td></td></tr><tr><td>Pockels cell</td><td>Conoptics</td><td>350-80 LA-BK</td><td>EOM, used as fast switch and for power adjustment</td></tr><tr><td>Pockels cell driver</td><td>Conoptics</td><td>M302-RM</td><td>High voltage differential amplifier</td></tr><tr><td>Shutter</td><td>Vincent Associates</td><td>LS6ZM2</td><td>ALMgF&lt;sub&gt;2&lt;/sub&gt;-coated BeCu blades</td></tr><tr><td>Shutter controller</td><td>Custom built</td><td></td><td></td></tr><tr><td>Shutter driver</td><td>Vincent Associates</td><td>Uniblitz VCM D1</td><td></td></tr><tr><td>Slicer/Vibratome</td><td>Thermo Scientific</td><td>Microm HM 650 V</td><td></td></tr><tr><td>Uncaging software</td><td>Rapp OptoElectronic</td><td>UGA40 1.1.2.6 + NetCam</td><td></td></tr><tr><td>UV laser</td><td>Rapp OptoElectronic</td><td>DL-355/10</td><td>cw DPSSL Laser, 355 nm; 10 mW; CAUTION</td></tr><tr><td>UV laser scanning system</td><td>Rapp OptoElectronic</td><td>UGA 40</td><td>Galvanometric scanning system</td></tr><tr><td>&lt;strong&gt;Name of Compound&lt;/strong&gt;</td><td>&lt;strong&gt;Company&lt;/strong&gt;</td><td>&lt;strong&gt;Catalog Number&lt;/strong&gt;</td><td>&lt;strong&gt;Comments/&lt;br /&gt;Description&lt;/strong&gt;</td></tr><tr><td>CNQX</td><td>Sigma Aldrich</td><td>C-127</td><td>AMPA receptor antagonist; CAUTION</td></tr><tr><td>DL-AP5</td><td>Alfa Aesar</td><td>J64210</td><td>NMDA receptor antagonist; CAUTION</td></tr><tr><td>SBFI K&lt;sup&gt;+&lt;/sup&gt; salt</td><td>Teflabs</td><td>OO32</td><td></td></tr><tr><td>TTX</td><td>Ascent Scientific</td><td>Asc-055</td><td>Inhibitor of voltage-dependent Na+ channels; CAUTION</td></tr><tr><td>NMI-caged-L-Glutamate</td><td>Torcis</td><td>1490</td><td>Caged glutamate released upon UV absobtion</td></tr></tbody></table>

multi-photon intracellular sodium imaging combined with uv-mediated focal uncaging of glutamate in ca1 pyramidal neurons

多光子荧光显微镜，使脑细胞的形态和生理参数的分析，在完整的组织具有较高的空间和时间分辨率。再加上电，它被广泛用于研究活动相关的钙信号在小亚细胞如树突和树突棘。除了钙瞬变，突触活动也诱导突触后钠信号，其中仅略微理解的属性。在这里，我们描述了结合全细胞膜片钳和多光子成像钠在中央神经元细胞的微结构域的方法。此外，我们介绍了紫外（UV）谷氨酸 - 光诱导的解笼锁，这使得谷氨酸受体在组织中的可靠和焦活化的改性过程。为此，全细胞记录了上考纽Ammonis细分1（CA1）中的急性组织切片锥体神经元进行小鼠海马。神经元中装满通过膜片移液管中的钠敏感荧光染料SBFI和SBFI的多光子激发启用树突和相邻脊的可视化。建立由紫外线引起的焦解笼锁，几个参数，包括光的强度，体积受紫外线解笼锁梁，梁的定位以及笼形化合物的浓度进行了测试和优化。我们的研究结果表明，与笼谷氨酸（MNI-谷氨酸）局部灌注，其焦点紫外光解笼锁导致的内向电流和瞬态钠树突棘。时间过程和两个内向电流和钠的信号的振幅相关的解笼锁脉冲的持续时间。此外，我们的结果表明，细胞内的钠信号被阻断的阻断剂的存在下进行离子型谷氨酸受体，这表明它们是由潮虽然该通路钠介导的。总之，我们的方法提供了一个可靠的工具调查大鼠局灶性受体激活在完整的脑组织细胞内钠的信号。

在本研究中，我们证明了在急性小鼠海马脑组织切片CA1锥体神经元的钠依赖性荧光染料SBFI细胞钠动态的多光子显微镜的过程。此外，我们将展示如何将这些成像技术结合了神经活性化合物的激光扫描为基础的解笼锁（ 如笼谷氨酸）及其精确靶向细胞微域。 中通过膜片吸管神经元SBFI使整个小区的可视化包括精细枝晶和通过使用多光子激发相邻的棘（ 图6A，B，D和图7A）。 <img alt="图6" fo:content-width="5in" src="/files/ftp_upload/52038/52038fig6highres.jpg" width="500" /> 图6钠信号，诱导笼谷氨酸的光解突触电流（A）美心通过补丁吸管（PP）的装载SBFIàCA1锥体神经元的人的投影图像。该框表示放大显示B.唱片中的区域表示吸管为笼谷氨酸的局部灌注的位置和方向（B）的堆叠与相邻的树突枝状晶体的光学切片的高功率最大突出。光学部分的行栈Z-对齐和反褶积。红十字会表示解笼锁束的靶区域。橙色虚线描绘的兴趣从该荧光发射被记录的区域。该框表示离开了放大显示在D（C）面积:诱导笼谷氨酸（由黄色闪烁表示）的光解钠信号（ 上排 ）和体细胞内向电流（ 下行 ）。红色的线代表一个合适的实验数据。灰色区域表示的期间内的解笼锁闪光灯（300毫秒）阻塞SBFI荧光成像权:没有预灌注笼谷氨酸，同样的紫外线闪光灯既不诱发SBFI排放量的变化，也不是一个内向电流（D）左 :树突和邻近的高功率形象刺的划定B.除了与倒灰度值相同的图像。虚线表示的兴趣，其中荧光发射被记录的区域。红十字会表示解笼锁梁的本地化右:诱发笼谷氨酸紫外线光解瞬态钠。蓝色曲线:在枝晶钠的信号;红色曲线:平均从毗邻解笼锁点3刺响应;绿色线:场均三遥远刺的回应。 <a href="https://jove.unicartagenaproxy.elogim.com/files/ftp_upload/52038/52038fig6highres.jpg" target="_blank">请点击这里了解这个数字的放大版本。</a> 后局部灌注笼谷氨酸盐，施加紫外线闪光灯接近枝状晶体导致在SBFI的荧光发射瞬时下降，增加细胞内钠浓度（ 图6C，D和图7B）。同时，一个向内的电流被记录在体细胞（ 图6C和图7B）。增加UV闪光的持续时间造成了增加两者引起内向电流和钠的信号（数据未显示）的幅度，表明该系统是完全在其动态范围，并且既不解笼锁，也不细胞反应被饱和。紫外线闪光灯的相同或更长的持续时间的应用到片所未曾预灌注笼谷氨酸，从不引起变化SBFI荧光也不内向电流，这表明这些信号是由于谷氨酸解笼锁（ 图6C）。此外，这些结果表明，不相互依赖的imaging-及解笼锁部件是观察Ú升气管我们的实验条件。钠的信号也可以在树突（ 图6D）检测。虽然我们并没有试图实现单棘只有谷氨酸解笼锁的刺激，峰值幅度往往是在刺靠近解笼锁点略高，而棘渐行渐远呈现几乎相同的荧光变化父枝晶（ 图6D）。 最后，我们研究了该途径为钠涌入树突和棘响应于谷氨酸解笼锁。为此，我们采用CNQX和APV，它们是选择性的阻断剂为钠透，离子型谷氨酸的AMPA-和NMDA亚型，分别的受体。我们的结果表明，谷氨酸诱导的细胞内钠的信号和被诱发体细胞的电流在这些阻滞剂（ 图7B）的存在下被删去。在洗出的阻断剂，该信号被恢复。这表明塔谷氨酸吨解笼锁启动对CA1区锥体神经元介导的钠涌入树突棘，导致细胞内钠瞬态和内向电流离子型谷氨酸受体。 <img alt="图7" fo:content-width="6in" src="/files/ftp_upload/52038/52038fig7highres.jpg" width="600" /> 诱发钠信号和内向电流图7的药理学特性。 （A）SBFI填充的CA1锥体神经元与膜片电极（PP）附着在体细胞（B）左:钠瞬态和在枝晶通过光激活笼谷氨酸诱导的体电流和附加棘中心:灌注用离子型谷氨酸受体拮抗剂CNQX和APV抑制两者的钠信号和诱导解笼锁的内向电流右:当洗出的阻断剂，该信号被恢复。红线代表中TS一个合适的实验数据。灰色区域表示该解笼锁闪光灯（300毫秒）阻塞SBFI荧光成像的时期。 <a href="https://jove.unicartagenaproxy.elogim.com/files/ftp_upload/52038/52038fig7highres.jpg" target="_blank">请点击这里了解这个数字的放大版本。</a>

Watch this Scientific Journal Video about Multi-photon Intracellular Sodium Imaging Combined with UV-mediated Focal Uncaging of Glutamate in CA1 Pyramidal Neurons at JoVE.com

Multi-photon Intracellular Sodium Imaging Combined with UV-mediated Focal Uncaging of Glutamate in CA1 Pyramidal Neurons

我们描述了焦紫外线引起的光激活的神经活性化合物与全细胞膜片钳和在枝晶和海马神经元的棘在小鼠脑的急性组织切片的细胞​​内钠瞬变多光子成像的组合。

多光子细胞内钠成像结合谷氨酸海马CA1锥体神经元的UV-介导的焦点解笼锁

Multi-photon fluorescence microscopy has enabled the analysis of morphological and physiological parameters of brain cells in the intact tissue with high ...

multi-photon-intracellular-sodium-imaging-combined-with-uv-mediated

Universidad de Cartagena UDEC

Research

JoVE Journal

Neuroscience

14.0K 次观看.  Heinrich Heine University Düsseldorf. 我们描述了焦紫外线引起的光激活的神经活性化合物与全细胞膜片钳和在枝晶和海马神经元的棘在小鼠脑的急性组织切片的细胞​​内钠瞬变多光子成像的组合。 

视频: Multi-photon Intracellular Sodium Imaging Combined with UV-mediated Focal Uncaging of Glutamate in CA1 Pyramidal Neurons

Vibrodissociation of Neurons from Rodent Brain Slices to Study Synaptic Transmission and Image Presynaptic Terminals

Mechanical dissociation of neurons from the central nervous system has the advantage that presynaptic boutons remain attached to the isolated neuron of interest. This allows for examination of synaptic transmission under conditions where the extracellular and postsynaptic intracellular environments can be well controlled. A vibration-based technique without the use of proteases, known as vibrodissociation, is the most popular technique for mechanical isolation. A micropipette, with the tip fire-polished to the shape of a small ball, is placed into a brain slice made from a P1-P21 rodent. The micropipette is vibrated parallel to the slice surface and lowered through the slice thickness resulting in the liberation of isolated neurons. The isolated neurons are ready for study within a few minutes of vibrodissociation. This technique has advantages over the use of primary neuronal cultures, brain slices and enzymatically isolated neurons including: rapid production of viable, relatively mature neurons suitable for electrophysiological and imaging studies; superior control of the extracellular environment free from the influence of neighboring cells; suitability for well-controlled pharmacological experiments using rapid drug application and total cell superfusion; and improved space-clamp in whole-cell recordings relative to neurons in slice or cell culture preparations. This preparation can be used to examine synaptic physiology, pharmacology, modulation and plasticity. Real-time imaging of both pre- and postsynaptic elements in the living cells and boutons is also possible using vibrodissociated neurons. Characterization of the molecular constituents of pre- and postsynaptic elements can also be achieved with immunological and imaging-based approaches.

This report demonstrates a technique for mechanical isolation of individual viable neurons retaining attached presynaptic boutons. Vibrodissociated neurons have the advantages of rapid production, excellent pharmacological control and improved space-clamp without influence from neighboring cells. This method can be used for imaging of synaptic elements and patch-clamp recording.

Vibrodissociation从啮齿动物脑切片的神经元，研究突触传递和图像突触前的码头

Mechanical dissociation of neurons from the central nervous system has the advantage that presynaptic boutons remain attached to the isolated neuron of ...

科研

神经科学

In vivo Neuronal Calcium Imaging in C. elegans

The nematode worm C. elegans is an ideal model organism for relatively simple, low cost neuronal imaging in vivo. Its small transparent body and simple, well-characterized nervous system allows identification and fluorescence imaging of any neuron within the intact animal. Simple immobilization techniques with minimal impact on the animal's physiology allow extended time-lapse imaging. The development of genetically-encoded calcium sensitive fluorophores such as cameleon 1 and GCaMP 2 allow in vivo imaging of neuronal calcium relating both cell physiology and neuronal activity. Numerous transgenic strains expressing these fluorophores in specific neurons are readily available or can be constructed using well-established techniques. Here, we describe detailed procedures for measuring calcium dynamics within a single neuron in vivo using both GCaMP and cameleon. We discuss advantages and disadvantages of both as well as various methods of sample preparation (animal immobilization) and image analysis. Finally, we present results from two experiments: 1) Using GCaMP to measure the sensory response of a specific neuron to an external electrical field and 2) Using cameleon to measure the physiological calcium response of a neuron to traumatic laser damage. Calcium imaging techniques such as these are used extensively in C. elegans and have been extended to measurements in freely moving animals, multiple neurons simultaneously and comparison across genetic backgrounds. C. elegans presents a robust and flexible system for in vivo neuronal imaging with advantages over other model systems in technical simplicity and cost.

In vivo Neuronal Calcium Imaging in C. elegans

With its small transparent body, well-documented neuroanatomy and a host of amenable genetic techniques and reagents, C. elegans makes an ideal model organism for in vivo neuronal imaging using relatively simple, low-cost techniques. Here we describe single neuron imaging within intact adult animals using genetically encoded fluorescent calcium indicators.

C.在体内神经元钙成像技术线虫

The nematode worm C. elegans is an ideal model organism  for relatively simple, low cost neuronal imaging in vivo. Its small transparent body and simple, ...

Simultaneous Electrophysiological Recording and Calcium Imaging of Suprachiasmatic Nucleus Neurons

Simultaneous electrophysiological and fluorescent imaging recording methods were used to study the role of changes of membrane potential or current in regulating the intracellular calcium concentration. Changing environmental conditions, such as the light-dark cycle, can modify neuronal and neural network activity and the expression of a family of circadian clock genes within the suprachiasmatic nucleus (SCN), the location of the master circadian clock in the mammalian brain. Excitatory synaptic transmission leads to an increase in the postsynaptic Ca2+ concentration that is believed to activate the signaling pathways that shifts the rhythmic expression of circadian clock genes. Hypothalamic slices containing the SCN were patch clamped using microelectrodes filled with an internal solution containing the calcium indicator bis-fura-2. After a seal was formed between the microelectrode and the SCN neuronal membrane, the membrane was ruptured using gentle suction and the calcium probe diffused into the neuron filling both the soma and dendrites. Quantitative ratiometric measurements of the intracellular calcium concentration were recorded simultaneously with membrane potential or current. Using these methods it is possible to study the role of changes of the intracellular calcium concentration produced by synaptic activity and action potential firing of individual neurons. In this presentation we demonstrate the methods to simultaneously record electrophysiological activity along with intracellular calcium from individual SCN neurons maintained in brain slices.

Procedures are described to perform simultaneous recordings of membrane potential or current and changes of intracellular calcium concentration. Suprachiasmatic nucleus neurons are filled with the calcium indicator bis-fura-2 using a patch clamp electrode in the whole cell patch clamp configuration.

Simultaneous electrophysiological and fluorescent imaging recording methods were used to study the role of changes of membrane potential or current in ...

Two-photon Calcium Imaging in Neuronal Dendrites in Brain Slices

Calcium (Ca2+) imaging is a powerful tool to investigate the spatiotemporal dynamics of intracellular Ca2+ signals in neuronal dendrites. Ca2+ fluctuations can occur through a variety of membrane and intracellular mechanisms and play a crucial role in the induction of synaptic plasticity and regulation of dendritic excitability. Hence, the ability to record different types of Ca2+ signals in dendritic branches is valuable for groups studying how dendrites integrate information. The advent of two-photon microscopy has made such studies significantly easier by solving the problems inherent to imaging in live tissue, such as light scattering and photodamage. Moreover, through combination of conventional electrophysiological techniques with two-photon Ca2+ imaging, it is possible to investigate local Ca2+ fluctuations in neuronal dendrites in parallel with recordings of synaptic activity in soma. Here, we describe how to use this method to study the dynamics of local Ca2+ transients (CaTs) in dendrites of GABAergic inhibitory interneurons. The method can be also applied to studying dendritic Ca2+ signaling in different neuronal types in acute brain slices.

We present a method combining whole-cell patch-clamp recordings and two-photon imaging to record Ca2+ transients in neuronal dendrites in acute brain slices.

脑切片神经元树突的双光子钙成像

Calcium (Ca2+) imaging is a powerful tool to investigate the spatiotemporal dynamics of intracellular Ca2+ signals in neuronal dendrites. Ca2+ ...

Ratiometric Calcium Imaging of Individual Neurons in Behaving Caenorhabditis Elegans

It has become increasingly clear that neural circuit activity in behaving animals differs substantially from that seen in anesthetized or immobilized animals. Highly sensitive, genetically encoded fluorescent reporters of Ca2+ have revolutionized the recording of cell and synaptic activity using non-invasive optical approaches in behaving animals. When combined with genetic and optogenetic techniques, the molecular mechanisms that modulate cell and circuit activity during different behavior states can be identified.
Here we describe methods for ratiometric Ca2+ imaging of single neurons in freely behaving Caenorhabditis elegans worms. We demonstrate a simple mounting technique that gently overlays worms growing on a standard Nematode Growth Media (NGM) agar block with a glass coverslip, permitting animals to be recorded at high-resolution during unrestricted movement and behavior. With this technique, we use the sensitive Ca2+ reporter GCaMP5 to record changes in intracellular Ca2+ in the serotonergic Hermaphrodite Specific Neurons (HSNs) as they drive egg-laying behavior. By co-expressing mCherry, a Ca2+-insensitive fluorescent protein, we can track the position of the HSN within ~ 1 &#181;m and correct for fluctuations in fluorescence caused by changes in focus or movement. Simultaneous, infrared brightfield imaging allows for behavior recording and animal tracking using a motorized stage. By integrating these microscopic techniques and data streams, we can record Ca2+ activity in the C. elegans egg-laying circuit as it progresses between inactive and active behavior states over tens of minutes.

Ratiometric Calcium Imaging of Individual Neurons in Behaving Caenorhabditis Elegans

This protocol describes the use of genetically encoded Ca2+ reporters to record changes in neural activity in behaving Caenorhabditis elegans worms. &#8195;

比率钙显像对单个神经元的行为线虫线虫

It has become increasingly clear that neural circuit activity in behaving animals differs substantially from that seen in anesthetized or immobilized ...

Single Synapse Indicators of Glutamate Release and Uptake in Acute Brain Slices from Normal and Huntington Mice

Synapses are highly compartmentalized functional units that operate independently on each other. In Huntington&#39;s disease (HD) and other neurodegenerative disorders, this independence might be compromised due to insufficient glutamate clearance and the resulting spill-in and spill-out effects. Altered astrocytic coverage of the presynaptic terminals and/or dendritic spines as well as a reduced size of glutamate transporter clusters at glutamate release sites have been implicated in the pathogenesis of diseases resulting in symptoms of dys-/hyperkinesia. However, the mechanisms leading to the dysfunction of glutamatergic synapses in HD are not well understood. Improving and applying synapse imaging we have obtained data shedding new light on the mechanisms impeding the initiation of movements. Here, we describe the principle elements of a relatively inexpensive approach to achieve single synapse resolution by using the new genetically encoded ultrafast glutamate sensor iGluu, wide-field optics, a scientific CMOS (sCMOS) camera, a 473 nm laser and a laser positioning system to evaluate the state of corticostriatal synapses in acute slices from age appropriate healthy or diseased mice. Glutamate transients were constructed from single or multiple pixels to obtain estimates of i) glutamate release based on the maximal elevation of the glutamate concentration [Glu] next to the active zone and ii) glutamate uptake as reflected in the time constant of decay (TauD) of the perisynaptic [Glu]. Differences in the resting bouton size and contrasting patterns of short-term plasticity served as criteria for the identification of corticostriatal terminals as belonging to the intratelencephalic (IT) or the pyramidal tract (PT) pathway. Using these methods, we discovered that in symptomatic HD mice ~40% of PT-type corticostriatal synapses exhibited insufficient glutamate clearance, suggesting that these synapses might be at risk to excitotoxic damage. The results underline the usefulness of TauD as a biomarker of dysfunctional synapses in Huntington mice with a hypokinetic phenotype.

We present a protocol to evaluate the balance between glutamate release and clearance at single corticostriatal glutamatergic synapses in acute slices from adult mice. This protocol uses the fluorescent sensor iGluu for glutamate detection, a sCMOS camera for signal acquisition and a device for focal laser illumination.

正常小鼠和亨廷顿小鼠急性脑切片中谷氨酸释放和服用的单突触指标

Synapses are highly compartmentalized functional units that operate independently on each other. In Huntington&#39;s disease (HD) and other ...

Quantifying Spontaneous Ca2+ Fluxes and their Downstream Effects in Primary Mouse Midbrain Neurons

Parkinson&#8217;s disease (PD) is a devastating neurodegenerative disorder caused by the degeneration of dopaminergic (DA) neurons. Excessive Ca2+ influx due to the abnormal activation of glutamate receptors results in DA excitotoxicity and has been identified as an important mechanism for DA neuron loss. In this study, we isolate, dissociate, and culture midbrain neurons from the mouse ventral mesencephalon (VM) of ED14 mouse embryos. We then infect the long-term primary mouse midbrain cultures with an adeno-associated virus (AAV) expressing a genetically encoded calcium indicator, GCaMP6f under control of the human neuron-specific synapsin promoter, hSyn. Using live confocal imaging, we show that cultured mouse midbrain neurons display spontaneous Ca2+ fluxes detected by AAV-hSyn-GCaMP6f. Bath application of glutamate to midbrain cultures causes abnormal elevations in intracellular Ca2+ within neurons and this is accompanied by caspase-3 activation in DA neurons, as demonstrated by immunostaining. The techniques to identify glutamate-mediated apoptosis in primary mouse DA neurons have important applications for the high content screening of drugs that preserve DA neuron health.

Quantifying Spontaneous Ca2+ Fluxes and their Downstream Effects in Primary Mouse Midbrain Neurons

Here we present a protocol to measure in vitro Ca2+ fluxes in midbrain neurons and their downstream effects on caspase-3 using primary mouse midbrain cultures. This model can be employed to study pathophysiologic changes related to abnormal Ca2+ activity in midbrain neurons, and to screen novel therapeutics for anti-apoptotic properties.

定量自发 Ca2+ 通量及其在原发性小鼠中脑神经元中的下游效应

Parkinson&#8217;s disease (PD) is a devastating neurodegenerative disorder caused by the degeneration of dopaminergic (DA) neurons. Excessive Ca2+ influx ...

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 ...

In Vivo Calcium Imaging of Neuronal Ensembles in Networks of Primary Sensory Neurons in Intact Dorsal Root Ganglia

Ca2+ imaging can be used as a proxy for cellular activity, including action potentials and various signaling mechanisms involving Ca2+ entry into the cytoplasm or the release of intracellular Ca2+ stores. Pirt-GCaMP3-based Ca2+ imaging of primary sensory neurons of the dorsal root ganglion (DRG) in mice offers the advantage of simultaneous measurement of a large number of cells. Up to 1,800 neurons can be monitored, allowing neuronal networks and somatosensory processes to be studied as an ensemble in their normal physiological context at a populational level in vivo. The large number of neurons monitored allows the detection of activity patterns that would be challenging to detect using other methods. Stimuli can be applied to the mouse hindpaw, allowing the direct effects of stimuli on the DRG neuron ensemble to be studied. The number of neurons producing Ca2+ transients as well as the amplitude of Ca2+ transients indicates sensitivity to specific sensory modalities. The diameter of neurons provides evidence of activated fiber types (non-noxious mechano vs. noxious pain fibers, A&#946;, A&#948;, and C fibers). Neurons expressing specific receptors can be genetically labeled with td-Tomato and specific Cre recombinases together with Pirt-GCaMP. Therefore, Pirt-GCaMP3 Ca2+ imaging of DRG provides a powerful tool and model for the analysis of specific sensory modalities and neuron subtypes acting as an ensemble at the populational level to study pain, itch, touch, and other somatosensory signals.

In Vivo Calcium Imaging of Neuronal Ensembles in Networks of Primary Sensory Neurons in Intact Dorsal Root Ganglia

This protocol describes the surgical exposure of the dorsal root ganglion (DRG) followed by GCaMP3 (genetically-encoded Ca2+ indicator; Green Fluorescent Protein-Calmodulin-M13 Protein 3) Ca2+ imaging of the neuronal ensembles using Pirt-GCaMP3 mice while applying a variety of stimuli to the ipsilateral hind paw.

体内 完整背根神经节中初级感觉神经元网络中神经元集合体的钙成像

Ca2+ imaging can be used as a proxy for cellular activity, including action potentials and various signaling mechanisms involving Ca2+ entry into the ...

Subcellular Imaging of Neuronal Calcium Handling In Vivo

Calcium (Ca2+) imaging has been largely used to examine neuronal activity, but it is becoming increasingly clear that subcellular Ca2+ handling is a crucial component of intracellular signaling. The visualization of subcellular Ca2+ dynamics in vivo,&#160;where neurons can be studied in their native, intact circuitry, has proven technically challenging in complex nervous systems. The transparency and relatively simple nervous system of the nematode Caenorhabditis elegans enable the cell-specific expression and in vivo visualization of fluorescent tags and indicators. Among these are fluorescent indicators that have been modified for use in the cytoplasm as well as various subcellular compartments, such as the mitochondria. This protocol enables non-ratiometric Ca2+ imaging in vivo with a subcellular resolution that permits the analysis of Ca2+ dynamics down to the level of individual dendritic spines and mitochondria. Here, two available genetically encoded indicators with different Ca2+ affinities are used to demonstrate the use of this protocol for measuring relative Ca2+ levels within the cytoplasm or mitochondrial matrix in a single pair of excitatory interneurons (AVA). Together with the genetic manipulations and longitudinal observations possible in C. elegans, this imaging protocol may be useful for answering questions regarding how Ca2+ handling regulates neuronal function and plasticity.

Subcellular Imaging of Neuronal Calcium Handling In Vivo

The current methods describe a non-ratiometric approach for high-resolution, sub-compartmental calcium imaging in vivo in Caenorhabditis elegans using readily available genetically encoded calcium indicators.

体内神经元钙处理的亚细胞成像

Calcium (Ca2+) imaging has been largely used to examine neuronal activity, but it is becoming increasingly clear that subcellular Ca2+ handling is a ...

多光子细胞内钠成像结合谷氨酸海马CA1锥体神经元的UV-介导的焦点解笼锁

摘要

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此视频中的章节

Vibrodissociation从啮齿动物脑切片的神经元，研究突触传递和图像突触前的码头

C.在体内神经元钙成像技术线虫

Simultaneous Electrophysiological Recording and Calcium Imaging of Suprachiasmatic Nucleus Neurons

脑切片神经元树突的双光子钙成像

比率钙显像对单个神经元的行为线虫线虫

正常小鼠和亨廷顿小鼠急性脑切片中谷氨酸释放和服用的单突触指标

定量自发 Ca²⁺ 通量及其在原发性小鼠中脑神经元中的下游效应

肌萎缩性侧索硬化症模型中突触功能的实时荧光测量

体内完整背根神经节中初级感觉神经元网络中神经元集合体的钙成像

体内神经元钙处理的亚细胞成像

多光子细胞内钠成像结合谷氨酸海马CA1锥体神经元的UV-介导的焦点解笼锁

摘要

探索更多视频

此视频中的章节

Vibrodissociation从啮齿动物脑切片的神经元，研究突触传递和图像突触前的码头

C.在体内神经元钙成像技术线虫

Simultaneous Electrophysiological Recording and Calcium Imaging of Suprachiasmatic Nucleus Neurons

脑切片神经元树突的双光子钙成像

比率钙显像对单个神经元的行为线虫线虫

正常小鼠和亨廷顿小鼠急性脑切片中谷氨酸释放和服用的单突触指标

定量自发 Ca2+ 通量及其在原发性小鼠中脑神经元中的下游效应

肌萎缩性侧索硬化症模型中突触功能的实时荧光测量

体内 完整背根神经节中初级感觉神经元网络中神经元集合体的钙成像

体内神经元钙处理的亚细胞成像

定量自发 Ca²⁺ 通量及其在原发性小鼠中脑神经元中的下游效应

体内完整背根神经节中初级感觉神经元网络中神经元集合体的钙成像